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Signaling and Signaling Pathways

Posted in Biological Networks, Gene Regulation and Evolution, Biomedical Measurement Science, Calcium Signaling, CANCER BIOLOGY & Innovations in Cancer Therapy, Cardiovascular Research, Cell Biology, Signaling & Cell Circuits, Curation methodology, Cytoskeleton, Developmental biology, Endocrine Diseases, Enzyme Induction, Epigenetics and Cardiovascular Risks, Evolutionary cognition, Explanatory, Frontiers in Cardiology and Cardiovascular Disorders, Gene Regulation, Human Immune System in Health and in Disease, Ionic Transporters Na+, Metabolomics, Na-K transport, Neurohumoral Transmission, Nitric Oxide in Health and Disease, Pharmaceutical Analytics, Pharmaceutical Drug Discovery, Pharmacotherapy and Cell Activity, Proteomics, Technology Advance Assessment of, tagged direct cell contact, distant effects, Intercellular (Paracellular) route, intercellular junctions, paracrine and juxtacrine, protein conformation, signaling, signaling pathways, surface charges on August 12, 2014| Leave a Comment »

Signaling and Signaling Pathways

Curator: Larry H. Bernstein, MD, FCAP

http://pharmaceuticalintelligence.com/8-9-2014/Signaling and Signaling Pathways

This portion of the discussion is a series of articles on signaling and signaling pathways. Many of the protein-protein interactions or protein-membrane interactions and associated regulatory features have been referred to previously, but the focus of the discussion or points made were different. I considered placing this after the discussion of proteins and how they play out their essential role, but this is quite a suitable place for a progression to what follows. This is introduced by material taken from Wikipedia, which will be followed by a series of mechanisms and examples from the current literature, which give insight into the developments in cell metabolism, with the later goal of separating views introduced by molecular biology and genomics from functional cellular dynamics that are not dependent on the classic view. The work is vast, and this discussion does not attempt to cover it in great depth. It is the first in a series.

Signaling and signaling pathways
Signaling transduction tutorial.
Carbohydrate metabolism
Lipid metabolism
Protein synthesis and degradation
Subcellular structure
Impairments in pathological states: endocrine disorders; stress hypermetabolism; cancer.

Signal transduction

(From Wikipedia, the free encyclopedia)
http://en.wikipedia.org/wiki/File:Signal_transduction_publications_graph.jpeg

Signal_transduction_pathways.svg

Signal transduction occurs when an extracellular signaling^[1] molecule activates a specific receptor located on the cell surface or inside the cell. In turn, this receptor triggers a biochemical chain of events inside the cell, creating a response.^[2] Depending on the cell, the response alters the cell’s metabolism, shape, gene expression, or ability to divide.^[3] The signal can be amplified at any step. Thus, one signaling molecule can cause many responses.^[4]

In 1970, Martin Rodbell examined the effects of glucagon on a rat’s liver cell membrane receptor. He noted that guanosine triphosphate disassociated glucagon from this receptor and stimulated the G-protein, which strongly influenced the cell’s metabolism. Thus, he deduced that the G-protein is a transducer that accepts glucagon molecules and affects the cell.^[5] For this, he shared the 1994 Nobel Prize in Physiology or Medicine with Alfred G. Gilman.

Signal_transduction_publications_graph

The earliest MEDLINE entry for “signal transduction” dates from 1972.^[6] Some early articles used the terms signal transmission and sensory transduction.^[7][8] In 2007, a total of 48,377 scientific papers—including 11,211 e review papers—were published on the subject. The term first appeared in a paper’s title in 1979.^[9][10] Widespread use of the term has been traced to a 1980 review article by Rodbell:^[5][11] Research papers focusing on signal transduction first appeared in large numbers in the late 1980s and early 1990s.^[12]

Notch-mediated juxtacrine signal between adjacent cells.

Signal transduction involves the binding of extracellular signaling molecules and ligands to cell-surface receptors that trigger events inside the cell. The combination of messenger with receptor causes a change in the conformation of the receptor, known as receptor activation. This activation is always the initial step (the cause) leading to the cell’s ultimate responses (effect) to the messenger. Despite the myriad of these ultimate responses, they are all directly due to changes in particular cell proteins. Intracellular signaling cascades can be started through cell-substratum interactions; examples are the integrin that binds ligands in the extracellular matrix and steroids.^[13] Most steroid hormones have receptors within the cytoplasm and act by stimulating the binding of their receptors to the promoter region of steroid-responsive genes.^[14] Examples of signaling molecules include the hormone melatonin,^[15] the neurotransmitter acetylcholine^[16] and the cytokine interferon γ.^[17]

Signal transduction cascades amplify the signal output

Various environmental stimuli exist that initiate signal transmission processes in multicellular organisms; examples include photons hitting cells in the retina of the eye,^[20] and odorants binding to odorant receptors in the nasal epithelium.^[21] Certain microbial molecules, such as viral nucleotides and protein antigens, can elicit an immune system response against invading pathogens mediated by signal transduction processes. This may occur independent of signal transduction stimulation by other molecules, as is the case for the toll-like receptor. It may occur with help from stimulatory molecules located at the cell surface of other cells, as with T-cell receptor signaling. Unicellular organisms may respond to environmental stimuli through the activation of signal transduction pathways. For example, slime molds secrete cyclic adenosine monophosphate upon starvation, stimulating individual cells in the immediate environment to aggregate,^[22] and yeast cells use mating factors to determine the mating types of other cells and to participate in sexual reproduction.^[23] Receptors can be roughly divided into two major classes: intracellular receptors and extracellular receptors.

Extracellular

Extracellular receptors are integral transmembrane proteins and make up most receptors. They span the plasma membrane of the cell, with one part of the receptor on the outside of the cell and the other on the inside. Signal transduction occurs as a result of a ligand binding to the outside; the molecule does not pass through the membrane. This binding stimulates a series of events inside the cell; different types of receptor stimulate different responses and receptors typically respond to only the binding of a specific ligand. Upon binding, the ligand induces a change in the conformation of the inside part of the receptor.^[24] These result in either the activation of an enzyme in the receptor or the exposure of a binding site for other intracellular signaling proteins within the cell, eventually propagating the signal through the cytoplasm.

In eukaryotic cells, most intracellular proteins activated by a ligand/receptor interaction possess an enzymatic activity; examples include tyrosine kinase and phosphatases. Some of them create second messengers such as cyclic AMP and IP₃, the latter controlling the release of intracellular calcium stores into the cytoplasm. Other activated proteins interact with adaptor proteins that facilitate signalling protein interactions and coordination of signalling complexes necessary to respond to a particular stimulus. Enzymes and adaptor proteins are both responsive to various second messenger molecules.

Many adaptor proteins and enzymes activated as part of signal transduction possess specialized protein domains that bind to specific secondary messenger molecules. For example, calcium ions bind to the EF hand domains of calmodulin, allowing it to bind and activate calmodulin-dependent kinase. PIP₃ and other phosphoinositides do the same thing to the Pleckstrin homology domains of proteins such as the kinase protein AKT.

G protein-coupled

G protein-coupled receptors (GPCRs) are a family of integral transmembrane proteins that possess seven transmembrane domains and are linked to a heterotrimeric G protein. Many receptors are in this family, including adrenergic receptors and chemokine receptors.

Arrestin binding to active GPCR kinase (GRK)-phosphorylated GPCRs blocks G protein coupling

Signal transduction by a GPCR begins with an inactive G protein coupled to the receptor; it exists as a heterotrimer consisting of Gα, Gβ, and Gγ.^[25] Once the GPCR recognizes a ligand, the conformation of the receptor changes to activate the G protein, causing Gα to bind a molecule of GTP and dissociate from the other two G-protein subunits. The dissociation exposes sites on the subunits that can interact with other molecules.^[26] The activated G protein subunits detach from the receptor and initiate signaling from many downstream effector proteins such as phospholipases and ion channels, the latter permitting the release of second messenger molecules.^[27] The total strength of signal amplification by a GPCR is determined by the lifetimes of the ligand-receptor complex and receptor-effector protein complex and the deactivation time of the activated receptor and effectors through intrinsic enzymatic activity.

A study was conducted where a point mutation was inserted into the gene encoding the chemokine receptor CXCR2; mutated cells underwent a malignant transformation due to the expression of CXCR2 in an active conformation despite the absence of chemokine-binding. This meant that chemokine receptors can contribute to cancer development.^[28]

Tyrosine and histidine kinase

Receptor tyrosine kinases (RTKs) are transmembrane proteins with an intracellular kinase domain and an extracellular domain that binds ligands; examples include growth factor receptors such as the insulin receptor.^[29] To perform signal transduction, RTKs need to form dimers in the plasma membrane;^[30] the dimer is stabilized by ligands binding to the receptor. The interaction between the cytoplasmic domains stimulates the autophosphorylation of tyrosines within the domains of the RTKs, causing conformational changes. Subsequent to this, the receptors’ kinase domains are activated, initiating phosphorylation signaling cascades of downstream cytoplasmic molecules that facilitate various cellular processes such as cell differentiation and metabolism.^[29]

As is the case with GPCRs, proteins that bind GTP play a major role in signal transduction from the activated RTK into the cell. In this case, the G proteins are members of the Ras, Rho, and Raf families, referred to collectively as small G proteins. They act as molecular switches usually tethered to membranes by isoprenyl groups linked to their carboxyl ends. Upon activation, they assign proteins to specific membrane subdomains where they participate in signaling. Activated RTKs in turn activate small G proteins that activate guanine nucleotide exchange factors such as SOS1. Once activated, these exchange factors can activate more small G proteins, thus amplifying the receptor’s initial signal. The mutation of certain RTK genes, as with that of GPCRs, can result in the expression of receptors that exist in a constitutively activate state; such mutated genes may act as oncogenes.^[31]

Histidine-specific protein kinases are structurally distinct from other protein kinases and are found in prokaryotes, fungi, and plants as part of a two-component signal transduction mechanism: a phosphate group from ATP is first added to a histidine residue within the kinase, then transferred to an aspartate residue on a receiver domain on a different protein or the kinase itself, thus activating the aspartate residue.^[32]

Integrin

integrin-mediated signal transduction

An overview of integrin-mediated signal transduction, adapted from Hehlgens et al. (2007).^[33]

Integrins are produced by a wide variety of cells; they play a role in cell attachment to other cells and the extracellular matrix and in the transduction of signals from extracellular matrix components such as fibronectin and collagen. Ligand binding to the extracellular domain of integrins changes the protein’s conformation, clustering it at the cell membrane to initiate signal transduction. Integrins lack kinase activity; hence, integrin-mediated signal transduction is achieved through a variety of intracellular protein kinases and adaptor molecules, the main coordinator being integrin-linked kinase.^[33] As shown in the picture to the right, cooperative integrin-RTK signalling determines the timing of cellular survival, apoptosis, proliferation, and differentiation.

Important differences exist between integrin-signalling in circulating blood cells and non-circulating cells such as epithelial cells; integrins of circulating cells are normally inactive. For example, cell membrane integrins on circulating leukocytes are maintained in an inactive state to avoid epithelial cell attachment; they are activated only in response to stimuli such as those received at the site of an inflammatory response. In a similar manner, integrins at the cell membrane of circulating platelets are normally kept inactive to avoid thrombosis. Epithelial cells (which are non-circulating) normally have active integrins at their cell membrane, helping maintain their stable adhesion to underlying stromal cells that provide signals to maintain normal functioning.^[34]

Toll gate

When activated, toll-like receptors (TLRs) take adapter molecules within the cytoplasm of cells in order to propagate a signal. Four adaptor molecules are known to be involved in signaling, which are Myd88, TIRAP, TRIF, and TRAM.^[35][36][37] These adapters activate other intracellular molecules such as IRAK1, IRAK4, TBK1^{[disambiguation needed]}, and IKKi that amplify the signal, eventually leading to the induction or suppression of genes that cause certain responses. Thousands of genes are activated by TLR signaling, implying that this method constitutes an important gateway for gene modulation.

Ligand-gated ion channel

A ligand-gated ion channel, upon binding with a ligand, changes conformation to open a channel in the cell membrane through which ions relaying signals can pass. An example of this mechanism is found in the receiving cell of a neural synapse. The influx of ions that occurs in response to the opening of these channels induces action potentials, such as those that travel along nerves, by depolarizing the membrane of post-synaptic cells, resulting in the opening of voltage-gated ion channels.

An example of an ion allowed into the cell during a ligand-gated ion channel opening is Ca²⁺; it acts as a second messenger initiating signal transduction cascades and altering the physiology of the responding cell. This results in amplification of the synapse response between synaptic cells by remodelling the dendritic spines involved in the synapse.

Ion transporters and channels in mammalian choroidal epithelium

Intracellular

Understanding these receptors and identifying their ligands and the resulting signal transduction pathways represent a major conceptual advance

intercellular signaling

conformational-rearrangements

membrane protein receptor binds with hormone

The multiple protein-dependent steps in signal transduction

Ca++ exchange

G protein-coupled

membrane_receptor_g protein

intracellular_receptor_steroid

Tyrosine and histidine kinase

insulin-receptor-and-and-insulin-receptor-signaling-pathway-irs

receptors-regulators

phosphorylation-cascade

Integrin

integrin-mediated signal transduction

An overview of integrin-mediated signal transduction, adapted from Hehlgens et al. (2007).^[33]

Platelet signaling pathways

Protein ubiquitylation

ubiquitylation-is-a-multistep-reaction.

https://pharmaceuticalintelligence.com/wp-content/uploads/2014/08/1741-7007-11-65-s1-macromolecular-juggling-by-ubiquitylation-enzymes.mp4

Important differences exist between integrin-signaling in circulating blood cells and non-circulating cells such as epithelial cells; integrins of circulating cells are normally inactive. For example, cell membrane integrins on circulating leukocytes are maintained in an inactive state to avoid epithelial cell attachment; they are activated only in response to stimuli such as those received at the site of an inflammatory response. In a similar manner, integrins at the cell membrane of circulating platelets are normally kept inactive to avoid thrombosis. Epithelial cells (which are non-circulating) normally have active integrins at their cell membrane, helping maintain their stable adhesion to underlying stromal cells that provide signals to maintain normal functioning.^[34]

Toll gate

SignalTrans

Ligand-gated ion channel

Ion transporters and channels in mammalian choroidal epithelium

Intracellular

Intracellular receptors, such as nuclear receptors and cytoplasmic receptors, are soluble proteins localized within their respective areas. The typical ligands for nuclear receptors are lipophilic hormones like the steroid hormones testosterone and progesterone and derivatives of vitamins A and D. To initiate signal transduction, the ligand must pass through the plasma membrane by passive diffusion. On binding with the receptor, the ligands pass through the nuclear membrane into the nucleus, enabling gene transcription and protein production.

Signal Transduction

Activated nuclear receptors attach to the DNA at receptor-specific hormone-responsive element (HRE) sequences, located in the promoter region of the genes activated by the hormone-receptor complex. Due to their enabling gene transcription, they are alternatively called inductors of gene expression. All hormones that act by regulation of gene expression have two consequences in their mechanism of action; their effects are produced after a characteristically long period of time and their effects persist for another long period of time, even after their concentration has been reduced to zero, due to a relatively slow turnover of most enzymes and proteins that would either deactivate or terminate ligand binding onto the receptor.

Signal transduction via these receptors involves little proteins, but the details of gene regulation by this method are not well-understood. Nucleic receptors have DNA-binding domains containing zinc fingers and a ligand-binding domain; the zinc fingers stabilize DNA binding by holding its phosphate backbone. DNA sequences that match the receptor are usually hexameric repeats of any kind; the sequences are similar but their orientation and distance differentiate them. The ligand-binding domain is additionally responsible for dimerization of nucleic receptors prior to binding and providing structures for transactivation used for communication with the translational apparatus.

signal-transduction-in-protease-signaling-

protein changes in biological mechanisms

Steroid receptors are a subclass of nuclear receptors located primarily within the cytosol; in the absence of steroids, they cling together in an aporeceptor complex containing chaperone or heatshock proteins (HSPs). The HSPs are necessary to activate the receptor by assisting the protein to fold in a way such that the signal sequence enabling its passage into the nucleus is accessible. Steroid receptors, on the other hand, may be repressive on gene expression when their transactivation domain is hidden; activity can be enhanced by phosphorylation of serine residues at their N-terminal as a result of another signal transduction pathway, a process called crosstalk.

Structure of the N-terminal domain of the yeast Hsp90 chaperone

Pincer movement of Hsp90 coupled to the ATPase cycle. NTD = N-terminal domain, MD = middle domain, CTD = C-terminal domain.

Retinoic acid receptors are another subset of nuclear receptors. They can be activated by an endocrine-synthesized ligand that entered the cell by diffusion, a ligand synthesised from a precursor like retinol brought to the cell through the bloodstream or a completely intracellularly synthesised ligand like prostaglandin. These receptors are located in the nucleus and are not accompanied by HSPs; they repress their gene by binding to their specific DNA sequence when no ligand binds to them, and vice versa.

Certain intracellular receptors of the immune system are cytoplasmic receptors; recently identified NOD-like receptors (NLRs) reside in the cytoplasm of some eukaryotic cells and interact with ligands using a leucine-rich repeat (LRR) motif similar to TLRs. Some of these molecules like NOD2 interact with RIP2 kinase that activates NF-κB signaling, whereas others like NALP3 interact with inflammatory caspases and initiate processing of particular cytokines like interleukin-1β.^[38][39]

Cell signaling

signaling pathjways map

Cell signalling is part of a complex system of communication that governs basic cellular activities and coordinates cell actions. The ability of cells to perceive and correctly respond to their microenvironment is the basis of development, tissue repair, and immunity as well as normal tissue homeostasis. Errors in cellular information processing are responsible for diseases such as cancer, autoimmunity, and diabetes. By understanding cell signalling, diseases may be treated effectively and, theoretically, artificial tissues may be created.

Traditional work in biology has focused on studying individual parts of cell signaling pathways. Systems biology research helps us to understand the underlying structure of cell signaling networks and how changes in these networks may affect the transmission and flow of information. Such networks are complex systems in their organization and may exhibit a number of emergent properties. Long-range allostery is often a significant component of cell signaling events.^[1]

Enzyme_Model allosterism

Classification

Signaling within, between, and among cells is subdivided into the following classifications:

Intracrine signals are produced by the target cell that stay within the target cell.
Autocrine signals are produced by the target cell, are secreted, and effect the target cell itself via receptors. Sometimes autocrine cells can target cells close by if they are the same type of cell as the emitting cell. An example of this are immune cells.
Juxtacrine signals target adjacent (touching) cells. These signals are transmitted along cell membranes via protein or lipid components integral to the membrane and are capable of affecting either the emitting cell or cells immediately adjacent.

transepithelial-electrogenic-ion-transport

calcium release calmodulin + ER

Ca++ exchange

Paracrine bidirectional cardiac fibroblast-myocyte crosstalk

Paracrine signals target cells in the vicinity of the emitting cell. Neurotransmitters represent an example.
Endocrine signals target distant cells. Endocrine cells produce hormones that travel through the blood to reach all parts of the body.

Notch-mediated juxtacrine signal between adjacent cells.

Some cell–cell communication requires direct cell–cell contact. Some cells can form gap junctions that connect their cytoplasm to the cytoplasm of adjacent cells. In cardiac muscle, gap junctions between adjacent cells allows for action potential propagation from the cardiac pacemaker region of the heart to spread and coordinately cause contraction of the heart.

The notch signaling mechanism is an example of juxtacrine signaling (also known as contact-dependent signaling) in which two adjacent cells must make physical contact in order to communicate. This requirement for direct contact allows for very precise control of cell differentiation during embryonic development. In the worm Caenorhabditis elegans, two cells of the developing gonad each have an equal chance of terminally differentiating or becoming a uterine precursor cell that continues to divide. The choice of which cell continues to divide is controlled by competition of cell surface signals. One cell will happen to produce more of a cell surface protein that activates the Notch receptor on the adjacent cell. This activates a feedback loop or system that reduces Notch expression in the cell that will differentiate and that increases Notch on the surface of the cell that continues as a stem cell.^[5]

Many cell signals are carried by molecules that are released by one cell and move to make contact with another cell. Endocrine signals are called hormones. Hormones are produced by endocrine cells and they travel through the blood to reach all parts of the body. Specificity of signaling can be controlled if only some cells can respond to a particular hormone. Paracrine signals such as retinoic acid target only cells in the vicinity of the emitting cell.^[6] Neurotransmitters represent another example of a paracrine signal. Some signaling molecules can function as both a hormone and a neurotransmitter. For example, epinephrine and norepinephrine can function as hormones when released from the adrenal gland and are transported to the heart by way of the blood stream. Norepinephrine can also be produced by neurons to function as a neurotransmitter within the brain.^[7] Estrogen can be released by the ovary and function as a hormone or act locally via paracrine or autocrine signaling.^[8] Active species of oxygen and nitric oxide can also act as cellular messengers. This process is dubbed redox signaling.

Signaling Pathways

Cell Signaling Biology

Michael J. Berridge

Module 2

Cell Signaling Pathways
The nine membrane-bound adenylyl cyclases (AC1–AC9) have a similar domain structure. The single polypeptide has a tandem repeat of six transmembrane domains (TM) with TM1- -TM6 in one repeat and TM7- -TM12 in the other. Each TM cassette is followed by large cytoplasmic domains (C1 and C2), which contain the catalytic regions that convert ATP into cyclic AMP. As shown in the lower panel, the C1 and C2 domains come together to form a heterodimer. The ATP-binding site is located at the interface between these two domains. The soluble AC10 isoform lacks the transmembrane regions, but it retains the C1 and C2 domains that are responsible for catalysis
www.cellsignallingbiology.org http://www.biochemj.org/csb/002/csb002.pdf

Resources:

Elucidate Target-Specific Pathways With a Suite of Cellular Assays

DiscoveRx® offers a comprehensive collection of cell-based pathway indicator assays designed to detect activation or inhibition of complex signal transduction pathways in response to compound treatment. Based on the proven PathHunter® technology, These biosensor cell lines allow you to measure distinct events within a variety of pathways involved in compound toxicity, cholesterol metabolism, antioxidant function, DNA damage and ER stress. In combination with our biosensor cell lines with fast and simple chemiluminescent detection, DiscoveRx Pathway Signaling assays will help you generate cellular pathway selectivity profiles of your compounds without relying on reporter gene assays or complex phenotypic screens. – See more at: http://www.discoverx.com/targets/signaling-pathways?gclid=CPPrxrrli8ACFSdp7AodO2IADQ#sthash.OhK3iKl4.dpuf

 GPCR Targets ,  Kinase Targets ,  Nuclear Receptors ,  Protease Targets ,  Epigenetic Targets ,  Signaling Pathways – See more at: http://www.discoverx.com/targets#sthash.KjwWEjjx.dpuf

inhibitors of signal transduction pathway

Inhibitors of MAPK Signaling Pathway

jak-stat

Nrf2 signaling in ARE-mediated coordinated activation of defensive genes

Regulation of AMPK

metabolic pathways

On these resource pages you can find signaling pathway diagrams, research overviews, relevant antibody products, publications, and other research resources organized by topic. The pathway diagrams associated with these topics have been assembled by CST scientists and outside experts to provide succinct and current overviews of selected signaling pathways. Please send suggestions for developing new pathways to info@cellsignal.com. Protein nodes in each pathway diagram are linked to specific antibody product information or, optionally, to protein-specific listings in the PhosphoSitePlus® database of post-translational modifications.

http://www.cellsignal.com/common/content/content.jsp?id=science-pathways
http://www.cellsignal.com/common/content/content.jsp?id=pathways-akt-signaling
http://www.cellsignal.com/common/content/content.jsp?id=pathways-mtor-signaling

PI3K / Akt Signaling Overview

The serine/threonine kinase Akt/PKB exists as three isoforms in mammals. Akt1 has a wide tissue distribution, whereas Akt2 is found predominantly in muscle and fat cells and Akt3 is expressed in testes and brain. Akt regulates multiple biological processes including cell survival, proliferation, growth, and glycogen metabolism. Various growth factors, hormones, and cytokines activate Akt by binding their cognate receptor tyrosine kinase (RTK), cytokine receptor, or GPCR and triggering activation of the lipid kinase PI3K, which generates PIP3 at the plasma membrane. Akt binds PIP3 through its pleckstrin homology (PH) domain, resulting in translocation of Akt to the membrane. Akt is activated through a dual phosphorylation mechanism. PDK1, which is also brought to the membrane through its PH domain, phosphorylates Akt within its activation loop at Thr308. A second phosphorylation at Ser473 within the carboxy terminus is also required for activity and is carried out by the mTOR-rictor complex, mTORC2.

PTEN, a lipid phosphatase that catalyzes the dephosphorylation of PIP3, is a major negative regulator of Akt signaling. Loss of PTEN function has been implicated in many human cancers. Akt activity is also negatively regulated by the phosphatases PP2A and PHLPP, as well as by the chemical modulators wortmannin and LY294002, both of which are inhibitors of PI3K.

Activated Akt phosphorylates a large number of downstream substrates containing the consensus sequence RXRXXS/T. One of its primary functions is to promote cell growth and protein synthesis through regulation of the mTOR signaling pathway. Akt directly phosphorylates and activates mTOR, as well as inhibits the mTOR inhibitor proteins PRAS40 and tuberin (TSC2). Combined, these actions promote cell growth and G1 cell cycle progression through signaling via p70 S6 Kinase and inhibition of 4E-BP1.

Phosphofructokinase mechanism

GSK-3 is a primary target of Akt and inhibitory phosphorylation of GSK-3α (Ser21) or GSK-3β (Ser9) has numerous cellular effects such as promoting glycogen metabolism, cell cycle progression, regulation of wnt signaling, and formation of neurofibrillary tangles in Alzheimers disease. Akt promotes cell survival directly by its ability to phosphorylate and inactivate several pro-apoptotic targets, including Bad, Bim, Bax, and the forkhead (FoxO1/3a) transcription factors. Akt also plays an important role in metabolism and insulin signaling. Insulin receptor signaling through Akt promotes Glut4 translocation through activation of AS160 and TBC1D1, resulting in increased glucose uptake. Akt regulates glycolysis through phosphorylation of PFK and hexokinase, and plays a significant role in aerobic glycolysis of cancer cells, also known as the Warburg Effect.

Aberrant Akt signaling is the underlying defect found in several pathologies. Akt is one of the most frequently activated kinases in human cancer as constitutively active Akt can promote unregulated cell proliferation. Abnormalities in Akt2 signaling can result in diabetes due to defects in glucose homeostasis. Akt is also a key player in cardiovascular disease through its role in cardiac growth, angiogenesis, and hypertrophy.

References

Robey RB, Hay N (2009) Is Akt the “Warburg kinase”?-Akt-energy metabolism interactions and oncogenesis. Cancer Biol. 19(1), 25–31.
Zhang S, Yu D (2010) PI(3)king apart PTEN’s role in cancer. Cancer Res. 16(17), 4325–30.
Zoncu R, Efeyan A, Sabatini DM (2011) mTOR: from growth signal integration to cancer, diabetes and ageing. Rev. Mol. Cell Biol. 12(1), 21–35.
Zhang X, Tang N, Hadden TJ, Rishi AK (2011) Akt, FoxO and regulation of apoptosis. Biophys. Acta 1813(11), 1978–86.
Kloet DE, Burgering BM (2011) The PKB/FOXO switch in aging and cancer. Biophys. Acta 1813(11), 1926–37.
Hers I, Vincent EE, Tavars JM (2011) Akt signalling in health and disease. Signal. 23(10), 1515–27.
Wang H, Zhang Q, Wen Q, Zheng Y, Lazarovici P, Philip L, Jiang H, Lin J, Zheng W (2012) Proline-rich Akt substrate of 40kDa (PRAS40): a novel downstream target of PI3k/Akt signaling pathway. Signal. 24(1), 17–24.
Dazert E, Hall MN (2011) mTOR signaling in disease. Opin. Cell Biol. 23(6), 744–55.
Bayley JP, Devilee P (2012) The Warburg effect in 2012. Curr Opin Oncol 24(1), 62–7.

mTOR Signaling Pathway

Akt mTOR pathway

The mammalian target of rapamycin (mTOR) is an atypical serine/threonine kinase that is present in two distinct complexes. mTOR complex 1 (mTORC1) is composed of mTOR, Raptor, GβL (mLST8), and Deptor and is partially inhibited by rapamycin. mTORC1 integrates multiple signals reflecting the availability of growth factors, nutrients, or energy to promote either cellular growth when conditions are favorable or catabolic processes during stress or when conditions are unfavorable. Growth factors and hormones (e.g. insulin) signal to mTORC1 via Akt, which inactivates TSC2 to prevent inhibition of mTORC1. Alternatively, low ATP levels lead to the AMPK-dependent activation of TSC2 and phosphorylation of raptor to reduce mTORC1 signaling. Amino acid availability is signaled to mTORC1 via a pathway involving the Rag and Ragulator (LAMTOR1-3) proteins. Active mTORC1 has a number of downstream biological effects including translation of mRNA via the phosphorylation of downstream targets (4E-BP1 and p70 S6 Kinase), suppression of autophagy (Atg13, ULK1), ribosome biogenesis, and activation of transcription leading to mitochondrial metabolism or adipogenesis. The mTOR complex 2 (mTORC2) is composed of mTOR, Rictor, GβL, Sin1, PRR5/Protor-1, and Deptor and promotes cellular survival by activating Akt. mTORC2 also regulates cytoskeletal dynamics by activating PKCα and regulates ion transport and growth via SGK1 phosphorylation. Aberrant mTOR signaling is involved in many disease states including cancer, cardiovascular disease, and metabolic disorders.

Selected Reviews:

Dowling RJ, Topisirovic I, Fonseca BD, Sonenberg N (2010) Dissecting the role of mTOR: lessons from mTOR inhibitors. Biochim. Biophys. Acta 1804(3), 433–9.
Dunlop EA, Tee AR (2009) Mammalian target of rapamycin complex 1: signalling inputs, substrates and feedback mechanisms. Cell. Signal. 21(6), 827–35.
Hoeffer CA, Klann E (2010) mTOR signaling: at the crossroads of plasticity, memory and disease. Trends Neurosci. 33(2), 67–75.
Laplante M, Sabatini DM (2009) mTOR signaling at a glance. J. Cell. Sci. 122(Pt 20), 3589–94.
Neufeld TP (2010) TOR-dependent control of autophagy: biting the hand that feeds. Curr. Opin. Cell Biol. 22(2), 157–68.
Zoncu R, Efeyan A, Sabatini DM (2011) mTOR: from growth signal integration to cancer, diabetes and ageing. Nat. Rev. Mol. Cell Biol. 12(1), 21–35.

We would like to thank Carson Thoreen and Prof. David Sabatini, Whitehead Institute for Biomedical Research, MIT, Cambridge, MA, for reviewing this diagram. revised November 2012

Protein Folding

conformational-rearrangements

Pincer movement of Hsp90 coupled to the ATPase cycle. NTD = N-terminal domain, MD = middle domain, CTD = C-terminal domain.

Heat Shock Proteins (HSPs) form seven families (small HSPs (sHSPs), HSP10, 40, 60, 70, 90, and 100) of molecular chaperone proteins that play a central role in the cellular resistance to stress and actin organization. They are involved in the proper folding of proteins and the recognition and refolding of misfolded proteins. HSP expression is induced by a variety of environmental stresses, including heat, hypoxia, nutrient deficiency, free radicals, toxins, ischemia, and UV radiation. HSP27 is a member of the sHSP family. It is phosphorylated at Ser15, Ser78, and Ser82 by MAPKAPK-2 as a result of the activation of the p38 MAP kinase pathway. Phosphorylation and increased concentration of HSP27 has been implicated in actin polymerization and reorganization. HSP70 and HSP90 interact with unfolded proteins to prevent irreversible aggregation and catalyze the refolding of their substrates in an ATP- and co-chaperone-dependent manner. HSP70 has a broad range of substrates including newly synthesized and denatured proteins, while HSP90 tends to have a more limited subset of substrates, most of which are signaling molecules. HSP70 and HSP90 are also essential for the maturation and inactivation of nuclear hormones and other signaling molecules.

References

Stenmark H (2009) Rab GTPases as coordinators of vesicle traffic. Rev. Mol. Cell Biol. 10(8), 513–25.
Horgan CP, McCaffrey MW (2009) The dynamic Rab11-FIPs. Soc. Trans. 37(Pt 5), 1032–6.
Evans CG, Chang L, Gestwicki JE (2010) Heat shock protein 70 (hsp70) as an emerging drug target. Med. Chem. 53(12), 4585–602.
Lanneau D, Wettstein G, Bonniaud P, Garrido C (2010) Heat shock proteins: cell protection through protein triage. ScientificWorldJournal 10, 1543–52.
Ghayour-Mobarhan M, Saber H, Ferns GA (2012) The potential role of heat shock protein 27 in cardiovascular disease. Chim. Acta 413(1-2), 15–24.
Horgan CP, McCaffrey MW (2011) Rab GTPases and microtubule motors. Soc. Trans. 39(5), 1202–6.
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The role and importance of transcription factors

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The role and importance of transcription factors

Larry H. Bernstein, MD, FCAP, Writer and Curator

http://pharmaceuticalintelligence.com/2014/8/05/The-role-and-importance-of-transcripton-factors

The following is a second in the 2^nd series that is focused on the topic of the impact of genomics and transcriptomics in the evolution of 21^st century of medicine, which shall have to be more efficient and more effective by the end of this decade, if the prediction for the funding of Medicare is expected to run out. Even so, Social Security was devised by none other than the Otto von Bismarck, who unified Germany, and United Kingdom has had a charity hospital care system begun to protect the widows of the ravages of war, and nursing was developed by Florence Nightengale as a result of the experience of war. It can only be concluded that the care for the elderly, the infirm, and those who have little resources to live on has a long history in western civilization, and it will not cease to exist as a public social obligation anytime soon. The 20^th century saw an explosive development of physics; organic, inorganic, biochemistry, and medicinal chemistry, and the elucidation of the genetic code and its mechanism of translation in plants, microorganisms, and eukaryotes. All of which occurred irrespective of the most horrendous wars that have reshaped the world map.

The following are the second portions of a puzzle in construction that is intended to move into deeper complexities introduced by proteomics, cell metabolism, metabolomics, and signaling. This is the only manner by which I can begin to appreciate what a wonder it is to view and live in this world with all its imperfections.

We have already visited the transcription process, by which an RNA sequence is read. This is essential for protein synthesis through the ordering of the amino acids in the primary structure. However, there are microRNAs and noncoding RNAs, and there are transcription factors. The transcription factors bind to chromatin, and the RNAs also have some role in regulating the transcription process. We shall examine this further.

RNA and the transcription the genetic code

Larry H. Bernstein, MD, FCAP, Writer and Curator
http://pharmaceuticalintelligence.com/2014/08/02/rna-and-the-transcription-of-the-genetic-code/

The role and importance of transcription factors?
Larry H. Bernstein, MD, FCAP, Writer and Curator
http://pharmaceuticalintelligence.com/2014/8/05/What-is-the-meaning-of-so-many-RNAs
What is the meaning of so many RNAs?

Larry H. Bernstein, MD, FCAP, Writer and Curator
http://pharmaceuticalintelligence.com/2014/8/05/What-is-the-meaning-of-so-many-RNAs

Pathology Emergence in the 21st Century
Larry Bernstein, MD, FCAP, Author and Curator
http://pharmaceuticalintelligence.com/2014/08/03/pathology-emergence-in-the-21st-century/
The Arnold Relman Challenge: US HealthCare Costs vs US HealthCare Outcomes

Larry H. Bernstein, MD, FCAP, Reviewer and Curator; and
Aviva Lev-Ari, PhD, RN, Curator
http://pharmaceuticalintelligence.com/2014/08/05/the-relman-challenge/

Quantifying transcription factor kinetics: At work or at play?

Posted online on September 11, 2013. (doi:10.3109/10409238.2013.833891)

Florian Mueller1,2, Timothy J. Stasevich3, Davide Mazza4, and James G. McNally5
1Institut Pasteur, Computational Imaging and Modeling Unit, CNRS, Paris, Fr
2Functional Imaging of Transcription, Institut de Biologie de l’Ecole Normale Supérieure, Paris, Fr
3Graduate School of Frontier Biosciences, Osaka University, Osaka, Jp
4Istituto Scientifico Ospedale San Raffaele, Centro di Imaging Sperimentale e Università Vita-Salute
San Raffaele, Milano, It, and
5Fluorescence Imaging Group, National Cancer Institute, NIH, Bethesda, MD, USA

Abstract

Transcription factors (TFs) interact dynamically in vivo with chromatin binding sites. Here we summarize and compare the four different techniques that are currently used to measure these kinetics in live cells, namely fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS), single molecule tracking (SMT) and competition ChIP (CC). We highlight the principles underlying each of these approaches as well as their advantages and disadvantages. A comparison of data from each of these techniques raises an important question: do measured transcription kinetics reflect biologically functional interactions at specific sites (i.e. working TFs) or do they reflect non-specific interactions (i.e. playing TFs)? To help resolve this dilemma we discuss five key unresolved biological questions related to the functionality of transient and prolonged binding events at both specific promoter response elements as well as non-specific sites. In support of functionality, we review data suggesting that TF residence times are tightly regulated, and that this regulation modulates transcriptional output at single genes. We argue that in addition to this site-specific regulatory role, TF residence times also determine the fraction of promoter targets occupied within a cell thereby impacting the functional status of cellular gene networks. Thus, TF residence times are key parameters that could influence transcription in multiple ways.

Keywords: Competition-ChIP, kinetic modeling, live-cell imaging, non-specific binding, specific binding, transcription, transcription factor dynamics http://informahealthcare.com/doi/abs/10.3109/10409238.2013.833891?goback=%2Egde_3795224_member_273907669#%2EUjYZ8jMt8mo%2Elinkedin

The Transcription Factor Titration Effect Dictates Level of Gene ExpressionCalifornia Institute of Technology

Robert C. Brewster, Franz M. Weinert, Hernan G. Garcia, Dan Song, Mattias Rydenfelt, and Rob Phillips CalTech

Cell Mar 13, 2014; 156:1312–1323,.

Models of transcription are often built around a picture of RNA polymerase and transcription factors (TFs) acting on a single copy of a promoter. However, most TFs are shared between multiple genes with varying binding affinities. Beyond that, genes often exist at high copy number—in multiple identical copies on the chromosome or on plasmids or viral vectors with copy numbers in the hundreds. Using a thermodynamic model, we characterize the interplay between TF copy number and the demand for that TF. We demonstrate the parameter-free predictive power of this model as a function of the copy number of the TF and the number and affinities of the available specific binding sites; such predictive control is important for the understanding of transcription and the desire to quantitatively design the output of genetic circuits. Finally, we use these experiments to dynamically measure plasmid copy number through the cell cycle.

Optimal reference genes for normalization of qRT-PCR data from archival formalin-fixed, paraffin-embedded breast tumors controlling for tumor cell content and decay of mRNA.

Tramm T, Sørensen BS, Overgaard J, Alsner J.

Diagn Mol Pathol. 2013 Sep;22(3):181-7. http://dx.doi.org:/10.1097/PDM.0b013e318285651e

Gene-expression analysis is increasingly performed on degraded mRNA from formalin-fixed, paraffin-embedded tissue (FFPE), giving the option of examining retrospective cohorts. The aim of this study was to select robust reference genes showing stable expression over time in FFPE, controlling for various content of tumor tissue and decay of mRNA because of variable length of storage of the tissue.

Sixteen reference genes were quantified by qRT-PCR in 40 FFPE breast tumor samples, stored for 1 to 29 years. Samples included 2 benign lesions and 38 carcinomas with varying tumor content. Stability of the reference genes were determined by the geNorm algorithm. mRNA was successfully extracted from all samples, and the 16 genes quantified in the majority of samples.

Results showed 14% loss of amplifiable mRNA per year, corresponding to a half-life of 4.6 years. The 4 most stable expressed genes were CALM2, RPL37A, ACTB, and RPLP0. Several of the other examined genes showed considerably instability over time (GAPDH, PSMC4, OAZ1, IPO8).

In conclusion, we identified 4 genes robustly expressed over time and independent of neoplastic tissue content in the FFPE block. PMID:23846446

Structures of Cas9 Endonucleases Reveal RNA-Mediated Conformational Activation

Martin Jinek ¹^,^*^,^†, Fuguo Jiang ²^,^*, David W. Taylor ³^,⁴^,^*, Samuel H. Sternberg ⁵^,^*, Emine Kaya ², et al.

¹Department of Biochemistry, University of Zurich, CH-8057 Zurich, Switzerland. ²Department of Molecular and Cell Biology,³Howard Hughes Medical Institute, ⁴California Institute for Quantitative Biosciences, ⁵Department of Chemistry, ⁶Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720,. ⁷The Laboratory for Molecular Infection Medicine Sweden, Umeå University, Umeå S-90187, Sweden. ⁸Helmholtz Centre for Infection Research, Department of Regulation in Infection Biology, D-38124 Braunschweig, Germany. ⁹Hannover Medical School, D-30625 Hannover, Germany. ¹⁰Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720.

↵‡ Present address: Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66 CH-4058 Basel, Switzerland.

↵§ Present address: Department of Agricultural and Biological Engineering, University of Florida, Gainesville, FL 32611, USA.

Science http://dx.doi.org:/10.1126/science.1247997

Type II CRISPR-Cas systems use an RNA-guided DNA endonuclease, Cas9,

to generate double-strand breaks in invasive DNA during an adaptive bacterial immune response.

Cas9 has been harnessed as a powerful tool for genome editing and gene regulation in many eukaryotic organisms.

Here, we report 2.6 and 2.2 Å resolution crystal structures of two major Cas9 enzymes subtypes,

revealing the structural core shared by all Cas9 family members.

The architectures of Cas9 enzymes define nucleic acid binding clefts, and

single-particle electron microscopy reconstructions show that the two structural lobes harboring these clefts undergo guide

RNA-induced reorientation to form a central channel where DNA substrates are bound.

The observation that extensive structural rearrangements occur before target DNA duplex binding

implicates guide RNA loading as a key step in Cas9 activation.

MicroRNA function in endothelial cells
Dr. Virginie Mattot
Angiogenesis, endothelium activation
Solving the mystery of an unknown target gene using microRNA Target Site Blockers

Dr. Virgine Mattot works in the team “Angiogenesis, endothelium activation and Cancer” directed by Dr. Fabrice Soncin at the Institut de Biologie de Lille in France where she studies the roles played by microRNAs in endothelial cells during physiological and pathological processes such as angiogenesis or endothelium activation. She has been using Target Site Blockers to investigate the role of microRNAs on putative targets which functions are yet unknown.

What is the main focus of the research conducted in your lab?

We are studying endothelial cell functions with a particular interest in angiogenesis and endothelium activation during physiological and tumoral vascular development.

How did your research lead to the study of microRNAs?

A few years ago, we identified

an endothelial cell-specific gene which
harbors a microRNA in its intronic sequence.

We have since been working on understanding the functions of

both this new gene and its intronic microRNA in endothelial cells.

What is the aim of your current project?

While we were searching for the functions of the intronic microRNA,

we identified an unknown gene as a putative target.

The aim of my project was to investigate if this unknown gene was actually a genuine target and if regulation of this gene by the microRNA was involved in endothelial cell function. We had already characterized the endothelial cell phenotype associated with the inhibition of our intronic microRNA. We then used miRCURY LNA™ Target Site Blockers to demonstrate

the expression of this unknown gene is actually controlled by this microRNA.
the microRNA regulates specific endothelial cell properties through regulation of this unknown gene.

How did you perform the experiments and analyze the results?

LNA™ enhanced target site blockers (TSB) for our microRNA were designed by Exiqon. We

transfected the TSBs into endothelial cells using our standard procedure and
analysed the induced phenotype.

As a control for these experiments, a mutated version of the TSB was designed by Exiqon and transfected into endothelial cells. We first verified that this TSB was functional by analyzing

the expression of the miRNA target against which the TSB was directed
we then showed the TSB induced similar phenotypes as those when we inhibited the microRNA in the same cells.

What do you find to be the main benefits/advantage of the LNA™ microRNA target site blockers from Exiqon?

Target Site Blockers are efficient tools to demonstrate the specific involvement of

putative microRNA targets in the function played by this microRNA.

What would be your advice to colleagues about getting started with microRNA functional analysis?

it is essential to perform both gain and loss of functions experiments.

Changing the core of transcription

Different members of the TAF family of proteins work in differentiated cells, such as motor neurons or brown fat cells, to control the expression of genes that are specific to each cell type.

Katherine A Jones
Jones. eLife 2014;3:e03575. http://dx.doi.org:/10.7554/eLife.03575

Related research articles: Herrera FJ, Yamaguchi T, Roelink H, Tjian R. 2014. Core promoter factor TAF9B regulates neuronal gene expression. eLife 3:e02559. http://dx.doi.org:/10.7554eLife.02559

Zhou H, Wan B, Grubisic I, Kaplan T, Tjian R. 2014. TAF7L modulates brown adipose tissue formation. eLife 3:e02811. Http://dx.doi.org:/10.7554/eLife.02811

Motor neurons (green) being grown in vitro

Image Motor neurons (green) being grown in vitro

In a developing organism, different genes are expressed at different times

the pattern of gene expression can often change abruptly.

Expressing a gene involves multiple steps:

the DNA must be transcribed into a molecule of messenger RNA,
which is then translated into a protein.

The mechanisms that start the transcription of protein-coding genes in rapidly growing cells are reasonably well understood: two types of proteins—

DNA-binding activators and general transcription factors—

cooperate to recruit an enzyme called RNA polymerase, which then transcribes the gene (Kadonaga, 2012).

These proteins bind to a region of the gene called the promoter, which is

upstream from the protein-coding region of the gene.

TATA-binding protein is a general transcription factor that

binds to certain sequences of DNA bases found within promoters

14 TATA-binding protein associated factors (TAFs) are included into two different protein complexes called TFIID and SAGA (Müller et al., 2010). which, in budding yeast, can recruit TATA-binding protein to gene promoters (Basehoar et al., 2004), but not all genes require all of the general transcription factors, and some genes require both TFIID and SAGA complexes.

Although the steps that are required to switch on genes when cells are rapidly dividing are fairly well known,

the same is not true for cells that are differentiating into specialised cell types.

In these cells, many transcription factors are downregulated and

the entire pattern of gene expression changes dramatically.

Moreover, certain TAFs are strongly up-regulated during differentiation. The core transcriptional machinery is essentially rebuilt at the genes that are expressed in differentiated cells.

Over the years Robert Tjian of the University of California Berkeley and co-workers have illuminated how individual TAFs can affect how a cell differentiates in different contexts (Figure 1). Now, in eLife, Francisco Herrera of UC Berkeley and co-workers—including Teppei Yamaguchi, Henk Roelink and Tjian—have identified a critical role for a TAF called TAF9B in the expression of genes in motor neurons (Herrera et al., 2014).

Herrera et al. found that TAF9B predominantly associates with the SAGA complex, rather than the TFIID complex, in the motor neuron cells. Mice in which the gene for TAF9B had been deleted had less neuronal tissue in the developing spinal cord. Moreover, the genes that are involved in forming the branches of neurons were not properly regu¬lated in these mice.

Recently, in another eLife paper, Tjian and co-workers at Berkeley, Fudan University and the Hebrew University of Jerusalem—including Haiying Zhou as first author, Bo Wan, Ivan Grubisic and Tommy Kaplan—reported that another TAF protein, called TAF7L, works as part of the TFIID complex to up-regulate genes that direct cells to become brown adipose tissue (Zhou et al., 2014).

TATA-binding protein associated factors

Figure 1. TATA-binding protein associated factors (TAFs) regulate transcription in specific cell types. TAF3, for example, works with another transcription factor to regulate the expression of genes that are critical for the differentiation of the endoderm in the early embryo (Liu et al., 2011). TAF3 also forms a complex with the TATA-related factor, TRF3, to regulate Myogenin and other muscle-specific genes to form myotubes (Deato et al., 2008). TAF7L interacts with another transcription factor to activate genes involved in the formation of adipocytes (‘fat cells’) and adipose tissue (Zhou et al., 2013; Zhou et al., 2014). Finally, TAF9B is a key regulator of transcription in motor neurons (Herrera et al., 2014). The names of some of the genes regulated by the TAFs are shown in brackets.

TAF9B

Deleting the gene for TAF9B in mouse embryonic stem cells revealed that this TAF

is not needed for the growth of stem cells, or
required for the expression of genes that prevent differentiation:

both of these processes are known to be highly-dependent upon the TFIID complex
(Pijnappel et al., 2013). However,

genes that would normally be expressed specifically in neurons were not
up-regulated when cells without the TAF9B gene started to specialise.

Herrera et al. identified numerous genes that can only be switched on when the TAF9B protein is present, which means that it joins a growing list of TAF proteins that are dedicated to controllingthe expression of genes in specialised cell types.

TAF9B activates neuron-specific genes by binding to sites that

reside outside of these genes’ core promoters.

Further, many of these sites were also bound by a master regulator of motor neuron-specific genes.

TAF7L

Whilst most of the fat tissue in humans is white adipose tissue, which contains cells that store fatty molecules, some is brown adipose tissue, or ‘brown fat’, that instead generates heat. When TAF7L promotes the differentiation of brown fat, it up-regulates genes that are targeted by a transcription

factor called PPAR-γ; last year it was shown that this transcription factor also promotes the differentiation of white adipose tissue (Zhou et al., 2013).
Mice without the TAF7L gene had 40% less brown fat than wild-type mice, and also grew too much skeletal muscle tissue. TAF7L was specifically required to activate genes that control how brown fat develops and functions. Thus TAF7L expression appears to shift the fate of a stem cell towards brown adipose tissue, potentially at the expense of skeletal muscle, as both cell types develop from the same group of stem cells.

When stem cells with less TAF7L than normal are differentiated in vitro, they yield more muscle than fat cells. Conversely, cells with an excess of TAF7L express brown fat-specific genes and switch off muscle-specific genes.

The work of Herrera et al. and Zhou et al. reinforces the idea that different TAFs

provide the flexibility needed to control gene expression in a tissue-specific manner, and
enable differentiating cells to change which genes they express rapidly.

However many interesting questions remain:

Which signals lead to the destruction of core transcription factors?
Are core promoter elements at tissue-specific genes designed to recognise variant TAFs?
What determines whether variant TAFs are incorporated within TFIID, SAGA, or other complexes?

Shortly after RNA polymerase II starts to transcribe a gene, it briefly pauses. Interestingly, a DNA sequence associated with this pausing, called the pause button, closely matches the sequences that bind to two subunits of TFIID (TAF6 and TAF9; Kadonaga, 2012). Consequently, TAF6 and TAF9 might be involved in pausing transcription, and if so, the variant TAF9B could play a similar role at motor neuron genes.

Molecular basis of transcription pausing

Jeffrey W. Roberts
Science 344, 1226 (2014); http://dx.doi.org:/10.1126/science.1255712
http://www.sciencemag.org/content/344/6189/1226.full.html

During RNA synthesis, RNA polymerase moves erratically along DNA, frequently
resting as it produces an RNA copy of the DNA sequence. Such pausing helps coordinate the appearance of a transcript with its utilization by cellular processes; to this end,

the movement of RNA polymerase is modulated by mechanisms that determine its rate. For example,
pausing is critical to regulatory activities of the enzyme such as the termination of transcription. It is also
essential during early modifications of eukaryotic RNA polymerase II that activate the enzyme for elongation.

Two reports analyzing transcription pausing on a global scale in Escherichia coli, by Larson et al. ( 1) and by Vvedenskaya et al. ( 2) on page 1285 of this issue, suggest

new functions of pausing and important aspects of its molecular basis.

The studies of Larson et al. and Vvedenskaya et al. follow decades of analysis of

bacterial transcription that has illuminated the molecular basis of polymerase pausing

events that serve critical regulatory functions.

A transcription pause specified by the DNA sequence synchronizes the translation of RNA into protein

with the transcription of leader regions of operons (groups of genes transcribed together) for amino acid biosynthesis;

this coordination controls amino acid synthesis in response to amino acid availability ( 3).

A protein induced pause occurs when the E. coli initiation factor σ70 restrains RNA polymerase by binding a second occurrence of the “–10” promoter element.

This paused polymerase provides a structure for engaging a transcription antiterminator (the bacteriophage λ Q protein) ( 4) that, in turn, inhibits transcription

pauses, including those essential for transcription termination.

Biochemical and structural analyses have identified an endpoint of the pausing process called the “elemental pause” in which the catalytic structure in the active site is distorted,

preventing further nucleotide addition ( 7).

The elemental paused state also involves distinct

conformational changes in the polymerase that may favor transcription termination
and allow the his and related pauses to be stabilized by RNA hairpins ( 8).

A consensus sequence for ubiquitous pauses was identified, with two important elements:

a preference for pyrimidine [mostly cytosine (C)] at the newly formed RNA end
followed by G to be incorporated next—just as found for the his pause; and a preference for G at position –10 of the RNA (10 nucleotides before the 3’ end)

Polymerase, paused

Polymerase, paused. During transcription, RNA exists in two states as RNA polymerase progresses: pretranslocated, just after the addition of the last nucleotide [here, cytosine (C)];

and posttranslocated, after all nucleic acids have shifted in register by one nucleotide relative

to the enzyme, exposing the active site for binding of the next substrate molecule [here, guanine (G)]. The pretranslocated state is dominant in the pause. The critical G-C base (RNA-DNA) pair at position –10 in the pretranslocated state and the nontemplate DNA strand G bound in the

polymerase in the posttranslocated state are marked with an asterisk.
Binding of G at position 􀀀1 to CRE only occurs in the posttranslocated state, which would thus

be favored over the pretranslocated state. Hence, if G binding inhibits pausing, then the rate-limiting paused structure must be in the pretranslocated state (a conclusion also made by Larson et al. from biochemical experiments).
This is an important insight into the sequence of protein–nucleic acid interactions that occur in pausing. Vvedenskaya et al. suggest that the actual role of the G binding site is to promote translocation and thus

inhibit pausing, to smooth out adventitious pauses in genomic DNA.
The studies by Larson et al. and Vvedenskaya et al. provide a refined and detailed analysis of DNA sequence–induced transcription pausing.
Processive Antitermination

Robert A. Weisberg1* and Max E. Gottesman2

Section on Microbial Genetics, Laboratory of Molecular Genetics, National Institute of Child Health and

Human Development, National Institutes of Health, Bethesda, Maryland 20892-2785,1 and

Institute of Cancer Research, Columbia University, New York, New York 100322

Journal Of Bacteriology, Jan. 1999; 181(2): 359–367.
After initiating synthesis of RNA at a promoter, RNA polymerase (RNAP) normally continues to elongate the transcript until it reaches a termination site. Important elements of termination sites are transcribed before polymerase translocation stops, and the resulting RNA is an active element of the termination pathway. Nascent transcripts of intrinsic sites can halt transcription without the assistance of additional factors, and

those of Rho-dependent sites recruit the Rho termination protein to the elongation complex. In both cases, RNAP, the transcript, and the template dissociate (reviewed in references 76 and 80).

Termination is rarely, if ever, completely efficient, and the expression of downstream genes can be controlled by altering the efficiency of terminator readthrough. Two distinct mechanisms of elongation control have been reported for bacterial RNA polymerases. In one, exemplified by attenuation of the his and trp operons of Salmonella typhimurium and Escherichia coli, respectively,

a single terminator is inactivated by interaction with an upstream sequence in the transcript, with a terminator-specific protein, or with a translating ribosome that follows closely behind RNAP (reviewed in references 35 and 104).

In a second, whose prototype is antitermination of phage l early transcription,

polymerase is stably modified to a terminator-resistant form after it leaves the promoter.

In this case, the modified enzyme not only transcribes through sequential downstream terminators,

but also it is less sensitive to the pause sites that normally delay transcript elongation.

Both pathways are widespread in nature, but in this minireview we consider only the second,

known as processive antitermination
(for previous reviews, see references 22, 23, 27, and 32).

The recent explosive growth in our understanding of transcription elongation (reviewed in references 57, 96, and 99) make this an especially appropriate time to survey regulatory elements that target the transcription elongation complex.

Antitermination in l is induced by two quite distinct mechanisms.

the result of interaction between l N protein and its targets in the early phage transcripts,
an interaction between the l Q protein and its target in the late phage promoter.

We describe the N mechanism first. Lambda N, a small basic protein of the arginine- rich motif (ARM) (Fig. 1) family of RNA binding proteins, binds to a 15-nucleotide (nt) stem-loop called BOXB (17) (Fig. 2).

FIG. 1. [not shown] (A) Alignment of phage N proteins and the HK022 Nun protein. The color groupings reflect the frequency of amino acid substitutions in evolutionarily related protein domains: an amino acid is more likely to be replaced by one in the same color group than by one in a different color group in related proteins (34).

The amino-proximal ARM regions were aligned by eye and according to the structures of the P22 and l ARMs complexed to their cognate nut sites (see text and Fig. 2), and the remainder of the proteins was aligned by ClustalW (38). The dots indicate gaps introduced to improve the alignment. Aside from the ARM regions, the

proteins fall into three very distantly related (or unrelated) families: (i) l and phage 21; (ii) P22, phage L, and HK97; and (iii) HK022 Nun.

FIG. 2. [not shown] BOXA and BOXB RNAs and their interaction with the ARM of their cognate N proteins. The amino acid-nucleotide interactions are shown to the left except for BOXB of phage 21, for which the structure of the complex is unknown. The sequences of BOXA and BOXA-BOXB spacer are shown to the right. The dots

to the left and right of the spacer sequences are for alignment. (A) l N-ARM-BOXB complex (adapted from reference 48 with permission of the publisher). Open circles, pentagons, and rectangles represent phosphates, riboses, and bases, respectively. Watson-Crick base pairs (????) are indicated. The zigzag line denotes a sheared

G z A base pair. Open circles, open rectangles, and arrowheads depict ionic, hydrophobic, and hydrogen-bonding interactions, respectively. Guanine-11, indicated by a bold rectangle, is extruded from the BOXB loop (see text). (B) P22 N-ARM-BOXB complex (adapted from reference 15 with permission of the publisher). Open

circles, pentagons, rectangles, and ovals represent phosphates, riboses, bases, and amino acids, respectively. The solid pentagons indicate riboses with a C29-endo pucker.

Base stacking ( ), intermolecular hydrogen bonding or electrostatic interactions (,—–), intermolecular hydrophobic or van der Waals interactions (4), intramolecular hydrogen bonds (– – – –) and Watson-Crick base pairs (?????) are indicated. Cytosine-11 is extruded from the loop (see text). Note that the amino-terminal amino acid

residue in the complex corresponds to Asn-14 in the complete protein (Fig. 1), and the displayed amino acids are numbered accordingly. (C) NUTL site of phage 21. The arrows indicate the inverted sequence repeats of BOXB.

FIG. 3. [not skown] HK022 put sites and folded PUT RNAs. (A) Alignment of putL and putR (43). The numbers give distances from the start sites of the PL and PR promoters, respectively, and the pairs of arrows indicate inverted sequence repeats. (B) Folded PUTL and PUTR RNAs. The structures, which were generated by energy

minimization as described (43), have been partially confirmed by genetic and biochemical studies (7, 43).
The active bacterial elongation complex consists of

core RNAP,
template, and
RNA product.

The 39 end of the RNA

is engaged in the active site of the enzyme,
The following ;8 nt are hybridized to the template strand of the DNA, and
the next ;9 nt remain closely associated with RNAP (64).
About 17 nt of the nontemplate DNA strand are separated from the template strand in the transcription bubble.

Elongation complexes can also contain NusA and/or NusG. These proteins, which

increase the stability of the N-mediated antitermination complex (see above),
have different effects on elongation.
NusA decreases and NusG increases the elongation rate, and
both proteins alter termination efficiency in a terminator-specific manner (13, 14, 86; see reference 76).

An elongation complex, unless located at a terminator, is extraordinarily stable,

even when translocation is prevented by removal of substrates.

Recent observations suggest that this stability depends mainly on

interactions between RNAP and the RNA-DNA hybrid as well as
between polymerase and the downstream duplex DNA template (63, 87).

Nascent RNA emerging from the hybrid region and upstream duplex DNA

do not appear to be required.

The strength of the RNA-DNA hybrid is believed to

assure the lateral stability of the complex.

Reducing the strength of the RNA-DNA bonds, for example

by incorporation of nucleotide analogs,
favors backsliding of RNAP on the template, with consequent
disengagement of the 39 RNA end from the active site, and
concerted retreat of the RNA-DNA hybrid region from the 39 end (65).

Such a disengaged complex retains its resistance to dissociation and

is capable of resuming elongation if the original or a newly created 39 end reengages with the active site (10, 44, 45, 65, 71, 95).

Intrinsic terminators consist of a guanine- and cytosine-rich RNA hairpin stem

immediately followed by a short uracil-rich segment
within which termination can occur.

If termination does not occur at this point,

polymerase continues to elongate the transcript with normal processivity
until it reaches the next terminator.

Neither the stem nor the uracil-rich segment

is sufficient for termination, although
either can transiently slow elongation.

The weakness of base pairing between rU and dA

destabilizes the RNA-DNA hybrid in the uracil-rich segment, and
this probably contributes to termination.

Formation of the hairpin stem as nascent terminator RNA emerges from polymerase

destabilizes the RNA-DNA hybrid and
interrupts contacts between the emerging nascent RNA and RNAP (62a).

It might also interfere with the stabilizing interactions between

RNAP and the hybrid or those between RNAP and
the downstream region of the template.

Cross-linking of nucleic acid to RNAP suggests that

both the downstream DNA and the nascent RNA
that emerges from the hybrid region, and
within which the terminator hairpin might form,
are located close to the same regions of the enzyme (64).

Conversely, modifications that render RNAP termination resistant

could prevent the terminator stem from destabilizing one or more of these targets,
at least while the 39 end of the RNA is within the uracil rich segment of the terminator.

The l N and Q proteins and HK022 PUT RNA

also suppress Rho-dependent terminators (43a, 79, 103) which,
in contrast to intrinsic terminators, lack a precisely determined termination point.

Rho is an RNA-dependent ATPase that binds to cytosine-rich, unstructured regions in nascent RNA and acts preferentially

to terminate elongation complexes that are paused at nearby downstream sites
(19, 29, 46, 47, 59, 60).

Rho possesses RNA-DNA helicase activity, and this activity is directional,

unwinding DNA paired to the 39 end of the RNA molecule (11, 90).
This corresponds to the location of the hybrid and of RNAP
in an active ternary elongation complex.

The ability of antiterminators to suppress Rho-dependent and -independent terminators

suggests that they prevent a step that is common to both classes.

Given the helicase activity of Rho, a likely candidate for this step is disruption of the RNA-DNA

hybrid. However, other candidates, such as destabilization of RNAP-template or RNAP-hybrid interactions, are also plausible.

Alternatively, the ability of N, Q, and PUT to suppress RNAP pausing (31, 43, 54, 74)

suggests that they prevent Rho-dependent termination
by accelerating polymerase away from Rho bound at upstream RNA sites.

This explanation raises the problem of why NusG,

which also accelerates polymerase,
enhances rather than suppresses Rho-dependent termination (see above).

Clearly, the molecular details of processive antitermination remain poorly understood despite the 30 years that have elapsed since its discovery.

System wide analyses have underestimated protein abundances and the importance of transcription in mammals

OPEN ACCESS

Jingyi Jessica Li1, 2, Peter J Bickel1 and Mark D Biggin3

1Department of Statistics, University of California, Berkeley, CA, USA

2Departments of Statistics and Human Genetics, University of California, Los Angeles, CA, USA

3Genomics Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA

Academic editor – Barbara Engelhardt http://dx.doi.org:/10.7717/peerj.270

Distributed under Creative-Commons CC-0

ABSTRACT

Large scale surveys in mammalian tissue culture cells suggest that the protein ex-

pressed at the median abundance is present at 8,000_16,000 molecules per cell and

that differences in mRNA expression between genes explain only 10_40% of the dif-

ferences in protein levels. We find, however, that these surveys have significantly un-

derestimated protein abundances and the relative importance of transcription.

Using individual measurements for 61 housekeeping proteins to rescale whole proteome

data from Schwanhausser et al. (2011), we find that the median protein detected is

expressed at 170,000 molecules per cell and that our corrected protein abundance

estimates show a higher correlation with mRNA abundances than do the uncorrected

protein data. In addition, we estimated the impact of further errors in mRNA and

protein abundances using direct experimental measurements of these errors.

The resulting analysis suggests that mRNA levels explain at least

56% of the differences in protein abundance for the 4,212 genes

detected by Schwanhausser et al. (2011), though because one major source of error

could not be estimated the true percent contribution should be higher.
We also employed a second, independent strategy to

determine the contribution of mRNA levels to protein expression.

The variance in translation rates directly measured by ribosome profiling is only 12%

of that inferred by Schwanhausser et al. (2011), and

the measured and inferred translation rates correlate poorly (R2 D 13).

Based on this, our second strategy suggests that

mRNA levels explain _81% of the variance in protein levels.

We also determined the percent contributions of

transcription,
RNA degradation,
translation
and protein degradation

to the variance in protein abundances using both of our strategies.

While the magnitudes of the two estimates vary, they both suggest that

transcription plays a more important role than the earlier studies implied and
translation a much smaller role.

Finally, the above estimates only apply to those genes whose mRNA and protein expression was detected. Based on a detailed analysis by Hebenstreit et al. (2012), we estimate that approximately

40% of genes in a given cell within a population express no mRNA.

Since there can be no translation in the absence of mRNA, we argue that

differences in translation rates can play no role in determining the expression levels for the _40% of genes that are non-expressed.

Subjects Bioinformatics, Computational Biology

Keywords Transcription, Translation, Mass spectrometry, Gene expression, Protein abundance

How to cite this article Li et al. (2014), System wide analyses have underestimated protein abundances and the importance of transcription in mammals. PeerJ 2:e270;

http://dx.doi.org:/10.7717/peerj.270

Assessing quality and completeness of human transcriptional regulatory pathways on a genome-wide scale

Evgeny Shmelkov1,2, Zuojian Tang2, Iannis Aifantis3, Alexander Statnikov2,4

Shmelkov et al. Biology Direct 2011, 6:15 http://www.biology-direct.com/content/6/1/15

Background: Pathway databases are becoming increasingly important and almost omnipresent in most types of biological and translational research. However, little is known about the quality and completeness of pathways stored in these databases. The present study conducts a comprehensive assessment of transcriptional regulatory pathways in humans for seven well-studied transcription factors: MYC, NOTCH1, BCL6, TP53, AR, STAT1, and RELA.

The employed benchmarking methodology first

involves integrating genome-wide binding with functional gene expression data to derive direct targets of transcription factors.
Then the lists of experimentally obtained direct targets are compared with relevant lists of transcriptional targets from 10 commonly used pathway databases.

Results: The results of this study show that for the majority of pathway databases,

the overlap between experimentally obtained target genes and targets reported in transcriptional regulatory pathway databases is surprisingly small and often is not statistically significant.

The only exception is MetaCore pathway database which yields statistically significant intersection with experimental results in 84% cases. Additionally, we suggest that

the lists of experimentally derived direct targets obtained in this study can be used to reveal new biological insight in transcriptional regulation and
suggest novel putative therapeutic targets in cancer.

Conclusions: Our study opens a debate on validity of using many popular pathway databases to obtain transcriptional regulatory targets. We conclude that the choice of pathway databases should be informed by solid scientific evidence and rigorous empirical evaluation.

Illustration of statistical methodology

Figure 2 Illustration of statistical methodology for comparison

between a gold-standard and a pathway database

Additional material

Additional file 1: Supplementary Information. Table S1: Functional gene expression data. Table 2: Transcription factor-DNA binding data. Table S3: Most confident direct transcriptional targets of each of the four transcription factors. These targets were obtained by overlapping several gold-standards obtained with different datasets for the same transcription factor. Table S4: Genes directly regulated by two or more of the three transcription factors: MYC, NOTCH1, and RELA. Figure S1: Comparison of gene sets of transcriptional targets derived from ten different pathway databases by Jaccard index. In case, where Jaccard index of an overlap could not be determined due to comparison of two empty gene lists, we assigned value 0. Cells are colored according to the Jaccard index, from white (Jaccard index equal to 0) to dark-orange (Jaccard index equal to 1). Each sub-figure gives results for a different transcription factor: (a) AR, (b) BCL6, (c) MYC, (d) NOTCH1, (e) RELA, (f) STAT1, (g) TP53

http://dx.doi.org:/10.1186/1745-6150-6-15

Cite this article as: Shmelkov et al.: Assessing quality and completeness of human transcriptional regulatory pathways on a genome-wide scale. Biology Direct 2011 6:15

The Functional Consequences of Variation in Transcription Factor Binding
Darren A. Cusanovich1, Bryan Pavlovic1,2, Jonathan K. Pritchard1,2,3*, Yoav Gilad1*

1 Department of Human Genetics, University of Chicago, 2 Howard Hughes Medical Institute, University of Chicago, Chicago,

Illinois, 3 Departments of Genetics and Biology and Howard Hughes Medical Institute, Stanford University, Stanford, California,

One goal of human genetics is to understand how the information for precise and dynamic gene expression programs is encoded in the genome. The interactions of transcription factors (TFs) with DNA regulatory elements clearly play an important role in determining gene expression outputs, yet the regulatory logic underlying functional transcription factor binding is poorly understood. Many studies have focused on characterizing the genomic locations of TF binding, yet it is unclear to what extent TF binding at any specific locus has functional consequences with respect to gene expression output.

To evaluate the context of functional TF binding we knocked down

59 TFs and chromatin modifiers in one HapMap lymphoblastoid cell line.
We identified genes whose expression was affected by the knockdowns.
We intersected the gene expression data with transcription factor binding data
(based on ChIP-seq and DNase-seq) within 10 kb of the transcription start sites

This combination of data allowed us to infer functional TF binding.

we found that only a small subset of genes bound by a factor were differentially expressed following the knockdown of that factor, suggesting that
most interactions between TF and chromatin do not result in measurable changes in gene expression levels of putative target genes.
functional TF binding is enriched in regulatory elements that harbor
- a large number of TF binding sites,
- at sites with predicted higher binding affinity, and
- at sites that are enriched in genomic regions annotated as ‘‘active enhancers.’’

Author Summary

An important question in genomics is to understand how a class of proteins called ‘‘transcription factors’’ controls the expression level of other genes in the genome in a cell type-specific manner – a process that is essential to human development. One major approach to this problem is to

study where these transcription factors bind in the genome, but this does not tell us about the effect of that binding on gene expression levels and it is generally accepted that much of the binding does not strongly influence gene expression. To address this issue, we artificially reduced the concentration of 59 different transcription factors in the cell and then examined which genes were impacted by the reduced transcription factor level. Our results implicate some attributes that might

influence what binding is functional, but they also suggest that a simple model of functional vs. non-functional binding may not suffice.

Citation: Cusanovich DA, Pavlovic B, Pritchard JK, Gilad Y (2014) The Functional Consequences of Variation in Transcription Factor Binding. PLoS Genet 10(3):e1004226. http://dx.doi.org:/10.1371/journal.pgen.1004226

Editor: Yitzhak Pilpel, Weizmann Institute of Science, Israel

Effect sizes for differentially expressed genes

Figure 2. Effect sizes for differentially expressed genes.

Boxplots of absolute Log2(fold-change) between knockdown arrays

and control arrays for all genes identified as differentially expressed in

each experiment. Outliers are not plotted. The gray bar indicates the

interquartile range across all genes differentially expressed in all

knockdowns. Boxplots are ordered by the number of genes differentially

expressed in each experiment. Outliers were not plotted.

http://dx.doi.org:/10.1371/journal.pgen.1004226.g002

Intersecting binding data and expression data for each knockdown

Figure 3. Intersecting binding data and expression data for each knockdown. (a) Example Venn diagrams showing the overlap of binding and differential expression for the knockdowns of HCST and IRF4 (the same genes as in Figure 1). (b) Boxplot summarizing the distribution of the fraction of all expressed genes that are bound by the targeted gene or downstream factors. (c) Boxplot summarizing the distribution of the fraction of

bound genes that are classified as differentially expressed, using an FDR of either 5% or 20%.

http://dx.doi.org:/10.1371/journal.pgen.1004226.g003

Degree of binding correlated with function

Figure 4. Degree of binding correlated with function. Boxplots comparing (a) the number of sites bound, and (b) the number of differentially expressed transcription factors binding events near functionally or non-functionally bound genes. We considered binding for siRNA-targeted factor and any factor differentially expressed in the knockdown. (c) Focusing only on genes differentially expressed in common between each pairwise set of knockdowns we tested for enrichments of functional binding (y-axis). Pairwise comparisons between knock-down experiments were binned by the fraction of differentially expressed transcription factors in common between the two experiments. For these boxplots, outliers were not plotted.

http://dx.doi.org:/10.1371/journal.pgen.1004226.g004

Distribution of functional binding about the TSS

Figure 5. Distribution of functional binding about the TSS. (a) A density plot of the distribution of bound sites within 10 kb of the TSS for both functional and non-functional genes. Inset is a zoom-in of the region +/21 kb from the TSS (b) Boxplots comparing the distances from the TSS to the binding sites for functionally bound genes and non-functionally bound genes. For the boxplots, 0.001 was added before log10 transforming

the distances and outliers were not plotted.

http://dx.doi.org:/10.1371/journal.pgen.1004226.g005

Magnitude and direction of differential expression after knockdown

Figure 6. Magnitude and direction of differential expression after knockdown. (a) Density plot of all Log2(fold-changes) between the knockdown arrays and controls for genes that are differentially expressed at 5% FDR in one of the knockdown experiments as well as bound by the targeted transcription factor. (b) Plot of the fraction of differentially expressed putative direct targets that were up-regulated in each of the knockdown experiments.

http://dx.doi.org:/10.1371/journal.pgen.1004226.g006

To test whether the number of paralogs or the degree of similarity with the closest paralog for each transcription factor knocked down might influence the number of genes differentially expressed in our experiments, we obtained definitions of paralogy and the calculations of percent identity for 29 different factors from Ensembl’s BioMart (http://useast.ensembl.org/biomart/martview/) [31]. We used genome build GRCh37.p13.

For each gene, we counted the number of paralogs classified as a ‘‘within_species_paralog’’. After selecting only genes considered a ‘‘within_species_paralog’’, we also assigned the maximum percent identity as the closest paralog.

To evaluate the effect that an independent assignment of target genes to regulatory regions might have on our analyses, we used the definition of target genes defined by Thurman et al. (ftp://ftp.ebi.ac.uk/pub/databases/…)

which use correlations in DNase hypersensitivity between distal and proximal regulatory regions across different cell types to link distal elements to putative target genes [38].

We intersected the midpoints of our called binding events (defined above) with these regulatory elements in order to assign our binding events to specific target genes and then re-analyzed the overlap between

binding and differential expression in our experiments.

PLOS Genetics 6 Mar 2014; 10 (3), e1004226

The essential biology of the endoplasmic reticulum stress response

for structural and computational biologists

Sadao Wakabayashi^a, Hiderou Yoshida^a,*

aDepartment of Molecular Biochemistry, Graduate School of Life Science,

University of Hyogo, Hyogo 678-1297, Japan

CSBJ Mar 2013; 6(7), e201303010, http://dx.doi.org/10.5936/csbj.201303010

Abstract: The endoplasmic reticulum (ER) stress response is a cytoprotective mechanism that maintains homeostasis of the ER by

upregulating the capacity of the ER in accordance with cellular demands.

If the ER stress response cannot function correctly, because of reasons such as aging, genetic mutation or environmental stress,

unfolded proteins accumulate in the ER and cause ER stress-induced apoptosis,
resulting in the onset of folding diseases,
- including Alzheimer’s disease and diabetes mellitus.

Although the mechanism of the ER stress response has been analyzed extensively by biochemists, cell biologists and molecular biologists, many aspects remain to be elucidated. For example,

it is unclear how sensor molecules detect ER stress, or
how cells choose the two opposite cell fates
(survival or apoptosis) during the ER stress response.

To resolve these critical issues, structural and computational approaches will be indispensable, although the mechanism of the ER stress response is complicated and difficult to understand holistically at a glance. Here, we provide a concise introduction to the mammalian ER stress response for structural and computational biologists.

The basic mechanism of the mammalian ER stress response

The mammalian ER stress response consists of three pathways: the ATF6, IRE1 and PERK pathways, of which the main functions are

augmentation of folding and ERAD capacity, and
translational attenuation, respectively.

Although these response pathways cross-talk with each other and have several branched subpathways, we focus on the main pathways in this section.

The ATF6 pathway regulates the transcriptional induction of ER chaperone genes
pATF6(P) is a sensor molecule comprising a type II transmembrane protein residing on the ER membrane (Figure 2).

When pATF6(P) detects ER stress,

the protein is transported to the Golgi apparatus through vesicular transport in a COP-II vesicle
and is sequentially cleaved by two proteases residing in the Golgi,
- namely site 1 protease (S1P) and site 2 protease (S2P)

The cytoplasmic portion of pATF6(P) (pATF6(N)) is

released from the Golgi membrane,
translocates into the nucleus,
binds to an enhancer element called the ER stress response element (ERSE),
and activates the transcription of ER chaperone genes,

including BiP, GRP94, calreticulin and protein disulfide isomerase (PDI)

The consensus nucleotide sequence of ERSE is CCAAT(N9)CCACG, and pATF6(N) recognizes both the CCACG portion and another transcription factor NF-Y,

which binds to the CCAAT portion

NF-Y is a general transcription factor required for

the transcription of various human genes

Figure 2. The ATF6 pathway. The sensor molecule pATF6(P) located on the ER membrane is transported to the Golgi apparatus by transport vesicles in response to ER stress. In the Golgi apparatus, pATF6(P) is sequentially cleaved by two proteases, S1P and S2P, resulting in release of the cytoplasmic portion pATF6(N) from the ER membrane. pATF6(N) translocates into the nucleus and activates transcription of ER chaperone genes through binding to the cis-acting enhancer ERSE.

Figure 3. The IRE1 pathway. In normal growth conditions, the sensor molecule IRE1 is an inactive monomer, whereas IRE1 forms an active oligomer in response to ER stress. Activated IRE1 converts unspliced XBP1 mRNA to mature mRNA by the cytoplasmic mRNA splicing. From mature XBP1 mRNA, an active transcription factor pXBP1(S) is translated and activates the transcription of ERAD genes through binding to the enhancer UPRE.

Figure 4. The PERK pathway. When PERK detects unfolded proteins in the ER, PERK phosphorylates eIF2α, resulting in translational attenuation and translational induction of ATF4. ATF4 activates the transcription of target genes encoding translation factors, anti-oxidation factors and a transcription factor CHOP. Other kinases such as PKR, GCN2 and HRI also phosphorylate eIF2α, and phosphorylated eIF2α is dephosphorylated by CReP, PP1C-GADD34 and p58IPK

Figure 7. Three functions of pXBP1(U). pXBP1(U) translated from XBP1(U) mRNA binds to pXBP1(S) and enhances its degradation. The CTR region of pXBP1(U) interacts with the ribosome tunnel and slows translation, while the HR2 region anchors XBP1(U) mRNA to the ER membrane, in order to enhance splicing of XBP1(U) mRNA by IRE1.

Figure 8. Major pathways of ER stress-induced apoptosis. ER stress induces apoptosis through various pathways, including transcriptional induction of CHOP by the PERK and ATF6 pathways, the IRE1-TRAF2 pathway and the caspase-12 pathway.

If cells are damaged by strong and sustained ER stress that they cannot deal with and ER stress still persists and hampers the survival of the organism, the ER stress response activates the apoptotic pathways and disposes of damaged cells from the body.

Computational simulation of response pathways to analyze the decision mechanism that determines cell fate (survival or apoptosis) provides a valuable analysis tool, although there have been few such studies to date.

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Pathology Emergence in the 21st Century

Posted in Bio Instrumentation in Experimental Life Sciences Research, Biological Networks, Gene Regulation and Evolution, Biomarkers & Medical Diagnostics, Biomedical Measurement Science, CANCER BIOLOGY & Innovations in Cancer Therapy, Cell Biology, Signaling & Cell Circuits, Cerebrovascular and Neurodegenerative Diseases, Chemical Biology and its relations to Metabolic Disease, Clinical Diagnostics, Computational Biology/Systems and Bioinformatics, Curation, Developmental biology, Experimental validation, Explanatory, Gene Regulation, Genomic Expression, Methylation, Oxidative phosphorylation, Technology Advance Assessment of, Warburg effect, tagged Cancer - General, inf;ammatory disease, molecular methods of discovery, patho;ogy, Regulation of gene expression on August 3, 2014| Leave a Comment »

Pathology Emergence in the 21st Century

Author and Curator: Larry Bernstein, MD, FCAP

This discussion follows the series on DNA and its replication, the code of life, and immediately follows a an up to date survey of RNA, it’s many discovered forms, their function, and the transcription of RNA, intermediate to protein synthesis. This will comprise a series of articles, including the chemistry, structure, and function of proteins, before turning to the “metabolome”. This discussion is about the development of the scientific profession of pathology in three notable phases. The first phase might be considered the gross anatomical discussion developed in Vienna, under Rokitansky, whose greatest student was the Hungarian pathologist, Semelweis, who began the insistence on handwashing prior to visiting the delivery room based on the observation that deliver was safer done by midwifes than by physicians. This was prior to the great discoveries in microbiology. The Rokitansky procedure is distinctive in removing organ systems in order to examine postmortem anatomical changes. His document on the pathology of the organs was monumental. It was refined by Rudolf Virchow, who removed one organ at a time, but he also had the now developing field of microscopic anatomy (also describing leukemia),

Rudolph Virchow 1821-1902

that would also be embellished by a generation of histologists who introduced staining techniques. The most remarkable anatomist to emerge in that period was John Hunter, the Scottish born anatomist and surgeon in London, who taught medical students from England and United States, and who was the physician for Sir David Hume. When he served in the 30 years war, he pulled wounded soldiers out of the mud to a clearing to care for their wounds.

The 20th century saw the introduction of a new medical school education system under advisement by the Carnegie Commission. [Abraham Flexner Report] This led to the teaching of basic sciences of anatomy, physiology, pharmacology, pathology, and later genetics and microbiology as prerequisite to clinical teaching. Moreover, teaching was done by formal systems of disease, which later developed into rotations in Obstetrics and Gynecology, Medicine, and Surgery, Pediatrics, to which endocrinology and neurology and psychiatry were added. The first great Medical School was actually Johns Hopkins, that paved the groundwork. Harvard, Cornell, Columbia, and NYU followed, as did the University of California, San Francisco, and the University of Chicago. This was a period dominated by many important discoveries, and a domination of the Nobel Prizes in Medicine and Physiology, Chemistry and Physics (rivalled only by Germany and United Kingdom). The Uniqueness of Pathology lies in its placement in the first year of medical school, in preparation for the formal study of medicine, with a foot in both basic sciences and the other in clinical practice. The pathologist received all tissues removed from surgical procedures, and performed autopsies to determine the cause of death and comorbid conditions. The development of a substantial knowledge of the kidney gave rise to the specialty of nephrology, and at the same time drew pathologists into a “phase” of molecular biology with the introduction of the electron microscope. However, the field of immunology developed, and the need for transfusion hastened the emergence of clinical antigen-antibody testing, the crossmatch, blood banking, and later, the emergence of organ transplantation. In addition, clinical microbiology became a part of clinical pathology, and included fungi and tick-borne diseases, and nemitodes.

We have entered the third phase of pathology with the completion of the human genetic code, and with the development of target pharmaceutical development in the 21st century.

Modern Techniques of Molecular Pathology

A look at clinical laboratory science and its expected progress over the next decade

Molecular Diagnostics 2013; 22 (2), p 35Promising forecasts project great expectations for medical sciences in the year 2013 and beyond. These predictions follow a decade after completion of the Human Genome Project, and are accompanied by immense breakthroughs in computational and applied mathematics. In my view, they are:

Genomic and allied -omics technology
Innovation in mathematical classification (complexity)
Nanotechnology
Synthetic chemistry from physics, organic and inorganic chemistry

It is not my intention to go deeply into the exponential group of these advanced and integrative sciences; rather, I want to raise awareness of an emerging new world that will open to the clinical laboratory scientist, and signal the need in the next generation of laboratory personnel to embrace knowledge domains that will be critical for their careers.

All of these breakthroughs are tied together by a search for personalized and integrative medicine. These breakthroughs will reinvent nutritional and pharmaceutical medicine as well as medical devices and restructure clinical laboratory and imaging applications to cardiology, oncology, radiology and anatomical pathology.

Metabolomics

What does metabolomics and metabolic profiling have to do with this? Metabolomics is the measurement of small molecules that interact with membrane receptors¹ that are involved with regulation of genomic transcription and cellular regulation and upregulation or downregulation of metabolic processes essential to health. As well, these small molecules may provide targets for disease treatments, and as they are investigated, also provide further “analytes for diagnosis and, moreover, prediction of short-term or long-term outcomes.”²

As a result, the laboratory will become a more significant factor in measuring health and disease and in guiding health or disease maintenance. As our population has reached increased age limits, the laboratory has been a contributor in the public health sphere, and will have a greater role as a result of:

Improved tie in with provision of information to not only the healthcare workers, but also the patient
Achieve turnaround times for critical results through better workflow
Greater control and access to a next generation of point-of-care technology integrated with the laboratory database, and a restructured electronic health record

Despite the hype about the “big data” revolution, this is achievable in the system proposed because there is a published model to achieve this.²

Familiar Methods

Either individually or grouped as a profile, metabolites are detected by nuclear magnetic resonance spectroscopy or mass spectrometry, providing a basis for uses of metabolome findings extended to the early detection and diagnosis of cancer and as both a predictive and pharmacodynamics marker of drug effect. We can expect it to become the link between the laboratory and the clinic. The methods used in genomics are microarrays, and for proteomics they are the already familiar chromatographic principles that species migrate at different rates through a column matrix based on their volatility, or carries out a separation as the molecules differ by their adsorption to and elution from a solid matrix, dependent on the binding to the matrix and solubility in the solvent eluate, modified by pH, ionic concentration, and specific conditions needed for recovery. Powerful mathematical tools are used to analyze the data.³

Cardiovascular Disease

Although coronary thrombosis is the final event in acute coronary syndromes, increasing evidence suggests that inflammation also plays a key role in development of atherosclerosis and its clinical manifestations, such as myocardial infarction, stroke and peripheral vascular disease. The inflammatory component was indicated by epidemiological studies of elevated serum levels of high sensitivity C-reactive protein. That eventually led to the demonstration of a benefit from reduction of CRP in individuals without characteristic lipidemia in a major clinical trial, which drew a relationship between diabetes, obesity and disordered inflammatory response in the causation of coronary artery disease, aortic valve and artery disease, carotid artery and peripheral vascular disease.

Cancer

Because cancer cells are known to possess a highly unique metabolic phenotype, development of specific biomarkers in oncology is possible and might be used in identifying fingerprints, profiles or signatures to detect the presence of cancer, determine prognosis and/or assess the pharmacodynamic effects of therapy.⁴

HDM2, a negative regulator of the tumor suppressor p53, is over-expressed in many cancers that retain wild-type p53. Consequently, the effectiveness of chemotherapies that induce p53 might be limited, and inhibitors of the HDM2-p53 interaction are being sought as tumor-selective drugs.⁵

Coagulation

Blood coagulation plays a key role among numerous mediating systems activated in inflammation. Receptors of the PAR family serve as sensors of serine proteinases of the blood clotting system in the target cells involved in inflammation. Activation of PAR_1 by thrombin and of PAR_2 by factor Xa leads to a rapid expression and exposure on the membrane of endothelial cells of both adhesive proteins that mediate an acute inflammatory reaction and of the tissue factor that initiates the blood coagulation cascade.

The details of evolving methods are avoided in order to build the argument that a very rapid expansion of discovery has been evolving depicting disease, disease mechanisms, disease associations, metabolic biomarkers, study of effects of diet and diet modification, and opportunities for targeted drug development.

Bernstein is CEO of Triplex Medical Science and CSO of Leaders in Pharmaceutical Intelligence. He has been involved in a collaborative project on reducing the noise that exists in complex data and developing a primary evidence-based classification since retiring from a career in pathology supning four decades.

References

1. Bernstein LH. Metabolomics, metabonomics and functional nutrition: The next step in nutritional metabolism and biotherapeutics. Journal of Pharmacy and Nutrition Sciences, 2012;2.

2. David G, Bernstein LH, Coifman RR. Generating evidence-based interpretation of hematology screens via anomaly characterization. The Open Clinical Chemistry Journal 2011;4: 10-16.

3. Grainger DJ. Megavariate statistics meets high data-density analytical methods: The future of medical diagnostics? IRTL Reviews 2003;1:1-6.

4. Spratlin JL, Serkova NJ, Eckhardt SG. Clinical applications of metabolomics in oncology: A review. Clin Cancer Res 2009;15; 15(2): 431-440.

5. Fischer PM, Lane DP. Small molecule inhibitors of thep53 suppressor HDM2: Have protein-protein interactions come of age as drug targets? Trends in Pharm Sci 2004;25(7):343-346.

Directions for Genomics in Personalized Medicine

Author: Larry H. Bernstein, MD, FCAP

Purpose

This discussion will identify the huge expansion of genomic technology in the search for biopharmacotherapeutic targets that continue to be explored involving different levels and interacting signaling pathways. There are several methods of analyzing gene expression that will be discussed. Great primary emphasis required investigation of combinations of mutations expressed in different cancer types.

James Watson has proposed a major hypothesis that expresses the need to focus on “central” “driver mutations” that correspond with the regulation of gene expression, cell proliferation, and cell metabolism with a critical rejection of antioxidant benefits. What hasn’t been know is why drug resistance develops and whether the cellular migration and aerobic glycolysis can be redirected after cell metastasis occurs. I attempt to bring out the complexities of current efforts.

Introduction

This discussion is a continuation of a previous discussion on the role of genomics if discovery of therapeutic targets for cancer, each somewhat different, but all related to:
The reversal of carcinoma by targeting a key driver of multiple signaling pathways that activate cell proliferation
Pinpointing a stage in a multistage process at which tumor progression links to changes in morphology from basal cells to invasive carcinoma with changes in polarity and loss of glandular architecture
Reversal of the carcinoma through using a small molecule that either is covalently bound to a nanoparticle delivery system that blocks or reverses tumor development
Synthesis of a small molecule that interacts with the translation of the genome either by substitution of a key driver molecule or by blocking at the mRNA stage of translation
Blocking more than one signaling pathway that are links to carcinogenesis and cellular proliferation and invasion

Difficulty of the problem

A problem expressed by James Watson is that the investigations that are ongoing

are following a pathway that is not driven by attacking the “primary” driver of carcinogenesis.

He uses the Myc gene as an example, as noted in the previous discussion. The problem may be more complicated than he envisions.

The most consistent problem in chemotherapy, irrespective of the design and the target has been cancer remission for a short time followed by recurrence, and then
switching to another drug, or combination chemotherapy.

It is common to “clean” the field at the time of resection using radiotherapy before chemotherapy.

But the goal is understood to be “palliation”, not cure.

This raises a serious issue in the hypothesis posed by Watson. The issue is

whether there is a core locus of genetic regulation that is common to carcinogenesis irrespective of tissue metabolic expression.
This is supported by the observation that tissue specific expression is lost in cancer cells by de-differentiation.

Historical Perspective

AEROBIC GLYCOLYSIS

In 1967 Otto Warburg published his view in a paper “The prime cause and prevention of cancer”.
There are primary and secondary causes of all diseases

plague – primary: plague bacillus
plague – secondary: filth, rats, and fleas

Otto Heinrich Warburg (Photo credit: Wikipedia)

cancer, above all diseases,

has countless seconday causes
primary – replacement of respiration of oxygen in normal body tissue by fermentation of glucose with conversion from obligate aerobic to anaerobic, as in bacterial cells

The cornerstone to understanding cancer is in study of the energetics of life

This thinking came out of decades of work in the Dahlem Institute Kaiser Wilhelm pre WWII and Max Planck Institute after WWII, supported by the Rockefeller Foundation.

The oxygen- and hydrogen-transferring enzymes were discovered and isolated.
The methods were elegant for that time, using a manometer that improved on the method used by Haldane, that did not allow the leakage of O2 or CO2.
The interest was initiated by the increased growth of Sea Urchin eggs after fertization, which turned out to be not comparable to the rapid growth of cancer cells.
Warburg used both normal and cancer tissue and measured the utilization of O2. He found
- that the normal tissue did not accumulate lactic acid.
- Cancer tissue generated lactic acid
- the rate of O2 consumption the same as normal tissue, but
- the rate of lactate formation far exceeded any tissue, except the retina.
- This was a discovery studied by “Pasteur” 60 years earlier (facultative aerobes), which he called thePasteur effect.
- Hematopoietic cells of bone marrow develop aerobic glycolysis when exposed to a low oxygen condition.

He then followed on an observation by Otto Meyerhoff (Embden-Myerhoff cycle) that in muscle

the consumption of one molecule of oxygen generates two molecules of lactate, but in aerobic glycolysis, the relationship disappears.
He expressed the effectiveness of respiration by the ‘Meyerhoff quotient’.
He found that cancer cells didn’t have a quotient of ’2′

The role of the allosteric enzyme phosphofructokinase (PFK) not then known, would tie together the glycolytic and gluconeogenic pathways.
He used a heavy metal ion chelator ethylcarbylamine to

sever the link and turn on aerobic glycolysis.

The explanation for this was provided years later by the work fleshed out by Lynen, Bucher, Lowry, Racker, and Sols.

The rate-limiting enzyme, PFK is regulated by the concentrations of ATP, ADP, and inorganic phosphate. The ethylcarbylamide was an ‘uncoupler’ of oxidative phosphorylation.

Warburg understood that when normal cells switched to aerobic glycolysis

it is a re-orientation of normal cell expression.
this provides the basis for the inference that neoplastic cells become more like each other than their cell of origin.
embryonic cells can be transformed into cancer cells under hypoxic conditions
re-exposure to higher oxygen did not cause reversion back to normal cells.

Warburg publically expressed the rejected view in 1954 (at age 83) that restriction of chemical wastes, food additives, and air pollution would substantially reduce cancer rates.

His emphasis on the impairment of respiration was inadequate.

the prevailing view today is loss of controlled growth of normal cells in cancer cells.

Otto Warburg: Cell Physiologist, Biochemist, and Eccentric. Hans Krebs, in collaboration with Roswitha Schmid. Clarendon Press, Oxford. 1991.ISBN 0-19-858171-8.

A multiphoton fluorescence microscope (MFM) is a specialized optical microscope.

The MFM uses pulsed long-wavelength light to excite fluorophores within the specimen being observed. The fluorophore absorbs the energy from two long-wavelength photons which must arrive simultaneously in order to excite an electron into a higher energy state, from which it can decay, emitting a fluorescence signal. It differs from traditional fluorescence microscopy in which the excitation wavelength is shorter than the emission wavelength, as the summed energies of two long-wavelength exciting photons will produce an emission wavelength shorter than the excitation wavelength.^[1]

Multiphoton fluorescence microscopy has similarities to confocal laser scanning microscopy. Both use focused laser beams scanned in a raster pattern to generate images, and both have an optical sectioning effect. Unlike confocal microscopes, multiphoton microscopes do not contain pinhole apertures, which give confocal microscopes their optical sectioning quality. The optical sectioning produced by multiphoton microscopes is a result of the point spread function formed where the pulsed laser beams coincide. The multiphoton point spread function is typically dumbbell-shaped (longer in the x-y plane), compared to the upright rugby-ball shaped point spread function of confocal microscopes. However, in many interesting cases the shape of the spot and its size can be designed to realize specific desired goals.^[2]

The longer wavelength, low energy (typically infra-red) excitation lasers of multiphoton microscopes are well-suited to use in imaging live cells as they cause less damage than short-wavelength lasers, so cells may be observed for longer periods with fewer toxic effects. Many researchers are currently working toward better and higher resolution multiphoton imaging developments.

Two-photon excitation microscopy is a fluorescence imaging technique that allows imaging of living tissue up to a very high depth, up to about one millimeter. Being a special variant of the multiphoton fluorescence microscope, it uses red-shifted excitation light which can also excite fluorescent dyes. However, for each excitation, two photons of infrared light are absorbed. Using infrared light minimizes scattering in the tissue. Due to the multiphoton absorption, the background signal is strongly suppressed. Both effects lead to an increased penetration depth for these microscopes. Two-photon excitation can be a superior alternative to confocal microscopy due to its deeper tissue penetration, efficient light detection, and reduced phototoxicity.^[1]

Two-photon excitation employs two-photon absorption, a concept first described by Maria Goeppert-Mayer (1906–1972) in her doctoral dissertation in 1931,^[2] and first observed in 1961 in a CaF₂:Eu²⁺ crystal using laser excitation by Wolfgang Kaiser.^[3] Isaac Abella showed in 1962 in cesium vapor that two-photon excitation of single atoms is possible.^[4]

The concept of two-photon excitation is based on the idea that two photons of comparably lower energy than needed for one photon excitation can also excite a fluorophore in one quantum event. Each photon carries approximately half the energy necessary to excite the molecule. An excitation results in the subsequent emission of a fluorescence photon, typically at a higher energy than either of the two excitatory photons. The probability of the near-simultaneous absorption of two photons is extremely low. Therefore a high flux of excitation photons is typically required, usually from a femtosecond laser. The purpose of employing the two-photon effect is that the axial spread of the point-spread-function is substantially lower than for single-photon excitation. As a result, the resolution along the z dimension is improved, allowing for thin optical sections to be cut. In addition, in many interesting cases the shape of the spot and its size can be designed to realize specific desired goals.^[5] Two-photon microscopes are less damaging to the sample than a single-photon confocal microscope.

The most commonly used fluorophores have excitation spectra in the 400–500 nm range, whereas the laser used to excite the two-photon fluorescence lies in the ~700–1000 nm (infrared) range. If the fluorophore absorbs two infrared photons simultaneously, it will absorb enough energy to be raised into the excited state. The fluorophore will then emit a single photon with a wavelength that depends on the type of fluorophore used (typically in the visible spectrum). Because two photons are absorbed during the excitation of the fluorophore, the probability for fluorescent emission from the fluorophores increases quadratically with the excitation intensity. Therefore, much more two-photon fluorescence is generated where the laser beam is tightly focused than where it is more diffuse. Effectively, excitation is restricted to the tiny focal volume (~1 femtoliter), resulting in a high degree of rejection of out-of-focus objects. This localization of excitation is the key advantage compared to single-photon excitation microscopes, which need to employ additional elements such as pinholes to reject out-of-focus fluorescence. The fluorescence from the sample is then collected by a high-sensitivity detector, such as a photomultiplier tube. This observed light intensity becomes one pixel in the eventual image; the focal point is scanned throughout a desired region of the sample to form all the pixels of the image. The scan head is typically composed of two mirrors, the angles of which can be rapidly altered with a galvanometer.

.Multiphoton microscopy: a potential “optical biopsy” tool for real-time evaluation of lung tumors without the need for exogenous contrast agents.

Jain M¹, Narula N, Aggarwal A, Stiles B, Shevchuk MM, Sterling J, Salamoon B, Chandel V, Webb WW, Altorki NK, Mukherjee S.
From the Departments of Urology (Dr Jain), Pathology and Laboratory Medicine (Drs Narula and Shevchuk), Biochemistry (Drs Aggarwal and Mukherjee, Mr Sterling, and Mr Salamoon), Thoracic Surgery (Drs Stiles and Altorki), and Surgery (Mr Chandel), Weill Cornell Medical College, New York, New York; and the School of Applied and Engineering Physics, Cornell University, Ithaca, New York (Dr Webb). Dr Aggarwal is now with the Department of Science, Borough of Manhattan Community College, New York.
Arch Pathol Lab Med. 2014 Aug;138(8):1037-47. http://dx.doi.org:/10.5858/arpa.2013-0122-OA. Epub 2013 Nov 7

Context.-Multiphoton microscopy (MPM) is an emerging, nonlinear, optical-biopsy technique, which can generate subcellular-resolution images from unprocessed and unstained tissue in real time.

Objective.-To assess the potential of MPM for lung tumor diagnosis. Design.-Fresh sections from tumor and adjacent nonneoplastic lung were imaged with MPM and then compared with corresponding hematoxylin-eosin slides.

Results.-Alveoli, bronchi, blood vessels, pleura, smokers’ macrophages, and lymphocytes were readily identified with MPM in nonneoplastic tissue. Atypical adenomatous hyperplasia (a preinvasive lesion) was identified in tissue adjacent to the tumor in one case. Of the 25 tumor specimens used for blinded pathologic diagnosis, 23 were diagnosable with MPM. Of these 23 cases, all but one adenocarcinoma (15 of 16; 94%) was correctly diagnosed on MPM, along with their histologic patterns. For squamous cell carcinoma, 4 of 7 specimens (57%) were correctly diagnosed. For the remaining 3 squamous cell carcinoma specimens, the solid pattern was correctly diagnosed in 2 additional cases (29%), but it was not possible to distinguish the squamous cell carcinoma from adenocarcinoma. The other squamous cell carcinoma specimen (1 of 7; 14%) was misdiagnosed as adenocarcinoma because of pseudogland formation. Invasive adenocarcinomas with acinar and solid pattern showed statistically significant increases in collagen. Interobserver agreement for collagen quantification (among 3 observers) was 80%.

Conclusions.-Our pilot study provides a proof of principle that MPM can differentiate neoplastic from nonneoplastic lung tissue and identify tumor subtypes. If confirmed in a future, larger study, we foresee real-time intraoperative applications of MPM, using miniaturized instruments for directing lung biopsies, assessing their adequacy for subsequent histopathologic analysis or banking, and evaluating surgical margins in limited lung resections. PMID: 24199831

Lung cancer is the most common cause of cancer-related mortality worldwide in both men and women, with 226 160 new cases and 160 340 deaths estimated in the United States alone in 2012.¹ Lung tumors are currently detected on chest radiography and computed tomography imaging, but definitive diagnosis, especially distinguishing the various subtypes of lung cancer, requires cytologic or histopathologic examination. Although considered the gold standard in establishing diagnosis, histopathology requires time-consuming tissue processing and can sometimes require repeat biopsies if the initial specimen was nondiagnostic. To overcome some of the obstacles associated with histopathologic processing, efforts have been made to develop high-resolution “optical biopsy” imaging techniques.

In this proof-of-principle pilot study, we explored the use of multiphoton microscopy (MPM) as a promising, new optical biopsy tool for the detection and diagnosis of lung tumors in real time.

Multiphoton microscopy relies on the simultaneous absorption of 2 or 3 low-energy (near-infrared) photons to cause a nonlinear excitation, equivalent to that created by a single photon of shorter wavelength light. By using 2-photon excitation in the 700- to 800-nm range, MPM enables both in vivo and ex vivo imaging of fresh, unprocessed, and unstained tissue at histologic resolution via generating intrinsic tissue emissions. Intrinsic tissue emission signals used in this study included autofluorescence and second harmonic generation (SHG).^2–4

Twenty-five adult subjects diagnosed with lung cancer and undergoing lobectomies at our institution participated in this Institutional Review Board–approved study.

An Olympus FluoView FV1000MPE imaging system (Olympus America, Center Valley, Pennsylvania) was used for all MPM imaging. For detailed description of MPM imaging conditions, please see Supplementary Methods (supplemental digital content for this article is available at www.archivesofpathology.org in the August 2014 table of contents). Briefly, fresh (unprocessed and unstained) specimens were excited using 780 nm light from a tunable titanium-sapphire laser (Mai Tai DeepSee, Spectra-Physics, Irvine, California). Three distinct intrinsic tissue-emission signals were collected using photomultiplier tubes and were then color coded by using MetaMorph (version 7.0, revision 4, Molecular Devices, Sunnyvale, California) as follows: (1) SHG (360–400 nm, color-coded red), a nonlinear scattering signal originating from tissue collagen; (2) short wavelength autofluorescence (420–490 nm, color-coded green), originating in part from reduced nicotinamide adenine dinucleotide and flavin adenine dinucleotide in normal epithelial, neoplastic, and inflammatory cells, and from elastin in the alveolar septa; and (3) long wavelength autofluorescence (550–650 nm, color-coded blue), originating in part from carbon-laden macrophages.

Arch Path MPM

http://www.archivesofpathology.org/na101/home/literatum/publisher/pinnacle/journals/content/arpa/2014/15432165-138.8/arpa.2013-0122-oa/20140721/images/medium/i1543-2165-138-8-1037-f01.gif

http://www.archivesofpathology.org/na101/home/literatum/publisher/pinnacle/journals/content/arpa/2014/15432165-138.8/arpa.2013-0122-oa/20140721/images/large/i1543-2165-138-8-1037-f01.jpeg

Figure 1. Comparative multiphoton microscopy (MPM) and hematoxylin-eosin images of nonneoplastic and smoker lung. A and B, Low-magnification images show lung parenchyma composed of alveoli (arrows) surrounded by pleura (arrowheads). Inset in MPM shows pleura with collagen (red) and elastin (green) components. C and D, High-magnification images show primarily elastin fibers, with some collagen in the septal wall (arrowheads) of the alveoli (arrows). E and F, Low-magnification images show bronchus (*) with cartilage (arrowheads) and a medium-sized blood vessel (arrows). G and H, High-magnification images show columnar lining of the bronchus (arrows) and underlying connective tissue (arrowheads). I and J, Alveoli filled with carbon-laden macrophages (arrows; blue in MPM) and noncarbon-laden macrophages (arrowheads; green in MPM). K and L, A collection of small lymphocytes (arrowheads and inset), along with smoker’s macrophages (arrows). Some loss and thickening of the alveolar septa is shown (I through L) (MPM, original magnifications ×48 [A and E], ×96 [A inset], ×300 [C, G, I, and K], ×600 [K inset]; hematoxylin-eosin, original magnifications ×40 [B, F] and ×200 [D, H, J, and L]).

To assess the diagnostic potential of MPM, a blinded analysis was conducted. The attending pulmonary pathologist and an attending general surgical pathologist first familiarized themselves to the histologic features seen on MPM images in both nonneoplastic and neoplastic (adenocarcinoma and squamous cell carcinoma [SCC]) lung tissue. Because, in our study, multiple images were acquired from different areas of a given tumor, we used some of those images for the training set and did not include them in the blinded test set. Subsequently, test MPM images from all lung tumor specimens were assessed in a blinded fashion and were categorized according to subtype and pattern using the routine histopathologic criteria.^9,10 These diagnoses were then correlated with diagnoses made by the same pathologist, based on the corresponding H&E sections prepared from the same specimens.

Visualizing Lung From Smoker With MPM
Using MPM to Identify Invasive and Preinvasive Adenocarcinoma

Figure 2, A through B, shows an example of a lesion with atypical adenomatous hyperplasia, which was an incidental finding on MPM in “tumor-free” lung tissue. It shows the proliferation of atypical pneumocytes, along the preexisting alveolar wall. Gaps between the cells (discontinuous layer of pneumocytes) support the diagnosis of atypical adenomatous hyperplasia. Adenocarcinoma of lung with lepidic-predominant pattern, in contrast, shows continuous proliferation of tumor cells along the alveolar wall.

Arch Path progression from atypical lesion to invasive adenocarcinoma

Figure 2. Comparative multiphoton microscopy and hematoxylin-eosin images showing progression from atypical lesion to various patterns of invasive adenocarcinoma of lung. A and B, Images of atypical adenomatous hyperplasia shows a focus of pneumocyte proliferation (cuboidal cells with gaps between them) along the alveolar wall (arrows and insets). C and D, Images of adenocarcinoma of lung with lepidic-predominant pattern (arrows) and a few clusters of free-floating tumor cells (arrowheads). E and F, Images of adenocarcinoma of lung with acinar-predominant pattern (arrows). G and H, Images showing solid pattern (arrows) with suggestion of gland formation (arrowheads). I and J, Images showing papillary pattern (papillae with fibrovascular core; arrows). K and L, Images showing micropapillary pattern, with complete destruction of normal lung parenchyma. The airspace shows small papillary clusters of tumor cells (arrows) lacking true fibrovascular cores (multiphoton microscopy, original magnifications ×300 [A, C, E, G, I, and K] and ×600 [inset]; hematoxylin-eosin, originalmagnifications ×200 [B, D, F, H, J, and L] ×400 [inset]).

Arch Path SCC

http://www.archivesofpathology.org/na101/home/literatum/publisher/pinnacle/journals/content/arpa/2014/15432165-138.8/arpa.2013-0122-oa/20140721/images/medium/i1543-2165-138-8-1037-f02.gif

http://www.archivesofpathology.org/na101/home/literatum/publisher/pinnacle/journals/content/arpa/2014/15432165-138.8/arpa.2013-0122-oa/20140721/images/large/i1543-2165-138-8-1037-f02.jpeg

Adenocarcinoma with a papillary-predominant pattern shows clear papillary projections composed of cuboidal to columnar cells, which line collagen-rich fibrovascular cores (Figure 2, I and J). Micropapillary adenocarcinoma, on the other hand, shows small papillary clusters of malignant cells within the airspace, with no true fibrovascular cores (Figure 2, K and L).

Using MPM to Identify SCC of Lung

Figure 3 shows high-magnification images acquired from the tumor mass in subjects with a diagnosis of SCC. Figure 3, A and B, shows sheets of malignant cells with a complete loss of the normal architecture of the lung parenchyma. These cells are seen as arranged in a pavementlike fashion with a high nuclear to cytoplasmic ratio (indicating a high-grade tumor). Pavementlike arrangement of cells is a characteristic of SCC that helps to differentiate it from the solid-predominant pattern of adenocarcinoma. We also observed an increased amount of stroma and mononuclear inflammatory cells surrounding the tumor (Figure 3, A, inset). These mononuclear inflammatory cells were confirmed as lymphocytes on H&E. This case was correctly diagnosed as SCC on MPM. Figure 3, C and D, shows images from the tumor mass of another subject, also diagnosed with SCC on H&E. However, it was misdiagnosed as adenocarcinoma on MPM, primarily because the central necrosis in the tumor nest was interpreted as gland formation. When the images were reanalyzed with knowledge of the SCC diagnosis, the pavementlike arrangement of cells was identified. We thus expect that, with a larger sample of SCC specimens and more experience, our ability to correctly diagnose SCC will improve significantly. Also, in the future, any specimen with a likely SCC diagnosis will be imaged over a larger area by using image tiling and by taking multiple stacks from various areas in the lesion, so as not to be misled by occasional, spurious, histologic features.

Figure 3. Comparative multiphoton microscopy and hematoxylin-eosin images of squamous cell carcinoma of the lung. A and B, Images of squamous cell carcinoma (SCC) of the lung showing sheets of malignant cells with high nuclear to cytoplasmic ratio (arrows), surrounded by lymphocytes (arrowheads) interspersed in collagen bundles (inset). C and D, Images of SCC of the lung showing pavementlike arrangement of the cells (arrows). Also shown is a nest of squamous cells with focal necrosis (arrowheads) forming pseudoglands, leading to a misdiagnosis of adenocarcinoma (multiphoton microscopy, original magnifications ×300 [A and C] and ×600 [inset]; hematoxylin-eosin, original magnifications ×200 [B and D]).

http://www.archivesofpathology.org/na101/home/literatum/publisher/pinnacle/journals/content/arpa/2014/15432165-138.8/arpa.2013-0122-oa/20140721/images/medium/i1543-2165-138-8-1037-f03.gif

http://www.archivesofpathology.org/na101/home/literatum/publisher/pinnacle/journals/content/arpa/2014/15432165-138.8/arpa.2013-0122-oa/20140721/images/large/i1543-2165-138-8-1037-f03.jpeg

Using MPM to Assess the Degree of Collagen in Lung Carcinoma

Previous studies have reported the amount of collagen as a prognostic factor in small, peripheral lung adenocarcinomas^5,6,8 and SCCs.⁷ In our study, we categorized adenocarcinomas into 3 groups:

(1) well-differentiated, adenocarcinomas with lepidic-predominant patterns;

(2) moderately differentiated, invasive adenocarcinomas with acinar-predominant patterns; and

(3) poorly differentiated, adenocarcinomas with solid-predominant patterns.

A well-preserved lung architecture (Figure 4, A through C), with slight alveolar septal thickening from collagen deposition, was seen in low-magnification images of a well-differentiated adenocarcinoma (with a lepidic-predominant pattern). In contrast, invasive adenocarcinomas with both acinar-predominant (Figure 4, D through F) and solid-predominant (Figure 4, G through I) patterns showed increases in collagen content.

Our proof-of-principle pilot study indicates the potential utility of MPM for differentiating nonneoplastic from neoplastic lung tissue in fresh, ex vivo specimens without the use of exogenous contrast agents. Furthermore, study pathologists successfully identified the histologic subtypes of tumor and recognized inflammatory cells, such as lymphocytes and smoker macrophages. We also performed collagen quantification in adenocarcinomas and demonstrated its correlation with the degree of differentiation.

Indeed, the diagnostic potential of MPM for differentiating malignant from benign/inflammatory lesions has been previously investigated in multiple organ systems in both animal models and human tissues.^3,12–19 Specifically, normal and diseased lung have been investigated in both small animals and in ex vivo human tissue using MPM.^20–26 However, most studies focused on extracellular matrix remodeling associated with lung pathologies.^22,23,25,26 To date, few studies have explored the potential of MPM for differentiating benign lesions from neoplastic ones.

. Our study is the first, to our knowledge, to present not only a detailed histology of normal human lung tissue obtained with MPM but also to show the histologic features that can be used to identify a variety of inflammatory, preneoplastic, and neoplastic lesions, in accordance with World Health Organization¹⁰ and International Association for the Study of Lung Cancer⁹ criteria. Furthermore, we demonstrated the ability of a pulmonary pathologist and a general surgical pathologist to differentiate between lesion subtypes in a blinded fashion with high reliability

The Human Genome Project

J. Craig Venter (Photo credit: Wikipedia)

The Human Genome Project, driven by Francis Collins at NIH, and by Craig Venterat the Institute for Genome Research (TIGR) had parallel projects to map the human chromosome, completed in 2003. It originally aimed to map the nucleotides contained in a human haploid reference genome (more than three billion). TIGR was the first complete genomic sequencing of a free living organism, Haemophilus influenzae, in 1995. This used a shotgun sequencing technique pioneered earlier, but which had never been used for a whole bacterium.
Venter broke away from the HGP and started Celera in 1998 because of resistance to the shotgun sequency method, and his team completed the genome sequence in three years – seven years’ less time than the HGP timetable (using the gene of Dr. Venter). TIGR eventually sequenced and analyzed more than 50 microbial genomes. Its bioinformatics group developed

pioneering software algorithms that were used to analyze these genomes,
including the automatic gene finder GLIMMER and
the sequence alignment program MUMmer.

In 2002, Venter created and personally funded the J. Craig Venter Institute (JCVI) Joint Technology Center (JTC), which specialized in high throughput sequencing. The JTC, in the top ranks of scientific institutions worldwide, sequenced nearly 100 million base pairs of DNA per day for its affiliated institutions (JCVI) .

He received his his Ph.D. degree in physiology and pharmacology from the University of California, San Diego in 1975 under biochemist Nathan O. Kaplan. A full professor at the State University of New York at Buffalo, he joined the National Institutes of Health in 1984. There he learned of a technique for rapidly identifying all of the mRNAs present in a cell and began to use it to identify human brain genes. The short cDNA sequence fragments discovered by this method are calledexpressed sequence tags (ESTs), a name coined by Anthony Kerlavage at TIGR.
Venter believed that shotgun sequencing was the fastest and most effective way to get useful human genome data. There was a belief that shotgun sequencing was less accurate than the clone-by-clone method chosen by the HGP, but the technique became widely accepted by the scientific community and is still the de facto standard used today.

References

Shreeve, James (2004). The Genome War: How Craig Venter Tried to Capture the Code of Life and Save the World. Knopf. ISBN 0375406298.
Sulston, John (2002). The Common Thread: A Story of Science, Politics, Ethics and the Human Genome. Joseph Henry Press. ISBN 0309084091.
“The Human Genome Project Race”. Center for Biomolecular Science & Engineering,UC Santa Cruz. Retrieved 20 March 2012.
Venter, J. Craig (2007). A Life Decoded: My Genome: My Life. Viking Adult. ISBN 0670063584.

Use of a Fluorophor Probe

An article has been discussed by Dr. Tilda Barilya on use of a sensitive fluorescent probe in the near IR spectrum at > 700 nm to identify malignant ovarian cells in-vivo in abdominal exploration

by tagging an overexpressed FR-α (folate-FITA)

The author makes the point that:

In ovarian cancer, the FR-α appears to constitute a good target because it is overexpressed in 90–95% of malignant tumors, especially serous carcinomas.
Targeting ligand, folate, is attractive as it is nontoxic, inexpensive and relatively easily conjugated to a fluorescent dye to create a tumor-specific fluorescent contrast agent.
The report is identified as “ the first in-human proof-of-principle of the use of intraoperative tumor-specific fluorescence imaging in staging and debulking surgery for ovarian cancer using the systemically administered targeted fluorescent agent folate-FITC.”

While this does invoke possibilities for prognosis, the decision to perform the surgery,

whether laparoscopic or open, is late in the discovery process. However,

it does suggest the possibility that the discovery and the treatment might be combined

if the biomarker itself had the fluorescence to identify the overexpression, but
it also is combined with a tag to block the overexpession. This hypothetical possibility is now expressed below.

http://pharmaceuticalintelligence.com/2013/01/19/ovarian-cancer-and-fluorescence-guided-surgery-a-report/

Gene Editing

Aviva Lev-Ari, PhD, RD

Dr. Aviva Lev-Ari reports that a new technique developed at MIT Broad Institute and the Rockefeller University can edit DNA in precise locations

taken from Science News titled “Editing Genome With High Precision: New Method to Insert Multiple Genes in Specific Locations, Delete Defective Genes”.

Using this system, scientists can alter

several genome sites simultaneously and
can achieve much greater control over where new genes are inserted

According to Feng Zhang, this is an improvement beyond splicing the gene in specific locations and

insertion of complexes difficult to assemble known as

transcription activator-like effector nucleases (TALENs).

The researchers create DNA-editing complexes
using naturally occurring bacterial protein-RNA systems
that recognize and snip viral DNA, including
a nuclease called Cas9 bound to short RNA sequences.
they target specific locations in the genome, and
when they encounter a match, Cas9 cuts the DNA.

This approach can be used either to

disrupt the function of a gene or
to replace it with a new one.
To replace the gene, a DNA template for the new gene has to be copied into the genome after the DNA is cut. The method is also very precise –
if there is a single base-pair difference between the RNA targeting sequence and the genome sequence, Cas9 is not activated.

In its first iteration, it appears comparable in efficiency to what

zinc finger nucleases and TALENs have to offer.

The research team has deposited the necessary genetic components with a nonprofit called Addgene, and they have also created a website with tips and tools for using this new technique.

The above story is reprinted from materials provided by Massachusetts Institute of Technology.

The original article was written by Anne Trafton. Le Cong, F. Ann Ran, David Cox, Shuailiang Lin, Robert Barretto, Naomi Habib, Patrick D. Hsu, Xuebing Wu, Wenyan Jiang, Luciano Marraffini, and Feng Zhang.

Multiplex Genome Engineering Using CRISPR/Cas Systems.
Science, 3 January 2013 DOI: 10.1126/science.1231143.
http://Science.com. Editing genome with high precision: New method to insert multiple genes in specific locations, delete defective genes. ScienceDaily. Retrieved January 20, 2013, from http://www.sciencedaily.com /releases/2013/01/130103143205.htm? goback=%2Egde_4346921_member_205356312.

Dr. Lev-Ari also reports on a study of early detection of breast cancer in “Mechanism involved in Breast Cancer Cell Growth: Function in Early Detection & Treatment“, by Dr. Rotem Karni and PhD student Vered Ben Hur at the Institute for Medical Research Israel-Canada of the Hebrew University.
http://pharmaceuticalintelligence.com/2013/01/17 /mechanism-involved-in-breast-cancer-cell-growth-function-in-early-detection-treatment/
These researchers have discovered a new mechanism by which breast cancer cells switch on their aggressive cancerous behavior. The discovery provides a valuable marker for the early diagnosis and follow-up treatment of malignant growths.
The method they use is

RNA splicing and insertion.
The information needed for the production of a mature protein is encoded in segments called exons .
In the splicing process, the non-coding segments of the RNA (introns) are spliced from the pre-mRNA and
the exons are joined together.

Alternative splicing is when a specific ”scene” (or exon) is either inserted or deleted from the movie (mRNA), thus changing its meaning.

Over 90 percent of the genes in our genome undergo alternative splicing of one or more of their exons, and
the resulting changes in the proteins encoded by these different mRNAs are required for normal function.
the normal process of alternative splicing is altered in cancer, and
”bad” protein forms are generated that aid cancer cell proliferation and survival.

The researchers reported in online Cell Reports that breast cancer cells

change the alternative splicing of an important enzyme, calledS6K1, which is
a protein involved in the transmission of information into the cell.
when this happens, breast cancer cells start to produce shorter versions of this enzyme and
these shorter versions transmit signals ordering the cells to grow, proliferate, survive and invade other tissues (otherwise proliferation is suppressed)

The application to biotherapeutics would be to ”reverse” the alternative splicing of S6K1 in cancer cells back to the normal situation as a novel anti-cancer therapy.

Imaging Mass Cytometry

This literature review shows how researchers used CyTOF mass cytometry to obtain spatial resolution of cell samples.

Doug Auld, Ph.D.

The authors used mass cytometry to measure heterogeneity in breast cancer tumors using FFPE breast cancer samples. [© Kheng Guan Toh – Fotolia.com]

The integration of mass spectrometry (specifically laser ablation of the sample in combination with inductively coupled plasma mass spectrometry) with flow cytometry instrumentation along with sensitive and rare earth metal labels

has enabled multiplexing of up to 32 cell markers (see Assay Drug Dev Technol 2011;9:567 commentary “Flow cytometry goes atomic;” CyTOF system sold by Fluidigm, formerly DVS Sciences).

This process employs typical immunocytochemistry techniques, but the antibodies are tagged with rare earth metal isotopes (predominately lanthanides)

that act as specific reporters of cellular proteins.

Comparison of these rare earth–labeled antibodies to typical fluorescent antibody labels supports that

these labels do not affect the specificity or sensitivity of the antibodies. In this article the authors* extend this method to obtain spatial resolution of cell samples.

mass cytometry to measure heterogeneity in breast cancer tumors using FFPE

is correlated with the position of the laser spot as it is scanned across the sample with 1 μm resolution.In the method, the signals from the rare earth reporters following laser ablation of the sample

The limit of detection is determined to be ∼500 molecules. The data can then be plotted based on the position of each ion spot for each rare earth reporter, and these images

are then overlaid to create a high-dimensional image that can be analyzed (Figure).

Measurement of a 0.5 mm × 0.5 mm area at 1 μm resolution takes ∼5 h. The system is capable of measuring 100 analytes simultaneously, but only 32 rare earth metal chelates are currently available. The authors applied this method

to measure heterogeneity in breast cancer tumors using formalin-fixed, paraffin-embedded (FFPE) breast cancer samples.

A total of 21 FFPE samples were analyzed using 32-plex imaging mass cytometry covering cell markers and phosphoproteins. Differences in expression even within the same tumor sample were noted, and

the subpopulations branch points often contained markers used for patient classification. Some exceptions occurred; for example,
Her2 was detected and confirmed in one triple-negative case.

This high-dimensional imaging should increase our understanding of tumor biology and pathologies.

*Abstract from Nature Methods 2014, Vol. 11: 403–406

Mass cytometry enables high-dimensional, single-cell analysis of cell type and state. In mass cytometry, rare earth metals are used as reporters on antibodies. Analysis of metal abundances using the mass cytometer

allows determination of marker expression in individual cells. Mass cytometry has previously been applied only to cell suspensions.

To gain spatial information, we have coupled

immunohistochemical and immunocytochemical methods
with high-resolution laser ablation to CyTOF mass cytometry.

This approach enables the simultaneous imaging of 32 proteins and protein modifications at subcellular resolution;

with the availability of additional isotopes, measurement of over 100 markers will be possible.

We applied imaging mass cytometry to human breast cancer samples, allowing delineation of cell subpopulations and cell–cell interactions and highlighting tumor heterogeneity. Imaging mass cytometry

complements existing imaging approaches.

It will enable basic studies of tissue heterogeneity and function and

support the transition of medicine toward individualized molecularly targeted diagnosis and therapies.

Preventing a cellular identity crisis

http://news.sciencemag.org/sites/default/files/styles/thumb_article_l/public/sn-criticalgenesH.jpg?itok=0wmD-o4a

Staying true.

neural progenitor cells keep their identities

Researchers have discovered a molecular signature in the genome that might help cells like these neural progenitor cells keep their identities throughout their lives.

By Mitch Leslie 31 July 2014

Cells rely on different ways to establish who they are and what they do. A novel mechanism

marks the identities of different kinds of cells in the human body—
and prevents them from transforming into another type altogether.

Scientists learned decades ago to read the basic genetic code by which cells convert a string of DNA bases into a protein’s amino acids. But for more than 10 years,

they’ve been trying to crack what’s known as the histone code, a more complex cipher embedded within organisms’ genomes.

Histones are the proteins that DNA coils around in chromosomes. Chemically tweaking histones in a variety of ways can

adjust the activity of genes, turning them up or down. For example,
cells shut off genes by attaching three methyl groups to a specific spot on a histone type known as H3.
affixing three methyl groups to another H3 location, a modification known as H3K4me3, has a different effect.

Cells typically add the H3K4me3 tags to histones in small sections of the genome, but researchers noticed that

sometimes the tag can sprawl across much larger areas,
modifying broad swaths of histones.

To find out whether these large blocks of histones carrying H3K4me3 tags convey a message in the histone code, molecular geneticist Anne Brunet of Stanford University in Palo Alto, California, and colleagues

traced their occurrence in more than 20 different cell types.

They found that the longest stretches pinpoint different sets of genes in different types of cells. As a result, the researchers realized

they could discriminate liver cells from, say, muscle cells or kidney cells
based only on the chromosomal locations of the largest H3K4me3 blocks. In addition,
they noticed that these stretches tended to mark genes that are crucial for a cell type’s function or
that help make it distinct. In embryonic stem cells, for instance,
they occur on genes that control the cells’ capacity to specialize.

The researchers further demonstrated that the labels mark cell identity genes by

using a technique called RNA interference (RNAi) in adult neural progenitor cells,
which can morph into any cell type in the brain.

As the researchers revealed online today in Cell,

they applied RNAi to dial down the genes that carried large blocks of H3K4me3 tags and
found that it impaired the cells’ ability to reproduce and to spawn neurons. However,
the progenitor cells could still divide normally if the researchers quieted genes that had only short sections of H3K4me3 tags or none at all.

The presence of long stretches of H3K4me3 markers

might help cells keep their identities for life.
“we’ve discovered a new signature,” Brunet says.

Although many other scientists have studied H3K4me3 tags, “the concept that this one mark

can distinguish all these cell types
the discovery could allow quick identification of cell types,
which would be useful in situations such as cancer diagnosis.

Posted in Biology

Gene Deletion Slows Aging and Reduces Cancer Risk

gene deletion

Source: © Gernot Krautberger – Fotolia.com

Scientists at the Wistar Institute say they have discovered that

mice lacking a specific protein live longer lives with fewer age-related illnesses.

The mice that lack the TRAP-1 protein, demonstrated less age-related tissue degeneration, obesity, and spontaneous tumor formation when compared to normal mice. Their findings could change how scientists view the metabolic networks within cells. In healthy cells,

TRAP-1 is an important regulator of metabolism and

regulates energy production in mitochondria, organelles that generate chemically useful energy for the cell.

In the mitochondria of cancer cells, TRAP-1 is universally overproduced.

The Wistar team’s report (“Deletion of the Mitochondrial Chaperone TRAP-1 Uncovers Global Reprogramming of Metabolic Networks”), which appears in Cell Reports, shows how ”

knockout” mice bred to lack the TRAP-1 protein compensate for this loss by switching to alternative cellular mechanisms for making energy.

“We see this astounding change in TRAP-1 knockout mice, where they show fewer signs of aging and are less likely to develop cancers,” said Dario C. Altieri. M.D., director of the Wistar Institute’s National Cancer Institute-designated Cancer Center. “Our findings provide

an unexpected explanation for how TRAP-1 and related proteins regulate metabolism within our cells.
we didn’t expect to see healthier mice with fewer tumors.

Dr. Altieri and his colleagues created the TRAP-1 knockout mice as part of their

ongoing investigation into the drug Gamitrinib, which targets the protein in the mitochondria of tumor cells.

TRAP-1 is a member of the heat shock 90 (HSP90) protein family, which are

chaperone proteins that guide the physical formation of other proteins and
serve a regulatory function within mitochondria.
Tumors use HSP90 proteins like TRAP-1 to help survive therapeutic attack.

In tumors,

the loss of TRAP-1 is devastating, triggering a host of catastrophic defects, including
metabolic problems that ultimately result in in death of the tumor cells,, BUT
Mice that lack TRAP-1 from the start have three weeks in the womb to compensate for the loss of the protein.”

In the knockout mice, the loss of TRAP-1

causes mitochondrial proteins to misfold, which then
triggers a compensatory response that causes cells to consume more oxygen and metabolize more sugar. which
causes mitochondria in knockout mice to produce deregulated levels of ATP,
the chemical used as an energy source to power all the everyday molecular reactions that allow a cell to function.

This increased mitochondrial activity actually creates a moderate boost in oxidative stress (free radical damage) and the associated DNA damage. While DNA damage may seem counterproductive to longevity and good health,

the low level of DNA damage actually reduces cell proliferation—slowing growth down to allow the cell’s natural repair mechanisms to take effect.

“TRAP-1^−/− mice are viable and showed reduced incidence of age-associated pathologies including – obesity, inflammatory tissue degeneration, dysplasia, and spontaneous tumor formation,- accompanied by

global upregulation of oxidative phosphorylation and glycolysis transcriptomes, causing

deregulated mitochondrial respiration,
oxidative stress,
impaired cell proliferation, and
a switch to glycolytic metabolism in vivo.

These data identify TRAP-1 as

a central regulator of mitochondrial bioenergetics, and
this pathway could contribute to metabolic rewiring in tumors.”

“Our findings strengthen the case

for targeting HSP90 in tumor cells, but it
may have implications for metabolism and longevity,” explained Dr. Altieri.

GEN News 8-1-2014

The role of the Wnt signaling pathway in cancer stem cells: prospects for drug development

Yong-Mi Kim, Michael Kahn
¹Children’s Hospital Los Angeles, Division of Hematology and Oncology, Department of Pediatrics and Pathology, ²Department of Biochemistry and Molecular Biology, Keck School of Medicine of University of Southern California, ³Norris Comprehensive Cancer Research Center, University of Southern California, Los Angeles, CA, USA

Research and Reports in Biochemistry July 2014; 4:1—12
http://dx.doi.org/10.2147/RRBC.S53823

Abstract: Cancer stem cells (CSCs), also known as tumor initiating cells are now considered to be

the root cause of most if not all cancers, evading treatment and giving rise to disease relapse.

They have become a central focus in new drug development.

Prospective identification,
understanding the key pathways that maintain CSCs, and
being able to target CSCs, particularly

if the normal stem cell population could be spared, could offer an incredible therapeutic advantage.

The Wnt signaling cascade is critically important in stem cell biology, both

in homeostatic maintenance of tissues and organs through their respective somatic stem cells and
in the CSC/tumor initiating cell population.

Aberrant Wnt signaling is associated with a wide array of tumor types. Therefore, the ability to

safely target the Wnt signaling pathway offers enormous promise to target CSCs. However,
just like the sword of Damocles, significant risks and concerns regarding targeting such a critical pathway in normal stem cell maintenance and tissue homeostasis remain ever present.

With this in mind, we review recent efforts in modulating the Wnt signaling cascade and critically analyze therapeutic approaches at various stages of development.

Keywords: beta-catenin, CBP, p300, wnt inhibition

A*STAR Scientists Pinpoint Genetic Changes that Spell Cancer: Fruit flies light the way for scientists to uncover genetic changes.

With a new approach, researchers may rapidly distinguish the range of

genetic changes that are causally linked to cancer (i.e.“driver” mutations)
versus those with limited impact on cancer progression.

This study published in the prestigious journal Genes & Development could pave the way

to design more targeted treatment against different cancer types, based on
the specific cancer-linked mutations present in the patient,
an advance in the development of personalized medicine.

Signaling pathways involved in tumour formation are conserved from fruit flies to humans. In fact, about 75 percent of known human disease genes have a recognizable match in the genome of fruit flies.
Leveraging on their genetic similarities, Dr Hector Herranz, a post-doctorate from the Dr. Stephen Cohen’s team developed an innovative strategy to genetically screen the whole fly genome for “cooperating” cancer genes.

These genes appear to have little or no impact on cancer.
However, they cooperate with other cancer genes, so that
the combination causes aggressive cancer, which
neither would cause alone.

In this study, the team was specifically looking for genes that

could cooperate withEGFR “driver” mutation,
a genetic change commonly associated with breast and lung cancers in humans.
SOCS5 (reported in this paper) is one of the several new “cooperating” cancer genes to be identified.

Already, there are indications that levels of SOCS5 expression are

reduced in breast cancer, and
patients with low levels of SOCS5 have poor prognosis.”

The IMCB team is preparing to explore the use of SOCS5 as a biomarker in diagnosis for cancer.

Probing What Fuels Cancer

http://genes&development.com

‘Altered cellular metabolism is a hallmark of cancer,’ says Dr Patrick Pollard, in the Nuffield Department of Clinical Medicine at Oxford.

Most cancer cells get the energy they need predominantly through

a high rate of glycolysis – allowing cancer cells deal with the low oxygen levels that tend to be present in a tumour. But
whether dysfunctional metabolism causes cancer, as Warburg believed, or is something that happens afterwards is a different question.
In the meantime, gene studies rapidly progressed and indicated that genetic changes occur in cancer.

DNA mutations spring up all the time in the body’s cells, but

most are quickly repaired.
Alternatively the cell might shut down or be killed off (apoptosis) before any damage is caused. However, the repair machinery is not perfect.
If changes occur that bypass parts of the repair machinery or sabotage it,
the cell can escape the body’s normal controls on growth and
DNA changes can begin to accumulate as the cell becomes cancerous.

Patrick believes certain changes in cells can’t always be accounted for by ‘genetics.’
He is now collaborating with Professor Tomoyoshi Soga’s large lab at Keio University in Japan, which has been at the forefront of developing the technology for metabolomics research over the past couple of decades.

The Japanese lab’s ability to

screen samples for thousands of compounds and metabolites at once, and
the access to tumour material and cell and animal models of disease
enables them to probe the metabolic changes that occur in cancer.

There is reason to believe that

dysfunctional cell metabolism is important in cancer.
genes with metabolic functions are associated with some cancers
changes in the function of a metabolic enzyme have been implicated in the development of gliomas.

These results have led to the idea that

some metabolic compounds, or metabolites, when they accumulate in cells, can cause changes to metabolic processes and set cells off on a path towards cancer.

Patrick Pollard and colleagues have now published a perspective article in the journal Frontiers in Molecular and Cellular Oncology that proposes

fumarate as such an ‘oncometabolite’. Fumarate is a standard compound involved in cellular metabolism.

The researchers summarize evidence that shows how

accumulation of fumarate when an enzyme goes wrong affects various biological pathways in the cell.
It shifts the balance of metabolic processes and disrupts the cell in ways that could favour development of cancer.

Patrick and colleagues write in their latest article that the shift in focus of cancer research to include cancer cell metabolism ‘has highlighted how woefully ignorant we are about the complexities and interrelationships of cellular metabolic pathways’.

NATURE GENETICS | BRIEF COMMUNICATION

Recurrent SMARCA4 mutations in small cell carcinoma of the ovary

Petar Jelinic, Jennifer J Mueller, Narciso Olvera, Fanny Dao, Sasinya N Scott, et al.

Nature Genetics (2014); 46: 424–426 http://dx.doi.org:/10.1038/ng.2922

Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is a rare, highly aggressive form of ovarian cancer primarily diagnosed in young women. We identified

inactivating biallelic SMARCA4 mutations in 100% of the 12 SCCOHT tumors examined.

Protein studies confirmed loss of SMARCA4 expression, suggesting a key role for the SWI/SNF chromatin-remodeling complex in SCCOHT.

At a glance

Figures

Figure 1: SMARCA4 mutations in SCCOHT and TCGA samples.close

SMARCA4 mutations in SCCOHT and TCGA samples.close

(a) Domain structure of the SMARCA4 protein (UniProt, SMCA4_HUMAN) overlaid with the alterations identified in 11 of the 12 SCCOHT cases in this study (case numbers in parentheses; case 103 with exon deletion is not shown). SNF2_N, SNF…

http://www.nature.com/ng/journal/v46/n5/carousel/ng.2922-F1.jpg

Figure 2: Analyses of the splice-site mutation in case 102.close

Analyses of the splice-site mutation in case 102.close

(a) Immunoblotting with antibody to the N terminus of SMARCA4. A high-grade serous ovarian cancer cell line (PEO4) and frozen tumor samples from two individuals with high-grade serous ovarian cancer (HGOC) were used as positive control…

http://www.nature.com/ng/journal/v46/n5/carousel/ng.2922-F2.jpg

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Download references

Primary authors

These authors contributed equally to this work.

Petar Jelinic & Jennifer J Mueller, Department of Surgery

Petar Jelinic, Jennifer J Mueller, Narciso Olvera, Fanny Dao & Douglas A Levine
Department of Surgery

Sasinya N Scott, Ronak Shah, Robert A Soslow & Michael F Berger
Department of Pathology,

JianJiong Gao & Nikolaus Schultz
Computational Biology Program,

Mithat Gonen Department of Epidemiology and Biostatistics, Memorial Sloan-Kettering Cancer Center, New York, New York, USA.

Corresponding author

Douglas A Levine

Supplementary information

Supplementary Figures

Supplementary Figure 1: Sequence analyses forSMARCA4 in SCCOHT cases. (715 KB)

Next-generation sequence coverage demonstrating identified variants (top panels) and validation through Sanger sequencing (bottom panels).

Supplementary Figure 2:SMARCA4 gene expression across TCGA tumors for cases with available mutation and RNA-seq data (RSEM). (366 KB)

A correlation is seen between inactivating SMARCA4 mutations and decreased gene expression across various solid tumors. A two-sided Student’s t test was used to compare samples with non-missense mutations and other samples without mutations or with only missense mutations. For all TCGA samples, the mean RNA-seq RSEM (2,050, s.d. of 1,760) was less in samples with non-missense mutations than in other samples without mutations or with only missense mutations (3,724, s.d. of 1,692; P = 8.7 × 10⁻⁴). For TCGA lung adenocarcinoma samples, the mean RNA-seq RSEM (601, s.d. of 370) was less in samples with non-missense mutations than in other samples without mutations or with only missense mutations (3,330, s.d. of 1,524; P = 2 × 10⁻⁸).

Supplementary Figure 3: Immunohistochemistry for SMARCA4 in SCCOHT cases. (566 KB)

High-grade serous ovarian carcinoma is used as a positive control. Case numbers are indicated in each panel. Immunohistochemistry results are provided in Supplementary Table 1. Note the intense staining of blood vessels and stromal cell nuclei as internal controls.

Supplementary Figure 4: Analysis of homozygous deletion in case 103. (109 KB)

Next-generation sequence coverage demonstrating that exons 25 and 26 are deleted. An electropherogram from Sanger sequencing of cDNA validating that the deletion retains an ORF from exon 24 to exon 27 (Panel A). One-step RT-PCR confirms that tumor tissue yields a single band with primers that span exons 24 and 27 (Panel B; *, nonspecific band). One-step RT-PCR with primers targeting regions upstream and downstream from the deletion site show equal expression, demonstrating continuation of transcription downstream from the deletion (Panel C).

Supplementary Figure 5: Analysis of splice-site variant in case 102. (90 KB)

One-step RT-PCR confirms that the exon-intron band is preferentially expressed over the exon-exon band in tumor tissue (Panel A). One-step RT-PCR with primers targeting regions upstream and downstream from the mutation site show equal expression, demonstrating continuation of transcription downstream from the mutation (Panel B). Immunoblots are shown in Figure 2b. The exon-exon primers detected weaker bands, reflecting loss of expression in tumor tissues compared with normal tissues in cases with splice-site mutations. The exon-intron primers demonstrated equivalent to greater expression of the retained intron in the tumor tissues. As SMARCA4 introns may be retained in non-cancer tissues, some intronic expression is expected in normal tissues. These data taken together indicate preferential intronic expression, as expected, in cDNA sequenced from tumor samples with splice-site mutations.

Supplementary Figure 6: Sequence analyses forSMARCA4 in the H1299 cell line. (134 KB)

An electropherogram from Sanger sequencing of genomic DNA validating a 69-nt deletion in the ORF of this control cell line that results in loss of protein expression, as shown inFigure 2b.

Supplementary Figure 7: SMARCA4 effect on cell proliferation. (131 KB)

SMARCA4 overexpression in H1299 cells. Representative immunoblot from three biologic replicates demonstrates a correlation between increased SMARCA4 and p21 expression (Panel A). Cell growth assessment in H1299 cells overexpressing SMARCA4. Mean cell counts from three biologic replicates (Panel B). Representative immunoblot confirmed SMARCA4 knockdown in 293T cells using shRNA. As a control, shNTC (Non-Targeting Control) was used (Panel C). XTT proliferation assay in 293T cells depleted of SMARCA4. Means represent three independent experiments (Panel D).

Supplementary Figure 8: Overall survival among lung adenocarcinoma TCGA cases based on inactivatingSMARCA4 (174 KB)

Median overall survival was 11.6 months among 6 patients with inactivating SMARCA4mutations compared with 44.6 months for 197 patients without inactivating mutations.

Supplementary Figure 9: Histopathological features of an SCCOHT. (979 KB)

The typical histopathological features of SCCOHT, including a combination of small neoplastic cells forming a pseudofollicular space and larger rhabdoid cells, are visible in a sample obtained from 1 of 12 tumors that were subjected to target capture and massively parallel DNA sequencing (hematoxylin and eosin).

PDF files

Supplementary Text and Figures (5,357 KB)

Supplementary Figures 1–9 and Supplementary Tables 1–5

CRISPR-Cas9 Foundational Technology originated at UC, Berkeley & UCSF, Broad Institute is developing Biotech Applications — Intellectual Property emerging as Legal Potential Dispute

Curator and Reporter: Aviva Lev-Ari, PhD, RN

http://pharmaceuticalintelligence.com/2014/06/18/crispr-cas9-foundational-technology-originated-at-uc-berkeley-ucsf-broad-institute-is-developing-biotech-applications-intellectual-property-emerging-as-legal-potential-dispute/

CRISPR-Cas9 Foundational Technology – The definition of “Prior Art” is at a very high stack, June 2014.

On 6/16/2014 Dr. Aviva Lev-Ari published the following two articles:

Lecture Contents delivered at Koch Institute for Integrative Cancer Research, Summer Symposium 2014: RNA Biology, Cancer and Therapeutic Implications, June 13, 2014 @MIT
http://pharmaceuticalintelligence.com/2014/06/16/lecture-contents-delivered-at-koch-institute-for-integrative-cancer-research-summer-symposium-2014-rna-biology-cancer-and-therapeutic-implications-june-13-2014-mit/
Prediction of the Winner RNA Technology, the FRONTIER of SCIENCE on RNA Biology, Cancer and Therapeutics & The Start Up Landscape in Boston
http://pharmaceuticalintelligence.com/2014/06/16/prediction-of-the-winner-rna-technology-the-frontier-of-science-on-rna-biology-cancer-and-therapeutics-the-start-up-landscape-in-boston/

Other related articles on CRISPR-Cas9 Technology published on this Open Access Online Scientific Journal include the following:

2:15 – 2:45, 6/13/2014, Jennifer Doudna “The biology of CRISPRs: from genome defense to genetic engineering”

http://pharmaceuticalintelligence.com/2014/06/13/215-245-6132014-jennifer-doudna-the-biology-of-crisprs-from-genome-defense-to-genetic-engineering/

Ribozymes and RNA Machines – Work of Jennifer A. Doudna

http://pharmaceuticalintelligence.com/2013/04/15/ribozymes-and-rna-machines-work-of-jennifer-a-doudna/

CRISPR @MIT – Genome Surgery

http://pharmaceuticalintelligence.com/2014/04/21/crispr-mit-genome-surgery/

Gene Therapy and the Genetic Study of Disease: @Berkeley and @UCSF – New DNA-editing technology spawns bold UC initiative as Crispr Goes Global

http://pharmaceuticalintelligence.com/2014/03/27/gene-therapy-and-the-genetic-study-of-disease-berkeley-and-ucsf-new-dna-editing-technology-spawns-bold-uc-initiative-as-crispr-goes-global/

Diagnosing Diseases & Gene Therapy: Precision Genome Editing and Cost-effective microRNA Profiling

http://pharmaceuticalintelligence.com/2013/03/28/diagnosing-diseases-gene-therapy-precision-genome-editing-and-cost-effective-microrna-profiling/

An expanded-DNA Biology from Scripps Research Institute: Beyond A-T and C-G: Applications for new Medicines and Nanotechnology

http://pharmaceuticalintelligence.com/2014/05/11/an-expanded-dna-biology-from-scripps-research-institute-beyond-a-t-and-c-g-applications-for-new-medicines-and-nanotechnology/

Evaluate your Cas9 Gene Editing Vectors: CRISPR/Cas Mediated Genome Engineering – Is your CRISPR gRNA optimized for your cell lines?

http://pharmaceuticalintelligence.com/2014/03/25/evaluate-your-cas9-gene-editing-vectors-crisprcas-mediated-genome-engineering-is-your-crispr-grna-optimized-for-your-cell-lines/

2:15 – 2:45, 6/13/2014, Jennifer Doudna “The biology of CRISPRs: from genome defense to genetic engineering”

http://pharmaceuticalintelligence.com/2014/06/13/215-245-6132014-jennifer-doudna-the-biology-of-crisprs-from-genome-defense-to-genetic-engineering/

About CRISPR “this technology will revolutionize biology in the same way PCR did,” Rudolf Jaenisch introducing Jennifer Doudna

Top CRISPR Related Publications

http://blog.appliedstemcell.com/top-crispr-related-publications/

Capturing key concepts of Prof. Jennifer Doudna’s Lecture @ KI Symposium:

acquired immunity in bacteria
three steps:

adaptation
biogenesis
interference

Big Pharma is using its venture cash to outsource early R&D to biotech

July 31, 2014 | By John Carroll

Analysts at Silicon Valley Bank have been crunching the numbers on biotech investing, and they have found that

a group of busy corporate venture arms has fundamentally changed the landscape for startups and
the entire field of early-stage drug development–
with some big implications for the current crop of industry upstarts.

Over the past two years corporate venture funding for biotech companies has surged back to 2008 levels, the bank’s analysts conclude, and

it now adds up to a much larger portion of the total amount of investment cash that’s available to biotechs.

Last year these corporate financing arms accounted for slightly

more than a third of all the cash that flowed into biotech, according to SVB. And
the corporate VCs have a big appetite for investing in early-stage rounds.

Courtesy of Silicon Valley Bank

Courtesy of Silicon Valley Bank

“I think we’ve reached a healthy level of funding in the sector right now,” says Jon Norris, the author of the report and managing director at Silicon Valley Bank. He adds that

with the IPO window still open to biotechs, a lot of early- or mid-stage companies are now choosing to jump through
to the public market rather than make a deal with pharma.

The IPO alternative has also made it possible to drive up the value of biotech assets, which now command record payments.

But that’s a trend that can’t run forever.

“This can’t run out too much longer into 2015,” says Norris. “I can see it starting to close.”

As the window shuts, he adds, you can expect to see the number of M&A deals rise. And the biggest biotech deals will likely be worth more, as big exits–defined as deals with an upfront of $75 million or more–jumped to an average record high of $549 million last year, a 10% spike over 2012.

Courtesy of Silicon Valley Bank 2

Courtesy of Silicon Valley Bank

In its analysis, Silicon Valley Bank concludes that the early-stage investment gamble now

amounts to a strategic move by the top Big Pharma companies to outsource a considerable portion of their early-stage R&D work,
priming the cash pump directly through their own venture arms as well as by investing in many of the new venture funds filling up with risk capital. And
the change-up is likely to continue to drive partnering as well as Big Pharma
forges a new round of development pacts and M&A deals with their venture colleagues involved in biotech.

“We’ve all seen over the last few years the pullback in overall R&D spending by pharma and biotech,” says Norris. “There’s a tendency for these (pharma) folks to outsource their innovation.”

Not surprisingly, experimental

cancer drugs are attracting the bulk of Big Pharma’s attention and corporate cash, followed by
platform technologies that generate new leads, metabolics, ophthalmology, cardiovascular, CNS, dermatology, GI and inflammation, says SVB.

The leading corporate venture investors in the industry include Novartis ($NVS),Astellas, Pfizer ($PFE), S.R. One ($GSK), Amgen ($AMGN) and J&J Development Corp. ($JNJ). And nearly 90% of top corporate investment deals are directed at Series A or B rounds. More than half of these new investments, says SVB, were in preclinical or Phase I companies.

Extensive Promoter-Centered Chromatin Interactions Provide a Topological Basis for Transcription Regulation
(Li G, Ruan X, Auerbach RK, Sandhu KS, et al.) Cell 2012; 148(1-2): 84-98. http://cell.com

http://FrontiersMolecularCellularOncology.com

Using genome-wide Chromatin Interaction Analysis with Paired-End-Tag sequencing (ChIA-PET),
mapped long-range chromatin interactions associated with RNA polymerase II in human cells
uncovered widespread promoter-centered intragenic, extragenic, and intergenic interactions.

These interactions further aggregated into higher-order clusters
proximal and distal genes were engaged through promoter-promoter interactions.
most genes with promoter-promoter interactions were active and transcribed cooperatively
some interacting promoters could influence each other implying combinatorial complexity of transcriptional controls.

Comparative analyses of different cell lines showed that

cell-specific chromatin interactions could provide structural frameworks for cell-specific transcription,
and suggested significant enrichment of enhancer-promoter interactions for cell-specific functions.
genetically-identified disease-associated noncoding elements were spatially engaged with corresponding genes through long-range interactions.

Overall, our study provides insights into transcription regulation by

three-dimensional chromatin interactions for both housekeeping and
cell-specific genes in human cells.

New Nucleoporin: Regulator of Transcriptional Repression and Beyond.

NJ Sarma and K Willis
Nucleus 2012; 3(6): 1–8; http://Nucleus.com © 2012 Landes Bioscience

Transcriptional regulation is a complex process that requires the integrated action of many multi-protein complexes.
The way in which a living cell coordinates the action of these complexes in time and space is still poorly understood.

nuclear pores, well known for their role in 3′ processing and export of transcripts, also participate in the control of transcriptional initiation.
nuclear pores interface with the well-described machinery that regulates initiation.

This work led to the discovery that

specific nucleoporins are required for binding of the repressor protein Mig1 to its site in target promoters.
Nuclear pores are involved in repressing, as well as activating, transcription.

Here we discuss in detail the main models explaining our result and consider what each implies about the roles that nuclear pores play in the regulation of gene expression.

Computational Design of Targeted Inhibitors of Polo-Like Kinase 1 ( lk1).

(KS Jani and DS Dalafave) Bioinformatics and Biology Insights 2012:6 23–31.
http://dx.doi.org:/10.4137/BBI.S8971

Computational design of small molecule putative inhibitors of Polo-like kinase 1 (Plk1) is presented. Plk1, which regulates the cell cycle, is often over expressed in cancers.

Down regulation of Plk1 has been shown to inhibit tumor progression.
Most kinase inhibitors interact with the ATP binding site on Plk1, which is highly conserved.
This makes the development of Plk1-specific inhibitors challenging, since different kinases have similar ATP sites.

However, Plk1 also contains a unique region called the polo-box domain (PBD), which is absent from other kinases.

the PBD site was used as a target for designed Plk1 putative inhibitors.
Common structural features of several experimentally known Plk1 ligands were first identified.
The findings were used to design small molecules that specifically bonded Plk1.
Drug likeness and possible toxicities of the molecules were investigated.
Molecules with no implied toxicities and optimal drug likeness values were used for docking studies.
Several molecules were identified that made stable complexes only with Plk1 and LYN kinases, but not with other kinases.
One molecule was found to bind exclusively the PBD site of Plk1.

Possible utilization of the designed molecules in drugs against cancers with over expressed Plk1 is discussed.

Conclusions

The previous discussions reviewed the status of an evolving personalized medicine multicentered and worldwide enterprise. It is also clear from these reports that the search for targeted drugs matched to a cancer profile or signature has identified several approaches that show great promise.

We know considerably more about metabolic pathways and linked changes in transcription that occur in neoplastic development.
There are several methods used to do highly accurate insertions in gene sequences that are linked to specific metabolic changes, and
some may have significant implications for therapeutics, if
- the link is a change that is associated with a driver mutation
- the link can be identified by a fluorescent or other probe
- the link is tied to a mRNA or peptide product that is a biomarker measured in the circulation
We have probes to genetic links to the control of many and interacting signaling pathways.
We know more about transcription through mRNA.
We are closer to the possibility that metabolic substrates, like ‘fumarate’ (a key intermediate in the TCA cycle), may provide a means to reverse regulate the neoplastic cells.
We may also find metabolic channels that drive the cells from proliferation to apoptosis or normal activity.

Summary

This discussion identified the huge expansion of genomic technology in the investigation of biopharmacotherapeutic targets that have been identified involving different levels and interacting signaling pathways. There are several methods of analyzing gene expression, and a primary emphasis is given to combinations of mutations expressed in different cancer types. There is a major hypothesis that expresses the need to focus on “central” “driver mutations” that correspond with the regulation of gene expression, cell proliferation, and cell metabolism. What hasn’t been know is why drug resistance develops and whether the cellular migration and aerobic glycolysis can be redirected after cell metastasis occurs.

mutation in the matched nucleotides

A slight mutation in the matched nucleotides can lead to chromosomal aberrations and unintentional genetic rearrangement. (Photo credit: Wikipedia)

Phosphofructokinase mechanism

Deutsch: Regulation der Phosphofructokinase (Photo credit: Wikipedia)

Additional Related articles

Unraveling the Human Genome: 6 Molecular Milestones(livescience.com)
The Role Of Microorganisms In Cancer Is Being Ignored By The Current Sequencing Strategies(3quarksdaily.com)
Biological Functions of Noncoding DNA(tginnovations.wordpress.com)
Retrovirus in the human genome is active in pluripotent stem cells(sciencedaily.com)
Consistent Personal Epigenetic ‘Signatures’ Discovered In Prostate Cancer Patients’ Metastases(medicalnewstoday.com)
Melanomas Often Mutate in Two Specific Ways, Shows Study(webpronews.com)

Universal Language: The Pistoia Alliance Takes on Indescribable Biology

By Aaron Krol

July 18, 2014 | The Pistoia Alliance, founded after a meeting between members of Pfizer, AstraZeneca, Novartis and GlaxoSmithKline, has come to resemble a United Nations of the life sciences industry. Now in its fifth year, the Alliance’s membership has grown to include nearly all the largest pharma companies (Eli Lilly is the only holdout in the top ten) plus a huge assortment of publishers, IT vendors, small biotechs and academic groups. It makes for a complicated network of business partners and competitors, but they do have some basic needs in common. In particular, the Pistoia Alliance exists to build IT architectures that serve the precompetitive stages of research and development.

“The key to the Pistoia Alliance is that, as time has gone by, most companies have figured out that you can’t go it alone,” says Sergio Rotstein, the Director of Research Business Technologies at Pfizer and a member of the Alliance’s board of directors. “Even the tightest of companies has opened up its walls quite a bit to collaboration… The idea of me asking my buddy from Merck, how did you solve that problem, and by the way would you mind giving me the solution — ten years ago, that would have gotten me laughed out of the room.”

The Pistoia Alliance has previously sponsored new methods for querying databases and the scientific literature, and a more effective algorithm for compressing and sharing genetic sequencing data. Over the past year, another Pistoia project, HELM, has entered the public domain after gradual development by an assortment of Alliance members. An open source language and set of editing tools for working with large biomolecules, HELM has already become a foundational part of research in at least three large pharmaceutical companies.

At the Bio-IT World Best Practices Awards this April, the HELM project won the Pistoia Alliance a top prize in the category of Informatics. These awards recognize advances in information technology and good management strategies at all levels of the biomedical industry. While the Best Practices Awards always seek to highlight programs that could be widely replicated, Bio-IT World rarely has the opportunity to single out a project that has been adopted so quickly across so many organizations as the Pistoia Alliance’s efforts around HELM.

A Loss for Words

HELM addresses a problem at the root level of drug discovery. Pharmaceutical and biotech companies are looking at increasingly complex molecules in the search for new therapeutics, testing out RNA- and peptide-based compounds that tap directly into cellular pathways. The trouble is that these large molecules, which are often hybrids of RNA, amino acids and other chemical structures, are difficult to concisely describe, even when their structures are perfectly known. They are too large and ungainly to represent atom-by-atom, but not uniform enough to be reduced to nucleotides and peptide chains.

“There have been a number of ways to represent small molecules,” says Rotstein. “That’s been the bread and butter of a number of companies for a long time, and that’s the realm of cheminformatics. And there’s been a lot of methodology for dealing with sequence-based entities, like genes and proteins, which is the realm of bioinformatics. The issue is that the types of molecules that we are targeting fall in between these two.”

This isn’t just a semantic issue; not having a standard language for biomolecules has practical consequences. It’s hard to register these molecules in databases, and even harder to conduct searches for them or share their structures with collaborators. The problem has recently come to a head, as growing knowledge of interlocking cellular systems has led researchers to therapies that increasingly resemble the body’s own tangled biology. “It follows the natural progression of science itself,” says Rotstein. “The application of peptides with unnatural amino acids, and the area of antibody -drug conjugates, has been growing a great deal over the past few years. A lot of the companies that traditionally worked in the small molecule space, nowadays are looking for a diverse portfolio.”

In 2008, Rotstein was part of an oligonucleotide unit at Pfizer that set out to build a new language to describe the compounds it was working with. The language would be similar to the small molecule notation SMILES (the Simplified Molecular-Input Line-Entry System), which renders a chemical structure as a continuous string of characters, while using symbols from the ASCII alphabet to resolve properties like where bonds occur and how molecules branch. Instead of using atoms as the smallest units in the chain, however, much larger groups — monomers like nucleotides and amino acids — would receive short, unique IDs that could be strung together into polymers. The amino acid cysteine, for instance, could be represented simply as “C.” New monomers would be registered with new IDs in a central database, and every ID would be linked to a complete description in small molecule notation.

oligonucleotide conplex

A complex oligonucleotide peptide conjugate, featuring amino acids, RNA, and other chemical structures. The molecule is rendered as both a monomer graph, and in HELM notation. Reproduced from the Journal of Chemical Information and Modeling with permission of the author

The language was called HELM, the Hierarchical Editing Language for Macromolecules: “hierarchical” because strings of monomers are built into simple polymers, which in turn are joined into complex polymers. HELM was easy to use and unambiguous, and was soon adopted in many more departments at Pfizer. For the first time, it was possible to quickly enter a new macromolecule in Pfizer’s registry, check for uniqueness, and receive a corporate ID to take the project forward.

A Living Language

At the same time that Rotstein’s team was developing HELM at Pfizer, other pharma companies and informatics vendors were struggling with the same problem. The software provider Accelrys (now BIOVIA), for instance, had modified the Molfile chemical table format to deal with hybrid macromolecules, in a system the company called the Self-Contained Sequence Representation (SCSR). There was a danger of proliferating standards, which would not only create redundant work at each company writing its own language, but also threaten the ability of these organizations to share information with each other.

Meanwhile, a member survey at the Pistoia Alliance flagged the representation of complex biomolecules as one of the industry’s top three non-competitive problems. Since Pfizer had already published a paper on HELM and built a software toolkit around the system, the company volunteered to make the entire program open source and continue its development with other members of the Alliance.

“We saw an opportunity for Pfizer,” says Rotstein. “If this did indeed become a standard, and the open source tools continued to evolve through contributions of the whole community, that would help us too.” All told, 24 companies sent volunteers to work on HELM, untangling the code from Pfizer’s internal systems, making it public, and extending the tools that serve the language.

The entire HELM project is now available on GitHub, and uses the permissive MIT open source license, which gives anyone the right to download and modify the code without requiring any contribution back to the project. That should encourage vendors to build commercial software on top of HELM, helping to foster compatibility across the industry.

The basic HELM toolkit includes search functions and uniqueness checks, as well as the HELM Editor, a platform for drawing chemical structures. The HELM Editor lets users plug in or draw monomers, then move up the scale to polymers made from those building blocks. It can be used simply as a translation tool, taking existing structures and giving them names in HELM notation, but Rotstein says it would also be a preferred platform for making new molecules from scratch.

HELM photo of siRNA

A screenshot from the HELM Editor, showing a siRNA molecule under construction. Image credit: Pistoia Alliance

Since HELM was released to the public last year, development has continued at various partner organizations. Roche was one of the first adopters, and has been relentlessly adding functionality to the toolkit. “Roche created a custom antibody-drawing capability on top of the HELM Editor, and it’s truly phenomenal,” says Rotstein. “They are now putting the finishing touches on that, and as soon as they’re done, they are pushing it right back out into the open source.”

He adds that Pfizer plans to start using Roche’s antibody drawing tool itself. “That tool alone will probably return our entire investment on externalizing HELM.”

Most recently, this month the Pistoia Alliance released Exchangeable HEL M, another big push for interoperability. While some basic monomers, like the natural amino acids and nucleotides, have universal IDs in HELM, most monomer IDs are unique to each user, stored in an internal database. That’s a necessary feature to make HELM flexible to the needs of every user, but it means that most molecules only make sense in the context of the databases against which they were designed.

Exchangeable HELM provides a file format that includes both the larger HELM sequence of a macromolecule, and separately, the chemical structure of each monomer inside it. That makes it easy for collaborators — say, a large pharma company and a CRO hired for a specific project — to send molecular structures back and forth. Exchangeable HELM also offers a tool to “translate” between databases, if two organizations have different internal IDs for the same monomer.

The Lingua Franca

So far, Pfizer, Roche, and Lundbeck are the largest drug companies to switch their systems over to HELM, and Rotstein says a “robust pipeline of other companies” is preparing to adopt the language. Meanwhile, vendors that serve the drug industry are preparing for a widespread change. NextMove Software and ChemAxon are both working in HELM, and even BIOVIA, which plans to continue using SCSR internally, has made its systems compatible with HELM to more easily share large molecules with clients and partners.

The adoption of HELM will be buoyed by public resources in the life sciences that are turning to the language as the obvious choice for representing complex molecules. One big supporter is the European Bioinformatics Institute, whose ubiquitous ChEMBL database of chemical compounds will include HELM notations in its next release.

Increasingly, says Rotstein, the Pistoia Alliance is speaking of a HELM ecosystem. “We want to have content providers that have structures in HELM format. We want vendors whose software can read and write HELM. We want companies that use HELM as their standard, we want CROs that can use HELM to exchange information with those companies, and next on our list are downstream things like scientific journals and regulatory agencies.” Large publishers and regulators would be especially important adopters, because they are such frequent and public ports of call for companies sending macromolecular structures outside their walls. If the FDA or Nature Publishing Group began accepting HELM structures, it could be a major convenience when applying for clinical trials and publications. “It would be much easier to just send a file that says, ‘here’s exactly what my structure is,’” says Rotstein, “rather than having to verbally explain the structure.”

Having HELM in place as a widely-shared language could also benefit other Pistoia Alliance projects. For example, the Controlled Substance Compliant Services Project is currently building a database of compounds that are regulated or restricted in various countries around the world, so companies can quickly refer to the local legislation affecting compounds they want to work with. If large biomolecules are subject to regulations, HELM would be a convenient way to make those policies searchable.

Like other Pistoia Alliance initiatives, HELM is designed to run smoothly in the background. Defining the structure of macromolecules in a standard format, is not a process that should offer any company an edge in drug discovery, but a basic feature at the foundation of the life sciences. In an ideal world, says Rotstein, “this should be a non-issue. The ability to represent these molecules, and get them in and out of our system so we can store them, search them, and run calculations on them, should be trivial.”

Pathology Practiced Todat

How doctors group non Hodgkin lymphomas

There are many different types of non Hodgkin lymphoma. Doctors estimate that there are more than 60 subtypes. Understanding how the different types of NHL are grouped, or classified, can be difficult. A variety of systems for classifying lymphomas have been used over the years. The latest is the World Health Organisation classification of 2008. We give a simple description of the groups on this page.

The pathologist will examine the cells to see

The gradeof your NHL
The type of cell affected(B cell or T cell)
What the cells look likeunder a microscope
Which proteins (markers) are on the surface of the lymphoma cells (immunohistochemistry)
If there are certain gene changes in the lymphoma cells (cytogenetics)

Grade of NHL

Doctors put non Hodgkin lymphomas into 2 groups depending on how quickly they are likely to grow and spread

Low grade (indolent) – these tend to grow very slowly
High grade (aggressive) – these tend to grow more quickly

The different grades of non Hodgkin lymphoma are treated in slightly different ways.

Type of white blood cell

One way of classifying NHL is by the type of white blood cells (lymphocytes) affected – B cells or T cells. Most people with NHL have B cell lymphomas.

What the lymphoma cells look like

Your doctor will be able to give your type of non Hodgkin lymphoma a name depending on the appearance of the lymphoma cells. These names are quite complicated. But they are useful to doctors because the different types can behave differently. Different treatments are used for the different types. So knowing the type helps the doctor know how to treat them. In the laboratory a pathologist looks at the cells to see if they are

Large or small
Grouped together in structures called follicles (follicular type) or spread out (diffuse type)

Low grade non Hodgkin lymphomas tend to have small cells that are grouped together.

Low grade (slowly growing) NHL

Low grade lymphomas tend to grow very slowly. Doctors call them indolent lymphomas. They include

Follicular lymphoma– the most common type
Mantle cell lymphoma
Marginal zone lymphoma
Small lymphocytic lymphoma(also called chronic lymphocytic leukaemia)
Lymphoplasmacytic NHL(including Waldenstrom’s macroglobulinaemia)
Skin lymphomas

Small lymphocytic lymphoma

Small lymphocytic lymphoma is also called chronic lymphocytic leukaemia (CLL). It makes up about 6 out of 100 lymphomas in the UK (6%). In theory, lymphoma is an illness that starts in the lymph nodes and leukaemia is an illness of the blood. But leukaemia and lymphoma have many similarities and often affect the body in similar ways. Chronic lymphocytic leukaemia is the term used for this condition if many of the abnormal cells are in the blood. Doctors call it small lymphocytic lymphoma when the disease involves the lymph nodes in particular.

The B-cell lymphomas are types of lymphoma affecting B cells. Lymphomas are “blood cancers” in the lymph glands. They develop more frequently in older adults and in immunocompromised individuals.

B-cell lymphomas include both Hodgkin’s lymphomas and most non-Hodgkins lymphomas. They are often divided into indolent (slow-growing) lymphomas and aggressive lymphomas. Indolent lymphomas respond rapidly to treatment and are kept under control (in remission) with long-term survival of many years, but are not cured. Aggressive lymphomas usually require intensive treatments, but have good prospects for a permanent cure.^[1]

Prognosis and treatment depends on the specific type of lymphoma as well as the stage and grade. Treatment includes radiation and chemotherapy. Early-stage indolent B-cell lymphomas can often be treated with radiation alone, with long-term non-reoccurrence. Early-stage aggressive disease is treated with chemotherapy and often radiation, with a 70-90% cure rate.^[1] Late-stage indolent lymphomas are sometimes left untreated and monitored until they progress. Late-stage aggressive disease is treated with chemotherapy, with cure rates of over 70%.^[1]

Small cell Lymphocytic lymphoma (overlaps with Chronic lymphocytic leukemia)

Indolent NHL. These types of lymphoma grow very slowly. As a result, people with indolent NHL may not need to start treatment when it is first diagnosed. They are followed closely, and treatment is only started when they develop symptoms or the disease begins to change; this is called watchful waiting. When indolent lymphoma is located only in one area (called localized disease, stages I and II; see the Stages section), radiation therapy may eliminate the NHL.

Subtyping

In addition to determining if the NHL is indolent or aggressive and whether it is B-cell, T-cell, of NK-cell lymphoma, it is very important to determine the subtype of NHL because each subtype can behave differently and may require different treatments. There are about 35 subtypes of NHL.

Small lymphocytic lymphoma. This type of lymphoma is very closely related to a disease called B-cell chronic lymphocytic leukemia (CLL), and about 5% of people with NHL have this subtype. It is considered an indolent lymphoma. Patients with small lymphocytic lymphoma may receive a combination of chemotherapy, monoclonal antibodies, and/or radiation therapy, or they may be followed closely with watchful waiting.

Lymphoma – B cell neoplasms

Non-Hodgkin Lymphoma

Cytogenetics
Reviewer: Nikhil Sangle, M.D., University of Utah and ARUP Laboratories (see Reviewers page)
Revised: 17 February 2011, last major update February 2011
Copyright: (c) 2001-2011, PathologyOutlines.com, Inc.

List of translocations
==============================================

Relatively common translocations are listed below
See each topic for more complete lists:

● t(1;14)(p32;q11): SCL (tal-1) and T cell receptor delta/alpha; preT ALL (15-30%)
● deletion of 11q23: CLL (10-20%)
● t(11;14)(q13;q32): bcl1/PRAD1 and IgH; mantle cell lymphoma (90%), B cell prolymphocytic leukemia (20%), myeloma (3%)
● Trisomy 12: B-CLL (30%)
● deletion 13q14: B -CLL (25-50%)
● t(14;19)(q32;q13): IgH and bcl3; B-CLL
● t(16;22);(q23;q11): cmaf and Ig lambda; multiple myeloma
● Trisomy 18: common in marginal zone lymphoma, MALT type

Lymphoma – B cell neoplasms

B cell lymphoma subtypes

Chronic lymphocytic leukemia – features that differ from SLL
Reviewer: Nikhil Sangle, M.D., University of Utah and ARUP Laboratories (see Reviewers page)
Revised: 20 September 2012, last major update February 2011
Copyright: (c) 2001-2012, PathologyOutlines.com, Inc

Terminology
==============================================

Leukemic disorder of CD5+ CD23+ tumor cells, usually B cell, that are small, round, low grade, with soccer ball appearance

Terminology
==============================================

Called CLL/chronic lymphocytic leukemia if leukemic involvement at diagnosis (5K or more of monoclonal B cell lymphocytosis per microliter)
● Less than 5K per microliter is termed monoclonal B lymphocytosis or possibly low stage CLL
● CLL with increased prolymphocytes (CLL/prolymphocytic leukemia): 10-55% prolymphocytes
● Prolymphocytic leukemia: >55% prolymphocytes

Lymphoma – B cell neoplasms

B cell lymphoma subtypes

Small lymphocytic lymphoma
Reviewer: Nikhil Sangle, M.D., University of Utah and ARUP Laboratories (see Reviewers page)
Revised: 6 February 2012, last major update February 2011
Copyright: (c) 2001-2012, PathologyOutlines.com, Inc

Definition
==============================================

Common low grade B cell lymphoma with pseudofollicles composed of mature lymphocytes resembling soccer balls in peripheral blood; cells are CD5+, CD23+

Clinical features
==============================================

Usually older patients (median age 60 years), 2/3 male, with disease in bone marrow, lymph nodes, spleen, liver
● Often presents with leukemia, although patients may be asymptomatic
● SLL may progress to blood (leukemic) involvement, but if so, there is usually less leukocytosis than cases with initial diagnosis of CLL
● Almost all cases are B cell origin
● Associated with hypogammaglobulinemia, monoclonal immunoglobulin spikes in some, infections; also autoantibodies to red blood cells and platelets causing hemolytic anemia and thrombocytopenia
● Median survival 4-6 years; indolent unless it transforms

Poor prognostic factors
==============================================

17p deletions, 11q22-23 deletion, non-mutated immunoglobulin genes, aberrant expression of CD2, CD7, CD10, CD13, CD33 or CD34 (Am J Clin Pathol 2003;119:824)

In 2013, the North American market was valued at $128.9 million and accounted for the largest share of the global digital pathology market, followed by Europe and Asia. The North American market is expected to grow at a healthy growth rate over the next five years. This high growth can be attributed to the favorable reimbursement scenario in the U.S. and the use of digital pathology to improve the quality of cancer diagnosis in Canada. However, lack of FDA approvals for digital pathology to be used for primary diagnosis acts as a major barrier for the North American market.

Other posts related to this discussion were published on this Open Source Online Scientific Journal from Leaders in Pharmaceutical Business Intelligence:

Big Data in Genomic Medicine, LHB
http://pharmaceuticalintelligence.com/2012/12/17/big-data-in-genomic-medicine/

A New Therapy for Melanoma, LHB
http://pharmaceuticalintelligence.com/2012/09/15/a-new-therapy-for-melanoma/

BRCA1 a tumour suppressor in breast and ovarian cancer – functions in transcription, ubiquitination and DNA repair, S Saha
http://pharmaceuticalintelligence.com/2012/12/04/brca1-a-tumour-suppressor-in-breast-and-ovarian-cancer-functions-in-transcription-ubiquitination-and-dna-repair/

Judging ‘Tumor response’-there is more food for thought, R Saxena
http://pharmaceuticalintelligence.com/2012/12/04/judging-the-tumor-response-there-is-more-food-for-thought/

Computational Genomics Center: New Unification of Computational Technologies at Stanford, A. Lev-Ari
http://pharmaceuticalintelligence.com/2012/12/03/computational-genomics-center-new-unification-of-computational-technologies-at-stanford/

Ovarian Cancer and fluorescence-guided surgery: A report, T. Barliya
http://pharmaceuticalintelligence.com/2013/01/19/ovarian-cancer-and-fluorescence-guided-surgery-a-report/

Personalized medicine gearing up to tackle cancer , R. Saxena
http://pharmaceuticalintelligence.com/2013/01/07/personalized-medicine-gearing-up-to-tackle-cancer/

Exploring the role of vitamin C in Cancer therapy, R. Saxena
http://pharmaceuticalintelligence.com/2013/01/15/exploring-the-role-of-vitamin-c-in-cancer-therapy/

Differentiation Therapy – Epigenetics Tackles Solid Tumors, SJ Williams
http://pharmaceuticalintelligence.com/2013/01/03/differentiation-therapy-epigenetics-tackles-solid-tumors/

Mechanism involved in Breast Cancer Cell Growth: Function in Early Detection & Treatment, A. Lev-Ari
http://pharmaceuticalintelligence.com/2013/01/17/mechanism-involved-in-breast-cancer-cell-growth-function-in-early-detection-treatment/

Personalized Medicine: Cancer Cell Biology and Minimally Invasive Surgery (MIS), A. Lev-Ari
http://pharmaceuticalintelligence.com/2012/12/01/personalized-medicine-cancer-cell-biology-and-minimally-invasive-surgery-mis/

Role of Primary Cilia in Ovarian Cancer, A. Awan
http://pharmaceuticalintelligence.com/2013/01/15/role-of-primary-cilia-in-ovarian-cancer-2/

The Molecular Pathology of Breast Cancer Progression, T. Bailiya`
http://pharmaceuticalintelligence.com/2013/01/10/the-molecular-pathology-of-breast-cancer-progression/

Stanniocalcin: A Cancer Biomarker, A. Awan
http://pharmaceuticalintelligence.com/2012/12/25/stanniocalcin-a-cancer-biomarker/

Nanotechnology, personalized medicine and DNA sequencing, T. Barliya
http://pharmaceuticalintelligence.com/2013/01/09/nanotechnology-personalized-medicine-and-dna-sequencing/

Gastric Cancer: Whole-genome reconstruction and mutational signatures, A. Lev-Ari
http://pharmaceuticalintelligence.com/2012/12/24/gastric-cancer-whole-genome-reconstruction-and-mutational-signatures-2/

Paradigm Shift in Human Genomics – Predictive Biomarkers and Personalized Medicine – Part 1, A. Lev-Ari
http://pharmaceuticalintelligence.com/2013/01/13/paradigm-shift-in-human-genomics-predictive-biomarkers-and-personalized-medicine-part-1/

LEADERS in Genome Sequencing of Genetic Mutations for Therapeutic Drug Selection in Cancer Personalized Treatment: Part 2, A. Lev-Ari
http://pharmaceuticalintelligence.com/2013/01/13/leaders-in-genome-sequencing-of-genetic-mutations-for-therapeutic-drug-selection-in-cancer-personalized-treatment-part-2/

Personalized Medicine: An Institute Profile – Coriell Institute for Medical Research: Part 3, A. Lev-Ari
http://pharmaceuticalintelligence.com/2013/01/13/personalized-medicine-an-institute-profile-coriell-institute-for-medical-research-part-3/

The Consumer Market for Personal DNA Sequencing: Part 4, A. Lev-Ari

http://pharmaceuticalintelligence.com/2013/01/13/consumer-market-for-personal-dna-sequencing-part-4/

Harnessing Personalized Medicine for Cancer Management, Prospects of Prevention and Cure: Opinions of Cancer Scientific Leaders @http://pharmaceuticalintelligence.com A. Lev-Ari
http://pharmaceuticalintelligence.com/2013/01/13/7000/

GSK for Personalized Medicine using Cancer Drugs needs Alacris systems biology model to determine the in silico effect of the inhibitor in its “virtual clinical trial” A Lev-Ari
http://pharmaceuticalintelligence.com/2012/11/14/gsk-for-personalized-medicine-using-cancer-drugs-needs-alacris-systems-biology-model-to-determine-the-in-silico-effect-of-the-inhibitor-in-its-virtual-clinical-trial/

Recurrent somatic mutations in chromatin-remodeling and ubiquitin ligase complex genes in serous endometrial tumors, S. Saha
http://pharmaceuticalintelligence.com/2012/11/19/recurrent-somatic-mutations-in-chromatin-remodeling-and-ubiquitin-ligase-complex-genes-in-serous-endometrial-tumors/

Metabolic drivers in aggressive brain tumors, pkandala
http://pharmaceuticalintelligence.com/2012/11/11/metabolic-drivers-in-aggressive-brain-tumors/

Personalized medicine-based cure for cancer might not be far away, R. Saxena
http://pharmaceuticalintelligence.com/2012/11/20/personalized-medicine-based-cure-for-cancer-might-not-be-far-away/

Response to Multiple Cancer Drugs through Regulation of TGF-β Receptor Signaling: a MED12 Control, A. Lev-Ari
http://pharmaceuticalintelligence.com/2012/11/21/response-to-multiple-cancer-drugs-through-regulation-of-tgf-%CE%B2-receptor-signaling-a-med12-control/

Human Variome Project: encyclopedic catalog of sequence variants indexed to the human genome sequence, A. Lev-Ari
http://pharmaceuticalintelligence.com/2012/11/24/human-variome-project-encyclopedic-catalog-of-sequence-variants-indexed-to-the-human-genome-sequence/

Prostate Cancer Cells: Histone Deacetylase Inhibitors Induce Epithelial-to-Mesenchymal Transition, SJ Williams
http://pharmaceuticalintelligence.com/2012/11/30/histone-deacetylase-inhibitors-induce-epithelial-to-mesenchymal-transition-in-prostate-cancer-cells/

Tumor Imaging and Targeting: Predicting Tumor Response to Treatment: Where we stand?, R. Saxena
http://pharmaceuticalintelligence.com/2012/12/13/imaging-and-targeting-the-tumor-predicting-tumor-response-where-we-stand/

Nanotechnology: Detecting and Treating metastatic cancer in the lymph node, T. Barliya
http://pharmaceuticalintelligence.com/2012/12/19/nanotechnology-detecting-and-treating-metastatic-cancer-in-the-lymph-node/

Heroes in Medical Research: Barnett Rosenberg and the Discovery of Cisplatin, SJ Williams
http://pharmaceuticalintelligence.com/2013/01/12/heroes-in-medical-research-barnett-rosenberg-and-the-discovery-of-cisplatin/

Inspiration From Dr. Maureen Cronin’s Achievements in Applying Genomic Sequencing to Cancer Diagnostics, A. Lev-Ari
http://pharmaceuticalintelligence.com/2013/01/10/inspiration-from-dr-maureen-cronins-achievements-in-applying-genomic-sequencing-to-cancer-diagnostics/

The “Cancer establishments” examined by James Watson, co-discoverer of DNA w/Crick, 4/1953, A. Lev-Ari
http://pharmaceuticalintelligence.com/2013/01/09/the-cancer-establishments-examined-by-james-watson-co-discover-of-dna-wcrick-41953/

Nanotech Therapy for Breast Cancer. T. Barlyia
http://pharmaceuticalintelligence.com/2012/12/09/naotech-therapy-for-breast-cancer/

Dasatinib in Combination With Other Drugs for Advanced, Recurrent Ovarian Cancer, pkandala
http://pharmaceuticalintelligence.com/2012/12/08/dasatinib-in-combination-with-other-drugs-for-advanced-recurrent-ovarian-cancer/

Squeezing Ovarian Cancer Cells to Predict Metastatic Potential: Cell Stiffness as Possible Biomarker, pkandala
http://pharmaceuticalintelligence.com/2012/12/08/squeezing-ovarian-cancer-cells-to-predict-metastatic-potential-cell-stiffness-as-possible-biomarker/

Hypothesis – following on James Watson, LHB

http://pharmaceuticalintelligence.com/2013/01/27/novel-cancer-h…ts-are-harmful/

Otto Warburg, A Giant of Modern Cellular Biology, LHB
http://pharmaceuticalintelligence.com/2012/11/02/otto-warburg-a-giant-of-modern-cellular-biology/

Is the Warburg Effect the cause or the effect of cancer: A 21st Century View? LHB
http://pharmaceuticalintelligence.com/2012/10/17/is-the-warburg-effect-the-cause-or-the-effect-of-cancer-a-21st-century-view/

Remembering a Great Scientist among Mentors, LHB
http://pharmaceuticalintelligence.com/2013/01/26/remembering-a-great-scientist-among-mentors/

Portrait of a great scientist and mentor: Nathan Oram Kaplan, LHB
http://pharmaceuticalintelligence.com/2013/01/26/portrait-of-a-great-scientist-and-mentor-nathan-oram-kaplan/

Predicting Tumor Response, Progression, and Time to Recurrence, LHB
http://pharmaceuticalintelligence.com/2012/12/20/predicting-tumor-response-progression-and-time-to-recurrence/

Directions for genomics in personalized medicine, LHB
http://pharmaceuticalintelligence.com/2013/01/27/directions-for-genomics-in-personalized-medicine/

How mobile elements in “Junk” DNA promote cancer. Part 1: Transposon-mediated tumorigenesis, Sjwilliams
http://pharmaceuticalintelligence.com/2012/10/31/how-mobile-elements-in-junk-dna-prote-cacner-part1-transposon-mediated-tumorigenesis/

Novel Cancer Hypothesis Suggests Antioxidants Are Harmful, LHB
http://pharmaceuticalintelligence.com/2013/01/27/novel-cancer-hypothesis-suggests-antioxidants-are-harmful/

Mitochondria: Origin from oxygen free environment, role in aerobic glycolysis, metabolic adaptation, LHB
http://pharmaceuticalintelligence.com/2012/09/26/mitochondria-origin-from-oxygen-free-environment-role-in-aerobic-glycolysis-metabolic-adaptation/

Advances in Separations Technology for the “OMICs” and Clarification of Therapeutic Targets, LHB
http://pharmaceuticalintelligence.com/2012/10/22/advances-in-separations-technology-for-the-omics-and-clarification-of-therapeutic-targets/

Cancer Innovations from across the Web, LHB
http://pharmaceuticalintelligence.com/2012/11/02/cancer-innovations-from-across-the-web/

Mitochondrial Damage and Repair under Oxidative Stress, LHB
http://pharmaceuticalintelligence.com/2012/10/28/mitochondrial-damage-and-repair-under-oxidative-stress/

Mitochondria: More than just the “powerhouse of the cell” R. Saxena
http://pharmaceuticalintelligence.com/2012/07/09/mitochondria-more-than-just-the-powerhouse-of-the-cell/

Mitochondria and Cancer: An overview of mechanisms, R. Saxena
http://pharmaceuticalintelligence.com/2012/09/01/mitochondria-and-cancer-an-overview/

Mitochondrial fission and fusion: potential therapeutic targets? R. Saxena
http://pharmaceuticalintelligence.com/2012/10/31/mitochondrial-fission-and-fusion-potential-therapeutic-target/

Mitochondrial mutation analysis might be “1-step” away, R. Saxena
http://pharmaceuticalintelligence.com/2012/08/14/mitochondrial-mutation-analysis-might-be-1-step-away/

β Integrin emerges as an important player in mitochondrial dysfunction associated Gastric Cancer, R. Saxena
http://pharmaceuticalintelligence.com/2012/09/10/%CE%B2-integrin-emerges-as-an-important-player-in-mitochondrial-dysfunction-associated-gastric-cancer/

mRNA interference with cancer expression, LHB
http://pharmaceuticalintelligence.com/2012/10/26/mrna-interference-with-cancer-expression/

What can we expect of tumor therapeutic response? LHB
http://pharmaceuticalintelligence.com/2012/12/05/what-can-we-expect-of-tumor-therapeutic-response/

Expanding the Genetic Alphabet and linking the genome to the metabolome, LHB
http://pharmaceuticalintelligence.com/2012/09/24/expanding-the-genetic-alphabet-and-linking-the-genome-to-the-metabolome/

Breast Cancer, drug resistance, and biopharmaceutical targets, LHB
http://pharmaceuticalintelligence.com/2012/09/18/breast-cancer-drug-resistance-and-biopharmaceutical-targets/

Breast Cancer: Genomic Profiling to Predict Survival: Combination of Histopathology and Gene Expression Analysis, A. Lev-Ari
http://pharmaceuticalintelligence.com/2012/12/24/breast-cancer-genomic-profiling-to-predict-survival-combination-of-histopathology-and-gene-expression-analysis/

Ubiquinin-Proteosome pathway, autophagy, the mitochondrion, proteolysis and cell apoptosis, LHB
http://pharmaceuticalintelligence.com/2012/10/30/ubiquinin-proteosome-pathway-autophagy-the-mitochondrion-proteolysis-and-cell-apoptosis/

Identification of Biomarkers that are Related to the Actin Cytoskeleton, LHB
http://pharmaceuticalintelligence.com/2012/12/10/identification-of-biomarkers-that-are-related-to-the-actin-cytoskeleton/

Nitric Oxide has a ubiquitous role in the regulation of glycolysis -with a concomitant influence on mitochondrial function, LHB
http://pharmaceuticalintelligence.com/2012/09/16/nitric-oxide-has-a-ubiquitous-role-in-the-regulation-of-glycolysis-with-a-concomitant-influence-on-mitochondrial-function/

Genomic Analysis: FLUIDIGM Technology in the Life Science and Agricultural Biotechnology, A. Lev-Ari http://pharmaceuticalintelligence.com/2012/08/22/genomic-analysis-fluidigm-technology-in-the-life-science-and-agricultural-biotechnology/

Reporter: Aviva Lev-Ari, PhD, RN

Crohn’s disease driven by inflammation – not genetics, reports study Aug. 15, 2012

Inflammation — not genetic susceptibility —

drives the growth of intestinal bacteria and invasive E. coli linked to Crohn’s disease (CD), reports a new Cornell study.

Scientists have long wondered about the role of bacteria in CD. Recent studies have shown marked changes in the composition of the intestinal bacteria in people

with CD, leading researchers to ask: Are microbial abnormalities a direct consequence of genetic abnormalities linked to Crohn’s and precede and initiate inflammation, or does intestinal inflammation bring on the bugs?

This study also reports that a common therapy directed against intestinal inflammation decreases dysbiosis. In addition, the study found that

the lack of a receptor that helps recruit T cells, which are needed for cell-mediated immunity, to the gut also decreases inflammation and dysbiosis, offering a new option for therapeutic intervention.Inflammation, in fact,
drives microbial imbalances (dysbiosis) and

the proliferation of a specific type of E. coli that is adherent, invasive and found in the ileum, reported Cornell researchers July 31 in PLoS (7[7]).

CD is a chronic debilitating inflammatory bowel disease that involves a complex interaction of

host genes,
the immune system,
the intestinal microbiome and
the environment.

To mirror the complex nature of the disease, Simpson’s team designed a study that

incorporated inflammatory triggers related to relapse of CD and ileal inflammation.

The team focused on ileal dysbiosis, which is prevalent in 70 percent of CD cases and

used a variety of contemporary techniques to generate a comprehensive picture of the composition and spatial distribution of the ileal microbiome.

Particular attention was paid to pinpointing

the number,
pathotype and
location

of E. coli associated with intestinal inflammation in people, dogs and mice.

The findings demonstrate that

inflammation drives ileal dysbiosis and proliferation of CD-associated adherent invasive E. coli.

the host genotype and therapeutically blocking inflammation both impact the onset and extent of ileal dysbiosis.

The investigation leveraged the knowledge and resources of researchers in the labs of Erik Denker, Dwight Bowman and Sean McDonough labs. Building on findings in patients with Crohn’s disease evaluated by Dr. Ellen Scherl’s group at Weill Cornell Medical College, this collaboration shed new light on this debilitating disease.

This work was supported by NewYork-Presbyterian Hospital/Weill Cornell Medical Center, the Jill Roberts Center for Inflammatory Bowel Disease and the National Institutes of Health.

http://www.news.cornell.edu/stories/Aug12/Inflammation.html

Functional Proteomics Related to Energy Metabolism of Synaptosomes

from iTRAQ-Based Quantitative Proteomics Analysis Revealed Alterations of Carbohydrate Metabolism Pathways and Mitochondrial Proteins in a Male Sterile Cybrid Pummelo

Bei-Bei Zheng †, Yan-Ni Fang †, Zhi-Yong Pan †, Li Sun †, Xiu-Xin Deng †, Jude W. Grosser ‡, andWen-Wu Guo *†

^† Key Laboratory of Horticultural Plant Biology (Ministry of Education), Huazhong Agricultural University, Wuhan 430070, China
^‡ Citrus Research and Education Center, University of Florida, 700 Experiment Station Road, Lake Alfred, Florida 33850, United States

Proteome Res., May 13, 2014, 13(6), pp 2998–3015 http://dx.doi.org:/10.1021/pr500126g

Plant Biochemistry

Comprehensive and quantitative proteomic information on citrus floral bud is significant for understanding

male sterility of the cybrid pummelo (G1+HBP) with nuclear genome of HBP and foreign mitochondrial genome of G1.

Scanning electron microscopy and transmission electron microscopy analyses of the anthers showed that

the development of pollen wall in G1+HBP was severely defective with a lack of exine and sporopollenin formation.

Proteomic analysis was used to identify the differentially expressed proteins between male sterile G1+HBP and fertile type (HBP)

with the aim to clarify their potential roles in another development and male sterility.

On the basis of iTRAQ quantitative proteomics, we identified 2235 high-confidence protein groups, 666 of which showed

differentially expressed profiles in one or more stages.

Proteins up- or down-regulated in G1+HBP were mainly involved in

carbohydrate and energy metabolism (e.g., pyruvate dehydrogenase, isocitrate dehydrogenase, ATP synthase, and malate dehydrogenase),
nucleotide binding (RNA-binding proteins),
protein synthesis and degradation (e.g., ribosome proteins and proteasome subunits).

Additionally, the proteins located in mitochondria also showed changed expression patterns. These findings provide a valuable inventory of proteins involved in floral bud development and contribute to elucidate the mechanism of cytoplasmic male sterility in the cybrid pummelo.

Keywords: cybrid; male sterility; mitochondria; proteome; transcriptome; primary metabolites

BIMSB Proteomics / Metabolomics

Overview

Within the past decades biochemical data of single processes, metabolic and signaling pathways were collected and advances in technology

led to improvements of sensitivity and resolution of bioanalytical techniques.

These achievements build the bases for the so called ‘genome wide biochemistry’. High throughput techniques are the tool for large scale ‘-omics’ studies

allowing the obtainment of a nearly complete picture of a determinate cell state, concerning its metabolites, proteins and transcripts.

However, a single level study of a living organism cannot give a complete understanding of the mechanisms regulating biological functions.
The integration of transcriptomics, proteomics and metabolomics data with existing knowledge allows connecting biological processes which were treated as independent so far. In this context the aim of our group is

to apply metabolomics and proteomics techniques for absolute quantification and
to analyze turnover rates of proteins and metabolites using stable isotopes. In addition,
the development of data analysis workflows and integrative strategies are in the focus of our interest.

The central metabolism is the principal source of energy and building blocks for cell growth and survival. It is highly flexible and adjusted to the physiological program of the cell, organ and organism. In a healthy state

cellular metabolism is tightly regulated to guarantee physiological function but also efficient usage of available recourses.

Metabolic dys-regulations are cause or response to many diseases. An impaired metabolic activity can lead to

the loss of the physiological activity, cell damage or inefficient substrate usage. However,
the underlying mechanisms leading to metabolic dys-functions are not well understood.

The regulation of metabolism is complex, because

it acts at all biological layers – transcriptional, translational and post-translational.

Thus the metabolic activity of a cell, organ or organism inherits the information of regulatory layers in a multidimensional manner. I guess only the use of integrative mathematical approaches will enable us to decode such complex information.

In this regard, decoding the metabolic composition of biofluids e.g. blood serum

may allow to determine a systems status, to identify diseases, predict drug responsiveness and to follow the success of medical treatments. This is a step towards personalized medicine.

http://www.mdc-berlin.de/20902775/en/research/core_facilities/cf_massspectromety_bimsb

Coordination of bacterial proteome with metabolism by cyclic AMP signaling

Conghui You, Hiroyuki Okano, Sheng Hui, Zhongge Zhang, Minsu Kim, et al.

Nature (15 August 2013); 500, 301–306 http://dx.doi.org:/10.1038/nature12446

The cyclic AMP (cAMP)-dependent catabolite repression effect in Escherichia coli is among the most intensely studied regulatory processes in biology. However,

the physiological function(s) of cAMP signalling and its molecular triggers remain elusive.

Here we use a quantitative physiological approach to show that

cAMP signalling tightly coordinates the expression of catabolic proteins with biosynthetic and ribosomal proteins,
in accordance with the cellular metabolic needs during exponential growth.

The expression of carbon catabolic genes increased linearly

with decreasing growth rates upon limitation of carbon influx,
but decreased linearly with decreasing growth rate upon limitation of nitrogen or sulphur influx.

In contrast, the expression of biosynthetic genes showed the opposite linear growth-rate dependence as the catabolic genes. A coarse-grained mathematical model provides a quantitative framework for understanding and predicting

gene expression responses to catabolic and anabolic limitations.

A scheme of integral feedback control featuring the inhibition of cAMP signalling by metabolic precursors is proposed and validated. These results reveal a key physiological role of

cAMP-dependent catabolite repression: to ensure that proteomic resources are spent on distinct metabolic sectors as needed
in different nutrient environments.

Our findings underscore the power of quantitative physiology in unravelling the underlying functions of complex molecular signalling networks.

Exosomes from bone marrow mesenchymal stem cells contain a microRNA that promotes dormancy in metastatic breast cancer cells

Makiko Ono¹, Nobuyoshi Kosaka¹, Naoomi Tominaga¹, Yusuke Yoshioka¹, Fumitaka Takeshita¹, et al.
Sci. Signal., July 2014; 7(332), p. ra63 http://dx.doi.org:/10.1126/scisignal.2005231

Breast cancer patients often develop metastatic disease years after resection of the primary tumor. The patients are asymptomatic because the disseminated cells appear to become dormant and are undetectable. Because the proliferation of these cells is slowed, dormant cells are often unresponsive to traditional chemotherapies that exploit the rapid cell cycling of most cancer cells. We generated a bone marrow–metastatic human breast cancer cell line (BM2) by tracking and isolating fluorescent-labeled MDA-MB-231 cells that disseminated to the bone marrow in mice. Coculturing BM2 cells with bone marrow mesenchymal stem cells (BM-MSCs) isolated from human donors revealed that BM-MSCs suppressed the proliferation of BM2 cells, decreased the abundance of stem cell–like surface markers, inhibited their invasion through Matrigel Transwells, and decreased their sensitivity to docetaxel, a common chemotherapy agent. Acquisition of these dormant phenotypes in BM2 cells was also observed by culturing the cells in BM-MSC–conditioned medium or with exosomes isolated from BM-MSC cultures, which were taken up by BM2 cells. Among various microRNAs (miRNAs) increased in BM-MSC–derived exosomes compared with those from adult fibroblasts, overexpression of miR-23b in BM2 cells induced dormant phenotypes through the suppression of a target gene, MARCKS, which encodes a protein that promotes cell cycling and motility. Metastatic breast cancer cells in patient bone marrow had increased miR-23b and decreasedMARCKS expression. Together, these findings suggest that exosomal transfer of miRNAs from the bone marrow may promote breast cancer cell dormancy in a metastatic niche.

Citation:

Ono, N. Kosaka, N. Tominaga, Y. Yoshioka, F. Takeshita, R. Takahashi, M. Yoshida, H. Tsuda, K. Tamura, and T. Ochiya, Exosomes from bone marrow mesenchymal stem cells contain a microRNA that promotes dormancy in metastatic breast cancer cells. Sci. Signal.7, ra63 (2014).

Poly(ADP-ribose) polymerase-dependent energy depletion occurs through inhibition of glycolysis.

Andrabi SA¹, Umanah GK², Chang C³, Stevens DA⁴, Karuppagounder SS², Gagné JP⁵, Poirier GG⁵, Dawson VL⁶, Dawson TM⁷.

Proc Natl Acad Sci U S A. 2014 Jul 15; 111(28):10209-14. http://dx.doi.org:/10.1073/pnas.1405158111

Excessive poly(ADP-ribose) (PAR) polymerase-1 (PARP-1) activation kills cells via a cell-death process designated “parthanatos” in which PAR induces the mitochondrial release and nuclear translocation of apoptosis-inducing factor to initiate chromatinolysis and cell death. Accompanying the formation of PAR are the reduction of cellular NAD(+) and energetic collapse, which have been thought to be caused by the consumption of cellular NAD(+) by PARP-1. Here we show that the bioenergetic collapse following PARP-1 activation is not dependent on NAD(+) depletion. Instead PARP-1 activation initiates glycolytic defects via PAR-dependent inhibition of hexokinase, which precedes the NAD(+) depletion in N-methyl-N-nitroso-N-nitroguanidine (MNNG)-treated cortical neurons. Mitochondrial defects are observed shortly after PARP-1 activation and are mediated largely through defective glycolysis, because supplementation of the mitochondrial substrates pyruvate and glutamine reverse the PARP-1-mediated mitochondrial dysfunction. Depleting neurons of NAD(+) with FK866, a highly specific noncompetitive inhibitor of nicotinamide phosphoribosyltransferase, does not alter glycolysis or mitochondrial function. Hexokinase, the first regulatory enzyme to initiate glycolysis by converting glucose to glucose-6-phosphate, contains a strong PAR-binding motif. PAR binds to hexokinase and inhibits hexokinase activity in MNNG-treated cortical neurons. Preventing PAR formation with PAR glycohydrolase prevents the PAR-dependent inhibition of hexokinase. These results indicate that bioenergetic collapse induced by overactivation of PARP-1 is caused by PAR-dependent inhibition of glycolysis through inhibition of hexokinase.

PMID:24987120 PMCID: PMC4104885 [Available on 2015/1/15]

Aim24 stabilizes respiratory chain supercomplexes and is required for efficient respiration

Deckers M¹, Balleininger M¹, Vukotic M¹, Römpler K¹, Bareth B¹, Juris L¹, Dudek J².

FEBS Lett. 2014 Jun 10. pii: S0014-5793(14)00458-X. http://dx.doi.org:/10.1016/j.febslet.2014.06.006

The mitochondrial respiratory chain is essential for the conversion of energy derived from the oxidation of metabolites into the membrane potential, which drives the synthesis of ATP. The electron transporting complexes bc₁ complex and the cytochrome c oxidase assemble into large supercomplexes, allowing efficient energy transduction. Currently, we have only limited information about what determines the structure of the supercomplex. Here, we characterize Aim24 in baker’s yeast as a protein, which is integrated in the mitochondrial inner membrane and is required for the structural integrity of the supercomplex. Deletion of AIM24 strongly affects activity of the respiratory chain and induces a growth defect on non-fermentable medium. Our data indicate that Aim24 has a function in stabilizing the respiratory chain supercomplexes. PMID: 24928273

KEYWORDS: Aim24; Membrane protein; Metabolism; Mitochondria; Respiration; Supercomplex

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Scientific Curation Fostering Expert Networks and Open Innovation: Lessons from Clive Thompson and others

Posted in Computational Biology/Systems and Bioinformatics, Curation, Curation methodology, Dialectic, Discovery process, Experimental validation, Explanatory, Health Economics and Outcomes Research, HealthCare IT, Open Access Journals, Pharmaceutical Industry Competitive Intelligence, Scientist: Career considerations, Scoop.it, Statistical Methods for Research Evaluation, Technology Advance Assessment of, Theoretic convergence, Uncategorized, tagged #science2_0, @science 2_0, Aviva Lev-Ari, collaboration, Conditions and Diseases, Content Curation, Curation, curation communities, Curation methodology, Curator, health, Larry H. Bernstein, medical literature, medical news, medical reporting, medicine, Open Access Online Scientific Journal, Open Access Publishing, Open data, science, Scientific literature, scientific networks, scientific news, Social network on July 17, 2014| Leave a Comment »

Scientific Curation Fostering Expert Networks and Open Innovation: Lessons from Clive Thompson

Curators and Writer: Stephen J. Williams, Ph.D. with input from Curators Larry H. Bernstein, MD, FCAP, Dr. Justin D. Pearlman, MD, PhD, FACC and Dr. Aviva Lev-Ari, PhD, RN

(this discussion is in a three part series including:

Using Scientific Content Curation as a Method for Validation and Biocuration

Using Scientific Content Curation as a Method for Open Innovation)

Every month I get my Wired Magazine (yes in hard print, I still like to turn pages manually plus I don’t mind if I get grease or wing sauce on my magazine rather than on my e-reader) but I always love reading articles written by Clive Thompson. He has a certain flair for understanding the techno world we live in and the human/technology interaction, writing about interesting ways in which we almost inadvertently integrate new technologies into our day-to-day living, generating new entrepreneurship, new value. He also writes extensively about tech and entrepreneurship.

October 2013 Wired article by Clive Thompson, entitled “How Successful Networks Nurture Good Ideas: Thinking Out Loud”, describes how the voluminous writings, postings, tweets, and sharing on social media is fostering connections between people and ideas which, previously, had not existed. The article was generated from Clive Thompson’s book Smarter Than you Think: How Technology is Changing Our Minds for the Better.Tom Peters also commented about the article in his blog (see here).

Clive gives a wonderful example of Ory Okolloh, a young Kenyan-born law student who, after becoming frustrated with the lack of coverage of problems back home, started a blog about Kenyan politics. Her blog not only got interest from movie producers who were documenting female bloggers but also gained the interest of fellow Kenyans who, during the upheaval after the 2007 Kenyan elections, helped Ory to develop a Google map for reporting of violence (http://www.ushahidi.com/, which eventually became a global organization using open-source technology to affect crises-management. There are a multitude of examples how networks and the conversations within these circles are fostering new ideas. As Clive states in the article:

Our ideas are PRODUCTS OF OUR ENVIRONMENT.

They are influenced by the conversations around us.

However the article got me thinking of how Science 2.0 and the internet is changing how scientists contribute, share, and make connections to produce new and transformative ideas.

But HOW MUCH Knowledge is OUT THERE?

Clive’s article listed some amazing facts about the mountains of posts, tweets, words etc. out on the internet EVERY DAY, all of which exemplifies the problem:

154.6 billion EMAILS per DAY
400 million TWEETS per DAY
1 million BLOG POSTS (including this one) per DAY
2 million COMMENTS on WordPress per DAY
16 million WORDS on Facebook per DAY
TOTAL 52 TRILLION WORDS per DAY

As he estimates this would be 520 million books per DAY (book with average 100,000 words).

A LOT of INFO. But as he suggests it is not the volume but how we create and share this information which is critical as the science fiction writer Theodore Sturgeon noted “Ninety percent of everything is crap” AKA Sturgeon’s Law.

Internet live stats show how congested the internet is each day (http://www.internetlivestats.com/). Needless to say Clive’s numbers are a bit off. As of the writing of this article:

2.9 billion internet users
981 million websites (only 25,000 hacked today)
128 billion emails
385 million Tweets
> 2.7 million BLOG posts today (including this one)

The Good, The Bad, and the Ugly of the Scientific Internet (The Wild West?)

So how many science blogs are out there? Well back in 2008 “grrlscientist” asked this question and turned up a total of 19,881 blogs however most were “pseudoscience” blogs, not written by Ph.D or MD level scientists. A deeper search on Technorati using the search term “scientist PhD” turned up about 2,000 written by trained scientists.

So granted, there is a lot of

….. when it comes to scientific information on the internet!

I had recently re-posted, on this site, a great example of how bad science and medicine can get propagated throughout the internet:

http://pharmaceuticalintelligence.com/2014/06/17/the-gonzalez-protocol-worse-than-useless-for-pancreatic-cancer/

and in a Nature Report:Stem cells: Taking a stand against pseudoscience

http://www.nature.com/news/stem-cells-taking-a-stand-against-pseudoscience-1.15408

Drs.Elena Cattaneo and Gilberto Corbellini document their long, hard fight against false and invalidated medical claims made by some “clinicians” about the utility and medical benefits of certain stem-cell therapies, sacrificing their time to debunk medical pseudoscience.

Using Curation and Science 2.0 to build Trusted, Expert Networks of Scientists and Clinicians

Establishing networks of trusted colleagues has been a cornerstone of the scientific discourse for centuries. For example, in the mid-1640s, the Royal Society began as:

“a meeting of natural philosophers to discuss promoting knowledge of the

natural world through observation and experiment”, i.e. science.

The Society met weekly to witness experiments and discuss what we

would now call scientific topics. The first Curator of Experiments

was Robert Hooke.”

– from The History of the Royal Society

The Royal Society of London for Improving Natural Knowledge.

(photo credit: Royal Society)

(Although one wonders why they met “in-cognito”)

Indeed as discussed in “Science 2.0/Brainstorming” by the originators of OpenWetWare, an open-source science-notebook software designed to foster open-innovation, the new search and aggregation tools are making it easier to find, contribute, and share information to interested individuals. This paradigm is the basis for the shift from Science 1.0 to Science 2.0. Science 2.0 is attempting to remedy current drawbacks which are hindering rapid and open scientific collaboration and discourse including:

Slow time frame of current publishing methods: reviews can take years to fashion leading to outdated material
Level of information dissemination is currently one dimensional: peer-review, highly polished work, conferences
Current publishing does not encourage open feedback and review
Published articles edited for print do not take advantage of new web-based features including tagging, search-engine features, interactive multimedia, no hyperlinks
Published data and methodology incomplete
Published data not available in formats which can be readably accessible across platforms: gene lists are now mandated to be supplied as files however other data does not have to be supplied in file format

(put in here a brief blurb of summary of problems and why curation could help)

Curation in the Sciences: View from Scientific Content Curators Larry H. Bernstein, MD, FCAP, Dr. Justin D. Pearlman, MD, PhD, FACC and Dr. Aviva Lev-Ari, PhD, RN

Curation is an active filtering of the web’s and peer reviewed literature found by such means – immense amount of relevant and irrelevant content. As a result content may be disruptive. However, in doing good curation, one does more than simply assign value by presentation of creative work in any category. Great curators comment and share experience across content, authors and themes. Great curators may see patterns others don’t, or may challenge or debate complex and apparently conflicting points of view. Answers to specifically focused questions comes from the hard work of many in laboratory settings creatively establishing answers to definitive questions, each a part of the larger knowledge-base of reference. There are those rare “Einstein’s” who imagine a whole universe, unlike the three blind men of the Sufi tale. One held the tail, the other the trunk, the other the ear, and they all said this is an elephant!
In my reading, I learn that the optimal ratio of curation to creation may be as high as 90% curation to 10% creation. Creating content is expensive. Curation, by comparison, is much less expensive.

– Larry H. Bernstein, MD, FCAP

Curation is Uniquely Distinguished by the Historical Exploratory Ties that Bind –Larry H. Bernstein, MD, FCAP

The explosion of information by numerous media, hardcopy and electronic, written and video, has created difficulties tracking topics and tying together relevant but separated discoveries, ideas, and potential applications. Some methods to help assimilate diverse sources of knowledge include a content expert preparing a textbook summary, a panel of experts leading a discussion or think tank, and conventions moderating presentations by researchers. Each of those methods has value and an audience, but they also have limitations, particularly with respect to timeliness and pushing the edge. In the electronic data age, there is a need for further innovation, to make synthesis, stimulating associations, synergy and contrasts available to audiences in a more timely and less formal manner. Hence the birth of curation. Key components of curation include expert identification of data, ideas and innovations of interest, expert interpretation of the original research results, integration with context, digesting, highlighting, correlating and presenting in novel light.

– Justin D Pearlman, MD, PhD, FACC from The Voice of Content Consultant on The Methodology of Curation in Cardiovascular Original Research: Cases in Methodology Design for Content Co-Curation The Art of Scientific & Medical Curation

In Power of Analogy: Curation in Music, Music Critique as a Curation and Curation of Medical Research Findings – A Comparison, Drs. Larry Bernstein and Aviva Lev-Ari likens the medical and scientific curation process to curation of musical works into a thematic program:

Work of Original Music Curation and Performance:

Music Review and Critique as a Curation

Work of Original Expression what is the methodology of Curation in the context of Medical Research Findings Exposition of Synthesis and Interpretation of the significance of the results to Clinical Care

… leading to new, curated, and collaborative works by networks of experts to generate (in this case) ebooks on most significant trends and interpretations of scientific knowledge as relates to medical practice.

In Summary: How Scientific Content Curation Can Help

Given the aforementioned problems of:

I. the complex and rapid deluge of scientific information

II. the need for a collaborative, open environment to produce transformative innovation

III. need for alternative ways to disseminate scientific findings

CURATION MAY OFFER SOLUTIONS

I. Curation exists beyond the review: curation decreases time for assessment of current trends adding multiple insights, analyses WITH an underlying METHODOLOGY (discussed below) while NOT acting as mere reiteration, regurgitation

II. Curation providing insights from WHOLE scientific community on multiple WEB 2.0 platforms

III. Curation makes use of new computational and Web-based tools to provide interoperability of data, reporting of findings (shown in Examples below)

Therefore a discussion is given on methodologies, definitions of best practices, and tools developed to assist the content curation community in this endeavor.

Methodology in Scientific Content Curation as Envisioned by Aviva lev-Ari, PhD, RN

At Leaders in Pharmaceutical Business Intelligence, site owner and chief editor Aviva lev-Ari, PhD, RN has been developing a strategy “for the facilitation of Global access to Biomedical knowledge rather than the access to sheer search results on Scientific subject matters in the Life Sciences and Medicine”. According to Aviva, “for the methodology to attain this complex goal it is to be dealing with popularization of ORIGINAL Scientific Research via Content Curation of Scientific Research Results by Experts, Authors, Writers using the critical thinking process of expert interpretation of the original research results.” The following post:

Cardiovascular Original Research: Cases in Methodology Design for Content Curation and Co-Curation

http://pharmaceuticalintelligence.com/2013/07/29/cardiovascular-original-research-cases-in-methodology-design-for-content-curation-and-co-curation/

demonstrate two examples how content co-curation attempts to achieve this aim and develop networks of scientist and clinician curators to aid in the active discussion of scientific and medical findings, and use scientific content curation as a means for critique offering a “new architecture for knowledge”. Indeed, popular search engines such as Google, Yahoo, or even scientific search engines such as NCBI’s PubMed and the OVID search engine rely on keywords and Boolean algorithms …

which has created a need for more context-driven scientific search and discourse.

In Science and Curation: the New Practice of Web 2.0, Célya Gruson-Daniel (@HackYourPhd) states:

To address this need, human intermediaries, empowered by the participatory wave of web 2.0, naturally started narrowing down the information and providing an angle of analysis and some context. They are bloggers, regular Internet users or community managers – a new type of profession dedicated to the web 2.0. A new use of the web has emerged, through which the information, once produced, is collectively spread and filtered by Internet users who create hierarchies of information.

.. where Célya considers curation an essential practice to manage open science and this new style of research.

As mentioned above in her article, Dr. Lev-Ari represents two examples of how content curation expanded thought, discussion, and eventually new ideas.

Curator edifies content through analytic process = NEW form of writing and organizations leading to new interconnections of ideas = NEW INSIGHTS

i) Evidence: curation methodology leading to new insights for biomarkers

Same as #1 but multiple players (experts) each bringing unique insights, perspectives, skills yielding new research = NEW LINE of CRITICAL THINKING

ii) Evidence: co-curation methodology among cardiovascular experts leading to cardiovascular series ebooks

The Life Cycle of Science 2.0. Due to Web 2.0, new paradigms of scientific collaboration are rapidly emerging. Originally, scientific discovery were performed by individual laboratories or “scientific silos” where the main method of communication was peer-reviewed publication, meeting presentation, and ultimately news outlets and multimedia. In this digital era, data was organized for literature search and biocurated databases. In an era of social media, Web 2.0, a group of scientifically and medically trained “curators” organize the piles of data of digitally generated data and fit data into an organizational structure which can be shared, communicated, and analyzed in a holistic approach, launching new ideas due to changes in organization structure of data and data analytics.

The result, in this case, is a collaborative written work above the scope of the review. Currently review articles are written by experts in the field and summarize the state of a research are. However, using collaborative, trusted networks of experts, the result is a real-time synopsis and analysis of the field with the goal in mind to

INCREASE THE SCIENTIFIC CURRENCY.

For detailed description of methodology please see Cardiovascular Original Research: Cases in Methodology Design for Content Co-Curation The Art of Scientific & Medical Curation

In her paper, Curating e-Science Data, Maureen Pennock, from The British Library, emphasized the importance of using a diligent, validated, and reproducible, and cost-effective methodology for curation by e-science communities over the ‘Grid’:

“The digital data deluge will have profound repercussions for the infrastructure of research and beyond. Data from a wide variety of new and existing sources will need to be annotated with metadata, then archived and curated so that both the data and the programmes used to transform the data can be reproduced for use in the future. The data represent a new foundation for new research, science, knowledge and discovery”

— JISC Senior Management Briefing Paper, The Data Deluge (2004)

As she states proper data and content curation is important for:

Post-analysis
Data and research result reuse for new research
Validation
Preservation of data in newer formats to prolong life-cycle of research results

However she laments the lack of

Funding for such efforts
Training
Organizational support
Monitoring
Established procedures

Tatiana Aders wrote a nice article based on an interview with Microsoft’s Robert Scoble, where he emphasized the need for curation in a world where “Twitter is the replacement of the Associated Press Wire Machine” and new technologic platforms are knocking out old platforms at a rapid pace. In addition he notes that curation is also a social art form where primary concerns are to understand an audience and a niche.

Indeed, part of the reason the need for curation is unmet, as writes Mark Carrigan, is the lack of appreciation by academics of the utility of tools such as Pinterest, Storify, and Pearl Trees to effectively communicate and build collaborative networks.

And teacher Nancy White, in her article Understanding Content Curation on her blog Innovations in Education, shows examples of how curation in an educational tool for students and teachers by demonstrating students need to CONTEXTUALIZE what the collect to add enhanced value, using higher mental processes such as:

Knowledge
Comprehension
Application
Analysis
Synthesis
Evaluation

A GREAT table about the differences between Collecting and Curating by Nancy White at http://d20innovation.d20blogs.org/2012/07/07/understanding-content-curation/

University of Massachusetts Medical School has aggregated some useful curation tools at http://esciencelibrary.umassmed.edu/data_curation

Although many tools are related to biocuration and building databases but the common idea is curating data with indexing, analyses, and contextual value to provide for an audience to generate NETWORKS OF NEW IDEAS.

See here for a curation of how networks fosters knowledge, by Erika Harrison on ScoopIt

(http://www.scoop.it/t/mobilizing-knowledge-through-complex-networks)

“Nowadays, any organization should employ network scientists/analysts who are able to map and analyze complex systems that are of importance to the organization (e.g. the organization itself, its activities, a country’s economic activities, transportation networks, research networks).”

– Andrea Carafa insight from World Economic Forum New Champions 2012 “Power of Networks”

Creating Content Curation Communities: Breaking Down the Silos!

An article by Dr. Dana Rotman “Facilitating Scientific Collaborations Through Content Curation Communities” highlights how scientific information resources, traditionally created and maintained by paid professionals, are being crowdsourced to professionals and nonprofessionals in which she termed “content curation communities”, consisting of professionals and nonprofessional volunteers who create, curate, and maintain the various scientific database tools we use such as Encyclopedia of Life, ChemSpider (for Slideshare see here), biowikipedia etc. Although very useful and openly available, these projects create their own challenges such as

information integration (various types of data and formats)
social integration (marginalized by scientific communities, no funding, no recognition)

The authors set forth some ways to overcome these challenges of the content curation community including:

standardization in practices
visualization to document contributions
emphasizing role of information professionals in content curation communities
maintaining quality control to increase respectability
recognizing participation to professional communities
proposing funding/national meeting – Data Intensive Collaboration in Science and Engineering Workshop

A few great presentations and papers from the 2012 DICOSE meeting are found below

Judith M. Brown, Robert Biddle, Stevenson Gossage, Jeff Wilson & Steven Greenspan. Collaboratively Analyzing Large Data Sets using Multitouch Surfaces. (PDF) NotesForBrown

Bill Howe, Cecilia Aragon, David Beck, Jeffrey P. Gardner, Ed Lazowska, Tanya McEwen. Supporting Data-Intensive Collaboration via Campus eScience Centers. (PDF) NotesForHowe

Kerk F. Kee & Larry D. Browning. Challenges of Scientist-Developers and Adopters of Existing Cyberinfrastructure Tools for Data-Intensive Collaboration, Computational Simulation, and Interdisciplinary Projects in Early e-Science in the U.S.. (PDF) NotesForKee

Ben Li. The mirages of big data. (PDF) NotesForLi ReflectionsByBen

Betsy Rolland & Charlotte P. Lee. Post-Doctoral Researchers’ Use of Preexisting Data in Cancer Epidemiology Research. (PDF) NoteForRolland

Dana Rotman, Jennifer Preece, Derek Hansen & Kezia Procita. Facilitating scientific collaboration through content curation communities. (PDF) NotesForRotman

Nicholas M. Weber & Karen S. Baker. System Slack in Cyberinfrastructure Development: Mind the Gaps. (PDF) NotesForWeber

Indeed, the movement of Science 2.0 from Science 1.0 had originated because these “silos” had frustrated many scientists, resulting in changes in the area of publishing (Open Access) but also communication of protocols (online protocol sites and notebooks like OpenWetWare and BioProtocols Online) and data and material registries (CGAP and tumor banks). Some examples are given below.

Open Science Case Studies in Curation

1. Open Science Project from Digital Curation Center

This project looked at what motivates researchers to work in an open manner with regard to their data, results and protocols, and whether advantages are delivered by working in this way.

The case studies consider the benefits and barriers to using ‘open science’ methods, and were carried out between November 2009 and April 2010 and published in the report Open to All? Case studies of openness in research. The Appendices to the main report (pdf) include a literature review, a framework for characterizing openness, a list of examples, and the interview schedule and topics. Some of the case study participants kindly agreed to us publishing the transcripts. This zip archive contains transcripts of interviews with researchers in astronomy, bioinformatics, chemistry, and language technology.

see: Pennock, M. (2006). “Curating e-Science Data”. DCC Briefing Papers: Introduction to Curation. Edinburgh: Digital Curation Centre. Handle: 1842/3330. Available online: http://www.dcc.ac.uk/resources/briefing-papers/introduction-curation– See more at: http://www.dcc.ac.uk/resources/briefing-papers/introduction-curation/curating-e-science-data#sthash.RdkPNi9F.dpuf

2. cBIO -cBio’s biological data curation group developed and operates using a methodology called CIMS, the Curation Information Management System. CIMS is a comprehensive curation and quality control process that efficiently extracts information from publications.

3. NIH Topic Maps – This website provides a database and web-based interface for searching and discovering the types of research awarded by the NIH. The database uses automated, computer generated categories from a statistical analysis known as topic modeling.

4. SciKnowMine (USC)- We propose to create a framework to support biocuration called SciKnowMine (after ‘Scientific Knowledge Mine’), cyberinfrastructure that supports biocuration through the automated mining of text, images, and other amenable media at the scale of the entire literature.

OpenWetWare– OpenWetWare is an effort to promote the sharing of information, know-how, and wisdom among researchers and groups who are working in biology & biological engineering. Learn more about us. If you would like edit access, would be interested in helping out, or want your lab website hosted on OpenWetWare, pleasejoin us. OpenWetWare is managed by the BioBricks Foundation. They also have a wiki about Science 2.0.

6. LabTrove: a lightweight, web based, laboratory “blog” as a route towards a marked up record of work in a bioscience research laboratory. Authors in PLOS One article, from University of Southampton, report the development of an open, scientific lab notebook using a blogging strategy to share information.

7. OpenScience Project– The OpenScience project is dedicated to writing and releasing free and Open Source scientific software. We are a group of scientists, mathematicians and engineers who want to encourage a collaborative environment in which science can be pursued by anyone who is inspired to discover something new about the natural world.

8. Open Science Grid is a multi-disciplinary partnership to federate local, regional, community and national cyberinfrastructures to meet the needs of research and academic communities at all scales.

9. Some ongoing biomedical knowledge (curation) projects at ISI

IICurate
This project is concerned with developing a curation and documentation system for information integration in collaboration with the II Group at ISI as part of the BIRN.

BioScholar
It’s primary purpose is to provide software for experimental biomedical scientists that would permit a single scientific worker (at the level of a graduate student or postdoctoral worker) to design, construct and manage a shared knowledge repository for a research group derived on a local store of PDF files. This project is funded by NIGMS from 2008-2012 ( RO1-GM083871).

10. Tools useful for scientific content curation

Research Analytic and Curation Tools from University of Queensland

Thomson Reuters information curation services for pharma industry

Microblogs as a way to communicate information about HPV infection among clinicians and patients; use of Chinese microblog SinaWeibo as a communication tool

VIVO for scientific communities– In order to connect this information about research activities across institutions and make it available to others, taking into account smaller players in the research landscape and addressing their need for specific information (for example, by proving non-conventional research objects), the open source software VIVO that provides research information as linked open data (LOD) is used in many countries. So-called VIVO harvesters collect research information that is freely available on the web, and convert the data collected in conformity with LOD standards. The VIVO ontology builds on prevalent LOD namespaces and, depending on the needs of the specialist community concerned, can be expanded.

11. Examples of scientific curation in different areas of Science/Pharma/Biotech/Education

From Science 2.0 to Pharma 3.0 Q&A with Hervé Basset

http://digimind.com/blog/experts/pharma-3-0/

Hervé Basset, specialist librarian in the pharmaceutical industry and owner of the blog “Science Intelligence“, to talk about the inspiration behind his recent book entitled “From Science 2.0 to Pharma 3.0″, published by Chandos Publishing and available on Amazon and how health care companies need a social media strategy to communicate and convince the health-care consumer, not just the practicioner.

Thomson Reuters and NuMedii Launch Ground-Breaking Initiative to Identify Drugs for Repurposing. Companies leverage content, Big Data analytics and expertise to improve success of drug discovery

Content Curation as a Context for Teaching and Learning in Science

#OZeLIVE Feb2014

http://www.youtube.com/watch?v=Ty-ugUA4az0

Creative Commons license

DigCCur: A graduate level program initiated by University of North Carolina to instruct the future digital curators in science and other subjects

Syracuse University offering a program in eScience and digital curation

Curation Tips from TED talks and tech experts

Steven Rosenbaum from Curation Nation

http://www.youtube.com/watch?v=HpncJd1v1k4

Pawan Deshpande form Curata on how content curation communities evolve and what makes a good content curation:

http://www.youtube.com/watch?v=QENhIU9YZyA

How the Internet of Things is Promoting the Curation Effort

Update by Stephen J. Williams, PhD 3/01/19

Up till now, curation efforts like wikis (Wikipedia, Wikimedicine, Wormbase, GenBank, etc.) have been supported by a largely voluntary army of citizens, scientists, and data enthusiasts. I am sure all have seen the requests for donations to help keep Wikipedia and its other related projects up and running. One of the obscure sister projects of Wikipedia, Wikidata, wants to curate and represent all information in such a way in which both machines, computers, and humans can converse in. About an army of 4 million have Wiki entries and maintain these databases.

Enter the Age of the Personal Digital Assistants (Hellooo Alexa!)

In a March 2019 WIRED article “Encyclopedia Automata: Where Alexa Gets Its Information” senior WIRED writer Tom Simonite reports on the need for new types of data structure as well as how curated databases are so important for the new fields of AI as well as enabling personal digital assistants like Alexa or Google Assistant decipher meaning of the user.

As Mr. Simonite noted, many of our libraries of knowledge are encoded in an “ancient technology largely opaque to machines-prose.” Search engines like Google do not have a problem with a question asked in prose as they just have to find relevant links to pages. Yet this is a problem for Google Assistant, for instance, as machines can’t quickly extract meaning from the internet’s mess of “predicates, complements, sentences, and paragraphs. It requires a guide.”

Enter Wikidata. According to founder Denny Vrandecic,

Language depends on knowing a lot of common sense, which computers don’t have access to

A wikidata entry (of which there are about 60 million) codes every concept and item with a numeric code, the QID code number. These codes are integrated with tags (like tags you use on Twitter as handles or tags in WordPress used for Search Engine Optimization) so computers can identify patterns of recognition between these codes.

Now human entry into these databases are critical as we add new facts and in particular meaning to each of these items. Else, machines have problems deciphering our meaning like Apple’s Siri, where they had complained of dumb algorithms to interpret requests.

The knowledge of future machines could be shaped by you and me, not just tech companies and PhDs.

But this effort needs money

Wikimedia’s executive director, Katherine Maher, had prodded and cajoled these megacorporations for tapping the free resources of Wiki’s. In response, Amazon and Facebook had donated millions for the Wikimedia projects. Google recently gave 3.1 million USD$ in donations.

Future postings on the relevance and application of scientific curation will include:

Using Scientific Content Curation as a Method for Validation and Biocuration

Using Scientific Content Curation as a Method for Open Innovation

Other posts on this site related to Content Curation and Methodology include:

The growing importance of content curation

Data Curation is for Big Data what Data Integration is for Small Data

6 Steps to More Effective Content Curation

Stem Cells and Cardiac Repair: Content Curation & Scientific Reporting

Cancer Research: Curations and Reporting

Cardiovascular Diseases and Pharmacological Therapy: Curations

Cardiovascular Original Research: Cases in Methodology Design for Content Co-Curation The Art of Scientific & Medical Curation

Exploring the Impact of Content Curation on Business Goals in 2013

Power of Analogy: Curation in Music, Music Critique as a Curation and Curation of Medical Research Findings – A Comparison

conceived: NEW Definition for Co-Curation in Medical Research

The Young Surgeon and The Retired Pathologist: On Science, Medicine and HealthCare Policy – The Best Writers Among the WRITERS

Reconstructed Science Communication for Open Access Online Scientific Curation

Read Full Post »

Commentary on Biomarkers for Genetics and Genomics of Cardiovascular Disease: Views by Larry H Bernstein, MD, FCAP

Posted in Acute Myocardial Infarction, Biological Networks, Gene Regulation and Evolution, Biomarkers & Medical Diagnostics, Cardiomyopathy, Cardiovascular Research, Cell Biology, Signaling & Cell Circuits, Curation, Discovery process, Disease Biology, Small Molecules in Development of Therapeutic Drugs, Experimental validation, Explanatory, Frontiers in Cardiology and Cardiovascular Disorders, Gene Regulation, Genome Biology, HTN, Metabolomics, Origins of Cardiovascular Disease, Pharmaceutical Analytics, Population Health Management, Genetics & Pharmaceutical, RNA Biology, Cancer and Therapeutics, Technology Transfer: Biotech and Pharmaceutical, Transcriptomics, Translational Research, Translational Science, tagged cardiovascular diseases, circulating microRNA, microRNAs, plasm biomarkers on July 16, 2014| Leave a Comment »

Commentary on Biomarkers for Genetics and Genomics of Cardiovascular Disease: : Views by Larry H Bernstein, MD, FCAP

Author: Larry H Bernstein, MD, FCAP

This review has examined a compendium of well regarded documents drawn from 248 articles in Circulation Cardiovascular Genetics from March 2010 to March 2013. The large amount of evidence obtained from large population studies identifying Genome Wide Analysis Studies (GWAS) examines a host of cardiac and vascular diseases in which there is association between specific single nucleotide peptides (SNPs), and gene loci, that may play or have no significant role in developing heart disease. It certainly is evidence of the role that the American Heart Association has is in supporting the leading research today for tomorrow’s patients. It is too early to sort them out, but it speaks to a large volume of discovery in this area.

It raises another issue that we have been confronted with mostly since the second half of the 20th century. What is that issue? The issue, it appears to me, is the vast improvements in analytical technology so that “imprecision” is far less likely to be a confounder in biological measurements and this lends access to far better accuracy? But from that question arises another! Accuracy only refers to what is measured, but does it give us better ability to explain a complex and dynamic process? In other words, what is what we are looking at representative of in manageable events? I think that this is the most important idea that should come out of the recent criticism of the trajectory that molecular genetics been on in the last 5 years.

It was still in an era that “BIG’ science was not the normal. One could spend an enormous effort at stepwise purification of a protein or enzyme, or other biomolecule starting with a slurry made from 100 lbs of “chicken heart”, for example. These separations were based on negative charges on the molecules and positive charges on the column, and the molecules of no interest were eluted by gradient elution. Much was learned about large scale preparation from small scale trials. But this work was not undertaken without the intent to carry out a number of investigations to understand the “functionality” of a link in a metabolic pathway. The studies that followed the purification required kinetic investigation with a coenzyme, or with a synthetically modified coenzyme, amino acid sequencing, NMR studies, etc. You could not put together a “mechanism” without having the minimum amount of necessary information for a reliable account. It is probably this requirement that led to today’s “BIG” science, that is founded upon multiple methods, now large data bases, and teams of investigators across institutions and continents. The acquisition of knowledge has been astounding, but the integration of knowledge has not caught up.

However, let’s see if we can sort out the most meaningful signals from what I too am beginning to call the “noisy channel”. As often happens, important areas of research are opened up that are followed by significant discovery and, in the long run, many other dead end publications that have no lasting significance. In order to do justice to the work, I’ll pick through documents I find interesting, keeping in mind there is a hidden layer of complexity of which only sufficient information leads to a better understanding. As much literature calls attention to, much of what ails us has nothing to do with classical Mendelian genetics, and has a postgenomic component.

The most fascinating aspect of this is the withering “dark matter” of the genome. While that component may be silent or expressed, the understanding comes at a higher observed order. The dark became light! The expression became subtle, like weak bond interactions. The underlying organization is a component of the adaptive ability of an organism or individual in an environment with plants and animals in a changing climate, at particular altitudes, with given water supplies, with disease vectors, and with endogenous sources of essential nutrients. This brings into focus the regulatory role of the genome as just as important a factor as transmission of the genetic code, especially in somatic cell populations.

The remainder of this discussion deals specifically with my observations on cardiovascular genomics. The following conclusion is appropriate, if incomplete, at this time on circulating miRNAs, particularly miR-133a:

elevated levels of circulating miR-133a in patients with cardiovascular diseases originate mainly from the injured myocardium.
Circulating miR-133a can be used as a marker for cardiomyocyte death, and
it may have functions in cardiovascular diseases.

Circulation: Cardiovascular Genetics. 2011;4:446-454.

Strikingly, in plasma from

acute myocardial infarction patients, cardiac myocyte–associated miR-208b and -499 were highly elevated, 1600-fold (P<0.005) and 100-fold (P<0.0005), respectively, as compared with control subjects. Receiver operating characteristic curve analysis revealed an area under the curve of 0.94 (P<10−10) for miR-208b and 0.92 (P<10−9) for miR-499. BothmicroRNAs correlated with plasma troponin T, indicating release of microRNAs from injured cardiomyocytes.
In patients with acute heart failure, only miR-499 was significantly elevated (2-fold), whereas
no significant changes in microRNAs studied could be observed in diastolic dysfunction.

Remarkably, plasma microRNA levels were not affected by a wide range of clinical confounders, including

age,
sex,
body mass index,
kidney function,
systolic blood pressure, and
white blood cell count.

This is miRNA with a different twist. It appears that there are 3 types found in AMI(133a, 208b, 409). But type 409 alone is increased with acute heart failure (no mention of chronic cardiomyopathy and no effect of estimated GFR, or of age).

If the problem was just of AMI, then we have to know what this brings to the table. As it is the hs-troponins have yet to be shown to effectively not only increase the high sensitivity of the tests, but to decrease the confusion generated by the elevation. The enormous improvement of a test that may be superior to the hs-ctn’s is for the patient with very indeterminiate shortness of breath, a nondefinitive ECG, and in a prodromal phase of AMI. This happened in the past, and it may happen now, and it may account for many cases of silent MI that were found at autopsy.

Cited by Plasma microRNAs serve as biomarkers of therapeutic efficacy and disease progression in hypertension-induced heart failure Eur J Heart Fail. 2013;0:hft018v1-hft018,

 Circulating microRNAs as diagnostic biomarkers for cardiovascular diseases Am. J. Physiol. Heart Circ. Physiol.. 2012;303:H1085-H1095,

Circulation Editors’ Picks: Most Read Articles in Cardiovascular Genetics Circulation. 2012;126:e163-e169,

MicroRNAs in Patients on Chronic Hemodialysis (MINOS Study) CJASN. 2012;7:619-623,

Novel techniques and targets in cardiovascular microRNA research Cardiovasc Res. 2012;93:545-554,

Microparticles: major transport vehicles for distinct microRNAs in circulationCardiovasc Res. 2012;93:633-644,

Profiling of circulating microRNAs: from single biomarkers to re-wired networksCardiovasc Res. 2012;93:555-562,

Small but smart–microRNAs in the centre of inflammatory processes during cardiovascular diseases, the metabolic syndrome, and ageing Cardiovasc Res. 2012;93:605-613,

Circulation: Heart Failure Editors’ Picks: Most Important Papers in Pathophysiology and Genetics Circ Heart Fail. 2012;5:e32-e49

Use of Circulating MicroRNAs to Diagnose Acute Myocardial Infarction Clin. Chem. 2012;58:559-567,

Circulating microRNAs to identify human heart failure Eur J Heart Fail. 2012;14:118-119,

Next Steps in Cardiovascular Disease Genomic Research–Sequencing, Epigenetics, and Transcriptomics Clin. Chem. 2012;58:113-126,

Most Read in Cardiovascular Genetics on Biomarkers, Inherited Cardiomyopathies and Arrhythmias, Metabolomics, and GenomicsCirc Cardiovasc Genet. 2011;4:e24-e30,

MicroRNA-126 modulates endothelial SDF-1 expression and mobilization of Sca-1+/Lin- progenitor cells in ischaemia Cardiovasc Res. 2011;92:449-455,

The use of genomics for treatment is another matter, and has several factors, e.g., age, residual function after AMI, comorbidities

Sonic Hedgehog-Modified Human CD34+ Cells Preserve Cardiac Function After Acute Myocardial Infarction. Circ. Res.. 2012;111:312-321

This is a lot of interesting work that opens as many questions as it answers. The observations are real, and they lead to questions relating to the heart and the circulation. Maybe it will generate answers to very tough issues concerning hypertension, renal disease and the heart. It is far too early to tell. It appears that we are about to hear a cacophony of miR’s in a symphony on cardiac and circulatory diseases not be be pieced together soon. But we have many more tools at our disposal than we did when Karmen discovered and made a distinction between

Aspartate and Alanine aminotransferases in the late 1950s, followed in the 1960s by
Creatine phosphokinase, the
MB-isoenzyme of CK by Sobel, Shell and Kjeckshus,
isoenzyme-1 of lactate dehydrogenase, and later the
Troponins,

leading to the programs to “reduce the extent of infarct damage”.

Then came the

and B-type natriuretic peptides (BNP),

which are still not fully understood in their role in congestive heart failure and inrenal disease.

One item strikes the imagination as a fruitful area of further study. Genetic Determinants of Potassium Sensitivity and Hypertension. Integrated Computational and Experimental Analysis of the Neuroendocrine Transcriptome in Genetic Hypertension Identifies Novel Control Points for the Cardiometabolic Syndrome

Essential hypertension, a common complex disease, displays substantial genetic influence. Contemporary methods to dissect the genetic basis of complex diseases such as the genomewide association study are powerful, yet a large gap exists betweens the fraction of population trait variance explained by such associations and total disease heritability.

Revised 7/17/2014

Gene expression profiles associated with acute myocardial infarction and risk of cardiovascular deathJ Kim, NGhasemzadeh, DJEapen, NC Chung, JD Storey,AAQuyyumi and GGibsonKim et al. Genome Medicine 2014, 6:40http://genomemedicine.com/content/6/5/40

Abstract

Background: Genetic risk scores have been developed for coronary artery disease and atherosclerosis, but are not predictive of adverse cardiovascular events. We asked whether peripheral blood expression profiles may be predictive of acute myocardial infarction (AMI) and/or cardiovascular death.

Methods: Peripheral blood samples from 338 subjects aged 62 ± 11 years with coronary artery disease (CAD) were analyzed in two phases (discovery N = 175, and replication N = 163), and followed for a mean 2.4 years for cardiovascular death. Gene expression was measured on Illumina HT-12 microarrays with two different normalization procedures to control technical and biological covariates. Whole genome genotyping was used to support comparative genome-wide association studies of gene expression. Analysis of variance was combined with receiver operating curve and survival analysis to define a transcriptional signature of cardiovascular death.

Results: In both phases, there was significant differential expression between healthy and AMI groups with overall down-regulation of genes involved in T-lymphocyte signaling and up-regulation of inflammatory genes. Expression quantitative trait loci analysis provided evidence for altered local genetic regulation of transcript abundance in AMI samples. On follow-up there were 31 cardiovascular deaths. A principal component (PC1) score capturing covariance of 238 genes that were differentially expressed between deceased and survivors in the discovery phase significantly predicted risk of cardiovascular death in the replication and combined samples (hazard ratio = 8.5, P< 0.0001) and improved the C-statistic (area under the curve 0.82 to 0.91, P= 0.03) after adjustment for traditional covariates.

Conclusions: A specific blood gene expression profile is associated with a significant risk of death in Caucasian subjects with CAD. This comprises a subset of transcripts that are also altered in expression during acute myocardial infarction.

MicroRNA References

Lecture Contents delivered at Koch Institute for Integrative Cancer Research, Summer Symposium 2014: RNA Biology, Cancer and Therapeutic Implications, June 13, 2014 @MIT Curator of Lecture Contents: Aviva Lev-Ari, PhD, RN https://pharmaceuticalintelligence.com/wp-admin/post.php?post=23174&action=edit

3:15 – 3:45, 6/13/2014, Laurie Boyer “Long non-coding RNAs: molecular regulators of cell fate”
http://pharmaceuticalintelligence.com/2014/06/13/315-345-2014-laurie-boyer-long-non-coding-rnas-molecular-regulators-of-cell-fate/

Plasma microRNAs serve as biomarkers of therapeutic efficacy and disease progression in hypertension-induced heart failure. Dickinson BA, Semus HM, Montgomery RL, Stack C, Latimer PA, et al. Eur J Heart Fail. 2013 Jun; 15(6):650-9. http://dx.doi.org:/10.1093/eurjhf/hft018

Circulating microRNAs – Biomarkers or mediators of cardiovascular disease? S Fichtlscherer, AM Zeiher, S Dimmeler. Arteriosclerosis, Thrombosis, and Vascular Biology.2011; 31:2383-2390.
http://dx.doi.org:/10.1161/ATVBAHA.111.226696

Circulating microRNAs as diagnostic biomarkers for cardiovascular diseases. AJ Tijsen, YM Pinto, and EE Creemers. Am J Physiol Heart Circ Physiol 303: H1085–H1095, 2012. http://dx.doi.org:/10.1152/ajpheart.00191.2012.

MicroRNAs in Patients on Chronic Hemodialysis (MINOS Study). Emilian C, Goretti E, Prospert F, Pouthier D, Duhoux P, et al. Clin J Am Soc Nephrol (CJASN)2012; 7: 619-623. http://dx.doi.org:/10.2215/CJN.10471011

Plasma microRNAs serve as biomarkers of therapeutic efficacy and disease progression in hypertension-induced heart failure.BA Dickinson, HM Semus, RL Montgomery, C Stack, PA Latimer, et al.
Eur J Heart Fail 2013 Jun 6;15(6):650-9. http://www.pubfacts.com/detail/23388090/Plasma-microRNAs-serve-as-biomarkers-of-therapeutic-efficacy-and-disease-progression-in-hypertension

Circulating MicroRNAs: Novel Biomarkers and Extracellular Communicators in Cardiovascular Disease? Esther E. Creemers, Anke J. Tijsen, Yigal M. Pinto. Circulation Research. 2012; 110: 483-495 http://dx.doi.org:/10.1161/CIRCRESAHA.111.247452

Novel techniques and targets in cardiovascular microRNA research. Dangwal S, Bang C, Thum T.Cardiovasc Res. 2012 Mar 15; 93(4):545-54. http://dx.doi.org:/10.1093/cvr/cvr297

Microparticles: major transport vehicles for distinct microRNAs in circulation. Diehl P, Fricke A, Sander L, Stamm J, Bassler N, Htun N, et al. Cardiovasc Res. 2012 Mar 15; 93(4):633-44. http://dx.doi.org:/10.1093/cvr/cvs007.

Profiling of circulating microRNAs: from single biomarkers to re-wired networks. A Zampetaki, P Willeit, I Drozdov, S Kiechl and M Mayr. Cardiovasc Res 2012; 93 (4): 555-562. http://dx.doi.org:/10.1093/cvr/cvr266

Small but smart–microRNAs in the centre of inflammatory processes during cardiovascular diseases, the metabolic syndrome, and ageing. Schroen B, Heymans S. Cardiovasc Res. 2012; 93(4):605-613. http://dx.doi.org:/10.1093/cvr/cvr268

Therapeutic Inhibition of miR-208a Improves Cardiac Function and Survival During Heart Failure. RL Montgomery, TG Hullinger, HM Semus, BA Dickinson, AG Seto, et al.
http://dx.doi.org:/10.1161/CIRCULATIONAHA.111.030932

Circulating microRNAs to identify human heart failure. Seto AG, van Rooij E.
Eur J Heart Fail. 2012;14(2):118-119. http://dx.doi.org:/10.1093/eurjhf/hfr179.

Use of Circulating MicroRNAs to Diagnose Acute Myocardial Infarction. Y Devaux, M Vausort, E Goretti, PV Nazarov, F Azuaje. Clin Chem. 2012; 58:559-567. http://dx.doi.org:/10.1373/clinchem.2011.173823

Next Steps in Cardiovascular Disease Genomic Research–Sequencing, Epigenetics, and Transcriptomics RB Schnabel, A Baccarelli, H Lin, PT Ellinor, and EJ Benjamin.
Clin Chem . 2012 Jan; 58(1): 113–126. http://dx.doi.org:/10.1373/clinchem.2011.170423

MicroRNA-133 Modulates the {beta}1-Adrenergic Receptor Transduction Cascade. A Castaldi, T Zaglia, V Di Mauro, P Carullo, G Viggiani, et al. Circ. Res..2014; 115:273-283.
http://dx.doi.org:/10.1161/CIRCRESAHA.115.303252

Development of microRNA therapeutics is coming of age. E van Rooij, S Kauppinen. EMBOMol Med.. 2014; 6:851-864. http://dx.doi.org:/10.15252/emmm.201100899

Pitx2-microRNA pathway that delimits sinoatrial node development and inhibits predisposition to atrial fibrillation. J Wang, Y Bai, N Li, W Ye, M Zhang,et al. PNAS 2014; 111: 9181-9186.
www.pnas.org/lookup/suppl/doi:10.1073/pnas.1405411111/-/DCSupplemental.

MicroRNA-126 modulates endothelial SDF-1 expression and mobilization of Sca-1+/Lin- progenitor cells in ischaemia Cardiovasc Res. 2011; 92:449-455,
http://dx.doi.org:/10.1093/cvr/cvr227

The use of genomics for treatment is another matter, and has several factors, e.g., age, residual function after AMI, comorbidities

Sonic Hedgehog-Modified Human CD34+ Cells Preserve Cardiac Function After Acute Myocardial Infarction.Mackie AR, Klyachko E, Thorne T, Schultz KM, Millay M, Ito A. Circulation Research (IF: 11.86). 05/2012; 111(3):312-21. http://dx.doi.org:/10.1161/CIRCRESAHA.112.266015

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Malnutrition in India, High Newborn Death Rate and Stunting of Children Age Under Five Years

Posted in Biomarkers & Medical Diagnostics, Biomedical Measurement Science, Bone Disease and Musculoskeletal Disease, Cerebrovascular and Neurodegenerative Diseases, Chemical Biology and its relations to Metabolic Disease, Curation, Discovery process, Experimental validation, Explanatory, Frontiers in Cardiology and Cardiovascular Disorders, Liver & Digestive Diseases Research, Metabolomics, Nutrigenomics, Nutrition, Origins of Cardiovascular Disease, Population Health Management, Nutrition and Phytochemistry, Proteomics, tagged Childhood malnutrition, Ganges River, Himalayan Plateau, hyperhomocysteinemia, Population sanitation, Protein-energy malnutrition, S deficiency, stunting of growth, Transthyretin on July 15, 2014| Leave a Comment »

Malnutrition in India, High Newborn Death Rate and Stunting of Children Age Under Five Years

Curator: Larry H Bernstein, MD, FCAP

A lead report in the New York Times focuses on a major public health problem in India today, with the irony of high growth rate and malnutrition and stunting of children under age 5 years that occurs in the majority and wealthy Hindu population, but not to any comparable degree in the Muslim population or in Bangladesh. This is prevalent along the Ganges River, which crosses India below the Himalaya Mountains. The inference is that the problem is perhaps solely related to poor sanitation, which is to a large degree indisputable, and the disease is related to the gut microbiome (not so stated), that leaves an intestinal mucosa with flattened epithelia, and no observation is made of the submucosal thymic-derived T-cell lymphocyte population, the largest in the human body.

Moreover, I might point out that the turnover of the intestinal epithelium with its large surface area is very high under normal metabolic circumstances. The result is that the children are malnourished, and they have visceral protein losses as well as somatic protein loss (stunted growth, probably affecting both skeletal muscle and the metaphyseal growth plates of long bones). This is not quite stated this way.

The irony is that they have sufficient food supply, except that if there is a diarrhea or intestinal malabsorption at an early age, the children just might not eat, except for perhaps soft foods. So it is not explicitly cleat that their is sufficient animal protein in the diet, which has a S:N ratio that is roughly twice that of an exclusively plant diet. The distinction is made between marasmus and kwashiorkor in that in kwashiorkor the protein deficiency is in the visceral compartment. Consequently, there is a reprioriotization of the liver to synthesize acute phase proteins with a decline in albumin, transthyretin, and retinol-binding protein. This is not insignificant, even though there may also be an inflammatory state, as from repeated infections.

I certainly would be interested in seeing data from the ongoing study that measures the serum protein analytes, and also a measurement of serum red cell Hb, serum cysteine, homocysteine, and glutathione, and perhaps a muscle biopsy.

I go directly to the article at this point.

Poor Sanitation in India May Afflict Well-Fed Children With Malnutrition

By GARDINER HARRIS JULY 13, 2014
http://www.nytimes.com/2014/07/15/world/asia/poor-sanitation-in-india-may-afflict-well-fed-children-with-malnutrition.html

SHEOHAR DISTRICT, India — He wore thick black eyeliner to ward off the evil eye, but Vivek, a tiny 1-year-old living in a village of mud huts and diminutive people, had nonetheless fallen victim to India’s great scourge of malnutrition.

His parents seemed to be doing all the right things. His mother still breast-fed him. His family had six goats, access to fresh buffalo milk and a hut filled with hundreds of pounds of wheat and potatoes. The economy of the state where he lives has for years grown faster than almost any other. His mother said she fed him as much as he would eat and took him four times to doctors, who diagnosed malnutrition. Just before Vivek was born in this green landscape of small plots and grazing water buffalo near the Nepali border, the family even got electricity.

So why was Vivek malnourished?

‘Bihar grew at 12% last 7 years’

Abhay Singh, TNN | Feb 15, 2014, 02.15AM IST

Bihar’s average annual growth rate has been 12% in the last seven fiscal years

The report has taken 1999-2006 as the cut-off period to highlight spectacular Bihar turnaround story achieved under CM Nitish Kumar.

PATNA: Bihar’s average annual growth rate has been 12% in the last seven fiscal years, one of the highest among all Indian states, on the back of high growth rate achieved in the agriculture and allied sectors. Besides, advancement has also been made in healthcare and education.

The state’s Economic Survey Report for 2013-14, which was tabled in the assembly on Friday, has concluded this. The summary of the report said, “During 1990-91 to 2005-06, the state’s income at constant prices grew at an annual rate of 5.7%.” It said after that the economy witnessed a turnaround and grew at an annual rate of 12%. “The rate of growth achieved by the economy during 2006-13 is not only much higher, but also one of the highest among all Indian states.”

The report has taken 1999-2006 as the cut-off period to highlight spectacular Bihar turnaround story achieved under CM Nitish Kumar.

Poor Sanitation Linked to Malnutrition in India

New research on malnutrition, which leads to childhood stunting, suggests that a root cause may be an abundance of human waste polluting soil and water, rather than a scarcity of food.

SANITATION – bathing in Ganges River contaminated by human waste

Like almost everyone else in their village, Vivek and his family have no toilet, and the district where they live has the highest concentration of people who defecate outdoors. As a result, children are exposed to a bacterial brew that often sickens them, leaving them unable to attain a healthy body weight no matter how much food they eat.

“These children’s bodies divert energy and nutrients away from growth and brain development to prioritize infection-fighting survival,” said Jean Humphrey, a professor of human nutrition at Johns Hopkins Bloomberg School of Public Health. “When this happens during the first two years of life, children become stunted. What’s particularly disturbing is that the lost height and intelligence are permanent.”

Two years ago, Unicef, the World Health Organization and the World Bank released a major report on child malnutrition that focused entirely on a lack of food. Sanitation was not mentioned. Now, Unicef officials and those from other major charitable organizations said in interviews that they believe that poor sanitation may cause more than half of the world’s stunting problems.

“Our realization about the connection between stunting and sanitation is just emerging,” said Sue Coates, chief of water, sanitation and hygiene at Unicef India. “At this point, it is still just an hypothesis, but it is an incredibly exciting and important one because of its potential impact.”

This research has quietly swept through many of the world’s nutrition and donor organizations in part because it resolves a great mystery: Why are Indian children so much more malnourished than their poorer counterparts in sub-Saharan Africa?

A child raised in India is far more likely to be malnourished than one from the Democratic Republic of Congo, Zimbabwe or Somalia, the planet’s poorest countries. Stunting affects 65 million Indian children under the age of 5, including a third of children from the country’s richest families.

This disconnect between wealth and malnutrition is so striking that economists have concluded that economic growth does almost nothing to reduce malnutrition.

Half of India’s population, or at least 620 million people, defecate outdoors. And while this share has declined slightly in the past decade, an analysis of census data shows that rapid population growth has meant that most Indians are being exposed to more human waste than ever before.

In Sheohar, for instance, a toilet-building program between 2001 and 2011 decreased the share of households without toilets to 80 percent from 87 percent, but population growth meant that exposure to human waste rose by half.

“The difference in average height between Indian and African children can be explained entirely by differing concentrations of open defecation,” said Dean Spears, an economist at the Delhi School of Economics. “There are far more people defecating outside in India more closely to one another’s children and homes than there are in Africa or anywhere else in the world.”

SANITATION-children defecate outside – 162 million malnourished and stunted

Not only does stunting contribute to the deaths of a million children under the age of 5 each year, but those who survive suffer cognitive deficits and are poorer and sicker than children not affected by stunting. They also may face increased risks for adult illnesses like diabetes, heart attacks and strokes.

“India’s stunting problem represents the largest loss of human potential in any country in history, and it affects 20 times more people in India alone than H.I.V./AIDS does around the world,” said Ramanan Laxminarayan, vice president for research and policy at the Public Health Foundation of India.

India is an increasingly risky place to raise children. The country’s sanitation and air quality are among the worst in the world. Parasitic diseases and infections like tuberculosis, often linked with poor sanitation, are most common in India. More than one in four newborn deaths occur in India.

Open defecation has long been an issue in India. Some ancient Hindu texts advised people to relieve themselves far from home, a practice that Gandhi sought to curb.

“The cause of many of our diseases is the condition of our lavatories and our bad habit of disposing of excreta anywhere and everywhere,” Gandhi wrote in 1925.

SANITATION-disposing of excreta anywhere and everywhere

Other developing countries have made huge strides in improving sanitation. Just 1 percent of Chinese and 3 percent of Bangladeshis relieve themselves outside compared with half of Indians. Attitudes may be just as important as access to toilets. Constructing and maintaining tens of millions of toilets in India would cost untold billions, a price many voters see no need to pay — a recent survey found that many people prefer going to the bathroom outside.

Few rural households build the sort of inexpensive latrines that have all but eliminated outdoor waste in neighboring Bangladesh.

“We need a cultural revolution in this country to completely change people’s attitudes toward sanitation and hygiene,” said Jairam Ramesh, an economist and former sanitation minister.

India’s government has for decades tried to resolve the country’s stubborn malnutrition problems by distributing vast stores of subsidized food. But more and better food has largely failed to reverse early stunting, studies have repeatedly shown.

India now spends about $26 billion annually on food and jobs programs, and less than $400 million on improving sanitation — a ratio of more than 60 to 1.

Lack of food is still an important contributor to malnutrition for some children, and some researchers say the field’s sudden embrace of sanitation has been overdone. “In South Asia, a more important factor driving stunting is diet quality,” said Zulfiqar A. Bhutta, a director of the Center for Global Child Health at the Hospital for Sick Children in Toronto.

Studies are underway in Bangladesh, Kenya and Zimbabwe to assess the share of stunting attributable to poor sanitation. “Is it 50 percent? Ninety percent? That’s a question worth answering,” said Dr. Stephen Luby, a professor of medicine at Stanford University who is overseeing a trial in Bangladesh that is expected to report its results in 2016. “In the meantime, I think we can all agree that it’s not a good idea to raise children surrounded by poop.”

Better sanitation in the West during the 19th and early 20th centuries led to huge improvements in health long before the advent of vaccines and antibiotics, and researchers have long known that childhood environments play a crucial role in child death and adult height.

The present research on gut diseases in children has focused on a condition resulting from repeated bacterial infections that flatten intestinal linings, reducing by a third the ability to absorb nutrients. A recent study of starving children found that they lacked the crucial gut bacteria needed to digest food.

In a little-discussed but surprising finding, Muslim children in India are 17 percent more likely to survive infancy than Hindus, even though Muslims are generally poorer and less educated. This enormous difference in infant mortality is explained by the fact that Muslims are far more likely to use latrines and live next to others also using latrines, a recent analysis found.

So widespread housing discrimination that confines many Muslims to separate slums may protect their children from increased exposure to the higher levels of waste in Hindu communities and, as a result, save thousands of Indian Muslim babies from death each year.

SANITATION-one in 4 newborn deaths related to sanitation

Discussion:

The coexistence of poor sanitation, where has a very large cultural barrier, with serious protein-energy malnutrition, is a toxic mix. There is the comparison with the Muslim population at the adjoining border of the Ganges River outflow in Bangladesh. One might also look at the catholic Portuguese population in Goa, the Jewish population in Mumbai and Kochi, and the nearby Catholic population. There is no malnutrition in those populations, or in the Siiks. This is undoubtedly a cultural phenomenon of ancient origin. (The migration of the jews and of the catholics to Kochi occurred around the Indian Ocean at the time of Christ. The catholic population in Goa was from Portugal.

I don’t think we have enough of the story here. The Ganges river flows centrally across India, and is not far from the Himalayas. This has some significance in the sufficiency of animal protein availability, and most importantly, of what I might expect of the tissue S:N ratio, which is critical for availability of methionine, S-adenosyl methionine, and mitochondrial energy reactions. These are also mediated by transsulfuration reactions and by cystathionine beta-synthase. Detailed discussions are available elsewhere. It has been pointed out by Vernon Young and Yve Ingenbleek that sulfur is insufficient in the soil where there is not a lava flow of volcanic ash, which could be the case here. So it is at best not a good geographic situation, even before compounding the issue.

The relationship to heart attack and stroke is established for elevated homocysteine.

Homocysteine and Vascular Disease
STEVEN E . S. MINER , M.D. , DAVID E .C. COLE *, M.D. , PHD. AND DUNCAN J . STEWART, M.D.
Cardiology Rounds A U G U S T 1 9 9 6 ; I(5)

Homocysteine is a naturally occurring, sulfur-containing amino acid. Continuously formed and catabolized in vivo, its metabolism is dependent on a complex interaction of genetics and physiology (Fig. 1). Its relevance is based on the increasing recognition of the correlation between elevated levels of homocysteine and human disease.

Table 1
Selected Determinants of Plasma Homocysteine*
1. Genetic
• Cystathionine-beta-synthase:
heterozygote mutations 0.5-1.5% {451}
• Methionine synthase: rare
• MTHFR: heterozygote mutations
approximately 50% {403}
2. Physiologic
• age: Hcy increases with increasing age {336}
• sex: pre-and post-menopausal women
have lower levels than men {247}
• diet: related to methionine and vitamin cofactor
(folate, vitamins B6 and B12) intake {437}
• alcohol: relationship unclear {375}
3. Pathologic
• vitamin deficiency: increased homocysteine
concentrations {10}
• renal disease: increase correlated
with increasing serum creatinine {81}
• transplantation: increased levels {149, 435}
• post stroke: transiently decreased levels {341}
• severe psoriasis: elevated levels {438}
4. Medications
• oral contraceptives/hormone replacement:
decreased levels {269}
• corticosteriods: increased {159}
• cyclosporine: increased {393}
• smoking: increased {336}

Abstracts of Interest
Serum total homocysteine and coronary heart disease in middleaged
British men.
IJ PERRY, H REFSUM, RW MORRIS, SB EBRAHIM, PM UELAND, AG SHAPER.
D E PA RTMENT OF PRIMARY CARE & POPULATION SCIENCES, ROYAL FREE
H O S P I TAL SCHOOL OF MEDICINE, LONDON, AND DEPA RTMENT OF CLINICAL
B I O L O G Y, UNIVERSITY OF BERGEN, NORWAY.
Serum total homocysteine (tHcy) levels are inversely associated with dietary intake of folic acid and B vitamins. Raised tHcy levels have been linked with coronary heart disease (CHD). We have examined the association between tHcy concentration and the subsequent risk of CHD, using a nested case control study design, within a prospective study of cardiovascular disease in British men. tHcy concentration was measured in serum samples, stored at entry to the study, from 110 incident cases of myocardial infarction and 118 controls. Cases were randomly sampled from events which occured after the first five years of follow-up. Cases and controls were frequency matched by town and age group. Levels of homocysteine [geometric mean (95% CI)] were significantly higher in cases than controls: homocysteine 13.5 (12.6 – 14.3) μmol/L vs 11.9 (11.3 – 12.6) μmol/L; p=0.005. There was a graded increase in the relative risk (odds ratio; OR) of CHD in the 2nd, 3rd and 4th quartile of tHcy (OR 1.4, 1.9, 2.2; trend p=0.006) relative to the first quartile. Adjustment for age, town, social class, body mass index, smoking, physical activity, alcohol intake, hypertensive status, serum cholesterol, and serum creatinine did not attenuate this association, (OR 2.1, 2.3, 2.7; trend p=0.04). tHcy levels were higher at baseline in men with evidence of pre-existing CHD and (as expected) adjustment for this factor attenuated the linear association between tHcy and subsequent events, trend p=0.07. The findings suggest that homocysteine is an independent risk factor for CHD
with no threshold level.
Reprinted from Heart, Volume 75 /Number 5 (Supplement 1), May 1996.
Homocysteine and Coronary Atherosclerosis
ELLEN L. MAYER, MD, DONALD W. JACOBSEN, PHD, KILLIAN ROBINSON, MD,
FACC, CLEVELAND, OHIO
The conventional risk factors for premature coronary artery disease include smoking, hyperlipidemia, hypertension, diabetes and a positive family history. However, many patients have precocious atherosclerosis without having any of these standard risk factors. Identification of other markers that increase the risk of coronary disease may improve our understanding of the pathophysiologic mechanisms of this disorder and allow the development of new preventive or therapeutic measures. An elevated plasma homocysteine level has recently received greater attention as an important risk factor for vascular disease, including coronary atherosclerosis. This review discusses the biochemistry of homocysteine and the related metabolic importance of folate, vitamin B6 (pyridoxine) and B12 (cobalamin) as well as a number of essential enzymes. The major factors that influence homocysteine concentration are genetic, nutritional and pathologic.
There is a large body of experimental and clinical evidence for high plasma homocysteine to be a risk factor for vascular disease, including coronary atherosclerosis.
Excerpted from Journal of the American College of Cardiology 1996;27:517-27

An important meta-analysis by Boushey et al in 1995 further quantified the magnitude of risk. In their analysis of all major studies available at that time, they found a linear, independent risk for increments in homocysteine. There were no levels above or below which an incremental rise in homocysteine did not affect cardiovascular risk. Specifically, every 5 μmol/L increment in homocysteine was found to be associated with odds ratios of 1.6 for m e n ; (95% Cl 1.4-1.7) and 1.8 for women; (95% CI 1.3-1.9) for coronary artery disease.

Cystathionine beta synthase (CBS) catalyzes the reaction taking homocysteine to cystathionine. This enzyme requires pyridoxine as a co-factor and is an integral part of the transsulfuration or
pyridoxine – dependent pathway. 33 distinct mutations have been identified with heterozygosity occurring at a prevalence of 0.5-1.5%. The majority of heterozygotes will have normal fasting homocysteine levels, but can be detected with a methionine load test.

Hyperhomocysteinemia is a Biomarker of Sulfur-Deficiency in Human Morbidities

Yves Ingenbleek
Laboratory of Nutrition, University Louis Pasteur Strasbourg, France
The Open Clinical Chemistry Journal, 2009, 2, 49-60

Abstract: Methionine (Met) is crucially involved in the synthesis of S-compounds endowed with molecular, structural and functional properties of survival value. Dietary Met may undergo transmethylation processes to release homocysteine (Hcy) which may either be regenerated to Met following remethylation (RM) pathways or catabolized along the transsulfuration
(TS) cascade. The activity of enzymes governing RM and TS pathways is depending on pyridoxine, folate and cobalamin bioavailability. Dietary restriction in any of these watersoluble B-vitamins may lead to hyperhomocysteinemia (HHcy) causing a panoply of cardiovascular disorders. Taken together, the vitamin triad only affords partial account of Hcy variance, prompting the search for additional causal factor(s). Body composition studies demonstrate that nitrogen (N) and sulfur (S) maintain tightly correlated concentrations in tissues of both healthy subjects and diseased patients. Any morbid condition characterized by insufficient N intake or assimilation, as seen in protein malnutrition or intestinal malabsorption, reduces body S accretion rates. Excessive urinary N-losses, as reported in acute or chronic inflammatory disorders, entail proportionate obligatory S-losses. As a result, lean body mass (LBM) undergoes downsizing and concomitant depletion of N and S body stores which depresses the activity of cystathionine-􀀁-synthase, thereby promoting upstream accumulation of Hcy and overstimulation of RM processes. HHcy thus appears as the dark side of efforts developed by S-deprived patients to safeguard Met homeostasis. Irrespective of vitamin-B status, Hcy values are negatively correlated with LBM shrinkage well identified by the serial measurement of plasma transthyretin (TTR). The S deprivation theory fulfills the gap and allows full causal coverage of the metabolic anomaly, hence providing together with vitamin-deficiencies an unifying overview of the main nutritional determinants implicated in HHcy epidemiology.

The Oxidative Stress of Hyperhomocysteinemia Results from Reduced Bioavailability of Sulfur-Containing Reductants

Yves Ingenbleek
Laboratory of Nutrition, Faculty of Pharmacy, University Louis Pasteur Strasbourg, France
The Open Clinical Chemistry Journal, 2011, 4, 34-44

Abstract: Vegetarian subjects consuming subnormal amounts of methionine (Met) are characterized by subclinical protein malnutrition causing reduction in size of their lean body mass (LBM) best identified by the serial measurement of plasma transthyretin (TTR). As a result, the transsulfuration pathway is depressed at cystathionine-beta-synthase (C-b-S) level triggering the upstream sequestration of homocysteine (Hcy) in biological fluids and promoting its conversion to Met. Maintenance of beneficial Met homeostasis is counterpoised by the drop of cysteine (Cys) and glutathione (GSH) values downstream to CbS causing in turn declining generation of hydrogen sulfide (H2S) from enzymatic sources. The biogenesis of H2S via non-enzymatic reduction is further inhibited in areas where earth’s crust is depleted in elemental sulfur (S8) and sulfate oxyanions. Combination of subclinical malnutrition and S8-deficiency thus maximizes the defective production of Cys, GSH and H2S reductants, explaining persistence of unabated oxidative burden. The clinical entity increases the risk of developing cardiovascular diseases (CVD) and stroke in underprivileged plant-eating populations regardless of Framingham criteria and vitamin-B status. Although unrecognized up to now, the nutritional disorder is one of the commonest worldwide, reaching top prevalence in populated regions of Southeastern Asia. Increased risk of hyperhomocysteinemia and oxidative stress may also affect individuals suffering from intestinal malabsorption or westernized communities
having adopted vegan dietary lifestyles.

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The Experience of a Patient with Thyroid Cancer

Posted in Best evidence, Clinical Diagnostics, Curation, Developmental biology, Disease Biology, Small Molecules in Development of Therapeutic Drugs, Electrophysiology, Endocrine Diseases, Experimental validation, Explanatory, Historical relevance, Immunodiagnostics, Inferential analysis, Innovation in Immunology Diagnostics, Interventional Oncology: Radiofrequency Ablation, Transarterial Chemoembolization, Microwave Ablation and Irreversible Electroporation (IRE), Medical Imaging Technology, Image Processing/Computing, MRI, CT, Nuclear Medicine, Ultra Sound, Nutrition, Patient Experience: Personal Memories of Invasive Medical Intervantion, Personalized and Precision Medicine & Genomic Research, Proteomics, Sensors & Analytics, Signaling, Technology Advance Assessment of, Translational Effectiveness, Translational Science, tagged Graves' disease, thyroid cancer, thyroid eye disease, TSH on July 14, 2014| Leave a Comment »

The Experience of a Patient with Thyroid Cancer

Interviewer and Curator: Larry H Bernstein, MD, FCAP

Thyroid cancer is usually a fairly innocuous disease, but it can present in different ways. There are are perhaps two main types – medullary, and follicular. But an anaplastic type is also a third uncommon type. It is speculative for me to suggest that the anaplastic type is a progression of either of the two main types. A RAS genotype coexists with the aggressive anaplastic carcinoma. Thyroid cancers are BRAF positive in genotype. The histological feature that is used to identify this neoplasm is the presence of “sammoma bodies”. It is more common in women, and less common in the elderly, and the incidence appears to have increased regionally in recent years. A recent paper suggests a common specific feature with breast cancer, which is unconfirmed.

When we consider thyroid disease, we start with euthyroid status, hypothyroid and hyperthyroid, all of which are related to the synthetic activity of the gland, that has a right and left lobe joined by a isthmus. In the midwestern US there is a deficiency of iodine, which leads to nodular thyroid goiter. The Mayo brothers pioneered in thyroid surgery at their clinic in Rochester, MN. This led to the insertion of iodine in table salt (Morton’s salt- “when it rains, it pours). Hyperthyroid status is over production of the hormone by an overactive gland. It is usually primary disease, called Grave’s Disease, after the physician who described it. I am not aware of the occurrence secondary to hyperactivity of the pituitary gland, which would result in both an increased thyroid stimulating hormone (TSH), thyrotropin, and elevated thyroid hormone, except by a primary neoplasm of thyrotropin secreting cells. The two hormones are under feedback control. This feedback is a valuable diagnostic indicator because the TSH is suppressed with Grave’s disease. The TSH assay is very accurate, and as the TSH falls, the TH increases, but the TH assay has never been as accurate as the TSH. The TH is transported in serum by three proteins: thyroxin-binding globulin (TBG), albumin, and trans-thy-retin (TTR), a quadruplex peptide with one subunit binding to retinol-binding protein (RPB), which transports retinol, vitamin A). The importance of TTR is not a subject for discussion here, but it has extremely important ties to metabolic disease that includes hyperhomocysteinemia and Alzheimer’s disease, as this protein is produced by both the liver and the choroid plexus, but the CP production declines in the elderly. The TTR metabolism is closely linked to total body sulfur, measured by K+ isotope measurement of lean body mass (fat free mass), and is a more accurate measure than use of urinary creatinine loss, which only measure the structural body mass, but not the visceral component.

There is another twist to the story in that thyroid hormone may be depressed over time secondary to an autoantibody to thyroid “peroxidase”, leading to destruction of the gland. The thyroid antibody that occurs has been recently reported to be a “peroxidase” antibody in common with the mammary gland. The disorder is denominated – Hashimoto’s thyroiditis. The presence of thyroid antibody may occur with Grave’s disease, with an occular protrusion with inflammation of the adductor muscles of eye movement. This is termed “exophthalmus”. However, thyroid eye disease is known to occur with hypo-, hyper-, and euthyroid status.

I here describe the long and difficult search to identify a confusing case.

Family history: Mother had thyroid cancer, surgically cured at Mayo Clinic. Sister had Hashimoto’s thyroiditis. Father had severe rheumatoid arthritis.

History of Illness. The patient is a male over 65 years age who attended a discussion group for several years and participated in supervised fitness exercises and did daily walks for 2-3 years prior to the discovery of the problem when he recalls, his voice was weak in making presentations to the discussion group (age 86 and over).

At the end of summer, 2013, he experienced shortness of breath and dizziness on walking. His physician had been concerned about the change of voice prior to this. He had a history of sleep apnea, and he was actively trying to lose weight. Cardiac and vascular examination of carotid and of peripheral circulation were unexpectedly excellent. Pulmonary studies were good.

A visit to an ENT physician did not explain the voice impairment. An unexpected low TSH result came back < 0.01, compared to a normal result 9 months earlier. This was the first indication of an active cyst or Grave’s disease. The patient was referred for ultrasound exam, and a thyroid panel was ordered. The result of the ultrasound was an enlarged right lobe with two large degenerate cysts, and a central small calcified cyst. The cyst was biopsied and it was malignant. It was BRAF pos and RAS negative.

He was referred to the nearest world-class academic center for further endocrine evaluation. The endocrinologist palpated a thyroid enlargement, and a biopsy was performed of the lymph nodes under a full scan of the neck. Surgery was scheduled and a surgeon skilled in endocrine surgery and cancer removed the thyroid, and noted that the right lobe compressed the recurrent laryngeal nerve. This was consistent with en ENT examination of the larynx that showed paralysis of the right larynx. The good news was that the prediction was that the nerve innovation was good, and would return.

There were a few involved lymph nodes in the removed specimen. The patient was put on synthroid. The next step was to schedule I131 radioiodine treatment by oral tablets. This required a preparatory diet of no salt or iodine intake prior to treatment. There was also a 5 day isolation for beta ray emission (which kills residual thyroid cells). The neck was scanned with a gamma scanned prior to induction of treatment, which required a dose of synthetic TSH and a low dose of I131. The patiemt is recovered for 14 days post treatment and has regained much energy.

There is a residual burden of the thyroid eye disease that requires special optical care because of loss of distance perception with diplopia. This is stable, but any surgical repair would have to wait for a year.

Notes from PathologyOutlines.com, Nathan Pernick, Editor-in-Chief

Thyroid gland

Reviewer: Zubair W. Baloch, M.D., Shahidul Islam, M.D., Ph.D., Ricardo R. Lastra, M.D., Michelle R. Pramick, M.D., Phillip A. Williams, M.D., MSC (see Reviewers page)

Revised: 11 July 2014, last major update IN PROGRESS
Copyright: (c) 2001-2014, PathologyOutlines.com, Inc.

Endocrine abnormalities and thyroid gland
Hyperthyroidism

Reviewer: Shahidul Islam, M.D., Ph.D.

General
=======================================================

Accelerated thyroid hormone biosynthesis and secretion by thyroid gland
Early symptoms: anxiety, palpitations, rapid pulse, fatigue, muscle weakness, tremor, weight loss, diarrhea, heat intolerance, warm skin, excessive perspiration, menstrual changes, hand tremor
Ocular changes: wide staring gaze and lid lag due to sympathetic overstimulation of levator palpebrae superioris

Thyrotoxicosis: hypermetabolic clinical syndrome due to elevated serum T3 or T4

Types
=======================================================

Primary hyperthyroidism: intrinsic thyroid abnormality
- Low TSH, high free T4, normal TRH stimulation test
Secondary hyperthyroidism: high TSH, abnormal TRH stimulation test
Subclinical hyperthyroidism: low TSH (< 0.1 µIU/ml), normal T3 and T4 (Eur J Endocrinol 2005;152:1), no clinical hyperthyroidism
- May be due to exogenous thyroid hormone (Hormones (Athens) 2006;5:119)
- Patients have increased risk of coronary heart disease (Int J Cardiol 2008;125:41)
T3 hyperthyroidism: 1-4%ofhyperthyroid patients
- Low TSH, high free T3, normal free T4
- Associated with early treatment of hyperthyroidism with antithyroid drugs
T4 hyperthyroidism:highT4, normal T3
- Due to primary hyperthyroidism causes, also iodine, amiodarone (Arch Pathol Lab Med 2003;127:e275), pregnancy (2%)

Graves’ disease (85%)

Micro images
=======================================================

Diffuse hyperplasia of thyroid gland

Additional references
=======================================================

Med J Aust 2004;180:186, eMedicine #1, #2, Wikipedia

Hashimoto’s thyroiditis

General
=======================================================

Autoimmune disease with goiter, elevated circulating anti-thyroid peroxidase and anti-thyroglobulin antibodies
First described by Hakaru Hashimoto in 1912 (World J Surg 2008;32:688)

Epidemiology
=======================================================

90-95% in women, usually 45-65 years old; clusters in families
More common in Whites than Blacks or Japanese (Am J Clin Pathol 1994;101:698)
Most common cause of sporadic goiter in children in iodine sufficient areas (J Pediatr Endocrinol Metab 2007;20:1199)

Clinical features

Clinical features
=======================================================

Adults present with painless, gradual thyroid failure due to autoimmune destruction, may initially have transient hyperthyroidism
Children have variable hypothyroidism and reversion to euthyroidism so must monitor thyroid function (Clin Endocrinol (Oxf) 2009;71:451)

Associated with HLA-DR5 (goitrous form), HLA-DR3 (atrophic form)
May coexist with SLE, rheumatoid arthritis, Sjögren’s syndrome, pernicious anemia, type 2 diabetes, Graves’ disease, chronic active hepatitis, adrenal insufficiency, MALT lymphoma of gastrointestinal tract (80:1 relative risk), other B cell lymphomas
Associated with well differentiated thyroid cancer (J Am Coll Surg 2007;204:764)
May evolve into thyroid lymphoma (J Clin Pathol 2008;61:438)

Laboratory
=======================================================

Autoantibodies include:
- Anti-TSH (specific for Hashimoto’s and Graves’ disease)
- Anti-thyroglobulin (less sensitive but similar specificity as anti-thyroid peroxidase, Clin Chem Lab Med 2006;44:837)
- Anti-thyroid peroxidase (previously called antimicrosomal antibody, sensitive but not specific as 20% of adult women without disease have these antibodies); anti-iodine transporter (rare)
- Note: anti-TSH antibodies block the TSH receptor in Hashimoto’s disease but stimulate the TSH receptor in Graves’ disease

Papillary carcinoma

75-80% of thyroid carcinomas
Occult tumors in 6% at autopsy (1 to 10 mm), 46% multicentric, 14% with nodal metastases (Am J Clin Pathol 1988;90:72)
Occult tumors in up to 24% with other thyroid disease, but with male predominance (Mod Pathol 1996;9:816)

Epidemiology
=======================================================

Usually women (70%) of reproductive age

Clinical features
=======================================================

Usually presents as painless nodule or mass in neck or cervical node; usually cold on scan
Usually diagnosed by FNA
At presentation, 67% in thyroid only, 13% in thyroid and cervical nodes, 20% in nodes only
Parathyroid gland invasion often occurs early (Arch Pathol Lab Med 2002;126:1511)
Nodal involvement is often not clinically apparent due to small size and similar consistency; nodal metastases may undergo cystic change and resemble branchial cleft cysts (The Internet Journal of Nuclear Medicine 2007;4:2)
Proteomic analysis of serum may be useful in future for screening or followup (Arch Otolaryngol Head Neck Surg 2008;134:198)
Lymph nodes:
- Measurement of thyroglobulin in FNA from lymph nodes in patients with history of papillary thyroid carcinoma is useful in detecting recurrent disease, especially if specimen is or likely will be inadequate for evaluation (Cytojournal 2008;5:1)
Risk factors:
- Ionizing radiation before age 20 (for acne – Surgery 1991;110:691, tonsillitis, tinea capitis, enlarged thymus)
- Post-Chernobyl (particularly children, Endocr Pathol 2006;17:307) or after exposure to nuclear explosions at Marshall Islands (J Epidemiol 2003;13:99)
- Hashimoto’s thyroiditis (possibly, Cancer 1995;76:2312)
- Familial adenomatous polyposis (World J Surg 1998;22:738)
- Is familial in 4.5%, similar prognosis as sporadic disease (Surgery 2009;145:100)

Prognostic factors
=======================================================

10 year survival is 98%, similar to general population (versus 92% for follicular carcinoma); 100% if under age 20, even with nodal metastases
Cervical nodal involvement does NOT affect prognosis
5-20% have local recurrences, 10-15% have distant metastases (lung, bones, CNS)
Poorer prognosis:
- Age 40+ or elderly, male (possibly), local invasion (associated with higher incidence of nodal metastases, Arch Pathol Lab Med 1998;122:166), distant metastases (other sites worse than lung, Surgery 2008;143:35), large tumor size, multicentricity, tall cell, columnar or diffuse sclerosing variants
- Poorly differentiated, anaplastic or squamous foci

added July 14, 2014

Summary – Intraoperative laryngeal nerve monitoring
Objectives: The aim of this study was to stimulate the recurrent laryngeal nerve during thyroidectomy or parathyroidectomy and to record the muscle responses in an attempt to predict postoperative vocal fold mobility.
Patients and methods: Intraoperative recurrent laryngeal nerve monitoring during general anaesthesia was performed by using an electrode-bearing endotracheal tube (nerve integrity monitor EMG endotracheal tube [Medtronic Xomed, Jacksonville, Flo, USA]). Two hundred and fifteen recurrent laryngeal nerves from 141 patients undergoing total thyroidectomy (n = 74),
hemithyroidectomy (n = 63), or parathyroidectomy (n = 4) were prospectively monitored. In each case, the muscle potential was recorded after stimulation of the recurrent laryngeal nerve by a monopolar probe.
Results: The nerve stimulation threshold before and after dissection that induced a muscle response of at least 100 V ranged from 0.1 to 0.85 mA (mean 0.4 mA). The supramaximal stimulation intensity was defined as 1 mA. The amplitude of muscle response varied considerably from one patient to another, but the similarity of the muscle response at supramaximal intensity between pre- and postdissection and between postdissection at the proximal and distal exposed
portions of the nerve was correlated with normal postoperative vocal fold function. Inversely, alteration of the muscle response indicated a considerable risk of recurrent laryngeal nerve palsy, but was not predictive of whether or not this lesion would be permanent. http://dx.doi.org:/10.1016/j.anorl.2011.09.003

Summary – Prognostic impact of tumour multifocality in thyroid papillary microcarcinoma
European Annals of Otorhinolaryngology, Head and Neck diseases (2012) 129, 175—178

Objective: The objective of this study was to evaluate the prognostic impact of tumour multifocality in papillary thyroid microcarcinoma (PTMC).
Methods: All patients who underwent total thyroidectomy and central neck dissection for PTMC in our institution between 1990 and 2007 were included in this retrospective study. Statistical correlations between tumour multifocality and various clinical or pathological prognostic parameters were assessed by univariate and multivariate analyses.
Results: A total of 160 patients (133 women and 27 men; mean age: 47.8 ± 13.7 years) were included in this study. Tumour multifocality was demonstrated in 59 (37%) patients. Central neck metastatic lymph node involvement was identified in 46 (28%) patients. No statistical correlation was demonstrated between tumour multifocality and the following factors: age, gender, tumour size, extension beyond the thyroid, metastatic central neck lymph node involvement and risk of recurrence. A tumour diameter greater than 5 mm was associated with a higher risk of recurrence (P = 0.008).
Conclusion: Tumour multifocality does not appear to have a prognostic impact in PTMC. http://dx.doi.org:/10.1016/j.anorl.2011.11.003

Positron emission tomography thyroid carcinoma
European Annals of Otorhinolaryngology, Head and Neck diseases (2012) 129, 251—256

Objectives: Recurrence is observed in 15—20% of patients under surveillance following treatment of differentiated thyroid cancer (DTC). However, due to cell dedifferentiation, the recurrence may be iodine-negative, thereby compromising detection. For this reason, new methods of exploration are indispensable to enable localization of such recurrences. The purpose of this work is to review the contribution of positron emission tomography—computed tomography (PET-CT) in the exploration of iodine-negative recurrent DTC.
Method: A comprehensive review and discussion of the medical literature was carried out.
Results: Depending on the report, the sensitivity of PET-CT ranged from 70% to 85%, with up to 90% specificity. However, the large number of false negatives, which can reach 40%, is the
disadvantage of this examination. PET-CT results lead to change in the therapeutic strategy in approximately 50% of patients with isolated raised serum thyroglobulin levels, and surgical exploration of a precise anatomical area in the neck.
Conclusion: As post-treatment recurrence of a DTC can affect patient survival, a thorough diagnostic work-up is required in these cases. Where thyroglobulin levels are elevated with no uptake on 131-iodine scans, PET-CT can be a useful complementary exploration, especially for localizing the site of recurrence.
http://dx.doi.org:/10.1016/j.anorl.2012.01.003
French ENT Society (SFORL) practice guidelines for lymph-node management in adult differentiated thyroid carcinoma
European Annals of Otorhinolaryngology, Head and Neck diseases (2012) 129, 197—206

Cervical and mediastinal lymph-node management differentiated thyroid carcinoma of the follicular epithelium (DTC) remains controversial. Depending on the situation, pre-operative staging and indications for and extent of lymph-node dissection are still matters of debate, even in case of palpable nodes found on primary surgery. Procedural indications for adenectomy, selective neck dissection, and anatomic regional extension of dissection are not clearly defined.

Questions raised:

• what is lymph-node involvement in DTC?
• what is the prognostic value of lymph-node invasion: for
recurrence, and for survival?

• what baseline assessment is required ahead of treatment
of papillary thyroid carcinoma to assess possible lymphnode
involvement?

• what are the principles of lymph-node surgery?
Central and lateral dissection, and dissection extended to the mediastinum;
• what is the iatrogenesis in cN0 and cN+ neck?
• what is the impact of central and lateral neck dissection on recurrence, survival, secondary treatment and surveillance in cN0 and cN+ ?
• in cN0 patients, when neck dissection is considered, what lymph-node regions should be indicated?
http://www.orlfrance.org/ download.php?id=159.

Molecular Diagnosis for Indeterminate Thyroid Nodules on Fine Needle Aspiration
Expert Rev Mol Diagn. 2013;13(6):613-62

Somatic mutation testing, mRNA gene expression platforms, protein immunocytochemistry and miRNA panels have improved the diagnostic accuracy of indeterminate thyroid nodules, and although no test is perfectly accurate, in the authors’ opinion, these methods will most certainly become an important part of the diagnostic tools for clinicians and cytopathologists in the future.

Several point mutations and gene rearrangements have been identified in thyroid cancer. The most common somatic mutation in differentiated thyroid cancer has been studied as a potential tool to enhance the diagnostic accuracy of indeterminate FNA lesions – BRAF. This mutation occurs in papillary, poorly differentiated and anaplastic thyroid cancer and causes a V600E substitution in the BRAF protein, which results in neoplastic progression by aberrant activation of the MAPK pathway. The BRAF V600E mutation, along with RET/PTC rearrangements, are a hallmark of thyroid cancer and a vast majority of indeterminate thyroid nodules harboring either one of these two mutations are malignant on final pathology.

The RAS proto-oncogene encodes three different membrane associated GTP proteins: HRAS, KRAS and NRAS. Mutation of these domains causes increased signal transduction through both the MAPK and the PI3K/AKT pathways. These mutations are highly prevalent in FTC and in the follicular variant of papillary thyroid cancer (40–50%) and seldom detected in the classic variant papillary thyroid cancer (10%). RAS mutations have also been identified in benign FA; however, it is unclear whether RAS-positive FA have a higher chance of progression to cancer.

Recurrence detection in differentiated thyroid cancer patients..
Clinical endocrinology, Vol. 72, No. 4. (10 September 2009), pp. 558-563, doi:10.1111/j.1365-2265.2009.03693.x

There was a correlation between TgAb level and recurrence (p = 0.032).
). Recurrence was found in 37.5% of 24 TgAb+/Tg- patients who showed a gradually increasing tendency in serial measurements of TgAb. Sixteen cervical foci (21.1%) missed on neck USG and 17 lesions (22.4%) located outside the neck were additionally detected with PET/CT in TgAb+ patients.

Solving the mystery of iodine uptake
Science 20 June 2014: Vol. 344 no. 6190 p. 1355 http://dx.doi.org:/10.1126/science.344.6190.1355-a

The cell membrane protein NIS (sodium/iodine symporter) transports iodine into thyroid cells, but because iodine concentrations outside of the cell are so low, how it does so is a mystery. The key? Moving two sodium ions along with the iodine ion, Nicola et al found. NIS also does not bind sodium very tightly, but the high concentrations of sodium outside the cell allow one sodium ion to bind. This binding increases the affinity of NIS for a second sodium ion and also for iodine. With the three ions bound, NIS changes its conformation so that it opens to the inside of the cell, where the sodium concentration is low enough for NIS to release its sodium ions. When the sodium goes away, so does NIS’s affinity for iodine, leading NIS to release it.

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A new relationship identified in preterm stress and development of autism or schizophrenia

Posted in Biological Networks, Gene Regulation and Evolution, Biomarkers & Medical Diagnostics, Cell Biology, Signaling & Cell Circuits, Computational Biology/Systems and Bioinformatics, Curation, Curation methodology, Developmental biology, Discovery process, Enzyme Induction, Experimental validation, Explanatory, Gene Regulation, Genome Biology, Inferential analysis, Innovations in Neurophysiology & Neuropsychology, Placenta, Proteomics, Technology Advance Assessment of, Theoretic convergence, Transcriptomics, Translational Science, tagged androsteinedione, autism spectrum disorder, brain development, Enzyme Induction, Gene Regulation, gestation, Histone, histone modification, Hsd17b3, mitochondrial function, O-linked-N-acetylglucosamine transferase, OGT, pregnancy, Schizophrenia, sex difference, testosterone, Transcriptomics, weaning on July 10, 2014| Leave a Comment »

Larry H Bernstein, MD, FCAP, Reporter and Curator

http://pharmaceyticalinnovation.com/7/10/2014/A new relationship identified in preterm stress and development of autism or schizophrenia/

This is a fascinating study. It is of considerable interest because it deals with several items that need to be addressed with respect to neurodevelopmental disruptive disorders. It leaves open some aspects that are known, but not subject to investigation in the experiments. Then there is also no reporting of some associations that are known at the time of deveopment of these disorders – autism spectrum, and schizophrenia. Of course, I don’t know how it would be possible to also look at prediction of a possible relationship to later development of mood disorders.

The placenta functions as an endocrine organ in the conversion of androsteinedione to testosterone during pregnancy, which is delivered to the fetus.
The conversion is by a known enzymatic pathway – and there is a sex difference in the depression of testosterone in males, females not affected.
There is a greater susceptibility of males to autism and schizophrenia than of females, which I as reader, had not known, but if this is true, it would lend some credence to a biological advantage to protect the females of animal species, and might raise some interest into what relationship it has to protecting multitasking for females.
It is well known that the twin studies that have been carried out determined that in identical twins, there is discordance as a rule. Those studies are old, and they did not examine whether the other identical twin might be anywhere on the autism spectrum disorder (not then termed “spectrum”.
However, there is a clear effect of stress on “gene expression”, and in this case we are looking at enzymation suppression at the placental level affecting trascriptional activity in the male fetus. The same genetic signature exists in the male genetic profile, so we are not looking at a clear somatic mutation in this study.
There is also much less specific an association with the MTHFR gene mutation at either one or two loci. This would have to be looked at as a possible separate post translational somatic mutation.
Whether there is another component expressed later in the function of the zinc metalloproteinase under stress in the affected subject is worth considering, but can’t be commented on with respect to the study.

Penn Team Links Placental Marker of Prenatal Stress to Neurodevelopmental Problems

By Ilene Schneider July 8, 2014

When a woman experiences a stressful event early in pregnancy, the risk that her child will develop autism spectrum disorders or schizophrenia increases. The way in which maternal stress is transmitted to the brain of the developing fetus, leading to these problems in neurodevelopment, is poorly understood.

New findings by University of Pennsylvania School of Veterinary Medicine scientists suggest that an enzyme found in the placenta is likely playing an important role. This enzyme, O-linked-N-acetylglucosamine transferase, or OGT, translates maternal stress into a reprogramming signal for the brain before birth. The study was supported by the National Institute of Mental Health.

“By manipulating this one gene, we were able to recapitulate many aspects of early prenatal stress,” said Tracy L. Bale, senior author on the paper and a professor in the Department of Animal Biology at Penn Vet. “OGT seems to be serving a role as the ‘canary in the coal mine,’ offering a readout of mom’s stress to change the baby’s developing brain. Bale, who also holds an appointment in the Department of Psychiatry, co-authored tha paper with postdoctoral researcher Christopher L. Howerton, for PNAS.

OGT is known to play a role in gene expression through chromatin remodeling, a process that makes some genes more or less available to be converted into proteins. In a study published last year in PNAS, Bale’s lab found that placentas from male mice pups had lower levels of OGT than those from female pups, and placentas from mothers that had been exposed to stress early in gestation had lower overall levels of OGT than placentas from the mothers’ unstressed counterparts.

“People think that the placenta only serves to promote blood flow between a mom and her baby, but that’s really not all it’s doing,” Bale said. “It’s a very dynamic endocrine tissue and it’s sex-specific, and we’ve shown that tampering with it can dramatically affect a baby’s developing brain.”

To elucidate how reduced levels of OGT might be transmitting signals through the placenta to a fetus, Bale and Howerton bred mice that partially or fully lacked OGT in the placenta. They then compared these transgenic mice to animals that had been subjected to mild stressors during early gestation, such as predator odor, unfamiliar objects or unusual noises, during the first week of their pregnancies.

The researchers performed a genome-wide search for genes that were affected by the altered levels of OGT and were also affected by exposure to early prenatal stress using a specific activational histone mark and found a broad swath of common gene expression patterns.

They chose to focus on one particular differentially regulated gene called Hsd17b3, which encodes an enzyme that converts androstenedione, a steroid hormone, to testosterone. The researchers found this gene to be particularly interesting in part because neurodevelopmental disorders such as autism and schizophrenia have strong gender biases, where they either predominantly affect males or present earlier in males.

Placentas associated with male mice pups born to stressed mothers had reduced levels of the enzyme Hsd17b3, and, as a result, had higher levels of androstenedione and lower levels of testosterone than normal mice.

“This could mean that, with early prenatal stress, males have less masculinization,” Bale said. “This is important because autism tends to be thought of as the brain in a hypermasculinized state, and schizophrenia is thought of as a hypomasculinized state. It makes sense that there is something about this process of testosterone synthesis that is being disrupted.”

Furthermore, the mice born to mothers with disrupted OGT looked like the offspring of stressed mothers in other ways. Although they were born at a normal weight, their growth slowed at weaning. Their body weight as adults was 10 to 20 percent lower than control mice.

Because of the key role that that the hypothalamus plays in controlling growth and many other critical survival functions, the Penn Vet researchers then screened the mouse genome for genes with differential expression in the hypothalamus, comparing normal mice, mice with reduced OGT and mice born to stressed mothers.

They identified several gene sets related to the structure and function of mitochrondria, the powerhouses of cells that are responsible for producing energy. And indeed, when compared by an enzymatic assay that examines mitochondria biogenesis, both the mice born to stressed mothers and mice born to mothers with reduced OGT had dramatically reduced mitochondrial function in their hypothalamus compared to normal mice. These studies were done in collaboration with Narayan Avadhani’s lab at Penn Vet. Such reduced function could explain why the growth patterns of mice appeared similar until weaning, at which point energy demands go up.

“If you have a really bad furnace you might be okay if temperatures are mild,” Bale said. “But, if it’s very cold, it can’t meet demand. It could be the same for these mice. If you’re in a litter close to your siblings and mom, you don’t need to produce a lot of heat, but once you wean you have an extra demand for producing heat. They’re just not keeping up.”

Bale points out that mitochondrial dysfunction in the brain has been reported in both schizophrenia and autism patients. In future work, Bale hopes to identify a suite of maternal plasma stress biomarkers that could signal an increased risk of neurodevelopmental disease for the baby.

“With that kind of a signature, we’d have a way to detect at-risk pregnancies and think about ways to intervene much earlier than waiting to look at the term placenta,” she said.

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Update on mitochondrial function, respiration, and associated disorders

Posted in Anaerobic Glycolysis, Ca2+ triggered activation, Ca2+ triggered activation, Cell Biology, Signaling & Cell Circuits, Curation, Discovery process, Disease Biology, Small Molecules in Development of Therapeutic Drugs, Experimental validation, Explanatory, Inferential analysis, Metabolomics, Na-K-ATPase, Nephrology, Neurohumoral Transmission, Oxidative phosphorylation, Phosphorylation, Proteomics, Translational Science, tagged 5HT1F, alpha ketoglutarate, ATP synthase, brown adipose tissue (BAT), C. Elegans, ca-uniporter (MCU), Ca2+-regulation, Cancer - General, CLL, coupled respiration, Glut4 transporter, inner membrane, K-depolarization, mitochondria, mTOR, NADPH, neurons, OCR, Oxidative phosphorylation, pyruvate -rate limiting, ROS, Sestrin2, TOR, UCP1 on July 8, 2014| Leave a Comment »

Larry H. Benstein, MD, FCAP, Gurator and writer

http://pharmaceuticalintelligence.com/7/8/2014/Update on mitochondrial function, respiration, and associated disorders

This is a condensed account of very recent published work on respiration and disturbed mitochondrail function. We know that their is an equilibrium between respiration and autophagy in eukaryotic cells. The Krebs Cycle produces 32 ATPs in oxidative phosphorylation, which is far more efficient than glycolysis. There is also a different contribution of mitochondrial metabolism, in the balance, between tissues that are synthetic and those that are catabolic. This is a subject long understood, essential for cellular energetics, and not adequately explored.

Gain-of-Function Mutant p53 Promotes Cell Growth and Cancer Cell Metabolism via Inhibition of AMPK Activation.

Zhou G¹, Wang J², Zhao M², Xie TX², Tanaka N², et al.
Mol Cell. 2014 Jun 19;54(6):960-974. doi: 10.1016/j.molcel.2014.04.024.

Many mutant p53 proteins (mutp53s) exert oncogenic gain-of-function (GOF) properties, but the mechanisms mediating these functions remain poorly defined.

We show here that GOF mutp53s inhibit AMP-activated protein kinase (AMPK) signaling in head and neck cancer cells.

Conversely, downregulation of GOF mutp53s enhances AMPK activation under energy stress, decreasing the activity of the anabolic factors acetyl-CoA carboxylase and ribosomal protein S6 and inhibiting aerobic glycolytic potential and invasive cell growth.

Under conditions of energy stress, GOF mutp53s, but not wild-type p53, preferentially bind to the AMPKα subunit and inhibit AMPK activation.

Given the importance of AMPK as an energy sensor and tumor suppressor that inhibits anabolic metabolism, our findings reveal that direct inhibition of AMPK activation is an important mechanism through which mutp53s can gain oncogenic function. PMID:24857548

Investigating and Targeting Chronic Lymphocytic Leukemia Metabolism with the HIV Protease Inhibitor Ritonavir and Metformin.

Adekola KU, Aydemir SD, Ma S, Zhou Z, Rosen ST, Shanmugam M.
Leuk Lymphoma. 2014 May 14:1-23.

Chronic Lymphocytic Leukemia (CLL) remains fatal due to the development of resistance to existing therapies. Targeting abnormal glucose metabolism sensitizes various cancer cells to chemotherapy and/or elicits toxicity.

Examination of glucose dependency in CLL demonstrated variable sensitivity to glucose deprivation. Further evaluation of metabolic dependencies of CLL cells resistant to glucose deprivation revealed increased engagement of fatty acid oxidation upon glucose withdrawal.

Investigation of glucose transporter expression in CLL reveals up-regulation of glucose transporter GLUT4. Treatment of CLL cells with HIV protease inhibitor ritonavir, that inhibits GLUT4, elicits toxicity similar to that elicited upon glucose-deprivation.

CLL cells resistant to ritonavir are sensitized by co-treatment with metformin, potentially targeting compensatory mitochondrial complex 1 activity. Ritonavir and metformin have been administered in humans for treatment of diabetes in HIV patients, demonstrating the tolerance of this combination in humans. Our studies strongly substantiate further investigation of FDA approved ritonavir and metformin for CLL.

KEYWORDS: Basic Biology; Chemotherapeutic approaches; Lymphoid Leukemia; Signal transduction PMID: 24828872

Utilizing hydrogen sulfide as a novel anti-cancer agent by targeting cancer glycolysis and pH imbalance.

Lee ZW¹, Teo XY, Tay EY, Tan CH, Hagen T, Moore PK, Deng LW.
Br J Pharmacol. 2014 May 15. doi: 10.1111/bph.12773

Many disparate studies have reported the ambiguous role of hydrogen sulfide (H₂ S) in cell survival. The present study investigated the effect of H₂ S on viability of cancer and non-cancer cells.

Cancer and non-cancer cells were exposed to H₂ S (using sodium hydrosulfide, NaHS and GYY4137) and cell viability was examined by crystal violet assay. We then examined cancer cellular glycolysis process by in vitro enzymatic assays and pH regulator activity. Lastly, intracellular pH (pHi) was determined by ratiometric pHi measurement using BCECF staining.

Continuous, but not single, exposure to H₂ S decreased cell survival more effectively in cancer cells, as compared to non-cancer cells. Slow H₂ S-releasing donor, GYY4137, significantly increased glycolysis leading to overproduction of lactate. H₂ S also decreased anion exchanger and sodium/proton exchanger activity. The combination of increased metabolic acid production and defective pH regulation resulted in an uncontrolled intracellular acidification leading to cancer cell death. In contrast, no significant intracellular acidification or cell death was observed in non-cancer cells.

Low and continuous exposure to H₂ S targets metabolic processes and pH homeostasis in cancer cells, potentially serving as a novel and selective anti-cancer strategy.

KEYWORDS: cancer cell death; cancer glucose metabolism; hydrogen sulfide; pH homeostasis PMID: 24827113

Agonism of the 5-Hydroxytryptamine 1F Receptor Promotes Mitochondrial Biogenesis and Recovery from Acute Kidney Injury

Garrett SM, Whitaker RM, Beeson CC, and Schnellmann RG

Center for Cell Death, Injury, and Regeneration, Department of Drug Discovery and Biomedical Sciences, Medical University of South Carolina, Charleston, South Carolina (S.M.G., R.M.W., C.C.B., R.G.S.); and Ralph H. Johnson Veterans Affairs Medical Center, Charleston, South Carolina (R.G.S.)
Address correspondence to: Dr. Rick G. Schnellmann, Department of Drug Discovery and Biomedical Sciences, MUSC, Charleston, SC 29425.
E-mail: schnell@musc.edu

Many acute and chronic conditions, such as acute kidney injury, chronic kidney disease, heart failure, and liver disease, involve mitochondrial dysfunction. Although we have provided evidence that drug-induced stimulation of mitochondrial biogenesis (MB) accelerates mitochondrial and cellular repair, leading to recovery of organ function, only a limited number of chemicals have been identified that induce MB.

The goal of this study was to assess the role of the 5-hydroxytryptamine 1F (5-HT_1F) receptor in MB. Immunoblot and quantitative polymerase chain reaction analyses revealed 5-HT_1F receptor expression in renal proximal tubule cells (RPTC). A MB screening assay demonstrated that two selective 5-HT_1F receptor agonists,

LY334370 (4-fluoro-N-[3-(1-methyl-4-piperidinyl)-1H-indol-5-yl]benzamide) and
LY344864 (N-[(3R)-3-(dimethylamino)-2,3,4,9-tetrahydro-1H-carbazol-6-yl]-4-fluorobenzamide; 1–100 nM)

increased carbonylcyanide-p-trifluoromethoxyphenylhydrazone–uncoupled oxygen consumption in RPTC, and

validation studies confirmed both agonists increased mitochondrial proteins in vitro.
[e.g., ATP synthase β, cytochrome c oxidase 1 (Cox1), and NADH dehydrogenase (ubiquinone) 1β subcomplex subunit 8 (NDUFB8)]

Small interfering RNA knockdown of the 5-HT_1F receptor

blocked agonist-induced MB.

Furthermore, LY344864 increased

peroxisome proliferator–activated receptor (PPAR) coactivator 1-α, Cox1, and
NDUFB8 transcript levels and
mitochondrial DNA (mtDNA) copy number

in murine renal cortex, heart, and liver.

Finally, LY344864 accelerated recovery of renal function, as indicated by

decreased blood urea nitrogen and kidney injury molecule 1 and
increased mtDNA copy number

following ischemia/reperfusion-induced acute kidney injury (AKI).

In summary, these studies reveal that

the 5-HT_1F receptor is linked to MB, 5-HT_1F receptor agonism promotes MB in vitro and in vivo, and

5-HT_1F receptor agonism promotes recovery from AKI injury.

Induction of MB through 5-HT_1F receptor agonism represents a new target and approach to treat mitochondrial organ dysfunction.

Footnotes

Portions of this work have been presented previously: Garrett SM, Wills LP, and Schnellmann RG (2012) Serotonin (5-HT) 1F receptor agonism as a potential treatment for acceleration of recovery from acute kidney injury.American Society of Nephrology Annual Meeting; 2012 Nov 1–4; San Diego, CA.
dx.doi.org/10.1124/jpet.114.214700.

Ca2+ regulation of mitochondrial function in neurons.

Rueda CB¹, Llorente-Folch I¹, Amigo I¹, Contreras L¹, González-Sánchez P¹, Martínez-Valero P¹, Juaristi I¹, Pardo B¹, Del Arco A², Satrústegui J³

Biochim Biophys Acta. 2014 May 10. pii: S0005-2728(14)00126-1.
doi: 10.1016/j.bbabio.2014.04.010.

Calcium is thought to regulate respiration but it is unclear whether this is dependent on the increase in ATP demand caused by any Ca²⁺ signal or to Ca²⁺ itself.

[Na⁺]_i, [Ca²⁺]_i and [ATP]_i dynamics in intact neurons exposed to different workloads in the absence and presence of Ca²⁺ clearly showed that

Ca²⁺-stimulation of coupled respiration is required to maintain [ATP]_i levels.

Ca²⁺ may regulate respiration by

activating metabolite transport in mitochondria from outer face of the inner mitochondrial membrane, or
after Ca²⁺ entry in mitochondria through the calcium uniporter (MCU).

Two Ca²⁺-regulated mitochondrial metabolite transporters are expressed in neurons,

the aspartate-glutamate exchanger ARALAR/AGC1/Slc25a12, a component of the malate-aspartate shuttle, with a Kd for Ca²⁺ activation of 300nM, and
the ATP-Mg/Pi exchanger SCaMC-3/Slc25a23, with S_0.5 for Ca²⁺ of 300nM and 3.4μM, respectively.

The lack of SCaMC-3 results in a smaller Ca²⁺-dependent stimulation of respiration only at high workloads, as caused by veratridine, whereas

the lack of ARALAR reduced by 46% basal OCR in intact neurons using glucose as energy source and the Ca²⁺-dependent responses to all workloads (veratridine, K⁺-depolarization, carbachol).

The lack of ARALAR caused a reduction of about 65-70% in the response to the high workload imposed by veratridine, and

completely suppressed the OCR responses to moderate (K⁺-depolarization) and small (carbachol) workloads,
effects reverted by pyruvate supply.

For K⁺-depolarization, this occurs in spite of the presence of large [Ca²⁺]_mit signals and increased reduction of mitochondrial NAD(P)H.

These results show that ARALAR-MAS is a major contributor of Ca²⁺-stimulated respiration in neurons

by providing increased pyruvate supply to mitochondria.

In its absence and under moderate workloads, matrix Ca²⁺ is unable to stimulate pyruvate metabolism and entry in mitochondria suggesting a limited role of MCU in these conditions.

KEYWORDS: ATP-Mg/Pi transporter; Aspartate–glutamate transporter; Calcium; Calcium-regulated transport; Mitochondrion; Neuronal respiration PMID: 24820519

Sestrin2 inhibits uncoupling protein 1 expression through suppressing reactive oxygen species.

Ro SH¹, Nam M², Jang I¹, Park HW¹, Park H¹, Semple IA¹, Kim M¹, et al.
Proc Natl Acad Sci U S A. 2014 May 27;111(21):7849-54.
doi: 10.1073/pnas.1401787111.

Uncoupling protein 1 (Ucp1), which is localized in the mitochondrial inner membrane of mammalian brown adipose tissue (BAT), generates heat by uncoupling oxidative phosphorylation. Upon cold exposure or nutritional abundance, sympathetic neurons stimulate BAT to express Ucp1 to induce energy dissipation and thermogenesis. Accordingly, increased Ucp1 expression reduces obesity in mice and is correlated with leanness in humans.

Despite this significance, there is currently a limited understanding of how Ucp1 expression is physiologically regulated at the molecular level. Here, we describe the involvement of Sestrin2 and reactive oxygen species (ROS) in regulation of Ucp1 expression. Transgenic overexpression of Sestrin2 in adipose tissues inhibited both basal and cold-induced Ucp1 expression in interscapular BAT, culminating in decreased thermogenesis and increased fat accumulation.

Endogenous Sestrin2 is also important for suppressing Ucp1 expression because BAT from Sestrin2(-/-) mice exhibited a highly elevated level of Ucp1 expression. The redox-inactive mutant of Sestrin2 was incapable of regulating Ucp1 expression, suggesting that Sestrin2 inhibits Ucp1 expression primarily through reducing ROS accumulation.

Consistently, ROS-suppressing antioxidant chemicals, such as butylated hydroxyanisole and N-acetylcysteine, inhibited cold- or cAMP-induced Ucp1 expression as well. p38 MAPK, a signaling mediator required for cAMP-induced Ucp1 expression, was inhibited by either Sestrin2 overexpression or antioxidant treatments.

Taken together, these results suggest that Sestrin2 and antioxidants inhibit Ucp1 expression through suppressing ROS-mediated p38 MAPK activation, implying a critical role of ROS in proper BAT metabolism.

KEYWORDS: aging; homeostasis; mouse; β-adrenergic signaling PMID: 24825887 PMCID: PMC4040599

Mitochondrial EF4 links respiratory dysfunction and cytoplasmic translation in Caenorhabditis elegans.

Yang F¹, Gao Y¹, Li Z², Chen L³, Xia Z⁴, Xu T⁵, Qin Y⁶Biochim Biophys Acta. 2014 May 15. pii: S0005-2728(14)00499-X.
doi: 10.1016/j.bbabio.2014.05.353.

How animals coordinate cellular bioenergetics in response to stress conditions is an essential question related to aging, obesity and cancer. Elongation factor 4 (EF4/LEPA) is a highly conserved protein that promotes protein synthesis under stress conditions, whereas its function in metazoans remains unknown.

Here, we show that, in Caenorhabditis elegans, the mitochondria-localized CeEF4 (referred to as mtEF4) affects mitochondrial functions, especially at low temperature (15°C).

At worms’ optimum growing temperature (20°C), mtef4 deletion leads to self-brood size reduction, growth delay and mitochondrial dysfunction.

Transcriptomic analyses show that mtef4 deletion induces retrograde pathways, including mitochondrial biogenesis and cytoplasmic translation reorganization.

At low temperature (15°C), mtef4 deletion reduces mitochondrial translation and disrupts the assembly of respiratory chain supercomplexes containing complex IV.

These observations are indicative of the important roles of mtEF4 in mitochondrial functions and adaptation to stressful conditions.

KEYWORDS: C. elegans; EF4(LepA/GUF1); Mitochondrial dysfunction; Retrograde pathways; Translation PMID: 24837196

The metabolite α-ketoglutarate extends lifespan by inhibiting ATP synthase and TOR.

Chin RM¹, Fu X², Pai MY³, Vergnes L⁴, Hwang H⁵, Deng G⁶, Diep S², et al.
Nature 2014 Jun 19;509(7505):397-401. doi: 10.1038/nature13264.

Metabolism and ageing are intimately linked. Compared with ad libitum feeding, dietary restriction consistently extends lifespan and delays age-related diseases in evolutionarily diverse organisms. Similar conditions of nutrient limitation and genetic or pharmacological perturbations of nutrient or energy metabolism also have longevity benefits.

Recently, several metabolites have been identified that modulate ageing; however, the molecular mechanisms underlying this are largely undefined. Here we show that α-ketoglutarate (α-KG), a tricarboxylic acid cycle intermediate, extends the lifespan of adult Caenorhabditis elegans.

ATP synthase subunit β is identified as a novel binding protein of α-KG using a small-molecule target identification strategy termed drug affinity responsive target stability (DARTS). The ATP synthase, also known as complex V of the mitochondrial electron transport chain, is the main cellular energy-generating machinery and is highly conserved throughout evolution.

Although complete loss of mitochondrial function is detrimental, partial suppression of the electron transport chain has been shown to extend C. elegans lifespan.

We show that α-KG inhibits ATP synthase and, similar to ATP synthase knockdown, inhibition by α-KG leads to reduced ATP content, decreased oxygen consumption, and increased autophagy in both C. elegans and mammalian cells.

We provide evidence that the lifespan increase by α-KG requires ATP synthase subunit β and is dependent on target of rapamycin (TOR) downstream.

Endogenous α-KG levels are increased on starvation and α-KG does not extend the lifespan of dietary-restricted animals, indicating that α-KG is a key metabolite that mediates longevity by dietary restriction.

Our analyses uncover new molecular links between a common metabolite, a universal cellular energy generator and dietary restriction in the regulation of organismal lifespan, thus suggesting new strategies for the prevention and treatment of ageing and age-related diseases.

PMID: 24828042

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Omega-3 fatty acids, depleting the source, and protein insufficiency in renal disease

Posted in Affordable Care Act, Cerebrovascular and Neurodegenerative Diseases, Chemical Biology and its relations to Metabolic Disease, Curation, Discovery process, Evolutionary cognition, Explanatory, Health Economics and Outcomes Research, Health Law & Patient Safety, Indigent Nutrition, Innovations in Neurophysiology & Neuropsychology, Liver & Digestive Diseases Research, Medicare and Medicaid, Metabolomics, Nephrology, Neurodegenerative Diseases, Nutrigenomics, Nutrition, Nutritional Supplements: Atherogenesis, lipid metabolism, Population Health Management, Nutrition and Phytochemistry, Proteomics, Skilled Nursing Facilities, Uninsured and Underinsured, tagged fish, memory loss, muscle wasting, omega-3 PUFA, sarcopenia, stage 3 & 4 renal disease on July 6, 2014| Leave a Comment »

Larry H. Bernstein, MD, FCAP, Curator

http://pharmaceuticalinnovation/6/7/2014/Omega-3 fatty acids, depleting the source, and protein insufficiency in renal disease

This article is concerned only with updating the importance of key nutrients for maintenance of health. Nutritional losses are associated with memory loss, impaired immunity, and loss of lean body mass.

Low levels of omega-3 fatty acids may cause memory problems

Disease and Conditions, General Diet • Tags: Alzheimer’s disease, American Academy of Neurology, Docosahexaenoic acid, Magnetic resonance imaging, Neurology, Omega-3 fatty acid, United States Environmental Protection Agency, University of California Los Angeles

09 Mar 2012

ST. PAUL, Minn. – A diet lacking in omega-3 fatty acids, nutrients commonly found in fish, may cause your brain to age faster and lose some of its memory and thinking abilities, according to a study published in the February 28, 2012, print issue of Neurology®, the medical journal of the American Academy of Neurology. Omega-3 fatty acids include the nutrients called docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA).

salmon dinner

“People with lower blood levels of omega-3 fatty acids had lower brain volumes that were equivalent to about two years of structural brain aging,” said study author Zaldy S. Tan, MD, MPH, of the Easton Center for Alzheimer’s Disease Research and the Division of Geriatrics, University of California at Los Angeles.

For the study, 1,575 people with an average age of 67 and free of dementia underwent MRI brain scans. They were also given tests that measured mental function, body mass and the omega-3 fatty acid levels in their red blood cells.

The researchers found that people whose DHA levels were among the bottom 25 percent of the participants had lower brain volume compared to people who had higher DHA levels. Similarly, participants with levels of all omega-3 fatty acids in the bottom 25 percent also scored lower on tests of visual memory and executive function, such as problem solving and multi-tasking and abstract thinking.

Potentially Helpful Omega-3 Fatty Acids Can Cross to Brain: Study(news.health.com)
Omega 3 FA (wedishnutrition.wordpress.com)
Omega Fatty Acids: The Science Behind Fish Oil- Guest post from Beauty by the Geeks (antisensescienceblog.wordpress.com)
Omega 3 Fatty Acids Slow Brain Aging? (futurepundit.com)
Why is Omega-3 Essential to the Human Body?(horningchiropracticcenter.wordpress.com)
Proof that omega-3 supplements can reach the brain(nyrnaturalnews.com)
Low Levels of Omega-3 Fatty Acids May Cause Memory Problems(feedproxy.google.com)

Mechanisms of muscle wasting in chronic kidney disease.

Xiaonan H Wang, William E Mitch

Nature Reviews Nephrology (Impact Factor: 7.94). 07/2014; DOI: 10.1038/nrneph.2014.112

Source: PubMed

ABSTRACT In patients with chronic kidney disease (CKD), loss of cellular proteins increases the risks of morbidity and mortality. Persistence of muscle protein catabolism in CKD results in striking losses of muscle proteins as whole-body protein turnover is great; even small but persistent imbalances between protein synthesis and degradation cause substantial protein loss. No reliable methods to prevent CKD-induced muscle wasting currently exist, but mechanisms that control cellular protein turnover have been identified, suggesting that therapeutic strategies will be developed to suppress or block protein loss. Catabolic pathways that cause protein wasting include activation of the ubiquitin-proteasome system (UPS), caspase-3, lysosomes and myostatin (a negative regulator of skeletal muscle growth). These pathways can be initiated by complications associated with CKD, such as metabolic acidosis, defective insulin signalling, inflammation, increased angiotensin II levels, abnormal appetite regulation and impaired microRNA responses. Inflammation stimulates cellular signalling pathways that activate myostatin, which accelerates UPS-mediated catabolism. Blocking this pathway can prevent loss of muscle proteins. Myostatin inhibition could yield new therapeutic directions for blocking muscle protein wasting in CKD or disorders associated with its complications.

We’re Fishing the Oceans Dry. It’s Time to Reconsider Fish Farms.

Food and Agriculture Organization of the United Nations -State of World Fisheries and Aquaculture 2014

Aquaculture has gotten much greener, with American innovators leading the way.

— Text by Maddie Oatman; video by Brett Brownell

| Wed Jul. 2, 2014 6:00 AM EDT MotherJones.com

When I meet Kenny Belov mid-morning at San Francisco’s Fisherman’s Wharf, the boats that would normally be out at sea chasing salmon sit tethered to their docks. The steady breeze coursing through the bay belies choppier conditions farther out—so rough that the local fishermen threw in the towel for the fifth morning in a row. Belov scans the horizon as he explains this, feet away from the warehouse of his sustainable seafood company, TwoXSea. Because his business hinges on what local fishermen can bring in, he’s used to coping with wild fish shortages.

If we continue to fish at the current pace, some scientists predict we’ll be facing oceans devoid of edible marine creatures by 2050.

But unlike these fishermen, Belov has a stash of treasure in his warehouse, as he soon shows me: a golf-cart-size container of plump trout, their glossy bodies still taut from rigor mortis. The night before, Belov drove north to Humboldt to help “chill kill” the fish by submerging them live into barrels of slushy ice water. Belov can count on shipments of these McFarland Springs trout every week—because he helped grow them himself on a farm.

For many consumers, aquaculture lost its appeal after unappetizing news spread about commercial fish farms—like fish feed’s pressure on wild resources, overflowing waste, toxic buildup in the water, and displacement of natural species. But consider this: Our appetite for seafood continues to rise. Globally, we’ve hungered for 3.2 percent more seafood every year for the last five decades, double the rate of our population. Yet more than four-fifths of the world’s wild fisheries are overexploited or fully exploited (yielding the most fish possible with no expected room for growth). Only 3 percent of stocks are considered underexploited—meaning they have any significant room for expansion. If we continue to fish at the current pace, some scientists predict we’ll be facing oceans devoid of edible marine creatures by 2050.

Aquaculture could come to the rescue. The Food and Agriculture Organization of the United Nations predicts that farmed fish will soon surpass wild-caught; by 2030, aquaculture may produce more than 60 percent of fish we consume as food.

Food and Agriculture Organization of the United Nations “State of World Fisheries and Aquaculture” 2014 report

One of the most pressing concerns about aquaculture, though, is that many farmed fish are raised on a diet of 15 million tons a year of smaller bait fish—species like anchovies and menhaden. These bait—also known as forage fish—are ground up and converted into a substance called fishmeal. It takes roughly five pounds of them to produce one pound of farmed salmon. Bait fish are also used for nonfood products like pet food, makeup, farm animal feed, and fish oil supplements.

Forage fish are a “finite resource that’s been fully utilized.”

It may appear as though the ocean enjoys endless schools of these tiny fish, but they too have been mismanaged, and their populations are prone to collapse. They’re a “finite resource that’s been fully utilized,” says Mike Rust of NOAA’s fisheries arm. Which is disturbing, considering that researchers like those at Oceana argue that forage fish may play an outsize role in maintaining the ocean’s ecological balance, including by contributing to the abundance of bigger predatory fish.

And that’s where Belov’s trout come in: Though he swears no one can taste the difference, his fish are vegetarians. That means those five pounds of forage fish can rest easy at sea. It also means that the trout don’t consume some of the other rendered animal proteins in normal fishmeal pellets: bone meal, feather meal, blood meal, and chicken byproducts.

Belov and McFarland Springs’ owner David McFarland were inspired to switch to vegetarian feed in part by Rick Barrows, a USDA researcher. About six years ago, recounts Barrows, several USDA studies confirmed that fish rely on nutrients—vitamins, minerals, fatty acids, and protein—rather than fishmeal or fish oil, to thrive. If those nutrients could be found in other products, including purely plant-based substances, then aquaculture might not be so dependent on feeding fish other smaller fish.

Barrows and team began to test about 50 potential materials a year, and now have a database of 140 that anyone can browse through online. Belov was one of their first commercial partners. The plant-based food fed to McFarland Springs’ trout consists of a hearty blend of marine algae, freshwater micro algae, vitamins, minerals, flax, flax oil, corn, and nut waste. The resulting complete protein means the trout’s omega 3s are high and their omega 6s are low—a ratio that’s said to enhance anti-inflammatory properties. And “they don’t have the concentration of heavy metals that come from the bait fish,” Belov says. I took one of his rosy fillets home and turned it into trout lox; find the recipe here.

McFarland Springs manages the trout’s waste by funneling it out into a natural sagebrush pasture where it composts the soil.

Belov’s fish feed includes California nuts that are too broken or disfigured to be sold.

Barrows thinks region-specific material for this type of feed offers the most potential. For instance, his team learned that around 5 percent of California nuts can’t be sold because they’re broken or disfigured. They realized they could repurpose excess nut parts for the trout feed; the nut bits helped round out the complete protein. Lately, Barrows has become especially excited about turning barley surplus from the beer industry—which comes at a cheap price in Montana, where he’s based—into a feed-grade concentrate for trout feed.

“You can get just as much growth rate out of fishmeal-free feeds as fishmeal,” says Barrows. And his lab has proven as much with eight different fish species: cobia, Florida pompano, coho salmon, Atlantic salmon, walleye, yellowtail, and White seabass.

But the price difference still stands in the way for many fish farmers. Belov pays slightly more than $1/pound for his plant-based feed, whereas fishmeal pellets average around $0.71/pound. He sells his trout for $6.95/pound, about a dollar more than conventional. But he’s well positioned in the affluent Bay Area, and he usually sells out of his McFarland Springs trout well before the end of each week. As innovation continues in the realm of plant-based feeds, he’s hopeful, along with Barrows, that the price of the pellets will continue to drop.

Here in the United States, we consume plenty of farmed fish already, but only 5 percent of it is sourced domestically. “If we didn’t import so much farmed seafood,” implored Four Fish author Paul Greenberg in a recent New York Times op-ed, “we might develop a viable, sustainable aquaculture sector of our own.” It doesn’t just boil down to economics: The locations we generally export from, like China and South Asia, don’t have near the stringent environmental and health regulations as the US. “Growing more seafood at home would help with trade deficit, but also we could control the safety more,” says Barrows.

Though our current aquaculture sector is relatively tiny, US farmers are in a better position to innovate, because we have a sophisticated animal nutrition research center and feed sector, says NOAA’s Rust. “We’re the leading technical country in the world on feed.”

Belov wasn’t always open to aquaculture, and he still feels that fish—such as some salmon—with healthy wild fisheries attached to them should never be farmed. That way, environmentally responsible fishermen can stay in business. His long-term strategy for sustainable seafood? Draw from the “amazing [wild] fisheries that exist, and then you backfill with intelligent aquaculture, and yes, you can feed the planet with sustainable marine products.” Which may take more work, but as he puts it, “We depleted the ocean. It wasn’t anybody else’s fault. So it’s our job to fix it.”

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Archive for the ‘Explanatory’ Category

Signaling and Signaling Pathways

Signaling and Signaling Pathways

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Pathology Emergence in the 21st Century

Pathology Emergence in the 21st Century

“Our findings strengthen the case

GEN News 8-1-2014

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Commentary on Biomarkers for Genetics and Genomics of Cardiovascular Disease: Views by Larry H Bernstein, MD, FCAP

Commentary on Biomarkers for Genetics and Genomics of Cardiovascular Disease: : Views by Larry H Bernstein, MD, FCAP

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Malnutrition in India, High Newborn Death Rate and Stunting of Children Age Under Five Years

Malnutrition in India, High Newborn Death Rate and Stunting of Children Age Under Five Years

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The Experience of a Patient with Thyroid Cancer

The Experience of a Patient with Thyroid Cancer

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A new relationship identified in preterm stress and development of autism or schizophrenia

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Update on mitochondrial function, respiration, and associated disorders

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Omega-3 fatty acids, depleting the source, and protein insufficiency in renal disease

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Archive for the ‘Explanatory’ Category

Signaling and Signaling Pathways

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Pathology Emergence in the 21st Century

“Our findings strengthen the case

GEN News 8-1-2014

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Scientific Curation Fostering Expert Networks and Open Innovation: Lessons from Clive Thompson

Our ideas are PRODUCTS OF OUR ENVIRONMENT.

They are influenced by the conversations around us.

But HOW MUCH Knowledge is OUT THERE?

The Good, The Bad, and the Ugly of the Scientific Internet (The Wild West?)

So granted, there is a lot of

….. when it comes to scientific information on the internet!

Using Curation and Science 2.0 to build Trusted, Expert Networks of Scientists and Clinicians

In Summary: How Scientific Content Curation Can Help

CURATION MAY OFFER SOLUTIONS

Methodology in Scientific Content Curation as Envisioned by Aviva lev-Ari, PhD, RN

Cardiovascular Original Research: Cases in Methodology Design for Content Curation and Co-Curation

which has created a need for more context-driven scientific search and discourse.

INCREASE THE SCIENTIFIC CURRENCY.

Creating Content Curation Communities: Breaking Down the Silos!

Open Science Case Studies in Curation

How the Internet of Things is Promoting the Curation Effort

But this effort needs money

Future postings on the relevance and application of scientific curation will include:

Using Scientific Content Curation as a Method for Validation and Biocuration

Using Scientific Content Curation as a Method for Open Innovation

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Commentary on Biomarkers for Genetics and Genomics of Cardiovascular Disease: : Views by Larry H Bernstein, MD, FCAP

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Malnutrition in India, High Newborn Death Rate and Stunting of Children Age Under Five Years

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The Experience of a Patient with Thyroid Cancer

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