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Higher alcohols significantly influence the quality and flavor profiles of Chinese Baijiu. ILV1-encoded threonine deaminase, LEU1-encoded α-isopropylmalate dehydrogenase, and LEU2-encoded β-isopropylmalate dehydrogenase are involved in the production of higher alcohols. In this work, ILV1, LEU1, and LEU2 deletions in α-type haploid, a-type haploid, and diploid Saccharomyces cerevisiae strains and ILV1, LEU1, and LEU2 single-allele deletions in diploid strains were constructed to examine the effects of these alterations on the metabolism of higher alcohols. Results showed that different genetic engineering strategies influence carbon flux and higher alcohol metabolism in different manners. Compared with the parental diploid strain, the ILV1 double-allele-deletion diploid mutant produced lower concentrations of n-propanol, active amyl alcohol, and 2-phenylethanol by 30.33, 35.58, and 11.71%, respectively. Moreover, the production of isobutanol and isoamyl alcohol increased by 326.39 and 57.6%, respectively. The LEU1 double-allele-deletion diploid mutant exhibited 14.09% increased n-propanol, 33.74% decreased isoamyl alcohol, and 13.21% decreased 2-phenylethanol production, which were similar to those of the LEU2 mutant. Furthermore, the LEU1 and LEU2 double-allele-deletion diploid mutants exhibited 41.72 and 52.18% increased isobutanol production, respectively. The effects of ILV1, LEU1, and LEU2 deletions on the production of higher alcohols by α-type and a-type haploid strains were similar to those of double-allele deletion in diploid strains. Moreover, the isobutanol production of the ILV1 single-allele-deletion diploid strain increased by 27.76%. Variations in higher alcohol production by the mutants are due to the carbon flux changes in yeast metabolism. This study could provide a valuable reference for further research on higher alcohol metabolism and future optimization of yeast strains for alcoholic beverages.

Acetate esters and higher alcohols greatly influence the quality and flavor profiles of Chinese Baijiu (Chinese liquor). Various mutants have been constructed to investigate the interactions of ATF1 overexpression, IAH1 deletion, and BAT2 deletion on the production of acetate esters and higher alcohols. The results showed that the overexpression of ATF1 under the control of the PGK1 promoter with BAT2 and IAH1 double-gene deletion led to a higher production of acetate esters and a lower production of higher alcohols than the overexpression of ATF1 with IAH1 deletion or overexpression of ATF1 with BAT2 deletion. Moreover, deletion of IAH1 in ATF1 overexpression strains effectively increased the production of isobutyl acetate and isoamyl acetate by reducing the hydrolysis of acetate esters. The decline in the production of higher alcohol by the ATF1 overexpression strains with BAT2 deletion is due to the interaction of ATF1 overexpression and BAT2 deletion. Mutants with varying abilities of producing acetate esters and higher alcohols were developed by genetic engineering. These strains have great potential for industrial application.

Type 2 diabetes mellitus (T2DM) is commonly observed in heart failure with preserved ejection fraction (HFpEF) patients. Despite its growing prevalence, HFpEF is frequently underdiagnosed. The aim of our study is to apply machine learning algorithms for identifying HFpEF in patients with T2DM. A total of 1,444 patients with T2DM who met the criteria were consecutively enrolled. Least absolute shrinkage and selection operator (LASSO) technique was applied for feature selection to identify key clinical variables. All patients were randomly divided into a training set and a test set at a ratio of 7:3. Extreme gradient boosting (XGBoost), random forest, K-nearest neighbors, support vector machine (SVM), light gradient boosting machine, decision tree and logistic regression were used to establish diagnostic models. The diagnostic performance of models was evaluated by the area under the receiver operating characteristic curve (AUC), precision, accuracy, F1 score, and Brier score. Calibration curve and decision curve analysis (DCA) were used to assess the accuracy and clinical validity of the model. Patients were divided into HFpEF group and non-HFpEF group. XGBoost model (precision 0.812, accuracy 0.770, sensitivity 0.719, AUC 0.852, F1 score 0.741, Brier score 0.148) and SVM model (precision 0.784, accuracy 0.765, sensitivity 0.681, AUC 0.857, F1 score 0.745, Brier score 0.166) had the highest diagnostic performance. However, the calibration curve of the SVM model depart from the line of perfect calibration which confirmed poor calibration. Therefore, XGBoost was demonstrated to be best ML model in identifying HFpEF in patients with T2DM. Rankings of variable importance based on the Gain metric showed that B-type natriuretic peptide over 100 pg/mL had the highest importance score, followed by albumin, E/e', age and high-sensitivity cardiac troponin T. This study found XGBoost to be the optimal machine learning algorithm in identifying HFpEF in T2DM patients. Additionally, the model demonstrated substantial clinical utility, as assessed by DCA.

Diacetyl and higher alcohols, as the crucial flavour compounds, have been found to associate with the sensory properties of the wine. The genes ILV2, ILV3 and ILV5 in isoleucine–valine (ILV) biosynthetic pathway related to diacetyl and higher alcohols have been reported in Saccharomyces cerevisiae, whereas the regulatory mechanism in Saccharomyces uvarum is still unclear. The current study explored that ILV2 deletion and ILV3 or/and ILV5 overexpression in S. uvarum resulted in significant changes in the diacetyl and higher alcohols production. And of all the mutant strains, 3QV2 + V35 (ILV2 triple-gene-deletion with ILV3 + ILV5 overexpression), in which the diacetyl, isobutanol, and isoamyl alcohol contents were reduced by 67.56%, 25.46% and 21.05%, respectively, presented the most effective effect on regulation of diacetyl and higher alcohols production in wine. This study provided a valuable reference for further research on diacetyl and higher alcohol metabolism in S. uvarum and future optimization of yeast strains for wine and other alcoholic beverages.

Skeletal myoblast transplantation seems a promising approach for the repair of myocardial infarction (MI). However, the low engraftment efficacy and impaired angiogenic ability limit the clinical efficiency of the myoblasts. Gene engineering with angiogenic growth factors promotes angiogenesis and enhances engraftment of transplanted skeletal myoblasts, leading to improved infarction recovery in myocardial ischemia. The present study evaluated the therapeutic effects of hepatocyte growth factor (HGF) gene-engineered skeletal myoblasts on tissue regeneration and restoration of heart function in a rat MI model. The skeletal myoblasts were isolated, expanded, and transduced with adenovirus carrying the HGF gene (Ad-HGF). Male SD rats underwent ligation of the left anterior descending coronary artery. After 2 weeks, the surviving rats were randomized into four groups and treated with skeletal myoblasts by direct injection into the myocardium. The survival and engraftment of skeletal myoblasts were determined by real-time PCR and in situ hybridization. The cardiac function with hemodynamic index and left ventricular architecture were monitored; The adenovirus-mediated-HGF gene transfection increases the HGF expression and promotes the proliferation of skeletal myoblasts in vitro. Transplantation of HGF-engineered skeletal myoblasts results in reduced infarct size and collagen deposition, increased vessel density, and improved cardiac function in a rat MI model. HGF gene modification also increases the myocardial levels of HGF, VEGF, and Bcl-2 and enhances the survival and engraftment of skeletal myoblasts. HGF engineering improves the regenerative effect of skeletal myoblasts on MI by enhancing their survival and engraftment ability.

Maltose metabolism of baker’s yeast ( Saccharomyces cerevisiae ) in lean dough is suppressed by the glucose effect, which negatively affects dough fermentation. In this study, differences and interactions among SNF4 (encoding for the regulatory subunit of Snf1 kinase) overexpression and REG1 and REG2 (which encodes for the regulatory subunits of the type I protein phosphatase) deletions in maltose metabolism of baker’s yeast were investigated using various mutants. Results revealed that SNF4 overexpression and REG1 and REG2 deletions effectively alleviated glucose repression at different levels, thereby enhancing maltose metabolism and leavening ability to varying degrees. SNF4 overexpression combined with REG1 / REG2 deletions further enhanced the increases in glucose derepression and maltose metabolism. The overexpressed SNF4 with deleted REG1 and REG2 mutant Δ REG1 Δ REG2  + SNF4 displayed the highest maltose metabolism and strongest leavening ability under the test conditions. Such baker’s yeast strains had excellent potential applications.

Background Tup1 is a general transcriptional repressor of diverse gene families coordinately controlled by glucose repression, mating type, and other mechanisms in Saccharomyces cerevisiae . Several functional domains of Tup1 have been identified, each of which has differing effects on transcriptional repression. In this study, we aim to investigate the role of Tup1 and its domains in maltose metabolism of industrial baker’s yeast. To this end, a battery of in-frame truncations in the TUP1 gene coding region were performed in the industrial baker’s yeasts with different genetic background, and the maltose metabolism, leavening ability, MAL gene expression levels, and growth characteristics were investigated. Results The results suggest that the TUP1 gene is essential to maltose metabolism in industrial baker’s yeast. Importantly, different domains of Tup1 play different roles in glucose repression and maltose metabolism of industrial baker’s yeast cells. The Ssn6 interaction, N-terminal repression and C-terminal repression domains might play roles in the regulation of MAL transcription by Tup1 for maltose metabolism of baker’s yeast. The WD region lacking the first repeat could influence the regulation of maltose metabolism directly, rather than indirectly through glucose repression. Conclusions These findings lay a foundation for the optimization of industrial baker’s yeast strains for accelerated maltose metabolism and facilitate future research on glucose repression in other sugar metabolism.

Acetate esters and higher alcohols significantly determine the flavor profiles of Chinese Baijiu (Chinese liquor). The aminotransferase encoded by BAT2 and alcohol acetyltransferases encoded by ATF1, ATF2, and Lg-ATF1 are involved in the production of branched-chain alcohols and synthesis of acetate esters, respectively. In this study, the effects of ATF1, ATF2, and Lg-ATF1 overexpressions with BAT2 deletion were explored in Chinese Baijiu yeast. The differences among these effects were also investigated. Results showed that the productions of acetate esters by mutant overexpressing ATF1 with BAT2 deletion and that overexpressing ATF2 with BAT2 deletion were 1353.45 and 73.40 mg/L, respectively, which were 43.16- and 2.34-fold higher than that by the original strain. Compared with mutant overexpressing ATF2 with BAT2 deletion, mutant overexpressing ATF1 with BAT2 deletion exhibited 48.17% decreased higher alcohol productivity. The production of higher alcohols in mutant overexpressing ATF2 with BAT2 deletion was similar to that in the BAT2 deletion mutant. Furthermore, no significant difference was observed between the BAT2 deletion mutant and the mutant overexpressing Lg-ATF1 with BAT2 deletion in terms of acetate ester and higher alcohol production. The mutants that have varying capacities for acetate ester and higher alcohol production can be potentially developed and applied.

Background Trehalose is related to several types of stress responses, especially freezing response in baker’s yeast ( Saccharomyces cerevisiae ). It is desirable to manipulate trehalose-related genes to create yeast strains that better tolerate freezing-thaw stress with improved fermentation capacity, which are in high demand in the baking industry. Results The strain overexpressing MAL62 gene showed increased trehalose content and cell viability after prefermention-freezing and long-term frozen. Deletion of NTH1 in combination of MAL62 overexpression further strengthens freezing tolerance and improves the leavening ability after freezing-thaw stress. Conclusions The mutants of the industrial baker’s yeast with enhanced freezing tolerance and leavening ability in lean dough were developed by genetic engineering. These strains had excellent potential industrial applications.

Background Although epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKIs) have become the standard care of patients with advanced EGFR-mutant non-small cell lung cancer (NSCLC), development of acquired resistance is inevitable. A secondary mutation of threonine 790 (T790M) is associated with approximately half of the cases of acquired resistance. Strategies or agents to overcome this type of resistance are still limited. In this study, enhanced antitumor effect of AT-101, a-pan-Bcl-2 inhibitor, on gefitinib was explored in NSCLC with T790M mutation. Methods The effect of cotreatment with AT-101 and gefitinib on the viability of NSCLC cell lines harboring acquired T790M mutation was investigated using the MTT assay. The cellular apoptosis of NSCLC cells after cotreatment with AT-101 and gefitinib was assessed by FITC-annexin V/PI assay and Western blots analysis. The potential underlying mechanisms of the enhanced therapeutic effect for AT-101 was also studied using Western blots analysis. The in vivo anti-cancer efficacy of the combination with AT-101 and gefitinib was examined in a mouse xenograft model. Results In this study, we found that treatment with AT-101 in combination with gefitinib significantly inhibited cell proliferation, as well as promoted apoptosis of EGFR TKIs resistant lung cancer cells. The apoptotic effects of the use of AT-101 was related to the blocking of antiapoptotic protein: Bcl-2, Bcl-xl, and Mcl-1 and downregrulation of the molecules in EGFR pathway. The observed enhancements of tumor growth suppression in xenografts supported the reverse effect of AT-101 in NSCLC with T790M mutation, which has been found in in vitro studies before. Conclusions AT-101 enhances gefitinib sensitivity in NSCLC with EGFR T790M mutations. The addition of AT-101 to gefitinib is a promising strategy to overcome EGFR TKIs resistance in NSCLC with EGFR T790M mutations.