The pmrA gene, a yeast PMR1 homologue, was isolated from Aspergillus niger. Sequence analysis of the pmrA cDNA and the genomic DNA revealed that two introns exist in the coding region, and that an open reading frame in the cDNA encodes a polypeptide of 1056 amino acids containing all the conserved regions present in P-type Ca2+-ATPases. The predicted pmrA protein exhibited a high degree of sequence similarity to the Pmr1 proteins from yeasts and mammalians (50-59% identity). The expression of the pmrA cDNA partially restored the growth defect of Yarrowia lipolytica pmr1 null mutant on EGTA-containing medium. This indicates that the A. niger pmrA gene encodes a functional homologue of the yeast P-type Ca2+-ATPase involved in the secretory pathway. An A. niger pmrA null mutant exhibited growth retardation on EGTA-containing medium and the growth defect was overcome by adding Ca2+ or Mn2+ into the medium. This suggests an involvement of the pmrA protein in Ca2+ and Mn2+ homeostasis in A. niger.
A gene homologous to Saccharomyces cerevisiae MNN9 has been cloned and characterized in the methylotrophic yeast Hansenula polymorpha. This gene was cloned from a H. polymorpha genomic DNA library using the S. cerevisiae MNN9 gene as a probe. The H. polymorpha MNN9 homologue (HpMNN9) contained a 1062 bp open reading frame encoding a predicted protein of 354 amino acids. The deduced amino acid sequence showed 58% and 51% identity, respectively, with the S. cerevisiae and Candida albicans Mnn9 proteins. Disruption of HpMNN9 leads to phenotypic effects suggestive of cell wall defects, including detergent sensitivity and hygromycin B sensitivity. The hygromycin B sensitivity of S. cerevisiae mnn9 null mutant was complemented in the presence of the HpMNN9 gene. The DNA sequence of the H. polymorpha homologue has been submitted to GenBank with the Accession No. AF264786.
Transposon-associated transposase B (TnpB) is deemed an ancestral protein for type V, Cas12 family members, and the closest ancestor to UnCas12f1 due to its high sequence similarity. Previously, we reported a set of engineered guide RNAs supporting high indel efficiency for Cas12f1 in human cells. Here, we suggest a new technology whereby the engineered gRNAs also manifest highly efficiency programmable endonuclease activity for TnpB, termed TaRGET (TnpB-augment RNA-based Genome Editing Technology). Having this feature in mind, we established TnpB-based adenine base editors. A codon-optimized Tad-Tad mutant (V106W, D108Q) dimer fused to the C-terminus of dTnpB (D354A) showed the highest levels of A-to-G conversion. The limited targetable sites for TaRGET-ABE were expanded by either developing several PAM variants of TnpB or by engineering TnpB and optimizing deaminases at PAM-distal and PAM-proximal sites, respectively. When delivered by AAV, the TaRGET-ABE showed potent A-to-G conversion rates in human cells. Collectively, the TaRGET-ABE will contribute to improving precise genome-editing tools that can be delivered by AAV, thereby harnessing the development of CRISPR-based gene therapy.
Purpose: Currently, aging-related sarcopenia is considered a serious problem. However, few studies have addressed the treatment or prevention of sarcopenia using <i>Gryllus bimaculatus</i> . Therefore the present study was carried out to determine whether <i>Gryllus bimaculatus</i> has an ameliorative effect on sarcopenia and to study its applicability as a food for sarcopenia in the elderly or in patients with this condition.Methods: The contents of moisture, crude ash, crude protein, crude fat, carbohydrate and minerals were analyzed. The amino acid and fatty acid compositions were analyzed using an automatic amino acid analyzer and gas chromatography, respectively. Cell viability was measured using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Myostatin expression was investigated in C2C12 cells using a luciferase reporter assay after treatment with a <i>Gryllus bimaculatus</i> extract.Results: The <i>Gryllus bimaculatus</i> powder (GBP), protein extract of <i>Gryllus bimaculatus</i> (PEGB), <i>Gryllus bimaculatus</i> egg (GBE), and egg protein extract of <i>Gryllus bimaculatus</i> (EPGB) were high in essential amino acids, especially branched chain amino acids, and unsaturated fatty acids, and low in carbohydrates. Moreover, <i>Gryllus bimaculatus</i> appeared to have ameliorative effects on sarcopenia by suppressing the mRNA expression of myostatin in C2C12 cells.Conclusion: It is expected that <i>Gryllus bimaculatus</i> will be positive for the development of food materials or to prevent sarcopenia in the elderly or in patients with this condition.
Author contribution SH designed, interpreted data, and wrote the manuscript. SHK performed experiment of research project and SMK helped to wrote the manuscript. Korea Basic Science Institute supported experiment equipment. All tables and figures are created by the author's Author contribution JSH designed, interpreted data and wrote the manuscript. SHK performed experiment of research project. Korea Basic Science Institute supported experiment equipment. All tables and figures are created by the author's.
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