The molecular mechanism of verbascoside(OC1) in promoting acetylcholine(ACh) release in the pathogenesis of Alzheimer's disease(AD) was studied. Adrenal pheochromocytoma cells(PC12) of rats induced by β-amyloid protein(1-42)(Aβ_(1-42)) were used as AD models in vitro and were divided into control group, model group(Aβ_(1-42) 10 μmol·L~(-1)), OC1 treatment group(2 and 10 μg·mL~(-1)). The effect of OC1 on phosphorylated proteins in AD models was analyzed by whole protein phosphorylation quantitative omics, and the selectivity of OC1 for calcium channel subtypes was virtually screened in combination with computer-aided drug design. The fluorescence probe Fluo-3/AM was used to detect Ca~(2+) concentration in cells. Western blot analysis was performed to detect the effects of OC1 on the expression of phosphorylated calmodulin-dependent protein kinase Ⅱ(p-CaMKⅡ, Thr286) and synaptic vesicle-related proteins, and UPLC/Q Exactive MS was used to detect the effects of OC1 on ACh release in AD models. The effects of OC1 on acetylcholine esterase(AChE) activity in AD models were detected. The results showed that the differentially modified proteins in the model group and the OC1 treatment group were related to calcium channel activation at three levels: GO classification, KEGG pathway, and protein domain. The results of molecular docking revealed the dominant role of L-type calcium channels. Fluo-3/AM fluorescence intensity decreased under the presence of Ca~(2+) chelating agent ethylene glycol tetraacetic acid(EGTA), L-type calcium channel blocker verapamil, and N-type calcium channel blocker conotoxin, and the effect of verapamil was stronger than that of conotoxin. This confirmed that OC1 promoted extracellular Ca~(2+) influx mainly through its interaction with L-type calcium channel protein. In addition, proteomic analysis and Western blot results showed that the expression of p-CaMKⅡ and downstream vesicle-related proteins was up-regulated after OC1 treatment, indicating that OC1 acted on vesicle-related proteins by activating CaMKⅡ and participated in synaptic remodeling and transmitter release, thus affecting learning and memory. OC1 also decreased the activity of AChE and prolonged the action time of ACh in synaptic gaps.
Animals ; Rats ; Glucosides/administration & dosage* ; Acetylcholine/metabolism* ; Alzheimer Disease/genetics* ; PC12 Cells ; Phenols/chemistry* ; Neurotransmitter Agents/metabolism* ; Drugs, Chinese Herbal ; Calcium-Calmodulin-Dependent Protein Kinase Type 2/genetics* ; Humans ; Phosphorylation/drug effects* ; Calcium/metabolism* ; Polyphenols

Objective To explore the value of droplet digital polymerase chain reaction(ddPCR)in the etiological diagnosis of severe acute pancreatitis(SAP)patients with suspected bloodstream infection(BSI).Methods SAP patients admitted to the department of critical care medicine in a hospital July to September 2022 were enrolled.When BSI was suspected,venous blood was collected for both ddPCR detection and blood culture(BC)with antimi-crobial susceptibility testing(AST)simultaneously.The time required for two detection methods was recorded,and the detection results of ddPCR and BC were compared.The etiological diagnostic efficacy of ddPCR was calculated,and the correlation between the value of pathogen load detected by ddPCR and the level of infection parameters was explored.Results A total of 22 patients were included in the analysis,and 52 venous blood specimens were collec-ted for detection.BC revealed 17 positive specimens(32.7％)and 29 pathogenic strains,while ddPCR showed 41 positive specimens(78.8％)and 73 pathogenic strains.Detection time required for ddPCR was significantly lower than that of BC([0.16±0.03]days vs[5.92±1.20]days,P＜0.001).Within the detection range of ddPCR and taking BC results as the gold standard,the sensitivity and specificity of ddPCR were 80.0％and 28.6％,respective-ly.With the combined assessment of BSI based on non-blood specimen microbial evidence within a week,the sensi-tivity and specificity of ddPCR detection increased to 91.9％and 76.9％,respectively.ddPCR detected resistance genes of blaKPC,blaNDM/IMP,VanA/VanM,and mecA from 19,9,6,and 5 specimens,respectively.Correlation analysis showed a positive correlation between pathogen load and levels of C-reactive protein as well as procalcitonin(r=0.347,0.414,P＜0.05).Conclusion As a supplementary detection method for BC in BSI diagnosis,ddPCR has the advantages of higher sensitivity and shorter detection time,and is worthy of further exploration in clinical application.

Tuberous sclerosis complex(TSC)is a rare genetic disease that can lead to benign dysplasia in multiple organs such as the skin, brain, eyes, oral cavity, heart, lungs, kidneys, liver, and bones. Its main symptoms include epilepsy, intellectual disabilities, skin depigmentation, and facial angiofibromas, whilst incidence is approximately 1 in 10 000 to 1 in 6000 newborns. This case presents a middle-aged woman who initially manifested with epilepsy and nodular depigmentation. Later, she developed a lower abdominal mass, elevated creatinine, and severe anemia. Based on clinical features and whole exome sequencing, the primary diagnosis was confirmed as TSC. Laboratory and imaging examinations revealed that the lower abdominal mass originated from the uterus. CT-guided biopsy pathology and surgical pathology suggested a combination of leiomyoma and abscess. With the involvement of multiple organs and various complications beyond the main diagnosis, the diagnostic and therapeutic process for this patient highlights the importance of rigorous clinical thinking and multidisciplinary collaboration in the diagnosis and treatment of rare and challenging diseases.

Objective:To construct recombinant lentivirus and adenovirus which regulate the expression of c-Cbl gene and evaluate their efficacy.Methods:The interference lentivirus and overexpressed adenovirus targeting human c-Cbl gene were constructed by gene recombination technology.Quantitative PCR and western blotting were used to detect the expression changes in c-Cbl gene and its transcription after leukemia cells(HL60,THP1)were infected by virus.Results:Three recombinant interfering lentiviral vectors targeting human c-Cbl genes to successfully constructed and were identified by DNA sequencing,and the titers of the packaged viruses were all greater than 1 x 108 TU/ml.Among them,shRNA-2 lentivirus had the highest interference efficiency,and the expression of c-Cbl gene and CBL protein were decreased about 95％and 60％respectively after leukemia cells were infected with shRNA-2;In addition,the recombinant overexpression adenovirus targeting human c-Cbl gene was packaged successfully with the virus titer greater than 1 x 109 TU/ml.When leukemia cells were infected with adenovirus,the expression of c-Cbl gene and CBL protein were up-regulated about 10 times and 1.5 times respectively.Conclusion:Both recombinant interfering lentivirus and overexpression adenovirus can efficiently infect leukemia cells and affect the expressions of c-Cbl gene and CBL protein.It will lay a preliminary foundation for the subsequent study on the function of c-Cbl gene in tumor cells.

Objective:To evaluate the safety and efficiency of robotic visualization system(RVS)-assisted microsurgical re-construction of the reproductive tract in male rats and the satisfaction of the surgeons.Methods:We randomly divided 8 adult male SD rats into an experimental and a control group,the former treated by RVS-assisted microsurgical vasoepididymostomy(VE)or vaso-vasostomy(VV),and the latter by VE or VV under the standard operating microscope(SOM).We compared the operation time,me-chanical patency and anastomosis leakage immediately after surgery,and the surgeons'satisfaction between the two groups.Results:No statistically significant difference was observed the operation time between the experimental and the control groups,and no anasto-mosis leakage occurred after VV in either group.The rate of mechanical patency immediately after surgery was 100％in both groups,and that of anastomosis leakage after VE was 16.7％in the experimental group and 14.3％in the control.Compared with the control group,the experimental group achieved dramatically higher scores on visual comfort(3.00±0.76 vs 4.00±0.53,P＜0.05),neck/back comfort(2.75±1.16 vs 4.38±1.06,P＜0.01)and man-machine interaction(3.88±1.55 va 4.88±0.35,P＜0.05).There were no statistically significant differences in the scores on image definition and operating room suitability between the two groups.Conclusion:RVS can be used in microsurgical reconstruction of the reproductive tract in male rats and,with its advantages over SOM in ergonomic design and image definition,has a potential application value in male reproductive system micosurgery.

Objective:To evaluate the safety and efficiency of China-made Toumai Robot-assisted laparoscopic radical prosta-tectomy(LRP).Methods:This study included 40 cases of PCa treated from January 2023 to May 2023 by robot-assisted LRP with preservation of the bladder neck and maximal functional urethral length,15 cases with the assistance of Toumai Robot(the TMR group)and the other 25 with the assistance of da Vinci Robot as controls(the DVR group).We recorded the docking time,laparo-scopic surgery time,vesico-urethral anastomosis time,intraoperative blood loss and postoperative urinary continence,and compared them between the two groups.Results:Operations were successfully completed in all the cases.No statistically significant differ-ences were observed between the TMR and DVR groups in the docking time(6 min vs 5 min,P＞0.05)or intraoperative blood loss(200 ml vs 150 ml,P＞0.05).The TMR group,compared with the DVR group,showed a significantly longer median laparoscopic surgery time(146 min vs 130 min,P＜0.05)and median vesico-urethral anastomosis time(19 min vs 16 min,P＜0.05).There were no statistically significant differences between the TMR and DVR groups in the rates of urinary continence recovery immediately af-ter surgery(60.0％[9/15]vs 64.0％[16/25],P＞0.05)or at 1 month(80.0％[12/15])vs(76.0％[19/25],P＞0.05),3 months(93.3％[14/15])vs(92.0％[23/25],P＞0.05)and 6 months postoperatively(100％[15/15])vs(96％[24/25],P＞0.05).Conclusion:China-made Toumai? Robot surgical system is safe and reliable for laparoscopic radical prosta-tectomy,with satisfactory postoperative recovery of urinary continence.

AIM To explore the protective effects of Tongxie Yaofang on the intestinal mucosa of rats with ulcerative colitis(UC)due to Liver Depression and Spleen Deficiency.METHODS The rats were randomly divided into the blank group,the model group,the mesalazine group(0.4 g/kg),the high-dose,medium-dose and low-dose Tongxie Yaofang groups(20.8,10.4,5.2 g/kg).The rat models were induced by DNBS/ethanol solution enema,restraint stress and inappropriate diet.All rats had their serum levels of IL-8 and TNF-α detected by ELISA;their colonic pathological changes observed by HE staining;and their colonic TLR4,MyD88 and NF-κB p65 mRNA and protein expressions detected by RT-qPCR,Western blot and immunohistochemistry method.RESULTS Compared with the model group,all theother groups intervened with the medical agents displayed lower serum levels of TNF-a and IL-8(P＜0.05,P＜0.01);decreased colonic expressions of MyD88 and NF-κB p65 mRNA and protein(P＜0.05,P＜0.01).The mesalazine group and the high-dose and medium-dose Tongxie Yaofang groups shared decreased colonic expression of TLR4 mRNA and protein(P＜0.05,P＜0.01);and the low-dose Tongxie Yaofang group demonstrated decreased colonic TLR4 protein expression(P＜0.05).CONCLUSION Tongxie Yaofang may inhibit the inflammatory reaction of the rats and repair their intestinal mucosal barrier damage via TLR4/MyD88/NF-κB signal pathway.

Objective To investigate the clinical characteristics and prognosis of pneumococcal meningitis(PM),and drug sensitivity of Streptococcus pneumoniae(SP)isolates in Chinese children.Methods A retrospective analysis was conducted on clinical information,laboratory data,and microbiological data of 160 hospitalized children under 15 years old with PM from January 2019 to December 2020 in 33 tertiary hospitals across the country.Results Among the 160 children with PM,there were 103 males and 57 females.The age ranged from 15 days to 15 years,with 109 cases(68.1% )aged 3 months to under 3 years.SP strains were isolated from 95 cases(59.4% )in cerebrospinal fluid cultures and from 57 cases(35.6% )in blood cultures.The positive rates of SP detection by cerebrospinal fluid metagenomic next-generation sequencing and cerebrospinal fluid SP antigen testing were 40% (35/87)and 27% (21/78),respectively.Fifty-five cases(34.4% )had one or more risk factors for purulent meningitis,113 cases(70.6% )had one or more extra-cranial infectious foci,and 18 cases(11.3% )had underlying diseases.The most common clinical symptoms were fever(147 cases,91.9% ),followed by lethargy(98 cases,61.3% )and vomiting(61 cases,38.1% ).Sixty-nine cases(43.1% )experienced intracranial complications during hospitalization,with subdural effusion and/or empyema being the most common complication[43 cases(26.9% )],followed by hydrocephalus in 24 cases(15.0% ),brain abscess in 23 cases(14.4% ),and cerebral hemorrhage in 8 cases(5.0% ).Subdural effusion and/or empyema and hydrocephalus mainly occurred in children under 1 year old,with rates of 91% (39/43)and 83% (20/24),respectively.SP strains exhibited complete sensitivity to vancomycin(100% ,75/75),linezolid(100% ,56/56),and meropenem(100% ,6/6).High sensitivity rates were also observed for levofloxacin(81% ,22/27),moxifloxacin(82% ,14/17),rifampicin(96% ,25/26),and chloramphenicol(91% ,21/23).However,low sensitivity rates were found for penicillin(16% ,11/68)and clindamycin(6% ,1/17),and SP strains were completely resistant to erythromycin(100% ,31/31).The rates of discharge with cure and improvement were 22.5% (36/160)and 66.2% (106/160),respectively,while 18 cases(11.3% )had adverse outcomes.Conclusions Pediatric PM is more common in children aged 3 months to under 3 years.Intracranial complications are more frequently observed in children under 1 year old.Fever is the most common clinical manifestation of PM,and subdural effusion/emphysema and hydrocephalus are the most frequent complications.Non-culture detection methods for cerebrospinal fluid can improve pathogen detection rates.Adverse outcomes can be noted in more than 10% of PM cases.SP strains are high sensitivity to vancomycin,linezolid,meropenem,levofloxacin,moxifloxacin,rifampicin,and chloramphenicol.[Chinese Journal of Contemporary Pediatrics,2024,26(2):131-138]