A Real-Time Detection Device for the Rapid Quantification of Skin Casual Sebum Using the Oil Red O Staining Method.

The human skin sebum suggests that it (along with other epidermal surface lipids) plays a role in skin barrier formation, the moderation of cutaneous inflammation, and antimicrobial defense. Various methods have been developed for collecting and measuring skin sebum. We tested methods of detection using "color intensity", by staining the skin casual sebum. This process was conducted in three steps; first, the selection of materials for sebum collection; second, staining the collected sebum; third, the development of a device that can measure the level of stained sebum. A plastic film was used to effectively collect sebum that increased with the replacement time of the sebum. In addition, the collected sebum was stained with Oil Red O (ORO) and checked with RGB; as a result, the R2 value was higher than 0.9. It was also confirmed that the correlation value was higher than 0.9 in the comparison result with Sebumeter®, which is a common standard technology. Finally, it was confirmed that the R2 value was higher than 0.9 in the detection value using the sensor. In conclusion, we have proven the proof of concept (PoC) for this method, and we would like to introduce an effective sebum measurement method that differs from the existing method.

Genome-wide analysis of DNA methylation patterns in horse.

BACKGROUND: DNA methylation is an epigenetic regulatory mechanism that plays an essential role in mediating biological processes and determining phenotypic plasticity in organisms. Although the horse reference genome and whole transcriptome data are publically available the global DNA methylation data are yet to be known. RESULTS: We report the first genome-wide DNA methylation characteristics data from skeletal muscle, heart, lung, and cerebrum tissues of thoroughbred (TH) and Jeju (JH) horses, an indigenous Korea breed, respectively by methyl-DNA immunoprecipitation sequencing. The analysis of the DNA methylation patterns indicated that the average methylation density was the lowest in the promoter region, while the density in the coding DNA sequence region was the highest. Among repeat elements, a relatively high density of methylation was observed in long interspersed nuclear elements compared to short interspersed nuclear elements or long terminal repeat elements. We also successfully identified differential methylated regions through a comparative analysis of corresponding tissues from TH and JH, indicating that the gene body regions showed a high methylation density. CONCLUSIONS: We provide report the first DNA methylation landscape and differentially methylated genomic regions (DMRs) of thoroughbred and Jeju horses, providing comprehensive DMRs maps of the DNA methylome. These data are invaluable resource to better understanding of epigenetics in the horse providing information for the further biological function analyses.

Epigenome-wide Association Study for Tic Disorders in Children: A Preliminary Study in Korean Population.

Objective: : Tic disorders can affect the quality of life in both childhood and adolescence. Many factors are involved in the etiology of tic disorders, and the genetic and epigenetic factors of tic disorders are considered complex and heterogeneous. Methods: : In this study, the differentially methylated regions (DMRs) between normal controls (n = 24; aged 6-15; 7 females) and patients with tic disorders (n = 16; aged 6-15; 5 females) were analyzed. We performed an epigenome-wide association study of tic disorders in Korean children. The tics were assessed using Yale Global Tic Severity Scale. The DNA methylation data consisted of 726,945 cytosine phosphate guanine (CpG) sites, assessed using the Illumina Infinium MethylationEPIC (850k) BeadChip. The DNA methylation data of the 40 participants were retrieved, and DMRs between the four groups based on sex and tic disorder were identified. From 28 male and 16 female samples, 37 and 38 DMRs were identified, respectively. We analyzed the enriched terms and visualized the network, heatmap, and upset plot. Results: : In male, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed hypomethylated patterns in the ligand, receptor, and second signal transductors of the PI3K-Akt and MAPK signaling pathway (most cells were indicated as green color), and in female, the opposite patterns were revealed (most cells were indicated as red color). Five mental disorder-related enriched terms were identified in the network analysis. Conclusion: : Here, we provide insights into the epigenetic mechanisms of tic disorders. Abnormal DNA methylation patterns are associated with mental disorder-related symptoms.

Exploring oral bacterial compositional network in two oral disease groups using a convergent approach of NGS-molecular diagnostics.

BACKGROUND: Since most of the commonly known oral diseases are explained in link with balance of microbial community, an accurate bacterial taxonomy profiling for determining bacterial compositional network is essential. However, compared to intestinal microbiome, research data pool related to oral microbiome is small, and general 16S rRNA screening method has a taxonomy misclassification issue in confirming complex bacterial composition at the species level. OBJECTIVE: Present study aimed to explore bacterial compositional networks at the species level within saliva of 39 oral disease patients (Dental Caries group: n = 26 and Periodontitis group: n = 13) through comparison with public Korean-specific healthy oral microbiome data. METHODS: Here, we applied comprehensive molecular diagnostics based on qRT-PCR and Sanger sequencing methods to complement the technical limitations of NGS-based 16S V3-V4 amplicon sequencing technology. RESULTS: As a result of microbiome profiling at the genus level, relative frequencies of many nitrate-reducing bacteria within each oral disease group were found to be significantly low compared to the healthy group. In addition, the molecular diagnostics-based bacterial identification method allowed the determination of the correct taxonomy of screened primary colonizers (Streptococcus and Actinomyces unclassification clusters) for each oral disease. Finally, as with the results of microbiome profiling at the genus level, many core-species classified within the saliva of each oral disease group were also related to nitrate-reduction, and it was estimated that various pathogens associated with each disease formed a bacterial network with the core-species. CONCLUSION: Our study introduced a novel approach that can compensate for the difficulty of identifying an accurate bacterial compositional network at the species level due to unclear taxonomy classification by using the convergent approach of NGS-molecular diagnostics. Ultimately, we suggest that our experimental approach and results could be potential reference materials for researchers who intend to prevent oral disease by determining the correlation between oral health and bacterial compositional network according to the changes in the relative frequency for nitrate-reducing species.

Understanding the bacterial compositional network associations between oral and gut microbiome within healthy Koreans.

Oral microbial ecosystem could influence intestinal diseases, but there have been insufficient studies demonstrating the association of microbial composition between the oral cavity and the intestinal system. Thus, we aimed to investigate the compositional network within the oral microbiome related to gut enterotype from saliva and stool samples collected from 112 healthy Korean subjects. Here, we performed bacterial 16S amplicon sequencing from clinical samples. Then, we determined oral microbiome type related to individual's gut enterotype for healthy Korean. The co-occurrence analysis was performed to interactivity prediction of microbiome within saliva samples. As a result, it could be classified into two Korean oral microbiome types (KO) and four oral-gut-associated microbiome types (KOGA) according to distribution and significant differences of oral microflora. The co-occurrence analysis showed various bacterial compositional networks linked around Streptococcus and Haemophilus within healthy subjects. The present study was first approach in healthy Koreans to identify the oral microbiome types related to the gut microbiome and investigate their characteristics. Hence, we suggest that our results could be potential healthy control data for identifying differences in microbial composition between healthy people and oral disease patients and studying microbial association with the gut microbial environment (oral-gut microbiome axis).

Alterations in gastric and gut microbiota following sleeve gastrectomy in high-fat diet-induced obese rats.

Obesity is considered a high-risk disease and a global epidemic, and the number of obese patients is rising at an alarming rate worldwide. High-fat diet-induced dysbiosis of the intestinal microbiota is considered an essential factor related to obesity. Bariatric surgery induces a sharp decrease in fat content and effectively improves the metabolism of obese individuals. Herein, we aimed to investigate the effects of a high-fat diet-induced obesity and the alterations in gastric and intestinal microbiota resulting from sleeve gastrectomy on clinical outcomes. We performed 16S sequencing of gastric and fecal samples obtained from rats in three treatment groups: normal chow diet, high-fat diet (HFD), and sleeve gastrectomy after HDF for 14 weeks. The area under the curve of fasting glucose and the levels of leptin and low-density lipoproteins were significantly different between groups. Microbial taxa that were highly correlated with several clinical parameters were identified for each group. Glyoxylate and dicarboxylate, taurine and hypotaurine, butanoate, nitrogen, and pyrimidine metabolism and aminoacyl-transfer ribonucleic acid biosynthesis were affected by bariatric surgery and were significantly associated with changes in the composition of gastric and fecal microbiomes. Connectivity and co-occurrence were higher in fecal samples than in gastric tissues. Our results elucidated the positive effects of sleeve gastrectomy in obesity and shed light on changes in the microbiomes of gastric and fecal samples.

Whole transcriptome analyses of six thoroughbred horses before and after exercise using RNA-Seq.

BACKGROUND: Thoroughbred horses are the most expensive domestic animals, and their running ability and knowledge about their muscle-related diseases are important in animal genetics. While the horse reference genome is available, there has been no large-scale functional annotation of the genome using expressed genes derived from transcriptomes. RESULTS: We present a large-scale analysis of whole transcriptome data. We sequenced the whole mRNA from the blood and muscle tissues of six thoroughbred horses before and after exercise. By comparing current genome annotations, we identified 32,361 unigene clusters spanning 51.83 Mb that contained 11,933 (36.87%) annotated genes. More than 60% (20,428) of the unigene clusters did not match any current equine gene model. We also identified 189,973 single nucleotide variations (SNVs) from the sequences aligned against the horse reference genome. Most SNVs (171,558 SNVs; 90.31%) were novel when compared with over 1.1 million equine SNPs from two SNP databases. Using differential expression analysis, we further identified a number of exercise-regulated genes: 62 up-regulated and 80 down-regulated genes in the blood, and 878 up-regulated and 285 down-regulated genes in the muscle. Six of 28 previously-known exercise-related genes were over-expressed in the muscle after exercise. Among the differentially expressed genes, there were 91 transcription factor-encoding genes, which included 56 functionally unknown transcription factor candidates that are probably associated with an early regulatory exercise mechanism. In addition, we found interesting RNA expression patterns where different alternative splicing forms of the same gene showed reversed expressions before and after exercising. CONCLUSION: The first sequencing-based horse transcriptome data, extensive analyses results, deferentially expressed genes before and after exercise, and candidate genes that are related to the exercise are provided in this study.

Molecular characterization of alternative transcripts of the horse BMAL1 gene.

The horse BMAL1 gene encodes the brain and muscle Arnt-like protein 1, which is a key regulator of circadian rhythmic systems in most organs and cells. The first exon of the horse-specific BMAL1 gene is produced by an exonization event of LINE3 (CR1) and SINE (MIR) was detected by bioinformatic analysis. Alternative variants generated by cassette exon event in various horse tissues were also detected by RT-PCR amplification and sequencing. The cDNA sequences of the horse transcripts (BMAL1a, BMAL1b) contain additional 21 bp and 71 bp fragments relative to horse BMAL1. Quantitative real-time RT-PCR was performed to compare the expression patterns between transcript variants in various horse tissues. The results of these experiments showed splice variants that were widely expressed in most tissues. Furthermore, they were highly expressed in cerebellum, heart, and kidney.

Genomic structure and expression analyses of the PYGM gene in the thoroughbred horse.

Muscle glycogen Phosphorylase (PYGM) has been shown to catalyze the degradation of glycogen to glucose-1-phosphate. The PYGM gene can contribute to providing energy to the body by disassembling the glycogen in muscle. Here, we analyzed the genomic structure and expression of the PYGM gene in the thoroughbred horse. The PYGM gene, containing several transposable elements (MIRs, LINEs, and MERs), was highly conserved in mammalian genomes. In order to understand the expression of the horse PYGM gene, we performed quantitative RT-PCR using 11 thoroughbred horse tissue samples. The horse PYGM gene was broadly expressed in all tissues tested. In particular, the highest expression of the horse PYGM gene was observed in skeletal muscle tissue relative to the other tissues. Interestingly, the horse PYGM gene contains fewer mobile elements than its human ortholog, resulting in an increase in the structural stability of the PYGM gene sequence. This study provides insights into the genomic structure of the horse PYGM gene that may be useful in future studies of its association with exercise capability.

Quantitative analysis of alternative transcripts of human PCDH11X/Y genes.

The Protocadherin 11X/Y (PCDH11X/Y) gene pair has been proposed as a carrier of the variation relating to cerebral asymmetry and psychosis on the ground that the Y gene was generated by duplication at 6 million years (close to the chimpanzee-human separation) and there is a case for an X/Y determinant of cerebral asymmetry. The present article investigated the patterns of alternative splicing and expression of the PCDH11X/Y genes. Twelve alternative transcripts of PCDH11X/Y genes were presently identified by in silico analysis. To investigate the biological roles of alternative transcripts of PCDH11X/Y genes, the transcripts were analyzed by real-time reverse transcription-polymerase chain reaction amplification. A total of 31 normal tissues including 11 different regions of human brain were used to investigate a wide spectrum of expression profiles. Dominant expression patterns were identified in several tissues (Tx1-fetal liver; Tx3-adult brain; Tx4-adult brain and kidney; Tx5-bone marrow; Ty1-fetal brain; Ty2-adrenal gland). Tx4 transcripts showed specific expression patterns in olfactory tissues. The findings can guide functional investigation of neuropsychiatric disorders.

Identification and molecular characterization of PERV gamma1 long terminal repeats.

Porcine endogenous retroviruses (PERVs) gamma1 in the pig genome have the potential to act as harmful factors in xenotransplantation (pig-to-human). Long terminal repeats (LTRs) are known to be strong promoter elements that could control the transcription activity of PERV elements and the adjacent functional genes. To investigate the transcribed PERV gamma1 LTR elements in pig tissues, bioin-formatic and experimental approaches were conducted. Using RT-PCR amplification and sequencing approaches, 69 different transcribed LTR elements were identified. And 69 LTR elements could be divided into six groups (15 subgroups) by internal variation including tandem repeated sequences, insertion and deletion (INDEL). Remarkably, all internal variations were indentified in U3 region of LTR elements. Taken together, the identification and characterization of various PERV LTR transcripts allow us to extend our knowledge of PERV and its transcriptional study.

Selection of internal reference genes for SYBR green qRT-PCR studies of rhesus monkey (Macaca mulatta) tissues.

BACKGROUND: The rhesus monkey (Macaca mulatta) is a valuable and widely used model animal for biomedical research. However, quantitative analyses of rhesus gene expression profiles under diverse experimental conditions are limited by a shortage of suitable internal controls for the normalization of mRNA levels. In this study, we used a systematic approach for the selection of potential reference genes in the rhesus monkey and compared their suitability to that of the corresponding genes in humans. RESULTS: Eight housekeeping genes (HKGs) (GAPDH, SDHA, ACTB, RPL13A, RPL32, UBA52, PGK1Y, and YWHAZ) from rhesus monkeys and humans were selected to test for normalization of expression levels in six different tissue types (brain, colon, kidney, liver, lung, and stomach). Their stability and suitability as reference genes were validated by geNorm, NormFinder and BestKeeper programs. Intriguingly, RPL13A and RPL32 were selected as ideal reference genes only in rhesus monkeys. CONCLUSION: The results clearly indicated the necessity of using different reference genes for normalization of expression levels between rhesus monkeys and humans in various tissues.

Cooperative exonization of MaLR and AluJo elements contributed an alternative promoter and novel splice variants of RNF19.

The RNF19 protein, which contains RING-finger and IBR motifs, acts as an E3 ubiquitin ligase localized to Lewy bodies. RNF19 is located on human chromosome 8q22.2, has a 4.4-kb transcript, and is ubiquitously expressed in various tissues. Here, we identified an alternative RNF19 promoter region and alternative RNF19 transcripts derived from MaLR (mammalian apparent LTR-retrotransposon) and AluJo elements. Comparative analyses indicated human-specific expression of the MaLR- and AluJo-related transcripts. From the expression analysis of 72 tissue samples including human normal, tumor, and primate tissues, three different isoforms (V1, V2, and V3) of MaLR-derived transcripts were identified. Quantitative RT-PCR analysis showed a dominant expression pattern of the V2 MaLR-derived transcript. A reporter gene assay for MaLR element promoter activity indicated that pGL2-RNF19/MaLR in the forward orientation is capable of driving luciferase gene expression in Cos7 and HCT116 cells. These findings suggest that RNF19 has acquired a new promoter and alternative exons via continuous retrotransposition events of MaLR and AluJo elements during mammalian and primate evolution, respectively.

Molecular characterization of the HERV-W env gene in humans and primates: expression, FISH, phylogeny, and evolution.

We characterized the human endogenous retrovirus (HERV-W) family in humans and primates. In silico expression data indicated that 22 complete HERV-W families from human chromosomes 1-3, 5-8, 10-12, 15, 19, and X are randomly expressed in various tissues. Quantitative real-time RT-PCR analysis of the HERV-W env gene derived from human chromosome 7q21.2 indicated predominant expression in the human placenta. Several copies of repeat sequences (SINE, LINE, LTR, simple repeat) were detected within the complete or processed pseudo HERV-W of the human, chimpanzee, and rhesus monkey. Compared to other regions (5'LTR, Gag, Gag-Pol, Env, 3'LTR), the repeat family has been mainly integrated into the region spanning the 5'LTRs of Gag (1398 bp) and Pol (3242 bp). FISH detected the HERV-W probe (fosWE1) derived from a gorilla fosmid library in the metaphase chromosomes of all primates (five hominoids, three Old World monkeys, two New World monkeys, and one prosimian), but not in Tupaia. This finding was supported by molecular clock and phylogeny data using the divergence values of the complete HERV-W LTR elements. The data suggested that the HERV-W family was integrated into the primate genome approximately 63 million years (Myr) ago, and evolved independently during the course of primate radiation.

Profiling and identification of pregnancy-associated circulating microRNAs in dairy cattle.

Holstein is one among the dairy cattle which provide higher milk yields than most other cattle breeds. Lack of high-accuracy, reliable methods for early detection of cattle pregnancy reduces overall productivity and constitutes a high economic burden to the dairy industry. The circulating microRNAs (miRNAs) in exosomes could provide information and serve as potential biomarkers for livestock health and disease. However, the complexity of miRNA in response to cattle early pregnancy remains unknown. Hence, we collected blood samples of three healthy dairy cows of normal and 30 days of pregnancy, in order to further characterize the miRNA transcriptome profile. A high-throughput RNA-Seq approach detected 794 known and 2154 novel circulating miRNAs in six libraries. A total of 29 miRNAs in the 30 days of pregnancy group showed significant differences compared to the normal group. Further, bta-miR-450b, bta-miR-146b, bta-miR-26b and bta-miR-27b were up-regulated which shown to be involved in preeclampsia, immune response and mammary gland development. GO enrichment analysis showed these target genes were involved in the metabolic process, signal transducer activity, and membrane etc., while KEGG analysis showed that these genes were enriched in membrane trafficking, chromosome and associated proteins, exosome and G protein-coupled receptors pathways. These results provide an experimental basis to reveal the potential role of miRNAs as biomarkers in early diagnosis of pregnancy and other molecular functions.

Data on transcriptome profiling of circulating microRNAs in dairy cattle.

MicroRNA (miRNA) are found in numerous biofluids including blood and are considered a new class of biomarkers. The data presented here are related to the research article entitled "Profiling and identification of pregnancy-associated circulating microRNAs in dairy cattle" (Markkandan et al. 2018). In the cited article, we sequenced the circulating microRNAs of the three healthy dairy cows of normal and 30 days of pregnancy (DOP) using Illumina RNA-Seq. Differentially expressed genes (DEG) analysis between normal and pregnant samples showed perturbations in miRNA expression. Herein, we made a comparison of DEGs at normal and 60 DOP libraries. The analysis results showed that 147 known miRNAs were differently expressed at 60 DOP groups when compared to the normal group. In addition, stage specific miRNAs were also predicted.

Identification of transposable elements fused in the exonic region of the olive flounder genome.

Transposable elements (TEs) are mobile genetic sequences that comprise a large portion of vertebrate genomes. The olive flounder (Paralichthys olivaceus) is a valuable marine resource in East Asia. The scope of most genomic studies on the olive flounder is limited to its immunology as their focus is the prevention of mass mortality of this species. Thus, for a broader understanding of the species, its genomic information is consistently in demand. Transcripts sequences were acquired from transcriptome analysis using gill tissues of 12 olive flounders. Distribution of TEs inserted in exonic region of the olive flounder genome was analyzed using RepeatMasker ( http://www.repeatmasker.org/ ). We found 1140 TEs in the exonic region of the genome and long interspersed nuclear elements (LINEs) and long terminal repeats (LTRs) insertions occurred with forward orientation preferences. Transposons belonging to the hAt, Gypsy, and LINE 1 (L1) subfamilies were the most abundant DNA transposons, LTRs, and long interspersed elements (LINEs), respectively. Finally, we carried out a gene ontology analysis to determine the function of TE-fused genes. These results provide some genomic information about TEs that is useful for future research on changes in properties and functions of genes by TEs in the olive flounder genome.

Gene expression profiles alteration after infection of virus, bacteria, and parasite in the Olive flounder (Paralichthys olivaceus).

Olive flounder (Paralichthys olivaceus) is one of economically valuable fish species in the East Asia. In comparison with its economic importance, available genomic information of the olive flounder is very limited. The mass mortality caused by variety of pathogens (virus, bacteria and parasites) is main problem in aquaculture industry, including in olive flounder culture. In this study, we carried out transcriptome analysis using the olive flounder gill tissues after infection of three types of pathogens (Virus; Viral hemorrhagic septicemia virus, Bacteria; Streptococcus parauberis, and Parasite; Miamiensis avidus), respectively. As a result, we identified total 12,415 differentially expressed genes (DEG) from viral infection, 1,754 from bacterial infection, and 795 from parasite infection, respectively. To investigate the effects of pathogenic infection on immune response, we analyzed Gene ontology (GO) enrichment analysis with DEGs and sorted immune-related GO terms per three pathogen groups. Especially, we verified various GO terms, and genes in these terms showed down-regulated expression pattern. In addition, we identified 67 common genes (10 up-regulated and 57 down-regulated) present in three pathogen infection groups. Our goals are to provide plenty of genomic knowledge about olive flounder transcripts for further research and report genes, which were changed in their expression after specific pathogen infection.

Gene structure variation in segmental duplication block C of human chromosome 7q 11.23 during primate evolution.

Segmental duplication, or low-copy repeat (LCR) event, occurs during primate evolution and is an important source of genomic diversity, including gain or loss of gene function. The human chromosome 7q 11.23 is related to the William-Beuren syndrome and contains large region-specific LCRs composed of blocks A, B, and C that have different copy numbers in humans and different primates. We analyzed the structure of POM121, NSUN5, FKBP6, and TRIM50 genes in the LCRs of block C. Based on computational analysis, POM121B created by a segmental duplication acquired a new exonic region, whereas NSUN5B (NSUN5C) showed structural variation by integration of HERV-K LTR after duplication from the original NSUN5 gene. The TRIM50 gene originally consists of seven exons, whereas the duplicated TRIM73 and TRIM74 genes present five exons because of homologous recombination-mediated deletion. In addition, independent duplication events of the FKBP6 gene generated two pseudogenes at different genomic locations. In summary, these clustered genes are created by segmental duplication, indicating that they show dynamic evolutionary events, leading to structure variation in the primate genome.