Isolation of Bovine Milk Exosome Using Electrophoretic Oscillation Assisted Tangential Flow Filtration with Antifouling of Micro-ultrafiltration Membrane Filters.

Tangent flow-driven ultrafiltration (TF-UF) is an efficient isolation process of milk exosomes without morphological deformation. However, the TF-UF approach with micro-ultrafiltration SiNx membrane filters suffers from the clogging and fouling of micro-ultrafiltration membrane filter pores with large bioparticles. Thus, it is limited in the long term, continuous isolation of large quantities of exosomes. In this work, we introduced electrophoretic oscillation (EPO) in the TF-UF approach to remove pore clogging and fouling of with micro-ultrafiltration SiNx membrane filters by large bioparticles. As a result, the combined EPO-assisted TF (EPOTF) filtration can isolate large quantities of bovine milk exosomes without deformation. Furthermore, several morphological and biological analyses confirmed that the EPOTF filtration approach could isolate the milk exosomes in high concentrations with high purity and intact morphology. In addition, the uptake test of fluorescent-labeled exosomes by the keratinocyte cells visualized the biological function of purified exosomes. Hence, compared to the TF-UF process, the EPOTF filtration produced a higher yield of bovine milk exosomes without stopping the filtering process for over 200 h. Therefore, this isolation process enables scalable and continuous production of morphologically intact exosomes from bovine milk, suggesting that high-quality exosome purification is possible for future applications such as drug nanocarriers, diagnosis, and treatments.

The newly developed single nucleotide polymorphism (SNP) markers for a potentially medicinal plant, Crepidiastrum denticulatum (Asteraceae), inferred from complete chloroplast genome data.

Medicinal effects of Crepidiastrum denticulatum have been previously reported. However, the genomic resources of this species and its applications have not been studied. In this study, based on the next generation sequencing method (Miseq sequencing system), we characterize the chloroplast genome of C. denticulatum which contains a large single copy (84,112 bp) and a small single copy (18,519 bp), separated by two inverted repeat regions (25,074 bp). This genome consists of 80 protein-coding gene, 30 tRNAs, and four rRNAs. Notably, the trnT_GGU is pseudogenized because of a small insertion within the coding region. Comparative genomic analysis reveals a high similarity among Asteraceae taxa. However, the junctions between LSC, SSC, and IRs locate in different positions within rps19 and ycf1 among examined species. Also, we describe a newly developed single nucleotide polymorphism (SNP) marker for C. denticulatum based on amplification-refractory mutation system (ARMS) technique. The markers, inferred from SNP in rbcL and matK genes, show effectiveness to recognize C. denticulatum from other related taxa through simple PCR protocol. The chloroplast genome-based molecular markers are effective to distinguish a potentially medicinal species, C. denticulatum, from other related taxa. Additionally, the complete chloroplast genome of C. denticulatum provides initial genomic data for further studies on phylogenomics, population genetics, and evolutionary history of Crepidiastrum as well as other taxa in Asteraceae.

Autophagy Activation by Crepidiastrum Denticulatum Extract Attenuates Environmental Pollutant-Induced Damage in Dermal Fibroblasts.

Pollution-induced skin damage results in oxidative stress; cellular toxicity; inflammation; and, ultimately, premature skin aging. Previous studies suggest that the activation of autophagy can protect oxidation-induced cellular damage and aging-like changes in skin. In order to develop new anti-pollution ingredients, this study screened various kinds of natural extracts to measure their autophagy activation efficacy in cultured dermal fibroblast. The stimulation of autophagy flux by the selected extracts was further confirmed both by the expression of proteins associated with the autophagy signals and by electron microscope. Crepidiastrum denticulatum (CD) extract treated cells showed the highest autophagic vacuole formation in the non-cytotoxic range. The phosphorylation of adenosine monophosphate kinase (AMPK), but not the inhibition of mammalian target of rapamycin (mTOR), was observed by CD-extract treatment. Its anti-pollution effects were further evaluated with model compounds, benzo[a]pyrene (BaP) and cadmium chloride (CdCl2), and a CD extract treatment resulted in both the protection of cytotoxicity and a reduction of proinflammatory cytokines. These results suggest that the autophagy activators can be a new protection regimen for anti-pollution. Therefore, CD extract can be used for anti-inflammatory and anti-pollution cosmetic ingredients.

Growth hormone-releasing peptide-biotin conjugate stimulates myocytes differentiation through insulin-like growth factor-1 and collagen type I.

Based on the potential beneficial effects of growth hormone releasing peptide (GHRP)-6 on muscle functions, a newly synthesized GHRP-6-biotin conjugate was tested on cultured myoblast cells. Increased expression of myogenic marker proteins was observed in GHRP-6-biotin conjugate-treated cells. Additionally, increased expression levels of insulin-like growth factor-1 and collagen type I were observed. Furthermore, GHRP-6-biotin conjugate-treated cells showed increased metabolic activity, as indicated by increased concentrations of energy metabolites, such as ATP and lactate, and increased enzymatic activity of lactate dehydrogenase and creatine kinase. Finally, binding protein analysis suggested few candidate proteins, including desmin, actin, and zinc finger protein 691 as potential targets for GHRP6-biotin conjugate action. These results suggest that the newly synthesized GHRP-6-biotin conjugate has myogenic stimulating activity through, at least in part, by stimulating collagen type I synthesis and several key proteins. Practical applications of the GHRP-6-biotin conjugate could include improving muscle condition.