Objective To evaluate the reversal effect of a cholinergic receptor agonist on acantholysis in pemphigus,and to investigate its mechanism.Methods Human HaCaT keratinocytes were co-cultured with pemphigus vulgaris immunoglobulin G (PV-IgG) to establish a cell model of pemphigus,then classified into two groups to be incubated with the cholinergic receptor agonist carbachol for 12 hours (test group) or remain untreated (control group).Cell dissociation assay was performed to quantitatively estimate the reversal effect of carbachol on acantholysis,and immunofluorescence assay to qualitatively assess the changes of desmosomal proteins.Radio-immunoprecipitation assay (RIPA) lysis buffer and Triton X-100 were used to lyse HaCaT cells to obtain total proteins and cytoplasmic proteins,and Western blot was conducted to determine the expression levels of adhesion-related proteins desmoglein 3 (Dsg3) and plakoglobin (PG) on the surface of HaCaT cells,as well as the phosphorylation levels of p38 mitogen activated protein kinase (p38 MAPK) and epidermal growth factor receptor (EGFR) at different time points.Quantitative polymerase chain reaction (qPCR) was performed to detect the mRNA expressions of the above surface proteins,and coimmunoprecipitation assay to qualitatively evaluate the interaction between Dsg3 and PG.Results The number of cell debris was significantly lower in the test group than in the control group (18.67 ± 2.52 vs.46.67 ± 2.03,t =11.22,P＜0.01).Immunofluorescence assay showed that carbachol could reverse the internalization of desmosomal molecules induced by PV-IgG.In the pemphigus cell model,the levels of total Dsg3 and PG as well as non-desmosomal Dsg3 were decreased,while the level of non-desmosomal PG increased,and the interaction between Dsg3 and PG was attenuated.When the pemphigus cell model was co-cultured with carbachol,these above changes were reversed.Carbachol also increased the mRNA levels (expressed as 2-△△Ct) of Dsg3 and PG from 1.428 ± 0.215 and 1.563 ± 0.247 in the control group to 4.974 ± 0.948 (t =3.65,P =0.01) and 13.420 ± 1.715 (t =6.85,P ＜ 0.01) in the test group respectively.In phosphorylation assay,carbachol inhibited the phosphorylation of EGFR,but had no significant effect on that of p38 MAPK.Conclusions The cholinergic receptor agonist carbachol can reverse acantholysis in pemphigus,likely by inhibiting the internalization of Dsg3 and PG,enhancing their expressions and interaction,and suppressing the phosphorylation of the key signaling molecule for acantholysis,EGFR.

Objective To construct a lentiviral vector delivering the Nicastrin (NCT) gene-targeted short hairpin RNA (shRNA) and determine gene-silencing efficiency of the vector in the human immortalized keratinocyte cell line HaCaT,and to construct a NCT gene-silenced HaCaT cell model to lay an experimental foundation for subsequently studying effects of NCT gene silencing on biological behavior of keratinocytes.Methods Three NCT gene-targeted shRNAs were designed and inserted into the pGLV3/ H1/GFP + Puro vector to construct three recombinant plasmids,which were then confirmed by sequencing.Recombinant plasmids combined with lentivirus packaging plasmids were co-transfected into 293T cells to obtain lentivirus particles,and the virus titer was determined.Cultured HaCaT cells were divided into 3 groups:blank group receiving no treatment,negative control group infected with the empty vector LV3-shNC,interference groups infected with lentivirus NCT-shRNA1,-shRNA2,-shRNA3,respectively.Flow cytometry was performed to determine transfection efficiency,and real-time fluorescence-based quantitative PCR (qRT-PCR) and Western blot analysis were conducted to determine efficiency of target gene silencing in HaCaT cells,so as to select the most efficient interference sequence.Results Sequencing analysis indicated that recombinant lentiviral vector NCT-shRNA was constructed successfully.After co-transfection of recombinant plasmids and lentivirus packaging plasmids into 293T cells,the titer of recombinant lentivirus particles was about 109 TU/ml.Flow cytometry showed that the transfection efficiency was greater than 95％.qRT-PCR revealed that the NCT mRNA expression was obviously down-regulated in the interference group compared with the negative control group,and NCT-shRNA1 was the most efficient sequence with interference efficiency being 75％.Western blot analysis showed that the inhibition rate of NCT protein expression was 71.7％ in the shRNA1 group compared with the negative control group.Conclusion The most efficient NCT-shRNA interference sequence is screened out,and the recombinant lentiviral vector NCT-shRNA and an NCT gene-silenced HaCaT cell model are both constructed successfully.

Objective　To improve the understanding and provide a reasonable plan for clinical diagnosis and treatment of TRE(TSC-related epilepsy,TRE),by analyzing the clinical and genetic characteristics of TRE as well as the treatment effect about medication and surgery.Methods　We analyzed the video EEG,imaging,pathology,genetic characteristics,and treatment effect of 10 patients retrospectively.These patients with refractory epilepsy were diagnosed and treated by our epilepsy center from November 2014 to November 2019.The clinical and genetic characteristics of TRE were summarized.Results　There were 3 females,7 males,4 patients with seizures before 3 years old,5 patients with seizures from 3 to 6 years old and 1 at 19 years old.There were 5 cases of spastic seizures and 5 cases of focal seizures.The Video EEG showed that 7 patients have epileptiform discharge site located on one side brain hemisphere and 3 patients about both side.Imaging results showed 4 cases of unilateral subventricular or subcortical nodules,5 cases of bilateral abnormalities,and 1 case of Dandy-Walker deformity.Pathology showed brain tissue section of 4 patients with six-story structure disappearance and immature neurons appearance.Genetic testing revealed 5 cases of TSC1 mutations,5 cases of TSC2 mutations,7 cases of family mutations,and 4 cases of surgical patients were nonsense mutations.The treatment effect about 3 patients of Rapamycin improved than before significantly.Conclusion　TRE patients usually fall ill in early age,more males than females,mainly spastic and focal seizures.The Video EEG showed epileptiform discharge site located on one side brain hemisphere.Gene testing is useful for treatment.The type of mutation may be related to the degree and location of brain lesions and the origin of the discharge,but there is no significant correlation between the discharge site recorded on the EEG and the abnormal imaging site.Therefore,genetic testing should be considered when clinical treatment is not improvement.

A case of limbic encephalitis with positive anti-leucine-rich glioma inactivated 1 protein (LGI1) antibody and anti-myelin oligodendrocyte glycoprotein (MOG) antibody was reported. The patient was a middle-aged male with a history of retinal vein occlusion. The main symptoms were temporal lobe epilepsy, facial arm dystonia, autonomic nerve dysfunction. Magnetic resonance imaging showed long T 2 signal in the right hippocampus without enhancement and normal perfusion. Electroencephalogram showed paroxysmal slow wave and sharp slow wave in interictal period. Blood anti-MOG antibody, blood and cerebrospinal fluid anti-LGI1 antibody were double positive. The main diagnosis was limbic encephalitis. After treatment with hormone and gamma globulin, the symptoms were improved and double antibodies were turned negative. Anti-LGI1/MOG double positive cases are rare, and the clinical manifestations and imaging manifestations of double positive antibody cases are not completely consistent with those with each single antibody, with different characteristics. This report can help clinicians enhance awareness.

Objective To investigate medication adherence and its influencing factors in community-based hypertensive patients in the Pearl River Delta of Guangdong Province, and lay the foundation for the development of targeted compliance interventions. Methods Between July 2015 and October 2016, a multi-stage cluster sampling method was used to conduct a community-based diagnostic survey in the Pearl River Delta region.A total of 1 829 community-based hypertension patients in this survey population were used to investigate medication adherence, the factors of which were derived through a multi-factor logistics regression analysis.Results The average medication-adherence score of hypertensive patients surveyed in this study was (4.6 ± 1.8), and patients with good medication adherence accounted for 62.82%(1 149/1 829).There were significant differences in medication adherence among patients according to their age, household registration types, marital status, level of education, employment status, medical payment methods,per capita monthly household income,drinking status,and family history of hypertension (P<0.05).Multivariate logistic regression analysis found that medication adherence was affected substantially by the type of household registration:local household types (OR= 0.537, 95% CI: 0.415-0.695); education level: college,bachelor and above (OR=2.139,95% CI:1.100-4.160); employment status: self-paying (OR=0.591, 95% CI: 0.376-0.930); and a family history of hypertension (OR= 1.279, 95% CI: 1.012-1.617).Conclusions It is necessary to pay more attention to medication adherence in patients with hypertension in communities.Given the influencing factors and characteristics illuminated by this study,it is suggested that various measures be taken to prevent and intervene in poor medication adherence, to improve the curative effect of hypertensive patients in communities.

Objective:To study the clinical and laboratory characteristics of neonatal isolated sulfite oxidase deficiency (ISOD).Methods:An infant with neonatal ISOD admitted to our hospital was retrospectively analyzed. Using key words "isolated sulfite oxidase deficiency", "SUOX gene", "Infant, newborn", databases including CNKI, Wanfang database, National library and literature center of science and technology, China science paper online, PubMed, Web of Science and EMBASE (up to January 2021) were searched and literature review was conducted. The clinical manifestations, laboratory results, treatment and prognosis were analyzed.Results:Our patient was a full-term male infant with eye movement disorder, refractory seizures, feeding difficulties, increased muscle tone, developmental retardation and microcephaly. Urine sulfite paper-strip test was positive. Uric acid was normal. Whole exon sequencing (WES) revealed SUOX c.475G>T and c.1201A>G compound heterozygous mutations. Cranial MRI showed multiple encephalomalacia and brain atrophy at 5-month of age. The infant died at 8-month. In the literature review, a total of 29 articles and 32 cases of neonatal ISOD were found. 87.5% of the cases developed symptoms within 1-week after birth. All had convulsive seizures. Some of them had feeding difficulties, muscle tone changes, developmental retardation, microcephaly and ectopia lentis. Cranial imaging showed white matter cystic lesions and brain atrophy. Laboratory examination showed elevated urinary sulfite and S-sulfocysteine. Uric acid and xanthine/hypoxanthine were normal. Blood homocysteine was decreased. 23 cases received genetic testing and all of them had SUOX mutations. The treatment was mainly symptomatic relief and supportive treatment. During follow-up, 15 cases died, 13 cases survived and 4 cases were unknown. All the surviving children had drug-resistant convulsions and developmental retardation.Conclusions:Neonatal ISOD may present with refractory convulsions, feeding difficulties and developmental retardation. Cystic white matter changes and brain atrophy may be seen on cranial imaging. Elevated urinary sulfites, decreased blood homocysteine and normal uric acid are important clues for diagnosis. Genetic testing is helpful for early diagnosis.

Objective:To prepare a fluorescent probe Cetuximab-IRDye800CW targeting epidermal growth factor receptor (EGFR) and investigate its application value in surgical navigation of glioblastoma (GBM).Methods:The fluorescence properties of Cetuximab-IRDye800CW were determined by fluorescence spectrophotometer. The specificity of Cetuximab-IRDye800CW bound to GBM cells was verified by Western blot. The competitive binding method of enzyme-linked immunosorbent assay (ELISA) was used to prove whether the probe could achieve tumor targeting by binding to EGFR. Subcutaneous models of 6 nude mice of GBM were divided into experimental group ( n=3; injected with Cetuximab-IRDye800CW) and control group ( n=3; injected with IRDye800CW), and images were obtained at 5 min, 24 h, 48 h and 72 h after injection. Differences of mean fluorescence intensity (MFI) and tumor to background ratio (TBR) between experimental group and control group were compared. In situ models of GBM nude mice were established ( n=6), and MRI and intraoperative navigation were conducted, which were compared with pathological distribution. Independent-sample t test was used to analyze the data. Results:The maximum emission wavelength of Cetuximab-IRDye800CW was 820 nm, which could be received by near infrared fluorescence imaging equipment. Western blot showed that Cetuximab-IRDye800CW was only bound to GBM cells. The competitive binding of ELISA showed that Cetuximab-IRdye800CW could achieve tumor targeting by binding with EGFR. At 5 min, 24 h, 48 h and 72 h after injection of fluorescent materials, the MFI values of experimental group were 109.00±3.81, 73.36±9.93, 55.24±8.82, 37.71±6.11, which were higher than those of control group (91.32±4.17, 42.91±5.39, 25.08±6.05, 8.33±1.00; t values: 4.36-9.40, P values: 0.011-0.049). The TBR of experimental group was higher than that of control group at 24 h and 48 h after injection (24 h: 2.40±0.28 vs 1.57±0.07, t=4.94, P=0.039; 48 h: 2.07±0.12 vs 1.22±0.08, t=9.85, P=0.010). GBM in situ model was successfully constructed and verified by MRI, and the tumor was visualized under the fluorescence device navigation. Pathological distribution of the tumor with HE staining was consistent with fluorescence imaging. Conclusion:Cetuximab-IRDye800CW has fluorescence imaging capability and can identify tumor boundaries in intraoperative navigation of GBM, which has potential clinical application value.

Objective To understand the differences in the demand,preference,and tendency for elderly care services between urban and rural areas in the Pearl River Delta(PRD),and to provide reference for the planning and balanced allocation of elderly care resources in urban and rural areas.Methods Using the multi-stage stratified random sampling method,we selected 7 community health service centers in 2 prefecture-level cities in the PRD and conducted a questionnaire survey on the elderly care service demand,preference,and tendency among 1919 regular residents aged 60 years and above who attended the centers.Results A total of 641 urban elderly residents(33.4%)and 1278 rural elderly residents(66.6%)were surveyed in the PRD.The urban and rural elderly residents showed differences in the child number(χ2 =43.379,P<0.001),willingness to purchase socialized elderly care services(χ2 =104.141,P<0.001),and attitudes to the concept of raising child to avoid elderly hardship(χ2 =65.632,P<0.001).The proportion(71.8%)of rural elderly residents who prefer family-based elderly care was higher than that(57.1%)of urban elderly residents(χ2 =41.373,P<0.001).The proportion(62.2%)of urban elderly residents clearly expressing their willingness to choose institutions for elderly care was higher than that(44.0%)of rural elderly residents(χ2 =57.007,P<0.001).Compared with family-based elderly care,the willingness to choose institutional or community-based in-house elderly care was low among the urban elderly residents with surplus monthly household income or balanced income and expenditure;urban males,those with college education background or above,and those who purchased so-cialized elderly care services tended to prefer community-based in-house elderly care.In rural areas,the elderly residents who had local household registry were prone to choose institutional or community-based in-house elderly care,while those who had more than one child and those who were satisfied with the current living conditions were less willing to choose community-based in-house elderly care.Conclusions It is suggested that the urban-rural differences in the elderly care service demand,preference and tendency should be fully considered in the planning and allocation of urban and rural elderly care resources.Efforts remain to be made to develop diversified social elderly care services tailored to the characteristics of urban and rural areas.

The mechanical microenvironment of cells plays a critical role in regulating the physiological function of cells. Cells in vivo are often subjected to a variety of mechanical forces from their mechanical micro-environment, such as shear, tension, and compression. At the same time, cells can adhere to the extracellular matrix (ECM) through adhesion molecules (such as integrin-ligand binding), and further sense the stiffness of the ECM. Cell mechanics mainly studies the properties and behavior of living cells under mechanical forces, and how they relate to cell functions. This review summarized the advances in cell mechanics in 2022, focusing on integrin-ligand interactions and the effects of matrix stiffness and mechanical forces on cell physiological behavior and morphogenesis.

Objective:To examine the efficacy of robot-assisted laparoscopic modified ventral onlay lingual mucosal graft for complex ureteral stricture.Methods:The clinical data of 8 patients with ureteral stricture admitted to the Department of Urology, General Hospital of Southern Theater Command from May to October 2022 were retrospectively analyzed. There were 6 males and 2 females, aged (45.1±10.2) years (range: 34 to 64 years), body mass index (24.6±2.0) kg/m 2 (range: 20.7 to 26.6 kg/m 2). Five cases on the left side, 3 cases on the right side, the length of the ureteral structure was (3.1±0.7) cm (range: 2.2 to 4.5 cm). The value of preoperative serum creatinine was (113.8±22.3) μmol/L (range: 96 to 15 μmol/L). Before excising the structure segment, the titched anastomosed part of the dorsal wall of the ureter, and then the posteriorly augmented anastomotic, the remaining ventral side was augmented with a onlay lingual mucosa graft, then the omentum flap was used to wrap the reconstructed ureteral segment. The lingual mucosa graft with a length of 2.5 to 5.0 cm and a width of 1.0 to 1.5 cm was cut according to the actual structure. The surgery information of the patient, complications, and recent follow-up were recorded. Results:The operation under robot-assisted laparoscopy was performed successfully in the 8 patients without conversion to open surgery. The duration of the operation was (226.9±22.8) minutes (range: 210 to 255 minutes), estimated blood loss was (93.8±25.9) ml (range: 75 to 150 ml), the retention time of the postoperative drainage tube was (4.8±1.3) days (range: 3 to 7 days), and the duration of postoperative hospitalization was (11.1±3.6) days (range: 9 to 14 days). One week after the operation, the patient could pronounce correctly, enunciate clearly, and eat normally. Double J tubes were removed 4 to 8 weeks after the operation. The follow-up time in this group was 3 to 9 months, the follow-up patients underwent imaging and other examinations, which showed a significant improvement in hydronephrosis on the affected side, and the value of renal pelvic separation on the affected side was (1.4±0.8) cm (range: 0 to 2.3 cm). The serum creatinine value was (100.1±24.9) μmol/L (range: 76 to 155 μmol/L). Three months after the operation, the ureteroscopy showed that the ureter was smooth and the mucosa was normal.Conclusions:Robot-assisted laparoscopic ureteroplasty with a lingual mucosal graft is a safe and feasible operation for complex ureteral stricture without serious complications, which provides a surgical option for repairing ureteral stricture.