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Functional Correlates of Signaling Pathways

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

 

We here move on to a number of specific, key published work on signaling, and look at the possible therapeutic applications to disease states.

Scripps Research Professor Wolfram Ruf and colleagues have identified a key connection between

  • the signaling pathways and the immune system spiraling out of control involving
  • the coagulation system and vascular endothelium that,
  • if disrupted may be a target for sepsis. (Science Daily, Feb 29, 2008).

It may be caused by a bacterial infection that enters the bloodstream, but

  • we now recognize the same cascade not triggered by bacterial invasion.

The acute respiratory distress syndrome (ARDS) has been defined as

  • a severe form of acute lung injury featuring
  • pulmonary inflammation and increased capillary leak.

ARDS is associated with a high mortality rate and accounts for 100,000 deaths annually in the United States. ARDS may arise in a number of clinical situations, especially in patients with sepsis. A well-described pathophysiological model of ARDS is one form of

  • the acute lung inflammation mediated by
  1. neutrophils,
  2. cytokines, and
  3. oxidant stress.

Neutrophils are major effect cells at the frontier of

  • innate immune responses, and they play
  • a critical role in host defense against invading microorganisms.

The tissue injury appears to be related to

  • proteases and toxic reactive oxygen radicals
  • released from activated neutrophils.

In addition, neutrophils can produce cytokines and chemokines that

enhance the acute inflammatory response.

Neutrophil accumulation in the lung plays a pivotal role in the pathogenesis of acute lung injury during sepsis. Directed movement of neutrophils is

  • mediated by a group of chemoattractants,
  • especially CXC chemokines.

Local lung production of CXC chemokines is intensified during experimental sepsis induced by cecal ligation and puncture (CLP).

Sepsis, Multi-organ Dysfunction Syndrome, and Septic Shock: A Conundrum of Signaling Pathways Cascading Out of Control

https://pharmaceuticalintelligence.com/2012/10/13/sepsis-multi-organ-dysfunction-syndrome-and-septic-shock-a-conundrum-of-signaling-pathways-cascading-out-of-control/

Integrins and extracellular matrix in mechanotransduction

ligand binding of integrins

ligand binding of integrins

Integrins are a family of cell surface receptors which

mediate cell–matrix and cell–cell adhesions.

Among other functions they provide an important

mechanical link between the cells external and intracellular environments while

the adhesions that they form also have critical roles in cellular signal-transduction.

Cell–matrix contacts occur at zones in the cell surface where

adhesion receptors cluster and when activated

the receptors bind to ligands in the extracellular matrix.

The extracellular matrix surrounds the cells of tissues and forms the

structural support of tissue which is particularly important in connective tissues.

Cells attach to the extracellular matrix through

specific cell-surface receptors and molecules

including integrins and transmembrane proteoglycans.

The integrin family of αβ heterodimeric receptors act as

cell adhesion molecules

connecting the ECM to the actin cytoskeleton.

The actin cytoskeleton is involved in the regulation of

1.cell motility,

2.cell polarity,

3.cell growth, and

4.cell survival.

The combination of αβ subunits determines

binding specificity and

signaling properties.

Both α and β integrin subunits contain two separate tails, which

penetrate the plasma membrane and possess small cytoplasmic domains which facilitate

the signaling functions of the receptor.

There is some evidence that the β subunit is the principal site for

binding of cytoskeletal and signaling molecules,

whereas the α subunit has a regulatory role. The integrin tails

link the ECM to the actin cytoskeleton within the cell and with cytoplasmic proteins,

such as talin, tensin, and filamin. The extracellular domains of integrin receptors bind the ECM ligands.

binding of integrins depends on ECM divalent cations ch19

binding of integrins depends on ECM divalent cations ch19

integrin coupled to F-actin via linker

integrin coupled to F-actin via linker

http://www.nature.com/nrm/journal/vaop/ncurrent/images/nrm3896-f4.jpg

Schematic of the ‘focal adhesion clutch’ on stiff (a) versus soft (b) extracellular matrix (ECM). In all cases, integrins are coupled to F-actin via linker proteins (for example, talin and vinculin). The linker proteins move backwards (as indicated by the small arrows) as F-actin also moves backwards, under pushing forces from actin polymerization and/or pulling forces from myosin II activity. This mechanism transfers force from actin to integrins, which pull on the ECM. A stiff ECM (a) resists this force so that the bound integrins remain immobile. A compliant matrix (b) deforms under this force (as indicated by the compressed ECM labelled as deformed matrix) so that the bound integrins can also move backwards. Their movement reduces the net loading rate on all the force-bearing elements, which results in altered cellular responses

The ECM is a complex mixture of matrix molecules, including –

  • glycoproteins, collagens, laminins, glycosaminoglycans, proteoglycans,
  • and nonmatrix proteins, – including growth factors

The integrin receptor formed from the binding of α and β subunits is

  • shaped like a globular head supported by two rod-like legs (Figure 1).

Most of the contact between the two subunits occurs in the head region, with

  • the intracellular tails of the subunits forming the legs of the receptor.

Integrin recognition of ligands is not constitutive but

  • is regulated by alteration of integrin affinity for ligand binding.

For integrin binding to ligands to occur

  • the integrin must be primed and activated, both of which involve
  • conformational changes to the receptor.

Linking integrin conformation to function

Figure  Integrin binding to extracellular matrix (ECM). Conformational changes to integrin structure and clustering of subunits which allow enhanced function of the receptor.

Integrins work alongside other proteins such as

cadherins,

immunoglobulin superfamily

cell adhesion molecules,

selectins, and

syndecans

to mediate

cell–cell and

cell–matrix interactions and communication.

Activation of adhesion receptors triggers the formation of matrix contacts in which

bound matrix components,

adhesion receptors,

and associated intracellular cytoskeletal and signaling molecules

form large functional, localized multiprotein complexes.

Cell–matrix contacts are important in a variety of different cell and

tissue properties including

1.embryonic development,

2.inflammatory responses,

3.wound healing,

4.and adult tissue homeostasis.

Integrin extracellular binding activity is regulated from inside the cell and binding to the ECM induces signals that are transmitted into the cell. This bidirectional signaling requires

dynamic,

spatially, and

temporally regulated formation and

disassembly of multiprotein complexes that

form around the short cytoplasmic tails of integrins.

Ligand binding to integrin family members leads to clustering of integrin molecules in the plasma membrane and recruitment of actin filaments and intracellular signaling molecules to the cytoplasmic domain of the integrins. This forms focal adhesion complexes which are able to maintain

not only adhesion to the ECM

but are involved in complex signaling pathways

which include establishing

1.cell polarity,

2.directed cell migration, and

3.maintaining cell growth and survival.

Initial activation through integrin adhesion to matrix recruits up to around 50 diverse signaling molecules

to assemble the focal adhesion complex

which is capable of responding to environmental stimuli efficiently.

Mapping of the integrin

adhesome binding and signaling interactions

a network of 156 components linked together which can be modified by 690 interactions.

Genetic programming occurs with the binding of integrins to the ECM

Signal transduction pathway activation arising from integrin-ECM binding results in

  • changes in gene expression of cells and
  • leads to alterations in cell and tissue function.

Various different effects can arise depending on the

1.cell type,

2.matrix composition, and

3.integrins activated

It has been suggested that integrin-type I collagen interaction is necessary for

  • the phosphorylation and activation of osteoblast-specific transcription factors
  • present in committed osteoprogenitor cells.

During mechanical loading/stimulation of chondrocytes there is an

  1. influx of ions across the cell membrane resulting from
  2. activation of mechanosensitive ion channels
  3. which can be inhibited by subunit-specific anti-integrin blocking antibodies or RGD peptides.

Using these strategies it was identified that

  • α5β1 integrin is a major mechanoreceptor in articular chondrocyte
  • responses to mechanical loading/stimulation.

Osteoarthritic chondrocytes show a depolarization response to 0.33 Hz stimulation

  • in contrast to the hyperpolarization response of normal chondrocytes.

The mechanotransduction pathway in chondrocytes derived from normal and osteoarthritic cartilage

  • both involve recognition of the mechanical stimulus
  • by integrin receptors resulting in
  • the activation of integrin signaling pathways
  • leading to the generation of a cytokine loop.

Normal and osteoarthritic chondrocytes show differences

  • at multiple stages of the mechanotransduction cascade.
Signaling pathways activated in chondrocytes

Signaling pathways activated in chondrocytes

http://dx.doi.org/10.1016/j.matbio.2014.08.007

Chondrocyte integrins are important mediators of cell–matrix interactions in cartilage

  • by regulating the response of the cells to signals from the ECM that
  1. control cell proliferation,
  2. survival,
  3. differentiation,
  4. matrix remodeling.

Integrins participate in development and maintenance of the tissue but also

  • in pathological processes related to matrix destruction, where
  • they likely play a role in the progression of OA.

Cellular adaptation to mechanical stress: role of integrins, Rho, cytoskeletal tension and mechanosensitive ion channels

Cells exhibited four types of mechanical responses:

(1) an immediate viscoelastic response;

(2) early adaptive behavior characterized by pulse-to-pulse attenuation in response to oscillatory forces;

(3) later adaptive cell stiffening with sustained (>15 second) static stresses; and

(4) a large-scale repositioning response with prolonged (>1 minute) stress.

Importantly, these adaptation responses differed biochemically.

The immediate and early responses were affected by

chemically dissipating cytoskeletal prestress (isometric tension), whereas

the later adaptive response was not.

The repositioning response was prevented by

inhibiting tension through interference with Rho signaling,

similar to the case of the immediate and early responses, but it was also prevented by

blocking mechanosensitive ion channels or

by inhibiting Src tyrosine kinases.

All adaptive responses were suppressed by cooling cells to 4°C to slow biochemical remodeling. Thus, cells use multiple mechanisms to sense and respond to static and dynamic changes in the level of mechanical stress applied to integrins.

Microtubule-Stimulated ADP Release, ATP Binding, and Force Generation In Transport Kinesins

All three classes of molecular motor proteins are now known to be

  • large protein families with diverse cellular functions.

Both the kinesin family and the myosin family have been defined and their proteins grouped into subfamilies. Finally, the elusive cytoplasmic version of dynein was identified and a multigene family of flagellar and cytoplasmic dyneins defined. Members of a given motor protein family share

  • significant homology in their motor domains with the defining member,
  • kinesin, dynein or myosin; but they also contain
  • unique protein domains that are specialized for interaction with different cargoes.

This large number of motor proteins may reflect

  • the number of cellular functions that require force generation or movement,
  • ranging from mitosis to morphogenesis to transport of vesicles.

Kinesins are a large family of microtubule (MT)-based motors that play important roles in many cellular activities including

mitosis,

motility, and

intracellular transport

Their involvement in a range of pathological processes

  • also highlights their significance as therapeutic targets and
  • the importance of understanding the molecular basis of their function

They are defined by their motor domains that contain both

  • the microtubule (MT) and
  • ATP binding sites.

Three ATP binding motifs—

  1. the P-loop,
  2. switch I,
  3. switch II–

are highly conserved among

  1. kinesins,
  2. myosin motors, and
  • small GTPases.

They share a conserved mode of MT binding such that

  • MT binding,
  • ATP binding, and
  • hydrolysis

are functionally coupled for efficient MT-based work.

The interior of a cell is a hive of activity, filled with

  • proteins and other items moving from one location to another.

A network of filaments called microtubules forms tracks

  • along which so-called motor proteins carry these items.

Kinesins are one group of motor proteins, and a typical kinesin protein has

  • one end (called the ‘motor domain’) that can attach itself to the microtubules.

The other end links to the cargo being carried, and a ‘neck’ connects the two. When two of these proteins work together,

  • flexible regions of the neck allow the two motor domains to move past one another,
  • which enable the kinesin to essentially walk along a microtubule in a stepwise manner.

Although the two kinesins have been thought to move along the microtubule tracks in different ways, Atherton et al. find that the core mechanism used by their motor domains is the same.

When a motor domain binds to the microtubule, its shape changes,

  • first stimulating release of the breakdown products of ATP from the previous cycle.

This release makes room for a new ATP molecule to bind. The structural changes caused by ATP binding

  • produce larger changes in the flexible neck region that
  • enable individual motor domains within a kinesin pair to
  • co-ordinate their movement and move in a consistent direction.

The major and largely invariant point of contact between kinesin motor domains and the MT is helix-α4,

  • which lies at the tubulin intradimer interface.

The conformational changes in functionally important regions of each motor domain are described,

  • starting with the nucleotide-binding site,
  • from which all other conformational changes emanate.

The nucleotide-binding site (Figure 2) has three major elements:

(1) the P-loop (brown) is visible in all our reconstructions;

(2) loop9 (yellow, contains switch I) undergoes major conformational changes through the ATPase cycle; and

(3) loop11 (red, contains switch II) that connects strand-β7 to helix-α4, the conformation and flexibility of which is

  • determined by MT binding and motor nucleotide state.

Movement and extension of helix-α6 controls neck linker docking

the N-terminus of helix-α6 is closely associated with elements of the nucleotide binding site suggesting that

  • its conformation alters in response to different nucleotide states.

Further,

  • because the orientation of helix-α6 with respect to helix-α4 controls neck linker docking and
  • because helix-α4 is held against the MT during the ATPase cycle,
    • conformational changes in helix-α6 control movement of the neck linker.

Mechanical amplification and force generation involves conformational changes across the motor domain

A key conformational change in the motor domain following Mg-ATP binding is

  • peeling of the central β-sheet from the C-terminus of helix-α4 increasing their separation;
  • this is required to accommodate rotation of helix-α6 and consequent neck linker docking

ATP binding draws loop11 and loop9 closer together; causing

(1) tilting of most of the motor domain not contacting the MT towards the nucleotide-binding site,

(2) rotation, translation, and extension of helix-α6 which we propose contributes to force generation, and

(3) allows neck linker docking and biases movement of the 2nd head towards the MT plus end.

In both motors, microtubule binding promotes

ordered conformations of conserved loops that

stimulate ADP release,

enhance microtubule affinity and

prime the catalytic site for ATP binding.

ATP binding causes only small shifts of these nucleotide-coordinating loops but induces

large conformational changes elsewhere that

allow force generation and

neck linker docking towards the microtubule plus end.

The study presents evidence provide evidence for a conserved ATP-driven

mechanism for kinesins and

reveals the critical mechanistic contribution of the microtubule interface.

Phosphorylation at endothelial cell–cell junctions: Implications for VE-cadherin function

This review summarizes the role of VE-cadherin phosphorylation in the regulation of endothelial cell–cell junctions and highlights how this affects vascular permeability and leukocyte extravasation.

The vascular endothelium is the inner lining of blood vessels and

forms a physical barrier between the vessel lumen and surrounding tissue;

controlling the extravasation of fluids,

plasma proteins and leukocytes.

Changes in the permeability of the endothelium are tightly regulated. Under basal physiological conditions, there is a continuous transfer of substances across the capillary beds. In addition the endothelium can mediate inducible,

transient hyperpermeability

in response to stimulation with inflammatory mediators,

which takes place primarily in post-capillary venules

However, when severe, inflammation may result in dysfunction of the endothelial barrier

  • in various parts of the vascular tree, including large veins, arterioles and capillaries.

Dysregulated permeability is observed in various pathological conditions, such as

  • tumor-induced angiogenesis,
  • cerebrovascular accident and
  • atherosclerosis.

Two fundamentally different pathways regulate endothelial permeability,

  1. the transcellular and
  2. paracellular pathways.

Solutes and cells can pass through the body of endothelial cells via the transcellular pathway, which includes

  • vesicular transport systems,
  • fenestrae, and
  • biochemical transporters.

The paracellular route is controlled by

  • the coordinated opening and closing of endothelial junctions and
  • thereby regulates traffic across the intercellular spaces between endothelial cells.

Endothelial cells are connected by

tight, gap and

adherens junctions,

of which the latter, and particularly the adherens junction component,

vascular endothelial (VE)-cadherin,

are of central importance for the initiation and stabilization of cell–cell contacts.

Although multiple adhesion molecules are localized at endothelial junctions,

  • blocking the adhesive function of VE-cadherin using antibodies
  • is sufficient to disrupt endothelial junctions and
  • to increase endothelial monolayer permeability both in vitro and in vivo.

Like other cadherins, VE-cadherin mediates adhesion via

  • homophilic, calcium-dependent interactions.

This cell–cell adhesion

is strengthened by binding of cytoplasmic proteins, the catenins,

to the C-terminus of VE-cadherin.

VE-cadherin can directly bind

  • β-catenin and plakoglobin, which
  • both associate with the actin binding protein α-catenin.

Initially, α-catenin was thought to directly anchor cadherins to the actin cytoskeleton, but recently it became clear that

  • α-catenin cannot bind to both β-catenin and actin simultaneously.

Numerous lines of evidence indicate that p120-catenin

  • promotes VE-cadherin surface expression and stability at the plasma membrane.

Different models are proposed that describe how

  • p120-catenin regulates cadherin membrane dynamics, including the hypothesis
  • that p120-catenin functions as a ‘cap’ that prevents the interaction of VE-cadherin
  • with the endocytic membrane trafficking machinery.

In addition, p120-catenin might regulate VE-cadherin internalization

  • through interactions with small GTPases.

Cytoplasmic p120-catenin, which is not bound to VE-cadherin, has been shown to

decrease RhoA activity,

elevate active Rac1 and Cdc42, and thereby is thought

to regulate actin cytoskeleton organization and membrane trafficking.

The intact cadherin-catenin complex is required for proper functioning of the adherens junction.

Several mechanisms may be involved in the

  • regulation of the organization and function of the cadherin–catenin complex, including
  1. endocytosis of the complex,
  2. VE-cadherin cleavage and
  3. actin cytoskeleton reorganization.

The remainder of this review primarily focuses on the

role of tyrosine phosphorylation in the control of VE-cadherin-mediated cell–cell adhesion.

Regulation of the adhesive function of VE-cadherin by tyrosine phosphorylation

It is a widely accepted concept that tyrosine phosphorylation of

  • components of the VE–cadherin-catenin complex
  • Correlates with the weakening of cell–cell adhesion.

A general idea has emerged that

tyrosine phosphorylation of the VE-cadherin complex

leads to the uncoupling of VE-cadherin from the actin cytoskeleton

through dissociation of catenins from the cadherin.

However, tyrosine phosphorylation of VE-cadherin

  • is required for efficient transmigration of leukocytes.

This suggests that VE-cadherin-mediated cell–cell contacts

1.are not just pushed open by the migrating leukocytes, but play

2.a more active role in the transmigration process.

A schematic overview of leukocyte adhesion-induced signals leading to VE-cadherin phosphorylation

Regulation of the integrity of endothelial cell–cell contacts by phosphorylation of VE-cadherin.

Regulation of the integrity of endothelial cell–cell contacts by phosphorylation of VE-cadherin

Regulation of the integrity of endothelial cell–cell contacts by phosphorylation of VE-cadherin

N-glycosylation status of E-cadherin controls cytoskeletal dynamics through the organization of distinct β-catenin- and γ-catenin-containing AJs

N-glycosylation of E-cadherin has been shown to inhibit cell–cell adhesion.

Specifically, our recent studies have provided evidence that

  • the reduction of E-cadherin N-glycosylation
  • promoted the recruitment of stabilizing components,
  • vinculin and serine/ threonine protein phosphatase 2A (PP2A), to adherens junctions (AJs)
  • and enhanced the association of AJs with the actin cytoskeleton.

Here, we examined the details of how

N-glycosylation of E-cadherin affected the molecular organization of AJs and their cytoskeletal interactions.

Using the hypoglycosylated E-cadherin variant, V13, we show that

V13/β-catenin complexes preferentially interacted with PP2A and with the microtubule motor protein dynein.

This correlated with dephosphorylation of the microtubule-associated protein tau, suggesting that

increased association of PP2A with V13-containing AJs promoted their tethering to microtubules.

These studies provide the first mechanistic insights into how N-glycosylation of E-cadherin drives changes in AJ composition through

  • the assembly of distinct β-catenin- and γ-catenin-containing scaffolds that impact the interaction with different cytoskeletal components

Cytoskeletal Basis of Ion Channel Function in Cardiac Muscle

MacKinnon. Fig 1  Ion channels exhibit three basic properties

MacKinnon. Fig 1 Ion channels exhibit three basic properties

In order to contract and accommodate the repetitive morphological changes induced by the cardiac cycle, cardiomyocytes

depend on their highly evolved and specialized cytoskeletal apparatus.

Defects in components of the cytoskeleton, in the long term,

affect the ability of the cell to compensate at both functional and structural levels.

In addition to the structural remodeling,

the myocardium becomes increasingly susceptible to altered electrical activity leading to arrhythmogenesis.

The development of arrhythmias secondary to structural remodeling defects has been noted, although the detailed molecular mechanisms are still elusive.

subjects with severe left ventricular chamber dilation such as in DCM can have left bundle branch block (LBBB), while right bundle branch block (RBBB) is more characteristic of right ventricular failure.  LBBB and RBBB have both been repeatedly associated with AV block in heart failure.

The impact of volume overload on structural and electro-cardiographic alterations has been noted in cardiomyopathy patients treated with left ventricular assist device (LVAD) therapy, which puts the heart at mechanical rest.

In LVAD-treated subjects,

QRS- and both QT- and QTc duration decreased,

suggesting that QRS- and QT-duration are significantly influenced by mechanical load and

that the shortening of the action potential duration contributes to the improved contractile performance after LVAD support.

An early postoperative period study after cardiac unloading therapy in 17 HF patients showed that in the first two weeks after LVAD implantation,

HF was associated with a relatively high incidence of ventricular arrhythmias associated with QTc interval prolongation.

In addition, a recent retrospective study of 100 adult patients with advanced HF, treated with an axial-flow HeartMate LVAD suggested that

  • the rate of new-onset monomorphic ventricular tachycardia (MVT) was increased in LVAD treated patients compared to patients given only medical treatment,

The myocardium is exposed to severe and continuous biomechanical stress during each contraction-relaxation cycle. When fiber tension remains uncompensated or simply unbalanced,

it may represent a trigger for arrhythmogenesis caused by cytoskeletal stretching,

which ultimately leads to altered ion channel localization, and subsequent action potential and conduction alterations.

Cytoskeletal proteins not only provide the backbone of the cellular structure, but they also

maintain the shape and flexibility of the different sub-cellular compartments, including the

1.plasma membrane,

2.the double lipid layer, which defines the boundaries of the cell and where

ion channels are mainly localized.

The interaction between the sarcomere, which is the basic for the passive force during diastole and for the restoring force during systole.

Sarcomeric Proteins and Ion Channels

besides fiber stretch associated with mechanical and hemodynamic impairment, cytoskeletal alterations due to primary genetic defects or indirectly to alterations in response to cellular injury can potentially

1.affect ion channel anchoring, and trafficking, as well as

2.functional regulation by second messenger pathways,

3.causing an imbalance in cardiac ionic homeostasis that will trigger arrhythmogenesis.

Intense investigation of

the sarcomeric actin network,

the Z-line structure, and

chaperone molecules docking in the plasma membrane,

has shed new light on the molecular basis of

  • cytoskeletal interactions in regulating ion channels

Actin disruption using cytochalasin D, an agent that interferes with actin polymerization, increased Na+ channel activity in 90% of excised patches tested within 2 min, which indicated that

the integrity of the filamentous actin (F-actin) network was essential for the maintenance of normal Na+ channel function

These data were the first to support a role for the cytoskeleton in cardiac arrhythmias.

Molecular interactions between the cytoskeleton and ion channels

The figure illustrates the interactions between the ion channels on the sarcolemma, and the sarcomere in cardiac myocytes. Note that the Z-line is connected to the cardiac T-tubules. The diagram illustrates the complex protein-protein interactions that occur between structural components of the cytoskeleton and ion channels. The cytoskeleton is involved in regulating the metabolism of ion channels, modifying their expression, localization, and electrical properties.

sarcomere structure

sarcomere structure

It is important to be aware of the enormous variety of clinical presentations that derive from distinct variants in the same pool of genetic factors. Knowledge of these variants could facilitate tailoring the therapy of choice for each patient. In particular,

the recent findings of structural and functional links between

the cytoskeleton and ion channels

could expand the therapeutic interventions in

arrhythmia management in structurally abnormal myocardium, where aberrant binding

between cytoskeletal proteins can directly or indirectly alter ion channel function.

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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

Curator: Aviva Lev-Ari, PhD, RN

UPDATED on 11/6/2018

Which biological systems should be engineered?

To solve real-world problems using emerging abilities in synthetic biology, research must focus on a few ambitious goals, argues Dan Fletcher, Professor of bioengineering and biophysics, and chair of the Department of Bioengineering at the University of California, Berkeley, USA. He is also a Chan Zuckerberg Biohub Investigator.
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Artificial blood cells. Blood transfusions are crucial in treatments for everything from transplant surgery and cardiovascular procedures to car accidents, pregnancy-related complications and childhood malaria (see go.nature.com/2ozbfwt). In the United States alone, 36,000 units of red blood cells and 7,000 units of platelets are needed every day (see go.nature.com/2ycr2wo).

But maintaining an adequate supply of blood from voluntary donors can be challenging, especially in low- and middle-income countries. To complicate matters, blood from donors must be checked extensively to prevent the spread of infectious diseases, and can be kept for only a limited time — 42 days or 5 days for platelets alone. What if blood cells could be assembled from purified or synthesized components on demand?

In principle, cell-like compartments could be made that have the oxygen-carrying capacity of red blood cells or the clotting ability of platelets. The compartments would need to be built with molecules on their surfaces to protect the compartments from the immune system, resembling those on a normal blood cell. Other surface molecules would be needed to detect signals and trigger a response.

In the case of artificial platelets, that signal might be the protein collagen, to which circulating platelets are exposed when a blood vessel ruptures5. Such compartments would also need to be able to release certain molecules, such as factor V or the von Willebrand clotting factor. This could happen by building in a rudimentary form of exocytosis, for example, whereby a membrane-bound sac containing the molecule would be released by fusing with the compartment’s outer membrane.

It is already possible to encapsulate cytoplasmic components from living cells in membrane compartments6,7. Now a major challenge is developing ways to insert desired protein receptors into the lipid membrane8, along with reconstituting receptor signalling.

Red blood cells and platelets are good candidates for the first functionally useful synthetic cellular system because they lack nuclei. Complex functions such as nuclear transport, protein synthesis and protein trafficking wouldn’t have to be replicated. If successful, we might look back with horror on the current practice of bleeding one person to treat another.

Micrograph of red blood cells, 3 T-lymphocytes and activated platelets

Human blood as viewed under a scanning electron microscope.Credit: Dennis Kunkel Microscopy/SPL

Designer immune cells. Immunotherapy is currently offering new hope for people with cancer by shaping how the immune system responds to tumours. Cancer cells often turn off the immune response that would otherwise destroy them. The use of therapeutic antibodies to stop this process has drastically increased survival rates for people with multiple cancers, including those of the skin, blood and lung9. Similarly successful is the technique of adoptive T-cell transfer. In this, a patient’s T cells or those of a donor are engineered to express a receptor that targets a protein (antigen) on the surface of tumour cells, resulting in the T cells killing the cancerous cells (called CAR-T therapies)10. All of this has opened the door to cleverly rewiring the downstream signalling that results in the destruction of tumour cells by white blood cells11.

What if researchers went a step further and tried to create synthetic cells capable of moving towards, binding to and eliminating tumour cells?

In principle, untethered from evolutionary pressures, such cells could be designed to accomplish all sorts of tasks — from killing specific tumour cells and pathogens to removing brain amyloid plaques or cholesterol deposits. If mass production of artificial immune cells were possible, it might even lessen the need to tailor treatments to individuals — cutting costs and increasing accessibility.

To ensure that healthy cells are not targeted for destruction, engineers would also need to design complex signal-processing systems and safeguards. The designer immune cells would need to be capable of detecting and moving towards a chemical signal or tumour. (Reconstituting the complex process of cell motility is itself a major challenge, from the delivery of energy-generating ATP molecules to the assembly of actin and myosin motors that enable movement.)

Researchers have already made cell-like compartments that can change shape12, and have installed signalling circuits within them13. These could eventually be used to control movement and mediate responses to external signals.

Smart delivery vehicles. The relative ease of exposing cells in the lab to drugs, as well as introducing new proteins and engineering genomes, belies how hard it is to deliver molecules to specific locations inside living organisms. One of the biggest challenges in most therapies is getting molecules to the right place in the right cell at the right time.

Harnessing the natural proclivity of viruses to deliver DNA and RNA molecules into cells has been successful14. But virus size limits cargo size, and viruses don’t necessarily infect the cell types researchers and clinicians are aiming at. Antibody-targeted synthetic vesicles have improved the delivery of drugs to some tumours. But getting the drug close to the tumour generally depends on the vesicles leaking from the patient’s circulatory system, so results have been mixed.

Could ‘smart’ delivery vehicles containing therapeutic cargo be designed to sense where they are in the body and move the cargo to where it needs to go, such as across the blood–brain barrier?

This has long been a dream of those in drug delivery. The challenges are similar to those of constructing artificial blood and immune cells: encapsulating defined components in a membrane, incorporating receptors into that membrane, and designing signal-processing systems to control movement and trigger release of the vehicle’s contents.

The development of immune-cell ‘backpacks’ is an exciting step in the right direction. In this, particles containing therapeutic molecules are tethered to immune cells, exploiting the motility and targeting ability of the cells to carry the molecules to particular locations15.

A minimal chassis for expression. In each of the previous examples, the engineered cell-like system could conceivably be built to function over hours or days, without the need for additional protein production and regulation through gene expression. For many other tasks, however, such as the continuous production of insulin in the body, it will be crucial to have the ability to express proteins, upregulate or downregulate certain genes, and carry out functions for longer periods.

Engineering a ‘minimal chassis’ that is capable of sustained gene expression and functional homeostasis would be an invaluable starting point for building synthetic cells that produce proteins, form tissues and remain viable for months to years. This would require detailed understanding and incorporation of metabolic pathways, trafficking systems and nuclear import and export — an admittedly tall order.

It is already possible to synthesize DNA in the lab, whether through chemically reacting bases or using biological enzymes or large-scale assembly in a cell16. But we do not yet know how to ‘boot up’ DNA and turn a synthetic genome into a functional system in the absence of a live cell.

Since the early 2000s, biologists have achieved gene expression in synthetic compartments loaded with cytoplasmic extract17. And genetic circuits of increasing complexity (in which the expression of one protein results in the production or degradation of another) are now the subject of extensive research. Still to be accomplished are: long-lived gene expression, basic protein trafficking and energy production reminiscent of live cells.

End Quote

SOURCE

https://www.nature.com/articles/d41586-018-07291-3?utm_source=briefing-dy&utm_medium=email&utm_campaign=briefing&utm_content=20181106

 

UPDATED on 10/14/2013

Genetics of Atherosclerotic Plaque in Patients with Chronic Coronary Artery Disease

372/3:15 Genetic influence on LpPLA2 activity at baseline as evaluated in the exome chip-enriched GWAS study among ~13600 patients with chronic coronary artery disease in the STABILITY (STabilisation of Atherosclerotic plaque By Initiation of darapLadIb TherapY) trial. L. Warren, L. Li, D. Fraser, J. Aponte, A. Yeo, R. Davies, C. Macphee, L. Hegg, L. Tarka, C. Held, R. Stewart, L. Wallentin, H. White, M. Nelson, D. Waterworth.

Genetic influence on LpPLA2 activity at baseline as evaluated in the exome chip-enrichedGWASstudy among ~13600 patients with chronic coronary artery disease in the STABILITY (STabilisation of Atherosclerotic plaque By Initiation of darapLadIb TherapY) trial.

L. Warren1, L. Li1, D. Fraser1, J. Aponte1, A. Yeo2, R. Davies3, C. Macphee3, L. Hegg3,

L. Tarka3, C. Held4, R. Stewart5, L. Wallentin4, H. White5, M. Nelson1, D.

Waterworth3.

1) GlaxoSmithKline, Res Triangle Park, NC;

2) GlaxoSmithKline, Stevenage, UK;

3) GlaxoSmithKline, Upper Merion, Pennsylvania, USA;

4) Uppsala Clinical Research Center, Department of Medical Sciences, Uppsala University, Uppsala, Sweden;

5) 5Green Lane Cardiovascular Service, Auckland Cty Hospital, Auckland, New Zealand.

STABILITY is an ongoing phase III cardiovascular outcomes study that compares the effects of darapladib enteric coated (EC) tablets, 160 mg versus placebo, when added to the standard of care, on the incidence of major adverse cardiovascular events (MACE) in subjects with chronic coronary heart disease (CHD). Blood samples for determination of the LpPLA2 activity level in plasma and for extraction of DNA was obtained at randomization. To identify genetic variants that may predict response to darapladib, we genotyped ~900K common and low frequency coding variations using Illumina OmniExpress GWAS plus exome chip in advance of study completion. Among the 15828 Intent-to-Treat recruited subjects, 13674 (86%) provided informed consent for genetic analysis. Our pharmacogenetic (PGx) analysis group is comprised of subjects from 39 countries on five continents, including 10139 Whites of European heritage, 1682 Asians of East Asian or Japanese heritage, 414 Asians of Central/South Asian heritage, 268 Blacks, 1027 Hispanics and 144 others. Here we report association analysis of baseline levels of LpPLA2 to support future PGx analysis of drug response post trial completion. Among the 911375 variants genotyped, 213540 (23%) were rare (MAF < 0.5%).

Our analyses were focused on the drug target, LpPLA2 enzyme activity measured at baseline. GWAS analysis of LpPLA2 activity adjusting for age, gender and top 20 principle component scores identified 58 variants surpassing GWAS-significant threshold (5e-08).

Genome-wide stepwise regression analyses identified multiple independent associations from PLA2G7, CELSR2, APOB, KIF6, and APOE, reflecting the dependency of LpPLA2 on LDL-cholesterol levels. Most notably, several low frequency and rare coding variants in PLA2G7 were identified to be strongly associated with LpPLA2 activity. They are V279F (MAF=1.0%, P= 1.7e-108), a previously known association, and four novel associations due to I1317N (MAF=0.05%, P=4.9e-8), Q287X (MAF=0.05%, P=1.6e-7), T278M (MAF=0.02%, P=7.6e-5) and L389S (MAF=0.04%, P=4.3e-4).

All these variants had enzyme activity lowering effects and each appeared to be specific to certain ethnicity. Our comprehensive PGx analyses of baseline data has already provided great insight into common and rare coding genetic variants associated with drug target and related traits and this knowledge will be invaluable in facilitating future PGx investigation of darapladib response.

SOURCE

http://www.ashg.org/2013meeting/pdf/46025_Platform_bookmark%20for%20Web%20Final%20from%20AGS.pdf

Synthetic Biology: On Advanced Genome Interpretation for

  • Gene Variants and
  • Pathways,
  • Inversion Polymorphism,
  • Passenger Deletions,
  • De Novo Mutations,
  • Whole Genome Sequencing w/Linkage Analysis

What is the Genetic Base of Atherosclerosis and Loss of Arterial Elasticity with Aging?

In a recent publication by my colleague, Stephen J. Williams, Ph.D. on  5/15/2013 titled

Finding the Genetic Links in Common Disease:  Caveats of Whole Genome Sequencing Studies

https://pharmaceuticalintelligence.com/2013/05/15/finding-the-genetic-links-in-common-disease-caveats-of-whole-genome-sequencing-studies/

we learned that:

  • Groups of variants in the same gene confirmed link between APOC3 and higher risk for early-onset heart attack
  • No other significant gene variants linked with heart disease

APOC3 – apolipoprotein C-III – Potential Relevance to the Human Aging Process

Main reason for selection
Entry selected based on indirect or inconclusive evidence linking the gene product to ageing in humans or in one or more model systems
Description
APOC3 is involved in fat metabolism and may delay the catabolism of triglyceride-rich particles. Changes in APOC3 expression levels have been reported in aged mice [1754]. Results from mice suggest that FOXO1 may regulate the expression of APOC3 [1743]. Polymorphisms in the human APOC3 gene and promoter have been associated with lipoprotein profile, cardiovascular health, insulin (INS) sensitivity, and longevity [1756]. Therefore, APOC3 may impact on some age-related diseases, though its exact role in human ageing remains to be determined.

Cytogenetic information

Cytogenetic band
11q23.1-q2
Location
116,205,833 bp to 116,208,997 bp
Orientation
Plus strand

Display region using the UCSC Genome Browser

Protein information

Gene Ontology
Process: GO:0006869; lipid transport
GO:0016042; lipid catabolic process
GO:0042157; lipoprotein metabolic process
Function: GO:0005319; lipid transporter activity
Cellular component: GO:0005576; extracellular region
GO:0042627; chylomicron

Protein interactions and network

No interactions in records.

Retrieve sequences for APOC3

Promoter
Promoter
ORF
ORF
CDS
CDS

Homologues in model organisms

Bos taurus
APOC3_BOVI
Mus musculus
Apoc3
Pan troglodytes
APOC3

In other databases

AnAge
This species has an entry in AnAge

Selected references

  • [2125] Pollin et al. (2008) A null mutation in human APOC3 confers a favorable plasma lipid profile and apparent cardioprotection.PubMed
  • [1756] Atzmon et al. (2006) Lipoprotein genotype and conserved pathway for exceptional longevity in humansPubMed
  • [1755] Araki and Goto (2004) Dietary restriction in aged mice can partially restore impaired metabolism of apolipoprotein A-IV and C-IIIPubMed
  • [1743] Altomonte et al. (2004) Foxo1 mediates insulin action on apoC-III and triglyceride metabolismPubMed
  • [1754] Araki et al. (2004) Impaired lipid metabolism in aged mice as revealed by fasting-induced expression of apolipoprotein mRNAs in the liver and changes in serum lipidsPubMed
  • [1753] Panza et al. (2004) Vascular genetic factors and human longevityPubMed
  • [1752] Anisimov et al. (2001) Age-associated accumulation of the apolipoprotein C-III gene T-455C polymorphism C 

http://genomics.senescence.info/genes/entry.php?hgnc=APOC3

Apolipoprotein C-III is a protein component of very low density lipoprotein (VLDL). APOC3 inhibitslipoprotein lipase and hepatic lipase; it is thought to inhibit hepatic uptake[1] of triglyceride-rich particles. The APOA1, APOC3 and APOA4 genes are closely linked in both rat and human genomes. The A-I and A-IV genes are transcribed from the same strand, while the A-1 and C-III genes are convergently transcribed. An increase in apoC-III levels induces the development of hypertriglyceridemia.

Clinical significance

Two novel susceptibility haplotypes (specifically, P2-S2-X1 and P1-S2-X1) have been discovered in ApoAI-CIII-AIV gene cluster on chromosome 11q23; these confer approximately threefold higher risk ofcoronary heart disease in normal[2] as well as non-insulin diabetes mellitus.[3]Apo-CIII delays the catabolism of triglyceride rich particles. Elevations of Apo-CIII found in genetic variation studies may predispose patients to non-alcoholic fatty liver disease.

  1. ^ Mendivil CO, Zheng C, Furtado J, Lel J, Sacks FM (2009). “Metabolism of VLDL and LDL containing apolipoprotein C-III and not other small apolipoproteins – R2”.Arteriosclerosis, Thrombosis and Vascular Biology 30 (2): 239–45. doi:10.1161/ATVBAHA.109.197830PMC 2818784PMID 19910636.
  2. ^ Singh PP, Singh M, Kaur TP, Grewal SS (2007). “A novel haplotype in ApoAI-CIII-AIV gene region is detrimental to Northwest Indians with coronary heart disease”. Int J Cardiol 130 (3): e93–5. doi:10.1016/j.ijcard.2007.07.029PMID 17825930.
  3. ^ Singh PP, Singh M, Gaur S, Grewal SS (2007). “The ApoAI-CIII-AIV gene cluster and its relation to lipid levels in type 2 diabetes mellitus and coronary heart disease: determination of a novel susceptible haplotype”. Diab Vasc Dis Res 4 (2): 124–29. doi:10.3132/dvdr.2007.030PMID 17654446.

In 2013 we reported on the discovery that there is a

Genetic Associations with Valvular Calcification and Aortic Stenosis

N Engl J Med 2013; 368:503-512

February 7, 2013DOI: 10.1056/NEJMoa1109034

METHODS

We determined genomewide associations with the presence of aortic-valve calcification (among 6942 participants) and mitral annular calcification (among 3795 participants), as detected by computed tomographic (CT) scanning; the study population for this analysis included persons of white European ancestry from three cohorts participating in the Cohorts for Heart and Aging Research in Genomic Epidemiology consortium (discovery population). Findings were replicated in independent cohorts of persons with either CT-detected valvular calcification or clinical aortic stenosis.

CONCLUSIONS

Genetic variation in the LPA locus, mediated by Lp(a) levels, is associated with aortic-valve calcification across multiple ethnic groups and with incident clinical aortic stenosis. (Funded by the National Heart, Lung, and Blood Institute and others.)

SOURCE:

N Engl J Med 2013; 368:503-512

Related Research by Author & Curator of this article:

Artherogenesis: Predictor of CVD – the Smaller and Denser LDL Particles

Cardiovascular Biomarkers

Genetics of Conduction Disease: Atrioventricular (AV) Conduction Disease (block): Gene Mutations – Transcription, Excitability, and Energy Homeostasis

Genomics & Genetics of Cardiovascular Disease Diagnoses: A Literature Survey of AHA’s Circulation Cardiovascular Genetics, 3/2010 – 3/2013

Hypertriglyceridemia concurrent Hyperlipidemia: Vertical Density Gradient Ultracentrifugation a Better Test to Prevent Undertreatment of High-Risk Cardiac Patients

Hypertension and Vascular Compliance: 2013 Thought Frontier – An Arterial Elasticity Focus

Personalized Cardiovascular Genetic Medicine at Partners HealthCare and Harvard Medical School

Genomics Orientations for Individualized Medicine Volume One

Market Readiness Pulse for Advanced Genome Interpretation and Individualized Medicine

We present below the MARKET LEADER in Interpretation of the Genomics Computations Results in the emerging new ERA of Medicine:  Genomic Medicine, Knome.com and its home grown software power house.

A second Case study in the  Advanced Genome Interpretation and Individualized Medicine presented following the Market Leader, is the Genome-Phenome Analyzer by SimulConsult, A Simultaneous Consult On Your Patient’s Diagnosis, Chestnut Hill, MA

 

2012: The Year When Genomic Medicine Started Paying Off

Luke Timmerman

An excerpt of an interesting article mentioning Knome [emphasis ours]…

Remember a couple of years ago when people commemorated the 10-year anniversary of the first draft human genome sequencing? The storyline then, in 200, was that we all went off to genome camp and only came home with a lousy T-shirt. Society, we were told, invested huge scientific resources in deciphering the code of life, and there wasn’t much of a payoff in the form of customized, personalized medicine.

That was an easy conclusion to reach then, when personalized medicine advocates could only point to a couple of effective targeted cancer drugs—Genentech’s Herceptin and Novartis’ Gleevec—and a couple of diagnostics. But that’s changing. My inbox the past week has been full of analyst reports from medical meetings, which mostly alerted readers to mere “incremental” advances with a number of genomic-based medicines and diagnostics. But that’s a matter of focusing on the trees, not the forest. This past year, we witnessed some really impressive progress from the early days of “clinical genomics” or “medical genomics.” The investment in deep understanding of genomics and biology is starting to look visionary.

The movement toward clinical genomics gathered steam back in June at the American Society of Clinical Oncology annual meeting. One of the hidden gem stories from ASCO was about little companies like Cambridge, MA-based Foundation Medicine and Cambridge, MA-based Knome that started seeing a surprising surge in demand from physicians for their services to help turn genomic data into medical information. The New York Times wrote a great story a month later about a young genomics researcher at Washington University in St. Louis who got cancer, had access to incredibly rich information about his tumors, and—after some wrestling with his insurance company—ended up getting a targeted drug nobody would have thought to prescribe without that information. And last month, I checked back on Stanford University researcher Mike Snyder, who made headlines this year using a smorgasbord of “omics” tools to correctly diagnose himself early with Type 2 diabetes, and then monitor his progress back into a healthy state–read the entire article

http://www.knome.com/knome-blog/2012-the-year-when-genomic-medicine-started-paying-off/

Knome and Real Time Genomics Ink Deal to Integrate and Sell the RTG Variant Platform on knoSYS™100 System

Partnership to bring accurate and fast genome analysis to translational researchers

CAMBRIDGE, MA –  May 6, 2013 – Knome Inc., the genome interpretation company, and Real Time Genomics, Inc., the genome analytics company, today announced that the Real Time Genomics (RTG) Variant platform will be integrated into every shipment of the knoSYS™100 interpretation system. The agreement enables customers to easily purchase the RTG analytics engine as an upgrade to the system. The product will combine two world-class commercial platforms to deliver end-to-end genome analytics and interpretation with superior accuracy and speed. Financial terms of the agreement were not disclosed.

“In the past year demand for genome interpretation has surged as translational researchers and clinicians adopt sequencing for human disease discovery and diagnosis,” said Wolfgang Daum, CEO of Knome. “Concomitant with that demand is the need for accurate and easy-to-use industrial grade analysis that meets expectations of clinical accuracy. The RTG platform is both incredibly fast and truly differentiating to customers doing family studies, and we are excited to add such a powerful platform to the knoSYS ecosystem.”

The partnership simplifies the purchasing process by allowing knoSYS customers to purchase the RTG platform directly from Knome sales representatives.

“The Knome system is a perfect complementary channel to further expand our commercial effort to bring the RTG platform to market,” said Steve Lombardi, CEO of Real Time Genomics. “Knome has built a recognizable brand around human clinical genome interpretation, and by delivering the RTG platform within their system, both companies are simplifying genomics to help customers understand human disease and guide clinical actions.”

About Knome

Knome Inc. (www.knome.com) is a leading provider of human genome interpretation systems and services. We help clients in two dozen countries identify the genetic basis of disease, tumor growth, and drug response. Designed to accelerate and industrialize the process of interpreting whole genomes, Knome’s big data technologies are helping to pave the healthcare industry’s transition to molecular-based, precision medicine.

About Real Time Genomics

Real Time Genomics (www.realtimegenomics.com) has a passion for genomics.  The company offers software tools and applications for the extraction of unique value from genomes.  Its competency lies in applying the combination of its patented core technology and deep computational expertise in algorithms to solve problems in next generation genomic analysis.  Real Time Genomics is a private San Francisco based company backed by investment from Catamount Ventures, Lightspeed Venture Partners, and GeneValue Ltd.

http://www.knome.com/knome-blog/knome-and-real-time-genomics-ink-deal-to-integrate-and-sell-the-rtg-variant-platform-on-knosys100-system/

Direct-to-Consumer Genomics Reinvents Itself

Malorye Allison

An excerpt of an interesting article mentioning Knome [emphasis ours]:

Cambridge, Massachusetts–based Knome made one of the splashiest entries into the field, but has now turned entirely to contract research. The company began providing DTC whole-genome sequencing to independently wealthy individuals at a time when the price was still sky high. The company’s first client, Dan Stoicescu, was a former biotech entrepreneur who paid $350,000 to have his genome sequenced in 2008 so he could review it “like a stock portfolio” as new genetic discoveries unfolded4. About a year later, the company was auctioning off a genome, with such frills as a dinner with renowned Harvard genomics researcher George Church, at a starting price of $68,000; at the time, a full-genome sequence came at the price of $99,000, indicating that the cost of genome sequencing has been plummeting steadily.

Now, the company’s model is very different. “We stopped working with the ‘wealthy healthy’ in 2010,” says Jonas Lee, Knome’s chief marketing officer. “The model changed as sequencing changed.” The new emphasis, he says, is now on using Knome’s technology and technical expertise for genome interpretation. Knome’s customers are researchers, pharmaceutical companies and medical institutions, such as Johns Hopkins University School of Medicine in Baltimore, which in January signed the company up to interpret 1,000 genomes for a study of genetic variants underlying asthma in African American and African Caribbean populations.

Knome is trying to advance the clinical use of genomics, working with groups that “want to be prepared for what’s ahead,” Lee says. “We work with at least 50 academic institutions and 20 pharmaceutical companies looking at variants and drug response.” Cancer and idiopathic genetic diseases are the first sweet spots for genomic sequencing, he says. Although cancer genomics has been hot for a while, a recent string of discoveries of Mendelian diseases5 made by whole-genome sequencing has lit up that field, too. Lee is also confident, however, that “chronic diseases like heart disease are right behind those.” The company also provides software tools. The price for its KnomeDiscovery sequencing and analysis service starts at about $12,000 per sample–read the entire article here.

http://www.knome.com/knome-blog/direct-to-consumer-genomics-reinvents-itself/

Regenesis: How Synthetic Biology Will Reinvent Nature and Ourselves

VIEW VIDEO

http://www.colbertnation.com/the-colbert-report-videos/419824/october-04-2012/george-church

 

Knome Software Makes Sense of the Genome

The startup’s software takes raw genome data and creates a usable report for doctors.

DNA decoder: Knome’s software can tease out medically relevant changes in DNA that could disrupt individual gene function or even a whole molecular pathway, as is highlighted here—certain mutations in the BRCA2 gene, which affects the function of many other genes, can be associated with an increased risk of breast cancer.

A genome analysis company called Knome is introducing software that could help doctors and other medical professionals identify genetic variations within a patient’s genome that are linked to diseases or drug response. This new product, available for now only to select medical institutions, is a patient-focused spin on Knome’s existing products aimed at researchers and pharmaceutical companies. The Knome software turns a patient’s raw genome sequence into a medically relevant report on disease risks and drug metabolism. The software can be run within a clinic’s own network—rather than in the cloud, as is the case with some genome-interpretation services—which keeps the information private.

Advances in DNA sequencing technology have sharply reduced the amount of time and money required to identify all three billion base pairs of DNA in a person’s genome. But the use of genomic information for medical decisions is still limited because the process creates such large volumes of data. Less than five years ago, Knome, based in Cambridge, Massachusetts, made headlines by offering what seemed then like a low price—$350,000—for a genome sequencing and profiling package. The same service now costs just a few thousand dollars.

Today, genome profiling has two main uses in the clinic. It’s part of the search for the cause of rare genetic diseases, and it generates tumor-specific profiles to help doctors discover the weaknesses of a patient’s particular cancer. But within a few years, the technique could move beyond rare diseases and cancer. The information gleaned from a patient’s genome could explain the origin of specific disease, could help save costs by allowing doctors to pretreat future diseases, or could improve the effectiveness and safety of medications by allowing doctors to prescribe drugs that are tuned to a person’s ability to metabolize drugs.

But teasing out the relevant genetic information from a patient’s genome is not trivial. To find the particular genetic variant that causes a specific disease or drug response can require expertise from many disciplines—from genetics to statistics to software engineering—and a lot of time. In any given patient’s genome, millions of places in that genome will differ from the standard of reference. The vast majority of these differences, or variants, will be unrelated to a patient’s medical condition, but determining that can take between 20 minutes and two hours for each variant, says Heidi Rehm, a clinical geneticist who directs the Laboratory for Molecular Medicine at Partners Healthcare Center for Personalized Genetic Medicine in Boston, and who will soon serve on the clinical advisory board of Knome. “If you scale that to … millions of variants, it becomes impossible.”

A software package like Knome’s can help whittle down the list based on factors such as disease type, the pattern of inheritance in a family, and the effects of given mutations on genes. Other companies have introduced Web- or cloud-based services to perform such an analysis, but Knome’s software suite can operate within a hospital’s network, which is critically important for privacy-concerned hospitals.

The greatest benefit of the widespread adoption of genomics in the clinic will come from the “clinical intelligence” doctors gain from networks of patient data, says Martin Tolar, CEO of Knome. Information about the association between certain genetic variants and disease or drug response could be anonymized—that is, no specific patient could be tied to the data—and shared among large hospital networks. Knome’s software will make it easy to share that kind of information, says Tolar.

“In the future, you could be in the situation where your physician will be able to pull the most appropriate information for your specific case that actually leads to recommendations about drugs and so forth,” he says.

http://www.technologyreview.com/news/428179/knome-software-makes-sense-of-the-genome/

An End-to-end Human Genome Interpretation System

The knoSYS™100 seamlessly integrates an interpretation application (knoSOFT) and informatics engine (kGAP) with a high-performance grid computer. Designed for whole genome, exome, and targeted NGS data, the knoSYS™100 helps labs quickly go “from reads to reports.”


 


Advanced Interpretation and Reporting Software

The knoSYS™100 ships with knoSOFT, an advanced application for managing sequence data through the informatics pipeline, filtering variants, running gene panels, classifying/interpreting variants, and reporting results.

knoSOFT has powerful and scalable multi-sample comparison features–capable of performing family studies, tumor/normal studies, and large case-control comparisons of hundreds of whole genomes.

Multiple simultaneous users (10) are supported, including technicians running sequence data through informatics pipeline, developers creating next-generation gene panels, geneticists researching causal variants, and production staff processing gene panels.

http://www.knome.com/knosys-100-overview/

Publications

View our collection of journal articles and genome research papers written by Knome employees, Knome board members, and other industry experts.

Publications by Knome employees and board members

The Top Two Axes of Variation of the Combined Dataset (MS, BD, PD, and IBD)

21 Aug 2012

Discerning the Ancestry of European Americans in Genetic Association Studies

Co-authored by Dr. David Goldstein, Clinical and Scientific board member for Knome

Author summary: Genetic association studies analyze both phenotypes (such as disease status) and genotypes (at sites of DNA variation) of a given set of individuals. … more

Pedigree and genetic risk prediction workflow

20 Aug 2012

Phased Whole-Genome Genetic Risk in a Family Quartet Using a Major Allele Reference Sequence

Co-authored by Dr. George Church and Dr. Heidi Rehm, Clinical and Scientific Board Members for Knome

Author summary: An individual’s genetic profile plays an important role in determining risk for disease and response to medical therapy. The development of technologies that facilitate rapid whole-genome sequencing will provide unprecedented power in the estimation of disease risk. Here we develop methods to characterize genetic determinants of disease risk and … more

20 Aug 2012

A Genome-Wide Investigation of SNPs and CNVs in Schizophrenia

Co-authored by Dr. David Goldstein, Clinical and Scientific board member for Knome

Author summary: Schizophrenia is a highly heritable disease. While the drugs commonly used to treat schizophrenia offer important relief from some symptoms, other symptoms are not well treated, and the drugs cause serious adverse effects in many individuals. This has fueled intense interest over the years in identifying genetic contributors to … more

fetchObject

20 Aug 2012

Whole-Genome Sequencing of a Single Proband Together with Linkage Analysis Identifies a Mendelian Disease Gene

Co-authored by Dr. David Goldstein, Clinical and Scientific board member for Knome

Author summary: Metachondromatosis (MC) is an autosomal dominant condition characterized by exostoses (osteochondromas), commonly of the hands and feet, and enchondromas of long bone metaphyses and iliac crests. MC exostoses may regress or even resolve over time, and short stature … more

19 Aug 2012

Exploring Concordance and Discordance for Return of Incidental Findings from Clinical Sequencing Co-authored by Dr. Heidi Rehm, Clinical and Scientific board member for Knome

Introduction: There is an increasing consensus that whole-exome sequencing (WES) and whole-genome sequencing (WGS) will continue to improve in accuracy and decline in price and that the use of these technologies will eventually become an integral part of clinical medicine.1–7 … more

Publications by industry experts and thought-leaders

22 Aug 2012

Rate of De Novo Mutations and the Importance of Father’s Age to Disease Risk

Augustine Kong, Michael L. Frigge, Gisli Masson, Soren Besenbacher, Patrick Sulem, Gisli Magnusson, Sigurjon A. Gudjonsson, Asgeir Sigurdsson, Aslaug Jonasdottir, Adalbjorg Jonasdottir, Wendy S. W. Wong, Gunnar Sigurdsson, G. Bragi Walters, Stacy Steinberg, Hannes Helgason, Gudmar Thorleifsson, Daniel F. Gudbjartsson, Agnar Helgason, Olafur Th. Magnusson, Unnur Thorsteinsdottir, & Kari Stefansson

Abstract: Mutations generate sequence diversity and provide a substrate for selection. The rate of de novo mutations is therefore of major importance to evolution. Here we conduct a study of genome-wide mutation rates by sequencing the entire genomes of 78 … more

15 Aug 2012

Passenger Deletions Generate Therapeutic Vulnerabilities in Cancer

Florian L. Muller, Simona Colla, Elisa Aquilanti, Veronica E. Manzo, Giannicola Genovese, Jaclyn Lee, Daniel Eisenson, Rujuta Narurkar, Pingna Deng, Luigi Nezi, Michelle A. Lee, Baoli Hu, Jian Hu, Ergun Sahin, Derrick Ong, Eliot Fletcher-Sananikone, Dennis Ho, Lawrence Kwong, Cameron Brennan, Y. Alan Wang, Lynda Chin, & Ronald A. DePinho

Abstract: Inactivation of tumour-suppressor genes by homozygous deletion is a prototypic event in the cancer genome, yet such deletions often encompass neighbouring genes. We propose that homozygous deletions in such passenger genes can expose cancer-specific therapeutic vulnerabilities when the collaterally … more

1 Jul 2012

Structural Diversity and African Origin of the 17q21.31 Inversion Polymorphism

Karyn Meltz Steinberg, Francesca Antonacci, Peter H Sudmant, Jeffrey M Kidd, Catarina D Campbell, Laura Vives, Maika Malig, Laura Scheinfeldt, William Beggs, Muntaser Ibrahim, Godfrey Lema, Thomas B Nyambo, Sabah A Omar, Jean-Marie Bodo, Alain Froment, Michael P Donnelly, Kenneth K Kidd, Sarah A Tishkoff, & Evan E Eichler

Abstract: The 17q21.31 inversion polymorphism exists either as direct (H1) or inverted (H2) haplotypes with differential predispositions to disease and selection. We investigated its genetic diversity in 2,700 individuals, with an emphasis on African populations. We characterize eight structural haplotypes … more

http://www.knome.com/publications/

knome’s Systems & Software

Technical specifications

Connections and communications

Two networks: 40-Gigabit Infiniband QDR via a Mellanox Switch for storage traffic and HP ProCurve switch for network traffic

High performance computing cluster

Four nodes, each node with two 8-core/16 thread, 2.4Ghz, 64 bit Intel® Xeon® E5-2660 processor with 20MB cache, 128GB of DDR3 ECC 1600 memory; 2x2TB SATA drives (7,200RPM)

Metadata server

2x2TB 3.5″ drives with 6GB/sec SATA, RAID 1 and 2x300GB SSD (RAID 1)

Object storage server

Lustre array: Two 12x4TB arrays of 12 3.5″ drives with 6GB/sec serial SATA channels, each OSS powered by a 6-core Intel Xeon 64-bit processor running at 20GHz with 32GB RAM.

knoSYS_server

96TB total, 64TB useable storage (redundancy for failure tolerance). Expandable 384TB total.

Data sources

Reference genome GRCh37 (HG19)

dbSNP, v137

Condel (SIFT and PolyPhen-2)

HPO

OMIM

Exome Variant server, with allelisms and allele frequencies

1000 Genomes, with allelisms and allele frequencies

Human Gene Mutation db (HGMD)

Phastcons 46, mammalian conservation

PhyloP

Input/output formats

Input formats: kGAP accepts Illumina FASTQ and VCF 4.1 files as inputs

Output formats: annotated VCF files

Electrical and operating requirements

Line voltage: 110V to 120V AC, 200-240V (single phase)

Frequency: 50Hz to 60Hz

Current: 30A, RoSH compliant

Connection: NEMA L5-30

Operating temperature: 50° to 95° F

UPS included

Maximum operating altitude: 10,000 feet

Power consumption: 2,800 VA (peak)

Size and weight

Height 49.2 Inches (1250 mm)
Width 30.7 Inches (780 mm)
Depth 47.6 Inches (1210 mm)
Weight 394 lbs (179 kg)

Noise generation and heat dissipation

Enclosure provides 28dB of acoustic noise reduction; system suitable for placing in working lab environment

7200w of active heat dissipation

Included in the package

knoSYS™100 hardware

Knome software: knoSOFT, kGAP

Operating system: Linux (CentOS 6.3)

http://www.knome.com/knosys-100-specifications/

Our research services group uses a set of advanced software tools designed for whole genome and exome interpretation. These tools are also available to our clients through our knomeBASE informatics service. In addition to various scripts, libraries, and conversion utilities, these tools include knomeVARIANTS and knomePATHWAYS.

knomeVARIANTS

Genome_software_knomeVARIANTS

knome VARIANTS is a query kit that lets users search for candidate causal variants in studied genomes. It includes a query interface (see above), scripting libraries, and data conversion utilities.

Users select cases and controls, input a putative inheritance mode, and add sensible filter criteria (variant functional class, rarity/novelty, location in prior candidate regions, etc.) to automatically generate a sorted short-list of leading candidates. The application includes a SQL query interface to let users query the database as they wish, including by complex or novel sets of criteria.

In addition to querying, the application lets users export subsets of the database for viewing in MS Excel. Subsets can be output that target common research foci, including the following:

  • Sites implicated in phenotypes, regardless of subject genotypes
  • Sites where at least one studied genome mismatches the reference
  • Sites where a particular set of one or more genomes, but no other genomes, show a novel variant
  • Sites in phenotype-implicated genes
  • Sites with nonsense, frameshift, splice-site, or read-through variants, relative to reference
  • Sites where some but not all subject genome were called

knomePATHWAYS

Genome_software_knomePATHWAYS

knomePATHWAYS is a visualization tool that overlays variants found in each sample genome onto known gene interaction networks in order to help spot functional interactions between variants in distinct genes, and pathways enriched for variants in cases versus controls, differential drug responder groups, etc.

knomePATHWAYS integrates reference data from many sources, including GO, HPRD, and MsigDB (which includes KEGG and Reactome data). The application is particularly helpful in addressing higher-order questions, such as finding candidate genes and protein pathways, that are not readily addressed from tabular annotation data alone.

http://www.knome.com/interpretation-toolkit/

Genome-Phenome Analyzer by SimulConsult

A Simultaneous Consult On Your Patient’s Diagnosis

Clinicians can get a “simultaneous consult” about their patient’s diagnosis using SimulConsult’s diagnostic decision support software.

Using the free “phenome” version, medical professionals can enter patient findings into the software and get an initial differential diagnosis and suggestions about other useful findings, including tests.  The database used by the software has > 4,000 diagnoses, most complete for genetics and neurology.  It includes all genes in GeneTests and all diseases in GeneReviews.  The information about diseases is entered by clinicians, referenced to the literature and peer-reviewed by experts.  The software takes into account pertinent negatives, temporal information, and cost of tests, information ignored in other diagnostic approaches.  It transforms medical diagnosis by lowering costs, reducing errors and eliminating the medical diagnostic odysseys experienced by far too many patients and their families.

http://www.simulconsult.com/index.html

Using the “genome-phenome analyzer” version, a lab can combine a genome variant table with the phenotypic data entered by the referring clinician, thereby using the full power of genome + phenome to arrive at a diagnosis in seconds.  An innovative measure of pertinence of genes focuses attention on the genes accounting for the clinical picture, even if more than one gene is involved.  The referring clinician can use the results in the free phenome version of the software, for example adding information from confirmatory tests or adding new findings that develop over time.  For details, click here.

http://www.simulconsult.com/genome/index.html

Michael M. Segal MD, PhD, Founder,Chairman and Chief Scientist.  Dr. Segal did his undergraduate work at Harvard and his MD and PhD at Columbia, where his thesis project outlined rules for the types of chemical synapses that will form in a nervous system.  After his residency in pediatric neurology at Columbia, he moved to Harvard Medical School, where he joined the faculty and developed the microisland system for studying small numbers of brain neurons in culture.  Using this system, he developed a simplified model of epilepsy, work that won him national and international young investigator awards, and set the stage for later work on the molecular mechanism of attention deficit disorder.  Dr. Segal has a long history of interest in computers, and patterned the SimulConsult software after the way that experienced clinicians actually think about diagnosis.  He is on the Electronic Communication Committee of the Child Neurology Society and the Scientific Program Committee of the American Medical Informatics Association.

http://www.simulconsult.com/company/management.html

Read Full Post »


Curator/Reporter Aviral Vatsa PhD, MBBS

Based on: A review by (Wink et al., 2011)

This post is in continuation to Part 1 by the same title.

In part one I covered the basics of role of redox chemistry in immune reactions, the phagosome cauldron, and how bacteria bacteria, virus and parasites trigger the complex pathway of NO production and its downstream effects. While we move further in this post, the previous post can be accessed here.

REDOX REGULATION OF IMMUNE FUNCTION

Regulation of the redox immunomodulators—NO/RNS and ROS

In addition to eradicating pathogens, NO/RNS and ROS and their chemical interactions act as effective immunomodulators that regulate many cellular metabolic pathways and tissue repair and proinflammatory pathways. Figure 3 shows these pathways.

Figure 3. Schematic overview of interactive connections between NO and ROS-mediated metabolic pathways. Credit: (Wink et al., 2011)

Regulation of iNOS enzyme activity is critical to NO production. Factors such as the availability of arginine, BH4, NADPH, and superoxide affect iNOS activity and thus NO production. In the absence of arginine and BH4 iNOS becomes a O2_/H2O2 generator (Vásquez-Vivar et al., 1999). Hence metabolic pathways that control arginine and BH4 play a role in determining the NO/superoxide balance. Arginine levels in cells depend on various factors such as type of uptake mechanisms that determine its spatial presence in various compartments and enzymatic systems. As shown in Fig3 Arginine is the sole substrate for iNOS and arginase. Arginase is another key enzyme in immunemodulation. AG is also regulated by NOS and NOX activities. NOHA, a product of NOS, inhibits AG, and O2–increases AG activity. Importantly, high AG activity is associated with elevated ROS and low NO fluxes. NO antagonises NOX2 assembly that in turn leads to reduction in O2_ production. NO also inhibits COX2 activity thus reducing ROS production. Thus, as NO levels decline, oxidative mechanisms increase. Oxidative and nitrosative stress can also decrease intracellular GSH (reduced form) levels, resulting in a reduced antioxidant capability of the cell.

Immune-associated redox pathways regulate other important metabolic cell functions that have the potential for widespread impact on cells, organs, and organisms. These pathways, such as mediated via methionine and polyamines, are critical for DNA stabilization, cell proliferation, and membrane channel activity, all of which are also involved in immune-mediated repair processes.

NO levels dictate the immune signaling pathway

NO/RNS and ROS actively control innate and adaptive immune signaling by participating in induction, maintenance, and/or termination of proinflammatory and anti-inflammatory signaling. As in pathogen eradication, the temporal and spatial concentration profiles of NO are key factors in determining immune-mediated processes.

Brune and coworkers (Messmer et al., 1994) first demonstrated that p53 expression was associated with the concentrations of NO that led to apoptosis in macrophages. Subsequent studies linked NO concentration profiles with expression of other key signaling proteins such as HIF-1α and Akt-P (Ridnour et al., 2008; Thomas et al., 2008). Various levels of NO concentrations trigger different pathways and expectedly this concentration-dependent profile varies with distance from the NO source.NO is highly diffucible and this characteristic can result in 1000 fold reduction in concentration within one cell length distance travelled from the source of production. Time course studies have also shown alteration in effects of same levels of NO over time e.g. NO-mediated ERK-P levels initially increased rapidly on exposure to NO donors and then decreased with continued NO exposure (Thomas et al., 2004), however HIF-1α levels remained high as long as NO levels were elevated. Thus some of the important factors that play critical role in NO effects are: distance from source, NO concentrations, duration of exposure, bioavailability of NO, and production/absence of other redox molecules.

Figure and legend credits: (Wink et al., 2011)

Fig 4: The effect of steady-state flux of NO on signal transduction mechanisms.

This diagram represents the level of sustained NO that is required to activate specific pathways in tumor cells. Similar effects have been seen on endothelial cells. These data were generated by treating tumor or endothelial cells with the NO donor DETANO (NOC-18) for 24 h and then measuring the appropriate outcome measures (for example, p53 activation). Various concentrations of DETANO that correspond to cellular levels of NO are: 40–60 μM DETANO = 50 nM NO; 80–120 μM DETANO = 100 nM NO; 500 μM DETANO = 400 nM NO; and 1 mM DETANO = 1 μM NO. The diagram represents the effect of diffusion of NO with distance from the point source (an activated murine macrophage producing iNOS) in vitro (Petri dish) generating 1 μM NO or more. Thus, reactants or cells located at a specific distance from the point source (i.e., iNOS, represented by star) would be exposed to a level of NO that governs a specific subset of physiological or pathophysiological reactions. The x-axis represents the different zone of NO-mediated events that is experienced at a specific distance from a source iNOS producing >1 μM. Note: Akt activation is regulated by NO at two different sites and by two different concentration levels of NO.

Species-specific NO production

The relationship of NO and immunoregulation has been established on the basis of studies on tumor cell lines or rodent macrophages, which are readily available sources of NO. However in humans the levels of protein expression for NOS enzymes and the immune induction required for such levels of expression are quite different than in rodents (Weinberg, 1998). This difference is most likely due to the human iNOS promotor rather than the activity of iNOS itself. There is a significant mismatch between the promoters of humans and rodents and that is likely to account for the notable differences in the regulation of gene induction between them. The combined data on rodent versus human NO and O2– production strongly suggest that in general, ROS production is a predominant feature of activated human macrophages, neutrophils, and monocytes, and the equivalent murine immune cells generate a combination of O2– and NO and in some cases, favor NO production. These differences may be crucial to understanding how immune responses are regulated in a species-specific manner. This is particularly useful, as pathogen challenges change constantly.

The next post in this series will cover the following topics:

The impact of NO signaling on an innate immune response—classical activation

NO and proinflammatory genes

NO and regulation of anti-inflammatory pathways

NO impact on adaptive immunity—immunosuppression and tissue-restoration response

NO and revascularization

Acute versus chronic inflammatory disease

Bibliography

1. Wink, D. A. et al. Nitric oxide and redox mechanisms in the immune response. J Leukoc Biol 89, 873–891 (2011).

2. Vásquez-Vivar, J. et al. Tetrahydrobiopterin-dependent inhibition of superoxide generation from neuronal nitric oxide synthase. J. Biol. Chem. 274, 26736–26742 (1999).

3. Messmer, U. K., Ankarcrona, M., Nicotera, P. & Brüne, B. p53 expression in nitric oxide-induced apoptosis. FEBS Lett. 355, 23–26 (1994).

4. Ridnour, L. A. et al. Molecular mechanisms for discrete nitric oxide levels in cancer. Nitric Oxide 19, 73–76 (2008).

5. Thomas, D. D. et al. The chemical biology of nitric oxide: implications in cellular signaling. Free Radic. Biol. Med. 45, 18–31 (2008).

6. Thomas, D. D. et al. Hypoxic inducible factor 1alpha, extracellular signal-regulated kinase, and p53 are regulated by distinct threshold concentrations of nitric oxide. Proc. Natl. Acad. Sci. U.S.A. 101, 8894–8899 (2004).

7. Weinberg, J. B. Nitric oxide production and nitric oxide synthase type 2 expression by human mononuclear phagocytes: a review. Mol. Med. 4, 557–591 (1998).

Further reading on NO:

Nitric Oxide in bone metabolism July 16, 2012

Author: Aviral Vatsa PhD, MBBS

https://pharmaceuticalintelligence.com/2012/07/16/nitric-oxide-in-bone-metabolism/?goback=%2Egde_4346921_member_134751669

Nitric Oxide production in Systemic sclerosis July 25, 2012

Curator: Aviral Vatsa, PhD, MBBS

https://pharmaceuticalintelligence.com/2012/07/25/nitric-oxide-production-in-systemic-sclerosis/?goback=%2Egde_4346921_member_138370383

Nitric Oxide Signalling Pathways August 22, 2012 by

Curator/ Author: Aviral Vatsa, PhD, MBBS

https://pharmaceuticalintelligence.com/2012/08/22/nitric-oxide-signalling-pathways/?goback=%2Egde_4346921_member_151245569

Nitric Oxide: a short historic perspective August 5, 2012

Author/Curator: Aviral Vatsa PhD, MBBS

https://pharmaceuticalintelligence.com/2012/08/05/nitric-oxide-a-short-historic-perspective-7/

Nitric Oxide: Chemistry and function August 10, 2012

Curator/Author: Aviral Vatsa PhD, MBBS

https://pharmaceuticalintelligence.com/2012/08/10/nitric-oxide-chemistry-and-function/?goback=%2Egde_4346921_member_145137865

Nitric Oxide and Platelet Aggregation August 16, 2012 by

Author: Dr. Venkat S. Karra, Ph.D.

https://pharmaceuticalintelligence.com/2012/08/16/no-and-platelet-aggregation/?goback=%2Egde_4346921_member_147475405

The rationale and use of inhaled NO in Pulmonary Artery Hypertension and Right Sided Heart Failure August 20, 2012

Author: Larry Bernstein, MD

https://pharmaceuticalintelligence.com/2012/08/20/the-rationale-and-use-of-inhaled-no-in-pulmonary-artery-hypertension-and-right-sided-heart-failure/

Nitric Oxide: The Nobel Prize in Physiology or Medicine 1998 Robert F. Furchgott, Louis J. Ignarro, Ferid Murad August 16, 2012

Reporter: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2012/08/16/nitric-oxide-the-nobel-prize-in-physiology-or-medicine-1998-robert-f-furchgott-louis-j-ignarro-ferid-murad/

Coronary Artery Disease – Medical Devices Solutions: From First-In-Man Stent Implantation, via Medical Ethical Dilemmas to Drug Eluting Stents August 13, 2012

Author: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2012/08/13/coronary-artery-disease-medical-devices-solutions-from-first-in-man-stent-implantation-via-medical-ethical-dilemmas-to-drug-eluting-stents/

Nano-particles as Synthetic Platelets to Stop Internal Bleeding Resulting from Trauma

August 22, 2012

Reported by: Dr. V. S. Karra, Ph.D.

https://pharmaceuticalintelligence.com/2012/08/22/nano-particles-as-synthetic-platelets-to-stop-internal-bleeding-resulting-from-trauma/

Cardiovascular Disease (CVD) and the Role of agent alternatives in endothelial Nitric Oxide Synthase (eNOS) Activation and Nitric Oxide Production July 19, 2012

Curator and Research Study Originator: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2012/07/19/cardiovascular-disease-cvd-and-the-role-of-agent-alternatives-in-endothelial-nitric-oxide-synthase-enos-activation-and-nitric-oxide-production/

Macrovascular Disease – Therapeutic Potential of cEPCs: Reduction Methods for CV Risk

July 2, 2012

An Investigation of the Potential of circulating Endothelial Progenitor Cells (cEPCs) as a Therapeutic Target for Pharmacological Therapy Design for Cardiovascular Risk Reduction: A New Multimarker Biomarker Discovery

Curator: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2012/07/02/macrovascular-disease-therapeutic-potential-of-cepcs-reduction-methods-for-cv-risk/

Bone remodelling in a nutshell June 22, 2012

Author: Aviral Vatsa, Ph.D., MBBS

https://pharmaceuticalintelligence.com/2012/06/22/bone-remodelling-in-a-nutshell/

Targeted delivery of therapeutics to bone and connective tissues: current status and challenges- Part, September  

Author: Aviral Vatsa, PhD, September 23, 2012

https://pharmaceuticalintelligence.com/2012/09/23/targeted-delivery-of-therapeutics-to-bone-and-connective-tissues-current-status-and-challenges-part-i/

Calcium dependent NOS induction by sex hormones: Estrogen

Curator: S. Saha, PhD, October 3, 2012

https://pharmaceuticalintelligence.com/2012/10/03/calcium-dependent-nos-induction-by-sex-hormones/

Nitric Oxide and Platelet Aggregation,

Author V. Karra, PhD, August 16, 2012

https://pharmaceuticalintelligence.com/2012/08/16/no-and-platelet-aggregation/

Bystolic’s generic Nebivolol – positive effect on circulating Endothelial Progenitor Cells endogenous augmentation

Curator: Aviva Lev-Ari, PhD, July 16, 2012

https://pharmaceuticalintelligence.com/?s=Nebivolol

Endothelin Receptors in Cardiovascular Diseases: The Role of eNOS Stimulation

Author: Aviva Lev-Ari, PhD, 10/4/2012

https://pharmaceuticalintelligence.com/2012/10/04/endothelin-receptors-in-cardiovascular-diseases-the-role-of-enos-stimulation/

Inhibition of ET-1, ETA and ETA-ETB, Induction of NO production, stimulation of eNOS and Treatment Regime with PPAR-gamma agonists (TZD): cEPCs Endogenous Augmentation for Cardiovascular Risk Reduction – A Bibliography

Curator: Aviva Lev-Ari, 10/4/2012.

https://pharmaceuticalintelligence.com/2012/10/04/inhibition-of-et-1-eta-and-eta-etb-induction-of-no-production-and-stimulation-of-enos-and-treatment-regime-with-ppar-gamma-agonists-tzd-cepcs-endogenous-augmentation-for-cardiovascular-risk-reduc/

Nitric Oxide Nutritional remedies for hypertension and atherosclerosis. It’s 12 am: do you know where your electrons are?

Author and Reporter: Meg Baker, 10/7/2012.

https://pharmaceuticalintelligence.com/2012/10/07/no-nutritional-remedies-for-hypertension-and-atherosclerosis-its-12-am-do-you-know-where-your-electrons-are/

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Curator and Reporter: Aviral Vatsa PhD, MBBS

Based on: A review by Wink et al., 2011

This is the first part of a two part post

Nitric oxide (NO), reactive nitrogen species (RNS) and reactive oxygen species (ROS) perform dual roles as immunotoxins and immunomodulators. An incoming immune signal initiates NO and ROS production both for tackling the pathogens and modulating the downstream immune response via complex signaling pathways. The complexity of these interactions is a reflection of involvement of redox chemistry in biological setting (fig. 1)

Fig 1. Image credit: (Wink et al., 2011)

Previous studies have highlighted the role of NO in immunity. It was shown that macrophages released a substance that had antitumor and antipathogen activity and required arginine for its production (Hibbs et al., 1987, 1988). Hibbs and coworkers further strengthened the connection between immunity and NO by demonstrating that IL2 mediated immune activation increased NO levels in patients and promoted tumor eradication in mice (Hibbs et al., 1992; Yim et al., 1995).

In 1980s a number of authors showed the direct evidence that macrophages made nitrite, nitrates and nitrosamines. It was also shown that NO generated by macrophages could kill leukemia cells (Stuehr and Nathan, 1989). Collectively these studies along with others demonstrated the important role NO plays in immunity and lay the path for further research in understanding the role of redox molecules in immunity.

NO is produced by different forms of nitric oxide synthase (NOS) enzymes such as eNOS (endothelial), iNOS (inducible) and nNOS (neuronal). The constitutive forms of eNOS generally produce NO in short bursts and in calcium dependent manner. The inducible form produces NO for longer durations and is calcium independent. In immunity, iNOS plays a vital role. NO production by iNOS can occur over a wide range of concentrations from as little as nM to as much as µM. This wide range of NO concentrations provide iNOS with a unique flexibility to be functionally effective in various conditions and micro-environements and thus provide different temporal and concentration profiles of NO, that can be highly efficient in dealing with immune challenges.

Redox reactions in immune responses

NO/RNS and ROS are two categories of molecules that bring about immune regulation and ‘killing’ of pathogens. These molecules can perform independently or in combination with each other. NO reacts directly with transition metals in heme or cobalamine, with non-heme iron, or with reactive radicals (Wink and Mitchell, 1998). The last reactivity also imparts it a powerful antioxidant capability. NO can thus act directly as a powerful antioxidant and prevent injury initiated by ROS (Wink et al., 1999). On the other hand, NO does not react directly with thiols or other nucleophiles but requires activation with superoxide to generate RNS. The RNS species then cause nitrosative and oxidative stress (Wink and Mitchell, 1998).

The variety of functions achieved by NO can be understood if one looks at certain chemical concepts. NO and NO2 are lipophilic and thus can migrate through cells, thus widening potential target profiles. ONOO-, a RNS, reacts rapidly with CO2 that shortens its half life to <10 ms. The anionic form and short half life limits its mobility across membranes. When NO levels are higher than superoxide levels, the CO2-OONOintermediate is converted to NO2 and N2O3 and changes the redox profile from an oxidative to a nitrosative microenvironment. The interaction of NO and ROS determines the bioavailability of NO and proximity of RNS generation to superoxide source, thus defining a reaction profile. The ROS also consumes NO to generate NO2 and N2O3 as well as nitrite in certain locations. The combination of these reactions in different micro-environments provides a vast repertoire of reaction profiles for NO/RNS and ROS entities.

The Phagosome ‘cauldron’

The phagosome provides an ‘isolated’ environment for the cell to carry out foreign body ‘destruction’. ROS, NO and RNS interact to bring about redox reactions. The concentration of NO in a phagosome can depend on the kind of NOS in the vicinity and its activity and other localised cellular factors. NO and is metabolites such as nitrites and nitrates along with ROS combine forces to kill pathogens in the acidic environment of the phagosome as depicted in the figure 2 below.

Fig 2. The NO chemistry of the phagosome. (image credit: (Wink et al., 2011)

This diagram depicts the different nitrogen oxide and ROS chemistry that can occur within the phagosome to fight pathogens. The presence of NOX2 in the phagosomes serves two purposes: one is to focus the nitrite accumulation through scavenging mechanisms, and the second provides peroxide as a source of ROS or FA generation. The nitrite (NO2−) formed in the acidic environment provides nitrosative stress with NO/NO2/N2O3. The combined acidic nature and the ability to form multiple RNS and ROS within the acidic environment of the phagosome provide the immune response with multiple chemical options with which it can combat bacteria.

Bacteria

There are various ways in which NO combines forces with other molecules to bring about bacterial killing. Here are few examples

E.coli: It appears to be resistant to individual action of NO/RNS and H2O2 /ROS. However, when combined together, H2O2 plus NO mediate a dramatic, three-log increase in cytotoxicity, as opposed to 50% killing by NO alone or H2O2 alone. This indicates that these bacteria are highly susceptible to their synergistic action.

Staphylococcus: The combined presence of NO and peroxide in staphylococcal infections imparts protective effect. However, when these bacteria are first exposed to peroxide and then to NO there is increased toxicity. Hence a sequential exposure to superoxide/ROS and then NO is a potent tool in eradicating staphylococcal bacteria.

Mycobacterium tuberculosis: These bacterium are sensitive to NO and RNS, but in this case, NO2 is the toxic species. A phagosome microenvironment consisting of ROS combined with acidic nitrite generates NO2/N2O3/NO, which is essential for pathogen eradication by the alveolar macrophage. Overall, NO has a dual function; it participates directly in killing an organism, and/or it disarms a pathway used by that organism to elude other immune responses.

Parasites

Many human parasites have demonstrated the initiation of the immune response via the induction of iNOS, that then leads to expulsion of the parasite. The parasites include Plasmodia(malaria), Leishmania(leishmaniasis), and Toxoplasma(toxoplasmosis). Severe cases of malaria have been related with increased production of NO. High levels of NO production are however protective in these cases as NO was shown to kill the parasites (Rockett et al., 1991; Gyan et al., 1994). Leishmania is an intracellualr parasite that resides in the mamalian macrophages. NO upregulation via iNOS induction is the primary pathway involved in containing its infestation. A critical aspect of NO metabolism is that NOHA inhibits AG activity, thereby limiting the growth of parasites and bacteria including Leishmania, Trypanosoma, Schistosoma, HelicobacterMycobacterium, and Salmonella, and is distinct from the effects of RNS. Toxoplasma gondii is also an intracellular parasite that elicits NO mediated response. INOS knockout mice have shown more severe inflammatory lesions in the CNS that their wild type counterparts, in response to toxoplasma exposure. This indicates the CNS preventative role of iNOS in toxoplasmosis (Silva et al., 2009).

Virus

Viral replication can be checked by increased production of NO by induction of iNOS (HIV-1, coxsackievirus, influenza A and B, rhino virus, CMV, vaccinia virus, ectromelia virus, human herpesvirus-1, and human parainfluenza virus type 3) (Xu et al., 2006). NO can reduce viral load, reduce latency and reduce viral replication. One of the main mechanisms as to how NO participates in viral eradication involves the nitrosation of critical cysteines within key proteins required for viral infection, transcription, and maturation stages. For example, viral proteases or even the host caspases that contain cysteines in their active site are involved in the maturation of the virus. The nitrosative stress environment produced by iNOS may serve to protect against some viruses by inhibiting viral infectivity, replication, and maturation.

To be continued in part 2 …

Bibliography

Gyan, B., Troye-Blomberg, M., Perlmann, P., Björkman, A., 1994. Human monocytes cultured with and without interferon-gamma inhibit Plasmodium falciparum parasite growth in vitro via secretion of reactive nitrogen intermediates. Parasite Immunol. 16, 371–3

Hibbs, J.B., Jr, Taintor, R.R., Vavrin, Z., 1987. Macrophage cytotoxicity: role for L-arginine deiminase and imino nitrogen oxidation to nitrite. Science 235, 473–476.

Hibbs, J.B., Jr, Taintor, R.R., Vavrin, Z., Rachlin, E.M., 1988. Nitric oxide: a cytotoxic activated macrophage effector molecule. Biochem. Biophys. Res. Commun. 157, 87–94.

Hibbs, J.B., Jr, Westenfelder, C., Taintor, R., Vavrin, Z., Kablitz, C., Baranowski, R.L., Ward, J.H., Menlove, R.L., McMurry, M.P., Kushner, J.P., 1992. Evidence for cytokine-inducible nitric oxide synthesis from L-arginine in patients receiving interleu

Rockett, K.A., Awburn, M.M., Cowden, W.B., Clark, I.A., 1991. Killing of Plasmodium falciparum in vitro by nitric oxide derivatives. Infect Immun 59, 3280–3283.

Stuehr, D.J., Nathan, C.F., 1989. Nitric oxide. A macrophage product responsible for cytostasis and respiratory inhibition in tumor target cells. J. Exp. Med. 169, 1543–1555.

Wink, D.A., Hines, H.B., Cheng, R.Y.S., Switzer, C.H., Flores-Santana, W., Vitek, M.P., Ridnour, L.A., Colton, C.A., 2011. Nitric oxide and redox mechanisms in the immune response. J Leukoc Biol 89, 873–891.

Wink, D.A., Mitchell, J.B., 1998. Chemical biology of nitric oxide: Insights into regulatory, cytotoxic, and cytoprotective mechanisms of nitric oxide. Free Radic. Biol. Med. 25, 434–456.

Wink, D.A., Vodovotz, Y., Grisham, M.B., DeGraff, W., Cook, J.C., Pacelli, R., Krishna, M., Mitchell, J.B., 1999. Antioxidant effects of nitric oxide. Meth. Enzymol. 301, 413–424.

Xu, W., Zheng, S., Dweik, R.A., Erzurum, S.C., 2006. Role of epithelial nitric oxide in airway viral infection. Free Radic. Biol. Med. 41, 19–28.

Yim, C.Y., McGregor, J.R., Kwon, O.D., Bastian, N.R., Rees, M., Mori, M., Hibbs, J.B., Jr, Samlowski, W.E., 1995. Nitric oxide synthesis contributes to IL-2-induced antitumor responses against intraperitoneal Meth A tumor. J. Immunol. 155, 4382–4390.

Further reading on NO:

Nitric Oxide in bone metabolism July 16, 2012

Author: Aviral Vatsa PhD, MBBS

https://pharmaceuticalintelligence.com/2012/07/16/nitric-oxide-in-bone-metabolism/?goback=%2Egde_4346921_member_134751669

Nitric Oxide production in Systemic sclerosis July 25, 2012

Curator: Aviral Vatsa, PhD, MBBS

https://pharmaceuticalintelligence.com/2012/07/25/nitric-oxide-production-in-systemic-sclerosis/?goback=%2Egde_4346921_member_138370383

Nitric Oxide Signalling Pathways August 22, 2012 by

Curator/ Author: Aviral Vatsa, PhD, MBBS

https://pharmaceuticalintelligence.com/2012/08/22/nitric-oxide-signalling-pathways/?goback=%2Egde_4346921_member_151245569

Nitric Oxide: a short historic perspective August 5, 2012

Author/Curator: Aviral Vatsa PhD, MBBS

https://pharmaceuticalintelligence.com/2012/08/05/nitric-oxide-a-short-historic-perspective-7/

Nitric Oxide: Chemistry and function August 10, 2012

Curator/Author: Aviral Vatsa PhD, MBBS

https://pharmaceuticalintelligence.com/2012/08/10/nitric-oxide-chemistry-and-function/?goback=%2Egde_4346921_member_145137865

Nitric Oxide and Platelet Aggregation August 16, 2012 by

Author: Dr. Venkat S. Karra, Ph.D.

https://pharmaceuticalintelligence.com/2012/08/16/no-and-platelet-aggregation/?goback=%2Egde_4346921_member_147475405

The rationale and use of inhaled NO in Pulmonary Artery Hypertension and Right Sided Heart Failure August 20, 2012

Author: Larry Bernstein, MD

http://pharmaceuticalintelligence.com/2012/08/20/the-rationale-and-use-of-inhaled-no-in-pulmonary-artery-hypertension-and-right-sided-heart-failure/

Nitric Oxide: The Nobel Prize in Physiology or Medicine 1998 Robert F. Furchgott, Louis J. Ignarro, Ferid Murad August 16, 2012

Reporter: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2012/08/16/nitric-oxide-the-nobel-prize-in-physiology-or-medicine-1998-robert-f-furchgott-louis-j-ignarro-ferid-murad/

Coronary Artery Disease – Medical Devices Solutions: From First-In-Man Stent Implantation, via Medical Ethical Dilemmas to Drug Eluting Stents August 13, 2012

Author: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2012/08/13/coronary-artery-disease-medical-devices-solutions-from-first-in-man-stent-implantation-via-medical-ethical-dilemmas-to-drug-eluting-stents/

Nano-particles as Synthetic Platelets to Stop Internal Bleeding Resulting from Trauma

August 22, 2012

Reported by: Dr. V. S. Karra, Ph.D.

https://pharmaceuticalintelligence.com/2012/08/22/nano-particles-as-synthetic-platelets-to-stop-internal-bleeding-resulting-from-trauma/

Cardiovascular Disease (CVD) and the Role of agent alternatives in endothelial Nitric Oxide Synthase (eNOS) Activation and Nitric Oxide Production July 19, 2012

Curator and Research Study Originator: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2012/07/19/cardiovascular-disease-cvd-and-the-role-of-agent-alternatives-in-endothelial-nitric-oxide-synthase-enos-activation-and-nitric-oxide-production/

Macrovascular Disease – Therapeutic Potential of cEPCs: Reduction Methods for CV Risk

July 2, 2012

An Investigation of the Potential of circulating Endothelial Progenitor Cells (cEPCs) as a Therapeutic Target for Pharmacological Therapy Design for Cardiovascular Risk Reduction: A New Multimarker Biomarker Discovery

Curator: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2012/07/02/macrovascular-disease-therapeutic-potential-of-cepcs-reduction-methods-for-cv-risk/

Bone remodelling in a nutshell June 22, 2012

Author: Aviral Vatsa, Ph.D., MBBS

https://pharmaceuticalintelligence.com/2012/06/22/bone-remodelling-in-a-nutshell/

Targeted delivery of therapeutics to bone and connective tissues: current status and challenges- Part, September  

Author: Aviral Vatsa, PhD, September 23, 2012

https://pharmaceuticalintelligence.com/2012/09/23/targeted-delivery-of-therapeutics-to-bone-and-connective-tissues-current-status-and-challenges-part-i/

Calcium dependent NOS induction by sex hormones: Estrogen

Curator: S. Saha, PhD, October 3, 2012

https://pharmaceuticalintelligence.com/2012/10/03/calcium-dependent-nos-induction-by-sex-hormones/

Nitric Oxide and Platelet Aggregation,

Author V. Karra, PhD, August 16, 2012

https://pharmaceuticalintelligence.com/2012/08/16/no-and-platelet-aggregation/

Bystolic’s generic Nebivolol – positive effect on circulating Endothelial Progenitor Cells endogenous augmentation

Curator: Aviva Lev-Ari, PhD, July 16, 2012

https://pharmaceuticalintelligence.com/?s=Nebivolol

Endothelin Receptors in Cardiovascular Diseases: The Role of eNOS Stimulation

Author: Aviva Lev-Ari, PhD, 10/4/2012

https://pharmaceuticalintelligence.com/2012/10/04/endothelin-receptors-in-cardiovascular-diseases-the-role-of-enos-stimulation/

Inhibition of ET-1, ETA and ETA-ETB, Induction of NO production, stimulation of eNOS and Treatment Regime with PPAR-gamma agonists (TZD): cEPCs Endogenous Augmentation for Cardiovascular Risk Reduction – A Bibliography

Curator: Aviva Lev-Ari, 10/4/2012.

https://pharmaceuticalintelligence.com/2012/10/04/inhibition-of-et-1-eta-and-eta-etb-induction-of-no-production-and-stimulation-of-enos-and-treatment-regime-with-ppar-gamma-agonists-tzd-cepcs-endogenous-augmentation-for-cardiovascular-risk-reduc/

Nitric Oxide Nutritional remedies for hypertension and atherosclerosis. It’s 12 am: do you know where your electrons are?

Author and Reporter: Meg Baker, 10/7/2012.

https://pharmaceuticalintelligence.com/2012/10/07/no-nutritional-remedies-for-hypertension-and-atherosclerosis-its-12-am-do-you-know-where-your-electrons-are/

 

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Curated/reported by : Aviral Vatsa PhD, MBBS

Based on : S Moncada et al

It was in 1980 that Furchgott & Zawadzki first described endothelium- dependent relaxation of the blood vessels by acetylcholine. Further studies in 1984 revealed that other factors such as bradykinin, histamine and 5-hydroxytryptamine release endothelium derived relaxing factor (EDRF), which can modulate vessel tone. EDRF was shown to stimulate soluble guanylate cyclase and was inhibited by haemoglobin. In 1986 it was demonstrated that superoxide (O2) anions mediated EDRF inactivation and that the inhibitors of EDRF generated superoxide (O2) anions in solution as a mean to inhibit EDRF. It was later established that all compounds that inhibit EDRF have one property in common, redox activity, which accounted for their inhibitory action on EDRF. One exception was haemoglobin, which inactivates EDRF by binding to it. In 1987 Furchgott proposed that EDRF might be nitric oxide (NO) based on a study of the transient relaxations of endothelium-denuded rings of rabbit aorta to ‘acidified’ inorganic nitrite (NO) solutions and the observations that superoxide dismutase (SOD, which removes O2) protected EDRF. Till then NO was not known to be produced in mammalian cells. In 1988 Palmer et al could detect NO production both biologically and chemically by chemiluminescence. The following year in 1989 the enzyme responsible for NO production, NO synthase, was discovered and L-arginine:NO pathway was proposed.

Roles of L-arginine:NO pathway

By 1987 it was proposed that NO is generated in tissues other than endothelium. Hibbs et al and Marletta et al proposed that NO was generated by macrophages. Moreover release of EDRF was demonstrated in cerebellar cells following activation with N-methyl-D- aspartate (NMDA ). Both noradrenergic and cholinergic responses are ‘controlled’ by the nitrergic system so that the release of NO (e.g., during electrical field stimulation) counteracts and dominates the response to the noradrenergic or cholinergic stimulus (Cellek & Moncada, 1997). Mechanism of penile erection was unveiled by the studies on nitrergic neurotransmission that led to therapeutic intervention. Selective damage of nitrergic nerves in disease states was proposed as a potent mechanism of pathophysiology. Broadly three areas of research based on three isoforms of NOS came into being;

  • cardiovascular
  • nervous
  • immunology

Identification of NG-monomethyl-L-arginine (L-NMMA) as an inhibitor of the synthesis of NO lay the basis of future research into investigating the role of NO in biological systems. In 1989 it was demonstrated that intravenous infusion of L-NMMA resulted in increase in blood pressure that was reversible by infusing L-arginine. NO was thus implicated in constantly maintaining blood vessel tone. eNOS knockout studies showed a hypertensive phenotypes in the animal models and over expression of eNOS led to lowering of the blood pressure. Furthermore, eNOS activation was attributed to phosphorylation of a specific tyrosine residue in the enzyme.

NO and Mitochondria 

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

NO reacts with some of the complexes of the respiratory chain, and inhibits mitochondrial respiration – this is a well accepted notion. Initially it was believed that the target for NO was soluble guanylate cyclase, which in vasculature would lead to elevation of cGMP that eventually results in NO mediated vasodilatation and platelet aggregation inhibition. In 1994, another potential target, cytochrome c oxidase, for inhibitory effects of NO was discovered. This was a reversible effect, in competition with oxygen concentrations. Increases in NO production were also shown to inhibit cellular respiration irreversibly by selectively inhibiting complex I . Hence in 2002 it was proposed that this might be a mechanism through which cell pathology was initiated in certain conditions. Furthermore, NO was proposed to be implicated in the activation of the grp78-dependent stress response , via modulating calcium-related interaction between mitochondria and endoplasmic reticulum . This host defence mechanism might also have role in vasculature. Further evidence was provided in 2003 to link the role of NO in mitochondrogenesis and thus indicating that NO might be involved in the regulation of the balance between glycolysis and oxidative phosphorylation in cells.

NO and Pathophysiology

Lack of NO: By 2000, NO was established as a haemostatic regulator in the vasculature. Its absence was implicated in pathological states such as hypertension and vasospasm. These pathophysiological states share a common beginning of endothelial dysfunction, which has low NO production as one of its characterstic features. This dysfunction has been observed prior to the appearance of cardiovascular disease in predisposed subjects with family history of essential hypertension and atherosclerosis. The most likely mechanism for endothelial dysfunction is that of a reduced bioavailability of NO . The mechanism of this aspect is discussed elsewhere on this site. Protection against reduction of NO bio-availability in the vasculature is a vital therapeutic target and is extensively explored. This can be achieved by the use of antioxidants and/or augmentation of eNOS expression. In 2003 statins were shown to increase the production of endothelial NO in endothelial cell cultures and in animals by the reduction of oxidative stress or by increasing the coupling of the eNOS. It was way back in 1994 that oestrogen was shown to increase both the activity and expression of eNOS. In addition, more recently in 2003, oestrogen was shown to reduce the breakdown of available NO.

Excess of NO: In 2000 it was shown that NO produced from iNOS in vasculature is involved in extensive vasodilatation in septic shock. Later it was demonstrated that inhibition of mitochondrial respiration is an important component of the NO-induced tissue damage. This inhibition of respiration, which is initially NO-dependent and reversible, becomes persistent with time as a result of oxidative stress . Such metabolic hypoxic states where in tissues cannot utilise available oxygen due to NO, could also contribute to other inflammatory and degenerative conditions. An obvious therapeutic target for reducing NO production in such conditions would be L-NMMA. L-NMM was tested in a clinical trial for septic shock in 2004. The results were however disappointing probably due to the blanket reduction in NO production from other NOS enzymes there by having deleterious effects on the treatment group. More specific inhibitors for NOS forms are being investigated for in different disease states.

In conclusion, the L-arginine: NO pathway has had a major impact in many areas of research, specially vascular biology. A lot has been understood about this pathway and its interactions, therapeutic targets are being aggressively investigated, but further investigations are required to delineate further the role of NO in human health and disease.

Further Reading

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1760731/?tool=pubmed

Nitric Oxide and Platelet Aggregation

Inhaled NO in Pulmonary Artery Hypertension and Right Sided Heart Failure

Cardiovascular Disease (CVD) and the Role of agent alternatives in endothelial Nitric Oxide Synthase (eNOS) Activation and Nitric Oxide Production

Nitric Oxide in bone metabolism

Nitric oxide and signalling pathways

Rationale of NO use in hypertension and heart failure

Interaction of Nitric Oxide and Prostacyclin in Vascular Endothelium

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

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Curator/ Author: Aviral Vatsa, PhD, MBBS

In continuation with the previous posts that dealt with short history and chemistry of nitric oxide (NO), here I will try to highlight the pathways involved in NO chemical signalling.

NO is a very small molecule, with a short half life (<5 sec). It diffuses rapidly to its surroundings and is metabolised to nitrites and nitrates. It can travel short distances, a few micrometers, before it is oxidised. Although it was previously believed that NO can only exert its effect for a very short time as other nitrogen oxides were believed to be biologically inert. Recent data suggests that other NO containing compounds such as S- or N-nitrosoproteins and iron-nitrosyl complexes can be reduced back to produce NO. These NO containing compounds can serve as storage and can reach distant tissues via blood circulation, remote from their place of origin. Hence NO can have both paracrine and ‘endocrine’ effects.

Intracellularly the oxidants present in the cytosol determine the amount of bioacitivity that NO performs. NO can travel roughly 100 microns from NOS enzymes where it is produced. NOS enzymes on the other hand are localised to specific sub-cellular areas, which have relevant proteins in the vicinity as targets for signalling.

NO signalling occurs primarily via three mechanisms (according to Martínez-Ruiz et al):

  1. Classical: This occurs via soluble guanylyl cyclase (sGC). Once NO is produced by NOS it diffuses to sGC intracellularly or even in other cells. SGC is highly sensitive for NO, even nanomolar amounts of NO activates sGC, thus making it a potent target for NO in signalling pathways. sGC in turn increases the conversion of GTP to cGMP. cGMP further mediates the regulation of contractile proteins and gene expression pathways via cGMP-activated protein kinases (PKGs). cGMPs cause confirmational changes in PKGs. Signalling by cGMP is terminated by the action of phosphodiestrases (PDEs). PDEs have become major therapeutic targets in the upcoming exciting research projects.
  2. Less classical: Within the mitochondria NO can compete with O2 and inhibit cytochrome c oxidase (CcO) enzyme. This is a reversible inhibition that depends on O2and NO concentrations and can occur at physiological levels of NO. Various studies have demonstrated that endogenously generated NO can inhibit respiration or that NOS inhibitors can increase respiration at cellular, tissue or whole animal level. Although the exact mechanism of CcO inhibition of NO is still debated, NO-CcO interaction is considered important signalling step in a variety of functions such as inhibition of mitochondrial oxidative phosphorylation, apoptosis and reactive oxygen species (ROS) generation. Interestingly, at higher concentration (~1nM) NO can cause irreversible inhibition of cellular oxidation by reversible and/or irreversible damage to the mitochondrial iron–sulfur centers,In addition to the above mentioned pathways, NO (along with AMP, ROS and O2), can also activate AMP- activated protein kinase (AMPK), an enzyme that plays a central role in regulating intracellular energy metabolism. NO can also regulate hypoxia inducible factor (HIF), an O2-dependent transcription factor that plays a key role in cell adaptation to hypoxia .
  3. Non- classical: S-nitrosylation or S-nitrosation is the covalent insertion of NO into thiol groups such as of cysteine residues of proteins. It is precise, reversible, and spatiotemporally restricted post translational modification. This chemical activity is dependent upon the reactivity between nitrosylating agent (a small molecule) and the target (protein residue). It might appear that this generic interaction results in non-specific, wide spread chemical activity with various proteins. However, three factors might determine the regulation of specificity of s-nitrosylation for signalling purposes:
  • Subcellular compartmentalisation: high concentrations of nitrosylating agents are required in the vicinity of target residues, thus making it a specific activity.
  • Site specificity: certain cysteine residues are more reactive in specific protein microenvironments than others, thus favouring their modification. As a result under physiological conditions only a specific number of cysteine residues would be modified, but under higher NO levels even the slow reacting ones would be modified. Increased impetus in research in this area to determine protein specificity to s-nitrosylation provides huge potential in discovering new therapeutic targets.
  • Denitrosylation: different rates of denitrosylation result in s-nitrosylation specificity.

Other modifications in non classical NO mechanisms include S-glutathionylation and tyrosine nitration

Peroxynitrite: It is one of the important reactive nitrogen species that has immense biological relevance. NO reacts with superoxide to form peroxynitrite. Production of peroxynitrite depletes the bioactivty of NO in physiological systems. Peroxynitrite can diffuse through membranes and react with cellular components such as mitochondrial proteins, DNA, lipids, thiols, and amino acid residues. Peroxynitrite can modify proteins such as haemoglobin, myoglobin and cytochrome c. it can alter calcium homeostasis and promote mitochondrial signalling of cell death. However, NO itself in low concentrations have protective action on mitochondrial signalling of cell death.

More details about various aspects of NO signalling can be obtained from the following references.

The post is based on the following Sources:

  1. http://www.sciencedirect.com/science/article/pii/S089158491100236Xhttp://dx.doi.org/10.1016/j.freeradbiomed.2011.04.010
  2. http://content.karger.com/produktedb/produkte.asp?doi=338150Cardiology 2012;122:55-68 (DOI: 10.1159/000338150)
  3. http://content.onlinejacc.org/article.aspx?articleid=1137266 J Am Coll Cardiol. 2006;47(3):580-581. doi:10.1016/j.jacc.2005.11.016
  4. http://goo.gl/y6oY3

 

In addition, other aspects of NO involvement in biological systems in humans are covered in the following posts on this site:

  1. Nitric Oxide and Platelet Aggregation
  2. Inhaled NO in Pulmonary Artery Hypertension and Right Sided Heart Failure
  3. Cardiovascular Disease (CVD) and the Role of agent alternatives in endothelial Nitric Oxide Synthase (eNOS) Activation and Nitric Oxide Production
  4. Nitric Oxide in bone metabolism

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