Objective　To conduct a genotypic analysis of dengue virus in Gengma County, Yunnan Province, located in the China-Myanmar border region in 2023, and provide a reference for developing effective clinical diagnosis and treatment strategies for dengue fever in the region. Methods Serum samples were collected from hospitalized patients testing positive for dengue NS1 antigen, and dengue virus nucleic acid was detected using real-time quantitative PCR (qPCR). Nucleic acid-positive samples underwent cell culture and sequencing analysis of the dengue virus E gene to determine the origin of the virus. Results Specific nucleic acid detection was performed in 86 NS1-positive serum samples, 75 samples tested positive for dengue virus serotype 1 (DENV-1), 6 samples for serotype 2 (DENV-2), and 5 samples were DENV-negative. Twelve DENV-1 and four DENV-2 strains were obtained by blind cell culture transmission for 3 generations. Sequencing of E gene sequences (1 485 bp) of 16 strains showed that the nucleotide similarity of 12 DENV-1 E gene sequences ranged from 99.3% to 100.0%, with amino acid homology of 99.8% to 100.0%. For the 4 DENV-2 E gene sequences, the nucleotide similarity ranged from 99.7% to 99.9%, and amino acid homology ranged from 99.0% to 99.8%. Phylogenetic analysis showed that 12 DENV-1 strains belonged to genotype I, while 4 DENV-2 strains belonged to the Asian-II genotype. Conclusion The dengue fever outbreak in Gengma County, Yunnan Province, China, the China-Myanmar border area, was mainly caused by dengue virus 1 genotype I and dengue virus 2 genotype Asian-II. Both dengue virus 1 and 2 may have similar original strains from Guangzhou, China in 2023. It was recommended that local relative departments should strengthen the monitoring of dengue fever.

L-citrulline is a nonprotein amino acid that plays an important role in human health and has great market demand. Although microbial cell factories have been widely used for biosynthesis, there are still challenges such as genetic instability and low efficiency in the biosynthesis of L-citrulline. In this study, an efficient, plasmid-free, non-inducible L-citrulline-producing strain of Escherichia coli BL21(DE3) was engineered by combined strategies. Firstly, a chassis strain capable of synthesizing L-citrulline was constructed by block of L-citrulline degradation and removal of feedback inhibition, with the L-citrulline titer of 0.43 g/L. Secondly, a push-pull-restrain strategy was employed to enhance the L-citrulline biosynthesis, which realized the L-citrulline titer of 6.0 g/L. Thirdly, the NADPH synthesis and L-citrulline transport were strengthened to promote the synthesis efficiency, which achieved the L-citrulline titer of 11.6 g/L. Finally, fed-batch fermentation was performed with the engineered strain in a 3 L fermenter, in which the L-citrulline titer reached 44.9 g/L. This study lays the foundation for the industrial production of L-citrulline and provides insights for the modification of other amino acid metabolic networks.
Citrulline/biosynthesis* ; Escherichia coli/genetics* ; Metabolic Engineering/methods* ; Fermentation ; NADP/biosynthesis*

Objective:To investigate the impact of abnormal patterns of 75 g oral glucose tolerance test (OGTT) in the second trimester on the risk of large for gestational age (LGA) newborn deliveries.Methods:General clinical data and OGTT results of 66 290 pregnant women who received regular prenatal care and delivered in Guangdong Maternal and Child Health Hospital from December 24, 2016 to July 26, 2022 were collected. According to the results of OGTT, the pregnant women were divided into 8 groups: normal blood glucose group (normal fasting blood glucose, 1-hour and 2-hour after oral glucose, 54 518 cases), gestational diabetes mellitus (GDM) 0 group (only abnormal fasting blood glucose, 1 430 cases), GDM 1 group (only abnormal blood glucose at 1-hour after oral glucose, 2 150 cases), GDM 2 group (only abnormal blood glucose at 2-hour after oral glucose, 3 736 cases), GDM 0+1 group (both fasting blood glucose and 1-hour after oral glucose were abnormal, 371 cases), GDM 0+2 group (both fasting blood glucose and 2-hour after oral glucose were abnormal, 280 cases), GDM 1+2 group (abnormal blood glucose at 1-hour and 2-hour after oral glucose, 2 981 cases) and GDM 0+1+2 group (abnormal fasting blood glucose, 1-hour and 2-hour after oral glucose, 824 cases). Multivariate logistic regression was used to analyze the effects of different abnormal OGTT patterns on LGA. In addition, the blood glucose measurements at the three time points of OGTT were combined and used as continuous variables in the receiver operating characteristic (ROC) curve to evaluate the predictive value of each blood glucose measurement mode for LGA and the area under the curve (AUC) was compared.Results:(1) Multivariate logistic regression analysis showed that the risks of LGA were significantly increased in GDM 0 group ( OR=1.76, 95% CI: 1.50-2.08; P<0.001), GDM 0+1 group ( OR=2.29, 95% CI: 1.72-3.04; P<0.001), and GDM 0+1+2 group ( OR=1.98, 95% CI: 1.61-2.43; P<0.001). (2) ROC curve analysis showed that fasting blood glucose, 1-hour after oral glucose, 2-hour after oral glucose, fasting+1-hour after oral glucose, fasting+2-hour after oral glucose, 1-hour+2-hour after oral glucose, and fasting+1-hour+2-hour after oral glucose had certain predictive value for LGA (all P<0.001). The AUC of fasting blood glucose measurement was higher than that of 2-hour blood glucose measurement in predicting LGA, and the difference was statistically significant ( P<0.05). There was no significant difference in the AUC between fasting blood glucose and other blood glucose measurement modes for predicting LGA (all P>0.05). Conclusions:In the abnormal OGTT patterns, pregnant women with abnormal fasting blood glucose, abnormal fasting+1-hour after oral glucose, and abnormal fasting+1-hour+2-hour after oral glucose have an increased risk of LGA. Fasting blood glucose measurement is of great significance for the prediction of LGA, and could be used as an optimal indicator to evaluate the risk of LGA in clinical practice.

Objective:To clarify the transitional components in the blood of compound Banlangen Granules; To explore the mechanism of drugs in the treatment of epidemic encephalitis B, hepatitis and parotitis.Methods:The transitional components in blood of compound Banlangen Granules were analyzed by ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS/MS). The regulatory targets and pathways of compound Banlangen Granules in the treatment of epidemic encephalitis B, hepatitis and parotitis were analyzed based on UPLC-MS/MS and network pharmacology.Results:A total of 9 blood components were identified, of which 8 were prototype components, including sucrose, o-aminobenzoic acid, uridine, adenosine, guanosine, indole-3-acetonitrile-2 murine-S-β-D-glucopyranoside and salicylic acid. Through network pharmacological analysis, it was concluded that compound Banlangen Granules may treat epidemic encephalitis B, hepatitis and parotitis by regulating lipid and atherosclerosis, insulin resistance, IL-17 and other signal pathways.Conclusion:The 9 blood components of compound Banlangen Granules may treat epidemic encephalitis B, hepatitis and parotitis by regulating lipid and atherosclerosis, insulin resistance, IL-17 and other signal pathways.

Bacillus subtilis is recognized as a generally-regarded-as-safe strain, and has been widely used in the biosynthesis of high value-added products, including N-acetylneuraminic acid (NeuAc) which is widely used as a nutraceutical and a pharmaceutical intermediate. Biosensors responding to target products are widely used in dynamic regulation and high-throughput screening in metabolic engineering to improve the efficiency of biosynthesis. However, B. subtilis lacks biosensors that can efficiently respond to NeuAc. This study first tested and optimized the transport capacity of NeuAc transporters, and obtained a series of strains with different transport capacities for testing NeuAc-responsive biosensors. Subsequently, the binding site sequence of Bbr_NanR responding to NeuAc was inserted into different sites of the constitutive promoter of B. subtilis, and active hybrid promoters were obtained. Next, by introducing and optimizing the expression of Bbr_NanR in B. subtilis with NeuAc transport capacity, we obtained an NeuAc-responsive biosensor with wide dynamic range and higher activation fold. Among them, P535-N2 can sensitively respond to changes in intracellular NeuAc concentration, with the largest dynamic range (180-20 245) AU/OD. P566-N2 shows a 122-fold of activation, which is 2 times of the reported NeuAc-responsive biosensor in B. subtilis. The NeuAc-responsive biosensor developed in this study can be used to screen enzyme mutants and B. subtilis strains with high NeuAc production efficiency, providing an efficient and sensitive analysis and regulation tool for biosynthesis of NeuAc in B. subtilis.
N-Acetylneuraminic Acid/metabolism* ; Bacillus subtilis/metabolism* ; Promoter Regions, Genetic/genetics* ; Binding Sites ; Biosensing Techniques

Fermentation engineering is an industrial process that uses the transformation of microorganisms or other cells to produce a specific product in a specific bioreactor. Fermentation engineering has developed from an ancient food fermentation relying solely on experience accumulation to an important production mode of food, agriculture, medicine, chemical industry and other means of production and life. It has become a key technology to support the sustainable development of human beings, and is inseparable from the continuous progress of interdisciplinary technology. The interdisciplinary integration and the continuous upward movement of China's global industrial chain will inevitably put forward higher requirements for the cultivation of fermentation engineering composite talents in the new situation. In order to constantly improve the interdisciplinary fermentation engineering compound talent training system, in recent years, the research lab has been refining and improving the concept of talent training, and actively deepening the reform of talent training system. Systematic research and practice have been carried out around the aspects of training program, enrollment system, teacher background, subject setting, scientific research practice, evaluation system, etc., which has promoted the technological progress of fermentation engineering and related supporting industries, and contributed an important force to the transformation of China from a big fermentation country to a powerful fermentation country.
Agriculture ; China ; Fermentation ; Humans ; Industry

Objective:To explore the expressions of programmed death-1 (PD-1) and programmed death-ligand 1 (PD-L1) and clinicopathological characteristics in post-transplant lymphoproliferative disorder (PTLD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT) in children, with the aim of clarifying whether checkpoint inhibition of PD-1/PD-L1 inhibitors may serve as a therapy option.Methods:The clinical data of 13 cases of PTLD after allo-HSCT pathologically confirmed in Shenzhen Children′s Hospital from January 1, 2012 to December 30, 2019 were retrospectively analyzed.The detection was performed by immunohistochemical staining by MaxVision? method, Epstein-Barr virus(EBV) in situ hybridization and lymphoma gene rearrangement.The relationship between the expression of PD-1 and PD-L1 and the clinicopathological characteristics of PTLD were analyzed.Results:The expression of PD-1 was not correlated with gender, age, primary diseases, histopathological types, transplantation mode and the expression of EBV in situ hybridization (all P>0.05). The expression of PD-L1 was correlated with histopathological types ( P<0.05). Furthermore, the expression rate of PD-L1 on severe β-thalassemia was significantly higher than that of severe aplastic anemia [90.0%(9/10 cases) vs. 66.7%(2/3 cases)] and monomorphic PTLD was higher than that of polymorphic PTLD [100.0%(2/2 cases) vs. 83.3%(5/6 cases)]. Moreover, the positive PTLD in EBV was higher than the negative PTLD in EBV [90.9%(10/11 cases) vs. 50.0%(1/2 cases)]. The positive rates of PD-1 and PD-L1 in 13 cases with PTLD were 46.2%(6/13 cases) and 61.5%(8/13 cases) in tumor cells, 92.3% (12/13 cases) and 76.9% (10/13 cases) in microenvironmental cells, and 84.6%(11/13 cases) in EBV, respectively. Conclusions:PD-L1 has a higher positive rate in tumor cells with monomorphic PTLD; and routine staining for PD-1 and PD-L1 can be performed in all types of PTLD when standard immunotherapy and chemotherapy are ineffective.

Objective:To investigate the effect of miR-200c-3p on the proliferation and apoptosis of nephroblastoma SK-NEP-1 cell and its mechanism.Methods:From January 2015 to August 2019, nephroblastoma tissue and peritumoral tissue of 30 patients in Shenzhen Children′s hospital were collected.The experimental group of mimic negative control, miR-200c-3p and miR-200c-3p inhibitor was set up.The expression of miR-200c-3p in 30 paired nephroblastoma tissues and adjacent kidney tissues were detected by real-time fluorescence quantitative PCR.The differential expression of miR-200c-3p was also detected in SK-NEP-1 and 293FT cell lines.The effects of miR-200c and miR-200c-3p inhibitor on the proliferation, cell cycle distribution and apoptosis of SK-NEP-1 cells were detected by cell counting kit-8(CCK-8)and flow cytometry assays, respectively.Xenograft tumors were generated by peri-renal adipose tissue injection to assess the effect of miR-200c-3p on tumor growth in vivo.The pathological morphology of xenograft tumors was observed by HE staining.The proliferation index of Ki-67 were detected by immunohistochemistry.Western blot method was used to detect the B-cell lymphoma 2(Bcl-2), Bcl-2 related X protein (Bax) and cleaved cysteinyl aspartate specific proteinase-3(Caspase-3)expression level on xenograft tumors. Results:The expression level of miR-200c-3p in nephroblastoma(0.420±0.587)was significantly lower than that in matched normal kidney(1.500±0.504)( t=8.613, P<0.001). The expression level of miR-200c-3p in SK-NEP-1 cells (0.363±0.006) was significantly lower than that in human embryonic kidney 293FT cells (0.807±0.186) ( t=4.136, P<0.05). The group and time interaction results of CCK-8 proved that miR-200c-3p could inhibit the proliferation of SK-NEP-1 cells( F=16.81, P<0.001). The flow cytometer test of cell cycle and apoptosis showed that miR-200c-3p could block in G0/G1 and S phase( t=-7.770, P<0.01; t=11.501, P<0.001). Moreover, it increased the early apoptosis rate and decreased the late apoptosis rate ( t=-22.270, P<0.001; t=4.612, P<0.01). A orthotopic transplantation assay was employed to evaluate the effect of miR-200c-3p and miR-200c-3p inhibitor on the proliferation of SK-NEP-1 cells.The final volume of tumor miR-200c-3p group [(0.419±0.16) cm 3]was significantly lower than those in the control group [(2.469±0.914) cm 3, t=0.507, P<0.001]. However, the miR-200c-3p inhibitor group had no significant difference [(1.627±0.189) cm 3; t=2.209, P=0.052]. miR-200c-3p overexpression upregulated expression levels of apoptotic proteins cleaved Caspase-3 and Bax ( t=-47.000, -82.730, all P<0.001), but downregulated the expression level of anti-apoptotic protein Bcl-2( t=53.740, P<0.001). Conclusions:The overexpression of miR-200c-3p can inhibit the proliferation, promote the apoptosis of the SK-NEP-1 cells and partially inhibited tumorigenicity of nude mouse acted as a tumor suppressor gene.

Objective:To summarized the experience of 149 cases of critical pediatric cardiac surgery in a single-center during the epidemic period, for providing a safe and feasible management strategy.Methods:Based on the epidemiological characteristics of COVID-19, a strategy consisting of 14 days of isolation was established for the arrangement of cardiac surgery in children during the epidemic period. Retrospective analysis of clinical data of 149 cases of critical cardiac surgery performed from January 23, 2020 to March 20, 2020 under the guidance of this strategy. The primary composite endpoint was death and suspected or confirmed COVID-19.Results:The median age of the children undergoing surgery was 136 days; 73(49.0%) cases were male. Twenty-one cases (14.1%) came from Shanghai, one case (0.7%) came from Hubei Province, and 127 cases (85.2%) came from areas other than Hubei and Shanghai. One patient (0.7%) outside Shanghai who had been isolated for less than 14 days carried emergency surgery under special protection; other 148 patients (99.3%) underwent elective early repair procedure. One patient (0.7%) died, and no COVID-19 was confirmed or suspected.Conclusion:During the COVID-19 epidemic, pediatric cardiac surgery can be safely performed using a specific management strategy, which can be used as a reference when major public health events occur.

Objective    To analyze the outcomes of surgical repair for mixed total anomalous pulmonary venous connection (TAPVC). Methods    Between 2006 and 2018, a total of 51 patients with mixed TAPVC underwent surgery in our hospital. Patients with such associated anomalies as single ventricle and tetralogy of Fallot were excluded. There were 35 males and 16 females with a median age of 102.0 (59.0, 181.0) days and a median weight of 5.0 (4.1, 6.4) kg. Patients were divided into three categories based on the anatomy: "3+1" pattern (n=38, three pulmonary veins drained at one site, and the other drained at the opposite site); "2+2" pattern (n=9, the pulmonary veins from each lung joined to form a confluence and drained at separate sites); bizarre pattern (n=4, the anatomy could not be classified into the above two patterns). Results    There was no in-hospital death. The median follow-up was 41.0 (18.0, 86.5) months. Postoperative pulmonary venous obstruction occurred in 10 patients. Kaplan-Meier survival curves showed no statistically significant difference in postoperative pulmonary venous obstruction among the three groups (P=0.239). Cox risk regression showed that preoperative pulmonary venous obstruction was significantly associated with postoperative pulmonary venous obstruction (P=0.024). Conclusion    Mixed TAPVC has various anatomic morphologies and requires individualized surgery.