Analysis of 16S rRNA gene sequencing data for the taxonomic characterization of the vaginal and the fecal microbial communities in Hanwoo.

OBJECTIVE: The study of Hanwoo (Korean native cattle) has mainly been focused on meat quality and productivity. Recently the field of microbiome research has increased dramatically. However, the information on the microbiome in Hanwoo is still insufficient, especially relationship between vagina and feces. Therefore, the purpose of this study is to examine the microbial community characteristics by analyzing the 16S rRNA sequencing data of Hanwoo vagina and feces, as well as to confirm the difference and correlation between vaginal and fecal microorganisms. As a result, the goal is to investigate if fecal microbiome can be used to predict vaginal microbiome. METHODS: A total of 31 clinically healthy Hanwoo that delivered healthy calves more than once in Cheongju, South Korea were enrolled in this study. During the breeding season, we collected vaginal and fecal samples and sequenced the microbial 16S rRNA genes V3-V4 hypervariable regions from microbial DNA of samples. RESULTS: The results revealed that the phylum-level microorganisms with the largest relative distribution were Firmicutes, Actinobacteria, Bacteroidetes, and Proteobacteria in the vagina, and Firmicutes, Bacteroidetes, and Spirochaetes in the feces, respectively. In the analysis of alpha, beta diversity, and effect size measurements (LefSe), the results showed significant differences between the vaginal and fecal samples. We also identified the function of these differentially abundant microorganisms by functional annotation analyses. But there is no significant correlation between vaginal and fecal microbiome. CONCLUSION: There is a significant difference between vaginal and fecal microbiome, but no significant correlation. Therefore, it is difficult to interrelate vaginal microbiome as fecal microbiome in Hanwoo. In a further study, it will be necessary to identify the genetic relationship of the entire microorganism between vagina and feces through the whole metagenome sequencing analysis and meta-transcriptome analysis to figure out their relationship.

Insight into the Candidate Genes and Enriched Pathways Associated with Height, Length, Length to Height Ratio and Body-Weight of Korean Indigenous Breed, Jindo Dog Using Gene Set Enrichment-Based GWAS Analysis.

As a companion and hunting dog, height, length, length to height ratio (LHR) and body-weight are the vital economic traits for Jindo dog. Human selection and targeted breeding have produced an extraordinary diversity in these traits. Therefore, the identification of causative markers, genes and pathways that help us to understand the genetic basis of this variability is essential for their selection purposes. Here, we performed a genome-wide association study (GWAS) combined with enrichment analysis on 757 dogs using 118,879 SNPs. The genomic heritability (h2) was 0.33 for height and 0.28 for weight trait in Jindo. At p-value < 5 × 10-5, ten, six, thirteen and eleven SNPs on different chromosomes were significantly associated with height, length, LHR and body-weight traits, respectively. Based on our results, HHIP, LCORL and NCAPG for height, IGFI and FGFR3 for length, DLK1 and EFEMP1 for LHR and PTPN2, IGFI and RASAL2 for weight can be the potential candidate genes because of the significant SNPs located in their intronic or upstream regions. The gene-set enrichment analysis highlighted here nine and seven overlapping significant (p < 0.05) gene ontology (GO) terms and pathways among traits. Interestingly, the highlighted pathways were related to hormone synthesis, secretion and signalling were generally involved in the metabolism, growth and development process. Our data provide an insight into the significant genes and pathways if verified further, which will have a significant effect on the breeding of the Jindo dog's population.

Genome-Wide Association Study Identifies 12 Loci Associated with Body Weight at Age 8 Weeks in Korean Native Chickens.

Meat from Korean native chickens (KNCs) has high consumer demand; however, slow growth performance and high variation in body weight (BW) of KNCs remain an issue. Genome-wide association study (GWAS) is a powerful method to identify quantitative trait-associated genomic loci. A GWAS, based on a large-scale KNC population, is needed to identify underlying genetic mechanisms related to its growth traits. To identify BW-associated genomic regions, we performed a GWAS using the chicken 60K single nucleotide polymorphism (SNP) panel for 1328 KNCs. BW was measured at 8 weeks of age, from 2018 to 2020. Twelve SNPs were associated with BW at the suggestive significance level (p < 2.95 × 10-5) and located near or within 11 candidate genes, including WDR37, KCNIP4, SLIT2, PPARGC1A, MYOCD and ADGRA3. Gene set enrichment analysis based on the GWAS results at p < 0.05 (1680 SNPs) showed that 32 Gene Ontology terms and two Kyoto Encyclopedia of Genes and Genomes pathways, including regulation of transcription, motor activity, the mitogen-activated protein kinase signaling pathway, and tight junction, were significantly enriched (p < 0.05) for BW-associated genes. These pathways are involved in cell growth and development, related to BW gain. The identified SNPs are potential biomarkers in KNC breeding.

A Gene-Set Enrichment and Protein-Protein Interaction Network-Based GWAS with Regulatory SNPs Identifies Candidate Genes and Pathways Associated with Carcass Traits in Hanwoo Cattle.

Non-synonymous SNPs and protein coding SNPs within the promoter region of genes (regulatory SNPs) might have a significant effect on carcass traits. Imputed sequence level data of 10,215 Hanwoo bulls, annotated and filtered to include only regulatory SNPs (450,062 SNPs), were used in a genome-wide association study (GWAS) to identify loci associated with backfat thickness (BFT), carcass weight (CWT), eye muscle area (EMA), and marbling score (MS). A total of 15, 176, and 1 SNPs were found to be significantly associated (p < 1.11 × 10-7) with BFT, CWT, and EMA, respectively. The significant loci were BTA4 (CWT), BTA6 (CWT), BTA14 (CWT and EMA), and BTA19 (BFT). BayesR estimated that 1.1%~1.9% of the SNPs contributed to more than 0.01% of the phenotypic variance. So, the GWAS was complemented by a gene-set enrichment (GSEA) and protein-protein interaction network (PPIN) analysis in identifying the pathways affecting carcass traits. At p < 0.005 (~2,261 SNPs), 25 GO and 18 KEGG categories, including calcium signaling, cell proliferation, and folate biosynthesis, were found to be enriched through GSEA. The PPIN analysis showed enrichment for 81 candidate genes involved in various pathways, including the PI3K-AKT, calcium, and FoxO signaling pathways. Our finding provides insight into the effects of regulatory SNPs on carcass traits.

Isolation and characterization of a novel endo-beta-1, 4-glucanase from a metagenomic library of the black-goat rumen

ABSTRACT The various types of lignocellulosic biomass found in plants comprise the most abundant renewable bioresources on Earth. In this study, the ruminal microbial ecosystem of black goats was explored because of their strong ability to digest lignocellulosic forage. A metagenomic fosmid library containing 115,200 clones was prepared from the black-goat rumen and screened for a novel cellulolytic enzyme. The KG35 gene, containing a novel glycosyl hydrolase family 5 cellulase domain, was isolated and functionally characterized. The novel glycosyl hydrolase family 5 cellulase gene is composed of a 963-bp open reading frame encoding a protein of 320 amino acid residues (35.1 kDa). The deduced amino acid sequence showed the highest sequence identity (58%) for sequences from the glycosyl hydrolase family 5 cellulases. The novel glycosyl hydrolase family 5 cellulase gene was overexpressed in Escherichia coli. Substrate specificity analysis revealed that this recombinant glycosyl hydrolase family 5 cellulase functions as an endo-β-1,4-glucanase. The recombinant KG35 endo-β-1,4-glucanase showed optimal activity within the range of 30-50 °C at a pH of 6-7. The thermostability was retained and the pH was stable in the range of 30-50 °C at a pH of 5-7.

Isolation and characterization of a novel endo-ß-1,4-glucanase from a metagenomic library of the black-goat rumen.

The various types of lignocellulosic biomass found in plants comprise the most abundant renewable bioresources on Earth. In this study, the ruminal microbial ecosystem of black goats was explored because of their strong ability to digest lignocellulosic forage. A metagenomic fosmid library containing 115,200 clones was prepared from the black-goat rumen and screened for a novel cellulolytic enzyme. The KG35 gene, containing a novel glycosyl hydrolase family 5 cellulase domain, was isolated and functionally characterized. The novel glycosyl hydrolase family 5 cellulase gene is composed of a 963-bp open reading frame encoding a protein of 320 amino acid residues (35.1kDa). The deduced amino acid sequence showed the highest sequence identity (58%) for sequences from the glycosyl hydrolase family 5 cellulases. The novel glycosyl hydrolase family 5 cellulase gene was overexpressed in Escherichia coli. Substrate specificity analysis revealed that this recombinant glycosyl hydrolase family 5 cellulase functions as an endo-ß-1,4-glucanase. The recombinant KG35 endo-ß-1,4-glucanase showed optimal activity within the range of 30-50°C at a pH of 6-7. The thermostability was retained and the pH was stable in the range of 30-50°C at a pH of 5-7.

Isolation and characterization of a novel glycosyl hydrolase family 74 (GH74) cellulase from the black goat rumen metagenomic library.

This study aimed to isolate and characterize a novel cellulolytic enzyme from black goat rumen by using a culture-independent approach. A metagenomic fosmid library was constructed from black goat rumen contents and screened for a novel cellulase. The KG37 gene encoding a protein of 858 amino acid residues (92.7 kDa) was isolated. The deduced protein contained a glycosyl hydrolase family 74 (GH74) domain and showed 77% sequence identity to two endo-1,4-ß-glucanases from Fibrobacter succinogenes. The novel GH74 cellulase gene was overexpressed in Escherichia coli, and its protein product was functionally characterized. The recombinant GH74 cellulase showed a broad substrate spectrum. The enzyme exhibited its optimum activity at pH 5.0 and temperature range of 20-50 °C. The enzyme was thermally stable at pH 5.0 and at a temperature of 20-40 °C. The novel GH74 cellulase can be practically exploited to convert lignocellulosic biomass to value-added products in various industrial applications in future.

Neuronal genes for subcutaneous fat thickness in human and pig are identified by local genomic sequencing and combined SNP association study.

Obesity represents a major global public health problem that increases the risk for cardiovascular or metabolic disease. The pigs represent an exceptional biomedical model related to energy metabolism and obesity in humans. To pinpoint causal genetic factors for a common form of obesity, we conducted local genomic de novo sequencing, 18.2 Mb, of a porcine QTL region affecting fatness traits, and carried out SNP association studies for backfat thickness and intramuscular fat content in pigs. In order to relate the association studies in pigs to human obesity, we performed a targeted genome wide association study for subcutaneous fat thickness in a cohort population of 8,842 Korean individuals. These combined association studies in human and pig revealed a significant SNP located in a gene family with sequence similarity 73, member A (FAM73A) associated with subscapular skin-fold thickness in humans (rs4121165, GC-corrected p-value  = 0.0000175) and with backfat thickness in pigs (ASGA0029495, p-value  = 0.000031). Our combined association studies also suggest that eight neuronal genes are responsible for subcutaneous fat thickness: NEGR1, SLC44A5, PDE4B, LPHN2, ELTD1, ST6GALNAC3, ST6GALNAC5, and TTLL7. These results provide strong support for a major involvement of the CNS in the genetic predisposition to a common form of obesity.

Proteomic analysis of oxidative stress-induced neuronal cell death by using two-dimensional fluorescence difference gel electrophoresis.

Oxidative stress has been implicated in a number of neurological disorders, including cerebral ischemia and neuro-degenerative diseases. Comprehensive proteomic studies were carried out using an immortalized mouse hippocampal cell line, HT22, exhibiting oxidative stress-mediated cell death upon glutamate treatment. Two-dimensional fluorescence difference gel electrophoresis (2D DIGE) of subcellular organelle fractions revealed that significant numbers of proteins showed quantitative changes during HT22 cell death, among which a total of 51 proteins were identified by mass spectrometry. The identified proteins indicate that HT22 cell death occurs through perturbations in mitochondrial function, changes in translational elongation machinery, and translocation of proteins across subcellular organelles. This list of proteins may shed light on oxidative stress-mediated neuronal cell death.

Proteomic analysis of bovine omental, subcutaneous and intramuscular preadipocytes during in vitro adipogenic differentiation.

Given the substantial rise in obesity, depot-specific fat accumulation and its associated diseases like diabetes, it is important to understand the molecular basis of depot-specific adipocyte differentiation. Many studies have successfully exploited the adipocyte differentiation, but most of them were not related to depot-specificity, particularly using freshly isolated primary preadipocytes. Using 2-dimensional polyacrylamide gel electrophoresis coupled with sequencing mass spectrometry, we searched and compared the proteins differentially expressed in undifferentiated and differentiated preadipocytes from bovine omental, subcutaneous and intramuscular adipose depots. Our proteome mapping strategy to identify differentially expressed intracellular proteins during adipogenic conversion revealed 65 different proteins that were found to be common for the three depots. Further, we validated the differential expression for a subset of proteins by immunoblotting analyses. The results demonstrated that many structural proteins were down-regulated during differentiation of preadipocytes from all the depots. Most up-regulated proteins like Ubiquinol-cytochrome-c reductase complex core protein I (UQCRC1), ATP synthase D chain, Superoxide dismutase (SOD), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Sulfotransferase 1A1 (SULT1A1), Carnitine O-palmitoyltransferase 2 (CPT2) and Heat-shock protein beta 1 (HSPB1) across the three depots were found to be associated with lipid metabolism and metabolic activity. Further, all the up-regulated proteins were found to have higher protein expression in omental than subcutaneous or intramuscular depots.

Trimeric structure of PRL-1 phosphatase reveals an active enzyme conformation and regulation mechanisms.

The PRL phosphatases, which constitute a subfamily of the protein tyrosine phosphatases (PTPs), are implicated in oncogenic and metastatic processes. Here, we report the crystal structure of human PRL-1 determined at 2.7A resolution. The crystal structure reveals the shallow active-site pocket with highly hydrophobic character. A structural comparison with the previously determined NMR structure of PRL-3 exhibits significant differences in the active-site region. In the PRL-1 structure, a sulfate ion is bound to the active-site, providing stabilizing interactions to maintain the canonically found active conformation of PTPs, whereas the NMR structure exhibits an open conformation of the active-site. We also found that PRL-1 forms a trimer in the crystal and the trimer exists in the membrane fraction of cells, suggesting the possible biological regulation of PRL-1 activity by oligomerization. The detailed structural information on the active enzyme conformation and regulation of PRL-1 provides the structural basis for the development of potential inhibitors of PRL enzymes.

Reference map of soluble proteins from Salmonella enterica serovar Enteritidis by two-dimensional electrophoresis.

Protein identification by peptide mass fingerprinting using matrix-assisted laser desorption ionization time of fight (MALDI-TOF) mass spectrometry (MS) can analyze unambiguously identity of the spots from a 2-dimensional electrophoresis (2-DE) gel. This study developed a technique for 2-DE of Salmonella enterica serovar Enteritidis (S. enteritidis) by improving the dissolution conditions by 2-DE using a pH 4 - 7 immobilized pH gradient (IPG) strip. This report examines the protein components from the patterns of the S. enteritidis protein. The most abundant protein displayed a great number of clusters within the pH 4.5 - 7 range with a molecular mass ranging from 35-80 kDa. Some of these spots were identified as metabolic related enzymes. The protein fraction was also analyzed using an immobilized pH gradient strip. Different proteins were identified on the spot according to the elongation factors. In addition, this study showed that the 2-DE analysis of S. enteritidis provides useful information regarding the S. enteritidis proteome, and this approach might provide a strategy for identifying bacterial proteins using a proteome technology.