Myriocin

The fungal metabolite from which fingolimod — a pioneering oral disease-modifying multiple sclerosis drug — was derived.

Myriocin systematic name 2-Amino-3,4-dihydroxy-2-(hydroxymethyl)-14-oxoicos-6-enoic acid, is a metabolite of the fungusIsaria sinclairii and a sphingoid base analog with immunosuppressive properties. Its chemical derivative fingolimod, systematic name 2-amino-2-[2-(4-octylphenyl)ethyl]propane-1,3-diol, was approved on 22 September 2010 by the US Food and Drug Administration for the treatment of multiple sclerosis (MS) — a chronic autoimmune disease that affects millions of people worldwide. It is the first orally administered disease-modifying treatment for MS to be approved in the United States and uses a unique strategy to disrupt the disease's attack on the central nervous system (CNS).

One clue that Isaria sinclairii might yield a useful drug came from traditional Chinese medicine — it has been used for centuries as an “elixir for eternal youth”. Another clue was Isaria sinclairii's ability to attack other organisms — in this case, insects — with a chemical arsenal. In 1994, a team of Japanese scientists was the first to extract and observe the immunosuppressive effects of myriocin ¹ . Soon after this, a program of derivatization to improve effectiveness and reduce toxicity resulted in FTY720, or fingolimod ² .

Fifteen years later, extensive research exploring the therapeutic potential of fingolimod has now led to its US regulatory approval for the treatment of relapsing forms of MS ³ . During this period, the key to fingolimod's mode of action was uncovered — modulation of the cell-surface receptors for sphingosine-1-phosphate (S1P)⁴ . Because of its structural similarity to sphingosine, fingolimod can also be phosphorylated at its aminodiol head group by sphingosine kinases ⁵ . Phosphoro-fingolimod acts as a functional antagonist of S1P₁receptors, internalizing them. It is thought that this leads to the retention of certain immune cells in the lymph nodes, thereby preventing them from migration into the CNS and involvement in the autoimmune attacks characteristic of MS ³ .

Immune system modulation is not myriocin's only useful contribution to biochemistry — it can also deplete sphingolipids from cells, because it inhibits the enzyme serine palmitoyltransferase⁷ which catalyses the formation of sphingosine, a precursor of sphingolipids.

Lipidomics Gateway (27 October 2010) [doi:10.1038/lipidmaps.2010.34]

Mouse macrophage lipids: All systems go

By combining lipidomic with transcriptomic methods, researchers have uncovered a whole-system picture of immunologically- and pharmacologically-induced lipid perturbations in mouse macrophages.

The ultimate goal of the LIPID MAPS consortium is to improve understanding of lipid metabolism and the active role that lipids play in human disease, which could aid the development of new therapies. The first step is to define the lipidome of the mouse macrophage — a cell with important roles in innate and adaptive immunity, and cardiovascular and inflammatory disease.

Now, Edward A. Dennis and colleagues from the consortium, writing in the Journal of Biological Chemistry, reveal the most complete lipids “parts list” to date for the mouse macrophage. By harnessing advanced mass spectrometry techniques to accurately measure the levels of lipid molecules, and coupling these with gene-transcription analysis, they present snapshots through time of the lipidomic response to immune-system and pharmacological perturbations. The research uncovers both expected and unexpected changes, including evidence of cross-talk between different lipid categories.

To analyse the effects of an immune-system perturbation on cellular lipid levels, the researchers used Kdo2 Lipid A (KLA), the active component of an inflammatory lipopolysaccharide. This ligand mimics aspects of bacterial infection by specifically acting on Toll-like receptor 4. The team also tested compactin — a drug which inhibits cholesterol biosysnthesis — both with and without KLA stimulation.

Over 400 lipid species were analysed by six research teams using liquid chromatography-mass spectrometry techniques, applying a different protocol for each major lipid category. In addition, to investigate how and why lipid levels might change, the researchers also measured the cellular mRNA of over 20,000 genes, including those that encode lipid-synthesizing proteins. Both mRNA and lipid measurements were taken at time intervals following perturbation by KLA and compactin.

Using statistical techniques, the researchers calculated correlations between lipids and genes over time and were able to distinguish between the effects of KLA and compactin. Many genes coding for lipid biosynthetic proteins were differently expressed, correlating with associated lipid concentrations.

One pathway significantly altered by KLA was the arachidonate oxidation pathway, in which cyclooxygenase 2 activity, prostaglandins and secondary metabolites increased. However, unsaturated fatty acid levels decreased, correlating with down-regulated elongase and desaturase genes. Also decreased were acyl CoAs, which have a key position in the lipid synthetic system, and their associated genes.

Glycerolipids, glycerophospholipids, sphingolipids and sterol esters were all remodelled by KLA. In particular, the authors found evidence for de novo synthesis of sphingolipids and for sterol synthesis via acetyl CoA. Saturated and monounsaturated cholesteryl esters and their associated enzymes also increased, suggesting that these species might accumulate due to TLR4 activation. Because oxysterols promote sphingomyelin biosynthesis, the authors suggest that elevated sphingomyelins might be due to cross-talk between the two pathways. Galactoceramides, which promote phagocytosis by macrophages, were also elevated. Interestingly, increases in saturated and monounsaturated phosphatidic acid and phosphatidylinositol lipid species were seen as late as 24 hours following KLA stimulation.

As expected, compactin, which inhibits the conversion of HMG-CoA to mevalonic acid, blocked the biosynthesis of cholesterol, but did not prevent accumulation of cholesterol owing to lipoprotein particle degradation. It also slowed the KLA-induced increase in CoQ. Unexpectedly, however, compactin appeared to increase two inflammatory eicosanoids — PGD₂ and PGE₂ — and their synthetic enzymes. To explain this finding, the authors propose cross-talk between sterols and eicosanoids via a new, as yet undiscovered, pathway.

Overall, the paper highlights the growing maturity of the lipidomics field, showing how developments in mass spectrometry are allowing the accurate measurement of an ever-growing number of lipid molecules, and that such data can reveal not just concentrations of individual lipids, but processes involving the whole system.

Lipidomics Gateway (27 October 2010) [doi:10.1038/lipidmaps.2010.32]

Synaptic size regulation: Tweeking PI to get a bouton

By controlling the localization of an actin-binding protein, PI(4,5)P₂ controls synapse growth in Drosophila.

During the larval stages of Drosophila melanogaster development, there is a neuromuscular junction (NMJ) growth spurt to accommodate large increases in body wall muscle area. Using a genetic approach, Patrik Verstreken and colleagues report in Proceedings of the National Academy of Sciences of the USA that the phosphoinositide lipidPI(4,5)P₂ helps to maintain NMJ size and morphology by regulating the actin-binding protein WASP.

Phosphoinositide (PI) lipids play numerous roles in the regulation of synaptic transmission, including the formation of new synaptic vesicles at neuronal membranes. By employing a PI(4,5)P₂-binding protein domain (PH) from phospholipase C, which causes a dominant negative effect of reduced PI(4,5)P₂ availability, the authors were able to test the effect of PI(4,5)P₂ depletion in a Drosophila larval system of NMJ growth. Indeed, they saw decreased stimulation-induced synaptic vesicle cycling, consistent with PI(4,5)P₂depletion. This depletion, as well as knockdown of the kinases immediately upstream of PI(4,5)P₂, increased the total branch length and the number of satellite boutons — knob-like projections of neurons. Increasing PI(4,5)P₂ levels by mutating a PI phosphatase restored the NMJ overgrowth caused by PH domain overexpression.

Because mutations in the WASP protein also show enlarged NMJs, the authors wanted to know if expressing the PH domain to deplete PI(4,5)P₂ in mutants lacking the WASP protein (wsp) would have an additive effect on NMJ overgrowth. This was not the case, suggesting that PI(4,5)P₂ controls NMJ growth in a WASP-dependent manner. This regulation was dependent on the known PI(4,5)P₂-binding domain of WASP. The WASP- and PI(4,5)P₂-mediated effects on NMJ function are likely not completely overlapping, as the authors saw differences in vesicle cycling and neurotransmitter release between the two mutant systems.

Monitoring the localization of WASP, the authors determined that PI(4,5)P₂ is involved in localizing or stabilizing WASP. Consistent with a role for WASP in regulating actin branching and for the actin cytoskeleton in NMJ development, the authors found that PI(4,5)P₂ depletion, like wsp mutants, accumulates actin in patches in NMJs. The actin patches, labelled with the actin-binding protein Moesin tagged with GFP, correlate with NMJ growth. The results suggest that unbranched actin, induced during WASP inhibition by PI(4,5)P₂-depletion, supports new bouton sprouting.

It is thought that WASP restricts NMJ growth by cooperating with its binding partner, Nervous Wreck (NWK). Testing various single and double mutant combinations of wsp, nwk, and wit, the gene encoding a bone morphogenetic protein receptor that NWK inhibits, the authors determined that PI(4,5)P₂ regulates NMJ morphology by acting in parallel to NWK and WIT. A similar epistasis analysis using tweek mutants, which show reduced boutonic PI(4,5)P₂ availability, suggests that NWK, WSP and PI(4,5)P₂ act in a Tweek-dependent pathway to regulate NMJ growth.

These results suggest that Tweek regulates NMJ growth by signaling through the two parallel pathways: the NWK/WIT system of synaptic BMP signaling and the WSP/PI(4,5)P₂ pathway that regulates the actin cytoskeleton.

Related highlights:

Jul 10: Membrane trafficking: PIP₂ calcium let-out cause
Direct activation of TRPML channels by PI(3,5)P₂ controls Ca²⁺ release and membrane fusion and fission events.

Apr 10: Cytokinesis: Where PIP splits, fatty acid stops
Phosphatidylinositol-3-phosphate controls cytokinesis by recruiting protein machinery required for abscission, whereas palmitate induces cytokinetic failure.

Mirella Bucci

 

Lipidomics Gateway (27 October 2010) [doi:10.1038/lipidmaps.2010.33]

Bacterial lipid rafts discovered

In eukaryotic cells, membrane proteins involved in signal transduction are often localized into microdomains that are enriched in particular lipid species. Commonly referred to as lipid rafts, these microdomains are important for cellular function, and their disruption can cause defects in signal transduction processes. In bacteria, the heterogeneous distribution of several proteins in the cytoplasmic membrane has hinted at the existence of functional membrane microdomains, but whether these foci are true lipid rafts has remained unclear. Now, writing in Genes & Development, López and Kolter describe the identification and characterization of bacterial lipid rafts that are important for the signalling pathways that regulate biofilm formation in Bacillus subtilis.

López and Kolter had previously discovered that the antifungal agent nystatin can trigger B. subtilis biofilm formation by activating a signalling pathway involving the membrane histidine kinase KinC and the transcription factor Spo0A. This observation was somewhat curious, given that nystatin inhibits fungal growth by binding to ergosterol in the membrane, but ergosterol is not produced by bacteria. Therefore, the authors used bioinformatics to identify known or putative B. subtilis genes that might be involved in the synthesis of ergosterol-like lipids. They identified yisP, which encodes a functional squalene synthase; the deletion of yisPblocked nystatin-dependent biofilm formation. Liquid chromatography–mass spectrometry analysis of lipid extracts from wild-type and ΔyisP strains revealed two peaks that were absent in the deletion mutant. Furthermore, addition of exogenous squalene-derived molecules rescued nystatin-dependent biofilm formation in the ΔyisP mutant.

To investigate whether the lipid species produced by YisP might organize KinC into membrane microdomains, the authors compared the constituents of the detergent-resistant membrane (DRM) and detergent-sensitive membrane (DSM) fractions from B. subtilis extracts. Using SDS-PAGE, they observed substantially different protein profiles in the two fractions, with KinC only present in the DRM fraction. Importantly, the number and intensity of the protein bands in the DRM fraction was greatly decreased in extracts from the ΔyisP mutant.

In eukaryotes, the protein flotillin 1 is a common constituent of lipid rafts and is thought to have roles in raft formation and enhancing signal transduction. Interestingly, flotillin-like proteins are encoded by most bacteria. Accordingly, López and Kolter observed that the yuaG gene product (which has 39% sequence identity with flotillin 1) was heterogeneously distributed into approximately six distinct foci in the B. subtilis membrane, leading them to rename the encoded protein FloT. Furthermore, they found that KinC colocalized with FloT in these foci and that treatment of the cells with zaragozic acid — a squalene synthase inhibitor that inhibits the activity of YisP, thereby blocking the formation of the membrane microdomains — led to a gradual loss of the foci as FloT diffused throughout the membrane. Finally, the authors observed distinct membrane foci formed by FloT homologues in the membranes of Staphylococcus aureus and Escherichia coli.

Taken together, these findings suggest that functional lipid rafts that coordinate cellular signalling pathways exist in bacterial as well as eukaryotic membranes. Furthermore, these findings might explain the remarkable reduction in the incidence of post-operative bacterial infections in patients receiving statins to decrease high cholesterol levels. Therefore, disrupting lipid rafts might prove to be an effective approach to antibacterial therapy.

Nature Reviews Microbiology 8, 756 (November 2010) [doi:10.1038/nrmicro2455]

Virology: Severing the bud

Although many viruses use the host endosomal complex required for transport (ESCRT) to mediate budding and scission, influenza is thought to use an ESCRT-independent mechanism that was not clear. Lamb and colleagues now reveal that influenza budding and scission depends on the ion channel protein matrix 2 (M2).

M2 is part of the viral envelope, which also contains the viral glycoproteins haemagglutinin and neuraminidase; these glycoproteins associate with M1, which recruits M2, thereby incorporating it in the virion. M2 is known to be involved in the viral life cycle through its ion channel activities, and recent studies indicate that it might also affect viral budding. Using an in vitro reconstitution system, the authors found that M2 affects membrane curvature in a cholesterol-dependent manner and can induce vesicle formation in giant unilamellar vesicles (GUVs) in the presence of low or intermediate (17 mol %) cholesterol levels through its amphipathic helix. Reconstitution of GUVs with this amphipathic helix alone could also induce vesicle formation in the presence of 17 mol % cholesterol (which is comparable to the cholesterol concentration in areas of the plasma membrane), confirming the role of the M2 amphipathic helix in budding and scission.

Because GUVs have only one lipid phase (that is, lipids are spread homogeneously), they do not accurately mimic the plasma membrane, so the authors examined whether M2 can also induce budding in phase-separated GUVs. At low cholesterol levels, the M2 amphipathic helix bound the lipid-disordered phase of the GUV, clustering at the phase boundary, and induced excision of the lipid-ordered phase, initiating budding. Similar observations were made using full-length M2 in plasma membrane spheres, indicating that M2 induces budding by modifying the line tension between lipid phases.

So what is the role of M2 in viral budding in vivo? Electron microscopy showed that, in cells infected with wild-type virus, M2 localizes at the base of budding virions, where scission occurs. Interestingly, influenza with a mutated M2 amphipathic helix could bud but showed impaired scission, resulting in a string of attached viral particles. This mutated M2 localized at the constrictions between incompletely budded virions, suggesting that M2 is not required for budding but is necessary for scission.

On the basis of their findings and previous work, the authors propose a model for M2-mediated influenza budding. They suggest that budding is initiated by haemagglutinin, which clusters at lipid rafts and associates with M1. In turn, M1 recruits M2, which, in the cholesterol-rich environment of the rafts, stabilizes the site of budding until other proteins are recruited. When the pool of haemagglutinin is depleted by the assembling virions, M2 can move to the lipid phase boundary (that is, between the virion and the plasma membrane), where cholesterol levels are low, and mediate scission through its amphipathic helix.

Nature Reviews Microbiology 8, 757 (November 2010) [doi:10.1038/nrmicro2458]

Cytoskeleton: Filopodia self-assemble

Filopodia are finger-like projections made up of bundled actin filaments that have many biological roles, including cell migration. How filopodia are formed has been difficult to determine, as current experimental systems do not recapitulate aspects of filopodium biology. Kirschner and colleagues now address this question using an in vitro model system in which filopodia can self-assemble in the absence of a pre-existing cytoskeletal network.

To examine how filopodia are formed, the authors incubated frog egg extracts with lipid bilayers (and not liposomes, as had been done previously) to recapitulate actin nucleation at membranes in vitro. Dense, long structures grew from the bilayer surface that were made up of bundled actin filaments. Actin polymerization occurred at the tip of the structures, where, similarly to filopodia, proteins such as vasodilator-stimulated phosphoprotein (VASP), neural Wiskott–Aldrich syndrome protein (NWASP), CDC42 and the formin diaphanous 2 (DIA2), localized. Moreover, the kinetics of actin monomer addition at the tip were similar to those of filopodium growth in vivo. Together, these findings indicate that these structures (which the authors term filopodium-like structures (FLSs)) resemble filopodia and mimic filopodium formation in vivo.

So how do filopodia assemble? Using FLSs as an in vitro model system, the authors could not detect preformed domains on the membranes, indicating that filopodium assembly is not template driven. Instead, filopodia self-assemble on permissive membrane surfaces enriched in phosphatidylinositol (4,5)-bisphosphate. Specifically, transducer of CDC42-dependent actin assembly 1 (TOCA1; also known as FNBP1L), which interacts with membrane lipids through its F-BAR domain, is recruited early to sites where FLSs later form. This in turn recruits NWASP, followed by the actin-related protein 2/3 (ARP2/3) complex, actin, VASP, DIA2 and the bundling protein fascin. Interestingly, ARP2/3 complex-driven actin polymerization is necessary for the initiation of FLS formation but is not strictly required for elongation. Instead, a significant reduction of FLS elongation was seen only when both ARP2/3 and diaphanous-related formins were inhibited, suggesting that they both have roles in this process.

On the basis of their findings, the authors propose that filopodia self-assemble in a step-wise manner: negatively charged membranes signal the recruitment of F-BAR domain superfamily proteins, such as TOCA1, which in turn recruit nucleation-promoting factors, such as NWASP, leading to the recruitment of the ARP2/3 complex and actin, thereby initiating FLS formation.

Rachel David

Nature Reviews Molecular Cell Biology 11, 756-757, (November 2010) [doi:10.1038/nrm2991]

Jasmonate perception by inositol-phosphate-potentiated COI1–JAZ co-receptor

Jasmonates are a family of plant hormones that regulate plant growth, development and responses to stress. The F-box protein CORONATINE INSENSITIVE 1 (COI1) mediates jasmonate signalling by promoting hormone-dependent ubiquitylation and degradation of transcriptional repressor JAZ proteins. Despite its importance, the mechanism of jasmonate perception remains unclear. Here we present structural and pharmacological data to show that the true Arabidopsis jasmonate receptor is a complex of both COI1 and JAZ. COI1 contains an open pocket that recognizes the bioactive hormone (3R,7S)-jasmonoyl-l-isoleucine (JA-Ile) with high specificity. High-affinity hormone binding requires a bipartite JAZ degron sequence consisting of a conserved α-helix for COI1 docking and a loop region to trap the hormone in its binding pocket. In addition, we identify a third critical component of the jasmonate co-receptor complex, inositol pentakisphosphate, which interacts with both COI1 and JAZ adjacent to the ligand. Our results unravel the mechanism of jasmonate perception and highlight the ability of F-box proteins to evolve as multi-component signalling hubs.

 

Nature advance online publication 6 October 2010 | doi:10.1038/nature09430; Received 30 June 2010; Accepted 19 August 2010; Published online 6 October 2010

The herbicide ketoclomazone inhibits 1-deoxy-d-xylulose 5-phosphate synthase in the 2-C-methyl-d-erythritol 4-phosphate pathway and shows antibacterial activity against Haemophilus influenzae

Two distinct metabolic pathways have been elucidated for the formation of isopentenyl diphosphate and dimethylallyl diphosphate, essential metabolic precursors for isoprenoid biosynthesis: the mevalonate pathway, found ubiquitously in mammals, and the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway, found in most bacteria. As the MEP pathway is absent from mammals, all MEP pathway enzymes represent effective targets for the development of antibacterial drugs. In this study, we found that a herbicide, ketoclomazone, exhibited antibacterial activity against a pathogenic bacterium, Haemophilus influenzae, with an MIC value of 12.5 μg ml⁻¹ and that antibacterial activity was suppressed by adding 1-deoxyxylulose, a free alcohol of 1-deoxy-d-xylulose 5-phosphate (DXP). DXP is an MEP pathway intermediate synthesized from pyruvate and d-glyceraldehyde 3-phosphate (d-GAP) by the action of DXP synthase. Thus, we investigated the enzyme kinetics of DXP synthase of H. influenzae(HiDXS) to elucidate an inhibitory mechanism of ketoclomazone on HiDXS. The dxs gene was cloned from H. influenzae and overexpressed in Escherichia coli, and the enzyme was purified to homogeneity. The purified HiDXS was a soluble dimeric 70-kDa protein. Steady-state kinetic constants for HiDXS were calculated, and Lineweaver–Burk plots were consistent with a ping-pong bi bi mechanism. The kinetics of inhibition by ketoclomazone suggested that ketoclomazone binds to an unidentified inhibitor-binding site that differs from both the pyruvate-binding site and the d-GAP-binding site on DXP synthase. These data reveal the inhibitory mechanism of ketoclomazone on DXP synthase.

The Journal of Antibiotics (2010) 63, 583–588; doi:10.1038/ja.2010.100; published online 1 September 2010

Structure–activity relationships of 11 new congeners of the SMTP plasminogen modulator

The fungal metabolite Stachybotrys microspora triprenyl phenols (SMTPs) are small-molecule plasminogen modulators that enhance plasminogen activation. The SMTP molecule consists of a tricyclic γ-lactam moiety, an isoprene side-chain and an N-linked side-chain. Previous investigations have demonstrated that the N-linked side-chain is crucial for its activity. In this study, we have isolated 11 new SMTP congeners with a variety of N-linked side-chain structures, to investigate structure–activity relationships. Active compounds included congeners with a carboxyl or a sulfonic acid group in the N-linked side-chain, whereas not all the congeners with a carboxyl group were active. Of these congeners, that with methionine or tyrosine as the N-linked side-chain moiety was more active than that with an aliphatic amino acid. Congeners without ionizable group in the N-linked side-chain were essentially inactive.

The Journal of Antibiotics (2010) 63, 589–593; doi:10.1038/ja.2010.101; published online 15 September 2010

Organic Reaction Mechanisms

All most all of the reactions that occurs in metabolic pathways are enzymatically catalyzed organic reactions. Various mechanisms enzymes have at that their disposal for catalyzing reactions: Acid-Base catalysis, covalent catalysis, metal ion catalysis, electrostatic catalysis, proximity and orientation effects, and transition state binding. Yet few enzymes alter the chemical mechanisms of these reactions, so much can be learned about enzymatic mechanisms from the study of nonenzymatic model reactions.

Christopher Walsh has classified biochemical reactions into four categories:

1-Group Transfer Reactions,

2-Oxidations and Reductions

3-Eliminations, isomerizations and rearrangements

4-Reactions that make or break carbon-carbon bonds.

A-Chemical Logic:

A covalent bond consists of an electron pair shared  between two atoms. In breaking such a bond, the electron pair can either remain with one of the atoms-Heterolytic bond cleavage or separate such that one electron accompanies each of the atoms- Homolytic bond cleavage.

Homolytic bond cleavage usually produces unstable radicals, occurs mostly in oxidation-reduction reactions.

Heterolytic  C-H bond cleavage involves either cabanion and protein H+ formation or carbocation (carbonium ion) and hydride ion H- formation. Since hydride ions are highly reactive species and carbon atoms are slightly more electonegative than hydrogen atoms, bond cleavage in which the electron pair remains with the carbon atom is the predominant mode of C-H bond breaking in biochemical systems. Hydride ion abstraction occurs only if the hydride is transferred directly to an acceptor such as NAD+ or NADP+.

Metabolic Pathways

Metabolic pathways are series of consecutive enzymatic reactions that produce specific products. Their reactants, intermediates and products are referred to as metabolites. Since an organism utilizes many metabolites, it has many metabolic pathways.

Each reaction in metabolic pathways are catalyzed by a distinct enzyme, of which there are more than 2000 known. At first glance, this network seems hopelessly complex.

The reaction pathways that comprise metabolism are often divided into two categories. Those involved in degradation- Catabolism and those involved in biosynthesis- Anabolism.

In Catabolism pathways, complex metabolites are exergonically broken down to simpler products.The free energy released during these processes is conserved by the synthesis of ATP from ADP and phosphate or by the reduction of the coenzyme NADP+ to NADPH.

ATP and NADPH are the major free energy sources for anabolic pathways.

A striking characteristic of degradative metabolism is that it converts a large number of diverse substances (carbohydrates,lipids and proteins) to common intermediates. These intermediates are then further metabolized in a central oxidative pathway that terminates in a few end products. 

Proteins into amino acids; carbohydrates into glucose and lipids into fatty acids & glycerol, then to common intermediate acetyl coenzyme A. This is followed by the oxidation of the acetyl group to CO2 and H2O by the sequential actions of  citric acid cycle, the electron transport chain and oxidative phosphorylation.

Biosynthesis carries out the opposite process. Relatively few metabolities, mainly pyruvate, acetyl Co A and the Citric acid cycle intermediates, serve as starting materials for a host of varied biosynthetic products.

Four principal characteristics of metabolic pathways stem from their function of generating products for use by the cell:

1- Metabolic pathways are irreversible:

They are highly exergonic(have large negative free energy changes), so their reactions go to completion. This characteristic provides the pathway with direction. Consequently, if two metabolites are metabolically interconvertible , the pathway from the first to the second must differ from the pathways from the second back to the first.

The reason for this difference is that if the route from the first metabolite to the second is exergonic, free energy must be supplied in order to bring it “back up the hill”. This required a different pathway for at least some of the reaction steps. The existence of independent interconversion routes is an important property of metabolic pathways because it allows independent control of the rate of the two processes.

If metabolite 2 is required by the cell, it is necessary to “turn off” the pathway from 2 to 1 while “turning on “ the pathway from 1 to 2. Such independent control would be impossible without different pathways.

2- Every metabolic pathway has a first committed step:

Although metabolic pathways are irreversible, most of their component reactions function close to equilibrium. Early in each pathway, however, there is generally an irreversible (exergonic) reaction that “commits” the intermediate it produces to continue down the pathway.

3-All metabolic pathways are regulated:

In order to exert control on the flux of metabolites through a metabolic pathways, it is necessary to regulate its rate limiting step. The first committed step, being irreversible, functions too slowly to permit its substrates and products to equilibrate. Since most of the other reactions in a pathway function close to equilibrium , the first committed step is often its rate limiting step. Most metabolic pathways are therefore controlled by regulating the enzyme that catalyze their first committed steps. This is the most efficient way to exert control because it prevents the unnecessary synthesis of metabolites further along the pathway when they are not required.

4-Metabolic pathways in eukaryotic cells occur in specific cellular locations

The synthesis of metabolites in specific membrane bounded subcellular compartments makes their transport between these compartments a vital component of eukaryotic metabolism. Biological membranes are selectively permeable to metabolites because of the presence of in membranes of specific transport proteins.

For example, ATP is generated in the mitochondria but much of it is utilized in the cytosol.

The synthesis and utilization of acetyl-CoA is also compartmentalized . This metabolic intermediate is utilized in the cytosolic synthesis of fatty acids, but is synthesized in mitochondria.

Yet there is  no transport protein for acetyl CoA in the mitochondrial membrane.

Introduction to Metabolism

Living organisms are not at equilibrium. Rather they require a continuous influx of free energy to maintain order in a universe bent on maximizing disorder.

Metabolism is the overall process through which living systems acquire and utilize the free energy they need to  carry out their various functions. They do so by coupling the exergonic reactions of nutrient oxidation to the endergonic processes required to maintain the living state such as the performance of mechanical work, the active transport of molecules against gradients and the biosynthesis of complex molecules.

How do living things acquire this necessary free energy?

And what is the nature of the energy coupling process?

Phototrophs- plants and certain bacteria acquire free energy from the sun through photosynthesis, a process in which light energy powers the endergonic reactions of CO2 and H2O to form carbohydrates and O2.

Chemotrophs-obtains their free energy by oxidizing organic compounds(carbohydrates, lipids, proteins) obtained from other organisms, ultimately phototrophs.

This free energy is most often coupled to endergonic reactions through the intermediate synthesis of “high energy” phosphate compounds such as adenosine triphosphate- ATP. In addition to being completely oxidized, nutrients are broken down in a series of metabolic reactions to common intermediates that are used as precursors in the synthesis of other biological molecules.

A remarkable property of living systems is that, despite the complexity of their internal processes, they maintain a steady state. This is strikingly demonstrated by the observation that, over a 40 year time span, a normal human adult consume literally tons of nutrients and imbibes over 20,000 L of water, but does so without significant weight change. This steady state is maintained by a sophisticated set of metabolic controls.

Use of recombinant CFP-10 protein for a skin test specific for Mycobacterium tuberculosis infection

Xueqiong Wu*, Shisheng Feng, Haiqing Duan, Lingxia Zhang, Junxian Zhang, Yourong Yang,
Yan Liang and Yingchang Shi

According to the World Health Organization, Mycobacterium tuberculosis has infected approximately one thirt of the world population and more than 8 million new cases of tuberculosis worldwide was reported in 2002. It is estimated that 45% of Chines population are latently infected with MTB, among which 10% latently infected individuals may become activated later in their lives. Proper identification of latently infected individuals for prophylactic chemotherapy is needed to prevent future reactivation of MTB infection and to control TB epidemics in China. At present, the purified protein derivative PPD  also known as the tuberculin skin test based on the delayed type hypersensitivity DTH reaction is widely used to detect MTB infection. However, the specificity of PPD skin test is low, because the antigens in PPD are common to many mycobacterial species including Mycobacterium bovis Bacills Calmette Guerin BCG, the vaccine strain being used widely for immunization against MTB infection. As a result the PPD skin test cannot be used to definitively identify MTB infection.

Mycobacterial antigens that can distinguish the MTB specific DTH reaction from that induced by the BCG vaccination are highly desirable and their utility in skin test specific for TB diagnosis needs be demonstrated.

The genetic region of difference 1- RD1 is present in the genomes of MTB and M.bovis but it is absent in all strains of M.bovis BCG, as well as most non tuberculosis mycobacteria NTM including Mycobacterium avium and Mycobacterium intracellulare. Both CFP-10 and ESAT-6 antigens are encoded by Rv3874 and Rv3875 genes, respectively, in one operon within the RD1 region and are expressed simultaneously at a similar ration. Because of their absence in BCG and NTM strains, ESAT-6 and CFP-10 have been extensively investigated as MTB specific antigens and shown to have great potential as the specific antigens for the diagnosis of MTB infection in humans. The immune responses found in experimental animals and humans to the recombinant CFP-10 or ESAT-6 proteins are similar to those found for the CFP-10 or ESAT-6 peptides. However, there were differences in immune responses of human or cattle to individual CFP-10 and ESAT-6 antigens, determined by ELISPOT assay, 18% people tested were positive for CFP-10 peptides, 30% were positive for ESAT-6 peptides, 33% were positive for both peptides and 34% were positive for the ESAT-6/CFP-10 fusion protein; 39% subjects had either a positive result for a peptide or a positive result for the fusion protein. The difference may be explained by polymorphism in the human leucocyte antigen HLA type in the population and difference of MTB stains infected in different countries. However, the use of a single antigen in the skin test may exclude those who do not have memory T cells specific of rESAT-6 even after exposure to MTB due to their unique HLA type. In this study, we evaluated the utility of rCFP-10 protein as a stimulating antigen to improve the sensitivity of rESAT-6 skin test or to find a better antigen that ESAT-6 for differential diagnosis of MTB infection.

MATERIALS AND METHODS

Bacterial Strains and Products

Escherichia coli strain BL21(DE3) was grown in Luria Bertani-LB liquid and solid media Mycobacterium reference stains including MTB(H37Rv), M.bovis, and M.bovis BCG Danish were used. Stock culture of MTB, M.bovis and M.bovis BCG Danish were grown on Lowenstein-Jensen slants at 37 degree C for 4 weeks and then trasferred to Sauton liquid medium at 37 degree C without shaking for 4 weeks. PPD produced from MTB(50 IU/ml) was used.

Gene Cloning, expression and protein purification

The MTB CFP-10 cloning, expression and purification were performed with standard procedures described by Sambrook et al. Briefly, the gene encoding the MTB CFP-10 protein was amplified by PCR using two primers. The forward primer contained a kpn1 restriction enzyme recognition site: -

5’-CGAGATCTGGGTACCGACGACGACGACAAGATGGCAGAGATGAAGACCGA-3’.  The reverse primer contained an EcoR1 restriction enzyme recognition site:-

5’-CGGAATTCT CAGAAGCCCATTTGCGAG G-3’.

PCR amplification is performed by 30 cycles of denaturation at 94 degree C for 30s, primer annealing at 55 degree C for 30s, extension at 68 degree C for 1 min and final extension at 72 degree C for 2 min. PCR products were digested by the restrictive enzyme Kpn1 and EcoR1. The resulting fragments were ligated with T4 DNA ligase into a Kpn-1 and EcoR-1 digested pET-32a plasmid vector containing the kanamycin resistant gene. The recombinant plasmids were transferred into E.coli BL21(DE3) and kanamycin resistant transformants were selected on LB agar plate containing kanamycin (50 microgram/ ml) and 5-bromo-4-choro-3-indolyl Beta D-galactopyranoside (X-Gal) (20 microgram/ml). The plasmid from one kanamycin resistant transformant was sequenced , using the same primers as for PCR amplification. Sequences were compared with that registered in the GenBank database by Basic Local Alignment Search Tool BLAST analysis.

The expression of rCFP-10 as an N-termnal 6xhistidine-tagged protein was induced by 1.0mM Isopropyl-b-D-thiogalactopyranoside(IPTG), in solube form after incubation at 30 degree C for 4 h. Soluble rCFP-10 was purified by metal chelate affinity chromotography under the native condition, the fusion tag was then cleaved with enterokinase and the resulting tag-free rCFP-10 was further purified by affinity chromatography according to the purification procedure provided by the expression vector manufacturer.

The freshly prepared rCFP-10 protein was suspended in 0.1mM phophate buffered saline(PBS, pH=7.0) and the solution was filtered by passing it through the Acrodisc Syringe Filter (0.45 micro meter) with low protein binding. The protein concentration was determined spectrophotometrically. The final protein solution was aliquoted into small glass ampules, each contained 1 mg of purified rCFP-10 protein. The sample were then lyphilized sealed and stored at –20 degree C. The protein was reconstituted in 1 ml saline before use.

Determination of molecular mass and purity

The molecular mass of purified rCFP-10 protein was determined by 15% sodium dodecyl sulphate polyacrylamide gel electrophoresis(SDS-PAGE). The molecular masses of the protein standards ranged from 6 to 97.4 kDa. Gels were stained with Coomassie blue. The molecular mass and purity of rCFP-10 protein were analyzed and calculated with TotalLab v1.11. The molecular mass of purified rCFP-10 protein was further confirmed by mass spectrometry.

Guinea pig sensitization and skin tests

38 NIH white guinea pig weighing 250 to 300 g were used and maintained under specific pathogen free conditions. There was a female to male ration of 1:1 . In the first experiment, 8 guinea pig(4 male and 4 femaile) were sensitized by hypodermic injection of 5 mg autoclaved MTB in 0.1ml of saline in the groin once weekly for four weeks. In the second experiment, 6 guinea pigs were sensitized by peritoneal injection of 100 live MTB in 0.5 ml saline and the skin test on these animals was performed 5 weeks after senitization. In the third experiment, 24 guinea pigs were divided into 3 groups and sensitized by hypodermic injection of 0.1 ml live BCG-Danish Vaccine(group 1), 5 mg autoclaved MTB (group2) or 5 mg autoclaved M.bovis (group 3) in animal’s groin area once weekly for four weeks.

For skin testing, guinea pigs were shaved on the back and injected intradermally with 0.8 to 1.2 microgram of the purified rCFP-10 protein in 0.1 ml of PBS or 0.1 ml (5 lU) of PPD or 1.0 microgram of the purified rESAT-6 protein  0.1 ml of PBS as a control for four to eight weeks following sensitization. The diameters of both axes of skin erythema were independently measured and recorded at 24,48 and 72 h after antigen injection. Results were expressed as means of diameter in millimeters of erythema.

RESULTS

Cloning, expression and purification of CFP-10

The nucleotide sequence encoding the rCFP-10 had 100% homologous identity with CFP-10 sequence reported in GenBank database. The molecular mass obtained from rCFP-10 fusion protein as determined by SDS-PAGE was 30.8 kDa. In order to determine an ideal condition for rCFP-10 protein expression, we compared the levels of expression under various IPTG concentrations and different induction time and temperatures. The results showed that IPTG concentrations had no effect on the level of the expression, whereas the induction time and temperature affected the expression level significantly. The amount of rCFP-10 protein that existed in soluble form was at 35% to 50% of the total soluble proteins when the induction took place under 1.0 mM IPTG at 30 degree C for 4 h. The purified rCFP-10 protein showed only one band when analyzed by 15% SDS PAGE with a purity of more than 90%. The molecular mass of the purified rCFP-10 protein was 14.8 kDa as determined by SDS-PAGE and 13.8 kDa by mass spectrometry.

DTH reactivity to rCFP-10 in guinea pigs sensitized with killed MTB

Different doses of rCFP-10 protein were tested for their ability to produce DTH responses in guinea pigs sensitized with killed MTB. In the first study, each of the seven guinea pigs(1 female was dead during the immunization) was injected intradermally at five sites with PBS as the negative control, PPD (5 lU) as the positive control and 3 doses of rCFP-10(0.8, 1.0 and 1.2 microgram). All three doses of rCFP-10 antigen were able to induce positive skin reaction at a similar level as compared to the positive control PPD at 24 and 48 h after injection as defined by an erythema response greater than 5 mm in diameter. The difference in skin reaction was  not statistically significant. The positive responses measured at the 24 h time point were generally stronger than that at 48h for all groups, though there was no statistically significant difference. By 72 hrs, the reaction to PPD had decreased markedly as compared to the reactions at the previous time points. All reaction to rCFP-10 reduced to less than 5 mm in diameter and were therefore considered negative.

DISCUSSION

It has been shown that ESAT-6 could be used as an antigen in skin test for the detection of MTB infection. However, the population with different HLA type may recognize different epitopes. CFP-10 may induce DTH responses in people who do not respond to ESAT-6, overcoming the problems related to genetic restriction in antigen recognition and improving the sensitivity of skin test. Thus the ability of CFP-10 to elicit DTH responsive in experimental animals sensitized with various strains of MTB complex should be evaluated to prepare a new antigen of skin test.

 

African Journal of Biotechnology Vol. 9(42), pp. 7180-7185, 18 October, 2010
Available online at http://www.academicjournals.org/AJB
ISSN 1684–5315 ©2010 Academic Journals

Regulatory Oversight and Safety of Probiotic Use

Veena Venugopalan, Kimberly A. Shriner, and Annie Wong-Beringer

Abstract:
Depending on intended use of probiotic(drug vs. dietary supplement), regulatory requirements differs greatly. For dietary supplements, premarketing demonstration of safety and efficacy and approval by the Food and Drug Administration are not required; only premarket notification is required. Saccharomyces boulardii is a probiotic regulated as a dietar supplement intended for use by the general healthy population, not as a drug to prevent, treat or mitigate disease. However, since recent increases in incidence and severity of Clostridium difficile infection, probiotics have been used to treat recurrent and/or refractory disease in hospitalized patients. Saccharomyces fungemia secondary to use of the probiotic has been described for patients who are critically ill, are receiving nutrition enterally, or have a central venous catheter. Before use of a probiotic is considered for hospitalized patients, careful assessment of risk versus benefit must be made. To ensure patient safety, probiotics should be properly handled during administration.
Probiotics are defined by the Food and Agriculture Organization of the World Health Organization as live microorganisms that, when administered in adequate amounts confer a health benefit on the host. The term probiotic can be subcategorized to include probiotic drugs, probiotic foods(e.g. foods, food ingredients and dietary supplements), direct fed microbials (probiotics for animal use) and designer probiotics (genetically modified probiotics). In the US, probiotic products are marketed to a generally healthy population as foods or dietary supplements.
Recent increases in the incidence and severity of Clostridium difficile infection (CDI)have led some clinicians to consider use of probiotics as "drugs", either alone or in combination with traditional antimicrobial agents for the prevention and treatment of CDI. Several recent reviews have summarized results from clinical studies evaluating the efficacy of probiotics in diarrheal illness. The goal is to highlight the current regulatory oversight for probiotics in US, identify potential risk situations associated with their administration and offer suggestions on practical aspects of probiotic administration to ensure patient safety.
Accordingly to the Food and Drug Administration definition, a drug is an article intended for use as a drug, then it must undergo the regulatory process as a drug, which is similar to that of any new therapeutic agent.




http://www.cdc.gov/eid/content/16/11/1661.htm

Emory Joins Network

Emory University researchers recently joined the Florida Node Alliance of the National Institute on Drug Abuse's Clinical Trials Network. CTN works to improve substance abuse treatment by linking researchers with care providers. Emory was chosen for its researchers' previous experience in working with HIV-positive crack cocaine users. The first network-related study will evaluate an intervention intended to link and retain HIV-infected substance abusers in care and treatment, and eventually to decrease their viral load and risk of transmission. 

Atlanta Journal-Constitution     (10.07.10)

New HIV Cases Top One Thousand Again

New diagnoses of HIV in Australia reached 1,050 in 2009, the continuation of a decade-long climb and the highest figure in almost two decades.

"It's fair to say over the last decade there was a substantial increase and we are starting to stabilize out, just recently," said Dr. David Wilson, of the National Center in HIV Epidemiology and Clinical Research.

The climb in HIV diagnoses comes amid mixed success in addressing other STDs. Chlamydia diagnoses rose 4 percent in 2009 to 62,613. At the same time, rates of gonorrhea and syphilis improved as did the incidence of hepatitis C. Among those 15 to 19 years of age, a drop in injecting drug use is credited with lowering cases of hepatitis C by 80 percent in the last five years.

Wilson attributed the rise in HIV diagnoses to the fact that improved treatment has made the disease "not as scary as it was in the 1980s."

While HIV diagnoses are increasing, mortality from HIV has declined since the 1990s. In 2009, nine deaths in Australia were attributed to AIDS, down from 26 in 2008.

"We are in an era where we are seeing the lowest deaths associated with HIV than we have seen in history," Wilson said.

About 25 percent of the HIV-positive population is 55 or older, compared to 2.5 percent in 1985 and the 44 percent expected in 10 years, Wilson said.
Australian Associated Press     (10.18.10):: Danny Rose

Fighting HIV Among Inmates

he National Institutes of Health (NIH) is funding its first initiative to fight HIV in correctional facilities with $50 million in grants over the next five years.

The research grants, awarded by the NIH's National Institute on Drug Abuse (NIDA), will be distributed to organizations in several states, including Illinois.

"If you don't tackle these hard-to-reach populations, there's enough of them to sustain the epidemic," said Dr. Jacques Normand, director of NIDA's AIDS research program.

In Illinois, $7 million will cover three main initiatives: revamping HIV testing in prisons, examining the use of telemedicine for HIV-positive inmates, and keeping closer ties with HIV-positive inmates once they leave prison.

Illinois prison officials will use the grant money to establish routine HIV testing on those entering the system unless an inmate opts out. Cook County Jail will switch to an opt-out approach this month.

The switch to routine testing is consistent with CDC guidelines and is expected to slash the number of inmates who are unaware of their HIV status, said Dr. Jeremy Young, an infectious-disease specialist with the University of Illinois at Chicago (UIC) and one of three lead investigators on the project.

In the telemedicine research project, investigators will examine a two-month-old pilot project involving UIC and the Illinois Department of Corrections. While a nurse is in the room with a patient, an HIV specialist examines the patient with the help of a camera and remote stethoscope.

"It's basically just like a live visit," Young said.

To help keep track of HIV-positive inmates when they leave prison, the grant provides for additional case managers who help inmates secure mental health services, drug counseling, and participation in the state's AIDS Drug Assistance Program.

Chicago Sun-Times     (10.12.10):: Monifa Thomas

1 in 22 Blacks Will Get HIV, CDC Report Says

A new CDC report estimating the lifetime risk of HIV diagnosis for several populations found great disparities by racial/ethnic groups. Based on HIV surveillance, vital statistics, and census data from 37 states and Puerto Rico for 2007, an estimated 4.65 percent of blacks/African Americans would receive an HIV diagnosis during their lifetime, or 1 in 22, according to the new report.

The 1 in 22 risk was more than twice the estimated lifetime risk of HIV diagnosis for Hispanics/Latinos (1.92 percent, or 1 in 52) and eight times that of whites (0.59 percent, or 1 in 170), the report found.

The estimates of lifetime risk of HIV diagnosis are not representative of all HIV diagnoses in the United States. However, the data also were not considered unusual. A report published in 2008 found a similar high estimated lifetime risk of HIV diagnosis for blacks.

The new report, "Estimated Lifetime Risk for Diagnosis of HIV Infection Among Hispanics/Latinos - 37 States and Puerto Rico, 2007," was published in Morbidity and Mortality Weekly Report (2010;59(40):1297-1301).

Colorado State University Gets Funding to Validate Tuberculosis Treatment Test

The US Food and Drug Administration awarded Colorado State University almost $500,000 to continue studies on a test that measures molecules in urine to determine whether TB treatment is working. The researchers look for large decreases in more than 50 small molecules present in the urine at the time of diagnosis. At present, doctors cannot tell whether treatment is effective until about two months after a patient has been taking antituberculosis drugs. According to principal investigator John Belisle, the researchers have identified a series of metabolites in TB patients’ urine that disappear or decrease in abundance when the patients respond to antituberculosis treatment.  The grant would enable the scientists to continue working on the test so it can be used in clinical trials for new TB treatments, as well as predict whether or not patients will redevelop TB after completing treatment.
Colorado State University, www.news.colostate.edu, October 11, 2010, by Dell Rae Moellenberg, DellRae.Moellenberg@ColoState.EDU,

University of Georgia Gets Part of $2.9 Million Federal Grant to Study TB

A scientist at the University of Georgia is among the recipients of a $2.9 million US Food and Drug Administration grant for TB research. Frederick D. Quinn of the College of Veterinary Medicine hopes to develop an improved diagnostic test for latent TB. While about 9 - 14 million people have active TB disease globally, 2 billion have latent TB infection, Quinn said. The grant will be divided among six researchers, and Quinn’s share is $742,498 over two years.
Associated Press, October 8, 2010

Notch and EGFR pathway interaction

Notch and EGFR pathway interaction regulates neural stem cell number and
self-renewal

Specialized cellular microenvironments, or ‘niches’, modulate stem cell properties, including cell number, self-renewal and fate decisions1, 2. In the
adult brain, niches that maintain a source of neural stem cells (NSCs) and neural
progenitor cells (NPCs) are the subventricular zone (SVZ) of the lateral
ventricle and the dentate gyrus of the hippocampus3, 4, 5. The size of the NSC
population of the SVZ at any time is the result of several ongoing processes,
including self-renewal, cell differentiation, and cell death.
Maintaining the balance between NSCs and NPCs in the SVZ niche is critical to



Nature 467, 323-327 (16 September 2010) |
doi:10.1038/nature09347;

Small but dominant RNA

As Mendel observed over a century ago, the manifestation of a phenotype can crucially depend on the dominant-recessive relationship between alleles at a given locus. A study of plant self-incompatibility suggests a new mechanism by which such dominant-recessive relationships arise: in this system, a small RNA encoded by the dominant allele acts in trans to epigenetically silence the expression of the recessive allele.
A plant uses a 'self-incompatibility'mechanism to reject fertilization by pollen that is genetically too similar to itself. In Brasssica species, this trait is controlled by three tightly linked, multiallelic loci and particularly by the gene encoding the male-specific protein S locus protein 11 (SP11) and its female-specific receptor S receptor kinase SRK and SRK alleles known as the S haplotye/





Nature Reviews Genetics 11, 670-671 (October 2010) |
doi:10.1038/nrg2875

Predicting functional modules

The precise spatiotemporal patterns of gene expression that are required during the development of multicellular organisms are largely governed by the binding of transcription factors to cis-regulatory sequences. Although many methods have been successful in identifying specific cis-regulatory modules (CRMs), it remains difficult to systematically describe the location and function of
CRMs in a specific developmental process. This paper presents a novel computational approach that tackles this challenge and has led to the most
complete, quantitative description to date of the control network for fruitfly embryo anteroposterior patterning.
Kazemian and colleagues developed a regression-based model that estimates
the potential of a genomic region to control the spatially restricted
expression of a nearby gene, which they term the 'pattern-generating



Nature Reviews Genetics 11, 669 (October 2010) | doi:10.1038/nrg2872

Tumor cells engineered to codisplay on their surface 4-1BBL and LIGHT

Tumor cells engineered to codisplay on their surface 4-1BBL and LIGHT
costimulatory proteins as a novel vaccine approach for cancer immunotherapy

Primary tumor cells genetically modified to express a collection of immunological
ligands on their surface may have the utility as therapeutic autologous cancer
vaccines. However, genetic modification of primary tumor cells is not only cost,
labor and time intensive, but also has safety repercussions. As an alternative,
we developed the ProtEx technology that involves generation of immunological
ligands with core streptavidin (SA) and their display on biotinylated cells in a
rapid and efficient manner. We herein demonstrate that TC-1 tumor cells can be
rapidly and efficiently engineered to codisplay on their surface two costimulatory proteins, SA-4-1BBL and SA-LIGHT, simultaneously. Vaccination with irradiated TC-1 cells codisplaying both chimeric proteins showed 100% efficacy in a prophylactic and >55% efficacy in a therapeutic tumor setting. In
contrast, vaccination with TC-1 cells


Cancer Gene Therapy 17, 730-741 (October 2010) |
doi:10.1038/cgt.2010.29

Inhibition of follicular T-helper

Inhibition of follicular T-helper cells by CD8+ regulatory T cells is essential
for self tolerance

The ability to produce vigorous immune responses that spare self tissues and
organs depends on the elimination of autoreactive T and B cells. However,
purging of immature and mature self-reactive T and B cells is incomplete
and may also require the involvement of cells programmed to suppress immune
responses1. Regulatory T cells (Treg ) belonging to the CD4+ T-cell subset may
have a role in preventing untoward inflammatory responses, but T-cell
subsets programmed to inhibit the development of autoantibody formation
and systemic-lupus-erythematosus-like disease have not yet been defined2. Here
we delineate a CD8+ regulatory T-cell



Nature 467, 328-332 (16 September 2010) |
doi:10.1038/nature09370;

Vitamin D and disease

A recent study demonstrates the power of combining chromatin immunoprecipitation followed by sequencing (ChIP–seq) with genome-wide
association (GWA) study data sets to explore the molecular basis of complex disease.
Ramagopalan and colleagues used ChIP–seq to produce high-resolution
maps of the genomic binding of the vitamin D receptor (VDR) — a ligand-
activated transcription factor — in human lymphoblastoid cell lines, with
and without active ligand. In the presence of ligand, they identified 2,776
binding sites, many of which are in regions associated with active chromatin,
consistent with the expected role of VDR at gene regulatory elements.Vitamin D has been linked to several diseases, particularly autoimmune diseases, but the basis for the link is unclear. The authors compared their ChIP–seq data with GWA study data sets for 47 common traits and found significant enrichment of VDR sites in associated genomic intervals for multiple sclerosis and type 1 diabetes, along with other autoimmune diseases, cancers and traits such as height.



Nature Reviews Genetics 11, 670 (October 2010) | doi:10.1038/nrg2873

In vitro assembly of cubic RNA-

In vitro assembly of cubic RNA-based scaffolds designed in silico



The organization of biological materials into versatile three-dimensional
assemblies could be used to build multifunctional therapeutic scaffolds for
use in nanomedicine. Here, we report a strategy to design three-dimensional
nanoscale scaffolds that can be self-assembled from RNA with precise
control over their shape, size and composition. These cubic nanoscaffolds
are only ~13 nm in diameter and are composed of short oligonucleotides,
making them amenable to chemical synthesis, point modifications and
further functionalization. Nanocube assembly is verified by gel assays,
dynamic light scattering and cryogenic electron microscopy. Formation of
functional RNA nanocubes is also demonstrated by incorporation of a
light-up fluorescent RNA aptamer that is optimally active only upon full RNA
assembly. Moreover, we show that the RNA nanoscaffolds can self-assemble in
isothermal conditions (37 °C) during in


Nature Nanotechnology 5, 676 - 682 (2010)
Published online: 29 August 2010 | doi:10.1038/nnano.2010.160

Baculovirus-transduced bone marrow mesenchymal

Baculovirus-transduced bone marrow mesenchymal stem cells for systemic cancer
therapy


Adult stem cells may serve as powerful cellular vehicles to deliver therapeutic
genes for cancer therapy. In such applications, effective and safe transduction to load stem cells with genes of interest is essential. To examine whether  baculovirus can be used to fulfill this task, we tested a range of baculoviral
vectors in human bone marrow mesenchymal stem cells (MSCs). A
vector using the human cytomegalovirus immediate-early gene promoter to drive
transgene expression and the woodchuck hepatitis virus posttranscriptional
regulatory element to enhance translation was able to transduce MSCs
with efficiency close to 80%. Following the observation that baculoviral
transduction did not significantly affect surface marker expression of the stem
cells, we tested the feasibility of using baculovirus-transduced MSCs for
targeted cancer therapy. We transduced cells with a baculoviral vector harboring
the herpes simplex virus thymidine kinase gene, and performed tail vein
injection of the transduced cells into



Cancer Gene Therapy 17, 721-729 (October 2010) |
doi:10.1038/cgt.2010.32

CTL responses to human papillomavirus oncoproteins

Hepatitis B surface antigen fusions delivered by DNA vaccination elicit CTL
responses to human papillomavirus oncoproteins associated with tumor protection








We describe the construction and evaluation of a recombinant hepatitis B
surface antigen (HBsAg)-vectored DNA vaccine encoding the E7 and E6 tumor-
associated oncoproteins of human papillomavirus (HPV) type 16. We show
the induction of effector and memory cytotoxic T lymphocyte responses to E7
and E6 class I-restricted epitopes after a single immunization, which were
associated with tumor prevention and therapy. The findings vindicate the use of
a HBsAg-based DNA vaccine as a vehicle to elicit responses to co-encoded tumor
antigens, and have specific implications for the development of a therapeutic
vaccine for HPV-associated squamous carcinomas.




Cancer Gene Therapy 17, 708-720 (October 2010) |
doi:10.1038/cgt.2010.27

Inflaming resistance to Tarceva

A team at Cold Spring Harbor Laboratory has found that in cancer, inflammation-driven IL-6 signaling can cause resistance to drugs that inhibit epidermal growth factor receptor, such as Tarceva erlotinib and Iressa gefitinib.1 The data suggest that blocking IL-6 could help treat drug-resistant
cancers, a hypothesis Alder Biopharmaceuticals Inc. may put to the test in a Phase IIa trial of its anti-IL-6 antibody, ALD518.
About 80% of patients who respond to small molecule inhibitors of epidermal growth factor receptor (EGFR) carry oncogenic mutations in the kinase domain of the receptor that render them sensitive to EGFR inhibition.2 However, all patients will eventually develop resistance to these drugs.
Half of resistant cases stem from secondary mutations within EGFR or amplification of c-Met proto- oncogene (MET; HGFR). In the other half of cases, the mechanisms underlying resistance are unknown.
Thus, a team led by Raffaella Sordella, assistant professor at Cold Spring Harbor Laboratory, sought to identify new mechanisms of drug resistance. The researchers started by selecting EGFR mutant cell lines that were resistant to Tarceva but lacked any of the known mutations that confer resistance.
“We noticed these erlotinib-resistant cells had a dramatically different morphology than erlotinib- sensitive cells,” Sordella told SciBX. “We performed gene expression profiling to understand what was different about them.”
Her team found that transforming growth factor-β (TGFB; TGFβ) was induced in resistant cells,explaining their difference in morphology. Moreover, IL-6 secretion from these cells was induced more than 10-fold, and the increase required TGFβ.
Adding IL-6 to erlotinib-sensitive cells increased their resistance to the drug compared with that of cells not given IL-6.
Sordella's team also examined non–small cell lung cancer (NSCLC) samples isolated from Tarceva-naïve patients and found that a subpopulation of cells had high levels of TGFβ and IL-6. That finding suggests some tumor cells may be intrinsically resistant to Tarceva.
Tarceva is marketed by Astellas Pharma Inc. and Roche's Genentech Inc. unit, whereas Iressa is marketed by AstraZeneca plc.



Cain, C. SciBX 3(35); doi:10.1038/scibx.2010.1056
Published online Sep. 9 2010

Y variants tip the chromatin balance

A new study has shed light on the mystery of how the
Y chromosome of male fruitflies can contribute to
phenotypic differences, despite the fact that this
chromosome has little sequence polymorphism in its
protein-coding genes.
Lemos et al. built on previous findings that Y
chromosomes in Drosophila melanogaster show
extensive variation in repeat sequences, which is known to contribute to
differences in Y chromosome heterochromatin. Such heterochromatin
differences have previously been shown to contribute to Y-linked regulatory
variation (YRV) of gene expression. The new evidence suggests that repeat
polymorphism on the D. melanogaster Y chromosome mediates its effects
through altering global chromatin dynamics.






Nature Reviews Genetics 11, 670-671 (October 2010) |
doi:10.1038/nrg2874

requirement for ELMO1

Apoptosis and the subsequent clearance
of dying cells occurs throughout
development and adult life in many
tissues. Failure to promptly clear
apoptotic cells has been linked to many
diseases1, 2, 3. ELMO1 is an evolutionarily
conserved cytoplasmic engulfment
protein that functions downstream of the
phosphatidylserine receptor BAI1, and,
along with DOCK1 and the GTPase RAC1,



Nature 467, 333-337 (16 September 2010) |
doi:10.1038/nature09356; Received 24 March 2010; Accepted 12 July
2010

VE-cadherin inhibits angiogenesis

Vascular endothelial-specific cadherin
(VE-cadherin) is an endothelial
cell-specific adhesion molecule,
localized at cell–cell contact sites. It is
involved in physiological and
pathological angiogenesis. In this study,
we showed that in vitro a soluble
N-terminal fragment of VE-cadherin
(EC1–3) corresponding to cadherin 1–3
ectodomains inhibited vascular
endothelial growth factor-stimulated
endothelial cell proliferation and
capillary tube structure formation in the
matrigel model. In vivo, EC1–3 was
tested in a murine colon cancer model.
EC1–3-expressing colon cancer C51 cells
were subcutaneously grafted into nude
mice, and tumor growth and
angiogenesis were evaluated. At day 33,
the mean volume of the tumors
developed was reduced (510±104 versus
990±120 mm 3 for control). Similarly,
injection of EC1–3 virus-producing cells
into established C51 tumors resulted in


Cancer Gene Therapy 17, 700-707 (October 2010) |
doi:10.1038/cgt.2010.26

widespread and critical impact of batch effects in high-throughput data

High-throughput technologies are widely used, for example to
assay genetic variants, gene and protein expression, and
epigenetic modifications. One often overlooked complication
with such studies is batch effects, which occur because
measurements are affected by laboratory conditions, reagent
lots and personnel differences. This becomes a major problem
when batch effects are correlated with an outcome of interest
and lead to incorrect conclusions. Using both published studies
and our own analyses, we argue that batch effects (as well as
other technical and biological artefacts) are widespread and
critical to address. We review experimental and computational
approaches for doing so.



Nature Reviews Genetics 11, 733-739 (October 2010) |
doi:10.1038/nrg2825

Inhibitors of leucine-rich repeat kinase-2 protect against models of Parkinson's disease

Leucine-rich repeat kinase-2 (LRRK2)
mutations are a common cause of
Parkinson's disease. Here we identify
inhibitors of LRRK2 kinase that are
protective in in vitro and in vivo models
of LRRK2-induced neurodegeneration.
These results establish that LRRK2-
induced degeneration of neurons in vivo
is kinase dependent and that LRRK2
kinase inhibition provides a potential
new neuroprotective paradigm for the
treatment of Parkinson's disease.









Nature Medicine 16, 998 - 1000 (2010)
Published online: 22 August 2010 | doi:10.1038/nm.2199

Skin patch flu vaccine shows promise

An influenza vaccine delivered through the skin by a
dissolving microneedle patch has generated immune
responses that are comparable with those triggered
by intramuscular injection. Moreover, the delivery
system conferred enhanced clearance of the virus in
mice challenged with a lethal dose of influenza.
Using a moulding process, Sullivan and colleagues
fabricated the microneedle patch from
polyvinylpyrrolidone (PVP) — a water-soluble
polymer widely used in medicine — that
encapsulated a dried form of an inactivated influenza
vaccine. When the patch is applied to skin the
microneedles (650 μm in length) penetrate across
the epidermis and dissolve within minutes to release
at least 80% of the vaccine load. An added advantage
of using this delivery method is that the depth of penetration is optimized

Averting inflammation by targeting the cytokine environment

Cytokines are key instigators and regulators of immune
responses and therefore hold great potential as targets for new
therapeutic strategies. However, the selection of which
cytokines to target, and in particular the identification of which
cytokines regulate the rate-limiting steps of disease pathways, is
crucial to the success of such strategies. Moreover, balancing
the need for ablating pathological inflammatory responses and
simultaneously maintaining the ability to control infectious
agents is a key consideration. Recent advances in our
understanding of cytokine networks, as well as technical
progress in blocking cytokines in vivo, are likely to be a source
for new drugs that can control chronic inflammatory diseases.




Nature Reviews Drug Discovery 9, 703-718 (September 2010) |
doi:10.1038/nrd2805

Blocking BTK in B-cell disorders

The aberrant activation of B cells plays a central role
in the pathogenesis of various autoimmune diseases
and B-cell lymphomas. Now, Honigberg and
colleagues describe an orally available, potent and
selective inhibitor of the B-cell receptor (BCR)
signalling molecule Bruton's tyrosine kinase (BTK), which shows promising
activity in mouse models of autoimmunity and in spontaneously occuring
B-cell lymphomas in dogs.
BTK is a well-defined downstream component of the BCR signalling cascade
with an essential role in B-cell development and activation. BTK mutations in
humans cause X-linked agammaglobulinaemia, which is characterized by
severe B cell-specific defects including the complete absence of B cells in the
periphery. Clinical studies with rituximab, a regulatory approved
CD20-specific monoclonal antibody, have shown that B-cell depletion is
efficacious in autoimmune diseases such as rheumatoid arthritis. Rituximab
is also approved for the treatment of non-Hodgkin's lymphoma (NHL).

Nature Reviews Drug Discovery 9, 681 (September 2010) |
doi:10.1038/nrd3262

MICU1 encodes a mitochondrial EF hand protein required for Ca 2+ uptake

Mitochondrial calcium uptake has a
central role in cell physiology by
stimulating ATP production, shaping
cytosolic calcium transients and
regulating cell death. The biophysical
properties of mitochondrial calcium
uptake have been studied in detail, but
the underlying proteins remain elusive.
Here we use an integrative strategy to
predict human genes involved in
mitochondrial calcium entry based on
clues from comparative physiology,
evolutionary genomics and organelle
proteomics. RNA interference against 13
top candidates highlighted one gene,
CBARA1, that we call hereafter


Nature 467, 291-296 doi:10.1038/nature09358;

Epigenetic memory in induced pluripotent stem cells

Somatic cell nuclear transfer and transcription-factor-based reprogramming revert adult cells to an embryonic state, and yield pluripotent stem cells that can generate all tissues. Through different mechanisms and kinetics, these two
reprogramming methods reset genomic methylation, an epigenetic modification of DNA that influences gene expression, leading us to hypothesize that the resulting pluripotent stem cells might have different properties. Here we
observe that low-passage induced pluripotent stem cells (iPSCs) derived by factor-based reprogramming of adult murine tissues harbour residual DNA methylation signatures characteristic of their somatic tissue of origin, which
favours their differentiation along lineages related to the donor cell, while restricting alternative cell fates. Such an ‘epigenetic memory’ of the donor tissue could be reset by differentiation and serial reprogramming, or by treatment of
iPSCs with chromatin-modifying drugs. In contrast, the differentiation and methylation of nuclear-transfer-derived pluripotent stem cells were more similar to classical embryonic stem cells than were iPSCs. Our data indicate that
nuclear transfer is more effective at establishing the ground state of pluripotency than factor-based reprogramming, which can leave an epigenetic memory of the tissue of origin that may influence efforts at directed differentiation for
applications in disease modelling or treatment.




Nature 467 , 285–290 (16 September 2010)doi:10.1038/nature09342