The Ebola Virus matrix protein, VP40, requires phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) for extensive oligomerization at the plasma membrane and viral egress

VP40 is one of eight proteins encoded by the Ebola Virus (EBOV) and serves as the primary matrix protein, forming virus like particles (VLPs) from mammalian cells without the need for other EBOV proteins. While VP40 is required for viral assembly and budding from host cells during infection, the mechanisms that target VP40 to the plasma membrane are not well understood. Phosphatidylserine is required for VP40 plasma membrane binding, VP40 hexamer formation, and VLP egress, However, PS also becomes exposed on the outer membrane leaflet at sites of VP40 budding, raising the question of how VP40 maintains an interaction with the plasma membrane inner leaflet when PS is flipped to the opposite side. To address this question, cellular and in vitro assays were employed to determine if phosphoinositides are important for efficient VP40 localization to the plasma membrane. Cellular studies demonstrated that PI(4,5)P2 was an important component of VP40 assembly at the plasma membrane and subsequent virus like particle formation. Additionally, PI(4,5)P2 was required for formation of extensive oligomers of VP40, suggesting PS and PI(4,5)P2 have different roles in VP40 assembly where PS regulates formation of hexamers from VP40 dimers and PI(4,5)P2 stabilizes and/or induces extensive VP40 oligomerization at the plasma membrane

Associations between residual feed intake and apparent nutrient digestibility, in vitro methane-producing activity, and volatile fatty acid concentrations in growing beef cattle

The objectives of this study were to examine the relationship between residual feed intake (RFI) and DM and nutrient digestibility, in vitro methane production, and volatile fatty acid (VFA) concentrations in growing beef cattle. Residual feed intake was measured in growing Santa Gertrudis steers (Study 1; n = 57; initial BW = 291.1 ± 33.8 kg) and Brangus heifers (Study 2; n = 468; initial BW = 271.4 ± 26.1 kg) fed a high-roughage-based diet (ME = 2.1 Mcal/kg DM) for 70 d in a Calan-gate feeding barn. Animals were ranked by RFI based on performance and feed intake measured from day 0 to 70 (Study 1) or day 56 (Study 2) of the trial, and 20 animals with the lowest and highest RFI were identified for subsequent collections of fecal and feed refusal samples for DM and nutrient digestibility analysis. In Study 2, rumen fluid and feces were collected for in vitro methane-producing activity (MPA) and VFA analysis in trials 2, 3, and 4. Residual feed intake classification did not affect BW or BW gain (P \u3e 0.05), but low-RFI steers and heifers both consumed 19% less (P \u3c 0.01) DMI compared with high-RFI animals. Steers with low RFI tended (P \u3c 0.1) to have higher DM digestibility (DMD) compared with high-RFI steers (70.3 vs. 66.5 ± 1.6% DM). Heifers with low RFI had 4% higher DMD (76.3 vs. 73.3 ± 1.0% DM) and 4 to 5% higher (P \u3c 0.01) CP, NDF, and ADF digestibility compared with heifers with high RFI. Low-RFI heifers emitted 14% less (P \u3c 0.01) methane (% GE intake; GEI) calculated according to Blaxter and Clapperton (1965) as modified by Wilkerson et al. (1995), and tended (P = 0.09) to have a higher rumen acetate:propionate ratio than heifers with high RFI (GEI = 5.58 vs. 6.51 ± 0.08%; A:P ratio = 5.02 vs. 4.82 ± 0.14%). Stepwise regression analysis revealed that apparent nutrient digestibilities (DMD and NDF digestibility) for Study 1 and Study 2 accounted for an additional 8 and 6%, respectively, of the variation in intake unaccounted for by ADG and mid-test BW0.75. When DMD, NDF digestibility, and total ruminal VFA were added to the base model for Study 2, trials 2, 3, and 4, the R2 increased from 0.33 to 0.47, explaining an additional 15% of the variation in DMI unrelated to growth and body size. On the basis of the results of these studies, differences in observed phenotypic RFI in growing beef animals may be a result of inter-animal variation in apparent nutrient digestibility and ruminal VFA concentrations

Prions Adhere to Soil Minerals and Remain Infectious

An unidentified environmental reservoir of infectivity contributes to the natural transmission of prion diseases (transmissible spongiform encephalopathies [TSEs]) in sheep, deer, and elk. Prion infectivity may enter soil environments via shedding from diseased animals and decomposition of infected carcasses. Burial of TSE-infected cattle, sheep, and deer as a means of disposal has resulted in unintentional introduction of prions into subsurface environments. We examined the potential for soil to serve as a TSE reservoir by studying the interaction of the disease-associated prion protein (PrP(Sc)) with common soil minerals. In this study, we demonstrated substantial PrP(Sc) adsorption to two clay minerals, quartz, and four whole soil samples. We quantified the PrP(Sc)-binding capacities of each mineral. Furthermore, we observed that PrP(Sc) desorbed from montmorillonite clay was cleaved at an N-terminal site and the interaction between PrP(Sc) and Mte was strong, making desorption of the protein difficult. Despite cleavage and avid binding, PrP(Sc) bound to Mte remained infectious. Results from our study suggest that PrP(Sc) released into soil environments may be preserved in a bioavailable form, perpetuating prion disease epizootics and exposing other species to the infectious agent

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

Germline genetic variants in men with prostate cancer and one or more additional cancers

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/138930/1/cncr30817.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/138930/2/cncr30817_am.pd

Cytonuclear discordance in the Florida Everglades invasive Burmese python (Python bivittatus) population reveals possible hybridization with the Indian python (P. molurus)

The invasive Burmese python (Python bivittatus) has been reproducing in the Florida Everglades since the 1980s. These giant constrictor snakes have caused a precipitous decline in small mammal populations in southern Florida following escapes or releases from the commercial pet trade. To better understand the invasion pathway and genetic composition of the population, two mitochondrial (mtDNA) loci across 1,398 base pairs were sequenced on 426 snakes and 22 microsatellites were assessed on 389 snakes. Concatenated mtDNA sequences produced six haplotypes with an average nucleotide and haplotype diversity of π = 0.002 and h = 0.097, respectively. Samples collected in Florida from morphologically identified P. bivittatus snakes were similar to published cytochrome oxidase 1 and cytochrome b sequences from both P. bivittatus and Python molurus and were highly divergent (genetic distances of 5.4% and 4.3%, respectively). The average number of microsatellite alleles and expected heterozygosity were NA = 5.50 and HE = 0.60, respectively. Nuclear Bayesian assignment tests supported two genetically distinct groups and an admixed group, not geographically differentiated. The effective population size (NE = 315.1) was lower than expected for a population this large, but reflected the low genetic diversity overall. The patterns of genetic diversity between mtDNA and microsatellites were disparate, indicating nuclear introgression of separate mtDNA lineages corresponding to cytonuclear discordance. The introgression likely occurred prior to the invasion, but genetic information on the native range and commercial trade is needed for verification. Our finding that the Florida python population is comprised of distinct lineages suggests greater standing variation for adaptation and the potential for broader areas of suitable habitat in the invaded range

Cellular Target Deconvolution of Small Molecules Using a Selection-Based Genetic Screening Platform

Small-molecule drug target identification is an essential and often rate-limiting step in phenotypic drug discovery and remains a major challenge. Here, we report a novel platform for target identification of activators of signaling pathways by leveraging the power of a clustered regularly interspaced short palindromic repeats (CRISPR) knockout library. This platform links the expression of a suicide gene to the small-molecule-activated signaling pathway to create a selection system. With this system, loss-of-function screening using a CRISPR single-guide (sg) RNA library positively enriches cells in which the target has been knocked out. The identities of the drug targets and other essential genes required for the activity of small molecules of interest are then uncovered by sequencing. We tested this platform on BDW568, a newly discovered type-I interferon signaling activator, and identified stimulator of interferon genes (STING) as its target and carboxylesterase 1 (CES1) to be a key metabolizing enzyme required to activate BDW568 for target engagement. The platform we present here can be a general method applicable for target identification for a wide range of small molecules that activate different signaling pathways

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

A bacterial quorum-sensing precursor induces mortality in the marine coccolithophore, Emiliania huxleyi

© The Author(s), 2016. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Frontiers in Microbiology 7 (2016): 59, doi:10.3389/fmicb.2016.00059.Interactions between phytoplankton and bacteria play a central role in mediating biogeochemical cycling and food web structure in the ocean. However, deciphering the chemical drivers of these interspecies interactions remains challenging. Here, we report the isolation of 2-heptyl-4-quinolone (HHQ), released by Pseudoalteromonas piscicida, a marine gamma-proteobacteria previously reported to induce phytoplankton mortality through a hitherto unknown algicidal mechanism. HHQ functions as both an antibiotic and a bacterial signaling molecule in cell–cell communication in clinical infection models. Co-culture of the bloom-forming coccolithophore, Emiliania huxleyi with both live P. piscicida and cell-free filtrates caused a significant decrease in algal growth. Investigations of the P. piscicida exometabolome revealed HHQ, at nanomolar concentrations, induced mortality in three strains of E. huxleyi. Mortality of E. huxleyi in response to HHQ occurred slowly, implying static growth rather than a singular loss event (e.g., rapid cell lysis). In contrast, the marine chlorophyte, Dunaliella tertiolecta and diatom, Phaeodactylum tricornutum were unaffected by HHQ exposures. These results suggest that HHQ mediates the type of inter-domain interactions that cause shifts in phytoplankton population dynamics. These chemically mediated interactions, and other like it, ultimately influence large-scale oceanographic processes.This research was support through funding from the Gordon and Betty Moore Foundation through Grant GBMF3301 to MJ and TM; NIH grant from the National Institute of Allergy and Infectious Disease (NIAID – 1R21Al119311-01) to TM and KW; the National Science Foundation (OCE – 1313747) and US National Institute of Environmental Health Science (P01-ES021921) through the Oceans and Human Health Program to BM. Additional financial support was provided to TM from the Flatley Discovery Lab