Differential Gene Expression and Biological Analyses of Primary Hepatocytes Following D-Chiro-Inositol Supplement.

Dietary supplements have improved the prevention of insulin resistance and metabolic diseases, which became a research hotspot in food science and nutrition. Obesity and insulin resistance, caused by a high-fat diet, eventually result in severe metabolic diseases, can be prevented with the dietary supplement D-chiro-inositol (DCI). In this work, we isolated mice primary hepatocytes with palmitic acid stimulation and DCI was applied to compare and contrast its effects of in primary hepatocyte biology. Before and after intervention with DCI, we used RNA-Seq technology to establish a primary hepatocyte transcriptome gene profile. We found that both PA and DCI cause a wide variation in gene expression. Particularly, we found that DCI plays critical role in this model by acting on glycolysis and gluconeogenesis. Overall, we generated extensive transcripts from primary hepatocytes and uncovered new functions and gene targets for DCI.

Characterization of the complete mitochondrial genome of Coprinellus micaceus, a wild saprobic mushroom in China.

The Glistening Inkcap (Coprinellus micaceus) is a wild saprobic mushroom in China. In this study, we assembled and annotated its complete mitochondrial genome using raw data sequenced through Illumina NovaSeq 6000 platform (Illumina, San Diego, CA). The length of the C. micaceus mitochondrial genome is 65,450 bp with 33.05% GC content. Totally, 41 genes, including 14 protein-coding genes, 25 tRNAs and 2 rRNAs were identified in the mitochondrial genome. Phylogenetic analysis showed that the mitochondrial genome relationship between C. micaceus and Coprinopsis cinerea was the closest.

Purification and characterization of a novel protease-resistant GH27 α-galactosidase from Hericium erinaceus.

A novel 57-kDa acidic α-galactosidase designated as HEG has been purified from the dry fruiting bodies of Hericium erinaceus. The isolation protocol involved ion-exchange chromatography and gel filtration on a Superdex75 column. The purification fold and specific activity were 1251 and 46 units/mg, respectively. A BLAST search of internal peptide sequences obtained by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis suggested that the enzyme belonged to the GH27 family. The activity of the enzyme reached its maximum at a pH of 6.0 or at 60 °C. The enzyme was stable within an acidic pH range of 2.2-7.0 and in a narrow temperature range. The enzyme was strongly inhibited by Zn2+, Fe3+, Ag+ ions and SDS. The Lineweaver-Burk plot suggested that the mode of inhibition by galactose and melibiose were of a mixed type. N-bromosuccinimide drastically decreased the activity of the enzyme, whereas diethylpyrocarbonate and carbodiimide strengthened the activity slightly. Moreover, the isolated enzyme displayed remarkable resistance to acid proteases, neutral proteases and pepsin. The enzyme could also hydrolyse oligosaccharides and polysaccharides. In addition, acidic protease promoted the hydrolysis of RFOs by HEG. The Km values of the enzyme towards pNPGal, raffinose and stachyose were 0.36 mM, 40.07 mM and 54.71 mM, respectively. These favourable properties increase the potential of the enzyme in the food industry and animal feed applications.

A Comparative Proteome Approach Reveals Metabolic Changes Associated with Flammulina velutipes Mycelia in Response to Cold and Light Stress.

In some industrial processes, cold and light stresses are recognized as two important environmental triggers for the transformation of mycelia into fruit-bodies via intermediate primordia in Flammulina velutipes cultivation. To gain insights into the mechanism of regulation of F. velutipes mycelia in response to cold and light stress, proteins expressed abundantly and characteristically at particular stress states were investigated by using the isobaric tags for the relative and absolute quantitation labeling technique. Among the 1046 nonredundant proteins identified with a high degree of confidence, 264 proteins, which were detected as differentially expressed proteins, were associated with 176 specific KEGG pathways. In-depth data analysis revealed that the regulatory network underlying the cold and light response mechanisms of F. velutipes mycelia was complex and multifaceted, as it included varied functions such as rapid energy supply, the biosynthesis of lysine, phenylalanine, tyrosine, and γ-aminobutyric acid, the calcium signal transduction process, dynein-dependent actin and microtubule cytoskeleton formation, autolysis, oxidative stress adaptation, pigment secretion, tissue and organ morphogenesis, and other interesting stress-related processes. Insights into the proteins might shed light on an intuitive understanding of the cold and light stress response mechanism underlying the fruiting processes of F. velutipes. Furthermore, the data might also provide further insights into the stress response mechanism of macro-fungi and valuable information for scientific improvement of some mushroom cultivation techniques in practice.

Comparative Proteome Reveals Metabolic Changes during the Fruiting Process in Flammulina velutipes.

Understanding the molecular mechanisms regulating the fruiting process in macro-fungi, especially industrially cultivated mushrooms, has long been a goal in mycological research. To gain insights into the events accompanying the transformation of mycelia into fruit-bodies in Flammulina velutipes, proteins expressed characteristically and abundantly at primordium and fruit-body stages were investigated by using the iTRAQ labeling technique. Among the 171 differentially expressed proteins, a total of 68 displayed up-regulated expression levels that were associated with 84 specific KEGG pathways. Some up-regulated proteins, such as pyruvate carboxylase, aldehyde dehydrogenase, fatty acid synthase, aspartate aminotransferase, 2-cysteine peroxiredoxin, FDS protein, translation elongation factor 1-alpha, mitogen-activated protein kinases (MAPKs), and heat-shock protein 70 that are involved in carbohydrate metabolism, carotenoid formation, the TCA cycle, MAPK signaling pathway, and the biosynthesis of fatty acids and branched-chain amino acids, could serve as potential stage-specific biomarkers to study the fruiting process in F. velutipes. Knowledge of the proteins might provide valuable evidence to better understand the molecular mechanisms of fruit-body initiation and development in basidiomycete fungi. Furthermore, this study also offers valuable evidence for yield improvement and quality control of super golden-needle mushroom in practice.

iTRAQ-Based Comparative Proteomics Analysis of the Fruiting Dikaryon and the Non-fruiting Monokaryon of Flammulina velutipes.

Flammulina velutipes is a potentially excellent fungus to study basic mechanisms of basidiomycete mycelium biology. To provide a better understanding of the mechanism of hyphae growth and fruit-body formation, the biological functions of the differentially abundant proteins between the fruiting dikaryon and the non-fruiting monokaryon of F. velutipes were investigated at the proteomic level using iTRAQ-coupled two-dimensional liquid chromatography tandem mass spectrometry technique. Among the 1198 proteins identified with high confidence, a total of 472 proteins were detected differentially abundant at least one of the mycelium development stages. In-depth data analysis revealed that differentially expressed proteins were influenced a variety of cellular processes, particularly metabolic processes. Functional pathway analysis indicated that 63 up-regulated proteins at only the fruiting dikaryon (Fv13) stage were mainly distributed in 51 specific Kyoto Encyclopedia of Genes and Genome pathways, such as amino acids biosynthesis and metabolism, signaling pathway, and central carbon metabolism. These up-regulated proteins could possibly serve as potential biomarkers to study the mycelium development pathways as well as provide new insights on the mycelium heterogenic compatibility and fruit-body formation mechanisms of basidiomycetes.

Immunomodulatory and antioxidative activity of Cordyceps militaris polysaccharides in mice.

To evaluate the immune activation and reactive oxygen species scavenging activity of Cordyceps militaris polysaccharides (CMP) in vivo, 24 male and 24 female Kunming mice were randomly divided into four groups. The mice in the four experimental groups were administered 0 (normal control), 50, 100, or 200mg/kg/d body weight CMP via gavage. After 30 days, the viscera index, leukocyte count, differential leukocyte count, immunoglobulin (IgG) levels, and biochemical parameters were measured. The effect of CMP on the expression of tumor necrosis (TNF)-α, interferon (IFN)-γ, and interleukin (IL)-1ß in the spleens of experimental mice was investigated by real-time polymerase chain reaction. The results showed that the administration of CMP improved the immune function in mice, significantly increased the spleen and thymus indices, the spleen lymphocyte activity, the total quantity of white blood cells, and IgG function in mice serum. CMP exhibited significant antioxidative activity in mice, and decreased malondialdehyde levels in vivo. CMP upregulated the expression of TNF-α, IFN-γ, and IL-1ß mRNA in high-dose groups compared to that observed for the control mice. We can thus conclude that CMP effectively improved the immune function through protection against oxidative stress. CMP thus shows potential for development as drugs and health supplements.