A novel starch-active lytic polysaccharide monooxygenase discovered with bioinformatics screening and its application in textile desizing.

BACKGROUND: Lytic polysaccharide monooxygenases (LPMOs) catalyzing the oxidative cleavage of different types of polysaccharides have potential to be used in various industries. However, AA13 family LPMOs which specifically catalyze starch substrates have relatively less members than AA9 and AA10 families to limit their application range. Amylase has been used in enzymatic desizing treatment of cotton fabric for semicentury which urgently need for new assistant enzymes to improve reaction efficiency and reduce cost so as to promote their application in the textile industry. RESULTS: A total of 380 unannotated new genes which probably encode AA13 family LPMOs were discovered by the Hidden Markov model scanning in this study. Ten of them have been successfully heterologous overexpressed. AlLPMO13 with the highest activity has been purified and determined its optimum pH and temperature as pH 5.0 and 50 °C. It also showed various oxidative activities on different substrates (modified corn starch > amylose > amylopectin > corn starch). The results of enzymatic textile desizing application showed that the best combination of amylase (5 g/L), AlLPMO13 (5 mg/L), and H2O2 (3 g/L) made the desizing level and the capillary effects increased by 3 grades and more than 20%, respectively, compared with the results treated by only amylase. CONCLUSION: The Hidden Markov model constructed basing on 34 AA13 family LPMOs was proved to be a valid bioinformatics tool for discovering novel starch-active LPMOs. The novel enzyme AlLPMO13 has strong development potential in the enzymatic textile industry both concerning on economy and on application effect.

[Gene-gene/gene-environment interaction of transforming growth factor-ß signaling pathway and the risk of non-syndromic oral clefts].

OBJECTIVE: To explore the association between polymorphisms of transforming growth factor-ß (TGF-ß) signaling pathway and non-syndromic cleft lip with or without cleft palate (NSCL/P) among Asian populations, while considering gene-gene interaction and gene-environment interaction. METHODS: A total of 1 038 Asian NSCL/P case-parent trios were ascertained from an international consortium, which conducted a genome-wide association study using a case-parent trio design to investigate the genes affec-ting risk to NSCL/P. After stringent quality control measures, 343 single nucleotide polymorphism (SNP) spanning across 10 pivotal genes in the TGF-ß signaling pathway were selected from the original genome-wide association study(GWAS) dataset for further analysis. The transmission disequilibrium test (TDT) was used to test for SNP effects. The conditional Logistic regression models were used to test for gene-gene interaction and gene-environment interaction. Environmental factors collected for the study included smoking during pregnancy, passive smoking during pregnancy, alcohol intake during pregnancy, and vitamin use during pregnancy. Due to the low rates of exposure to smoking during pregnancy and alcohol consumption during pregnancy (<3%), only the interaction between maternal smoking during pregnancy and multivitamin supplementation during pregnancy was analyzed. The threshold for statistical significance was rigorously set at P =1.46×10-4, applying Bonferroni correction to account for multiple testing. RESULTS: A total of 23 SNPs in 4 genes yielded nominal association with NSCL/P (P<0.05), but none of these associations was statistically significant after Bonferroni' s multiple test correction. However, there were 6 pairs of SNPs rs4939874 (SMAD2) and rs1864615 (TGFBR2), rs2796813 (TGFB2) and rs2132298 (TGFBR2), rs4147358 (SMAD3) and rs1346907 (TGFBR2), rs4939874 (SMAD2) and rs1019855 (TGFBR2), rs4939874 (SMAD2) and rs12490466 (TGFBR2), rs2009112 (TGFB2) and rs4075748 (TGFBR2) showed statistically significant SNP-SNP interaction (P<1.46×10-4). In contrast, the analysis of gene-environment interactions did not yield any significant results after being corrected by multiple testing. CONCLUSION: The comprehensive evaluation of SNP associations and interactions within the TGF-ß signaling pathway did not yield any direct associations with NSCL/P risk in Asian populations. However, the significant gene-gene interactions identified suggest that the genetic architecture influencing NSCL/P risk may involve interactions between genes within the TGF-ß signaling pathway. These findings underscore the necessity for further investigations to unravel these results and further explore the underlying biological mechanisms.

Evidence of the folate-mediated one-carbon metabolism pathway genes in controlling the non-syndromic oral clefts risks.

The folate-mediated one-carbon metabolism pathway is thought to play an important role in the etiology of non-syndromic oral clefts (NSOFC), although none of the genes in this pathway has shown significant signals in genome-wide association studies (GWAS). Recent evidence indicated that enhanced understanding could be gained by aggregating multiple SNPs effect simultaneously into polygenic risk score (PRS) to assess its association with disease risks. This study is aimed to assess the association between the genetic effect of folate-mediated one-carbon metabolism pathway and NSOFC risks using PRS based on a case-parent trio design. A total of 297 SNPs mapped from 18 genes in the folate-mediated one-carbon metabolism pathway were aggregated from a GWAS of 2458 case-parent trios recruited from an international consortium. We found a PRS based on the folate-mediated one-carbon metabolism pathway was significant among all NSOFC trios (OR = 1.95, 95% CI: 1.66-2.28, p = 2.39 × 10-16 ), as well as two major subtypes, non-syndromic cleft lip with or without cleft palate (NSCL/P) trios (OR = 1.71, 95% CI: 1.50-1.96, p = 7.66 × 10-15 ) and non-syndromic cleft palate only (NSCPO) trios (OR = 1.51, 95% CI: 1.36-1.68, p = 2.1 × 10-14 ). Similar results were also observed in further subgroup analyses stratified into Asian and European trios. The averaged PRS of the folate-mediated one-carbon metabolism pathway varied between the NSOFC case group and its comparison group (p < 0.05) with higher average PRS in the cases. Moreover, the top 5% pathway PRS group had 2.25 (95% CI: 1.85-2.73) times increased NSOFC risk, also 3.09 (95% CI: 2.50-3.81) and 2.06 (95% CI: 1.39-3.02) times increased risk of NSCL/P and NSCPO compared to the remainder of the distribution. The results of our study confirmed the folate-mediated one-carbon metabolism pathway was important in controlling risk to NSOFC and this study enhanced evidence towards understanding the genetic risks of NSOFC.

Gender disparities in the mediating role of symptom knowledge level in reducing acute coronary syndrome (ACS) decision delay: Findings from a community-based study in China.

BACKGROUND: Implementing training programs to educate patients on the prodromal symptoms of acute coronary syndrome (ACS) may assist patients in accurately recognizing these symptoms, and ultimately decrease their time delay in seeking emergency medical services (EMS). However, the effectiveness of this approach remains uncertain, particularly among the Chinese population. METHODS: A cross-sectional study was conducted within 22 communities in Beijing, China between 2015 and 2018, with a total of 1099 participants recruited. The study utilized a standardized questionnaire to evaluate the presence of intentional decision delay in turning to EMS under a hypothetical chest pain, the participants' knowledge of ACS prodromal symptoms, and whether they had ever received any training programs aimed at increasing their symptom knowledge. Mediation analysis was performed with regression models and bootstrapping methods, and gender difference was further analyzed through moderated mediation analysis. RESULTS: A total of 1099 participants (58.2% female, median [IQR] age 34 [20]) were included in the study. The results of the mediation analysis indicated that training programs were associated with a decrease risk in decision delay, with increased knowledge playing a mediating role (mediation effect/total effect = 36.59%, P < 0.0001). Gender modified this mediation effect, with it being observed only in the male group. Specifically, training programs were not found to significantly decrease decision delay among females (P > 0.05), even though they did improve women's knowledge of ACS prodromal symptoms (ß = 0.57, P = 0.012). CONCLUSION: The results suggested a relationship between prior training programs and reduced decision delay, with increased knowledge of prodromal symptoms of ACS serving as a mediator. However, the effect was only observed in male participants and not in female participants. This highlights the notion that mere transfer of knowledge regarding ACS prodromal symptoms may not be sufficient to mitigate decision delay in the female population. Further research is needed to corroborate these results and to gain deeper insights into the gender-specific barriers encountered in this study.

Wound complications and bleeding with new oral anticoagulants in patients undergoing total joint arthroplasty: A systematic review and meta-analysis of randomized controlled trials.

AIMS: The results of associations between new oral anticoagulants (NOACs) and wound complications after total joint arthroplasty remain inconsistent. We conducted a systematic review and meta-analysis of randomized controlled trials to make comparisons with low molecular weight heparins (LMWH) on the clinical outcomes of total wound complications, together with other efficacy and safety endpoints to further evaluate the safety and efficacy of NOACs. METHODS: This meta-analysis was conducted based on a published protocol (PROSPERO: CRD42019140841). We searched for available articles in PubMed, Embase and Cochrane Library through Jun 62 021. Random-effects meta-analyses, including subgroup analyses, were conducted to estimate the pooled relative risk (RR) and 95% confidence interval (CI) for specific doses of NOACs. RESULTS: We retrieved 1683 studies, of which 20 were eligible for inclusion. We found that apixaban was associated with a lower incidence of total wound complications compared with LMWH (RR = 0.81; 95% CI: 0.65-1.00), while dabigatran and rivaroxaban did not increase the risk of total wound complications. In addition, apixaban was associated with a reduction in the risk of major/clinically relevant nonmajor bleeding events compared to LMWH (RR = 0.80, 95% CI: 0.65-0.99), while rivaroxaban increased the risk for major/clinically relevant nonmajor bleeding events (RR = 1.23, 95% CI: 1.02-1.50). Moreover, all 4 NOACs were associated with lower incidences of major venous thromboembolism compared with LMWH. CONCLUSION: A lower risk of wound complications was detected for apixaban, while dabigatran and rivaroxaban did not increase the risk when compared with LMWH. The efficacy of 4 NOACs was broadly similar.

Development and validation of a questionnaire-based risk scoring system to identify individuals at high risk for gastric cancer in Chinese populations.

OBJECTIVE: This study aimed to develop and validate a risk scoring system to identify high-risk individuals carrying malignant lesions in stomach for tailored gastric cancer screening. METHODS: A gastric cancer risk scoring system (GC-RSS) was developed based on questionnaire-based predictors for gastric cancer derived from systematic literature review. To assess the capability of this system for discrimination, risk scores for 8,214 and 7,235 outpatient subjects accepting endoscopic examination in two endoscopy centers, and 32,630 participants in a community-based cohort in China were calculated to plot receiver operating characteristic curves and generate area under the curve (AUC). To evaluate the performance of GC-RSS, the screening proportion, sensitivity and detection rate ratio compared to universal screening were used under different risk score cutoff values. RESULTS: GC-RSS comprised nine predictors including advanced age, male gender, low body mass index (<18.5 kg/m2), family history of gastric cancer, cigarette smoking, consumption of alcohol, preference for salty food, irregularity of meals and consumption of preserved food. This tool performed well in determining the risk of malignant gastric lesions with AUCs of 0.763, 0.706 and 0.696 in three validation sets. When subjects with risk scores ≥5 were evaluated with endoscopy, nearly 50% of these endoscopies could be saved with a detection rate of over 1.5 times achieved. When the cutoff was set at 8, only about 10% of subjects with the highest risk would be offered endoscopy, and detection rates for gastric cancer could be increased 2-4 fold compared to universal screening. CONCLUSIONS: An effective questionnaire-based GC-RSS was developed and validated. This tool may play an important role in establishing a tailored screening strategy for gastric cancer in China.

High copy number and highly stable Escherichia coli-Bacillus subtilis shuttle plasmids based on pWB980.

BACKGROUND: pWB980 derived from pUB110 is a promising expression vector in Bacillus for its high copy number and high stability. However, the low transformation rate of recombinant plasmids to the wild cells limited the application of it. On the basis of pWB980, constructing an E. coli-B. subtilis shuttle plasmid could facilitate the transformation rate to Bacillus cells. Because the insertion site for E. coli replication origin sequence (ori) is not unique in pWB980, in order to investigate the best insertion site, eight shuttle plasmids (pUC980-1 ~ pUC980-8) containing all possible insertion sites and directions were constructed. RESULTS: The results showed that all the selected insertion sites could be used to construct shuttle plasmid but some sites required a specific direction. And different insertion sites led to different properties of the shuttle plasmids. The best shuttle plasmids pUC980-1 and pUC980-2, which showed copies more than 450 per cell and segregational stabilities up to 98%, were selected for heterologous expressions of an alkaline pectate lyase gene pelN, an alkaline protease spro1 and a pullulanase gene pulA11, respectively. The highest extracellular activities of PelN, Spro1 and PulA11 were up to 5200 U/mL, 21,537 U/mL and 504 U/mL correspondingly after 54 h, 60 h and 48 h fermentation in a 10 L fermentor. Notably, PelN and Spro1 showed remarkably higher yields in Bacillus than previous reports. CONCLUSION: The optimum ori insertion site was the upstream region of BA3-1 in pWB980 which resulted in shuttle plasmids with higher copy numbers and higher stabilities. The novel shuttle plasmids pUC980-1 and pUC980-2 will be promising expression vectors in B. subtilis. Moreover, the ori insertion mechanism revealed in this work could provide theoretical guidance for further studies of pWB980 and constructions of other shuttle plasmids.

Enhanced Heterologous Production of Glycosyltransferase UGT76G1 by Co-Expression of Endogenous prpD and malK in Escherichia coli and Its Transglycosylation Application in Production of Rebaudioside.

Steviol glycosides (SGs) with zero calories and high-intensity sweetness are the best substitutes of sugar for the human diet. Uridine diphosphate dependent glycosyltransferase (UGT) UGT76G1, as a key enzyme for the biosynthesis of SGs with a low heterologous expression level, hinders its application. In this study, a suitable fusion partner, Smt3, was found to enhance the soluble expression of UGT76G1 by 60%. Additionally, a novel strategy to improve the expression of Smt3-UGT76G1 was performed, which co-expressed endogenous genes prpD and malK in Escherichia coli. Notably, this is the first report of constructing an efficient E. coli expression system by regulating prpD and malK expression, which remarkably improved the expression of Smt3-UGT76G1 by 200% as a consequence. Using the high-expression strain E. coli BL21 (DE3) M/P-3-S32U produced 1.97 g/L of Smt3-UGT76G1 with a yield rate of 61.6 mg/L/h by fed-batch fermentation in a 10 L fermenter. The final yield of rebadioside A (Reb A) and rebadioside M (Reb M) reached 4.8 g/L and 1.8 g/L, respectively, when catalyzed by Smt3-UGT76G1 in the practical UDP-glucose regeneration transformation system in vitro. This study not only carried out low-cost biotransformation of SGs but also provided a novel strategy for improving expression of heterologous proteins in E. coli.

Ethanol effects on the overexpression of heterologous catalase in Escherichia coli BL21 (DE3).

A novel method involving ethanol-induced increase in the heterologous recombinant protein expression in E. coli cells was commonly used in recent studies. However, the detailed mechanism of this method is still to be revealed. This work used comparative transcriptomic analysis and numerous experiments to uncover the mechanism of ethanol effects on the expression of heterologous catalase in the recombinant strain E. coli BL21 (pET26b-katA). The key regulatory genes malK and prpD were found to have the most significant effects on the expression of heterologous catalase. Thus, the maltose ABC transporter and carbon metabolism from propanoate metabolism to citrate cycle were found to be the main regulatory pathways activated by ethanol to enhance the synthesis of heterologous proteins. Based on these mechanisms, a universally applicable E. coli expression host strain for improving the expression of heterologous proteins might be constructed.

Construction of a plasmid interspecific transfer system in Bacillus species with the counter-selectable marker mazF.

Bacillus sp. strains as attractive hosts for the production of heterologous secretory proteins usually play important roles in bio-industry. However, low transformation efficiency of exogenous plasmids limited the application of Bacillus species. Here, a novel plasmid interspecific transfer system, with high transformation efficiency, high positive rate, and convenient manipulation, has been successfully constructed. A high electrotransformation efficiency strain Bacillus subtilis F-168 containing the counter-selectable marker mazF was used as the plasmid donor strain in this transfer method. A shuttled plasmid pBE980 and its recombinant plasmids pBE980::pulA and pBE980::HSPA were successfully transferred into the recipient Bacillus strains (Bacillus amyloliquefaciens 66, Bacillus licheniformis 124 and Bacillus megaterium 258) by this method. After co-culturing the donor cells (OD600nm = 1.3-1.7) and the recipient cells (OD600nm = 0.5-0.9) for 24 h in 22 °C, more than 1.0 × 105 positive transformants were obtained and a interspecific transformation efficiency of 1.0 × 10-3. It would provide a new approach for genetic manipulation in Bacillus strains and accelerate the research progress of the wild Bacillus strains in bio-industry.

High level extracellular production of a truncated alkaline ß-mannanase from alkaliphilic Bacillus sp. N16-5 in Escherichia coli by the optimization of induction condition and fed-batch fermentation.

To improve the extracellular production of alkaline ß-mannanase from alkaliphilic Bacillus sp. N16-5 in Escherichia coli, two truncated recombinant mannanases (32a-ManAR2 and 22b-ManAR2) were obtained. Compared with the full-length mannanases (32a-ManAR1 and 22b-ManAR1), the truncated mannanases not only showed higher secretion rate, but also exhibited higher thermostability and alkalistability. The K m value (11 mg/mL) of 32a-ManAR2 was higher than that (1.46 mg/mL) of 32a-ManAR1. The specific activity of 22b-ManAR2 was 2.7 times higher than that of 22b-ManAR1. 22b-ManAR2 showed the highest k cat/K m value of 602.7 ml/mg s. The parameters of induction for recombinant mannanase production of E. coli BL21 (pET32a-manAR2) and E. coli BL21 (pET22b-manAR2) were subsequently optimized. The yield of soluble mannanase was found to be enhanced with lower induction temperature (25 °C), lower IPTG concentration (0.01-0.05 mM), and Triton X-100 supplement (0.1 %) in a shake flask. Moreover, a one-time feeding strategy and Triton X-100 supplement were applied in production of 22b-ManAR2 in a 10 L fermentor. The productivity of the total soluble mannanase reached 9284.64 U/mL with the extracellular rate of 74 % at 46 h of fermentation, which was the highest productive level of alkaline ß-mannanase in recombinant E. coli to date.

Direct cloning, expression of a thermostable xylanase gene from the metagenomic DNA of cow dung compost and enzymatic production of xylooligosaccharides from corncob.

OBJECTIVES: To acquire a thermostable xylanase, that is suitable for xylooligosaccharide production from pretreated corncobs, the metagenomic method was used to obtain the gene from an uncultured environmental microorganism. RESULTS: A thermostable xylanase-encoding gene (xyn10CD18) was cloned directly from the metagenomic DNA of cow dung compost. When xyn10CD18 was expressed in Bacillus megaterium MS941, extracellular xylansae activity at 106 IU/ml was achieved. The purified recombinant Xyn10CD18 was optimally active at pH 7 and 75 °C as measured over 10 min. It retained over 55% of its initial activity at 70 °C and pH 7 after 24 h. Its action on birchwood xylan for 18 h liberated xylooligosaccharides with 2°-4° of polymerization, with xylobiose and xylotetraose as the main products. When pretreated corncobs were hydrolyzed by Xyn10CD18 for 18 h, the xylooligosaccharides (DP 2-4) products increased to 80% and the xylose was just increased by 3%. CONCLUSION: Xyn10CD18 is a thermostable endoxylanase and is a promising candidate for biomass conversion and xylooligosaccharide production.

Studies on properties of the xylan­binding domain and linker sequence of xylanase XynG1­1 from Paenibacillus campinasensis G1­1.

Xylanase XynG1-1 from Paenibacillus campinasensis G1-1 consists of a catalytic domain (CD), a family 6_36 carbohydrate-binding module which is a xylan-binding domain (XBD), and a linker sequence (LS)between them. The structure of XynG1-3 from Bacillus pumilus G1-3 consists only of a CD. To investigate the functions and properties of the XBD and LS of XynG1-1, two truncated forms (XynG1-1CDL, XynG1-1CD) and three fusion derivatives (XynG1-3CDL, XynG1-3CDX and XynG1-3CDLX) were constructed and biochemically characterized. The optimum conditions for the catalytic activity of mutants of XynG1-1 and XynG1-3 were 60 °C and pH 7.0, and 55 °C and pH 8.0, respectively, the same as for the corresponding wild-type enzymes. XynGs with an XBD were stable over a broad temperature (30-80 °C)and pH range (4.0-11.0), respectively, on incubation for 3 h. Kinetic parameters (Km, kcat, kcat/Km) of XynGs were determined with soluble birchwood xylan and insoluble oat spelt xylan as substrates. XynGs with the XBD showed better affinities toward, and more efficient catalysis of hydrolysis of the insoluble substrate. The XBD had positive effects on thermostability and pH stability and a crucial function in the ability of the enzyme to bind and hydrolyze insoluble substrate. The LS had little effect on the overall stability of the xylanase and no relationship with affinities for soluble and insoluble substrates or catalytic efficiency.

Improvement of alkali stability and thermostability of Paenibacillus campinasensis Family-11 xylanase by directed evolution and site-directed mutagenesis.

The extreme process condition of high temperature and high alkali limits the applications of most of natural xylanases in pulp and paper industry. Recently, various methods of protein engineering have been used to improve the thermal and alkalic tolerance of xylanases. In this work, directed evolution and site-directed mutagenesis were performed to obtain a mutant xylanase improved both on alkali stability and thermostability from the native Paenibacillus campinasensis Family-11 xylanase (XynG1-1). Mutant XynG1-1B43 (V90R/P172H) with two units increased in the optimum pH (pH 7.0-pH 9.0) and significant improvement on alkali stability was selected from the second round of epPCR library. And the further thermoduric mutant XynG1-1B43cc16 (V90R/P172H/T84C-T182C/D16Y) with 10 °C increased in the optimum temperature (60-70 °C) was then obtained by introducing a disulfide bridge (T84C-T182C) and a single amino acid substitution (D16Y) to XynG1-1B43 using site-directed mutagenesis. XynG1-1B43cc16 also showed higher thermostability and catalytic efficiency (k cat /K m ) than that of wild-type (XynG1-1) and XynG1-1B43. The attractive improved properties make XynG1-1B43cc16 more suitable for bioleaching of cotton stalk pulp under the extreme process condition of high temperature (70 °C) and high alkali (pH 9.0).

Development and external validation of a quantitative diagnostic model for malignant gastric lesions in clinical opportunistic screening: A multicenter real-world study.

BACKGROUND: Clinical opportunistic screening is a cost-effective cancer screening modality. This study aimed to establish an easy-to-use diagnostic model serving as a risk stratification tool for identification of individuals with malignant gastric lesions for opportunistic screening. METHODS: We developed a questionnaire-based diagnostic model using a joint dataset including two clinical cohorts from northern and southern China. The cohorts consisted of 17,360 outpatients who had undergone upper gastrointestinal endoscopic examination in endoscopic clinics. The final model was derived based on unconditional logistic regression, and predictors were selected according to the Akaike information criterion. External validation was carried out with 32,614 participants from a community-based randomized controlled trial. RESULTS: This questionnaire-based diagnostic model for malignant gastric lesions had eight predictors, including advanced age, male gender, family history of gastric cancer, low body mass index, unexplained weight loss, consumption of leftover food, consumption of preserved food, and epigastric pain. This model showed high discriminative power in the development set with an area under the receiver operating characteristic curve (AUC) of 0.791 (95% confidence interval [CI]: 0.750-0.831). External validation of the model in the general population generated an AUC of 0.696 (95% CI: 0.570-0.822). This model showed an ideal ability for enriching prevalent malignant gastric lesions when applied to various scenarios. CONCLUSION: This easy-to-use questionnaire-based model for diagnosis of prevalent malignant gastric lesions may serve as an effective prescreening tool in clinical opportunistic screening for gastric cancer.

High-level production of pullulan and its biosynthesis regulation in Aureobasidium pullulans BL06.

Pullulan has many potential applications in the food, pharmaceutical, cosmetic and environmental industries. However, the yield and molecular properties of pullulan produced by various strains still need to be promoted to fit the application needs. A novel yeast-like strain Aureobasidium pullulans BL06 producing high molecular weight (Mw) pullulan (3.3 × 106 Da) was isolated and identified in this study. The remarkable Mw of pullulan produced by A. pullulans BL06 was the highest level ever reported thus far. To further regulate the biosynthesis of pullulan in A. pullulans BL06, three gene knockout strains A. pullulans BL06 ΔPMAs, A. pullulans BL06 Δmel, and A. pullulans BL06 ΔPMAsΔmel, were constructed. The results showed that A. pullulans BL06 ΔPMAs could produce 140.2 g/L of moderate Mw (1.3 × 105 Da) pullulan after 120 h of fermentation. The highest yield level of pullulan to date could vastly reduce its production cost and expand its application scope and potential. The application experiments in food preservation showed that the moderate-Mw pullulan obtained in this work could reduce the weight loss of celery cabbages and mangos by 12.5% and 22%, respectively. Thus, the novel strains A. pullulans BL06 and A. pullulans BL06 ΔPMAs possessed unlimited development prospects in pullulan production at various Mw ranges and pullulan applications in multiple fields.

Trehalose Production Using Three Extracellular Enzymes Produced via One-Step Fermentation of an Engineered Bacillus subtilis Strain.

At present, the double-enzyme catalyzed method using maltooligosyltrehalose synthase (MTSase) and maltooligosyltrehalose trehalohydrolase (MTHase) is the mainstream technology for industrial trehalose production. However, MTSase and MTHase are prepared mainly using the heterologous expression in the engineered Escherichia coli strains so far. In this study, we first proved that the addition of 3 U/g neutral pullulanase PulA could enhance the trehalose conversion rate by 2.46 times in the double-enzyme catalyzed system. Then, a CBM68 domain was used to successfully assist the secretory expression of MTSase and MTHase from Arthrobacter ramosus S34 in Bacillus subtilis SCK6. At the basis, an engineered strain B. subtilis PSH02 (amyE::pulA/pHT43-C68-ARS/pMC68-ARH), which co-expressed MTSase, MTHase, and PulA, was constructed. After the 24 h fermentation of B. subtilis PSH02, the optimum ratio of the extracellular multi-enzymes was obtained to make the highest trehalose conversion rate of 80% from 100 g/L maltodextrin. The high passage stability and multi-enzyme preservation stability made B. subtilis PSH02 an excellent industrial production strain. Moreover, trehalose production using these extracellular enzymes produced via the one-step fermentation of B. subtilis PSH02 would greatly simplify the procedure for multi-enzyme preparation and be expected to reduce production costs.

Absence of Lugol staining indicates initiation of esophageal squamous cell carcinoma: A combined genomic and epidemiologic study.

The genomic characteristics during the carcinogenic process of esophageal squamous cell carcinoma (ESCC) remain largely unknown. We report here the genomic characteristics of 106 esophageal tissues of various stages from a population-based screening cohort in China ("Endoscopic Screening for Esophageal Cancer in China" trial) and 57 ESCC tissues from a local hospital. A significant increase in somatic mutation and copy number alterations is observed in the non-dysplastic Lugol unstaining lesions (ND-LULs). Extensive clonal expansion has emerged in the ND-LULs to an extent similar to that in higher-stage lesions. The burden of genomic alterations correlates with the size of LULs in the ND-LULs. 8-year follow-up shows that ND-LULs harbor an increased risk of progression to ESCC (adjusted IRR6-10 mm vs. none = 4.66, adjusted IRR>10 mm vs. none = 40.70), and the risk is correlated with LUL size for both non-dysplastic and dysplastic lesions. Lugol unstaining can be the initial stage in the carcinogenic process of ESCC.

A pedigree-based cohort to study the genetic risk factors for cardiometabolic diseases: study design, baseline characteristics and preliminary results.

Background: We initiated the Fujian Tulou Pedigree-based Cohort (FTPC) as the integration of extended pedigrees and prospective cohort to clarify the genetic and environmental risk factors of cardiometabolic diseases. Methods: FTPC was carried out in Nanjing County, Fujian Province, China from August 2015 to December 2017 to recruit probands with the same surnames and then enroll their first-degree and more distant relatives. The participants were asked to complete questionnaire interview, physical examination, and blood collection. According to the local genealogical booklets and family registry, we reconstructed extended pedigrees to estimate the heritability of cardiometabolic traits. The follow-up of FTPC is scheduled every 5 years in the future. Results: The baseline survey interviewed 2,727 individuals in two clans. A total of 1,563 adult subjects who completed all baseline examinations were used to reconstruct pedigrees and 452 extended pedigrees were finally identified, including one seven-generation pedigree, two five-generation pedigrees, 23 four-generation pedigrees, 186 three-generation pedigrees, and 240 two-generation pedigrees. The average age of the participants was 57.4 years, with 43.6% being males. The prevalence of hypertension, diabetes and dyslipidemia in FTPC were 49.2, 10.0, and 45.2%, respectively. Based on the pedigree structure, the heritability of systolic blood pressure, diastolic blood pressure, fast blood glucose, total cholesterol, triglyceride, high-density lipoprotein, and low-density lipoprotein was estimated at 0.379, 0.306, 0.386, 0.452, 0.568, 0.852, and 0.387, respectively. Conclusion: As an extended pedigree cohort in China, FTPC will provide an important source to study both genetic and environmental risk factors prospectively.

A pyruvate-centered metabolic regulation mechanism for the enhanced expression of exogenous genes in Escherichia coli.

A novel Escherichia coli efficient expression system had been constructed in our previous study. The system was based on the overexpression of endogenous genes prpD and malK to enhance the expression of exogenous genes. In this study, a general regulatory mechanism of prpD and malK was first revealed through transcriptome analysis and many experimental verifications. We surprisingly proved that overexpression of malK could up-regulate the expression of prpD and propanoate metabolism, which leads to increased expression of exogenous genes. More importantly, the overexpression of prpD or malK could arouse a complex set of pyruvate-centered metabolic networks that mainly increase the energy supply (ATP), by-product recycling (acetate), and amino acids for the efficient expression of exogenous genes. This novel theory for promoting the efficient expression of exogenous genes will be useful in a wide range of fields. It also opens up a new perspective on the regulation of metabolism in E. coli cells.