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The RNA-binding protein TRBP is a central component of the Dicer complex. Despite a decade of biochemical and structural studies, the essential functionality of TRBP in microRNA (miRNA) biogenesis remains unknown. Here we show that TRBP is an integral cofactor for time-efficient Dicer processing in RNA-crowded environments. We competed for Dicer processing of pre-miRNA with a large amount of cellular RNA species and found that Dicer-TRBP, but not Dicer alone, remains resilient. To apprehend the mechanism of this substrate selectivity, we use single-molecule fluorescence. The real-time observation reveals that TRBP acts as a gatekeeper, precluding Dicer from engaging with pre-miRNA-like substrates. TRBP acquires the selectivity using the PAZ domain of Dicer, whereas Dicer moderates the RNA-binding affinity of TRBP for fast turnover. This coordinated action between TRBP and Dicer accomplishes an efficient way of discarding pre-miRNA-like substrates. The RNA binding protein TRBP is a component of the Dicer complex but its role in microRNA biogenesis remains poorly understood. Here the authors use a crowded RNA environment and single-molecule imaging to show that TRBP acts as a gatekeeper to prevent Dicer engagement with pre miRNA-like substrates.

Infections with Mycobacterium tuberculosis are substantially increasing on a worldwide scale and new antibiotics are urgently needed to combat concomitantly emerging drug-resistant mycobacterial strains. The diarylquinoline TMC207 is a highly promising drug candidate for treatment of tuberculosis. This compound kills M. tuberculosis by binding to a new target, mycobacterial ATP synthase. In this study we used biochemical assays and binding studies to characterize the interaction between TMC207 and ATP synthase. We show that TMC207 acts independent of the proton motive force and does not compete with protons for a common binding site. The drug is active on mycobacterial ATP synthesis at neutral and acidic pH with no significant change in affinity between pH 5.25 and pH 7.5, indicating that the protonated form of TMC207 is the active drug entity. The interaction of TMC207 with ATP synthase can be explained by a one-site binding mechanism, the drug molecule thus binds to a defined binding site on ATP synthase. TMC207 affinity for its target decreases with increasing ionic strength, suggesting that electrostatic forces play a significant role in drug binding. Our results are consistent with previous docking studies and provide experimental support for a predicted function of TMC207 in mimicking key residues in the proton transfer chain and blocking rotary movement of subunit c during catalysis. Furthermore, the high affinity of TMC207 at low proton motive force and low pH values may in part explain the exceptional ability of this compound to efficiently kill mycobacteria in different microenvironments.

The last decade has witnessed a remarkable increase in our ability to measure genetic information. Advancements of sequencing technologies are challenging the existing methods of data storage and analysis. While methods to cope with the data deluge are progressing, many biologists have lagged behind due to the fast pace of computational advancements and tools available to address their scientific questions. Future generations of biologists must be more computationally aware and capable. This means they should be trained to give them the computational skills to keep pace with technological developments. Here, we propose a model that bridges experimental and bioinformatics concepts using the Oxford Nanopore Technologies (ONT) sequencing platform. We provide both a guide to begin to empower the new generation of educators, scientists, and students in performing long-read assembly of bacterial and bacteriophage genomes and a standalone virtual machine containing all the required software and learning materials for the course.

Objective: To study factors associated with toxicity, final dose, and efficacy of methotrexate (MTX) in patients with rheumatoid arthritis (RA). Methods: Data were used from a randomised clinical 48 week trial on 411 patients with RA all treated with MTX, comparing folates and placebo. Logistic regression was used to study the relation between baseline variables and various dependent factors, including hepatotoxicity (alanine aminotransferase ⩾3×upper limit of normal), MTX withdrawal, final MTX dose ⩾15 mg/week, and MTX efficacy. Results: Addition of folates to MTX treatment was strongly related to the lack of hepatotoxicity. Next to this, high body mass index was related to the occurrence of hepatotoxicity. Prior gastrointestinal (GI) events and younger age were related to the adverse event, diarrhoea. Hepatotoxicity and GI adverse events were the main reason for MTX withdrawal, which in turn was associated with the absence of folate supplementation, body mass index, prior GI events, and female sex. Renal function (creatinine clearance ⩾50 ml/min) was not associated with toxicity. Reaching a final dose of MTX of ⩾15 mg/week was related to folate supplementation and the absence of prior GI events. Efficacy of MTX treatment was associated with low disease activity at baseline, male sex, use of non-steroidal anti-inflammatory drugs (NSAIDs), and lower creatinine clearance. Conclusions: MTX toxicity, final dose, and efficacy are influenced by folate supplementation. Baseline characteristics predicting the outcome of MTX treatment are mainly prior GI events, body mass index, sex, use of NSAIDs, and creatinine clearance.

Objectives:To compare the efficacy and tolerability of allopurinol 300–600 mg/day versus benzbromarone 100–200 mg/day used to attain a target serum urate concentration (sUr) ⩽0.30 mmol/l (5 mg/dl).Methods:A randomised, controlled, open-label, multicentre trial in gout patients with renal function defined as a calculated creatinine clearance ⩾50 ml/min. Patients were treated with 300 mg allopurinol or 100 mg benzbromarone once a day (stage 1). If sUr ⩽0.30 mmol/l was not attained after 2 months, the dose was doubled to allopurinol 300 mg twice a day or benzbromarone 200 mg once a day (stage 2). The primary end point was treatment success in either of the two stages, defined as clinical tolerability and attainment of biochemical target sUr.Results:Sixty-five patients were enrolled in stage 1; 36 received allopurinol and 29 received benzbromarone. Fifty-five patients (85%) were analysed at stage 1: the success rates were 8/31 (26%) and 13/25 (52%), respectively, and the difference was −0.26 (95% CI from −0.486 to −0.005), p = 0.049. At stage 2, the success rates were 21/27 (78%) and 18/23 (78%), respectively, and the difference was −0.005 (95% CI from −0.223 to 0.220), p = 1.00. Two patients stopped receiving allopurinol and three stopped receiving benzbromarone because of adverse drug reactions.Conclusions:Increasing the allopurinol dose from 300 to 600 mg/day and the benzbromarone dose from 100 to 200 mg/day according to the target sUr produced significantly higher success rates (both 78% successful in attaining sUr ⩽0.30 mmol/l). No significant differences in treatment success between benzbromarone and allopurinol were found after dose escalation.Trial registration number:ISRCTN49563848).

Abstract ATP synthase is a validated drug target for the treatment of tuberculosis, and ATP synthase inhibitors are promising candidate drugs for the treatment of infections caused by other slow-growing mycobacteria, such as Mycobacterium leprae and Mycobacterium ulcerans. ATP synthase is an essential enzyme in the energy metabolism of Mycobacterium tuberculosis; however, no biochemical data are available to characterize the role of ATP synthase in slow-growing mycobacterial strains. Here, we show that inverted membrane vesicles from the slow-growing model strain Mycobacterium bovis BCG are active in ATP synthesis, but ATP synthase displays no detectable ATP hydrolysis activity and does not set up a proton-motive force (PMF) using ATP as a substrate. Treatment with methanol as well as PMF activation unmasked the ATP hydrolysis activity, indicating that the intrinsic subunit ɛ and inhibitory ADP are responsible for the suppression of hydrolytic activity. These results suggest that the enzyme is needed for the synthesis of ATP, not for the maintenance of the PMF. For the development of new antimycobacterial drugs acting on ATP synthase, screening for ATP synthesis inhibitors, but not for ATP hydrolysis blockers, can be regarded as a promising strategy.

Background At present, there are no prognostic parameters unequivocally predicting treatment failure in early rheumatoid arthritis (RA) patients. We investigated whether baseline ultrasonography (US) findings of joints, when added to baseline clinical, laboratory, and radiographical data, could improve prediction of failure to achieve Disease Activity Score assessing 28 joints (DAS28) remission (<2.6) at 1 year in newly diagnosed RA patients. Methods A multicentre cohort of newly diagnosed RA patients was followed prospectively for 1 year. US of the hands, wrists, and feet was performed at baseline. Clinical, laboratory, and radiographical parameters were recorded. Primary analysis was the prediction by logistic regression of the absence of DAS28 remission 12 months after diagnosis and start of therapy. Results Of 194 patients included, 174 were used for the analysis, with complete data available for 159. In a multivariate model with baseline DAS28 (odds ratio (OR) 1.6, 95% confidence interval (CI) 1.2–2.2), the presence of rheumatoid factor (OR 2.3, 95% CI 1.1–5.1), and type of monitoring strategy (OR 0.2, 95% CI 0.05–0.85), the addition of baseline US results for joints (OR 0.96, 95% CI 0.89–1.04) did not significantly improve the prediction of failure to achieve DAS28 remission (likelihood ratio test, 1.04; p  = 0.31). Conclusion In an early RA population, adding baseline ultrasonography of the hands, wrists, and feet to commonly available baseline characteristics did not improve prediction of failure to achieve DAS28 remission at 12 months. Trial registration Clinicaltrials.gov, NCT01752309 . Registered on 19 December 2012.

Prokaryotes adapt to challenges from mobile genetic elements by integrating spacers derived from foreign DNA in the CRISPR array 1 . Spacer insertion is carried out by the Cas1–Cas2 integrase complex 2 – 4 . A substantial fraction of CRISPR–Cas systems use a Fe–S cluster containing Cas4 nuclease to ensure that spacers are acquired from DNA flanked by a protospacer adjacent motif (PAM) 5 , 6 and inserted into the CRISPR array unidirectionally, so that the transcribed CRISPR RNA can guide target searching in a PAM-dependent manner. Here we provide a high-resolution mechanistic explanation for the Cas4-assisted PAM selection, spacer biogenesis and directional integration by type I-G CRISPR in Geobacter sulfurreducens , in which Cas4 is naturally fused with Cas1, forming Cas4/Cas1. During biogenesis, only DNA duplexes possessing a PAM-embedded 3′-overhang trigger Cas4/Cas1–Cas2 assembly. During this process, the PAM overhang is specifically recognized and sequestered, but is not cleaved by Cas4. This ‘molecular constipation’ prevents the PAM-side prespacer from participating in integration. Lacking such sequestration, the non-PAM overhang is trimmed by host nucleases and integrated to the leader-side CRISPR repeat. Half-integration subsequently triggers PAM cleavage and Cas4 dissociation, allowing spacer-side integration. Overall, the intricate molecular interaction between Cas4 and Cas1–Cas2 selects PAM-containing prespacers for integration and couples the timing of PAM processing with the stepwise integration to establish directionality. Structures of the Cas4–Cas1–Cas2 complex from Geobacter sulfurreducens show that a 3′-overhang in the protospacer adjacent motif is required for complex assembly and spacer insertion into the CRISPR array.

OBJECTIVE To study the influence of sulphasalazine (SSZ), methotrexate (MTX), and the combination (COMBI) of both on plasma homocysteine and to study the relation between plasma homocysteine and their clinical effects. METHODS 105 patients with early rheumatoid arthritis (RA) were randomised between SSZ (2–3 g/day), MTX (7.5–15 mg/week), and the COMBI (same dose range) and evaluated double blindly during 52 weeks. Plasma homocysteine, serum folate concentrations, and vitamin B12 were measured. The influence of the C677T mutation of the enzyme methylenetetrahydrofolatereductase (MTHFR) gene was analysed. RESULTS A slight trend towards increased efficacy and an increased occurrence of minor gastrointestinal toxicity was present in the COMBI group, no differences existed clinically between SSZ and MTX. Only a slight and temporary increase in plasma homocysteine was found in the SSZ group, in contrast with the persistent rise in the MTX group and the even greater increase in the COMBI patients. Patients homozygous for the mutation in the MTHFR gene had significantly higher baseline homocysteine, heterozygous MTHFR genotype induced a significantly higher plasma homocysteine at week 52 compared with no mutation. No correlation was found between clinical efficacy variables and homocysteine. Patients with gastrointestinal toxicity had a significantly greater increase in homocysteine. CONCLUSION A persistent increase in plasma homocysteine concentrations was observed in patients treated with MTX alone and more pronounced in combination with SSZ, in contrast with SSZ alone. An increase in plasma homocysteine is related to the C677T mutation in MTHFR. A relation in the change in homocysteine concentrations with (gastrointestinal) toxicity was found, no relation with clinical efficacy existed.

Objectives: To investigate the effect of higher weekly maintenance dose methotrexate (MTX) (⩾25 mg/week) on plasma homocysteine concentrations in adults with RA. Methods: Patients with RA were treated with high doses of MTX with adjuvant folic acid. Plasma homocysteine was determined at baseline and 1, 2, 4, 8, 12, and 48 hours after subcutaneous MTX administration. Maximum homocysteine concentrations after MTX administration were compared with baseline concentrations. Results: Fifteen patients with RA (11 women) were included, with a median age of 61 years (range 31–72) and median disease duration 7 years (range 2–32). Median MTX dose was 30 mg (range 25–40). All patients received folic acid supplementation (5–30 mg/week). Median plasma homocysteine concentration at baseline was 10.1 μmol/l (range 6.6–12.7; normal 6–15). Homocysteine concentrations increased after MTX administration by a median of 2.5 μmol/l (range 0.7–5.1). Median maximum plasma homocysteine was significantly higher than at baseline. Peak homocysteine was reached after 12 hours. No relation between serum folate concentrations and plasma homocysteine concentrations was found. Conclusions: In patients with RA higher MTX doses with adjuvant folic acid do not increase baseline concentrations of homocysteine. An intermittent significant rise in plasma homocysteine occurs in the 48 hours after MTX administration.