bluuebunny/crossref_metadata_2025_split huggingface dataset:
Single-Particle Detection of Transcription following Rotavirus Entry
Authors: Salgado Eric N. , Upadhyayula Srigokul , Harrison Stephen C. 
Published: July 2017

ABSTRACT

Infectious rotavirus particles are triple-layered, icosahedral assemblies. The outer layer proteins, VP4 (cleaved to VP8* and VP5*) and VP7, surround a transcriptionally competent, double-layer particle (DLP), which they deliver into the cytosol. During entry of rhesus rotavirus, VP8* interacts with cell surface gangliosides, allowing engulfment into a membrane vesicle by a clathrin-independent process. Escape into the cytosol and outer-layer shedding depend on interaction of a hydrophobic surface on VP5* with the membrane bilayer and on a large-scale conformational change. We report here experiments that detect the fate of released DLPs and their efficiency in initiating RNA synthesis. By replacing the outer layer with fluorescently tagged, recombinant proteins and also tagging the DLP, we distinguished particles that have lost their outer layer and entered the cytosol (uncoated) from those still within membrane vesicles. We used fluorescent
in situ
hybridization with probes for nascent transcripts to determine how soon after uncoating transcription began and what fraction of the uncoated particles were active in initiating RNA synthesis. We detected RNA synthesis by uncoated particles as early as 15 min after adding virus. The uncoating efficiency was 20 to 50%; of the uncoated particles, about 10 to 15% synthesized detectable RNA. In the format of our experiments, about 10% of the added particles attached to the cell surface, giving an overall ratio of added particles to RNA-synthesizing particles of between 250:1 and 500:1, in good agreement with the ratio of particles to focus-forming units determined by infectivity assays. Thus, RNA synthesis by even a single, uncoated particle can initiate infection in a cell.

IMPORTANCE
The pathways by which a virus enters a cell transform its packaged genome into an active one. Contemporary fluorescence microscopy can detect individual virus particles as they enter cells, allowing us to map their multistep entry pathways. Rotaviruses, like most viruses that lack membranes of their own, disrupt or perforate the intracellular, membrane-enclosed compartment into which they become engulfed following attachment to a cell surface, in order to gain access to the cell interior. The properties of rotavirus particles make it possible to determine molecular mechanisms for these entry steps. In the work described here, we have asked the following question: what fraction of the rotavirus particles that penetrate into the cell make new viral RNA? We find that of the cell-attached particles, between 20 and 50% ultimately penetrate, and of these, about 10% make RNA. RNA synthesis by even a single virus particle can initiate a productive infection.
Partially Uncleaved Alphavirus Replicase Forms Spherule Structures in the Presence and Absence of RNA Template
Authors: Hellström Kirsi , Kallio Katri , Utt Age , Quirin Tania , Jokitalo Eija , Merits Andres , Ahola Tero 
Published: July 2017

ABSTRACT
Alphaviruses are positive-strand RNA viruses expressing their replicase as a polyprotein, P1234, which is cleaved to four final products, nonstructural proteins nsP1 to nsP4. The replicase proteins together with viral RNA and host factors form membrane invaginations termed spherules, which act as the replication complexes producing progeny RNAs. We have previously shown that the wild-type alphavirus replicase requires a functional RNA template and active polymerase to generate spherule structures. However, we now find that specific partially processed forms of the replicase proteins alone can give rise to membrane invaginations in the absence of RNA or replication. The minimal requirement for spherule formation was the expression of properly cleaved nsP4, together with either uncleaved P123 or with the combination of nsP1 and uncleaved P23. These inactive spherules were morphologically less regular than replication-induced spherules. In the presence of template, nsP1 plus uncleaved P23 plus nsP4 could efficiently assemble active replication spherules producing both negative-sense and positive-sense RNA strands. P23 alone did not have membrane affinity, but could be recruited to membrane sites in the presence of nsP1 and nsP4. These results define the set of viral components required for alphavirus replication complex assembly and suggest the possibility that it could be reconstituted from separately expressed nonstructural proteins.

IMPORTANCE
All positive-strand RNA viruses extensively modify host cell membranes to serve as efficient platforms for viral RNA replication. Alphaviruses and several other groups induce protective membrane invaginations (spherules) as their genome factories. Most positive-strand viruses produce their replicase as a polyprotein precursor, which is further processed through precise and regulated cleavages. We show here that specific cleavage intermediates of the alphavirus replicase can give rise to spherule structures in the absence of viral RNA. In the presence of template RNA, the same intermediates yield active replication complexes. Thus, partially cleaved replicase proteins play key roles that connect replication complex assembly, membrane deformation, and the different stages of RNA synthesis.
Increased, Durable B-Cell and ADCC Responses Associated with T-Helper Cell Responses to HIV-1 Envelope in Macaques Vaccinated with gp140 Occluded at the CD4 Receptor Binding Site
Authors: Bogers Willy M. J. M. , Barnett Susan W. , Oostermeijer Herman , Nieuwenhuis Ivonne G. , Beenhakker Niels , Mortier Daniella , Mooij Petra , Koopman Gerrit , Remarque Edmund , Martin Gregoire , Lai Rachel Pei-Jen , Dey Antu K. , Sun Yide , Burke Brian , Ferrari Guido , Montefiori David , Martin Loic , Davis David , Srivastava Indresh , Heeney Jonathan L. 
Published: July 2017

ABSTRACTStrategies are needed to improve the immunogenicity of HIV-1 envelope (Env) antigens (Ag) for more long-lived, efficacious HIV-1 vaccine-induced B-cell responses. HIV-1 Env gp140 (native or uncleaved molecules) or gp120 monomeric proteins elicit relatively poor B-cell responses which are short-lived. We hypothesized that Env engagement of the CD4 receptor on T-helper cells results in anergic effects on T-cell recruitment and consequently a lack of strong, robust, and durable B-memory responses. To test this hypothesis, we occluded the CD4 binding site (CD4bs) of gp140 by stable cross-linking with a 3-kDa CD4 miniprotein mimetic, serving to block ligation of gp140 on CD4+T cells while preserving CD4-inducible (CDi) neutralizing epitopes targeted by antibody-dependent cellular cytotoxicity (ADCC) effector responses. Importantly, immunization of rhesus macaques consistently gave superior B-cell (P< 0.001) response kinetics and superior ADCC (P< 0.014) in a group receiving the CD4bs-occluded vaccine compared to those of animals immunized with gp140. Of the cytokines examined, Ag-specific interleukin-4 (IL-4) T-helper enzyme-linked immunosorbent spot (ELISpot) assays of the CD4bs-occluded group increased earlier (P= 0.025) during the inductive phase. Importantly, CD4bs-occluded gp140 antigen induced superior B-cell and ADCC responses, and the elevated B-cell responses proved to be remarkably durable, lasting more than 60 weeks postimmunization.IMPORTANCEAttempts to develop HIV vaccines capable of inducing potent and durable B-cell responses have been unsuccessful until now. Antigen-specific B-cell development and affinity maturation occurs in germinal centers in lymphoid follicles through a critical interaction between B cells and T follicular helper cells. The HIV envelope binds the CD4 receptor on T cells as soluble shed antigen or as antigen-antibody complexes, causing impairment in the activation of these specialized CD4-positive T cells. We proposed that CD4-binding impairment is partly responsible for the relatively poor B-cell responses to HIV envelope-based vaccines. To test this hypothesis, we blocked the CD4 binding site of the envelope antigen and compared it to currently used unblocked envelope protein. We found superior and durable B-cell responses in macaques vaccinated with an occluded CD4 binding site on the HIV envelope antigen, demonstrating a potentially important new direction in future design of new HIV vaccines.
Multicentre, randomised clinical trial of paediatric concussion assessment of rest and exertion (PedCARE): a study to determine when to resume physical activities following concussion in children
Authors: Ledoux Andrée-Anne , Barrowman Nicholas J , Boutis Kathy , Davis Adrienne , Reid Sarah , Sangha Gurinder , Farion Ken J , Belanger Kevin , Tremblay Mark S , Yeates Keith Owen , DeMatteo Carol , Reed Nick , Zemek Roger 
Published: July 2017

IntroductionRest until symptom-free, followed by a progressive stepwise return to activities, is often prescribed in the management of paediatric concussions. Recent evidence suggests prolonged rest may hinder recovery, and early resumption of physical activity may be associated with more rapid recovery postconcussion. The primary objective is to determine whether the early reintroduction of non-contact physical activity beginning 72 hours postinjury reduces postconcussive symptoms at 2 weeks in children following an acute concussion as compared with a rest until asymptomatic protocol.Methods and analysisThis study is a randomised clinical trial across three Canadian academic paediatric emergency departments. A total of 350 participants, aged 10–17.99 years, who present within 48 hours of an acute concussion, will be recruited and randomly assigned to either the study intervention protocol (resumption of physical activity 72 hours postconcussion even if experiencing symptoms) or physical rest until fully asymptomatic. Participants will document their daily physical and cognitive activities. Follow-up questionnaires will be completed at 1, 2 and 4 weeks postinjury. Compliance with the intervention will be measured using an accelerometer (24 hours/day for 14 days). Symptoms will be measured using the validated Health and Behaviour Inventory. A linear multivariable model, adjusting for site and prognostically important covariates, will be tested to determine differences between groups. The proposed protocol adheres to the RCT-CONSORT guidelines.DiscussionThis trial will determine if early resumption of non-contact physical activity following concussion reduces the burden of concussion and will provide healthcare professionals with the evidence by which to recommend the best timing of reintroducing physical activities.Trial registration numberTrial identifier (Clinicaltrials.gov)NCT02893969.
Roles of autophagy and metabolism in pancreatic cancer cell adaptation to environmental challenges
Authors: Maertin Sandrina , Elperin Jason M. , Lotshaw Ethan , Sendler Matthias , Speakman Steven D. , Takakura Kazuki , Reicher Benjamin M. , Mareninova Olga A. , Grippo Paul J. , Mayerle Julia , Lerch Markus M. , Gukovskaya Anna S. 
Published: July 2017

Pancreatic ductal adenocarcinoma (PDAC) displays extensive and poorly vascularized desmoplastic stromal reaction, and therefore, pancreatic cancer (PaCa) cells are confronted with nutrient deprivation and hypoxia. Here, we investigate the roles of autophagy and metabolism in PaCa cell adaptation to environmental stresses, amino acid (AA) depletion, and hypoxia. It is known that in healthy cells, basal autophagy is at a low level, but it is greatly activated by environmental stresses. By contrast, we find that in PaCa cells, basal autophagic activity is relatively high, but AA depletion and hypoxia activate autophagy only weakly or not at all, due to their failure to inhibit mechanistic target of rapamycin. Basal, but not stress-induced, autophagy is necessary for PaCa cell proliferation, and AA supply is even more critical to maintain PaCa cell growth. To gain insight into the underlying mechanisms, we analyzed the effects of autophagy inhibition and AA depletion on PaCa cell metabolism. PaCa cells display mixed oxidative/glycolytic metabolism, with oxidative phosphorylation (OXPHOS) predominant. Both autophagy inhibition and AA depletion dramatically decreased OXPHOS; furthermore, pharmacologic inhibitors of OXPHOS suppressed PaCa cell proliferation. The data indicate that the maintenance of OXPHOS is a key mechanism through which autophagy and AA supply support PaCa cell growth. We find that the expression of oncogenic activation mutation in GTPase Kras markedly promotes basal autophagy and stimulates OXPHOS through an autophagy-dependent mechanism. The results suggest that approaches aimed to suppress OXPHOS, particularly through limiting AA supply, could be beneficial in treating PDAC. NEW & NOTEWORTHY Cancer cells in the highly desmoplastic pancreatic ductal adenocarcinoma confront nutrient [i.e., amino acids (AA)] deprivation and hypoxia, but how pancreatic cancer (PaCa) cells adapt to these conditions is poorly understood. This study provides evidence that the maintenance of mitochondrial function, in particular, oxidative phosphorylation (OXPHOS), is a key mechanism that supports PaCa cell growth, both in normal conditions and under the environmental stresses. OXPHOS in PaCa cells critically depends on autophagy and AA supply. Furthermore, the oncogenic activation mutation in GTPase Kras upregulates OXPHOS through an autophagy-dependent mechanism.
Augmentation of cGMP/PKG pathway and colonic motility by hydrogen sulfide
Authors: Nalli Ancy D. , Bhattacharya Sayak , Wang Hongxia , Kendig Derek M. , Grider John R. , Murthy Karnam S. 
Published: July 2017

Hydrogen sulfide (H2S), like nitric oxide (NO), causes smooth muscle relaxation, but unlike NO, does not stimulate soluble guanylyl cyclase (sGC) activity and generate cyclic guanosine 5′-monophosphate (cGMP). The aim of this study was to investigate the interplay between NO and H2S in colonic smooth muscle. In colonic smooth muscle from rabbit, mouse, and human, l-cysteine, substrate of cystathionine-γ-lyase (CSE), or NaHS, an H2S donor, inhibited phosphodiesterase 5 (PDE5) activity and augmented the increase in cGMP levels, IP3 receptor phosphorylation at Ser1756 (measured as a proxy for PKG activation), and muscle relaxation in response to NO donor S-nitrosoglutathione (GSNO), suggesting augmentation of cGMP/PKG pathway by H2S. The inhibitory effect of l-cysteine, but not NaHS, on PDE5 activity was blocked in cells transfected with CSE siRNA or treated with CSE inhibitor d,l-propargylglycine (dl-PPG), suggesting activation of CSE and generation of H2S in response to l-cysteine. H2S levels were increased in response to l-cysteine, and the effect of l-cysteine was augmented by GSNO in a cGMP-dependent protein kinase-sensitive manner, suggesting augmentation of CSE/H2S by cGMP/PKG pathway. As a result, GSNO-induced relaxation was inhibited by dl-PPG. In flat-sheet preparation of colon, l-cysteine augmented calcitonin gene-related peptide release in response to mucosal stimulation, and in intact segments, l-cysteine increased the velocity of pellet propulsion. These results demonstrate that in colonic smooth muscle, there is a novel interplay between NO and H2S. NO generates H2S via cGMP/PKG pathway, and H2S, in turn, inhibits PDE5 activity and augments NO-induced cGMP levels. In the intact colon, H2S promotes colonic transit. NEW & NOTEWORTHY Hydrogen sulfide (H2S) and nitric oxide (NO) are important regulators of gastrointestinal motility. The studies herein provide the cross talk between NO and H2S signaling to mediate smooth muscle relaxation and colonic transit. H2S inhibits phosphodiesterase 5 activity to augment cGMP levels in response to NO, which, in turn, via cGMP/PKG pathway, generates H2S. These studies suggest that interventions targeted at restoring NO and H2S homeostasis within the smooth muscle may provide novel therapeutic approaches to mitigate motility disorders.
Effects of β-hydroxy-β-methylbutyrate on skeletal muscle mitochondrial content and dynamics, and lipids after 10 days of bed rest in older adults
Authors: Standley Robert A. , Distefano Giovanna , Pereira Suzette L. , Tian Min , Kelly Owen J. , Coen Paul M. , Deutz Nicolaas E. P. , Wolfe Robert R. , Goodpaster Bret H. 
Published: July 2017

Loss of muscle mass during periods of disuse likely has negative health consequences for older adults. We have previously shown that β-hydroxy-β-methylbutyrate (HMB) supplementation during 10 days of strict bed rest (BR) attenuates the loss of lean mass in older adults. To elucidate potential molecular mechanisms of HMB effects on muscle during BR and resistance training rehabilitation (RT), we examined mediators of skeletal muscle mitochondrial dynamics, autophagy and atrophy, and intramyocellular lipids. Nineteen older adults (60–76 yr) completed 10 days BR followed by 8-wk RT rehabilitation. Subjects were randomized to either HMB (3 g/day HMB; n = 11) or control (CON; n = 8) groups. Skeletal muscle cross-sectional area (CSA) was determined by histology from percutaneous vastus lateralis biopsies. We measured protein markers of mitochondrial content [oxidative phosphorylation (OXPHOS)], fusion and fission (MFN2, OPA1, FIS1, and DRP1), autophagy (Beclin1, LC3B, and BNIP3), and atrophy [poly-ubiquinated proteins (poly-ub)] by Western blot. Fatty acid composition of several lipid classes in skeletal muscle was measured by infusion-MS analysis. Poly-ub proteins and OXPHOS complex I increased in both groups following BR ( P < 0.05, main effect for time), and muscle triglyceride content tended to increase following BR in the HMB group ( P = 0.055). RT rehabilitation increased OXPHOS complex II protein ( P < 0.05), and total OXPHOS content tended ( P = 0.0504) to be higher in HMB group. In addition, higher levels of DRP1 and MFN2 were maintained in the HMB group after RT ( P < 0.05). BNIP3 and poly-ub proteins were significantly reduced following rehabilitation in both groups ( P < 0.05). Collectively, these data suggest that HMB influences mitochondrial dynamics and lipid metabolism during disuse atrophy and rehabilitation. NEW & NOTEWORTHY Mitochondrial content and dynamics remained unchanged over 10 days of BR in older adults. HMB stimulated intramuscular lipid storage as triacylglycerol following 10 days of bed rest (BR) and maintained higher mitochondrial OXPHOS content and dynamics during the 8-wk resistance exercise rehabilitation program.
Circular RNA Expression Profiles Alter Significantly in Mouse Brain After Transient Focal Ischemia
Authors: Mehta Suresh L. , Pandi Gopal , Vemuganti Raghu 
Published: July 2017

Background and Purpose—
Circular RNAs (circRNAs) are a novel class of noncoding RNAs formed from many protein-coding genes by backsplicing. Although their physiological functions are not yet completely defined, they are thought to control transcription, translation, and microRNA levels. We investigated whether stroke changes the circRNAs expression profile in the mouse brain.

Methods—
Male C57BL/6J mice were subjected to transient middle cerebral artery occlusion, and circRNA expression profile was evaluated in the penumbral cortex at 6, 12, and 24 hours of reperfusion using circRNA microarrays and real-time PCR. Bioinformatics analysis was conducted to identify microRNA binding sites, transcription factor binding, and gene ontology of circRNAs altered after ischemia.

Results—
One thousand three-hundred twenty circRNAs were expressed at detectable levels mostly from exonic (1064) regions of the genes in the cerebral cortex of sham animals. Of those, 283 were altered (>2-fold) at least at one of the reperfusion time points, whereas 16 were altered at all 3 time points of reperfusion after transient middle cerebral artery occlusion compared with sham. Postischemic changes in circRNAs identified by microarray analysis were confirmed by real-time PCR. Bioinformatics showed that these 16 circRNAs contain binding sites for many microRNAs. Promoter analysis showed that the circRNAs altered after stroke might be controlled by a set of transcription factors. The major biological and molecular functions controlled by circRNAs altered after transient middle cerebral artery occlusion are biological regulation, metabolic process, cell communication, and binding to proteins, ions, and nucleic acids.

Conclusions—
This is a first study that shows that stroke alters the expression of circRNAs with possible functional implication to poststroke pathophysiology.
The Best of Both Worlds: A Decision-Making Framework for Combining Traditional and Contemporary Forecast Systems
Authors: Plotz Roan D. , Chambers Lynda E. , Finn Charlotte K. 
Published: July 2017

AbstractIn most countries, national meteorological services either generate or have access to seasonal climate forecasts. However, in a number of regions, the uptake of these forecasts by local communities can be limited, with the locals instead relying on traditional knowledge to make their climate forecasts. Both approaches to seasonal climate forecasting have benefits, and the incorporation of traditional forecast methods into contemporary forecast systems can lead to forecasts that are locally relevant and better trusted by the users. This in turn could significantly improve the communication and application of climate information, especially to remote communities. A number of different methodologies have been proposed for combining these forecasts. Through considering the benefits and limitations of each approach, practical recommendations are provided on selecting a method, in the form of a decision framework, that takes into consideration both user and provider needs. The framework comprises four main decision points: 1) consideration of the level of involvement of traditional-knowledge experts or the community that is required, 2) existing levels of traditional knowledge of climate forecasting and its level of cultural sensitivity, 3) the availability of long-term data—both traditional-knowledge and contemporary-forecast components, and 4) the level of resourcing available. No one method is suitable for everyone and every situation; however, the decision framework helps to select the most appropriate method for a given situation.
Scale Dependence of Midlatitude Air–Sea Interaction
Authors: Bishop Stuart P. , Small R. Justin , Bryan Frank O. , Tomas Robert A. 
Published: July 2017

Abstract
It has traditionally been thought that midlatitude sea surface temperature (SST) variability is predominantly driven by variations in air–sea surface heat fluxes (SHFs) associated with synoptic weather variability. Here it is shown that in regions marked by the highest climatological SST gradients and SHF loss to the atmosphere, the variability in SST and SHF at monthly and longer time scales is driven by internal ocean processes, termed here “oceanic weather.” This is shown within the context of an energy balance model of coupled air–sea interaction that includes both stochastic forcing for the atmosphere and ocean. The functional form of the lagged correlation between SST and SHF allows us to discriminate between variability that is driven by atmospheric versus oceanic weather. Observations show that the lagged functional relationship of SST–SHF and SST tendency–SHF correlation is indicative of ocean-driven SST variability in the western boundary currents (WBCs) and the Antarctic Circumpolar Current (ACC). By applying spatial and temporal smoothing, thereby dampening the signature SST anomalies generated by eddy stirring, it is shown that the oceanic influence on SST variability increases with time scale but decreases with increasing spatial scale. The scale at which SST variability in the WBCs and the ACC transitions from ocean to atmosphere driven occurs at scales less than 500 km. This transition scale highlights the need to resolve mesoscale eddies in coupled climate models to adequately simulate the variability of air–sea interaction. Away from strong SST fronts the lagged functional relationships are indicative of the traditional paradigm of atmospherically driven SST variability.