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During the COVID-19 pandemic, immunosuppressed patients showed prolonged SARS-CoV-2 infections, with several studies reporting the accumulation of mutations in the viral genome. The weakened immune system present in these individuals, along with the effect of antiviral therapies, are thought to create a favourable environment for intra-host viral evolution and have been linked to the emergence of new viral variants which strongly challenged containment measures and some therapeutic treatments. To assess whether impaired immunity could lead to the increased instability of viral genomes, longitudinal nasopharyngeal swabs were collected from eight immunocompromised patients and fourteen non-immunocompromised subjects, all undergoing SARS-CoV-2 infection. Intra-host viral evolution was compared between the two groups through deep sequencing, exploiting a probe-based enrichment method to minimise the possibility of artefactual mutations commonly generated in amplicon-based methods, which heavily rely on PCR amplification. Although, as expected, immunocompromised patients experienced significantly longer infections, the acquisition of novel intra-host viral mutations was similar between the two groups. Moreover, a thorough analysis of viral quasispecies showed that the variability of viral populations in the two groups is comparable not only at the consensus level, but also when considering low-frequency mutations. This study suggests that a compromised immune system alone does not affect SARS-CoV-2 within-host genomic variability. [ABSTRACT FROM AUTHOR]

Motivation Defining the full domain of protein functions belonging to an organism is a complex challenge that is due to the huge heterogeneity of the taxonomy, where single or small groups of species can bear unique functional characteristics. FunTaxIS-lite provides a solution to this challenge by determining taxon-based constraints on Gene Ontology (GO) terms, which specify the functions that an organism can or cannot perform. The tool employs a set of rules to generate and spread the constraints across both the taxon hierarchy and the GO graph. Results The taxon-based constraints produced by FunTaxIS-lite extend those provided by the Gene Ontology Consortium by an average of 300%. The implementation of these rules significantly reduces errors in function predictions made by automatic algorithms and can assist in correcting inconsistent protein annotations in databases. Availability and implementation FunTaxIS-lite is available on https://www.medcomp.medicina.unipd.it/funtaxis-lite and from https://github.com/MedCompUnipd/FunTaxIS-lite. [ABSTRACT FROM AUTHOR]

Abstract Background Predicting protein function has become increasingly demanding in the era of next generation sequencing technology. The task to assign a curator-reviewed function to every single sequence is impracticable. Bioinformatics tools, easy to use and able to provide automatic and reliable annotations at a genomic scale, are necessary and urgent. In this scenario, the Gene Ontology has provided the means to standardize the annotation classification with a structured vocabulary which can be easily exploited by computational methods. Results Argot2 is a web-based function prediction tool able to annotate nucleic or protein sequences from small datasets up to entire genomes. It accepts as input a list of sequences in FASTA format, which are processed using BLAST and HMMER searches vs UniProKB and Pfam databases respectively; these sequences are then annotated with GO terms retrieved from the UniProtKB-GOA database and the terms are weighted using the e-values from BLAST and HMMER. The weighted GO terms are processed according to both their semantic similarity relations described by the Gene Ontology and their associated score. The algorithm is based on the original idea developed in a previous tool called Argot. The entire engine has been completely rewritten to improve both accuracy and computational efficiency, thus allowing for the annotation of complete genomes. Conclusions The revised algorithm has been already employed and successfully tested during in-house genome projects of grape and apple, and has proven to have a high precision and recall in all our benchmark conditions. It has also been successfully compared with Blast2GO, one of the methods most commonly employed for sequence annotation. The server is freely accessible at http://www.medcomp.medicina.unipd.it/Argot2.

The DNA secondary structures that deviate from the classic Watson and Crick base pairing are increasingly being reported to form transiently in the cell and regulate specific cellular mechanisms. Human viruses are cell parasites that have evolved mechanisms shared with the host cell to support their own replication and spreading. Contrary to human host cells, viruses display a diverse array of nucleic acid types, which include DNA or RNA in single-stranded or double-stranded conformations. This heterogeneity improves the possible occurrence of non-canonical nucleic acid structures. We have previously shown that human virus genomes are enriched in G-rich sequences that fold in four-stranded nucleic acid secondary structures, the G-quadruplexes.Here, by extensive bioinformatics analysis on all available genomes, we showed that human viruses are enriched in highly conserved multiple A (and T or U) tracts, with such an array that they could in principle form quadruplex structures. By circular dichroism, NMR, and Taq polymerase stop assays, we proved that, while A/T/U-quadruplexes do not form, these tracts still display biological significance, as they invariably trigger polymerase pausing within two bases from the A/T/U tract. "A" bases display the strongest effect. Most of the identified A-tracts are in the coding strand, both at the DNA and RNA levels, suggesting their possible relevance during viral translation. This study expands on the presence and mechanism of nucleic acid secondary structures in human viruses and provides a new direction for antiviral research. [ABSTRACT FROM AUTHOR]

The aims of the present study were to obtain sleep quality and sleep timing information in a group of university students and to evaluate the effects of a circadian hygiene education initiative. All students of the University of Padova (approximately 64,000) were contacted by e-mail (major campaigns in October 2019 and October 2020) and directed to an ad hoc website for collection of demographics and sleep quality/timing information. Participants (n = 5,740) received one of two sets of circadian hygiene advice ("A regular life" or "Bright days and dark nights"). Every month, they were then asked how easy it had been to comply and provided with the advice again. At any even month from joining, they completed the sleep quality/timing questionnaires again. Information on academic performance was obtained post hoc , together with representative samples of lecture (n = 5,972) and examination (n = 1,800) timings, plus lecture attendances (n = 25,302). Fifty-two percent of students had poor sleep quality, and 82% showed signs of social jetlag. Those who joined in October 2020, after several months of lockdown and distance learning, had better sleep quality, less social jetlag, and later sleep habits. Over approximately a year, the "Bright days and dark nights" advice resulted in significantly earlier get-up times compared with the "A regular life" advice. Similarly, it also resulted in a trend toward earlier midsleep (i.e., the midpoint, expressed as clock time, between sleep onset and sleep offset) and toward a decrease in the latency between wake-up and get-up time, with no impact on sleep duration. Significant changes in most sleep quality and sleep timing variables (i.e., fewer night awakenings, less social jetlag, and delayed sleep timing during lock-down) were observed in both advice groups over approximately a year, mostly in association with pandemic-related events characterizing 2020. Early chronotype students had better academic performances compared with their later chronotype counterparts. In a multivariate model, sleep quality, chronotype and study subject (science and technology, health and medical, or social and humanities) were independent predictors of academic performance. Taken together, these results underlie the importance of designing circadian-friendly university timetables. [ABSTRACT FROM AUTHOR]

Abstract Background In the area of protein structure prediction, recently a lot of effort has gone into the development of Model Quality Assessment Programs (MQAPs). MQAPs distinguish high quality protein structure models from inferior models. Here, we propose a new method to use an MQAP to improve the quality of models. With a given target sequence and template structure, we construct a number of different alignments and corresponding models for the sequence. The quality of these models is scored with an MQAP and used to choose the most promising model. An SVM-based selection scheme is suggested for combining MQAP partial potentials, in order to optimize for improved model selection. Results The approach has been tested on a representative set of proteins. The ability of the method to improve models was validated by comparing the MQAP-selected structures to the native structures with the model quality evaluation program TM-score. Using the SVM-based model selection, a significant increase in model quality is obtained (as shown with a Wilcoxon signed rank test yielding p-values below 10-15). The average increase in TMscore is 0.016, the maximum observed increase in TM-score is 0.29. Conclusion In template-based protein structure prediction alignment is known to be a bottleneck limiting the overall model quality. Here we show that a combination of systematic alignment variation and modern model scoring functions can significantly improve the quality of alignment-based models.

Abstract Background We describe the development, validation, and use of a highly redundant 120,000 oligonucleotide microarray (MuscleChip) containing 4,601 probe sets representing 1,150 known genes expressed in muscle and 2,075 EST clusters from a non-normalized subtracted muscle EST sequencing project (28,074 EST sequences). This set included 369 novel EST clusters showing no match to previously characterized proteins in any database. Each probe set was designed to contain 20–32 25 mer oligonucleotides (10–16 paired perfect match and mismatch probe pairs per gene), with each probe evaluated for hybridization kinetics (Tm) and similarity to other sequences. The 120,000 oligonucleotides were synthesized by photolithography and light-activated chemistry on each microarray. Results Hybridization of human muscle cRNAs to this MuscleChip (33 samples) showed a correlation of 0.6 between the number of ESTs sequenced in each cluster and hybridization intensity. Out of 369 novel EST clusters not showing any similarity to previously characterized proteins, we focused on 250 EST clusters that were represented by robust probe sets on the MuscleChip fulfilling all stringent rules. 102 (41%) were found to be consistently "present" by analysis of hybridization to human muscle RNA, of which 40 ESTs (39%) could be genome anchored to potential transcription units in the human genome sequence. 19 ESTs of the 40 ESTs were furthermore computer-predicted as exons by one or more than three gene identification algorithms. Conclusion Our analysis found 40 transcriptionally validated, genome-anchored novel EST clusters to be expressed in human muscle. As most of these ESTs were low copy clusters (duplex and triplex) in the original 28,000 EST project, the identification of these as significantly expressed is a robust validation of the transcript units that permits subsequent focus on the novel proteins encoded by these genes.

Ras‐selective lethal small molecule 3 (RSL3), a drug candidate prototype for cancer chemotherapy, triggers ferroptosis by inactivating the glutathione peroxidase glutathione peroxidase 4 (GPx4). Here, we report the purification of the protein indispensable for GPx4 inactivation by RSL3. Mass spectrometric analysis identified 14‐3‐3 isoforms as candidates, and recombinant human 14‐3‐3ε confirms the identification. The function of 14‐3‐3ε is redox‐regulated. Moreover, overexpression or silencing of the gene coding for 14‐3‐3ε consistently controls the inactivation of GPx4 by RSL3. The interaction of GPx4 with a redox‐regulated adaptor protein operating in cell signaling further contributes to frame it within redox‐regulated pathways of cell survival and death and opens new therapeutic perspectives. [ABSTRACT FROM AUTHOR]

Motivation G-quadruplexes (G4s) are non-canonical nucleic acid conformations that are widespread in all kingdoms of life and are emerging as important regulators both in RNA and DNA. Recently, two new higher-order architectures have been reported: adjacent interacting G4s and G4s with stable long loops forming stem-loop structures. As there are no specialized tools to identify these conformations, we developed QPARSE. Results QPARSE can exhaustively search for degenerate potential quadruplex-forming sequences (PQSs) containing bulges and/or mismatches at genomic level, as well as either multimeric or long-looped PQS (MPQS and LLPQS, respectively). While its assessment versus known reference datasets is comparable with the state-of-the-art, what is more interesting is its performance in the identification of MPQS and LLPQS that present algorithms are not designed to search for. We report a comprehensive analysis of MPQS in human gene promoters and the analysis of LLPQS on three experimentally validated case studies from HIV-1, BCL2 and hTERT. Availability and implementation QPARSE is freely accessible on the web at http://www.medcomp.medicina.unipd.it/qparse/index or downloadable from github as a python 2.7 program https://github.com/B3rse/qparse Supplementary information Supplementary data are available at Bioinformatics online. [ABSTRACT FROM AUTHOR]

G-quadruplexes are non-canonical nucleic-acid structures that control transcription, replication, and recombination in organisms. G-quadruplexes are present in eukaryotes, prokaryotes, and viruses. In the latter, mounting evidence indicates their key biological activity. Since data on viruses are scattered, we here present a comprehensive analysis of potential quadruplex-forming sequences (PQS) in the genome of all known viruses that can infect humans. We show that occurrence and location of PQSs are features characteristic of each virus class and family. Our statistical analysis proves that their presence within the viral genome is orderly arranged, as indicated by the possibility to correctly assign up to two-thirds of viruses to their exact class based on the PQS classification. For each virus we provide: i) the list of all PQS present in the genome (positive and negative strands), ii) their position in the viral genome, iii) the degree of conservation among strains of each PQS in its genome context, iv) the statistical significance of PQS abundance. This information is accessible from a database to allow the easy navigation of the results: . The availability of these data will greatly expedite research on G-quadruplex in viruses, with the possibility to accelerate finding therapeutic opportunities to numerous and some fearsome human diseases. [ABSTRACT FROM AUTHOR]

Aims Arrhythmogenic cardiomyopathy (AC) is an inherited heart disease characterized by life-threatening ventricular arrhythmias and fibro-fatty replacement of the myocardium. More than 60% of AC patients show pathogenic mutations in genes encoding for desmosomal proteins. By focusing our attention on the AC8 form, linked to the junctional protein desmoplakin (DSP), we present here a zebrafish model of DSP deficiency, exploited to identify early changes of cell signalling in the cardiac region. Methods and results To obtain an embryonic model of Dsp deficiency, we first confirmed the orthologous correspondence of zebrafish Dsp genes (dspa and dspb) to the human DSP counterpart. Then, we verified their cardiac expression, at embryonic and adult stages, and subsequently we targeted them by antisense morpholino strategy, confirming specific and disruptive effects on desmosomes, like those identified in AC patients. Finally, we exploited our Dsp-deficient models for an in vivo cell signalling screen, using pathway-specific reporter transgenes. Out of nine considered, three pathways (Wnt/β-catenin, TGFβ/Smad3, and Hippo/YAP-TAZ) were significantly altered, with Wnt as the most dramatically affected. Interestingly, under persistent Dsp deficiency, Wnt signalling is rescuable both by a genetic and a pharmacological approach. Conclusion Our data point to Wnt/β-catenin as the final common pathway underlying different desmosomal AC forms and support the zebrafish as a suitable model for detecting early signalling pathways involved in the pathogenesis of DSP-associated diseases, possibly responsive to pharmacological or genetic rescue. [ABSTRACT FROM AUTHOR]

Salinity tolerance has been extensively investigated in recent years due to its agricultural importance. Several features, such as the regulation of ionic transporters and metabolic adjustments, have been identified as salt tolerance hallmarks. Nevertheless, due to the complexity of the trait, the results achieved to date have met with limited success in improving the salt tolerance of rice plants when tested in the field, thus suggesting that a better understanding of the tolerance mechanisms is still required. In this work, differences between two varieties of rice with contrasting salt sensitivities were revealed by the imaging of photosynthetic parameters, ion content analysis and a transcriptomic approach. The transcriptomic analysis conducted on tolerant plants supported the setting up of an adaptive program consisting of sodium distribution preferentially limited to the roots and older leaves, and in the activation of regulatory mechanisms of photosynthesis in the new leaves. As a result, plants resumed grow even under prolonged saline stress. In contrast, in the sensitive variety, RNA-seq analysis revealed a misleading response, ending in senescence and cell death. The physiological response at the cellular level was investigated by measuring the intracellular profile of H2O2 in the roots, using a fluorescent probe. In the roots of tolerant plants, a quick response was observed with an increase in H2O2 production within 5 min after salt treatment. The expression analysis of some of the genes involved in perception, signal transduction and salt stress response confirmed their early induction in the roots of tolerant plants compared to sensitive ones. By inhibiting the synthesis of apoplastic H2O2, a reduction in the expression of these genes was detected. Our results indicate that quick H2O2 signaling in the roots is part of a coordinated response that leads to adaptation instead of senescence in salt-treated rice plants. [ABSTRACT FROM AUTHOR]

Neisseria meningitidis is a human-specific pathogen known for its capability to cause sepsis and meningitis. Here we report the availability of 2 draft genome sequences obtained from patients infected during the same epidemic outbreak. Both bacterial isolates belong to serogroup C, but their genome sequences show local and remarkable differences compared with each other or with the reference genome of strain FAM18. doi: 10.1128/JB.00789-10

The development of functional peripheral ganglia requires a balance of specification of both neuronal and glial components. In the developing dorsal root ganglia (DRGs), these components form from partially-restricted bipotent neuroglial precursors derived from the neural crest. Work in mouse and chick has identified several factors, including Delta/Notch signaling, required for specification of a balance of these components. We have previously shown in zebrafish that the Sry-related HMG domain transcription factor, Sox10, plays an unexpected, but crucial, role in sensory neuron fate specification in vivo. In the same study we described a novel Sox10 mutant allele, sox10baz1, in which sensory neuron numbers are elevated above those of wild-types. Here we investigate the origin of this neurogenic phenotype. We demonstrate that the supernumerary neurons are sensory neurons, and that enteric and sympathetic neurons are almost absent just as in classical sox10 null alleles; peripheral glial development is also severely abrogated in a manner similar to other sox10 mutant alleles. Examination of proliferation and apoptosis in the developing DRG reveals very low levels of both processes in wild-type and sox10baz1, excluding changes in the balance of these as an explanation for the overproduction of sensory neurons. Using chemical inhibition of Delta-Notch-Notch signaling we demonstrate that in embryonic zebrafish, as in mouse and chick, lateral inhibition during the phase of trunk DRG development is required to achieve a balance between glial and neuronal numbers. Importantly, however, we show that this mechanism is insufficient to explain quantitative aspects of the baz1 phenotype. The Sox10(baz1) protein shows a single amino acid substitution in the DNA binding HMG domain; structural analysis indicates that this change is likely to result in reduced flexibility in the HMG domain, consistent with sequence-specific modification of Sox10 binding to DNA. Unlike other Sox10 mutant proteins, Sox10(baz1) retains an ability to drive neurogenin1 transcription. We show that overexpression of neurogenin1 is sufficient to produce supernumerary DRG sensory neurons in a wild-type background, and can rescue the sensory neuron phenotype of sox10 morphants in a manner closely resembling the baz1 phenotype. We conclude that an imbalance of neuronal and glial fate specification results from the Sox10(baz1) protein’s unique ability to drive sensory neuron specification whilst failing to drive glial development. The sox10baz1 phenotype reveals for the first time that a Notch-dependent lateral inhibition mechanism is not sufficient to fully explain the balance of neurons and glia in the developing DRGs, and that a second Sox10-dependent mechanism is necessary. Sox10 is thus a key transcription factor in achieving the balance of sensory neuronal and glial fates. [ABSTRACT FROM AUTHOR]

Introduction: The diagnosis of infectious diseases is among the most successful areas of application of new generation sequencing technologies. The field has seen the development of numerous experimental and analytical approaches for the detection and the fine description of pathogenic and non-pathogenic microorganisms. Areas covered: Without claiming to be exhaustive with respect to all applications and methods developed over the years, this review focuses on the advantages and the issues brought by the new technologies, with an eye in particular to third generation sequencing methods. Both experimental procedures and algorithmic strategies are presented, following the most relevant publications which have led to progress in our ability of detecting infectious agents. Expert commentary: The technical advance brought by third generation sequencing platforms has the potential to significantly expand the range of diagnostic tools that will be available to clinicians. Nonetheless, the implementation of these technologies in clinical practice is still far from being actionable and will temporally follow the path undertaken by second generation methods, which still require the setup of standardized pipelines in both wet and dry laboratory procedures. [ABSTRACT FROM PUBLISHER]