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Here, we report the determination of acidity constants of eight amino amides through the use of Nuclear Magnetic Resonance (NMR) to carry out the selective crystallization of diprotonated (a), monoprotonated (b), and neutral species (c) derived from these compounds. Crystallographic studies of compounds 2a, 6a, 7a, 8a, 2b, 3b, 6b and 2c revealed that the presence of substituents at the C8 and C10 positions determines the conformation of the molecule regardless of its degree of protonation. Although diprotonated species (a) have the same conformation and similar hydrogen bonding patterns, the supramolecular structures of these compounds are different. Furthermore, chloride ions and solvent molecules play an outstanding role in stabilizing the supramolecular structure of these compounds. This phenomenon restricts the π⋅⋅⋅π interactions between benzimidazole groups and consequently limits the possibility of aggregation in amino amides 1–8. [ABSTRACT FROM AUTHOR]

• Trichlorozincate compounds were selectively synthesized at controlled pH. • Δδ/pH studies revealed the presence of various zn anionic species in solution. • Hydrogen bond interactions contribute to the stabilization of the N→Zn bonds. • Solvent promotes the supramolecular stabilization of trichlorozincates. Herein, we report the synthesis of (2-((2-ammoniopropanamido)methyl)-1 H -benzimidazol- κ N3) trichlorozincate (1-) 1a , (2-(1-(2-ammoniopropanamido)ethyl)-1 H -benzimidazole- κ N3)trichlorozincate (1-) 2a , and (2-((2-ammonio-3-methylbutanamido)methyl)-1 H -benzimidazole- κ N3)trichlorozincate (-1) 3a in a controlled pH environment (pH = 4.0). NMR studies reveal that the N→Zn bond is stronger in 1a and 3a than 2a and that the acid–base equilibria of the amino amides in the presence of Zn(II) result from the formation of various anionic species within the system (Cl−, Zn n Cl m 2−). Crystallography studies show that intramolecular C H•••Cl interactions play a relevant role in the strength of the coordination bond. Furthermore, the cocrystallization of solvent molecules is essential to stabilize the supramolecular structure of 1a and 2a. The infrared spectroscopy corroborated the presence of solvent molecules giving rise to complex hydrogen-bond networks. Moreover, the vibrational spectra indicate that the C10-H10•••Cl3 interactions preferentially occur through the charge transfer phenomena n (Cl3)→σ* C10 H10. Additionally, unlike 2a , the displacement in the scissor vibration of the methylene group between 1a and 3a corroborates that the charge transfer phenomena n (Cl3)→σ*C10 H10 dominate the C10-H10•••Cl3 interaction. [ABSTRACT FROM AUTHOR]

To fine map a quantitative trait locus (QTL) affecting milk production traits previously associated with microsatellite RM188, we implemented an interval mapping analysis by using microsatellite markers in a large Israeli Holstein half-sib sire family, and linkage disequilibrium (LD) mapping in a large set of US Holstein bulls. Interval mapping located the target QTL to the near vicinity of RM188. For the LD mapping, we identified 42 single nucleotide polymorphisms (SNP) in 15 genes in a 12-Mb region on bovine chromosome 4. A total of 24 tag SNP were genotyped in 882 bulls belonging to the University of California Davis archival collection of Holstein bull DNA samples with predicted transmitted ability phenotypes. Marker-to-marker LD analysis revealed 2 LD blocks, with intrablock r² values of 0.10 and 0.46, respectively; outside the blocks, r² values ranged from 0.002 to 0.23. A standard additive/ dominance model using the generalized linear model procedure of SAS and the regression module of HelixTree software were used to test marker-trait associations. Single nucleotide polymorphism 9 on ARL4A, SNP10 on XR_027435.1, SNP12 on ETV1, SNP21 on SNX13, and SNP24 were significantly associated with milk production traits. We propose the interval encompassing ARL4A and SNX13 genes as a candidate region in bovine chromosome 4 for a concordant QTL related to milk protein traits in dairy cattle. Functional studies are needed to confirm this result. [ABSTRACT FROM AUTHOR]

The high growth (HG) mouse mutation is a 460 Kb deletion of chromosome 10 which causes a 30–50% increase in growth in the homozygous animal. We have shotgun sequenced six bacterial artificial chromosomes which span the length of the deletion to an average depth of 13.2× to generate a 649,868 bp sequence. Sequence analysis revealed the presence of three genes, suppressor of cytokine signaling-2 (Socs-2), caspase and RIP adaptor with death domain (Raidd/Cradd), and viral encoded semaphorin receptor (Plexin C1, viral encoded semaphorin receptor). The two deletion breakpoints lie in within the second introns of both Socs-2 and Plexin C1, resulting in the formation of a novel expressed fusion transcript between Socs-2 and Plexin C1 in HG mice. Expression of the fusion transcript, the presence of four splice variants of Raidd/Cradd and the exon structure of Socs-2 were illustrated using polymerase chain reaction. Genomic comparisons of the mouse and human sequence were used to verify the sequence assembly. [Copyright &y& Elsevier]

The annotation of animal genomes plays an important role in elucidating molecular mechanisms behind the genetic control of economically important traits. Here, we employed long‐read sequencing technology, Oxford Nanopore Technology, to annotate the pig transcriptome across 17 tissues from two Yorkshire littermate pigs. More than 9.8 million reads were obtained from a single flow cell, and 69 781 unique transcripts at 50 108 loci were identified. Of these transcripts, 16 255 were found to be novel isoforms, and 22 344 were found at loci that were novel and unannotated in the Ensembl (release 102) and NCBI (release 106) annotations. Novel transcripts were mostly expressed in cerebellum, followed by lung, liver, spleen, and hypothalamus. By comparing the unannotated transcripts to existing databases, there were 21 285 (95.3%) transcripts matched to the NT database (v5) and 13 676 (61.2%) matched to the NR database (v5). Moreover, there were 4324 (19.4%) transcripts matched to the SwissProt database (v5), corresponding to 11 356 proteins. Tissue‐specific gene expression analyses showed that 9749 transcripts were highly tissue‐specific, and cerebellum contained the most tissue‐specific transcripts. As the same samples were used for the annotation of cis‐regulatory elements in the pig genome, the transcriptome annotation generated by this study provides an additional and complementary annotation resource for the Functional Annotation of Animal Genomes effort to comprehensively annotate the pig genome. [ABSTRACT FROM AUTHOR]

The technological properties of milk have significant importance for the dairy industry. Citrate, a normal constituent of milk, forms one of the main buffer systems that regulate the equilibrium between Ca2+ and H+ ions. Higher-than-normal citrate content is associated with poor coagulation properties of milk. To identify the genes responsible for the variation of citrate content in milk in dairy cattle, the metabolic steps involved in citrate and fatty acid synthesis pathways in ruminant mammary tissue using RNA sequencing were studied. Genetic markers that could influence milk citrate content in Holstein cows were used in a marker-trait association study to establish the relationship between 74 single nucleotide polymorphisms (SNP) in 20 candidate genes and citrate content in 250 Holstein cows. This analysis revealed 6 SNP in key metabolic pathway genes [isocitrate dehydrogenase 1 (NADP+), soluble (IDH1); pyruvate dehydrogenase (lipoamide) β (PDHB); pyruvate kinase (PKM2); and solute carrier family 25 (mitochondrial carrier; citrate transporter), member 1 (SLC25A1)] significantly associated with increased milk citrate content. The amount of the phenotypic variation explained by the 6 SNP ranged from 10.1 to 13.7%. Also, genotype-combination analysis revealed the highest phenotypic variation was explained combining IDH1_23211, PDHB_5562, and SLC25A1_4446 genotypes. This specific genotype combination explained 21.3% of the phenotypic variation. The largest citrate associated effect was in the 3' untranslated region of the SLC25A1 gene, which is responsible for the transport of citrate across the mitochondrial inner membrane. This study provides an approach using RNA sequencing, metabolic pathway analysis, and association studies to identify genetic variation in functional target genes determining complex trait phenotypes. [ABSTRACT FROM AUTHOR]

High-throughput sequencing of RNA (RNA-Seq) was developed primarily to analyze global gene expression in different tissues. However, it also is an efficient way to discover coding SNPs. The objective of this study was to perform a SNP discovery analysis in the milk transcriptome using RNA-Seq. Seven milk samples from Holstein cows were analyzed by sequencing cDNAs using the Illumina Genome Analyzer system. We detected 19,175 genes expressed in milk samples corresponding to approximately 70% of the total number of genes analyzed. The SNP detection analysis revealed 100,734 SNPs in Holstein samples, and a large number of those corresponded to differences between the Holstein breed and the Hereford bovine genome assembly Btau4.0. The number of polymorphic SNPs within Holstein cows was 33,045. The accuracy of RNA-Seq SNP discovery was tested by comparing SNPs detected in a set of 42 candidate genes expressed in milk that had been resequenced earlier using Sanger sequencing technology. Seventy of 86 SNPs were detected using both RNA-Seq and Sanger sequencing technologies. The KASPar Genotyping System was used to validate unique SNPs found by RNA-Seq but not observed by Sanger technology. Our results confirm that analyzing the transcriptome using RNA-Seq technology is an efficient and cost-effective method to identify SNPs in transcribed regions. This study creates guidelines to maximize the accuracy of SNP discovery and prevention of false-positive SNP detection, and provides more than 33,000 SNPs located in coding regions of genes expressed during lactation that can be used to develop genotyping platforms to perform marker-trait association studies in Holstein cattle. [ABSTRACT FROM AUTHOR]

Here, we report the synthesis of six organic-inorganic hybrid compounds from amino amides and tetrachlorozincate anions to study the processes underlying the modulation of hydrogen bonds and π∙∙∙π interactions in these salts. Thus, 2-((2-ammoniopropanamido)-methyl)-1 H -benzimidazole-3-ium tetrachlorozincate 7 , 2-(1-(2-ammoniopropanamido)-ethyl)-1 H -benzimidazole-3-ium tetrachlorozincate 8 , 2-((2-ammonio-3-methylbutanamido)methyl)-1 H -benzimidazole-3-ium tetrachlorozincate 9 , 2-(1-(2-ammonio-3-methylbutanamido)-2-methylpropyl)-1 H -benzimidazole-3-ium tetrachlorozincate 10 , 2-((2-ammonio-4-methylpentanamido)methyl)-1 H -benzimidazole-3-ium tetrachlorozincate 11 , and 2-(1-(2-ammonio-4-methylpentanamido)-3-methylbutyl)-1 H -benzimidazoel-3-ium tetrachlorozincate 12 were obtained from the corresponding hydrochlorides (1 – 6). Crystallographic experiments using compounds 7 – 12 revealed that the presence of bulky substituents situated on C10 and C13 reduces the efficacy of stacking phenomena. Moreover, the ZnCl 4 2− ∙∙∙H–N hydrogen bonds induce the polarization of the imidazole ring and the π∙∙∙π interactions occur through the " dipole-induced dipole " mechanism. In the case of N–H∙∙∙solvent interactions, the stacking phenomena occur preferentially through the " induced dipole-induced dipole " mechanism. Image 1 • Tetrachlorozincate anions promote π-stacking interactions in amino amides 7 – 12. • Steric phenomena restrict π∙∙∙π interactions in amino amides 7 – 12. • Hydrogen bond nature has a relevant roll in the presence of π∙∙∙π interactions. • Solvent promote bifurcated hydrogen bonds interactions in amino amides 7 – 12. [ABSTRACT FROM AUTHOR]

Equine maternal recognition of pregnancy (MRP) is a process whose signal remains unknown. During MRP the conceptus and endometrium communicate to attenuate prostaglandin F2α (PGF) secretion, sparing the corpus luteum and maintaining progesterone production. Recognition of a mobile conceptus by the endometrium is critical by days 14–16 post-ovulation (PO), when endometrium produces PGF, initiating luteolysis. The objective of this study was to evaluate endometrial gene expression changes based upon pregnancy status via RNA sequencing. This experiment utilized a cross-over design with each mare serving as both a pregnant and non-mated control on days nine, 11, and 13 PO (n = 3/status/day). Mares were randomly assigned to collection day and pregnancy confirmed by terminal uterine lavage at the time of endometrial biopsy. Total RNA was isolated and libraries prepared using Illumina TruSeq RNA sample preparation kit. Reads were mapped and annotated using HISAT2 and Stringtie. Expression values were evaluated with DESEQ2 (P ≤ 0.05 indicated significance). On day nine, 11, and 13 there were 1435, 1435 and 916 significant transcripts, respectively. Multiple genes with splice variants had different expression patterns within the same day. These are the first data to evaluate the endometrial transcriptome during MRP on days nine, 11, and 13. [ABSTRACT FROM AUTHOR]

Mastitis is a very costly and common disease in the dairy industry. The study of the transcriptome from healthy and mastitic milk somatic cell samples using RNA-Sequencing technology can provide measurements of transcript levels associated with the immune response to the infection. The objective of this study was to characterize the Holstein milk somatic cell transcriptome from 6 cows to determine host response to intramammary infections. RNA-Sequencing was performed on 2 samples from each cow from 2 separate quarters, one classified as healthy (n = 6) and one as mastitic (n = 6). In total, 449 genes were differentially expressed between the healthy and mastitic quarters (false discovery rate <0.05, fold change >±2). Among the differentially expressed genes, the most expressed genes based on reads per kilobase per million mapped reads (RPKM) in the healthy group were associated with milk components (CSN2 and CSN3), and in the mastitic group they were associated with immunity (B2M and CD74). In silico functional analysis was performed using the list of 449 differentially expressed genes, which identified 36 significantly enriched metabolic pathways (false discovery rate <0.01), some of which were associated with the immune system, such as cytokine-cytokine interaction and cell adhesion molecules. Seven functional candidate genes were selected, based on the criteria of being highly differentially expressed between healthy and mastitic groups and significantly enriched in metabolic pathways that are relevant to the inflammatory process (GLYCAM1 , B2M , CD74 , BoLA-DRA , FCER1G , SDS , and NFKBIA). Last, we identified the differentially expressed genes that are located in quantitative trait locus regions previously known to be associated with mastitis, specifically clinical mastitis, somatic cell count, and somatic cell score. It was concluded that multiple genes within quantitative trait locus regions could potentially affect host response to mastitis-causing agents, making some cows more susceptible to intramammary infections. The identification of potential candidate genes with functional, statistical, biological, and positional relevance associated with host defense to infection will contribute to a better understanding of the underlying genetic architecture associated with mastitis. This in turn will improve the sustainability of agricultural practices by facilitating the selection of cows with improved host defense leading to increased resistance to mastitis. [ABSTRACT FROM AUTHOR]

Progenitor cells of the tendon proper and peritenon have unique properties that could impact their utilization in tendon repair strategies. While a few markers have been found to aid in distinguishing progenitors cells from each region, there is great value in identifying more markers. In this study, we hypothesized that RNAseq could be used to improve our understanding of those markers that define these cell types. Transcriptome profiles were generated for pools of mouse Achilles tendon progenitor cells from both regions and catalogues of potential markers were generated. Moreover, common (e.g., glycoprotein, signaling, and proteinaceous extracellular matrix) and unique (e.g., cartilage development versus angiogenesis and muscle contraction) biological processes and molecular functions were described for progenitors from each region. Real‐time quantitative PCR of a subset of genes was used to gain insight into the heterogeneity amongst individual progenitor colonies from each region. Markers like Scx, Mkx, Thbs4, and Wnt10a were consistently able to distinguish tendon proper progenitors from peritenon progenitors; expression variability for other genes suggested greater cell type complexity for potential peritenon progenitor markers. This is the first effort to define Achilles tendon progenitor markers by region. Further efforts to investigate the value of these cataloged markers are required by screening more individual colonies of progenitors for more markers. Clinical Significance: Findings from this study advance efforts in the discernment of cell type specific markers for tendon proper and peritenon progenitor cells; insight into marker sets could improve tracking and sorting strategies for these cells for future therapeutic strategies. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1409–1418, 2019. [ABSTRACT FROM AUTHOR]

Summary: Progesterone signaling and uterine function are crucial in terms of pregnancy establishment. To investigate how the uterine tissue and its secretion changes in relation to puberty, we sampled tissue and uterine fluid from six pre‐ and six post‐pubertal Brahman heifers. Post‐pubertal heifers were sampled in the luteal phase. Gene expression of the uterine tissue was investigated with RNA‐sequencing, whereas the uterine fluid was used for protein profiling with mass spectrometry. A total of 4034 genes were differentially expressed (DE) at a nominal P‐value of 0.05, and 26 genes were significantly DE after Bonferroni correction (P < 3.1 × 10−6). We also identified 79 proteins (out of 230 proteins) that were DE (P < 1 × 10−5) in the uterine fluid. When we compared proteomics and transcriptome results, four DE proteins were identified as being encoded by DE genes: OVGP1, GRP, CAP1 and HBA. Except for CAP1, the other three had lower expression post‐puberty. The function of these four genes hypothetically related to preparation of the uterus for a potential pregnancy is discussed in the context of puberty. All DE genes and proteins were also used in pathway and ontology enrichment analyses to investigate overall function. The DE genes were enriched for terms related to ribosomal activity. Transcription factors that were deemed key regulators of DE genes are also reported. Transcription factors ZNF567,ZNF775,RELA,PIAS2,LHX4,SOX2,MEF2C,ZNF354C,HMG20A,TCF7L2,ZNF420,HIC1,GTF3A and two novel genes had the highest regulatory impact factor scores. These data can help to understand how puberty influences uterine function. [ABSTRACT FROM AUTHOR]

Epizootic bovine abortion (EBA), or "foothill abortion," is the leading cause of beef cattle abortion in California and has also been reported in Nevada and Oregon. In the 1970s, the soft-shelled tick Ornithodoros coriaceus,or "pajaroello tick," was confirmed as the disease-transmitting vector. In 2005, a novel Deltaproteobacterium was discovered as the etiologic agent of EBA (aoEBA), recently named Pajaroellobacter abortibovis. This organism cannot be grown in culture using traditional microbiological techniques; it can only be grown in experimentally-infected severe combined immunodeficient (SCID) mice. The objectives of this study were to perform a de novo genome assembly for P. abortibovis and identify and validate potential antigenic proteins as candidates for future recombinant vaccine development. DNA and RNA were extracted from spleen tissue collected from experimentally-infected SCID mice following exposure to P. abortibovis. This combination of mouse and bacterial DNA was sequenced and aligned to the mouse genome. Mouse sequences were subtracted from the sequence pool and the remaining sequences were de novo assembled at 50x coverage into a 1.82 Mbp complete closed circular Deltaproteobacterial genome containing 2250 putative protein-coding sequences. Phylogenetic analysis of P. abortibovis predicts that this bacterium is most closely related to the organisms of the order Myxococcales, referred to as Myxobacteria. In silico prediction of vaccine candidates was performed using a reverse vaccinology approach resulting in the identification and ranking of the top 10 candidate proteins that are likely to be antigenic. Immunologic testing of these candidate proteins confirmed antigenicity of seven of the nine expressed protein candidates using serum from P. abortibovis immunized mice. [ABSTRACT FROM AUTHOR]

To understand genes, pathways, and networks related to puberty, we characterized the transcriptome of two tissues: the pituitary gland and ovaries. Samples were harvested from pre- and postpubertal Brahman heifers (same age group). Brahman heifers (Bos indicus) are older at puberty compared with Bos taurus, a productivity issue. With RNA sequencing, we identified differentially expressed (DEx) genes and important transcription factors (TF) and predicted coexpression networks. The number of DEx genes detected in the pituitary gland was 284 (P < 0.05), and VWC2L was the most DEx gene (fold change = 4.12, P = 0.01). The gene VWC2L promotes bone mineralization through transforming growth factor-β (TGFβ) signaling. Further studies of the link between bone mineralization and puberty could target VWC2L. In ovaries, 3,871 genes were DEx (P < 0.05). Four highly DEx genes were noteworthy for their function: SLC6A13 (a γ-aminobutyric acid [GABA] transporter), OXT (oxytocin), and NPY (neuropeptide Y) and its receptor NPY2R. These genes had higher ovarian expression in postpubertal heifers. The GABA and its receptors and transporters were expressed in the ovaries of many mammals, suggesting a role for this pathway beyond the brain. The OXT pathway has been known to influence the timing of puberty in rats, via modulation of GnRH. The effects of NPY at the hypothalamus, pituitary gland, and ovaries have been documented. Neuropeptide Y and its receptors are known factors in the release of GnRH, similar to OXT and GABA, although their roles in ovarian tissue are less clear. Pathways previously related to puberty such as TGFβ signaling (P = 6.71 × 10-5), Wnt signaling (P = 4.1 × 10-2), and peroxisome proliferator-activated receptor (PPAR) signaling (P = 4.84 × 10-2) were enriched in our data set. Seven genes were identified as key TF in both tissues: HIC2, ZIC4, ZNF219, ZSCAN26, LHX1, OLIG1, and a novel gene. An ovarian subnetwork created with TF and significant ovarian DEx genes revealed five zinc fingers as regulators: ZNF507, ZNF12, ZNF512, ZNF184, and ZNF432. Recent work of hypothalamic gene expression also pointed to zinc fingers as TF for bovine puberty. Although some zinc fingers may be ubiquitously expressed, the identification of DEx genes in common across tissues points to key regulators of puberty. The hypothalamus and pituitary gland had eight DEx genes in common. The hypothalamus and ovaries had 89 DEx genes in common. The pituitary gland and ovaries had 48 DEx genes in common. Our study confirmed the complexity of puberty and suggested further investigation on genes that code zinc fingers. [ABSTRACT FROM AUTHOR]

Puberty onset is a developmental process influenced by genetic determinants, environment, and nutrition. Mutations and regulatory gene networks constitute the molecular basis for the genetic determinants of puberty onset. The emerging knowledge of these genetic determinants presents opportunities for innovation in the breeding of early pubertal cattle. This paper presents new data on hypothalamic gene expression related to puberty in Bos indicus (Brahman) in age- and weight-matched heifers. Six postpubertal heifers were compared with 6 prepubertal heifers using whole-genome RNA sequencing methodology for quantification of global gene expression in the hypothalamus. Five transcription factors (TF) with potential regulatory roles in the hypothalamus were identified in this experiment: E2F8, NFAT5, SIX5, ZBTB38, and ZNF605. These TF genes were significantly differentially expressed in the hypothalamus of postpubertal versus prepubertal heifers and were also identified as significant according to the applied regulatory impact factor metric (P < 0.05). Two of these 5 TF, ZBTB38 and ZNF605, were zinc fingers, belonging to a gene family previously reported to have a central regulatory role in mammalian puberty. The SIX5 gene belongs to the family of homologues of Drosophila sine oculis (SIX) genes implicated in transcriptional regulation of gonadotrope gene expression. Tumor-related genes such as E2F8 and NFAT5 are known to affect basic cellular processes that are relevant in both cancer and developmental processes. Mutations in NFAT5 were associated with puberty in humans. Mutations in these TF, together with other genetic determinants previously discovered, could be used in genomic selection to predict the genetic merit of cattle (i.e., the likelihood of the offspring presenting earlier than average puberty for Brahman). Knowledge of key mutations involved in genetic traits is an advantage for genomic prediction because it can increase its accuracy. [ABSTRACT FROM AUTHOR]

Calorie restriction (CR) consistently extends longevity and delays age-related diseases across several animal models. We have previously shown that different dietary fat sources can modulate life span and mitochondrial ultrastructure, function and membrane fatty acid composition in mice maintained on a 40% CR. In particular, animals consuming lard as the main fat source (CR-Lard) lived longer than CR mice consuming diets with soybean oil (CR-Soy) or fish oil (CR-Fish) as the predominant lipid source. In the present work, a transcriptomic analysis in the liver and skeletal muscle was performed in order to elucidate possible mechanisms underlying the changes in energy metabolism and longevity induced by dietary fat in CR mice. After 8 months of CR, transcription downstream of several mediators of inflammation was inhibited in liver. In contrast, proinflammatory signaling was increased in the CR-Fish versus other CR groups. Dietary fish oil induced a gene expression pattern consistent with increased transcriptional regulation by several cytokines (TNF, GM-CSF, TGF-β) and sex hormones when compared to the other CR groups. The CR-Fish also had lower expression of genes involved in fatty acid biosynthesis and increased expression of mitochondrial and peroxisomal fatty acid β-oxidation genes than the other CR diet groups. Our data suggest that a diet high in n-3 PUFA, partially reverts CR-related changes in gene expression of key processes, such as inflammation and steroid hormone signaling, and this may mitigate life span extension with CR in mice consuming diets high in fish oil. [ABSTRACT FROM AUTHOR]

A variety of proteases release hundreds of endogenous peptide fragments from intact bovine milk proteins. Mass spectrometry-based peptidomics allows for high throughput sequence assignment of a large number of these peptides. Mastitis is known to result in increased protease activity in the mammary gland. Therefore, we hypothesized that subclinically mastitic milks would contain higher concentrations of released peptides. In this work, milks were sampled from three cows and, for each, one healthy and one subclinically mastitic teat were sampled for milk. Peptides were analyzed by nano-liquid chromatography quadrupole time of flight tandem mass spectrometry and identified with database searching. In total, 682 peptides were identified. The total number of released peptides increased 146% from healthy to subclinically mastitic milks ( p < 0.05), and the total abundance of released peptides also increased significantly ( p < 0.05). Bioinformatic analysis of enzyme cleavage revealed increases in activity of cathepsin D and elastase ( p < 0.05) with subclinical mastitis. [ABSTRACT FROM AUTHOR]

Background Mouse chromosome 2 is linked to growth and body fat phenotypes in many mouse crosses. With the goal to identify the underlying genes regulating growth and body fat on mouse chromosome 2, we developed five overlapping subcongenic strains that contained CAST/EiJ donor regions in a C57BL/6Jhg/hg background (hg is a spontaneous deletion of 500 Kb on mouse chromosome 10). To fine map QTL on distal mouse chromosome 2 a total of 1,712 F2 mice from the five subcongenic strains, plus 278 F2 mice from the HG2D founder congenic strain were phenotyped and analyzed. Interval mapping (IM) and composite IM (CIM) were performed on body weight and body fat traits on a combination of SNP and microsatellite markers, which generated a high-density genotyping panel. Results Phenotypic analysis and interval mapping of total fat mass identified two QTL on distal mouse chromosome 2. One QTL between 150 and 161 Mb, Fatq2a, and the second between 173.3 and 175.6 Mb, Fatq2b. The two QTL reside in different congenic strains with significant total fat differences between homozygous cast/cast and b6/b6 littermates. Both of these QTL were previously identified only as a single QTL affecting body fat, Fatq2. Furthermore, through a novel approach referred here as replicated CIM, Fatq2b was mapped to the Gnas imprinted locus. Conclusions The integration of subcongenic strains, high-density genotyping, and CIM succesfully partitioned two previously linked QTL 20 Mb apart, and the strongest QTL, Fatq2b, was fine mapped to a ∼2.3 Mb region interval encompassing the Gnas imprinted locus. [ABSTRACT FROM AUTHOR]

The objective of this study was to examine five different sources of RNA, namely mammary gland tissue (MGT), milk somatic cells (SC), laser microdissected mammary epithelial cells (LCMEC), milk fat globules (MFG) and antibody-captured milk mammary epithelial cells (mMEC) to analyze the bovine mammary gland transcriptome using RNA-Sequencing. Our results provide a comparison between different sampling methods (invasive and non-invasive) to define the transcriptome of mammary gland tissue and milk cells. This information will be of value to investigators in choosing the most appropriate sampling method for different research applications to study specific physiological states during lactation. One of the simplest procedures to study the transcriptome associated with milk appears to be the isolation of total RNA directly from SC or MFG released into milk during lactation. Our results indicate that the SC and MFG transcriptome are representative of MGT and LCMEC and can be used as effective and alternative samples to study mammary gland expression without the need to perform a tissue biopsy. [ABSTRACT FROM AUTHOR]

Puberty is a complex physiological event by which animals mature into an adult capable of sexual reproduction. In order to enhance our understanding of the genes and regulatory pathways and networks involved in puberty, we characterized the transcriptome of five reproductive tissues (i.e. hypothalamus, pituitary gland, ovary, uterus, and endometrium) as well as tissues known to be relevant to growth and metabolism needed to achieve puberty (i.e., longissimus dorsi muscle, adipose, and liver). These tissues were collected from pre- and post-pubertal Brangus heifers (3/8 Brahman; Bos indicus x 5/8 Angus; Bos taurus) derived from a population of cattle used to identify quantitative trait loci associated with fertility traits (i.e., age of first observed corpus luteum (ACL), first service conception (FSC), and heifer pregnancy (HPG)). In order to exploit the power of complementary omics analyses, pre- and post-puberty co-expression gene networks were constructed by combining the results from genome-wide association studies (GWAS), RNA-Seq, and bovine transcription factors. Eight tissues among pre-pubertal and post-pubertal Brangus heifers revealed 1,515 differentially expressed and 943 tissue-specific genes within the 17,832 genes confirmed by RNA-Seq analysis. The hypothalamus experienced the most notable up-regulation of genes via puberty (i.e., 204 out of 275 genes). Combining the results of GWAS and RNA-Seq, we identified 25 loci containing a single nucleotide polymorphism (SNP) associated with ACL, FSC, and (or) HPG. Seventeen of these SNP were within a gene and 13 of the genes were expressed in uterus or endometrium. Multi-tissue omics analyses revealed 2,450 co-expressed genes relative to puberty. The pre-pubertal network had 372,861 connections whereas the post-pubertal network had 328,357 connections. A sub-network from this process revealed key transcriptional regulators (i.e., PITX2, FOXA1, DACH2, PROP1, SIX6, etc.). Results from these multi-tissue omics analyses improve understanding of the number of genes and their complex interactions for puberty in cattle. [ABSTRACT FROM AUTHOR]

We assessed the relationships of polar bears (Ursus maritimus), brown bears (U. arctos), and black bears (U. americanus) with high throughput genomic sequencing data with an average coverage of 25× for each species. A total of 1.4 billion 100-bp paired-end reads were assembled using the polar bear and annotated giant panda (Ailuropoda melanoleuca) genome sequences as references. We identified 13.8 million single nucleotide polymorphisms (SNP) in the 3 species aligned to the polar bear genome. These data indicate that polar bears and brown bears share more SNP with each other than either does with black bears. Concatenation and coalescence-based analysis of consensus sequences of approximately 1 million base pairs of ultraconserved elements in the nuclear genome resulted in a phylogeny with black bears as the sister group to brown and polar bears, and all brown bears are in a separate clade from polar bears. Genotypes for 162 SNP loci of 336 bears from Alaska and Montana showed that the species are genetically differentiated and there is geographic population structure of brown and black bears but not polar bears. [ABSTRACT FROM AUTHOR]

Abstract: The genomic architecture and expression of the Igf-1 gene are complex yielding multiple IGF-I transcript isoforms with putative functional contributions to growth and metabolism. Using RNA-seq on different tissues, physiological states, and species, the breadth of transcripts expressed was determined. Tissues from pre- and post-pubertal heifers and mature mice were collected and the transcript isoforms characterized. Three different IGF-I isoforms were detected in heifers with Class 1 transcripts most abundantly expressed. The pituitary reduced IGF-I expression post-pubertally whereas the uterus increased expression. Murine IGF-I transcript expression was more diverse utilizing multiple exons, start sites, and 3′UTRs. The RNA-seq methodology to characterize expression profiles permits assessment of the transcript isoforms yielding insight into functional roles of each transcript. [Copyright &y& Elsevier]

Background Studies of normal human mammary gland development and function have mostly relied on cell culture, limited surgical specimens, and rodent models. Although RNA extracted from human milk has been used to assay the mammary transcriptome non-invasively, this assay has not been adequately validated in primates. Thus, the objectives of the current study were to assess the suitability of lactating rhesus macaques as a model for lactating humans and to determine whether RNA extracted from milk fractions is representative of RNA extracted from mammary tissue for the purpose of studying the transcriptome of milk-producing cells. Results We confirmed that macaque milk contains cytoplasmic crescents and that ample high-quality RNA can be obtained for sequencing. Using RNA sequencing, RNA extracted from macaque milk fat and milk cell fractions more accurately represented RNA from mammary epithelial cells (cells that produce milk) than did RNA from whole mammary tissue. Mammary epithelium-specific transcripts were more abundant in macaque milk fat, whereas adipose or stroma-specific transcripts were more abundant in mammary tissue. Functional analyses confirmed the validity of milk as a source of RNA from milk-producing mammary epithelial cells. Conclusions RNA extracted from the milk fat during lactation accurately portrayed the RNA profile of milk-producing mammary epithelial cells in a non-human primate. However, this sample type clearly requires protocols that minimize RNA degradation. Overall, we validated the use of RNA extracted from human and macaque milk and provided evidence to support the use of lactating macaques as a model for human lactation. [ABSTRACT FROM AUTHOR]

Measures of heifer fertility are economi-cally relevant traits for beef production systems and knowledge of candidate genes could be incorporated into future genomic selection strategies. Ten traits related to growth and fertility were measured in 890 Brangus heif-ers (3/8 Brahman x 5/8 Angus, from 67 sires). These traits were: BW and hip height adjusted to 205 and 365 d of age, postweaning ADG, yearling assessment of carcass traits (i.e., back fat thickness, intramuscular fat, and LM area), as well as heifer pregnancy and first service con-ception (FSC). These fertility traits were collected from controlled breeding seasons initiated with estrous syn-chronization and Al targeting heifers to calve by 24 mo of age. The BovineSNP50 BeadChip was used to ascertain 53,692 SNP genotypes for -802 heifers. Associations of genotypes and phenotypes were performed and SNP effects were estimated for each trait. Minimally associ-ated SNP (P < 0.05) and their effects across the 10 traits formed the basis for an association weight matrix and its derived gene network related to FSC (57.3% success and heritability = 0.06 ± 0.05). These analyses yielded 1,555 important SNP, which inferred genes linked by 113,873 correlations within a network. Specifically, 1,386 SNP were nodes and the 5,132 strongest correlations (|r| > 0.90) were edges. The network was filtered with genes queried from a transcriptome resource created from deep sequencing of RNA (i.e., RNA-Seq) from the hypothala-mus of a prepubertal and a postpubertal Brangus heifer. The remaining hypothalamic-influenced network con-tained 978 genes connected by 2,560 edges or predicted gene interactions. This hypothalamic gene network was enriched with genes involved in axon guidance, which is a pathway known to influence pulsatile release of LHRH. There were 5 transcription factors with 21 or more connections: ZMAT3, STAT6, RFX4, PLAGL1, and NR6A1 for FSC. The SNP that identified these genes were intragenic and were on chromosomes 1, 5, 9, and 11. Chromosome 5 harbored both STAT6 and RFX4. The large number of interactions and genes observed with network analyses of multiple sources of genomic data (i.e., GWAS and RNA-Seq) support the concept of FSC being a polygenic trait. [ABSTRACT FROM AUTHOR]

The objective was to identify single nucleotide polymorphisms (SNP) associated to fertility in cows raised under a subtropical environment. Re-sequencing of nine genes associated to GH-IGF endocrine pathway, which are located in bovine chromosomes 5, 16 and 20, identified 73 SNP useful for associative genetic studies; however, only seven resulted as polymorphic and unique to the Romosinuano breed. Then, DNA samples were extracted from 129 beef heifers and used to determine genotypes corresponding to each SNP. Mixed model analysis identified one SNP from the PAPP-A2 gene (C/T, rs110490898) as predictor (P<0.05) of reproductive performance. The allele T was the most favorable allele (P<0.05), because it was associated with lower age at first calving (-37.1 ± 14.4 d) and age at second calving (-65.43 ± 30.8 d). In the contrast analysis, the linear term resulted as significant and quadratic term as non significant, which suggested an additive effect of the alleles. These results provide evidence to support the PAPP-A2 gene as a candidate gene associated to reproductive performance in heifers and first-calf cows from the Romosinuano breed, raised under a subtropical environment. [ABSTRACT FROM AUTHOR]

El objetivo fue identificar polimorfismos de un solo nucleótido (SNP) asociados a la fertilidad en hembras bovinas criadas en subtropico. La re-secuenciación de nueve genes relacionados al eje endocrino GH-IGF, localizados en los cromosomas 5, 16 y 20 del bovino, identificó 73 SNP útiles para estudios genéticos asociativos; sin embargo, sólo siete resultaron polimórficos y exclusivos de la raza Romosinuano. Muestras de ADN se extrajeron de 129 vaquillas Romosinuano y usadas para determinar los genotipos correspondientes a cada SNP. Un análisis de modelos mixtos identificó únicamente a un polimorfismo del gen PAPP-A2 (C/T, rs110490898) como predictor (P<0.05) del comportamiento reproductivo. El alelo T fue el más favorable (P<0.05) ya que éste se asoció a una reducción tanto en la edad al primer parto (-37.1 ± 14.4 días), como en la edad al segundo parto (-65.43 ± 30.8 días). En el análisis de contrastes el término lineal resultó significativo (P<0.05), pero no el quadrático, lo cual sugiere un efecto aditivo de los alelos. Los resultados proporcionan evidencia para proponer al gen PAPP-A2, como candidato asociado al comportamiento reproductivo en vaquillas y vacas primerizas de la raza Romosinuano. [ABSTRACT FROM AUTHOR]

The growth endocrine axis influences reproduction. The objectives of this study were to evaluate population genetic characteristics of SNP genotypes within genes of the GH-IGF axis in straightbred and crossbred Angus, Brahman, and Romosinuano heifers (n = 650) and to test the association of these genotypes with measures of reproduction. These objectives were achieved using 73 SNP within 7 genes on chromosome 5 and the pregnancy-associated plasma protein A2 (PAPP-A2) and GH-receptor genes, which map to chromosomes 16 and 20, respectively. The SNP were elucidated by resequencing conserved regions of each gene by using DNA from familial-unrelated cattle of a multibreed discovery population. A multiplex SNP assay yielded 59 biallelic SNP useful for evaluating genetic identity and distance. Specifically, the divergence of straightbred Brahman cattle was approximately 15.5% from 5 Bos taurus-influenced breed groups. In the straightbred groups used as a validation population, only 3 SNP had minor allele frequencies >10%. These SNP were in the genes PAPP-A2 (ss115492449-A/C and ss115492450-G/T within intron 10) and signal transducers and activators of transcription 2 (STAT2; ss252841035-A/G within the 5′ untranslated region), and they met the conditions of Hardy-Weinberg equilibrium (P > 0.31). The other 56 SNP were useful for assigning each animal into ancestral clusters (n = 3 proportions) to account for population stratification in genotype to phenotype association analyses. The 2 SNP in the PA PP-A 2 gene influenced (P < 0.05) traits indicative of first-calf heifer rebreeding (i.e., calving interval, days to calving, and pregnancy rate). A STAT2 SNP genotype (i.e., GG) × primary ancestral cluster interaction (P < 0.05) suggested heifers primarily of B. taurus ancestry had a reduction of approximately 16.4 + 0.1% in calving interval and days to calving relative to heifers clustering primarily as Bus indicus ancestry. Even though additional research is needed to delineate the allelic variation attributed to genes of the GH-IGF axis, results of this study provide support for STAT2 and PA PP-A2 as potential candidate genes associated with first-calf heifer rebreeding traits. [ABSTRACT FROM AUTHOR]

This study examines the genes coding for enzymes involved in bovine milk oligosaccharide metabolism by comparing the oligosaccharide profiles with the expressions of glycosylation-related genes. Fresh milk samples (n = 32) were collected from four Holstein and Jersey cows at days 1, 15, 90 and 250 of lactation and free milk oligosaccharide profiles were analyzed. RNA was extracted from milk somatic cells at days 15 and 250 of lactation (n = 12) and gene expression analysis was conducted by RNA-Sequencing. A list was created of 121 glycosylation-related genes involved in oligosaccharide metabolism pathways in bovine by analyzing the oligosaccharide profiles and performing an extensive literature search. No significant differences were observed in either oligosaccharide profiles or expressions of glycosylation-related genes between Holstein and Jersey cows. The highest concentrations of free oligosaccharides were observed in the colostrum samples and a sharp decrease was observed in the concentration of free oligosaccharides on day 15, followed by progressive decrease on days 90 and 250. Ninety-two glycosylation-related genes were expressed in milk somatic cells. Most of these genes exhibited higher expression in day 250 samples indicating increases in net glycosylation-related metabolism in spite of decreases in free milk oligosaccharides in late lactation milk. Even though fucosylated free oligosaccharides were not identified, gene expression indicated the likely presence of fucosylated oligosaccharides in bovine milk. Fucosidase genes were expressed in milk and a possible explanation for not detecting fucosylated free oligosaccharides is the degradation of large fucosylated free oligosaccharides by the fucosidases. Detailed characterization of enzymes encoded by the 92 glycosylation-related genes identified in this study will provide the basic knowledge for metabolic network analysis of oligosaccharides in mammalian milk. These candidate genes will guide the design of a targeted breeding strategy to optimize the content of beneficial oligosaccharides in bovine milk. [ABSTRACT FROM AUTHOR]

Background: The identification of novel genes is critical to understanding the molecular basis of body weight. Towards this goal, we have identified secretogranin V (Scg5; also referred to as Sgne1), as a candidate gene for growth traits. Results: Through a combination of DNA microarray analysis and quantitative PCR we identified a strong expression quantitative trait locus (eQTL) regulating Scg5 expression in two mouse chromosome 2 congenic strains and three additional F2 intercrosses. More importantly, the eQTL was coincident with a body weight QTL in congenic mice and Scg5 expression was negatively correlated with body weight in two of the F2 intercrosses. Analysis of haplotype blocks and genomic sequencing of Scg5 in high (C3H/HeJ, DBA/2J, BALB/cByJ, CAST/EiJ) and low (C57BL/6J) expressing strains revealed mutations unique to C57BL/6J and possibly responsible for the difference in mRNA abundance. To evaluate the functional consequence of Scg5 overexpression we measured the pituitary levels of 7B2 protein and PCSK2 activity and found both to be increased. In spite of this increase, the level of pituitary α-MSH, a PCSK2 processing product, was unaltered. Conclusion: Together, these data support a role for Scg5 in the modulation of body weight. [ABSTRACT FROM AUTHOR]

Maternal recognition of pregnancy (MRP) in the mare is not well defined. In a non-pregnant mare, prostaglandin F2α (PGF) is released on day 14 post-ovulation (PO) to cause luteal regression, resulting in loss of progesterone production. Equine MRP occurs prior to day 14 to halt PGF production. Studies have failed to identify a gene candidate for MRP, so attention has turned to small, non-coding RNAs. The objective of this study was to evaluate small RNA (<200 nucleotides) content in endometrium during MRP. Mares were used in a cross-over design with each having a pregnant and non-mated cycle. Each mare was randomly assigned to collection day 11 or 13 PO (n = 3/day) and endometrial biopsies were obtained. Total RNA was isolated and sequencing libraries were prepared using a small RNA library preparation kit and sequenced on a HiSeq 2000. EquCab3 was used as the reference genome and DESeq2 was used for statistical analysis. On day 11, 419 ncRNAs, representing miRNA, snRNA, snoRNA, scaRNA, and vaultRNA, were different between pregnancy statuses, but none on day 13. Equine endometrial ncRNAs with unknown structure and function were also identified. This study is the first to describe ncRNA transcriptome in equine endometrium. Identifying targets of these ncRNAs could lead to determining MRP. [ABSTRACT FROM AUTHOR]