Identification of genes differentially expressed in myogenin knock-down bovine muscle satellite cells during differentiation through RNA sequencing analysis.

BACKGROUND: The expression of myogenic regulatory factors (MRFs) consisting of MyoD, Myf5, myogenin (MyoG) and MRF4 characterizes various phases of skeletal muscle development including myoblast proliferation, cell-cycle exit, cell fusion and the maturation of myotubes to form myofibers. Although it is well known that the function of MyoG cannot be compensated for other MRFs, the molecular mechanism by which MyoG controls muscle cell differentiation is still unclear. Therefore, in this study, RNA-Seq technology was applied to profile changes in gene expression in response to MyoG knock-down (MyoGkd) in primary bovine muscle satellite cells (MSCs). RESULTS: About 61-64% of the reads of over 42 million total reads were mapped to more than 13,000 genes in the reference bovine genome. RNA-Seq analysis identified 8,469 unique genes that were differentially expressed in MyoGkd. Among these genes, 230 were up-regulated and 224 were down-regulated by at least four-fold. DAVID Functional Annotation Cluster (FAC) and pathway analysis of all up- and down-regulated genes identified overrepresentation for cell cycle and division, DNA replication, mitosis, organelle lumen, nucleoplasm and cytosol, phosphate metabolic process, phosphoprotein phosphatase activity, cytoskeleton and cell morphogenesis, signifying the functional implication of these processes and pathways during skeletal muscle development. The RNA-Seq data was validated by real time RT-PCR analysis for eight out of ten genes as well as five marker genes investigated. CONCLUSIONS: This study is the first RNA-Seq based gene expression analysis of MyoGkd undertaken in primary bovine MSCs. Computational analysis of the differentially expressed genes has identified the significance of genes such as SAP30-like (SAP30L), Protein lyl-1 (LYL1), various matrix metalloproteinases, and several glycogenes in myogenesis. The results of the present study widen our knowledge of the molecular basis of skeletal muscle development and reveal the vital regulatory role of MyoG in retaining muscle cell differentiation.

Antibacterial properties of a pre-formulated recombinant phage endolysin, SAL-1.

To evaluate the phage endolysin SAL-1 as a therapeutic agent for Staphylococcus aureus infections, the in vitro and in vivo antibacterial properties of a pre-formulation containing recombinant SAL-1 as an active pharmaceutical ingredient were investigated. The stable pre-formulation (designated SAL200) uniquely included calcium ions and Poloxamer 188 as enhancing and stabilising ingredients, respectively. SAL-1 was successfully produced with no extraneous amino acids by decreasing the culture temperature and was highly purified using a two-step chromatography procedure consisting of ion exchange and hydrophobic interaction chromatography. SAL200 exhibited rapid and effective bactericidal activity against encapsulated and biofilm-forming S. aureus as well as against planktonic S. aureus cells. In addition, SAL200 demonstrated increased effectiveness in the serum environment, with a significantly reduced minimum bactericidal concentration compared with that determined in culture medium. In in vitro antibacterial tests performed against 425 clinical isolates [including 336 meticillin-resistant S. aureus (MRSA) isolates and 1 vancomycin-intermediate S. aureus isolate], collected from 421 patients and four animals, SAL200 exhibited obvious antibacterial activity against all S. aureus isolates tested. Intravenous injection of SAL200 in a mouse model of MRSA infection prolonged the viability of mice and significantly reduced bacterial counts in the bloodstream and splenic tissue. The results presented in this article strongly support SAL200 as a highly potent bactericidal agent against MRSA with an adequate pharmaceutical formulation.