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

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

Peer-to-Peer energy trading in micro/mini-grids for local energy communities: A review and case study of Nepal

Distributed Energy Resources (DERs) are being integrated into the power market by customers rather than large scale energy suppliers, thereby slowly transforming the centralized, unidirectional market to a decentralized, bidirectional market and transitioning customers into prosumers. Various system architectures are used in the real field to coordinate the energy distribution in the micro/ mini-grids integrated with DERs, all of which have their strengths, weaknesses and challenges. Peer-to-peer (P2P) is an emerging architecture in the field of electrical energy trading and Distributed Generation (DG) management that can be applied in local energy markets. This paper focuses on P2P energy trading, with an in-depth discussion on its various operating algorithms, their principles, characteristics, features and scope through state of art review on P2P. Furthermore, the energy system of Nepal is used as a case study in this paper, and the micro/mini-grids of Nepal and their associated challenges, constraints and opportunities for improvement are discussed. Finally, an energy trading model is proposed to address the problems occurring in the specific case of Nepalese energy market

Micro Health Project

Community health diagnosis is a comprehensive assessment of health status of the community in relation to its social, physical and biological environment. The purpose of community health diagnosis is to define existing problems, determine available resources and set priorities for planning, implementing and evaluating health action, by and for the community. The community health diagnosis program began on 4th September 2015 and continued till 13th September 2015 in ward no 1 and 5 Rupakot VDC, Kaski, Nepal. The program was organized in following phases: data collection, data analysis, first community presentation, prioritization of need and planning of micro health project (MHP), implementation and evaluation of MHP, and final community presentation. On the basis of the observed and the felt needs of the community, we found the real needs and prioritized them as follows. For community: Proper water purification, information about common diseases, KAP on diseases, knowledge on TB and DOTS. For school-going children: Education on environmental sanitation, education on personal hygiene - teeth brushing and hand washing, adolescent health education. We launched micro health project (MHP) on these topics, conducting school-based as well as community-based programs.  Journal of Gandaki Medical College Vol. 10, No. 1, 2017, Page: 59-6

Role of Alfuzosin in Ureteral Stent Related Urinary Symptoms Score: A Comparative Study

Background: Ureteral stent placement is a routine urological procedure. However, patients inserted with ureteral stent exhibited increased urinary symptoms that compromise patients’ quality of life. Objective: To assess the efficacy of alpha blockers (Alfuzosin) in the management of ureteral stent related urinary symptoms. Methods: Total of 60 patients after ureteral stent insertion was randomly divided into two equal groups; 30 in alfuzosin group and the remaining 30 in control group. Urinary symptoms questionnaire was filled after two weeks and results were statistically analyzed. Results: Urinary symptoms like urgency, frequency and flank pain were significantly less in the alfuzosin group when compared with control group. Conclusion: Alpha blocker (Alfuzosin) was found to be effective in reducing ureteral stent related urinary symptoms. Journal of Gandaki Medical College Vol. 10, No. 1, 2017, page: 28-3

Cognitive dysfunction in naturally occurring canine idiopathic epilepsy

Globally, epilepsy is a common serious brain disorder. In addition to seizure activity, epilepsy is associated with cognitive impairments including static cognitive impairments present at onset, progressive seizure-induced impairments and co-morbid dementia. Epilepsy occurs naturally in domestic dogs but its impact on canine cognition has yet to be studied, despite canine cognitive dysfunction (CCD) recognised as a spontaneous model of dementia. Here we use data from a psychometrically validated tool, the canine cognitive dysfunction rating (CCDR) scale, to compare cognitive dysfunction in dogs diagnosed with idiopathic epilepsy (IE) with controls while accounting for age. An online cross-sectional study resulted in a sample of 4051 dogs, of which n = 286 had been diagnosed with IE. Four factors were significantly associated with a diagnosis of CCD (above the diagnostic cut-off of CCDR ≥50): (i) epilepsy diagnosis: dogs with epilepsy were at higher risk; (ii) age: older dogs were at higher risk; (iii) weight: lighter dogs (kg) were at higher risk; (iv) training history: dogs with more exposure to training activities were at lower risk. Impairments in memory were most common in dogs with IE, but progression of impairments was not observed compared to controls. A significant interaction between epilepsy and age was identified, with IE dogs exhibiting a higher risk of CCD at a young age, while control dogs followed the expected pattern of low-risk throughout middle age, with risk increasing exponentially in geriatric years. Within the IE sub-population, dogs with a history of cluster seizures and high seizure frequency had higher CCDR scores. The age of onset, nature and progression of cognitive impairment in the current IE dogs appear divergent from those classically seen in CCD. Longitudinal monitoring of cognitive function from seizure onset is required to further characterise these impairments