Detecting RNA base methylations in single cells by in situ hybridization

Methylated bases in tRNA, rRNA and mRNA control a variety of cellular processes, including protein synthesis, antimicrobial resistance and gene expression. Currently, bulk methods that report the average methylation state of ~104–107 cells are used to detect these modifications, obscuring potentially important biological information. Here, we use in situ hybridization of Molecular Beacons for single-cell detection of three methylations (m62A, m1G and m3U) that destabilize Watson–Crick base pairs. Our method—methylation-sensitive RNA fluorescence in situ hybridization—detects single methylations of rRNA, quantifies antibiotic-resistant bacteria in mixtures of cells and simultaneously detects multiple methylations using multicolor fluorescence imaging

A Rieske oxygenase/epoxide hydrolase-catalysed reaction cascade creates oxygen heterocycles in mupirocin biosynthesis

\ua9 2018, The Author(s), under exclusive licence to Springer Nature Limited.Oxygen heterocycles—in particular, tetrahydropyrans (THPs) and tetrahydrofurans—are common structural features of many biologically active polyketide natural products. Mupirocin is a clinically important antibiotic isolated from Pseudomonas fluorescens and is assembled on a THP ring, which is essential for bioactivity. However, the biosynthesis of this moiety has remained elusive. Here, we show an oxidative enzyme-catalysed cascade that generates the THP ring of mupirocin. Rieske non-haem oxygenase (MupW)-catalysed selective oxidation of the C8–C16 single bond in a complex acyclic precursor is combined with an epoxide hydrolase (MupZ) to catalyse the subsequent regioselective ring formation to give the hydroxylated THP. In the absence of MupZ, a five-membered tetrahydrofuran ring is isolated, and model studies are consistent with cyclization occurring via an epoxide intermediate. High-resolution X-ray crystallographic studies, molecular modelling and mutagenesis experiments of MupZ provide insights into THP ring formation proceeding via an anti-Baldwin 6-endo-tet cyclization

The bristol sponge microbiome collection: A unique repository of deep-sea microorganisms and associated natural products

\ua9 2020 by the authors. Licensee MDPI, Basel, Switzerland. The deep ocean is the largest habitat for life on Earth, though the microorganisms that occupy this unique environmental niche remain largely unexplored. Due to the significant logistical and operational challenges associated with accessing the deep ocean, bioprospecting programmes that seek to generate novel products from marine organisms have, to date, focused predominantly on samples recovered from shallow seas. For this reason, the deep ocean remains a largely untapped resource of novel microbiological life and associated natural products. Here we report the establishment of the Bristol Sponge Microbiome Collection (BISECT), a unique repository of deep-sea microorganisms and associated metabolites isolated from the microbiota of marine sponges, recovered from previously unsurveyed regions of the mid Atlantic Ocean, at depths of 0.3–3 km. An integrated biodiscovery pipeline comprising molecular, genetic, bioinformatic and analytical tools is also described, which is being applied to interrogate this collection. The potential of this approach is illustrated using data reporting our initial efforts to identify antimicrobial natural product lead compounds. Prospects for the use of BISECT to address allied pharmaceutical needs, along with mechanisms of access to the collection are also discussed

Cysteine Methylation Controls Radical Generation in the Cfr Radical AdoMet rRNA Methyltransferase

The 'radical S-adenosyl-L-methionine (AdoMet)' enzyme Cfr methylates adenosine 2503 of the 23S rRNA in the peptidyltransferase centre (P-site) of the bacterial ribosome. This modification protects host bacteria, notably methicillin-resistant Staphylococcus aureus (MRSA), from numerous antibiotics, including agents (e.g. linezolid, retapamulin) that were developed to treat such organisms. Cfr contains a single [4Fe-4S] cluster that binds two separate molecules of AdoMet during the reaction cycle. These are used sequentially to first methylate a cysteine residue, Cys338; and subsequently generate an oxidative radical intermediate that facilitates methyl transfer to the unreactive C8 (and/or C2) carbon centres of adenosine 2503. How the Cfr active site, with its single [4Fe-4S] cluster, catalyses these two distinct activities that each utilise AdoMet as a substrate remains to be established. Here, we use absorbance and electron paramagnetic resonance (EPR) spectroscopy to investigate the interactions of AdoMet with the [4Fe-4S] clusters of wild-type Cfr and a Cys338 Ala mutant, which is unable to accept a methyl group. Cfr binds AdoMet with high (∼ 10 µM) affinity notwithstanding the absence of the RNA cosubstrate. In wild-type Cfr, where Cys338 is methylated, AdoMet binding leads to rapid oxidation of the [4Fe-4S] cluster and production of 5'-deoxyadenosine (DOA). In contrast, while Cys338 Ala Cfr binds AdoMet with equivalent affinity, oxidation of the [4Fe-4S] cluster is not observed. Our results indicate that the presence of a methyl group on Cfr Cys338 is a key determinant of the activity of the enzyme towards AdoMet, thus enabling a single active site to support two distinct modes of AdoMet cleavage

Impact of early haemodynamic goal-directed therapy in patients undergoing emergency surgery: an open prospective, randomised trial

Haemodynamic goal-directed therapies (GDT) may improve outcome following elective major surgery. So far, few data exist regarding haemodynamic optimization during emergency surgery. In this randomized, controlled trial, 50 surgical patients with hypovolemic or septic conditions were enrolled and we compared two algorithms of GDTs based either on conventional parameters and pressure pulse variation (control group) or on cardiac index, global end-diastolic volume index and stroke volume variation as derived from the PiCCO monitoring system (optimized group). Postoperative outcome was estimated by a composite index including major complications and by the Sequential Organ Failure Assessment (SOFA) Score within the first 3 days after surgery (POD1, POD2 and POD3). Data from 43 patients were analyzed (control group, N = 23; optimized group, N = 20). Similar amounts of fluid were given in the two groups. Intraoperatively, dobutamine was given in 45 % optimized patients but in no control patients. Major complications occurred more frequently in the optimized group [19 (95 %) versus 10 (40 %) in the control group, P < 0.001]. Likewise, SOFA scores were higher in the optimized group on POD1 (10.2 ± 2.5 versus 6.6 ± 2.2 in the control group, P = 0.001), POD2 (8.4 ± 2.6 vs 5.0 ± 2.4 in the control group, P = 0.002) and POD 3 (5.2 ± 3.6 and 2.2 ± 1.3 in the control group, P = 0.01). There was no significant difference in hospital mortality (13 % in the control group and 25 % in the optimized group). Haemodynamic optimization based on volumetric and flow PiCCO-derived parameters was associated with a less favorable postoperative outcome compared with a conventional GDT protocol during emergency surgery