• Asian-Australasian Journal of Animal Sciences
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• v.28 no.6
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• pp.870-875
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• 2015

The purpose of this study was to investigate the alternative splicing in equine cordon-bleu WH2 repeat protein-like 1 (COBLL1) gene that was identified in horse muscle and blood leukocytes, and to predict functional consequences of alternative splicing by bioinformatics analysis. In a previous study, RNA-seq analysis predicted the presence of alternative spliced isoforms of equine COBLL1, namely COBLL1a as a long form and COBLL1b as a short form. In this study, we validated two isoforms of COBLL1 transcripts in horse tissues by the real-time polymerase chain reaction, and cloned them for Sanger sequencing. The sequencing results showed that the alternative splicing occurs at exon 9. Prediction of protein structure of these isoforms revealed three putative phosphorylation sites at the amino acid sequences encoded in exon 9, which is deleted in COBLL1b. In expression analysis, it was found that COBLL1b was expressed ubiquitously and equivalently in all the analyzed tissues, whereas COBLL1a showed strong expression in kidney, spinal cord and lung, moderate expression in heart and skeletal muscle, and low expression in thyroid and colon. In muscle, both COBLL1a and COBLL1b expression decreased after exercise. It is assumed that the regulation of COBLL1 expression may be important for regulating glucose level or switching of energy source, possibly through an insulin signaling pathway, in muscle after exercise. Further study is warranted to reveal the functional importance of COBLL1 on athletic performance in race horses.

• Proceedings of the Korean Society for Applied Microbiology Conference
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• 2004.06a
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• pp.60-61
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• 2004

Metabolic engineering is now a well established discipline, used extensively to determine and execute rational strategies of strain development to improve the performance of micro-organisms employed in industrial fermentations. The basic principle of this approach is that performance of the microbial catalyst should be adequately characterised metabolically so as to clearlyidentify the metabolic network constraints, thereby identifying the most probable targets for genetic engineering and the extent to which improvements can be realistically achieved. In order to harness correctly this potential, it is clear that the physiological analysis of each strain studied needs to be undertaken under conditions as close as possible to the physico-chemical environment in which the strain evolves within the full-scale process. Furthermore, this analysis needs to be undertaken throughoutthe entire fermentation so as to take into account the changing environment in an essentially dynamic situation in which metabolic stress is accentuated by the microbial activity itself, leading to increasingly important stress response at a metabolic level. All too often these industrial fermentation constraints are overlooked, leading to identification of targets whose validity within the industrial context is at best limited. Thus the conceptual error is linked to experimental design rather than inadequate methodology. New tools are becoming available which open up new possibilities in metabolic engineering and the characterisation of complex metabolic networks. Traditionally metabolic analysis was targeted towards pre-identified genes and their corresponding enzymatic activities within pre-selected metabolic pathways. Those pathways not included at the onset were intrinsically removed from the network giving a fundamentally localised vision of pathway functionality. New tools from genome research extend this reductive approach so as to include the global characteristics of a given biological model which can now be seen as an integrated functional unit rather than a specific sub-group of biochemical reactions, thereby facilitating the resolution of complexnetworks whose exact composition cannot be estimated at the onset. This global overview of whole cell physiology enables new targets to be identified which would classically not have been suspected previously. Of course, as with all powerful analytical tools, post-genomic technology must be used carefully so as to avoid expensive errors. This is not always the case and the data obtained need to be examined carefully to avoid embarking on the study of artefacts due to poor understanding of cell biology. These basic developments and the underlying concepts will be illustrated with examples from the author's laboratory concerning the industrial production of commodity chemicals using a number of industrially important bacteria. The different levels of possibleinvestigation and the extent to which the data can be extrapolated will be highlighted together with the extent to which realistic yield targets can be attained. Genetic engineering strategies and the performance of the resulting strains will be examined within the context of the prevailing experimental conditions encountered in the industrial fermentor. Examples used will include the production of amino acids, vitamins and polysaccharides. In each case metabolic constraints can be identified and the extent to which performance can be enhanced predicted

• Mycobiology
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• v.46 no.3
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• pp.224-235
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• 2018

Temperature is an important environmental factor that can greatly influence the cultivation of Auricularia cornea. In this study, lignin peroxidase, laccase, manganese peroxidase, and cellulose in A. cornea fruiting bodies were tested under five different temperatures ($20^{\circ}C$, $25^{\circ}C$, $30^{\circ}C$, $35^{\circ}C$, and $40^{\circ}C$) in three different culture periods (10 days, 20 days and 30 days). In addition, the V4 region of bacterial 16S rRNA genes in the substrate of A. cornea cultivated for 30 days at different temperatures were sequenced using next-generation sequencing technology to explore the structure and diversity of bacterial communities in the substrate. Temperature and culture days had a significant effect on the activities of the four enzymes, and changes in activity were not synchronized with changes in temperature and culture days. Overall, we obtained 487,694 sequences from 15 samples and assigned them to 16 bacterial phyla. Bacterial community composition and structure in the substrate changed when the temperature was above $35^{\circ}C$. The relative abundances of some bacteria were significantly affected by temperature. A total of 35 genera at five temperatures in the substrate were correlated, and 41 functional pathways were predicted in the study. Bacterial genes associated with the membrane transport pathway had the highest average abundance (16.16%), and this increased at $35^{\circ}C$ and $40^{\circ}C$. Generally, different temperatures had impacts on the physiological activity of A. cornea and the bacterial community in the substrate; therefore, the data presented herein should facilitate cultivation of A. cornea.

• Korean Journal of Poultry Science
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• v.48 no.3
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• pp.111-122
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• 2021

Recently, interleukin 34 (IL-34) was identified as the second functional ligand for macrophage colony-stimulating factor receptor (M-CSFR). IL-34 functions similarly to M-CSF through its binding to the M-CSFR. There is still insufficient information on IL-34 in chickens, which has until now been reported only through predicted sequences and not through experimental research. Thus, to confirm its expression and to determine its potent biological activity, several chicken lines and cell lines were used. Cloning of recombinant chicken IL-34 and M-CSF genes was performed to investigate their modulatory effects on proinflammatory cytokine expression in vitro. The expression levels of IL-34, M-CSF, and M-CSFR genes were upregulated in broiler chickens with leg dysfunction (cause unknown). However, IL-34 was downregulated in most pathogen-stimulated tissues. M-CSFR expression was enhanced by recombinant IL-34 and M-CSF proteins in vitro. IFN-γ expression was enhanced by recombinant IL-34, but not by M-CSF. However, IL-12 expression was not regulated in any of the treated cells, and IL-1β was decreased in all tissues. These results indicate that IL-34 and M-CSF have roles in both the classical and alternative macrophage activation pathways. Collectively, our findings demonstrate the expression of IL-34 in chickens for pathogenic trials, both in vitro and in vivo. Our results suggest that the IL-34 protein plays a role in both pro- and anti-inflammatory functions in macrophages. Therefore, further research is needed to determine the cytokines or chemokines that can be induced by IL-34 and to further elucidate the functions of IL-34 in the inflammatory pathway.

• Animal Bioscience
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• v.36 no.9
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• pp.1336-1349
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• 2023

Objective: The study was conducted to screen differentially expressed miRNAs in sows at early pregnancy by high-throughput sequencing and explore its mechanism of action on embryo implantation. Methods: The blood serum of pregnant and non-pregnant Landrace×Yorkshire sows were collected 14 days after artificial insemination, and exosomal miRNAs were purified for high throughput miRNA sequencing. The expression patterns of 10 differentially expressed (DE) miRNAs were validated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The qRT-PCR quantified the abundance of serum exosomal miR-192 in pregnant and control sows, and the diagnostic power was assessed by receiver operating characteristic (ROC) analysis. The target genes of DE miRNAs were predicted with bioinformatics software, and the functional and pathway enrichment analysis was performed on gene ontology and the Kyoto encyclopedia of genes and genomes terms. Furthermore, a luciferase reporter system was used to identify the target relation between miR-192 and integrin alpha 4 (ITGA4), a gene influencing embryo implantation in pigs. Finally, the expression levels of miRNAs and the target gene ITGA4 were analyzed by qRT-PCR, and western blot, with the proliferation of BeWo cells detected by cell counting kit-8 (CCK-8). Results: A total of 221 known miRNAs were detected in the libraries of the pregnant and non-pregnant sows, of which 55 were up-regulated and 67 were down-regulated in the pregnant individuals compared with the non-pregnant controls. From these, the expression patterns of 10 DE miRNAs were validated. The qRT-PCR analysis further confirmed a significantly higher expression of miR-192 in the serum exosomes extracted from pregnant sows, when compared to controls. The ROC analysis revealed that miR-192 provided excellent diagnostic accuracy for pregnancy (area under the ROC curve [AUC]=0.843; p>0.001). The dual-luciferase reporter assay indicated that miR-192 directly targeted ITGA4. The protein expression of ITGA4 was reduced in cells that overexpressed miR-192. Overexpression of miR-192 resulted in the decreased proliferation of BeWo cells and regulated the expression of cell cycle-related genes. Conclusion: Serum exosomal miR-192 could serve as a potential biomarker for early pregnancy in pigs. miR-192 targeted ITGA4 gene directly, and miR-192 can regulate cellular proliferation.