Global trends of waste-to-energy (WtE) technologies in carbon neutral perspective: Bibliometric analysis.

Waste-to-energy (WtE) technology is at the forefront of low-carbon municipal solid waste (MSW) treatment. MSW has been favoured by researchers in recent years due to its high potential to dispose of resources with WtE technology, which contributes to the carbon neutrality goal. However, there is a lack of research that integrates MSW WtE treatment from a global perspective and explores its future direction. Bibliometric methods are widely used because of their advantages in qualitative and quantitative literature information analysis. A comprehensive search was conducted in the Web of Science (WOS) Core Collection database, covering the period from 1990-2022, resulting in the collection of 702 articles. Subsequently, bibliometric software such as CiteSpace, VOSviewer, and Bibliometrix, were jointly employed for co-occurrence, co-citation, and cluster analyses, providing an in-depth qualitative and quantitative analysis of the research hotspots and development trends of WtE technology for MSW treatment. The research findings indicate a rapid growth in studies on carbon emission reduction through WtE technology for MSW treatment since 2015, with these related articles accounting for 50% of articles. Globally, China, the United States, Italy, and other countries were active research regions, with Chinese research institutions making the largest contributions. However, contributions from developing countries are limited. Furthermore, this study systematically elaborates on the research hotspots in this field. Finally, this study identified some frontier research hotspots and directions. Research on WtE technology primarily focuses on technological methods and policy management, particularly from the carbon neutrality perspective, emphasis WtE technology sustainability in reducing carbon emissions and achieving carbon neutrality goals. Promoting the use of assisted decision-making models in the MSW management process, and focusing on the conversion of food waste into valuable energy. It is hoped that these research directions will provide new ideas for the balanced and rapid development of WtE technology.

Association between the triglyceride-glucose index and left ventricular global longitudinal strain in patients with coronary heart disease in Jilin Province, China: a cross-sectional study.

BACKGROUND: This study aimed to investigate the association between the triglyceride-glucose (TyG) index and left ventricular global longitudinal strain (GLS) in patients with coronary heart disease and to examine the role of left ventricular GLS in detecting early changes in cardiac function in patients with coronary heart disease in the subclinical stage. METHODS: A cross-sectional study involving 178 participants with symptomatic coronary artery disease excluding myocardial infarction or left ventricular dysfunction was conducted in Jilin Province, China. Basic clinical, biochemical, and echocardiographic data were obtained from all participants. Myocardial strain parameters were compared between patients with higher TyG index and those with lower TyG index, and the association between the gradually elevated TyG index and on subclinical cardiac function in patients with coronary heart disease was evaluated. RESULTS: The GLS of left ventricle was lower in the higher TyG index group than in the lower TyG index group. As the TyG index increases, the GLS progressively decreases. The results remained stable after adjusting for confounding factors. CONCLUSIONS: A higher TyG index maybe independently associated with subclinical left ventricular dysfunction in patients with coronary heart disease.

Diabetes in intraductal papillary mucinous neoplasm of the pancreas and its association with malignancy.

BACKGROUND: Intraductal papillary mucinous neoplasms (IPMNs) have the potential to become malignant. Few studies have focused on the prevalence of the diabetes mellitus (DM) in IPMNs and its association with malignancy. We evaluated the association between DM and malignant IPMNs in this study. METHODS: A total of 226 patients with pathologically confirmed IPMNs were included. Demographic data, serum biochemical data, and imaging findings were collected. The malignant IPMNs were defined as those with high-grade dysplasia and associated invasive carcinoma. The association between DM and malignant IPMNs was studied using logistic regression analysis. RESULTS: The prevalence of DM was 17.7% for all type IPMNs and 31.7% for malignant IPMNs. Compared to patients without DM, those with DM had a 3.6-fold (odds ratio [OR]: 3.62; 95% confidence interval [CI]: 1.26-10.44) higher risk of invasive carcinoma and 2.5-fold (OR: 2.48; 95% CI: 1.06-5.77) higher risk of malignant IPMNs. Similar results were observed in main pancreatic duct (MPD) involved IPMNs. New-onset DM was associated with a 4.13-fold (OR: 4.13, 95% CI: 1.27-13.36) higher risk of invasive carcinoma. CONCLUSION: DM is associated with a higher risk of malignant IPMNs and invasive carcinoma, especially in patients with MPD-involved IPMNs.

Effects of dairy processing sludge and derived biochar on greenhouse gas emissions from Danish and Irish soils.

Globally, to ensure food security bio-based fertilizers must replace a percentage of chemical fertilizers. Such replacement must be deemed sustainable from agronomic and greenhouse gas (GHG) emission perspectives. For agronomic performance several controlled protocols are in place but not for testing GHG emissions. Herein, a pre-screening tool is presented to examine GHG emissions from bio-waste as fertilizers. The various treatments examined are as follows: soil with added mineral nitrogen (N, 140 kg N ha-1) fertilizer (MF), the same amount of MF combined with dairy processing sludge (DS), sludge-derived biochar produced at 450 °C (BC450) and 700 °C (BC700) and untreated control (CK). These treatments were combined with Danish (sandy loam) or Irish (clay loam) soils, with carbon dioxide (CO2), methane (CH4) and nitrous oxide (N2O) emissions and soil inorganic-N contents measured on selected days. During the incubation, biochar mitigated N2O emissions by regulating denitrification. BC450 reduced N2O emissions from Danish soil by 95.5% and BC700 by 97.7% compared to emissions with the sludge application, and for Irish soil, the N2O reductions were 93.6% and 32.3%, respectively. For both soils, biochar reduced CO2 emissions by 50% as compared to the sludge. The lower N2O reduction potential of BC700 for Irish soil could be due to the high soil organic carbon and clay content and pyrolysis temperature. For the same reasons emissions of N2O and CO2 from Irish soil were significantly higher than from Danish soil. The temporal variation in N2O emissions was correlated with soil inorganic-N contents. The CH4 emissions across treatments were not significantly different. This study developed a simple and cost-effective pre-screening method to evaluate the GHG emission potential of new bio-waste before its field application and guide the development of national emission inventories, towards achieving the goals of circular economy and the European Green Deal.

Development of a base editor for protein evolution via in situ mutation in vivo.

Protein evolution has significantly enhanced the development of life science. However, it is difficult to achieve in vitro evolution of some special proteins because of difficulties with heterologous expression, purification, and function detection. To achieve protein evolution via in situ mutation in vivo, we developed a base editor by fusing nCas with a cytidine deaminase in Bacillus subtilis through genome integration. The base editor introduced a cytidine-to-thymidine mutation of approximately 100% across a 5 nt editable window, which was much higher than those of other base editors. The editable window was expanded to 8 nt by extending the length of sgRNA, and conversion efficiency could be regulated by changing culture conditions, which was suitable for constructing a mutant protein library efficiently in vivo. As proof-of-concept, the Sec-translocase complex and bacitracin-resistance-related protein BceB were successfully evolved in vivo using the base editor. A Sec mutant with 3.6-fold translocation efficiency and the BceB mutants with different sensitivity to bacitracin were obtained. As the construction of the base editor does not rely on any additional or host-dependent factors, such base editors (BEs) may be readily constructed and applicable to a wide range of bacteria for protein evolution via in situ mutation.

Understanding the relative contributions of fungi and bacteria led nitrous oxide emissions in an acidic soil amended with industrial waste.

Amendment of fertilized arable soil with alkaline industrial waste has the potential to ameliorate soil acidification whilst also improving crop yield. Another co-benefit is nitrous oxide (N2O) emission abatement but the contribution of fungi and bacteria involved in this process remains unclear. Two incubation experiments were conducted to: 1) examine how amendment of acidic soils with a mixture of phosphorus tailings mixture and insoluble potassium-containing rocks (PT) affect N2O emissions and 2) understand the microbial mechanisms and relative contributions of fungi and bacteria responsible for N2O emissions. In the first incubation experiment, the four treatments consisted of: i) the study control, ii) urea, iii) PT amendment and iv) PT amendment plus urea. Results showed that the PT amendment significantly increased soil pH from 4.8 to above 6.0, and reduced N2O emissions by 65.7%. PT-amended soils had higher N2 emissions and faster O2 consumption. The PT amendment significantly increased extracellular enzyme activities of leucine aminopeptidase and N-Acetyl-ß-glucosaminidase, while it significantly decreased activities of ß-1, 4-glucosidase and ß-cellobiosidase. Two antibiotics (cycloheximide and streptomycin) combined with substrate-induced respiration method were used in the second incubation experiment. Compared to soil with urea, urea with PT amendment raised soil bacteria-related N2O from 9.2% to 18.8% while decreasing fungi-related N2O from 50.5% to 43.2%. These findings suggest that the N2O emissions from acidic soils can be considerably mitigated by the application of alkaline industrial wastes. The contribution of fungi should be considered when designing and applying N2O mitigation strategies in acidic soils. DATA AVAILABILITY: Data will be made available on request.

Luteolin alleviated neutrophilic asthma by inhibiting IL-36γ secretion-mediated MAPK pathways.

CONTEXT: Luteolin can affect multiple biological functions, such as anti-inflammatory, antioxidant and immune enhancement processes. Luteolin can inhibit inflammation of T2-high asthma, but its role in neutrophilic asthma has been insufficently studied. OBJECTIVE: This study determines the effect of luteolin on IL-36γ secretion-mediated MAPK pathway signalling in neutrophilic asthma. MATERIALS AND METHODS: The asthma model was established by using ovalbumin/lipopolysaccharide (OVA/LPS). Female 6-8-week-old C57BL/6 mice were divided into control, asthma, luteolin (20 mg/kg) and asthma + luteolin (20 mg/kg) groups. To explore the mechanism of anti-inflammatory effects of luteolin in neutrophilic asthma, Beas-2B cells were treated with luteolin (20 µmol/L), LPS (100 ng/mL), recombinant human IL-36γ protein (rhIL-36γ; 100 ng/mL) or IL-36γ siRNA. RESULTS: IL-36γ secretion and MAPK/IL-1ß signalling were significantly increased in the asthma mouse model compared with the control (p < 0.05). However, the levels of IL-36γ secretion and MAPK/IL-1ß signalling were reduced by luteolin (p < 0.05). In addition, luteolin inhibited IL-36γ and MAPK/IL-1ß levels after LPS (100 ng/mL) stimulation of Beas-2B cells (p < 0.05). We found that in Beas-2B cells, luteolin inhibited activation of the MAPK pathway and IL-1ß secretion following stimulation with rhIL-36γ (100 ng/mL; p < 0.05). Finally, IL-1ß and phosphorylated MAPK levels were found to be lower in the IL-36γ siRNA + LPS (100 ng/mL) group than in the nonspecific control (NC) siRNA + LPS group (p < 0.05). DISCUSSION AND CONCLUSIONS: Luteolin alleviated neutrophilic asthma by inhibiting IL-36γ secretion-mediated MAPK pathways. These findings provided a theoretical basis for the application of luteolin in the treatment of neutrophilic asthma.

Polyoxometalate-Incorporated Metal-Organic Network as a Heterogeneous Catalyst for Selective Oxidation of Aryl Alkenes.

Selective oxidation of aryl alkenes is important for chemical synthesis reactions, in which the key lies in the rational design of efficient catalysts. Herein, four polyoxometalate (POM)-incorporated metal-organic networks, with the formulas of [Co(ttb)(H2O)3]2[SiMo12O40]·2H2O (1), [Co(ttb)(H2O)2]2[SiW12O40]·8H2O (2), [Zn(Httb)(H2ttb)][BW12O40]·9H2O (3) and {[Zn(H2O)3(ttb)]4[Zn3(H2O)6]}[H3SiW10.5Zn1.5O40]2·24H2O (4) (ttb = 1,3,5-tri(1,2,4-triazol-1-ylmethyl)-2,4,6-trimethylbenzene), were hydrothermally synthesized and structurally characterized. Structural analysis showed that compound 1 consists of a POM-encapsulated three-dimensional (3-D) supramolecular framework; compound 2 is composed of a POM-supported 3-D coordination network; and compounds 3-4 show POM-incorporated 3-D supramolecular networks. Using selective catalytic oxidation of styrene as the model reaction, compounds 1-4 as heterogeneous catalysts display excellent performance with the double advantages of high styrene conversion and benzaldehyde selectivity owing to the synergistic effect among POM anions and transition metal (TM) centers. Among them, compound 1 exhibits the highest performance with ca. 96% styrene conversion and ca. 99% benzaldehyde selectivity in 3 h. In addition, compound 1 also displays excellent substrate compatibility, good reusability, and structural stability. Thus, a plausible reaction pathway for the selective oxidation of styrene is proposed. This study on the structure-function relationship paves a way for the rational design of POM-based heterogeneous catalysts for important catalysis applications.

The associations between serum high-density lipoprotein cholesterol levels and malignant behavior in pancreatic neuroendocrine neoplasms.

BACKGROUND: The role of serum high-density lipoprotein cholesterol (HDL-c) in tumorigenesis are observed in several endocrine-related cancers. However, its role in pancreatic neuroendocrine neoplasms (PNENs) has not been understood. In the current study, the relationship between HDL-c levels and malignant behavior in PNENs was explored. METHODS: One hundred ninety-seven patients with histopathology confirmed PNENs were included. PNENs were divided into three grades (G1, G2 and G3) as 2017 WHO classification based on ki67 index and mitosis count. The demographic data, clinical information, tumor morphological and pathological features (organs invasion, lymph node metastasis, vascular invasion and perineural invasion), and serum tumor biomarkers were collected. The relationships between HDL-c levels and malignant behaviors in PNENs were analyzed using logistic regression analysis. Models were also developed for the identification of high grade PNENs. RESULTS: The levels of serum HDL-c in G2/G3 tumor were significantly lower than that in G1 tumor (P = 0.031). However, no such difference was found between G3 and G1/G2. The proportions of low HDL-c (≤ 0.9 mmol/L) were higher in high-grade PNENs (G2/G3 or G3) than those in low-grade (G1 or G1/G2) (29.0 vs 15.2%, P = 0.032; 37.0 vs 20.5%, P = 0.023). The risk of G2/G3 tumors in patients with high serum HDL-c levels was decreased (odds ratio (OR) = 0.35, 95% confidence interval (CI): 0.12-0.99). Similarly, the risk of G3 PNENs increased in patients with low HDL-c levels (OR = 2.51, 95%CI:1.12-5.60). HDL-c level was also associated with a high ki67 index (> 55%) (OR = 0.10, 95%CI: 0.02-0.51) and neuroendocrine carcinoma G3 (OR = 0.21, 95%CI: 0.06-0.80). The area under the curve (AUC) of HDL-c + tumor size + age was 0.85 (95% CI: 0.79-0.91) in identifying G2/G3 PNENs, and HDL-c (> 0.9 mmol/L) + tumor size + age had an AUC of 0.77 (95% CI: 0.70-0.84) in identifying G3 PNENs. HDL-c level was associated with lymph node metastasis (OR = 0.24, 95%CI:0.08-0.99). CONCLUSION: Serum HDL-c levels were significantly associated with malignant behaviors in PNENs, in particular to tumor grade and lymph node metastasis.

The association between hemoglobin level and osteoporosis in a Chinese population with environmental lead and cadmium exposure.

Low hemoglobin (Hb) level or anemia is associated with osteoporosis and bone fracture. Cadmium (Cd) and lead (Pb) exposure are also risk factors of osteoporosis and anemia. However, the role of anemia in Cd/Pb related bone loss remains unclear. The aim of present study was to investigate the association between Hb level and bone loss in a population with environmental lead and cadmium exposure. One hundred and ninety-four women and 108 men with different levels of Cd/Pb exposure were included in our study. The Cd/Pb exposure was determined using graphite-furnace atomic absorption spectrometry. Forearm bone mineral density (BMD) was determined by peripheral dual-energy X-ray absortiometry. Hb concentration was determined using an automatic blood cellcounter. A logistic model was established to predict the risk of osteoporosis. The BMDs of women that had the highest quartile BCd and BPb were markedly lower than that with the lowest quartile (p < 0.05). The BMD and the prevalence of osteoporosis in men with anemia were lower and higher than that with normal Hb (p < 0.05), respectively. In men, age, BPb and anemia were independent risk factors for osteoporosis. The odds ratio (OR) of men with anemia was 11.28 (95%confidence interval (CI):1.94-65.54) and 19.56 (95%CI: 2.98-128.78) compared to those with normal Hb after adjusting for potential cofounders. No such association was found in women. The area under the curve was 0.88 (95%CI: 0.82-0.96) in predicting osteoporosis using the logistic model in men. Linear discriminant analysis also showed that 90.7% of osteoporosis was correctly classified. Our data show that anemia is associated with incident of osteoporosis in men but not in women that environmentally exposed to Pb and Cd.

Exploration of key residues and conformational change of anti-terminator protein GlpP for ligand and RNA binding.

Anti-terminator protein GlpP regulates gene expression of glycerol uptake operon at post-transcriptional level in a number of bacteria. By now, the molecular dynamics details of ligand and RNA binding by GlpP are still obscure. In this study, we employed the molecular dynamic (MD) simulation and constructed a functional verification platform of GlpP to resolve these puzzles. By combining molecular docking, MD simulation and alanine scanning mutagenesis, a ligand binding pocket consisting of R14, R104 and R157 was identified. Among these residues with positive charge, R14 was dominant for binding glycerol-3-phosphate (G3P). Moreover, the "parallel to crossed" conformational change of the predicted RNA binding region was observed in MD simulation. In this process, the interaction between R104 and E129 was crucial to trigger the conformational change. To further verify this speculation, three ligand independent mutants were obtained by error-prone PCR. The MD simulation indicated that the conformational change happened in all the three mutants, confirming the "parallel to crossed" conformational change endowed GlpP the activity of binding RNA. In recent years, as a potable biological part, anti-terminator was more and more widely used to regulate gene expression in metabolic engineering and synthetic biology. The work in this study deepened our understanding to the typical anti-terminator GlpP, contributing to the further engineering and application of this type of regulator.

Low high-density lipoprotein cholesterol levels are associated with malignant intraductal papillary mucinous neoplasms: A multicenter study.

BACKGROUND: Intraductal papillary mucinous neoplasms (IPMNs) can potentially undergo malignant transformation. Studies have shown that high-density lipoprotein cholesterol (HDL-c) was associated with the risk of cancer. In this study, the association between HDL-c and the incidence of malignancy in IPMNs was investigated. MATERIALS AND METHODS: 226 patients with histologically proven IPMNs who underwent surgery were included in the present study. Patients were assigned to a training group (n = 151) and validation group (n = 75). Patients' demographic information, clinical data, and histopathological evaluation findings were obtained from medical records. Malignant IPMNs were defined as lesions that showed high grade dysplasia and invasive carcinoma. Logistic regression analyses were used to show the association between HDL-c and malignant IPMNs. Receiver operating characteristic (ROC) curves were generated to analyze predictive performance. RESULTS: The prevalence of low HDL-c levels was higher in patients with malignant IPMNs than in those with non-malignant IPMNs (P < 0.01) in both the training group and validation group. The prevalence of malignant IPMNs decreased with an increase in HDL-c levels both in patients with all types of IPMNs, as well as in those with branch-duct IPMNs (BD-IPMNs).Logistic analysis showed that low HDL-c levels were associated with malignant IPMNs (odds ratio (OR) = 20.56, 95 % confidence interval (CI): 2.58-163.64, P < 0.01) in all types of IPMNs and BD-IPMNs (OR = 17.6, 95 %CI: 1.16-268.46, P = 0.02 ).The predictive performance of mural nodules plus low HDL-c levels was higher than that of mural nodules alone or mural nodules plus cyst size for the identification of malignant BD-IPMNs. CONCLUSIONS: HDL-c levels may serve a potential biomarker for identifying malignant IPMNs and improve the predictive ability of malignancy in BD-IPMNs.

Differentiation between non-hypervascular pancreatic neuroendocrine tumors and mass-forming pancreatitis using contrast-enhanced computed tomography.

BACKGROUND: Non-hypervascular pancreatic neuroendocrine tumors (PNETs) showed slight or iso-enhancement in contrast-enhanced computed tomography (CE-CT), which shared similar imaging findings with mass-forming pancreatitis (MFPs). PURPOSE: To explore the value of CT imaging features in differentiating the two diseases. MATERIAL AND METHODS: Fifty-one patients with histologically proved MFPs (n = 27) or non-hypervascular PNETs (n = 24) were included. Two radiologists reviewed CT imaging findings and clinical features. Logistic regression analysis was performed to identify relevant features in differentiating non-hypervascular PNETs and MFPs. Receiver operating characteristic (ROC) curve analysis was used to show the performance of the optimal parameters in differentiating non-hypervascular PNETs and MFPs. RESULTS: A well-defined margin was more common in non-hypervascular PNETs (P < 0.05) than that in MFPs. MFPs often occurred in older people (P < 0.01) and the head-neck of the pancreas compared with non-hypervascular PNETs (P < 0.05). Metastases only presented in non-hypervascular PNETs (P < 0.05). CT values at venous phase and delay phase of MFPs were higher (P = 0.010 and P = 0.029) than those in non-hypervascular PNETs. Logistic analysis showed gender, tumor margin, CT values at venous phase, and tumor components were independent predictors in differentiating the two lesions. The area under the curve (AUC) was 0.938 with a sensitivity of 87.5% and specificity of 92.6% for combined predicators. CONCLUSION: Gender, tumor margin, CT values at venous phase, and tumor components were useful predicators in differentiating non-hypervascular PNETs and MFPs.

The association between life-time dietary cadmium intake from rice and chronic kidney disease.

The association between internal cadmium exposure and chronic kidney disease (CKD) has been investigated before. However, few studies have shown the association between dietary cadmium intake and CKD. In this study, we show the association between life-time dietary cadmium intake and CKD based on a follow-up study. At baseline, we collected blood and urine samples for assays of cadmium and renal effect biomarkers. A questionnaire and food survey was given to each subject to collect diet and lifestyle information for the estimation of cadmium intake. Dietary cadmium, cadmium in blood and urine were regarded as exposure markers. Life-time dietary cadmium intake was estimated based on an individual's daily cadmium intake and exposure time. At follow-up, 467 persons (163 men and 304 women) were finally included. CKD at follow-up was considered if the estimated glomerular filtration rate (eGFR) was less than 60 mL/min/1.73 m2. The eGFR level in subjects in the highest quartile of total dietary cadmium intake (>9.34 g) was significantly lower than in those with a moderate or low intake (p < 0.01). eGFR was negatively associated with total dietary cadmium intake (ß = -0.42, 95% confidence interval (CI): -0.77 to -0.07) after adjustment with confounders. Logistic regression further showed that the risk of CKD in subjects with a high total dietary cadmium intake (>2.2 g) was higher than in those with a low intake (odds ratio (OR) = 18.16, 95%CI: 1.75-188.85). A similar association was found between the baseline urinary albumin (UALB) level and CKD incidence. A predictive model based on UALB and life-time dietary cadmium intake showed an acceptable performance (the area under the curve was 0.77 (95%CI: 0.65-0.88)). Our data show that high dietary cadmium exposure was associated with CKD after controlling for renal tubular dysfunction and internal cadmium exposure.

Surface engineering of a Pantoea agglomerans-derived phenylalanine aminomutase for the improvement of (S)-ß-phenylalanine biosynthesis.

A Pantoea agglomerans-derived phenylalanine aminomutase (PaPAM) was engineered to improve the biocatalytic synthesis of (S)-ß-phenylalanine, which is an important precursor of pharmaceuticals and peptidomimetics. A semi-rational design strategy based on a combination of surface-amino-acid engineering and the amino acid preference of the thermozyme was applied to counteract the enzyme trade-off between improving its activity and stability. The surface glycine, lysine and serine of PaPAM were mutated to alanine, arginine and alanine, respectively. A K340R mutant was screened with a 2.23-fold increased activity and 2.12-fold improved half-life at 50 °C over those of the wild-type PaPAM. These improvements resulted from the more stable enzymatic conformation as well as the more rigid inner loop in K340R. When tested in a whole-cell biocatalytic reaction, the (S)-ß-phenylalanine volumetric productivity of K340R reached 0.47 g/L·h (1.4-fold greater than that of the wild-type PaPAM), and the conversion rate was improved by 17% compared to that of the wild-type PaPAM. The enzymatic properties of K340R and the resulting (S)-ß-phenylalanine production are among the highest reported, and the results indicate that the described strategy is potent for engineering enzymatic stability and activity of PAM.

High-level extracellular production of recombinant nattokinase in Bacillus subtilis WB800 by multiple tandem promoters.

BACKGROUND: Nattokinase (NK), which is a member of the subtilisin family, is a potent fibrinolytic enzyme that might be useful for thrombosis therapy. Extensive work has been done to improve its production for the food industry. The aim of our study was to enhance NK production by tandem promoters in Bacillus subtilis WB800. RESULTS: Six recombinant strains harboring different plasmids with a single promoter (PP43, PHpaII, PBcaprE, PgsiB, PyxiE or PluxS) were constructed, and the analysis of the fibrinolytic activity showed that PP43 and PHpaII exhibited a higher expression activity than that of the others. The NK yield that was mediated by PP43 and PHpaII reached 140.5 ± 3.9 FU/ml and 110.8 ± 3.6 FU/ml, respectively. These promoters were arranged in tandem to enhance the expression level of NK, and our results indicated that the arrangement of promoters in tandem has intrinsic effects on the NK expression level. As the number of repetitive PP43 or PHpaII increased, the expression level of NK was enhanced up to the triple-promoter, but did not increase unconditionally. In addition, the repetitive core region of PP43 or PHpaII could effectively enhance NK production. Eight triple-promoters with PP43 and PHpaII in different orders were constructed, and the highest yield of NK finally reached 264.2 ± 7.0 FU/ml, which was mediated by the promoter PHpaII-PHpaII-PP43. The scale-up production of NK that was promoted by PHpaII-PHpaII-PP43 was also carried out in a 5-L fermenter, and the NK activity reached 816.7 ± 30.0 FU/mL. CONCLUSIONS: Our studies demonstrated that NK was efficiently overproduced by tandem promoters in Bacillus subtilis. The highest fibrinolytic activity was promoted by PHpaII-PHpaII-PP43, which was much higher than that had been reported in previous studies. These multiple tandem promoters were used successfully to control NK expression and might be useful for improving the expression level of the other genes.

Improvement of the acid resistance, catalytic efficiency, and thermostability of nattokinase by multisite-directed mutagenesis.

Nattokinase (NK) is a serine protease of the subtilisin family; as a potent fibrinolytic enzyme, it is potentially useful for thrombosis therapy. For NK to be applied as an oral medicine for the treatment of cardiovascular diseases, it must overcome the extremely acidic environments of the gastrointestinal tract despite its limited acidic stability. In this study, three strategies were adopted to improve the acid resistance of NK: (a) Surface charge engineering, (b) sequence alignment, and (c) mutation based on the literature. Eleven variants were constructed and four single-point mutations were screened out for their distinctive catalytic properties: Q59E increased the specific activity; S78T improved the acid stability; Y217K enhanced the acid and thermal stabilities; and N218D improved the thermostability. Based on these observations, multipoint variants were constructed and characterized, and one variant with better acid stability, catalytic efficiency, and thermostability was obtained. Molecular dynamics simulation was carried out to clarify the molecular mechanism of the increased stability of S78T and Y217K mutants under acidic conditions. This study explored effective strategies to engineer acid resistance of NK; moreover, the NK variants with better catalytic properties found in this study have potential applications for the medical industry.

Metallochaperone function of the self-subunit swapping chaperone involved in the maturation of subunit-fused cobalt-type nitrile hydratase.

The transition metal (iron or cobalt) is a mandatory part that constitutes the catalytic center of nitrile hydratase (NHase). The incorporation of the cobalt ion into cobalt-containing NHase (Co-NHase) was reported to depend on self-subunit swapping and the activator of the Co-NHase acts as a self-subunit swapping chaperone for subunit exchange. Here we discovered that the activator acting as a metallochaperone transferred the cobalt ion into subunit-fused Co-NHase. We successfully isolated two activators, P14K and NhlE, which were the activators of NHases from Pseudomonas putida NRRL-18668 and the activator of low-molecular-mass NHase from Rhodococcus rhodochrous J1, respectively. Cobalt content determination demonstrated that NhlE and P14K were two cobalt-containing proteins. Substitution of the amino acids involved in the C-terminus of the activators affected the activity of the two NHases, indicating that the potential cobalt-binding sites might be located at the flexible C-terminal region. The cobalt-free NHases could be activated by either of the two activators, and both the two activators activated their cognate NHase more efficiently than did the noncognate ones. This study provided insights into the maturation of subunit-fused NHases and confirmed the metallochaperone function of the self-subunit swapping chaperone.

Development of a novel strategy for robust synthetic bacterial promoters based on a stepwise evolution targeting the spacer region of the core promoter in Bacillus subtilis.

BACKGROUND: Promoter evolution by synthetic promoter library (SPL) is a powerful approach to development of functional synthetic promoters to synthetic biology. However, it requires much tedious and time-consuming screenings because of the plethora of different variants in SPL. Actually, a large proportion of mutants in the SPL are significantly lower in strength, which contributes only to fabrication of a promoter library with a continuum of strength. Thus, to effectively obtain the evolved synthetic promoter exhibiting higher strength, it is essential to develop novel strategies to construct mutant library targeting the pivotal region rather than the arbitrary region of the template promoter. In this study, a strategy termed stepwise evolution targeting the spacer of core promoter (SETarSCoP) was established in Bacillus subtilis to effectively evolve the strength of bacterial promoter. RESULTS: The native promoter, PsrfA, from B. subtilis, which exhibits higher strength than the strong promoter P43, was set as the parental template. According to the comparison of conservation of the spacer sequences between - 35 box and - 10 box among a set of strong and weak native promoter, it revealed that 7-bp sequence immediately upstream of the - 10 box featured in the regulation of promoter strength. Based on the conservative feature, two rounds of consecutive evolution were performed targeting the hot region of PsrfA. In the first round, a primary promoter mutation library (pPML) was constructed by mutagenesis targeting the 3-bp sequence immediately upstream of the - 10 box of the PsrfA. Subsequently, four evolved mutants from pPML were selected to construction of four secondary promoter mutation libraries (sPMLs) based on mutagenesis of the 4-bp sequence upstream of the first-round target. After the consecutive two-step evolution, the mutant PBH4 was identified and verified to be a highly evolved synthetic promoter. The strength of PBH4 was higher than PsrfA by approximately 3 times. Moreover, PBH4 also exhibited broad suitability for different cargo proteins, such as ß-glucuronidase and nattokinase. The proof-of-principle test showed that SETarSCoP successfully evolved both constitutive and inducible promoters. CONCLUSION: Comparing with the commonly used SPL strategy, SETarSCoP facilitates the evolution process to obtain strength-evolved synthetic bacterial promoter through fabrication and screening of small-scale mutation libraries. This strategy will be a promising method to evolve diverse bacterial promoters to expand the toolbox for synthetic biology.

Characterization of cysteine sulfinic acid decarboxylase from Tribolium castaneum and its application in the production of ß-alanine.

ß-alanine is a precursor for the production of pharmaceuticals and food additives that is produced by chemical methods in industry. As concerns about the environment and energy are increasing, biocatalysis using L-aspartate-α-decarboxylase (ADC) to convert L-aspartate to ß-alanine has great potential. Many studies have focused on the catalytic activity of ADC, but these researches were limited to the prokaryotic enzymes. In this study, the gene encoding cysteine sulfinic acid decarboxylase from Tribolium castaneum (TcCSADC) was synthesized and overexpressed in Escherichia coli, and the enzyme was purified and characterized for the first time. It could use L-aspartate as its substrate, and the specific activity was 4.83 µmol/min/mg, which was much higher than that of ADCs from prokaryotes. A homology modeling assay indicated that TcCSADC had a dimer structure. Based on the evolutionary information from thermophilic bacteria, twenty-three variants were constructed to attempt to improve its abilities that transform L-aspartate to ß-alanine. One mutant, G369A, was screened that had improved thermal stability. An analysis of the suitability of the catalytic process showed that the up to 162 g/L ß-alanine could be produced using cells expressing the recombinant G369A variant, which is the highest yield to date. The CSADC from T. castaneum has important value for studies of the mechanism of ADCs and CSADCs from eukaryotes, and the engineered strain containing the G369A variant has great potential for the industrial production of ß-alanine.