Budding of Marburgvirus is associated with filopodia

Viruses exploit the cytoskeleton of host cells to transport their components and spread to neighbouring cells. Here we show that the actin cytoskeleton is involved in the release of Marburgvirus (MARV) particles. We found that peripherally located nucleocapsids and envelope precursors of MARV are located either at the tip or at the side of filopodial actin bundles. Importantly, viral budding was almost exclusively detected at filopodia. Inhibiting actin polymerization in MARV-infected cells significantly diminished the amount of viral particles released into the medium. This suggested that dynamic polymerization of actin in filopodia is essential for efficient release of MARV. The viral matrix protein VP40 plays a key role in the release of MARV particles and we found that the intracellular localization of recombinant VP40 and its release in form of virus-like particles were strongly influenced by overexpression or inhibition of myosin 10 and Cdc42, proteins important in filopodia formation and function. We suggest that VP40, which is capable of interacting with viral nucleocapsids, provides an interface of MARV subviral particles and filopodia. As filopodia are in close contact with neighbouring cells, usurpation of these structures may facilitate spread of MARV to adjacent cells.     

Resistance to friend virus-induced erythroleukemia in W/W(v) mice is caused by a spleen-specific defect which results in a severe reduction in target cells and a lack of Sf-Stk expression

The characteristic progression and specificity of Friend virus for the erythroid lineage have allowed for the identification of a number of host-encoded loci that are required for disease progression. Several of these loci, including the Friend virus susceptibility gene 2 (Fv2), dominant white spotting gene (W), and Steel gene (Sl), regulate the initial polyclonal expansion of infected erythroid progenitor cells. W and Sl encode the Kit receptor tyrosine kinase and its ligand, stem cell factor, respectively. W mutant mice are severely anemic, and earlier work suggested that this defect in erythroid differentiation is the cause for the resistance to Friend virus-induced erythroleukemia. Here we show that in bone marrow, W/W(v) mice have near normal numbers of target cells and the initial infection of bone marrow occurs normally in vivo. In contrast, spleen cells from W/W(v) mice infected both in vitro and in vivo with Friend virus failed to give rise to erythropoietin-independent colonies at any time following Friend virus infection, suggesting that mutation of the Kit receptor specifically affects target cells in the spleen, rendering the mutant mice resistant to the development of Friend virus-induced erythroleukemia. In addition, we show that the Kit+ pathogenic targets of Friend virus in the spleen are distinct from the pathogenic targets in bone marrow and this population of spleen target cells is markedly decreased in W/W(v) mice and these cells fail to express Sf-Stk. These results also underscore the unique nature of the spleen microenvironment in its role in supporting the progression of acute leukemia in Friend virus-infected mice.     

Ter-Seq: A high-throughput method to stabilize transient ternary complexes and measure associated kinetics

Regulation of biological processes by proteins often involves the formation of transient, multimeric complexes whose characterization is mechanistically important but challenging. The bacterial toxin CcdB binds and poisons DNA Gyrase. The corresponding antitoxin CcdA extracts CcdB from its complex with Gyrase through the formation of a transient ternary complex, thus rejuvenating Gyrase. We describe a high throughput methodology called Ter-Seq to stabilize probable ternary complexes and measure associated kinetics using the CcdA-CcdB-GyrA14 ternary complex as a model system. The method involves screening a yeast surface display (YSD) saturation mutagenesis library of one partner (CcdB) for mutants that show enhanced ternary complex formation. We also isolated CcdB mutants that were either resistant or sensitive to rejuvenation, and used surface plasmon resonance (SPR) with purified proteins to validate the kinetics measured using the surface display. Positions, where CcdB mutations lead to slower rejuvenation rates, are largely involved in CcdA-binding, though there were several notable exceptions suggesting allostery. Mutations at these positions reduce the affinity towards CcdA, thereby slowing down the rejuvenation process. Mutations at GyrA14-interacting positions significantly enhanced rejuvenation rates, either due to reduced affinity or complete loss of CcdB binding to GyrA14. We examined the effect of different parameters (CcdA affinity, GyrA14 affinity, surface accessibilities, evolutionary conservation) on the rate of rejuvenation. Finally, we further validated the Ter-Seq results by monitoring the kinetics of ternary complex formation for individual CcdB mutants in solution by fluorescence resonance energy transfer (FRET) studies.     

The Molecular Basis for Modulation of Human Vγ9Vδ2 T Cell Responses by CD277/Butyrophilin-3 (BTN3A)-specific Antibodies

Human Vγ9Vδ2 T cells are well known for their rapid and potent response to infection and tumorigenesis when in the presence of endogenous or exogenous phosphoisoprenoids. However, the molecular mechanisms behind the activation of this γδ T cell population remains unclear. Evidence pointing to a role for the CD277/butyrophilin-3 (BTN3A) molecules in this response led us to investigate the structures of these molecules and their modifications upon binding to an agonist antibody (20.1) that mimics phosphoisoprenoid-mediated Vγ9Vδ2 activation and an antagonist antibody (103.2) that inhibits this reactivity. We find that the three BTN3A isoforms: BTN3A1, BTN3A2, and BTN3A3, have high structural homology to the B7 superfamily of proteins and exist as V-shaped homodimers in solution, associating through the membrane proximal C-type Ig domain. The 20.1 and 103.2 antibodies bind to separate epitopes on the BTN3A Ig-V domain with high affinity but likely with different valencies based on their binding orientation. These structures directly complement functional studies of this system that demonstrate that BTN3A1 is necessary for Vγ9Vδ2 activation and begin to unravel the extracellular events that occur during stimulation through the Vγ9Vδ2 T cell receptor.     

Structural and Functional Properties of Exopolysaccharide Excreted by a Novel <Emphasis Type="Italic">Bacillus anthracis</Emphasis> (Strain PFAB2) of Hot Spring Origin

Exopolysaccharide produced by a unique avirulent Bacillus anthracis strain PFAB2 of hot spring origin has been characterized and its functional properties are investigated which is a first report. Maximum yield of EPS is 7.66 g/l with 2% glucose and 1% peptone as optimum carbon and nitrogen source respectively. The EPS is found to be a homopolymer consisting of only glucose as principle monosaccharide component. Through ¹H NMR study, different dextran-like proton peaks are observed. Molecular weight of the EPS resembles low molecular weight bacterial origin polysaccharides. Melting transition of the EPS has started after 276 °C which indicates good thermal stability. The EPS also shows potent antioxidant activity in terms of DPPH and ABTS mediated free radical scavenging property compared to standard ascorbic acid. Emulsifying property of the EPS is also observed and has shown good emulsification of vegetable oils. The polysaccharide forms a thermo resistant gel during the heating phase, with G′ higher than G″ indicating excellent shear-thinning behaviour and viscoelastic nature of the EPS.     

Decreasing mortality and hospitalizations with rising costs related to gastric cancer in the USA: an epidemiological perspective

Background

There is no convincing data on the trends of hospitalizations, mortality, cost, and demographic variations associated with inpatient admissions for gastric cancer in the USA. The aim of this study was to use a national database of US hospitals to evaluate the trends associated with gastric cancer.

Methods

We analyzed the National Inpatient Sample (NIS) database for all patients in whom gastric cancer (ICD-9 code: 151.0, 151.1, 151.2, 151.3, 151.4, 151.5, 151.6, 151.8, 151.9) was the principal discharge diagnosis during the period, 2003–2014. The NIS is the largest publicly available all-payer inpatient care database in the US. It contains data from approximately eight million hospital stays each year. The statistical significance of the difference in the number of hospital discharges, length of stay, and hospital costs over the study period was determined by regression analysis.

Results

In 2003, there were 23,921 admissions with a principal discharge diagnosis of gastric cancer as compared to 21,540 in 2014 (P?<?0.01). The mean length of stay for gastric cancer decreased by 17% between 2003 and 2014 from 10.9?days to 8.95?days (P?<?0.01). However, during this period, the mean hospital charges increased significantly by 21% from $ 75,341 per patient in 2003 to $ 91,385 per patient in 2014 (P?<?0.001). There was a more significant reduction in mortality over a period of 11?years from 2428 (10.15%) in 2003 to 1345 (6.24%) in 2014 (P?<?0.01). The aggregate charges (i.e., “national bill”) for gastric cancer increased significantly from 1.79 bn $ to 1. 96 bn $ (P?<?0.001), despite decrease in hospitalization (inflation adjusted).

Conclusion

Although the number of inpatient admissions for gastric cancer have decreased over the past decade, the healthcare burden and cost related to it has increased significantly. Inpatient mortality is decreasing which is consistent with overall decrease in gastric cancer-related deaths. Cost increase associated with gastric cancer contributed significantly to the national healthcare bill.

     

Effect of lithium on hepatic drug-metabolizing enzymes of protein-deficient rats

Protein deficiency was produced by feeding synthetic 8%-protein diet. Lithium carbonate at the dose level of 1.1g/kg diet was administered to normal and protein-deficient rats for a period of one mo. A significant inhibition in the levels of cytochrome (cyt) P₄₅₀, cyt b₅, glutathione (GSH), glutathione S-transferase (GST) and glutathione peroxidase (GPx), but an increase in γ-glutamyl transpeptidase (γ-GT), was observed in low-protein LP-fed rats. Lithium treatment to normal rats caused no significant change in the activities of cyt P₄₅₀, cyt b₅, GST, and GSH levels, whereas there was elevation in the activities of γ-GT and GPx and suppression in glutathione reductase (GRd) activity. Lithium administration to LP-fed rats resulted in significant increases in the hepatic γ-GT and GPx activities.