Plasma membrane association facilitates conformational changes in the Marburg virus protein VP40 dimer

Filovirus infections cause hemorrhagic fever in humans and non-human primates that often results in high fatality rates. The Marburg virus is a lipid-enveloped virus from the Filoviridae family and is closely related to the Ebola virus. The viral matrix layer underneath the lipid envelope is formed by the matrix protein VP40 (VP40), which is also involved in other functions during the viral life-cycle. As in the Ebola virus VP40 (eVP40), the recently determined X-ray crystal structure of the Marburg virus VP40 (mVP40) features loops containing cationic residues that form a lipid binding basic patch. However, the mVP40 basic patch is significantly flatter with a more extended surface than in eVP40, suggesting the possibility of differences in the plasma membrane interactions and phospholipid specificity between the VP40 dimers. In this paper, we report on molecular dynamics simulations that investigate the roles of various residues and lipid types in PM association as well as the conformational changes of the mVP40 dimer facilitated by membrane association. We compared the structural changes of the mVP40 dimer with the mVP40 dimer in both lipid free and membrane associated conditions. Despite the significant structural differences in the crystal structure, the Marburg VP40 dimer is found to adopt a configuration very similar to the Ebola VP40 dimer after associating with the membrane. This conformational rearrangement upon lipid binding allows Marburg VP40 to localize and stabilize at the membrane surface in a manner similar to the Ebola VP40 dimer. Consideration of the structural information in its lipid-interacting condition may be important in targeting mVP40 for novel drugs to inhibit viral budding from the plasma membrane

Graphene-VP40 interactions and potential disruption of the Ebola virus matrix filaments

Ebola virus infections cause hemorrhagic fever that often results in very high fatality rates. In addition to exploring vaccines, development of drugs is also essential for treating the disease and preventing the spread of the infection. The Ebola virus matrix protein VP40 exists in various conformational and oligomeric forms and is a potential pharmacological target for disrupting the virus life-cycle. Here we explored graphene-VP40 interactions using molecular dynamics simulations and graphene pelleting assays. We found that graphene sheets associate strongly with VP40 at various interfaces. We also found that the graphene is able to disrupt the C-terminal domain (CTD-CTD) interface of VP40 hexamers. This VP40 hexamer-hexamer interface is crucial in forming the Ebola viral matrix and disruption of this interface may provide a method to use graphene or similar nanoparticle based solutions as a disinfectant that can significantly reduce the spread of the disease and prevent an Ebola epidemic

Detection of lipid-induced structural changes of the Marburg virus matrix protein VP40 using hydrogen/deuterium exchange-mass spectrometry

Marburg virus (MARV) is a lipid-enveloped virus from the Filoviridae family containing a negative sense RNA genome. One of the seven MARV genes encodes the matrix protein VP40, which forms a matrix layer beneath the plasma membrane inner leaflet to facilitate budding from the host cell. MARV VP40 (mVP40) has been shown to be a dimeric peripheral protein with a broad and flat basic surface that can associate with anionic phospholipids such as phosphatidylserine. Although a number of mVP40 cationic residues have been shown to facilitate binding to membranes containing anionic lipids, much less is known on how mVP40 assembles to form the matrix layer following membrane binding. Here we have used hydrogen/deuterium exchange (HDX) mass spectrometry to determine the solvent accessibility of mVP40 residues in the absence and presence of phosphatidylserine and phosphatidylinositol 4,5-bisphosphate. HDX analysis demonstrates that two basic loops in the mVP40 C-terminal domain make important contributions to anionic membrane binding and also reveals a potential oligomerization interface in the C-terminal domain as well as a conserved oligomerization interface in the mVP40 N-terminal domain. Lipid binding assays confirm the role of the two basic patches elucidated with HD/X measurements, whereas molecular dynamics simulations and membrane insertion measurements complement these studies to demonstrate that mVP40 does not appreciably insert into the hydrocarbon region of anionic membranes in contrast to the matrix protein from Ebola virus. Taken together, we propose a model by which association of the mVP40 dimer with the anionic plasma membrane facilitates assembly of mVP40 oligomers

Ceftazidime-avibactam has potent sterilizing activity against highly drug-resistant tuberculosis.

There are currently many patients with multidrug-resistant and extensively drug-resistant tuberculosis. Ongoing transmission of the highly drug-resistant strains and high mortality despite treatment remain problematic. The current strategy of drug discovery and development takes up to a decade to bring a new drug to clinical use. We embarked on a strategy to screen all antibiotics in current use and examined them for use in tuberculosis. We found that ceftazidime-avibactam, which is already used in the clinic for multidrug-resistant Gram-negative bacillary infections, markedly killed rapidly growing, intracellular, and semidormant Mycobacterium tuberculosis in the hollow fiber system model. Moreover, multidrug-resistant and extensively drug-resistant clinical isolates demonstrated good ceftazidime-avibactam susceptibility profiles and were inhibited by clinically achievable concentrations. Resistance arose because of mutations in the transpeptidase domain of the penicillin-binding protein PonA1, suggesting that the drug kills M. tuberculosis bacilli via interference with cell wall remodeling. We identified concentrations (exposure targets) for optimal effect in tuberculosis, which we used with susceptibility results in computer-aided clinical trial simulations to identify doses for immediate clinical use as salvage therapy for adults and young children. Moreover, this work provides a roadmap for efficient and timely evaluation of antibiotics and optimization of clinically relevant dosing regimens

Cysteine mutations in the ebolavirus matrix protein VP40 promote phosphatidylserine binding by increasing the flexibility of a lipid-binding loop

Ebolavirus (EBOV) is a negative-sense RNA virus that causes severe hemorrhagic fever in humans. The matrix protein VP40 facilitates viral budding by binding to lipids in the host cell plasma membrane and driving the formation of filamentous, pleomorphic virus particles. The C-terminal domain of VP40 contains two highly-conserved cysteine residues at positions 311 and 314, but their role in the viral life cycle is unknown. We therefore investigated the properties of VP40 mutants in which the conserved cysteine residues were replaced with alanine. The C311A mutation significantly increased the affinity of VP40 for membranes containing phosphatidylserine (PS), resulting in the assembly of longer virus-like particles (VLPs) compared to wild-type VP40. The C314A mutation also increased the affinity of VP40 for membranes containing PS, albeit to a lesser degree than C311A. The double mutant behaved in a similar manner to the individual mutants. Computer modeling revealed that both cysteine residues restrain a loop segment containing lysine residues that interact with the plasma membrane, but Cys311 has the dominant role. Accordingly, the C311A mutation increases the flexibility of this membrane-binding loop, changes the profile of hydrogen bonding within VP40 and therefore binds to PS with greater affinity. This is the first evidence that mutations in VP40 can increase its affinity for biological membranes and modify the length of Ebola VLPs. The Cys311 and Cys314 residues therefore play an important role in dynamic interactions at the plasma membrane by modulating the ability of VP40 to bind PS

Adapting Livestock Production Systems to Climate Change in Nepal: Challenges and Opportunities

To assess climate change impacts and identify challenges and opportunities for livestock climate change adaption, we conducted a comprehensive study in the Thulokhola watershed of Nuwakot district in Nepal from June 1, 2011 to January 31, 2013. We established nine community livestock groups (CLGs) consisting of 51 members and trained the CLG members in daily livestock record keeping and monitoring surface water quality. Monthly fecal samples from 50 cattle, 50 goats, and 50 buffaloes were collected for the determination of gastrointestinal parasites. Soil and fodder samples were also collected and analyzed. Group discussions, Participatory Rural Appraisals, and full-fledged household survey of 97 households were done. A survey of 41 water sources in the watershed was also conducted. While 85.3 % of the water sources have either dried up or decreased in flow in recent years, drought conditions had great toll on agricultural production. Prevalence rates of helminthes on goats, cattle, and buffalo was 53.8%, 31.32%, and 23.52%, respectively, and animal deaths were remarkably high. Declining pregnancy rates on livestock along with waning supply of fodder and forages and poor soil quality were additional major problems. Although local communities have undertaken several measures including adding new breed, destocking, purchasing fodder and forages, and planting grasses for livestock climate change adaptation, the problems of animal health, breeding conditions, soil fertility, forest degradation, increasing women workload, and water shortages were largely unaddressed. Opportunities for livestock climate change adaptation in Nepal include agroforestry intervention, groundwater utilization, rainwater harvesting, enhancing feed efficiency, and community capacity-building

A qualitative study of enablers and barriers influencing the incorporation of social accountability values into organisational culture: a perspective from two medical schools

Background: Definitions of social accountability describe the obligation of medical schools to direct education, research and service activities towards addressing the priority health concerns of the population they serve. While such statements give some direction as to how the goal might be reached, it does not identify what factors might facilitate or hinder its achievement. This study set out to identify and explore enablers and barriers influencing the incorporation of social accountability values into medical schools. Methods: Semi structured interviews of fourteen senior staff in Bar Ilan and Leeds medical schools were undertaken following a literature review. Participants were recruited by purposive sampling in order to identify factors perceived to play a part in the workings of each institution. Results: Academic prestige was seen as a key barrier that was dependent on research priorities and student selection. The role of champions was considered to be vital to tackle staff perceptions and facilitate progress. Including practical community experience for students was felt to be a relevant way in which the curriculum could be designed through engagement with local partners. Conclusions: Successful adoption of social accountability values requires addressing concerns around potential negative impacts on academic prestige and standards. Identifying and supporting credible social accountability champions to disseminate the values throughout research and education departments in medical and other faculties is also necessary, including mapping onto existing work streams and research agendas. Demonstrating the contribution the institution can make to local health improvement and regional development by a consideration of its economic footprint may also be valuable

Analysis of Risk Factors for Incisional Hernias and its Management

Introduction: Incisional hernia is a common problem after abdominal surgery. Patients present with pain, swelling and intestinal obstruction. It may be repaired by either anatomical suturing or mesh repair. Methods: It is a prospective observational study conducted in Western Regional Hospital and Fewa City Hospital, Pokhara from 2013 to 2016. A total of consecutive 100 patients admitted in these hospitals during the study period were included. Results: Incisional hernia is more common in females (M : F = 1 : 3.8), and in 30 - 50 years age group (60%). Major risk factors were wound infection (30%), overweight (25%), and postoperative cough (10%). It is found to be more associated with gynecological (65%), than gastrointestinal operations, and more so with lower abdominal midline incision (65%). It is found to occur mostly within one year (60%) of primary surgery than later. Even 24% of the patients had first symptom within six months. Mesh repair (92%) was the preferred standard surgical treatment for incisional hernia. Conclusion: Overweight females of age range between 30 - 50 years with history of gynecological operations by lower abdominal midline incision are more prone to develop incisional hernia. This incidence increases when there is wound infection. Mesh repair is the choice of operation for incisional hernia. J-GMC-N | Volume 11 | Issue 01 | January-June 2018, Page: 16-2