The synthesis of fine chemicals using multi-enzyme cascade reactions is a recent hot research topic in the field of biocatalysis. The traditional chemical synthesis methods were replaced by constructing in vitro multi-enzyme cascades, then the green synthesis of a variety of bifunctional chemicals can be achieved. This article summarizes the construction strategies of different types of multi-enzyme cascade reactions and their characteristics. In addition, the general methods for recruiting enzymes used in cascade reactions, as well as the regeneration of coenzyme such as NAD(P)H or ATP and their application in multi-enzyme cascade reactions are summarized. Finally, we illustrate the application of multi-enzyme cascades in the synthesis of six bifunctional chemicals, including ω-amino fatty acids, alkyl lactams, α, ω-dicarboxylic acids, α, ω-diamines, α, ω-diols, and ω-amino alcohols.
Amino Acids ; Biocatalysis ; Amino Alcohols ; Coenzymes/metabolism* ; Diamines

Objective To investigate the value of interleukin-6 (IL-6) combined with Model for End-stage Liver Disease (MELD) score in predicting the prognosis of patients with hepatitis B virus (HBV)-related acute-on-chronic liver failure (HBV-ACLF). Methods A total of 86 patients with HBV-ACLF who were hospitalized in The First Affiliated Hospital of Soochow University from January 2015 to December 2018 were enrolled, and according to their survival status after follow-up for 90 days, they were divided into death group with 50 patients and survival group with 36 patients. ELISA was used to measure the serum level of IL-6, and a statistical analysis was performed for general information. The t -test was used for comparison of normally distributed continuous data between groups, and the Mann-Whitney U test was used for comparison of non-normally distributed continuous data between two groups; the chi-square test was used for comparison of categorical data between two groups. A Pearson correlation analysis was performed for IL-6 and other variables; a binary logistic regression analysis was used to investigate the independent risk factors for the prognosis of patients with HBV-ACLF; the receiver operating characteristic (ROC) curve was used to assess the value of IL-6 combined with MELD score in predicting the prognosis of HBV-ACLF. Results There were significant differences between the death group and the survival group in hematocrit ( t =2.413), platelet count ( t =6.584), total bilirubin (TBil) ( t =-8.070), prothrombin time (PT) ( U =77.500), international standardized ratio ( U =102.000), HBV DNA ( t =-2.767), IL-6 ( t =-16.543), and MELD score ( t =-8.192), and the death group had a significantly higher level of IL-6 than the survival group (27.13±12.18 pg/mL vs 9.72±5.56 pg/mL, P < 0.001). The Pearson correlation analysis showed that IL-6 was positively correlated with TBil and PT ( r =0.579 and 0.681, both P < 0.001). The binary logistic regression analysis showed that IL-6 (odds ratio[ OR ]=1.480, 95% confidence interval [ CI ]: 1.196~1.833, P =0.007) and MELD score ( OR =1.128, 95% CI : 1.033~1.231, P < 0.001) were independent risk factors for the death of HBV-ACLF patients within 90 days. IL-6 combined with MELD score had an area under the ROC curve (AUC) of 0.891 (95% CI : 0.778~0.999), with a higher AUC than IL-6 (AUC=0.838, 95% CI : 0.687~0.989) or MELD score (AUC=0.783, 95% CI : 0.634~0.933). IL-6 combined with MELD score had a significantly higher value than IL-6 alone in predicting the prognosis of patients with HBV-ACLF ( Z =-2.257, P =0.024). Conclusion IL-6 combined with MELD score can be used as a good model for predicting the short-term prognosis of patients with HBV-ACLF.

ObjectiveTo investigate the role of coagulation function parameters and platelet indices in thrombotic events in patients with hepatitis B virus-associated acute-on-chronic liver failure (HBV-ACLF). MethodsA total of 56 patients with HBV-ACLF who were hospitalized in The First Affiliated Hospital of Soochow University from January 2015 to December 2019 were enrolled and divided into thrombotic complication (TC) group with 24 patients and non-thrombotic complication (NTC) group with 32 patients. A retrospective analysis was performed for their general clinical data on admission, and the patients were observed in terms of the changes in coagulation function, platelet count (PLT), and the platelet function-related index mean platelet volume (MPV) on days 1-7 after admission. The t-test was used for comparison of normally distributed continuous data between two groups, and the Mann-Whitney U rank sum test was used for comparison of non-normally distributed continuous data between two groups; the chi-square test was used for comparison of categorical data between two groups. A repeated measures analysis of variance was used to compare coagulation markers within and between groups at different time points. ResultsOn admission, the TC group had a significantly younger age than the NTC group ［31.5 (29.0-34.0) years vs 48.5 (36.0-50.7) years, Z=-2.637, P=0.008］. On the day of admission, there was no significant difference in MPV between the TC group and the NTC group (P ＞0.05), while on days 2-7 after admission, there was a significant difference in MPV between the two groups (t=-2.696、-2.742、-2.894、-4.174、-3.945、-4.716,all P ＜0.01). In the TC group, MPV reached the peak value on day 5 of admission, with a mean value of 13.90±1.12 fl, which was higher than the range of normal values. On admission, all patients had a mean prothrombin time (PT) of 28.8±7.2 s, a mean activated partial thromboplastin time (APTT) of 50.5±8.7 s, and a mean international normalized ratio (INR) of 2.6±0.7, which were higher than normal values; all patients had a mean fibrinogen (Fb) level of 1.16±0.3 g/L and a mean PLT of (107.7±26.5)×109/L, which were lower than normal values. There were no significant differences in PT, APTT, Fb, INR, and PLT between the TC group and the NTC group (all P ＞0.05). ConclusionCoagulation disorder in patients with liver failure is more of a low-equilibrium state, which is complex and heterogeneous and requires individualized treatment. For patients with HBV-ACLF, the development of thrombotic events may be more associated with platelet function than PLT or conventional coagulation markers.

Objective:To explore the significance of the clinical target volume (CTV) dose optimization in the upper and middle neck in protecting the laryngopharynx, anterior and posterior rings during intensity-modulated radiotherapy (IMRT) and multimodal imaging system for nasopharyngeal carcinoma.Methods:Clinical data of 298 nasopharyngeal carcinoma patients admitted to Jiangsu Cancer Hospital from 2016 to 2018 were retrospectively analyzed. According to the following five strategies of CTV dose optimization in the upper and middle neck: group A, complete optimization of bilateral cervical lymph nodes (CLNs), that is, the CTV doses of bilateral CLNs were 50.4 Gy; group B, complete optimization of unilateral CLNs, that is, the CTV dose of unilateral CLNs was 50.4 Gy and the contralateral CLNs was 60 Gy; group C, incomplete optimization of bilateral CLNs, that is, the CTV doses of bilateral CLNs were 50.4 Gy, while the suspicious positive CLNs were selectively boosted to 60 Gy; group D, incomplete optimization of unilateral CLNs, that is, the CTV dose of unilateral CLNs was 50.4 Gy and the suspicious positive CLNs were selectively boosted to 60 Gy, and the CTV dose of contralateral side was 60 Gy; group E: no optimization, that is, the CTV doses of bilateral CLNs were 60 Gy.Results:Among 298 patients, 215 patients received dose optimization and 83 cases did not receive dose optimization. In the dose optimization schemes, 114 cases were assigned in group A, 36 cases in group B, 60 cases in group C and 5 cases in group D. The median (range) follow-up time was 28.5(6.0-46.3) months. The overall survival rate was 95.6%, the progression-free survival rate was 84.2% and the locoregional control rate of CLNs was 98.0%. Local relapse of CLNs occurred in six patients, including 1 case of retropharyngeal lymph node, 4 cases of Ⅱ area and 1 case of Ⅳ area. The P values of average dose of laryngopharynx in group A, group B, group C and group D compared with that in group E were<0.001, 0.016, 0.001 and 0.572, respectively. The P values of the average dose of the anterior ring in group A, group B, group C and group D compared with that in group E were<0.001, 0.011, <0.001 and 0.805, respectively. The P values of the average dose of the posterior ring in group A, group B, group C and group D compared with that in group E were<0.001, 0.004, <0.001 and 0.252, respectively.Conclusions:Combined with the metastatic rules of CLNs and multimodal imaging system, it is safe to optimize the CTV dose of the upper and middle neck during IMRT in nasopharyngeal carcinoma patients, which can significantly reduce the doses of laryngopharynx, anterior and posterior rings, thereby providing evidence for reducing the CTV dose in the upper and middle neck.

ObjectiveTo investigate the protective effect of fecal microbiota transplantation (FMT) on mice with acute-on-chronic liver failure (ACLF) and its effect on intestinal flora. MethodsA total of 40 mice were randomly divided into control group (CON group), model group (MOD group), FMT group (feces of the mice in the CON group were used as fecal microbiota donor), and FMT model group (ANFMT group, with feces of the mice in the MOD group as fecal microbiota donor), with 10 mice in each group. All mice were observed in terms of body weight, death, liver histopathology, and changes in aspartate aminotransferase (AST), alanine aminotransferase (ALT), and intestinal flora. A one-way analysis of variance was used for comparison of normally distributed continuous data between multiple groups, and the SNK-q test was used for further comparison between two groups. ResultsCompared with the CON group, the MOD group had a significant reduction in body weight and significant increases in AST and ALT (all P＜0.05), as well as large patchy necrosis of hepatocytes, significant increases in Verrucomicrobia, Akkermansia, and Erysipelatoclostridium, and significant reductions in Dubosiella and Duncaniella (all P＜0.05). Compared with the CON group, the ANFMT group had a significant increase in AST (P＜0.05), hepatocyte swelling and mild ballooning degeneration, significant increases in Unclassified and Faecalibaculum, and significant reductions in Patescibacteria, Deferribacteres, Muribaculum, Candidatus_Saccharimonas, Rikenella, Odoribacter, Mucispirillum, and Lachnospiraceae_unclassified (all P＜0.05). Compared with the MOD group, the FMT group had significant reductions in AST and ALT (both P＜0.05), mild hepatocellular necrosis and marked ballooning degeneration, significant increases in Paramuribaculum and Bilophila, and significant reductions in Firmicutes, Rikenella, and Absiella (all P＜0.05). ConclusionIntestinal flora disturbance is observed in ACLF mice, and dysbacteriosis may lead to liver injury. FMT can alleviate liver inflammation in ACLF mice and thus exert a protective effect.

ObjectiveTo investigate the association between mean platelet volume (MPV) and prognosis of hepatitis B virus-associated acute-on-chronic liver failure (HBV-ACLF) and the value of MPV in prognostic evaluation. MethodsA retrospective analysis was performed for the clinical data of 37 patients with hepatitis B cirrhosis, 44 patients with HBV-ACLF, and 27 patients with chronic hepatitis B, who were admitted to Department of Infectious Diseases, The First Affiliated Hospital of Soochow University, from January 2015 to December 2019, as well as 24 individuals who underwent physical examination during the same period of time. Related clinical data included sex, age, total bilirubin (TBil), creatinine (Cr), blood urea nitrogen (BUN), albumin (Alb), blood ammonia (NH3), routine blood test results, Model for End-Stage Liver Disease (MELD) score, and blood coagulation parameters. According to prognosis, the patients with HBV-ACLF patients were divided into improvement group with 19 patients and non-improvement group with 25 patients, and the patients were followed up for 3 months. The Kruskal-Wallis H test was used for comparison between multiple groups, and the Wilcoxon rank sum test was used for comparison within each group， the Mann-Whitney U test was used for comparison between two groups； a binary logistic regression analysis was used to investigate the independent influencing factors for prognosis, and the receiver operating characteristic (ROC) curve was used to predict the accuracy of variables. ResultsThere were significant differences in MPV, platelet count, prothrombin time (PT), international normalized ratio (INR), Alb, BUN, TBil, and MELD score between the liver cirrhosis group, the HBV-ACLF group, the chronic hepatitis group, and the healthy group (χ2=39.031， 75.837, 59.894, 56.033, 22.760, 83.353 and 70.299, all P＜0.001). Further comparison of MPV between two groups showed that the HBV-ACLF group had a significantly higher MPV than the healthy group and the chronic hepatitis group (Z=9.076 and 4.435, both corrected P＜0.001), and the liver cirrhosis group had a significantly higher MPV than the healthy group (Z=2.646, corrected P=0.049). For the patients with HBV-ACLF, there were significant differences in MPV, age, PT, INR, and MELD score between the improvement group and the non-improvement group (Z=-3.710， -2.726， -2.678， -2.322 and -2.610, all P＜0.05), and further binary logistic regression analysis showed that MPV (odds ratio ［OR］=1.175, 95% confidence interval ［CI］: 1.067-2.756, P=0.026) and MELD score (OR=1.643, 95% CI: 1.021-2.644, P=0.041) were independent influencing factors for the prognosis of HBV-ACLF. MPV alone, MELD score alone, and MPV combined with MELD score had an area under the ROC curve of 0.742, 0.731, and 0.791, respectively, in predicting the prognosis of HBV-ACLF, and MPV combined with MELD score had relatively high sensitivity (0.72) and specificity (0.895). ConclusionMPV is an independent influencing factor for the prognosis of HBV-ACLF and has a good value in predicting prognosis. MPV combined with MELD score has advantages over MPV or MELD score alone in predicting the prognosis of HBV-ACLF.

Objective:To explore the clinical characteristics and establish a corresponding prognostic scoring model in patients with early-stage clinical features of hepatitis B-induced acute-on-chronic liver failure (HBV-ACLF).Methods:Clinical characteristics of 725 cases with hepatitis B-related acute-on-chronic hepatic dysfunction (HBV-ACHD) were retrospectively analyzed using Chinese group on the study of severe hepatitis B (COSSH). The independent risk factors associated with 90-day prognosis to establish a prognostic scoring model was analyzed by multivariate Cox regression, and was validated by 500 internal and 390 external HBV-ACHD patients.Results:Among 725 cases with HBV-ACHD, 76.8% were male, 96.8% had cirrhosis base,66.5% had complications of ascites, 4.1% had coagulation failure in respect to organ failure, and 9.2% had 90-day mortality rate. Multivariate Cox regression analysis showed that TBil, WBC and ALP were the best predictors of 90-day mortality rate in HBV-ACHD patients. The established scoring model was COSS-HACHADs = 0.75 × ln(WBC) + 0.57 × ln(TBil)-0.94 × ln(ALP) +10. The area under the receiver operating characteristic curve (AUROC) of subjects was significantly higher than MELD, MELD-Na, CTP and CLIF-C ADs( P < 0.05). An analysis of 500 and 390 cases of internal random selection group and external group had similar verified results. Conclusion:HBV-ACHD patients are a group of people with decompensated cirrhosis combined with small number of organ failure, and the 90-day mortality rate is 9.2%. COSSH-ACHDs have a higher predictive effect on HBV-ACHD patients' 90-day prognosis, and thus provide evidence-based medicine for early clinical diagnosis and treatment.

Industrial enzymes have become the core "chip" for bio-manufacturing technology. Design and development of novel and efficient enzymes is the key to the development of industrial biotechnology. The scientific basis for the innovative design of industrial catalysts is an in-depth analysis of the structure-activity relationship between enzymes and substrates, as well as their regulatory mechanisms. With the development of bioinformatics and computational technology, the catalytic mechanism of the enzyme can be solved by various calculation methods. Subsequently, the specific regions of the structure can be rationally reconstructed to improve the catalytic performance, which will further promote the industrial application of the target enzyme. Computational simulation and rational design based on the analysis of the structure-activity relationship have become the crucial technology for the preparation of high-efficiency industrial enzymes. This review provides a brief introduction and discussion on various calculation methods and design strategies as well as future trends.
Biocatalysis ; Biotechnology ; Enzymes ; chemistry ; metabolism ; Metabolic Engineering ; Protein Engineering ; Structure-Activity Relationship

Objective: To analyze the subjective and objective misdiagnostic reasons of brucellosis in Suzhou City. Methods: The clinical data of inpatients with brucellosis from the department of infectious diseases, the First Affiliated Hospital of Soochow University from 2016 to 2018 were collected. The main onset methods, the first consultation department, the diagnosis and treatment process, and the main diagnosis were analyzed. Simultaneously, a questionnaire survey was conducted among doctors from different departments in Suzhou City. To investigate the knowledge and diagnosis of brucellosis by electronic questionnaire, and analyze the results. Results: Totally 24 brucellosis cases were collected. Fever (13 cases, 54.17%), lumbago/arthralgia (6 cases, 25.00%) or both (3 cases, 12.50%) were the main initial symptoms, while a patient with fever, arthralgia and orchitis(1 case, 4.17%) and another with purpura (1 case, 4.17%) were also admitted. The 24 patients were distributed among 10 different departments, in which the department of infectious diseases (6 cases, 25.00%) and orthopedics (5 cases, 20.83%) dominated. Brucellosis was considered in only 2 patients (8.33%) before pathogen results, and the main suspected diagnosis were malignant hematopathy/lymphoma (7 cases, 29.17%), sepsis (5 cases, 20.83%), and tuberculosis (4 cases, 16.67%). Totally 124 questionnaires were collected and analyzed. We divided them into 2 groups: department of infectious diseases (52/124, 41.94%) and other departments (72/124, 58.06%). The proportion of answer "yes" for the question Suzhou is the epidemic area of brucellosis, question whether brucellosis is considered in suspected patients, question whether the epidemiology is collected and question how and where to conduct the serological test was 38.71% (48/124), 32.26% (40/124), 53.23% (66/124) and 29.84% (37/124), respectively. The positive answer in the department of infectious diseases (24/52, 46.15%) was significantly higher than those of others (16/72, 22.22%, χ²=7.913, P < 0.05). The respondents thought the serological test could not be conducted, because of that the non-pasture doctors were not familiar with the test procedure, which was complex (93/124, 75.00%) and the disease was not considered (85/124, 68.55%). And the respondents thought measures helpful to reduce misdiagnosis should include setting up serological test or simplifying the procedure (96/124, 77.42%), massed learning organized by related administrations (95/124, 76.61%) and learning by books, papers and classes (72/124, 58.06%). Conclusion There is obvious clinical misdiagnosis of brucellosis in Suzhou City, which may be caused by Suzhou doctors not paying enough attention to brucellosis, being unfamiliar with the inspection process and complicated inspection process.

Racemases have been applied for the synthesis of enantiomerically pure compounds through the deracemization methods. Mandelate racemase from Pseudomonas putida was the only enzyme that catalyzes the interconversion of mandelate enantiomers. Using genome mining approaches, we identified 9 mandelate racemases (MRs). A novel racemase named ArMR with higher activity and better soluble protein expression, was isolated from Agrobacterium radiobacter. ArMR displayed the optimum catalytic activity at 50 ℃, pH 7.8 in Tris-HCl buffer. The half-life of ArMR at 50, 40 and 30 ℃ was 0.17, 27.2 and 70.7 h, respectively. KM parameter of ArMR towards (R)- and (S)-mandelic acid was 1.44 and 0.81 mmol/L, respectively; the corresponding kcat value was 410 s⁻¹ and 218 s⁻¹. In addition, KM of ArMR towards (R)- and (S)-2-chloro mandelic acid was 6.48 and 6.37 mmol/L, and the corresponding kcat value 0.22 s⁻¹ and 0.23 s⁻¹, respectively. Meanwhile, Mg²⁺ and Mn²⁺ could activate the enzyme whereas Zn²⁺ inactivated the enzyme completely. Discovery of more novel MRs provides supports further research and development of these racemases.