Background: Assisted Reproductive Technologies (ART) use can increase the risk of congenital overgrowth syndromes, such as large offspring syndrome (LOS) in ruminants. Epigenetic variations are known to influence gene expression and differentially methylated regions (DMRs) were previously determined to be associated with LOS in cattle. We observed DMRs overlapping tRNA clusters which could affect tRNA abundance and be associated with tissue specificity or overgrowth. Variations in tRNA expression have been identified in several disease pathways suggesting an important role in the regulation of biological processes. Understanding the role of tRNA expression in cattle offers an opportunity to reveal mechanisms of regulation at the translational level. We analyzed tRNA expression in the skeletal muscle and liver tissues of day 105 artificial insemination-conceived, ART-conceived with a normal body weight, and ART-conceived bovine fetuses with a body weight above the 97^th percentile compared to Control-AI. Results: Despite the centrality of tRNAs to translation, in silico predictions have revealed dramatic differences in the number of tRNA genes between humans and cattle (597 vs 1,659). Consistent with reports in human, only a fraction of predicted tRNA genes are expressed. We detected the expression of 474 and 487 bovine tRNA genes in the muscle and liver with the remainder being unexpressed. 193 and 198 unique tRNA sequences were expressed in all treatment groups within muscle and liver respectively. In addition, an average of 193 tRNA sequences were expressed within the same treatment group in different tissues. Some tRNA isodecoders were differentially expressed between treatment groups. In the skeletal muscle and liver, we categorized 11 tRNA isoacceptors with undetected expression as well as an isodecoder that was unexpressed in the liver (Ser^GGA). Our results identified variation in the proportion of tRNA gene copies expressed between tissues and differences in the highest contributing tRNA anticodon within an amino acid family due to treatment and tissue type. Out of all amino acid families, roughly half of the most highly expressed tRNA isoacceptors correlated to their most frequent codon in the bovine genome. Conclusion: Although the number of bovine tRNA genes is nearly triple of that of the tRNA genes in human, there is a shared occurrence of transcriptionally inactive tRNA genes in both species. We detected differential expression of tRNA genes as well as tissue- and treatment- specific tRNA transcripts with unique sequence variations that could modulate translation during protein homeostasis or cellular stress, and give rise to regulatory products targeting genes related to overgrowth in the skeletal muscle and/or tumor development in the liver of LOS individuals. While the absence of certain isodecoders may be relieved by wobble base pairing, missing tRNA species could increase the likelihood of mistranslation or mRNA degradation.

Bovine respiratory disease complex (BRDC) is a multifactorial syndrome that involves complex interactions between environment, bacterial/viral pathogens, and the host. Mannheimia haemolytica is the most significant bacterial pathogen associated with BRDC. This study investigated the virulence of a M. haemolytica serotype 1 capsular-deficient (Δ cap ) mutant and a M. haemolytica lipopolysaccharide (LPS)-sialylation-deficient (Δ neuA , cytidine monophosphate-sialic acid synthetase) mutant in a calf lung challenge model. Twelve colostrum-deprived calves were divided into three groups (four calves per group) and intratracheally administered inoculum of M. haemolytica wild-type (WT), M. haemolytica Δ cap , or M. haemolytica Δ neuA strains (~5 × 10 ⁸ CFU per animal). Animals were observed for signs of pneumonia and were humanely euthanized 2 to 3 days post-bacterial challenge. Lungs were examined for gross pulmonary lesions, histopathological changes, and bacterial culture. Calves administered WT M. haemolytica exhibited severe lung lesions characterized by extensive consolidation and hemorrhage. In contrast, calves administered M. haemolytica Δ cap or M. haemolytica Δ neuA mutants displayed significantly reduced lung lesions ( P < 0.05). The most severely affected lung lobes were the right cranial and right middle lobes, with ~50% consolidation. The WT group exhibited significantly higher lung tissue bacterial loads than either of the groups receiving the mutant strains ( P < 0.05). The reduced clinical signs, pneumonic lung lesions, and bacterial recovery in the lungs of the calves challenged with either the Δ cap or the Δ neuA M. haemolytica mutant strains indicated that these mutations were significantly less virulent than the parent strain. IMPORTANCE: While Mannheimia haemolytica leukotoxin is well recognized as a major virulence factor, the roles of other possible virulence factors, such as capsule and sialic acid (sialylation of LPS) in M. haemolytica , have not been investigated in animal models. This study revealed that the abolishment of capsule (Δ cap ) or LPS sialylation (Δ neuA ) significantly reduced the virulence of each mutant in calf lung challenges. These results demonstrate that both M. haemolytica capsule and sialylated LPS are important virulence factors that play a key role in the evasion of host defenses.

Beckwith-Wiedemann Syndrome (BWS) is the most common epigenetic overgrowth syndrome, caused by epigenetic alterations on chromosome 11p15. In ∼50% of patients with BWS, the imprinted region KvDMR1 (IC2) is hypomethylated. Nearly all children with BWS develop organ overgrowth and up to 28% develop cancer during childhood. The global epigenetic alterations beyond the 11p15 region in BWS are not currently known. Uncovering these alterations at the methylome, transcriptome, and chromatin architecture levels are necessary steps to improve the diagnosis and understanding of patients with BWS. Here we characterized the complete epigenetic profiles of BWS IC2 individuals together with the animal model of BWS, bovine large offspring syndrome (LOS). A novel finding of this research is the identification of two molecular subgroups of BWS IC2 individuals. Genome-wide alternations were detected for DNA methylation, transcript abundance, alternative splicing events of RNA, chromosome compartments, and topologically associating domains (TADs) in BWS and LOS, with shared alterations identified between species. Altered chromosome compartments and TADs were correlated with differentially expressed genes in BWS and LOS. Together, we highlight genes and genomic regions that have the potential to serve as targets for biomarker development to improve current molecular diagnostic methodologies for BWS.

Nicrophorusis a genus of beetles that bury and transform small vertebrate carcasses into a brood ball coated with their oral and anal secretions to prevent decay and that will serve as a food source for their young.Nicrophorus pustulatusis an unusual species with the ability to overtake brood of other burying beetles and whose secretions, unlike otherNicrophorusspecies, has been reported not to exhibit antimicrobial properties. This work aims to better understand how the presence or absence of a food source influences the expression of genes involved in the feeding process ofN.pustulatus. To achieve that, total RNA was extracted from pooled samples of salivary gland tissue fromN.pustulatusand sequenced using an Illumina platform. The resulting reads were used to assemble ade novotranscriptome using Trinity. Duplicates with more than 95% similarity were removed to obtain a “unigene” set. Annotation of the unigene set was done using the Trinotate pipeline. Transcript abundance was determined using Kallisto and differential gene expression analysis was performed using edgeR. A total of 651 genes were found to be differentially expressed, including 390 upregulated and 261 downregulated genes in fed insects compared to starved. Several genes upregulated in fed beetles are associated with the insect immune response and detoxification processes with only one transcript encoding for the antimicrobial peptide (AMP) defensin. These results confirm thatN.pustulatusdoes not upregulate the production of genes encoding AMPs during feeding. This study provides a snapshot of the changes in gene expression in the salivary glands ofN.pustulatusfollowing feeding while providing a well described transcriptome for the further analysis of this unique burying beetle.

Small non-coding RNAs, such as microRNAs (miRNA) and tRNA-derived fragments (tRF), are known to be involved in post-transcriptional gene regulation. Research has provided evidence that small RNAs may influence immune development in calves. Bovine leukosis is a disease in cattle caused by Bovine Leukemia Virus (BLV) that leads to increased susceptibility to opportunistic pathogens. No research has addressed the potential influence that a maternal BLV infection may have on gene regulation through the differential expression of miRNAs or tRFs in progeny. Blood samples from 14-day old Holstein calves born to BLV-infected dams were collected. Antibodies for BLV were assessed using ELISA and levels of BLV provirus were assessed using qPCR. Total RNA was extracted from whole blood samples for small RNA sequencing. Five miRNAs (bta-miR-1, bta-miR-206, bta-miR-133a, bta-miR-133b, and bta-miR-2450d) and five tRFs (tRF-36-8JZ8RN58X2NF79E, tRF-20-0PF05B2I, tRF-27-W4R951KHZKK, tRF-22-S3M8309NF, and tRF-26-M87SFR2W9J0) were dysregulated in calves born to BLV-infected dams. The miRNAs appear to be involved in the gene regulation of immunological responses and muscle development. The tRF subtypes and parental tRNA profiles in calves born to infected dams appear to be consistent with previous publications in adult cattle with BLV infection. These findings offer insight into how maternal BLV infection status may impact the development of offspring.

Small noncoding RNAs play critical regulatory roles in development across organisms. This study profiled microRNAs (miRNAs) and tRNA-derived fragments (tRFs) during bovine conceptus elongation. Elongating conceptuses were obtained via superovulation of eight Angus heifers. Twenty samples from ovoid (OV, n = 6; 0.5–3 mm), tubular (TUB, n = 7; 5–15 mm), and filamentous (FIL, n = 7; 20–34 mm) stages underwent small RNA sequencing. Differential expression of miRNAs and tRFs was analyzed using DESeq2, accounting for donor-sire effects. No tRFs showed differential abundance across any pairwise comparisons. For miRNAs, the expressions of six miRNAs were upregulated in OV versus TUB conceptuses (padj < 0.05), including four let-7 family members (bta-let-7g, bta-let-7f, bta-let-7a-5p, and bta-let-7c) and two additional miRNAs (bta-miR-224 and bta-miR-449a). Furthermore, there were 3 miRNAs differently abundant between the ovoid and filamentous transition (padj < 0.04), including two members of the let7 family (bta-let-7g and bta-let-7f) and bta-miR-449a. Predicted targets of these differentially abundant miRNAs were identified using miRanda. Enrichment analyses of the targeted genes included pathways regulating cellular proliferation, pathways in cancer, and immune-related pathways. The let-7 family, along with miR-449a and miR-224, are candidate regulators of the balance between cellular proliferation and differentiation during elongation, based on their differential abundance and in silico target predictions.