Rotavirus core shell protein sites that regulate intra-particle polymerase activity.

IMPORTANCE: Rotaviruses are important causes of severe gastroenteritis in young children. A characteristic feature of rotaviruses is that they copy ribonucleic acid (RNA) inside of the viral particle. In fact, the viral polymerase (VP1) only functions when it is connected to the viral inner core shell protein (VP2). Here, we employed a biochemical assay to identify which sites of VP2 are critical for regulating VP1 activity. Specifically, we engineered VP2 proteins to contain amino acid changes at structurally defined sites and assayed them for their capacity to support VP1 function in a test tube. Through this work, we were able to identify several VP2 residues that appeared to regulate the activity of the polymerase, positively and negatively. These results are important because they help explain how rotavirus synthesizes its RNA while inside of particles and they identify targets for the future rational design of drugs to prevent rotavirus disease.

Flexibility of the Rotavirus NSP2 C-Terminal Region Supports Factory Formation via Liquid-Liquid Phase Separation.

Many viruses sequester the materials needed for their replication into discrete subcellular factories. For rotaviruses (RVs), these factories are called viroplasms, and they are formed in the host cell cytosol via the process of liquid-liquid phase separation (LLPS). The nonstructural protein 2 (NSP2) and its binding partner, nonstructural protein 5 (NSP5), are critical for viroplasm biogenesis. Yet it is not fully understood how NSP2 and NSP5 cooperate to form factories. The C-terminal region (CTR) of NSP2 (residues 291 to 317) is flexible, allowing it to participate in domain-swapping interactions that promote interoctamer interactions and, presumably, viroplasm formation. Molecular dynamics simulations showed that a lysine-to-glutamic acid change at position 294 (K294E) reduces NSP2 CTR flexibility in silico. To test the impact of reduced NSP2 CTR flexibility during infection, we engineered a mutant RV bearing this change (rRV-NSP2K294E). Single-cycle growth assays revealed a >1.2-log reduction in endpoint titers for rRV-NSP2K294E versus the wild-type control (rRV-WT). Using immunofluorescence assays, we found that rRV-NSP2K294E formed smaller, more numerous viroplasms than rRV-WT. Live-cell imaging experiments confirmed these results and revealed that rRV-NSP2K294E factories had delayed fusion kinetics. Moreover, NSP2K294E and several other CTR mutants formed fewer viroplasm-like structures in NSP5 coexpressing cells than did control NSP2WT. Finally, NSP2K294E exhibited defects in its capacity to induce LLPS droplet formation in vitro when incubated alongside NSP5. These results underscore the importance of NSP2 CTR flexibility in supporting the biogenesis of RV factories. IMPORTANCE Viruses often condense the materials needed for their replication into discrete intracellular factories. For rotaviruses, agents of severe gastroenteritis in children, factory formation is mediated in part by an octameric protein called NSP2. A flexible C-terminal region of NSP2 has been proposed to link several NSP2 octamers together, a feature that might be important for factory formation. Here, we created a change in NSP2 that reduced C-terminal flexibility and analyzed the impact on rotavirus factories. We found that the change caused the formation of smaller and more numerous factories that could not readily fuse together like those of the wild-type virus. The altered NSP2 protein also had a reduced capacity to form factory-like condensates in a test tube. Together, these results add to our growing understanding of how NSP2 supports rotavirus factory formation-a key step of viral replication.

GRK2 mediates ß-arrestin interactions with 5-HT2 receptors for JC polyomavirus endocytosis.

JC polyomavirus (JCPyV) infects the majority of the population, establishing a lifelong, asymptomatic infection in the kidney of healthy individuals. People that become severely immunocompromised may experience JCPyV reactivation, which can cause progressive multifocal leukoencephalopathy (PML), a neurodegenerative disease. Due to a lack of therapeutic options, PML results in fatality or significant debilitation among affected individuals. Cellular internalization of JCPyV is mediated by serotonin 5-hydroxytryptamine subfamily 2 receptors (5-HT2Rs) via clathrin-mediated endocytosis. The JCPyV entry process requires the clathrin-scaffolding proteins ß-arrestin, adaptor protein 2 (AP2), and dynamin. Further, a ß-arrestin interacting domain, the Ala-Ser-Lys (ASK) motif, within the C-terminus of 5-HT2AR is important for JCPyV internalization and infection. Interestingly, 5-HT2R subtypes A, B, and C equally support JCPyV entry and infection, and all subtypes contain an ASK motif, suggesting a conserved mechanism for viral entry. However, the role of the 5-HT2R ASK motifs and the activation of ß-arrestin-associated proteins during internalization has not been fully elucidated. Through mutagenesis, the ASK motifs within 5-HT2BR and 5-HT2CR were identified as critical for JCPyV internalization and infectivity. Further, utilizing biochemical pulldown techniques, mutagenesis of the ASK motifs in 5-HT2BR and 5-HT2CR resulted in reduced ß-arrestin binding. Utilizing small-molecule chemical inhibitors and RNA interference, G-protein receptor kinase 2 (GRK2) was determined to be required for JCPyV internalization and infection by mediating interactions between ß-arrestin and the ASK motif of 5-HT2Rs. These findings demonstrate that GRK2 and ß-arrestin interactions with 5-HT2Rs are critical for JCPyV entry by clathrin-mediated endocytosis and resultant infection.IMPORTANCE As intracellular parasites, viruses require a host cell to replicate and cause disease. Therefore, virus-host interactions contribute to viral pathogenesis. JC polyomavirus (JCPyV) infects most of the population, establishing a lifelong asymptomatic infection within the kidney. Under conditions of severe immunosuppression JCPyV may spread to the central nervous system, causing the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML). Individuals living with HIV or undergoing immunomodulatory therapies are at risk for developing PML. The mechanisms of how JCPyV uses specific receptors on the surface of host cells to initiate internalization and infection is a poorly understood process. We have further identified cellular proteins involved in JCPyV internalization and infection and elucidated their specific interactions that are responsible for activation of receptors. Collectively, these findings illuminate how viruses usurp cellular receptors during infection, contributing to current development efforts for therapeutic options for the treatment or prevention of PML.

Parasite-host specificity: A cross-infection study of the parasite Ophryocystis elektroscirrha.

Many parasites are constrained to only one or a few hosts, showing host specificity. It remains unclear why some parasites are specialists and other parasites are generalists. The parasite Ophryocystis elektroscirrha (OE) is a neogregarine protozoan thought to be restricted to monarch butterflies, Danaus plexippus (Nymphaliae) and D. gilippus. Recently, we found OE-like spores in other Lepidoptera, specifically in three noctuid moths: Helicoverpa armigera, H. assulta and H. punctigera, as well as another nymphalid, Parthenos sylvia. To our knowledge, this is the first report of OE-like parasite infections in species other than the genus Danaus. In sequencing 558 bp of 18S rRNA, we found the genetic similarity between OE from D. plexippus and OE-like parasite from the moths H. armigera and H. punctigera to be 95.2%. When we conducted cross-species infection experiments, we could not infect the moths with OE from D. plexippus, but OE-like parasite from H. armigera did infect D. plexippus and a closely related moth species Heliothis virescens. Interestingly, we did not find the OE-like parasite in the H. armigera population from Spain. Inter-population infection experiments with H. armigera demonstrated a higher sensitivity to OE-like infection in the population from Spain compared to the populations from Australia and China. These results suggest geographic variation in OE-like susceptibility and coevolution between parasite and host. Our findings give important new insights into the prevalence and host specificity of OE and OE-like parasites, and provide opportunities to study parasite transmission over spatial and temporal scales.

JCPyV-Induced MAPK Signaling Activates Transcription Factors during Infection.

JC polyomavirus (JCPyV), a ubiquitous human pathogen, is the etiological agent of the fatal neurodegenerative disease progressive multifocal leukoencephalopathy (PML). Like most viruses, JCPyV infection requires the activation of host-cell signaling pathways in order to promote viral replication processes. Previous works have established the necessity of the extracellular signal-regulated kinase (ERK), the terminal core kinase of the mitogen-activated protein kinase (MAPK) cascade (MAPK-ERK) for facilitating transcription of the JCPyV genome. However, the underlying mechanisms by which the MAPK-ERK pathway becomes activated and induces viral transcription are poorly understood. Treatment of cells with siRNAs specific for Raf and MAP kinase kinase (MEK) targets proteins in the MAPK-ERK cascade, significantly reducing JCPyV infection. MEK, the dual-specificity kinase responsible for the phosphorylation of ERK, is phosphorylated at times congruent with early events in the virus infectious cycle. Moreover, a MAPK-specific signaling array revealed that transcription factors downstream of the MAPK cascade, including cMyc and SMAD4, are upregulated within infected cells. Confocal microscopy analysis demonstrated that cMyc and SMAD4 shuttle to the nucleus during infection, and nuclear localization is reduced when ERK is inhibited. These findings suggest that JCPyV induction of the MAPK-ERK pathway is mediated by Raf and MEK and leads to the activation of downstream transcription factors during infection. This study further defines the role of the MAPK cascade during JCPyV infection and the downstream signaling consequences, illuminating kinases as potential therapeutic targets for viral infection.

Dietary Changes Albertan Women Make During Pregnancy: Thematic Analysis of Self-reported Changes and Reasons.

PURPOSE: To explore dietary changes Albertan women make during pregnancy, reasons they make changes, and alignment with prenatal nutrition recommendations. METHODS: Women up to 6 months postpartum were recruited in public health centres and Primary Care Networks. Qualitative data were collected through a self-administered survey including 2 open-ended questions that asked about changes made to food/beverage intake during pregnancy and why these changes were made. RESULTS: A majority (n = 577) of the 737 women completing the survey described changes they made to their food/beverage intake during pregnancy and 193 respondents provided reasons for these changes. Increased intake of fruits/vegetables, meat, milk, and their alternatives (n = 600); limiting or avoidance of foods/beverages known to be harmful during pregnancy (n = 445); and increased food/fluid intake or meal/snack frequency (n = 405) were commonly reported dietary changes. Motivations relating to health and to control physiological changes/manage health conditions were the most frequent reasons provided. CONCLUSIONS: Women make diverse dietary changes and have various motivations for food choices during pregnancy. A majority make dietary changes to support a healthy pregnancy. However, the motivation to control discomforts and respond to hunger and thirst sensations reflect a stronger influencer on women's choices than is currently addressed in prenatal nutrition messages.

Rotavirus NSP2: A Master Orchestrator of Early Viral Particle Assembly.

Rotaviruses (RVs) are 11-segmented, double-stranded (ds) RNA viruses and important causes of acute gastroenteritis in humans and other animal species. Early RV particle assembly is a multi-step process that includes the assortment, packaging and replication of the 11 genome segments in close connection with capsid morphogenesis. This process occurs inside virally induced, cytosolic, membrane-less organelles called viroplasms. While many viral and cellular proteins play roles during early RV assembly, the octameric nonstructural protein 2 (NSP2) has emerged as a master orchestrator of this key stage of the viral replication cycle. NSP2 is critical for viroplasm biogenesis as well as for the selective RNA-RNA interactions that underpin the assortment of 11 viral genome segments. Moreover, NSP2's associated enzymatic activities might serve to maintain nucleotide pools for use during viral genome replication, a process that is concurrent with early particle assembly. The goal of this review article is to summarize the available data about the structures, functions and interactions of RV NSP2 while also drawing attention to important unanswered questions in the field.

Ecological immunology: do sexual attraction and immunity trade-off through a desaturase?

Given the limited availability of resources in nature, sexual attractiveness may trade off with immunocompetence, as the immunocompetence handicap hypothesis (ICHH) posits. In invertebrates, a direct link between trade-offs through hormonal/molecular effectors in sexual signals and immunity has not been found so far. Here, we assessed how variation in sexual signals affected parasite infection in two sex pheromone selected lines of the moth Chloridea virescens: an attractive line with a low ratio of 16:Ald/Z11-16:Ald and an unattractive line with a high ratio. When infecting these lines with an apicomplexan parasite, we found that the attractive Low line was significantly more susceptible to the parasite infection than the unattractive High line. Since the ratio difference between these two lines is determined by a delta-11-desturase, we hypothesized that this desaturase may have a dual role, i.e., in the quality of the sexual signal as well as an involvement in immune response, comparable to testosterone in vertebrates. However, when we used CRISPR/cas9 to knockout delta-11-desturase in the attractive Low line, we found that the pheromonal phenotype did change to that of the High line, but the infection susceptibility did not. Notably, when checking the genomic location of delta-11-desaturase in the C. virescens, we found that mucin is adjacent to delta-11-desaturase. When comparing the mucin sequences in both lines, we found four nonsynonymous SNPs in the coding sequence, as well as intronic variation between the two lines. These differences suggest that genetic hitchhiking may explain the variation in susceptibility to parasitic infection.

Arc1 : a regulator of triglyceride homeostasis in male Drosophila.

Achieving metabolic homeostasis is necessary for survival, and many genes are required to control organismal metabolism. A genetic screen in Drosophila larvae identified putative fat storage genes including Arc1 . Arc1 has been shown to act in neurons to regulate larval lipid storage; however, whether Arc1 functions to regulate adult metabolism is unknown. Arc1 esm18 males store more fat than controls while both groups eat similar amounts. Arc1 esm18 flies express more brummer lipase and less of the glycolytic enzyme triose phosphate isomerase, which may contribute to excess fat observed in these mutants. These results suggest that Arc1 regulates adult Drosophila lipid homeostasis.

Optimizing skin tightening in aesthetics in men.

The field of cosmetic dermatology has recently witnessed unbridled growth in the past several years. Part of this has been due to the increasing popularity of aesthetic treatments in men, who represent a growing patient population. Men tend to have higher levels of collagen density and greater skin thickness, but these begin to decrease earlier on. They can also more frequently have severe photodamage. Their clinical presentations can affect the selection of treatments. Physicians should be familiar with the subtle differences between treating men and women. Early studies and literature are beginning to shed more light on these important distinctions. We review the notable differences in facial aging, pathophysiology, and patient selection and discuss available treatment options with these factors in mind.

Recognizing Salinity Threats in the Climate Crisis.

Climate change is causing habitat salinity to transform at unprecedented rates across the globe. While much of the research on climate change has focused on rapid shifts in temperature, far less attention has focused on the effects of changes in environmental salinity. Consequently, predictive studies on the physiological, evolutionary, and migratory responses of organisms and populations to the threats of salinity change are relatively lacking. This omission represents a major oversight, given that salinity is among the most important factors that define biogeographic boundaries in aquatic habitats. In this perspective, we briefly touch on responses of organisms and populations to rapid changes in salinity occurring on contemporary time scales. We then discuss factors that might confer resilience to certain taxa, enabling them to survive rapid salinity shifts. Next, we consider approaches for predicting how geographic distributions will shift in response to salinity change. Finally, we identify additional data that are needed to make better predictions in the future. Future studies on climate change should account for the multiple environmental factors that are rapidly changing, especially habitat salinity.

High-throughput sequencing of faeces provides evidence for dispersal of parasites and pathogens by migratory waterbirds.

Examination of faecal material has demonstrated how a broad range of organisms are distributed by bird movements. Such research has largely focused on dispersal of plant seeds by frugivores and of freshwater organisms by waterbirds. However, with few exceptions (e.g. avian influenza, Ebola virus), there is a dearth of evidence for transport of parasites and pathogens. High-throughput sequencing methods now provide a powerful means of addressing this knowledge gap by elucidating faecal contents in unprecedented detail. We collected faeces excreted by a range of migratory waterbirds in south-west Spain and pooled faecal DNA to create libraries reflective of feeding behavior. We created sets of libraries using high-throughput metagenomic and amplicon sequencing. For the latter we employed two sets of primers to broadly target the V4 region of the 18S rRNA gene (one set amplifying the region across all eukaryotes, the other excluding amplification of metazoans). Libraries revealed a wide diversity of eukaryotes, including parasites of the faecal producers themselves, parasites of food items, or those incidentally ingested. We also detected novel microbial eukaryotic taxa and found that parasite assemblage profiles were relatively distinct. Comparing the performance of the methods used supports their joint use for future studies of diversity and abundance. Because viable stages of many parasites are likely to be present in faeces, our results suggest significant levels of bird-mediated dispersal of parasites (both from avian and other hosts). Our methods revealed much hidden biodiversity, and allowed identification of the individuals who produced the faecal samples to species level, facilitating the study of interaction networks.

Exploring wavepacket dynamics behind strong-field momentum-dependent photodissociation in CH(2)BrI(+).

The ultrafast photodissociation dynamics of CH(2)BrI(+) into CH(2)Br(+) + I is studied using high level ab initio electronic structure calculations in conjunction with integration of the time-dependent Schrödinger equation and compared with measured pump-probe signals. These pump-probe measurements provide evidence for momentum-dependent dissociation, which is interpreted using two theoretical models. The first is based on DFT and TD-DFT calculations neglecting spin-orbit coupling, while the other, more rigorous model employs a larger number of coupled multi-configurational potentials obtained by means of CASSCF calculations. The latter model highlights the role of spin-orbit coupling between ionic electronic states as well as the effect of strong fields in the quantum dynamics including Stark-shifts and multi-photon excitation.

Peptide-TLR-7/8a conjugate vaccines chemically programmed for nanoparticle self-assembly enhance CD8 T-cell immunity to tumor antigens.

Personalized cancer vaccines targeting patient-specific neoantigens are a promising cancer treatment modality; however, neoantigen physicochemical variability can present challenges to manufacturing personalized cancer vaccines in an optimal format for inducing anticancer T cells. Here, we developed a vaccine platform (SNP-7/8a) based on charge-modified peptide-TLR-7/8a conjugates that are chemically programmed to self-assemble into nanoparticles of uniform size (~20 nm) irrespective of the peptide antigen composition. This approach provided precise loading of diverse peptide neoantigens linked to TLR-7/8a (adjuvant) in nanoparticles, which increased uptake by and activation of antigen-presenting cells that promote T-cell immunity. Vaccination of mice with SNP-7/8a using predicted neoantigens (n = 179) from three tumor models induced CD8 T cells against ~50% of neoantigens with high predicted MHC-I binding affinity and led to enhanced tumor clearance. SNP-7/8a delivering in silico-designed mock neoantigens also induced CD8 T cells in nonhuman primates. Altogether, SNP-7/8a is a generalizable approach for codelivering peptide antigens and adjuvants in nanoparticles for inducing anticancer T-cell immunity.

Adolescent comprehension of emergency contraception in New York City.

OBJECTIVE: To estimate comprehension of the over-the-counter emergency contraception label among female adolescents aged 12 through 17 years, and to compare the results with a similar study that focused on adults. METHODS: Surveys were administered to female adolescents in New York City in public venues such as malls, movie theaters, and parks. Participants were asked to read the emergency contraception (levonorgestrel) label before answering survey questions. Comparisons were made in SPSS version 13.0 using chi tests of independence and Fisher exact tests for sparse data. RESULTS: One thousand eighty-five girls between the ages of 12 and 17 participated in the study. Overall, adolescents demonstrated high comprehension of the key points of emergency contraception: (1) that it is a method of preventing pregnancy 92% (confidence interval [CI] 91-94%); (2) that it has to be taken within the first 72 hours after unprotected intercourse 83% (CI 83-87%); (3) that if you are already pregnant emergency contraception will not be effective 87% (CI 85-89%); (4) that emergency contraception will not protect against human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS) 95% (CI 94-96%); and (5) that emergency contraception should not be used as a method of long-term birth control 85% (CI 83-87%). CONCLUSION: After reading the emergency contraception (levonorgestrel) label, female adolescents aged 12 to 17 understood the information necessary to use emergency contraception safely and effectively as well as their adult counterparts. LEVEL OF EVIDENCE: III.

A programme for Health Impact Assessment in Brighton and Hove.

HIA is based on the theory of health determinants, which recognizes that well-being is determined by a wide range of economic, social and environmental factors, by heredity and medical intervention. The intended HIA procedure represents a new approach to the evaluation of all local authority policies in order to assess their potential health impacts and to improve the quality of governmental decisions, through recommendations to enhance predicted positive health impacts and minimize negative ones.

A novel splice variant of Pmel17 expressed by human melanocytes and melanoma cells lacking some of the internal repeats.

Pmel17 is a approximately 100 kDa pigment cell specific glycoprotein that plays a crucial part in the morphogenesis of melanosome precursors. Anti-Pmel17 immunoprecipitates from metabolically pulse labeled melanoma cells and melanocytes contain, in addition to full-length Pmel17, a glycoprotein that migrates with a lower relative molecular weight. Here we show that this glycoprotein is encoded by an mRNA that results from alternative splicing of the human Pmel17 gene from which a cryptic intron is excised. Immunoprecipitation recapture experiments showed that this glycoprotein contained both the N- and C-termini of full-length Pmel17. Sequence analysis of cDNA corresponding to the alternatively spliced form reveals the loss of three of 10 imperfect direct repeats from the central region of the lumenal domain. The product of the splice variant is processed with similar kinetics to full-length Pmel17, and localizes similarly to late endosomes when expressed ectopically in nonpigment cells. We speculate that truncation of the repeat region within Pmel17 alters either fibrillogenic activity or the interaction of Pmel17 with melanin intermediates. The expression of an alternatively spliced product may furthermore affect the cohort of peptides generated for recognition of melanoma cells by tumor-directed T lymphocytes.

Health and safety implications of virtual reality: a review of empirical evidence.

For the last 10 years a number of papers have been written that discuss human factors issues associated with virtual reality (VR). The nature of these papers has gradually evolved from speculation and anecdotal report to empirical research. Despite developments in VR technology, some participants still experience health and safety problems associated with VR use-termed VR-induced symptoms and effects (VRISE). The key concern from the literature is VR-induced sickness, experienced by a large proportion of VR participants, but for the majority these effects are mild and subside quickly. This paper makes a number of recommendations regarding the future direction of research into health and safety implications of VR, including the need to take into account the way in which VR is being used when conducting empirical research: first, to ensure that studies consider both effects and their consequences, second, to ensure that empirical trials reflect the actual likely context of VR use; third, to consider interactions between effects: and finally, to consider ways in which effects can be managed.

Examining the impact of spiritual care in long-term care.

This project examines the effects of spiritual care on chronically ill and aging populations and those who care for them by studying the development of a comprehensive Pastoral Care Program at Episcopal Communities & Services (ECS), a nonprofit that owns two Continuing Care Retirement Communities (CCRC) in Southern California (in 2010 ECS operated three communities). The study includes the vision, methodology, and specific steps taken to implement this spiritual care program and methods to measure its efficacy. Data is analyzed from satisfaction surveys conducted the year before the program's introduction and surveys taken 2 and 4 years after the institution of the Pastoral Care Program, along with anecdotal findings. Results indicated that spiritual awareness and satisfaction increased throughout the resident population after the Pastoral Care Program's establishment and that satisfaction levels continued to improve as the program developed over time. This study suggests that spiritual support (both religious and nonreligious) is a vital factor in well-being and quality of life at the end of life and that transdisciplinary palliative care is needed in long-term care settings to address spiritual and psychosocial needs.

Pulse-shaping multiphoton FRET microscopy.

Fluorescence Resonance Energy Transfer (FRET) microscopy is a commonly-used technique to study problems in biophysics that range from uncovering cellular signaling pathways to detecting conformational changes in single biomolecules. Unfortunately, excitation and emission spectral overlap between the fluorophores create challenges in quantitative FRET studies. It has been shown previously that quantitative FRET stoichiometry can be performed by selective excitation of donor and acceptor fluorophores. Extending this approach to two-photon FRET applications is difficult when conventional femtosecond laser sources are used due to their limited bandwidth and slow tuning response time. Extremely broadband titanium:sapphire lasers enable the simultaneous excitation of both donor and acceptor for two-photon FRET, but do so without selectivity. Here we present a novel two-photon FRET microscopy technique that employs pulse-shaping to perform selective excitation of fluorophores in live cells and detect FRET between them. Pulse-shaping via multiphoton intrapulse interference can tailor the excitation pulses to achieve selective excitation. This technique overcomes the limitation of conventional femtosecond lasers to allow rapid switching between selective excitation of the donor and acceptor fluorophores. We apply the method to live cells expressing the fluorescent proteins mCerulean and mCherry, demonstrating selective excitation of fluorophores via pulse-shaping and the detection of two-photon FRET. This work paves the way for two-photon FRET stoichiometry.