Syntaxin 3 SPI-2 dependent crosstalk facilitates the division of Salmonella containing vacuole

Intracellular membrane fusion is mediated by membrane-bridging complexes of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). SNARE proteins are one of the key players in vesicular transport. Several reports shed light on intracellular bacteria modulating host SNARE machinery to establish infection successfully. The critical SNAREs in macrophages responsible for phagosome maturation are Syntaxin 3 (STX3) and Syntaxin 4 (STX4). Reports also suggest that Salmonella actively modulates its vacuole membrane composition to escape lysosomal fusion. Salmonella containing vacuole (SCV) harbours recycling endosomal SNARE Syntaxin 12 (STX12). However, the role of host SNAREs in SCV biogenesis and pathogenesis remains unclear. Upon knockdown of STX3, we observed a reduction in bacterial proliferation, which is concomitantly restored upon the overexpression of STX3. Live-cell imaging of Salmonella-infected cells showed that STX3 localises to the SCV membranes and thus might help in the fusion of SCV with intracellular vesicles to acquire membrane for its division. We also found the interaction STX3-SCV was abrogated when we infected with SPI-2 encoded Type 3 secretion system (T3SS) apparatus mutant (STM ∆ssaV) but not with SPI-1 encoded T3SS apparatus mutant (STM ∆invC). These observations were also consistent in the mice model of Salmonella infection. Together, these results shed light on the effector molecules secreted through T3SS encoded by SPI-2, possibly involved in interaction with host SNARE STX3, which is essential to maintain the division of Salmonella in SCV and help to maintain a single bacterium per vacuole.     

Absence of detectable XMRV and other MLV-related viruses in healthy blood donors in the United States

Background

Preliminary studies in chronic fatigue syndrome (CFS) patients and XMRV infected animals demonstrated plasma viremia and infection of blood cells with XMRV, indicating the potential risk for transfusion transmission. XMRV and MLV-related virus gene sequences have also been detected in 4–6% of healthy individuals including blood donors in the U.S. These results imply that millions of persons in the U.S. may be carrying the nucleic acid sequences of XMRV and/or MLV-related viruses, which is a serious public health and blood safety concern.

Methodology/Principal Findings

To gain evidence of XMRV or MLV-related virus infection in the U.S. blood donors, 110 plasma samples and 71 PBMC samples from blood donors at the NIH blood bank were screened for XMRV and MLV-related virus infection. We employed highly sensitive assays, including nested PCR and real-time PCR, as well as co-culture of plasma with highly sensitive indicator DERSE cells. Using these assays, none of the samples were positive for XMRV or MLV-related virus.

Conclusions/Significance

Our results are consistent with those from several other studies, and demonstrate the absence of XMRV or MLV-related viruses in the U.S. blood donors that we studied.     

Live sequence charts to model medical information

Background

Consensus exists that several bariatric surgery procedures produce a rapid improvement of glucose homeostasis in obese diabetic patients, improvement apparently uncorrelated with the degree of eventual weight loss after surgery. Several hypotheses have been suggested to account for these results: among these, the anti-incretin, the ghrelin and the lower-intestinal dumping hypotheses have been discussed in the literature. Since no clear-cut experimental results are so far available to confirm or disprove any of these hypotheses, in the present work a mathematical model of the glucose-insulin-incretin system has been built, capable of expressing these three postulated mechanisms. The model has been populated with critically evaluated parameter values from the literature, and simulations under the three scenarios have been compared.

Results

The modeling results seem to indicate that the suppression of ghrelin release is unlikely to determine major changes in short-term glucose control. The possible existence of an anti-incretin hormone would be supported if an experimental increase of GIP concentrations were evident post-surgery. Given that, on the contrary, collected evidence suggests that GIP concentrations decrease post-surgery, the lower-intestinal dumping hypothesis would seem to describe the mechanism most likely to produce the observed normalization of Type 2 Diabetes Mellitus (T2DM) after bariatric surgery.

Conclusions

The proposed model can help discriminate among competing hypotheses in a context where definitive data are not available and mechanisms are still not clear.     

Alpha-adrenoceptor-mediated coronary vasoconstriction is augmented during exercise in experimental diabetes mellitus.

This study tested whether alpha-adrenoceptor-mediated coronary vasoconstriction is augmented during exercise in diabetes mellitus. Experiments were conducted in dogs instrumented with catheters in the aorta and coronary sinus and with a flow transducer around the circumflex coronary artery. Diabetes was induced with alloxan monohydrate (n = 8, 40 mg/kg i.v.). Arterial plasma glucose concentration increased from 4.7 +/- 0.2 mM in nondiabetic, control dogs (n = 8) to 21.4 +/- 1.9 mM 1 wk after alloxan injection. Coronary blood flow, myocardial oxygen consumption (MVo(2)), aortic pressure, and heart rate were measured at rest and during graded treadmill exercise before and after infusion of the alpha-adrenoceptor antagonist phentolamine (1 mg/kg iv). In untreated diabetic dogs, exercise increased MVo(2) 2.7-fold, coronary blood flow 2.2-fold, and heart rate 2.3-fold. Coronary venous Po(2) fell as MVo(2) increased during exercise. After alpha-adrenoceptor blockade, exercise increased MVo(2) 3.1-fold, coronary blood flow 2.7-fold, and heart rate 2.1-fold. Relative to untreated diabetic dogs, alpha-adrenoceptor blockade significantly decreased the slope of the relationship between coronary venous Po(2) and MVo(2). The difference between the untreated and phentolamine-treated slopes was greater in the diabetic dogs than in the nondiabetic dogs. In addition, the decrease in coronary blood flow to intracoronary norepinephrine infusion was significantly augmented in anesthetized, open-chest, beta-adrenoceptor-blocked diabetic dogs compared with the nondiabetic dogs. These findings demonstrate that alpha-adrenoceptor-mediated coronary vasoconstriction is augmented in alloxan-induced diabetic dogs during physiological increases in MVo(2).     

Protective effect of aqueous extract of Phyllanthus fraternus against bromobenzene induced changes on cytosolic glutathione S-transferase isozymes in rat liver

The aim of this study was to investigate beneficial effect of aqueous extract of Phyllanthus fraternus (AEPF) on bromobenzene (BB) induced changes on cytosolic glutathione S-transferase (GST) isozymes in rat liver. Administration of BB significantly decreased the activity of GST, however, prior administration of AEPF prevented the BB induced decrease in GST activity. Further the cytosolic GSTs were purified from 3 groups of animals (control, BB and AEPF+BB administered) and resolved into three protein bands on SDS-PAGE. Densitometric analysis showed a significant decrease in BB group compared to control. Further, 2D PAGE analysis resolved these proteins into 8 bands which were identified as five isozymes of alpha, two of Mu and one of theta by MALDI-TOF MS and also observed decreased levels of isozymes in BB group. However, on prior administration of AEPF significantly prevented the BB induced decrease in GSTs and restored to normal levels.     

High-definition Fourier Transform Infrared (FT-IR) Spectroscopic Imaging of Human Tissue Sections towards Improving Pathology

High-definition Fourier Transform Infrared (FT-IR) spectroscopic imaging is an emerging approach to obtain detailed images that have associated biochemical information. FT-IR imaging of tissue is based on the principle that different regions of the mid-infrared are absorbed by different chemical bonds (e.g., C=O, C-H, N-H) within cells or tissue that can then be related to the presence and composition of biomolecules (e.g., lipids, DNA, glycogen, protein, collagen). In an FT-IR image, every pixel within the image comprises an entire Infrared (IR) spectrum that can give information on the biochemical status of the cells that can then be exploited for cell-type or disease-type classification. In this paper, we show: how to obtain IR images from human tissues using an FT-IR system, how to modify existing instrumentation to allow for high-definition imaging capabilities, and how to visualize FT-IR images. We then present some applications of FT-IR for pathology using the liver and kidney as examples. FT-IR imaging holds exciting applications in providing a novel route to obtain biochemical information from cells and tissue in an entirely label-free non-perturbing route towards giving new insight into biomolecular changes as part of disease processes. Additionally, this biochemical information can potentially allow for objective and automated analysis of certain aspects of disease diagnosis.     

Rab9A is required for delivery of cargo from recycling endosomes to melanosomes

Melanosomes are a type of lysosome‐related organelle that is commonly defective in Hermansky–Pudlak syndrome. Biogenesis of melanosomes is regulated by BLOC‐1, ‐2, ‐3, or AP‐1, ‐3 complexes, which mediate cargo transport from recycling endosomes to melanosomes. Although several Rab GTPases have been shown to regulate these trafficking steps, the precise role of Rab9A remains unknown. Here, we found that a cohort of Rab9A associates with the melanosomes and its knockdown in melanocytes results in hypopigmented melanosomes due to mistargeting of melanosomal proteins to lysosomes. In addition, the Rab9A‐depletion phenotype resembles Rab38/32‐inactivated or BLOC‐3‐deficient melanocytes, suggesting that Rab9A works in line with BLOC‐3 and Rab38/32 during melanosome cargo transport. Furthermore, silencing of Rab9A, Rab38/32 or its effector VARP, or BLOC‐3‐deficiency in melanocytes decreased the length of STX13‐positive recycling endosomal tubules and targeted the SNARE to lysosomes. This result indicates a defect in directing recycling endosomal tubules to melanosomes. Thus, Rab9A and its co‐regulatory GTPases control STX13‐mediated cargo delivery to maturing melanosomes.