Integration of network pharmacology, transcriptomics and molecular docking reveals two novel hypoglycemic components in snow chrysanthemum.

Our previous studies uncovered the glucose-lowering properties of snow chrysanthemum tea, however, the active ingredients and underlying mechanisms were yet to be uncovered. Flavonoids are the most active and abundant components in snow chrysanthemum tea. In this study, we treated leptin-deficient diabetic ob/ob or high-fat diet (HFD)-induced C57BL/6 J obese mice with or without total flavonoids of snow chrysanthemum (TFSC) for 14 weeks. Results indicated that TFSC ameliorated dyslipidemia and fatty liver, thereby reducing hyperlipidemia. Further mechanism experiments, including RNA-seq and experimental validation, revealed TFSC improved glycolipid metabolism primarily by activating the AMPK/Sirt1/PPARγ pathway. Additionally, by integrating UPLC, network pharmacology, transcriptomics, and experimental validation, we identified two novel hypoglycemic compounds, sulfuretin and leptosidin, in TFSC. Treatment with 12.5 µmol/L sulfuretin obviously stimulated cellular glucose consumption, and sulfuretin (3.125, 6.25 and 12.5 µmol/L) significantly mitigated glucose uptake damage and reliably facilitated glucose consumption in insulin-resistant HepG2 cells. Remarkably, sulfuretin interacted with the ligand-binding pocket of PPARγ via three hydrogen bond interactions with the residues LYS-367, GLN-286 and TYR-477. Furthermore, a concentration of 12.5 µmol/L sulfuretin effectively upregulated the expression of PPARγ, exhibiting a comparable potency to a renowned PPARγ agonist at 20 µmol/L. Taken together, our findings have identified two new hypoglycemic compounds and revealed their mechanisms, which significantly expands people's understanding of the active components in snow chrysanthemum that have hypoglycemic effects.

Therapy Follows Diagnosis: Old and New Approaches for the Treatment of Acute Porphyrias, What We Know and What We Should Know.

Heme, iron protoporphyrin IX, is one of life's most central molecules. Hence, availability of the enzymatic machinery necessary for its synthesis is crucial for every cell. Consequently, inborn errors of porphyrin metabolism that compromise normal synthesis, namely the family of porphyrias, undermine normal cellular metabolism given that heme has functions in catalytic centers, signal transduction and functional regulation and its synthesis is fully integrated into the center of intermediary metabolism. Very often, diagnosis of porphyrias is difficult and therefore delayed. Therapy can be as complicated. Over the last 50 years, several strategies have been developed: because of its integration with other parts of intermediary metabolism, the infusion of glucose (glucose effect) was one of the first attempts to counterbalance the dysregulation of porphyrin synthesis in porphyrias. Since heme synthesis is impaired, infusional replacement of heme was the next important therapeutic step. Recently, siRNA technology has been introduced in order to downregulate 5-ALA-synthase 1, which contributes to the patho-physiology of these diseases. Moreover, other novel therapies using enzyme protein replacement, mRNA techniques or proteostasis regulators are being developed.

Purification and anticancer activity of glutaminase and urease-free l-asparaginase from novel endophyte Chaetomium sp.

l-Asparaginase catalyzes the hydrolysis of asparagine into aspartic acid and ammonia. The present work elaborates the isolation and identification of a novel endophytic fungal isolate producing l-glutaminase and urease-free l-asparaginase. Cell growth and enzyme production were investigated for large production. The isolated endophytic fungi were identified at molecular levels and a phylogenetic tree was constructed. The enzyme synthesis was evaluated by cultivating the isolated microorganisms in potato dextrose agar medium. Out of 27 isolated endophytes, nine were producing "l-glutaminase and urease-free l-asparaginase." l-Asparaginase from Chaetomium sp. exhibited superior enzyme activity than from the other isolates. Observed optimal conditions for l-asparaginase activity were 25 min of incubation time, 0.5 mg of enzyme source, 40°C of temperature, and pH 7.0. l-Asparaginase from Chaetomium sp. exhibited anticancer activity on human blood cancer (MOLT-4) cells. The current study has demonstrated the production of contaminant-free l-asparaginase enzyme from endophytic fungal species. The results showed that: (a) maximum enzyme activity was observed for l-asparaginase from Chaetomium sp., (b) concentration of glucose in the medium as a carbon source suppressed the enzyme production. Chaetomium sp. is a novel source for "l-glutaminase and urease-free l-asparaginase," which may play a major role in pharmacotherapy.

A Thi2p Regulatory Network Controls the Post-glucose Effect of Xylose Utilization in Saccharomyces cerevisiae.

The complete and efficient utilization of both glucose and xylose is necessary for the economically viable production of biofuels and chemicals using lignocellulosic feedstocks. Although recently obtained recombinant Saccharomyces cerevisiae strains metabolize xylose well when xylose is the sole carbon source in the medium (henceforth referred to as "X stage"), their xylose consumption rate is significantly reduced during the xylose-only consumption phase of glucose-xylose co-fermentation ("GX stage"). This post-glucose effect seriously decreases overall fermentation efficiency. We showed in previous work that THI2 deletion can alleviate this post-glucose effect, but the underlying mechanisms were ill-defined. In the present study, we profiled the transcriptome of a thi2Δ strain growing at the GX stage. Thi2p in GX stage cells regulates genes involved in the cell cycle, stress tolerance, and cell viability. Importantly, the regulation of Thi2p differs from a previous regulatory network that functions when glucose is the sole carbon source, which suggests that the function of Thi2p depends on the carbon source. Modeling research seeking to optimize metabolic engineering via TFs should account for this important carbon source difference. Building on our initial study, we confirmed that several identified factors did indeed increase fermentation efficiency. Specifically, overexpressing STT4, RGI2, and TFC3 increases specific xylose utilization rate of the strain by 36.9, 29.7, 42.8%, respectively, in the GX stage of anaerobic fermentation. Our study thus illustrates a promising strategy for the rational engineering of yeast for lignocellulosic ethanol production.

Disruption of the transcription factors Thi2p and Nrm1p alleviates the post-glucose effect on xylose utilization in Saccharomyces cerevisiae.

BACKGROUND: The recombinant Saccharomyces cerevisiae strains that acquired the ability to utilize xylose through metabolic and evolutionary engineering exhibit good performance when xylose is the sole carbon source in the medium (designated the X stage in the present work). However, the xylose consumption rate of strains is generally low after glucose depletion during glucose-xylose co-fermentation, despite the presence of xylose in the medium (designated the GX stage in the present work). Glucose fermentation appears to reduce the capacity of these strains to "recognize" xylose during the GX stage, a phenomenon termed the post-glucose effect on xylose metabolism. RESULTS: Two independent xylose-fermenting S. cerevisiae strains derived from a haploid laboratory strain and a diploid industrial strain were used in the present study. Their common characteristics were investigated to reveal the mechanism underlying the post-glucose effect and to develop methods to alleviate this effect. Both strains showed lower growth and specific xylose consumption rates during the GX stage than during the X stage. Glycolysis, the pentose phosphate pathway, and translation-related gene expression were reduced; meanwhile, genes in the tricarboxylic acid cycle and glyoxylic acid cycle demonstrated higher expression during the GX stage than during the X stage. The effects of 11 transcription factors (TFs) whose expression levels significantly differed between the GX and X stages in both strains were investigated. Knockout of THI2 promoted ribosome synthesis, and the growth rate, specific xylose utilization rate, and specific ethanol production rate of the strain increased by 17.4, 26.8, and 32.4%, respectively, in the GX stage. Overexpression of the ribosome-related genes RPL9A, RPL7B, and RPL7A also enhanced xylose utilization in a corresponding manner. Furthermore, the overexpression of NRM1, which is related to the cell cycle, increased the growth rate by 8.7%, the xylose utilization rate by 30.0%, and the ethanol production rate by 76.6%. CONCLUSIONS: The TFs Thi2p and Nrm1p exerted unexpected effects on the post-glucose effect, enhancing ribosome synthesis and altering the cell cycle, respectively. The results of this study will aid in maintaining highly efficient xylose metabolism during glucose-xylose co-fermentation, which is utilized for lignocellulosic bioethanol production.

Increased diazinon hydrolysis to 2-isopropyl-6-methyl-4-pyrimidinol in liquid medium by a specific Streptomyces mixed culture.

Actinobacteria identified as Streptomyces spp. were evaluated for their ability to remove diazinon as the only carbon source from a liquid medium. Single cultures of Streptomyces strains were exposed to diazinon at a concentration of 50 mg L(-1). After 96 h incubation, six of the eight cultures grew and five strains showed an increase in their total protein concentrations and changes in their protein profile. Up to 32% of the diazinon was removed by the single Streptomyces cultures. A compatibility assay showed that the different Streptomyces species were not antagonistic. Twenty-six mixed cultures were then prepared. Diazinon removal was increased when mixed cultures were used, and maximum diazinon removal of 62% was observed when the Streptomyces spp. strains AC5, AC9, GA11 and ISP13 were mixed; this was defined as the selected mixed culture (SMC). Diazinon removal was positively influenced by the addition of glucose into the liquid medium. Our study showed a diazinon degradation rate of 0.025 h(-1), half-life of 28 h(-1) and 2-isopropyl-6-methyl-4-pyrimidinol (IMHP) production of 0.143 mg L h(-1). Rapid diazinon hydrolysis to IMHP was associated with a decrease in the pH of the medium as a consequence of microbial glucose metabolism and organic acid exudation. Moreover, the SMC of Streptomyces was able to remove IMHP. This work constitutes a new, if not the only, report on diazinon degradation by mixed cultures of Streptomyces spp. Given the high levels of diazinon removal, the SMC formed by four Streptomyces strains has the potential to be used to treat the diazinon present in environmental matrices.

Impact of hypoglycemia on patients with type 2 diabetes mellitus and their quality of life, work productivity, and medication adherence.

BACKGROUND: The purpose of this study was to determine the characteristics of adults with type 2 diabetes mellitus (T2DM) that correlate with greater risk of hypoglycemia and determine the impact of hypoglycemia on health-related quality of life, work productivity, and medication adherence from a patient perspective. METHODS: Data from a large web-based survey were retrospectively analyzed. Adults with a diagnosis of T2DM taking antihyperglycemic agents were included in the analysis. Participants with knowledge of their hypoglycemic history were divided into three groups: those experiencing recent hypoglycemia (previous 3 months), those experiencing nonrecent hypoglycemia, and those never experiencing hypoglycemia. RESULTS: Of the participants with T2DM taking antihyperglycemic agents who were knowledgeable of their hypoglycemia history, 55.7% had ever experienced hypoglycemia. Of those, 52.7% had recent hypoglycemia. Compared with those who never experienced hypoglycemia, those who experienced hypoglycemia tended to: be younger; be more aware of their glycated hemoglobin (HbA1c) levels; have higher HbA1c levels; have a higher body mass index; have higher Charlson Comorbidity Index scores; be on insulin, sulfonylureas, and/or glucagon-like peptide-1 agonists; and be less adherent to their antihyperglycemic agents. Hypoglycemia interfered with social activities, caused more missed work (absenteeism), more impairment while at work (presenteeism), and decreased overall work productivity compared with patients who had never experienced hypoglycemia. Overall health-related quality of life, as determined by the Short Form-36 health questionnaire, was negatively impacted by hypoglycemia. Both Physical and Mental Summary scores were significantly lower for the recent hypoglycemia and nonrecent hypoglycemia groups compared with the never hypoglycemia group. CONCLUSION: Hypoglycemia can negatively impact many aspects of life. Greater awareness of those who are at risk for developing hypoglycemia can lead to the development of measures (eg, patient and physician education) to prevent future hypoglycemia episodes.

Effect of glucose on the expression of c-myc gene in cultured RINm5F cell

The study was designed to examine the effect of glucose on the expression of c-myc gene in cultured RINm5F cells. After monolayer culture was established in RPMI 1640 media supplemented with 10% fetal calf serum (FCS), the cells were cultured in various concentrations of glucose and 1 or 10% FCS for another 24 hours. A mRNA was extracted from the cultured cells by a single step method, and Northern analysis was done to detect RNA band. A 0.5 kilobase single band was detected as c-myc mRNA. The expression of c-myc gene mRNA was reduced with increased concentration of glucose with 1% FCS. However, supplementation of 10% FCS abolished the effect of glucose on expression of c-myc gene. These findings suggested that glucose in conjunction with other growth promoting factors played an important role in expression of oncogene and cell growth in RINm5F cells.

Hatching of Meloidogyne incognita Eggs in the Neutral Carbohydrate Fraction of Root Exudates of Gnotobiotically Grown Alfalfa.

Meloidogyne incognita eggs were hatched in soil sterilized by gamma kradiation and wetted with root exudates from alfalfa plants in different stages of development and subjected to various levels of clipping. Carbohydrate components of the exudates were identified by gas chromatography-mass spectrometry. Although significant stimulation of hatch was detected in exudates of seedling and flowering plants, the practical importance of the increase is doubtful as hatch in distilled water was always greater than 50%. Hatch did not differ among exudate samples from clipped plants. Incubation of eggs in soil moistened with 10 to 10(3) M solutions of glucose did not result in increased hatching over that in distilled water.