• Animal Bioscience
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• v.37 no.3
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• pp.509-521
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• 2024

Objective: It was shown that microRNAs (miRNAs) play an important role in milk protein synthesis. However, the post-transcriptional regulation of casein expression by exogenous miRNA (xeno-miRNAs) in ruminants remains unclear. This study explores the regulatory roles of alfalfa xeno-miR162 on casein synthesis in bovine mammary epithelial cells (bMECs). Methods: The effects of alfalfa xenomiR-162 and G protein subunit gamma 11 (GNG11) on proliferation and milk protein metabolism of bMECs were detected by 5-Ethynyl-2'-Deoxyuridine (EdU) staining, flow cytometry, cell counting kit-8 (CCK-8), enzyme-linked immunosorbent assay, quantitative real-time polymerase chain reaction (qRT-PCR), and Western blot. Dual-luciferase reporter assay was used to verify the targeting relationship between GNG11 and xenomiR-162. Results: Results showed that over-expression of xenomiR-162 inhibited cell proliferation but promoted apoptosis, which also up-regulated the expression of several casein coding genes, including CSN1S1, CSN1S2, and CSN3, while decreasing the expression of CSN2. Furthermore, the targeting relationship between GNG11 and xenomiR-162 was determined, and it was confirmed that GNG11 silencing also inhibited cell proliferation but promoted apoptosis and reduced the expression of casein coding genes and genes related to the mammalian target of rapamycin (mTOR) pathway. Conclusion: Alfalfa xenomiR-162 appears to regulate bMECs proliferation and milk protein synthesis via GNG11 in the mTOR pathway, suggesting that this xeno-miRNA could be harnessed to modulate CSN3 expression in dairy cows, and increase κ-casein contents in milk.

• Molecules and Cells
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• v.39 no.7
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• pp.543-549
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• 2016

MicroRNAs (miRNAs) have been reported to be involved in many neurodegenerative diseases. The present study focused on the role of hsa-miR-144-3p in one of the neuro-degenerative diseases, Parkinson's disease (PD). Our study showed a remarkable down-regulation of miR-144-3p expression in 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP)-treated SH-SY5Y cells. MiR-144-3p was then overexpressed and silenced in human SH-SY5Y cells by miRNA-mimics and miRNA-inhibitor transfections, respectively. Furthermore, ${\beta}$-amyloid precursor protein (APP) was identified as a target gene of miR-144-3p via a luciferase reporter assay. We found that miR-144-3p overexpression significantly inhibited the protein expression of APP. Since mitochondrial dysfunction has been shown to be one of the major pathological events in PD, we also focused on the role of miR-144-3p and APP in regulating mitochondrial functions. Our study demonstrated that up-regulation of miR-144-3p increased expression of the key genes involved in maintaining mitochondrial function, including peroxisome proliferator-activated receptor ${\gamma}$ coactivator-$1{\alpha}$(PGC-$1{\alpha}$), nuclear respiratory factor 1 (NRF-1) and mitochondrial transcription factor A (TFAM). Moreover, there was also a significant increase in cellular ATP, cell viability and the relative copy number of mtDNA in the presence of miR-144-3p overexpression. In contrast, miR-144-3p silencing showed opposite effects. We also found that APP overexpression significantly decreased ATP level, cell viability, the relative copy number of mtDNA and the expression of these three genes, which reversed the effects of miR-144-3p overexpression. Taken together, these results show that miR-144-3p plays an important role in maintaining mitochondrial function, and its target gene APP is also involved in this process.

• The Journal of Korean Medicine
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• v.31 no.4
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• pp.79-100
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• 2010

Objective: Production of ROS from glucose toxicity results in injury of pancreatic $\beta$-cells in diabetes models. This study was undertaken to examine the influence of Lespedeza Cuneata extract (LCE) on cytoprotective effects on glucose toxicity, insulin secretion and gene expression in RIN-m5F cells. Methods: First, we measured LCE's antioxidant activity by DPPH free radical-scavenging activity and SOD activity. After the various concentrations of LCE were added to the RIN-m5F cells, we measured cell viability with glucose stimulation by MTT assay and glucose-stimulated insulin secretion. We analyzed gene expression with Agilent whole mouse genome 44K oligo DNA microarray and searched for related pathways in KEGG (Kyoto Encyclopedia of Genes and Genomes). Lastly we measured INS-1, INS-2, INS-R, IRS-1, IRS-2, IRS-3, GLP-1R, and GLP-2R mRNA expression by real time RT-PCR. Results: Free radical-scavenging activity, SOD activity and insulin secretion increased dependent on LCE concentration, but LCE did not show considerable cytoprotective effect on RIN-m5F cells. More than twice expressed gene was 6362 in Oligo DNA chip. In KEGG, the most related pathway was the metabolic pathway. In the insulin signaling pathway, up expressed genes were Irs1, Mapk8, Akt1, and Lipe and down expressed genes were Rhoq, Fbp2, Prkar2b, Gck, and Prkag1. In real time RT-PCR, IRS-2, and IRS-3 expression increased significantly compared to the control group on LCE $12{\mu}g/m{\ell}$ concentration and GCK expression decreased significantly compared to the control group. Conclusions: These results show that LCE encourages insulin secretion and insulin metabolism by complicated gene mechanisms. Further mechanism study and clinical study seem to be necessary about Lespedeza Cuneata.

• Journal of Microbiology and Biotechnology
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• v.13 no.6
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• pp.942-948
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• 2003

Soil samples were obtained from 5 sites contaminated with polycyclic aromatic hydrocarbons (PAHs). These soil samples were cultured in using phenanthrene as a sole carbon and energy source, and 36 strains of phenanthrene-degrading bacteria were isolated from 3 sites. Most of them degraded 500 ppm of phenanthrene within 8 to 10 days, and these isolates could degrade a few other PAHs other than phenanthrene. Their genotypes were determined by restriction digests of the l6S rRNA genes [amplified ribosomal DNA restriction analysis (ARDRA)]. It was found that all the phenanthrene degrading isolates were included in 4 ARDRA types, and they showed a strict site endemism. l6S rDNAs of 12 strains selected from different sites were sequenced, and they were all confirmed as Sphingomonas strains. Their l6S rDNA sequences were compared for phylogenetic analysis; their sequence showed a similar result to ARDRA typing, thus indicating that these heterotrophic soil bacteria are not regionally mixed. In addition, it was found that the microbial diversity among sampling sites could be monitored by l6S rDNA PCR-RFLP pattern alone, which is simpler and easier to perform, without l6S rDNA sequence analysis.

• Journal of Microbiology and Biotechnology
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• v.24 no.6
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• pp.843-853
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• 2014

In the context of artificial groundwater recharge, a reactive soil column at pilot-scale (4.5 m depth and 3 m in diameter) fed by treated wastewater was designed to evaluate soil filtration ability. Here, as a part of this project, the impact of treated wastewater filtration on soil bacterial communities and the soil's biological ability for wastewater treatment as well as the relevance of the use of multi-bioindicators were studied as a function of depth and time. Biomass; bacterial 16S rRNA gene diversity fingerprints; potential nitrifying, denitrifying, and sulfate-reducing activities; and functional gene (amo, nir, nar, and dsr) detection were analyzed to highlight the real and potential microbial activity and diversity within the soil column. These bioindicators show that topsoil (0 to 20 cm depth) was the more active and the more impacted by treated wastewater filtration. Nitrification was the main activity in the pilot. No sulfate-reducing activity or dsr genes were detected during the first 6 months of wastewater application. Denitrification was also absent, but genes of denitrifying bacteria were detected, suggesting that the denitrifying process may occur rapidly if adequate chemical conditions are favored within the soil column. Results also underline that a dry period (20 days without any wastewater supply) significantly impacted soil bacterial diversity, leading to a decrease of enzyme activities and biomass. Finally, our work shows that treated wastewater filtration leads to a modification of the bacterial genetic and functional structures in topsoil.

• Journal of Life Science
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• v.22 no.1
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• pp.62-73
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• 2012

This study was carried out to investigate the genetic relationship of Flammulina velutipes with other species. The ribosomal DNA cluster containing 4 rRNA genes from F. velutipes 4154 were sequenced. The length of the rDNA cluster sequence was estimated at 7,403 bp long and consisted of 1,806 bp of SSU rDNA, 245 bp of ITS 1 region, 159 bp of 5.8S rDNA, 308 bp of ITS 2 region, 3,402 bp of LSU rDNA, 1,400 bp of IGS 1 region, and 83 bp of 5S rDNA. The F. velutipes 4154 genes were contained in the rDNA cluster of F. velutipes in the order of SSU rDNA - ITS 1 - 5.8S rDNA - ITS 2 - LSU rDNA - IGS 1 - 5S rDNA. The phylogenetic relationships of 20 strains of Tricholomataceae and Physalacriaceae were analyzed by conducting distance analysis using the Neighbor-joining (NJ) method. The 20 strains used in this study were divided into three groups and the strains of the genus Flammulina were related very closely to strains of Physalacria bambusae.

• Journal of Microbiology and Biotechnology
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• v.12 no.5
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• pp.801-806
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• 2002

A multiplex competitive polymerase chain reaction (PCR) method was developed for the rapid identification and quantification of Leuconostoc mesnteroides and Lactobacillus plantarum populations which are the key microorganisms in kimchi fermentation. The strain-specific primers were designed to selectively amplify the target genes encoding 165 rRNA of L. plantarum and dextransucrase of L. mesenteroides. There was a linear relationship between the band intensity of PCR products and the number of colony forming units of each model organism. The PCR quantification method was compared with a traditional plate-counting method f3r the enumeration of the two lactic acid bacteria in a mixed suspension culture and also applied to a real food system, namely, watery kimchi. The population dynamics of the two model organisms in the mixed culture were reliably predictable by the competitive PCR analysis.

• Journal of Microbiology and Biotechnology
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• v.25 no.5
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• pp.709-717
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• 2015

Airborne bacteria from hog farms may have detrimental impacts on human health, particularly in terms of antibiotic resistance and pathogen zoonosis. Despite human health risks, very little is known about the composition and diversity of airborne bacteria from hog farms and hog-related spray fields. We used pyrosequencing analysis of 16S rRNA genes to compare airborne bacterial communities in a North Carolina hog farm and lagoon spray field. In addition, we isolated and identified antibiotic-resistant bacteria from both air samples. Based on 16S rRNA gene pyrosequence analysis, Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria were the dominant phyla in airborne bacterial communities from both hog farm and spray field sites. Within the Firmicutes genera, Clostridium spp. were more abundant in the hog farm, whereas Staphylococcus spp. were higher in the spray field. The presence of opportunitic pathogens, including several Staphylococcus species and Propionibacterium acnes, was detected in both bioaerosol communities based on phylogenetic analysis. The isolation and identification of antibiotic-resistant bacteria from air samples also showed similar results with dominance of Actinobacteria and Proteobacteria in both hog farm and spray field air. Thus, the existence of opportunistic pathogens and antibiotic resistant bacteria in airborne communities evidences potential health risks to farmers and other residents from swine bioaerosol exposure.

• Food Science of Animal Resources
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• v.35 no.3
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• pp.382-388
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• 2015

A duplex real-time polymerase chain reaction (PCR) based assay for the detection of porcine and horse meat in sausages was designed by using EvaGreen fluorescent dye. Primers were selected from mitochondrial 12S rRNA and 16S rRNA genes which are powerful regions for identification of horse and porcine meat. DNA from reference samples and industrial products was successfully extracted using the GIDAGEN® Multi-Fast DNA Isolation Kit. Genomes were identified based on their specific melting peaks (Mp) which are 82.5℃ and 78℃ for horse and porcine, respectively. The assay used in this study allowed the detection of as little as 0.0001% level of horse meat and 0.001% level of porcine meat in the experimental admixtures. These findings indicate that EvaGreen based duplex realtime PCR is a potentially sensitive, reliable, rapid and accurate assay for the detection of meat species adulterated with porcine and horse meats.

• Journal of Microbiology and Biotechnology
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• v.28 no.2
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• pp.227-235
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• 2018

Foodborne illness represents a major threat to public health and is frequently attributed to pathogenic microorganisms on fresh produce. Recurrent outbreaks often come from vegetables that are grown close to or within the ground. Therefore, the first step to understanding the public health risk of microorganisms on fresh vegetables is to identify and describe microbial communities. We investigated the phyllospheres on Chinese cabbage (Brassica rapa subsp. pekinensis, N = 54). 16S rRNA gene amplicon sequencing targeting the V5-V6 region of 16S rRNA genes was conducted by employing the Illumina MiSeq system. Sequence quality was assessed, and phylogenetic assessments were performed using the RDP classifier implemented in QIIME with a bootstrap cutoff of 80%. Principal coordinate analysis was performed using a weighted Fast UniFrac matrix. The average number of sequence reads generated per sample was 34,584. At the phylum level, bacterial communities were composed primarily of Proteobacteria and Bacteroidetes. The most abundant genera on Chinese cabbages were Chryseobacterium, Aurantimonadaceae_g, Sphingomonas, and Pseudomonas. Diverse potential pathogens, such as Pantoea, Erwinia, Klebsiella, Yersinia, Bacillus, Staphylococcus, Salmonella, and Clostridium were also detected from the samples. Although further epidemiological studies will be required to determine whether the detected potential pathogens are associated with foodborne illness, our results imply that a metagenomic approach can be used to detect pathogenic bacteria on fresh vegetables.