Homozygosity Haplotype and Whole-Exome Sequencing Analysis to Identify Potentially Functional Rare Variants Involved in Multiple Sclerosis among Sardinian Families

Multiple Sclerosis (MS) is a complex multifactorial autoimmune disease, whose sex- and age-adjusted prevalence in Sardinia (Italy) is among the highest worldwide. To date, 233 loci were associated with MS and almost 20% of risk heritability is attributable to common genetic variants, but many low-frequency and rare variants remain to be discovered. Here, we aimed to contribute to the understanding of the genetic basis of MS by investigating potentially functional rare variants. To this end, we analyzed thirteen multiplex Sardinian families with Immunochip genotyping data. For five families, Whole Exome Sequencing (WES) data were also available. Firstly, we performed a non-parametric Homozygosity Haplotype analysis for identifying the Region from Common Ancestor (RCA). Then, on these potential disease-linked RCA, we searched for the presence of rare variants shared by the affected individuals by analyzing WES data. We found: (i) a variant (43181034 T > G) in the splicing region on exon 27 of CUL9; (ii) a variant (50245517 A > C) in the splicing region on exon 16 of ATP9A; (iii) a non-synonymous variant (43223539 A > C), on exon 9 of TTBK1; (iv) a non-synonymous variant (42976917 A > C) on exon 9 of PPP2R5D; and v) a variant (109859349-109859354) in 3′UTR of MYO16.     

A Trust‐Based Pact in Research Biobanks. From Theory to Practice

Traditional Informed Consent is becoming increasingly inadequate, especially in the context of research biobanks. How much information is needed by patients for their consent to be truly informed? How does the quality of the information they receive match up to the quality of the information they ought to receive? How can information be conveyed fairly about future, non‐predictable lines of research? To circumvent these difficulties, some scholars have proposed that current consent guidelines should be reassessed, with trust being used as a guiding principle instead of information. Here, we analyse one of these proposals, based on a Participation Pact, which is already being offered to patients at the Istituto Europeo di Oncologia, a comprehensive cancer hospital in Milan, Italy.     

Influences of temperature and threshold effect of NaCl concentration on Alpias vulpinus OCT

Thermodynamic, circular dichroism (CD), and activity measurements have been used to characterize the different conformational states and the effects of NaCl concentrations (0.0-3.0 M) on thermal unfolding of ornithine carbamoyltransferase (OCT) from Alopias vulpinus. Furthermore conformational changes in whole enzyme structure have been monitored by titration of SH-groups. OCT unfolding process follows an irreversible two-state mechanism with a first-order kinetic of denaturation, without breaking-point. NaCl shows very little stabilization effects at low concentration and its action become very important over 1.5 M concentration. The presence of 3.0 M NaCl completely avoids OCT unfolding at 60, 64 and 66 degrees C. Kinetic and thermodynamic parameters are strongly influenced by the presence of high NaCl concentration. Our experiments showed that NaCl stabilization process involved changes in preferential binding, in electrostatic and van der Waals interactions and exposure of buried site and SH-groups. During thermal denaturation, UV-vis and CD spectroscopy show that high salts concentration preserves OCT activity, avoiding exposure of hydrophobic site and destruction of secondary and tertiary structure elements.     

Mechanisms of zoonotic severe acute respiratory syndrome coronavirus host range expansion in human airway epithelium

In 2003, severe acute respiratory syndrome coronavirus (SARS-CoV) emerged and caused over 8,000 human cases of infection and more than 700 deaths worldwide. Zoonotic SARS-CoV likely evolved to infect humans by a series of transmission events between humans and animals for sale in China. Using synthetic biology, we engineered the spike protein (S) from a civet strain, SZ16, into our epidemic strain infectious clone, creating the chimeric virus icSZ16-S, which was infectious but yielded progeny viruses incapable of propagating in vitro. After introducing a K479N mutation within the S receptor binding domain (RBD) of SZ16, the recombinant virus (icSZ16-S K479N) replicated in Vero cells but was severely debilitated in growth. The in vitro evolution of icSZ16-S K479N on human airway epithelial (HAE) cells produced two viruses (icSZ16-S K479N D8 and D22) with enhanced growth on HAE cells and on delayed brain tumor cells expressing the SARS-CoV receptor, human angiotensin I converting enzyme 2 (hACE2). The icSZ16-S K479N D8 and D22 virus RBDs contained mutations in ACE2 contact residues, Y442F and L472F, that remodeled S interactions with hACE2. Further, these viruses were neutralized by a human monoclonal antibody (MAb), S230.15, but the parent icSZ16-S K479N strain was eight times more resistant than the mutants. These data suggest that the human adaptation of zoonotic SARS-CoV strains may select for some variants that are highly susceptible to select MAbs that bind to RBDs. The epidemic, icSZ16-S K479N, and icSZ16-S K479N D22 viruses replicate similarly in the BALB/c mouse lung, highlighting the potential use of these zoonotic spike SARS-CoVs to assess vaccine or serotherapy efficacy in vivo.     

Exome sequences and multi‐environment field trials elucidate the genetic basis of adaptation in barley

Broadening the genetic base of crops is crucial for developing varieties to respond to global agricultural challenges such as climate change. Here, we analysed a diverse panel of 371 domesticated lines of the model crop barley to explore the genetics of crop adaptation. We first collected exome sequence data and phenotypes of key life history traits from contrasting multi‐environment common garden trials. Then we applied refined statistical methods, including some based on exomic haplotype states, for genotype‐by‐environment (G×E) modelling. Sub‐populations defined from exomic profiles were coincident with barley's biology, geography and history, and explained a high proportion of trial phenotypic variance. Clear G×E interactions indicated adaptation profiles that varied for landraces and cultivars. Exploration of circadian clock‐related genes, associated with the environmentally adaptive days to heading trait (crucial for the crop's spread from the Fertile Crescent), illustrated complexities in G×E effect directions, and the importance of latitudinally based genic context in the expression of large‐effect alleles. Our analysis supports a gene‐level scientific understanding of crop adaption and leads to practical opportunities for crop improvement, allowing the prioritisation of genomic regions and particular sets of lines for breeding efforts seeking to cope with climate change and other stresses.     

Clinical Efficacy and Safety of Ezetimibe on Major Cardiovascular Endpoints: Systematic Review and Meta-Analysis of Randomized Controlled Trials

Background

Randomized clinical trials (RCTs) about Ezetimibe''s efficacy on patient-oriented outcomes have given discordant results. The aim of this study was to determine the net effect of Ezetimibe and of the widely marketed combination, Ezetimibe+simvastatin, on mortality and morbidity outcomes.

Methods and Findings

We searched for RCT on Ezetimibe using MEDLINE, CCTR, EMBASE, ClinicalTrials.gov databases up to December 2013, Merck and Novartis online registers, and personal communications. Two authors independently selected trials fulfilling these criteria: RCTs comparing Ezetimibe±statin or another lipid-lowering drug against placebo, or against the same lipid-lowering drug at the same dosage, with a follow-up at least 24 weeks and one or more of these outcomes: all-cause mortality, cardiovascular (CV) mortality, stroke, myocardial infarction (MI), cancer, serious adverse events (SAEs); we assessed the risk of bias using the Cochrane checklist. We extracted the data for major clinical events as a dichotomous measure, with the patient the unit of analysis. Pooled analysis was done with random and fixed effect based models. Trials comparing Ezetimibe plus a lipid-lowering drug against the same lipidlowering drug representing the net effect of Ezetimibe, showed a nonsignificant tendency toward damage for cancer, MI, stroke and SAEs. Ezetimibe+simvastatin vs. simvastatin alone showed a stronger tendency towards a higher risk for all-cause death (2.52; 0.65-9.74), CV death (3.04; 0.48-19.21), non-CV death (3.03; 0.12-73.50), MI (1.91; 0.42-8.70), stroke (2.38; 0.46-12.35), cancer (RR 11.11; 0.62-198.29), and SAEs (1.45; 0.95-2.23). Limitations include small numbers of events and inadequate power of the pooling. Trials comparing Ezetimibe+simvastatin vs placebo showed non-significant effects: MI (0.81; 0.66-1.00 p = 0.051), all-cause death (1.02; 0.95-1.09), CV death (0.91; 0.80-1.04), non-CV death (108; 0.99-1.18), stroke (0.86; 0.72-1.04), cancer (1.18; 0.80-1.74), SAEs (1.01; 0.96-1.06).

Conclusions

Ezetimibe±simvastatin had inconsistent effects on important outcomes. No firm conclusions are possible, but findings indicative of damage suggest much more selective use of Ezetimibe±simvastatin.     

Urinary proteomics for the study of genetic kidney diseases

Despite their low prevalence, genetic kidney diseases (GKD) still represent a serious health problem. They often lead to kidney failure and to the consequent need of dialysis or kidney transplant. To date, reliable diagnosis requires laborious genetic tests and/or a renal biopsy. Moreover, only scant and non-specific markers exist for prognostic purposes. Biomarkers assayed in an easily available and low-cost sample, such as urine, would be highly valuable. Urinary proteomics can provide clues related to their development through the identification of differentially expressed proteins codified by the affected genes, or other dis-regulated species, in total or fractionated urine, providing novel mechanistic insights. In this review, the authors summarize and discuss the results of the main proteomic investigations on GKD urine samples and in urinary extracellular vesicles.     

VS411 Reduced Immune Activation and HIV-1 RNA Levels in 28 Days: Randomized Proof-of-Concept Study for AntiViral-HyperActivation Limiting Therapeutics

Background

A new class of antiretrovirals, AntiViral-HyperActivation Limiting Therapeutics (AV-HALTs), has been proposed as a disease-modifying therapy to both reduce Human Immunodeficiency Virus Type 1 (HIV-1) RNA levels and the excessive immune activation now recognized as the major driver of not only the continual loss of CD4⁺ T cells and progression to Acquired Immunodeficiency Syndrome (AIDS), but also of the emergence of both AIDS-defining and non-AIDS events that negatively impact upon morbidity and mortality despite successful (ie, fully suppressive) therapy. VS411, the first-in-class AV-HALT, combined low-dose, slow-release didanosine with low-dose hydroxycarbamide to accomplish both objectives with a favorable toxicity profile during short-term administration. Five dose combinations were administered as VS411 to test the AV-HALT Proof-of-Concept in HIV-1-infected subjects.

Methods

Multinational, double-blind, 28-day Phase 2a dose-ranging Proof-of-Concept study of antiviral activity, immunological parameters, safety, and genotypic resistance in 58 evaluable antiretroviral-naïve HIV-1-infected adults. Randomization and allocation to study arms were carried out by a central computer system. Results were analyzed by ANOVA, Kruskal-Wallis, ANCOVA, and two-tailed paired t tests.

Results

VS411 was well-tolerated, produced significant reductions of HIV-1 RNA levels, increased CD4⁺ T cell counts, and led to significant, rapid, unprecedented reductions of immune activation markers after 28 days despite incomplete viral suppression and without inhibiting HIV-1-specific immune responses. The didanosine 200 mg/HC 900 mg once-daily formulation demonstrated the greatest antiviral efficacy (HIV-1 RNA: −1.47 log₁₀ copies/mL; CD4⁺ T cell count: +135 cells/mm³) and fewest adverse events.

Conclusions

VS411 successfully established the Proof-of-Concept that AV-HALTs can combine antiviral efficacy with rapid, potentially beneficial reductions in the excessive immune system activation associated with HIV-1 disease. Rapid reductions in markers of immune system hyperactivation and cellular proliferation were obtained despite the fact that VS411 did not attain maximal suppression of HIV RNA, suggesting this effect was due to the HALT component.

Trial Registration

ITEudraCT 2007-002460-98