Roles of the Methylcitrate and Methylmalonyl-COA Pathways in Mycobacterial Metabolism and Pathogenesis

Mycobacterium tuberculosis has been a human pathogen for the history of mankind, but we are only now beginning to understand how it is able to survive and persist indefinitely in the host. Understanding carbon metabolism of the pathogen during infection is key, not only as a source of potential drug targets, but also for elucidating the environment in vivo, so that drugs can be tested under relevant conditions. Studies have revealed that, during infection, M. tuberculosis relies on gluconeogenic carbon sources rather than sugars. Fatty acids, cholesterol, and amino acids have all been demonstrated as usable carbon sources in vitro and can all generate propionyl-CoA. The methylcitrate cycle, which, in M. tuberculosis, uses a bifunctional isocitrate lyase/methylisocitrate lyase (ICL/MCL), is one of the two routes for metabolism of propionyl-CoA. A mutant strain of M. tuberculosis lacking the ICL/MCL was rapidly cleared from the lungs of infected mice. However, the upstream enzymes of this pathway have been demonstrated to be dispensable for infection and survival in the mouse model. The methylmalonyl- CoA route of propionyl-CoA utilization can be activated in vitro by addition of the vitamin B12 cofactor of the methylmalonyl-CoA mutase. This route may buffer the loss of the methylcitrate cycle in vivo, depending on B12 availability or production in the host. The work here examines the relative use of the methylcitrate cycle and methylmalonyl-CoA pathways in M. tuberculosis and in the related, nonpathogenic species, M. smegmatis, using genetic mutants of either or both of the metabolic routes. It is shown here that, as for M. tuberculosis, M. smegmatis preferentially uses the methylcitrate cycle for growth on propionate. In the absence of the methylcitrate cycle M. smegmatis, in contrast to M. tuberculosis, can eventually endogenously activate the methylmalonyl-CoA pathway in vitro, presumably through B12 synthesis. In mutants of both species, lacking both pathways, the use of other carbon sources in the media is inhibited in the presence of propionate. This dominant inhibition implies the accumulation of toxic metabolites derived from the inability to metabolize propionate, as has been suggested by previous studies. To detect propionate-derived intermediates, metabolite analysis by targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used in this study. Accumulation of these metabolites under propionate exposure was identified in a strain of M. smegmatis impaired in both metabolic routes, but not in the wild-type. These studies also revealed similar accumulation under glucose growth, where the mutant strain displayed a slight growth defect, and also under no-carbon conditions, where the mutant demonstrated a survival defect compared to wild-type. These findings suggest a role of the propionate pathways for endogenously derived propionyl-CoA as well as during starvationinduced amino acid and/or fatty acid mobilization. The M. tuberculosis mutant strains generated here were tested in the mouse infection model. The methylmalonyl-CoA mutase was found to be individually dispensable for growth in vivo. However, a strain with the additional deletion of the methylcitrate cycle was attenuated during the early stage of infection and caused less tissue pathology, even after the bacterial burden reached wild-type levels. While propionate metabolism may not be required per se for in vivo growth, the suggested accumulation of toxic intermediates, demonstrated here in M. smegmatis, may indicate a required role for ICL/MCL in M. tuberculosis for detoxification of propionyl-CoA in vivo

Local temperature-sensitive mechanisms are important mediators of limb tissue hyperemia in the heat-stressed human at rest and during small muscle mass exercise.

Limb tissue and systemic blood flow increases with heat stress, but the underlying mechanisms remain poorly understood. Here, we tested the hypothesis that heat stress-induced increases in limb tissue perfusion are primarily mediated by local temperature-sensitive mechanisms. Leg and systemic temperatures and hemodynamics were measured at rest and during incremental single-legged knee extensor exercise in 15 males exposed to 1 h of either systemic passive heat-stress with simultaneous cooling of a single leg (n=8) or isolated leg heating or cooling (n=7). Systemic heat-stress increased core, skin and heated leg blood (Tb) temperatures, cardiac output and heated leg blood flow (LBF, 0.6 ± 0.1 l.min(-1); P0.05). Increased heated leg deep tissue BF was closely related to Tb (R(2) = 0.50; P0.05), despite unchanged systemic temperatures and hemodynamics. During incremental exercise, heated LBF was consistently maintained ~ 0.6 l.min(-1) higher than that in the cooled leg (P<0.01), with LBF and vascular conductance in both legs showing a strong correlation with their respective local Tb (R(2) = 0.85 and 0.95, P<0.05). We conclude that local temperature-sensitive mechanisms are important mediators in limb tissue perfusion regulation both at rest and during small-muscle mass exercise in hyperthermic humans.The invasive study was partially funded by Gatorade Sports Science Institute, PepsiCo

Study of spatial-temporal variations in the green Noctiluca scintillans and diatom blooms in the Arabian Sea using MODIS data

Phytoplankton blooms of green Noctiluca scintillans (a dinoflagellate) and diatom, which appear in the Northern and Central Arabian Sea during winter are far-reaching and persistent. Generation of phytoplankton species images revealed a massive winter bloom with huge spatial extent in 2015. In contrast to this, the classified species images for 2013 indicated relatively weaker bloom with respect to its spread. A plot of total number of pixels classified as diatom and Noctiluca scintillans for different years revealed a cyclic pattern of the spread. The report deals with an approach to forecast the bloom / productivity of the oceanic waters in the Northern-Central Arabian Sea in a qualitative way making use of the systematic pattern of its distribution across the year

Design, Implementation and Evaluation of a Configurable NoC for AcENoCs FPGA Accelerated Emulation Platform

The heterogenous nature and the demand for extensive parallel processing in modern applications have resulted in widespread use of Multicore System-on-Chip (SoC) architectures. The emerging Network-on-Chip (NoC) architecture provides an energy-efficient and scalable communication solution for Multicore SoCs, serving as a powerful replacement for traditional bus-based solutions. The key to successful realization of such architectures is a flexible, fast and robust emulation platform for fast design space exploration. In this research, we present the design and evaluation of a highly configurable NoC used in AcENoCs (Accelerated Emulation platform for NoCs), a flexible and cycle accurate field programmable gate array (FPGA) emulation platform for validating NoC architectures. Along with the implementation details, we also discuss the various design optimizations and tradeoffs, and assess the performance improvements of AcENoCs over existing simulators and emulators. We design a hardware library consisting of routers and links using verilog hardware description language (HDL). The router is parameterized and has a configurable number of physical ports, virtual channels (VCs) and pipeline depth. A packet switched NoC is constructed by connecting the routers in either 2D-Mesh or 2D-Torus topology. The NoC is integrated in the AcENoCs platform and prototyped on Xilinx Virtex-5 FPGA. The NoC was evaluated under various synthetic and realistic workloads generated by AcENoCs' traffic generators implemented on the Xilinx MicroBlaze embedded processor. In order to validate the NoC design, performance metrics like average latency and throughput were measured and compared against the results obtained using standard network simulators. FPGA implementation of the NoC using Xilinx tools indicated a 76% LUT utilization for a 5x5 2D-Mesh network. A VC allocator was found to be the single largest consumer of hardware resources within a router. The router design synthesized at a frequency of 135MHz, 124MHz and 109MHz for 3-port, 4-port and 5-port configurations, respectively. The operational frequency of the router in the AcENoCs environment was limited only by the software execution latency even though the hardware itself could be clocked at a much higher rate. An AcENoCs emulator showed speedup improvements of 10000-12000X over HDL simulators and 5-15X over software simulators, without sacrificing cycle accuracy

Preliminary X-ray crystallographic analysis of B-carbonic anhydrase psCA3 from Pseudomonas aeruginosa

Pseudomonas aeruginosa is a Gram-negative bacterium that causes life-threatening infections in susceptible individuals and is resistant to most clinically available antimicrobials. Genomic and proteomic studies have identified three genes, pa0102, pa2053 and pa4676, in P. aeruginosa PAO1 encoding three functional [beta]-carbonic anhydrases ([beta]-CAs): psCA1, psCA2 and psCA3, respectively. These [beta]-CAs could serve as novel antimicrobial drug targets for this pathogen. X-ray crystallographic structural studies have been initiated to characterize the structure and function of these proteins. This communication describes the production of two crystal forms (A and B) of [beta]-CA psCA3. Form A diffracted to a resolution of 2.9 A; it belonged to space group P212121, with unit-cell parameters a = 81.9, b = 84.9, c = 124.2 A, and had a calculated Matthews coefficient of 2.23 A3 Da-1 assuming four molecules in the crystallographic asymmetric unit. Form B diffracted to a resolution of 3.0 A; it belonged to space group P21212, with unit-cell parameters a = 69.9, b = 77.7, c = 88.5 A, and had a calculated Matthews coefficient of 2.48 A3 Da-1 assuming two molecules in the crystallographic asymmetric unit. Preliminary molecular-replacement solutions have been determined with the PHENIX AutoMR wizard and refinement of both crystal forms is currently in progress.Peer reviewedMicrobiology and Molecular Genetic

Structure-activity relationships for analogs of the tuberculosis drug bedaquiline with the naphthalene unit replaced by bicyclic heterocycles

Replacing the naphthalene C-unit of the anti-tuberculosis drug bedaquiline with a range of bicyclic heterocycles of widely differing lipophilicity gave analogs with a 4.5-fold range in clogP values. The biological results for these compounds indicate on average a lower clogP limit of about 5.0 in this series for retention of potent inhibitory activity (MIC90s) against M.tb in culture. Some of the compounds also showed a significant reduction in inhibition of hERG channel potassium current compared with bedaquiline, but there was no common structural feature that distinguished these

In vitro Evaluation of Novel Sustained Release Microspheres of Glipizide Prepared by the Emulsion Solvent Diffusion-Evaporation Method

The objective of the current investigation is to reduce dosing frequency and improve patient compliance by designing and systematically evaluating sustained release microspheres of Glipizide. An anti-diabetic drug, Glipizide, is delivered through the microparticulate system using ethyl cellulose as the controlled release polymer. Microspheres were developed by the emulsion solvent diffusion-evaporation technique by using the modified ethanol,-dichloromethane co-solvent system. The polymer mixture of ethyl cellulose and Eudragit® S100 was used in different ratios (1:0, 1:1, 2:3, 1:4 and 0:1) to formulate batches F1 to F5. The resulting microspheres were evaluated for particle size, densities, flow properties, morphology, recovery yield, drug content, and in vitro drug release behavior. The formulated microspheres were discrete, spherical with relatively smooth surface, and with good flow properties. Among different formulations, the fabricated microspheres of batch F3 had shown the optimum percent drug encapsulation of microspheres and the sustained release of the Glipizide for about 12 h. Release pattern of Glipizide from microspheres of batch F3 followed Korsmeyers-peppas model and zero-order release kinetic model. The value of ‘n’ was found to be 0.960, which indicates that the drug release was followed by anomalous (non-fickian) diffusion. The data obtained thus suggest that a microparticulate system can be successfully designed for sustained delivery of Glipizide and to improve dosage form characteristics for easy formulation

Mechanisms for the control of local tissue blood flow during thermal interventions: influence of temperature-dependent ATP release from human blood and endothelial cells

© 2016 The Authors. Local tissue perfusion changes with alterations in temperature during heating and cooling, but the thermosensitivity of the vascular ATP signalling mechanisms for control of blood flow during thermal interventions remains unknown. Here we tested the hypotheses that the release of the vasodilator mediator ATP from human erythrocytes, but not from endothelial cells or other blood constituents, is sensitive to both increases and reductions in temperature and that increasing intravascular ATP availability with ATP infusion would potentiate thermal hyperaemia in limb tissues. We first measured blood temperature (Tb), brachial artery blood flow (BABF) and plasma [ATP] during passive arm heating and cooling in healthy males and found they increased by 3.0 ± 1.2 ºC, 105 ± 25 ml min-1 °C-1 and 2-fold (all P<0.05) with heating, but decreased or remained unchanged with cooling. In additional males, intrabrachial artery ATP infusion increased skin and deep tissue perfusion to levels equal or above thermal hyperaemia. In isolated erythrocyte samples exposed to different temperatures, ATP release increased 1.9-fold from 33°C to 39°C (P<0.05) and declined by ~50% at 20°C (P<0.05), but no changes were observed in cultured human endothelial cells, plasma or serum samples. In conclusion, increases in plasma [ATP] and skin and deep tissue perfusion with limb heating are associated with elevations in ATP release from erythrocytes, but not from endothelial cells or other blood constituents. Erythrocyte ATP release is also sensitive to temperature reductions, suggesting erythrocytes may function as thermal sensors and ATP signalling generators for tissue perfusion control during thermal interventions