A Two-Phase Innate Host Response to Alphavirus Infection Identified by mRNP-Tagging In Vivo

A concept fundamental to viral pathogenesis is that infection induces specific changes within the host cell, within specific tissues, or within the entire animal. These changes are reflected in a cascade of altered transcription patterns evident during infection. However, elucidation of this cascade in vivo has been limited by a general inability to distinguish changes occurring in the minority of infected cells from those in surrounding uninfected cells. To circumvent this inherent limitation of traditional gene expression profiling methods, an innovative mRNP-tagging technique was implemented to isolate host mRNA specifically from infected cells in vitro as well as in vivo following Venezuelan equine encephalitis virus (VEE) infection. This technique facilitated a direct characterization of the host defense response specifically within the first cells infected with VEE, while simultaneous total RNA analysis assessed the collective response of both the infected and uninfected cells. The result was a unique, multifaceted profile of the early response to VEE infection in primary dendritic cells, as well as in the draining lymph node, the initially targeted tissue in the mouse model. A dynamic environment of complex interactions was revealed, and suggested a two-step innate response in which activation of a subset of host genes in infected cells subsequently leads to activation of the surrounding uninfected cells. Our findings suggest that the application of viral mRNP-tagging systems, as introduced here, will facilitate a much more detailed understanding of the highly coordinated host response to infectious agents

Impact of Sleep and Circadian Disruption on Energy Balance and Diabetes: A Summary of Workshop Discussions

A workshop was held at the National Institute for Diabetes and Digestive and Kidney Diseases with a focus on the impact of sleep and circadian disruption on energy balance and diabetes. The workshop identified a number of key principles for research in this area and a number of specific opportunities. Studies in this area would be facilitated by active collaboration between investigators in sleep/circadian research and investigators in metabolism/diabetes. There is a need to translate the elegant findings from basic research into improving the metabolic health of the American public. There is also a need for investigators studying the impact of sleep/circadian disruption in humans to move beyond measurements of insulin and glucose and conduct more in-depth phenotyping. There is also a need for the assessments of sleep and circadian rhythms as well as assessments for sleep-disordered breathing to be incorporated into all ongoing cohort studies related to diabetes risk. Studies in humans need to complement the elegant short-term laboratory-based human studies of simulated short sleep and shift work etc. with studies in subjects in the general population with these disorders. It is conceivable that chronic adaptations occur, and if so, the mechanisms by which they occur needs to be identified and understood. Particular areas of opportunity that are ready for translation are studies to address whether CPAP treatment of patients with pre-diabetes and obstructive sleep apnea (OSA) prevents or delays the onset of diabetes and whether temporal restricted feeding has the same impact on obesity rates in humans as it does in mice

Tissue Type-Specific Expression of the dsRNA-Binding Protein 76 and Genome-Wide Elucidation of Its Target mRNAs

Background: RNA-binding proteins accompany all steps in the life of mRNAs and provide dynamic gene regulatory functions for rapid adjustment to changing extra-or intracellular conditions. The association of RNA-binding proteins with their targets is regulated through changing subcellular distribution, post-translational modification or association with other proteins. Methodology: We demonstrate that the dsRNA binding protein 76 (DRBP76), synonymous with nuclear factor 90, displays inherently distinct tissue type-specific subcellular distribution in the normal human central nervous system and in malignant brain tumors of glial origin. Altered subcellular localization and isoform distribution in malignant glioma indicate that tumor-specific changes in DRBP76-related gene products and their regulatory functions may contribute to the formation and/or maintenance of these tumors. To identify endogenous mRNA targets of DRBP76, we performed RNA-immunoprecipitation and genome-wide microarray analyses in HEK293 cells, and identified specific classes of transcripts encoding critical functions in cellular metabolism. Significance: Our data suggest that physiologic DRBP76 expression, isoform distribution and subcellular localization are profoundly altered upon malignant transformation. Thus, the functional role of DRBP76 in co- or post-transcriptional gene regulation may contribute to the neoplastic phenotype

Neuropathological and Genetic Correlates of Survival and Dementia Onset in Synucleinopathies: A Retrospective Analysis

Background Great heterogeneity exists in survival and the interval between onset of motor symptoms and dementia symptoms across synucleinopathies. We aimed to identify genetic and pathological markers that have the strongest association with these features of clinical heterogeneity in synucleinopathies. Methods In this retrospective study, we examined symptom onset, and genetic and neuropathological data from a cohort of patients with Lewy body disorders with autopsy-confirmed α synucleinopathy (as of Oct 1, 2015) who were previously included in other studies from five academic institutions in five cities in the USA. We used histopathology techniques and markers to assess the burden of tau neurofibrillary tangles, neuritic plaques, α-synuclein inclusions, and other pathological changes in cortical regions. These samples were graded on an ordinal scale and genotyped for variants associated with synucleinopathies. We assessed the interval from onset of motor symptoms to onset of dementia, and overall survival in groups with varying levels of comorbid Alzheimer\u27s disease pathology according to US National Institute on Aging–Alzheimer\u27s Association neuropathological criteria, and used multivariate regression to control for age at death and sex. Findings On the basis of data from 213 patients who had been followed up to autopsy and met inclusion criteria of Lewy body disorder with autopsy-confirmed α synucleinopathy, we identified 49 (23%) patients with no Alzheimer\u27s disease neuropathology, 56 (26%) with low-level Alzheimer\u27s disease neuropathology, 45 (21%) with intermediate-level Alzheimer\u27s disease neuropathology, and 63 (30%) with high-level Alzheimer\u27s disease neuropathology. As levels of Alzheimer\u27s disease neuropathology increased, cerebral α-synuclein scores were higher, and the interval between onset of motor and dementia symptoms and disease duration was shorter (p \u3c 0·0001 for all comparisons). Multivariate regression showed independent negative associations of cerebral tau neurofibrillary tangles score with the interval between onset of motor and dementia symptoms (β −4·0, 95% CI −5·5 to −2·6; p \u3c 0·0001; R 2 0·22, p \u3c 0·0001) and with survival (–2·0, −3·2 to −0·8; 0·003; 0·15, \u3c 0·0001) in models that included age at death, sex, cerebral neuritic plaque scores, cerebral α-synuclein scores, presence of cerebrovascular disease, MAPT haplotype, and APOE genotype as covariates. Interpretation Alzheimer\u27s disease neuropathology is common in synucleinopathies and confers a worse prognosis for each increasing level of neuropathological change. Cerebral neurofibrillary tangles burden, in addition to α-synuclein pathology and amyloid plaque pathology, are the strongest pathological predictors of a shorter interval between onset of motor and dementia symptoms and survival. Diagnostic criteria based on reliable biomarkers for Alzheimer\u27s disease neuropathology in synucleinopathies should help to identify the most appropriate patients for clinical trials of emerging therapies targeting tau, amyloid-β or α synuclein, and to stratify them by level of Alzheimer\u27s disease neuropathology

The Primarily Undergraduate Nanomaterials Cooperative: A New Model for Supporting Collaborative Research at Small Institutions on a National Scale

The Primarily Undergraduate Nanomaterials Cooperative (PUNC) is an organization for research-active faculty studying nanomaterials at Primarily Undergraduate Institutions (PUIs), where undergraduate teaching and research go hand-in-hand. In this perspective, we outline the differences in maintaining an active research group at a PUI compared to an R1 institution. We also discuss the work of PUNC, which focuses on community building, instrument sharing, and facilitating new collaborations. Currently consisting of 37 members from across the United States, PUNC has created an online community consisting of its Web site (nanocooperative.org), a weekly online summer group meeting program for faculty and students, and a Discord server for informal conversations. Additionally, in-person symposia at ACS conferences and PUNC-specific conferences are planned for the future. It is our hope that in the years to come PUNC will be seen as a model organization for community building and research support at primarily undergraduate institutions

The Yeast La Related Protein Slf1p Is a Key Activator of Translation during the Oxidative Stress Response

The mechanisms by which RNA-binding proteins control the translation of subsets of mRNAs are not yet clear. Slf1p and Sro9p are atypical-La motif containing proteins which are members of a superfamily of RNA-binding proteins conserved in eukaryotes. RIP-Seq analysis of these two yeast proteins identified overlapping and distinct sets of mRNA targets, including highly translated mRNAs such as those encoding ribosomal proteins. In paralell, transcriptome analysis of slf1Δ and sro9Δ mutant strains indicated altered gene expression in similar functional classes of mRNAs following loss of each factor. The loss of SLF1 had a greater impact on the transcriptome, and in particular, revealed changes in genes involved in the oxidative stress response. slf1Δ cells are more sensitive to oxidants and RIP-Seq analysis of oxidatively stressed cells enriched Slf1p targets encoding antioxidants and other proteins required for oxidant tolerance. To quantify these effects at the protein level, we used label-free mass spectrometry to compare the proteomes of wild-type and slf1Δ strains following oxidative stress. This analysis identified several proteins which are normally induced in response to hydrogen peroxide, but where this increase is attenuated in the slf1Δ mutant. Importantly, a significant number of the mRNAs encoding these targets were also identified as Slf1p-mRNA targets. We show that Slf1p remains associated with the few translating ribosomes following hydrogen peroxide stress and that Slf1p co-immunoprecipitates ribosomes and members of the eIF4E/eIF4G/Pab1p ‘closed loop’ complex suggesting that Slf1p interacts with actively translated mRNAs following stress. Finally, mutational analysis of SLF1 revealed a novel ribosome interacting domain in Slf1p, independent of its RNA binding La-motif. Together, our results indicate that Slf1p mediates a translational response to oxidative stress via mRNA-specific translational control

The geochemical cycling of reactive chlorine through the marine troposphere

Heterogeneous reactions involving sea‐salt aerosol in the marine troposphere are the major global source for volatile inorganic chlorine. We measured reactant and product species hypothesized to be associated with these chemical transformations as a function of phase, particle size, and altitude over the North Atlantic Ocean during the summer of 1988. Concentrations of HCl were typically less than 1.0 ppbv near the sea surface and decreased with altitude and with distance from the U.S. east coast. Concentrations of Cl volatilized from aerosols were generally equivalent to the corresponding concentrations of HCl and ranged from less than detection limits to 125 nmol m−3 STP. Highest absolute and percentage losses of particulate Cl were typically associated with elevated concentrations of anthropogenic combustion products. Concentrations of product nss SO42− and N03− in coarse aerosol fractions indicate that on average only 38% of measured Cl− deficits could be accounted for by the combined effects of acid‐base desorption and reactions involving nonacidic N gases. We hypothesize a mechanism for the Cl loss initiated by reaction of O3 at sea‐salt aerosol surfaces, generating Cl2 followed by rapid photochemical conversion of Cl2 to HCl via Cl atoms (Cl˙) and eventual recapture of HCl by the aerosol. Simulations with a zero‐dimension (0‐D) photochemical model suggest that oxidation by Cl˙ may be an important tropospheric sink for dimethyl sulfide and hydrocarbons. Under low‐NOx conditions, the rapid cycling of reactive Cl would provide a catalytic loss mechanism for O3, which would possibly explain the low O3 concentrations often observed above the world\u27s oceans

Target cell-specific synaptic dynamics of excitatory to inhibitory neuron connections in supragranular layers of human neocortex.

Rodent studies have demonstrated that synaptic dynamics from excitatory to inhibitory neuron types are often dependent on the target cell type. However, these target cell-specific properties have not been well investigated in human cortex, where there are major technical challenges in reliably obtaining healthy tissue, conducting multiple patch-clamp recordings on inhibitory cell types, and identifying those cell types. Here, we take advantage of newly developed methods for human neurosurgical tissue analysis with multiple patch-clamp recordings, post-hoc fluorescent in situ hybridization (FISH), machine learning-based cell type classification and prospective GABAergic AAV-based labeling to investigate synaptic properties between pyramidal neurons and PVALB- vs. SST-positive interneurons. We find that there are robust molecular differences in synapse-associated genes between these neuron types, and that individual presynaptic pyramidal neurons evoke postsynaptic responses with heterogeneous synaptic dynamics in different postsynaptic cell types. Using molecular identification with FISH and classifiers based on transcriptomically identified PVALB neurons analyzed by Patch-seq, we find that PVALB neurons typically show depressing synaptic characteristics, whereas other interneuron types including SST-positive neurons show facilitating characteristics. Together, these data support the existence of target cell-specific synaptic properties in human cortex that are similar to rodent, thereby indicating evolutionary conservation of local circuit connectivity motifs from excitatory to inhibitory neurons and their synaptic dynamics

Platelet-rich plasma in Achilles tendon healing 2 (PATH-2) trial: statistical analysis plan for a multicentre, double-blinded, parallel-group, placebo-controlled randomised clinical trial.

BACKGROUND: There has been a recent steep growth in platelet-rich plasma (PRP) use for musculoskeletal conditions, but findings from high quality clinical trial data are lacking in the literature. Here, we describe the statistical analysis plan (SAP) for the Platelet-rich plasma in Achilles Tendon Healing 2 (PATH-2) trial. METHODS: PATH-2 is a pragmatic, parallel-group, multi-centre, double-blinded, randomised, placebo-controlled, superiority trial. The study aims to evaluate the clinical efficacy of PRP in acute Achilles tendon rupture in terms of muscle-tendon function. Patients are identified in the orthopaedic/trauma outpatient clinic. The primary outcome is muscle-tendon work capacity from the Heel Rise Endurance Test result, expressed as the Limb Symmetry Index (work, in joules), at 24 weeks post randomisation. Multivariate linear regression adjusting for the stratification factors (centre and age) and additional prognostic factors will be used to investigate the adjusted effect of the intervention. The analysis will be by modified intention-to-treat. Sensitivity analysis will assess the internal validity of the trial results by performing a per-protocol analysis. Safety will be summarised by treatment arm for all patients who started treatment. Secondary patient-reported outcome measures will be analysed using linear mixed effects models to allow all data collected at all follow-up points to be considered. Missing data will be summarised and reported by treatment arm. Missing data imputation will be performed, if appropriate. DISCUSSION: The PATH-2 trial will be reported in accordance with the CONSORT statement. This SAP publication will avoid bias arising from prior knowledge of the study results. Any changes or deviations from the current SAP will be described and justified in the final report. TRIAL REGISTRATION: ISRCTN registry: ISRCTN54992179 , assigned 12 January 2015. ClinicalTrials.gov: NCT02302664, received 18 November 2014. UK Clinical Research Network Study Portfolio Database: ID 17850

The RNA binding protein HuR differentially regulates unique subsets of mRNAs in estrogen receptor negative and estrogen receptor positive breast cancer

<p>Abstract</p> <p>Background</p> <p>The discordance between steady-state levels of mRNAs and protein has been attributed to posttranscriptional control mechanisms affecting mRNA stability and translation. Traditional methods of genome wide microarray analysis, profiling steady-state levels of mRNA, may miss important mRNA targets owing to significant posttranscriptional gene regulation by RNA binding proteins (RBPs).</p> <p>Methods</p> <p>The ribonomic approach, utilizing RNA immunoprecipitation hybridized to microarray (RIP-Chip), provides global identification of putative endogenous mRNA targets of different RBPs. HuR is an RBP that binds to the AU-rich elements (ARE) of labile mRNAs, such as proto-oncogenes, facilitating their translation into protein. HuR has been shown to play a role in cancer progression and elevated levels of cytoplasmic HuR directly correlate with increased invasiveness and poor prognosis for many cancers, including those of the breast. HuR has been described to control genes in several of the acquired capabilities of cancer and has been hypothesized to be a tumor-maintenance gene, allowing for cancers to proliferate once they are established.</p> <p>Results</p> <p>We used HuR RIP-Chip as a comprehensive and systematic method to survey breast cancer target genes in both MCF-7 (estrogen receptor positive, ER+) and MDA-MB-231 (estrogen receptor negative, ER-) breast cancer cell lines. We identified unique subsets of HuR-associated mRNAs found individually or in both cell types. Two novel HuR targets, <it>CD9 </it>and <it>CALM2 </it>mRNAs, were identified and validated by quantitative RT-PCR and biotin pull-down analysis.</p> <p>Conclusion</p> <p>This is the first report of a side-by-side genome-wide comparison of HuR-associated targets in wild type ER+ and ER- breast cancer. We found distinct, differentially expressed subsets of cancer related genes in ER+ and ER- breast cancer cell lines, and noted that the differential regulation of two cancer-related genes by HuR was contingent upon the cellular environment.</p