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Unlocking the full dimensionality of single-cell RNA sequencing data (scRNAseq) is the next frontier to a richer, fuller understanding of cell biology. We introduce q-diffusion, a framework for capturing the coexpression structure of an entire library of genes, improving on state-of-the-art analysis tools. The method is demonstrated via three case studies. In the first, q-diffusion helps gain statistical significance for differential effects on patient outcomes when analyzing the CALGB/SWOG 80405 randomized phase III clinical trial, suggesting precision guidance for the treatment of metastatic colorectal cancer. Secondly, q-diffusion is benchmarked against existing scRNAseq classification methods using an in vitro PBMC dataset, in which the proposed method discriminates IFN-γ stimulation more accurately. The same case study demonstrates improvements in unsupervised cell clustering with the recent Tabula Sapiens human atlas. Finally, a local distributional segmentation approach for spatial scRNAseq, driven by q-diffusion, yields interpretable structures of human cortical tissue. The q-diffusion method uses the full dimensionality of gene coexpression in transcriptomics and benchmarking shows improvment of scRNAseq analyses. [ABSTRACT FROM AUTHOR]

This study aimed to identify genes related to acetic acid tolerance and elucidate the tolerance mechanism in yeast Saccharomyces cerevisiae as an appropriate eukaryote model. In this study, one tolerant strain exposed to acetic acid stress was isolated by screening a transposon-mediated mutant library and the disrupted gene was identified to be RGD1. In addition, this phenotype related to acetic acid tolerance was confirmed by deletion and overexpressing of this corresponding gene. This mutant strain showed activation of Hog1p and increased expression of the MSN2/4 and STRE-mediated genes such as CTT1 and HSP12 in response to acetic acid stress. [ABSTRACT FROM AUTHOR]

Atopic dermatitis and autoimmune diseases are highly heritable conditions that may co-occur from an early age. The primary study is a national administrative cohort study involving 499,428 children born in 2002, tracked until 2017. Atopic dermatitis was defined as five or more principal diagnoses of atopic dermatitis and two or more topical steroid prescriptions. We estimated the risks for the occurrence of 41 autoimmune diseases, controlling for risk factors. In addition, we sourced a gene library from the National Library of Medicine to conduct a comprehensive gene ontology. We used Gene Weaver to identify gene set similarity and clustering, and used GeneMania to generate a network for shared genes. Exposed and unexposed groups included 39,832 and 159,328 children, respectively. During a mean follow-up of 12 years, the exposed group had an increased risk of autoimmune disease (hazard ratio, 1.27 [95 % confidence interval, 1.23–1.32]) compared to the unexposed group. The hazard ratios of autoimmune illnesses consistently increased with two- and five years lag times and alternative atopic dermatitis definitions. Shared genes between atopic dermatitis and autoimmune diseases were associated with comorbidities such as asthma, bronchiolitis, and specific infections. Genetic interactions of these shared genes revealed clustering in Th1, Th2, Th17, and non-classifiable pathways. Atopic dermatitis was significantly associated with an increased risk of subsequent autoimmune disease. we identified the genetically associated disease in atopic dermatitis patients comorbid with autoimmune disease and demonstrated a genetic network between atopic dermatitis and autoimmune diseases. [Display omitted] [ABSTRACT FROM AUTHOR]

Filamentous fungi of the genus Aspergillus include producers of industrially important organic acids, enzymes, and secondary metabolites, as well as pathogens of many plants and animals. Novel genes in the Aspergillus genome are potentially crucial for the fermentation and drug industries (e.g., agrochemicals and antifungal drugs). A research approach based on classical genetics is effective for identifying functionally unknown genes. During analyses based on classical genetics, mutations must be identified easily and quickly. Herein, we report the development of a cosmid-based plasmid pTOCK1 and the use of a genomic library of Aspergillus nidulans constructed using pTOCK1. The cosmid-based genomic library was used for convenient auxotrophic mutants (pyroA and pabaB), as well as mutants with abnormal colony morphology (gfsA) and yellow conidia (yA), to obtain library clones complementary to these phenotypes. The complementary strain could be obtained through a single transformation, and the cosmid could be rescued. Thus, our cosmid library system can be used to identify the causative gene in a mutant strain. [ABSTRACT FROM AUTHOR]

Gallbladder cancer (GBC) is a very aggressive malignant neoplasm of the biliary tract with a poor prognosis. There are no specific therapies for the treatment of GBC or early diagnosis tools; for this reason, the development of strategies and technologies that facilitate or allow an early diagnosis of GBC continues to be decisive. Phage display is a robust technique used for the production of monoclonal antibodies (mAbs) involving (1) the generation of gene libraries, (2) the screening and selection of isoforms related to an immobilized antigen, and (3) the in vitro maturation of the affinity of the antibody for the antigen. This research aimed to construct a human immune library from PBMCs of GBC patients and the isolation of scFv-phage clones with specificity against the larger extracellular loop belonging to claudin 18.2, which is an important biomarker overexpressed in GBC as well as gastric cancer. The immune-library-denominated GALLBLA1 was constructed from seven GBC patients and has a diversity of 6.12 × 1010 pfu mL−1. After three rounds of panning, we were able to identify clones with specificity against claudin 18.2. GALLBLA1 can contribute to the selection, isolation, and recombinant production of new human mAbs candidates for the treatment of gastrointestinal cancers. [ABSTRACT FROM AUTHOR]

SUMMARY: By conducting hierarchical clustering along a sliding window, we generated haplotypes across hundreds of re‐sequenced genomes in a few hours. We leveraged our method to define cryptic introgressions underlying disease resistance in tomato (Solanum lycopersicum L.) and to discover resistant germplasm in the tomato seed bank. The genomes of 9 accessions with early blight (Alternaria linariae) disease resistance were newly sequenced and analyzed together with published sequences for 770 tomato and wild species accessions, most of which are available in germplasm collections. Identification of common ancestral haplotypes among resistant germplasm enabled rapid fine mapping of recently discovered quantitative trait loci (QTL) conferring resistance and the identification of possible causal variants. The source of the early blight QTL EB‐9 was traced to a vintage tomato named 'Devon Surprise'. Another QTL, EB‐5, as well as resistance to bacterial spot disease (Xanthomonas spp.), was traced to Hawaii 7998. A genomic survey of all accessions forecasted EB‐9‐derived resistance in several heirloom tomatoes, accessions of S. lycopersicum var. cerasiforme, and S. pimpinellifolium PI 37009. Our haplotype‐based predictions were validated by screening the accessions against the causal pathogen. There was little evidence of EB‐5 prevalence in surveyed contemporary germplasm, presenting an opportunity to bolster tomato disease resistance by adding this rare locus. Our work demonstrates practical insights that can be derived from the efficient processing of large genome‐scale datasets, including rapid functional prediction of disease resistance QTL in diverse genetic backgrounds. Finally, our work finds more efficient ways to leverage public genetic resources for crop improvement. Significance Statement: We introduce a powerful windowed haplotype classification method that can efficiently detect local homology across genomes and predict biological traits in germplasm. We co‐analyzed hundreds of genomic sequences to define and predict introgressions conferring resistance to early blight disease of tomato. These predictions were validated in planta with pathology experiments, demonstrating the functional prediction of quantitative resistance of breeding and research significance. Our haplotype clustering and prediction method is broadly applicable to crops with large genomic datasets. [ABSTRACT FROM AUTHOR]

Background: Ehlers-Danlos syndrome Type IV (aka Vascular Ehlers Danlos, or vEDS) is a dominantly inherited mutation in the Collagen 3A1 gene (COL3A1). The disease is characterized by tissue friability and age-related susceptibility to arterial aneurysm, dissection and rupture as well as uterine and bowl tears. These clinical manifestations result in major surgical intervention and decreased life expectancy. Understanding how mutations in COL3A1 impact the structure and function of the extracellular matrix (ECM) is important to managing the disease and finding treatments. Results: Skin fibroblasts from vEDS subjects heterozygous for the p.G588S pathogenic variant in the COL3A1 gene and a normal individual were cultured and studied. Proteomics analysis identified dozens of upregulated proteins related to extracellular matrix dysregulation that is characteristic of fibrosis. Gene expression libraries from cultured primary fibroblasts were screened for messenger RNA (mRNA) markers of ECM degradation. The proteomics and targeted gene expression array results were largely consistent with dysregulation of the extracellular matrix in vEDS. The data show upregulation of multiple Collagen proteins and genes, other ECM components, and enzymes related to ECM processing and turn-over. vEDS fibroblasts expressed significantly more cross linked C-Telopeptide of Collagen III (CTXIII) than normal fibroblasts, indicative of Collagen III degradation and turn-over. Further, the expression and activity of Lysyl Oxidase (LOX), an enzyme that initiates covalent cross-linking of soluble collagen and elastin into protease resistant fibers, is elevated in vEDS fibroblasts compared to normal fibroblasts. Conclusion: Together, these findings suggest dysregulated ECM deposition and processing, reminiscent of a state of fibrosis. Therapeutics that target the dysregulated ECM proteins or help replace damaged tissue may improve clinical outcomes. [ABSTRACT FROM AUTHOR]

Large gene libraries are frequently created in Escherichia coli plasmids, which can induce cell toxicity and expression instability due to the high gene dosage. To address these limitations, gene libraries can be integrated in a single copy into the bacterial chromosome. Here, we describe an efficient system for the massive integration (MAIN) of large gene libraries in the E. coli chromosome that generates in‐frame gene fusions that are expressed stably. MAIN uses a thermosensitive integrative plasmid that is linearized in vivo to promote extensive integration of the gene library via homologous recombination. Positive and negative selections efficiently remove bacteria lacking gene integration in the target site. We tested MAIN with a library of 107 VHH genes that encode nanobodies (Nbs). The integration of VHH genes into a custom target locus of the E. coli chromosome enabled stable expression and surface display of the Nbs. Next‐generation DNA sequencing confirmed that MAIN preserved the diversity of the gene library after integration. Finally, we screened the integrated library to select Nbs that bind a specific antigen using magnetic and fluorescence‐activated cell sorting. This allowed us to identify Nbs binding the epidermal growth factor receptor that were not previously isolated in a similar screening of a multicopy plasmid library. Our results demonstrate that MAIN enables large gene library integration into the E. coli chromosome, creating stably expressed in‐frame fusions for functional screening. [ABSTRACT FROM AUTHOR]

Plant defensins are a large family of small cationic proteins with diverse functions and mechanisms of action, most of which assert antifungal activity against a broad spectrum of fungi. The partial mechanism of action has been resolved for a small number of members of plant defensins, and studies have revealed that many act by more than one mechanism. The plant defensin Ppdef1 has a unique sequence and long loop 5 with fungicidal activity against a range of human fungal pathogens, but little is known about its mechanism of action. We screened the S. cerevisiae non-essential gene deletion library and identified the involvement of the mitochondria in the mechanism of action of Ppdef1. Further analysis revealed that the hyperpolarisation of the mitochondrial membrane potential (MMP) activates ROS production, vacuolar fusion and cell death and is an important step in the mechanism of action of Ppdef1, and it is likely that a similar mechanism acts in Trichophyton rubrum. [ABSTRACT FROM AUTHOR]

5-Aminovaleric acid (5-AVA), 5-hydroxyvalerate (5HV), copolymer P(3HB- co -5HV) of 3 -hydroxybutyrate (3HB) and 5HV were produced from L-lysine as a substrate by recombinant Halomonas bluephagenesis constructed based on codon optimization, deletions of competitive pathway and L-lysine export protein, and three copies of davBA genes encoding L-lysine monooxygenase (DavB) and 5-aminovaleramide amidohydrolase (DavA) inserted into its genome to form H. bluephagenesis YF117Δ gabT 1+2 , which produced 16.4 g L−1 and 67.4 g L−1 5-AVA in flask cultures and in 7 L bioreactor, respectively. It was able to de novo synthesize 5-AVA from glucose by L-lysine-overproducing H. bluephagenesis TD226. Corn steep liquor was used instead of yeast extract for cost reduction during the 5-AVA production. Using promoter engineering based on P porin mutant library for downstream genes, H. bluephagenesis YF117 harboring pSEVA341-P porin 42 - yqhD EC produced 6 g L−1 5HV in shake flask growth , while H. bluephagenesis YF117 harboring pSEVA341-P porin 42 - yqhD EC -P porin 278 - phaC RE -abfT synthesized 42 wt% P(3HB- co -4.8 mol% 5HV) under the same condition. Thus, H. bluephagenesis was successfully engineered to produce 5-AVA and 5HV in supernatant and intracellular P(3HB- co -5HV) utilizing L-lysine as the substrate. [Display omitted] • 5-Aminovaleric acid (5-AVA), 5-hydroxyvalerate (5HV), and P(3HB- co -5HV) were produced by Halomonas bluephagenesis. • The engineered strain YF117Δ gabT 1+2 , produced 16.4and 67.4 g L−1 5-AVA in flask cultures and in 7 L bioreactor, respectively. • It was able to de novo synthesize around 600 mg L−1 5-AVA from glucose by L-lysine- overproducing H. bluephagenesis TD226. • H. bluephagenesis YF117 harboring pSEVA341-P porin 42 - yqhD EC produced 6 g L−1 5HV in shake flasks. • YF117 harboring pSEVA341-P porin 42 - yqhD EC -P porin 278 - phaC RE - abfT synthesized 42 wt% P(3HB- co -4.8 mol% 5HV). [ABSTRACT FROM AUTHOR]

Objectives: The data presented in this study were collected with the aim of obtaining the complete genomes of specific strains of Bacillus bacteria, namely, Bacillus licheniformis T5. This strain was chosen based on its enzymatic activities, particularly amylolytic activity. In this study, nanopore sequencing technology was employed to obtain the genome sequences of this strain. It is important to note that these data represent a focused objective within a larger research context, which involves exploring the biochemical features of promising Bacilli strains and investigating the relationship between enzymatic activity, phenotypic features, and the microorganism's genome. Data description: In this study, the whole-genome sequence was obtained from one Bacillus strain, Bacillus licheniformis T5, isolated from soil samples in Kazakhstan. Sample preparation and genomic DNA library construction were performed according to the Ligation sequencing gDNA kit (SQK-LSK109) protocol and NEBNext module. The prepared library was sequenced on a MinION instrument (Oxford Nanopore Technologies nanopore sequencer with a maximum throughput of up to 30 billion nucleotides per run and no limit on read length), using a flow cell for nanopore sequencing FLO-MIN106D. The genome de novo assembly was performed using the long sequencing reads generated by MinION Oxford Nanopore platform. Finally, one circular contig was obtained harboring a length of 4,247,430 bp with 46.16% G + C content and the mean contig 428X coverage. B. licheniformis T5 genome assembly annotation revealed 5391 protein-coding sequences, 81 tRNAs, 51 repeat regions, 24 rRNAs, 3 virulence factors and 53 antibiotic resistance genes. This sequence encompasses the complete genetic information of the strain, including genes, regulatory elements, and noncoding regions. The data reveal important insights into the genetic characteristics, phenotypic traits, and enzymatic activity of this Bacillus strain. The findings of this study have particular value to researchers interested in microbial biology, biotechnology, and antimicrobial studies. The genomic sequence offers a foundation for understanding the genetic basis of traits such as endospore formation, alkaline tolerance, temperature range for growth, nutrient utilization, and enzymatic activities. These insights can contribute to the development of novel biotechnological applications, such as the production of enzymes for industrial purposes. Overall, this study provides valuable insights into the genetic characteristics, phenotypic traits, and enzymatic activities of the Bacillus licheniformis T5 strain. The acquired genomic sequences contribute to a better understanding of this strain and have implications for various research fields, such as microbiology, biotechnology, and antimicrobial studies. [ABSTRACT FROM AUTHOR]

Simple sequence repeat (SSR) markers are valuable genetic and genomic research tools and are extensively used in major crops. However, a paucity of available molecular markers in lentils (Lens culinaris Medik.) has significantly hindered genomic studies in this vital legume crop. In this study, we developed 33 new SSR markers for lentils using an enriched genomic library and tested their polymorphism in 10 lentil cultivars. We found that 16 (48.5%) SSR markers were polymorphic. The expected heterozygosity values of the polymorphic SSR markers ranged from 0.095 to 0.820, while observed heterozygosity values varied between 0.100 and 1.00. In addition, we tested the transferability of 86 SSR markers, including the 33 newly developed ones, to other legume species. Transferability rates of lentil SSR markers to other species varied between 13.76% (Vicia sativa L.) and 2.58% (Phaseolus vulgaris L. and Trifolium pratense L.). These new SSR markers could be used in further molecular breeding, population genetics, and genetic mapping studies in lentils and other legume species. [ABSTRACT FROM AUTHOR]

A critical knowledge gap in freshwater plant research is the lack of genetic tools necessary to answer fundamental questions about their demographic histories, adaptation and phylogenetic relationships. One example of this is Typha, a global genus of freshwater plants foundational to wetlands that also is becoming an increasingly problematic biological invader in numerous regions worldwide; although important insights have been discovered for this genus, existing markers are insufficient to answer fundamental questions about their demographic histories, adaptation and phylogenetic relationships; to identify introduced and hybrid lineages; and to examine patterns of hybridisation and introgression.We optimised a library preparation and data processing protocol to develop genome–wide nuclear and plastid resources for studying the evolutionary history, genetic structure and diversity, hybridisation, local adaptation, invasiveness, and geographical expansion dynamics of Typha.We sequenced 140 Typha samples and identified ~120 K nuclear single nucleotide polymorphisms (SNPs) that differentiate T. angustifolia, T. domingensis and T. latifolia, and retrieved their plastome sequences. We observed genetic introgression among the three species.Following a fast, straightforward and cost‐efficient genomic library preparation protocol, we produced a suite of genome‐wide resources to facilitate investigations into the taxonomy and population genetics of Typha and to advance the genomic understanding of wetland plants.The protocol described, the updated chromosome‐level genome assembly of T. latifolia, the catalogue of species‐specific SNPs, and the chloroplast sequences produced in this study comprise permanent resources that can be applied to study the genetic composition of multiple populations and hybrid zones, and will be incorporated into future studies of Typha, an ecologically important and globally invasive macrophyte. [ABSTRACT FROM AUTHOR]

There is a need for novel chemical matter for phenotypic and target-based screens to find starting points for drug discovery programmes in neglected infectious diseases and non-hormonal contraceptives that disproportionately affect Low- and Middle-Income Countries (LMICs). In some disease areas multiple screens of corporate and other libraries have been carried out, giving rise to some valuable starting points and leading to preclinical candidates. Whilst in other disease areas, little screening has been carried out. Much screening against pathogens has been conducted phenotypically as there are few robustly validated protein targets. However, many of the active compound series identified share the same molecular targets. To address the need for new chemical material, in this article we describe the design of a new library, designed for screening in drug discovery programmes for neglected infectious diseases. The compounds have been selected from the Enamine REAL (REadily AccessibLe) library, a virtual library which contains approximately 4.5 billion molecules. The molecules theoretically can be synthesized quickly using commercially available intermediates and building blocks. The vast majority of these have not been prepared before, so this is a source of novel compounds. In this paper we describe the design of a diverse library of 30,000 compounds from this collection (graphical abstract). The new library will be made available to laboratories working in neglected infectious diseases, subject to a review process. The project has been supported by the Bill & Melinda Gates Foundation and the Wellcome Trust (Wellcome). Author summary: Screening of diverse compound libraries is a powerful way to find chemical start points for drug discovery programmes. There is a lack of such chemical libraries available for neglected infectious diseases and other neglected areas such as non-hormonal contraception. In this paper we describe the design of such a library of 30,000 compounds (graphical abstract). The aim was to develop a library of diverse chemical matter unlikely to have been screened in these disease areas. The compounds were selected from the REAL (REadily AccessibLe) Enamine library, a virtual library of 4.5 billion compounds. The methodology for selection of the compounds is described, along with a detailed analysis of the physicochemical properties of the compounds and comparison to other libraries that we have assembled. This shows that the library contains hit/lead-like compounds suitable as chemical start points, and whilst it covers a similar space to some other libraries, the material within the library is mainly different to other libraries. Copies of the library will be made available for screening; the process for gaining access to this library is described. [ABSTRACT FROM AUTHOR]

This is the first comprehensive study to reveal genetic variation and population structure at genome level in six Anatolian (Anatolian Black, East Anatolian Red, South Anatolian Red, South Anatolian Yellow, Turkish Grey Steppe, and Zavot) and two cosmopolitan (Brown Swiss and Holstein Friesian) cattle breeds reared in Türkiye. Being 20 samples from each population, a total of 160 blood samples retrieved from representative herds were utilized to generate genomic libraries by ddRADseq method. Genomic libraries sequenced by Illumina HiSeq X Ten instrument revealed a total of 211,119 bi-allelic SNPs with high call rate. Compared to cosmopolitan cattle breeds, a higher genetic variation was observed in native Turkish cattle with an average of 0.380 observed heterozygosity. Genetic distances were comparatively low between native cattle breeds, whereas the highest genetic distance (0.064) was detected between South Anatolian Yellow and Brown Swiss. Population structure analyses showed that the native Turkish and cosmopolitan cattle breeds were clearly different from each other according to their phylogenetic origin. Besides, a high level of genetic admixture was detected among Anatolian Black, Turkish Grey Steppe, South Anatolian Red, and South Anatolian Yellow, whereas East Anatolian Red and Zavot were distinct from the other native and cosmopolitan cattle breeds. TreeMix algorithm under the assumption of one and two migration events revealed a migration route from Anatolian clade to Anatolian Black, while a second migration edge was drawn from Brown Swiss to East Anatolian Red. This study demonstrates the importance of national conservation studies in the native breeds whose population size has dramatically decreased. In addition, SNP arrays and next-generation sequencing platforms are recommended for future studies to reveal the genetic variation of other local Turkish livestock species to arrange effective conservation programs. [ABSTRACT FROM AUTHOR]

Background: Monoclonal antibody (mAb)-based immunotherapies have achieved promising outcomes in the treatment of immunological and oncological indications. CD19 is considered one of the most qualified antigens in the treatment of B-cell neoplasms. VHHs (nanobodies) are known for their physicochemical advantages over conventional mAbs rendering them suitable therapeutics and diagnostic tools. Herein, we aimed to isolate CD19-specific VHHs from a novel immune library using phage display. Methods: An immune VHH gene library was constructed. Using phage display and after five biopanning rounds, two monoclonal CD19-specific VHHs were isolated. The selected VHHs were expressed, purified, and characterized in terms of their affinity, specificity, sensitivity, and ability to target CD19-positive cell lines. Moreover, in silico analyses were employed for further characterization. Results: A VHH library was developed, and because the outputs of the 4th biopanning round exhibited the most favorable characteristics, a panel of random VHHs was selected from them. Ultimately, two of the most favorable VHHs were selected and DNA sequenced (designated as GR37 and GR41). Precise experiments indicated that GR37 and GR41 exhibited considerable specificity, sensitivity, and affinity (1.15 × 107 M−1 and 2.08 × 107 M−1, respectively) to CD19. Flow cytometric analyses revealed that GR37 and GR41 could bind CD19 on the surface of cell lines expressing the antigen. Moreover, in silico experiments predicted that both VHHs target epitopes that are distinct from that targeted by the CD19-specific single-chain variable fragment (scFv) FMC63. Conclusion: The selected VHHs can be used as potential targeting tools for the development of CD19-based immunotherapeutics. [ABSTRACT FROM AUTHOR]

Background: Goat rumen microbial communities are perceived as one of the most potential biochemical reservoirs of multi-functional enzymes, which are applicable to enhance wide array of bioprocesses such as the hydrolysis of cellulose and hemi-cellulose into fermentable sugar for biofuel and other value-added biochemical production. Even though, the limited understanding of rumen microbial genetic diversity and the absence of effective screening culture methods have impeded the full utilization of these potential enzymes. In this study, we applied culture independent metagenomics sequencing approach to isolate, and identify microbial communities in goat rumen, meanwhile, clone and functionally characterize novel cellulase and xylanase genes in goat rumen bacterial communities. Results: Bacterial DNA samples were extracted from goat rumen fluid. Three genomic libraries were sequenced using Illumina HiSeq 2000 for paired-end 100-bp (PE100) and Illumina HiSeq 2500 for paired-end 125-bp (PE125). A total of 435gb raw reads were generated. Taxonomic analysis using Graphlan revealed that Fibrobacter, Prevotella, and Ruminococcus are the most abundant genera of bacteria in goat rumen. SPAdes assembly and prodigal annotation were performed. The contigs were also annotated using the DOE-JGI pipeline. In total, 117,502 CAZymes, comprising endoglucanases, exoglucanases, beta-glucosidases, xylosidases, and xylanases, were detected in all three samples. Two genes with predicted cellulolytic/xylanolytic activities were cloned and expressed in E. coli BL21(DE3). The endoglucanases and xylanase enzymatic activities of the recombinant proteins were confirmed using substrate plate assay and dinitrosalicylic acid (DNS) analysis. The 3D structures of endoglucanase A and endo-1,4-beta xylanase was predicted using the Swiss Model. Based on the 3D structure analysis, the two enzymes isolated from goat's rumen metagenome are unique with only 56–59% similarities to those homologous proteins in protein data bank (PDB) meanwhile, the structures of the enzymes also displayed greater stability, and higher catalytic activity. Conclusions: In summary, this study provided the database resources of bacterial metagenomes from goat's rumen fluid, including gene sequences with annotated functions and methods for gene isolation and over-expression of cellulolytic enzymes; and a wealth of genes in the metabolic pathways affecting food and nutrition of ruminant animals. [ABSTRACT FROM AUTHOR]

During infection, bacteriophages produce diverse gene products to overcome bacterial antiphage defenses, to outcompete other phages, and to take over cellular processes. Even in the best-studied model phages, the roles of most phage-encoded gene products are unknown, and the phage population represents a largely untapped reservoir of novel gene functions. Considering the sheer size of this population, experimental screening methods are needed to sort through the enormous collection of available sequences and identify gene products that can modulate bacterial behavior for downstream functional characterization. Here, we describe the construction of a plasmid-based overexpression library of 94 genes encoded by Hammy, a Cluster K mycobacteriophage closely related to those infecting clinically important mycobacteria. The arrayed library was systematically screened in a plate-based cytotoxicity assay, identifying a diverse set of 24 gene products (representing ~25% of the Hammy genome) capable of inhibiting growth of the host bacterium Mycobacterium smegmatis. Half of these are related to growth inhibitors previously identified in related phage Waterfoul, supporting their functional conservation; the other genes represent novel additions to the list of known antimycobacterial growth inhibitors. This work, conducted as part of the HHMI-supported Science Education Alliance Gene-function Exploration by a Network of Emerging Scientists (SEA-GENES) project, highlights the value of parallel, comprehensive overexpression screens in exploring genome-wide patterns of phage gene function and novel interactions between phages and their hosts. [ABSTRACT FROM AUTHOR]

The article discusses about "UC Eclipse," a photoperiod-insensitive strawberry cultivar developed by the University of California, Davis, for summer-plant production. Topics include the cultivar's characteristics, breeding origin, field testing results, and disease resistance, positioning "UC Eclipse" as a valuable addition to meet the demand for fresh strawberries in the summer-plant segment.

Opisthorchis felineus is a food-borne trematode which causes opisthorchiosis and affects mainly the liver and bile ducts of the liver with a possible risk of bile duct carcinogenesis resulting in cholangiocarcinoma. In Russia, O. felineus is mainly endemic in Western Siberia (Ob and Irtysh river basins) and occurs throughout the Volga, Kama, Don, and Dnepr river basins. The prevalence, intensity, and clinical significance of human infections and the incidence of cholangiocarcinoma vary geographically in endemic regions. Currently, there is substantial evidence on genetic variation of O. felineus, but information on the population genetic structure is so far very scarce. Because microsatellite DNA of this parasite is not available, we for the first time isolated sufficient microsatellite loci to examine the genetic diversity and population structure of O. felineus, using multiple nuclear loci approach. A total of ten highly polymorphic microsatellite loci from a constructed enriched genomic DNA library were characterized, using 29 samples representing huge O. felineus metapopulation extended in latitude over 5000 km from Middle Europe to Western Siberia. At least three populations can be discerned as result of analysis of the microsatellite loci genetic diversity. Based on the results for the first time, a hypothesis was put forward about the formation of a modern habitat of O. felineus. [ABSTRACT FROM AUTHOR]

The function of regulatory elements is highly dependent on the cellular context, and thus for understanding the function of elements associated with psychiatric diseases these would ideally be studied in neurons in a living brain. Massively Parallel Reporter Assays (MPRAs) are molecular genetic tools that enable functional screening of hundreds of predefined sequences in a single experiment. These assays have not yet been adapted to query specific cell types in vivo in a complex tissue like the mouse brain. Here, using a test-case 3′UTR MPRA library with genomic elements containing variants from autism patients, we developed a method to achieve reproducible measurements of element effects in vivo in a cell type-specific manner, using excitatory cortical neurons and striatal medium spiny neurons as test cases. This targeted technique should enable robust, functional annotation of genetic elements in the cellular contexts most relevant to psychiatric disease. A Cre-dependent massively parallel reporter assay allows for cell-type specific assessment of the functional effects of non-coding elements in vivo, using excitatory cortical neurons and striatal medium spiny neurons as test cases. [ABSTRACT FROM AUTHOR]

Barcoded mutant libraries are a powerful tool for elucidating gene function in microbes, particularly when screened in multiple growth conditions. Here, we screened a pooled CRISPR interference library of the model cyanobacterium Synechocystis sp. PCC 6803 in 11 bioreactor-controlled conditions, spanning multiple light regimes and carbon sources. This gene repression library contained 21,705 individual mutants with high redundancy over all open reading frames and noncoding RNAs. Comparison of the derived gene fitness scores revealed multiple instances of gene repression being beneficial in 1 condition while generally detrimental in others, particularly for genes within light harvesting and conversion, such as antennae components at high light and PSII subunits during photoheterotrophy. Suboptimal regulation of such genes likely represents a tradeoff of reduced growth speed for enhanced robustness to perturbation. The extensive data set assigns condition-specific importance to many previously unannotated genes and suggests additional functions for central metabolic enzymes. Phosphoribulokinase, glyceraldehyde-3-phosphate dehydrogenase, and the small protein CP12 were critical for mixotrophy and photoheterotrophy, which implicates the ternary complex as important for redirecting metabolic flux in these conditions in addition to inactivation of the Calvin cycle in the dark. To predict the potency of sgRNA sequences, we applied machine learning on sgRNA sequences and gene repression data, which showed the importance of C enrichment and T depletion proximal to the PAM site. Fitness data for all genes in all conditions are compiled in an interactive web application. CRISPR interference screens of Synechocystis in different growth regimes allowed mapping of condition-specific gene fitness. [ABSTRACT FROM AUTHOR]

Lao Pa Koi (LPK) chicken is a popular fighting breed in Thailand, prized for (its unique characteristics acquired by selective breeding), and a valuable model for exploring the genetic diversity and admixture of red junglefowls and domestic chickens. In this study, genetic structure and diversity of LPK chicken were assessed using 28 microsatellite markers and mitochondrial DNA (mtDNA) D-loop sequences, and the findings were compared to a gene pool library from "The Siam Chicken Bioresource Project". High genetic variability was observed in LPK chickens using mtDNA D-loop haplotype analysis, and six haplotypes were identified. Microsatellite data revealed 182 alleles, with an average of 6.5 alleles per locus. These results confirmed the occurrence of genetic admixture of red junglefowl and Thai domestic chickens in LPK chicken breed. A maximum entropy modeling approach was used to analyze the spatial suitability and to assess the adaptive evolution of LPK chickens in diverse local environments. The model identified 82.52% of the area studied as unsuitable, and 9.34%, 7.11%, and 2.02% of the area indicated moderate, low, and high suitability, respectively. The highest contribution rate to land suitability for LPK chickens was found at an elevation of 100–250 m, suggesting the importance of elevation for their potential distribution. The results of this study provide valuable insights into the genetic origin of LPK chicken breed and identify resources for future genetic improvement. [ABSTRACT FROM AUTHOR]

Klebsiella pneumoniae is a ubiquitous human pathogen, and its clinical treatment faces two major challenges: multidrug resistance and the pathogenesis of hypervirulent K. pneumoniae. The discovery and study of conditionally essential (CE) genes that can function as potential antimicrobial targets has always been a research concern due to their restriction in the development of novel antibiotics. However, the lack of essential functional genomic data has hampered the study of the mechanisms of essential genes related to antimicrobial susceptibility. In this study, we developed a pooled CE genes mobile clustered regularly interspaced short palindromic repeat (CRISPR) interference screening method (Mobile-CRISPRi-seq) for K. pneumoniae to identify genes that play critical roles in antimicrobial fitness in vitro and host immunity in vivo. Targeting 870 predicted CE genes in K. pneumoniae, Mobile-CRISPRi-seq uncovered the depletion of tetrahydrofolate synthesis pathway genes folB and folP under trimethoprim pressure. Our screening also identified genes waaE and fldA related to polymyxin and ß-lactam susceptibility by applying a screening strategy based on Mobile-CRISPRi-seq and comparative genomics. Furthermore, using a mouse infection model and Mobile-CRISPRi-seq, multiple virulence genes were identified, and among these genes, pal, yciS, and ribB were demonstrated to contribute to the pathogenesis of K. pneumoniae. This study provides a simple, rapid, and effective platform for screening potential antimicrobial targets and virulence genes in K. pneumoniae, and this broadly applicable system can be expanded for high-throughput functional gene study in multiple pathogenic bacteria, especially in gram-negative bacteria. [ABSTRACT FROM AUTHOR]

Coral disease is one of the major threats to coral assemblages globally and can significantly influence the coral microbiome as a result of climatic and anthropogenic stressors. Coral disease surveys were conducted at three patch reefs in the northern Red Sea of Hurghada coast. Surveys occurred during a heatwave event in August 2021, when the average surface water temperature reached about 29 °C. This study compared the differences in bacterial communities associated with healthy and diseased coral tissues of nine reefbuilding coral genera collected from the surveyed reefs. The five most common coral diseases identified were black band disease (BBD), white syndrome (WS), growth anomalies (GA), pink line syndrome (PLS), and skeletal eroding band (SEB). In general, the microbial abundance in infected parts of corals was higher than that in healthy parts. Bacteria associated with the black band disease on Platygyra lamellina showed that the number of total bacteria found in the tissue were higher than that in the mucus of the corals. Furthermore, the cultural Vibrio populations (TCBS) of tissue samples were higher than that in mucus samples, and the Vibrio population of the infected coral tissue and mucus was greater than the healthy tissue and mucus. Bacterial 16S rRNA gene clone libraries derived from 12 cultivable isolates of coral mucus and tissue revealed a distinct partitioning of bacterial genera into healthy and diseased samples. Species identified from the healthy samples were dominated by Paracoccus yeei, Staphylococcus aureus, and Acinetobacter sp., while bacteria associated with BBD-affected coral samples were Acinetobacter sp., Desulfovibrio sp., Bacillus farraginis, Vibrio hepatarius, Vibrio brasiliensis, Arcobacter sp., and Micromonospora sp. The present study provides a baseline assessment of coral disease incidence and associated microbial communities in a northern Red Sea region, which is expected to rise as a consequence of increased frequency and severity of climatic and non-climatic stressors. [ABSTRACT FROM AUTHOR]

Preassembly refers to an evolutionary mechanism that complements current theory. It proposes that genes and gene fragments, formed over the eons among libraries of noncoding genes, ultimately contributed to coding sequences. Previous publications of ours on preassembly have helped explain many enigmas that beset modern biology; for example, the Cambrian explosion ("one of the most remarkable and puzzling events in the history of life," S. J. Gould), and the appearance of flowering plants with their sparsity of precursors (an "abominable mystery," C. R. Darwin). The rapid evolutionary development of the domestic cat and human intelligence is also viewed as evidence for a mechanism supplementing neo-Darwinism. The current account centers on convergent evolution as interpreted via DNA preassembly. Convergent evolution refers to the development of near-identical traits in organisms that lack a common ancestor. A prime example is the remarkable similarity between the eyes of humans and octopuses. When early ancestors of these species split apart on the evolution tree, hundreds of millions of years ago, only eyespots existed. Clearly, convergent eyes in the two organisms evolved independently. Current evolutionary belief says that the eyes appeared after Nature had first screened hundreds of mutations, one at a time, and that this multistep and random process produced, fortuitously, two near identical structures. Preassembly, on the other hand, proposes primeval genes, or DNA segments, that had been collected over vast time periods within huge libraries of noncoding DNA (i.e., "preassembled genes"). Ultimately, these DNA units were incorporated into functional genes, a process possibly aided by genetic site-scanning. Menger, F. M. (2017). Molecular Lamarckism. On the evolution of human intelligence. World Futures, 73, 89–103. Menger, F. M. (2020). An alternative view of evolution, how DNA was altered over geological time. Molecules, 25, 5081. Menger, F. M., & Rizvi, S. A. A. (2021). Evolution of complexity. Molecular aspects of preassembly. Molecules, 26, 6618. [ABSTRACT FROM AUTHOR]

We sought to explore the hypothesis that host factors required for HIV-1 replication also play a role in latency reversal. Using a CRISPR gene library of putative HIV dependency factors, we performed a screen to identify genes required for latency reactivation. We identified several HIV-1 dependency factors that play a key role in HIV-1 latency reactivation including ELL, UBE2M, TBL1XR1, HDAC3, AMBRA1, and ALYREF. The knockout of Cyclin T1 (CCNT1), a component of the P-TEFb complex that is important for transcription elongation, was the top hit in the screen and had the largest effect on HIV latency reversal with a wide variety of latency reversal agents. Moreover, CCNT1 knockout prevents latency reactivation in a primary CD4+ T cell model of HIV latency without affecting the activation of these cells. RNA sequencing data showed that CCNT1 regulates HIV-1 proviral genes to a larger extent than any other host gene and had no significant effects on RNA transcripts in primary T cells after activation. We conclude that CCNT1 function is non-essential in T cells but is absolutely required for HIV latency reversal. [ABSTRACT FROM AUTHOR]

The broad-banded velvetchin Hapalogenys analis is an economically and scientifically important global temperate and tropical nearshore marine fish. To understand the genetic evolution mechanism of H. analis, simplified genome sequencing analysis was conducted on 82 samples of H. analis from three populations (Akashi, Zhoushan, and Shantou) in the northwestern Pacific Ocean using double-digest restriction site-associated DNA sequencing. Genetic characteristics were identified using single-nucleotide polymorphism loci, and a small fragment library (250-450 bp long) was constructed for double-end sequencing. Single-nucleotide polymorphisms (SNPs) were detected using Stacks software and analyzed. The phylogenetic tree was constructed to analyze the population structure characteristics and the genetic evolutionary relationships between individuals in different regions at the genetic level. The sequencing library constructed for 82 samples of H. analis using double-digest restriction site-associated DNA sequencing possessed an average of 7,931,195 sequencing sequences and an average of 1,141,560,806 bases in the 82 samples. A total of 3,204,106 SNP loci were obtained from the three populations of H. analis, indicating rich genetic diversity. The transition to transversion ratio of SNPs was >1.5, indicating transformational reversal bias in H. analis. The observed heterozygosity of the various populations was 0.186-0.199, with an expected heterozygosity of 0.253-0.268. High diversity in all three populations was detected using nucleotide diversity analysis. AS and ZS had the highest degree of differentiation. The H. analis samples shared two gene libraries; some populations were closely related, whereas others had a distant genetic relationship. The results of genetic statistics, genetic differentiation, population structure, principal component, phylogenetic, and genetic relationship analyses supported independent clustering of the AS population, which can be used as a new genetic resource for conserving H. analis. These results provide a reference basis for comparative genomics studies of H. analis and rational utilization of Hapalogenys resources. [ABSTRACT FROM AUTHOR]

The emerging lung pathogen Mycobacterium abscessus is understudied for its virulence determinants and molecular targets for diagnosis and therapeutics. Here, we report a comprehensive secretome (600 proteins) of this species, which was identified using a multipronged strategy based on genetic/genomic, proteomic, and bioinformatic approaches. In-solution digested bottom-up proteomics from various growth phases identified a total of 517 proteins, while 2D-GE proteomics identified 33 proteins. A reporter-gene-fusion-based genomic library that was custom-generated in this study enabled the detection of 23 secretory proteins. A genome-wide survey for N-terminal signal sequences using bioinformatic tools (Psortb 2.0 and SignalP 3.0) combined with a strategy of the subtraction of lipoproteins and proteins containing multiple transmembrane domains yielded 116 secretory proteins. A homology search against the M. tuberculosis database identified nine additional secretory protein homologs that lacked a secretory signal sequence. Considering the little overlap (80 proteins) among the different approaches used, this study emphasized the importance of using a multipronged strategy for a comprehensive understanding of the secretome. Notably, the majority of the secreted proteins identified (over 50%) turned out to be "orphans" (those with no known functional homologs). The revelation of these species-specific orphan proteins offers a hitherto unexplored repertoire of potential targets for diagnostic, therapeutic, and vaccine research in this emerging lung pathogen. [ABSTRACT FROM AUTHOR]

Plant viruses are of significant threat to agricultural productivity worldwide, causing substantial economic losses and impacting food security. Understanding the molecular interactions between plant viruses and their hosts is essential for developing effective strategies for virus control and crop protection. In this study, we employed homology modeling and an in silico approach to investigate interactions between chilli host protein, chloroplastic photosystem II protein D1 with the coat protein of cucumber mosaic virus (CMV). CMV coat protein sequences were retrieved from genomic data bank to identify putative host targets. Subsequently, we utilized established protein-protein interaction prediction algorithms and resources to predict potential interactions between CMV proteins and host proteins. This in silico study provides a foundation for further experimental investigations, offering potential targets for the development of novel antiviral strategies and crop protection measures. From the obtained docking scores, it was presumed that CMV CP and Photosystem II protein D1 are interacting and established structural-functional relation provided a basis to propose probable hostvirus interactions in terms of virus infection and symptom development. [ABSTRACT FROM AUTHOR]

Introduction: Seagrass roots harbour diverse assemblages of microorganisms that likely benefit the growth and survival of meadows. Yet, restoration efforts rarely consider their effect on developing seagrass seedlings. Sediment origin should determine the types of rhizosphere and root-colonising (rhizoplane) microorganisms and thus the performance of seedlings during restoration, particularly for slow growing climax species like Posidonia. Recent Posidonia restoration attempts in South Australia used commercially sourced 'play pit sand' for seedling propagation and planting, but have been impacted by high mortality. More natural substrates like seagrass meadow sediment have not been previously investigated for suitability over conventional substrates with regard to seedling growth and survival. Methods: To assess the relevance of seagrass associated microorganisms in the growth of Posidonia angustifolia seedlings, we investigated the bacterial communities from tank-raised seedlings propagated in autoclave treated and untreated 'play pit sand' and meadow mix (comprising a 1:3 ratio of natural meadow sediment and beach sand) over a 12-week period. Autoclave treatment was adopted in order to diminish the bacterial load prior to planting and thus inform their contribution (if any) on early seedling growth. Samples for bacterial community analysis and seedling growth measurements (numbers and total length of roots/leaves, root diameter, seedling weight, starch reserves) were taken at 4 and 12 weeks. Bacterial assemblages were surveyed from DNA extracts from bulk and rhizosphere sediments and root tissues, as well as from swabs of P. angustifolia fruit, established meadow sediment and water samples prior to seedling propagation, by constructing Illumina 16S rRNA gene libraries. Results: While most growth measurements did not vary significantly between sediment type or treatment, proportional growth of roots versus leaves (as expressed as a pseudo root:shoot ratio) was significantly related to treatment, sediment type and seed length. Seedlings from meadow mix invested more in leaves, regardless of treatment, when compared to play sand. Autoclave treatment increased investment in roots for play sand but increased the investment in leaves for meadow mix. Bacterial communities differed significantly between sediments and between sample types (bulk, rhizosphere and roots), with the roots from meadow mix seedlings containing an increased abundance of various potentially beneficial bacterial taxa. Discussion: While such changes appear to affect the early development of seedlings, bacterial community dynamics are also likely coupled to changes in nutrient availability. Further research is thus required to disentangle host seedling growth-nutrient-bacterial community dynamics with the view to identifying microbes that may support the growth and vigour of seedlings under different nutrient conditions as part of future restoration efforts. [ABSTRACT FROM AUTHOR]

Background: Rheumatoid arthritis (RA) is one of the most common chronic inflammatory autoimmune diseases. However, the underlying molecular mechanisms of its pathogenesis are unknown. This study aimed to identify the common biomarkers of ferroptosis and pyroptosis in RA and screen potential drugs.Methods: The RA-related differentially expressed genes (DEGs) in GSE55235 were screened by R software and intersected with ferroptosis and pyroptosis gene libraries to obtain differentially expressed ferroptosis-related genes (DEFRGs) and differentially expressed pyroptosis-related genes (DEPRGs). We performed Gene Ontology (GO), Kyoto Encyclopedia of the Genome (KEGG), ClueGO, and Protein-Protein Interaction (PPI) analysis for DEFRGs and DEPRGs and validated them by machine learning. The microRNA/transcription factor (TF)-hub genes regulatory network was further constructed. The key gene was validated using the GSE77298 validation set, cellular validation was performed in in vitro experiments, and immune infiltration analysis was performed using CIBERSORT. Network pharmacology was used to find key gene-targeting drugs, followed by molecular docking and molecular dynamics simulations to analyze the binding stability between small-molecule drugs and large-molecule proteins.Results: Three hub genes (CASP8, PTGS2, and JUN) were screened via bioinformatics, and the key gene (CASP8) was validated and obtained through the validation set, and the diagnostic efficacy was verified to be excellent through the receiver operating characteristic (ROC) curves. The ferroptosis and pyroptosis phenotypes were constructed by fibroblast-like synoviocytes (FLS), and caspase-8 was detected and validated as a common biomarker for ferroptosis and pyroptosis in RA, and quercetin can reduce caspase-8 levels. Quercetin was found to be a potential target drug for caspase-8 by network pharmacology, and the stability of their binding was further verified using molecular docking and molecular dynamics simulations.Conclusion: Caspase-8 is an important biomarker for ferroptosis and pyroptosis in RA, and quercetin is a potential therapy for RA via targeting caspase-8 through ferroptosis and pyroptosis. [ABSTRACT FROM AUTHOR]

Most experiments studying bacterial microbiomes rely on the PCR amplification of all or part of the gene for the 16S rRNA subunit, which serves as a biomarker for identifying and quantifying the various taxa present in a microbiome sample. Several computational methods exist for analyzing 16S amplicon sequencing. However, the most-used bioinformatics tools cannot produce high quality genus-level or species-level taxonomic calls and may underestimate the potential accuracy of these calls. We used 16S sequencing data from mock bacterial communities to evaluate the sensitivity and specificity of several bioinformatics pipelines and genomic reference libraries used for microbiome analyses, concentrating on measuring the accuracy of species-level taxonomic assignments of 16S amplicon reads. We evaluated the tools DADA2, QIIME 2, Mothur, PathoScope 2, and Kraken 2 in conjunction with reference libraries from Greengenes, SILVA, Kraken 2, and RefSeq. Profiling tools were compared using publicly available mock community data from several sources, comprising 136 samples with varied species richness and evenness, several different amplified regions within the 16S rRNA gene, and both DNA spike-ins and cDNA from collections of plated cells. PathoScope 2 and Kraken 2, both tools designed for whole-genome metagenomics, outperformed DADA2, QIIME 2 using the DADA2 plugin, and Mothur, which are theoretically specialized for 16S analyses. Evaluations of reference libraries identified the SILVA and RefSeq/Kraken 2 Standard libraries as superior in accuracy compared to Greengenes. These findings support PathoScope and Kraken 2 as fully capable, competitive options for genus- and species-level 16S amplicon sequencing data analysis, whole genome sequencing, and metagenomics data tools. [ABSTRACT FROM AUTHOR]

The efficiency of AST is quantified by measuring the relative ratio of viable cells between the adherent and suspension cell populations using CellTiter Glo® 2.0 cell viability assay (Promega, G9241). Melanoma primary tumor and lung metastasis tissue dissociation GFP-positive B16F10 cells were isolated from the primary sites and lungs. Paired-end sequencing was performed using NovaSeq 5000/6000 S1 Rgt Kit v1.5 (200 cycles) on a NovaSeq600 (Illunmina) sequencer. scRNA-seq analysis of primary tumor cells and CTCs The Illumina outputs of human and mouse samples were mapped to the human reference genome (GRCh38) using Cell Ranger 6.0.0 (10X Genomics). Cell viability and ROS detection assay In vitro cell viability was assessed using the CellTiter Glo® 2.0 cell viability assay (Promega, G9241) and LIVE/DEAD® cell imaging kit (ThermoFisher, R37601) according to the manufacturer's protocols. [Extracted from the article]

Almost 40% of infertile men cases are classified as idiopathic when tested negative to the current diagnostic routine based on the screening of karyotype, Y chromosome microdeletions and CFTR mutations in men with azoospermia or oligozoospermia. Rare monogenic forms of infertility are not routinely evaluated. In this study we aim to investigate the unknown potential genetic causes in couples with pure male idiopathic infertility by applying variant prioritization to whole exome sequencing (WES) in a cohort of 99 idiopathic Italian patients. The ad-hoc manually curated gene library prioritizes genes already known to be associated with more common and rare syndromic and non-syndromic male infertility forms. Twelve monogenic cases (12.1%) were identified in the whole cohort of patients. Of these, three patients had variants related to mild androgen insensitivity syndrome, two in genes related to hypogonadotropic hypogonadism, and six in genes related to spermatogenic failure, while one patient is mutant in PKD1. These results suggest that NGS combined with our manually curated pipeline for variant prioritization and classification can uncover a considerable number of Mendelian causes of infertility even in a small cohort of patients. [ABSTRACT FROM AUTHOR]

Insect pathogenic fungi, also known as entomopathogenic fungi, are one of the largest insect pathogenic microorganism communities, represented by Beauveria spp. and Metarhizium spp. Entomopathogenic fungi have been proved to be a great substitute for chemical pesticide in agriculture. In fact, a lot of functional genes were also already characterized in entomopathogenic fungi, but more depth of exploration is still needed to reveal their complicated pathogenic mechanism to insects. Metarhizium rileyi (Nomuraea rileyi) is a great potential biocontrol fungus that can parasitize more than 40 distinct species (mainly Lepidoptera: Noctuidae) to cause large-scale infectious diseases within insect population. In this study, a comparative analysis of transcriptome profile was performed with topical inoculation and hemolymph injection to character the infectious pattern of M. rileyi. Appressorium and multiple hydrolases are indispensable constituents to break the insect host primary cuticle defense in entomopathogenic fungi. Within our transcriptome data, numerous transcripts related to destruction of insect cuticle rather growth regulations were obtained. Most importantly, some unreported ribosomal protein genes and novel unannotated protein (hypothetical protein) genes were proved to participate in the course of pathogenic regulation. Our current data provide a higher efficiency gene library for virulence factors screen in M. rileyi, and this library may be also useful for furnishing valuable information on entomopathogenic fungal pathogenic mechanisms to host. [ABSTRACT FROM AUTHOR]

Key message: Brassica orphan gene BrFLM, identified by two allelic mutants, was involved in leafy head formation in Chinese cabbage. Leafy head formation is a unique agronomic trait of Chinese cabbage that determines its yield and quality. In our previous study, an EMS mutagenesis Chinese cabbage mutant library was constructed using the heading Chinese cabbage double haploid (DH) line FT as the wild-type. Here, we screened two extremely similar leafy head deficiency mutants lfm-1 and lfm-2 with geotropic growth leaves from the library to investigate the gene(s) related to leafy head formation. Reciprocal crossing results showed that these two mutants were allelic. We utilized lfm-1 to identify the mutant gene(s). Genetic analysis showed that the mutated trait was controlled by a single nuclear gene Brlfm. Mutmap analysis showed that Brlfm was located on chromosome A05, and BraA05g012440.3C or BraA05g021450.3C were the candidate gene. Kompetitive allele-specific PCR analysis eliminated BraA05g012440.3C from the candidates. Sanger sequencing identified an SNP from G to A at the 271st nucleotide on BraA05g021450.3C. The sequencing of lfm-2 detected another non-synonymous SNP (G to A) located at the 266st nucleotide on BraA05g021450.3C, which verified its function on leafy head formation. We blasted BraA05g021450.3C on database and found that it belongs to a Brassica orphan gene encoding an unknown 13.74 kDa protein, named BrLFM. Subcellular localization showed that BrLFM was located in the nucleus. These findings reveal that BrLFM is involved in leafy head formation in Chinese cabbage. [ABSTRACT FROM AUTHOR]

Objective: Pancreatic ductal adenocarcinoma (PDAC) is a highly malignant gastrointestinal cancer with a 5-year survival rate of only 9%. Of PDAC patients, 15%-20% are eligible for radical surgery. Gemcitabine is an important chemotherapeutic agent for patients with PDAC; however, the efficacy of gemcitabine is limited due to resistance. Therefore, reducing gemcitabine resistance is essential for improving survival of patients with PDAC. Identifying the key target that determines gemcitabine resistance in PDAC and reversing gemcitabine resistance using target inhibitors in combination with gemcitabine are crucial steps in the quest to improve survival prognosis in patients with PDAC. Methods: We constructed a human genome-wide CRISPRa / dCas 9 overexpression library in PDAC cell lines to screen key targets of drug resistance based on sgRNA abundance and enrichment. Then, co-IP, ChIP, ChIP-seq, transcriptome sequencing, and qPCR were used to determine the specific mechanism by which phospholipase D1 (PLD1) confers resistance to gemcitabine. Results: PLD1 combines with nucleophosmin 1 (NPM1) and triggers NPM1 nuclear translocation, where NPM1 acts as a transcription factor to upregulate interleukin 7 receptor (IL7R) expression. Upon interleukin 7 (IL-7) binding, IL7R activates the JAK1/STAT5 signaling pathway to increase the expression of the anti-apoptotic protein, BCL-2, and induce gemcitabine resistance. The PLD1 inhibitor, Vu0155069, targets PLD1 to induce apoptosis in gemcitabine-resistant PDAC cells. Conclusions: PLD1 is an enzyme that has a critical role in PDAC-associated gemcitabine resistance through a non-enzymatic interaction with NPM1, further promoting the downstream JAK1/STAT5/Bcl-2 pathway. Inhibiting any of the participants of this pathway can increase gemcitabine sensitivity. [ABSTRACT FROM AUTHOR]

Motivation Droplet-based single-cell RNA sequencing (scRNA-seq) is widely used in biomedical research for interrogating the transcriptomes of single cells on a large scale. Pooling and processing cells from different samples together can reduce costs and batch effects. To pool cells, they are often first labeled with hashtag oligonucleotides (HTOs). These HTOs are sequenced alongside the cells' RNA in the droplets and subsequently used to computationally assign each droplet to its sample of origin, a process referred to as demultiplexing. Accurate demultiplexing is crucial but can be challenging due to background HTOs, low-quality cells/cell debris, and multiplets. Results A new demultiplexing method based on negative binomial regression mixture models is introduced. The method, called demuxmix, implements two significant improvements. First, demuxmix's probabilistic classification framework provides error probabilities for droplet assignments that can be used to discard uncertain droplets and inform about the quality of the HTO data and the success of the demultiplexing process. Second, demuxmix utilizes the positive association between detected genes in the RNA library and HTO counts to explain parts of the variance in the HTO data resulting in improved droplet assignments. The improved performance of demuxmix compared with existing demultiplexing methods is assessed using real and simulated data. Finally, the feasibility of accurately demultiplexing experimental designs where non-labeled cells are pooled with labeled cells is demonstrated. Availability and implementation R/Bioconductor package demuxmix (https://doi.org/doi:10.18129/B9.bioc.demuxmix) [ABSTRACT FROM AUTHOR]

Large, randomized libraries are a key technology for many biotechnological applications. While genetic diversity is the main parameter most libraries direct their resources on, less focus is devoted to ensuring functional IN-frame expression. This study describes a faster and more efficient system based on a split β-lactamase complementation for removal of OFF-frame clones and increase of functional diversity, suitable for construction of randomized libraries. The gene of interest is inserted between two fragments of the β-lactamase gene, conferring resistance to β-lactam drugs only upon expression of an inserted IN-frame gene without stop codons or frameshifts. The preinduction-free system was capable of eliminating OFF-frame clones in starting mixtures of as little as 1% IN-frame clones and enriching to about 70% IN-frame clones, even when their starting rate was as low as 0.001%. The curation system was verified by constructing a single-domain antibody phage display library using trinucleotide phosphoramidites for randomizing a complementary determining region, while eliminating OFF-frame clones and maximizing functional diversity. • Generation of libraries with randomized genes for various applications. • Goal is large, diverse libraries, but can result in many OFF-frame genes. • Improved, faster, robust version of split β-lactamase IN-frame enrichment system. • High enrichment rate, efficient, greater yield of IN-frame clones in shorter time. • Use of Nanopore and Sanger sequencing to prove enrichment efficiency. [ABSTRACT FROM AUTHOR]

Nematomorpha (hairworms) is a phylum of parasitic ecdysozoans, best known for infecting arthropods and guiding their hosts toward water, where the parasite can complete its life cycle. Over 350 species of nematomorphs have been described, yet molecular data for the group remain scarce. The few available mitochondrial genomes of nematomorphs are enriched with long inverted repeats, which are embedded in the coding sequences of their genes—a remarkably unusual feature exclusive to this phylum. Here, we obtain and annotate the repeats in the mitochondrial genome of another nematomorph species—Parachordodes pustulosus. Using genomic and transcriptomic libraries, we investigate the impact of inverted repeats on the read coverage of the mitochondrial genome. Pronounced drops in the read coverage coincide with regions containing long inverted repeats, denoting the 'blind spots' of short-fragment sequencing libraries. Phylogenetic inference with the novel data reveals multiple disagreements between the traditional system of Nematomorpha and molecular data, rendering several genera paraphyletic, including Parachordodes. [ABSTRACT FROM AUTHOR]

The article focuses on the development of a genetic population suppression system called Ifegenia in the malaria vector Anopheles gambiae. It mentions by disrupting a female-essential gene, the system achieves complete genetic sexing through heritable daughter gynecide, allowing for sustained population suppression.

The Na+/H+ antiporter NhaC family protein is a kind of Na+/H+ exchanger from the ion transporter (IT) superfamily, which has mainly been identified in the halophilic bacteria of Bacillus. However, little is known about the Na+/H+ antiporter NhaC family of proteins in the extremely halophilic archaea. In this study, two Na+/H+ antiporter genes, nhaC1 and nhaC2, were screened from the genome of Natronorubrum daqingense based on the gene library and complementation of salt-sensitive Escherichia coli KNabc. A clone vector pUC18 containing nhaC1 or nhaC2 could make KNabc tolerate 0.6 M/0.7 M NaCl or 30 mM/40 mM LiCl and a pH of up to 8.5/9.5, respectively. Functional analysis shows that the Na+(K+, Li+)/H+ antiport activities of NhaC1 and NhaC2 are both pH-dependent in the range of pH 7.0–10.0, and the optimal pH is 9.5. Phylogenetic analysis shows that both NhaC1 and NhaC2 belong to the Na+/H+ antiporter NhaC family of proteins and are significantly distant from the identified NhaC proteins from Bacillus. In summary, we have identified two Na+(K+, Li+)/H+ antiporters from N. daqingense. [ABSTRACT FROM AUTHOR]

The article discusses the formation of snake scales, highlighting the role of somitic positional information and reaction-diffusion mechanisms. By studying wild-type and scaleless snakes, researchers uncover the coordination between these factors in establishing the precise hexagonal pattern of scales and its importance for snake locomotion.

The potential nitrogen-fixing bacterial diversity in the rhizospheric soil of the native switchgrass (Panicum virgatum L.) from Tall Grass Prairies of Northern Oklahoma was studied using a partial region of nitrogenase structural gene—nifH. Eleven clone libraries constructed from nifH amplicons gave 407 good-quality sequences. More than 70% of sequences showed similarity of nifH with uncultured bacteria (< 98%). The dominance of sequences affiliated with Deltaproteobacterial nifH was observed, followed by Betaproteobacterial nifH sequences. The nifH gene library was dominated by the genera Geobacter, Rhizobacter, Paenibacillus, and Azoarcus. Sequences affiliated with rhizobia, such as Bradyrhizobium, Methylocystis, Ensifer, etc., were also in the rhizosphere in small numbers. From Deltaproteobacteria, five genera, namely Geobacter, Pelobacter, Geomonas, Desulfovibrio, and Anaeromyxobacter, contributed to 48% of the total sequences suggesting the dominance of group Deltaproteobacteria in the rhizosphere of native switchgrass. Considering the percent similarity of the nifH sequences with cultivated bacteria, this study demonstrated the presence of novel bacterial species in switchgrass rhizospheric soil from Tall Grass Prairie. [ABSTRACT FROM AUTHOR]

During the interaction between Brassica napus and Sclerotinia sclerotiorum, the expression of JA (jasmonic acid) synthesis-related genes increases, while JA signal transduction is inhibited. It is speculated that the effector proteins secreted by S. sclerotiorum may directly or indirectly interact with JAR1 protein, reducing its activity and thus inhibiting the defense system of B. napus dependent on JA pathway. The full-length sequence of the differentially expressed gene BnJarl was obtained by transcriptome sequencing in the early stage of this research. In order to further reveal the mechanism of BnJarl gene involved in the interaction between B. napus and S. sclerotiorum, this study analyzed and predicted the bioinformatics of BnJAR1 protein, constructed a yeast expression gene cDNA library of B. napus-S. sclerotiorum interaction, and used BnJar1 as bait protein to screen the library by yeast two-hybrid. Through the study, 5 interacting proteins from B. napus were obtained: BnAOC2, BnCOP9, BnDcoH, BnNIT2, and BnTSJT1; and 2 interacting proteins from S. sclerotiorum: SsMFS and SsRho3. The relevant results contributed to our research on the interaction between B. napus and S. sclerotiorum, exploration of pathogenic or resistance-related genes, and on further elucidation of their mechanisms. [ABSTRACT FROM AUTHOR]

The use of microorganisms for efficient conversion of 5-hydroxymethylfurfural (HMF) to more valuable chemical materials has received extensive attention. However, HMF can seriously impede the growth of microorganisms. In order to overcome the problem, it is of great significance to explore the mechanism of microbial tolerance to HMF. A total of 294 genes has been found to be associated with HMF tolerance in Saccharomyces cerevisiae by whole gene knockout library scanning and SGAtool analysis. In this study, KEGG and GO enrichment analysis on Cytoscape identified ASP2, GRH1, PIB1, PSD2, VID24, VMA21, VPS21, VPS51 in cytoplasmic vesicles pathway as HMF tolerance-related genes. Among them, VID24 is involved in the tolerance regulation of HMF based on spot-test validation. According to the subcellular structure observation, after 60 Mm HMF treatment for 3 h, the proportion of damaged endoplasmic reticulum (ER) cells, damaged vacuole mitochondrial cells in VID24 gene knockout strain respectively increased by 3.92% and 29.83% compared with standard strain BY4741. In summary, gene VID24 plays an important role in protecting ER in cellular level and maintaining the stability of vacuole structures. These mechanisms provide a theoretical basis for HMF modification in strains. [ABSTRACT FROM AUTHOR]

Single-cell gene expression analysis using sequencing (scRNA-seq) has gained increased attention in the past decades for studying cellular transcriptional programs and their heterogeneity in an unbiased manner, and novel protocols allow the simultaneous measurement of gene expression, T-cell receptor clonality and cell surface protein expression. In this article, we describe the methods to isolate scRNA/TCR-seq-compatible CD4+ T cells from murine tissues, such as skin, spleen, and lymph nodes. We describe the processing of cells and quality control parameters during library preparation, protocols for multiplexing of samples, and strategies for sequencing. Moreover, we describe a step-by-step bioinformatic analysis pipeline from sequencing data generated using these protocols. This includes quality control, preprocessing of sequencing data and demultiplexing of individual samples. We perform quantification of gene expression and extraction of T-cell receptor alpha and beta chain sequences, followed by quality control and doublet detection, and methods for harmonization and integration of datasets. Next, we describe the identification of highly variable genes and dimensionality reduction, clustering and pseudotemporal ordering of data, and we demonstrate how to visualize the results with interactive and reproducible dashboards. We will combine different analytic R-based frameworks such as Bioconductor and Seurat, illustrating how these can be interoperable to optimally analyze scRNA/TCR-seq data of CD4+ T cells from murine tissues. [ABSTRACT FROM AUTHOR]

Introduction: Anaerobic oxidation of methane (AOM) is hypothesized to occur through reverse hydrogenotrophic methanogenesis in marine sediments because sulfate reducers pull hydrogen concentrations so low that reverse hydrogenotrophic methanogenesis is exergonic. If true, hydrogenotrophic methanogenesis can theoretically co-occur with sulfate reduction if the organic matter is so labile that fermenters produce more hydrogen than sulfate reducers can consume, causing hydrogen concentrations to rise. Finding accumulation of biologically-produced methane in sulfate-containing organic-rich sediments would therefore support the theory that AOM occurs through reverse hydrogenotrophic methanogenesis since it would signal the absence of net AOM in the presence of sulfate. Methods: 16S rRNA gene libraries were compared to geochemistry and incubations in high depth-resolution sediment cores collected from organic-rich Cape Lookout Bight, North Carolina. Results: We found that methane began to accumulate while sulfate is still abundant (6-8 mM). Methane-cycling archaea ANME-1, Methanosarciniales, and Methanomicrobiales also increased at these depths. Incubations showed that methane production in the upper 16 cm in sulfate-rich sediments was biotic since it could be inhibited by 2-bromoethanosulfonoic acid (BES). Discussion: We conclude that methanogens mediate biological methane production in these organic-rich sediments at sulfate concentrations that inhibit methanogenesis in sediments with less labile organic matter, and that methane accumulation and growth of methanogens can occur under these conditions as well. Our data supports the theory that H2 concentrations, rather than the co-occurrence of sulfate and methane, control whether methanogenesis or AOM via reverse hydrogenotrophic methanogenesis occurs. We hypothesize that the high amount of labile organic matter at this site prevents AOM, allowing methane accumulation when sulfate is low but still present in mM concentrations. [ABSTRACT FROM AUTHOR]

The addition of sulfur (S) to organic matter to form organosulfur compounds is generally thought to protect organic matter from microbial degradation and promote its preservation. While most microbial sulfur cycling occurs in sulfate-rich sediments above the sulfate-methane transition zone, recently discovered active sulfur cycling in deeper sulfate-poor environments may have a yet-unquantified impact on the mineralization of organic matter. Here we investigated the fate of buried S-compounds down to 10-m sediment depth representing the entire ~13.5 kya history of the sulfate-rich alpine Lake Cadagno. Chemical profiles of sulfate and sulfur reveal that these oxidized species are depleted at the sediment surface with the concomitant formation of iron sulfide minerals. An underlying aquifer provides a second source of sulfate and other oxidants to the deepest and oldest sediment layers generating an inverse redox gradient. At both sulfate depletion zones, isotopes of chromium-reducible sulfur (CRS) and humic-bound sulfur are highly negative (−30 to −65 per mil) compared to background sulfate suggesting ongoing microbial sulfur cycling. Interestingly, humic-bound S from intermittent sediment layers within the sulfate-depleted methanogenesis zone consistently exhibits a lower δ34S than CRS in lacustrine deposits but a higher δ34S than CRS in terrestrial deposits, which could possibly be due to different reactivities of organic matter types (lacustrine versus terrestrial origin) to sulfide or the ability of microorganisms to form/degrade organic S. Although sulfate concentrations are extremely low between the sulfate depletion zones, dsrB gene libraries reveal a huge potential for microbial sulfur reduction throughout the sediment column. [ABSTRACT FROM AUTHOR]