Expressing the human proteome for affinity proteomics: optimising expression of soluble protein domains and in vivo biotinylation

The generation of affinity reagents to large numbers of human proteins depends on the ability to express the target proteins as high-quality antigens. The Structural Genomics Consortium (SGC) focuses on the production and structure determination of human proteins. In a 7-year period, the SGC has deposited crystal structures of >800 human protein domains, and has additionally expressed and purified a similar number of protein domains that have not yet been crystallised. The targets include a diversity of protein domains, with an attempt to provide high coverage of protein families. The family approach provides an excellent basis for characterising the selectivity of affinity reagents. We present a summary of the approaches used to generate purified human proteins or protein domains, a test case demonstrating the ability to rapidly generate new proteins, and an optimisation study on the modification of >70 proteins by biotinylation in vivo. These results provide a unique synergy between large-scale structural projects and the recent efforts to produce a wide coverage of affinity reagents to the human proteome

Specific inhibition of diverse pathogens in human cells by synthetic microRNA-like oligonucleotides inferred from RNAi screens

Systematic genetic perturbation screening in human cells remains technically challenging. Typically, large libraries of chemically synthesized siRNA oligonucleotides are used, each designed to degrade a specific cellular mRNA via the RNA interference (RNAi) mechanism. Here, we report on data from three genome-wide siRNA screens, conducted to uncover host factors required for infection of human cells by two bacterial and one viral pathogen. We find that the majority of phenotypic effects of siRNAs are unrelated to the intended “on-target” mechanism, defined by full complementarity of the 21-nt siRNA sequence to a target mRNA. Instead, phenotypes are largely dictated by “off-target” effects resulting from partial complementarity of siRNAs to multiple mRNAs via the “seed” region (i.e., nucleotides 2–8), reminiscent of the way specificity is determined for endogenous microRNAs. Quantitative analysis enabled the prediction of seeds that strongly and specifically block infection, independent of the intended on-target effect. This prediction was confirmed experimentally by designing oligos that do not have any on-target sequence match at all, yet can strongly reproduce the predicted phenotypes. Our results suggest that published RNAi screens have primarily, and unintentionally, screened the sequence space of microRNA seeds instead of the intended on-target space of protein-coding genes. This helps to explain why previously published RNAi screens have exhibited relatively little overlap. Our analysis suggests a possible way of identifying “seed reagents” for controlling phenotypes of interest and establishes a general strategy for extracting valuable untapped information from past and future RNAi screens

Albiglutide and cardiovascular outcomes in patients with type 2 diabetes and cardiovascular disease (Harmony Outcomes): a double-blind, randomised placebo-controlled trial

Background: Glucagon-like peptide 1 receptor agonists differ in chemical structure, duration of action, and in their effects on clinical outcomes. The cardiovascular effects of once-weekly albiglutide in type 2 diabetes are unknown. We aimed to determine the safety and efficacy of albiglutide in preventing cardiovascular death, myocardial infarction, or stroke. Methods: We did a double-blind, randomised, placebo-controlled trial in 610 sites across 28 countries. We randomly assigned patients aged 40 years and older with type 2 diabetes and cardiovascular disease (at a 1:1 ratio) to groups that either received a subcutaneous injection of albiglutide (30–50 mg, based on glycaemic response and tolerability) or of a matched volume of placebo once a week, in addition to their standard care. Investigators used an interactive voice or web response system to obtain treatment assignment, and patients and all study investigators were masked to their treatment allocation. We hypothesised that albiglutide would be non-inferior to placebo for the primary outcome of the first occurrence of cardiovascular death, myocardial infarction, or stroke, which was assessed in the intention-to-treat population. If non-inferiority was confirmed by an upper limit of the 95% CI for a hazard ratio of less than 1·30, closed testing for superiority was prespecified. This study is registered with ClinicalTrials.gov, number NCT02465515. Findings: Patients were screened between July 1, 2015, and Nov 24, 2016. 10 793 patients were screened and 9463 participants were enrolled and randomly assigned to groups: 4731 patients were assigned to receive albiglutide and 4732 patients to receive placebo. On Nov 8, 2017, it was determined that 611 primary endpoints and a median follow-up of at least 1·5 years had accrued, and participants returned for a final visit and discontinuation from study treatment; the last patient visit was on March 12, 2018. These 9463 patients, the intention-to-treat population, were evaluated for a median duration of 1·6 years and were assessed for the primary outcome. The primary composite outcome occurred in 338 (7%) of 4731 patients at an incidence rate of 4·6 events per 100 person-years in the albiglutide group and in 428 (9%) of 4732 patients at an incidence rate of 5·9 events per 100 person-years in the placebo group (hazard ratio 0·78, 95% CI 0·68–0·90), which indicated that albiglutide was superior to placebo (p<0·0001 for non-inferiority; p=0·0006 for superiority). The incidence of acute pancreatitis (ten patients in the albiglutide group and seven patients in the placebo group), pancreatic cancer (six patients in the albiglutide group and five patients in the placebo group), medullary thyroid carcinoma (zero patients in both groups), and other serious adverse events did not differ between the two groups. There were three (<1%) deaths in the placebo group that were assessed by investigators, who were masked to study drug assignment, to be treatment-related and two (<1%) deaths in the albiglutide group. Interpretation: In patients with type 2 diabetes and cardiovascular disease, albiglutide was superior to placebo with respect to major adverse cardiovascular events. Evidence-based glucagon-like peptide 1 receptor agonists should therefore be considered as part of a comprehensive strategy to reduce the risk of cardiovascular events in patients with type 2 diabetes. Funding: GlaxoSmithKline

The SIB Swiss Institute of Bioinformatics' resources: focus on curated databases

The SIB Swiss Institute of Bioinformatics (www.isb-sib.ch) provides world-class bioinformatics databases, software tools, services and training to the international life science community in academia and industry. These solutions allow life scientists to turn the exponentially growing amount of data into knowledge. Here, we provide an overview of SIB's resources and competence areas, with a strong focus on curated databases and SIB's most popular and widely used resources. In particular, SIB's Bioinformatics resource portal ExPASy features over 150 resources, including UniProtKB/Swiss-Prot, ENZYME, PROSITE, neXtProt, STRING, UniCarbKB, SugarBindDB, SwissRegulon, EPD, arrayMap, Bgee, SWISS-MODEL Repository, OMA, OrthoDB and other databases, which are briefly described in this article

Synergistic Effect of Cigarette Smoke and Bacterial Induced Chronic Obstructive Pulmonary Disease Type Airway Inflammation on Promotion of K-ras Mutant Lung Cancer

Lung cancer (LC), specifically K-ras mutant lung cancer, primarily caused by cigarette smoking (CS), is the leading cause of cancer death worldwide. Previous epidemiologic studies have shown a strong association between LC and COPD (chronic obstructive pulmonary disease), an inflammatory disease of the airways caused by CS. These facts suggest a link between COPD-related airway inflammation and LC promotion. NTHi is the most common colonizing bacteria in the lower respiratory tract of patients with COPD and could be the cause of the promoting persistent airway inflammation after CS smoking. We further studied the effect of combined CS and NTHi exposure in the induction of COPD phenotype and promotion of LC. Briefly, 6-week old C57BL/6J and K-ras mutant mice (CC-LR) were exposed to NTHi lysate once a week for 8 weeks and to cigarette smoke (CS) daily for 2 hours/day, 5 days/week for 8 weeks and studied at the age of 14 weeks. The CS caused a mild macrophage dominant airway inflammation, leading to a 2.3-fold increase in lung tumor burden. These results suggest that CS exposure and colonization of smoke-injured airways induce an inflammatory and structural COPD phenotype recapitulating Human COPD, providing a microenvironment promoting K-ras mutant LC. This project was completed with contributions from Seyed Moghaddam from the MD Anderson Cancer Center - Pulmonary Medicine.Honors CollegeBiology and Biochemistry, Department o

immersion - émersion

International audienc

Specific inhibition of diverse pathogens in human cells by synthetic microRNA-like oligonucleotides inferred from RNAi screens

Systematic genetic perturbation screening in human cells remains technically challenging. Typically, large libraries of chemically synthesized siRNA oligonucleotides are used, each designed to degrade a specific cellular mRNA via the RNA interference (RNAi) mechanism. Here, we report on data from three genome-wide siRNA screens, conducted to uncover host factors required for infection of human cells by two bacterial and one viral pathogen. We find that the majority of phenotypic effects of siRNAs are unrelated to the intended “on-target” mechanism, defined by full complementarity of the 21-nt siRNA sequence to a target mRNA. Instead, phenotypes are largely dictated by “off-target” effects resulting from partial complementarity of siRNAs to multiple mRNAs via the “seed” region (i.e., nucleotides 2–8), reminiscent of the way specificity is determined for endogenous microRNAs. Quantitative analysis enabled the prediction of seeds that strongly and specifically block infection, independent of the intended on-target effect. This prediction was confirmed experimentally by designing oligos that do not have any on-target sequence match at all, yet can strongly reproduce the predicted phenotypes. Our results suggest that published RNAi screens have primarily, and unintentionally, screened the sequence space of microRNA seeds instead of the intended on-target space of protein-coding genes. This helps to explain why previously published RNAi screens have exhibited relatively little overlap. Our analysis suggests a possible way of identifying “seed reagents” for controlling phenotypes of interest and establishes a general strategy for extracting valuable untapped information from past and future RNAi screens