Cloning, expression and characterization of novel fungal endoglucanases

Global energy consumption is projected to double between 1980 and 2020. Furthermore, about 65% of present energy consumption is derived by burning nonrenewable fossil fuels that increase atmospheric levels of CO2 , the major greenhouse gas implicated in global warming. It is therefore important that carbon neutral alternative energy sources be developed. For transportation fuels bioethanol derived from renewable energy sources is considered among the most attractive alternatives. Presently, most bioethanol is produced from corn kernels and sugar cane sucrose, with the United State and Brazil being the world's major producers. Cellulose is another possible feedstock that can be used to produce bioethanol. Compared with the currently used feedstocks, cellulose is very abundant in nature and its use for biofuels production would not compete with the food or animal feed industries. In order to use cellulose for bioethanol production, the cellulose must be converted into the fermentable sugar glucose. Currently, the high cost of converting the cellulose into glucose is a major impediment to using cellulose as a feedstock for the production of bioethanol. The goal of my research was to address this issue by discovering new fungal enzymes that could improve the cellulose hydrolyzing efficiency of existing commercial cellulase systems. In this study I identified, cloned, functionally expressed and characterized six endoglucanases, AfumEgl2010, AnidEgl2020, FgraEgl2010, FgraEgl1020, FgraEgl2020 and NcraEgl2010. Three of these endoglucanases, AfumEgl2010, AnidEgl2020, FgraEgl2010, were characterized in detail in this study. Only AfumEgl2010 harbours a carbohydrate-binding module (CBM). All three of the endoglucanases that were subject to detailed characterization show highest activity at pH 5.0. The temperature optima of these three endoglucanases were determined to be 40°C, 60°C and 70°C. All of them were stable during 30 minutes pre-incubation at 60°C. The kinetic parameters of these three endoglucanases and four other endoglucanases, the Trichoderma reesei (T. reesei ) Eg2/Cel5A and ApulSEQ15654, StheSEQ13822, GtraSEQ630 (previously identified by undergraduate student Christopher St-Francois) were determined at 37°C and the pH optima of each endoglucanase. The Km values ranged from 2.0 to 29 mg/ml. The Vmax values ranged from 5.7 to 41 omole/mg/min. The degree of synergism when these endoglucanases were combined with the T. reesei Cbh1/Ce17A was also determined

The impact of gum-chewing on postoperative ileus following gynecological cancer surgery: A systematic review and meta-analysis of randomized controlled trials

ObjectiveTo assess the effect and safety of gum-chewing on the prevention of postoperative ileus after gynecological cancer surgery.MethodsWe conducted a systematic review of randomized controlled trials (RCTs) published between 2000 and 2022 in English and Chinese, using the EBSCO, Web of Science, Scopus, Cochrane Central Register of Controlled Trials (Cochrane database), PubMed, Medline (via Ovid), Chinese National Knowledge Infrastructure (CNKI), China Science and Technology Journal Database, and Wan Fang databases. A total of 837 studies were screened using Endnote software, and those that met the inclusion criteria were selected for analysis. The main outcome of interest was the incidence of postoperative ileus, and secondary outcomes included time to first flatus, time to first bowel movement, and length of hospital stay.ResultsTwo authors extracted data and performed quality assessment independently. The review included six RCTs with a total of 669 patients. Compared with routine care, gum-chewing could significantly reduce the incidence of postoperative ileus (RR 0.46, 95% CI: 0.30, 0.72, P=0.0006), shorten the time to first flatus (WMD -9.58, 95% CI: -15.04, -4.12, P=0.0006), first bowel movement (WMD -11.31, 95% CI: -21.05, -1.56, P=0.02), and the length of hospital stay (WMD -1.53, 95% CI: -2.08, -0.98, P<0.00001).ConclusionsGum-chewing is associated with early recovery of gastrointestinal function after gynecological cancer surgery and may be an effective and harmless intervention to prevent postoperative ileus.Systemaic review registrationhttps://www.crd.york.ac.uk/prospero/#searchadvanced, identifier CRD42022384346

High persistence rate of hepatitis B virus in a hydrodynamic injection-based transfection model in C3H/HeN mice

AIM: To optimize the viral persistence rate in a hydrodynamic injection (HI) based hepatitis B virus (HBV) transfection mouse model.METHODS: (1) 5-6-wk-old male C3H/HeN and C57BL/6 mice were hydrodynamically injected with 10 μg endotoxin-free pAAV/HBV1.2 plasmid DNA via the tail vein. Hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg) and HBV DNA, both in the serum and liver, were detected at different time points post HI by ELISA, immunohistochemical staining or quantitative polymerase chain reaction (PCR); (2) male C3H/HeN and C57BL/6 mice, either hydrodynamically injected mice at 10 wk post HI or naïve mice, were all immunized subcutaneously with 5 μg HBsAg formulated in complete Freund's adjuvant three times at a 2-wk interval. Two weeks after the final immunization, splenocytes were isolated for T cell function analysis by ELISPOT assay; and (3) five weeks post HI, C3H/HeN mice were intragastrically administered 0.1 mg/kg entecavir once a day for 14 d, or were intraperitoneally injected with 1 mg/kg interferon (IFN)-α twice a week for 2 wk, or were treated with PBS as controls. The sera were collected and assayed for HBV DNA on days 0, 7 and 14 after drug treatment.RESULTS: (1) Approximately 90% (22/25) of the injected C3H/HeN mice were still HBsAg-positive at 46 wk post HI, whereas HBsAg in C57BL/6 mice were completely cleared at 24 wk. Serum levels of HBeAg in C3H/HeN mice were higher than those in C57BL/6 mice from 4 wk to 46 wk. HBV DNA levels in the hydrodynamically injected C3H/HeN mice were higher than those in the C57BL/6 mice, both in the serum (from 4 wk to 46 wk) and in the liver (detected at 8 wk and 46 wk post HI). Histology showed that hepatitis B core antigen and HBsAg were expressed longer in the liver of C3H/HeN mice than in C57BL/6; (2) HBsAg specific T cell responses after HBsAg vaccination in hydrodynamically injected C3H/HeN and C57BL/6 mice, or naive control mice were detected by ELISPOT assay. After stimulation with HBsAg, the frequencies of IFN-γ producing splenocytes in the hydrodynamically injected C3H/HeN mice were significantly lower than those in hydrodynamically injected C57BL/6 mice, control C3H/HeN and control C57BL/6 mice, which were 0, 17 ± 7, 18 ± 10, and 41 ± 10 SFCs/10(6) splenocytes, respectively, and the mean spot sizes showed the same pattern. Even just stimulated with PMA and ionomysin, T-cell responses elicited in the vaccinated control C3H/HeN were much higher than those in hydrodynamically injected C3H/HeN mice; and (3) For drug treatment experiments on the hydrodynamically injected C3H/HeN mice, serum HBV DNA levels in the entecavir treatment group declined (131.2 folds, P &lt; 0.01) on day 7 after treatment and kept going down. In the group of IFN-α treatment, serum HBV DNA levels declined to a lowest point (6.42 folds, P &lt; 0.05) on 7 d after treatment and then rebounded.CONCLUSION: We have developed a novel HI-based HBV transfection model using C3H/HeN mice, which had a higher HBV persistence rate than the classic C57BL/6 mouse model.</p

Label-free quantitative proteomic analysis of molting-related proteins of Trichinella spiralis intestinal infective larvae

International audienceAbstractMolting is a key step for body-size expansion and environmental adaptation of parasitic nematodes, and it is extremely important for Trichinella spiralis growth and development, but the molting mechanism is not fully understood. In this work, label-free LC–MS/MS was used to determine the proteome differences between T. spiralis muscle larvae (ML) at the encapsulated stage and intestinal infective larvae (IIL) at the molting stage. The results showed that a total of 2885 T. spiralis proteins were identified, 323 of which were differentially expressed. These proteins were involved in cuticle structural elements, regulation of cuticle synthesis, remodeling and degradation, and hormonal regulation of molting. These differential proteins were also involved in diverse intracellular pathways, such as fatty acid biosynthesis, arachidonic acid metabolism, and mucin type O-glycan biosynthesis. qPCR results showed that five T. spiralis genes (cuticle collagen 14, putative DOMON domain-containing protein, glutamine synthetase, cathepsin F and NADP-dependent isocitrate dehydrogenase) had significantly higher transcriptional levels in 10 h IIL than ML (P < 0.05), which were similar to their protein expression levels, suggesting that they might be T. spiralis molting-related genes. Identification and characterization of T. spiralis molting-related proteins will be helpful for developing vaccines and new drugs against the early enteral stage of T. spiralis

Identification of DAPK1 Promoter Hypermethylation as a Biomarker for Intra-Epithelial Lesion and Cervical Cancer: A Meta-Analysis of Published Studies, TCGA, and GEO Datasets

Background: Promoter hypermethylation in death-associated protein kinase 1 (DAPK1) gene has been long linked to cervical neoplasia, but the established results remained controversial. Here, we performed a meta-analysis to assess the associations of DAPK1 promoter hypermethylation with low-grade intra-epithelial lesion (HSIL), high-grade intra-epithelial lesion (HSIL), cervical cancer (CC), and clinicopathological features of CC.Methods: Published studies with qualitative methylation data were initially searched from PubMed, Web of Science, EMBASE, and China National Knowledge Infrastructure databases (up to March 2018). Then, quantitative methylation datasets, retrieved from the Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases, were pooled to validate the results of published studies.Results: In a meta-analysis of 37 published studies, DAPK1 promoter hypermethylation progressively increased the risk of LSIL by 2.41-fold (P = 0.012), HSIL by 7.62-fold (P &lt; 0.001), and CC by 23.17-fold (P &lt; 0.001). Summary receiver operating characteristic curves suggested a potential diagnostic value of DAPK1 promoter hypermethylation in CC, with a large area-under-the-curve of 0.83, a high specificity of 97%, and a moderate sensitivity of 59%. There were significant impacts of DAPK1 promoter hypermethylation on histological type (odds ratio (OR) = 3.53, P &lt; 0.001) and FIGO stage of CC (OR = 2.15, P = 0.003). Then, a pooled analysis of nine TCGA and GEO datasets, covering 13 CPG sites within DAPK1 promoter, identified eight CC-associated sites, six sites with diagnostic values for CC (pooled specificities: 74–90%; pooled sensitivities: 70–81%), nine loci associated with the histological type of CC, and all 13 loci with down-regulated effects on DAPK1 mRNA expression.Conclusion: The meta-analysis suggests that DAPK1 promoter hypermethylation is significantly associated with the disease severity of cervical neoplasia. DAPK1 methylation detection exhibits a promising ability to discriminate CC from cancer-free controls

Essential oil from Chenopodium ambrosioides L. induces mitochondrial-mediated pathway and endoplasmic reticulum stress-related apoptosis in human liver cancer SMMC-7721 cells

Purpose: To evaluate the cytotoxic effect of essential oil derived from Chenopodium ambrosioides L. in Sichuan Province on human liver cancer SMMC-7721 cells, as well as its possible molecular mechanisms.Methods: Cytotoxicity was characterized by MTT assay and transmission electron microscopy (TEM) of SMMC-7721 cells ultrastructure. The apoptotic effect of the essential oil was evaluated by changes in mitochondrial membrane potential and Western blot assay.Results: MTT assay data indicate that the essential oil was cytotoxic to SMMC-7721 cells, while TEN revealed that there were vacuoles and nucleus fragmentation in the SMMC-7721 cell cytosol, cell swelling, and a large amount of leakage. Mitochondrial membrane potential assay and Western Blot data indicate that the essential oil induced cell apoptosis.Conclusion: The essential oil of Chenopodium ambrosioides L. in Sichuan Province seems to induce apoptosis of human liver cancer SMMC-7721 cells via the mitochondrial-mediated pathway and endoplasmic reticulum stress. Thus, this plant requires further investigation as a potential source of ananti-liver cancer drug. Keywords: Chenopodium ambrosioides Essential oil; Anti-tumor activity, Liver cancer Apoptosis, SMMC-7721 cell

Trichostatin A relieves anxiety-and depression-like symptoms in APP/PS1 mice

Background:Cognitive deficits and behavioral disorders such as anxiety and depression are common manifestations of Alzheimer’s disease (AD). Our previous work demonstrated that Trichostatin A (TSA) could alleviate neuroinflammatory plaques and improve cognitive disorders. AD, anxiety, and depression are all associated with microglial inflammation. However, whether TSA could attenuate anxiety- and depression-like behaviors in APP/PS1 mice through anti-inflammatory signaling is still unclearly.Methods:In the present study, all mice were subjected to the open field, elevated plus maze, and forced swim tests to assess anxiety- and depression-related behaviors after TSA administration. To understand the possible mechanisms underlying the behavioral effects observed, CST7 was measured in the hippocampus of mice and LPS-treated BV2 microglia.Results:The results of this study indicated that TSA administration relieved the behaviors of depression and anxiety in APP/PS1 mice, and decreased CST7 levels in the hippocampus of APP/PS1 mice and LPS-induced BV2 cells.Conclusion:Overall, these findings support the idea that TSA might be beneficial for reducing neurobehavioral disorders in AD and this could be due to suppression of CST7-related microglial inflammation