Cationic amino acid transporters and Salmonella Typhimurium ArgT collectively regulate arginine availability towards intracellular Salmonella growth

Cationic amino acid transporters (mCAT1 and mCAT2B) regulate the arginine availability in macrophages. How in the infected cell a pathogen can alter the arginine metabolism of the host remains to be understood. We reveal here a novel mechanism by which Salmonella exploit mCAT1 and mCAT2B to acquire host arginine towards its own intracellular growth within antigen presenting cells. We demonstrate that Salmonella infected bone marrow derived macrophages and dendritic cells show enhanced arginine uptake and increased expression of mCAT1 and mCAT2B. We show that the mCAT1 transporter is in close proximity to Salmonella containing vacuole (SCV) specifically by live intracellular Salmonella in order to access the macrophage cytosolic arginine pool. Further, Lysosome associated membrane protein 1, a marker of SCV, also was found to colocalize with mCAT1 in the Salmonella infected cell. The intra vacuolar Salmonella then acquire the host arginine via its own arginine transporter, ArgT for growth. The argT knockout strain was unable to acquire host arginine and was attenuated in growth in both macrophages and in mice model of infection. Together, these data reveal survival strategies by which virulent Salmonella adapt to the harsh conditions prevailing in the infected host cells.     

Activated pericyte attenuates endothelial functions: nitric oxide-cGMP rescues activated pericyte-associated endothelial dysfunctions.

Hepatic stellate cells are liver-specific pericytes and exist in close proximity with endothelial cells. The activation of liver pericytes is intrinsic to liver pathogenesis, and leads to endothelial dysfunction, including the low bioavailability of nitric oxide (NO). However, the role of nitric oxide in pericyte-endothelium cross-talk has not yet been elucidated. This work examines the cellular mechanism of action of NO in pericyte-mediated endothelial dysfunction. We used in vitro coculture and conditioned medium systems to study the effects of activated liver pericytes on endothelial function, and an egg yolk vascular bed model was used to study the effects of activated pericytes on angiogenesis. This study also demonstrates that activated pericytes attenuate the migration, proliferation, permeability, and NO production of endothelial cells. Our results demonstrate that activated pericytes restrict angiogenesis in egg yolk vascular bed models, and NO supplementation recovers 70% of the inhibition. Our results also demonstrate that supplementation with NO, sildenafil citrate (phosphodiesterase inhibitor), and 8-bromo-cGMP (cGMP analog) partially recovers activated-pericyte-mediated endothelium dysfunction. We conclude that NO-cGMP alleviates activated-pericyte-associated endothelial dysfunction, including angiogenesis, in a cGMP-dependent manner.     

Large-scale purification of choline acetyltransferase and production of highly specific antisera

Choline acetyltransferase (ChAT) was purified by immunoaffinity chromatography using a covalently immobilized monoclonal antibody. In a two-step procedure, 10 kg porcine brain yielded 750 micrograms active enzyme of apparent homogeneity. This amount of ChAT was purified routinely. The purification factor was 18,000 and the yield of activity 4.3%. The affinity resin was stable under the experimental conditions applied and was used many times. The highly purified enzyme was subsequently employed to obtain a specific anti-ChAT antiserum of high titer.     

Human spleen tyrosine kinase (Syk) recombinant expression systems for high-throughput assays

Spleen tyrosine kinase (Syk) is an important non-receptor tyrosine kinase and its aberrant regulation is associated with a variety of allergic disorders and autoimmune diseases. To identify small molecule inhibitors of Syk in high-throughput assays, recombinant Syk protein is needed in bulk quantity. We studied the expression of recombinant human Syk in three heterologous systems: E. coli, baculovirus expression vector system (BEVS), and the cellular slime mold Dictyostelium discoideum (Dd). Syk activity was higher in the BEVS as compared to the Dd expression host, whereas in E. coli, no activity was observed under our assay conditions. Purified Syk kinase domain protein from BEVS showed concentration dependent inhibition with OXSI-2, a known Syk inhibitor. Molecular modeling and docking studies were performed to understand the binding mode and critical interactions of the inhibitor with catalytic domain of Syk. The BEVS generated Syk kinase domain showed stability upon multiple freeze-thaw cycles and exhibited significantly higher levels of tyrosine phosphorylation at pTyr⁵²⁵/Tyr⁵²⁶ in the Syk activation loop. Based on our data, we conclude that BEVS is the ideal host to produce an active and stable enzyme, which can be successfully employed for screening of Syk inhibitors in a high-throughput system.     

Operationally determined chemical speciation of barium and chromium in drilling fluid wastes by sequential extraction

ABSTRACT

A study has been conducted to determine the influence of pH on the speciation and distribution of barium and chromium in drilling mud. Samples in equilibrium under controlled conditions were subjected to sequential extraction procedure to fractionate the heavy metals into the designated forms of exchangeable, adsorbed, organically bound, carbonate and residual phases. This provides an insight into the potential availability of the heavy metals for possible release into groundwaters and/or surface waters. The major portion of both the metals studied was found in the carbonate and residual forms with the relative distribution depending on the pH. Generally, decreasing pH caused a shift from the residual form towards the carbonate or organic forms of metals. The occurrence of metals in more stable organic, carbonate and residual forms in drilling mud, coupled with no significant release to the aqueous phase upon varying the pH indicates the resistance of these metals to remobilisation from drilling mud.     

Etiology of Severe Febrile Illness in Low- and Middle-Income Countries: A Systematic Review

Background

With apparent declines in malaria worldwide during the last decade and more widespread use of malaria rapid diagnostic tests, healthcare workers in low-resource areas face a growing proportion of febrile patients without malaria. We sought to describe current knowledge and identify information gaps of the etiology severe febrile illness in low-and middle-income countries.

Methods and Findings

We conducted a systematic review of studies conducted in low-and-middle income countries 1980–2013 that prospectively assessed consecutive febrile patients admitted to hospital using rigorous laboratory-based case definitions. We found 45 eligible studies describing 54,578 patients; 9,771 (17.9%) had a positive result for ≥1 pathogen meeting diagnostic criteria. There were no eligible studies identified from Southern and Middle Africa, Eastern Asia, Oceania, Latin American and Caribbean regions, and the European region. The median (range) number of diagnostic tests meeting our confirmed laboratory case definitions was 2 (1 to 11) per study. Of diagnostic tests, 5,052 (10.3%) of 49,143 had confirmed bacterial or fungal bloodstream infection; 709 (3.8%) of 18,142 had bacterial zoonosis; 3,488 (28.5%) of 12,245 had malaria; and 1,804 (17.4%) of 10,389 had a viral infection.

Conclusions

We demonstrate a wide range of pathogens associated with severe febrile illness and highlight the substantial information gaps regarding the geographic distribution and role of common pathogens. High quality severe febrile illness etiology research that is comprehensive with respect to pathogens and geographically representative is needed.