A 4-α-Glucanotransferase from Thermus thermophilus HB8: Secretory Expression and Characterization.

4-α-glucanotransferase (4GT, EC 2.4.1.25) catalyzes the breakdown of the α-1,4 glycosidic bonds of the starch main chain and forms new α-1,4 glycosidic bonds in the side chain, which is often used to optimize the physical and chemical properties of starch and to improve the quality of starch-based food. However, the low enzyme activity of 4GT limits its production and widespread application. Herein, the 4GT gene encoding 500 amino acids from Thermus thermophilus HB8 was cloned and expressed in Escherichia coli. The purified 4GT exhibited maximum activity at pH 7.0 and 60 °C and had a good stability at pH 6.0-8.0 and 30-60 °C. It was confirmed that 4GT possessed the catalytic function of extending the branch length of potato starch. Furthermore, the 4GT gene was successfully expressed extracellularly in Bacillus subtilis. Then, the enzyme yield of 4GT increased by 4.1 times through screening of different plasmids and hosts. Additionally, the fermentation conditions were optimized to enhance 4GT extracellular enzyme yield. Finally, a recombinant Bacillus subtilis with 299.9 U/mL enzyme yield of 4GT was obtained under the optimized fermentation process. In conclusion, this study provides a valuable reference for characterization and expression of food-grade enzymes.

Environmental exposure to perfluoroalkyl substances in early pregnancy, maternal glucose homeostasis and the risk of gestational diabetes: A prospective cohort study.

BACKGROUND: Humans are widely exposed to environmental perfluoroalkyl substances (PFAS), which may affect glucose homeostasis. However, research linking PFAS exposure to glucose homeostasis during pregnancy is limited and the results were inconsistent. We aimed to investigate the association between PFAS exposure and glucose homeostasis in pregnancy in a large prospective cohort. METHODS: A total of 2747 pregnant women who participated in the Shanghai Birth Cohort, had blood samples in early pregnancy and completed a 75 g oral glucose tolerance test (OGTT) at 24-28 gestational weeks were included. 10 PFAS were determined by high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS-MS) in the plasma samples in early pregnancy. Logistic regression was used to explore the associations between PFAS concentrations and gestational diabetes mellitus (GDM), while multiple linear regression was used to model the associations between PFAS and OGTT fasting, 1-h and 2-h glucose levels. Potential confounders were adjusted. Bayesian kernel machine regression (BKMR) and a quantile-based g-computation approach (qgcomp) were employed to explore the joint and independent effects of PFAS on glucose homeostasis. RESULTS: The incidence of GDM was 11.8%. One log-unit increment in plasma concentrations in early pregnancy was associated with an increased risk of GDM for perfluorobutane sulfonate (PFBS) (adjusted odd ratio (aOR) = 1.23, 95% confidence interval (95% CI): 1.05, 1.44) and perfluoroheptanoic acid (PFHpA) (aOR = 1.25, 95% CI: 1.07, 1.46). Perfluorooctane sulfonic acid (PFOS), perfluorononanoic acid (PFNA), perfluorodecanoic acid (PFDA), perfluorohexanesulfonate (PFHxS) and PFHpA were positively correlated with 1-h and 2-h glucose levels. Results of the mixed exposure model showed that the joint effects of PFAS were significantly associated with abnormal glucose homeostasis; In the BKMR model, PFAS mixture exposure was positively associated with the GDM incidence, 1-h and 2-h glucose levels and negatively correlated with FBG level. A similar trend could be observed in qgcomp and the positive correlation between PFAS and 2-h glucose level was significant (ß = 0.12, 95% CI: 0.04, 0.20). PFOS, PFNA and PFHpA may be the main contributors after controlling for other PFAS congeners. PFOS was significantly correlated with GDM incidence and 2-h glucose level, and PFHpA was significantly associated with FBG and 2-h glucose levels. The above associations were more prominent among women with a normal prepregnant BMI. CONCLUSIONS: Environmental exposure to PFAS may affect glucose homeostasis in pregnancy and increase the risk of GDM, especially in normal weight women.

MiR-195-3p inhibits cell proliferation in cervical cancer by targeting BCDIN3D.

BACKGROUND: Cervical cancer (CC) is one of the most prevailing cancers among females. Accumulated studies concentrated on the regulatory role of micro RNA in cancers. This research is to explore the potential role of mir-195-3p in cervical cancer progression. METHODS: Bioinformatics tools were used to investigate differential expression of mir-195-3p and BCDIN3D in cervical cancer. RNA expression patterns of both mir-195-3p and BCDIN3D were detected by RT-PCR in CC cell lines. The protein expression of BCDIN3D was measured by Western Blot. Hela and Siha cell lines were transfected with mir-195-3p inhibitors, mir-195-3p mimics and BCDIN3D si-RNA, si-NC. Luciferase reporter assays were adopted to confirm the binding. The interplays between mir-195-3p and BCDIN3D were explored in CC cell lines. CCK-8 assays checked how mir-195-3p regulated cell proliferation and Ki67 was examined by Western blot for its protein expressions as a biomarker for CC cell proliferation. RESULTS: MiR-195-3p was downregulated while BCDIN3D was upregulated in cervical cancer cell lines. The binding was confirmed via Luciferase Assay. RT-PCR suggested that upregulation of mir-195-3p inhibited BCDIN3D and downregulation of BCDIN3D in return induced mir-195-3p. CCK-8 pointed out that overexpression of mir-195-3p inhibited the cell viability. Ki67 protein expression was inhibited by miR-195-3p mimics or silence of BCDIN3D. CONCLUSION: The present research led us to a conclusion that mir-195-3p might inhibit cervical cancer cell proliferation and was reversely regulated by BCDIN3D. This suggests that miR-195-3p mimics/ BCDIN3D si-RNA might be used in the treatments of cervical cancer in the future after various animal assays and clinical trials.

CD97 Is Decreased in Preeclamptic Placentas and Promotes Human Trophoblast Invasion Through PI3K/Akt/mTOR Signaling Pathway.

Preeclampsia (PE) is a pregnancy disorder leading to the morbidity and mortality. Despite the development of the understanding of etiology, the only effective treatment of PE is the delivery of the placenta. An improved mastery on the regulation of trophoblast invasion could be meaningful to alleviate the disease burden of PE. Relative expression of CD97 in PE and normal placental tissues was evaluated by quantitative real-time polymerase chain reaction, immunohistology, and Western blot. The CD97 siRNA and expression vector was transfected to cultured human trophoblast HTR-8/SVneo, and the cell invasion as well as the protein expression in PI3K/Akt/mTOR signaling pathway were evaluated. Expression of CD97 is significantly downregulated in PE placental tissues compared to normal controls. The Si-CD97 inhibits HTR-8/SVneo trophoblast cells invasion, as well as the activation of PI3K/Akt/mTOR signaling pathway. In accordance, overexpression of CD97 promotes trophoblast cell invasion. In addition, CD97 regulates FOXC2 expression and showed similar effects on PI3K/Akt/mTOR signaling pathway as specific FOXC2 inhibitor. In short, this study demonstrated the downregulation of CD97 expression in preeclamptic placentas. Further mechanism investigation revealed that CD97 promoted trophoblast invasion by targeting FOXC2 via PI3K/Akt/mTOR signaling pathway, laying the foundation for the development of PE intervention strategy by targeting CD97 in placentation and pathogenesis of PE.

CD97 Is Decreased in Preeclamptic Placentas and Promotes Human Trophoblast Invasion Through PI3K/Akt/mTOR Signaling Pathway.

Preeclampsia (PE) is a pregnancy disorder leading to the morbidity and mortality. Despite the development of the understanding of etiology, the only effective treatment of PE is the delivery of the placenta. An improved mastery on the regulation of trophoblast invasion could be meaningful to alleviate the disease burden of PE. Relative expression of CD97 in PE and normal placental tissues was evaluated by quantitative real-time polymerase chain reaction, immunohistology, and Western blot. The CD97 siRNA and expression vector was transfected to cultured human trophoblast HTR-8/SVneo, and the cell invasion as well as the protein expression in PI3K/Akt/mTOR signaling pathway were evaluated. Expression of CD97 is significantly downregulated in PE placental tissues compared to normal controls. The Si-CD97 inhibits HTR-8/SVneo trophoblast cells invasion, as well as the activation of PI3K/Akt/mTOR signaling pathway. In accordance, overexpression of CD97 promotes trophoblast cell invasion. In addition, CD97 regulates FOXC2 expression and showed similar effects on PI3K/Akt/mTOR signaling pathway as specific FOXC2 inhibitor. In short, this study demonstrated the downregulation of CD97 expression in preeclamptic placentas. Further mechanism investigation revealed that CD97 promoted trophoblast invasion by targeting FOXC2 via PI3K/Akt/mTOR signaling pathway, laying the foundation for the development of PE intervention strategy by targeting CD97 in placentation and pathogenesis of PE.

Transplant of insulin-like growth factor-1 expressing bone marrow stem cells improves functional regeneration of injured rat uterus by NF-κB pathway.

To investigate the potential beneficial effect of insulin-like growth factor-1 (IGF-1) in BMSC transplantation therapy of uterus injury and the underlying molecular mechanisms, rat BMSCs were isolated and cultured. The relative expressions of IGF-1 and IL-10 were determined by RT-PCR and immunoblotting. The secretory IL-10 and released E2 were measured using ELISA kits. The relative vWF and α-SMA expressions were determined by immunohistochemistry. The direct binding of NF-κB subunit p50 with IL-10 promoter was analysed by chromatin immunoprecipitation assay. The regulation of IL-10 expression by p50 was interrogated by luciferase reporter assay. Our data demonstrated that IGF-1 expression in BMSCs induced IL-10 expression and secretion, which was further enhanced by E2-PLGA. IGF-1 overexpression improved BMSCs transplantation therapy in rat uterus injury. We further demonstrated that both inhibition and knockdown of p50 abolished IGF-1-induced expression and secretion of IL-10 in BMSCs, which consequently compromised the IGF-1 conferred therapeutic benefits against uterus injury. Furthermore, we elucidated that p50 regulated IL-10 expression via direct association with its promoter. Our data suggested that transplantation of IGF-1 overexpressing BMSCs improved functional regeneration of injured uterus by inducing IL-10 expression and secretion via activation of NF-κB signalling.

MicroRNA-29 facilitates transplantation of bone marrow-derived mesenchymal stem cells to alleviate pelvic floor dysfunction by repressing elastin.

BACKGROUND: Pelvic floor dysfunction (PFD) is a condition affecting many women worldwide, with symptoms including stress urinary incontinence (SUI) and pelvic organ prolapse (POP). We have previously demonstrated stable elastin-expressing bone marrow-derived mesenchymal stem cells (BMSCs) attenuated PFD in rats, and aim to further study the effect of microRNA-29a-3p regulation on elastin expression and efficacy of BMSC transplantation therapy. METHODS: We inhibited endogenous microRNA-29a-3p in BMSCs and investigated its effect on elastin expression by RT-PCR and Western blot. MicroRNA-29-inhibited BMSCs were then transplanted into PFD rats, accompanied by sustained release of bFGF using formulated bFGF in poly (lactic-co-glycolic acid) (PLGA) nanoparticles (NP), followed by evaluation of urodynamic tests. RESULTS: MicroRNA-29a-3p inhibition resulted in upregulated expression and secretion of elastin in in vitro culture of BMSCs. After co-injection with PLGA-loaded bFGF NP into the PFD rats in vivo, microRNA-29a-3p-inhibited BMSCs significantly improved the urodynamic test results. CONCLUSIONS: Our multidisciplinary study, combining microRNA biology, genetically engineered BMSCs, and nanoparticle technology, provides an excellent stem cell-based therapy for repairing connective tissues and treating PFD.

Transplantation of bone marrow-derived mesenchymal stem cells expressing elastin alleviates pelvic floor dysfunction.

BACKGROUND: Pelvic floor dysfunction (PFD) is a group of clinical conditions including stress urinary incontinence (SUI) and pelvic organ prolapse (POP). The abnormality of collagen and elastin metabolism in pelvic connective tissues is implicated in SUI and POP. METHODS: To reconstitute the connective tissues with normal distribution of collagen and elastin, we transduced elastin to bone marrow-derived mesenchymal stem cells (BMSC). Elastin-expressing BMSCs were then differentiated to fibroblasts using bFGF, which produced collagen and elastin. To achieve the sustained release of bFGF, we formulated bFGF in poly (lactic-co-glycolic acid) (PLGA) nanoparticles (NP). RESULTS: In an in vitro cell culture system of 7 days, when no additional bFGF was administrated, the initial PLGA-loaded bFGF NP induced prolonged production of collagen and elastin from elastin-expressing BMSCs. In vivo, co-injection of PLGA-loaded bFGF NP and elastin-expressing BMSCs into the PFD rats significantly improved the outcome of urodynamic tests. Together, these results provided an efficient model of connective tissue engineering using BMSC and injectable PLGA-loaded growth factors. CONCLUSIONS: Our results provided the first instance of a multidisciplinary approach, combining both stem cell and nanoparticle technologies, for the treatment of PFD.

Prognostic significance of histone methyltransferase enhancer of zeste homolog 2 in patients with cervical squamous cell carcinoma.

Histone methyltransferase enhancer of zeste homolog 2 (EZH2) has been reported to be associated with certain malignant phenotypes in cervical cancer. However, clinicopathological parameters and clinical outcomes of EZH2 in cervical cancer, particularly in cervical squamous cell carcinoma (CSCC) remain largely unknown. The retrospective cohort comprising of 117 consecutive patients with CSCC was incorporated into a tissue microarray which also included 23 paired normal tissues. Immunohistochemical analysis was performed to evaluate the correlation between EZH2 expression and clinicopathological implications. Aberrant overexpression of EZH2 was frequently observed in CSCCs as compared with adjacent normal tissues (P=0.0005). Expression of EZH2 is associated with poor tumor differentiation grade (P=0.020) and lymphovascular invasion (P=0.012). Univariate analysis revealed that the patients with CSCC whose tumors exhibited higher EZH2 levels had inferior overall survival (OS) compared to those whose tumors expressed lower EZH2 (log rank P=0.004). In the multivariate analysis, EZH2 expression was an independent predictor of OS (hazard ratio = 1.836, 95% confidence interval: 1.090-2.993, P=0.022). EZH2 overexpression is common in the development of CSCC and is a promising prognostic predictor for patients with CSCC.

MicroRNA-150 predicts a favorable prognosis in patients with epithelial ovarian cancer, and inhibits cell invasion and metastasis by suppressing transcriptional repressor ZEB1.

MicroRNA (miR)-150 has been reported to be dramatically downregulated in human epithelial ovarian cancer (EOC) tissues and patients' serum compared to normal controls. This study aimed to investigate clinical significance and molecular mechanisms of miR-150 in EOC. In the current study, quantitative real-time PCR analysis showed that miR-150 was significantly downregulated in human EOC tissues compared to normal tissue samples. Then, we demonstrated the significant associations of miR-150 downregulation with aggressive clinicopathological features of EOC patients, including high clinical stage and pathological grade, and shorter overall and progression-free survivals. More importantly, the multivariate analysis identified miR-150 expression as an independent prognostic biomarker in EOC. After that, luciferase reporter assays demonstrated that Zinc Finger E-Box Binding Homeobox 1 (ZEB1), a crucial regulator of epithelial-to-mesenchymal transition (EMT), was a direct target of miR-150 in EOC cells. Moreover, we found that the ectopic expression of miR-150 could efficiently inhibit cell proliferation, invasion and metastasis by suppressing the expression of ZEB1. Furthermore, we also observed a significantly negative correlation between miR-150 and ZEB1 mRNA expression in EOC tissues (rs = -0.45, P<0.001). In conclusion, these findings offer the convincing evidence that aberrant expression of miR-150 may play a role in tumor progression and prognosis in patients with EOC. Moreover, our data reveal that miR-150 may function as a tumor suppressor and modulate EOC cell proliferation, and invasion by directly and negatively regulating ZEB1, implying the re-expression of miR-150 might be a potential therapeutic strategy for EOC.