Construction of a Zygosaccharomyces rouxii strain overexpressing the QOR gene for increased HDMF production.

4-Hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF) is a flavor compound widely found in natural products and is used in food as a flavor-enhancing agent. Quinone oxidoreductase (QOR) was verified as a key enzyme to synthesize HDMF in strawberry, while its impact on HDMF production by Zygosaccharomyces rouxii was still unknown. The QOR gene was cloned and overexpressed in Z. rouxii, and its impact on HDMF production by Z. rouxii was then further analyzed. At the same time, it is expected to obtain engineered strains of Z. rouxii with high HDMF production. The results showed that the engineered strains of Z. rouxii exhibit different levels of QOR gene expression and HDMF production; among them, the QOR6 strain exhibiting the highest gene expression level and HDMF production was named as ZrQOR. The HDMF production of the ZrQOR strain was significantly higher than that of wild-type Z. rouxii at 3 and 5 days of culture, with 1.41-fold and 1.08-fold increases, respectively. At 3 days of fermentation, the highest HDMF yield of ZrQOR strain was obtained (2.75 mg/L), 2 days ahead of the reported highest HDMF production by Z. rouxii. At 3, 5, and 7 days, QOR gene expression was 4.8-fold, 3.3-fold, and 5.6-fold higher in the ZrQOR strain than in the wild-type Z. rouxii, respectively. Therefore, overexpression of the QOR gene facilitates HDMF synthesis. The genetic stability of the 0-20 generation ZrQOR strain was stable, and there was no significant difference in colony shape, QOR expression, or HDMF production compared to the wild type. In this study, the genetic engineering Z. rouxii strain was used to improve HDMF production. This research has laid the groundwork for further industrial production of HDMF via microbial synthesis.

Blood Glucose Meter Buying Behavior of Diabetic Patients: Factors Influencing Purchase.

Objective: To understand the blood glucose meter buying behavior of type 2 diabetic patients with poor glycemic control (two or more HbA1c ≥ 8% during visits in one year) and identify factors influencing it. Methods: A survey was conducted among 585 diabetic patients with poor glycemic control who were treated in the outpatient or inpatient clinics of the Department of Endocrinology, Taizhou Hospital, Zhejiang Province from June 2020 to May 2021. The questionnaire collected general information and clinical data, and assessed blood glucose meter buying behavior. Chi-square test was used to compare the essential characteristics and clinical data between buyers and non-buyers of blood glucose meters. Additionally, stepwise logistic regression was used to analyze the factors influencing purchase. Results: Of the 585 questionnaires distributed, 527 (90.09%) valid questionnaires were collected. Of the 527 respondents, 285 (54.08%) had purchased blood glucose meters. Not receiving insulin therapy (OR: 1.77, 95% CI: 1.13-2.77) and unawareness of self-monitoring of blood glucose (OR: 19.46, 95% CI: 12.51-30.26) were risk factors for non-purchase. Conclusion: There is a need to actively increase the purchase of glucose meters among diabetic patients, by educating them about the importance of self-monitoring of blood glucose.

Molecular mechanism of the response of Zygosaccharomyces rouxii to D-fructose stress by the glutathione metabolism pathway.

Zygosaccharomyces rouxii produces high levels of 4-hydroxy-2,5-dimethyl-3(2H)-furanone in YPD medium supplemented with 120 g/L D-fructose and 180 g/L NaCl after 5 d. D-fructose has a stress effect on Z. rouxii, and GSH-Px is a main enzyme involved in the defense of Z. rouxii against oxygen stress according to our previous report. In order to further explore the molecular mechanism of the glutathione metabolism pathway in Z. rouxii in response to D-fructose stress, changes in the expression of genes and proteins involved in the synthesis of glutathione precursor amino acids and enzymes were observed. In addition, changes in the intermediates related to glutathione synthesis in Z. rouxii were reported. The results indicated that some gene-encoding enzymes involved in the glutamate, cysteine and glycine biosynthesis pathways and key genes involved in glutathione synthesis were upregulated. The expression levels of other genes, except SHMT, were consistent with the qRT-PCR results. The contents of γ-glutamylcysteine and glutathione amide in the D-fructose group were higher than those in the control group. In the D-fructose stress groups, the metabolic flux towards glutathione synthesis was increased. These results might provide more in-depth and detailed theoretical support for the oxidative stress defense mechanism of Z. rouxii under D-fructose stress.

Molecular mechanisms of furanone production through the EMP and PP pathways in Zygosaccharomyces rouxii with D-fructose addition.

4-Hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF) and 4-hydroxy-2(or 5)-ethyl-5(or 2)-methyl-3(2H)-furanone (HEMF) are important aroma chemicals in fermented foods. In this study, transcriptomics, qRT-PCR and enzymology methods were used to study the molecular mechanisms of furanone production through the Embden-Meyerhof-Parnas (EMP) and Pentose Phosphate (PP) pathways in Zygosaccharomyces rouxii based on the results of our previous study. The results indicated that D-fructose addition could significantly enhance Z. rouxii biomass production. In addition, HDMF and HEMF production was increased as a result of D-fructose addition based on HPLC analysis. The significant pathways for furanone synthesis were EMP (zro00010) and PP (zro00030) based on KEGG analysis. At the mRNA level, the differentially expressed genes involved in HDMF and HEMF biosynthesis were HK, PFK1, G6PI, FBA, TPI, 6GPL, TKT, and 6PGDH. Transient overexpression of FBA and 6PGDH in Z. rouxii was significantly increased during furanone production. FBA can regulate the accumulation of dihydroxyacetone phosphate (DHAP), which is one of the precursors of HDMF, while 6PGDH can regulate the accumulation of ribulose-5-phosphate, a precursor of HEMF. In addition, the activities of PFK1, FBA, and 6PGDH were significantly correlated with furanone production. LC-MS/MS results indicated that the primary metabolites for furanone synthesis in the EMP and PP pathways gradually increased with the consumption of D-fructose. These data demonstrate that D-fructose addition can be used to generate furanones through the EMP and PP pathways in Z. rouxii.

A new understanding: Gene expression, cell characteristic and antioxidant enzymes of Zygosaccharomyces rouxii under the D-fructose regulation.

Zygosaccharomyces rouxii is a well-known salt-tolerant yeast. In our previous study, it was interesting that Z. rouxii could produce higher levels of 4-hydroxy-2, 5-dimethyl-3(2 H)-furanone in 120 g/L D-fructose and 180 g/L NaCl involved YPD medium at 5 d. In order to explore the resistance and furanone production mechanisms of Z. rouxii under D-fructose regulation, a comparative transcriptomics method in Z. rouxii was to set to find differentially expressed genes, the physiological and biochemical indexes (growth and cell morphology, lipid peroxidation and relative electrical conductivity, the antioxidant enzymes activity), and the expression of oxidoreductase activity genes. The results indicated that a larger number of different expressed genes at transcriptome analysis, such as the series antioxidant enzymes were related to the resistance characteristics. Research had confirmed that the living cell numbers and cell areas of D-fructose regulation group were significantly lower than the controls at the initial stage, while those higher than of the controls at the late stage. During the fermentation period, the lipid peroxidation and the relative electrical conductivity of the yeast cell membrane were increased. And also the D-fructose regulation group present lower inhibition superoxide anion ability. The activity of CAT in the D-fructose regulation group was always higher than that of the control group. Only the activity of GSH-Px was found to be significantly increased at 1 d except for other enzymes activities. Most of the oxidoreductase activity genes, such as especially the GSH-Px gene under D-fructose regulation conditions were expressed at higher levels than those of control groups. Combining the levels of transcription and enzymes activity data, those could understand that exogenous D-fructose had a stress effect on Z. rouxii at the early stage of culture. With the fermentation time progress, it was no longer a stressor substance for the Z. rouxii, and changed the nutrient to promote growth of Z. rouxii in the later stages. During the whole process, GSH-Px was the main defense enzyme and CAT was the sustained defense enzyme. Therefore, the experimental results might provide effective mechanisms in Z. rouxii for practical application of furanone production in the industry under exogenous D-fructose regulation.

[Genetic characterization analysis on epidemic rubella virus strains isolated in Liaoning from 2007 to 2012].

To analyze the genetic characterization of epidemic rubella virus strains isolated in Liaoning from 2007-2012, a total of 145 rubella virus strains were isolated using Vero/Slam cell line from the patients' throat swabs during rubella outbreaks and sporadics cases in Liaoning Province from 2007 to 2012. Fragments of 945 nucleotides containing 1E gene from 145 rubella virus isolates were amplified by RT-PCR, the PCR products were sequenced and analyzed. Based on the 739 nucleotides of 1E gene, the phylogenetic trees were constructed with 32 WHO rubella reference strains of 13 genotypes downloaded from GenBank and 145 rubella virus strains. The results showed that the 145 rubella virus strains in 2007 -2012 belonged to genotype 1E, nucleotide acids and amino acids similarities were 97.2%-100.0% and 97.6%-100.0%, respectively. Compared to the 1E reference strains(Rvi/ Dezhou.CHN/02, RVi/MYS/01), the nucleotide acids and amino acids similarities were 96.6%-99.2% and 98.2%-100.0%, respectively except for one amino acid change (Val246-Ala246) of RVi/Shenyang. Liaoning. CHN/13.11/13, and Asp262-Asn262 of RVi/Shenyang. Liaoning. CHN/13.11/4 and RVi/Liaoyang. Liaoning. CHN/26. 11/2. there had no change found in the important antigenic epitope sites, the hemagglutination inhibition and neutralization epitopes of the other rubella viruses. All the 145 strains isolated had the same amino acid change (Leu338--Phe338) in E1 protein. These findings suggested that genotype 1E of rubella virus was the predominant genotype in Liaoning province. the rubella prevailed in recent six years was mainly caused by rubella viruses genotype 1E with multi-transmission routes.

Air pollution and children's respiratory symptoms in six cities of Northern China.

OBJECTIVE: The associations between air pollution and children's respiratory health in the high pollution range have not yet been clearly characterized. We evaluated the effects of outdoor air pollution on respiratory morbidity in children selected from multiple sites in a heavy industrial province of northeastern China. METHODS: The study included 11,860 children aged 3-12 years, selected from 18 districts of 6 cities in Liaoning province, the participation rate is 89.9%. Informed consent and written responses to surveys about children's historic and current health status, personal and household characteristics, and other information were obtained from parents. A two-stage regression approach was applied in data analyses. RESULTS: There were wide gradients for TSP (188-689 µg/m(3)), SO(2) (14-140 µg/m(3) and NO(2) (29-94 µg/m(3)) across the 18 districts of 6 cities. The three air pollutants significantly increased the prevalence of persistent cough (21-28%), persistent phlegm (21-30%) and current asthma (39-56%) for each interquartile range increment (172 µg/m(3) for TSP, 69 µg/m(3) for SO(2), 30 µg/m(3) for NO(2)), showing larger between-city effects than within-city. Rates of respiratory symptoms were significantly higher for children with younger age, atopy, respiratory disease in early age, family history of asthma or chronic bronchitis, and tobacco smoke exposure. CONCLUSION: The high levels of outdoor air pollution in north China are positively associated with children's respiratory symptoms, the associations with TSP appear to be stronger than SO(2) and NO(2).