Inside Out Computational Redesign of Cavities for Improving Thermostability and Catalytic Activity of Rhizomucor Miehei Lipase.

Cavities are created by hydrophobic interactions between residue side chain atoms during the folding of enzymes. Redesigning cavities can improve the thermostability and catalytic activity of the enzyme; however, the synergistic effect of cavities remains unclear. In this study, Rhizomucor miehei lipase (RML) was used as a model to explore volume fluctuation and spatial distribution changes of the internal cavities, which could reveal the roles of internal cavities in the thermostability and catalytic activity. We present an inside out cavity engineering (CE) strategy based on computational techniques to explore how changes in the volumes and spatial distribution of cavities affect the thermostability and catalytic activity of the enzyme. We obtained 12 single-point mutants, among which the melting temperatures (Tm) of 8 mutants showed an increase of more than 2°C. Sixteen multipoint mutations were further designed by spatial distribution rearrangement of internal cavities. The Tm of the most stable triple variant, with mutations including T21V (a change of T to V at position 21), S27A, and T198L (T21V/S27A/T198L), was elevated by 11.0°C, together with a 28.7-fold increase in the half-life at 65°C and a specific activity increase of 9.9-fold (up to 5,828 U mg-1), one of the highest lipase activities reported. The possible mechanism of decreased volumes and spatial rearrangement of the internal cavities improved the stability of the enzyme, optimizing the outer substrate tunnel to improve the catalytic efficiency. Overall, the inside out computational redesign of cavities method could help to deeply understand the effect of cavities on enzymatic stability and activity, which would be beneficial for protein engineering efforts to optimize natural enzymes. IMPORTANCE In the present study, R. miehei lipase, which is widely used in various industries, provides an opportunity to explore the effects of internal cavities on the thermostability and catalytic activity of enzymes. Here, we execute high hydrostatic pressure molecular dynamics (HP-MD) simulations to screen the critical internal cavity and reshape the internal cavities through site-directed mutation. We show that as the global internal cavity volume decreases, cavity rearrangement can improve the stability of the protein while optimizing the substrate channel to improve the catalytic efficiency. Our results provide significant insights into understanding the mechanism of action of the internal cavity. Our strategy is expected to be applied to other enzymes to promote increases in thermostability and catalytic activity.

TRPC1 exacerbate metastasis in gastric cancer via ciRS-7/miR-135a-5p/TRPC1 axis.

Metastasis is frequently occurred in end-stage GC. Nevertheless, the initiation and progression of metastasis in GC remains unclear. The transient receptor potential canonical (TRPC) has been confirmed to be crucial for metastasis in many kinds of tumors, including GC. However, the molecular mechanisms regulating TRPC1 is unclear. Therefore, we investigated the role and mechanisms of TRPC1 in GC metastasis. We first evaluated the role of TRPC1 in GC by searching the public database, and tested the expression of TRPC1 in 50 paired GC tissues by qRT-PCR and IHC assays. Then, we generated BGC-823-shTRPC1 cells and MKN-45-TRPC1 cells to investigate the effects of TRPC1 on metastasis in vitro. For the mechanism study, we applied luciferase reporter assay, RNA pull-down assay, as well as RIP assay to validate the interation of ciRS-7, miR-135a-5p and TRPC1 in GC cells. This study, we showed that TRPC1 exacerbate EMT in gastric cancer via ciRS-7/miR-135a-5p/TRPC1 axis, and target TRPC1 could be beneficial for end-stage GC patients.

Metabolic engineering to improve the biomanufacturing efficiency of acetic acid bacteria: advances and prospects.

With the high tolerance for acetic acid and abundant multifunctional enzymes, acetic acid bacteria (AAB), as valuable biocatalysts, exhibit great advantages during industrial acetic acid production and value-added chemical fermentation. However, low biomass and a low production rates arising from acid stress remains major hurdles in industrial processes. Engineering AAB with excellent properties is expected to obtain economically viable production and facilitates their biotechnological applications. Here, the investigation of acetic acid-tolerance mechanisms and metabolic features is discussed, and effective targets are provided for the metabolic engineering of AAB. Next, we review the advances in improving AAB and compare these advances with improvement to other model acid-tolerant microorganisms. Furthermore, future directions involving the combination of systems biology and synthetic biology to achieve efficient biomanufacturing in AAB are highlighted.

Early classic hemofiltration exhibits no benefits in severe acute pancreatitis with early organ failure: a retrospective case-matched study.

Continuous venovenous hemofiltration (CVVH) is an important organ supportive technique. This study aimed to evaluate the impact of early classic CVVH on the outcomes of severe acute pancreatitis (SAP) patients with early organ failure (EOF). Between 2008 and 2012, a total of 44 SAP patients with EOF were admitted to our department. The 44 patients were classified into two groups according to whether they received early classic CVVH (2 L/h, initiated within 24 h after admission): 25 patients received early CVVH (ECVVH group), and 19 patients did not receive early CVVH (control group). The two groups were matched for age and Acute Physiology and Chronic Health Evaluation II scores. The severity of organ dysfunctions was evaluated by Sequential Organ Failure Assessment (SOFA) scores. Each group included 19 patients. The baseline characters between the two groups were balanced. The SOFA scores in the ECVVH group increased compared with those in the control group. The time to weaning from mechanical ventilation was significantly longer in the ECVVH group (log-rank test: χ(2) = 4.007, P = 0.045). Renal support was also significantly prolonged in the ECVVH group (the number of patients receiving CVVH 72 h after admission: 10 vs. 3, respectively, P = 0.038). Nine patients died in the ECVVH group versus six patients in the control group (P = 0.508). In conclusion, our study failed to prove that early classic CVVH had any benefits on the outcomes of SAP patients with EOF. Unexpectedly, early classic CVVH worsened organ functional capacity. However, it is possible that CVVH using advanced techniques may be beneficial in SAP patients with EOF.

Effect of umbilical cord mesenchymal stem cells on treatment of severe acute pancreatitis in rats.

BACKGROUND AIMS: The aim of this study was to investigate the effect of umbilical cord mesenchymal stem cells (UCMSCs) on severe acute pancreatitis (SAP) in rats. METHODS: SAP was established in rats by retrograde pancreatic duct injection of sodium taurocholate. In one group, 5 × 10(6) cells/kg of UCMSC suspension was injected into the tail vein 0 h, 1 h, 6 h and 12 h after the induction of SAP. In other groups, different doses of UCMSC suspension (5 × 10(4) cells/kg, 5 × 10(5) cells/kg, 5 × 10(6) cells/kg or 1 × 10(7) cells/kg) were administered at 1 h. Serum amylase was assayed at 12 h. Mortality, ascites, serum tumor necrosis factor-α, interferon-γ (assayed using enzyme-linked immunosorbent assay) and the wet-dry weight of the pancreas gland were assessed at 48 h. Pathologic changes of pancreatic and pulmonary tissues were observed. RESULTS: Mortality in rats receiving 5 × 10(6) cells/kg of UCMSCs at 0 h was 10% compared with 58% in the SAP control group. Ascites, serum amylase and wet-dry pancreatic weight significantly decreased, and production of tumor necrosis factor-α and interferon-γ were reduced. Pathologic injuries of pancreatic and pulmonary tissues were markedly alleviated. Administration of UCMSCs (5 × 10(5) cells/kg, 5 × 10(6) cells/kg or 1 × 10(7) cells/kg) at 1 h or 5 × 10(6) cells/kg at 6 h significantly reduced the severity of SAP. The effect was less marked at 12 h and with lower concentrations of UCMSCs. CONCLUSIONS: UCMSCs significantly decreased pancreatic injury caused by SAP in a time-dependent and dose-dependent way.

Co-expression of two fibrolytic enzyme genes in CHO cells and transgenic mice.

Cellulose is the main non-starch polysaccharides (NSP) in plant cell walls and acts as anti-nutritional factor in animal feed. However, monogastric animals do not synthesize enzymes that cleave such plant structural polysaccharides and thus waste of resources and pollute the environment. We described the vectors construction and co-expressions of a multi-functional cellulase EGX (with the activities of exo-ß-1,4-glucanase, endo-ß-1,4-glucanase, and endo-ß-1,4-xylanase activities) from mollusca, Ampullaria crossean and a ß-glucosidase BGL1 from Asperjillus niger in CHO cells and the transgenic mice. The recombinant enzymes were synthesised, secreted by the direction of pig PSP signal peptide and functionally active in the eukaryote systems including both of CHO cells and transgenic mice by RT-PCR analysis, western blot analysis and cellulolytic enzymes activities assays. Expressions were salivary glands-specific dependent under the control of pig PSP promoter in transgenic mice. 2A peptide was used as the self-cleaving sequence to mediate co-expression of the fusion genes and the cleavage efficiency was very high both in vitro and in vivo according to the western blot analysis. In summary, we have demonstrated that the single ORF containing EGX and BGL1 were co-expressed by 2A peptide in CHO cells and transgenic mice. It presents a viable technology for efficient disruption of plant cell wall and liberation of nutrients. To our knowledge, this is the first report using 2A sequence to produce multiple cellulases in mammalian cells and transgenic animals.

Isothermal Compressibility Perturbation as a Protein Design Principle for T1 Lipase Stability-Activity Trade-Off Counteracting.

Given the widely existing stability-activity trade-off in enzyme evolution, it is still a goal to obtain enzymes embracing both high activity and stability. Herein, we employed an isothermal compressibility (ßT) perturbation engineering (ICPE) strategy to comprehensively understand the stability-activity seesaw-like mechanism. The stability and activity of mutants derived from ICPE uncovered a high Pearson correlation (r = 0.93) in a prototypical enzyme T1 lipase. The best variant A186L/L188M/A190Y exhibited a high Tm value up to 78.70 °C, catalytic activity of 474.04 U/mg, and a 73.33% increase in dimethyl sulfoxide resistance compared to the wild type, one of the highest comprehensive performances reported to date. The elastic activation mechanism mediated by conformational change with a ΔßT range of -6.81 × 10-6 to -1.90 × 10-6 bar-1 may account for the balancing of stability and activity to achieve better performing enzymes. The ICPE strategy deepens our understanding of stability-activity trade-off and boosts its applications in enzyme engineering.

CircLONP2 Accelerates Esophageal Squamous Cell Carcinoma Progression via Direct MiR-27b-3p-ZEB1 Axis.

Circular RNAs (circRNAs) are important mediators in esophageal squamous cell carcinoma (ESCC) carcinogenesis. We aim to explore the functions and mechanisms of circLONP2 in ESCC progression. The circLONP2 level was evaluated in ESCC samples and cell lines. The role and mechanisms of circLONP2 in ESCC proliferation and migration were demonstrated in vitro. We found that circLONP2 was upregulated in human ESCC and predicts poor overall survival (OS) and disease-free survival (DFS). CircLONP2 promotes ESCC aggressiveness by directly interacting with miR-27b-3p, thus upregulating the expression levels of its target gene ZEB1 by suppressing miR-27b-3p activity. Therefore, we demonstrated that circLONP2/miR-27b-3p/ZEB1 axis promotes ESCC metastasis via regulating epithelial-to-mesenchymal transition (EMT)-related proteins. CircLONP2 may serve as an oncogenic circRNA and as a prognostic biomarker in ESCC progression.

A longitudinal cohort study on benefit finding evolution in Chinese women breast cancer survivals.

Even though the prevalence of benefit finding (BF) has been empirically shown to exist among breast cancer (BC) survivals, how does benefit finding evolve over time remains inadequately investigated. The objective of this cohort study is to examine how BF evolves over time among Chinese breast cancer survivals and determine the demographic, medical and psychosocial factors that can sustain BF increase over time. Participants were 486 women with different stages of breast cancer (stages I, II and III) followed from completion of primary treatment. Analysis were performed on the data collected during 2014-2019. During the assessment, each participant completed self-report questionnaires of characteristics and benefit finding at six time points with the interval of 6 months since BC diagnosis. The relationships between demographic, medical and psychosocial characteristics and benefit finding evolution over time were examined using mixed models. Participants reported mixed results on the evolving patterns of benefit finding: 28% reported an upward trend in BF scoring over time, 49% instead reported an downward trend, and the remaining 23% reported no obvious change. Our study has shown that some well-known covariates of benefit finding, e.g. education, income, and social support, are not associated with BF trends. In comparison, levels of spirituality and disease coping at diagnosis can more reliably predict BF evolution over time. Identifying the sustaining factors of benefit finding in the experience of breast cancer is the key to design effective psycho clinical solutions for patients' long-term post-traumatic growth. As time goes by, breast cancer patients may experience less benefit finding. Our results strongly indicate that benefit finding can be sustained and increased by encouraging attempts at meaning-making and active disease coping during breast cancer treatment. To the best of our knowledge, this study is among the first to examine the evolution trends of benefit finding over time on breast cancer survivals and determine their psychosocial predictors in developing countries.

Stem signatures associated antibodies yield early diagnosis and precise prognosis predication of patients with non-small cell lung cancer.

BACKGROUND: This study was designed to detect patients with early NSCLC with tentatively using the stem signatures associated autoantibodies (AAbs), and to evaluate its latent values in the early diagnosis and precise prognosis prediction. METHODS: The serum concentrations of selective antibodies were quantitated by enzyme-linked immunosorbent assay (ELISA), and a total of 458 cases were enrolled (training set = 401; validation set = 57). TCGA databases were used to analyze the distinct expressions and prognostic values of related genes. The optimal cut-off values were 11.60 U/ml for P53, 4.90 U/ml for MAGEA1, 3.85 U/ml for SOX2, and 7.05U/ml for PGP9.5. RESULTS: We found that the stem signatures associated antibodies of MAGEA1, PGP9.5, SOX2, and TP53 exhibited high expressions in NSCLC, negatively correlating with the overall survival (OS) (P < 0.05). In the test groups, the diagnosis sensitivity of P53, PGP9.5, SOX2, and MAGEA1 reached to 21.5%, 39.0%, 50.3%, and 35.0%, respectively, and the specificity reached to 98.7%, 99.4%, 92.2%, and 97.4%. The four candidates' panel gave a sensitivity of 71.8% with a specificity of 89%. In the validation group, the detection of the four antibodies in early diagnosis of NSCLC also exhibited high specificity and sensitivity, further consolidating their potential application. CONCLUSIONS: The detection regarding stem signatures associated antibodies could be used as effective tools in early NSCLC diagnosis, but not for localized screening of cancers, and their abnormal expression was in accordance with poorer survival.

Remodeling gut microbiota by Clostridium butyricum (C.butyricum) attenuates intestinal injury in burned mice.

BACKGROUND: The dysbiosis of gastrointestinal microbiome is an important reason for burn-induced intestinal injury. Clostridium butyricum (C.butyricum) and its production butyrate are beneficial for the homeostasis of intestinal microflora and suppression of inflammatory response. PURPOSE: The roles of C.butyricum and butyrate in burn-induced intestinal injury were explored. The effects of oral administration of C.butyricum on intestinal injury were observed in burned mice. MATERIALS AND METHODS: The skin surface of mice was exposed to 95 °C water to induce a burn injury. Then the intestinal microbiome structure, abundance of C.butyricum and level of butyrate were respectively observed. The correction between intestinal permeability indicated by FITC dextran level and abundance of C.butyricum or level of butyrate was analyzed. C.butyricum was cultured and orally administrated to burned mice. The levels of butyrate, FITC dextran and pro-inflammatory cytokines, including interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α) were respectively measured. RESULTS: Burn injury altered the intestinal microbiome structure of mice, and especially decreased the abundance of C.butyricum and level of butyrate. Both the abundance of C.butyricum and the level of butyrate were negatively correlated with the intestinal permeability. Oral administration of C.butyricum increased the level of butyrate, decreased levels of TNF-α and IL-6, and suppressed intestinal damage in burn-injured mice. CONCLUSION: Oral administration of C.butyricum significantly alleviated the intestinal damage induced by burn injury. The therapeutic effects of C.butyricum and butyrate on burn injury should be further explored, which deserves further investigation.

The continual reassessment method for multiple toxicity grades: a bayesian model selection approach.

Grade information has been considered in Yuan et al. (2007) wherein they proposed a Quasi-CRM method to incorporate the grade toxicity information in phase I trials. A potential problem with the Quasi-CRM model is that the choice of skeleton may dramatically vary the performance of the CRM model, which results in similar consequences for the Quasi-CRM model. In this paper, we propose a new model by utilizing bayesian model selection approach--Robust Quasi-CRM model--to tackle the above-mentioned pitfall with the Quasi-CRM model. The Robust Quasi-CRM model literally inherits the BMA-CRM model proposed by Yin and Yuan (2009) to consider a parallel of skeletons for Quasi-CRM. The superior performance of Robust Quasi-CRM model was demonstrated by extensive simulation studies. We conclude that the proposed method can be freely used in real practice.

MicroRNA-149 inhibits proliferation and cell cycle progression through the targeting of ZBTB2 in human gastric cancer.

An increasing body of evidence indicates that miR-149 can both suppress and promote tumor growth depending on the tumor type. However, the role of miR-149 in the progression of gastric cancer (GC) remains unknown. Here we report that miR-149 is a tumor suppressor in human gastric cancer. miR-149 expression is decreased in GC cell lines and clinical specimens in comparison to normal gastric epithelial cell and tissues, respectively. The expression levels of miR-149 also correlate with the differentiation degree of GC cells and tissues. Moreover, ectopic expression of miR-149 in gastric cancer cells inhibits proliferation and cell cycle progression by down-regulating ZBTB2, a potent repressor of the ARF-HDM2-p53-p21 pathway, with a potential binding site for miR-149 in its mRNA's 3'UTR. It is also found that ZBTB2 expression increases in GC cells and tissues compared to normal gastric epithelial cell and tissues, respectively. Silencing of ZBTB2 leads to suppression of cell growth and cell cycle arrest in G0/G1 phase, indicating that ZBTB2 may act as an oncogene in GC. Furthermore, transfection of miR-149 mimics into gastric cancer cells induces down-regulation of ZBTB2 and HDM2, and up-regulation of ARF, p53, and p21 compared to the controls. In summary, our data suggest that miR-149 functions as a tumor suppressor in human gastric cancer by, at least partially through, targeting ZBTB2.