The Cleavable Carboxyl-Terminus of the Small Coat Protein of Cowpea Mosaic Virus Is Involved in RNA Encapsidation

AbstractThe site of cleavage of the small coat protein of cowpea mosaic virus has been precisely mapped and the proteolysis has been shown to result in the loss of 24 amino acids from the carboxyl-terminus of the protein. A series of premature termination and deletion mutants was constructed to investigate the role or roles of these carboxyl-terminal amino acids in the viral replication cycle. Mutants containing premature termination codons at or downstream of the cleavage site were viable but reverted to wild-type after a single passage through cowpea plants, indicating that the carboxyl-terminal amino acids are important. Mutants with the equivalent deletions were genetically stable and shown to be debilitated with respect to virus accumulation. The specific infectivity of preparations of a deletion mutant (DM4) lacking all 24 amino acids was 6-fold less than that of a wild-type preparation. This was shown to be a result of DM4 preparations containing a much increased percentage (73%) of empty (RNA-free) particles, a finding that implicates the cleavable carboxyl-terminal residues in the packaging of the virion RNAs

Structures Illuminate Cardiac Ion Channel Functions in Health and in Long QT Syndrome

The cardiac action potential is critical to the production of a synchronized heartbeat. This electrical impulse is governed by the intricate activity of cardiac ion channels, among them the cardiac voltage-gated potassium (Kv) channels KCNQ1 and hERG as well as the voltage-gated sodium (Nav) channel encoded by SCN5A. Each channel performs a highly distinct function, despite sharing a common topology and structural components. These three channels are also the primary proteins mutated in congenital long QT syndrome (LQTS), a genetic condition that predisposes to cardiac arrhythmia and sudden cardiac death due to impaired repolarization of the action potential and has a particular proclivity for reentrant ventricular arrhythmias. Recent cryo-electron microscopy structures of human KCNQ1 and hERG, along with the rat homolog of SCN5A and other mammalian sodium channels, provide atomic-level insight into the structure and function of these proteins that advance our understanding of their distinct functions in the cardiac action potential, as well as the molecular basis of LQTS. In this review, the gating, regulation, LQTS mechanisms, and pharmacological properties of KCNQ1, hERG, and SCN5A are discussed in light of these recent structural findings

Peripheral Administration of the Soluble TNF Inhibitor XPro1595 Modifies Brain Immune Cell Profiles, Decreases Beta-Amyloid Plaque Load, and Rescues Impaired Long-Term Potentiation in 5xFAD Mice

Clinical and animal model studies have implicated inflammation and peripheral immune cell responses in the pathophysiology of Alzheimer’s disease (AD). Peripheral immune cells including T cells circulate in the cerebrospinal fluid (CSF) of healthy adults and are found in the brains of AD patients and AD rodent models. Blocking entry of peripheral macrophages into the CNS was reported to increase amyloid burden in an AD mouse model. To assess inflammation in the 5xFAD (Tg) mouse model, we first quantified central and immune cell profiles in the deep cervical lymph nodes and spleen. In the brains of Tg mice, activated (MHCII+, CD45high, and Ly6Chigh) myeloid-derived CD11b+ immune cells are decreased while CD3+ T cells are increased as a function of age relative to non-Tg mice. These immunological changes along with evidence of increased mRNA levels for several cytokines suggest that immune regulation and trafficking patterns are altered in Tg mice. Levels of soluble Tumor Necrosis Factor (sTNF) modulate blood-brain barrier (BBB) permeability and are increased in CSF and brain parenchyma post-mortem in AD subjects and Tg mice. We report here that in vivo peripheral administration of XPro1595, a novel biologic that sequesters sTNF into inactive heterotrimers, reduced the age-dependent increase in activated immune cells in Tg mice, while decreasing the overall number of CD4+ T cells. In addition, XPro1595 treatment in vivo rescued impaired long-term potentiation (LTP) measured in brain slices in association with decreased Aβ plaques in the subiculum. Selective targeting of sTNF may modulate brain immune cell infiltration, and prevent or delay neuronal dysfunction in AD

Functional and emotional outcomes after transient ischaemic attack: A 12-month prospective controlled cohort study

Background: Symptoms of transient ischemic attack are believed to fully resolve within 24 h of onset. Emerging evidence suggests that there may be prolonged functional and psychological impact, although studies have not been able to robustly identify whether these are the effect of transient ischemic attack or changes usually associated with ageing. We describe trajectories of disability and risk of anxiety and depression among patients seen at transient ischemic attack clinics over 12 months, compared to healthy controls. Methods: Thirty transient ischemic attack clinics across England participated. A total of 1320 participants were included: 373 diagnosed with transient ischemic attack, 186 with minor stroke, 310 with “possible transient ischemic attack,” 213 with another condition mimicking a transient ischemic attack and 238 controls recruited from primary care providers. Participants completed questionnaires after diagnosis then after 3, 6 and 12 months. Outcomes were the Nottingham Extended Activities of Daily Living Scale and the Hospital Anxiety and Depression Scale. Mixed effects regression was used to estimate group differences and trajectories. Results: At baseline, confirmed transient ischemic attack patients scored 1.31 HADS-Anxiety points (s.e. = 0.28; p < 0.001), 0.51 HADS-Depression points (s.e. = 0.26; p = 0.056), and 2.6 NEADL points (s.e. = 1.1; p = 0.020) worse than controls. At 12 months, the deficits were 0.78 (s.e. = 0.30; p = 0.008), 0.97 (s.e. = 0.23; p < 0.001), and 0.96 (s.e. = 0.92; p = 0.294) respectively. Differences among patients diagnosed with minor stroke were like or worse than transient ischemic attack patients. Conclusions: Transient ischemic attack clinic patients may have functional and emotional impairments compared to the general population irrespective of final diagnosis. The presence of emotional symptoms or risk of developing anxiety or depression did not always fully recover and may increase

ARL3 mutations cause Joubert syndrome by disrupting ciliary protein composition

Joubert syndrome (JBTS) is a genetically heterogeneous autosomal recessive neurodevelopmental ciliopathy. We investigated further the underlying genetic etiology of Joubert syndrome by studying two unrelated families in whom JBTS was not associated with pathogenic variants in known JBTSrelated genes. Combined autozygosity mapping of both families highlighted a candidate locus on chromosome 10 (chr10: 101569997-109106128 (hg 19)), and exome sequencing revealed two missense variants in ARL3 within the candidate locus. The encoded protein, ADP Ribosylation Factor-Like GTPase 3, ARL3, is a small GTP-binding protein that is involved in directing lipid-modified proteins into the cilium in a GTP-dependent manner. Both missense variants replace the highly conserved Arg149 residue, which we show to be necessary for the interaction with its guanine nucleotide exchange factor ARL13B, such that the mutant protein is associated with reduced INPP5E and NPHP3 localisation in cilia. We propose that ARL3 provides a potential hub in the network of encoded ciliopathy genes, whereby perturbation of ARL3 results in the mislocalisation of multiple ciliary proteins due to abnormal displacement of lipidated protein cargo

Transcriptome analysis identifies an ASD-Like phenotype in oligodendrocytes and microglia from C58/J amygdala that is dependent on sex and sociability

Background Autism Spectrum Disorder (ASD) is a group of neurodevelopmental disorders with higher incidence in males and is characterized by atypical verbal/nonverbal communication, restricted interests that can be accompanied by repetitive behavior, and disturbances in social behavior. This study investigated brain mechanisms that contribute to sociability deficits and sex differences in an ASD animal model. Methods Sociability was measured in C58/J and C57BL/6J mice using the 3-chamber social choice test. Bulk RNA-Seq and snRNA-Seq identified transcriptional changes in C58/J and C57BL/6J amygdala within which DMRseq was used to measure differentially methylated regions in amygdala.ResultsC58/J mice displayed divergent social strata in the 3-chamber test. Transcriptional and pathway signatures revealed immune-related biological processes differ between C58/J and C57BL/6J amygdala. Hypermethylated and hypomethylated genes were identified in C58/J versus C57BL/6J amygdala. snRNA-Seq data in C58/J amygdala identified differential transcriptional signatures within oligodendrocytes and microglia characterized by increased ASD risk gene expression and predicted impaired myelination that was dependent on sex and sociability. RNA velocity, gene regulatory network, and cell communication analysis showed diminished oligodendrocyte/microglia differentiation. Findings were verified using Bulk RNA-Seq and demonstrated oxytocin’s beneficial effects on myelin gene expression. Limitations Our findings are significant. However, limitations can be noted. The cellular mechanisms linking reduced oligodendrocyte differentiation and reduced myelination to an ASD phenotype in C58/J mice need further investigation. Additional snRNA-Seq and spatial studies would determine if effects in oligodendrocytes/microglia are unique to amygdala or if this occurs in other brain regions. Oxytocin’s effects need further examination to understand its’ potential as an ASD therapeutic.ConclusionsOur work demonstrates the C58/J mouse model’s utility in evaluating the influence of sex and sociability on the transcriptome in concomitant brain regions involved in ASD. Our single-nucleus transcriptome analysis elucidates potential pathological roles of oligodendrocytes and microglia in ASD. This investigation provides details regarding regulatory features disrupted in these cell types, including transcriptional gene dysregulation, aberrant cell differentiation, altered gene regulatory networks, and changes to key pathways that promote microglia/oligodendrocyte differentiation. Our studies provide insight into interactions between genetic risk and epigenetic processes associated with divergent affiliative behavior and lack of positive sociability

Randomized Trial of Four Financial-Incentive Programs for Smoking Cessation

Background Financial incentives promote many health behaviors, but effective ways to deliver health incentives remain uncertain. Methods We randomly assigned CVS Caremark employees and their relatives and friends to one of four incentive programs or to usual care for smoking cessation. Two of the incentive programs targeted individuals, and two targeted groups of six participants. One of the individual-oriented programs and one of the group-oriented programs entailed rewards of approximately $800 for smoking cessation; the others entailed refundable deposits of$150 plus $650 in reward payments for successful participants. Usual care included informational resources and free smoking-cessation aids. Results Overall, 2538 participants were enrolled. Of those assigned to reward-based programs, 90.0% accepted this assignment, as compared with 13.7% of those assigned to deposit-based programs (P Conclusions Reward-based programs were much more commonly accepted than deposit-based programs, leading to higher rates of sustained abstinence from smoking. Group-oriented incentive programs were no more effective than individual-oriented programs

Predicting mortality in sick African children: the FEAST Paediatric Emergency Triage (PET) Score.

BACKGROUND: Mortality in paediatric emergency care units in Africa often occurs within the first 24 h of admission and remains high. Alongside effective triage systems, a practical clinical bedside risk score to identify those at greatest risk could contribute to reducing mortality. METHODS: Data collected during the Fluid As Expansive Supportive Therapy (FEAST) trial, a multi-centre trial involving 3,170 severely ill African children, were analysed to identify clinical and laboratory prognostic factors for mortality. Multivariable Cox regression was used to build a model in this derivation dataset based on clinical parameters that could be quickly and easily assessed at the bedside. A score developed from the model coefficients was externally validated in two admissions datasets from Kilifi District Hospital, Kenya, and compared to published risk scores using Area Under the Receiver Operating Curve (AUROC) and Hosmer-Lemeshow tests. The Net Reclassification Index (NRI) was used to identify additional laboratory prognostic factors. RESULTS: A risk score using 8 clinical variables (temperature, heart rate, capillary refill time, conscious level, severe pallor, respiratory distress, lung crepitations, and weak pulse volume) was developed. The score ranged from 0-10 and had an AUROC of 0.82 (95 % CI, 0.77-0.87) in the FEAST trial derivation set. In the independent validation datasets, the score had an AUROC of 0.77 (95 % CI, 0.72-0.82) amongst admissions to a paediatric high dependency ward and 0.86 (95 % CI, 0.82-0.89) amongst general paediatric admissions. This discriminative ability was similar to, or better than other risk scores in the validation datasets. NRI identified lactate, blood urea nitrogen, and pH to be important prognostic laboratory variables that could add information to the clinical score. CONCLUSIONS: Eight clinical prognostic factors that could be rapidly assessed by healthcare staff for triage were combined to create the FEAST Paediatric Emergency Triage (PET) score and externally validated. The score discriminated those at highest risk of fatal outcome at the point of hospital admission and compared well to other published risk scores. Further laboratory tests were also identified as prognostic factors which could be added if resources were available or as indices of severity for comparison between centres in future research studies

Vaginal Colonization with Staphylococcus aureus in Healthy Women: A Review of Four Studies

Four studies assessed the frequency of vaginal Staphylococcus aureus colonization in healthy women and associated risk factors. An association was found between S. aureus vaginal colonization and colonization at the labia minora and the anterior nares. Significant risk factors associated with an increased risk of vaginal S. aureus in at least one study were a history of genital herpes simplex infection, insertion of tampons without an applicator, and the use of Rely (Procter & Gamble) tampons. The use of systemic antibiotics within 2 weeks of the vaginal culture decreased the risk of recovery of S. aureus. The overall frequency of vaginal S. aureus in the 808 women in the four studies was 9.2%

Clinical and pharmacogenetic predictors of circulating atorvastatin and rosuvastatin concentrations in routine clinical care

Background-A barrier to statin therapy is myopathy associated with elevated systemic drug exposure. Our objective was to examine the association between clinical and pharmacogenetic variables and statin concentrations in patients. Methods and Results-In total, 299 patients taking atorvastatin or rosuvastatin were prospectively recruited at an outpatient referral center. The contribution of clinical variables and transporter gene polymorphisms to statin concentration was assessed using multiple linear regression. We observed 45-fold variation in statin concentration among patients taking the same dose. After adjustment for sex, age, body mass index, ethnicity, dose, and time from last dose, SLCO1B1 c.521T\u3eC (P\u3c0.001) and ABCG2 c.421C\u3eA (P\u3c0.01) were important to rosuvastatin concentration (adjusted R2=0.56 for the final model). Atorvastatin concentration was associated with SLCO1B1 c.388A\u3eG (P\u3c0.01) and c.521T\u3eC (P\u3c0.05) and 4β-hydroxycholesterol, a CYP3A activity marker (adjusted R2=0.47). A second cohort of 579 patients from primary and specialty care databases were retrospectively genotyped. In this cohort, genotypes associated with statin concentration were not differently distributed among dosing groups, implying providers had not yet optimized each patient\u27s risk-benefit ratio. Nearly 50% of patients in routine practice taking the highest doses were predicted to have statin concentrations greater than the 90th percentile. Conclusions-Interindividual variability in statin exposure in patients is associated with uptake and efflux transporter polymorphisms. An algorithm incorporating genomic and clinical variables to avoid high atorvastatin and rosuvastatin levels is described; further study will determine whether this approach reduces incidence of statin myopathy. © 2013 American Heart Association, Inc