CRHP Finder, a webtool for the detection of clarithromycin resistance in Helicobacter pylori from whole-genome sequencing data.

BACKGROUND: Resistance to clarithromycin in Helicobacter pylori (H pylori) is mediated by mutations in the domain V of the 23S rRNA gene (A2142G, A2143G, A2142C). Other polymorphisms in the 23S rRNA gene have been reported to cause low-level clarithromycin resistance but their importance is still under debate. In this study, we aimed to develop and evaluate the CRHP Finder webtool for detection of the most common mutations mediating clarithromycin resistance from whole-genome sequencing (WGS) data. Moreover, we included an analysis of 23 H pylori strains from Danish patients between January 2017 and September 2019 in Copenhagen, Denmark. MATERIALS AND METHODS: The CRHP Finder detects the fraction of each of the four nucleotides in nucleotide positions 2142, 2143, 2182, 2244 and 2712 of the 23S rRNA gene in H pylori (E coli numbering) by aligning raw sequencing reads (fastq format) with k-mer alignment (KMA). The nucleotide distribution in each position is compared to previously described point mutations mediating clarithromycin resistance in H pylori, and a genotypic prediction of the clarithromycin resistance phenotype is presented as output. For validation of the CRHP webtool, 137 fastq paired-end sequencing datasets originating from a well-characterized strain collection of H pylori were analyzed. RESULTS: The CRHP Finder correctly identified all resistance mutations reported in the sequencing data of 137 H pylori strains. In the 23 Danish H pylori strains, CRHP Finder detected A2143G (13%) in all resistant strains, and T2182C (13%) and C2244T (4,3%) nucleotide exchanges in only susceptible strains. CONCLUSION: In this study, we present the validation of the first webtool for H pylori resistance prediction based on the detection of 23S rRNA mutations (A2142C, A2142G, A2143G, T2182C, C2244T, T2712C) from WGS data of H pylori.

Vascular Imaging of Matrix Metalloproteinase Activity as an Informative Preclinical Biomarker of Drug-induced Vascular Injury.

Lack of biomarkers specific to and either predictive or diagnostic of drug-induced vascular injury (DIVI) continues to be a major obstacle during drug development. Biomarkers derived from physiologic responses to vessel injury, such as inflammation and vascular remodeling, could make good candidates; however, they characteristically lack specificity for vasculature. We evaluated whether vascular remodeling-associated protease activity, as well as changes to vessel permeability resulting from DIVI, could be visualized ex vivo in affected vessels, thereby allowing for visual monitoring of the pathology to address specificity. We found that visualization of matrix metalloproteinase activation accompanied by increased vascular leakage in the mesentery of rats treated with agents known to induce vascular injury correlated well with incidence and severity of histopathological findings and associated inflammation as well as with circulating levels of tissue inhibitors of metalloproteinase 1 and neutrophil gelatinase-associated lipocalin. The weight of evidence approach reported here shows promise as a composite DIVI preclinical tool by means of complementing noninvasive monitoring of circulating biomarkers of inflammation with direct imaging of affected vasculature and thus lending specificity to its interpretation. These findings are supportive of a potential strategy that relies on translational imaging tools in conjunction with circulating biomarker data for high-specificity monitoring of VI both preclinically and clinically.

Characterization of ADME properties of [(14)C]asunaprevir (BMS-650032) in humans.

1. Asunaprevir (ASV, BMS-650032), a highly selective and potent NS3 protease inhibitor, is currently under development for the treatment of chronic hepatic C virus infection. This study describes in vivo biotransformation in humans and the identification of metabolic enzymes of ASV. 2. Following a single oral dose of [(14)C]ASV to humans, the majority of radioactivity (>73% of the dose) was excreted in feces with <1% of the dose recovered in urine. Drug-related radioactivity readily appeared in circulation and the plasma radioactivity was mainly attributed to ASV. A few minor metabolites were observed in human plasma and are not expected to contribute to the pharmacological activity because of low levels. The area under the curve (AUC) values of each circulating metabolite in humans were well below their levels in animals used in the long-term toxicological studies. In bile and feces, intact ASV was a prominent radioactive peak suggesting that both metabolism and direct excretion played important roles in ASV clearance. 3. The primary metabolic pathways of ASV were hydroxylation, sulfonamide hydrolysis and the loss of isoquinoline. In vitro studies with human cDNA expressed CYP enzymes and with human liver microsomes (HLM) in the presence of selective chemical inhibitors demonstrated that ASV was primarily catalyzed by CYP3A4 and CYP3A5.

Inhibition of influenza virus replication via small molecules that induce the formation of higher-order nucleoprotein oligomers.

Influenza nucleoprotein (NP) plays multiple roles in the virus life cycle, including an essential function in viral replication as an integral component of the ribonucleoprotein complex, associating with viral RNA and polymerase within the viral core. The multifunctional nature of NP makes it an attractive target for antiviral intervention, and inhibitors targeting this protein have recently been reported. In a parallel effort, we discovered a structurally similar series of influenza replication inhibitors and show that they interfere with NP-dependent processes via formation of higher-order NP oligomers. Support for this unique mechanism is provided by site-directed mutagenesis studies, biophysical characterization of the oligomeric ligand:NP complex, and an X-ray cocrystal structure of an NP dimer of trimers (or hexamer) comprising three NP_A:NP_B dimeric subunits. Each NP_A:NP_B dimeric subunit contains two ligands that bridge two composite, protein-spanning binding sites in an antiparallel orientation to form a stable quaternary complex. Optimization of the initial screening hit produced an analog that protects mice from influenza-induced weight loss and mortality by reducing viral titers to undetectable levels throughout the course of treatment.

SpanSeq: similarity-based sequence data splitting method for improved development and assessment of deep learning projects.

The use of deep learning models in computational biology has increased massively in recent years, and it is expected to continue with the current advances in the fields such as Natural Language Processing. These models, although able to draw complex relations between input and target, are also inclined to learn noisy deviations from the pool of data used during their development. In order to assess their performance on unseen data (their capacity to generalize), it is common to split the available data randomly into development (train/validation) and test sets. This procedure, although standard, has been shown to produce dubious assessments of generalization due to the existing similarity between samples in the databases used. In this work, we present SpanSeq, a database partition method for machine learning that can scale to most biological sequences (genes, proteins and genomes) in order to avoid data leakage between sets. We also explore the effect of not restraining similarity between sets by reproducing the development of two state-of-the-art models on bioinformatics, not only confirming the consequences of randomly splitting databases on the model assessment, but expanding those repercussions to the model development. SpanSeq is available at https://github.com/genomicepidemiology/SpanSeq.

A novel ring oxidation of 4- or 5-substituted 2H-oxazole to corresponding 2-oxazolone catalyzed by cytosolic aldehyde oxidase.

The ring oxidation of 2H-oxazole, or C2-unsubstituted oxazole, to 2-oxazolone, a cyclic carbamate, was observed on various 4- or 5-substituted oxazoles. Using 5-(3-bromophenyl)oxazole as a model compound, its 2-oxazolone metabolite M1 was fully characterized by liquid chromatography/tandem mass spectrometry and nuclear magnetic resonance. The reaction mainly occurred in the liver cytosolic fraction without the requirement of cytochrome P450 enzymes and cofactor NADPH. Investigations into the mechanism of formation of 2-oxazolone using various chemical inhibitors indicated that the reaction was primarily catalyzed by aldehyde oxidase and not by xanthine oxidase. In addition, cytosol incubation of 5-(3-bromophenyl)oxazole in the medium containing H2¹8O led to the ¹8O incorporation into M1, substantiating the reaction mechanism of a typical molybdenum hydroxylase. The rank order of liver cytosols for the 2-oxazolone formation was mouse > monkey ≫ rat and human liver cytosol, whereas M1 was not formed in dog liver cytosol. Because the reaction was observed with a number of 4- or 5-substituted 2H-oxazoles in mouse liver cytosols, 2H-oxazoles represent a new substrate chemotype for ring oxidation catalyzed by aldehyde oxidase.

Pressure dependent aerosol formation from the cyclohexene gas-phase ozonolysis in the presence and absence of sulfur dioxide: a new perspective on the stabilisation of the initial clusters.

The ozonolysis of cyclohexene is studied with respect to the pressure dependent formation of stable gas-phase products and secondary organic aerosol (SOA) as well as the influence of the presence of SO(2). In addition the rate coefficient for the initial reaction cyclohexene + O(3) was determined at 295 K. The observed increase in CO and ethene yields at low pressures and the absence of ketene in the product spectrum confirm previously proposed reaction pathways forming these decomposition products. An enhanced ethene formation at pressures below 300 mbar coincides with drastically decreased aerosol yields pointing to a high influence on SOA formation of chemical activation driven dynamics in the vinylhydroperoxide channel. The static reactor experiments at 450 mbar in the presence of SO(2) in the present study showed a similar sensitivity of additional particle formation to H(2)SO(4) number densities as found in near-atmospheric flow reactor experiments [Sipiläet al., Science, 2010, 327, 1243], a surprising result with regard to the very different experimental approaches. At low pressures (around 40 mbar) no significant new particle formation is observed even at high H(2)SO(4) concentrations. These findings indicate that the collisional stabilisation of initial clusters is an important aspect for SOA formation processes involving sulfuric acid and organic compounds. The results may have implications for geo-engineering strategies based on stratospheric sulfur injection, but caution is mandatory when room temperature laboratory results are extrapolated to stratospheric conditions.

ResFinder - an open online resource for identification of antimicrobial resistance genes in next-generation sequencing data and prediction of phenotypes from genotypes.

Antimicrobial resistance (AMR) is one of the most important health threats globally. The ability to accurately identify resistant bacterial isolates and the individual antimicrobial resistance genes (ARGs) is essential for understanding the evolution and emergence of AMR and to provide appropriate treatment. The rapid developments in next-generation sequencing technologies have made this technology available to researchers and microbiologists at routine laboratories around the world. However, tools available for those with limited experience with bioinformatics are lacking, especially to enable researchers and microbiologists in low- and middle-income countries (LMICs) to perform their own studies. The CGE-tools (Center for Genomic Epidemiology) including ResFinder (https://cge.cbs.dtu.dk/services/ResFinder/) was developed to provide freely available easy to use online bioinformatic tools allowing inexperienced researchers and microbiologists to perform simple bioinformatic analyses. The main purpose was and is to provide these solutions for people involved in frontline diagnosis especially in LMICs. Since its original publication in 2012, ResFinder has undergone a number of improvements including improvement of the code and databases, inclusion of point mutations for selected bacterial species and predictions of phenotypes also for selected species. As of 28 September 2021, 820 803 analyses have been performed using ResFinder from 61 776 IP-addresses in 171 countries. ResFinder clearly fulfills a need for several people around the globe and we hope to be able to continue to provide this service free of charge in the future. We also hope and expect to provide further improvements including phenotypic predictions for additional bacterial species.

Quality of life of persons with transfemoral amputation: Comparison of socket prostheses and osseointegrated prostheses.

BACKGROUND: Until recently, no study had compared the quality of life of persons with transfemoral amputation treated with osseointegration to socket prosthesis users. OBJECTIVES: Comparison of quality of life in two types of prostheses users: a cohort of patients with osseointegration and patients equipped with a socket prosthesis who were group-matched for age, body mass index and mobility grade. STUDY DESIGN: A cross-sectional study that compared METHODS:: The quality of life of 39 participants (22 in the osseointegration group and 17 in the socket prosthesis group) was measured using the Questionnaire for Persons with Transfemoral Amputation (Q-TFA) and European Questionnaire 5-dimension 3-level (EQ-5D-3L) surveys. RESULTS: Compared with the socket prosthesis group, the osseointegration group had a significantly higher 'Global' score (p = 0.022) and a significantly lower 'Problem' score (p < 0.001) of the Q-TFA. The 'Mobility' (p = 0.051) and 'Use' scores (p = 0.146) of the Q-TFA, the EQ-5D-3L index (p = 0.723), and EQ-5D visual analog scale (p = 0.497) showed no significant differences between groups. CONCLUSIONS: Patients with osseointegration experienced less prosthesis-associated problems than socket prosthesis users and had a higher prosthesis-associated quality of life when assessed with the Q-TFA. General quality of life, as assessed with the EQ-5D-3L, was not different between groups.

Cas13b-dependent and Cas13b-independent RNA knockdown of viral sequences in mosquito cells following guide RNA expression.

Aedes aegypti and Aedes albopictus mosquitoes are vectors of the RNA viruses chikungunya (CHIKV) and dengue that currently have no specific therapeutic treatments. The development of new methods to generate virus-refractory mosquitoes would be beneficial. Cas13b is an enzyme that uses RNA guides to target and cleave RNA molecules and has been reported to suppress RNA viruses in mammalian and plant cells. We investigated the potential use of the Prevotella sp. P5-125 Cas13b system to provide viral refractoriness in mosquito cells, using a virus-derived reporter and a CHIKV split replication system. Cas13b in combination with suitable guide RNAs could induce strong suppression of virus-derived reporter RNAs in insect cells. Surprisingly, the RNA guides alone (without Cas13b) also gave substantial suppression. Our study provides support for the potential use of Cas13b in mosquitoes, but also caution in interpreting CRISPR/Cas data as we show that guide RNAs can have Cas-independent effects.

Global monitoring of antimicrobial resistance based on metagenomics analyses of urban sewage.

Antimicrobial resistance (AMR) is a serious threat to global public health, but obtaining representative data on AMR for healthy human populations is difficult. Here, we use metagenomic analysis of untreated sewage to characterize the bacterial resistome from 79 sites in 60 countries. We find systematic differences in abundance and diversity of AMR genes between Europe/North-America/Oceania and Africa/Asia/South-America. Antimicrobial use data and bacterial taxonomy only explains a minor part of the AMR variation that we observe. We find no evidence for cross-selection between antimicrobial classes, or for effect of air travel between sites. However, AMR gene abundance strongly correlates with socio-economic, health and environmental factors, which we use to predict AMR gene abundances in all countries in the world. Our findings suggest that global AMR gene diversity and abundance vary by region, and that improving sanitation and health could potentially limit the global burden of AMR. We propose metagenomic analysis of sewage as an ethically acceptable and economically feasible approach for continuous global surveillance and prediction of AMR.

Fluorine substitution can block CYP3A4 metabolism-dependent inhibition: identification of (S)-N-[1-(4-fluoro-3- morpholin-4-ylphenyl)ethyl]-3- (4-fluorophenyl)acrylamide as an orally bioavailable KCNQ2 opener devoid of CYP3A4 metabolism-dependent inhibition.

The formation of a reactive intermediate was found to be responsible for CYP3A4 metabolism-dependent inhibition (MDI) observed with (S)-N-[1-(3-morpholin-4-ylphenyl)ethyl]-3-phenyl-acrylamide (1). Structure-3A4 MDI relationship studies culminated in the discovery of a difluoro analogue, (S)-N-[1-(4-fluoro-3-morpholin-4-ylphenyl)ethyl]-3-(4-fluoro-phenyl)acrylamide (2), as an orally bioavailable KCNQ2 opener free of CYP3A4 MDI.

Screening and characterization of reactive compounds with in vitro peptide-trapping and liquid chromatography/high-resolution accurate mass spectrometry.

The present study describes a novel methodology for the detection of reactive compounds using in vitro peptide-trapping and liquid chromatography-high-resolution accurate mass spectrometry (LC-HRMS). Compounds that contain electrophilic groups can covalently bind to nucleophilic moieties in proteins and form adducts. Such adducts are thought to be associated with drug-mediated toxicity and therefore represent potential liabilities in drug discovery programs. In addition, reactive compounds identified in biological screening can be associated with data that can be misinterpreted if the reactive nature of the compound is not appreciated. In this work, to facilitate the triage of hits from high-throughput screening (HTS), a novel assay was developed to monitor the formation of covalent peptide adducts by compounds suspected to be chemically reactive. The assay consists of in vitro incubations of test compounds (under conditions of physiological pH) with synthetically prepared peptides presenting a variety of nucleophilic moieties such as cysteine, lysine, histidine, arginine, serine, and tyrosine. Reaction mixtures were analyzed using full-scan LC-HRMS, the data were interrogated using postacquisition data mining, and modified amino acids were identified by subsequent LC-HRMS/mass spectrometry. The study demonstrated that in vitro nucleophilic peptide trapping followed by LC-HRMS analysis is a useful approach for screening of intrinsically reactive compounds identified from HTS exercises, which are then removed from follow-up processes, thus obviating the generation of data from biochemical activity assays.

Labor epidural analgesia is independent risk factor for neonatal pyrexia.

OBJECTIVE: To explore whether epidural analgesia (EA) in labor is independent risk factor for neonatal pyrexia after controlling for intrapartum pyrexia. METHODS: Retrospective observational study of 480 consecutive term singleton infants born to mothers who received EA in labor (EA group) and 480 term infants delivered to mothers who did not receive EA (NEA group). RESULTS: Mothers in the EA group had significantly higher incidence of intrapartum pyrexia [54/480 (11%) vs. 4/480 (0.8%), OR = 15.1, p < 0.0001] and neonatal pyrexia [68/480 (14.2%) vs. 15/480 (3.1%), OR = 5.1, p < 0.0001]. Neonates in the EA group had a median duration of pyrexia of 1 h (maximum 5 h) with a peak temperature within 1 h. Stepwise logistic regression analysis showed that maternal EA was independent risk factor for neonatal pyrexia (>37.5°C) after controlling for intrapartum pyrexia (>37.9°C) and other confounders (OR = 3.44, CI = 1.9-6.3, p < 0.0001). Sepsis work-up was performed significantly more frequently in infants in the EA group [11.7% vs. 2.5%, OR= 5.2, CI = 2.7-9.7, p < 0.0001] with negative blood cultures. CONCLUSIONS: EA in labor is an independent risk factor for pyrexia in term neonates. It is unnecessary to investigate febrile offspring of mothers who have had epidurals unless pyrexia persists for longer than 5 h or other signs or risk factors for neonatal sepsis are present.