S-adenosyl-L-methionine-dependent 2′-O-methylati-on of the 3′-terminal nucleotide plays important roles in biogenesis of eukaryotic small non-coding RNAs, such as siRNAs, miRNAs and Piwi-interacting RNAs (piRNAs). Here we demonstrate that, in contrast to Mg2+/Mn2+-dependent plant and bacterial homologues, the Drosophila DmHen1 and human HsHEN1 piRNA methyltransferases require cobalt cations for their enzymatic activity in vitro. We also show for the first time the capacity of the animal Hen1 to catalyse the transfer of a variety of extended chemical groups from synthetic analogues of the AdoMet cofactor onto a wide range (22–80 nt) of single-stranded RNAs permitting their 3′-terminal functionalization and labelling. Moreover, we provide evidence that deletion of a small C-terminal region of the DmHen1 protein further increases its modification efficiency and abolishes a modest 3′-terminal nucleotide bias observed for the full-length protein. Finally, we show that fluorophore-tagged ssRNA molecules are successfully detected in fluorescence resonance energy transfer assays both individually and in a total RNA mixture. The presented DmHen1-assisted RNA labelling provides a solid basis for developing novel chemo-enzymatic approaches for in vitro studies and in vivo monitoring of single-stranded RNA pools.

Bedaquiline Drug Resistance Emergence Assessment in Multidrug-resistant-tuberculosis (MDR-TB) (DREAM) was a 5-year (2015–2019) phenotypic drug-resistance surveillance study across 11 countries. DREAM assessed the susceptibility of 5036 MDR-TB isolates of bedaquiline-treatment-naïve patients to bedaquiline and other anti-tuberculosis drugs by the 7H9 broth microdilution (BMD) and 7H10/7H11 agar dilution (AD) minimal inhibitory concentration (MIC) methods. Bedaquiline AD MIC quality control (QC) range for the H37Rv reference strain was unchanged, but the BMD MIC QC range (0.015–0.12 μg/ml) was adjusted compared with ranges from a multilaboratory, multicountry reproducibility study conforming to Clinical and Laboratory Standards Institute Tier-2 criteria. Epidemiological cut-off values of 0.12 μg/ml by BMD and 0.25 μg/ml by AD were consistent with previous bedaquiline breakpoints. An area of technical uncertainty or Intermediate category was set at 0.25 μg/ml and 0.5 μg/ml for BMD and AD, respectively. When applied to the 5036 MDR-TB isolates, bedaquiline-susceptible, intermediate and bedaquiline-resistant rates were 97.9%, 1.5% and 0.6%, respectively, for BMD, and 98.8%, 0.8% and 0.4% for AD. Resistance rates were: ofloxacin 35.1%, levofloxacin 34.2%, moxifloxacin 33.3%, 1.5% linezolid and 2% clofazimine. Phenotypic cross resistance between bedaquiline and clofazimine was 0.4% in MDR-TB and 1% in pre-extensively drug-resistant (pre-XDR-TB)/XDR-TB populations. Co-resistance to bedaquiline and linezolid, and clofazimine and linezolid, were 0.1% and 0.3%, respectively, in MDR-TB, and 0.2% and 0.4% in pre-XDR-TB/XDR-TB populations. Resistance rates to bedaquiline appear to be low in the bedaquiline-treatment-naïve population. No treatment-limiting patterns for cross-resistance and co-resistance have been identified with key TB drugs to date.

SETTING:Implementation of novel diagnostic assays in tuberculosis (TB) laboratory diagnosis requires effective management of time and resources.OBJECTIVE:To further develop and assess at multiple centres a time-and-motion (T&M) tool as an objective means for recording the actual time spent on running laboratory assays.DESIGN:Multicentre prospective study conducted in six European Union (EU) reference TB laboratories.RESULTS:A total of 1060 specimens were tested using four laboratory assays. The number of specimens per batch varied from one to 60; a total of 64 recordings were performed. Theoretical hands-on times per specimen (TTPS) in h:min:s for Xpert® MTB/RIF, mycobacterial interspersed repetitive unit-variable number of tandem repeats genotyping, Ziehl-Neelsen staining and manual fluorescence microscopy were respectively 00:33:02 ± 00:12:32, 00:13:34 ± 00:03:11, 00:09:54 ± 00:00:53 and 00:06:23 ± 00:01:36. Variations between laboratories were predominantly linked to the time spent on reporting and administrative procedures. Processing specimens in batches could help save time in highly automated assays (e.g., line-probe) (TTPS 00:14:00 vs. 00:09:45 for batches comprising 7 and 31 specimens, respectively).CONCLUSIONS:The T&M tool can be considered a universal and objective methodology contributing to workload assessment in TB diagnostic laboratories. Comparison of workload between laboratories could help laboratory managers justify their resource and personnel needs for the implementation of novel, time-saving, cost-effective technologies, as well as identify areas for improvement.

BackgroundFatty acids (FA) and their metabolites are closely related to some mechanisms involved in the development of uronephrolithiasis. The aim of this study was to evaluate the relationship between FA composition and type of kidney stones.Material/MethodsAbdominal adipose tissue fatty acid methyl esters of 71 men with nephrolithiasis were identified by GC/MS, and the type of kidney stones was identified using FTIR infrared spectroscopy. Patients were divided into groups according to diagnosis of metabolic syndrome (MS) and type of kidney stone. The composition of FA was compared within different groups of patients with different types of kidney stones and between the patients and healthy individuals (control group) (n=100).ResultsIndividuals with nephrolithiasis had a significantly higher level of monounsaturated fatty acids (MUFA) and a lower level of polyunsaturated fatty acids (PUFA) versus healthy individuals. Patients with MS had a higher level of 18: 1ω9 and a lower level of 16: 1ω7 than patients without MS. Individuals with nephrolithiasis, but without MS, had a higher level of saturated fatty acids (SFA) compared to controls. The level of PUFA was higher in the control group (p<0.0001) compared to individuals with uronephrolithiasis, with or without MS. PUFA, ω – 6 PUFA, and 18: 2ω6 were higher in patients with calcium-based kidney stones without MS versus patients with uric acid kidney stones with MS.ConclusionsThe levels of MUFA were significantly higher and the levels of PUFA were significantly lower in patients with uronephrolithiasis compared to controls.

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