Characterization of Shewanella oneidensis MtrC: a cell-surface decaheme cytochrome involved in respiratory electron transport to extracellular electron acceptors

MtrC is a decaheme c-type cytochrome associated with the outer cell membrane of Fe(III)-respiring species of the Shewanella genus. It is proposed to play a role in anaerobic respiration by mediating electron transfer to extracellular mineral oxides that can serve as terminal electron acceptors. The present work presents the first spectropotentiometric and voltammetric characterization of MtrC, using protein purified from Shewanella oneidensis MR-1. Potentiometric titrations, monitored by UV–vis absorption and electron paramagnetic resonance (EPR) spectroscopy, reveal that the hemes within MtrC titrate over a broad potential range spanning between approximately +100 and approximately -500 mV (vs. the standard hydrogen electrode). Across this potential window the UV–vis absorption spectra are characteristic of low-spin c-type hemes and the EPR spectra reveal broad, complex features that suggest the presence of magnetically spin-coupled low-spin c-hemes. Non-catalytic protein film voltammetry of MtrC demonstrates reversible electrochemistry over a potential window similar to that disclosed spectroscopically. The voltammetry also allows definition of kinetic properties of MtrC in direct electron exchange with a solid electrode surface and during reduction of a model Fe(III) substrate. Taken together, the data provide quantitative information on the potential domain in which MtrC can operate

Gut evacuation rate and grazing impact of the krill Thysanoessa raschii and T. inermis

Gut evacuation rates and ingestion rates were measured for the krill Thysanoessa raschii and T. inermis in Godthåbsfjord, SW Greenland. Combined with biomass of the krill community, the grazing potential on phytoplankton along the fjord was estimated. Gut evacuation rates were 3.9 and 2.3 h−1 for T. raschii and T. inermis, respectively. Ingestion rates were 12.2 ± 7.5 µg C mg C−1 day−1 (n = 4) for T. inermis and 4.9 ± 3.2 µg C mg C−1 day−1 (n = 4) for T. raschii, corresponding to daily rations of 1.2 and 0.5 % body carbon day−1. Clearance experiments conducted in parallel to the gut evacuation experiment gave similar results for ingestion rates and daily rations. Krill biomass was highest in the central part of the fjord’s length, with T. raschii dominating. Community grazing rates from krill and copepods were comparable; however, their combined impact was low, estimated as <1 % of phytoplankton standing stock being removed per day during this late spring study

Utilisation of an operative difficulty grading scale for laparoscopic cholecystectomy

Background A reliable system for grading operative difficulty of laparoscopic cholecystectomy would standardise description of findings and reporting of outcomes. The aim of this study was to validate a difficulty grading system (Nassar scale), testing its applicability and consistency in two large prospective datasets. Methods Patient and disease-related variables and 30-day outcomes were identified in two prospective cholecystectomy databases: the multi-centre prospective cohort of 8820 patients from the recent CholeS Study and the single-surgeon series containing 4089 patients. Operative data and patient outcomes were correlated with Nassar operative difficultly scale, using Kendall’s tau for dichotomous variables, or Jonckheere–Terpstra tests for continuous variables. A ROC curve analysis was performed, to quantify the predictive accuracy of the scale for each outcome, with continuous outcomes dichotomised, prior to analysis. Results A higher operative difficulty grade was consistently associated with worse outcomes for the patients in both the reference and CholeS cohorts. The median length of stay increased from 0 to 4 days, and the 30-day complication rate from 7.6 to 24.4% as the difficulty grade increased from 1 to 4/5 (both p < 0.001). In the CholeS cohort, a higher difficulty grade was found to be most strongly associated with conversion to open and 30-day mortality (AUROC = 0.903, 0.822, respectively). On multivariable analysis, the Nassar operative difficultly scale was found to be a significant independent predictor of operative duration, conversion to open surgery, 30-day complications and 30-day reintervention (all p < 0.001). Conclusion We have shown that an operative difficulty scale can standardise the description of operative findings by multiple grades of surgeons to facilitate audit, training assessment and research. It provides a tool for reporting operative findings, disease severity and technical difficulty and can be utilised in future research to reliably compare outcomes according to case mix and intra-operative difficulty

Modulation of the surface proteome through multiple ubiquitylation pathways in African Trypanosomes

Recently we identified multiple suramin-sensitivity genes with a genome wide screen in Trypanosoma brucei that includes the invariant surface glycoprotein ISG75, the adaptin-1 (AP-1) complex and two deubiquitylating enzymes (DUBs) orthologous to ScUbp15/HsHAUSP1 and pVHL-interacting DUB1 (type I), designated TbUsp7 and TbVdu1, respectively. Here we have examined the roles of these genes in trafficking of ISG75, which appears key to suramin uptake. We found that, while AP-1 does not influence ISG75 abundance, knockdown of TbUsp7 or TbVdu1 leads to reduced ISG75 abundance. Silencing TbVdu1 also reduced ISG65 abundance. TbVdu1 is a component of an evolutionarily conserved ubiquitylation switch and responsible for rapid receptor modulation, suggesting similar regulation of ISGs in T. brucei. Unexpectedly, TbUsp7 knockdown also blocked endocytosis. To integrate these observations we analysed the impact of TbUsp7 and TbVdu1 knockdown on the global proteome using SILAC. For TbVdu1, ISG65 and ISG75 are the only significantly modulated proteins, but for TbUsp7 a cohort of integral membrane proteins, including the acid phosphatase MBAP1, that is required for endocytosis, and additional ISG-related proteins are down-regulated. Furthermore, we find increased expression of the ESAG6/7 transferrin receptor and ESAG5, likely resulting from decreased endocytic activity. Therefore, multiple ubiquitylation pathways, with a complex interplay with trafficking pathways, control surface proteome expression in trypanosomes

Differentiating Embryonic Stem Cells Pass through ‘Temporal Windows’ That Mark Responsiveness to Exogenous and Paracrine Mesendoderm Inducing Signals

BACKGROUND: Mesendoderm induction during embryonic stem cell (ESC) differentiation in vitro is stimulated by the Transforming Growth Factor and Wingless (Wnt) families of growth factors. PRINCIPAL FINDINGS: We identified the periods during which Bone Morphogenetic Protein (BMP) 4, Wnt3a or Activin A were able to induce expression of the mesendoderm marker, Mixl1, in differentiating mouse ESCs. BMP4 and Wnt3a were required between differentiation day (d) 1.5 and 3 to most effectively induce Mixl1, whilst Activin A induced Mixl1 expression in ESC when added between d2 and d4, indicating a subtle difference in the requirement for Activin receptor signalling in this process. Stimulation of ESCs with these factors at earlier or later times resulted in little Mixl1 induction, suggesting that the differentiating ESCs passed through 'temporal windows' in which they sequentially gained and lost competence to respond to each growth factor. Inhibition of either Activin or Wnt signalling blocked Mixl1 induction by any of the three mesendoderm-inducing factors. Mixing experiments in which chimeric EBs were formed between growth factor-treated and untreated ESCs revealed that BMP, Activin and Wnt signalling all contributed to the propagation of paracrine mesendoderm inducing signals between adjacent cells. Finally, we demonstrated that the differentiating cells passed through 'exit gates' after which point they were no longer dependent on signalling from inducing molecules for Mixl1 expression. CONCLUSIONS: These studies suggest that differentiating ESCs are directed by an interconnected network of growth factors similar to those present in early embryos and that the timing of growth factor activity is critical for mesendoderm induction

The conserved dileucine- and tyrosine-based motifs in MLV and MPMV envelope glycoproteins are both important to regulate a common Env intracellular trafficking

BACKGROUND: Retrovirus particles emerge from the assembly of two structural protein components, Gag that is translated as a soluble protein in the cytoplasm of the host cells, and Env, a type I transmembrane protein. Because both components are translated in different intracellular compartments, elucidating the mechanisms of retrovirus assembly thus requires the study of their intracellular trafficking. RESULTS: We used a CD25 (Tac) chimera-based approach to study the trafficking of Moloney murine leukemia virus and Mason-Pfizer monkey virus Env proteins. We found that the cytoplasmic tails (CTs) of both Env conserved two major signals that control a complex intracellular trafficking. A dileucine-based motif controls the sorting of the chimeras from the trans-Golgi network (TGN) toward endosomal compartments. Env proteins then follow a retrograde transport to the TGN due to the action of a tyrosine-based motif. Mutation of either motif induces the mis-localization of the chimeric proteins and both motifs are found to mediate interactions of the viral CTs with clathrin adaptors. CONCLUSION: This data reveals the unexpected complexity of the intracellular trafficking of retrovirus Env proteins that cycle between the TGN and endosomes. Given that Gag proteins hijack endosomal host proteins, our work suggests that the endosomal pathway may be used by retroviruses to ensure proper encountering of viral structural Gag and Env proteins in cells, an essential step of virus assembly

Effects of temperature on the transmission of Yersinia Pestis by the flea, Xenopsylla Cheopis, in the late phase period

<p>Abstract</p> <p>Background</p> <p>Traditionally, efficient flea-borne transmission of <it>Yersinia pestis</it>, the causative agent of plague, was thought to be dependent on a process referred to as blockage in which biofilm-mediated growth of the bacteria physically blocks the flea gut, leading to the regurgitation of contaminated blood into the host. This process was previously shown to be temperature-regulated, with blockage failing at temperatures approaching 30°C; however, the abilities of fleas to transmit infections at different temperatures had not been adequately assessed. We infected colony-reared fleas of <it>Xenopsylla cheopis </it>with a wild type strain of <it>Y. pestis </it>and maintained them at 10, 23, 27, or 30°C. Naïve mice were exposed to groups of infected fleas beginning on day 7 post-infection (p.i.), and every 3-4 days thereafter until day 14 p.i. for fleas held at 10°C, or 28 days p.i. for fleas held at 23-30°C. Transmission was confirmed using <it>Y. pestis</it>-specific antigen or antibody detection assays on mouse tissues.</p> <p>Results</p> <p>Although no statistically significant differences in per flea transmission efficiencies were detected between 23 and 30°C, efficiencies were highest for fleas maintained at 23°C and they began to decline at 27 and 30°C by day 21 p.i. These declines coincided with declining median bacterial loads in fleas at 27 and 30°C. Survival and feeding rates of fleas also varied by temperature to suggest fleas at 27 and 30°C would be less likely to sustain transmission than fleas maintained at 23°C. Fleas held at 10°C transmitted <it>Y. pestis </it>infections, although flea survival was significantly reduced compared to that of uninfected fleas at this temperature. Median bacterial loads were significantly higher at 10°C than at the other temperatures.</p> <p>Conclusions</p> <p>Our results suggest that temperature does not significantly effect the per flea efficiency of <it>Y. pestis </it>transmission by <it>X. cheopis</it>, but that temperature is likely to influence the dynamics of <it>Y. pestis </it>flea-borne transmission, perhaps by affecting persistence of the bacteria in the flea gut or by influencing flea survival. Whether <it>Y. pestis </it>biofilm production is important for transmission at different temperatures remains unresolved, although our results support the hypothesis that blockage is not necessary for efficient transmission.</p

Emergence and Genetic Variation of Neuraminidase Stalk Deletions in Avian Influenza Viruses

When avian influenza viruses (AIVs) are transmitted from their reservoir hosts (wild waterfowl and shorebirds) to domestic bird species, they undergo genetic changes that have been linked to higher virulence and broader host range. Common genetic AIV modifications in viral proteins of poultry isolates are deletions in the stalk region of the neuraminidase (NA) and additions of glycosylation sites on the hemagglutinin (HA). Even though these NA deletion mutations occur in several AIV subtypes, they have not been analyzed comprehensively. In this study, 4,920 NA nucleotide sequences, 5,596 HA nucleotide and 4,702 HA amino acid sequences were analyzed to elucidate the widespread emergence of NA stalk deletions in gallinaceous hosts, the genetic polymorphism of the deletion patterns and association between the stalk deletions in NA and amino acid variants in HA. Forty-seven different NA stalk deletion patterns were identified in six NA subtypes, N1–N3 and N5–N7. An analysis that controlled for phylogenetic dependence due to shared ancestry showed that NA stalk deletions are statistically correlated with gallinaceous hosts and certain amino acid features on the HA protein. Those HA features included five glycosylation sites, one insertion and one deletion. The correlations between NA stalk deletions and HA features are HA-NA-subtype-specific. Our results demonstrate that stalk deletions in the NA proteins of AIV are relatively common. Understanding the NA stalk deletion and related HA features may be important for vaccine and drug development and could be useful in establishing effective early detection and warning systems for the poultry industry