Intrahepatic osteopontin signaling by CREBZF defines a checkpoint for steatosis-to-NASH progression.

BACKGROUND AND AIMS: NASH has emerged as a leading cause of chronic liver disease. However, the mechanisms that govern NASH fibrosis remain largely unknown. CREBZF is a CREB/ATF bZIP transcription factor that causes hepatic steatosis and metabolic defects in obesity. APPROACH AND RESULTS: Here, we show that CREBZF is a key mechanism of liver fibrosis checkpoint that promotes hepatocyte injury and exacerbates diet-induced NASH in mice. CREBZF deficiency attenuated liver injury, fibrosis, and inflammation in diet-induced mouse models of NASH. CREBZF increases HSC activation and fibrosis in a hepatocyte-autonomous manner by stimulating an extracellular matrix protein osteopontin, a key regulator of fibrosis. The inhibition of miR-6964-3p mediates CREBZF-induced production and secretion of osteopontin in hepatocytes. Adeno-associated virus -mediated rescue of osteopontin restored HSC activation, liver fibrosis, and NASH progression in CREBZF-deficient mice. Importantly, expression levels of CREBZF are increased in livers of diet-induced NASH mouse models and humans with NASH. CONCLUSIONS: Osteopontin signaling by CREBZF represents a previously unrecognized intrahepatic mechanism that triggers liver fibrosis and contributes to the severity of NASH.

Effects of silencing hsa_circ_0015326 on proliferation, migration, invasion, and apoptosis of epithelial ovarian cancer cells.

Although the treatment of ovarian cancer has made great progress, there are still many patients who are not timely detected and given targeted therapy due to unknown pathogenesis. Recent studies have found that hsa_circ_0015326 is upregulated in ovarian cancer and is involved in the proliferation, invasion, and migration of ovarian cancer cells. However, whether hsa_circ_0015326 can be used as a new target of ovarian cancer needs further investigation. Therefore, the effect of hsa_circ_0015326 on epithelial ovarian cancer was investigated in this study. At first, si-hsa_circ_0015326 lentivirus was transfected into epithelial ovarian cancer cells. Then real-time fluorescence quantitative PCR (qRT-PCR) was used to detect hsa_circ_0015326 level. The proliferation of ovarian cancer cells was detected by CCK-8 assay. The horizontal and vertical migration abilities of the cells were detected by wound-healing assay and Transwell assay, respectively. Transwell assay was also used to determine the invasion rate. As for the apoptosis rate, it was assessed by flow cytometry. As a result, the expression level of hsa_circ_0015326 in A2780 and SKOV3 was found to be higher than that in IOSE-80. However, after transfecting si-hsa_circ_0015326 and si-NC into the cells, the proliferation, migration, and invasion abilities of A2780 and SKOV3 cells in the si-hsa_circ_0015326 group were significantly reduced in comparison to those in the si-NC and mock groups, while their apoptosis rates were elevated. Collectively, silencing hsa_circ_0015326 bears the capability of inhibiting the proliferation, migration, and invasion of ovarian cancer cells while increasing apoptosis rate. It can be concluded that hsa_circ_0015326 promotes the malignant biological activities of epithelial ovarian cancer cells.

A comparative study to investigate the individual contribution of metabolic and physical interaction on volatiles formation in the mixed fermentation of Torulaspora delbrueckii and Saccharomyces cerevisiae.

It is well-known that the co-inoculation of Saccharomyces cerevisiae and non-Saccharomyces strains can modulate and improve the aromatic quality of wine through their multi-level interactions. However, the individual contribution of metabolic interaction (MI) and physical interaction (PI) on wine volatiles remains poorly understood. In this work, we utilized a double-compartment bioreactor to examine the aromatic effect of MI and PI by comparing the volatiles production in Torulaspora delbrueckii and Saccharomyces cerevisiae single fermentations to their mixed fermentations with or without physical separation. Results showed that the PI between T. delbrueckii and S. cerevisiae increased the production of most aroma compounds, especially for acetate esters and volatile fatty acids. In comparison, the MI only promoted a few volatile compounds, including ethyl decanoate, isoamyl acetate, and isobutanol. Noticeably, the MI significantly decreased the levels of ethyl dodecanoate, 2-phenylethyl alcohol, and decanoic acid, which exhibited opposite profiles in PI. Our results indicated that the PI was mainly responsible for the improved volatiles in T. delbrueckii/S. cerevisiae mixed fermentation, while the MI can be targeted to modulate the specific aroma compounds. A thorough understanding of the PI and MI aromatic effect will empower winemakers to accurately and directionally control the volatile profile of the wine, promoting the application of multi-starters to produce diverse styles of wines.

Anlotinib plus icotinib as a potential treatment option for EGFR-mutated advanced non-squamous non-small cell lung cancer with concurrent mutations: final analysis of the prospective phase 2, multicenter ALTER-L004 study.

BACKGROUND: Non-small cell lung cancer (NSCLC) patients with epidermal growth factor receptor (EGFR) mutation and concurrent mutations have a poor prognosis. This study aimed to examine anlotinib plus icotinib as a first-line treatment option for advanced NSCLC carrying EGFR mutation with or without concurrent mutations. METHODS: This phase 2, single-arm, multicenter trial (ClinicalTrials.gov NCT03736837) was performed at five hospitals in China from December 2018 to November 2020. Non-squamous NSCLC cases with EGFR-sensitizing mutations were treated with anlotinib and icotinib. The primary endpoint was progression-free survival (PFS). Secondary endpoints included the objective response rate (ORR), disease control rate (DCR), overall survival (OS), and toxicity. RESULTS: Sixty participants were enrolled, including 31 (52%) and 29 (48%) with concurrent mutations and pathogenic concurrent mutations, respectively. The median follow-up was 26.9 (range, 15.0-38.9) months. ORR and DCR were 68.5% and 98.2%, respectively. Median PFS was 15.1 (95%CI: 12.6-17.6) months which met the primary endpoint, median DoR was 13.5 (95%CI: 10.0-17.1) months, and median OS was 30.0 (95%CI: 25.5-34.5) months. Median PFS and OS in patients with pathogenic concurrent mutations were 15.6 (95%CI: 12.5-18.7) months and not reached (95%CI: 17.46 months to not reached), respectively. All patients experienced TRAEs, including 26 (43%) and 1 (1.7%) who had grade ≥ 3 and serious treatment-related adverse events (TRAEs). CONCLUSIONS: Anlotinib combined with icotinib was effective and well-tolerated as a first-line treatment option for EGFR mutation-positive advanced NSCLC with or without concurrent mutations. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT03736837.

Transcriptomics and metabolomics analysis of L-phenylalanine overproduction in Escherichia coli.

BACKGROUND: Highly efficient production of L-phenylalanine (L-Phe) in E. coli has been achieved by multiple rounds of random mutagenesis and modification of key genes of the shikimate (SHIK) and L-Phe branch pathways. In this study, we performed transcriptomic (16, 24 and 48 h) and metabolomic analyses (8, 16, 24, 32,40, and 48 h) based on time sequences in an engineered E. coli strain producing L-Phe, aiming to reveal the overall changes of metabolic activities during the fermentation process. RESULTS: The largest biomass increase rate and the highest production rate were seen at 16 h and 24 h of fermentation, respectively reaching 5.9 h-1 and 2.76 g/L/h, while the maximal L-Phe titer of 60 g/L was accumulated after 48 h of fermentation. The DEGs and metabolites involved in the EMP, PP, TCA, SHIIK and L-Phe-branch pathways showed significant differences at different stages of fermentation. Specifically, the significant upregulation of genes encoding rate-limiting enzymes (aroD and yidB) and key genes (aroF, pheA and aspC) pushed more carbon flux toward the L-Phe synthesis. The RIA changes of a number of important metabolites (DAHP, DHS, DHQ, Glu and PPN) enabled the adequate supply of precursors for high-yield L-Phe production. In addition, other genes related to Glc transport and phosphate metabolism increased the absorption of Glc and contributed to rerouting the carbon flux into the L-Phe-branch. CONCLUSIONS: Transcriptomic and metabolomic analyses of an L-Phe overproducing strain of E. coli confirmed that precursor supply was not a major limiting factor in this strain, whereas the rational distribution of metabolic fluxes was achieved by redistributing the carbon flux (for example, the expression intensity of the genes tyrB, aspC, aroL and aroF/G/H or the activity of these enzymes is increased to some extent), which is the optimal strategy for enhancing L-Phe production.

The eisosomes contribute to acid tolerance of yeast by maintaining cell membrane integrity.

Microbes have evolved multiple mechanisms to resist environmental stresses, which are regulated in complex and delicate ways. Though the role of cell membranes in acid resistance from the perspective of physicochemical properties and membrane proteins has been deeply studied, the function of eisosomes is still in its infancy. In this study, we firstly reported the dynamic changes of eisosomes under acid stress and the decreased acid tolerance of yeasts caused by eisosome disruption. Physiological indicators and non-targeted lipid profiling revealed that eisosome disruption caused changes in multiple lipids and imbalances in lipid homeostasis, which are responsible for membrane integrity damage. Thus the increased infiltration of carboxylic acids and the raised ROS levels were detected in strains with disrupted eisosome assembly, resulting in decreased cellular tolerance. The results here provide novel insights into the acid-resistant mechanism of yeasts from the perspective of the cell membrane subdomain, which has practical impacts on green biological manufacturing and food preservation.

Intracranial efficacy of alectinib in ALK-positive NSCLC patients with CNS metastases-a multicenter retrospective study.

BACKGROUND: Central nervous system (CNS) metastases in patients with ALK-positive non-small cell lung cancer (NSCLC) are a cause of substantial morbidity and mortality. Although alectinib had demonstrated promising intracranial efficacy in several clinical trials, data were limited on its CNS activity in real-world settings. METHODS: In this retrospective study, ALK-positive NSCLC patients with brain metastases (BM) or leptomeningeal metastases (LM) from six hospitals in China were divided into three cohorts based on the treatment history before the administration of alectinib. ALK-TKI-naive patients were enrolled in cohort 1, cohort 2 included patients who experienced intracranial progression with or without extracranial progression after treatment with crizotinib, and cohort 3 included patients who developed progression only in CNS following treatment with other second-generation ALK-TKIs. The definition and evaluation of intracranial and extracranial lesions were based on Response Evaluation Criteria in Solid Tumors version 1.1. RESULTS: Sixty-five patients were eligible and included in our study (cohort 1: 20, cohort 2: 32, cohort 3: 13). For the overall population and patients with uncontrolled CNS metastases, similar intracranial response in CNS target lesions was observed: cohort 1: 81.8% and 80%; cohort 2: 76.5% and 86.7%; cohort 3: 42.8% and 33.3%. For patients in these three cohorts, 75% (6/8), 78.6% (11/14), and 83.3% (5/6) were reported to have significant improvement in CNS-related symptoms respectively. The number of patients who were in need of mannitol or corticosteroids decreased remarkably after the treatment of alectinib (p < 0.001), and there was also a steep fall-over in the number of patients with ECOG ≥2 points before and after the administration of alectinib (p = 0.003). All patients (8/8) diagnosed with LM ± BM experienced substantial alleviation in CNS-related symptoms. In cohort 1 and cohort 2, no significant difference in CNS-time to progression was found between patients with symptomatic or asymptomatic BM when treated with alectinib alone. CONCLUSIONS: Our study substantiated the potent CNS activity of alectinib in real-world settings. Patients with symptomatic and asymptomatic BM could benefit from alectinib comparatively, which indicated that alectinib alone might defer the timing of local treatment. However, our results should be treated cautiously owing to limited sample size.

Adaptive immune responses and cytokine immune profiles in humans following prime and boost vaccination with the SARS-CoV-2 CoronaVac vaccine.

BACKGROUND: Adaptive immune response has been thought to play a key role in SARS-CoV-2 infection. The role of B cells, CD4+T, and CD8+T cells are different in vaccine-induced immune response, thus it is imperative to explore the functions and kinetics of adaptive immune response. We collected blood samples from unvaccinated and vaccinated individuals. To assess the mechanisms contributing to protective immunity of CoronaVac vaccines, we mapped the kinetics and durability of humoral and cellular immune responses after primary and boost vaccination with CoronaVac vaccine in different timepoints. MATERIALS AND METHODS: We separate PBMC and plasma from blood samples. The differentiation and function of RBD-spcific CD4+T and CD8+T cells were analyzed by flow cytometry and ELISA. Antibodies response was analyzed by ELISA. ELISPOT analysis was perfomed to detected the RBD-spcific memory B cells. CBA analysis was performed to detected the cytokine immune profiles. Graphpad prism 8 and Origin 2021 were used for statistical analysis. RESULTS: Vaccine-induced CD4+T cell responses to RBD were more prominent than CD8+T cell responses, and characterized by a predominant Th1 and weak Th17 helper response. CoronaVac vaccine triggered predominant IgG1 antibody response and effectively recalled specific antibodies to RBD protein after booster vaccination. Robust antigen-specific memory B cells were detected (p < 0.0001) following booster vaccination and maintained at 6 months (p < 0.0001) following primary vaccination. Vaccine-induced CD4+T cells correlated with CD8+T cells (r = 0.7147, 0.3258, p < 0.0001, p = 0.04), memory B cell responses (r = 0.7083, p < 0.0001), and IgG and IgA (r = 0.6168, 0.5519, p = 0.0006, 0.003) after vaccination. In addition, vaccine induced a broader and complex cytokine pattern in plasma at early stage. CONCLUSION: Taken together, these results highlight the potential role of B cell and T cell responses in vaccine-induced long-term immunity.

Enhanced production of amyrin in Yarrowia lipolytica using a combinatorial protein and metabolic engineering approach.

BACKGROUND: Amyrin is an important triterpenoid and precursor to a wide range of cosmetic, pharmaceutical and nutraceutical products. In this study, we metabolically engineered the oleaginous yeast, Yarrowia lipolytica to produce α- and ß-amyrin on simple sugar and waste cooking oil. RESULTS: We first validated the in vivo enzymatic activity of a multi-functional amyrin synthase (CrMAS) from Catharanthus roseus, by expressing its codon-optimized gene in Y. lipolytica and assayed for amyrins. To increase yield, prevailing genes in the mevalonate pathway, namely HMG1, ERG20, ERG9 and ERG1, were overexpressed singly and in combination to direct flux towards amyrin biosynthesis. By means of a semi-rational protein engineering approach, we augmented the catalytic activity of CrMAS and attained ~ 10-folds higher production level on glucose. When applied together, protein engineering with enhanced precursor supplies resulted in more than 20-folds increase in total amyrins. We also investigated the effects of different fermentation conditions in flask cultures, including temperature, volumetric oxygen mass transfer coefficient and carbon source types. The optimized fermentation condition attained titers of at least 100 mg/L α-amyrin and 20 mg/L ß-amyrin. CONCLUSIONS: The design workflow demonstrated herein is simple and remarkably effective in amplifying triterpenoid biosynthesis in the yeast Y. lipolytica.

Comparative transcriptome analysis reveals the key regulatory genes for higher alcohol formation by yeast at different α-amino nitrogen concentrations.

Higher alcohols are important flavor substance in alcoholic beverages. The content of α-amino nitrogen (α-AN) in the fermentation system affects the formation of higher alcohols by Saccharomyces cerevisiae. In this study, the effect of α-AN concentration on the higher alcohol productivity of yeast was explored, and the mechanism of this effect was investigated through metabolite and transcription sequence analyses. We screened 12 most likely genes and constructed the recombinant strain to evaluate the effect of each gene on high alcohol formation. Results showed that the AGP1, GDH1, and THR6 genes were important regulators of higher alcohol metabolism in S. cerevisiae. This study provided knowledge about the metabolic pathways of higher alcohols and gave an important reference for the breeding of S. cerevisiae with low-yield higher alcohols to deal with the fermentation system with different α-AN concentrations in the brewing industry.

Shading of the mother plant during seed development promotes subsequent seed germination in soybean.

The effect of shading during seed development on subsequent germination remains largely unknown. In this study, two soybean (Glycine max) seed production systems, monocropping (MC) and maize-soybean intercropping (IC), were employed to examine the effects of shading of the mother plant on subsequent seed germination. Compared to the MC soybean seeds, which received light, the developing IC seeds were exposed to shade resulting from the taller neighboring maize plants. The IC seeds germinated faster than the MC seeds, although there was no significant difference in the thickness of the seed coat. The concentration of soluble pro-anthocyanidin in the IC seed coat was significantly lower than that in the MC seed coat. Changes in the concentrations of several types of fatty acids in IC seeds were also observed, the nature of which were consistent with the effect on germination. The expression levels of genes involved in abscisic acid (ABA) biosynthesis were down-regulated in IC seeds, while the transcription levels of the genes related to gibberellin (GA) biosynthesis were up-regulated. This was consistently reflected in decreased ABA concentrations and increased active GA4 concentrations in IC seeds, resulting in an increased GA4/ABA ratio. Our results thus indicated that shading of the mother plant during seed development in soybean promoted subsequent germination by mediating the biosynthesis of pro-anthocyanidins, fatty acids, and phytohormones.

Improve the production of D-limonene by regulating the mevalonate pathway of Saccharomyces cerevisiae during alcoholic beverage fermentation.

D-Limonene, a cyclized monoterpene, possesses citrus-like olfactory property and multi-physiological functions, which can be used as a bioactive compound and flavor to improve the overall quality of alcoholic beverages. In our previous study, we established an orthogonal pathway of D-limonene synthesis by introducing neryl diphosphate synthase 1 (tNDPS1) and D-limonene synthase (tLS) in Saccharomyces cerevisiae. To further increase D-limonene formation, the metabolic flux of the mevalonate (MVA) pathway was enhanced by overexpressing the key genes tHMGR1, ERG12, IDI1, and IDI1WWW, respectively, or co-overexpressing. The results showed that strengthening the MVA pathway significantly improved D-limonene production, while the best strain yielded 62.31 mg/L D-limonene by co-expressing tHMGR1, ERG12, and IDI1WWW genes in alcoholic beverages. Furthermore, we also studied the effect of enhancing the MVA pathway on the growth and fermentation of engineered yeasts during alcoholic beverage fermentation. Besides, to further resolve the problem of yeast growth inhibition, we separately investigated transporter proteins of the high-yielding D-limonene yeasts and the parental strain under the stress of different D-limonene concentration, suggesting that the transporters of Aus1p, Pdr18p, Pdr5p, Pdr3p, Pdr11p, Pdr15p, Tpo1p, and Ste6p might play a more critical role in alleviating cytotoxicity and improving the tolerance to D-limonene. Finally, we verified the functions of three transporter proteins, finding that the transporter of Aus1p failed to transport D-limonene, and the others (Pdr5p and Pdr15p) could improve the tolerance of yeast to D-limonene. This study provided a valuable platform for other monoterpenes' biosynthesis in yeast during alcoholic beverage fermentation.

Engineering Saccharomyces cerevisiae for production of the valuable monoterpene d-limonene during Chinese Baijiu fermentation.

d-Limonene, a cyclic monoterpene, possesses citrus-like olfactory property and multi-physiological functions. In this study, the d-limonene synthase (tLS) from Citrus limon was codon-optimized and heterologously expressed in Saccharomyces cerevisiae. The metabolic flux of canonical pathway based on overexpressing endogenous geranyl diphosphate synthase gene (ERG20) and its variant ERG20F96W-N127W was strengthened for improvement d-limonene production in Chinese Baijiu. To further elevate production, we established an orthogonal pathway by introducing neryl diphosphate synthase 1 (tNDPS1) from Solanum lycopersicum. The results showed that expressing ERG20 and ERG20F96W-N127W could enhance d-limonene synthesis, while expressing heterologous NPP synthase gene significantly increase d-limonene formation. Furthermore, we constructed a tLS-tNDPS1 fusion protein, and the best strain yielded 9.8 mg/L d-limonene after optimizing the amino acid linker and fusion order, a 40% improvement over the free enzymes during Chinese Baijiu fermentation. Finally, under the optimized fermentation conditions, a maximum d-limonene content of 23.7 mg/L in strain AY12α-L9 was achieved, which was the highest reported production in Chinese Baijiu. In addition, we also investigated that the effect of d-limonene concentration on yeast growth and fermentation. This study provided a meaningful insight into the platform for other valuable monoterpenes biosynthesis in Chinese Baijiu fermentation.

Long noncoding RNA MEG3 plays a promoting role in the proliferation, invasion, and angiogenesis of lung adenocarcinoma cells through the AKT pathway.

The role of long noncoding RNA maternally expressed gene 3 (MEG3) in lung adenocarcinoma has not been explored entirely. In this study, it was demonstrated that the expression of MEG3 was enhanced in lung adenocarcinoma tissues from TCGA database and some specific cell lines, while the survival analysis showed that patients with higher MEG3 levels had lower survival probabilities, which suggested that MEG3 might serve as a lung adenocarcinoma promoter. In addition, the results suggested that the overexpression of MEG3 could promote the proliferation and invasion of lung adenocarcinoma cells. Furthermore, increased MEG3 expression could result in a notable increase in angiogenesis-related factors as well as in the capillary tube formation of endothelial cells, which indicates that the overexpression of MEG3 could promote the angiogenesis of lung adenocarcinoma. From a mechanistic perspective, the results obtained revealed that the upregulation of MEG3 could stimulate the AKT signaling pathway and consequently lead to the biological behaviors mentioned above. In summary, all the results obtained from this study indicated that MEG3 plays a promoting role in the tumorigenesis and angiogenesis of lung adenocarcinoma, which deserves special attention when considered as a potential therapeutic target for lung adenocarcinoma.

The therapeutic effect of verapamil in lipopolysaccharide-induced acute lung injury.

The objective of this study was to investigate the exact therapeutic effects of Verapamil on lipopolysaccharide (LPS)-induced acute lung injury (ALI) and the molecular mechanism involved, through using LPS-induced animal models as well as LPS-stimulated mouse primary peritoneal macrophages models. Our results demonstrated that Verapamil reduced LPS-induced pathological damage of the lung tissue, infiltration of inflammatory cells and the production of IL-1ß, TNF-α, and MCP-1 in the serum. The MPO activity, MDA content, lung wet/dry ratio and LDH activity were also attenuated by Verapamil. In addition, Verapamil attenuated LPS-induced inflammatory cytokine production and oxidative stress in primary murine peritoneal macrophages in vitro. Moreover, we confirmed that NF-κB/NLRP3 pathway was involved in the therapeutic effect of Verapamil against LPS-induced injury in vivo and in vitro. In conclusion, these findings indicate that Verapamil has a therapeutic effect on LPS-induced ALI in mice. The mechanism may be related to the inhibition of NF-κB and NLRP3 signaling pathways. Verapamil may be a potential therapeutic agent for the treatment of ALI.

Metabolomic analysis reveals metabolic characteristics of children with short stature caused by growth hormone deficiency.

The diagnosis of short stature (SS) is of widespread importance for later treatment. In the present paper, a metabolomic method was used to analyze the metabolic characteristics of SS children caused by endocrine metabolic diseases in order to understand the underlying biochemical mechanism and provide a potential intervention strategy for SS. According to the clinical diagnosis and family investigation, all patients with SS were confirmed to be due to the endocrine disorders, especially GH deficiency (GHD). A nuclear magnetic resonance (NMR)-based metabolomic analysis of serum was used to identify the metabolic changes in 45 SS children from the 35 healthy controls (HCs). The disturbed metabolic network related to SS was correspondingly derived from the differential metabolites. The SS children demonstrated higher serum levels of citrate, phenylalanine, creatinine, and tyrosine and lower serum levels of glucose, serine, betaine, inositol, lysine, glycerol, and glutamine compared with the HCs. The results demonstrated that the disturbed glucose metabolism and metabolism and biosynthesis of amino acids are typical metabolic features of SS, and the lower levels of lysine and glutamine are the metabolic characterization of the affected growth axes and stress state of SS, respectively. The significant changes of those serum metabolites are able to be regarded as potential biomarkers for the diagnosis of SS. Accordingly, supplemental betaine in dietary pattern, the improvement of glycometabolism, and endogenous replenishment of lysine and glutamine allow the possible treatment strategy for SS.

Effects of Vitamin A on Expressions of Apoptosis Genes Bax and Bcl-2 in Epithelial Cells of Corneal Tissues Induced by Benzalkonium Chloride in Mice with Dry Eye.

BACKGROUND The apoptosis of corneal epithelial cells participates in the pathological processes of dry eye, which is expected to be a treatment target for dry eye. The aim of this study was to investigate the effects of vitamin A (VA) on apoptosis of corneal epithelial cells in a mouse model with dry eye induced by benzalkonium chloride (BAC). MATERIAL AND METHODS We randomly divided 60 male BALB/c mice aged 8-10 weeks into 3 groups: the blank control group, the dry eye + vehicle group, and the dry eye + drug group. On the 7th day after the dry eye model successfully induced, the mouse eyeballs removed, and the mouse corneal tissues were isolated. The expression levels of Bax and Bcl-2 in corneal tissues were detected via reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. The apoptotic corneal epithelial cells were quantified using terminal deoxynucleotidyl transferase (TdT) deoxyuridine triphosphate (dUTP) nick-end labeling (TUNEL) staining technique. RESULTS VA suppressed the upregulation of the Bax gene at the mRNA and protein levels, and upregulated the expression of the Bcl-2 gene (P<0.05). TUNEL results revealed that the number of apoptotic epithelial cells in the dry eye group was 40 times larger as that in the blank control group. After the intervention of VA at an appropriate concentration, the number of apoptotic corneal epithelial cells was remarkably reduced to about 10 times that in the blank control group (P<0.05). CONCLUSIONS VA can inhibit upregulation of the expressions of Bax and Bcl-2 in the epithelial cells of mice with dry eye induced by BAC, so as to suppress the apoptosis of epithelial cells in mice with dry eye.

[Pharmacokinetics of four phenolic acids from Danshen-Chuanxiong drug pair in plasma and heart tissue of rats by UPLC-MS/MS].

This study is to investigate the compatibility mechanism of Danshen-Chuanxiong drug pair on the pharmacokinetics of four phenolic acids. A UPLC-MS/MS method for quantitative determination of salvianolic acid B( Sal B),rosmarinic acid( RA),lithospermic acid( LA) and ferulic acid( FA) in plasma and heart tissue of rats was established. After single salvianolic acids and Chuanxiong-extract or combined intravenous infusion was given to rats,plasma samples and heart tissues in different time were collected. The chromatographic separation was performed on a BEH C18 column using 0. 15% formic acid-acetonitrile as mobile phase for gradient elution. A triple-quadrupole tandem mass spectrometry equipped with an electrospray ionization source was used as detector operating on multiple-reaction monitoring( MRM) scanning in negative ionization mode. Full validation of UPLC method including calibration curves,accuracy,precision,repeatability and matrix effect was investigated to comply with quantitative analysis requirements for biological samples. There were significant differences in the major pharmacokinetic parameters of Sal B,FA and RA for intravenous infusion of salvianolic acids and Chuanxiong-extract or combined in rat plasma. The AUC of Sal B and FA were increased above 40% and100%,respectively. Their Vd and CL were dropped evidently. t1/2 and Vd of RA increased above 130%. The concentration of four phenolic acids were all increased obviously in heart tissue comparing with single infusion. These results demonstrated that the compatibility mechanism of Danshen-Chuanxiong drug pair showed synergistic effect.

DIAPH3 promoted the growth, migration and metastasis of hepatocellular carcinoma cells by activating beta-catenin/TCF signaling.

The enhanced ability of cancer cell migration and metastasis is the major cause for the cancer-related death of hepatocellular carcinoma (HCC). Better understanding the mechanisms for the motility of cancer cells will benefit the treatment. Diaphanous-related formin 3 (DIAPH3) has been reported to regulate the motility of cells by remodeling the cytoskeleton. However, the mechanism through which DIAPH3 regulated the motility of cancer cells remains largely unknown. In this study, we have shown that the expression of DIAPH3 was up-regulated in HCC. DIAPH3 positively regulated the growth, migration, colony formation, epithelia mesenchymal transition, and metastasis of HCC cells. Mechanically, DIAPH3 activated the beta-catenin/TCF signaling by binding HSP90 and disrupting the interaction between GSK3beta and HSP90. Taken together, our study demonstrated the oncogenic activity of DIAPH3 in the progression of HCC and suggested that PDIAPH3 might be a therapeutic target.

An oleaginous yeast platform for renewable 1-butanol synthesis based on a heterologous CoA-dependent pathway and an endogenous pathway.

BACKGROUND: Microbial biofuel production provides a promising sustainable alternative to fossil fuels. 1-Butanol is recognized as an advanced biofuel and is gaining attention as an ideal green replacement for gasoline. In this proof-of-principle study, the oleaginous yeast Yarrowia lipolytica was first engineered with a heterologous CoA-dependent pathway and an endogenous pathway, respectively. RESULTS: The co-overexpression of two heterologous genes ETR1 and EutE resulted in the production of 1-butanol at a concentration of 65 µg/L. Through the overexpression of multiple 1-butanol pathway genes, the titer was increased to 92 µg/L. Cofactor engineering through endogenous overexpression of a glyceraldehyde-3-phosphate dehydrogenase and a malate dehydrogenase further led to titer improvements to 121 µg/L and 110 µg/L, respectively. In addition, the presence of an endogenous 1-butanol production pathway and a gene involved in the regulation of 1-butanol production was successfully identified in Y. lipolytica. The highest titer of 123.0 mg/L was obtained through this endogenous route by combining a pathway gene overexpression strategy. CONCLUSIONS: This study represents the first report on 1-butanol biosynthesis in Y. lipolytica. The results obtained in this work lay the foundation for future engineering of the pathways to optimize 1-butanol production in Y. lipolytica.