The bacterial Type III toxin-antitoxin system, ToxIN, is a dynamic protein-RNA complex with stability-dependent antiviral abortive infection activity.

Bacteria have evolved numerous defense systems to protect themselves from viral (bacteriophage) infection. The ToxIN system of Pectobacterium atrosepticum is a Type III toxin-antitoxin complex and "altruistic suicide" anti-phage system, which kills phage-infected cells through the release of a ribonuclease toxin, ToxN. ToxN is counteracted by a co-transcribed antitoxic RNA pseudoknot, ToxI, which self-assembles with ToxN into an inactive 3 ToxI:3 ToxN complex in vitro. However it is not known whether this complex is predominant in vivo, or how the complex is disassembled following infection to trigger a lethal, "altruistic" response. In this study, we characterise ToxI turnover and folding, and explore the link between complex stability and anti-phage activity, with a view to understanding events that lead to ToxN-mediated suicide following phage infection. We present evidence that ToxN constantly cleaves fresh ToxI in vivo rather than staying associated with pre-processed antitoxin, and that the ToxI antitoxin can partially fold spontaneously using conserved nucleotides. We also show that reducing the stability of the ToxIN complex can increase the strength of the antiviral response in a phage-dependent manner. Based on this information, we propose a revised model for ToxN inhibition, complex assembly and activation by infecting bacteriophage

Broadening substrate specificity of a chain-extending ketosynthase through a single active-site mutation.

An in vitro model system based on a ketosynthase domain of the erythromycin polyketide synthase was used to probe the apparent substrate tolerance of ketosynthase domains of the mycolactone polyketide synthase. A specific residue change was identified that led to an emphatic increase in turnover of a range of substrates.BBSRC (BB/J007250/1)This is the final version of the article. It first appeared from Royal Society of Chemistry] via https://doi.org/10.1039/C6CC03501A

Modular type I polyketide synthase acyl carrier protein domains share a common N-terminally extended fold.

Acyl carrier protein (ACP) domains act as interaction hubs within modular polyketide synthase (PKS) systems, employing specific protein-protein interactions to present acyl substrates to a series of enzyme active sites. Many domains from the multimodular PKS that generates the toxin mycolactone display an unusually high degree of sequence similarity, implying that the few sites which vary may do so for functional reasons. When domain boundaries based on prior studies were used to prepare two isolated ACP segments from this system for studies of their interaction properties, one fragment adopted the expected tertiary structure, but the other failed to fold, despite sharing a sequence identity of 49%. Secondary structure prediction uncovered a previously undetected helical region (H0) that precedes the canonical helix-bundle ACP topology in both cases. This article reports the NMR solution structures of two N-terminally extended mycolactone mACP constructs, mH0ACPa and mH0ACPb, both of which possess an additional α-helix that behaves like a rigid component of the domain. The interactions of these species with a phosphopantetheinyl transferase and a ketoreductase domain are unaffected by the presence of H0, but a shorter construct that lacks the H0 region is shown to be substantially less thermostable than mH0ACPb. Bioinformatics analysis suggests that the extended H0-ACP motif is present in 98% of type I cis-acyltransferase PKS chain-extension modules. The polypeptide linker that connects an H0-ACP motif to the preceding domain must therefore be ~12 residues shorter than previously thought, imposing strict limits on ACP-mediated substrate delivery within and between PKS modules

Cis–trans isomerization at a proline opens the pore of a neurotransmitter-gated ion channel

5-Hydroxytryptamine type 3 (5-HT_3) receptors are members of the Cys-loop receptor superfamily. Neurotransmitter binding in these proteins triggers the opening (gating) of an ion channel by means of an as-yet-uncharacterized conformational change. Here we show that a specific proline (Pro 8*), located at the apex of the loop between the second and third transmembrane helices (M2–M3), can link binding to gating through a cis–trans isomerization of the protein backbone. Using unnatural amino acid mutagenesis, a series of proline analogues with varying preference for the cis conformer was incorporated at the 8* position. Proline analogues that strongly favour the trans conformer produced non-functional channels. Among the functional mutants there was a strong correlation between the intrinsic cis–trans energy gap of the proline analogue and the activation of the channel, suggesting that cis–trans isomerization of this single proline provides the switch that interconverts the open and closed states of the channel. Consistent with this proposal, nuclear magnetic resonance studies on an M2–M3 loop peptide reveal two distinct, structured forms. Our results thus confirm the structure of the M2–M3 loop and the critical role of Pro 8* in the 5-HT_3 receptor. In addition, they suggest that a molecular rearrangement at Pro 8* is the structural mechanism that opens the receptor pore

Discovery of Two Distant Type Ia Supernovae in the Hubble Deep Field North with the Advanced Camera for Surveys

We present observations of the first two supernovae discovered with the recently installed Advanced Camera for Surveys (ACS) on the Hubble Space Telescope. The supernovae were found in Wide Field Camera images of the Hubble Deep Field North taken with the F775W, F850LP, and G800L optical elements as part of the ACS guaranteed time observation program. Spectra extracted from the ACS G800L grism exposures confirm that the objects are Type Ia supernovae (SNe Ia) at redshifts z=0.47 and z=0.95. Follow-up HST observations have been conducted with ACS in F775W and F850LP and with NICMOS in the near-infrared F110W bandpass, yielding a total of 9 flux measurements in the 3 bandpasses over a period of 50 days in the observed frame. We discuss many of the important issues in doing accurate photometry with the ACS. We analyze the multi-band light curves using two different fitting methods to calibrate the supernovae luminosities and place them on the SNe Ia Hubble diagram. The resulting distances are consistent with the redshift-distance relation of the accelerating universe model, although evolving intergalactic grey dust remains as a less likely possibility. The relative ease with which these SNe Ia were found, confirmed, and monitored demonstrates the potential ACS holds for revolutionizing the field of high-redshift SNe Ia, and therefore of testing the accelerating universe cosmology and constraining the "epoch of deceleration".Comment: 11 pages, 8 embedded figures. Accepted for publication in Ap

Bromelain inhibits SARS-CoV-2 infection via targeting ACE-2, TMPRSS2, and spike protein

The new coronavirus, SARS-CoV-2, transmits rapidly from human-to-human resulting in the ongoing pandemic. SARS-CoV-2 infects angiotensin-converting enzyme 2 (ACE-2) expressing lung, heart, kidney, intestine, gall bladder, and testicular tissues of patients, leading to organ failure and sometimes death.1, 2 Currently, COVID-19 patients are treated with different agents, including favilavir, remdesivir, chloroquine, hydroxychloroquine, lopinavir, darunavir, and tocilizumab.3, 4 However, the safety and efficacy of those drugs against COVID-19 still need further confirmation by randomized clinical trials. Hence, there is an emergent need to repurpose the existing drugs or develop new virus-based and host-based antivirals against SARS-CoV-2. Bromelain is a cysteine protease isolated from pineapple stem and is used as a dietary supplement for treating patients with pain, inflammation,5 thrombosis,6 and cancerPeer Reviewe

Analysis of the natively unstructured RNA/protein-recognition core in the Escherichia coli RNA degradosome and its interactions with regulatory RNA/Hfq complexes.

The RNA degradosome is a multi-enzyme assembly that plays a central role in the RNA metabolism of Escherichia coli and numerous other bacterial species including pathogens. At the core of the assembly is the endoribonuclease RNase E, one of the largest E. coli proteins and also one that bears the greatest region predicted to be natively unstructured. This extensive unstructured region, situated in the C-terminal half of RNase E, is punctuated with conserved short linear motifs that recruit partner proteins, direct RNA interactions, and enable association with the cytoplasmic membrane. We have structurally characterized a subassembly of the degradosome-comprising a 248-residue segment of the natively unstructured part of RNase E, the DEAD-box helicase RhlB and the glycolytic enzyme enolase, and provide evidence that it serves as a flexible recognition centre that can co-recruit small regulatory RNA and the RNA chaperone Hfq. Our results support a model in which the degradosome captures substrates and regulatory RNAs through the recognition centre, facilitates pairing to cognate transcripts and presents the target to the ribonuclease active sites of the greater assembly for cooperative degradation or processing

Angiopoietin-Like4 Is a Novel Marker of COVID-19 Severity

IMPORTANCE: Vascular dysfunction and capillary leak are common in critically ill COVID-19 patients, but identification of endothelial pathways involved in COVID-19 pathogenesis has been limited. Angiopoietin-like 4 (ANGPTL4) is a protein secreted in response to hypoxic and nutrient-poor conditions that has a variety of biological effects including vascular injury and capillary leak. OBJECTIVES: To assess the role of ANGPTL4 in COVID-19-related outcomes. DESIGN SETTING AND PARTICIPANTS: Two hundred twenty-five COVID-19 ICU patients were enrolled from April 2020 to May 2021 in a prospective, multicenter cohort study from three different medical centers, University of Washington, University of Southern California and New York University. MAIN OUTCOMES AND MEASURES: Plasma ANGPTL4 was measured on days 1, 7, and 14 after ICU admission. We used previously published tissue proteomic data and lung single nucleus RNA (snRNA) sequencing data from specimens collected from COVID-19 patients to determine the tissues and cells that produce ANGPTL4. RESULTS: Higher plasma ANGPTL4 concentrations were significantly associated with worse hospital mortality (adjusted odds ratio per log CONCLUSIONS AND RELEVANCE: ANGPTL4 is expressed in pulmonary epithelial cells and fibroblasts and is associated with clinical prognosis in critically ill COVID-19 patients

The development and validation of a scoring tool to predict the operative duration of elective laparoscopic cholecystectomy

Background: The ability to accurately predict operative duration has the potential to optimise theatre efficiency and utilisation, thus reducing costs and increasing staff and patient satisfaction. With laparoscopic cholecystectomy being one of the most commonly performed procedures worldwide, a tool to predict operative duration could be extremely beneficial to healthcare organisations. Methods: Data collected from the CholeS study on patients undergoing cholecystectomy in UK and Irish hospitals between 04/2014 and 05/2014 were used to study operative duration. A multivariable binary logistic regression model was produced in order to identify significant independent predictors of long (> 90 min) operations. The resulting model was converted to a risk score, which was subsequently validated on second cohort of patients using ROC curves. Results: After exclusions, data were available for 7227 patients in the derivation (CholeS) cohort. The median operative duration was 60 min (interquartile range 45–85), with 17.7% of operations lasting longer than 90 min. Ten factors were found to be significant independent predictors of operative durations > 90 min, including ASA, age, previous surgical admissions, BMI, gallbladder wall thickness and CBD diameter. A risk score was then produced from these factors, and applied to a cohort of 2405 patients from a tertiary centre for external validation. This returned an area under the ROC curve of 0.708 (SE = 0.013, p  90 min increasing more than eightfold from 5.1 to 41.8% in the extremes of the score. Conclusion: The scoring tool produced in this study was found to be significantly predictive of long operative durations on validation in an external cohort. As such, the tool may have the potential to enable organisations to better organise theatre lists and deliver greater efficiencies in care