Epilepsy is a chronic neurological disorder affecting ~65 million individuals worldwide. Abnormal synaptic plasticity is one of the most important pathological features of this condition. We investigated how ubiquitin-specific peptidase 47 (USP47) influences synaptic plasticity and its link to epilepsy. We found that USP47 enhanced excitatory postsynaptic transmission and increased the density of total dendritic spines and the proportion of mature dendritic spines. Furthermore, USP47 inhibited the degradation of the ubiquitinated α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) subunit glutamate receptor 1 (GluR1), which is associated with synaptic plasticity. In addition, elevated levels of USP47 were found in epileptic mice, and USP47 knockdown reduced the frequency and duration of seizure-like events and alleviated epileptic seizures. To summarize, we present a new mechanism whereby USP47 regulates excitatory postsynaptic plasticity through the inhibition of ubiquitinated GluR1 degradation. Modulating USP47 may offer a potential approach for controlling seizures and modifying disease progression in future therapeutic strategies.
Animals ; Receptors, AMPA/metabolism* ; Neuronal Plasticity/physiology* ; Seizures/physiopathology* ; Disease Models, Animal ; Mice, Inbred C57BL ; Mice ; Ubiquitin Thiolesterase/genetics* ; Male ; Excitatory Postsynaptic Potentials/physiology* ; Ubiquitination ; Dendritic Spines/metabolism* ; Hippocampus/metabolism*

AIM:To investigate the effects of γ-synuclein on autophagy and apoptosis of colon cancer cells in-duced by endoplasmic reticulum stress,as well as the protective effect on the cells.METHODS:Gene expression profile chip analysis was performed to compare the cDNA expression profiles between human colon cancer HCT116 cells with γ-synu-clein knockdown and HCT116 cells with control siRNA,and to identify potential molecules related to autophagy and apop-tosis.In colon cancer cell lines,the functional effects of γ-synuclein on autophagy and apoptosis induced by thapsigargin(TG),an endoplasmic reticulum stress-inducing agent,were systematically explored by conducting immunofluorescence staining,Western blot,CCK-8 assay,flow cytometry,and transmission electron microscopy.Western blot was used to de-tect the expression of γ-synuclein protein,autophagy-related proteins[microtubule-associated protein 1 light chain 3(LC3),beclin-1,autophagy-related protein 5(ATG5)and ATG7],and apoptosis-related proteins[poly(ADP-ribose)polymerase(PARP),pro-caspase-3,and pro-caspase-9].To further analyze the mechanism of γ-synuclein in regulating autophagy and apoptosis,extracellular signal-regulated kinase(ERK)inhibitor PD98059,ERK inhibitor SP600125 and c-Jun N-terminal kinase(JNK)activator anisomycin were applied separately to test HCT116 cells transfected with γ-synu-clein siRNA.Subsequently,autophagy proteins,apoptosis proteins,and ERK and JNK pathway-related proteins were de-tected by Western blot.RESULTS:The TG-induced autophagy of colon cancer cells mainly occurred at the early stage(0～24 h),and apoptosis mainly occurred at the late stage(36～48 h).Endoplasmic reticulum stress up-regulated the ex-pression of γ-synuclein in colon cancer cells,which was associated with enhanced autophagy.γ-Synuclein promoted au-tophagy by activating ERK and JNK pathways at the early stage(0～24 h),and inhibited apoptosis by blocking JNK path-ways at the late stage(24～48 h)to protect HCT116 cells.In our model,γ-synuclein was observed to play a critical role in the transition from endoplasmic reticulum stress-induced autophagy to apoptosis.CONCLUSION:In the context of endo-plasmic reticulum stress,γ-synuclein promotes autophagy and inhibits apoptosis by regulating ERK and JNK signaling pathways,thus protecting colon cancer cells.This provides a potential idea for anti-tumor therapy.

OBJECTIVE To evalu ate the effects of perimenopausal one-day outpatient multidisciplinary model on the cognition of women to menopause hormone therapy （MHT）. METHODS The perimenopausal one-day outpatient service opened by the Third Affiliated Hospital of Chongqing Medical University in 2017 was introduced ，which was participated by doctors ，pharmacists， nutritionists，music psychotherapists ，nurses and other multidisciplinary members to provide systematic ，all-round，humanistic and face-to-face group science popularization and education ，practical experience and Q&A for women in perimenopause or about to enter perimenopause （called“students”）. The students who participated in one-day outpatient service in 2020 were selected as the survey object ， and the questionnaire was CMEI2020KPYJ（ZAMM）00206] designed to analyze the understanding of perimenopausal syndrome，awareness rate of MHT ，willingness to use and concerns. RESULTS A total of 295 students completed the questionnaire. Compared with before participation ， after participated in one-day outpatient service ，the cognition level of the students were all improved significantly ，including perimenopausal age （96.61％ vs. 99.32％，P＝0.037），the importance of perimenopausal health care knowledge （91.19％ vs. 96.95％，P＜0.001），whether the perimenopausal syndrome needs treatment （88.47％ vs. 99.32％，P＜0.001），willingness to use MHT （70.59％ vs. 94.48％ ，P＜0.001），MHT treatment timing （60.50％ vs. 95.17％ ，P＜0.001），reducing the risk of cardiovascular disease （51.68％ vs. 96.55％ ，P＜0.001），delaying aging （69.75％ vs. 97.59％ ，P＜0.001），preventing osteoporosis（65.13％ vs. 97.59％），P＜0.001）；the medical staff were higher than the non-medical staff （P＜0.05）. Totally 90％ of the students said they had gained knowledge about the prevention and treatment of common diseases ，nutrition and guidance， psychological regulation guidance hormone therapy related knowledges and exercise methods ；the proportion of the students who were willing to use MHT for 1 to 5 years（31.09％ vs. 47.93％）increased significantly. The proportion of the students who concerned about the risk of thrombosis （60.24％ vs. 36.81％），weight gain （59.64％ vs. 14.84％）and life-long dependence （52.41％ vs. 18.13％）was significantly reduced ，but that of the students who concerned about cancer risk was not diminished. From 2017 to 2020，the utilization rate of MHT was increased from 2.22％ to 62.16％ in non-medical staff among the students. CONCLUSIONS The multidisciplinary model of perimenopausal one-day outpatient service can improve the awareness of MHT among perimenopausal women ，eliminate misunderstandings ，and increase the utilization rate of MHT.

To explore the mode of"one-day outpatient"based continuous management and examine its feasibility and preliminary effects for improving menopausal syndrome and mood among menopausal women.Clinical intervention study was conducted in Obstetrics and Gynecology Center of the Third Affiliated Hospital of Chongqing Medical University from May 2020 to May 2021.The continuous management mode of "one-day outpatient service" for menopause was constructed in terms of multidisciplinary resources, including offline "one-day outpatient" health education, online 7-week group continuous intervention on "healthy lifestyle" and offline half-day focus group interview. Pre-and post-scores of the modified Kupperman scale and the positive/negative emotional scale (PANAS) were measured to compare the status of menopausal syndrome and emotional experience of Seventy-eight female participants (40-60 years old), meanwhile, before and after comparison of the blood lipid and body composition indexes of participants were also performed. Paired t test or Wilcoxon signed rank test were used.Results show that the pre-and post Kupperman scores were 14.65±8.51 vs 10.26±5.83 ( t=-5.59, P<0.01), and the positive emotional scores (pre- 28.53±5.85 vs post- 30.13±6.30) were improved ( t=2.59, P=0.012) and negative emotional scores [pre- 20.5(10) vs post- 17.0(7)] were decreased ( Z=-5.09, P<0.01). The triglyceride level of participants declined from (1.27±0.54) mmol/L to (1.09±0.38) mmol/L ( t=-2.45, P<0.05). In addition, the body mass index(pre- 22.52±2.34 vs post- 22.06±2.22), percentage of body fat (pre- 31.72±6.22 vs post- 30.91±6.52)and Visceral fat area(pre- 83.96±30.26 vs post- 79.66±29.71) were all improved ( t=-3.58, t=-2.57, t=-2.59, P<0.05). Therefore,the mode of"one-day outpatient"based continuous management can effectively improve menopausal syndrome and adverse mood, reduce patients′ blood lipid, improve the body composition, and maybe contribute to the prevention of long-term chronic diseases.

To explore the mode of"one-day outpatient"based continuous management and examine its feasibility and preliminary effects for improving menopausal syndrome and mood among menopausal women.Clinical intervention study was conducted in Obstetrics and Gynecology Center of the Third Affiliated Hospital of Chongqing Medical University from May 2020 to May 2021.The continuous management mode of "one-day outpatient service" for menopause was constructed in terms of multidisciplinary resources, including offline "one-day outpatient" health education, online 7-week group continuous intervention on "healthy lifestyle" and offline half-day focus group interview. Pre-and post-scores of the modified Kupperman scale and the positive/negative emotional scale (PANAS) were measured to compare the status of menopausal syndrome and emotional experience of Seventy-eight female participants (40-60 years old), meanwhile, before and after comparison of the blood lipid and body composition indexes of participants were also performed. Paired t test or Wilcoxon signed rank test were used.Results show that the pre-and post Kupperman scores were 14.65±8.51 vs 10.26±5.83 ( t=-5.59, P<0.01), and the positive emotional scores (pre- 28.53±5.85 vs post- 30.13±6.30) were improved ( t=2.59, P=0.012) and negative emotional scores [pre- 20.5(10) vs post- 17.0(7)] were decreased ( Z=-5.09, P<0.01). The triglyceride level of participants declined from (1.27±0.54) mmol/L to (1.09±0.38) mmol/L ( t=-2.45, P<0.05). In addition, the body mass index(pre- 22.52±2.34 vs post- 22.06±2.22), percentage of body fat (pre- 31.72±6.22 vs post- 30.91±6.52)and Visceral fat area(pre- 83.96±30.26 vs post- 79.66±29.71) were all improved ( t=-3.58, t=-2.57, t=-2.59, P<0.05). Therefore,the mode of"one-day outpatient"based continuous management can effectively improve menopausal syndrome and adverse mood, reduce patients′ blood lipid, improve the body composition, and maybe contribute to the prevention of long-term chronic diseases.

Objective: : To explore the effect of PD-1 gene knockout by CRISPR/Cas9 system on the proliferation and IFN-γ secretion in human T cells. Methods: : The sequence of sgRNA targeting PD-1 was designed. The PD-1-sgRNA and Cas9 mRNA were synthesized by T7 RNApolymerase in vitro, and then the mixture of PD-1-sgRNAand Cas9 mRNAwas delivered into activated T cells by nucleofection. The efficiency of gene knockout was confirmed by sequencing. The phenotypes of T lymphocytes and the expression of PD-1 after gene knockout were analyzed by Flow cytometry. The proliferation of T lymphocytes was calculated by trypan blue counting. The level of IFN-γ secreted by T lymphocytes was detected by ELISA. Results： ：PD-1-sgRNA and Cas9 mRNA were successfully synthesized in vitro and delivered into T cells by nucleofection. Sequencing technology confirmed that the PD-1 gene sequence was edited and the editing efficiency was 58.3%. The expression of PD-1 on T lymphocyte surface was down-regulated successfully by CRISPR/Cas9 system [(9.6±1.85)% vs (16.2±2.05)%, P<0.05]. The knockout of PD-1 gene did not affect the proliferation and phenotype of T lymphocytes(P<0.05); However, compared with the control group, the level of IFN-γ secreted by T lymphocytes in the PD-1sgRNA group was significantly increased (P<0.01). Conclusion: : CRISPR/Cas9 system can successfully ablate PD-1 gene in human T lymphocytes, which could block the negative regulation of PD-1／PD-L1 and further promote the IFN-γ secretion in T cells.

OBJECTIVETo construct a lentiviral vector carrying the γ-synuclein(SNCG) gene and establish a human colorectal carcinoma cell line SW1116 stably expressing this gene, and then investigate the inhibition of the growth and invasion capacity of SW1116 cells.

METHODSRNA interference fragment was designed according to the SNCG sequence (GenBank: No.NM003087.2), and then SNCG RNAi effective target genes were screened. After the Oligo DNA of target sequences was synthesized, the lentiviral vectors carrying LV-SNCG-RNAi-EGFP (RNAi group) and LV-SNCG-NC-EGFP (NC group) were constructed and packaged to produce lentivirus venom. The supernatants of different virus-producing cells were used to transfect SW1116 cells respectively. Wild SW1116 cells were used as blank control (CON group) EGFP fluorescence was detected by fluorescent microscopy and the differential expression of SNCG mRNA and protein was detected by real-time PCR and Western blot. CCK-8, soft agar assay and Transwell chamber were employed to estimate the inhibiting effect on growth and invasion of SW1116 respectively.

RESULTSRecombinant lentiviral vectors respectively carrying the SNCG-RNAi-EGFP and SNCG-NC-EGFP were successfully constructed and the supernatants of lentivirus could effectively infect SW1116 cells. The titer of the virus carrying LV-SNCG-RNAi-EGFP or LV-SNCG-NC-EGFP was 8×10(8) TU/ml. Real-time PCR and Western blot confirmed that compared with the NC group, SNCG-RNAi group had lower SNCG expression (1.009±0.161 vs. 0.114±0.030, P=0.009), and showed tremendous silencing effect as 76.8%(P<0.05). SNCG protein expression was also significantly reduced (RNAi:12.001±2.884, NC:32.443±4.731, CON:34.308±6.920, P<0.05). After SNCG knockdown, the number of proliferation cells was obviously reduced at 48, 72, 96 and 120 hours respectively(P=0.036). In soft agar assay, clones in RNAi group were smaller[RNAi:(0.582±0.103) mm, NC:(1.863±0.316) mm, CON:(1.749±0.525) mm]. Colony formation rate of RNAi group was down to (17.1±3.5)%, which was significantly lower than (36.5±4.3)% in NC group and (33.8±3.9)% in CON group. In migration test, the number of invasion cell was 37.4±9.3 in RNAi group, which was significantly less than 112.3±8.6 in NC group and 100±0.0 in CON group.

CONCLUSIONExpression of SNCG mRNA and protein plays an important role in the growth and the invasion capacity of SW1116 cells.

Cell Line, Tumor ; Cell Proliferation ; Colorectal Neoplasms ; pathology ; Genetic Vectors ; Humans ; Lentivirus ; RNA Interference ; RNA, Messenger ; RNA, Small Interfering ; genetics ; Real-Time Polymerase Chain Reaction ; Transfection ; gamma-Synuclein ; genetics

The purpose of this research was to study the effect of Xingxiong sodium chloride injection on cerebral ischemia-reperfusion injury in rats. Rats were divided into five groups, namely sham group, model group, ginkgolide injection group(3 mg/kg), Xingxiong sodium chloride injection high-dose group(14 mg/kg)and low dose group(7 mg/kg). Except for the sham group, animals in other groups were subjected to ischemia for 2h and reperfusion for 72 h by middle cerebral artery occlusion(MCAO)with thread technique, and then drugs were administered continuously by tail intravenous injection during reperfusion period for 3 days. The neurological deficit score and the histopathological score were evaluated; infarction ratio, brain water content and biochemical indexes of animals in each group were determined. Compared with model group, reduction of neurological deficit score, brain water content and infarction area were observed obviously at 7 mg/kg and 14 mg/kg of Xingxiong sodium chloride injection. Additionally, reduction of histopathological score, H2O2 and MDA content, increase of the level of GSH, GSH-Px, SOD and also the capacity of inhibition of superoxide anion radical(·O-2)and hydroxyl radial(·OH)were observed at 14 mg/kg. The results suggested that Xingxiong sodium chloride injection could effectively enhance the ability of anti-oxidation in the brain tissues, and protect brain from ischemia-reperfusion injury.

OBJECTIVETo construct γ-synuclein gene eukaryotic expression vector, and to study its effect on the invasion of colon cancer cell line SW1116 and the adhesion between SW1116 and human umbilical vein endothelial cells(HUVECs) in vitro.

METHODSTotal RNA was extracted from colon cancer cell line HT29 and the cDNA of γ-synuclein was amplified using RT-PCR. The digested fragment of cDNA coding sequence was linked to the eukaryotic expression vector pEGFP-C1 containing the GFP gene. After identification by sequence analysis, the recombinant plasmid was transfected into colon cancer cell line SW1116 via lipofectamine. The stable cell line was selected with G-418. The invasion in vitro was tested by Transwell invasion chamber assay. HUVECs were previously seeded onto 96-well plates before SW1116 cells seeded, and fluorescence intensity of GFP was detected to represent the amount of adhesion cells by ELISA.

RESULTSHuman γ-synuclein eukaryotic expression vector was successfully constructed, which was stably expressed in SW1116 cells and could translate the GFP-γ-synuclein protein in vitro. γ-synuclein facilitated SW1116 cell passing through matrigel and filter membrane(198.4±20.7 vs. 98.8±13.2, P<0.05) and elevated the adherence of SW1116 cells to HUVECs(3.08±0.36 vs. 1.22±0.21, P<0.05).

CONCLUSIONExpression of γ-synuclein can strengthen colon cancer cell SW1116 potentiality of invasion and metastasis in vitro.

Cell Adhesion ; Cell Line, Tumor ; Colonic Neoplasms ; pathology ; Genetic Vectors ; biosynthesis ; Human Umbilical Vein Endothelial Cells ; Humans ; Neoplasm Invasiveness ; Neoplasm Metastasis ; gamma-Synuclein ; genetics

Objective To explore the impact of iodine nutrition on 8-10 years old children after adjusting the iodine content in iodized salt in Hangzhou.Methods Twelve counties (areas,cities) were divided into urban,suburban and rural areas in Hangzhou.By population proportion survey (PPS),every county(area,city) was divided into east,west,south,north and middle districts; one school was selected in each district; forty children (half male and half female) aged 8-10 years old in each school were selected; family salt and urine samples of each student were collected.The levels of salt and urinary iodine were measured by picric sodium thiosulfate titrimetric (GB 13025.7-2012) and spectrophotometer method (WS/T 107-2006),respectively.Results Two thousand seven hundred and twenty-five household salt samples were collected.The median of salt iodine,the iodized salt coverage rate,the qualification rate of iodized salt and the consumption rate of qualified iodized salt were 24.00 mg/kg,4.35％(2 571/2 725),91.02％(2 340/2 571) and 85.87％(2 340/2 725),respectively.The medians of salt iodine in urban,suburb and rural areas were 24.10,22.12,24.30 mg/kg,respectively.A total of 2 664 children urine samples were collected.The median of urinary iodine (MUI) of the children was 177.24 μg/L.The MUIs in urban,suburb and rural areas were 175.00,178.55,178.00 μg/L,respectively; in male was 183.00 μg/L and female was 170.50 μg/L.When non-iodized and unqualified iodized salt were taken,the differences of urinary iodine within groups were statistically significant in urban,suburb and rural areas(x2 =18.652,14.686,all P ＜ 0.05).In rural area,the difference of urinary iodine of 8-10 years old children who ingested different types of iodized salt was statistically significant(x2 =39.07,P ＜ 0.05).Conclusion After adjusting the iodine content of salt in Hangzhou,the iodine-nutritional status of 8-10 years old students is at a appropriatelevel.