Bacterial diversity in semen from stallions in three European countries evaluated by 16S sequencing.

The microbiome plays a significant role in shaping the health and functioning of the systems it inhabits. The seminal microbiome of stallions has implications for the health of the reproductive tract, sperm quality during preservation and antibiotic use in semen extenders. Diverse bacteria are present on the external genital tract and a mix of commensal microorganisms populates various parts of the reproductive tract, influencing the seminal bacterial content. Other sources of bacteria include the environment, semen collection equipment, and personnel. The bacterial load can adversely affect sperm quality and fertility, particularly in artificial insemination, where semen is extended and stored before use. Antibiotics are frequently used to inhibit bacterial growth, but their effectiveness varies depending on the bacterial strains present. The aim of this study was to assess the bacterial diversity in semen from 37 healthy stallions across three European nations (Germany, Portugal, and Sweden) using 16S sequencing. Semen samples were collected from individual stallions at three AI centers; DNA extraction, sequencing, and bioinformatic analysis were performed. Differences in bacterial diversity among the stallions were seen; although bacterial phyla were shared across the regions, differences were observed at the genus level. Climate, husbandry practices, and individual variability likely contribute to these differences. These findings underscore the importance of tailoring antibiotic strategies for semen preservation based on regional bacterial profiles. The study presents a comprehensive approach to understanding the intricacies of the stallion seminal microbiome and its potential implications for reproductive technologies and animal health.

Haplotype-resolved genome of heterozygous African cassava cultivar TMEB117 (Manihot esculenta).

Cassava (Manihot esculenta Crantz) is a vital tropical root crop providing essential dietary energy to over 800 million people in tropical and subtropical regions. As a climate-resilient crop, its significance grows as the human population expands. However, yield improvement faces challenges from biotic and abiotic stress and limited breeding. Advanced sequencing and assembly techniques enabled the generation of a highly accurate, nearly complete, haplotype-resolved genome of the African cassava cultivar TMEB117. It is the most accurate cassava genome sequence to date with a base-level accuracy of QV > 64, N50 > 35 Mbp, and 98.9% BUSCO completeness. Over 60% of the genome comprises repetitive elements. We predicted over 45,000 gene models for both haplotypes. This achievement offers valuable insights into the heterozygosity genome organization of the cassava genome, with improved accuracy, completeness, and phased genomes. Due to its high susceptibility to African Cassava Mosaic Virus (ACMV) infections compared to other cassava varieties, TMEB117 provides an ideal reference for studying virus resistance mechanisms, including epigenetic variations and smallRNA expressions.

Microbial Profiling of Amniotic Fluid, Umbilical Blood and Placenta of the Foaling Mare.

The presence of a microbiome/microbiota in the placenta is hotly debated. In previous studies, the presence of bacteria in equine amniotic fluid and umbilical blood was independent of foal health. The objective of the present study was to determine if the same bacteria are present in the equine placenta as in amniotic fluid and umbilical blood. Samples were obtained from 24 parturient mares and foals. Placental bacterial DNA was extracted, and the microbiome was identified using 16S rRNA sequencing. All amniotic fluid samples contained some polymorphonucleocytes; bacteria were isolated from four samples. Aerobic or anaerobic growth was found in 18 and 3 umbilical blood samples, respectively. Serum amyloid A was <5 mg/L in all 24 samples, total WBC varied between 2900 and 10,700/µL, and fibrinogen varied between 0 and 5.16 g/L. In jugular blood, serum amyloid A was <5 mg/L in all 24 foals, total white blood count was 3200 to 8100/µL, and fibrinogen was 0.44 to 4.42 g/L. The diversity of bacterial microbiota was similar in all placental regions at the phylum level but differed at the genus level; the most abundant phyla were Proteobacteria (42-46.26%) and Actinobacteria (26.91-29.96%). In conclusion, bacteria were found in the fetal compartments and placenta of healthy equine pregnancies; however, we can neither prove nor disprove the hypothesis that the placenta has its own microbiome.

Antibiotics induced changes in nitrogen metabolism and antioxidative enzymes in mung bean (Vigna radiata).

Excessive use and release of antibiotics into the soil environment in the developing world have resulted in altered soil processes affecting terrestrial organisms and posing a serious threat to crop growth and productivity. The present study investigated the influence of exogenously applied oxytetracycline (OXY) and levofloxacin (LEV) on plant physiological responses, key enzymes involved in nitrogen metabolism (e.g., nitrate reductase, glutamine synthetase), nitrogen contents and oxidative stress response of mung bean (Vigna radiata). Plants were irrigated weekly with antibiotics containing water for exposing the plants to different concentrations i.e., 1, 10, 20, 50, and 100 mg L-1. Results showed a significant decrease in nitrate reductase activity in both antibiotic treatments and their mixtures and increased antioxidant enzymatic activities in plants. At lower concentrations of antibiotics (≤20 mg L-1), 53.9 % to 78.4 % increase in nitrogen content was observed in levofloxacin and mixtures compared to the control, resulting in an increase in the overall plant biomass. Higher antibiotic (≥50 mg L-1) concentration showed 58 % decrease in plant biomass content and an overall decrease in plant nitrogen content upon exposure to the mixtures. This was further complemented by 22 % to 42 % increase in glutamine synthetase activity observed in the plants treated with levofloxacin and mixtures. The application of low doses of antibiotics throughout the experiments resulted in lower toxicity symptoms in the plants. However, significantly higher malondialdehyde (MDA) concentrations at higher doses (20 mg L-1 and above) than the control showed that plants' tolerance against oxidative stress was conceded with increasing antibiotic concentrations. The toxicity trend was: levofloxacin > mixture > oxytetracycline.

Nano3P-seq: transcriptome-wide analysis of gene expression and tail dynamics using end-capture nanopore cDNA sequencing.

RNA polyadenylation plays a central role in RNA maturation, fate, and stability. In response to developmental cues, polyA tail lengths can vary, affecting the translation efficiency and stability of mRNAs. Here we develop Nanopore 3' end-capture sequencing (Nano3P-seq), a method that relies on nanopore cDNA sequencing to simultaneously quantify RNA abundance, tail composition, and tail length dynamics at per-read resolution. By employing a template-switching-based sequencing protocol, Nano3P-seq can sequence RNA molecule from its 3' end, regardless of its polyadenylation status, without the need for PCR amplification or ligation of RNA adapters. We demonstrate that Nano3P-seq provides quantitative estimates of RNA abundance and tail lengths, and captures a wide diversity of RNA biotypes. We find that, in addition to mRNA and long non-coding RNA, polyA tails can be identified in 16S mitochondrial ribosomal RNA in both mouse and zebrafish models. Moreover, we show that mRNA tail lengths are dynamically regulated during vertebrate embryogenesis at an isoform-specific level, correlating with mRNA decay. Finally, we demonstrate the ability of Nano3P-seq in capturing non-A bases within polyA tails of various lengths, and reveal their distribution during vertebrate embryogenesis. Overall, Nano3P-seq is a simple and robust method for accurately estimating transcript levels, tail lengths, and tail composition heterogeneity in individual reads, with minimal library preparation biases, both in the coding and non-coding transcriptome.

Long-read single-molecule RNA structure sequencing using nanopore.

RNA molecules can form secondary and tertiary structures that can regulate their localization and function. Using enzymatic or chemical probing together with high-throughput sequencing, secondary structure can be mapped across the entire transcriptome. However, a limiting factor is that only population averages can be obtained since each read is an independent measurement. Although long-read sequencing has recently been used to determine RNA structure, these methods still used aggregate signals across the strands to detect structure. Averaging across the population also means that only limited information about structural heterogeneity across molecules or dependencies within each molecule can be obtained. Here, we present Single-Molecule Structure sequencing (SMS-seq) that combines structural probing with native RNA sequencing to provide non-amplified, structural profiles of individual molecules with novel analysis methods. Our new approach using mutual information enabled single molecule structural interrogation. Each RNA is probed at numerous bases enabling the discovery of dependencies and heterogeneity of structural features. We also show that SMS-seq can capture tertiary interactions, dynamics of riboswitch ligand binding, and mRNA structural features.

Identification of Bull Semen Microbiome by 16S Sequencing and Possible Relationships with Fertility.

Reports on the use of 16S sequencing for the identification of bacteria in healthy animals are lacking. Bacterial contamination of bull semen can have a negative effect on the sperm quality. The aims of this study were threefold: to identify bacteria in the semen of healthy bulls using 16S sequencing; to investigate the differences in the bacterial community between individual bulls; and to establish if there was a relationship between the bacteria isolated and bull fertility. Semen from 18 bulls of known fertility was used for the DNA extraction and 16S sequencing; 107 bacterial genera were identified. The differences in the amplicon sequence variants (ASVs) and the numbers of genera between bulls were noted. Negative correlations (p < 0.05) between several bacterial genera with Curvibacter, Rikenellaceae RC9-gut-group and Dyella spp. were seen. Other negatively correlated bacteria were Cutibacterium, Ruminococcaceae UCG-005, Ruminococcaceae UCG-010 and Staphylococcus, all within the top 20 genera. Two genera, W5053 and Lawsonella, were enriched in bulls of low fertility; this is the first time that these bacteria have been reported in bull semen samples. The majority of the bacteria were environmental organisms or were species originating from the mucous membranes of animals and humans. The results of this study indicate that differences in the seminal microbiota of healthy bulls occur and might be correlated with fertility.

Transcriptome Analysis of Post-Mortem Brain Tissue Reveals Up-Regulation of the Complement Cascade in a Subgroup of Schizophrenia Patients.

Schizophrenia is a genetically complex neuropsychiatric disorder with largely unresolved mechanisms of pathology. Identification of genes and pathways associated with schizophrenia is important for understanding the development, progression and treatment of schizophrenia. In this study, pathways associated with schizophrenia were explored at the level of gene expression. The study included post-mortem brain tissue samples from 68 schizophrenia patients and 44 age and sex-matched control subjects. Whole transcriptome poly-A selected paired-end RNA sequencing was performed on tissue from the prefrontal cortex and orbitofrontal cortex. RNA expression differences were detected between case and control individuals, focusing both on single genes and pathways. The results were validated with RT-qPCR. Significant differential expression between patient and controls groups was found for 71 genes. Gene ontology analysis of differentially expressed genes revealed an up-regulation of multiple genes in immune response among the patients (corrected p-value = 0.004). Several genes in the category belong to the complement system, including C1R, C1S, C7, FCN3, SERPING1, C4A and CFI. The increased complement expression is primarily driven by a subgroup of patients with increased expression of immune/inflammatory response genes, pointing to important differences in disease etiology within the patient group. Weighted gene co-expression network analysis highlighted networks associated with both synaptic transmission and activation of the immune response. Our results demonstrate the importance of immune-related pathways in schizophrenia and provide evidence for elevated expression of the complement cascade as an important pathway in schizophrenia pathology.

Transcript Isoform-Specific Estimation of Poly(A) Tail Length by Nanopore Sequencing of Native RNA.

The poly(A) tail is a homopolymeric stretch of adenosine at the 3'-end of mature RNA transcripts and its length plays an important role in nuclear export, stability, and translational regulation of mRNA. Existing techniques for genome-wide estimation of poly(A) tail length are based on short-read sequencing. These methods are limited because they sequence a synthetic DNA copy of mRNA instead of the native transcripts. Furthermore, they can identify only a short segment of the transcript proximal to the poly(A) tail which makes it difficult to assign the measured poly(A) length uniquely to a single transcript isoform. With the introduction of native RNA sequencing by Oxford Nanopore Technologies, it is now possible to sequence full-length native RNA. A single long read contains both the transcript and the associated poly(A) tail, thereby making transcriptome-wide isoform-specific poly(A) tail length assessment feasible. We developed tailfindr-an R-based package for estimating poly(A) tail length from Oxford Nanopore sequencing data. In this chapter, we describe in detail the pipeline for transcript isoform-specific poly(A) tail profiling based on native RNA Nanopore sequencing-from library preparation to downstream data analysis with tailfindr.

Characterization of the nuclear and cytosolic transcriptomes in human brain tissue reveals new insights into the subcellular distribution of RNA transcripts.

Transcriptome analysis has mainly relied on analyzing RNA sequencing data from whole cells, overlooking the impact of subcellular RNA localization and its influence on our understanding of gene function, and interpretation of gene expression signatures in cells. Here, we separated cytosolic and nuclear RNA from human fetal and adult brain samples and performed a comprehensive analysis of cytosolic and nuclear transcriptomes. There are significant differences in RNA expression for protein-coding and lncRNA genes between cytosol and nucleus. We show that transcripts encoding the nuclear-encoded mitochondrial proteins are significantly enriched in the cytosol compared to the rest of protein-coding genes. Differential expression analysis between fetal and adult frontal cortex show that results obtained from the cytosolic RNA differ from results using nuclear RNA both at the level of transcript types and the number of differentially expressed genes. Our data provide a resource for the subcellular localization of thousands of RNA transcripts in the human brain and highlight differences in using the cytosolic or the nuclear transcriptomes for expression analysis.

Phytotoxicity of different antibiotics to rice and stress alleviation upon application of organic amendments.

Extensive use of antibiotic results in significant antibiotics pollution in the environment. Main objective of this study was to gain insight into potential impacts of antibiotics on plant physiological growth and nutritional composition, and stress alleviation through application of different organic amendments. Effects of five antibiotics (ciprofloxacin, levofloxacin, ofloxacin, amoxicillin and ampicillin) were observed in the presence of three organic amendments (rice husk, farmyard manure and poultry litter) with rice (Oryza sativa L.) as a model plant. Organic amendments were mixed with soil (@ 5 g kg-1) and after three weeks, antibiotics were applied (@10 mg kg-1) and plants were allowed to grow for four months. After which plants were harvested and physical growth parameters (root/shoot length, biomass) and nutritional composition (grain protein content, carbohydrates, phosphorous and iron) were monitored. It was observed that germination rate, seedling root/shoot length, seedling biomass and vigor index were negatively impacted. The application of organic amendments alleviated antibiotic stress on seedling dry biomass, length and vigor index by 1.8-, 3.1- and 2.5-folds, respectively as compared to the antibiotic controls. Concentrations of phosphorous, iron, carbohydrates and proteins were decreased by 5.3-, 1.3-, 1.4- and 1.6-folds upon application of antibiotics. Rice husk was the most effective treatment in case of physical growth parameters and alleviating antibiotics' induced genotoxicity. Whereas, poultry litter had the highest positive effect on nutritional composition of plants. In general, the application of organic amendments alleviated the phytotoxicity as well as genotoxicity in plants under antibiotics stress.

LPS-treatment of bovine endometrial epithelial cells causes differential DNA methylation of genes associated with inflammation and endometrial function.

BACKGROUND: Lipopolysaccharide (LPS) endotoxin stimulates pro-inflammatory pathways and is a key player in the pathological mechanisms involved in the development of endometritis. This study aimed to investigate LPS-induced DNA methylation changes in bovine endometrial epithelial cells (bEECs), which may affect endometrial function. Following in vitro culture, bEECs from three cows were either untreated (0) or exposed to 2 and 8 µg/mL LPS for 24 h. RESULTS: DNA samples extracted at 0 h and 24 h were sequenced using reduced representation bisulfite sequencing (RRBS). When comparing DNA methylation results at 24 h to time 0 h, a larger proportion of hypomethylated regions were identified in the LPS-treated groups, whereas the trend was opposite in controls. When comparing LPS groups to controls at 24 h, a total of 1291 differentially methylated regions (DMRs) were identified (55% hypomethylated and 45% hypermethylated). Integration of DNA methylation data obtained here with our previously published gene expression data obtained from the same samples showed a negative correlation (r = - 0.41 for gene promoter, r = - 0.22 for gene body regions, p < 0.05). Differential methylation analysis revealed that effects of LPS treatment were associated with methylation changes for genes involved in regulation of immune and inflammatory responses, cell adhesion, and external stimuli. Gene ontology and pathway analyses showed that most of the differentially methylated genes (DMGs) were associated with cell proliferation and apoptotic processes; and pathways such as calcium-, oxytocin- and MAPK-signaling pathways with recognized roles in innate immunity. Several DMGs were related to systemic inflammation and tissue re-modelling including HDAC4, IRAK1, AKT1, MAP3K6, Wnt7A and ADAMTS17. CONCLUSIONS: The present results show that LPS altered the DNA methylation patterns of bovine endometrial epithelial cells. This information, combined with our previously reported changes in gene expression related to endometrial function, confirm that LPS activates pro-inflammatory mechanisms leading to perturbed immune balance and cell adhesion processes in the endometrium.

Identification of Transmission Routes of Campylobacter and On-Farm Measures to Reduce Campylobacter in Chicken.

An in-depth analysis was performed on Swedish broiler producers that had delivered chickens with Campylobacter to slaughter over several years, in order to identify possible transmission routes and formulate effective measures to prevent chickens being colonized with Campylobacter. Between 2017 and 2019, 626 samples were collected at farm level and Campylobacter was isolated from 133 (21.2%). All C. jejuni and C. coli isolated from these samples were whole-genome sequenced, together with isolates from the corresponding cecum samples at slaughter (n = 256). Core genome multi-locus sequence typing (cgMLST) analysis, using schemes consisting of 1140 and 529 genes for C. jejuni and C. coli, respectively, revealed that nearby cattle, contaminated drinking water, water ponds, transport crates, and parent flocks were potential reservoirs of Campylobacter. A novel feature compared with previous studies is that measures were implemented and tested during the work. These contributed to a nationwide decrease in Campylobacter-positive flocks from 15.4% in 2016 to 4.6% in 2019, which is the lowest ever rate in Sweden. To conclude, there are different sources and routes of Campylobacter transmission to chickens from different broiler producers, and individual measures must be taken by each producer to prevent Campylobacter colonization of chickens.

The Genome of Setaria digitata: A Cattle Nematode Closely Related to Human Filarial Parasites.

Here we present the draft genome sequence of Setaria digitata, a parasitic nematode affecting cattle. Due to its similarity to Wuchereria bancrofti, the parasitic nematode that causes lymphatic filariasis in humans, S. digitata has been used as a model organism at the genomic level to find drug targets which can be used for the development of novel drugs and/or vaccines for human filariasis. Setaria digitata causes cerebrospinal nematodiasis in goats, sheep, and horses posing a serious threat to livestock in developing countries. The genome sequence of S. digitata will assist in finding candidate genes to use as drug targets in both S. digitata and W. bancrofti. The assembled draft genome is ∼90 Mb long and contains 8,974 genomic scaffolds with a G+C content of 31.73%.

Transcriptome analysis of fibroblasts from schizophrenia patients reveals differential expression of schizophrenia-related genes.

Schizophrenia is a complex neurodevelopmental disorder with high rate of morbidity and mortality. While the heritability rate is high, the precise etiology is still unknown. Although schizophrenia is a central nervous system disorder, studies using peripheral tissues have also been established to search for patient specific biomarkers and to increase understanding of schizophrenia etiology. Among all peripheral tissues, fibroblasts stand out as they are easy to obtain and culture. Furthermore, they keep genetic stability for long period and exhibit molecular similarities to cells from nervous system. Using a unique set of fibroblast samples from a genetically isolated population in northern Sweden, we performed whole transcriptome sequencing to compare differentially expressed genes in seven controls and nine patients. We found differential fibroblast expression between cases and controls for 48 genes, including eight genes previously implicated in schizophrenia or schizophrenia related pathways; HGF, PRRT2, EGR1, EGR3, C11orf87, TLR3, PLEKHH2 and PIK3CD. Weighted gene correlation network analysis identified three differentially co-expressed networks of genes significantly-associated with schizophrenia. All three modules were significantly suppressed in patients compared to control, with one module highly enriched in genes involved in synaptic plasticity, behavior and synaptic transmission. In conclusion, our results support the use of fibroblasts for identification of differentially expressed genes in schizophrenia and highlight dysregulation of synaptic networks as an important mechanism in schizophrenia.

In-silico pharmacophoric and molecular docking-based drug discovery against the Main Protease (Mpro) of SARS-CoV-2, a causative agent COVID-19.

COVID-19 (Coronavirus Disease 2019) caused by a novel 'SARS-CoV-2' virus resulted in public health emergencies across the world. An effective vaccine to cure this virus is not yet available, thus requires concerted efforts at various scales. In this study, we employed Computer-Aided Drug Design (CADD) based approach to identify the drug-like compounds - inhibiting the replication of the main protease (Mpro) of SARS-CoV-2. Our database search using an online tool "ZINC pharmer" retrieved ~1500 compounds based on pharmacophore features. Lipinski's rule was applied to further evaluate the drug-like compounds, followed by molecular docking-based screening, and the selection of screening ligand complex with Mpro based on S-score (higher than reference inhibitor) and root-mean-square deviation (RMSD) value (less than reference inhibitor) using AutoDock 4.2. Resultantly, ~200 compounds were identified having strong interaction with Mpro of SARS-CoV-2. After evaluating their binding energy using the AutoDock 4.2 software, three compounds (ZINC20291569, ZINC90403206, ZINC95480156) were identified that showed highest binding energy with Mpro of SARS-CoV-2 and strong inhibition effect than the N3 (reference inhibitor). A good binding energy, drug likeness and effective pharmacokinetic parameters suggest that these candidates have greater potential to stop the replication of SARS-CoV-2, hence might lead to the cure of COVID-19.

In silico Characterization of a Candidate Protein from Aphid Gelling Saliva with Potential for Aphid Control in Plants.

BACKGROUND: Sheath or gelling saliva, secreted during feeding by aphids, is a hard material that supports the piercing mouthparts and remains in the plant after feeding. Solidification or gelling of the saliva might be due to the composition of amino acids in the constituent proteins, many of which probably interact with plant defenses. OBJECTIVE: The complete complement of proteins in the gelling saliva are still unknown, although one sheath protein (SHP) has previously been identified as a potential candidate protein to control aphid feeding, but its structure and its physiochemical role remains obscure. The current study provides structural information and biochemical properties of the aphid sheath protein. METHODS: The Sheath protein encoding gene was amplified from cDNA of the pea aphid (Acyrthosiphon pisum) through PCR using specific gene primers. Sequence was in silico characterized by using EXPASY, Berkeley Drosophila Genome Project (BDGP) Neural Network Promoter Prediction, BioEdit, Mega7, ProtParam, Phyre server, 3D LigandSite SMART, MEME and GSDS programs, available online. RESULTS: BLASTp analysis revealed that the sequenced gene was identical (100%) to the sequence from Acyrthosiphon pisum, with 87% identity to Metpolophium dirhodum and 84% identity to Sitobion avenae. Phylogenetically monocot feeders such as M. dirhodum and S. avenae are in a sister taxa to dicot feeders. In silico analysis of the sequence revealed that sheath protein has a molecular weight of 144 kDa and 50% of the protein is composed of only six amino acids, i.e., threonine, serine, aspartic acid, glutamic acid, isoleucine and tyrosine. The computed IP value revealed that sheath protein is acidic in nature. Ligand binding sites for sheath protein were predicted on residues 1123 and 1125 (isoleucine and glutamine, respectively). Metallic heterogens are also present in sheath protein that are iron, zinc and magnesium, respectively. CONCLUSION: It is conceivable that variation in the salivary gene sequences may reveal important biological information of relevance to the insect-plant interaction. Further exploration of insect salivary proteins, their composition and structure will provide powerful information, especially when these proteins are interacting with plant proteins, and specific information about the sheath protein, which is interacting with plants at a molecular/cellular level, will be important to progress strategies aimed specifically against sucking pests such as aphids.

Field evaluation ratified by transcript and computational analyses unveils myco-protective role of SUGARWIN proteins in sugarcane.

Nine elite sugarcane genotypes (SPF-234, CPF-246, CPF-247, CPF-248, HSF-240, CP-77-400, S-2006-US-658, S-2003-US-127 and S-2006-US-633) were assessed for field level tolerance against Colletotrichum falcatum followed by quantitative expression and computational analyses of mycoprotective proteins. Plug inoculation method was used to assess level of tolerance of aforementioned genotypes while growing in the field. Genotype S-2006-US-658 was categorized as resistant whereas genotypes CPF-246, CPF-248, HSF-240, S-2003-US-127, S-2006-US-633 and CP-77-400 were categorized as moderately resistant and genotypes SPF-234, CPF-247 as moderately susceptible. Quantitative transcript analyses also revealed that the expression of mycoprotective genes (SUGARWIN1 and SUGARWIN2) was maximum in genotype CPF-246 whereas lowest in genotype SPF-234. Hence these mycoprotective proteins play some critical role in fungal pathogen protection as genotypes with higher expression are more tolerant compared to the genotypes with lower expression of mycoprotective proteins. In-silico interaction of these mycoprotective proteins with chitin, glucan, chitosan and mannan (the core constituents of fungal cell wall) also validated their role in disease susceptibility or resistance. These studies will prove a step forward in understanding mycoprotective proteins and can be employed to develop molecular markers for the selection and screening of red rot resistant sugarcane varieties resulting in enhanced productivity of this valuable cash crop.

Differential gene expression in bovine endometrial epithelial cells after challenge with LPS; specific implications for genes involved in embryo maternal interactions.

Lipopolysaccharide (LPS) expressed on the surface of Gram-negative bacteria activates pro-inflammatory pathways, dys-regulates the function of endometrial cells and is a key player in the mechanisms involved in endometritis. This study aimed to investigate the effects of LPS on bovine endometrial epithelial cells (bEEC) from whole transcriptome with a special focus on genes involved in embryo-maternal interactions. Following in vitro culture, bEEC from three cows were exposed to 0, 2, and 8 µg/mL LPS for 24h. RNA samples extracted at 0 and 24 hours were analyzed by RNA sequencing (RNA-seq). At 24h, 2035 differentially expressed genes (DEGs) were identified between controls and samples treated with 2 µg/mL LPS. Gene ontology analysis showed that over-expressed DEGs were associated to immune response, response to stress and external stimuli, catalytic activity, and cell cycle. Genes associated with cell membrane and cell adhesion pathways were under-expressed. LPS induced changes in expression of specific genes related to embryo-maternal interactions including under-expression of eight members of the cadherin superfamily, over-expression of six members of the mucin family, and differential expression of a large set of genes binding the above molecules and of more than 20 transcripts coding for cytokines and their receptors. Type I interferon-τ dependent genes were also over-expressed. From a sub-set of 19 genes, (biological replicates of bEEC from cows taken at time 6 (n = 3), 24 (n = 6) and 48 hours (n = 3), and 2 technical replicates per sample) differential gene expression was confirmed by RT2-qPCR (r2 between fold changes at 24 hours by RT2-qPCR and RNA-seq = 0.97). These results indicate that LPS affects the function of bEEC in many ways by differential transcription, glycolytic metabolism and oxidative stress. Many transcriptomic signatures related to implantation and embryo maternal interactions were strongly affected by LPS. These results pave the way for further studies to investigate the duration of these changes and their possible impact on endometrial function and fertility.

tailfindr: alignment-free poly(A) length measurement for Oxford Nanopore RNA and DNA sequencing.

Polyadenylation at the 3'-end is a major regulator of messenger RNA and its length is known to affect nuclear export, stability, and translation, among others. Only recently have strategies emerged that allow for genome-wide poly(A) length assessment. These methods identify genes connected to poly(A) tail measurements indirectly by short-read alignment to genetic 3'-ends. Concurrently, Oxford Nanopore Technologies (ONT) established full-length isoform-specific RNA sequencing containing the entire poly(A) tail. However, assessing poly(A) length through base-calling has so far not been possible due to the inability to resolve long homopolymeric stretches in ONT sequencing. Here we present tailfindr, an R package to estimate poly(A) tail length on ONT long-read sequencing data. tailfindr operates on unaligned, base-called data. It measures poly(A) tail length from both native RNA and DNA sequencing, which makes poly(A) tail studies by full-length cDNA approaches possible for the first time. We assess tailfindr's performance across different poly(A) lengths, demonstrating that tailfindr is a versatile tool providing poly(A) tail estimates across a wide range of sequencing conditions.