The nuclear egress complex of Epstein-Barr virus buds membranes through an oligomerization-driven mechanism.

During replication, herpesviral capsids are translocated from the nucleus into the cytoplasm by an unusual mechanism, termed nuclear egress, that involves capsid budding at the inner nuclear membrane. This process is mediated by the viral nuclear egress complex (NEC) that deforms the membrane around the capsid. Although the NEC is essential for capsid nuclear egress across all three subfamilies of the Herpesviridae, most studies to date have focused on the NEC homologs from alpha- and beta- but not gammaherpesviruses. Here, we report the crystal structure of the NEC from Epstein-Barr virus (EBV), a prototypical gammaherpesvirus. The structure resembles known structures of NEC homologs yet is conformationally dynamic. We also show that purified, recombinant EBV NEC buds synthetic membranes in vitro and forms membrane-bound coats of unknown geometry. However, unlike other NEC homologs, EBV NEC forms dimers in the crystals instead of hexamers. The dimeric interfaces observed in the EBV NEC crystals are similar to the hexameric interfaces observed in other NEC homologs. Moreover, mutations engineered to disrupt the dimeric interface reduce budding. Putting together these data, we propose that EBV NEC-mediated budding is driven by oligomerization into membrane-bound coats.

Nuclear Egress.

During viral replication, herpesviruses utilize a unique strategy, termed nuclear egress, to translocate capsids from the nucleus into the cytoplasm. This initial budding step transfers a newly formed capsid from within the nucleus, too large to fit through nuclear pores, through the inner nuclear membrane to the perinuclear space. The perinuclear enveloped virion must then fuse with the outer nuclear membrane to be released into the cytoplasm for further maturation, undergoing budding once again at the trans-Golgi network or early endosomes, and ultimately exit the cell non-lytically to spread infection. This first budding process is mediated by two conserved viral proteins, UL31 and UL34, that form a heterodimer called the nuclear egress complex (NEC). This review focuses on what we know about how the NEC mediates capsid transport to the perinuclear space, including steps prior to and after this budding event. Additionally, we discuss the involvement of other viral proteins in this process and how NEC-mediated budding may be regulated during infection.

A novel glucuronoxylan hydrolase produced by fermentation is safe as feed additive: toxicology and tolerance in broiler chickens.

The current study presents a safety evaluation of a novel glucuronoxylan hydrolase (EC 3.2.1.136) from Bacillus subtilis produced in Bacillus licheniformis. The glucuronoxylan hydrolase preparation did not exhibit irritative potential to the eye and skin when applied in in vitro models. The glucuronoxylan hydrolase preparation was non-mutagenic and non-clastogenic in in vitro tests. Oral administration of the glucuronoxylan hydrolase preparation to rats did not cause any adverse effect in a 90-days subchronic toxicity study. A tolerance study was performed with broiler chickens and confirmed that this glucuronoxylan hydrolase is safe for broiler chickens when fed at the maximum recommended dose, as well as at the 10 times higher dose. In conclusion, there are no safety concerns with using this novel glucuronoxylan hydrolase as a feed additive as it is toxicologically inert and the glucuronoxylan hydrolase is well tolerated by broiler chickens. The beneficial safety evaluation of glucuronoxylan hydrolase is consistent with the fact that this type of enzyme is ubiquitous in nature.

Metabolic footprinting for investigation of antifungal properties of Lactobacillus paracasei.

Lactic acid bacteria with antifungal properties are applied for biopreservation of food. In order to further our understanding of their antifungal mechanism, there is an ongoing search for bioactive molecules. With a focus on the metabolites formed, bioassay-guided fractionation and comprehensive screening have identified compounds as antifungal. Although these are active, the compounds have been found in concentrations that are too low to account for the observed antifungal effect. It has been hypothesized that the formation of metabolites and consumption of nutrients during bacterial fermentations form the basis for the antifungal effect, i.e., the composition of the exometabolome. To build a more comprehensive view of the chemical changes induced by bacterial fermentation and the effects on mold growth, a strategy for correlating the exometabolomic profiles with mold growth was applied. The antifungal properties were assessed by measuring mold growth of two Penicillium strains on cell-free ferments of three strains of Lactobacillus paracasei pre-fermented in a chemically defined medium. Exometabolomic profiling was performed by reversed-phase liquid chromatography in combination with mass spectrometry in electrospray positive and negative modes. By multivariate data analysis, the three strains of Lb. paracasei were readily distinguished by the relative difference of their exometabolomes. The relative differences correlated with the relative growth of the two Penicillium strains. Metabolic footprinting proved to be a supplement to bioassay-guided fractionation for investigation of antifungal properties of bacterial ferments. Additionally, three previously identified and three novel antifungal metabolites from Lb. paracasei and their potential precursors were detected and assigned using the strategy.

Glutathione serves an extracellular defence function to decrease arsenite accumulation and toxicity in yeast.

Arsenic is an environmental toxin and a worldwide health hazard. Since this metalloid is ubiquitous in nature, virtually all living organisms require systems for detoxification and tolerance acquisition. Here, we show that during chronic exposure to arsenite [As(III)], Saccharomyces cerevisiae (budding yeast) exports and accumulates the low-molecular-weight thiol molecule glutathione (GSH) outside of cells. Extracellular accumulation of the arsenite triglutathione complex As(GS)3 was also detected and direct transport assays demonstrate that As(GS)3 does not readily enter cells. Yeast cells with increased extracellular GSH levels accumulate less arsenic and display improved growth when challenged with As(III). Conversely, cells defective in export and extracellular accumulation of GSH are As(III) sensitive. Taken together, our data are consistent with a novel detoxification mechanism in which GSH is exported to protect yeast cells from arsenite toxicity by preventing its uptake.

Liquid chromatography-mass spectrometry for metabolic footprinting of co-cultures of lactic and propionic acid bacteria.

Co-cultures of specific lactic and propionic acid bacteria have been shown to have an antagonistic effect against yeast and moulds in dairy systems. In studies of these co-cultures by bioassay-guided fractionation and analysis, numerous compounds have been reported to inhibit yeast and moulds. Although active, the compounds do not account for the full effect observed. Instead, the inhibitory action in the co-culture is believed to be a result of synergy between known exo-metabolites, depletion of nutrients, and/or compounds not yet identified. Untargeted metabolomics or metabolic footprinting could be a potent approach to elucidation of the mechanism. The purpose of this review is to discuss the two pre-requisites for such a study--the compound classes expected in the co-cultures, and on the basis of these, the most suitable analytical technique(s). Ultrahigh-performance liquid chromatography (UPLC) coupled to high-resolution mass spectrometry (MS) via electrospray ionisation (ESI) operated in both positive and negative modes is regarded as the optimum instrumental technique. The applicability of a range of liquid chromatographic techniques ranging from ion-pair (IPC) and hydrophilic interaction (HILIC) to reversed-phase chromatography (RPC) is discussed in terms of the expected metabolome. Use of both HILIC and RPC is suggested, on account of the complementarity of these modes. The most promising strategy uses a combination of the two electrospray polarities and two modes of LC. The strategy recommended in this study does not include all compound classes, and suggestions for supplementary methods are listed.

Efficacy and safety profile of a subtilisin protease produced by fermentation in Bacillus licheniformis to be used as a feed additive.

The efficacy and safety of a novel Subtilisin protease from a Bacillus sp. produced in Bacillus licheniformis was investigated in broiler chickens, and a range of toxicological tests, respectively. The B. licheniformis production strain culture supernatant was not found cytotoxic in a Vero cell assay. Subtilisin was non-mutagenic and non-clastogenic in in-vitro tests, and did not exhibit irritating potential to the eye or skin in ex-vivo/in-vitro models. Oral administration of Subtilisin to rats did not cause any adverse effects in a 13-week sub-chronic toxicity study. In addition, a 35-day dose response broiler performance trial conducted with Subtilisin (30,000 and 60,000 NFP/kg diet), showed a significant linear improvement in both body weight gain and feed conversion ratio up to 35 days of protease supplementation. In conclusion, there are no safety concerns using this novel Subtilisin as a feed additive, and the protease is efficient in improving broiler growth performance, making it a good candidate for use as a feed additive.

Peer preferences and characteristics of same-group and cross-group social interactions among autistic and non-autistic adolescents.

LAY ABSTRACT: Autistic students often experience challenges in peer interactions, especially for young adolescents who are navigating the increased social expectations in secondary education. Previous research on the peer interactions of autistic adolescents mainly compared the social behaviors of autistic and non-autistic students and overlooked the peers in the social context. However, recent research has shown that the social challenges faced by autistic may not be solely contributed by their social differences, but a mismatch in the social communication styles between autistic and non-autistic people. As such, this study aimed to investigate the student-and-peer match in real-world peer interactions between six autistic and six non-autistic adolescents in an inclusive school club. We examined the odds of autistic and non-autistic students interacting with either an autistic peer, a non-autistic peer, or multiple peers, and the results showed that autistic students were more likely to interact with autistic peers then non-autistic peers. This preference for same-group peer interactions strengthened over the 5-month school club in both autistic and non-autistic students. We further found that same-group peer interactions, in both autistic and non-autistic students, were more likely to convey a social interest rather than a functional purpose or need, be sharing thoughts, experiences, or items rather than requesting help or objects, and be highly reciprocal than cross-group social behaviors. Collectively, our findings support that peer interaction outcomes may be determined by the match between the group memberships of the student and their peers, either autistic or non-autistic, rather than the student's autism diagnosis.

Safety and efficacy profile of a phytase produced by fermentation and used as a feed additive.

Enzymes can aid in optimal feed stock utilization when used as feed additives. A range of toxicological studies were performed to evaluate the safety profile of a novel phytase (phytase HM) from Citrobacter b raakii produced in Aspergillus oryzae. Phytase HM was found to be non-mutagenic and non-clastogenic in in vitro tests. Further, the phytase HM preparation did not exhibit irritative potential to the eye and skin when applied in in vitro models. A 13-week subchronic toxicity study with oral administration of phytase HM to rats did not show any adverse effects. Efficacy studies showed that the dietary supplementation of this phytase significantly improved growth performance and bone mineralization in broiler chickens and piglets fed P-deficient diets, and increased retention of phosphorus (P) and calcium (Ca), and phytate-P degradation in excreta of broiler chickens in a dose-dependent manner. In conclusion, there are no safety concerns using phytase HM as a feed additive and the phytase is well tolerated by broiler chickens and pigs. Further, phytase HM improves with high efficacy the growth performance in both broiler chickens and pigs.

Highly Basic Clusters in the Herpes Simplex Virus 1 Nuclear Egress Complex Drive Membrane Budding by Inducing Lipid Ordering.

During replication of herpesviruses, capsids escape from the nucleus into the cytoplasm by budding at the inner nuclear membrane. This unusual process is mediated by the viral nuclear egress complex (NEC) that deforms the membrane around the capsid by oligomerizing into a hexagonal, membrane-bound scaffold. Here, we found that highly basic membrane-proximal regions (MPRs) of the NEC alter lipid order by inserting into the lipid headgroups and promote negative Gaussian curvature. We also find that the electrostatic interactions between the MPRs and the membranes are essential for membrane deformation. One of the MPRs is phosphorylated by a viral kinase during infection, and the corresponding phosphomimicking mutations block capsid nuclear egress. We show that the same phosphomimicking mutations disrupt the NEC-membrane interactions and inhibit NEC-mediated budding in vitro, providing a biophysical explanation for the in vivo phenomenon. Our data suggest that the NEC generates negative membrane curvature by both lipid ordering and protein scaffolding and that phosphorylation acts as an off switch that inhibits the membrane-budding activity of the NEC to prevent capsid-less budding. IMPORTANCE Herpesviruses are large viruses that infect nearly all vertebrates and some invertebrates and cause lifelong infections in most of the world's population. During replication, herpesviruses export their capsids from the nucleus into the cytoplasm by an unusual mechanism in which the viral nuclear egress complex (NEC) deforms the nuclear membrane around the capsid. However, how membrane deformation is achieved is unclear. Here, we show that the NEC from herpes simplex virus 1, a prototypical herpesvirus, uses clusters of positive charges to bind membranes and order membrane lipids. Reducing the positive charge or introducing negative charges weakens the membrane deforming ability of the NEC. We propose that the virus employs electrostatics to deform nuclear membrane around the capsid and can control this process by changing the NEC charge through phosphorylation. Blocking NEC-membrane interactions could be exploited as a therapeutic strategy.

Evolutionary forces act on promoter length: identification of enriched cis-regulatory elements.

Transcription factors govern gene expression by binding to short DNA sequences called cis-regulatory elements. These sequences are typically located in promoters, which are regions of variable length upstream of the open reading frames of genes. Here, we report that promoter length and gene function are related in yeast, fungi, and plants. In particular, the promoters for stress-responsive genes are in general longer than those of other genes. Essential genes have, on the other hand, relatively short promoters. We utilize these findings in a novel method for identifying relevant cis-regulatory elements in a set of coexpressed genes. The method is shown to generate more accurate results and fewer false positives compared with other common procedures. Our results suggest that genes with complex transcriptional regulation tend to have longer promoters than genes responding to few signals. This phenomenon is present in all investigated species, indicating that evolution adjust promoter length according to gene function. Identification of cis-regulatory elements in Saccharomyces cerevisiae can be done with the web service located at http://enricher.zool.gu.se.

Genetic basis of arsenite and cadmium tolerance in Saccharomyces cerevisiae.

BACKGROUND: Arsenic and cadmium are widely distributed in nature and pose serious threats to the environment and human health. Exposure to these nonessential toxic metals may result in a variety of human diseases including cancer. However, arsenic and cadmium toxicity targets and the cellular systems contributing to tolerance acquisition are not fully known. RESULTS: To gain insight into metal action and cellular tolerance mechanisms, we carried out genome-wide screening of the Saccharomyces cerevisiae haploid and homozygous diploid deletion mutant collections and scored for reduced growth in the presence of arsenite or cadmium. Processes found to be required for tolerance to both metals included sulphur and glutathione biosynthesis, environmental sensing, mRNA synthesis and transcription, and vacuolar/endosomal transport and sorting. We also identified metal-specific defence processes. Arsenite-specific defence functions were related to cell cycle regulation, lipid and fatty acid metabolism, mitochondrial biogenesis, and the cytoskeleton whereas cadmium-specific defence functions were mainly related to sugar/carbohydrate metabolism, and metal-ion homeostasis and transport. Molecular evidence indicated that the cytoskeleton is targeted by arsenite and that phosphorylation of the Snf1p kinase is required for cadmium tolerance. CONCLUSION: This study has pin-pointed core functions that protect cells from arsenite and cadmium toxicity. It also emphasizes the existence of both common and specific defence systems. Since many of the yeast genes that confer tolerance to these agents have homologues in humans, similar biological processes may act in yeast and humans to prevent metal toxicity and carcinogenesis.

Characterization of the DNA-binding motif of the arsenic-responsive transcription factor Yap8p.

Saccharomyces cerevisiae uses several mechanisms for arsenic detoxification including the arsenate reductase Acr2p and the arsenite efflux protein Acr3p. ACR2 and ACR3 are transcribed in opposite directions from the same promoter and expression of these genes is regulated by the AP-1 (activator protein 1)-like transcription factor Yap8p. Yap8p has been shown to permanently associate with this promoter and to stimulate ACR2/ACR3 expression in response to arsenic. In the present study we characterized the DNA sequence that is targeted by Yap8p. We show that Yap8p binds to a pseudo-palindromic TGATTAATAATCA sequence that is related to, but distinct from, the sequence recognized by other fungal AP-1 proteins. Probing the promoter by mutational analysis, we confirm the importance of the TTAATAA core element and pin-point nucleotides that flank this element as crucial for Yap8p binding and in vivo activation of ACR3 expression. A genome-wide search for this element combined with global gene expression analysis indicates that the principal function of Yap8p is to control expression of ACR2 and ACR3. We conclude that Yap8p and other yeast AP-1 proteins require distinct DNA-binding motifs to induce gene expression and propose that this fact contributed towards a separation of function between AP-1 proteins during evolution.

The MAPK Hog1p modulates Fps1p-dependent arsenite uptake and tolerance in yeast.

Arsenic is widely distributed in nature and all organisms possess regulatory mechanisms to evade toxicity and acquire tolerance. Yet, little is known about arsenic sensing and signaling mechanisms or about their impact on tolerance and detoxification systems. Here, we describe a novel role of the S. cerevisiae mitogen-activated protein kinase Hog1p in protecting cells during exposure to arsenite and the related metalloid antimonite. Cells impaired in Hog1p function are metalloid hypersensitive, whereas cells with elevated Hog1p activity display improved tolerance. Hog1p is phosphorylated in response to arsenite and this phosphorylation requires Ssk1p and Pbs2p. Arsenite-activated Hog1p remains primarily cytoplasmic and does not mediate a major transcriptional response. Instead, hog1delta sensitivity is accompanied by elevated cellular arsenic levels and we demonstrate that increased arsenite influx is dependent on the aquaglyceroporin Fps1p. Fps1p is phosphorylated on threonine 231 in vivo and this phosphorylation critically affects Fps1p activity. Moreover, Hog1p is shown to affect Fps1p phosphorylation. Our data are the first to demonstrate Hog1p activation by metalloids and provides a mechanism by which this kinase contributes to tolerance acquisition. Understanding how arsenite/antimonite uptake and toxicity is modulated may prove of value for their use in medical therapy.

A subgroup of plant aquaporins facilitate the bi-directional diffusion of As(OH)3 and Sb(OH)3 across membranes.

BACKGROUND: Arsenic is a toxic and highly abundant metalloid that endangers human health through drinking water and the food chain. The most common forms of arsenic in the environment are arsenate (As(V)) and arsenite (As(III)). As(V) is a non-functional phosphate analog that enters the food chain via plant phosphate transporters. Inside cells, As(V) becomes reduced to As(III) for subsequent extrusion or compartmentation. Although much is known about As(III) transport and handling in microbes and mammals, the transport systems for As(III) have not yet been characterized in plants. RESULTS: Here we show that the Nodulin26-like Intrinsic Proteins (NIPs) AtNIP5;1 and AtNIP6;1 from Arabidopsis thaliana, OsNIP2;1 and OsNIP3;2 from Oryza sativa, and LjNIP5;1 and LjNIP6;1 from Lotus japonicus are bi-directional As(III) channels. Expression of these NIPs sensitized yeast cells to As(III) and antimonite (Sb(III)), and direct transport assays confirmed their ability to facilitate As(III) transport across cell membranes. On medium containing As(V), expression of the same NIPs improved yeast growth, probably due to increased As(III) efflux. Our data furthermore provide evidence that NIPs can discriminate between highly similar substrates and that they may have differential preferences in the direction of transport. A subgroup of As(III) permeable channels that group together in a phylogenetic tree required N-terminal truncation for functional expression in yeast. CONCLUSION: This is the first molecular identification of plant As(III) transport systems and we propose that metalloid transport through NIPs is a conserved and ancient feature. Our observations are potentially of great importance for improved remediation and tolerance of plants, and may provide a key to the development of low arsenic crops for food production.

Analysis of a gene panel for targeted sequencing of colorectal cancer samples.

Colorectal cancer (CRC) is a leading cause of death worldwide. Surgical intervention is a successful treatment for stage I patients, whereas other more advanced cases may require adjuvant chemotherapy. The selection of effective adjuvant treatments remains, however, challenging. Accurate patient stratification is necessary for the identification of the subset of patients likely responding to treatment, while sparing others from pernicious treatment. Targeted sequencing approaches may help in this regard, enabling rapid genetic investigation, and at the same time easily applicable in routine diagnosis. We propose a set of guidelines for the identification, including variant calling and filtering, of somatic mutations driving tumorigenesis in the absence of matched healthy tissue. We also discuss the inclusion criteria for the generation of our gene panel. Furthermore, we evaluate the prognostic impact of individual genes, using Cox regression models in the context of overall survival and disease-free survival. These analyses confirmed the role of commonly used biomarkers, and shed light on controversial genes such as CYP2C8. Applying those guidelines, we created a novel gene panel to investigate the onset and progression of CRC in 273 patients. Our comprehensive biomarker set includes 266 genes that may play a role in the progression through the different stages of the disease. Tracing the developmental state of the tumour, and its resistances, is instrumental in patient stratification and reliable decision making in precision clinical practice.

Quantitative transcriptome, proteome, and sulfur metabolite profiling of the Saccharomyces cerevisiae response to arsenite.

Arsenic is ubiquitously present in nature, and various mechanisms have evolved enabling cells to evade toxicity and acquire tolerance. Herein, we explored how Saccharomyces cerevisiae (budding yeast) respond to trivalent arsenic (arsenite) by quantitative transcriptome, proteome, and sulfur metabolite profiling. Arsenite exposure affected transcription of genes encoding functions related to protein biosynthesis, arsenic detoxification, oxidative stress defense, redox maintenance, and proteolytic activity. Importantly, we observed that nearly all components of the sulfate assimilation and glutathione biosynthesis pathways were induced at both gene and protein levels. Kinetic metabolic profiling evidenced a significant increase in the pools of sulfur metabolites as well as elevated cellular glutathione levels. Moreover, the flux in the sulfur assimilation pathway as well as the glutathione synthesis rate strongly increased with a concomitant reduction of sulfur incorporation into proteins. By combining comparative genomics and molecular analyses, we pinpointed transcription factors that mediate the core of the transcriptional response to arsenite. Taken together, our data reveal that arsenite-exposed cells channel a large part of assimilated sulfur into glutathione biosynthesis, and we provide evidence that the transcriptional regulators Yap1p and Met4p control this response in concert.

Transcriptional activation of metalloid tolerance genes in Saccharomyces cerevisiae requires the AP-1-like proteins Yap1p and Yap8p.

All organisms are equipped with systems for detoxification of the metalloids arsenic and antimony. Here, we show that two parallel pathways involving the AP-1-like proteins Yap1p and Yap8p are required for acquisition of metalloid tolerance in the budding yeast S. cerevisiae. Yap8p is demonstrated to reside in the nucleus where it mediates enhanced expression of the arsenic detoxification genes ACR2 and ACR3. Using chromatin immunoprecipitation assays, we show that Yap8p is associated with the ACR3 promoter in untreated as well as arsenic-exposed cells. Like for Yap1p, specific cysteine residues are critical for Yap8p function. We further show that metalloid exposure triggers nuclear accumulation of Yap1p and stimulates expression of antioxidant genes. Yap1p mutants that are unable to accumulate in the nucleus during H(2)O(2) treatment showed nearly normal nuclear retention in response to metalloid exposure. Thus, our data are the first to demonstrate that Yap1p is being regulated by metalloid stress and to indicate that this activation of Yap1p operates in a manner distinct from stress caused by chemical oxidants. We conclude that Yap1p and Yap8p mediate tolerance by controlling separate subsets of detoxification genes and propose that the two AP-1-like proteins respond to metalloids through distinct mechanisms.

Quantitative RT-PCR for MicroRNAs in Biofluids.

MicroRNAs in biofluids hold great promise as minimally invasive diagnostic biomarkers for a wide range of diseases and biological processes. One of the most sensitive technologies for detection and measuring expression levels of microRNA is quantitative RT-PCR. However, quantification of microRNA in biofluid samples is challenging in many ways. Biofluids contain low levels of RNA and high levels of inhibitors of enzymatic processes like reverse transcription and PCR. Furthermore, biofluids are susceptible to many preanalytical variables. Here we describe procedures developed to address these challenges, which include highly sensitive and accurate microRNA detection methods, combined with optimized protocols for sample handling and preparation, and extensive quality control (QC) procedures.

Genome-Wide Comparison of Next-Generation Sequencing and qPCR Platforms for microRNA Profiling in Serum.

This study compares next-generation sequencing (NGS) technologies that have been optimized specifically for biofluid samples, with more established qPCR-based methods for profiling microRNAs in biofluids. The same patient serum samples were analyzed by NGS and qPCR, and differences in the serum microRNA profile between HBV and HCV infected patients were investigated. While there was overall good agreement between NGS and qPCR, there were some differences between the platforms, highlighting the importance of validation.