A Practical Guide to Phage- and Robotics-assisted Near-continuous Evolution.

Robotics-accelerated Evolution techniques improve the reliability and speed of evolution using feedback control, improving the outcomes of protein and organism evolution experiments. In this article, we present a guide to setting up the hardware and software necessary to implement Phage- and Robotics-assisted Near-continuous Evolution (PRANCE). PRANCE combines fast phage-based molecular evolution with the ability to run hundreds of independent, feedback-controlled evolution experiments simultaneously. This paper will describe the hardware requirements and setup for PRANCE, including a liquid-handling instrument, a plate reader, auxiliary pumps, heaters, and 3D-printed containers. We describe how to configure the liquid handling robot to be compatible with Python-based open-source software. Finally, we provide suggestions for the first two experiments that can be conducted with a newly constructed PRANCE system that exercises its capabilities and validates that the system is ready to conduct multiplexed evolution. This guide is intended to serve as a handbook for navigating the considerable equipment setup associated with conducting robotics-accelerated evolution.

Metabolic adverse events associated with systemic corticosteroid therapy-a systematic review and meta-analysis.

OBJECTIVES: To assess the risk of new-onset or worsening hyperglycaemia, hypertension, weight gain and hyperlipidaemia with systemic corticosteroid therapy (CST) as reported in published randomised control trial (RCT) studies. DATA SOURCES: Literature search using MEDLINE, EMBASE, Cochrane library, Web of Science and Scopus STUDY ELIGIBILITY CRITERIA: Published articles on results of RCT with a systemic CST arm with numerical data presented on adverse effect (AE). PARTICIPANTS AND INTERVENTIONS: Reports of hyperglycaemia, hypertension, weight gain and hyperlipidaemia associated with systemic CST in patients or healthy volunteer's ≥17 years of age. STUDY APPRAISAL METHODS: Risk of bias tool, assessment at the level of AE and key study characteristics. RESULTS: A total of 5446 articles were screened to include 118 studies with 152 systemic CST arms (total participants=17 113 among which 8569 participants treated with CST). Pooled prevalence of hyperglycaemia in the CST arms within the studies was 10% (95% CI 7% to 14%), with the highest prevalence in respiratory illnesses at 22% (95% CI 9% to 35%). Pooled prevalence of severe hyperglycaemia, hypertension, weight gain and hyperlipidaemia within the corticosteroid arms was 5% (95% CI 2% to 9%), 6% (95% CI 4% to 8%), 13% (95% CI 8% to 18%), 8% (95% CI 4% to 17%), respectively. CST was significantly associated hyperglycaemia, hypertension and weight gain as noted in double-blinded placebo-controlled parallel-arms studies: OR of 2.13 (95% CI 1.66 to 2.72), 1.68 (95% CI 0.96 to 2.95) and 5.20 (95% CI 2.10 to 12.90), respectively. Intravenous therapy posed higher risk than oral therapy: OR of 2.39 (95% CI 1.16 to 4.91). LIMITATIONS: There was significant heterogeneity in the AE definitions and quality of AE reporting in the primary studies and patient populations in the studies. The impact of cumulative dose effect on incidental AE could not be calculated. CONCLUSIONS AND IMPLICATIONS OF KEY FINDINGS: Systemic CST use is associated with increased risk of metabolic AEs, which differs for each disease group and route of administration. PROSPERO REGISTRATION NUMBER: CRD42020161270.

TRIM7 Restricts Coxsackievirus and Norovirus Infection by Detecting the C-Terminal Glutamine Generated by 3C Protease Processing.

TRIM7 catalyzes the ubiquitination of multiple substrates with unrelated biological functions. This cross-reactivity is at odds with the specificity usually displayed by enzymes, including ubiquitin ligases. Here we show that TRIM7's extreme substrate promiscuity is due to a highly unusual binding mechanism, in which the PRYSPRY domain captures any ligand with a C-terminal helix that terminates in a hydrophobic residue followed by a glutamine. Many of the non-structural proteins found in RNA viruses contain C-terminal glutamines as a result of polyprotein cleavage by 3C protease. This viral processing strategy generates novel substrates for TRIM7 and explains its ability to inhibit Coxsackie virus and norovirus replication. In addition to viral proteins, cellular proteins such as glycogenin have evolved C-termini that make them a TRIM7 substrate. The 'helix-ΦQ' degron motif recognized by TRIM7 is reminiscent of the N-end degron system and is found in ~1% of cellular proteins. These features, together with TRIM7's restricted tissue expression and lack of immune regulation, suggest that viral restriction may not be its physiological function.