G Protein Subunit Dissociation and Translocation Regulate Cellular Response to Receptor Stimulation

We examined the role of G proteins in modulating the response of living cells to receptor activation. The response of an effector, phospholipase C-β to M3 muscarinic receptor activation was measured using sensors that detect the generation of inositol triphosphate or diacylglycerol. The recently discovered translocation of Gβγ from plasma membrane to endomembranes on receptor activation attenuated this response. A FRET based G protein sensor suggested that in contrast to translocating Gβγ, non-translocating Gβγ subunits do not dissociate from the αq subunit on receptor activation leading to prolonged retention of the heterotrimer state and an accentuated response. M3 receptors with tethered αq induced differential responses to receptor activation in cells with or without an endogenous translocation capable γ subunit. G protein heterotrimer dissociation and βγ translocation are thus unanticipated modulators of the intensity of a cell's response to an extracellular signal

Identification of gene targets against dormant phase Mycobacterium tuberculosis infections

<p>Abstract</p> <p>Background</p> <p><it>Mycobacterium tuberculosis</it>, the causative agent of tuberculosis (TB), infects approximately 2 billion people worldwide and is the leading cause of mortality due to infectious disease. Current TB therapy involves a regimen of four antibiotics taken over a six month period. Patient compliance, cost of drugs and increasing incidence of drug resistant <it>M. tuberculosis </it>strains have added urgency to the development of novel TB therapies. Eradication of TB is affected by the ability of the bacterium to survive up to decades in a dormant state primarily in hypoxic granulomas in the lung and to cause recurrent infections.</p> <p>Methods</p> <p>The availability of <it>M. tuberculosis </it>genome-wide DNA microarrays has lead to the publication of several gene expression studies under simulated dormancy conditions. However, no single model best replicates the conditions of human pathogenicity. In order to identify novel TB drug targets, we performed a meta-analysis of multiple published datasets from gene expression DNA microarray experiments that modeled infection leading to and including the dormant state, along with data from genome-wide insertional mutagenesis that examined gene essentiality.</p> <p>Results</p> <p>Based on the analysis of these data sets following normalization, several genome wide trends were identified and used to guide the selection of targets for therapeutic development. The trends included the significant up-regulation of genes controlled by <it>devR</it>, down-regulation of protein and ATP synthesis, and the adaptation of two-carbon metabolism to the hypoxic and nutrient limited environment of the granuloma. Promising targets for drug discovery were several regulatory elements (<it>devR/devS</it>, <it>relA</it>, <it>mprAB</it>), enzymes involved in redox balance and respiration, sulfur transport and fixation, pantothenate, isoprene, and NAD biosynthesis. The advantages and liabilities of each target are discussed in the context of enzymology, bacterial pathways, target tractability, and drug development.</p> <p>Conclusion</p> <p>Based on our bioinformatics analysis and additional discussion of in-depth biological rationale, several novel anti-TB targets have been proposed as potential opportunities to improve present therapeutic treatments for this disease.</p

Mycobacterium tuberculosis Transcriptional Adaptation, Growth Arrest and Dormancy Phenotype Development Is Triggered by Vitamin C

BACKGROUND: Tubercle bacilli are thought to persist in a dormant state during latent tuberculosis (TB) infection. Although little is known about the host factors that induce and maintain Mycobacterium tuberculosis (M. tb) within latent lesions, O(2) depletion, nutrient limitation and acidification are some of the stresses implicated in bacterial dormancy development/growth arrest. Adaptation to hypoxia and exposure to NO/CO is implemented through the DevRS/DosT two-component system which induces the dormancy regulon. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that vitamin C (ascorbic acid/AA) can serve as an additional signal to induce the DevR regulon. Physiological levels of AA scavenge O(2) and rapidly induce the DevR regulon at an estimated O(2) saturation of <30%. The kinetics and magnitude of the response suggests an initial involvement of DosT and a sustained DevS-mediated response during bacterial adaptation to increasing hypoxia. In addition to inducing DevR regulon mechanisms, vitamin C induces the expression of selected genes previously shown to be responsive to low pH and oxidative stress, triggers bacterial growth arrest and promotes dormancy phenotype development in M. tb grown in axenic culture and intracellularly in THP-1 cells. CONCLUSIONS/SIGNIFICANCE: Vitamin C mimics multiple intracellular stresses and has wide-ranging regulatory effects on gene expression and physiology of M. tb which leads to growth arrest and a 'dormant' drug-tolerant phenotype, but in a manner independent of the DevRS/DosT system. The 'AA-dormancy infection model' offers a potential alternative to other models of non-replicating persistence of M. tb and may be useful for investigating host-'dormant' M. tb interactions. Our findings offer a new perspective on the role of nutritional factors in TB and suggest a possible role for vitamin C in TB

Subcellular optogenetic inhibition of G proteins generates signaling gradients and cell migration

Cells sense gradients of extracellular cues and generate polarized responses such as cell migration and neurite initiation. There is static information on the intracellular signaling molecules involved in these responses, but how they dynamically orchestrate polarized cell behaviors is not well understood. A limitation has been the lack of methods to exert spatial and temporal control over specific signaling molecules inside a living cell. Here we introduce optogenetic tools that act downstream of native G protein–coupled receptor (GPCRs) and provide direct control over the activity of endogenous heterotrimeric G protein subunits. Light-triggered recruitment of a truncated regulator of G protein signaling (RGS) protein or a Gβγ-sequestering domain to a selected region on the plasma membrane results in localized inhibition of G protein signaling. In immune cells exposed to spatially uniform chemoattractants, these optogenetic tools allow us to create reversible gradients of signaling activity. Migratory responses generated by this approach show that a gradient of active G protein αi and βγ subunits is sufficient to generate directed cell migration. They also provide the most direct evidence so for a global inhibition pathway triggered by Gi signaling in directional sensing and adaptation. These optogenetic tools can be applied to interrogate the mechanistic basis of other GPCR-modulated cellular functions

Corticotropin Releasing Factor-Induced CREB Activation in Striatal Neurons Occurs via a Novel Gβγ Signaling Pathway

The peptide corticotropin-releasing factor (CRF) was initially identified as a critical component of the stress response. CRF exerts its cellular effects by binding to one of two cognate G-protein coupled receptors (GPCRs), CRF receptor 1 (CRFR1) or 2 (CRFR2). While these GPCRs were originally characterized as being coupled to Gαs, leading to downstream activation of adenylyl cyclase (AC) and subsequent increases in cAMP, it has since become clear that CRFRs couple to and activate numerous other downstream signaling cascades. In addition, CRF signaling influences the activity of many diverse brain regions, affecting a variety of behaviors. One of these regions is the striatum, including the nucleus accumbens (NAc). CRF exerts profound effects on striatal-dependent behaviors such as drug addiction, pair-bonding, and natural reward. Recent data indicate that at least some of these behaviors regulated by CRF are mediated through CRF activation of the transcription factor CREB. Thus, we aimed to elucidate the signaling pathway by which CRF activates CREB in striatal neurons. Here we describe a novel neuronal signaling pathway whereby CRF leads to a rapid Gβγ- and MEK-dependent increase in CREB phosphorylation. These data are the first descriptions of CRF leading to activation of a Gβγ-dependent signaling pathway in neurons, as well as the first description of Gβγ activation leading to downstream CREB phosphorylation in any cellular system. Additionally, these data provide additional insight into the mechanisms by which CRF can regulate neuronal function

Snake Bite in South Asia: A Review

Snake bite is one of the most neglected public health issues in poor rural communities living in the tropics. Because of serious misreporting, the true worldwide burden of snake bite is not known. South Asia is the world's most heavily affected region, due to its high population density, widespread agricultural activities, numerous venomous snake species and lack of functional snake bite control programs. Despite increasing knowledge of snake venoms' composition and mode of action, good understanding of clinical features of envenoming and sufficient production of antivenom by Indian manufacturers, snake bite management remains unsatisfactory in this region. Field diagnostic tests for snake species identification do not exist and treatment mainly relies on the administration of antivenoms that do not cover all of the important venomous snakes of the region. Care-givers need better training and supervision, and national guidelines should be fed by evidence-based data generated by well-designed research studies. Poorly informed rural populations often apply inappropriate first-aid measures and vital time is lost before the victim is transported to a treatment centre, where cost of treatment can constitute an additional hurdle. The deficiency of snake bite management in South Asia is multi-causal and requires joint collaborative efforts from researchers, antivenom manufacturers, policy makers, public health authorities and international funders

Search for neutral MSSM Higgs bosons decaying to a pair of tau leptons in pp collisions

Peer reviewe

Measurement of top quark–antiquark pair production in association with a W or Z boson in pp collisions at √s=8 TeV

Peer reviewe