OBJECTIVE：To provide reference for the c onstruction of subject diagnosis and treatment scheme in drug clinical trials. METHODS ：The subject diagnosis and treatment module was developed and implemented in our hospital on the basis of CTMS，and its effects were evaluated. RESULTS ：A subject diagnosis and treatment module was established in CTMS of our hospital. Within one year from the launch of the module in the middle of October ，2019，the overall number of subjects in the group showed an increasing trend ，and the overall mean dropout rate of subjects was 0.16％. The data interface of CTMS system ， hospital information system （HIS），laboratory information management system ，medical imaging information system had been established，so as to realize the synchronization of subject information （displaying subject identification in HIS system ）and the interaction of diagnosis and treatment information and billing data （patients and subjects were charged separately ）. Since the launch of the module ，the amount of data generated by the interface had been increasing ，and the number of departments producing the subject diagnosis and treatment business had been increasing month by month. Compared with subject diagnosis and treatment project based on HIS system ，the number of subject diagnosis and treatment business based on CTMS system was increased significantly（P＜0.05）. CONCLUSIONS ：The subject diagnosis and treatment module based on CTMS improves the efficiency of subject diagnosis and treatment project implementation and financial settlement ，and realizes the efficient implementation of drug clinical trial projects in large general hospitals.

L-asparaginase hydrolyzes L-asparagine to produce L-aspartic acid and ammonia. It is widely distributed in microorganisms, plants and serum of some rodents, and has important applications in the pharmaceutical and food industries. However, the poor thermal stability, low catalytic efficiency and low yield hampered the further application of L-asparaginase. In this paper, rational design and 5' untranslated region (5'UTR) design strategies were used to increase the specific enzyme activity and protein expression of L-asparaginase derived from Rhizomucor miehei (RmAsnase). The results showed that among the six mutants constructed through homology modeling combined with sequence alignment, the specific enzyme activity of the mutant A344E was 1.5 times higher than the wild type. Subsequently, a food-safe strain Bacillus subtilis 168/pMA5-A344E was constructed, and the UTR strategy was used for the construction of recombinant strain B. subtilis 168/pMA5 UTR-A344E. The enzyme activity of B. subtilis 168/pMA5 UTR-A344E was 7.2 times higher than that of B. subtilis 168/pMA5-A344E. The recombinant strain B. subtilis 168/pMA5 UTR-A344E was scaled up in 5 L fermenter, and the final yield of L-asparaginase was 489.1 U/mL, showing great potential for industrial application.
Asparaginase/genetics* ; Bacillus subtilis/genetics* ; Industrial Microbiology ; Protein Engineering ; Rhizomucor/enzymology* ; Sequence Alignment

2-Hydroxybutyric acid (2-HBA) is an important intermediate for synthesizing biodegradable materials and various medicines. Chemically synthesized racemized 2-HBA requires deracemization to obtain optically pure enantiomers for industrial application. In this study, we designed a cascade biosynthesis system in Escherichia coli BL21 by coexpressing L-threonine deaminase (TD), NAD-dependent L-lactate dehydrogenase (LDH) and formate dehydrogenase (FDH) for production of optically pure (S)-2-HBA from bulk chemical L-threonine (L-Thr). To coordinate the production rate and the consumption rate of the intermediate 2-oxobutyric acid in the multi-enzyme cascade catalytic reactions, we explored promoter engineering to regulate the expression levels of TD and FDH, and developed a recombinant strain P21285FDH-T7V7827 with a tunable system to achieve a coordinated multi-enzyme expression. The recombinant strain P21285FDH-T7V7827 was able to efficiently produce (S)-2-HBA with the highest titer of 143 g/L and a molar yield of 97% achieved within 16 hours. This titer was approximately 1.83 times than that of the highest yield reported to date, showing great potential for industrial application. Our results indicated that constructing a multi-enzyme-coordinated expression system in a single cell significantly contributed to the biosynthesis of hydroxyl acids.
Escherichia coli/genetics* ; Formate Dehydrogenases ; Hydroxybutyrates ; Threonine Dehydratase

Leucine dehydrogenase (LDH) is the key rate-limiting enzyme in the production of L-2-aminobutyric acid (L-2-ABA). In this study, we modified the C-terminal Loop region of this enzyme to improve the specific enzyme activity and stability for efficient synthesis of L-2-ABA. Using molecular dynamics simulation of LDH, we analyzed the change of root mean square fluctuation (RMSF), rationally designed the Loop region with greatly fluctuated RMSF, and obtained a mutant EsLDHD2 with a specific enzyme activity 23.2% higher than that of the wild type. Since the rate of the threonine deaminase-catalyzed reaction converting L-threonine into 2-ketobutyrate was so fast, the multi-enzyme cascade catalysis system became unbalanced. Therefore, the LDH and the formate dehydrogenase were double copied in a new construct E. coli BL21/pACYCDuet-RM. Compared with E. coli BL21/pACYCDuet-RO, the molar conversion rate of L-2-ABA increased by 74.6%. The whole cell biotransformation conditions were optimized and the optimal pH, temperature and substrate concentration were 7.5, 35 °C and 80 g/L, respectively. Under these conditions, the molar conversion rate was higher than 99%. Finally, 80 g and 40 g L-threonine were consecutively fed into a 1 L reaction mixture under the optimal conversion conditions, producing 97.9 g L-2-ABA. Thus, this strategy provides a green and efficient synthesis of L-2-ABA, and has great industrial application potential.
Aminobutyrates ; Escherichia coli/genetics* ; Leucine Dehydrogenase/genetics* ; Threonine Dehydratase

D-allulose-3-epimerase (DPEase) is the key enzyme for isomerization of D-fructose to D-allulose. In order to improve its thermal stability, short amphiphilic peptides (SAP) were fused to the N-terminal of DPEase. SDS-PAGE analysis showed that the heterologously expressed DPEase folded correctly in Bacillus subtilis, and the protein size was 33 kDa. After incubation at 40 °C for 48 h, the residual enzyme activity of SAP1-DSDPEase was 58%. To make the recombinant B. subtilis strain reusable, cells were immobilized with a composite carrier of sodium alginate (SA) and titanium dioxide (TiO2). The results showed that 2% SA, 2% CaCl2, 0.03% glutaraldehyde solution and a ratio of TiO2 to SA of 1:4 were optimal for immobilization. Under these conditions, up to 82% of the activity of immobilized cells could be retained. Compared with free cells, the optimal reaction temperature of immobilized cells remained unchanged at 80 °C but the thermal stability improved. After 10 consecutive cycles, the mechanical strength remained unchanged, while 58% of the enzyme activity could be retained, with a conversion rate of 28.8% achieved. This study demonstrated a simple approach for using SAPs to improve the thermal stability of recombinant enzymes. Moreover, addition of TiO2 into SA during immobilization was demonstrated to increase the mechanical strength and reduce cell leakage.
Bacillus subtilis/metabolism* ; Carbohydrate Epimerases/genetics* ; Enzyme Stability ; Enzymes, Immobilized/metabolism* ; Fructose ; Hydrogen-Ion Concentration ; Racemases and Epimerases ; Temperature

5-aminolevulinic acid (5-ALA) plays an important role in the fields of medicine and agriculture. 5-ALA can be produced by engineered Escherichia coli and Corynebacterium glutamicum. We systematically engineered the C4 metabolic pathway of C. glutamicum to further improve its ability to produce 5-ALA. Firstly, the hemA gene encoding 5-ALA synthase (ALAS) from Rhodobacter capsulatus and Rhodopseudomonas palustris were heterologously expressed in C. glutamicum, respectively. The RphemA gene of R. palustris which showed relatively high enzyme activity was selected. Screening of the optimal ribosome binding site sequence RBS5 significantly increased the activity of RphemA. The ALAS activity of the recombinant strain reached (221.87±3.10) U/mg and 5-ALA production increased by 14.3%. Subsequently, knocking out genes encoding α-ketoglutarate dehydrogenase inhibitor protein (odhI) and succinate dehydrogenase (sdhA) increased the flux of succinyl CoA towards the production of 5-ALA. Moreover, inhibiting the expression of hemB by means of sRNA reduced the degradation of 5-ALA, while overexpressing the cysteine/O-acetylserine transporter eamA increased the output efficiency of intracellular 5-ALA. Shake flask fermentation using the engineered strain C. glutamicum 13032/∆odhI/∆sdhA-sRNAhemB- RBS5RphemA-eamA resulted in a yield of 11.90 g/L, which was 57% higher than that of the original strain. Fed-batch fermentation using the engineered strain in a 5 L fermenter produced 25.05 g/L of 5-ALA within 48 h, which is the highest reported-to-date yield of 5-ALA from glucose.
Aminolevulinic Acid/metabolism* ; Corynebacterium glutamicum/metabolism* ; Fermentation ; Metabolic Engineering ; Rhodobacter capsulatus/enzymology* ; Rhodopseudomonas/enzymology*

Objective:To investigate the diagnostic values of interleukin-22 (IL-22), interferon-γ(IFN-γ)and macrophage migration inhibition factor (MIF) in pleural effusion for tuberculosis pleurisy.Methods:From April 2018 to May 2019, a total of 77 patients including 45 cases of tuberculous pleurisy, 19 cases of malignant pleurisy, 13 cases of parapneumonia and 13 cases of healthy control in Wuxi Fifth People′s Hospital were enrolled. The levels of IL-22, IFN-γ and MIF in plasma and pleural effusion were detected by enzyme linked immunosorbent assay (ELISA). Mann-Whitney U test was used for statistical analysis.The receiver operating characteristic (ROC) curve was used to evaluate the diagnostic values of IL-22, IFN-γ and MIF for tuberculous pleurisy. Results:The median levels of IL-22, IFN-γ, MIF and adenosine deaminase in 45 cases with pleural effusion in tuberculosis pleurisy group were 396.8 ng/L, 2 200.0 ng/L, 241.3 μg/L and 70.8 U/L, respectively, which were all significantly higher than 32 cases with non-tuberculosis pleurisy group, including 19 cases with malignant pleurisy and 13 cases with parapneumonia (52.8 ng/L, 232.3 ng/L, 179.6 μg/L and 17.0 U/L, respectively). The differences were all statistically significant ( U=179.000, 118.500, 287.000, 162.000, respectively, all P<0.05). The median levels of IL-22 and IFN-γ in plasma of tuberculosis pleurisy group were 20.0 ng/L and 45.9 ng/L, respectively, which were both higher than healthy control group (14.3 ng/L and 33.4 ng/L, respectively). The level of MIF was 96.2 μg/L, which was lower than healthy control (159.5 μg/L). The differences were all statistically significant ( U=74.000, 13.000 and 73.000, respectively, all P<0.05). The areas under ROC curve (AUC) of IL-22, IFN-γ and MIF in pleural effusion for the diagnosis of tuberculosis pleurisy were 0.876, 0.917 and 0.682, respectively.The sensitivities were 93.75%, 100.00% and 63.64%, respectively; the specificities were 82.22%, 91.11% and 65.85%, respectively. The median levels of IL-22 and IFN-γ in plasma in tuberculosis pleurisy group at two months of follow-up after anti-tuberculosis therapy were 16.0 ng/L and 33.9 ng/L, respectively, which were both lower than baseline (20.0 ng/L and 44.7 ng/L, respectively). The differences were both statistically significant ( U=2.156 and 2.221, respectively, both P<0.05). Conclusion:IFN-γ and IL-22 in pleural effusion could be used as effective indicators to identify tuberculous pleurisy, and the dynamic monitoring of IL-22 in patients′plasma could be an important biomarker in evaluating the efficacy of anti-tuberculosis treatment.

Pancreatic duct stone is a rare pancreatic disease in clinic, which is often associated with chronic pancreatitis, and could seriously damage the quality of life of patients, and even induce pancreatic cancer. The diagnosis is mainly based on imaging examination, and the treatment methods are diverse. It is necessary to follow the principle of individualized treatment and treat it as soon as possible. This article reviewed the etiology, mechanism, diagnosis, classification and treatment of the disease.

Luminal breast cancer is the most common subtype of breast cancer, representing more than 60% of all breast cancers. Endocrine resistance and late recurrence are two challenges in the treatment of luminal breast cancer. To overcome endocrine resistance in multiple levels, high-dose-fulvestrant can inhibit estrogen-receptor (ER)-dependent pathways, while targeted drugs can block ER-independent pathways.To reduce the risk of late recurrence in luminal breast cancer, recurrence prediction model should be formed. For patients with high risk of late recurrence, extended endocrine therapy, combination of ovarian function suppression (OFS) or vascular endothelial growth factor (VEGF) inhibitor could be utilized. Based on the challenges of the treatment, scientific research achievements can be used in clinical practice, and finally optimize the clinical treatment strategy.

Luminal breast cancer is the most common subtype of breast cancer, representing more than 60% of all breast cancers. Endocrine resistance and late recurrence are two challenges in the treatment of luminal breast cancer. To overcome endocrine resistance in multiple levels, high-dose-fulvestrant can inhibit estrogen-receptor (ER)-dependent pathways, while targeted drugs can block ER-independent pathways.To reduce the risk of late recurrence in luminal breast cancer, recurrence prediction model should be formed. For patients with high risk of late recurrence, extended endocrine therapy, combination of ovarian function suppression (OFS) or vascular endothelial growth factor (VEGF) inhibitor could be utilized. Based on the challenges of the treatment, scientific research achievements can be used in clinical practice, and finally optimize the clinical treatment strategy.