• 생물정신의학
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• 제29권1호
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• pp.9-14
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• 2022

Objectives Sleep is fundamental to maintaining homeostatic control and has behavioral and psychological effects on humans. To better understand the function and pathophysiology of sleep, specific gene expressions in reference to sleep deprivation have been studied. In this study, we investigated the gene expression of peripheral blood mononuclear cells after sleep deprivation to better understand the functional consequence of sleep. Methods In eight healthy men, 24 h sleep deprivation was induced. Blood was sampled at 14:00, before and after sleep deprivation. mRNA was isolated and analyzed via microarrays. cDNAs before and after sleep deprivation were coupled to Cy3 or Cy5, respectively, and normalized cDNAs were selected with a ratio greater than two as a significant gene. Results are expressed as mean. Results Among 41174 transcripts, 38852 genes were selected as reliable, and only a small minority (< 1%) of the genes were up-or down-regulated. Total six and eleven genes were selected as significant upregulated and downregulated genes, respectively. Protein tyrosine phosphatase receptor type O was most upregulated (6.9-fold), and low-density lipoprotein receptor-related protein 5-like protein showed the most substantial inhibition (0.06-fold). Conclusions This study showed significant associations between sleep deprivation and the immune system. Acute sleep deprivation affects pathways in proinflammatory cytokines as well as metabolic pathways of glutamate and purine, neurotransmitters related to sleep and wake cycle.

• Journal of Microbiology and Biotechnology
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• 제19권3호
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• pp.323-330
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• 2009

Nontuberculous mycobacteria (NTM) are a major cause of opportunistic infections in immunocompromised patients, making the reliable and rapid identification of NTM to the species level very important for the treatment of such patients. Therefore, this study evaluated the usefulness of the novel target genes tuf and tmRNA for the identification of NTM to the species level, using a PCRrestriction fragment length polymorphism analysis (PRA). A total of 44 reference strains and 17 clinical isolates of the genus Mycobacterium were used. The 741 bp or 744 bp tuf genes were amplified, restricted with two restriction enzymes (HaeIII/MboI), and sequenced. The tuf gene-PRA patterns were compared with those for the tmRNA (AvaII), hsp65 (HaeIII/HphI), rpoB (MspI/HaeIII), and 16S rRNA (HaeIII) genes. For the reference strains, the tuf gene-PRA yielded 43 HaeIII patterns, of which 35 (81.4%) showed unique patterns on the species level, whereas the tmRNA, hsp65, rpoB, and 16S rRNA-PRAs only showed 10 (23.3%), 32 (74.4%), 19 (44.2%), and 3 (7%) unique patterns after single digestion, respectively. The tuf gene-PRA produced a clear distinction between closely related NTM species, such as M. abscessus (557-84-58) and M. chelonae (477-84-80-58), and M. kansasii (141-136-80-63-58-54-51) and M. gastri (141-136-117-80-58-51). No difference was observed between the tuf-PRA patterns for the reference strains and clinical isolates. Thus, a diagnostic algorithm using a tuf gene-targeting PRA is a promising tool with more advantages than the previously used hsp65, rpoB, and 16S rRNA genes for the identification of NTM to the species level.

• Asian-Australasian Journal of Animal Sciences
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• 제33권12호
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• pp.2021-2030
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• 2020

Objective: Quantitative polymerase chain reaction (qPCR) has been extensively used in the field of mesenchymal stem cell (MSC) research to elucidate their characteristics and clinical potential by normalization of target genes against reference genes (RGs), which are believed to be stably expressed irrespective of various experimental conditions. However, the expression of RGs is also variable depending on the experimental conditions, which may lead to false or contradictory conclusions upon normalization. Due to the current lack of information for a clear list of stable RGs in bovine MSCs, we conducted this study to identify suitable RGs in bovine MSCs. Methods: The cycle threshold values of ten traditionally used RGs (18S ribosomal RNA [18S], beta-2-microglobulin [B2M], H2A histone family, member Z [H2A], peptidylprolyl isomerase A [PPIA], ribosomal protein 4 [RPL4], succinate dehydrogenase complex, subunit A [SDHA], beta actin [ACTB], glyceraldehyde-3-phosphate dehydrogenase [GAPDH], TATA box binding protein [TBP], and hypoxanthine phosphoribosyltrasnfrase1 [HPRT1]) in bovine bone marrow-derived MSCs (bBMMSCs) were validated for their stabilities using three types of RG evaluation algorithms (geNorm, Normfinder, and Bestkeeper). The effect of validated RGs was then verified by normalization of lineage-specific genes (fatty acid binding protein 4 [FABP4] and osteonectin [ON]) expressions during differentiations of bBMMSCs or POU class 5 homeobox 1 (OCT4) expression between bBMMSCs and dermal skins. Results: Based on the results obtained for the three most stable RGs from geNorm (TBP, RPL4, and H2A), Normfinder (TBP, RPL4, and SDHA), and Bestkeeper (TBP, RPL4, and SDHA), it was comprehensively determined that TBP and RPL4 were the most stable RGs in bBMMSCs. However, traditional RGs were suggested to be the least stable (18S) or moderately stable (GAPDH and ACTB) in bBMMSCs. Normalization of FABP4 or ON against TBP, RPL4, and 18S presented significant differences during differentiation of bBMMSCs. However, although significantly low expression of OCT4 was detected in dermal skins compared to that in bBMMSCs when TBP and RPL4 were used in normalization, normalization against 18S exhibited no significance. Conclusion: This study proposes that TBP and RPL4 were suitable as stable RGs for qPCR study in bovine MSCs.

• Asian Pacific Journal of Cancer Prevention
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• 제17권12호
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• pp.5229-5235
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• 2016

Prostate cancer (PCa) remains one of the most widespread and perplexing of all human malignancies. Assessment of gene expression is thought to have an important impact on cancer diagnosis, prognosis and therapeutic decisions. In this context, we explored combined expression of PCa related target genes AMACR and PCA3 in 126 formalin fixed paraffin embedded prostate tissues (FFPE) from Moroccan patients, using quantitative real time reverse transcription-PCR (RT-qPCR). This quantification required data normalization accomplished using stably expressed reference genes (RGs). A panel of twelve RG was assessed, data being analyzed using GenEx V6 based on geNorm, NormFinder and statistical methods. Accordingly, the hnRNP A1 gene was identified and selected as the most stably expressed RG for reliable and accurate gene expression quantification in prostate tissues. The ratios of both PCA3 and AMACR gene expression relative to that of the hnRNP A1 gene were calculated and the performance of each target gene for PCa diagnosis was evaluated using receiver-operating characteristics. PCA3 and AMACR mRNA quantification based on RT-qPCR may prove useful in PCa diagnosis. Of particular interesting, combining PCA3 and AMACR quantification improved PCa prediction by increasing sensitivity with retention of good specificity.

• Animal Bioscience
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• 제35권7호
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• pp.975-988
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• 2022

Objective: In this study, we aimed to identify long non-coding RNAs (lncRNAs) that play important roles in starvation stress, analyze their functions, and discover potential molecular targets to alleviate starvation stress to provide a theoretical reference for subsequent in-depth research. Methods: We generated a piglet starvation stress animal model. Nine Yorkshire weaned piglets were randomly divided into a long-term starvation stress group (starved for 72 h), short-term starvation stress group (starved for 48 h), and the control group. LncRNA libraries were constructed using high-throughput sequencing of piglet ileums. Results: We obtained 11,792 lncRNAs, among which, 2,500 lncRNAs were novel. In total, 509 differentially expressed (DE)lncRNAs were identified in this study. Target genes of DElncRNAs were predicted via cis and trans interactions, and functional and pathway analyses were performed. Gene ontology functions and Kyoto encyclopedia of genes and genomes analysis revealed that lncRNA-targeted genes mainly participated in metabolic pathways, cellular processes, immune system processes, digestive systems, and transport activities. To reveal the mechanism underlying starvation stress, the interaction network between lncRNAs and their targets was constructed based on 26 DElncRNAs and 72 DEmRNAs. We performed an interaction network analysis of 121 DElncRNA-DEmRNA pairs with a Pearson correlation coefficient greater than 0.99. Conclusion: We found that MSTRG.19894.13, MSTRG.16726.3, and MSTRG.12176.1 might play important roles in starvation stress. This study not only generated a library of enriched lncRNAs in piglets, but its outcomes also provide a strong foundation to screen key lncRNAs involved in starvation stress and a reference for subsequent in-depth research.

• The Plant Pathology Journal
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• 제38권4호
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• pp.287-295
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• 2022

Citrus tristeza virus (CTV) is efficiently transmitted in a semi-persistent manner by the brown citrus aphid (Toxoptera citricida (Kirkaldy)). Currently, the most sensitive method for detecting plant viruses in insect vectors is reverse-transcription quantitative polymerase chain reaction (RT-qPCR). In this study, the elongation factor-1 alpha (EF-1α) gene and acidic p0 ribosomal protein (RPAP0) gene were confirmed to be suitable reference genes for RT-qPCR normalization in viruliferous T. citricida aphids using the geNorm, NormFinder, and BestKeeper tools. Then the relative CTV titer in aphids (T. citricida) at different post-acquisition feeding times on healthy plants was quantified by RT-qPCR using EF-1α and RPAP0 as reference genes. The relative CTV titer retained in the aphids gradually decreased with increasing feeding time. During the first 0.5 h of feeding time on healthy plants, the remaining CTV titer in aphids showed about 80% rapid loss for the highly transmissible isolate CT11A and 40% loss for the poorly transmissible isolate CTLJ. The relative CTV titer in aphids during more than 12 h post-acquisition times for CT11A was significantly lower than at the other feeding times, which is similar to the trend found for CTLJ. To our knowledge, this is the first report about the relative titer variation of CTV remaining in T. citricida at different post-acquisition feeding times on healthy plants.

• International Journal of Industrial Entomology and Biomaterials
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• 제17권2호
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• pp.229-234
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• 2008

We used serial analysis of gene expression (SAGE) approach to derive a profile of expressed genes of the posterior silk glands (PSG) and to create a reference for understanding gene cluster related to the mechanism of silk protein synthesis in the silkworm, Bombyx mori. We constructed a 3' SAGE library from the PSG of the fifth instar larvae of the silkworm. In total we obtained 2,406 SAGE tags, of which 682 were unique tags. Sorted by tag count number, 27 (4%) unique tags were significantly more abundant genes (ten or more times), whereas 445 (65%) unique tags were detected as single copies. The annotation of 682 unique SAGE tags revealed that 462 (68%) of the SAGE tag sequences represented known genes, whereas 220 (32%) of the tag sequences had no matches in SAGE map and silkworm EST databases. Of the 682 SAGE tags, the most abundant tag sequences were that of the fibroin light chain gene and the silk protein P25. In addition, we compared two relative abundance results of the SAGE and the EST approaches to verify whether their transcript quantitative aspects are significant or not. The comparative results of relative abundances of the fibroin H-, L- chain and P25 glycoprotein genes indicated that the quantitative approach based on SAGE tags is effective for quantitative cataloging and comparison of expressed genes in same organs. The SAGE tag information reported in this study would be useful for researchers in the field to analyze genes associated with silk processing mechanisms of insects.

• Genomics & Informatics
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• 제9권3호
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• pp.102-113
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• 2011

C-terminal SRC kinase (CSK) is a ubiquitously expressed, cytosolic enzyme that phosphorylates and inactivates several SRC family protein tyrosine kinases. Recent genomewide association studies have implicated CSK in the regulation of blood pressure. The current study aim is to determine the blood pressure association of the genes regulated by CSK down-regulation. The CSK mRNA expression was downregulated in vascular smooth muscle cells using small interfering RNA (siRNA). CSK mRNA levels fell by 90% in cells that were treated with CSK siRNA; the RNA from these cells was examined by microarray using the Illumina HumanRef-8 v3 platform, which comprises 24,526 reference mRNA probes. On treatment with CSK siRNA, 19 genes were downregulated by more than 2-fold and 13 genes were upregulated by more than 2-fold. Three (CANX, SLC30A7, and HMOX1) of them revealed more than 3 fold differential expression. Interestingly, the HMOX1 SNPs were associated with diastolic blood pressure in the 7551 Koreans using Korea Association REsource data, and the result was supported by the other reports that HMOX1 linked to blood vessel maintenance. Among the remaining 29 differentially expressed genes, seven (SSBP1, CDH2, YWHAE, ME2, PFTK1, G3BP2, and TUFT1) revealed association with both systolic and diastolic blood pressures. The CDH2 gene was linked to blood pressures. Conclusively, we identified 32 differentially expressed genes which were regulated by CSK reduction, and two (HOMX1 and CDH2) of them might influence the blood pressure regulation through CSK pathway.

• 한국미생물생명공학회:학술대회논문집
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• 한국미생물생명공학회 2004년도 Annual Meeting BioExibition International Symposium
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• pp.258-265
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• 2004

Numerous genes of Escherichia coli have been shown to growth phase-dependent expression throughout growth. The global patterns of growth phase-dependent gene expression of E. coli throughout growth using oligonucleotide microarrays containing a nearly complete set of 4,289 annotated open reading frames. To determine the change of gene expression throughout growth, we compared RNAs taken from timecourses with common reference RNA, which is combined with equal amount of RNA pooled from each time point. The hierarchical clustering of the conditions in accordance with timecourse expression revealed that growth phases were clustered into four classes, consistent with known physiological growth status. We analyzed the differences of expression levels at genome level in both exponential and stationary growth phase cultures. Statistical analysis showed that 213 genes are shown to, growth phase-dependent expression. We also analyzed the expression of 256 known operons and 208 regulatory genes. To assess the global impact of RpoS, we identified 193 genes coregulated with rpoS and their expression levels were examined in the isogenic rpoS mutant. The results revealed that 99 of 193 were novel RpoS-dependent stationary phase-induced genes and the majority of those are functionally unknown. Our data provide that global changes and adjustments of gene expression are coordinately regulated by growth transition in E. coli.

• 대한바이러스학회지
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• 제28권2호
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• pp.165-174
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• 1998

Characterizations of the VP4 (P type) and VP7 (G type) genes of Korean isolates of bovine rotavirus were performed using RT-PCR/RFLP and nucleotide sequencing analysis. After RT-PCR amplification of partial length (1094bp) of the VP4 and full length (1062bp) of the VP7 genes, amplified PCR products were digested with restriction endonucleases and digestion patterns were compared with those of reference rotaviruses. With the VP4 genes, four RFLP (A-D) profiles were observed; three (A, Band C) were the same as those of bovine rotavirus NCDV (P[1]), IND (P[5]) and B223 (P[11]), respectively. Profile D was the same as that of porcine rotavirus OSU (P[7]). With the VP7 genes, five RFLP profiles (I-V) were observed; three of them (I, II and III) were the same as those of bovine rotavirus NCDV (G6), Cody 1-801 (G8), and B223 (G10), respectively. Profile IV and V were atypical to those of reference bovine rotaviruses used in this study. These two profiles were identified as G6 and G5, respectively, after analyzing and comparing the nucleotide sequences. The G typing analysis revealed that 61.9% (26/42) were G6, which included G6 subtype; 28.6% (12/42) were G5; 7.1% (3/42) were G10; 2.4% (1/42) were G8. The P typing analysis revealed that 54.8% (23/42) were P[5]; 28.6% (12/42) were P[7]; 11.8% (5/42) were [11]; 4.8% (2/42) were P[1]. Our results showed that G6/P[5] were the most prevalent rotaviruses in diarrheic calves in Korea. Also, this is the first report that G5/P[7] rotaviruses were identified from cattle with diarrhea.