Cholera toxin B subunit(CTB)can enhance antigen presentation and promote T cell pro-liferation,B cell differentiation and B cell isotype conversion.Moreover,woodchuck hepatitis virus post transcriptional regulatory element(WPRE)can enhance gene expression efficiency by optimi-zing RNA polyadenylation,denuclearization and/or translation.In order to construct porcine epi-demic diarrhea virus like particles(VLPs)chimerized with CTB and WPRE and evaluate their im-munogenicity,the G Ⅱ type PEDV S gene,combined with the elements promoting the protein ex-pression and enhancing immune effects,was synthesized by the company and cloned into pET32a(+).Af-ter double enzyme digestion and gel recovery,the gene named as TSCW was cloned into pFastBacl to construct the recombinant plasmid pFastBac-TSCW.pFastBac-TSCW was further transformed into DH10Bac competent cells to obtain recombinant bacmid Bacmid-TSCW.Subsequently,the Bacmid-TSCW was transfected into sf9 cells to obtain recombinant baculovirus BV-TSCW.After co-infection of BV-TSCW and BV-M into sf9 cells,viral like particles VLP-TSCW was obtained and used to immunize mice to evaluate its immunogenicity.The results showed that the recombi-nant plasmid pFastBac-TSCW and bacmid Bacmid-TSCW were successfully constructed.After transfection of sf9 cells with recombinant baculovirus,significant cytopathic effects were observed.PCR and Western blot results showed that the recombinant baculoviruses existed stably in sf9 cells and the target proteins was also expressed stably.In addition,the electron microscopy results showed that BV-TSCW and BV-M successfully assembled into viral like particles VLP-TSCW.Furthermore,ELISA results indicated that VLP-TSCW induced high level specific antibodies.The above results laid the foundation for further optimization,design and development of PEDV VLPs subunit vaccines.

The purpose of this study is to investigate the effect of TPCK trypsin on the proliferation pattern of porcine epidemic diarrhea virus in Vero cells.The TPCK trypsin and conventional tryp-sin were added for virus proliferation,and RT-qPCR technology was used to analyze the changes in virus adsorption and invasion in Vero cells.The replication ability of porcine epidemic diarrhea vi-rus in Vero cells was explored through growth curve drawing,IFA identification,and cell activity detection.The results showed that the optimal concentrations of TPCK trypsin and conventional trypsin were 1 mg/L and 6 mg/L,respectively.The virus showed a decreasing trend with the pro-longation of TPCK trypsin and conventional trypsin pretreatment time.Adding different pancreatic enzymes during the virus proliferation process did not promote the virus invasion in Vero cells.Af-ter 4 h of invasion,the virus particles of each group gradually increased.By plotting the growth curve,it was found that the virus content of the TPCK trypsin group reached its highest level at 24 h(lgTCID50=(6.30±0.14)/0.1 mL),followed by a decreasing trend at 36 h,and the fluorescence intensity produced at 24 h was higher than that of conventional trypsin.In summary,TPCK trypsin has a better promoting effect on the proliferation of porcine epidemic diarrhea virus in Vero cells.It provided theoretical basis for further research on the mechanism of TPCK trypsin affecting porcine epidemic diarrhea virus proliferation,and also provided data support for the isola-tion and purification of porcine epidemic diarrhea virus epidemic strains.

Attention Deficit Hyperactivity Disorder (ADHD) is a chronic neurodevelopmental disorder with an estimated prevalence of approximately 6.4% among children and adolescents aged 6 to 16 in China. The etiology and pathogenesis of ADHD remain incompletely understood, and conventional treatment methods often have limited effectiveness. With advances in research into the etiology of ADHD, nutritional intervention has emerged as a potential adjunctive treatment and has garnered widespread attention. Multiple studies have demonstrated the potential effectiveness of nutritional interventions for ADHD. This review aims to summarize the progress in nutritional interventions for pediatric ADHD, particularly focusing on nutrient-level interventions (including polyunsaturated fatty acids, minerals, and vitamins), nutrition-related factors (such as probiotics), as well as the combined use of multiple nutrients and dietary nutrition (including dietary patterns). We will analyze and discuss the effectiveness of different nutritional interventions to provide a valuable reference for clinical nutritional diagnosis and treatment of the disease.

Attention Deficit Hyperactivity Disorder (ADHD) is a chronic neurodevelopmental disorder with an estimated prevalence of approximately 6.4% among children and adolescents aged 6 to 16 in China. The etiology and pathogenesis of ADHD remain incompletely understood, and conventional treatment methods often have limited effectiveness. With advances in research into the etiology of ADHD, nutritional intervention has emerged as a potential adjunctive treatment and has garnered widespread attention. Multiple studies have demonstrated the potential effectiveness of nutritional interventions for ADHD. This review aims to summarize the progress in nutritional interventions for pediatric ADHD, particularly focusing on nutrient-level interventions (including polyunsaturated fatty acids, minerals, and vitamins), nutrition-related factors (such as probiotics), as well as the combined use of multiple nutrients and dietary nutrition (including dietary patterns). We will analyze and discuss the effectiveness of different nutritional interventions to provide a valuable reference for clinical nutritional diagnosis and treatment of the disease.

The purpose of this study is to investigate the effect of TPCK trypsin on the proliferation pattern of porcine epidemic diarrhea virus in Vero cells.The TPCK trypsin and conventional tryp-sin were added for virus proliferation,and RT-qPCR technology was used to analyze the changes in virus adsorption and invasion in Vero cells.The replication ability of porcine epidemic diarrhea vi-rus in Vero cells was explored through growth curve drawing,IFA identification,and cell activity detection.The results showed that the optimal concentrations of TPCK trypsin and conventional trypsin were 1 mg/L and 6 mg/L,respectively.The virus showed a decreasing trend with the pro-longation of TPCK trypsin and conventional trypsin pretreatment time.Adding different pancreatic enzymes during the virus proliferation process did not promote the virus invasion in Vero cells.Af-ter 4 h of invasion,the virus particles of each group gradually increased.By plotting the growth curve,it was found that the virus content of the TPCK trypsin group reached its highest level at 24 h(lgTCID50=(6.30±0.14)/0.1 mL),followed by a decreasing trend at 36 h,and the fluorescence intensity produced at 24 h was higher than that of conventional trypsin.In summary,TPCK trypsin has a better promoting effect on the proliferation of porcine epidemic diarrhea virus in Vero cells.It provided theoretical basis for further research on the mechanism of TPCK trypsin affecting porcine epidemic diarrhea virus proliferation,and also provided data support for the isola-tion and purification of porcine epidemic diarrhea virus epidemic strains.

Cholera toxin B subunit(CTB)can enhance antigen presentation and promote T cell pro-liferation,B cell differentiation and B cell isotype conversion.Moreover,woodchuck hepatitis virus post transcriptional regulatory element(WPRE)can enhance gene expression efficiency by optimi-zing RNA polyadenylation,denuclearization and/or translation.In order to construct porcine epi-demic diarrhea virus like particles(VLPs)chimerized with CTB and WPRE and evaluate their im-munogenicity,the G Ⅱ type PEDV S gene,combined with the elements promoting the protein ex-pression and enhancing immune effects,was synthesized by the company and cloned into pET32a(+).Af-ter double enzyme digestion and gel recovery,the gene named as TSCW was cloned into pFastBacl to construct the recombinant plasmid pFastBac-TSCW.pFastBac-TSCW was further transformed into DH10Bac competent cells to obtain recombinant bacmid Bacmid-TSCW.Subsequently,the Bacmid-TSCW was transfected into sf9 cells to obtain recombinant baculovirus BV-TSCW.After co-infection of BV-TSCW and BV-M into sf9 cells,viral like particles VLP-TSCW was obtained and used to immunize mice to evaluate its immunogenicity.The results showed that the recombi-nant plasmid pFastBac-TSCW and bacmid Bacmid-TSCW were successfully constructed.After transfection of sf9 cells with recombinant baculovirus,significant cytopathic effects were observed.PCR and Western blot results showed that the recombinant baculoviruses existed stably in sf9 cells and the target proteins was also expressed stably.In addition,the electron microscopy results showed that BV-TSCW and BV-M successfully assembled into viral like particles VLP-TSCW.Furthermore,ELISA results indicated that VLP-TSCW induced high level specific antibodies.The above results laid the foundation for further optimization,design and development of PEDV VLPs subunit vaccines.

By targeting the key gene csgD involved in the biofilm formation of Escherichia coli, we employed molecular docking and molecular dynamics simulation to screen the active components of Chinese medicine with inhibitory effects on the biofilm formation from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP). After the anti-biofilm properties of the active components were validated in vitro, data-independent acquisition (DIA) proteomics was employed to further identify the differential proteins involved in interfering with the biofilm formation of Escherichia coli. The mechanisms of inhibition were explored with consideration to the phenotype. Through virtual screening, we identified four candidate active components, including tannic acid, narirutin, salvianolic acid B, and rosmarinic acid. Among them, tannic acid demonstrated significant inhibitory effect on the biofilm formation of E. coli. The analysis of differential proteins, combined with relevant phenotype validation, suggested that tannic acid primarily affected E. coli by intervening in pilus assembly, succinic acid metabolism, and the quorum sensing system. This study provided a lead compound for the development of new drugs against biofilm-associated infections in the future.
Biofilms/drug effects* ; Escherichia coli/metabolism* ; Molecular Docking Simulation ; Drugs, Chinese Herbal/chemistry* ; Tannins/chemistry* ; Cinnamates/metabolism* ; Benzofurans/chemistry* ; Depsides/metabolism* ; Rosmarinic Acid ; Anti-Bacterial Agents/chemistry* ; Escherichia coli Proteins/genetics* ; Medicine, Chinese Traditional

Objective:To explore the mechanisms of regulation of miR-575 on the proliferation and invasion properties of non-small cell lung cancer cell (NSCLC).Methods:Real-time PCR was selected to detect the expression of miR-575 and BLID in differ ent NSCLC cell lines.CCK-8 assay was processed to measure the alternations of A549 cell proliferation at different time points after transfection of miR-575 mimic and miR-575 inhibitor.The invasion ability of A549 cells was evaluated by transwell.The targeting of BLID by miR-575 was predicted by Targetcan software and verified by dual-Luciferase assay.BLID protein expression level was detected by western blot.Results:miR-575 highly expressed in NSCLC cell lines,including A549,SPC-A1,H1299,H1650 (P＜0.001),miR-575 mimic could efficiently elevated the expression ofmiR-575 in A549 cells (P＜0.001),and strengthened the proliferation and invasion ability of NSCLC cells (P＜0.05),while,transfection of miR-575 inhibitor could down-regulate the expression ofmiR-575,and also inhibit the proliferation and invasion ability of NSCLC cells (P＜0.01).Targetscan software predicted that BLID might be the target gene of miR-575,and dual-luciferase assay revealed that miR-575 could obviously decrease the luciferase reaction of wild type BLID 3'UTR (P＜0.01),besides,miR-575 could down-regulate the protein expression ofBLID (P＜0.01).Real-time PCR results showed that NSCLC cell lines had lower level of BLID mRNA expression compared with 16HBE control cells (P＜0.001),and restore of BLID could markedly inhibited cell proliferation and invasion ability (P＜0.05),which could be reversed by miR-575 co-tranfection (P＜0.01).Conclusion:In NSCLC cells,the expression ofmiR-575 could promote cell proliferation and invasion ability by directly regulating downstream target tumor-suppressor gene BLID expression.

Objeetive To explore the situation of rotavirus infection and extraintestinal organe damage in children in Yueqing city.Methods Two hundred and eighty-seven cases with acute rotavirus gastroenteritis in our hospital were analyzed for prospective study from October 2011 to January 2013 by stool tests.Results Rotavirus infection was found to be more common in autumn and winter.There were 223 cases (17.7％) got extraintestinal organe damage,175 cases (60.80％) got myocardial lesion,and 78 cases (27.18％) got respiratory infection.At the same time,there were 51 cases (17.77％) and 21 cases (7.31％) got liver function lesion and convulsion respectively.Among the metabolic acidosis(48 cases),39 cases were combined with myocardial lesion.While non metabolic acidosis were 239 cases (81.25％),and 136 cases were combined with myocardial lesion.Therefore,the myocardial lesion was significant correlation with metabolic acidosis (P ＜ 0.01).Iron deficiency anemia was 123 cases and combined with 15 cases (12.19％) convulsion,while the convulsion prevalence rates of non iron deficiency anemia was 6 cases (3.65 ％).There was a statistically significant difference between the convulsion prevalence rates of iron deficiency anemia and that of non iron deficiency anemia (P ＜ 0.01).Conclusion Rotavirus diarrhea can lead to extraintestinal organe damage,and the clinical doctors should pay attention to them.