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The Role of Exosomes in Metabolic Regulation

Author: Larry H. Bernstein, MD, FCAP

 

On 9/25/2017, Aviva Lev-Ari, PhD, RN commissioned Dr. Larry H. Bernstein to write a short article on the following topic reported on 9/22/2017 in sciencemission.com

 

We are publishing, below the new article created by Larry H. Bernstein, MD, FCAP.

 

Background

During the period between 9/2015  and 6/2017 the Team at Leaders in Pharmaceutical Business Intelligence (LPBI)  has launched an R&D effort lead by Aviva Lev-Ari, PhD, RN in conjunction with SBH Sciences, Inc. headed by Dr. Raphael Nir.

This effort, also known as, “DrugDiscovery @LPBI Group”  has yielded several publications on EXOSOMES on this Open Access Online Scientific Journal. Among them are included the following:

 

QIAGEN – International Leader in NGS and RNA Sequencing, 10/08/2017

Reporter: Aviva Lev-Ari, PhD, RN

 

cell-free DNA (cfDNA) tests could become the ultimate “Molecular Stethoscope” that opens up a whole new way of practicing Medicine, 09/08/2017

Reporter: Aviva Lev-Ari, PhD, RN

 

Detecting Multiple Types of Cancer With a Single Blood Test (Human Exomes Galore), 07/02/2017

Reporter and Curator: Irina Robu, PhD

 

Exosomes: Natural Carriers for siRNA Delivery, 04/24/2017

Reporter: Aviva Lev-Ari, PhD, RN

 

One blood sample can be tested for a comprehensive array of cancer cell biomarkers: R&D at WPI, 01/05/2017

Curator: Marzan Khan, B.Sc

 

SBI’s Exosome Research Technologies, 12/29/2016

Reporter: Aviva Lev-Ari, PhD, RN

 

A novel 5-gene pancreatic adenocarcinoma classifier: Meta-analysis of transcriptome data – Clinical Genomics Research @BIDMC, 12/28/2016

Curator: Tilda Barliya, PhD

 

Liquid Biopsy Chip detects an array of metastatic cancer cell markers in blood – R&D @Worcester Polytechnic Institute, Micro and Nanotechnology Lab, 12/28/2016

Reporters: Tilda Barliya, PhD and Aviva Lev-Ari, PhD, RN

 

Exosomes – History and Promise, 04/28/2016

Reporter: Aviva Lev-Ari, PhD, RN

 

Exosomes, 11/17/2015

Curator: Larry H. Bernstein, MD, FCAP

 

Liquid Biopsy Assay May Predict Drug Resistance, 11/16/2015

Curator: Larry H. Bernstein, MD, FCAP

 

Glypican-1 identifies cancer exosomes, 10/31/2015

Curator: Larry H. Bernstein, MD, FCAP

 

Circulating Biomarkers World Congress, March 23-24, 2015, Boston: Exosomes, Microvesicles, Circulating DNA, Circulating RNA, Circulating Tumor Cells, Sample Preparation, 03/24/2015

Reporter: Aviva Lev-Ari, PhD, RN

 

Cambridge Healthtech Institute’s Second Annual Exosomes and Microvesicles as Biomarkers and Diagnostics Conference, March 16-17, 2015 in Cambridge, MA, 03/17, 2015

Reporter: Aviva Lev-Ari, PhD, RN

 

The newly created think-piece on the relationship between regulatory functions of Exosomes and Metabolic processes is developed conceptually, below.

 

The Role of Exosomes in Metabolic Regulation

Author: Larry H. Bernstein, MD, FCAP

We have had more than a half century of research into the genetic code and transcription leading to abundant work on RNA and proteomics. However, more recent work in the last two decades has identified RNA interference in siRNA. These molecules may be found in the circulation, but it has been a challenge to find their use in therapeutics. Exosomes were first discovered in the 1980s, but only recently there has been a huge amount of research into their origin, structure and function. Exosomes are 30–120 nm endocytic membrane-bound extracellular vesicles (EVs)(1-23) , and more specifically multiple vesicle bodies (MVBs) by a budding process from invagination of the outer cell membrane that carry microRNA (miRNA), and have structures composed of protein and lipids (1,23-27 ). EVs are the membrane vesicles secreted by eukaryotic cells for intracellular communication by transferring the proteins, lipids, and RNA under various physiologic conditions as well as during the disease stage. EVs also act as a signalosomes in many biological processes. Inward budding of the plasma membrane forms small vesicles that fuse. Intraluminal vesicles (ILVs) are formed by invagination of the limiting endosomal membrane during the maturation process of early endosome.

EVs are the MVBs secreted that serve in intracellular communication by transferring a cargo consisting of proteins, lipids, and RNA under various physiologic conditions (4, 23). Exosome-mediated miRNA transfer between cells is considered to be necessary for intercellular signaling and exosome-associated miRNAs in biofluids (23). Exosomes carry various molecular constituents of their cell of origin, including proteins, lipids, mRNAs, and microRNAs (miRNAs) (. They are released from many cell types, such as dendritic cells (DCs), lymphocytes, platelets, mast cells, epithelial cells, endothelial cells, and neurons, and can be found in most bodily fluids including blood, urine, saliva, amniotic fluid, breast milk, hydrothoracic fluid, and ascitic fluid, as well as in culture medium of most cell types.Exosomes have also been shown to be involved in noncoding RNA surveillance machinery in generating antibody diversity (24). There are also a vast number of long non-coding RNAs (lncRNAs) and enhancer RNAs (eRNAs) that accumulate R-loop structures upon RNA exosome ablation, thereby, resolving deleterious DNA/RNA hybrids arising from active enhancers and distal divergent eRNA-expressing elements (lncRNA-CSR) engaged in long-range DNA interactions (25). RNA exosomes are large multimeric 3′-5′ exo- and endonucleases representing the central RNA 3′-end processing factor and are implicated in processing, quality control, and turnover of both coding and noncoding RNAs. They are large macromolecular cages that channel RNA to the ribonuclease sites (29). A major interest has been developed to characterize of exosomal cargo, which includes numerous non-randomly packed proteins and nucleic acids (1). Moreover, exosomes play an active role in tumorigenesis, metastasis, and response to therapy through the transfer of oncogenes and onco-miRNAs between cancer cells and the tumor stroma. Blood cells and the vascular endothelium is also exosomal shedding, which has significance for cardiovascular,   neurologicological disorders, stroke, and antiphospholipid syndrome (1). Dysregulation of microRNAs and the affected pathways is seen in numerous pathologies their expression can reflect molecular processes of tumor onset and progression qualifying microRNAs as potential diagnostic and prognostic biomarkers (30).

Exosomes are secreted by many cells like B lymphocytes and dendritic cells of hematopoietic and non-hematopoietic origin viz. platelets, Schwann cells, neurons, mast cells, cytotoxic T cells, oligodendrocytes, intestinal epithelial cells were also found to be releasing exosomes (4). They are engaged in complex functions like persuading immune response as the exosomes secreted by antigen presenting cells activate T cells (4). They all have a common set of proteins e.g. Rab family of GTPases, Alix and ESCRT (required for transport) protein and they maintain their cytoskeleton dynamics and participate in membrane fusion. However, they are involved in retrovirus disease pathology as a result of recruitment of the host`s endosomal compartments in order to generate viral vesicles, and they can either spread or limit an infection based on the type of pathogen and its target cells (5).

Upon further consideration, it is understandable how this growing biological work on exosomes has enormous significance for laboratory diagnostics (1, 3, 5, 6, 11, 14, 15, 17-20, 23,30-41) . They are released from many cell types, such as dendritic cells (DCs), lymphocytes, platelets, mast cells, epithelial cells, endothelial cells, and neurons, and can be found in most bodily fluids including blood, urine, saliva, amniotic fluid, breast milk, thoracic and abdominal effusions, and ascitic fluid (1). The involvement of exosomes in disease is broad, and includes: cancer, autoimmune and infectious disease, hematologic disorders, neurodegenerative diseases, and cardiovascular disease. Proteins frequently identified in exosomes include membrane transporters and fusion proteins (e.g., GTPases, annexins, and flotillin), heat shock proteins (e.g., HSC70), tetraspanins (e.g., CD9, CD63, and CD81), MVB biogenesis proteins (e.g., alix and TSG101), and lipid-related proteins and phospholipases. The exosomal lipid composition has been thoroughly analyzed in exosomes secreted from several cell types including DCs and mast cells, reticulocytes, and B-lymphocytes (1). Dysregulation of microRNAs of pathways observed in numerous pathologies (5, 10, 12, 21, 27, 35, 37) including cancers (30), particularly, colon, pancreas, breast, liver, brain, lung (2, 6, 17-20, 30, 33-36, 38, 39). Following these considerations, it is important that we characterize the content of exosomal cargo to gain clues to their biogenesis, targeting, and cellular effects which may lead to identification of biomarkers for disease diagnosis, prognosis and response to treatment (42).

We might continue in pursuit of a particular noteworthy exosome, the NLRP3 inflammasome, which is activated by a variety of external or host-derived stimuli, thereby, initiating an inflammatory response through caspase-1 activation, resulting in inflammatory cytokine IL-1b maturation and secretion (43).
Inflammasomes are multi-protein signaling complexes that activate the inflammatory caspases and the maturation of interleukin-1b. The NLRP3 inflammasome is linked with human autoinflammatory and autoimmune diseases (44). This makes the NLRP3 inflammasome a promising target for anti-inflammatory therapies. The NLRP3 inflammasome is activated in response to a variety of signals that indicate tissue damage, metabolic stress, and infection (45). Upon activation, the NLRP3 inflammasome serves as a platform for activation of the cysteine protease caspase-1, which leads to the processing and secretion of the proinflammatory cytokines interleukin-1β (IL-1β) and IL-18. Heritable and acquired inflammatory diseases are both characterized by dysregulation of NLRP3 inflammasome activation (45).
Receptors of innate immunity recognize conserved moieties associated with either cellular damage [danger-associated molecular patterns (DAMPs)] or invading organisms [pathogen-associated molecular patterns (PAMPs)](45). Either chronic stimulation or overwhelming tissue damage is injurious and responsible for the pathology seen in a number of autoinflammatory and autoimmune disorders, such as arthritis and diabetes. The nucleotide-binding domain leucine-rich repeat (LRR)-containing receptors (NLRs) are PRRs are found intracellularly and they share a unique domain architecture. It consists of a central nucleotide binding and oligomerization domain called the NACHT domain that is located between an N-terminal effector domain and a C-terminal LRR domain (45). The NLR family members NLRP1, NLRP3, and NLRC4 are capable of forming multiprotein complexes called inflammasomes when activated.

The (NLRP3) inflammasome is important in chronic airway diseases such as asthma and chronic obstructive pulmonary disease because the activation results, in pro-IL-1β processing and the secretion of the proinflammatory cytokine IL-1β (46). It has been proposed that Activation of the NLRP3 inflammasome by invading pathogens may prove cell type-specific in exacerbations of airway inflammation in asthma (46). First, NLRP3 interacts with the adaptor protein ASC by sensing microbial pathogens and self-danger signals. Then pro-caspase-1 is recruited and the large protein complex called the NLRP3 inflammasome is formed. This is followed by autocleavage and activation of caspase-1, after which pro-IL-1β and pro-IL-18 are converted into their mature forms. Ion fluxes disrupt membrane integrity, and also mitochondrial damage both play key roles in NLRP3 inflammasome activation (47). Depletion of mitochondria as well as inhibitors that block mitochondrial respiration and ROS production prevented NLRP3 inflammasome activation. Futhermore, genetic ablation of VDAC channels (namely VDAC1 and VDAC3) that are located on the mitochondrial outer membrane and that are responsible for exchanging ions and metabolites with the cytoplasm, leads to diminished mitochondrial (mt) ROS production and inhibition of NLRP3 inflammasome activation (47). Inflammasome activation not only occurs in immune cells, primarily macrophages and dendritic cells, but also in kidney cells, specifically the renal tubular epithelium. The NLRP3 inflammasome is probably involved in the pathogenesis of acute kidney injury, chronic kidney disease, diabetic nephropathy and crystal-related nephropathy (48). The inflammasome also plays a role in autoimmune kidney disease. IL-1 blockade and two recently identified specific NLRP3 inflammasome blockers, MCC950 and β-hydroxybutyrate, may prove to have value in the treatment of inflammasome-mediated conditions.

Autophagosomes derived from tumor cells are referred to as defective ribosomal products in blebs (DRibbles). DRibbles mediate tumor regression by stimulating potent T-cell responses and, thus, have been used as therapeutic cancer vaccines in multiple preclinical cancer models (49). It has been found that DRibbles could induce a rapid differentiation of monocytes and DC precursor (pre-DC) cells into functional APCs (49). Consequently, DRibbles could potentially induce strong innate immune responses via multiple pattern recognition receptors. This explains why DRibbles might be excellent antigen carriers to induce adaptive immune responses to both tumor cells and viruses. This suggests that isolated autophagosomes (DRibbles) from antigen donor cells activate inflammasomes by providing the necessary signals required for IL-1β production.

The Hsp90 system is characterized by a cohort of co-chaperones that bind to Hsp90 and affect its function (50). The co-chaperones enable Hsp90 to chaperone structurally and functionally diverse client proteins. Sahasrabudhe et al. (50) show that the nature of the client protein dictates the contribution of a co-chaperone to its maturation. The study reveals the general importance of the cochaperone Sgt1 (50). In addition to Hsp90, we have to consider Hsp60. Adult cardiac myocytes release heat shock protein (HSP)60 in exosomes. Extracellular HSP60, when not in exosomes, causes cardiac myocyte apoptosis via the activation of Toll-like receptor 4. the protein content of cardiac exosomes differed significantly from other types of exosomes in the literature and contained cytosolic, sarcomeric, and mitochondrial proteins (21).

A new Protein Organic Solvent Precipitation (PROSPR) method efficiently isolates the EV repertoire from human biological samples. Proteomic profiling of PROSPR-enriched CNS EVs indicated that > 75 % of the proteins identified matched previously reported exosomal and microvesicle cargoes. In addition lipidomic characterization of enriched CNS vesicles identified previously reported EV-specific lipid families and novel lipid isoforms not previously detected in human EVs. The characterization of these structures from central nervous system (CNS) tissues is relevant to current neuroscience, especially to advance the understanding of neurodegeneration in amyotrophic lateral sclerosis (ALS), Parkinson’s disease (PD) and Alzheimer’s disease (AD)(15). In addition, study of EVs in brain will enable characterization of the degenerative posttranslational modifications (DPMs) occurring in those proteins.
Neurodegenerative disease is characterized by dysregulation because of NLRP3 inflammasome activation. Alzheimer’s disease (AD) and Parkinson’s disease (PD), both neurodegenerative diseases are associated with the NLRP3 inflammasome. PD is characterized by accumulation of Lewy bodies (LB) formed by a-synuclein (aSyn) aggregation. A recent study revealed that aSyn induces synthesis of pro-IL-1b by an interaction with TLR2 and activates NLRP3 inflammasome resulting in caspase-1 activation and IL-1b maturation in human primary monocytes (43). In addition mitophagy downregulates NLRP3 inflammasome activation by eliminating damaged mitochondria, blocking NLRP3 inflammasome activating signals. It is notable that in this aberrant activation mitophagy downregulates NLRP3 inflammasome activation by eliminating damaged mitochondria, blocking NLRP3 inflammasome activating signals (43).

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Blast crisis in myeloid leukemia and the activation of a microRNA-editing enzyme called ADAR1

Curator: Larry H. Bernstein, MD, FCAP

 

Fix to RNA-Editing Glitch May Defuse Blast Crisis

GEN News Highlights Jun 10, 2016   http://www.genengnews.com/gen-news-highlights/fix-to-rna-editing-glitch-may-defuse-blast-crisis/81252818/

The self-renewal of leukemia stem cells depends on the activation of a microRNA-editing enzyme called ADAR1. According to a new study, ADAR1 activation represents a unique therapeutic vulnerability in leukemia stem cells, which can give rise to blast crisis in chronic myeloid leukemia.     http://www.genengnews.com/Media/images/GENHighlight/thumb_Jun10_2016_ChronicMyeloidLeukemiaBloodCells6222419925.jpg

Few cancer mechanisms are as devastating as the generation of cancer stem cells, which arise in leukemia from white blood cell precursors. The mechanisms of this transition have been obscure, but the consequences are all too clear. Leukemia stem cells promote an aggressive, therapy-resistant form of disease called blast crisis.

Delving into the mechanisms by which leukemia stem cells are primed, a team of scientists at the University of California, San Diego (UCSD), uncovered a misfiring RNA-editing system. The main problem the scientists found was an enzyme called ADAR1 (adenosine deaminase acting on RNA1), which mediates post-transcriptional adenosine-to-inosine (A-to-I) RNA editing.

ADAR1 can edit the sequence of microRNAs (miRNAs), small pieces of genetic material. By swapping out just one miRNA building block for another, ADAR1 alters the carefully orchestrated system cells use to control which genes are turned on or off at which times.

ADAR1 is known to promote cancer progression and resistance to therapy. To study ADAR1, the UCSD team used human blast crisis chronic myeloid leukemia (CML) cells in the lab, and mice transplanted with these cells, to determine the enzyme’s role in governing leukemia stem cells.

The scientists, led by Catriona Jamieson, M.D., Ph.D., published their work June 9 in Cell Stem Cell, in an article entitled, “ADAR1 Activation Drives Leukemia Stem Cell Self-Renewal by Impairing Let-7 Biogenesis.” The article presented the first mechanistic link between pro-cancer inflammatory signals and RNA editing–driven reprogramming of precursor cells into leukemia stem cells.

The article describes how ADAR1-mediated A-to-I RNA editing is activated by Janus kinase 2 (JAK2) signaling and BCR-ABL1 signaling. Also, it indicated, in a model of blast crisis (BC) CML, that combined JAK2 and BCR-ABL1 inhibition prevents leukemia stem cell self-renewal commensurate with ADAR1 downregulation.

Essentially, the scientists were able to trace a series of molecular events: First, white blood cells with a leukemia-promoting gene mutation become more sensitive to signs of inflammation. That inflammatory response activates ADAR1. Then, hyper-ADAR1 editing slows down the miRNAs known as let-7. Ultimately, this activity increases cellular regeneration, or self-renewal, turning white blood cell precursors into leukemia stem cells.

“Lentiviral ADAR1 wild-type, but not an editing-defective ADAR1E912A mutant, induces self-renewal gene expression and impairs biogenesis of stem cell regulatory let-7 microRNAs,” wrote the author of the Cell Stem Cell article. “Combined RNA sequencing, qRT-PCR, CLIP-ADAR1, and pri-let-7 mutagenesis data suggest that ADAR1 promotes LSC generation via let-7 pri-microRNA editing andLIN28B upregulation.”

After learning how the ADAR1 system works, Dr. Jamieson’s team looked for a way to stop it. By inhibiting sensitivity to inflammation or inhibiting ADAR1 with a small-molecule tool compound, the researchers were able to counter ADAR1’s effect on leukemia stem cell self-renewal and restore let-7. Self-renewal of blast crisis CML cells was reduced by approximately 40% when treated with the small molecule called 8-Aza as compared to untreated cells.

“A small-molecule tool compound antagonizes ADAR1’s effect on LSC self-renewal in stromal co-cultures and restores let-7 biogenesis,” the study’s authors noted. “Thus, ADAR1 activation represents a unique therapeutic vulnerability in LSCs with active JAK2 signaling.”

“In this study, we showed that cancer stem cells co-opt a RNA editing system to clone themselves. What’s more, we found a method to dial it down,” said Dr. Catriona Jamieson. “Based on this research, we believe that detecting ADAR1 activity will be important for predicting cancer progression.

“In addition, inhibiting this enzyme represents a unique therapeutic vulnerability in cancer stem cells with active inflammatory signaling that may respond to pharmacologic inhibitors of inflammation sensitivity or selective ADAR1 inhibitors that are currently being developed.”

 

ADAR1 Activation Drives Leukemia Stem Cell Self-Renewal by Impairing Let-7 Biogenesis

Maria Anna Zipeto, Angela C. Court, Anil Sadarangani, Nathaniel P. Delos Santos, Larisa Balaian, Hye-Jung Chun, Gabriel Pineda, Sheldon R. Morris, Cayla N. Mason, Ifat Geron, Christian Barrett, Daniel J. Goff, Russell Wall, Maurizio Pellecchia, Mark Minden, Kelly A. Frazer, Marco A. Marra, Leslie A. Crews, Qingfei Jiang, Catriona H.M. Jamieson
Published online: June 9, 2016
  • JAK2 signaling activates ADAR1-mediated A-to-I RNA editing
  • JAK2 and BCR-ABL1 signaling converge on ADAR1 activation through STAT5a
  • ADAR1-mediated microRNA editing impairs let-7 biogenesis and enhances LSC self-renewal
  • JAK2 and BCR-ABL1 inhibition reduces ADAR1 expression and prevents LSC self-renewal

Post-transcriptional adenosine-to-inosine RNA editing mediated by adenosine deaminase acting on RNA1 (ADAR1) promotes cancer progression and therapeutic resistance. However, ADAR1 editase-dependent mechanisms governing leukemia stem cell (LSC) generation have not been elucidated. In blast crisis chronic myeloid leukemia (BC CML), we show that increased JAK2 signaling and BCR-ABL1 amplification activate ADAR1. In a humanized BC CML mouse model, combined JAK2 and BCR-ABL1 inhibition prevents LSC self-renewal commensurate with ADAR1 downregulation. Lentiviral ADAR1 wild-type, but not an editing-defective ADAR1E912A mutant, induces self-renewal gene expression and impairs biogenesis of stem cell regulatory let-7 microRNAs. Combined RNA sequencing, qRT-PCR, CLIP-ADAR1, and pri-let-7 mutagenesis data suggest that ADAR1 promotes LSC generation via let-7 pri-microRNA editing and LIN28Bupregulation. A small-molecule tool compound antagonizes ADAR1’s effect on LSC self-renewal in stromal co-cultures and restores let-7 biogenesis. Thus, ADAR1 activation represents a unique therapeutic vulnerability in LSCs with active JAK2 signaling.

 

Figure thumbnail fx1

 

 

https://ash.confex.com/ash/2015/webprogram/Paper85836.html

4014 Inflammatory Cytokine-Responsive ADAR1 Impairs Let-7 Biogenesis and Promotes Leukemia Stem Cell Generation

Chronic Myeloid Leukemia: Biology and Pathophysiology, excluding Therapy
Program: Oral and Poster Abstracts
Session: 631. Chronic Myeloid Leukemia: Biology and Pathophysiology, excluding Therapy: Poster III
Monday, December 7, 2015, 6:00 PM-8:00 PM
Hall A, Level 2 (Orange County Convention Center)

Maria Anna Zipeto, Ph.D1*, Angela Court Recart2*, Nathaniel Delos Santos3*, Qingfei Jiang, PhD4*, Leslie A Crews, PhD3* and Catriona HM Jamieson, MD, PhD3

1Sanford Consortium for Regenerative Medicine, University of California San Diego, La Jolla, CA
2University of California San Diego, LA JOLLA, CA
3Division of Regenerative Medicine, University of California, San Diego, La Jolla, CA
4University of California San Diego, La Jolla, CA

BackgroundIn advanced human malignancies, RNA sequencing (RNA-seq) has uncovered deregulation of adenosine deaminase acting on RNA (ADAR) editases that promote therapeutic resistance and leukemia stem cell (LSC) generation. Chronic myeloid leukemia (CML), an important paradigm for understanding LSC evolution, is initiated by BCR-ABL1 oncogene expression in hematopoietic stem cells (HSCs) but undergoes blast crisis (BC) transformation following aberrant self-renewal acquisition by myeloid progenitors harboring cytokine-responsive ADAR1 p150 overexpression. Emerging evidence suggests that adenosine to inosine editing at the level of primary (pri) or precursor (pre)-microRNA (miRNA), alters miRNA biogenesis and impairs biogenesis. However, relatively little is known about the role of inflammatory niche-driven ADAR1 miRNA editing in malignant reprogramming of progenitors into self-renewing LSCs.

Methods

Primary normal and CML progenitors were FACS-purified and RNA-Seq analysis as well as qRT-PCR validation were performed according to published methods (Jiang, 2013). MiRNAs were extracted from purified CD34+ cells derived from CP, BC CML and cord blood by RNeasy microKit (QIAGEN) and let-7 expression was evaluated by qRT-PCR using miScript Primer assay (QIAGEN). CD34+ cord blood (n=3) were transduced with lentiviral human JAK2, let-7a, wt-ADAR1 and mutant ADAR1, which lacks a functional deaminase domain.  Because STAT signaling triggers ADAR1 transcriptional activation and both BCR-ABL1 and JAK2 activate STAT5a, nanoproteomics analysis of STAT5a levels was performed.  Engrafted immunocompromised RAG2-/-γc-/- mice were treated with a JAK2 inhibitor, SAR302503, alone or in combination with a potent BCR-ABL1 TKI Dasatinib, for two weeks followed by FACS analysis of human progenitor engraftment in hematopoietic tissues and serial transplantation.

Results

RNA-seq and qRT-PCR analysis in FACS purified BC CML progenitors revealed an over-representation of inflammatory pathway activation and higher levels of JAK2-dependent inflammatory cytokine receptors, when compared to normal and chronic phase (CP) progenitors. Moreover, RNA-seq and qRT-PCR analysis showed decreased levels of mature let-7 family of stem cell regulatory miRNA in BC compared to normal and CP progenitors. Lentiviral human JAK2 transduction of CD34+ progenitors led to an increase of ADAR1 transcript levels and to a reduction in let-7 family members. Interestingly, lentiviral human JAK2 transduction of normal progenitors enhanced ADAR1 activity, as revealed by RNA editing-specific qRT-PCR and RNA-seq analysis. Moreover, qRT-PCR analysis of CD34+ progenitors transduced with wt-ADAR1, but not mutant ADAR1 lacking functional deaminase activity, reduced let-7 miRNA levels. These data suggested that ADAR1 impairs let-7 family biogenesis in a RNA editing dependent manner. Interestingly, RNA-seq analysis confirmed higher frequency of A-to-I editing events in pri- and pre-let-7 family members in CD34+ BC compared to CP progenitors, as well as normal progenitors transduced with human JAK2 and ADAR1-wt, but not mutant ADAR1. Lentiviral ADAR1 overexpression enhanced CP CML progenitor self-renewal and decreased levels of some members of the let-7 family. In contrast, lentiviral transduction of human let-7a significantly reduced self-renewal of progenitors. In vivo treatments with Dasatinib in combination with a JAK2 inhibitor, significantly reduced self-renewal of BCR-ABL1 expressing BC progenitors in the bone marrow thereby prolonging survival of serially transplanted mice. Finally, a reduction in ADAR1 p150 transcripts was also noted following combination treatment only suggesting a role for ADAR1 in CSC propagation.

Conclusion

This is the first demonstration that intrinsic BCR-ABL oncogenic signaling and extrinsic cytokines signaling through JAK2 converge on activation of ADAR1 that drives LSC generation by impairing let-7 miRNA biogenesis. Targeted reversal of ADAR1-mediated miRNA editing may enhance eradication of inflammatory niche resident cancer stem cells in a broad array of malignancies, including JAK2-driven myeloproliferative neoplasms.

Disclosures: Jamieson: J&J: Research Funding ; GSK: Research Funding .

Interferon Receptor Signaling in Malignancy: a Network of Cellular Pathways Defining Biological Outcomes

Interferons (IFNs) are cytokines with important anti-proliferative activity and exhibit key roles in immune surveillance against malignancies. Early work initiated over 3 decades ago led to the discovery of IFN receptor activated Jak-Stat pathways and provided important insights into mechanisms for transcriptional activation of interferon stimulated genes (ISGs) that mediate IFN-biological responses. Since then, additional evidence has established critical roles for other receptor activated signaling pathways in the induction of IFN-activities. These include MAPK pathways, mTOR cascades and PKC pathways. In addition, specific microRNAs (miRNAs) appear to play a significant role in the regulation of IFN-signaling responses. This review focuses on the emerging evidence for a model in which IFNs share signaling elements and pathways with growth factors and tumorigenic signals, but engage them in a distinctive manner to mediate anti-proliferative and antiviral responses.

Because of their antineoplastic, antiviral, and immunomodulatory properties, recombinant interferons (IFNs) have been used extensively in the treatment of various diseases in humans (1). IFNs have clinical activity against several malignancies and are actively used in the treatment of solid tumors such as malignant melanoma and renal cell carcinoma; and hematological malignancies, such as myeloproliferative neoplasms (MPNs) (1). In addition, IFNs play prominent roles in the treatment of viral syndromes, such as hepatitis B and C (2). In contrast to their beneficial therapeutic properties, IFNs have been also implicated in the pathophysiology of certain diseases in humans. In many cases this involvement reflects abnormal activation of the endogenous IFN system, which has important roles in various physiological processes. Diseases in which dysregulation of the Type I IFN system has been implicated as a pathogenetic mechanism include autoimmune disorders such as systemic lupus erythematosous (3), Sjogren’s syndrome (3,4), dermatomyositis (5) and systemic sclerosis (3, 4). In addition, Type II IFN (IFNγ) overproduction has been implicated in bone marrow failure syndromes, such as aplastic anemia (6). There is also recent evidence for opposing actions of distinct IFN subtypes in the pathophysiology of certain diseases. For instance, a recent study demonstrated that there is an inverse association between IFNβ and IFNγ gene expression in human leprosy, consistent with opposing functions between Type I and II IFNs in the pathophysiology of this disease (7). Thus, differential targeting of components of the IFN-system, to either promote or block induction of IFN-responses depending on the disease context, may be useful in the therapeutic management of various human illnesses. The emerging evidence for the complex regulation of the IFN-system underscores the need for a detailed understanding of the mechanisms of IFN-signaling in order to target IFN-responses effectively and selectively.

It took over 35 years from the original discovery of IFNs in 1957 to the discovery of Jak-Stat pathways (8). The identification of the functions of Jaks and Stats dramatically advanced our understanding of the mechanisms of IFN-signaling and had a broad impact on the cytokine research field as a whole, as it led to the identification of similar pathways from other cytokine receptors (8). Subsequently, several other IFN receptor (IFNR)-regulated pathways were identified (9). As discussed below, in recent years there has been accumulating evidence that beyond Stats, non-Stat pathways play important and essential roles in IFN-signaling. This has led to an evolution of our understanding of the complexity associated with IFN receptor activation and how interacting signaling networks determine the relevant IFN response.

Interferons and their functions

The interferons are classified in 3 major categories, Type I (α, β, ω, ε, τ, κ, ν); Type II (γ) and Type III IFNs (λ1, λ2, λ3) (1, 9, 10). The largest IFN-gene family is the group of Type I IFNs. This family includes 14 IFNα genes, one of which is a pseudogene, resulting in the expression of 13 IFNα protein subtypes (1, 9). There are 3 distinct IFNRs that are specific for the 3 different IFN types. All Type I IFN subtypes bind to and activate the Type I IFNR, while Type II and III IFNs bind to and activate the Type II and III IFNRs, respectively (911). It should be noted that although all the different Type I IFNs bind to and activate the Type I IFNR, differences in binding to the receptor may account for specific responses and biological effects (9). For instance, a recent study provided evidence that direct binding of mouse IFNβ to the Ifnar1 subunit, in the absence of Ifnar2, regulates engagement of signals that control expression of genes specifically induced by IFNβ, but not IFNα (12). This recent discovery followed original observations from the 90s that revealed differential interactions between the different subunits of the Type I IFN receptor in response to IFNβ binding as compared to IFNα binding and partially explained observed differences in functional responses between different Type I IFNs (9).

A common property of all IFNs, independently of type and subtype, is the induction of antiviral effects in vitro and in vivo (1). Because of their potent antiviral properties, IFNs constitute an important element of the immune defense against viral infections. There is emerging information indicating that specificity of the antiviral response is cell type dependent and/or reflects specific tissue expression of certain IFNs. As an example, a recent comparative analysis of the involvement of the Type I IFN system as compared to the Type III IFN system in antiviral protection against rotavirus infection of intestinal epithelial cells demonstrated an almost exclusive requirement for IFNλ (Type III IFN) (13). The antiviral effects of IFNα have led to the introduction of this cytokine in the treatment of hepatitis C and B in humans (2) and different viral genotypes have been associated with response or failure to IFN-therapy (14).

Most importantly, IFNs exhibit important antineoplastic effects, reflecting both direct antiproliferative responses mediated by IFNRs expressed on malignant cells, as well as indirect immunomodulatory effects (15). IFNα and its pegylated form (peg IFNα) have been widely used in the treatment of several neoplastic diseases, such as hairy cell leukemia (HCL), chronic myeloid leukemia (CML), cutaneous T cell lymphoma (CTCL), renal cell carcinoma (RCC), malignant melanoma, and myeloproliferative neoplasms (MPNs) (1, 16). Although the emergence of new targeted therapies and more effective agents have minimized the use of IFNs in the treatment of diseases like HCL and CML, IFNs are still used extensively in the treatment of melanoma, CTCL and MPNs (1, 16, 17). Notably, recent studies have provided evidence for long lasting molecular responses in patients with polycythemia vera (PV), essential thrombocytosis (ET) and myelofibrosis (MF) who were treated with IFNα (16). Beyond their inhibitory properties on malignant hematopoietic progenitors, IFNs are potent regulators of normal hematopoiesis (9) and contribute to the regulation of normal homeostasis in the human bone marrow (18). Related to its effects in the central nervous system, IFNβ has clinical activity in multiple sclerosis (MS) and has been used extensively for the treatment of patients with MS (19). The immunoregulatory properties of Type I IFNs include key roles in the control of innate and adaptive immune responses, as well as positive and negative effects on the activation of the inflammasome (15). Dysregulation of the Type I IFN response is seen in certain autoimmune diseases, such as Aicardi-Goutières syndrome (20). In fact, self-amplifying Type I IFN-production is a key pathophysiological mechanism in autoimmune syndromes (21). There is also emerging evidence that IFNλ may contribute to the IFN signature in autoimmune diseases (3).

Jak-Stat pathways

Jak kinases and DNA binding Stat-complexes

Tyrosine kinases of the Janus family (Jaks) are associated in unique combinations with different IFNRs and their functions are essential for IFN-inducible biological responses. Stats are transcriptional activators whose activation depends on tyrosine phosphorylation by Jaks (8, 9). In the case of the Type I IFN receptor, Tyk2 and Jak1 are constitutively associated with the IFNAR1 and IFNAR2 subunits, respectively (8, 9) (Fig. 1). For the Type II IFN receptor, Jak1 and Jak2 are associated with the IFNGR1 and IFNGR2 receptor subunits, respectively (8, 9) (Fig. 1). Finally, in the case of the Type III IFNR, Jak1 and Tyk2 are constitutively associated with the IFN-λR1 and IL-10R2 receptor chains, respectively (10) (Fig. 1). Upon engagement of the different IFNRs by the corresponding ligands, the kinase domains of the associated Jaks are activated and phosphorylate tyrosine residues in the intracellular domains of the receptor subunits that serve as recruitmenst sites for specific Stat proteins. Subsequently, the Jaks phosphorylate Stat proteins that form unique complexes and translocate to the nucleus where they bind to specific sequences in the promoters of ISGs to initiate transcription. A major Stat complex in IFN-signaling is the interferon stimulated gene factor 3 (ISGF3) complex. This IFN-inducible complex is composed or Stat1, Stat2 and IRF9 and regulates transcription by binding to IFN stimulated response elements (ISRE) in the promoters of a large group of IFN stimulated genes (ISGs) (8, 9). ISGF3 complexes are induced during engagement of the Type I and III IFN receptors, but not in response to activation of Type II IFN receptors (810) (Table 1). Beyond ISGF3, several other Stat-complexes involving different Stat homodimers or heterodimers are activated by IFNs and bind to IFNγ-activated (GAS) sequences in the promoters of groups of ISGs (8, 9). Such GAS binding complexes are induced by all different IFNs (I, II and III), although there is variability in the engagement and utilization of different Stats by the different IFN-receptors (Table 1). It should also be noted that engagement of certain Stats, such as Stat4 and Stat6, is cell type-specific and may be relevant for tissue specific functions (9). The significance of different Stat binding complexes in the induction of Type I and II IFN responses was in part addressed in a study in which Stat1 cooperative DNA binding was disrupted by generating knock-in mice expressing cooperativity-deficient STAT1 (22). As expected, Type II IFN-induced gene transcription and antibacterial responses were essentially lost in these mice, but Type I IFN-dependent recruitment of Stat1 to ISRE elements and antiviral responses were not affected (22), demonstrating the existence of important differences in Stat1 cooperative DNA binding between Type I and II IFN signaling.

Type I, II, III interferon receptors subunits, associated kinases of the Janus family, and effector Stat-pathways. Note: Stat:Stat reflects multiple potential Stat:Stat compexes, as outlined in Table 2.

Table 1

Different Stat-DNA binding complexes induced by Type I, II and III IFNs.

Serine phosphorylation of Stats

The nuclear translocation of Stat-proteins occurs after their activation, following phosphorylation on specific sites by Jak kinases (8, 9). It is well established that phosphorylation on tyrosine 701 is required for activation of Stat1 and phosphorylation on tyrosine 705 is required for activation of Stat3 (8, 9). Beyond tyrosine phosphorylation, phosphorylation on serine 727 in the Stat1 and Stat3 transactivation domains is required for full and optimal transcriptional activation of ISGs (8, 9). There is evidence that serine phosphorylation occurs after the phosphorylation of Stat1 on tyrosine 701 and that translocation to the nucleus and recruitment to the chromatin are essential in order for Stat1 to undergo serine 727 phosphorylation (23). Several IFN-dependent serine kinases for Stat1 have been described, raising the possibility that this phosphorylation occurs in a cell type specific manner. After the original demonstration that protein kinase C (PKC) delta (PKCδ) is a serine kinase for Stat1 and is required for optimal transcriptional activation in response to IFNα (24), extensive work has confirmed the role of this PKC isoform in the regulation of serine 727 phosphorylation in Stat1 and has been extended to different cellular systems (2529) (Table 2). In the Type II IFN system five different serine kinases for the transactivation domain (TAD) of Stat1/phosphorylation on serine 727 have been demonstrated in different cell systems.  …..

Serine phosphorylation of Stats

The nuclear translocation of Stat-proteins occurs after their activation, following phosphorylation on specific sites by Jak kinases (8, 9). It is well established that phosphorylation on tyrosine 701 is required for activation of Stat1 and phosphorylation on tyrosine 705 is required for activation of Stat3 (8, 9). Beyond tyrosine phosphorylation, phosphorylation on serine 727 in the Stat1 and Stat3 transactivation domains is required for full and optimal transcriptional activation of ISGs (8, 9). There is evidence that serine phosphorylation occurs after the phosphorylation of Stat1 on tyrosine 701 and that translocation to the nucleus and recruitment to the chromatin are essential in order for Stat1 to undergo serine 727 phosphorylation (23). Several IFN-dependent serine kinases for Stat1 have been described, raising the possibility that this phosphorylation occurs in a cell type specific manner. After the original demonstration that protein kinase C (PKC) delta (PKCδ) is a serine kinase for Stat1 and is required for optimal transcriptional activation in response to IFNα (24), extensive work has confirmed the role of this PKC isoform in the regulation of serine 727 phosphorylation in Stat1 and has been extended to different cellular systems (2529) (Table 2). In the Type II IFN system five different serine kinases for the transactivation domain (TAD) of Stat1/phosphorylation on serine 727 have been demonstrated in different cell systems. ….

Protein tyrosine phosphatases with regulatory effects on Jak-Stat pathways in IFN-signaling.
…….

MicroRNAs (miRs) and the IFN response

IFN-inducible JAK-STAT, MAPK and mTOR signaling cascades are also regulated potentially by microRNAs (miRs). miRs are important regulators of post-transcriptional events, leading to inhibition of mRNA translation or mRNA degradation (105). In recent years it has become apparent that the direct regulation of STAT activity by mIRs has profound effects on consequent gene expression, specifically in the context of cytokine-inducible events (106). Pertinent for this review of IFN-inducible STAT activation, miR-145, miR-146A and miR-221/222 target STAT1 and miR-221/222 target STAT2 (106). Numerous studies describe different miRs that target STAT3: mIR-17, miR-17-5p, mIR-17-3p, mIR-18a, miR-19b, mIR-92-1, miR-20b, Let-7a, miR-106a, miR-106-25, miR-106a-362 and miR-125b (106) (Fig. 4). mIR-132, miR-212 and miR-200a have been implicated in negatively regulating STAT4 expression in human NK cells (107) and miR-222 has been shown to regulate STAT5 expression (108). In addition, JAK-STAT signaling is affected by miR targeting of suppressors of cytokine signaling (SOCS) proteins. miR-122 and miR-155 targeting of SOCS1 releases the inhibition of STAT1 (and STAT5a/b) (109111), and mIR-19a regulation of SOCS1 and SOCS3 effectively prolongs activation of both STAT1 and STAT3 (112). There is also evidence that miR-155 targets the inositol phosphatase SHIP1, effectively prolonging/inducing IFN-γ expression (113). Much of the evidence associated with miRs prolonging JAK-STAT activation relates to cancer studies, where tumor-secreted miRs promote cell migration and angiogenesis by prolonging JAK-STAT activation (114). miR-145 targeting of SOCS7 affects nuclear translocation of STAT3 and has been associated with enhanced IFNβ production (115). Beyond inhibition of SOCS proteins, miRs may influence the expression of other inhibitory factors associated with JAK-STAT signaling, and miR-301a and miR-18a have been shown to inhibit PIAS3, a negative regulator of STAT3 activation (116). There is also the potential for STATS to directly regulate miR gene expression. STAT5 suppresses expression of miR15/16 (117) and there is evidence that there are potential STAT3 binding sites in the promoters of about 200 miRs (118). Viewed altogether, there is compelling evidence for miR-STAT interactions, yet few studies have considered the contributions of miRs to IFN-inducible JAK-STAT signaling.

Targeting and regulation of various proteins known to be involved in IFN-signaling by different miRNAs.  ….

Evolution of our understanding of IFN-signals and future perspectives

A substantial amount of knowledge has accumulated since the original discovery of the Jak-Stat pathway in the early 90s. It is now clear that several key signaling cascades are essential for the induction of Type I, II and III IFN-responses. The original view that IFN-signals can be transmitted from the cell surface to the nucleus in two simple steps involving tyrosine phosphorylation of Stat proteins (8) now appears somewhat simplistic, as it has been established that modifications of Jak-Stat signals by other pathways and/or simultaneous engagement of other essential complementary cellular cascades is essential for induction of ISG transcriptional activation, mRNA translation, protein expression and subsequent induction of IFN-responses. Such pathways include PKC and MAP kinase pathways and mTORC1 and mTORC2-dpendent signaling cascades.

Over the next decade our understanding of the mechanisms by which IFN-signals are induced will likely continue to evolve, with the anticipated outcome that it will be possible exploit this new knowledge for translational-therapeutic purposes. For instance, selective targeting of kinase-elements of the IFN-pathway with kinase inhibitors may be useful in the treatment of autoimmune diseases where dysregulated/excessive Type I IFN production contributes to the pathophysiology of disease. On the other hand, efforts to promote the induction of specific IFN-signals, may lead to novel, less toxic, therapeutic interventions for a variety of viral infectious diseases and neoplastic disorders.

Exploring the RNA World in Hematopoietic Cells Through the Lens of RNA-Binding Proteins

The discovery of microRNAs has renewed interest in post-transcriptional modes of regulation, fueling an emerging view of a rich RNA world within our cells that deserves further exploration. Much work has gone into elucidating genetic regulatory networks that orchestrate gene expression programs and direct cell fate decisions in the hematopoietic system. However, the focus has been to elucidate signaling pathways and transcriptional programs. To bring us one step closer to reverse engineering the molecular logic of cellular differentiation, it will be necessary to map post-transcriptional circuits as well and integrate them in the context of existing network models. In this regard, RNA-binding proteins (RBPs) may rival transcription factors as important regulators of cell fates and represent a tractable opportunity to connect the RNA world to the proteome. ChIP-seq has greatly facilitated genome-wide localization of DNA-binding proteins, helping us to understand genomic regulation at a systems level. Similarly, technological advances such as CLIP-seq allow transcriptome-wide mapping of RBP binding sites, aiding us to unravel post-transcriptional networks. Here, we review RBP-mediated post-transcriptional regulation, paying special attention to findings relevant to the immune system. As a prime example, we highlight the RBP Lin28B, which acts as a heterochronic switch between fetal and adult lymphopoiesis.

The basis of cellular differentiation and function can be represented as integrated circuits that are genetically programmed. Identification of the master regulators within these complex circuits that can switch on or off a genetic program will enable us to reprogram cells to suit biomedical needs. A remarkable example was the discovery by Takahashi and Yamanaka (1) that somatic cells could be reprogrammed into induced pluripotent stem (iPS) cells via the ectopic expression of four key transcription factors. Interestingly, a specific set of microRNAs (miRNAs) could also mediate this reprogramming (2, 3), revealing a powerful layer of post-transcriptional regulation that is able to override a pre-existing transcriptional program (4). Similarly, miR-9 and miR-124 were sufficient to mediate transdifferentiation of human fibroblasts into neurons (5). Accordingly, we are enamored by the RNA world and pay special attention in our investigations to regulatory non-coding RNAs (ncRNAs), particularly miRNAs and long non-coding RNAs (lncRNAs) and how they integrate with known genetic regulatory networks (Fig. 1). With the exception of certain ribozymes, regulatory RNAs generally do not work alone. Instead, they are physically organized as RNA-protein (RNP) complexes. Operationally, RNA-binding proteins (RBPs) and their interactome work in concert as post-transcriptional networks, or RNA regulons, in response to developmental and environmental cues (6). Inspired by this concept and other pioneering studies in the worm, we recently demonstrated that a single RBP Lin28 was sufficient to reprogram adult hematopoietic progenitors to adopt fetal-like properties (7). We discuss these and related findings, which begin to disentangle the complex functions of RBPs in the context of recent advances in post-transcriptional regulation, starting with the discovery of miRNAs.

Fig. 1

Updated model of gene regulation that integrates RBPs and ncRNAs

The Lin28/let-7 circuit: from worm development to lymphopoiesis

Inspiration from the worm

Working in C. elegans, Ambros and Horvitz (8) identified a set of genes that control developmental timing, a category that they termed heterochronic genes. Heterochrony is a term coined by evolutionary biologists and popularized by the worm community to denote events that either positively or negatively regulate developmental timing in multicellular organisms. The discovery of two heterochronic genes, lin-4 and lin-28, which encode a miRNA and RBP respectively, is particularly relevant to this review. The lineage (lin) mutants were previously identified and named because they displayed abnormalities in cell lineage differentiation. Furthermore, some of them were considered heterochronic, as adult mutants harbored immature characteristics (retarded phenotype) or, conversely, larval mutants displayed adult characteristics (precocious phenotype). It was not until 1993 that lin-4 was characterized molecularly, because contrary to popular expectations, the gene did not encode a protein but instead a small RNA now appreciated as the first miRNA to be discovered (9). The lin-4 miRNA acts in part by inhibiting the expression of the LIN-14 transcription factor through imperfect basepairing to sites in the 3′ untranslated region (UTR) of lin-14 mRNA (9, 10). However, it was not apparent initially whether lin-4 or lin-14 is evolutionarily conserved, potentially relegating these findings to be relevant only to the worm. Interestingly, Lin28, a gene conserved in mammals, was later identified to be a direct target of the lin-4 miRNA (11). Lin28 loss-of-function resulted in a precocious phenotype, whereas gain-of-function resulted in a retarded phenotype; thus, Lin28 acts as a heterochronic switch during C. elegans larval development (11).

The possibility that lin-4 may be an oddity of the worm was dissolved with the discovery of the second miRNA, again in C. elegans, let-7 (12). Unlike lin-4, the evolutionary conservation of let-7 from sea urchin to human was quickly appreciated (13). Importantly, expression analysis showed that let-7 expression is temporally regulated from molluscs to vertebrates in all three major clades of bilaterian animals, implying that its role as a developmental timekeeper is conserved (14). This established miRNAs as a field unto its own that has progressed rapidly with the identification of Drosha, Dgcr8, Dicer, and Argonaute (Ago) RBPs as core components of the miRNA pathway (15). Orthologs of lin-4were eventually found in mammals (mir-125a, -b-1, and -b-2) (16) along with hundreds of novel miRNAs from numerous organisms (17). We now recognize that miRNAs, in complex with the RBP Ago, frequently bind their cognate targets via imperfect complementarity to evolutionarily conserved sequences in 3′ UTRs (1820) and mediate post-transcriptional repression (21).

…..

One diverse group of RBPs appreciated to be important in the immune system, even before the discovery of miRNAs, is distinguished by their ability to bind to AU-rich elements (AREs) often found in 3′ UTRs of genes involved in inflammation, growth, and survival. Such RBPs are known as ARE-BPs and have been implicated in mRNA decay, alternative splicing, translation, as well as both alleviating and enhancing miRNA-mediated mRNA repression (104107). Genetic inactivation of several ARE-BPs have been linked to aberrant cytokine expression due to impaired ARE-mediated decay (5, 108111) (Table 1). In addition, deficiency of HuR and AUF1 has uncovered a pro-survival role for both in lymphocytes (112, 113), while ectopic expression of Tis11b (ZFP36L1) negatively regulates erythropoiesis by down-regulating Stat5b mRNA stability (114). The KH-type splicing regulatory protein (KSRP) originally identified as an alternative-splicing factor is a multi-functional RBP. It has been shown to associate with both Drosha and Dicer complexes to positively regulate the biogenesis of a subset of miRNAs including mir-155 and let-7 (73, 108, 115120). In addition, KSRP, like many other ARE-BPs, mediate selective decay of mRNAs by recruitment of exosome complexes to mRNA targets (121) and constitutes a prime example of a multi-functional RBP.

……

Biological processes involved in the development and function of the immune system require programmed changes in protein production and constitute prime candidates for post-transcriptional regulation. While the ENCODE project initially aimed to identify all functional elements in the human DNA sequence, recent discoveries centered around miRNAs and multi-tasking RBPs, such as Lin28, have highlighted the need for a similar systematic effort in mapping post-transcriptional functional elements within the transcriptome. Integration of genomic, transcriptomic, and proteomic data remains a daunting but necessary task to achieve understanding of the full impact of genetic programs and the enigmatic roles of regulatory RNAs. Mastering the science of (re)programming cell fates promises to unleash the potential of stem cells for Regenerative Medicine.

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Tumor Shrinking Triple Helices

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Tumor-Shrinking Triple-Helices

A braided structure and some adhesive hydrogel make therapeutic microRNAs both stable and sticky.

By Ruth Williams | April 1, 2016

http://www.the-scientist.com/?articles.view/articleNo/45576/title/Tumor-Shrinking-Triple-Helices

 

MicroRNAs (miRs) are small, noncoding ribonucleic acids that control the translation of target messenger RNAs (mRNAs). Given their roles in development, differentiation, and other cellular processes, misregulation of miRs can contribute to diseases such as cancer. Indeed, “they are recognized as important modulators of cancer progression,” says Natalie Artzi of Harvard Medical School.

In addition to occasionally promoting cancer pathology, miRs also hold the potential to treat it—either by restoring levels of suppressed miRs, or by repressing overactive ones using antisense miRs (antagomiRs). While miRs are promising therapeutic molecules, says Daniel Siegwart of the University of Texas Southwestern Medical Center in Dallas, their use “is currently hindered by at least two issues: nucleic acid instability in vivo, and the development of effective delivery systems to transport miRs into tumor cells.”

Artzi and her team have now addressed both of these issues in one fell swoop. They first assembled two therapeutic miRs—one antagomiR and one that replaced a deficient miR—together with a third miR, a complement of the replacement strand, into triple-helix structures, which increased molecular stability without affecting function. They then complexed these helices with dendrimers—large synthetic branching polymer particles—and mixed these complexes with dextran aldehyde to form an adhesive hydrogel. The gel could then be applied directly to the surface of tumors to deliver the therapeutic miRs into cells with high efficiency.

In mice with induced breast tumors, the triple-helix–hydrogel approach led to dramatic tumor shrinkage and extended life span: the animals survived approximately one month longer than those treated with standard-of-care chemotherapy drugs. Because the RNA-hydrogel mixture must be applied directly to the tumor, the approach will not be suitable for all cancers. But one potential application, says Siegwart, is that “the hydrogel could be applied by a surgeon after performing bulk tumor removal…[and] might kill remaining tumor cells that would otherwise cause tumor recurrence.” (Nature Materials, http://dx.doi.org:/10.1038/NMAT4497, 2015)

STICKING IT TO TUMORS: To deliver therapeutic microRNAs (miRs) to tumors, braids of three microRNAs (miRs)—an antisense strand that blocks a miR overactive in cancer, a strand that replaces a deficient miR, and a stabilizing strand (1)—are added to a dendrimer (2) and mixed with a hydrogel scaffold (3). When researchers introduced the sticky gel onto mouse mammary tumors (4), the malignancies shrank and the animals lived longer (5)© GEORGE RETSECK; J.CONDE ET AL., NATURE MATERIALS

 

miR DELIVERY SYSTEM VEHICLE DOSE TUMOR TARGETING APPLICABLE TUMOR TYPES
Nanoparticles Examples: gold particles, liposomes, peptide nucleic acids, or polymers Usually multiple injections Combining miRs with aptamers or antibodies can guide nanoparticles to target cells, but systemic delivery inevitably leads to some off-target dispersion. Multisite or blood cancers
RNA–triple-helix-hydrogel Dendrimer-dextran hydrogel One Adhesive hydrogel sticks miRs to tumor site with minimal dispersion to other tissues. Solid Tumors

 

Self-assembled RNA-triple-helix hydrogel scaffold for microRNA modulation in the tumour microenvironment

João CondeNuria OlivaMariana AtilanoHyun Seok Song & Natalie Artzi
Nature Materials15,353–363(2016)
                         http://dx.doi.org:/10.1038/nmat4497

The therapeutic potential of miRNA (miR) in cancer is limited by the lack of efficient delivery vehicles. Here, we show that a self-assembled dual-colour RNA-triple-helix structure comprising two miRNAs—a miR mimic (tumour suppressor miRNA) and an antagomiR (oncomiR inhibitor)—provides outstanding capability to synergistically abrogate tumours. Conjugation of RNA triple helices to dendrimers allows the formation of stable triplex nanoparticles, which form an RNA-triple-helix adhesive scaffold upon interaction with dextran aldehyde, the latter able to chemically interact and adhere to natural tissue amines in the tumour. We also show that the self-assembled RNA-triple-helix conjugates remain functional in vitro and in vivo, and that they lead to nearly 90% levels of tumour shrinkage two weeks post-gel implantation in a triple-negative breast cancer mouse model. Our findings suggest that the RNA-triple-helix hydrogels can be used as an efficient anticancer platform to locally modulate the expression of endogenous miRs in cancer.

 

Figure 1: Self-assembled RNA-triple-helix hydrogel nanoconjugates and scaffold for microRNA delivery.

Self-assembled RNA-triple-helix hydrogel nanoconjugates and scaffold for microRNA delivery.

a, Schematic showing the self-assembly process of three RNA strands to form a dual-colour RNA triple helix. The RNA triplex nanoparticles consist of stable two-pair FRET donor/quencher RNA oligonucleotides used for in vivo miRNA inhibit…

 

Figure 4: Proliferation, migration and survival of cancer cells as a function of RNA-triple-helix nanoparticles treatment.close

Proliferation, migration and survival of cancer cells as a function of RNA-triple-helix nanoparticles treatment.

a, miR-205 and miR-221 expression in breast cancer cells at 24, 48 and 72h of incubation (n = 3, statistical analysis performed with a two-tailed Students t-test, , P < 0.01). miRNA levels were normalized to the RNU6B reference gene

 

  1. Kasinski, A. L. & Slack, F. J. MicroRNAs en route to the clinic: Progress in validating and targeting microRNAs for cancer therapy. Nature Rev. Cancer 11, 849864 (2011).
  2. Li, Z. & Rana, T. M. Therapeutic targeting of microRNAs: Current status and future challenges. Nature Rev. Drug Discov. 13, 622638 (2014).
  3. Yin, H. et al. Non-viral vectors for gene-based therapy. Nature Rev. Genet. 15, 541555(2014).
  4. Conde, J., Edelman, E. R. & Artzi, N. Target-responsive DNA/RNA nanomaterials for microRNA sensing and inhibition: The jack-of-all-trades in cancer nanotheranostics? Adv. Drug Deliv. Rev. 81, 169183 (2015).
  5. Chen, Y. C., Gao, D. Y. & Huang, L. In vivo delivery of miRNAs for cancer therapy: Challenges and strategies. Adv. Drug Deliv. Rev. 81, 128141 (2015).
  6. Yin, P. T., Shah, B. P. & Lee, K. B. Combined magnetic nanoparticle-based microRNA and hyperthermia therapy to enhance apoptosis in brain cancer cells. Small 10, 41064112(2014).
  7. Hao, L. L., Patel, P. C., Alhasan, A. H., Giljohann, D. A. & Mirkin, C. A. Nucleic acid–gold nanoparticle conjugates as mimics of microRNA. Small 7, 31583162 (2011).
  8. Endo-Takahashi, Y. et al. Systemic delivery of miR-126 by miRNA-loaded bubble liposomes for the treatment of hindlimb ischemia. Sci. Rep. 4, 3883 (2014).
  9. Chen, Y. C., Zhu, X. D., Zhang, X. J., Liu, B. & Huang, L. Nanoparticles modified with tumor-targeting scFv deliver siRNA and miRNA for cancer therapy. Mol. Ther. 18, 16501656(2010).
  10. Anand, S. et al. MicroRNA-132-mediated loss of p120RasGAP activates the endothelium to facilitate pathological angiogenesis. Nature Med. 16, 909914 (2010).

 

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Proteomics, Metabolomics, Signaling Pathways, and Cell Regulation: a Compilation of Articles in the Journal http://pharmaceuticalintelligence.com


Compilation of References by Leaders in Pharmaceutical Business Intelligence in the Journal http://pharmaceuticalintelligence.com about
Proteomics, Metabolomics, Signaling Pathways, and Cell Regulation

Curator: Larry H Bernstein, MD, FCAP

Proteomics

  1. The Human Proteome Map Completed

Reporter and Curator: Larry H. Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2014/08/28/the-human-proteome-map-completed/

  1. Proteomics – The Pathway to Understanding and Decision-making in Medicine

Author and Curator, Larry H Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2014/06/24/proteomics-the-pathway-to-
understanding-and-decision-making-in-medicine/

3. Advances in Separations Technology for the “OMICs” and Clarification of Therapeutic Targets

Author and Curator, Larry H Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2012/10/22/advances-in-separations-technology-for-the-omics-and-clarification-         of-therapeutic-targets/

  1. Expanding the Genetic Alphabet and Linking the Genome to the Metabolome

Author and Curator, Larry H Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2012/09/24/expanding-the-genetic-alphabet-and-linking-the-genome-to-the-                metabolome/

5. Genomics, Proteomics and standards

Larry H Bernstein, MD, FCAP, Author and Curator

https://pharmaceuticalintelligence.com/2014/07/06/genomics-proteomics-and-standards/

6. Proteins and cellular adaptation to stress

Larry H Bernstein, MD, FCAP, Author and Curator

https://pharmaceuticalintelligence.com/2014/07/08/proteins-and-cellular-adaptation-to-stress/

 

Metabolomics

  1. Extracellular evaluation of intracellular flux in yeast cells

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

https://pharmaceuticalintelligence.com/2014/08/25/extracellular-evaluation-of-intracellular-flux-in-yeast-cells/

  1. Metabolomic analysis of two leukemia cell lines. I.

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

https://pharmaceuticalintelligence.com/2014/08/23/metabolomic-analysis-of-two-leukemia-cell-lines-_i/

  1. Metabolomic analysis of two leukemia cell lines. II.

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

https://pharmaceuticalintelligence.com/2014/08/24/metabolomic-analysis-of-two-leukemia-cell-lines-ii/

  1. Metabolomics, Metabonomics and Functional Nutrition: the next step in nutritional metabolism and biotherapeutics

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

https://pharmaceuticalintelligence.com/2014/08/22/metabolomics-metabonomics-and-functional-nutrition-the-next-step-          in-nutritional-metabolism-and-biotherapeutics/

  1. Buffering of genetic modules involved in tricarboxylic acid cycle metabolism provides homeomeostatic regulation

Larry H. Bernstein, MD, FCAP, Reviewer and curator

https://pharmaceuticalintelligence.com/2014/08/27/buffering-of-genetic-modules-involved-in-tricarboxylic-acid-cycle-              metabolism-provides-homeomeostatic-regulation/

Metabolic Pathways

  1. Pentose Shunt, Electron Transfer, Galactose, more Lipids in brief

Reviewer and Curator: Larry H. Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2014/08/21/pentose-shunt-electron-transfer-galactose-more-lipids-in-brief/

  1. Mitochondria: More than just the “powerhouse of the cell”

Ritu Saxena, PhD

https://pharmaceuticalintelligence.com/2012/07/09/mitochondria-more-than-just-the-powerhouse-of-the-cell/

  1. Mitochondrial fission and fusion: potential therapeutic targets?

Ritu saxena

https://pharmaceuticalintelligence.com/2012/10/31/mitochondrial-fission-and-fusion-potential-therapeutic-target/

4.  Mitochondrial mutation analysis might be “1-step” away

Ritu Saxena

https://pharmaceuticalintelligence.com/2012/08/14/mitochondrial-mutation-analysis-might-be-1-step-away/

  1. Selected References to Signaling and Metabolic Pathways in PharmaceuticalIntelligence.com

Curator: Larry H. Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2014/08/14/selected-references-to-signaling-and-metabolic-pathways-in-                     leaders-in-pharmaceutical-intelligence/

  1. Metabolic drivers in aggressive brain tumors

Prabodh Kandal, PhD

https://pharmaceuticalintelligence.com/2012/11/11/metabolic-drivers-in-aggressive-brain-tumors/

  1. Metabolite Identification Combining Genetic and Metabolic Information: Genetic association links unknown metabolites to functionally related genes

Writer and Curator, Aviva Lev-Ari, PhD, RD

https://pharmaceuticalintelligence.com/2012/10/22/metabolite-identification-combining-genetic-and-metabolic-                        information-genetic-association-links-unknown-metabolites-to-functionally-related-genes/

  1. Mitochondria: Origin from oxygen free environment, role in aerobic glycolysis, metabolic adaptation

Larry H Bernstein, MD, FCAP, author and curator

https://pharmaceuticalintelligence.com/2012/09/26/mitochondria-origin-from-oxygen-free-environment-role-in-aerobic-            glycolysis-metabolic-adaptation/

  1. Therapeutic Targets for Diabetes and Related Metabolic Disorders

Reporter, Aviva Lev-Ari, PhD, RD

https://pharmaceuticalintelligence.com/2012/08/20/therapeutic-targets-for-diabetes-and-related-metabolic-disorders/

10.  Buffering of genetic modules involved in tricarboxylic acid cycle metabolism provides homeomeostatic regulation

Larry H. Bernstein, MD, FCAP, Reviewer and curator

https://pharmaceuticalintelligence.com/2014/08/27/buffering-of-genetic-modules-involved-in-tricarboxylic-acid-cycle-              metabolism-provides-homeomeostatic-regulation/

11. The multi-step transfer of phosphate bond and hydrogen exchange energy

Larry H. Bernstein, MD, FCAP, Curator:

https://pharmaceuticalintelligence.com/2014/08/19/the-multi-step-transfer-of-phosphate-bond-and-hydrogen-                          exchange-energy/

12. Studies of Respiration Lead to Acetyl CoA

https://pharmaceuticalintelligence.com/2014/08/18/studies-of-respiration-lead-to-acetyl-coa/

13. Lipid Metabolism

Author and Curator: Larry H. Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2014/08/15/lipid-metabolism/

14. Carbohydrate Metabolism

Author and Curator: Larry H. Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2014/08/13/carbohydrate-metabolism/

15. Update on mitochondrial function, respiration, and associated disorders

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

https://pharmaceuticalintelligence.com/2014/07/08/update-on-mitochondrial-function-respiration-and-associated-                   disorders/

16. Prologue to Cancer – e-book Volume One – Where are we in this journey?

Author and Curator: Larry H. Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2014/04/13/prologue-to-cancer-ebook-4-where-are-we-in-this-journey/

17. Introduction – The Evolution of Cancer Therapy and Cancer Research: How We Got Here?

Author and Curator: Larry H. Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2014/04/04/introduction-the-evolution-of-cancer-therapy-and-cancer-research-          how-we-got-here/

18. Inhibition of the Cardiomyocyte-Specific Kinase TNNI3K

Author and Curator: Larry H. Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2013/11/01/inhibition-of-the-cardiomyocyte-specific-kinase-tnni3k/

19. The Binding of Oligonucleotides in DNA and 3-D Lattice Structures

Author and Curator: Larry H. Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2013/05/15/the-binding-of-oligonucleotides-in-dna-and-3-d-lattice-structures/

20. Mitochondrial Metabolism and Cardiac Function

Author and Curator: Larry H. Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2013/04/14/mitochondrial-metabolism-and-cardiac-function/

21. How Methionine Imbalance with Sulfur-Insufficiency Leads to Hyperhomocysteinemia

Curator: Larry H. Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2013/04/04/sulfur-deficiency-leads_to_hyperhomocysteinemia/

22. AMPK Is a Negative Regulator of the Warburg Effect and Suppresses Tumor Growth In Vivo

Author and Curator: Stephen J. Williams, PhD

https://pharmaceuticalintelligence.com/2013/03/12/ampk-is-a-negative-regulator-of-the-warburg-effect-and-suppresses-         tumor-growth-in-vivo/

23. A Second Look at the Transthyretin Nutrition Inflammatory Conundrum

Author and Curator: Larry H. Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2012/12/03/a-second-look-at-the-transthyretin-nutrition-inflammatory-                         conundrum/

24. Mitochondrial Damage and Repair under Oxidative Stress

Author and Curator: Larry H. Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2012/10/28/mitochondrial-damage-and-repair-under-oxidative-stress/

25. Nitric Oxide and Immune Responses: Part 2

Author and Curator: Aviral Vatsa, PhD, MBBS

https://pharmaceuticalintelligence.com/2012/10/28/nitric-oxide-and-immune-responses-part-2/

26. Overview of Posttranslational Modification (PTM)

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

https://pharmaceuticalintelligence.com/2014/07/29/overview-of-posttranslational-modification-ptm/

27. Malnutrition in India, high newborn death rate and stunting of children age under five years

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

https://pharmaceuticalintelligence.com/2014/07/15/malnutrition-in-india-high-newborn-death-rate-and-stunting-of-                   children-age-under-five-years/

28. Update on mitochondrial function, respiration, and associated disorders

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

https://pharmaceuticalintelligence.com/2014/07/08/update-on-mitochondrial-function-respiration-and-associated-                  disorders/

29. Omega-3 fatty acids, depleting the source, and protein insufficiency in renal disease

Larry H. Bernstein, MD, FCAP, Curator

https://pharmaceuticalintelligence.com/2014/07/06/omega-3-fatty-acids-depleting-the-source-and-protein-insufficiency-         in-renal-disease/

30. Introduction to e-Series A: Cardiovascular Diseases, Volume Four Part 2: Regenerative Medicine

Larry H. Bernstein, MD, FCAP, writer, and Aviva Lev- Ari, PhD, RN

https://pharmaceuticalintelligence.com/2014/04/27/larryhbernintroduction_to_cardiovascular_diseases-                                  translational_medicine-part_2/

31. Epilogue: Envisioning New Insights in Cancer Translational Biology
Series C: e-Books on Cancer & Oncology

Author & Curator: Larry H. Bernstein, MD, FCAP, Series C Content Consultant

https://pharmaceuticalintelligence.com/2014/03/29/epilogue-envisioning-new-insights/

32. Ca2+-Stimulated Exocytosis:  The Role of Calmodulin and Protein Kinase C in Ca2+ Regulation of Hormone                         and Neurotransmitter

Writer and Curator: Larry H Bernstein, MD, FCAP and
Curator and Content Editor: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/12/23/calmodulin-and-protein-kinase-c-drive-the-ca2-regulation-of-                    hormone-and-neurotransmitter-release-that-triggers-ca2-stimulated-exocy

33. Cardiac Contractility & Myocardial Performance: Therapeutic Implications of Ryanopathy (Calcium Release-                           related Contractile Dysfunction) and Catecholamine Responses

Author, and Content Consultant to e-SERIES A: Cardiovascular Diseases: Justin Pearlman, MD, PhD, FACC
Author and Curator: Larry H Bernstein, MD, FCAP
and Article Curator: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/08/28/cardiac-contractility-myocardium-performance-ventricular-arrhythmias-      and-non-ischemic-heart-failure-therapeutic-implications-for-cardiomyocyte-ryanopathy-calcium-release-related-                    contractile/

34. Role of Calcium, the Actin Skeleton, and Lipid Structures in Signaling and Cell Motility

Author and Curator: Larry H Bernstein, MD, FCAP Author: Stephen Williams, PhD, and Curator: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/08/26/role-of-calcium-the-actin-skeleton-and-lipid-structures-in-signaling-and-cell-motility/

35. Identification of Biomarkers that are Related to the Actin Cytoskeleton

Larry H Bernstein, MD, FCAP, Author and Curator

https://pharmaceuticalintelligence.com/2012/12/10/identification-of-biomarkers-that-are-related-to-the-actin-                           cytoskeleton/

36. Advanced Topics in Sepsis and the Cardiovascular System at its End Stage

Author: Larry H Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2013/08/18/advanced-topics-in-Sepsis-and-the-Cardiovascular-System-at-its-              End-Stage/

37. The Delicate Connection: IDO (Indolamine 2, 3 dehydrogenase) and Cancer Immunology

Demet Sag, PhD, Author and Curator

https://pharmaceuticalintelligence.com/2013/08/04/the-delicate-connection-ido-indolamine-2-3-dehydrogenase-and-               immunology/

38. IDO for Commitment of a Life Time: The Origins and Mechanisms of IDO, indolamine 2, 3-dioxygenase

Demet Sag, PhD, Author and Curator

https://pharmaceuticalintelligence.com/2013/08/04/ido-for-commitment-of-a-life-time-the-origins-and-mechanisms-of-             ido-indolamine-2-3-dioxygenase/

39. Confined Indolamine 2, 3 dioxygenase (IDO) Controls the Homeostasis of Immune Responses for Good and Bad

Curator: Demet Sag, PhD, CRA, GCP

https://pharmaceuticalintelligence.com/2013/07/31/confined-indolamine-2-3-dehydrogenase-controls-the-hemostasis-           of-immune-responses-for-good-and-bad/

40. Signaling Pathway that Makes Young Neurons Connect was discovered @ Scripps Research Institute

Reporter: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/06/26/signaling-pathway-that-makes-young-neurons-connect-was-                     discovered-scripps-research-institute/

41. Naked Mole Rats Cancer-Free

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

https://pharmaceuticalintelligence.com/2013/06/20/naked-mole-rats-cancer-free/

42. Late Onset of Alzheimer’s Disease and One-carbon Metabolism

Reporter and Curator: Dr. Sudipta Saha, Ph.D.

https://pharmaceuticalintelligence.com/2013/05/06/alzheimers-disease-and-one-carbon-metabolism/

43. Problems of vegetarianism

Reporter and Curator: Dr. Sudipta Saha, Ph.D.

https://pharmaceuticalintelligence.com/2013/04/22/problems-of-vegetarianism/

44.  Amyloidosis with Cardiomyopathy

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

https://pharmaceuticalintelligence.com/2013/03/31/amyloidosis-with-cardiomyopathy/

45. Liver endoplasmic reticulum stress and hepatosteatosis

Larry H Bernstein, MD, FACP

https://pharmaceuticalintelligence.com/2013/03/10/liver-endoplasmic-reticulum-stress-and-hepatosteatosis/

46. The Molecular Biology of Renal Disorders: Nitric Oxide – Part III

Curator and Author: Larry H Bernstein, MD, FACP

https://pharmaceuticalintelligence.com/2012/11/26/the-molecular-biology-of-renal-disorders/

47. Nitric Oxide Function in Coagulation – Part II

Curator and Author: Larry H. Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2012/11/26/nitric-oxide-function-in-coagulation/

48. Nitric Oxide, Platelets, Endothelium and Hemostasis

Curator and Author: Larry H Bernstein, MD, FACP

https://pharmaceuticalintelligence.com/2012/11/08/nitric-oxide-platelets-endothelium-and-hemostasis/

49. Interaction of Nitric Oxide and Prostacyclin in Vascular Endothelium

Curator and Author: Larry H Bernstein, MD, FACP

https://pharmaceuticalintelligence.com/2012/09/14/interaction-of-nitric-oxide-and-prostacyclin-in-vascular-endothelium/

50. Nitric Oxide and Immune Responses: Part 1

Curator and Author:  Aviral Vatsa PhD, MBBS

https://pharmaceuticalintelligence.com/2012/10/18/nitric-oxide-and-immune-responses-part-1/

51. Nitric Oxide and Immune Responses: Part 2

Curator and Author:  Aviral Vatsa PhD, MBBS

https://pharmaceuticalintelligence.com/2012/10/28/nitric-oxide-and-immune-responses-part-2/

52. Mitochondrial Damage and Repair under Oxidative Stress

Curator and Author: Larry H Bernstein, MD, FACP

https://pharmaceuticalintelligence.com/2012/10/28/mitochondrial-damage-and-repair-under-oxidative-stress/

53. Is the Warburg Effect the cause or the effect of cancer: A 21st Century View?

Curator and Author: Larry H Bernstein, MD, FACP

https://pharmaceuticalintelligence.com/2012/10/17/is-the-warburg-effect-the-cause-or-the-effect-of-cancer-a-21st-                 century-view/

54. Ubiquinin-Proteosome pathway, autophagy, the mitochondrion, proteolysis and cell apoptosis

Curator and Author: Larry H Bernstein, MD, FACP

https://pharmaceuticalintelligence.com/2012/10/30/ubiquinin-proteosome-pathway-autophagy-the-mitochondrion-                  proteolysis-and-cell-apoptosis/

55. Ubiquitin-Proteosome pathway, Autophagy, the Mitochondrion, Proteolysis and Cell Apoptosis: Part III

Curator and Author: Larry H Bernstein, MD, FACP

https://pharmaceuticalintelligence.com/2013/02/14/ubiquinin-proteosome-pathway-autophagy-the-mitochondrion-                   proteolysis-and-cell-apoptosis-reconsidered/

56. Nitric Oxide and iNOS have Key Roles in Kidney Diseases – Part II

Curator and Author: Larry H Bernstein, MD, FACP

https://pharmaceuticalintelligence.com/2012/11/26/nitric-oxide-and-inos-have-key-roles-in-kidney-diseases/

57. New Insights on Nitric Oxide donors – Part IV

Curator and Author: Larry H Bernstein, MD, FACP

https://pharmaceuticalintelligence.com/2012/11/26/new-insights-on-no-donors/

58. Crucial role of Nitric Oxide in Cancer

Curator and Author: Ritu Saxena, Ph.D.

https://pharmaceuticalintelligence.com/2012/10/16/crucial-role-of-nitric-oxide-in-cancer/

59. Nitric Oxide has a ubiquitous role in the regulation of glycolysis -with a concomitant influence on mitochondrial function

Curator and Author: Larry H Bernstein, MD, FACP

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

60. Targeting Mitochondrial-bound Hexokinase for Cancer Therapy

Curator and Author: Ziv Raviv, PhD, RN 04/06/2013

https://pharmaceuticalintelligence.com/2013/04/06/targeting-mitochondrial-bound-hexokinase-for-cancer-therapy/

61. Biochemistry of the Coagulation Cascade and Platelet Aggregation – Part I

Curator and Author: Larry H Bernstein, MD, FACP

https://pharmaceuticalintelligence.com/2012/11/26/biochemistry-of-the-coagulation-cascade-and-platelet-aggregation/

Genomics, Transcriptomics, and Epigenetics

  1. What is the meaning of so many RNAs?

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

https://pharmaceuticalintelligence.com/2014/08/06/what-is-the-meaning-of-so-many-rnas/

  1. RNA and the transcription the genetic code

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

https://pharmaceuticalintelligence.com/2014/08/02/rna-and-the-transcription-of-the-genetic-code/

  1. A Primer on DNA and DNA Replication

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

https://pharmaceuticalintelligence.com/2014/07/29/a_primer_on_dna_and_dna_replication/

4. Synthesizing Synthetic Biology: PLOS Collections

Reporter: Aviva Lev-Ari

https://pharmaceuticalintelligence.com/2012/08/17/synthesizing-synthetic-biology-plos-collections/

5. Pathology Emergence in the 21st Century

Author and Curator: Larry Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2014/08/03/pathology-emergence-in-the-21st-century/

6. RNA and the transcription the genetic code

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

https://pharmaceuticalintelligence.com/2014/08/02/rna-and-the-transcription-of-the-genetic-code/

7. A Great University engaged in Drug Discovery: University of Pittsburgh

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

https://pharmaceuticalintelligence.com/2014/07/15/a-great-university-engaged-in-drug-discovery/

8. microRNA called miRNA-142 involved in the process by which the immature cells in the bone  marrow give                              rise to all the types of blood cells, including immune cells and the oxygen-bearing red blood cells

Aviva Lev-Ari, PhD, RN, Author and Curator

https://pharmaceuticalintelligence.com/2014/07/24/microrna-called-mir-142-involved-in-the-process-by-which-the-                   immature-cells-in-the-bone-marrow-give-rise-to-all-the-types-of-blood-cells-including-immune-cells-and-the-oxygen-             bearing-red-blood-cells/

9. Genes, proteomes, and their interaction

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

https://pharmaceuticalintelligence.com/2014/07/28/genes-proteomes-and-their-interaction/

10. Regulation of somatic stem cell Function

Larry H. Bernstein, MD, FCAP, Writer and Curator    Aviva Lev-Ari, PhD, RN, Curator

https://pharmaceuticalintelligence.com/2014/07/29/regulation-of-somatic-stem-cell-function/

11. Scientists discover that pluripotency factor NANOG is also active in adult organisms

Larry H. Bernstein, MD, FCAP, Reporter

https://pharmaceuticalintelligence.com/2014/07/10/scientists-discover-that-pluripotency-factor-nanog-is-also-active-in-           adult-organisms/

12. Bzzz! Are fruitflies like us?

Larry H Bernstein, MD, FCAP, Author and Curator

https://pharmaceuticalintelligence.com/2014/07/07/bzzz-are-fruitflies-like-us/

13. Long Non-coding RNAs Can Encode Proteins After All

Larry H Bernstein, MD, FCAP, Reporter

https://pharmaceuticalintelligence.com/2014/06/29/long-non-coding-rnas-can-encode-proteins-after-all/

14. Michael Snyder @Stanford University sequenced the lymphoblastoid transcriptomes and developed an
allele-specific full-length transcriptome

Aviva Lev-Ari, PhD, RN, Author and Curator

https://pharmaceuticalintelligence.com/014/06/23/michael-snyder-stanford-university-sequenced-the-lymphoblastoid-            transcriptomes-and-developed-an-allele-specific-full-length-transcriptome/

15. Commentary on Biomarkers for Genetics and Genomics of Cardiovascular Disease: Views by Larry H                                     Bernstein, MD, FCAP

Author: Larry H Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2014/07/16/commentary-on-biomarkers-for-genetics-and-genomics-of-                        cardiovascular-disease-views-by-larry-h-bernstein-md-fcap/

16. Observations on Finding the Genetic Links in Common Disease: Whole Genomic Sequencing Studies

Author an curator: Larry H Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2013/05/18/observations-on-finding-the-genetic-links/

17. Silencing Cancers with Synthetic siRNAs

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

https://pharmaceuticalintelligence.com/2013/12/09/silencing-cancers-with-synthetic-sirnas/

18. Cardiometabolic Syndrome and the Genetics of Hypertension: The Neuroendocrine Transcriptome Control Points

Reporter: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/12/12/cardiometabolic-syndrome-and-the-genetics-of-hypertension-the-neuroendocrine-transcriptome-control-points/

19. Developments in the Genomics and Proteomics of Type 2 Diabetes Mellitus and Treatment Targets

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

https://pharmaceuticalintelligence.com/2013/12/08/developments-in-the-genomics-and-proteomics-of-type-2-diabetes-           mellitus-and-treatment-targets/

20. Loss of normal growth regulation

Larry H Bernstein, MD, FCAP, Curator

https://pharmaceuticalintelligence.com/2014/07/06/loss-of-normal-growth-regulation/

21. CT Angiography & TrueVision™ Metabolomics (Genomic Phenotyping) for new Therapeutic Targets to Atherosclerosis

Reporter: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/11/15/ct-angiography-truevision-metabolomics-genomic-phenotyping-for-           new-therapeutic-targets-to-atherosclerosis/

22.  CRACKING THE CODE OF HUMAN LIFE: The Birth of BioInformatics & Computational Genomics

Genomics Curator, Larry H Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2014/08/30/cracking-the-code-of-human-life-the-birth-of-bioinformatics-                      computational-genomics/

23. Big Data in Genomic Medicine

Author and Curator, Larry H Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2012/12/17/big-data-in-genomic-medicine/

24. From Genomics of Microorganisms to Translational Medicine

Author and Curator: Demet Sag, PhD

https://pharmaceuticalintelligence.com/2014/03/20/without-the-past-no-future-but-learn-and-move-genomics-of-                      microorganisms-to-translational-medicine/

25. Summary of Genomics and Medicine: Role in Cardiovascular Diseases

Author and Curator, Larry H Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2014/01/06/summary-of-genomics-and-medicine-role-in-cardiovascular-diseases/

 26. Genomic Promise for Neurodegenerative Diseases, Dementias, Autism Spectrum, Schizophrenia, and Serious                      Depression

Author and Curator, Larry H Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2013/02/19/genomic-promise-for-neurodegenerative-diseases-dementias-autism-        spectrum-schizophrenia-and-serious-depression/

 27.  BRCA1 a tumour suppressor in breast and ovarian cancer – functions in transcription, ubiquitination and DNA repair

Sudipta Saha, PhD

https://pharmaceuticalintelligence.com/2012/12/04/brca1-a-tumour-suppressor-in-breast-and-ovarian-cancer-functions-         in-transcription-ubiquitination-and-dna-repair/

28. Personalized medicine gearing up to tackle cancer

Ritu Saxena, PhD

https://pharmaceuticalintelligence.com/2013/01/07/personalized-medicine-gearing-up-to-tackle-cancer/

29. Differentiation Therapy – Epigenetics Tackles Solid Tumors

Stephen J Williams, PhD

      https://pharmaceuticalintelligence.com/2013/01/03/differentiation-therapy-epigenetics-tackles-solid-tumors/

30. Mechanism involved in Breast Cancer Cell Growth: Function in Early Detection & Treatment

     Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/01/17/mechanism-involved-in-breast-cancer-cell-growth-function-in-early-          detection-treatment/

31. The Molecular pathology of Breast Cancer Progression

Tilde Barliya, PhD

https://pharmaceuticalintelligence.com/2013/01/10/the-molecular-pathology-of-breast-cancer-progression

32. Gastric Cancer: Whole-genome reconstruction and mutational signatures

Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2012/12/24/gastric-cancer-whole-genome-reconstruction-and-mutational-                   signatures-2/

33. Paradigm Shift in Human Genomics – Predictive Biomarkers and Personalized Medicine –                                                       Part 1 (pharmaceuticalintelligence.com)

Aviva  Lev-Ari, PhD, RN

http://pharmaceuticalntelligence.com/2013/01/13/paradigm-shift-in-human-genomics-predictive-biomarkers-and-personalized-medicine-part-1/

34. LEADERS in Genome Sequencing of Genetic Mutations for Therapeutic Drug Selection in Cancer                                         Personalized Treatment: Part 2

A Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/01/13/leaders-in-genome-sequencing-of-genetic-mutations-for-therapeutic-       drug-selection-in-cancer-personalized-treatment-part-2/

35. Personalized Medicine: An Institute Profile – Coriell Institute for Medical Research: Part 3

Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/01/13/personalized-medicine-an-institute-profile-coriell-institute-for-medical-        research-part-3/

36. Harnessing Personalized Medicine for Cancer Management, Prospects of Prevention and Cure: Opinions of                           Cancer Scientific Leaders @http://pharmaceuticalintelligence.com

Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/01/13/7000/Harnessing_Personalized_Medicine_for_ Cancer_Management-      Prospects_of_Prevention_and_Cure/

37.  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”

Aviva Lev-Ari, PhD, RN

https://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/

38. Personalized medicine-based cure for cancer might not be far away

Ritu Saxena, PhD

  https://pharmaceuticalintelligence.com/2012/11/20/personalized-medicine-based-cure-for-cancer-might-not-be-far-away/

39. Human Variome Project: encyclopedic catalog of sequence variants indexed to the human genome sequence

Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2012/11/24/human-variome-project-encyclopedic-catalog-of-sequence-variants-         indexed-to-the-human-genome-sequence/

40. Inspiration From Dr. Maureen Cronin’s Achievements in Applying Genomic Sequencing to Cancer Diagnostics

Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/01/10/inspiration-from-dr-maureen-cronins-achievements-in-applying-                genomic-sequencing-to-cancer-diagnostics/

41. The “Cancer establishments” examined by James Watson, co-discoverer of DNA w/Crick, 4/1953

Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/01/09/the-cancer-establishments-examined-by-james-watson-co-discover-         of-dna-wcrick-41953/

42. What can we expect of tumor therapeutic response?

Author and curator: Larry H Bernstein, MD, FACP

https://pharmaceuticalintelligence.com/2012/12/05/what-can-we-expect-of-tumor-therapeutic-response/

43. Directions for genomics in personalized medicine

Author and Curator: Larry H. Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2013/01/27/directions-for-genomics-in-personalized-medicine/

44. How mobile elements in “Junk” DNA promote cancer. Part 1: Transposon-mediated tumorigenesis.

Stephen J Williams, PhD

https://pharmaceuticalintelligence.com/2012/10/31/how-mobile-elements-in-junk-dna-prote-cancer-part1-transposon-            mediated-tumorigenesis/

45. mRNA interference with cancer expression

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

 https://pharmaceuticalintelligence.com/2012/10/26/mrna-interference-with-cancer-expression/

46. Expanding the Genetic Alphabet and linking the genome to the metabolome

Aviva Lev-Ari, PhD, RD

https://pharmaceuticalintelligence.com/2012/09/24/expanding-the-genetic-alphabet-and-linking-the-genome-to-the-               metabolome/

47. Breast Cancer, drug resistance, and biopharmaceutical targets

Author and Curator: Larry H Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2012/09/18/breast-cancer-drug-resistance-and-biopharmaceutical-targets/

48.  Breast Cancer: Genomic profiling to predict Survival: Combination of Histopathology and Gene Expression                            Analysis

Aviva Lev-Ari, PhD, RD

https://pharmaceuticalintelligence.com/2012/12/24/breast-cancer-genomic-profiling-to-predict-survival-combination-of-           histopathology-and-gene-expression-analysis

49. Gastric Cancer: Whole-genome reconstruction and mutational signatures

Aviva  Lev-Ari, PhD, RD

https://pharmaceuticalintelligence.com/2012/12/24/gastric-cancer-whole-genome-reconstruction-and-mutational-                   signatures-2/

50. Genomic Analysis: FLUIDIGM Technology in the Life Science and Agricultural Biotechnology

Aviva Lev-Ari, PhD, RD

https://pharmaceuticalintelligence.com/2012/08/22/genomic-analysis-fluidigm-technology-in-the-life-science-and-                   agricultural-biotechnology/

51. 2013 Genomics: The Era Beyond the Sequencing Human Genome: Francis Collins, Craig Venter, Eric Lander, et al.

Aviva Lev-Ari, PhD, RD

https://pharmaceuticalintelligence.com/2013_Genomics

52. Paradigm Shift in Human Genomics – Predictive Biomarkers and Personalized Medicine – Part 1

Aviva Lev-Ari, PhD, RD

https://pharmaceuticalintelligence.com/Paradigm Shift in Human Genomics_/

Signaling Pathways

  1. Proteins and cellular adaptation to stress

Larry H Bernstein, MD, FCAP, Curator

https://pharmaceuticalintelligence.com/2014/07/08/proteins-and-cellular-adaptation-to-stress/

  1. A Synthesis of the Beauty and Complexity of How We View Cancer:
    Cancer Volume One – Summary

Author and Curator: Larry H. Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2014/03/26/a-synthesis-of-the-beauty-and-complexity-of-how-we-view-cancer/

  1. Recurrent somatic mutations in chromatin-remodeling and ubiquitin ligase complex genes in
    serous endometrial tumors

Sudipta Saha, PhD

https://pharmaceuticalintelligence.com/2012/11/19/recurrent-somatic-mutations-in-chromatin-remodeling-ad-ubiquitin-           ligase-complex-genes-in-serous-endometrial-tumors/

4.  Prostate Cancer Cells: Histone Deacetylase Inhibitors Induce Epithelial-to-Mesenchymal Transition

Stephen J Williams, PhD

https://pharmaceuticalintelligence.com/2012/11/30/histone-deacetylase-inhibitors-induce-epithelial-to-mesenchymal-              transition-in-prostate-cancer-cells/

5. Ubiquinin-Proteosome pathway, autophagy, the mitochondrion, proteolysis and cell apoptosis

Author and Curator: Larry H Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2012/10/30/ubiquinin-proteosome-pathway-autophagy-the-mitochondrion-                   proteolysis-and-cell-apoptosis/

6. Signaling and Signaling Pathways

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

https://pharmaceuticalintelligence.com/2014/08/12/signaling-and-signaling-pathways/

7.  Leptin signaling in mediating the cardiac hypertrophy associated with obesity

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

https://pharmaceuticalintelligence.com/2013/11/03/leptin-signaling-in-mediating-the-cardiac-hypertrophy-associated-            with-obesity/

  1. Sensors and Signaling in Oxidative Stress

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

https://pharmaceuticalintelligence.com/2013/11/01/sensors-and-signaling-in-oxidative-stress/

  1. The Final Considerations of the Role of Platelets and Platelet Endothelial Reactions in Atherosclerosis and Novel
    Treatments

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

https://pharmaceuticalintelligence.com/2013/10/15/the-final-considerations-of-the-role-of-platelets-and-platelet-                      endothelial-reactions-in-atherosclerosis-and-novel-treatments

10.   Platelets in Translational Research – Part 1

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

https://pharmaceuticalintelligence.com/2013/10/07/platelets-in-translational-research-1/

11.  Disruption of Calcium Homeostasis: Cardiomyocytes and Vascular Smooth Muscle Cells: The Cardiac and
Cardiovascular Calcium Signaling Mechanism

Author and Curator: Larry H Bernstein, MD, FCAP, Author, and Content Consultant to e-SERIES A:
Cardiovascular Diseases: Justin Pearlman, MD, PhD, FACC and Curator: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/09/12/disruption-of-calcium-homeostasis-cardiomyocytes-and-vascular-             smooth-muscle-cells-the-cardiac-and-cardiovascular-calcium-signaling-mechanism/

12. The Centrality of Ca(2+) Signaling and Cytoskeleton Involving Calmodulin Kinases and
Ryanodine Receptors in Cardiac Failure, Arterial Smooth Muscle, Post-ischemic Arrhythmia,
Similarities and Differences, and Pharmaceutical Targets

     Author and Curator: Larry H Bernstein, MD, FCAP, Author, and Content Consultant to
e-SERIES A: Cardiovascular Diseases: Justin Pearlman, MD, PhD, FACC and
Curator: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/09/08/the-centrality-of-ca2-signaling-and-cytoskeleton-involving-calmodulin-       kinases-and-ryanodine-receptors-in-cardiac-failure-arterial-smooth-muscle-post-ischemic-arrhythmia-similarities-and-           differen/

13.  Nitric Oxide Signalling Pathways

Aviral Vatsa, PhD, MBBS

https://pharmaceuticalintelligence.com/2012/08/22/nitric-oxide-signalling-pathways/

14. Immune activation, immunity, antibacterial activity

Larry H. Bernstein, MD, FCAP, Curator

https://pharmaceuticalintelligence.com/2014/07/06/immune-activation-immunity-antibacterial-activity/

15.  Regulation of somatic stem cell Function

Larry H. Bernstein, MD, FCAP, Writer and Curator    Aviva Lev-Ari, PhD, RN, Curator

https://pharmaceuticalintelligence.com/2014/07/29/regulation-of-somatic-stem-cell-function/

16. Scientists discover that pluripotency factor NANOG is also active in adult organisms

Larry H. Bernstein, MD, FCAP, Reporter

https://pharmaceuticalintelligence.com/2014/07/10/scientists-discover-that-pluripotency-factor-nanog-is-also-active-in-adult-organisms/

Read Full Post »

Summary of Translational Medicine – e-Series A: Cardiovascular Diseases, Volume Four – Part 1


Summary of Translational Medicine – e-Series A: Cardiovascular Diseases, Volume Four – Part 1

Author and Curator: Larry H Bernstein, MD, FCAP

and

Curator: Aviva Lev-Ari, PhD, RN

 

Part 1 of Volume 4 in the e-series A: Cardiovascular Diseases and Translational Medicine, provides a foundation for grasping a rapidly developing surging scientific endeavor that is transcending laboratory hypothesis testing and providing guidelines to:

  • Target genomes and multiple nucleotide sequences involved in either coding or in regulation that might have an impact on complex diseases, not necessarily genetic in nature.
  • Target signaling pathways that are demonstrably maladjusted, activated or suppressed in many common and complex diseases, or in their progression.
  • Enable a reduction in failure due to toxicities in the later stages of clinical drug trials as a result of this science-based understanding.
  • Enable a reduction in complications from the improvement of machanical devices that have already had an impact on the practice of interventional procedures in cardiology, cardiac surgery, and radiological imaging, as well as improving laboratory diagnostics at the molecular level.
  • Enable the discovery of new drugs in the continuing emergence of drug resistance.
  • Enable the construction of critical pathways and better guidelines for patient management based on population outcomes data, that will be critically dependent on computational methods and large data-bases.

What has been presented can be essentially viewed in the following Table:

 

Summary Table for TM - Part 1

Summary Table for TM – Part 1

 

 

 

There are some developments that deserve additional development:

1. The importance of mitochondrial function in the activity state of the mitochondria in cellular work (combustion) is understood, and impairments of function are identified in diseases of muscle, cardiac contraction, nerve conduction, ion transport, water balance, and the cytoskeleton – beyond the disordered metabolism in cancer.  A more detailed explanation of the energetics that was elucidated based on the electron transport chain might also be in order.

2. The processes that are enabling a more full application of technology to a host of problems in the environment we live in and in disease modification is growing rapidly, and will change the face of medicine and its allied health sciences.

 

Electron Transport and Bioenergetics

Deferred for metabolomics topic

Synthetic Biology

Introduction to Synthetic Biology and Metabolic Engineering

Kristala L. J. Prather: Part-1    <iBiology > iBioSeminars > Biophysics & Chemical Biology >

http://www.ibiology.org Lecturers generously donate their time to prepare these lectures. The project is funded by NSF and NIGMS, and is supported by the ASCB and HHMI.
Dr. Prather explains that synthetic biology involves applying engineering principles to biological systems to build “biological machines”.

Dr. Prather has received numerous awards both for her innovative research and for excellence in teaching.  Learn more about how Kris became a scientist at
Prather 1: Synthetic Biology and Metabolic Engineering  2/6/14IntroductionLecture Overview In the first part of her lecture, Dr. Prather explains that synthetic biology involves applying engineering principles to biological systems to build “biological machines”. The key material in building these machines is synthetic DNA. Synthetic DNA can be added in different combinations to biological hosts, such as bacteria, turning them into chemical factories that can produce small molecules of choice. In Part 2, Prather describes how her lab used design principles to engineer E. coli that produce glucaric acid from glucose. Glucaric acid is not naturally produced in bacteria, so Prather and her colleagues “bioprospected” enzymes from other organisms and expressed them in E. coli to build the needed enzymatic pathway. Prather walks us through the many steps of optimizing the timing, localization and levels of enzyme expression to produce the greatest yield. Speaker Bio: Kristala Jones Prather received her S.B. degree from the Massachusetts Institute of Technology and her PhD at the University of California, Berkeley both in chemical engineering. Upon graduation, Prather joined the Merck Research Labs for 4 years before returning to academia. Prather is now an Associate Professor of Chemical Engineering at MIT and an investigator with the multi-university Synthetic Biology Engineering Reseach Center (SynBERC). Her lab designs and constructs novel synthetic pathways in microorganisms converting them into tiny factories for the production of small molecules. Dr. Prather has received numerous awards both for her innovative research and for excellence in teaching.

VIEW VIDEOS

https://www.youtube.com/watch?feature=player_embedded&v=ndThuqVumAk#t=0

https://www.youtube.com/watch?feature=player_embedded&v=ndThuqVumAk#t=12

https://www.youtube.com/watch?feature=player_embedded&v=ndThuqVumAk#t=74

https://www.youtube.com/watch?feature=player_embedded&v=ndThuqVumAk#t=129

https://www.youtube.com/watch?feature=player_embedded&v=ndThuqVumAk#t=168

https://www.youtube.com/watch?feature=player_embedded&v=ndThuqVumAk

 

II. Regulatory Effects of Mammalian microRNAs

Calcium Cycling in Synthetic and Contractile Phasic or Tonic Vascular Smooth Muscle Cells

in INTECH
Current Basic and Pathological Approaches to
the Function of Muscle Cells and Tissues – From Molecules to HumansLarissa Lipskaia, Isabelle Limon, Regis Bobe and Roger Hajjar
Additional information is available at the end of the chapter
http://dx.doi.org/10.5772/48240
1. Introduction
Calcium ions (Ca ) are present in low concentrations in the cytosol (~100 nM) and in high concentrations (in mM range) in both the extracellular medium and intracellular stores (mainly sarco/endo/plasmic reticulum, SR). This differential allows the calcium ion messenger that carries information
as diverse as contraction, metabolism, apoptosis, proliferation and/or hypertrophic growth. The mechanisms responsible for generating a Ca signal greatly differ from one cell type to another.
In the different types of vascular smooth muscle cells (VSMC), enormous variations do exist with regard to the mechanisms responsible for generating Ca signal. In each VSMC phenotype (synthetic/proliferating and contractile [1], tonic or phasic), the Ca signaling system is adapted to its particular function and is due to the specific patterns of expression and regulation of Ca.
For instance, in contractile VSMCs, the initiation of contractile events is driven by mem- brane depolarization; and the principal entry-point for extracellular Ca is the voltage-operated L-type calcium channel (LTCC). In contrast, in synthetic/proliferating VSMCs, the principal way-in for extracellular Ca is the store-operated calcium (SOC) channel.
Whatever the cell type, the calcium signal consists of  limited elevations of cytosolic free calcium ions in time and space. The calcium pump, sarco/endoplasmic reticulum Ca ATPase (SERCA), has a critical role in determining the frequency of SR Ca release by upload into the sarcoplasmic
sensitivity of  SR calcium channels, Ryanodin Receptor, RyR and Inositol tri-Phosphate Receptor, IP3R.
Synthetic VSMCs have a fibroblast appearance, proliferate readily, and synthesize increased levels of various extracellular matrix components, particularly fibronectin, collagen types I and III, and tropoelastin [1].
Contractile VSMCs have a muscle-like or spindle-shaped appearance and well-developed contractile apparatus resulting from the expression and intracellular accumulation of thick and thin muscle filaments [1].
Schematic representation of Calcium Cycling in Contractile and Proliferating VSMCs

Schematic representation of Calcium Cycling in Contractile and Proliferating VSMCs

 

Figure 1. Schematic representation of Calcium Cycling in Contractile and Proliferating VSMCs.

Left panel: schematic representation of calcium cycling in quiescent /contractile VSMCs. Contractile re-sponse is initiated by extracellular Ca influx due to activation of Receptor Operated Ca (through phosphoinositol-coupled receptor) or to activation of L-Type Calcium channels (through an increase in luminal pressure). Small increase of cytosolic due IP3 binding to IP3R (puff) or RyR activation by LTCC or ROC-dependent Ca influx leads to large SR Ca IP3R or RyR clusters (“Ca -induced Ca SR calcium pumps (both SERCA2a and SERCA2b are expressed in quiescent VSMCs), maintaining high concentration of cytosolic Ca and setting the sensitivity of RyR or IP3R for the next spike.
Contraction of VSMCs occurs during oscillatory Ca transient.
Middle panel: schematic representa tion of atherosclerotic vessel wall. Contractile VSMC are located in the media layer, synthetic VSMC are located in sub-endothelial intima.
Right panel: schematic representation of calcium cycling in quiescent /contractile VSMCs. Agonist binding to phosphoinositol-coupled receptor leads to the activation of IP3R resulting in large increase in cytosolic Ca calcium pumps (only SERCA2b, having low turnover and low affinity to Ca depletion leads to translocation of SR Ca sensor STIM1 towards PM, resulting in extracellular Ca influx though opening of Store Operated Channel (CRAC). Resulted steady state Ca transient is critical for activation of proliferation-related transcription factors ‘NFAT).
Abbreviations: PLC – phospholipase C; PM – plasma membrane; PP2B – Ca /calmodulin-activated protein phosphatase 2B (calcineurin); ROC- receptor activated channel; IP3 – inositol-1,4,5-trisphosphate, IP3R – inositol-1,4,5- trisphosphate receptor; RyR – ryanodine receptor; NFAT – nuclear factor of activated T-lymphocytes; VSMC – vascular smooth muscle cells; SERCA – sarco(endo)plasmic reticulum Ca sarcoplasmic reticulum.

 

Time for New DNA Synthesis and Sequencing Cost Curves

By Rob Carlson

I’ll start with the productivity plot, as this one isn’t new. For a discussion of the substantial performance increase in sequencing compared to Moore’s Law, as well as the difficulty of finding this data, please see this post. If nothing else, keep two features of the plot in mind: 1) the consistency of the pace of Moore’s Law and 2) the inconsistency and pace of sequencing productivity. Illumina appears to be the primary driver, and beneficiary, of improvements in productivity at the moment, especially if you are looking at share prices. It looks like the recently announced NextSeq and Hiseq instruments will provide substantially higher productivities (hand waving, I would say the next datum will come in another order of magnitude higher), but I think I need a bit more data before officially putting another point on the plot.

 

cost-of-oligo-and-gene-synthesis

cost-of-oligo-and-gene-synthesis

Illumina’s instruments are now responsible for such a high percentage of sequencing output that the company is effectively setting prices for the entire industry. Illumina is being pushed by competition to increase performance, but this does not necessarily translate into lower prices. It doesn’t behoove Illumina to drop prices at this point, and we won’t see any substantial decrease until a serious competitor shows up and starts threatening Illumina’s market share. The absence of real competition is the primary reason sequencing prices have flattened out over the last couple of data points.

Note that the oligo prices above are for column-based synthesis, and that oligos synthesized on arrays are much less expensive. However, array synthesis comes with the usual caveat that the quality is generally lower, unless you are getting your DNA from Agilent, which probably means you are getting your dsDNA from Gen9.

Note also that the distinction between the price of oligos and the price of double-stranded sDNA is becoming less useful. Whether you are ordering from Life/Thermo or from your local academic facility, the cost of producing oligos is now, in most cases, independent of their length. That’s because the cost of capital (including rent, insurance, labor, etc) is now more significant than the cost of goods. Consequently, the price reflects the cost of capital rather than the cost of goods. Moreover, the cost of the columns, reagents, and shipping tubes is certainly more than the cost of the atoms in the sDNA you are ostensibly paying for. Once you get into longer oligos (substantially larger than 50-mers) this relationship breaks down and the sDNA is more expensive. But, at this point in time, most people aren’t going to use longer oligos to assemble genes unless they have a tricky job that doesn’t work using short oligos.

Looking forward, I suspect oligos aren’t going to get much cheaper unless someone sorts out how to either 1) replace the requisite human labor and thereby reduce the cost of capital, or 2) finally replace the phosphoramidite chemistry that the industry relies upon.

IDT’s gBlocks come at prices that are constant across quite substantial ranges in length. Moreover, part of the decrease in price for these products is embedded in the fact that you are buying smaller chunks of DNA that you then must assemble and integrate into your organism of choice.

Someone who has purchased and assembled an absolutely enormous amount of sDNA over the last decade, suggested that if prices fell by another order of magnitude, he could switch completely to outsourced assembly. This is a potentially interesting “tipping point”. However, what this person really needs is sDNA integrated in a particular way into a particular genome operating in a particular host. The integration and testing of the new genome in the host organism is where most of the cost is. Given the wide variety of emerging applications, and the growing array of hosts/chassis, it isn’t clear that any given technology or firm will be able to provide arbitrary synthetic sequences incorporated into arbitrary hosts.

 TrackBack URL: http://www.synthesis.cc/cgi-bin/mt/mt-t.cgi/397

 

Startup to Strengthen Synthetic Biology and Regenerative Medicine Industries with Cutting Edge Cell Products

28 Nov 2013 | PR Web

Dr. Jon Rowley and Dr. Uplaksh Kumar, Co-Founders of RoosterBio, Inc., a newly formed biotech startup located in Frederick, are paving the way for even more innovation in the rapidly growing fields of Synthetic Biology and Regenerative Medicine. Synthetic Biology combines engineering principles with basic science to build biological products, including regenerative medicines and cellular therapies. Regenerative medicine is a broad definition for innovative medical therapies that will enable the body to repair, replace, restore and regenerate damaged or diseased cells, tissues and organs. Regenerative therapies that are in clinical trials today may enable repair of damaged heart muscle following heart attack, replacement of skin for burn victims, restoration of movement after spinal cord injury, regeneration of pancreatic tissue for insulin production in diabetics and provide new treatments for Parkinson’s and Alzheimer’s diseases, to name just a few applications.

While the potential of the field is promising, the pace of development has been slow. One main reason for this is that the living cells required for these therapies are cost-prohibitive and not supplied at volumes that support many research and product development efforts. RoosterBio will manufacture large quantities of standardized primary cells at high quality and low cost, which will quicken the pace of scientific discovery and translation to the clinic. “Our goal is to accelerate the development of products that incorporate living cells by providing abundant, affordable and high quality materials to researchers that are developing and commercializing these regenerative technologies” says Dr. Rowley

 

Life at the Speed of Light

http://kcpw.org/?powerpress_pinw=92027-podcast

NHMU Lecture featuring – J. Craig Venter, Ph.D.
Founder, Chairman, and CEO – J. Craig Venter Institute; Co-Founder and CEO, Synthetic Genomics Inc.

J. Craig Venter, Ph.D., is Founder, Chairman, and CEO of the J. Craig Venter Institute (JVCI), a not-for-profit, research organization dedicated to human, microbial, plant, synthetic and environmental research. He is also Co-Founder and CEO of Synthetic Genomics Inc. (SGI), a privately-held company dedicated to commercializing genomic-driven solutions to address global needs.

In 1998, Dr. Venter founded Celera Genomics to sequence the human genome using new tools and techniques he and his team developed.  This research culminated with the February 2001 publication of the human genome in the journal, Science. Dr. Venter and his team at JVCI continue to blaze new trails in genomics.  They have sequenced and a created a bacterial cell constructed with synthetic DNA,  putting humankind at the threshold of a new phase of biological research.  Whereas, we could  previously read the genetic code (sequencing genomes), we can now write the genetic code for designing new species.

The science of synthetic genomics will have a profound impact on society, including new methods for chemical and energy production, human health and medical advances, clean water, and new food and nutritional products. One of the most prolific scientists of the 21st century for his numerous pioneering advances in genomics,  he  guides us through this emerging field, detailing its origins, current challenges, and the potential positive advances.

His work on synthetic biology truly embodies the theme of “pushing the boundaries of life.”  Essentially, Venter is seeking to “write the software of life” to create microbes designed by humans rather than only through evolution. The potential benefits and risks of this new technology are enormous. It also requires us to examine, both scientifically and philosophically, the question of “What is life?”

J Craig Venter wants to digitize DNA and transmit the signal to teleport organisms

https://pharmaceuticalintelligence.com/2013/11/01/j-craig-venter-wants-to-digitize-dna-and-transmit-the-signal-to-teleport-organisms/

2013 Genomics: The Era Beyond the Sequencing of the Human Genome: Francis Collins, Craig Venter, Eric Lander, et al.

https://pharmaceuticalintelligence.com/2013/02/11/2013-genomics-the-era-beyond-the-sequencing-human-genome-francis-collins-craig-venter-eric-lander-et-al/

Human Longevity Inc (HLI) – $70M in Financing of Venter’s New Integrative Omics and Clinical Bioinformatics

https://pharmaceuticalintelligence.com/2014/03/05/human-longevity-inc-hli-70m-in-financing-of-venters-new-integrative-omics-and-clinical-bioinformatics/

 

 

Where Will the Century of Biology Lead Us?

By Randall Mayes

A technology trend analyst offers an overview of synthetic biology, its potential applications, obstacles to its development, and prospects for public approval.

  • In addition to boosting the economy, synthetic biology projects currently in development could have profound implications for the future of manufacturing, sustainability, and medicine.
  • Before society can fully reap the benefits of synthetic biology, however, the field requires development and faces a series of hurdles in the process. Do researchers have the scientific know-how and technical capabilities to develop the field?

Biology + Engineering = Synthetic Biology

Bioengineers aim to build synthetic biological systems using compatible standardized parts that behave predictably. Bioengineers synthesize DNA parts—oligonucleotides composed of 50–100 base pairs—which make specialized components that ultimately make a biological system. As biology becomes a true engineering discipline, bioengineers will create genomes using mass-produced modular units similar to the microelectronics and computer industries.

Currently, bioengineering projects cost millions of dollars and take years to develop products. For synthetic biology to become a Schumpeterian revolution, smaller companies will need to be able to afford to use bioengineering concepts for industrial applications. This will require standardized and automated processes.

A major challenge to developing synthetic biology is the complexity of biological systems. When bioengineers assemble synthetic parts, they must prevent cross talk between signals in other biological pathways. Until researchers better understand these undesired interactions that nature has already worked out, applications such as gene therapy will have unwanted side effects. Scientists do not fully understand the effects of environmental and developmental interaction on gene expression. Currently, bioengineers must repeatedly use trial and error to create predictable systems.

Similar to physics, synthetic biology requires the ability to model systems and quantify relationships between variables in biological systems at the molecular level.

The second major challenge to ensuring the success of synthetic biology is the development of enabling technologies. With genomes having billions of nucleotides, this requires fast, powerful, and cost-efficient computers. Moore’s law, named for Intel co-founder Gordon Moore, posits that computing power progresses at a predictable rate and that the number of components in integrated circuits doubles each year until its limits are reached. Since Moore’s prediction, computer power has increased at an exponential rate while pricing has declined.

DNA sequencers and synthesizers are necessary to identify genes and make synthetic DNA sequences. Bioengineer Robert Carlson calculated that the capabilities of DNA sequencers and synthesizers have followed a pattern similar to computing. This pattern, referred to as the Carlson Curve, projects that scientists are approaching the ability to sequence a human genome for $1,000, perhaps in 2020. Carlson calculated that the costs of reading and writing new genes and genomes are falling by a factor of two every 18–24 months. (see recent Carlson comment on requirement to read and write for a variety of limiting  conditions).

Startup to Strengthen Synthetic Biology and Regenerative Medicine Industries with Cutting Edge Cell Products

https://pharmaceuticalintelligence.com/2013/11/28/startup-to-strengthen-synthetic-biology-and-regenerative-medicine-industries-with-cutting-edge-cell-products/

Synthetic Biology: On Advanced Genome Interpretation for Gene Variants and Pathways: What is the Genetic Base of Atherosclerosis and Loss of Arterial Elasticity with Aging

https://pharmaceuticalintelligence.com/2013/05/17/synthetic-biology-on-advanced-genome-interpretation-for-gene-variants-and-pathways-what-is-the-genetic-base-of-atherosclerosis-and-loss-of-arterial-elasticity-with-aging/

Synthesizing Synthetic Biology: PLOS Collections

https://pharmaceuticalintelligence.com/2012/08/17/synthesizing-synthetic-biology-plos-collections/

Capturing ten-color ultrasharp images of synthetic DNA structures resembling numerals 0 to 9

https://pharmaceuticalintelligence.com/2014/02/05/capturing-ten-color-ultrasharp-images-of-synthetic-dna-structures-resembling-numerals-0-to-9/

Silencing Cancers with Synthetic siRNAs

https://pharmaceuticalintelligence.com/2013/12/09/silencing-cancers-with-synthetic-sirnas/

Genomics Now—and Beyond the Bubble

Futurists have touted the twenty-first century as the century of biology based primarily on the promise of genomics. Medical researchers aim to use variations within genes as biomarkers for diseases, personalized treatments, and drug responses. Currently, we are experiencing a genomics bubble, but with advances in understanding biological complexity and the development of enabling technologies, synthetic biology is reviving optimism in many fields, particularly medicine.

BY MICHAEL BROOKS    17 APR, 2014     http://www.newstatesman.com/

Michael Brooks holds a PhD in quantum physics. He writes a weekly science column for the New Statesman, and his most recent book is The Secret Anarchy of Science.

The basic idea is that we take an organism – a bacterium, say – and re-engineer its genome so that it does something different. You might, for instance, make it ingest carbon dioxide from the atmosphere, process it and excrete crude oil.

That project is still under construction, but others, such as using synthesised DNA for data storage, have already been achieved. As evolution has proved, DNA is an extraordinarily stable medium that can preserve information for millions of years. In 2012, the Harvard geneticist George Church proved its potential by taking a book he had written, encoding it in a synthesised strand of DNA, and then making DNA sequencing machines read it back to him.

When we first started achieving such things it was costly and time-consuming and demanded extraordinary resources, such as those available to the millionaire biologist Craig Venter. Venter’s team spent most of the past two decades and tens of millions of dollars creating the first artificial organism, nicknamed “Synthia”. Using computer programs and robots that process the necessary chemicals, the team rebuilt the genome of the bacterium Mycoplasma mycoides from scratch. They also inserted a few watermarks and puzzles into the DNA sequence, partly as an identifying measure for safety’s sake, but mostly as a publicity stunt.

What they didn’t do was redesign the genome to do anything interesting. When the synthetic genome was inserted into an eviscerated bacterial cell, the new organism behaved exactly the same as its natural counterpart. Nevertheless, that Synthia, as Venter put it at the press conference to announce the research in 2010, was “the first self-replicating species we’ve had on the planet whose parent is a computer” made it a standout achievement.

Today, however, we have entered another era in synthetic biology and Venter faces stiff competition. The Steve Jobs to Venter’s Bill Gates is Jef Boeke, who researches yeast genetics at New York University.

Boeke wanted to redesign the yeast genome so that he could strip out various parts to see what they did. Because it took a private company a year to complete just a small part of the task, at a cost of $50,000, he realised he should go open-source. By teaching an undergraduate course on how to build a genome and teaming up with institutions all over the world, he has assembled a skilled workforce that, tinkering together, has made a synthetic chromosome for baker’s yeast.

 

Stepping into DIYbio and Synthetic Biology at ScienceHack

Posted April 22, 2014 by Heather McGaw and Kyrie Vala-Webb

We got a crash course on genetics and protein pathways, and then set out to design and build our own pathways using both the “Genomikon: Violacein Factory” kit and Synbiota platform. With Synbiota’s software, we dragged and dropped the enzymes to create the sequence that we were then going to build out. After a process of sketching ideas, mocking up pathways, and writing hypotheses, we were ready to start building!

The night stretched long, and at midnight we were forced to vacate the school. Not quite finished, we loaded our delicate bacteria, incubator, and boxes of gloves onto the bus and headed back to complete our bacterial transformation in one of our hotel rooms. Jammed in between the beds and the mini-fridge, we heat-shocked our bacteria in the hotel ice bucket. It was a surreal moment.

While waiting for our bacteria, we held an “unconference” where we explored bioethics, security and risk related to synthetic biology, 3D printing on Mars, patterns in juggling (with live demonstration!), and even did a Google Hangout with Rob Carlson. Every few hours, we would excitedly check in on our bacteria, looking for bacterial colonies and the purple hue characteristic of violacein.

Most impressive was the wildly successful and seamless integration of a diverse set of people: in a matter of hours, we were transformed from individual experts and practitioners in assorted fields into cohesive and passionate teams of DIY biologists and science hackers. The ability of everyone to connect and learn was a powerful experience, and over the course of just one weekend we were able to challenge each other and grow.

Returning to work on Monday, we were hungry for more. We wanted to find a way to bring the excitement and energy from the weekend into the studio and into the projects we’re working on. It struck us that there are strong parallels between design and DIYbio, and we knew there was an opportunity to bring some of the scientific approaches and curiosity into our studio.

 

 

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Myocardial Damage in Cardiovascular Disease: Circulating MicroRNA-208b and MicroRNA-499

Reporter: Aviva Lev-Ari, PhD, RN

Circulating MicroRNA-208b and MicroRNA-499 Reflect Myocardial Damage in Cardiovascular Disease

Maarten F. Corsten, MD, Robert Dennert, MD, Sylvia Jochems, BSc, Tatiana Kuznetsova, MD, PhD, Yvan Devaux, PhD, Leon Hofstra, MD, PhD, Daniel R. Wagner, MD, PhD, Jan A. Staessen, MD, PhD, Stephane Heymans, MD, PhD and Blanche Schroen, PhD

Author Affiliations

From the Center for Heart Failure Research (M.F.C., R.D., S.J., S.H., B.S.), Cardiovascular Research Institute, Maastricht, The Netherlands; the Division of Hypertension and Cardiovascular Rehabilitation (T.K., J.A.S.), Department of Cardiovascular Diseases, University of Leuven, Leuven, Belgium and Department of Epidemiology, Maastricht University Medical Center, Maastricht, The Netherlands; Centre de Recherche Public–Santé, Luxembourg (Y.D., D.R.W.), Luxembourg; Maastricht University Medical Center (L.H.), Maastricht, The Netherlands; and Centre Hospitalier Luxembourg (D.R.W.), Luxembourg.

Correspondence to Blanche Schroen, PhD, Center for Heart Failure Research, Cardiovascular Research Institute Maastricht, Universiteitssingel 50, 6229 ER Maastricht, The Netherlands. E-mail b.schroen@cardio.unimaas.nl

Drs Heymans and Schroen contributed equally to this work.

Abstract

Background— Small RNA molecules, called microRNAs, freely circulate in human plasma and correlate with varying pathologies. In this study, we explored their diagnostic potential in a selection of prevalent cardiovascular disorders.

Methods and Results— MicroRNAs were isolated from plasmas from well-characterized patients with varying degrees of cardiac damage:

(1) acute myocardial infarction,

(2) viral myocarditis,

(3) diastolic dysfunction, and

(4) acute heart failure.

Plasma levels of selected microRNAs, including heart-associated (miR-1, -133a, -208b, and -499), fibrosis-associated (miR-21 and miR-29b), and leukocyte-associated (miR-146, -155, and -223) candidates, were subsequently assessed using real-time polymerase chain reaction. 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<1010) for miR-208b and 0.92 (P<109) for miR-499. Both microRNAs correlated with plasma troponin T, indicating release of microRNAs from injured cardiomyocytes. In viral myocarditis, we observed a milder but significant elevation of these microRNAs, 30-fold and 6-fold, respectively. Plasma levels of leukocyte-expressed microRNAs were not significantly increased in acute myocardial infarction or viral myocarditis patients, despite elevated white blood cell counts. 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.

Conclusions— Cardiac damage initiates the detectable release of cardiomyocyte-specific microRNAs-208b and -499 into the circulation.

SOURCE:

Circulation: Cardiovascular Genetics. 2010; 3: 499-506

Published online before print October 4, 2010,

doi: 10.1161/ CIRCGENETICS.110.957415

 

 

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MicroRNA in Serum as Biomarker for Cardiovascular Pathologies: acute myocardial infarction, viral myocarditis,  diastolic dysfunction, and acute heart failure

Reporter: Aviva Lev-Ari, PhD, RN

Increased MicroRNA-1 and MicroRNA-133a Levels in Serum of Patients With Cardiovascular Disease Indicate Myocardial Damage

Yasuhide Kuwabara, MD, Koh Ono, MD, PhD, Takahiro Horie, MD, PhD, Hitoo Nishi, MD, PhD, Kazuya Nagao, MD, PhD, Minako Kinoshita, MD, PhD, Shin Watanabe, MD, PhD, Osamu Baba, MD, Yoji Kojima, MD, PhD, Satoshi Shizuta, MD, Masao Imai, MD,Toshihiro Tamura, MD, Toru Kita, MD, PhD and Takeshi Kimura, MD, PhD

Author Affiliations

From the Department of Cardiovascular Medicine, Graduate School of Medicine, Kyoto University, Kyoto, Japan (Y. Kuwabara, K.O., T.H., H.N., K.N., M.K., S.W., O.B., Y. Kojima, S.S., M.I., T.T., T. Kimura); and Kobe City Medical Center General Hospital, Kobe, Japan (T. Kita).

Correspondence to Koh Ono, MD, PhD, Department of Cardiovascular Medicine, Graduate School of Medicine, Kyoto University, 54 Shogoin-kawahara-cho, Sakyo-ku, Kyoto, Japan 606-8507. E-mail kohono@kuhp.kyoto-u.ac.jp

Abstract

Background—Recently, elevation of circulating muscle-specific microRNA (miRNA) levels has been reported in patients with acute myocardial infarction. However, it is still unclear from which part of the myocardium or under what conditions miRNAs are released into circulating blood. The purpose of this study was to identify the source of elevated levels of circulating miRNAs and their function in cardiovascular diseases.

Conclusions—These results suggest that elevated levels of circulating miRNA-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.

SOURCE:

Circulation: Cardiovascular Genetics. 2011; 4: 446-454

Published online before print June 2, 2011,

doi: 10.1161/ CIRCGENETICS.110.958975

 

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Genome Jigsaws

Genome Jigsaws (Photo credit: dullhunk)

Sequencing became the household name.  In 2000s, it was thought to be the key of the Pandora’s box for cure.  Then, after completion of Human Genome Projects showed that there are less number of genes than expected.  This outcome induce to originate yet another set of sequencing programs and collaborations around the world, such as Human Protein Project, Human Microorganisms Projects, ENCODE, Transcriptome Sequencing and Consortiums etc.

It is in humankind to believe in magic and illusion.  The strength of biological diversity and complex mechanism of expression may chalanges the set up of a simple but informative specific essay.  Thus, there is a new developing field to mash rules of biology with mathematical formulas to develop the best bioinformatics or also called computational biology.  Predicting transcription start or termination sites, exon boundaries, possible binding sites of transcription regulators for chromatin modification activities, like histone acetylates and enhancer- and insulator-associated factors based on the human genome sequence.  Deep in mind, this assumption supports that the sequence contains signatures for chromatin modifications essential for gene regulation and development.

There are three primary colors, red, yellow and blue, however, an artist can create many shades. Recently, scientists combining and organizing more data to make sense of our blueprint of life to transfer info generation to generation with the hope to cure diseases of human kind.

Analyzing genome and transcriptome open the door.  These studies suggested that all eukaryotic cells has a rich portfolio of RNAs. Among these long non-coding RNAs has impact on protein coding gene expression, regulating multiple processes even including epigenetic gene expression.

Epigenetics, stemness and non-coding RNAs  play a great role to manipulate and correct the gene expression not only at a proper cell type but also location and time within genome without disturbing the host.

Main concern is differentiation of embryonic stem cells under these epigenetics and influencers.  The best known post-transcriptional modifications, which include methylation, acetylation, ubiquination, and SUMOylation of lysine residues, methylation of arginine residues, and phosphorylation of serines, occur on histone tails. “Epi” means “top” or
“above” so this mechanism give a new direction to the genetic pathways as long as the organism live sometime and may lead into evolutions.  It is critical to show the complexity of
mechanism and relativity of a gene role with a single example for each. 

For example,  DNA methylation occurs mostly on cytosine residues on the CpG islands usually located on promoter regions that are associated with tissue-specific gene expression.  However, there are many other forms of DNA methylations, such as  monoallelic methylation in gene imprinting and inactivation of the X chromosome,  in repetitive elements, like transposons.  There are two main mechanisms but this is not our main topic.  Yet, Myc and hypoxia-inducible factor-1α versus certain methyl-CpG-binding proteins, such as MBD1,MBD2, MBD4, MeCP2, and Kaiso works differently.

Stemness is an important factor for an intervention to correct a pathological condition. In terms of epigenetics, regulation and non-coding RNA Vascular endothelial growth factor A (VEGF-A) is an interesting example for differentiation of endothelial cells and morphogenesis of the vascular system during development with several reasons, epigenetics, gene interactions, time and space.  Everything has to be just right, because neither less nor too much can fulfill the destiny to become a complete adult cell or an organism.   For example, both having only one VEGF-A allele and having two-fold excess of VEGF-A results in death during early embryogenesis, since mice can’t develop proper vascular network.  However, explaining diverse mechanisms and functions of VEGF-A is require more information with specific details.  VEGF-A plays many roles in many pathological cases, such as cancer, inflammation, retinopathies, and arthritis because VEGF-A has also function in epigenetic reprogramming of the promoter regions of Rex1 and Oct4 genes, that are critical for a stem cell. Preferred mechanism is anti-angiogeneic state but tumor cells prefer hypermethylation to induce pro-angiogeneic state, thus VEGF-A stimulates PIGF in tumour cells among many other factors.

Now, let’s turn around to observe development of a cell with Polycomb repressive complexes (PRCs) because they are important chromatin regulators of embryonic stem (ES) cell function.  Originally, RYBP shown to function  as transcriptional repressor in reporter assays from both in tissue culture cells and in fruit fly (Drosophila melanogaster ) and as a direct interactor with Ring1A during embryogenesis through methylation. In addition, RYBP in epigenetic resetting during preimplantation development through repression of germ line genes and PcG targets before formation of pluripotent epiblast cells.  However, I do believe that the most important element is efficient repression of endogenous retroviruses (murine endogenous retrovirus called MuERV class),  preimplantation containing zygotic genome activation stage and germ line specific genes. The selective repressor activity of  RYBP  is in the ES cell state. When RYBP−/− ES cells were analyzed by measuring gene expression during differentiation as embryo bodies formed from mutant and wild-type cells, the result presented that  expression of pluripotency genes Oct4 and Nanog was usually downregulated. However, RYBP is able to bind genomic regions independently of H3K27me3 and there is no relation between altered RYBP binding in Dnmt1-mutant cells to DNA methylation status. In sum, RYBP has a large value in undifferentiated ES cells and may affect or even reset epigenetic landscape during early developmental stages. These are the gaps filled by long non coding RNAs.

We learn more compelling information by comparing and contrasting what is normal and what is abnormal. As a result, pathology is a key learning canvas for basic mechanisms in molecular genetics. Then peppered with functional genomics completes the story for an edible outcome.  We generally refer this as a Translational Research.  For example, recent foundlings suggest that H19 contributes to cancer, including hepatocellular carcinoma (HCC) after reviewing Oncomine resource.  According to these observations, in most HCC cases there is a lower expression of  H19 level is compared to the liver. Thus, in vitro and in vivo studies were undertaken with classical genetic analyzes based on loss- and gain-of-function on H19 to characterize two outcomes depend on H19, that are the effects on gene expression and on HCC metastasis. First, the expression of H19 showed gene expression variation since H19 expression was low in tumor cells than peripheral tumor cells.  Second, the metastasis of cancer based on alteration of miR-200 pathway contributing mesenchymal-to-epithelial transition by H19. Therefore, H19 and miR-200 are targets to be utilized during molecular diagnostics development and establishing targeted therapies in cancer.

Long story short, there is a circle of life where everything is connected even though they look different.  As a result, when we see a sunflower or a baby we remember to smile, because life is still an act to puzzle human.

References and Further Readings:

 

Non-coding RNAs as regulators of gene expression and epigenetics” Cardiovascular Res 1 June 2011: 430-440.

Epigenetic regulation of key vascular genes and growth factors” Cardiovasc Res 1 June 2011: 441-446.

Epigenetic Regulation by Long Noncoding RNAs” Science 14 December 2012: 1435-1439.

Epigenetic control of embryonic stem cell fate” JEM 25 October 2010: 2287-2295.

Transcribed dark matter: meaning or myth?” Hum Mol Genet 15 October 2010: R162-R168.

Epigenetic activation of the MiR-200 family contributes to H19-mediated metastasis suppression in hepatocellular carcinoma” Carcinogenesis 1 March 2013: 577-586.

Vernalization-Mediated Epigenetic Silencing by a Long Intronic Noncoding RNA” Science 7 January 2011: 76-79.

Predicting the probability of H3K4me3 occupation at a base pair from the genome sequence context” Bioinformatics 1 May 2013: 1199-1205.

 

Further Readings specific to Embryonic Stem Cell Differentiation and Development :

“BMP Induces Cochlin Expression to Facilitate Self-renewal and Suppress Neural Differentiation of Mouse Embryonic Stem Cells” J. Biol. Chem. 2013 288:8053-8060

Abstract

“Regulation of DNA Methylation in Rheumatoid Arthritis Synoviocytes”  J. Immunol. 2013 190:1297-1303

Abstract

“DNA methylome signature in rheumatoid arthritis” Ann Rheum Dis 2013 72:110-117

Abstract

“The histone demethylase Kdm3a is essential to progression through differentiation” Nucleic Acids Res 2012 40:7219-7232

Abstract

“Targeted silencing of the oncogenic transcription factor SOX2 in breast cancer” Nucleic Acids Res 2012 40:6725-6740

Abstract

“Yin Yang 1 extends the Myc-related transcription factors network in embryonic stem cells” Nucleic Acids Res 2012 40:3403-3418

Abstract

“RYBP Represses Endogenous Retroviruses and Preimplantation- and Germ Line-Specific Genes in Mouse Embryonic Stem Cells” Mol. Cell. Biol. 2012 32:1139-1149

Abstract

“Polycomb Repressor Complex-2 Is a Novel Target for Mesothelioma Therapy” Clin. Cancer Res. 2012 18:77-90

Abstract

“OCT4 establishes and maintains nucleosome-depleted regions that provide additional layers of epigenetic regulation of its target genes” Proc. Natl. Acad. Sci. USA 2011 108:14497-14502

Abstract

“Genome-wide promoter DNA methylation dynamics of human hematopoietic progenitor cells during differentiation and aging” Blood 2011 117:e182-e189

Abstract

“The CHD3 Chromatin Remodeler PICKLE and Polycomb Group Proteins Antagonistically Regulate Meristem Activity in the Arabidopsis” RootPlant Cell 2011 23:1047-1060

Abstract

“Chromatin structure of pluripotent stem cells and induced pluripotent stem cells” Briefings in Functional Genomics 2011 10:37-49

Abstract

Abbreviations used:

DNMT       DNA methyl transferase

ES             embryonic stem

JmjC         Jumonji C

lincRNA     long ncRNA

ncRNA       noncoding RNA

PcG          Polycomb group

PRC          Polycomb repressive complex

PRE          Polycomb repressive element

Previous Posts on Stem Cells:

…  Aviva Lev-Ari, PhD, RN New Life – The Healing Promise of Stem Cells View … p://www.technioniit.com/2012/09/new-life-healing-promise-of-stem-cells.html       Diseases and conditions where stem cell treatment is promising or emerging. Source: Wikipedia Since the …

…  Aviva Lev-Ari, PhD, RN Stem cells create new heart cells in baby mice, but not in adults, study …  picture on the left shows green c-kit+ precursor stem cells within an infarct (lower right) in a

14 January 2013  by Dr. Sudipta Saha on Pharmaceutical Intelligence
…  and Curator: Dr. Sudipta Saha, Ph.D. Germline stem cells that produce oocytes in vitro and fertilization-competent eggs in …  from adult mouse ovaries. A fluorescence-activated cell sorting-based protocol has been standardized that can be used with adult …  compared to the ESC-derived or induced pluripotent stem cell-derived germline cells that are currently used as models for human …

…  PhD, RN The two leading therapy classes are: Cell-based Therapies for angiogenesis and myocardial …  Research Projects Stem Cell biology Embryonic stem cells in cardiovascular repairEarly differentiation of human endothelial …

…  Stem Cells with Unread Genome: microRNAs Author, Demet Sag, PhD Life is …  a coherent outcome. Thus, providing an engineered whole cell as a system of correction for “Stem Cell Therapy” may resolve unmet health problems.  Only 1% of the genome …

…  are not yet known. Some studies suggest a high rate of stem cell activity with differentiation of progenitors to cardiomyocytes. Other …

…  T-cells, said Dr. Margaret Goodell, director of the Stem Cells and Regenerative Medicine Center of Baylor College of Medicine. …  of pediatrics at BCM and a member of the Center for Cell and Gene Therapy at BCM, Texas Children¹s Hospital and The Methodist …  found that mice lacking the gene for this factor had a T-cell deficiency and in particular, too few of these early progenitor …

28 March 2013  by ritusaxena on Pharmaceutical Intelligence
…  and Curator: Ritu Saxena, Ph.D Although cancer stem cells constitute only a small percentage of the tumor burden, their …  after therapeutic target in cancer. The post on cancer stem cells published on the 22nd of March, 2013, describes the identity of CSCs, their functional characteristics, possible cell of origin and biomarkers. This post focuses on the therapeutic potential …

…  programs in the fields of personalized medicine, cell biology, cytogenetics, genotyping, and biobanking drive our …  by playing an important role in induced pluripotent stem (iPS) cell research. Induced pluripotent stem cells are powerful cells which can be made from skin or blood cells, and …

30 November 2012  by sjwilliamspa on Pharmaceutical Intelligence
…  seen in hematologic malignancies such as cutaneous T-cell lymphoma and peripheral T-cell lymphoma and little or no positive outcome …  resistance to chemotherapeutics, and similarity to cancer stem cells(6-10). Figure 1. HDACis led to the induction of EMT phemotype. (A …

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John Rinn - Genomic Garbage Man

John Rinn – Genomic Garbage Man (Photo credit: ChimpLearnGood)

DNA: One man’s trash is another man’s treasure, but there is no JUNK after all

Author: Demet Sag, PhD

One man’s trash is another man’s treasure, but there is no JUNK after all:

The JUNK has a meaning

 

Long non-coding RNAs recognized after transcriptome sequencing and studied more closely recently thanks to genomic tiling arrays, cDNA sequencing and RNA-Seq, which they have provided initial insights into the extent and depth of transcribed sequence across human and other genomes. How many are there in the genome? What are their mechanisms? How can we use them in molecular diagnostics and targeted therapies?  How do they effect the function in a disease? Is it possible to modulate gene expression at the level of stem cell to redirect the cell differentiation? These are the main questions that we are looking for.

In early 90s actually first lincRNA was described, Xist. The main function was dosage compensation. Then in 2000s FANTOM consortium project changed the perspective on these long transcripts. Then they are called natural antisense transcripts (NATs), because very large number of these transcripts is overlapping with, and is transcribed in the antisense direction, to protein-coding genes.  As a result of this study 11000 lincRNA discovered from full length cDNAs in mice. Later, yet another shift occur since these transcribed units are solely located in the introns or within “junk” DNA of protein-coding genes.  Another independent study quantified that about 40% of protein-coding genes express NATs. Proven that there is nothing junk about DNA. Then, it was found that there are 8000 lincRNAs and among these 4000 are determined since they provide cell identity with multi-exogenic, polyadenylated, capped, ether in the cytoplasm or in the nucleus. However, even more recent studies show that there are about 20,000 lincRNAs.  Furthermore, lincRNAs are classified under three distinct class: 1. Long-non-coding RNAs away from protein-coding genes, 2 NATs transcribed from the opposite strand of protein-coding genes, 3. Intronic lincRNAs expressed from within the introns of protein coding genes.

 

English: The human genome, categorized by func...

The human genome, categorized by function of each gene product, given both as number of genes and as percentage of all genes. (Photo credit: Wikipedia)

Their function is under study. However, keep in mind that they are redundant, so deleting or creating null mutations may or may not answer specific development questions. On the other hand, epigenetics, gene imprinting, and pathologies can be the best resource to identify their specific roles in biological functions and interactions.  Distinct gene regulation either as a cis or trans element, gene imprinting, modulating alternative splicing, nuclear organization, determining a chromatin structure are under study.  This will allow us to relate genome structure and function in health and disease better.  Identification of their function during biological responses require a long way to be completed due to complexity since lincRNAs also regulate microRNAs.  Regardless of many obstacles there is a progress.  Disregulation of these lincRNA mainly observed in several cancer types, prostate, breast, hepatocellular carcinoma, colorectal, glioma and melanoma, possibly more. Most of the studies are done in vitro. However, there are many great model organism work as well, such as mice, zebra fish, and worm.

It was also not surprising that their regulation possibly under control of hormones based on circadian clock of our body. So better to sleep eight hour a day is not a cliché.

 

Next topic will include understanding of lincRNA mechanisms and epigenetics followed by lincRNAs during disease and cellular genesis.

 

Mechanism, Genome and Genetics:

Long non-coding RNAs: insights into functions. Mercer TR, Dinger ME, Mattick JS Nat. Rev. Genet. 2009;10:155159. http://www.ncbi.nlm.nih.gov/pubmed/19188922

 

Long Noncoding RNAs: Past, Present, and Future” Genetics 1 March 2013: 651-669. http://www.genetics.org/content/193/3/651.abstract

 

“RNA-protein analysis using a conditional CRISPR nuclease” Proc. Natl. Acad. Sci. USA 2 April 2013: 5416-5421. http://www.pnas.org/content/110/14/5416.abstract

“Noncoding RNA and Polycomb recruitment” RNA 1 April 2013: 429-442. http://rnajournal.cshlp.org/content/19/4/429.abstract

 

“Emerging functional and mechanistic paradigms of mammalian long non-coding RNAs” Nucleic Acids Res 1 August 2012: 6391-6400. http://nar.oxfordjournals.org/content/40/14/6391.abstract

 

 

“Long noncoding RNAs regulate adipogenesis” Proc. Natl. Acad. Sci. USA 26 February 2013: 3387-3392. http://www.pnas.org/content/110/9/3387.abstract

 

“Circadian changes in long noncoding RNAs in the pineal gland” Proc. Natl. Acad. Sci. USA 14 August 2012: 13319-13324. http://www.pnas.org/content/109/33/13319.abstract

Animal and Development:

“Systematic identification of long noncoding RNAs expressed during zebrafish embryogenesis” Genome Res 1 March 2012: 577-591. http://genome.cshlp.org/content/22/3/577.abstract

 

“Genes for embryo development are packaged in blocks of multivalent chromatin in zebrafish sperm” Genome Res 1 April 2011: 578-589. http://genome.cshlp.org/content/21/4/578.abstract

Long noncoding RNAs in C. elegans” Genome Res 1 December 2012: 2529-2540. http://genome.cshlp.org/content/22/12/2529.abstract

A spatial and temporal map of C. elegans gene expression” Genome Res 1 February 2011: 325-341. http://genome.cshlp.org/content/21/2/325.abstract

 

“SFMBT1 functions with LSD1 to regulate expression of canonical histone genes and chromatin-related factors” Genes Dev. 1 April 2013: 749-766. http://genesdev.cshlp.org/content/27/7/749.abstract

Other related articles published on this Open Access Online Scientific Journal include the following:

…  therapies in a variety of animal models and contributed to regulatory CMC and IND-enabling safety and toxicology studies for inclusion in …  sequences sort a-cardiac and b-cytoplasmic actin messenger RNAs to different cytoplasmic compartments. J. Cell. Biol., …

…  and posttranscriptional mechanisms contributing to the regulatory network. We examined proinflammatory gene regulation in …  articles What about Circular RNAs? (pharmaceuticalintelligence.com) How Genes Function …

…  due to their broad scope and non-specificity in the human genome. “I am extremely pro-patent, but I simply believe that people …  believe that individuals have an innate right to their own genome, or to allow their doctor to look at that genome, just like the lungs or …

…  Intelligence https://pharmaceuticalintelligence.com/2013/03/02/genome-sequenc…of-the-healthy/ ‎ Key Issues in Genome Sequencing of Healthy Individuals Eric Topol, MD, Genomic Medicine I …  touching on important controversies in the use of whole genome …

6 February 2013  by Dr. Sudipta Saha on Pharmaceutical Intelligence
…  of recombination is highly uneven across the human genome, as in all studied organisms. Substantial recombination active regions …  this variation would require comparison of recombination genome-wide among many single genomes. Whole-genome amplification (WGA) of …

…  Lev-Ari, PhD, RN and Pnina G. Abir-Am, PhD Putting Genome Interpretation to the Test 01/30/2013 Ashley Yeager How well do methods for interpreting genome variation work? Ashley Yeager takes a look at a community experiment that is trying to assess just how useful genome interpretation tools in real-world situations. At the American …

…  genomes — through the end of this year, National Human Genome Research Institute estimates indicate. And in his book, The Creative …  the interpretation of an apparently healthy person’s genome and that of an individual who is already affected by a disease, whether …
Topics: Cardiovascular Pharmaceutical Genomics, Personalized Medicine & Genomic Research, Pharmaceutical R&D investment, CANCER BIOLOGY & Innovations in Cancer Therapy, Chemical Genetics, Cell Biology, Signaling & Cell Circuits, Computational Biology/Systems and Bioinformatics, Medical and Population Genetics, genome biology, Biological Networks, Gene Regulation and Evolution, Population Health Management, Genetics & Pharmaceut, human genome, National Institutes of Health, Scripps Research Institute, Proteomics, Bio Instrumentation in Experimental Life Sciences Resea, Massachusetts General Hospital, DNA, FDA Regulatory Affairs, Clinical Trials and IRB related issues, Biomarkers & Medical Diagnostics, metabolomics, Molecular Genetics & Pharmaceutical, Genomic Testing: Methodology for Diagnosis, Technology Transfer: Biotech and Pharmaceutical, Health Law & Patient Safety, Eric Topol, national human genome research institute, Encode, NIST

…  scary findings: the tale of John Lauerman’s whole genome sequencing FEBRUARY 15, 2012 Joe Thakuria draws John Lauerman’s blood for whole genome sequencing. By Madeleine Price Ball, licensed under …  scary findings: the tale of John Lauerman’s whole genome sequencing » Joe Thakuria draws John …

…  2: LEADERS in the Competitive Space of Genome Sequencing of Genetic Mutations for Therapeutic Drug Selection in …  Treatment https://pharmaceuticalintelligence.com/2013/01/13/leaders-in-genome-sequencing-of-genetic-mutations-for-therapeutic-drug-selection-in-cancer- …
Topics: Personalized Medicine & Genomic Research, Pharmaceutical R&D investment, Chemical Genetics, Computational Biology/Systems and Bioinformatics, Medical and Population Genetics, genome biology, Disease Biology, Small Molecules in Development of Ther, Population Health Management, Genetics & Pharmaceut, Cancer, Foundation Medicine, Proteomics, DNA, DNA Sequencing, Biomarkers & Medical Diagnostics, metabolomics, AstraZeneca, Molecular Genetics & Pharmaceutical, Nature Medicine, Stem Cells for Regenerative Medicine, Genomic Testing: Methodology for Diagnosis, Technology Transfer: Biotech and Pharmaceutical, Full genome sequencing, Genomic Endocrinology, Preimplantation Genetic Diagnosi, Interviews with Scientific Leaders, Pharmacogenomics, Drug Delivery Platform Technology, Digene, Yuri Milner

3 February 2013  by sjwilliamspa on Pharmaceutical Intelligence
Genome-Wide Detection of Single-Nucleotide and Copy-Number Variation of a Single …  of DNA replication and the ability to amplify a whole genome.  The amplicons are then sequenced either by whole-genome sequencing methods using Sanger-sequencing to verify any single …

…  Aviva Lev-Ari, RN Genome Biol. 2012 Dec 13;13(12):R115. [Epub ahead of print] Whole-genome reconstruction and mutational signatures in gastric cancer. Nagarajan …  read and DNA-PET sequencing to present the first whole-genome analysis of two gastric adenocarcinomas, one with chromosomal …

1 September 2012  by pkandala on Pharmaceutical Intelligence
…  by interpreting the mathematical patterns in the cancer genome. Researchers at the University of Oslo, Norway (UiO) have developed a …  Hospital and UiO. Finds the changed patterns in the genome There is much talk about finding the special cancer gene. In reality, …

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An illustration of the central dogma of molecu...

An illustration of the central dogma of molecular biology annotated with the processes ncRNAs are involved in. (Photo credit: Wikipedia)

X-ray structure of the tRNA Phe from yeast. Da...

X-ray structure of the tRNA Phe from yeast. Data was obtained by PDB 1ehz and rendered with PyMOL. violet: acceptor stem wine red: D-loop blue: anticodon loop orange: variable loop green: TPsiC-loop yellow: CCA-3′ of the acceptor stem grey: anticodon (Photo credit: Wikipedia)

 Our genome must be packed tightly to fit into the nucleus. Genome is the blue print of a living organism whether made up off a single or multiple cell.   Recently, the genome seen as a functional network of physical contacts within (cis) and between (trans) chromosomes.  It became necessary to map these physical DNA contacts at high-resolution with technologies such as the “chromosome conformation capture” (3C) and other 3C-related methods including 3C-Carbon Copy (5C) and Hi-C.  Yet, we all know that in vivo conformation, gene to gene interactions from a long distance, histones and 3D have an impact on gene regulation and expression.  The game is not just a sequence but functional genomics with a correct translation of sequence for development so that proper molecular diagnostics can be applied not only for prevention but also for monitoring the efficacy of the intervention. Thus, we can provide a targeted therapy for personalized medicine.

On the other hand, we still know very little about genome organization at the molecular level, although spatial genome organization can critically affect gene expression.  It is important to recognize who is there to be present and who is there to create the functional impact for regulation in a specific tissue and time.  In addition, mediation of these chromatin contacts based on a specific tissue is quite essential.  For example, during long-range control mechanism specific enhancers and distal promoters needed to be invited to a close physical proximity to each other by transcription factors that has been found at other loci.  Furthermore, chromatin-binding proteins such as the CCCTC-binding factor (CTCF) and cohesin seem to have critical roles in genome organization and gene expression.  Let’s not forget about epigenetics, since there are so many methods to regulate chromatin interactions like cytosine methylation, maternal gene, gradient level, post-translational modifications and non-coding RNAs.

The non-coding RNAs (ncRNAs) are silent but they have the 99% power because ncRNAs are a broad class of transcripts consisting of structural (rRNAs, tRNAs, snRNAs, snoRNAs, etc.), regulatory (miRNAs, piRNAs, etc.), and of sense/antisense transcripts.  Among these an interesting class is the latter group.   This class includes transcriptional “features” (eRNAs, tiRNAs), and a very large number of long non-coding RNAs (lncRNAs), length from 200 nt to 100 kb.  The magnificent future of lncRNAs comes from their production, as they can be transcribed nearby known protein-coding genes or from their introns. As a result, because of their intergenical production they are also called as “lincRNAs (long intergenical non-coding RNAs).  They are abundant and specific as microRNAs.  Hence, their inclusion into the biomarker list and assuming their roles during targeted therapy don’t require us to be a wizard but a functional genomicist knowing evolution, development and molecular genetics and plus signaling.

lincRNA can both activate and repress the gene either cis or trans acting to effect gene regulation will be discussed next.

As a result, one gene expression regulation needs from twenty to several hundred genes. As they say raising a child needs a village.

References:

“Functional demarcation of active and silent chromatin domains in human HOX loci by noncoding RNAs”.

Rinn JL, Kertesz M, Wang JK, Squazzo SL, Xu X, Brugmann SA, Goodnough LH, Helms JA, Farnham PJ, Segal E, Chang HY.  Cell. 2007 Jun 29; 129(7):1311-23.

“Long noncoding RNA as modular scaffold of histone modification complexes”

Tsai MC, Manor O, Wan Y, Mosammaparast N, Wang JK, Lan F, Shi Y, Segal E, Chang HYScience. 2010 Aug 6; 329(5992):689-93.

“Capturing Chromosome Conformation”.

Dekker J, Rippe K, Dekker M, Kleckner N.Science.2002;295:1306–1311.

“Chromosome Conformation Capture Carbon Copy (5C): a massively parallel solution for mapping interactions between genomic elements”.

Dostie J, Richmond TA, Arnaout RA, Selzer RR, Lee WL, Honan TA, Rubio ED, Krumm A, Lamb J, Nusbaum C, Green RD, Dekker J.Genome Res. 2006;16:1299–1309.

“Chromosome conformation capture carbon copy technology”.

Dostie J, Zhan Y, Dekker J. Curr. Protoc. Mol. Biol. 2007 Chapter 21, Unit 21 14.

“Comprehensive mapping of long-range interactions reveals folding principles of the human genome”.

Lieberman-Aiden E, van Berkum NL, Williams L, Imakaev M, Ragoczy T, Telling A, Amit I, Lajoie BR, Sabo PJ, Dorschner MO, Sandstrom R, Bernstein B, Bender MA, Groudine M, Gnirke A, Stamatoyannopoulos J, Mirny LA, Lander ES, Dekker J.  Science. 2009;326:289–293.

“Chromatin conformation signatures: ideal human disease biomarkers?”

Crutchley JL, Wang XQ, Ferraiuolo MA, Dostie J.Biomark. Med. 2010;4:611–629.

“Relationship between CAD risk genotype in the chromosome 9p21 locus and gene expression. Identification of eight new ANRIL splice variants”.

Folkersen L, Kyriakou T, Goel A, Peden J, Mälarstig A, Paulsson-Berne G, Hamsten A, Hugh Watkins, Franco-Cereceda A, Gabrielsen A, Eriksson P, PROCARDIS consortia

PLoS One. 2009 Nov 2; 4(11):e7677.

” A myelopoiesis-associated regulatory intergenic noncoding RNA transcript within the human HOXA cluster”.

Zhang X, Lian Z, Padden C, Gerstein MB, Rozowsky J, Snyder M, Gingeras TR, Kapranov P, Weissman SM, Newburger PE.  Blood. 2009 Mar 12; 113(11):2526-34.

Monk M.   Genes Dev. 1988 Aug; 2(8):921-5.

Hox genes specify vertebral types in the presomitic mesoderm

Marta Carapuço,1 Ana Nóvoa,1 Nicoletta Bobola,2 and Moisés Mallo1,3 .  Genes Dev. 2005 September 15; 19(18): 2116–2121.

Krumlauf R.  Cell. 1994 Jul 29; 78(2):191-201.

“Noncoding RNA synthesis and loss of Polycomb group repression accompanies the colinear activation of the human HOXA cluster”.

Sessa L, Breiling A, Lavorgna G, Silvestri L, Casari G, Orlando V.  RNA. 2007 Feb; 13(2):223-39.

“Functional demarcation of active and silent chromatin domains in human HOX loci by noncoding RNAs”.

Rinn JL, Kertesz M, Wang JK, Squazzo SL, Xu X, Brugmann SA, Goodnough LH, Helms JA, Farnham PJ, Segal E, Chang HY.  Cell. 2007 Jun 29; 129(7):1311-23.

“Long noncoding RNAs with enhancer-like function in human cells”.

Ørom UA, Derrien T, Beringer M, Gumireddy K, Gardini A, Bussotti G, Lai F, Zytnicki M, Notredame C, Huang Q, Guigo R, Shiekhattar R

“Histone modifications at human enhancers reflect global cell-type-specific gene expression”.

Heintzman ND, Hon GC, Hawkins RD, Kheradpour P, Stark A, Harp LF, Ye Z, Lee LK, Stuart RK, Ching CW, Ching KA, Antosiewicz-Bourget JE, Liu H, Zhang X, Green RD, Lobanenkov VV, Stewart R, Thomson JA, Crawford GE, Kellis M, Ren B.   Nature. 2009 May 7; 459(7243):108-12.

“Tiny RNAs associated with transcription start sites in animals”.

Taft RJ, Glazov EA, Cloonan N, Simons C, Stephen S, Faulkner GJ, Lassmann T, Forrest AR, Grimmond SM, Schroder K, Irvine K, Arakawa T, Nakamura M, Kubosaki A, Hayashida K, Kawazu C, Murata M, Nishiyori H, Fukuda S, Kawai J, Daub CO, Hume DA, Suzuki H, Orlando V, Carninci P, Hayashizaki Y, Mattick JS.  Nat Genet. 2009 May; 41(5):572-8.

“Chromatin modifications and their function”.

Kouzarides T.   Cell. 2007 Feb 23; 128(4):693-705.

Tripathi V, Ellis JD, Shen Z, Song DY, Pan Q, Watt AT, Freier SM, Bennett CF, Sharma A, Bubulya PA, Blencowe BJ, Prasanth SG, Prasanth KV.   Mol Cell. 2010 Sep 24; 39(6):925-38.

Selected Further Reading

“Small and long non-coding RNAs in cardiac homeostasis and regeneration”

Ounzain, S.; Crippa, S.; Pedrazzini, T.  BBA – Molecular Cell Research vol. 1833 issue 4 April, 2013. p. 923-933

“Regulatory mechanisms of long noncoding RNAs in vertebrate central nervous system development and function.” 

Knauss, J.L.; Sun, T.  “Neuroscience vol. 235 April 3, 2013. p. 200-214

“Comparative genomics reveals ‘novel’ Fur regulated sRNAs and coding genes in diverse proteobacteria.”

Sridhar, J.; Sabarinathan, R.; Gunasekaran, P.; Sekar, K.   Gene vol. 516 issue 2 March 10, 2013. p. 335-344 DOI: 10.1016/j.gene.2012.12.057. ISSN: 0378-1119.

miRNAs Regulate Expression and Function of Extracellular Matrix Molecules”

Rutnam, Z.J.; Wight, T.N.; Yang,  B.B.Matrixixix Biology vol. 32 issue 2 March 11, 2013. p. 74-85 DOI: 10.1016/j.matbio.2012.11.003. ISSN: 0945-053X.

Transcript profiling of microRNAs during the early development of the maize brace root via Solexa sequencing

Liu, P.; Yan, K.; Lei, Y.x.; Xu, R.; Zhang, Y.m.; Yang, G.d.; Huang, J.g.; Wu, C.A.; Zheng, C.C.Genomics vol. 101 issue 2 February, 2013. p. 149-156 DOI: 10.1016/j.ygeno.2012.11.004. ISSN: 0888-7543.

Regulatory mechanisms of long noncoding RNAs in vertebrate central nervous system development and function

Knauss, J.L.; Sun, T.  Neuroscience vol. 235 April 3, 2013. p. 200-214 DOI: 10.1016/j.neuroscience.2013.01.022. ISSN: 0306-4522.

“The dynamic biliary epithelia: Molecules, pathways, and disease”

O’Hara, Steven P.; Tabibian, James H.; Splinter, Patrick L.; LaRusso, Nicholas F. Journal of Hepatology vol. 58 issue 3 March, 2013. p. 575-582 DOI: 10.1016/j.jhep.2012.10.011. ISSN: 0168-8278

ABBREVIATIONS

3C = Chromosome conformation capture

rRNAs = Ribosomal RNAs

tRNAs = Transfer RNAs

snRNAs = Small nuclear RNAs

snoRNAs = Small nucleolar RNAs

miRNAs = MicroRNAs

piRNAs = Piwi-interacting RNAs

eRNAs = Enhancer RNAs

tiRNAs = Transcription initiation RNAs

spliRNAs = Splice-site RNAs

lincRNAs = Long intergenic non-coding RNAs

lncRNPs = Long non-coding ribonucleoprotein complexes

Igf2r = Insulin-like growth factor II receptor

HMTs = Histone methyl transferases

TSSs = Transcriptional start sites

TFs = Transcription factors

RNAi = RNA interference

PTMs = Post-translational modifications

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