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Age-associated changes in DNA methylation have been characterized across various animals, but not yet in amphibians, which are of particular interest because they include widely studied model organisms. In this study, we present clear evidence that the aquatic vertebrate species Xenopus tropicalis displays patterns of age-associated changes in DNA methylation. We have generated whole-genome bisulfite sequencing (WGBS) profiles from skin samples of nine frogs representing young, mature, and old adults and characterized the gene- and chromosome-scale DNA methylation changes with age. Many of the methylation features and changes we observe are consistent with what is known in mammalian species, suggesting that the mechanism of age-related changes is conserved. Moreover, we selected a few thousand age-associated CpG sites to build an assay based on targeted DNA methylation analysis (TBSseq) to expand our findings in future studies involving larger cohorts of individuals. Preliminary results of a pilot TBSeq experiment recapitulate the findings obtained with WGBS setting the basis for the development of an epigenetic clock assay. The results of this study will allow us to leverage the unique resources available for Xenopus to study how DNA methylation relates to other hallmarks of ageing.

Infectious bronchitis virus (IBV) is an avian pathogen from the Coronavirus family causing major health issues in poultry flocks worldwide. Because of its negative impact on health, performance, and bird welfare, commercial poultry are routinely vaccinated by administering live attenuated virus. However, field strains are capable of rapid adaptation and may evade vaccine-induced immunity. We set out to describe dynamics within and between lineages and assess potential escape from vaccine-induced immunity. We investigated a large nucleotide sequence database of over 1700 partial sequences of the S1 spike protein gene collected from clinical samples of Dutch chickens submitted to the laboratory of Royal GD between 2011 and 2020. Relative frequencies of the two major lineages GI-13 (793B) and GI-19 (QX) did not change in the investigated period, but we found a succession of distinct GI-19 sublineages. Analysis of dN/dS ratio over all sequences demonstrated episodic diversifying selection acting on multiple sites, some of which overlap predicted N-glycosylation motifs. We assessed several measures that would indicate divergence from vaccine strains, both in the overall database and in the two major lineages. However, the frequency of vaccine-homologous lineages did not decrease, no increase in genetic variation with time was detected, and the sequences did not grow more divergent from vaccine sequences in the examined time window. Concluding, our results show sublineage turnover within the GI-19 lineage and we demonstrate episodic diversifying selection acting on the partial sequence, but we cannot confirm nor rule out escape from vaccine-induced immunity. RESEARCH HIGHLIGHTS Succession of GI-19 IBV variants in broiler populations. IBV lineages overrepresented in either broiler, or layer production chickens. Ongoing episodic selection at the IBV S1 spike protein gene sequence. Several positively selected codons coincident with N-glycosylation motifs.

Funding: European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (Grant agreement No. 834103). Background : Previous work identifies conservative family behaviour amongst Pakistanis in the United Kingdom relative to natives, including earlier marriages, fewer dissolutions, and higher fertility. However, few studies have investigated how fertility and partnership are intertwined and interdependent. Objective : Our aims are, first, to identify differences between the family trajectories of Pakistanis and natives and, second, to determine if patterns are consistent across immigrant generations. Finally, we aim to identify how family trajectories vary across birth cohorts and education levels. Methods : We apply multichannel sequence analysis (MCSA) to data from the UK Household Longitudinal Study. We first use clustering methods to group similar individuals and then apply multinomial logistic regression to calculate the probability of belonging to a cluster based on individual characteristics. Results : The Pakistani population exhibits a higher likelihood of entering a direct marriage and having large families compared to natives. Cohabitation is rare amongst Pakistani population. These patterns have changed little between immigrant generations. Degree-level education is associated with a higher likelihood of adopting behaviours typical to ancestral natives; however, the effects are not large enough to indicate convergence. Contribution : We demonstrate the need to investigate partnership and fertility trajectories simultaneously and show the value of MCSA for identifying differences between migrant groups. The results improve our understanding of family formation patterns of Pakistani immigrants and their descendants in the United Kingdom. Publisher PDF

SUMMARY OBJECTIVE: The aim of this study was to evaluate the demographic data, molecular epidemiology, and in vitro antifungal susceptibility results of patients with Aspergillus isolated from various clinical specimens. METHODS: A total of 44 Aspergillus strains were studied. The definition of invasive aspergillosis in patients was made according to European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) criteria. Strains were phenotypically and molecularly identified. Demographic characteristics of patients and genotypes of strains were evaluated. Phylogenetic analysis was done by the The Unweighted Pair-Group Method with Arithmetic Mean (UPGMA). Antifungal susceptibility of strains was determined according to The Clinical and Laboratory Standards Institute (CLSI)-M61-Ed2 and The European Committee on Antimicrobial Susceptibility Testing (EUCAST). RESULTS: A total of 11 patients were classified as proven and 33 as probable invasive aspergillosis. There was a statistically significant difference in age groups, subdisease, neutropenic, and receiving chemotherapy between groups. A total of 23 strains were identified as Aspergillus fumigatus, 12 as Aspergillus niger, 6 as Aspergillus flavus, and 3 as Aspergillus terreus. Phylogenetic analysis revealed five different genotypes. No statistical difference was found in the comparisons between patients groups and genotype groups. There was a statistically significant difference between genotype groups and voriconazole, posaconazole, and itraconazole Minimum Inhibition Concentration (MIC). CONCLUSION: Accurate identification of strains and antifungal susceptibility studies should be performed due to azole and amphotericin B resistance. Genotyping studies are important in infection control due to identifying sources of infection and transmission routes.

Background and objectives: To extend work careers, it is important to focus on all working-aged people, including young adults. However, there is little knowledge about the stability of early careers and its life course social and health-related determinants. The aim of this study is to identify what kind of typical patterns of work participation and transitions between different statuses can be identified among young adults after their first entry into the labour market, and to examine whether the timing of entry together with parental and own socioeconomic position and health predict early work participation patterns.Methods: We used the Finnish Birth Cohort 1987 including data from several registers from all 59,476 children born in 1987 as well as their parents, followed until the end of 2015. We created sequences of monthly work participation statuses starting from the calendar month when a cohort member had their first at least six-month long spell in employment or entrepreneurship. Work participation statuses (8 categories) were collected for 60 months or until the end of the follow-up. We included altogether 51,871 individuals with an entry into paid employment, who could be followed for at least 12 subsequent months. Parental social and health-related covariates were included from national registers. We used first sequence analysis and cluster analysis to identify latent groups ofindividuals with similar work participation patterns. Next, we estimated a mixture Markov model that allows for identification of latent classes of labour market attachment, estimation of labour market transitions within classes, and prediction of class membership using childhood social and healthrelated determinants.Results: We observed that entry into the labour market as measured by six months in continuous employment was not a permanent entry for many, not only due to unemployment and health-related exits but also due to studies and parental leave. During the first five years, we observed a fairly linear relationship between the age at entry and early work participation pattern: individuals entering the labour market at a later age were more likely to be in continuous employment thereafter (all else equal). Regarding both own and parental social- and health-related childhood factors, latent classes show clear patterns of accumulated advantage and disadvantage: more advantaged background predicted exits due to studies or – when following a late entry – stable employment, while disadvantaged background factors predicted more unstable work and long-term exits from the labour market.Conclusions: There are varied work participation patterns and transitions between employment statuses among young employees. More in-depth understanding of these patterns and their determinants is important to plan targeted interventions and to promote more stable work participation among young adults.

PURPOSE Guidelines recommend somatic and germline testing for men with advanced prostate cancer (PCa). Barriers to widespread implementation result in underutilization of germline testing. Somatic testing alone risks missing pathogenic germline variants (PGVs). We sought to determine whether the addition of germline testing to tumor-only sequencing improves detection of PGVs in men with advanced PCa. Secondarily, we sought to define the added value of combining somatic and germline testing to optimize detection of clinically actionable alterations. PATIENTS AND METHODS We analyzed results of independent germline testing and tumor-only sequencing from 100 men with advanced PCa from a prospective clinical trial (ClinicalTrials.gov identifier: NCT03328091 ). The primary outcome was the proportion of PGVs not reported with tumor-only sequencing. The secondary outcome was the association of locus-specific loss of heterozygosity for PGVs in homologous recombination genes with clinical-genomic features. RESULTS In the 100 men who underwent germline testing and tumor-only sequencing, 24 PGVs were identified, 17 of which were clinically actionable, in 23 patients. Tumor-only sequencing failed to report four (17%) of the PGVs. One additional PGV (4.2%) had variant allele frequency on tumor-sequencing below the threshold for follow-up germline testing. When integrating tumor-only sequencing with germling testing results, 33% of patients harbored clinically actionable alterations. Rates of locus-specific loss of heterozygosity were higher for BRCA2 PGVs in castration-resistant PCa than PGVs in other homologous recombination genes in hormone-sensitive PCa ( P = .029). CONCLUSION Tumor-only sequencing failed to report more than 20% of PGVs in men with advanced PCa. These findings strongly support guideline recommendations for universal germline and somatic testing in this population. Combining tumor and germline sequencing doubled the chance of detecting a clinically actionable alteration.

The statistical inference of high-order Markov chains (MCs) for biological sequences is vital for molecular sequence analyses but can be hindered by the high dimensionality of free parameters. In the seminal article by Bühlmann and Wyner, variable length Markov chain (VLMC) model was proposed to embed the full-order MC in a sparse structured context tree. In the key procedure of tree pruning of their proposed context algorithm, the word count-based statistic for each branch was defined and compared with a fixed cutoff threshold calculated from a common chi-square distribution to prune the branch of the context tree. In this study, we find that the word counts for each branch are highly intercorrelated, resulting in non-negligible effects on the distribution of the statistic of interest. We demonstrate that the inferred context tree based on the original context algorithm by Bühlmann and Wyner, which uses a fixed cutoff threshold based on a common chi-square distribution, can be systematically biased and error prone. We denote the original context algorithm as VLMC-Biased (VLMC-B). To solve this problem, we propose a new context tree inference algorithm using an adaptive tree-pruning scheme, termed VLMC-Consistent (VLMC-C). The VLMC-C is founded on the consistent branch-specific mixed chi-square distributions calculated based on asymptotic normal distribution of multiple word patterns. We validate our theoretical branch-specific asymptotic distribution using simulated data. We compare VLMC-C with VLMC-B on context tree inference using both simulated and real genome sequence data and demonstrate that VLMC-C outperforms VLMC-B for both context tree reconstruction accuracy and model compression capacity.

Femoroacetabular impingement (FAI) has a strong clinical association with the development of hip osteoarthritis (OA); however, the pathobiological mechanisms underlying the transition from focal impingement to global joint degeneration remain poorly understood. The purpose of this study is to use whole-genome RNA sequencing to identify and subsequently validate differentially expressed genes (DEGs) in femoral head articular cartilage samples from patients with FAI and hip OA secondary to FAI. Thirty-seven patients were included in the study with whole-genome RNA sequencing performed on 10 gender-matched patients in the FAI and OA cohorts and the remaining specimens were used for validation analyses. We identified a total of 3531 DEGs between the FAI and OA cohorts with multiple targets for genes implicated in canonical OA pathways.Quantitative reverse transcription-polymerase chain reaction validation confirmed increased expression of FGF18 and WNT16 in the FAI samples, while there was increased expression of MMP13 and ADAMTS4 in the OA samples. Expression levels of FGF18 and WNT16 were also higher in FAI samples with mild cartilage damage compared to FAI samples with severe cartilage damage or OA cartilage. Our study further expands the knowledge regarding distinct genetic reprogramming in the cartilage between FAI and hip OA patients. We independently validated the results of the sequencing analysis and found increased expression of anabolic markers in patients with FAI and minimal histologic cartilage damage, suggesting that anabolic signaling may be increased in early FAI with a transition to catabolic and inflammatory gene expression as FAI progresses towards more severe hip OA.Clinical significance:Cam-typeFAI has a strong clinical association with hip OA; however, the cellular pathophysiology of disease progression remains poorly understood. Several previous studies have demonstrated increased expression of inflammatory markers in FAI cartilage samples, suggesting the involvement of these inflammatory pathways in the disease progression. Our study further expands the knowledge regarding distinct genetic reprogramming in the cartilage between FAI and hip OA patients. In addition to differences in inflammatory gene expression, we also identified differential expression in multiple pathways involved in hip OA progression.

RNA is a key regulator of almost every cellular process, and the structures adopted by RNA molecules are thought to be central to their functions. The recent fast-paced evolution of high-throughput sequencing-based RNA structure mapping methods has enabled the rapid in vivo structural interrogation of entire cellular transcriptomes. Collectively, these studies are shedding new light on the long underestimated complexity of the structural organization of the transcriptome - the RNA structurome. Moreover, recent analyses are challenging the view that the RNA structurome is a static entity by revealing how RNA molecules establish intricate networks of alternative intramolecular and intermolecular interactions and that these ensembles of RNA structures are dynamically regulated to finely tune RNA functions in living cells. This new understanding of how RNA can shape cell phenotypes has important implications for the development of RNA-targeted therapeutic strategies.

Acute myeloid leukaemia (AML) represents a set of heterogeneous myeloid malignancies, and hallmarks include mutations in epigenetic modifiers, transcription factors and kinasessup1-5/sup. The extent to which mutations in AML drive alterations in chromatin 3D structure and contribute to myeloid transformation is unclear. Here we use Hi-C and whole-genome sequencing to analyse 25 samples from patients with AML and 7 samples from healthy donors. Recurrent and subtype-specific alterations in A/B compartments, topologically associating domains and chromatin loops were identified. RNA sequencing, ATAC with sequencing and CUTamp;Tag for CTCF, H3K27ac and H3K27me3 in the same AML samples also revealed extensive and recurrent AML-specific promoter-enhancer and promoter-silencer loops. We validated the role of repressive loops on their target genes by CRISPR deletion and interference. Structural variation-induced enhancer-hijacking and silencer-hijacking events were further identified in AML samples. Hijacked enhancers play a part in AML cell growth, as demonstrated by CRISPR screening, whereas hijacked silencers have a downregulating role, as evidenced by CRISPR-interference-mediated de-repression. Finally, whole-genome bisulfite sequencing of 20 AML and normal samples revealed the delicate relationship between DNA methylation, CTCF binding and 3D genome structure. Treatment of AML cells with a DNA hypomethylating agent and triple knockdown of DNMT1, DNMT3A and DNMT3B enabled the manipulation of DNA methylation to revert 3D genome organization and gene expression. Overall, this study provides a resource for leukaemia studies and highlights the role of repressive loops and hijacked cis elements in human diseases.

Phage P1 was isolated from the abnormal fermented liquid using Lactobacillus plantarum (L. plantarum) IMAU10120. To date, genetic knowledge regarding L. plantarum phage diversity is still limited, and further in-depth sequencing analysis of isolated L. plantarum phages can fill this gap. Here, we investigated the whole genome sequence of L. plantarum phage P1, sequenced by Illumina HiSeq platform, to decipher its genomic characteristics and putative DNA packaging mechanism. It was revealed that phage P1 was 73,787 bp in length, which was composed of linear double-stranded DNA (dsDNA), and the GC content was 39.17%. Its genome contained 86 coding sequences for various functions, such as adsorption, injection, replication, assembly, and release. Moreover, it was observed that L. plantarum phage P1 utilized the 'cohesive ends' DNA packaging mechanism. This study furthered the genomic knowledge of L. plantarum phages and provided some basis for the control of L. plantarum phages in the dairy fermentation industry.

Epigenome-wide association studies (EWAS) are widely implemented in epidemiology, and the Illumina HumanMethylationEPIC BeadChip (EPIC) DNA microarray is the most-used technology. Recently, next-generation sequencing (NGS)-based methods, which assess DNA methylation at single-base resolution, have become more affordable and technically feasible. While the content of microarray technology is fixed, NGS-based approaches, such as the Roche Nimblegen, SeqCap Epi Enrichment System (SeqCap), offer the flexibility of targeting most CpGs in a gene. With the current usage of microarrays and emerging NGS-based technologies, it is important to establish whether data generated from the two platforms are comparable. We harnessed 112 samples from the Center for the Health Assessment of Mothers and Children of Salinas (CHAMACOS) birth cohort study and compared DNA methylation between the EPIC microarray and SeqCap for PON1 and nine additional candidate genes, by evaluating epigenomic coverage and correlations. We conducted multivariable linear regression and principal component analyses to assess the ability of the EPIC array and SeqCap to detect biological differences in gene methylation by the PON1−108 single nucleotide polymorphism. We found an overall high concordance (r = 0.84) between SeqCap and EPIC DNA methylation, among highly methylated and minimally methylated regions. However, substantial disagreement was present between the two methods in moderately methylated regions, with SeqCap measurements exhibiting greater within-site variation. Additionally, SeqCap did not capture PON1 SNP associated differences in DNA methylation that were evident with the EPIC array. Our findings indicate that microarrays perform well for analysing DNA methylation in large cohort studies but with limited coverage.

Background: Abnormal expression of miRNA is a common feature in many diseases. Some studies have also emphasized that miRNAs play an important role in asthma and allergic rhinitis (AR). This study attempts to reveal the differences between miRNAs expression and normal nasal mucosa in AR patients by microarray method so as to further understand the molecular mechanism of AR development. Method: MiRNA microarrays were used for analyzing six samples of the nasal mucosa of AR and six samples of nonallergic patients. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) of some differently expressed miRNAs was used to confirm the array results. Furthermore, pathway analysis was carried out. Results: The microarray identified that 64 miRNAs showed altered expression in the nasal mucosa of the AR group when compared with the control group. Moreover, the expression levels of ten miRNAs were significantly altered in the AR group. To verify the results of microarray, three differentially expressed miRNA were determined by RT-PCR, and the results also confirmed these changes. Ten differentially expressed miRNAs were present in the nasal mucosa of AR patients compared with the control group, and three differentially expressed miRNAs as miR-1244, miR- 4651, and miR-7641 were determined by RT-PCR. The results also confirmed the changes, indicating that they play important roles in the process of AR. Conclusion: MiR-1244, miR-4651, and miR-7641 may play important roles in the process of AR. Sequencing analysis indicated that three kinds of mutations existed in MAPK8 3’UTR, which may play a role in binding with miR-7641, and then influence the AR process. Single miRNA or, more probably, their sets hold the promise for their use as biomarkers of allergic rhinitis. They are also a promising target of future therapies.

Here the Human Pangenome Reference Consortium presents a first draft of the human pangenome reference. The pangenome contains 47 phased, diploid assemblies from a cohort of genetically diverse individuals1. These assemblies cover more than 99% of the expected sequence in each genome and are more than 99% accurate at the structural and base pair levels. Based on alignments of the assemblies, we generate a draft pangenome that captures known variants and haplotypes and reveals new alleles at structurally complex loci. We also add 119 million base pairs of euchromatic polymorphic sequences and 1,115 gene duplications relative to the existing reference GRCh38. Roughly 90 million of the additional base pairs are derived from structural variation. Using our draft pangenome to analyse short-read data reduced small variant discovery errors by 34% and increased the number of structural variants detectedper haplotype by 104% compared with GRCh38-based workflows, which enabled the typing of the vast majority of structural variant alleles per sample.

Background Mitochondrial genome sequences have become critical to the study of biodiversity. Genome skimming and other short-read based methods are the most common approaches, but they are not well-suited to scale up to multiplexing hundreds of samples. Here, we report on a new approach to sequence hundreds to thousands of complete mitochondrial genomes in parallel using long-amplicon sequencing. We amplified the mitochondrial genome of 677 specimens in two partially overlapping amplicons and implemented an asymmetric PCR-based indexing approach to multiplex 1,159 long amplicons together on a single PacBio SMRT Sequel II cell. We also tested this method on Oxford Nanopore Technologies (ONT) MinION R9.4 to assess if this method could be applied to other long-read technologies. We implemented several optimizations that make this method significantly more efficient than alternative mitochondrial genome sequencing methods. Results With the PacBio sequencing data we recovered at least one of the two fragments for 96% of samples (~ 80–90%) with mean coverage ~ 1,500x. The ONT data recovered less than 50% of input fragments likely due to low throughput and the design of the Barcoded Universal Primers which were optimized for PacBio sequencing. We compared a single mitochondrial gene alignment to half and full mitochondrial genomes and found, as expected, increased tree support with longer alignments, though whole mitochondrial genomes were not significantly better than half mitochondrial genomes. Conclusions This method can effectively capture thousands of long amplicons in a single run and be used to build more robust phylogenies quickly and effectively. We provide several recommendations for future users depending on the evolutionary scale of their system. A natural extension of this method is to collect multi-locus datasets consisting of mitochondrial genomes and several long nuclear loci at once.

There has been tremendous progress in phased genome assembly production by combining long-read data with parental information or linked-read data. Nevertheless, a typical phased genome assembly generated by trio-hifiasm still generates more than 140 gaps. We perform a detailed analysis of gaps, assembly breaks, and misorientations from 182 haploid assemblies obtained from a diversity panel of 77 unique human samples. Although trio-based approaches using HiFi are the current gold standard, chromosome-wide phasing accuracy is comparable when using Strand-seq instead of parental data. Importantly, the majority of assembly gaps cluster near the largest and most identical repeats (including segmental duplications [35.4%], satellite DNA [22.3%], or regions enriched in GA/AT-rich DNA [27.4%]). Consequently, 1513 protein-coding genes overlap assembly gaps in at least one haplotype, and 231 are recurrently disrupted or missing from five or more haplotypes. Furthermore, we estimate that 6-7 Mbp of DNA are misorientated per haplotype irrespective of whether trio-free or trio-based approaches are used. Of these misorientations, 81% correspond to bona fide large inversion polymorphisms in the human species, most of which are flanked by large segmental duplications. We also identify large-scale alignment discontinuities consistent with 11.9 Mbp of deletions and 161.4 Mbp of insertions per haploid genome. Although 99% of this variation corresponds to satellite DNA, we identify 230 regions of euchromatic DNA with frequent expansions and contractions, nearly half of which overlap with 197 protein-coding genes. Such variable and incompletely assembled regions are important targets for future algorithmic development and pangenome representation.

Bispecific antibodies (BsAbs) represent an emerging class of immunotherapy but inefficiency in the current BsAb discovery paradigm has limited their broad clinical availability. Here we report a high throughput, agnostic, single-cell-based BsAb functional screening pipeline, comprising molecular and cell engineering for efficient generation of BsAb library cells, followed by functional interrogation at the single-cell level to identify and sort positive clones and downstream sequence identification with single-cell PCR and sequencing and functionality characterization. Using a CD19xCD3 bispecific T cell engager (BiTE) as a model system, we demonstrate that our single cell platform possesses a high throughput screening efficiency of up to one and half million variant library cells per run and can isolate rare functional clones at low abundance of 0.008%. Using a complex CD19xCD3 BiTE-expressing cell library with approximately 22,300 unique variants comprising combinatorially varied scFvs, connecting linkers and VL/VH orientations, we have identified 98 unique clones including extremely rare ones (∼ 0.001% abundance). We also discovered BiTEs that exhibit novel properties contradictory to conventional wisdom, including harboring rigid scFv connecting peptide linkers yet with in vitro cytotoxicity comparable to that of clinically approved Blinatumomab. Through sequencing analyses on sorted BiTE clones, we discovered multiple design variable preferences for functionality including the CD19VL-VH– CD3VH-VL and CD19VH-VL–CD3VH-VL arrangements being the most favored orientation. Sequence analysis further interrogated the sequence composition of the CDRH3 domain in scFvs and identified amino acid residues conserved for function. We expect our single cell platform to not only significantly increase the development speed of high quality of new BsAb therapeutics for cancer and other disorders, but also enable identifying generalizable design principles for new BsAbs and other immunotherapeutics based on an in-depth understanding of the inter-relationships between sequence, structure, and function.

Background Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder that has been included in newborn screening programs. Current approaches to gene testing for CAH are facing challenges because of the complexity of the CYP21A2 locus and genetic heterogeneity of the disease. Methods A comprehensive analysis of CAH (CACAH) combining long-range locus-specific PCR and long-read sequencing (LRS) was developed to perform full sequence analysis of 5 common CAH candidate genes, including CYP21A2, CYP11B1, CYP17A1, HSD3B2, and StAR. In a blind retrospective study, the clinical utility of CACAH was evaluated in 37 samples by comparing to standard CAH testing using multiplex ligation-dependent probe amplification (MLPA) plus Sanger sequencing. Results Of the 37 clinical samples, a total of 69 pathogenic variants were identified, comprising 65 CYP21A2 variants, 2 HSD3B2 variants, and 2 CYP17A1 variants. For CYP21A2, the most frequent variant was c.518T > A (29.2%), followed by c.293−13C/A > G (21.5%). Compared with the current CAH testing using MLPA plus Sanger sequencing, the CACAH assay showed 100% specificity and 100% sensitivity, and precisely determined the junction sites of deletions/insertions and cis–trans configuration of multiple variants without analyzing family samples. Moreover, CACAH identified a case carrying 2 copies of CYP21A1 with the c.1451_1452delinsC variant on the same chromosome, which was not confirmed by MLPA plus Sanger sequencing. Conclusion LRS-based CACAH can determine all genotypes of CAH accurately and reliably in one assay, presenting a comprehensive approach for CAH genetic diagnosis and carrier screening.

The assay for transposase-accessible chromatin using sequencing (ATAC-seq) provides a simple and scalable way to detect the unique chromatin landscape associated with a cell type and how it may be altered by perturbation or disease. ATAC-seq requires a relatively small number of input cells and does not require a priori knowledge of the epigenetic marks or transcription factors governing the dynamics of the system. Here we describe an updated and optimized protocol for ATAC-seq, called Omni-ATAC, that is applicable across a broad range of cell and tissue types. The ATAC-seq workflow has five main steps: sample preparation, transposition, library preparation, sequencing and data analysis. This protocol details the steps to generate and sequence ATAC-seq libraries, with recommendations for sample preparation and downstream bioinformatic analysis. ATAC-seq libraries for roughly 12 samples can be generated in 10 h by someone familiar with basic molecular biology, and downstream sequencing analysis can be implemented using benchmarked pipelines by someone with basic bioinformatics skills and with access to a high-performance computing environment.

Motivation Single-cell sequencing methods provide previously impossible resolution into the transcriptome of individual cells. Cell hashing reduces single-cell sequencing costs by increasing capacity on droplet-based platforms. Cell hashing methods rely on demultiplexing algorithms to accurately classify droplets; however, assumptions underlying these algorithms limit accuracy of demultiplexing, ultimately impacting the quality of single-cell sequencing analyses. Results We present Bimodal Flexible Fitting (BFF) demultiplexing algorithms BFFcluster and BFFraw, a novel class of algorithms that rely on the single inviolable assumption that barcode count distributions are bimodal. We integrated these and other algorithms into cellhashR, a new R package that provides integrated QC and a single command to execute and compare multiple demultiplexing algorithms. We demonstrate that BFFcluster demultiplexing is both tunable and insensitive to issues with poorly behaved data that can confound other algorithms. Using two well-characterized reference datasets, we demonstrate that demultiplexing with BFF algorithms is accurate and consistent for both well-behaved and poorly behaved input data. Availability and implementation cellhashR is available as an R package at https://github.com/BimberLab/cellhashR. cellhashR version 1.0.3 was used for the analyses in this manuscript and is archived on Zenodo at https://www.doi.org/10.5281/zenodo.6402477. Supplementary information Supplementary data are available at Bioinformatics online.

L-ASPARAGINASE is an anti-neoplasm agent. This agent is classified as an enzyme compound. L-asparaginase is used in medicine as well as in the food industry. Microbial strains that are producing L-Asparaginase can be found in many habitats such as soil. The aim of this study was to find isolates producing L-asparaginase enzyme with high ability to inhibit cancer cells. Bacteria of the Actinobacter family were isolated from the soil samples around the roots of the plants in the actinomycet isolation medium. Then, the bacteria were isolated and extracted by screening in M9 medium. Isolates were identified using16S PCR sequencing method. Three isolates of ASP and B and C showed the highest enzyme production, which showed a molecular weight of 43 kDa and in terms of enzymatic activity112. 572 IU/ml, 99.706 IU/ml, 138.572 IU/ml respectively. These three isolates with sequence analysis was similar to Streptomyces flavoviridis and Streptomyces griseus and Streptomyces hypolyticus respectively. Isolate C showed good effects in inhibiting cell MCF-7, HepG-2. There are various isolates enzyme production power in nature and Actinobacter family has shown more ability to produce various enzymes and metabolites and the isolates isolated in this study are used for industrial enzyme production.

Since its initial release in 2000, the human reference genome has covered only the euchromatic fraction of the genome, leaving important heterochromatic regions unfinished. Addressing the remaining 8% of the genome, the Telomere-to-Telomere (T2T) Consortium presents a complete 3.055 billion–base pair sequence of a human genome, T2T-CHM13, that includes gapless assemblies for all chromosomes except Y, corrects errors in the prior references, and introduces nearly 200 million base pairs of sequence containing 1956 gene predictions, 99 of which are predicted to be protein coding. The completed regions include all centromeric satellite arrays, recent segmental duplications, and the short arms of all five acrocentric chromosomes, unlocking these complex regions of the genome to variational and functional studies.

Background Breast cancer (BC) treatments and related symptoms may affect return to work (RTW). The objective of this study was to investigate the impact of BC care pathways (timing and sequence of treatments) and related symptoms on RTW. Methods The study population included working-age women with BC who were enrolled in the French CONSTANCES cohort from 2012 to 2018. BC treatments, antidepressant/anxiolytic and antalgic drug deliveries (used as proxies of depression and pain, respectively) and statutory sick pay (used to estimate RTW and time to RTW) were assessed monthly using data from the French national healthcare system database. BC care pathways were identified with the sequence analysis method. Cox models with time-dependent covariates were used to investigate the impact of BC care pathways and related symptoms on RTW and time to RTW, after adjusting for age and socioeconomic characteristics. Results 73.2% (231/303) of women returned to work within 2 years after BC diagnosis. Five BC care pathway patterns were identified: (i) BC surgery only, (ii) BC surgery and radiotherapy, (iii) BC surgery and chemotherapy, (iv) BC surgery and chemotherapy and radiotherapy, and (v) BC surgery and long-term alternative chemotherapy/radiotherapy. The hazards ratios of non-RTW were significantly higher for women who received BC surgery and long-term alternative chemotherapy/radiotherapy and for > 55-year-old women. Time to RTW was significantly longer in women who received chemotherapy (patterns iii to v) and in women with antidepressant/anxiolytic and antalgic drug deliveries. Conclusion This study highlights the value of considering the dynamic, cumulative and temporal features of BC care pathways and related symptoms to facilitate the RTW of women with BC.

Cancer-associated fibroblasts (CAFs) were presumed absent in glioblastoma given the lack of brain fibroblasts. Serial trypsinization of glioblastoma specimens yielded cells with CAF morphology and single-cell transcriptomic profiles based on their lack of copy number variations (CNVs) and elevated individual cell CAF probability scores derived from the expression of 9 CAF markers and absence of 5 markers from non-CAF stromal cells sharing features with CAFs. Cells without CNVs and with high CAF probability scores were identified in single-cell RNA-Seq of 12 patient glioblastomas. Pseudotime reconstruction revealed that immature CAFs evolved into subtypes, with mature CAFs expressing actin alpha 2, smooth muscle (ACTA2). Spatial transcriptomics from 16 patient glioblastomas confirmed CAF proximity to mesenchymal glioblastoma stem cells (GSCs), endothelial cells, and M2 macrophages. CAFs were chemotactically attracted to GSCs, and CAFs enriched GSCs. We created a resource of inferred crosstalk by mapping expression of receptors to their cognate ligands, identifying PDGF and TGF-β as mediators of GSC effects on CAFs and osteopontin and HGF as mediators of CAF-induced GSC enrichment. CAFs induced M2 macrophage polarization by producing the extra domain A (EDA) fibronectin variant that binds macrophage TLR4. Supplementing GSC-derived xenografts with CAFs enhanced in vivo tumor growth. These findings are among the first to identify glioblastoma CAFs and their GSC interactions, making them an intriguing target.

The genomic sequence of the horse has been available since 2009, providing critical resources for discovering important genomic variants regarding both animal health and population structures. However, to fully understand the functional implications of these variants, detailed annotation of the horse genome is required. Currently, the horse genome is annotated using limited available RNA-seq data, as well as through comparative genomics by translating human and mouse genome annotation. While this approach has served the equine researchers well and led to various discoveries that would improve care and management of horses, many important questions remain unanswered. The limitation of the current annotation is two pronged. First, the heavy reliance on short-read sequencing-based RNA-seq data meant that alternate isoforms could not be accurately resolved. Second, epigenomic regulatory elements are crucial to detailed understanding of gene expression network but are yet to be systemically identified in the horse. Many of these regulatory elements, including enhancers, promoters, and insulators, are either not transcribed or transcribed at a very low level, necessitating alternate approaches to identify them. To solve above problems, the Functional Annotation of the Animal Genomes (FAANG) project proposed a systemic approach to tissue collection, phenotyping, and data generation, adopting the blueprint laid out by the Encyclopedia of DNA Elements (ENCODE) project. Here we detail the first comprehensive overview of gene expression and regulation in the horse, providing the equine research community an expanded set of genomics resources for studies of complex traits in the horse.Author SummaryFunctional annotation of a reference genome provides critical information that pertains the tissue-specific gene expression and regulation. Non-model organisms often rely on existing annotations of human and mouse genomes and the conservation between species for their genome annotation. This approach has limited power in annotating transcripts and regulatory elements that are less evolutionarily conserved. Such are the cases of alternatively spliced isoforms and enhancer elements. In a large-scale collaborated effort, Functional Annotation of Animal Genome (FAANG) aims to generate species-specific and tissue-aware functional annotation for farm animals. In this study, we present the overall annotation efforts and findings from the equine FAANG group. This integrated annotation for the horse genome provides, for the first time, a comprehensive overview of gene expression and regulation landscape in nine major equine tissues, as well as an analytical framework for further inclusion of other important tissues.

The current version of the human reference genome, GRCh38, contains a number of errors including 1.2 Mbp of falsely duplicated and 8.04 Mbp of collapsed regions. These errors impact the variant calling of 33 protein-coding genes, including 12 with medical relevance. Here, we present FixItFelix, an efficient remapping approach, together with a modified version of the GRCh38 reference genome that improves the subsequent analysis across these genes within minutes for an existing alignment file while maintaining the same coordinates. We showcase these improvements over multi-ethnic control samples, demonstrating improvements for population variant calling as well as eQTL studies.

MotivationCompositional analysis is based on the premise that a relatively small proportion of taxa are “differentially abundant”, while the ratios of the relative abundances of the remaining taxa remain unchanged. Most existing methods of compositional analysis such as ANCOM or ANCOM-BC use log-transformed data, but log-transformation of data with pervasive zero counts is problematic, and these methods cannot always control the false discovery rate (FDR). Further, high-throughput microbiome data such as 16S amplicon or metagenomic sequencing are subject to experimental biases that are introduced in every step of the experimental workflow. McLaren, Willis and Callahan [1] have recently proposed a model for how these biases affect relative abundance data.MethodsMotivated by [1], we show that the (log) odds ratios in a logistic regression comparing counts in two taxa are invariant to experimental biases. With this motivation, we propose LOCOM, a robust logistic regression approach to compositional analysis, that does not require pseudocounts. We use a Firth bias-corrected estimating function to account for sparse data. Inference is based on permutation to account for overdispersion and small sample sizes. Traits can be either binary or continuous, and adjustment for continuous and/or discrete confounding covariates is supported.ResultsOur simulations indicate that LOCOM always preserved FDR and had much improved sensitivity over existing methods. In contrast, ANCOM often had inflated FDR; ANCOM-BC largely controlled FDR but still had modest inflation occasionally; ALDEx2 generally had low sensitivity. LOCOM and ANCOM were robust to experimental biases in every situation, while ANCOM-BC and ALDEx2 had elevated FDR when biases at causal and non-causal taxa were differentially distributed. The flexibility of our method for a variety of microbiome studies is illustrated by the analysis of data from two microbiome studies.Availability and implementationOur R package LOCOM is available on GitHub athttps://github.com/yijuanhu/LOCOMin formats appropriate for Macintosh or Windows.

Little is known about the prevalence, functionality and ecological roles of temperate phages for members of the mycolic acid producing bacteria, the Mycolata. While many lytic phages infective for these organisms have been isolated, and assessed for their suitability for use as biological control agents of activated sludge foaming, no studies have investigated how temperate phages might be induced for this purpose. Bioinformatic analysis using the PHAge Search Tool (PHAST) on Mycolata whole genome sequence data in GenBank for members of the genera Gordonia, Mycobacterium, Nocardia, Rhodococcus, and Tsukamurella revealed 83% contained putative prophage DNA sequences. Subsequent prophage inductions using mitomycin C were conducted on 17 Mycolata strains. This led to the isolation and genome characterization of three novel Caudovirales temperate phages, namely GAL1, GMA1, and TPA4, induced from Gordonia alkanivorans, Gordonia malaquae, and Tsukamurella paurometabola, respectively. All possessed highly distinctive dsDNA genome sequences.

Genotyping single-nucleotide polymorphisms (SNPs) in microbiomes enables strain-level quantification. In this protocol, we describe a computational pipeline that performs fast and accurate SNP genotyping using metagenomic data. We first demonstrate how to use Maast to catalog SNPs from microbial genomes. Then we use GT-Pro to extract unique SNP-covering k-mers, optimize a data structure for storing these k-mers, and finally perform metagenotyping. For proof of concept, the protocol leverages public whole-genome sequences to metagenotype a synthetic community. For complete details on the use and execution of this protocol, please refer to Shi etal. (2022a)1 and Shi etal. (2022b).2.

SUMMARY OBJECTIVE: Retinitis pigmentosa is an inherited degenerative disorder causing severe retinal dystrophy and visual impairment, mainly with onset in the first or second decades. The next-generation sequencing has become an efficient tool to identify disease-causing mutations in retinitis pigmentosa. The aim of this retrospective study was to investigate novel gene variants and evaluate the utility of whole-exome sequencing in patients with retinitis pigmentosa. METHODS: The medical records of 20 patients with retinitis pigmentosa at Eskişehir City Hospital between September 2019 and February 2022 were analyzed retrospectively. Peripheral venous blood was obtained, followed by the extraction of genomic DNAs. The medical and ophthalmic histories were collected, and ophthalmological examinations were performed. Whole-exome sequencing was performed to determine the genetic etiology of the patients. RESULTS: The proportion of genetically solved cases was 75% (15/20) in the patients with retinitis pigmentosa. Molecular genetic testing identified 13 biallelic and 4 monoallelic mutations in known retinitis pigmentosa genes, including 11 novel variants. According to in silico prediction tools, nine variants were predicted as pathogenic or possibly pathogenic. We identified six previously reported mutations to be associated with retinitis pigmentosa. The age of onset of the patients ranged from 3 to 19, with a mean age of onset of 11.6. All patients had a loss of central vision. CONCLUSION: As the first study of the application of whole-exome sequencing among patients with retinitis pigmentosa in a Turkish cohort, our results may contribute to the characterization of the spectrum of variants related to retinitis pigmentosa in the Turkish population. Future population-based studies will enable us to reveal the detailed genetic epidemiology of retinitis pigmentosa.

Feed efficiency and energy balance are important traits underpinning profitability and environmental sustainability in animal production. They are complex traits, and our understanding of their underlying biology is currently limited. One measure of feed efficiency is residual feed intake (RFI), which is the difference between actual and predicted intake. Variation in RFI among individuals is attributable to the metabolic efficiency of energy utilization. High RFI (H_RFI) animals require more energy per unit of weight gain or milk produced compared with low RFI (L_RFI) animals. Energy balance (EB) is a closely related trait calculated very similarly to RFI. Cellular energy metabolism in mitochondria involves mitochondrial protein (MiP) encoded by both nuclear (NuMiP) and mitochondrial (MtMiP) genomes. We hypothesized that MiP genes are differentially expressed (DE) between H_RFI and L_RFI animal groups and similarly between negative and positive EB groups. Our study aimed to characterize MiP gene expression in white blood cells of H_RFI and L_RFI cows using RNA sequencing to identify genes and biological pathways associated with feed efficiency in dairy cattle. We used the top and bottom 14 cows ranked for RFI and EB out of 109 animals as H_RFI and L_RFI, and positive and negative EB groups, respectively. The gene expression counts across all nuclear and mitochondrial genes for animals in each group were used for differential gene expression analyses, weighted gene correlation network analysis, functional enrichment, and identification of hub genes. Out of 244 DE genes between RFI groups, 38 were MiP genes. The DE genes were enriched for the oxidative phosphorylation (OXPHOS) and ribosome pathways. The DE MiP genes were underexpressed in L_RFI (and negative EB) compared with the H_RFI (and positive EB) groups, suggestive of reduced mitochondrial activity in the L_RFI group. None of the MtMiP genes were among the DE MiP genes between the groups, which suggests a non-rate limiting role of MtMiP genes in feed efficiency and warrants further investigation. The role of MiP, particularly the NuMiP and OXPHOS pathways in RFI, was also supported by our gene correlation network analysis and the hub gene identification. We validated the findings in an independent data set. Overall, our study suggested that differences in feed efficiency in dairy cows may be linked to differences in cellular energy demand. This study broadens our knowledge of the biology of feed efficiency in dairy cattle.

La Trobe University Faculty of Science, Technology and Engineering Murray Darling Freshwater Research CentreMDFRC item.Routine microscopic examination of biomass samples from many activated sludge plants around the world reveals the presence of large numbers of both Gram positive and Gram negative coccoid cells often arranged in distinctive tetrads or sheets of tetrads. Three Gram negative tetrad cocci were successfully isolated into pure culture using micromanipulation from samples of biomass from plants in Tamworth, N.S.W., Australia, Verona, Italy and Macau. After extensive phenotypic characterisation and 16S rDNA sequence analysis, all isolates showed high overall similarity to each other, but were phylogenetically quite different to any previously sequenced bacterium. They have therefore been classified as three species of a new genus, the genus Amaricoccus in the a Proteobacteria in the domain Bacteria. None could accumulate polyphosphate granules, and production of intracellular poly-beta-hydroxybutyrate (PHB) granules depended on the carbon source used in the medium. A similar characterisation of the original isolate of the G-bacteria described by Cech and Hartman showed this organism also belonged to the same genus as the other three isolates, but as a distinct species. The practical implications of these observations are discussed.

Fetal Alcohol Syndrome (FAS) is the most serious form of Fetal Alcohol Spectrum Disorders (FASD) and the most prevalent neurodevelopmental disorder in North America. Patients with FAS may exhibit cognitive problems with working memory, manipulating information, and reduced executive functioning. Additionally, previous studies exhibited that stress responses are affected by prenatal alcohol consumption Gut microbiota compositions can also influence stress responses and memory, as several studies have shown strong relationships between the enteric gut system and the brain. However, few studies have examined how prenatal alcohol exposure’s effects on the gut microbiome and neuroinflammatory responses. For this study, pregnant HsdBlu:LE Long Evans rats were treated with either a dry diet, liquid diet, or liquid diet with alcohol. On day 28 and 42 after birth, three male and three female adolescent pups from each treatment group had their gut microbiome (fecal samples) analyzed through 16S rRNA amplicon sequencing. Brain histology staining of the cortex and hippocampus regions was also done to evaluate changes in the CNS through microglial counts and morphology analysis. There were no significant differences in alpha diversity of the fecal microbiome between groups of pups based on prenatal alcohol exposure (PAE), sex, age, the interaction of PAE and sex, or in the morphology of cortex microglia. However, analysis of beta diversity using Bray-Curtis dissimilarity and weighted UniFrac suggested distinct microbial communities between the treatment groups based on PAE and the interaction of PAE, sex, and the interaction between PAE and sex. Microglial count comparisons by PAE or sex were only statistically different in the cortex (p ≤ 0.005). The significance of this study suggests that there are some associations between the gut microbiome and CNS inflammation in rats with PAE. Based on these findings, 11 future studies may implement therapeutics such as antibiotics or probiotics to mitigate cognitive or neural symptoms of FASD affected individuals.

Single-cell genomics enables dissection of tumor heterogeneity and molecular underpinnings of drug response at an unprecedented resolution1-11. However, broad clinical application of these methods remains challenging, due to several practical and preanalytical challenges that are incompatible with typical clinical care workflows, namely the need for relatively large, fresh tissue inputs. In the present study, we show that multimodal, single-nucleus (sn)RNA/T cell receptor (TCR) sequencing, spatial transcriptomics and whole-genome sequencing (WGS) are feasible from small, frozen tissues that approximate routinely collected clinical specimens (for example, core needle biopsies). Compared with data from sample-matched fresh tissue, we find a similar quality in the biological outputs of snRNA/TCR-seq data, while reducing artifactual signals and compositional biases introduced by fresh tissue processing. Profiling sequentially collected melanoma samples from a patient treated in the KEYNOTE-001 trial12, we resolved cellular, genomic, spatial and clonotype dynamics that represent molecular patterns of heterogeneous intralesional evolution during anti-programmed cell death protein 1 therapy. To demonstrate applicability to banked biospecimens of rare diseases13, we generated a single-cell atlas of uveal melanoma liver metastasis with matched WGS data. These results show that single-cell genomics from archival, clinical specimens is feasible and provides a framework for translating these methods more broadly to the clinical arena.

Aeromonas phage AHP-1 was originally isolated from crucian carp (Carassius carassius) tissue. It was able to infect Aeromonas hydrophila and A. salmonicida. Genome sequence analysis revealed a 218,317-bp-long linear genome with an overall G + C content of 47.9%, 315 open reading frames (ORFs), and 25 tRNA sequences. Its genome was found to contain 67 unique ORFs (21.26%) that did not show any homology to previously characterized proteins. A comparative genome analysis suggested that its closest neighbors are unclassified phages belonging to the genus Tequatrovirus of the subfamily Tevenvirinae.

Since 2016, devastating bacterial blotch affecting the fruiting bodies of Agaricus bisporus, Cordyceps militaris, Flammulina filiformis, and Pleurotus ostreatus in China has caused severe economic losses. We isolated 102 bacterial strains and characterized them polyphasically. We identified the causal agent as Pseudomonas tolaasii and confirmed the pathogenicity of the strains. A host range test further confirmed the pathogen’s ability to infect multiple hosts. This is the first report in China of bacterial blotch in C. militaris caused by P. tolaasii. Whole-genome sequences were generated for three strains: Pt11 (6.48 Mb), Pt51 (6.63 Mb), and Pt53 (6.80 Mb), and pangenome analysis was performed with 13 other publicly accessible P. tolaasii genomes to determine their genetic diversity, virulence, antibiotic resistance, and mobile genetic elements. The pangenome of P. tolaasii is open, and many more gene families are likely to emerge with further genome sequencing. Multilocus sequence analysis using the sequences of four common housekeeping genes (glns, gyrB, rpoB, and rpoD) showed high genetic variability among the P. tolaasii strains, with 115 strains clustered into a monophyletic group. The P. tolaasii strains possess various genes for secretion systems, virulence factors, carbohydrate-active enzymes, toxins, secondary metabolites, and antimicrobial resistance genes that are associated with pathogenesis and adapted to different environments. The myriad of insertion sequences, integrons, prophages, and genome islands encoded in the strains may contribute to genome plasticity, virulence, and antibiotic resistance. These findings advance understanding of the determinants of virulence, which can be targeted for the effective control of bacterial blotch disease.

Bacterial diseases affecting wheat production in Uruguay are an issue of growing concern yet remain largely uninvestigated in the region. Surveys of 61 wheat fields carried out from 2017 to 2019 yielded a regional collection of 63 strains identified by 16S rRNA gene analysis as Xanthomonas spp. A real-time PCR protocol using species-specific primers previously reported allowed the identification of 44 strains as X. translucens, the causal agent of bacterial leaf streak (BLS) in wheat and other cereal crops. Multilocus sequence analysis (MLSA) of four housekeeping genes (dnaK, fyuA, gyrB, and rpoD) revealed that these strains were most closely related to X. translucens pv. undulosa, the pathovar that is most commonly associated with BLS of wheat. Multilocus sequence typing (MLST) was applied to examine the genetic diversity among X. translucens strains. Strains were assigned to four different sequence types, three of which have been previously reported globally. Additionally, 17 Xanthomonas strains not belonging to X. translucens were obtained from diseased wheat leaves. Phylogenetic analysis showed that these strains are closely related to Xanthomonas prunicola, and clustered together with previously uncharacterized Xanthomonas strains isolated from wheat in Minnesota, US. In planta pathogenicity assays carried out on a BLS susceptible wheat cultivar showed that X. translucens pv. undulosa strains caused brown necrosis symptoms typical of BLS, while non-translucens Xanthomonas sp. strains elicited an atypical symptom of dry necrosis. These findings suggest that local wheat fields are affected by X. translucens pv. undulosa, and by a new wheat pathogen within the Xanthomonas genus.

Resistance of grape Botrytis cinerea was analyzed based on PCR and sequencing technology, which provided a reasonable basis for guiding the species and usage of grape Botrytis cinerea. 104 monospora strains of grape B. cinerea were collected from the main grape-producing areas of China. After isolation and purification, the grape B. cinerea DNA was extracted, and PCR primers were designed according to the resistance mechanism of pyrimethene to pyrimethamines, pyridamines and diformolines. Sequence analysis showed that the resistance frequency of B. cinerea to pyrimidine was 22.22%-62.5%, the high-resistant strain frequency was 44.23%, and the mid-temperature zone was 62.5%. The total resistance rate of B. cinerea to pyrimidine was 42.60%. The resistance rate of B. cinerea to pyrimidine was 42.60%. Resistance frequencies of grape gray mold strains were significantly different. The resistance frequencies of grape gray mold strains to pyridimide were significant. The highest resistance frequencies of grape gray mold strains were 53.42% in the cool temperature zone. Only 3.85% of grape gray mold strains showed resistance to pyridimide. Therefore, the resistance of Grape B. cinerea to pyrimidine and pyrimidine is relatively common, and the resistance to enoylmorpholine is still in the initial stage.

This study aims to describe the utilization patterns of antipsychotic (AP) medication in patients with schizophrenia (SCZ), three years after initiating or reinitiating a given AP.Based on medico-administrative information on patients living in Quebec (Canada), this retrospective cohort study included 6444 patients with a previous diagnosis of SCZ initiating or reinitiating AP medication between January 1, 2012, and December 31, 2014, with continuous coverage by public drug insurance. For each day of follow-up (1092 days), patient was either exposed to one of the chosen categories of APs, or to none. This patient's sequence of AP exposure overtime has been referred to as the "antipsychotic utilization trajectory". These trajectories were analyzed using a State Sequence Analysis, an innovative approach which provides useful visual information on the continuation and discontinuation patterns of use over time.Clozapine and long-acting injectable second-generation APs had the best continuation and discontinuation patterns over 3 years among all other groups, including less switching of APs, while oral first-generation APs had the poorest patterns. These findings were comparable among incident and non-incident cohorts. Oral second-generation antipsychotics, excluding clozapine, had a poorer continuation and discontinuation pattern than long-acting injectable antipsychotics.State Sequence Analysis provides a clear representation of treatment adherence in comparison with dichotomous indicators of adherence or discontinuation. Consequently, this innovative method has shed light on the impact of the AP chosen to initiate or reinitiate treatment in SCZ, which has been identified as a key factor for long-term treatment continuation and discontinuation.

Post-transcriptional trimming and tailing of RNA 3′ ends play key roles in the processing and quality control of noncoding RNAs (ncRNAs). However, bioinformatic tools to examine changes in the RNA 3′ “tailome” are sparse and not standardized. Here we present Tailer, a bioinformatic pipeline in two parts that allows for robust quantification and analysis of tail information from next-generation sequencing experiments that preserve RNA 3′ end information. The first part of Tailer, Tailer-processing, uses genome annotation or reference FASTA gene sequences to quantify RNA 3′ ends from SAM-formatted alignment files or FASTQ sequence read files produced from sequencing experiments. The second part, Tailer-analysis, uses the output of Tailer-processing to identify statistically significant RNA targets of trimming and tailing and create graphs for data exploration. We apply Tailer to RNA 3′ end sequencing experiments from three published studies and find that it accurately and reproducibly recapitulates key findings. Thus, Tailer should be a useful and easily accessible tool to globally investigate tailing dynamics of nonpolyadenylated RNAs and conditions that perturb them.

Sheep and goats are among other herbivorous animals that serve as intermediate hosts (containing the larval stage Cysticercus tenuicollis) of Taenia hydatigena tapeworm. This infection can lead to serious complications or cause death. The genetic diversity and epidemiological significance of cysticercosis due to T. hydatigena is poorly understood. We examined 11,651 goats and 23,542 sheep slaughtered at the municipal abattoir in Makkah, for C. tenuicollis infection. The resulted DNA sequences were compared with previously available sequences from different hosts. Phylogenetic analysis and Pairwise nucleotide variations of cox1 gene were performed. Sheep and goats revealed infection rates of (4.95%) and (4.75%) respectively. DNA sequence analysis of all isolates from both sheep and goats showed that the total haplotypes number was 7. T. hydatigena population with high haplotypes diversity values. The nucleotide diversity was low, while Tajima’s D and Fu’s tests were negative (with no statistical significance). The present work will give valuable information regarding the prevalence and implementation of control and prevention measures of C. tenuicollis.

Objectives The main goal of this study was to accurately detect azole resistance in species of the Aspergillus fumigatus complex by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Methods Identification of isolates (n = 868) was done with MALDI-TOF MS using both commercial and in-house libraries. To determine azole susceptibility, the EUCAST E.Def. 9.3.2 method was applied as the reference standard. Identification of resistant isolates was confirmed by DNA sequence analysis. Protein spectra obtained by MALDI-TOF MS were analysed to differentiate species within the A. fumigatus complex and to detect azole-resistant A. fumigatus sensu stricto isolates. Results Correct discrimination of A. fumigatus sensu stricto from cryptic species was accomplished in 100% of the cases applying principal component analysis (PCA) to protein spectra generated by MALDI-TOF MS. Furthermore, a specific peak (4586 m/z) was found to be present only in cryptic species. The application of partial least squares (PLS) discriminant analysis allowed 98.43% (±0.038) discrimination between susceptible and azole-resistant A. fumigatus sensu stricto isolates. Finally, based on PLS and SVM, A. fumigatus sensu stricto isolates with different cyp51A gene mutations were correctly clustered in 91.5% of the cases. Conclusions MALDI-TOF MS combined with peak analysis is a novel tool that allows the differentiation of A. fumigatus sensu stricto from other species within the A. fumigatus complex, as well as the detection of azole-resistant A. fumigatus sensu stricto. Although further studies are still needed, the results reported here show the great potential of MALDI-TOF and machine learning for the rapid detection of azole-resistant Aspergillus fumigatus isolates from clinical origins.

Background: Treatment of the Covid-19 pandemic caused by the highly contagious and pathogenic SARS-CoV-2 is a global menace. Day by day, this pandemic is getting worse. Doctors, scientists and researchers across the world are urgently scrambling for a cure for novel corona virus and continuously working at break neck speed to develop vaccines or drugs. But to date, there are no specific drugs or vaccines available in the market to cope up with the virus. Objective: The present study helps us to elucidate 3D structures of SARS-CoV-2 proteins and also to identify natural compounds as potential inhibitors against COVID-19. Methods: The 3D structures of the proteins were constructed using Modeller 9.16 modeling tool. Modelled proteins were validated with PROCHECK by Ramachandran plot analysis. In this study, a small library of natural compounds (fifty compounds) was docked to the hACE2 binding site of the modelled surface glycoprotein of SARS-CoV-2 using AutoDock Vina to repurpose these inhibitors against SARS-CoV-2. Conceptual density functional theory calculations of the best eight compounds had been performed by Gaussian-09. Geometry optimizations for these molecules were done at M06-2X/ def2-TZVP level of theory. ADME parameters, pharmacokinetic properties and drug likeness of the compounds were analyzed using swissADME website. Results: In this study, we analysed the sequences of surface glycoprotein, nucleocapsid phosphoprotein and envelope protein obtained from different parts of the globe. We modelled all the different sequences of surface glycoprotein and envelop protein in order to derive 3D structure of a molecular target, which is essential for the development of therapeutics. Different electronic properties of the inhibitors have been calculated using DFT through M06-2X functional with def2-TZVP basis set. Docking result at the hACE2 binding site of all modelled surface glycoproteins of SARSCoV- 2 showed that all the eight inhibitors (actinomycin D, avellanin C, ichangin, kanglemycin A, obacunone, ursolic acid, ansamiotocin P-3 and isomitomycin A) studied here were many folds better compared to hydroxychloroquine which has been found to be effective to treat patients suffering from COVID-19. All the inhibitors meet most of the criteria of drug likeness assessment. Conclusion: We expect that eight compounds (actinomycin D, avellanin C, ichangin, kanglemycin A, obacunone, ursolic acid, ansamiotocin P-3 and isomitomycin A) can be used as potential inhibitors against SARS-CoV-2.

Syphilis infection during pregnancy causes perinatal complications and mother-to-child transmission if untreated. A newborn was delivered by emergent cesarean section due to non-reassuring fetal status at 34 weeks of gestation. The mother tested negative for rapid plasma reagin (RPR) and Treponema pallidum hemagglutination (TPHA) in early pregnancy. The newborn had a severe inflammatory reaction, thrombocytopenia, and elevated IgM as well as disseminated intravascular coagulation and multiple organ failure. The 16S ribosomal RNA gene sequence analysis of the amniotic fluid detected Treponema pallidum. The newborn tested positive for RPR, TPHA, and IgM fluorescent treponemal antibody-absorption in the blood, and thus, congenital syphilis was diagnosed. This is the first case that 16S ribosomal RNA gene sequencing of the amniotic fluid led to an early diagnosis of congenital syphilis in a newborn. The 16S rRNA gene sequencing may be a useful method for the early detection of the primary causative microbe of congenital infection.

Eugenol is an important phenylpropanoid in strawberry and the accumulation is affected by various abiotic stresses. In this study, the expression patterns of two transcription factors (TFs) (FaMYB10 and FaEOBII) and two synthetic genes (FaEGS1 and FaEGS2) involved in eugenol synthesis were characterized in Fragaria × ananassa cv. ‘Xiaobai’. Sequence analysis showed the four genes contained several cis-acting elements that were related to phytohormones and environmental stress signal responsiveness. FaEGS1 and FaEGS2 were clustered in the same group clade. The four genes were expressed in all analysed tissues and, in particular, expressed to higher relative expression levels in leaves. The expression of FaEGS1, FaEGS2, and FaEOBII was the highest in large green fruits, and FaMYB10 had the highest expression in red-ripening fruits. The amount of eugenol showed a stage-specific profile. The expression of these four genes was up-regulated by ethephon (ETH), dark, drought, heat, and salt treatments accompanied by the increase of eugenol production. Concentration and timing of cadmium treatment affected gene expression. Cadmium inhibited the expression of FaEGS1 and FaEOBII, but significantly promoted the expression of FaEGS2 and FaMYB10 and eugenol content. The results provide a basis for elucidating the role of these genes in response to abiotic stresses, and regulating the accumulation of eugenol, an important constituent of volatile compounds in many economically important and aromatic plants.

Although the availability of various sequencing technologies allows us to capture different genome properties at single-cell resolution, with the exception of a few co-assaying technologies, applying different sequencing assays on the same single cell is impossible. Single-cell alignment using optimal transport (SCOT) is an unsupervised algorithm that addresses this limitation by using optimal transport to align single-cell multiomics data. First, it preserves the local geometry by constructing a k-nearest neighbor (k-NN) graph for each data set (or domain) to capture the intra-domain distances. SCOT then finds a probabilistic coupling matrix that minimizes the discrepancy between the intra-domain distance matrices. Finally, it uses the coupling matrix to project one single-cell data set onto another through barycentric projection, thus aligning them. SCOT requires tuning only two hyperparameters and is robust to the choice of one. Furthermore, the Gromov-Wasserstein distance in the algorithm can guide SCOT's hyperparameter tuning in a fully unsupervised setting when no orthogonal alignment information is available. Thus, SCOT is a fast and accurate alignment method that provides a heuristic for hyperparameter selection in a real-world unsupervised single-cell data alignment scenario. We provide a tutorial for SCOT and make its source code publicly available on GitHub.

Thymoma is a rare malignant tumor that usually with an indolent presentation, which was falsely assumed to be benign previously. The tumor suppressor P53 (TP53) and EGFR gene mutate most frequently in human cancers. We tried to investigate the presence of TP53 and EGFR mutations among thymoma patients referred to an Indonesian referral respiratory hospital and to discuss its potential role in thymoma management and prognosis.Surgically resected tumor tissues were collected from thymoma patients and then underwent genomic analysis. PCR was performed on the extracted Paraffinized DNA to amplify exon 6 of TP53 and exons 18, 19, and 21 of EGFR. The evaluation of mutational status was done using direct sequencing and sequence analysis of purified PCR products.Among 27 collected samples, TP53 exon 6 mutation, namely missense mutation and nonsense mutation, was observed in two samples (7.4%). EGFR exon 18 mutation, namely E709K and nonsense mutation, was found in 2 samples (7.4%). An intronic mutation in EGFR exon 19 (3.7%) and exon 21 (3.7%) was observed in one sample.TP53 and EGFR mutations were not most frequent, so it seems that these genes are not involved in the pathogenesis of thymoma in Indonesian patients. Nevertheless, we found two samples with a significant mutation in p53 and EGFR genes, suggesting further research on thymoma prognostification and targeted therapy.

Cryptocaryon irritans, a holotrichous ciliate parasitic protozoan, can trigger marine white spot disease and cause substantial economic losses in mariculture. However, methods of preventing and curing the disease have negatively affect fish, human, other organisms, and the natural environment. The antiparasitic activity of some antimicrobial peptides (AMPs) has garnered extensive attention of scholars. In this study, we identified and characterised a novel antiparasitic peptide, named So-pis, from Sciaenops ocellatus. The sequence analysis, structural features, and tissue distribution suggested that So-pis is genetically related to the piscidins family. However, So-pis showed a relatively low overall conservation compared with other known piscidins. So-pis is abound in glycine residues (22.7 %) and it has a neutral isoelectric point, weak amphipathicity, relatively long α-helix, and high hydrophobicity. These key elements are responsible for its biological activity. Quantitative real-time polymerase chain reaction (qRT-PCR) data indicated that So-pis is a typically gill-expressed peptide. The expression of So-pis in the gill, skin, spleen, and head kidney could be regulated during C. irritans infection, thereby implicating a role of So-pis in immune defence against C. irritans. The synthetic So-pis had limited or no antimicrobial activity against bacterial and yeasts but exhibited potent antiparasitic activity against C. irritans in vitro. The activity of synthetic So-pis against erythrocytes was less potent than its antiparasitic activity against C. irritans. These results indicated that So-pis might be one of the crucial defence cytokines against C. irritans in the red drum. Cumulatively, our data suggested that So-pis might be a potential candidate for developing a novel, effective, and safe therapeutic agent against marine white spot disease.

Escherichia coli has traditionally been serotyped using antisera against the O and H antigens. However, a proportion of E. coli isolates are nonmotile and, in addition, some isolates do not react w...