Considerable escape of SARS-CoV-2 Omicron to antibody neutralization.

The SARS-CoV-2 Omicron variant was first identified in November 2021 in Botswana and South Africa1-3. It has since spread to many countries and is expected to rapidly become dominant worldwide. The lineage is characterized by the presence of around 32 mutations in spike-located mostly in the N-terminal domain and the receptor-binding domain-that may enhance viral fitness and enable antibody evasion. Here we isolated an infectious Omicron virus in Belgium from a traveller returning from Egypt. We examined its sensitivity to nine monoclonal antibodies that have been clinically approved or are in development4, and to antibodies present in 115 serum samples from COVID-19 vaccine recipients or individuals who have recovered from COVID-19. Omicron was completely or partially resistant to neutralization by all monoclonal antibodies tested. Sera from recipients of the Pfizer or AstraZeneca vaccine, sampled five months after complete vaccination, barely inhibited Omicron. Sera from COVID-19-convalescent patients collected 6 or 12 months after symptoms displayed low or no neutralizing activity against Omicron. Administration of a booster Pfizer dose as well as vaccination of previously infected individuals generated an anti-Omicron neutralizing response, with titres 6-fold to 23-fold lower against Omicron compared with those against Delta. Thus, Omicron escapes most therapeutic monoclonal antibodies and, to a large extent, vaccine-elicited antibodies. However, Omicron is neutralized by antibodies generated by a booster vaccine dose.

Reprohackathons: promoting reproducibility in bioinformatics through training.

MOTIVATION: The reproducibility crisis has highlighted the importance of improving the way bioinformatics data analyses are implemented, executed, and shared. To address this, various tools such as content versioning systems, workflow management systems, and software environment management systems have been developed. While these tools are becoming more widely used, there is still much work to be done to increase their adoption. The most effective way to ensure reproducibility becomes a standard part of most bioinformatics data analysis projects is to integrate it into the curriculum of bioinformatics Master's programs. RESULTS: In this article, we present the Reprohackathon, a Master's course that we have been running for the last 3 years at Université Paris-Saclay (France), and that has been attended by a total of 123 students. The course is divided into two parts. The first part includes lessons on the challenges related to reproducibility, content versioning systems, container management, and workflow systems. In the second part, students work on a data analysis project for 3-4 months, reanalyzing data from a previously published study. The Reprohackaton has taught us many valuable lessons, such as the fact that implementing reproducible analyses is a complex and challenging task that requires significant effort. However, providing in-depth teaching of the concepts and the tools during a Master's degree program greatly improves students' understanding and abilities in this area.

Robustness of Felsenstein's Versus Transfer Bootstrap Supports With Respect to Taxon Sampling.

The bootstrap method is based on resampling sequence alignments and re-estimating trees. Felsenstein's bootstrap proportions (FBP) are the most common approach to assess the reliability and robustness of sequence-based phylogenies. However, when increasing taxon sampling (i.e., the number of sequences) to hundreds or thousands of taxa, FBP tend to return low support for deep branches. The transfer bootstrap expectation (TBE) has been recently suggested as an alternative to FBP. TBE is measured using a continuous transfer index in [0,1] for each bootstrap tree, instead of the binary {0,1} index used in FBP to measure the presence/absence of the branch of interest. TBE has been shown to yield higher and more informative supports while inducing a very low number of falsely supported branches. Nonetheless, it has been argued that TBE must be used with care due to sampling issues, especially in datasets with a high number of closely related taxa. In this study, we conduct multiple experiments by varying taxon sampling and comparing FBP and TBE support values on different phylogenetic depths, using empirical datasets. Our results show that the main critique of TBE stands in extreme cases with shallow branches and highly unbalanced sampling among clades, but that TBE is still robust in most cases, while FBP is inescapably negatively impacted by high taxon sampling. We suggest guidelines and good practices in TBE (and FBP) computing and interpretation.

Deciphering RNA G-quadruplex function during the early steps of HIV-1 infection.

G-quadruplexes (G4s) are four-stranded nucleic acid structures formed by the stacking of G-tetrads. Here we investigated their formation and function during HIV-1 infection. Using bioinformatics and biophysics analyses we first searched for evolutionary conserved G4-forming sequences in HIV-1 genome. We identified 10 G4s with conservation rates higher than those of HIV-1 regulatory sequences such as RRE and TAR. We then used porphyrin-based G4-binders to probe the formation of the G4s during infection of human cells by native HIV-1. The G4-binders efficiently inhibited HIV-1 infectivity, which is attributed to the formation of G4 structures during HIV-1 replication. Using a qRT-PCR approach, we showed that the formation of viral G4s occurs during the first 2 h post-infection and their stabilization by the G4-binders prevents initiation of reverse transcription. We also used a G4-RNA pull-down approach, based on a G4-specific biotinylated probe, to allow the direct detection and identification of viral G4-RNA in infected cells. Most of the detected G4-RNAs contain crucial regulatory elements such as the PPT and cPPT sequences as well as the U3 region. Hence, these G4s would function in the early stages of infection when the viral RNA genome is being processed for the reverse transcription step.

APOBEC3F Is a Mutational Driver of the Human Monkeypox Virus Identified in the 2022 Outbreak.

BACKGROUND: On May 6, 2022, a powerful outbreak of monkeypox virus (MPXV) had been reported outside of Africa, with many continuing new cases being reported around the world. Analysis of mutations among the 2 different lineages present in the 2021 and 2022 outbreaks revealed the presence of G->A mutations occurring in the 5'GpA context, indicative of APOBEC3 cytidine deaminase activity. METHODS: By using a sensitive polymerase chain reaction (differential DNA denaturation PCR) method allowing differential amplification of AT-rich DNA, we analyzed the level of APOBEC3-induced MPXV editing in infected cells and in patients. RESULTS: We demonstrate that G->A hypermutated MPXV genomes can be recovered experimentally from APOBEC3 transfection followed by MPXV infection. Here, among the 7 human APOBEC3 cytidine deaminases (A3A-A3C, A3DE, A3F-A3H), only APOBEC3F was capable of extensively deaminating cytidine residues in MPXV genomes. Hyperedited genomes were also recovered in ∼42% of analyzed patients. Moreover, we demonstrate that substantial repair of these mutations occurs. Upon selection, corrected G->A mutations escaping drift loss contribute to the MPXV evolution observed in the current epidemic. CONCLUSIONS: Stochastic or transient overexpression of the APOBEC3F gene exposes the MPXV genome to a broad spectrum of mutations that may be modeling the mutational landscape after multiple cycles of viral replication.

COVID-Align: accurate online alignment of hCoV-19 genomes using a profile HMM.

MOTIVATION: The first cases of the COVID-19 pandemic emerged in December 2019. Until the end of February 2020, the number of available genomes was below 1000 and their multiple alignment was easily achieved using standard approaches. Subsequently, the availability of genomes has grown dramatically. Moreover, some genomes are of low quality with sequencing/assembly errors, making accurate re-alignment of all genomes nearly impossible on a daily basis. A more efficient, yet accurate approach was clearly required to pursue all subsequent bioinformatics analyses of this crucial data. RESULTS: hCoV-19 genomes are highly conserved, with very few indels and no recombination. This makes the profile HMM approach particularly well suited to align new genomes, add them to an existing alignment and filter problematic ones. Using a core of ∼2500 high quality genomes, we estimated a profile using HMMER, and implemented this profile in COVID-Align, a user-friendly interface to be used online or as standalone via Docker. The alignment of 1000 genomes requires ∼50 minutes on our cluster. Moreover, COVID-Align provides summary statistics, which can be used to determine the sequencing quality and evolutionary novelty of input genomes (e.g. number of new mutations and indels). AVAILABILITY AND IMPLEMENTATION: https://covalign.pasteur.cloud, hub.docker.com/r/evolbioinfo/covid-align. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

SpliceLauncher: a tool for detection, annotation and relative quantification of alternative junctions from RNAseq data.

SUMMARY: Alternative splicing is an important biological process widely analyzed in molecular diagnostic settings. Indeed, a variant can be pathogenic by splicing alteration and a suspected pathogenic variant (e.g. truncating variant) can be rescued by splicing. In this context, detecting and quantifying alternative splicing is challenging. We developed SpliceLauncher, a fast and easy to use open source tool that aims at detecting, annotating and quantifying alternative splice junctions at high resolution. AVAILABILITY AND IMPLEMENTATION: SpliceLauncher is available at https://github.com/raphaelleman/SpliceLauncher. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Prehospital management of acute respiratory distress in suspected COVID-19 patients.

BACKGROUND: In December 2019, coronavirus disease (COVID-19) emerged in China and became a world-wide pandemic in March 2020. Emergency services and intensive care units (ICUs) were faced with a novel disease with unknown clinical characteristics and presentations. Acute respiratory distress (ARD) was often the chief complaint for an EMS call. This retrospective study evaluated prehospital ARD management and identified factors associated with the need of prehospital mechanical ventilation (PMV) for suspected COVID-19 patients. METHODS: We included 256 consecutive patients with suspected COVID-19-related ARD that received prehospital care from a Paris Fire Brigade BLS or ALS team, from March 08 to April 18, 2020. We performed multivariate regression to identify factors predisposing to PMV. RESULTS: Of 256 patients (mean age 60 ± 18 years; 82 (32%) males), 77 (30%) had previous hypertension, 31 (12%) were obese, and 49 (19%) had diabetes mellitus. Nineteen patients (7%) required PMV. Logistic regression observed that a low initial pulse oximetry was associated with prehospital PMV (ORa = 0.86, 95%CI: 0.73-0.92; p = 0.004). CONCLUSIONS: This study showed that pulse oximetry might be a valuable marker for rapidly determining suspected COVID-19-patients requiring prehospital mechanical ventilation. Nevertheless, the impact of prehospital mechanical ventilation on COVID-19 patients outcome require further investigations.

NGPhylogeny.fr: new generation phylogenetic services for non-specialists.

Phylogeny.fr, created in 2008, has been designed to facilitate the execution of phylogenetic workflows, and is nowadays widely used. However, since its development, user needs have evolved, new tools and workflows have been published, and the number of jobs has increased dramatically, thus promoting new practices, which motivated its refactoring. We developed NGPhylogeny.fr to be more flexible in terms of tools and workflows, easily installable, and more scalable. It integrates numerous tools in their latest version (e.g. TNT, FastME, MrBayes, etc.) as well as new ones designed in the last ten years (e.g. PhyML, SMS, FastTree, trimAl, BOOSTER, etc.). These tools cover a large range of usage (sequence searching, multiple sequence alignment, model selection, tree inference and tree drawing) and a large panel of standard methods (distance, parsimony, maximum likelihood and Bayesian). They are integrated in workflows, which have been already configured ('One click'), can be customized ('Advanced'), or are built from scratch ('A la carte'). Workflows are managed and run by an underlying Galaxy workflow system, which makes workflows more scalable in terms of number of jobs and size of data. NGPhylogeny.fr is deployable on any server or personal computer, and is freely accessible at https://ngphylogeny.fr.

Genomic Epidemiology of 2015-2016 Zika Virus Outbreak in Cape Verde.

During 2015-2016, Cape Verde, an island nation off the coast of West Africa, experienced a Zika virus (ZIKV) outbreak involving 7,580 suspected Zika cases and 18 microcephaly cases. Analysis of the complete genomes of 3 ZIKV isolates from the outbreak indicated the strain was of the Asian (not African) lineage. The Cape Verde ZIKV sequences formed a distinct monophylogenetic group and possessed 1-2 (T659A, I756V) unique amino acid changes in the envelope protein. Phylogeographic and serologic evidence support earlier introduction of this lineage into Cape Verde, possibly from northeast Brazil, between June 2014 and August 2015, suggesting cryptic circulation of the virus before the initial wave of cases were detected in October 2015. These findings underscore the utility of genomic-scale epidemiology for outbreak investigations.

Distribution and asymptotic behavior of the phylogenetic transfer distance.

The transfer distance (TD) was introduced in the classification framework and studied in the context of phylogenetic tree matching. Recently, Lemoine et al. (Nature 556(7702):452-456, 2018. https://doi.org/10.1038/s41586-018-0043-0 ) showed that TD can be a powerful tool to assess the branch support on large phylogenies, thus providing a relevant alternative to Felsenstein's bootstrap. This distance allows a reference branch[Formula: see text] in a reference tree [Formula: see text] to be compared to a branch b from another tree T (typically a bootstrap tree), both on the same set of n taxa. The TD between these branches is the number of taxa that must be transferred from one side of b to the other in order to obtain [Formula: see text]. By taking the minimum TD from [Formula: see text] to all branches in T we define the transfer index, denoted by [Formula: see text], measuring the degree of agreement of T with [Formula: see text]. Let us consider a reference branch [Formula: see text] having p tips on its light side and define the transfer support (TS) as [Formula: see text]. Lemoine et al. (2018) used computer simulations to show that the TS defined in this manner is close to 0 for random "bootstrap" trees. In this paper, we demonstrate that result mathematically: when T is randomly drawn, TS converges in probability to 0 when n tends to [Formula: see text]. Moreover, we fully characterize the distribution of [Formula: see text] on caterpillar trees, indicating that the convergence is fast, and that even when n is small, moderate levels of branch support cannot appear by chance.

Molecular analyses of juvenile granulosa cell tumors bearing AKT1 mutations provide insights into tumor biology and therapeutic leads.

Juvenile granulosa cell tumors (JGCTs) of the ovary are pediatric neoplasms representing 5% of all granulosa cell tumors (GCTs). Most GCTs are of adult type (AGCTs) and bear a mutation in the FOXL2 gene. The molecular basis of JGCTs is poorly understood, although mutations in the GNAS gene have been reported. We have detected in-frame duplications within the oncogene AKT1 in >60% of the JGCTs studied. Here, to evaluate the functional impact of these duplications and the existence of potential co-driver alterations, we have sequenced the transcriptome of four JGCTs and compared them with control transcriptomes. A search for gene variants detected only private alterations probably unrelated with tumorigenesis, suggesting that tandem duplications are the best candidates to underlie tumor formation in the absence of GNAS alterations. We previously showed that the duplications were specific to JGCTs. However, the screening of eight AGCTs samples without FOXL2 mutation showed the existence of an AKT1 duplication in one case, also having a stromal luteoma. The analysis of RNA-Seq data pinpointed a series of differentially expressed genes, involved in cytokine and hormone signaling and cell division-related processes. Further analyses pointed to the existence of a possible dedifferentiation process and suggested that most of the transcriptomic dysregulation might be mediated by a limited set of transcription factors perturbed by AKT1 activation. Finally, we show that commercially available AKT inhibitors can modulate the in vitro activity of various mutated forms. These results shed light on the pathogenesis of JGCTs and provide therapeutic leads for a targeted treatment.

La pose de voie veineuse pédiatrique par les équipes de Smur adultes.

PAEDIATRIC VENOUS ACCESS BEING ESTABLISHED BY ADULT SMUR TEAMS: Paediatric perfusion during pre-hospital care is a major issue in the event of life-threatening emergencies. Access, often restricted, to backup specialising in paediatrics, implies the existence of practices on protocols for the adult mobile emergency and intensive care service (Smur) in partnership with paediatricians. Paediatric perfusion practices were assessed in these teams. The results show the presence of a paediatric protocol in the adult Smur teams is not very common. Nursing practices seem to be evolving towards the recommendations, thanks to technological advances such as the use of the intraosseous device.

Birth and expression evolution of mammalian microRNA genes.

MicroRNAs (miRNAs) are major post-transcriptional regulators of gene expression, yet their origins and functional evolution in mammals remain little understood due to the lack of appropriate comparative data. Using RNA sequencing, we have generated extensive and comparable miRNA data for five organs in six species that represent all main mammalian lineages and birds (the evolutionary outgroup) with the aim to unravel the evolution of mammalian miRNAs. Our analyses reveal an overall expansion of miRNA repertoires in mammals, with threefold accelerated birth rates of miRNA families in placentals and marsupials, facilitated by the de novo emergence of miRNAs in host gene introns. Generally, our analyses suggest a high rate of miRNA family turnover in mammals with many newly emerged miRNA families being lost soon after their formation. Selectively preserved mammalian miRNA families gradually evolved higher expression levels, as well as altered mature sequences and target gene repertoires, and were apparently mainly recruited to exert regulatory functions in nervous tissues. However, miRNAs that originated on the X chromosome evolved high expression levels and potentially diverse functions during spermatogenesis, including meiosis, through selectively driven duplication-divergence processes. Overall, our study thus provides detailed insights into the birth and evolution of mammalian miRNA genes and the associated selective forces.

Amplicon rearrangements during the extrachromosomal and intrachromosomal amplification process in a glioma.

The mechanisms of gene amplification in tumour cells are poorly understood and the relationship between extrachromosomal DNA molecules, named double minutes (dmins), and intrachromosomal homogeneously staining regions (hsr) is not documented at nucleotide resolution. Using fluorescent in situ hybridization and whole genome sequencing, we studied a xenografted human oligodendroglioma where the co-amplification of the EGFR and MYC loci was present in the form of dmins at early passages and of an hsr at later passages. The amplified regions underwent multiple rearrangements and deletions during the formation of the dmins and their transformation into hsr. In both forms of amplification, non-homologous end-joining and microhomology-mediated end-joining rather than replication repair mechanisms prevailed in fusions. Small fragments, some of a few tens of base pairs, were associated in contigs. They came from clusters of breakpoints localized hundreds of kilobases apart in the amplified regions. The characteristics of some pairs of junctions suggest that at least some fragments were not fused randomly but could result from the concomitant repair of neighbouring breakpoints during the interaction of remote DNA sequences. This characterization at nucleotide resolution of the transition between extra- and intrachromosome amplifications highlights a hitherto uncharacterized organization of the amplified regions suggesting the involvement of new mechanisms in their formation.

Lipocalin 2, the TNF-like receptor TWEAKR and its ligand TWEAK act downstream of NFAT1 to regulate breast cancer cell invasion.

NFAT1 is a transcription factor that elicits breast carcinoma cells to become invasive, thus contributing to metastasis. The molecular mechanisms by which NFAT1 operates in this respect are still poorly known. Here, we report that NFAT1 increases lipocalin 2 (LCN2) mRNA and protein expression by binding to specific sites in the LCN2 gene promoter region. We show that the LCN2 protein is required downstream of NFAT1 to increase breast cancer cell invasion. We demonstrate that the NFAT1-LCN2 axis is sufficient to regulate expression of the TNF-like receptor TWEAKR at the RNA level and of its ligand, TWEAK, at the protein level. We show, however, that TWEAKR mediates an anti-invasive effect in breast cancer cells whereas, depending on LCN2 expression, TWEAK has either anti- or pro-invasive capacities. Thus, we identify LCN2 and TWEAKR-TWEAK as crucial downstream effectors of NFAT1 that regulate breast cancer cell motility and invasive capacity.

Accurate Detection of Convergent Mutations in Large Protein Alignments With ConDor.

Evolutionary convergences are observed at all levels, from phenotype to DNA and protein sequences, and changes at these different levels tend to be correlated. Notably, convergent mutations can lead to convergent changes in phenotype, such as changes in metabolism, drug resistance, and other adaptations to changing environments. We propose a two-component approach to detect mutations subject to convergent evolution in protein alignments. The "Emergence" component selects mutations that emerge more often than expected, while the "Correlation" component selects mutations that correlate with the convergent phenotype under study. With regard to Emergence, a phylogeny deduced from the alignment is provided by the user and is used to simulate the evolution of each alignment position. These simulations allow us to estimate the expected number of mutations in a neutral model, which is compared to the observed number of mutations in the data studied. In Correlation, a comparative phylogenetic approach, is used to measure whether the presence of each of the observed mutations is correlated with the convergent phenotype. Each component can be used on its own, for example Emergence when no phenotype is available. Our method is implemented in a standalone workflow and a webserver, called ConDor. We evaluate the properties of ConDor using simulated data, and we apply it to three real datasets: sedge PEPC proteins, HIV reverse transcriptase, and fish rhodopsin. The results show that the two components of ConDor complement each other, with an overall accuracy that compares favorably to other available tools, especially on large datasets.

Integron cassettes integrate into bacterial genomes via widespread non-classical attG sites.

Integrons are genetic elements involved in bacterial adaptation which capture, shuffle and express genes encoding adaptive functions embedded in cassettes. These events are governed by the integron integrase through site-specific recombination between attC and attI integron sites. Using computational and molecular genetic approaches, here we demonstrate that the integrase also catalyses cassette integration into bacterial genomes outside of its known att sites. Once integrated, these cassettes can be expressed if located near bacterial promoters and can be excised at the integration point or outside, inducing chromosomal modifications in the latter case. Analysis of more than 5 × 105 independent integration events revealed a very large genomic integration landscape. We identified consensus recombination sequences, named attG sites, which differ greatly in sequence and structure from classical att sites. These results unveil an alternative route for dissemination of adaptive functions in bacteria and expand the role of integrons in bacterial evolution.

Analysis during chest compressions in out-of-hospital cardiac arrest patients, a cross/sectional study: The DEFI 2022 study.

AIMS: During out-of-hospital cardiac arrest (OHCA), an automatic external defibrillator (AED) analyzes the cardiac rhythm every two minutes; however, 80% of refibrillations occur within the first minute post-shock. We have implemented an algorithm for Analyzing cardiac rhythm While performing chest Compression (AWC). When AWC detects a shockable rhythm, it shortens the time between analyses to one minute. We investigated the effect of AWC on cardiopulmonary resuscitation quality. METHOD: In this cross-sectional study, we compared patients treated in 2022 with AWC, to a historical cohort from 2017. Inclusion criteria were OHCA patients with a shockable rhythm at the first analysis. Primary endpoint was the chest compression fraction (CCF). Secondary endpoints were cardiac rhythm evolution and survival, including survival analysis of non-prespecified subgroups. RESULTS: In 2017 and 2022, 355 and 377 OHCAs met the inclusion criteria, from which we analyzed the 285 first consecutive cases in each cohort. CCF increased in 2022 compared to 2017 (77% [72-80] vs 72% [67-76]; P < 0.001) and VF recurrences were shocked more promptly (53 s [32-69] vs 117 s [90-132]). Survival did not differ between 2017 and 2022 (adjusted hazard-ratio 0.96 [95% CI, 0.78-1.18]), but was higher in 2022 within the sub-group of OHCAs that occurred in a public place and within a short time from call to AED switch-on (adjusted hazard ratio 0.85[0.76-0.96]). CONCLUSIONS: OHCA patients treated with AWC had higher CCF, shorter time spent in ventricular fibrillation, but no survival difference, except for OHCA that occurred in public places with short intervention time.

Distinct evolution of SARS-CoV-2 Omicron XBB and BA.2.86/JN.1 lineages combining increased fitness and antibody evasion.

The unceasing circulation of SARS-CoV-2 leads to the continuous emergence of novel viral sublineages. Here, we isolated and characterized XBB.1, XBB.1.5, XBB.1.9.1, XBB.1.16.1, EG.5.1.1, EG.5.1.3, XBF, BA.2.86.1 and JN.1 variants, representing >80% of circulating variants in January 2024. The XBB subvariants carry few but recurrent mutations in the spike, whereas BA.2.86.1 and JN.1 harbor >30 additional changes. These variants replicated in IGROV-1 but no longer in Vero E6 and were not markedly fusogenic. They potently infected nasal epithelial cells, with EG.5.1.3 exhibiting the highest fitness. Antivirals remained active. Neutralizing antibody (NAb) responses from vaccinees and BA.1/BA.2-infected individuals were markedly lower compared to BA.1, without major differences between variants. An XBB breakthrough infection enhanced NAb responses against both XBB and BA.2.86 variants. JN.1 displayed lower affinity to ACE2 and higher immune evasion properties compared to BA.2.86.1. Thus, while distinct, the evolutionary trajectory of these variants combines increased fitness and antibody evasion.