Diabetic kidney disease(DKD)is one of the most important complications of diabetes.In recent years,domestic and foreign studies have found that mammalian target protein of rapamycin(mTOR)related signaling pathway is a classic pathway involved in the regulation of autophagy,which can achieve the therapeutic effect of DKD by targeting the autophagy pathway,and plays a crucial role in the prevention and treatment of DKD.In this paper,we reviewed the mechanism of mTOR-related signaling pathway targeted autophagy in the prevention and treatment of DKD,in order to provide a new reference and basis for clinical prevention and treatment of DKD.

3ʹ-Hydroxy-4ʹ-methoxy-2-hydroxy-5-bromochalcone (hereinafter referred to as C13) is a novel chalcone derivative obtained in the process of structural modification of DHMMF, the antitumor active compound of Resina Draconis, in our laboratory. In this study, we investigated the effects of C13 on the proliferation and apoptosis of human gastric cancer HGC-27 and AGS cells and its potential mechanism of action. Firstly, through methyl thiazolyl tetrazolium (MTT), colony formation assay, and 5-ethynyl-2'-deoxyuridine (EdU) staining, we found that C13 inhibited the proliferation ability of human gastric cancer HGC-27 and AGS cells. Using flow cytometry and Western blot, it was found that C13 induced apoptosis in human gastric cancer HGC-27 and AGS cells, and up-regulated the protein level of cleaved poly ADP-ribose polymerase (cleaved-PARP). The results of RNA sequencing analysis showed that the Erb-b2 receptor tyrosine kinase 4/phosphoinositide 3-kinases/AKT (ErbB4/PI3K/AKT) signaling pathway may be involved in anti-gastric cancer activity of C13. Finally, the results of immunoblotting assay showed that C13 treatment down-regulated the protein levels of ErbB4 and phospho-ErbB4, as well as down-regulated the phosphorylation levels of PI3K and AKT in human gastric cancer HGC-27 and AGS cells, which verified the results from RNA-seq analysis. In conclusion, C13 inhibited the proliferation and induced apoptosis of human gastric cancer cells, which may be related to the down-regulation of ErbB4/PI3K/AKT signaling pathway. This study may provide a candidate drug for the treatment of gastric cancer.

【Objective】 To explore the application value of transrectal contrast-enhanced ultrasound (CEUS) microangiography, elastography and conventional ultrasound-guided prostate cancer puncture biopsy in the diagnosis of prostate diseases, so as to provide reference for the early diagnosis of prostate cancer. 【Methods】 A total of 156 patients suspected of prostate cancer treated in our hospital during Jan.2021 and Dec.2022 were selected.The patients were divided into group A (n=52, conventional ultrasound), group B (n=49, elastography) and group C (n=55, CEUS microangiography) according to the puncture methods.The positive rate of puncture and diagnostic value of the three methods were analyzed. 【Results】 The positive rate of puncture was 24.18% in group B and 25.71% in group C, which was significantly higher than that in group A (13.15%, P<0.05).The diagnostic accuracy of prostate cancer was 93.88% in group B and 94.55% in group C, higher than that in group A (75.00%, P=0.002).In group B, the Emax and Emean of malignant lesions were (65.56±14.43) kPa and (59.59±11.02) kPa, respectively, which were higher than those of benign lesions (P<0.001).The difference in blood flow grade detected by CEUS microangiography was statistically significant between benign and malignant lesions, and grade 3 blood flow accounted for 95.65% of malignant lesions.The area under the receiver operating characteristic (ROC) curve (AUC) of Emean in elastography in the diagnosis of prostate cancer was 0.810 (95%CI: 0.690-0.930, P<0.05).The AUC of CEUS microangiography in the diagnosis of prostate cancer was 0.965 (95%CI: 0.913-1.000, P<0.05). 【Conclusion】 Compared with conventional ultrasound-guided prostate cancer puncture biopsy, CEUS microangiography and elastography guided prostate cancer puncture biopsy have better application value in the diagnosis of prostate diseases, which can provide semi-quantitative/quantitative parameter basis for the diagnosis of prostate diseases.

Objective: To investigate the marriage intention and its influencing factors among HIV-infected men who have sex with men (MSM), so as to provide the reference for formulating high-risk behavior intervention strategies and reducing the risk of HIV transmission among this population. Methods: Unmarried HIV-infected MSM were sampled from newly reported HIV/AIDS patients in Gongshu District of Hangzhou City from 2020 to 2022. Participants' sociodemographic information, family status and marriage intention were collected using questionnaire surveys. Factors affecting marriage intention were identified using a multivariable logistic regression model. Results: A total of 430 questionnaires were allocated and 400 valid questionnaires were recovered, with an effective recovery rate of 93.02%. The respondents had a mean age of (28.90±4.67) years, and included 271 cases with registered residence outside Hangzhou City (67.75%). There were 82 cases indicating that they would probably or definitely get married, accounting for 20.50%, including 46 cases (56.10%) aged 30 years and older, 67 cases (81.71%) engaged in commercial services/freelance work/worker and 53 cases (64.63%) being the only children. Multivariable logistic regression analysis showed that unmarried HIV-infected MSM who were aged 30 years and older (OR=2.751, 95%CI: 1.230-6.154), had a monthly income of <10 000 Yuan (5 000 to <10 000 Yuan, OR=7.010, 95%CI: 2.049-23.981; <5 000 Yuan, OR=5.229, 95%CI: 1.803-15.165), had a high school education and below (OR=5.935, 95%CI: 1.775-19.844), were the only children (OR=3.407, 95%CI: 1.468-7.910), came from families with economic conditions above the average level (OR=8.906, 95%CI: 2.070-38.308), did not disclose their sexual orientation (OR=3.298, 95%CI: 1.118-9.727), held a positive attitude towards government policies (OR=70.544, 95%CI: 14.583-341.237), had no homosexual intercourse in the past six months (OR=3.583, 95%CI: 1.278-10.044), and had not informed their sexual partners of their HIV status (OR=2.910, 95%CI: 1.228-6.897) were more willing to get married. Conclusion A certain proportion of unmarried HIV-infected MSM in Gongshu District are willing to get married, and their marriage intention is affected by age, educational level, family economic conditions, the only children or not, disclosing sexual orientation or not, having homosexual intercourse in the past six months or not, informing sexual partners of HIV status or not, and the attitude towards government policies.

Objective:Using retrogradely trans-monosynaptic tracing method mediated by rabies virus(RV)to ob-serve presynaptic neuronal distributions of vesicular glutamate transporter 2(VGLUT2)-positive neurons of the med-iodorsal thalamic nucleus(MD)in whole brain.Methods:The helper viruses of RV were injected into the right MD of VGLUT2-ires-Cre mice,and two weeks later RV was injected into the same area.After another one week,perfusion was taken and whole brain scanning was performed to observe the distributions of RV-labeled presynaptic neurons throughout the brain.Results:After RV virus was injected into the MD of VGLUT2-ires-Cre mice,dense presynaptic neurons were observed in the cortex and brainstem.RV labeled neurons were mostly distributed in primary motor cortex(M1),sec-ondary motor cortex(M2),medial prefrontal cortex,orbitofrontal cortex and insular cortex at the cortical level.Tha-lamic retrogradely labeled neurons were mostly found in reticular thalamic nucleus and lateral hypothalamic area,and retrogradely labeled neurons in the brainstem were mainly distributed in lateral parabrachial nucleus,periaqueductal grey matter and dorsal raphe nucleus.Conclusion:The results in the present experiment suggest that VGLUT2-positive neurons within the MD can receive ascending information transmission from the brainstem,or regulation from the reticu-lar nucleus of thalamus.As a higher-order thalamus,MD can also receive descending projections from the cortex,and is involved in a variety of functions.Our results provide morphological basis for the study of the function and the related neural circuit of the MD.

Objective:To observe the projections from tyrosine hydroxylase(TH)positive neurons in locus coerule-us(LC)to tachykinin-1(TAC1)neurons in paraventricular nucleus of the thalamus(PVT),and morphologically determine whether they are involved in transmission and modulation of nociceptive information.Methods:TAC1-ires-Cre mice were hybridized with Rosa26:CAG-LSL-tdTomato(Ai9)mice.And spared nerve injury(SNI)induced neu-ropathic pain model was established with TAC1-ires-Cre::Ai9 mice to observe the colocalization of TAC1 and Fos and the close appositions between TAC1/FOS double-labeled neurons and TH positive axonal terminals.The distribution of the TH positive neurons and FG retrogradely labeled neurons were observed in the LC after Fluorogold(FG)was injec-ted into the PVT.Finally,the coexistences of TH positive neurons and RV labeled neurons in the LC were observed after injection of RV-mediated retrograde tracing system.Results:TAC1 positive neurons were shown with red fluores-cence in TAC1-ires-Cre::Ai9 mice.TAC1/FOS double-labeled neurons were found in the PVT of the SNI model.Some TAC1/FOS double labeled neurons made close appositions with TH positive axonal terminals.FG retrogradely labeled neurons were observed in the LC after FG injected into the PVT,and some of the FG labeled neurons coexisted with TH positive neurons.Using RV retrograde transsynaptic tracing virus,the results showed that presynaptic neurons of TAC1 positive neurons in the PVT were found in the LC,and most of the presynaptic neurons were TH positive neu-rons.Conclusion:TH positive neurons in the LC project to TAC1 positive neurons of the PVT,forming LCTH+-PVTTAC1+neural circuit,which were activated by nociceptive information.It demonstrates that this pathway plays a role in pain transmission or regulation.

For the past 30 years,percutaneous balloon mitral valve dilatation has been performed under the guidance of X-rays and bedside ultrasound.However,there are still some cases of mitral valve stenosis in the large atrium where balloon dilation failed.Intraperitoneal ultrasound-guided percutaneous balloon mitral valve plasty is accurate and feasible,which can reduce the occurrence of complications and improve the success rate of such elderly complex cases.Two patients with severe mitral stenosis underwent percutaneous balloon mitral valve plasty guided by intracardiac ultrasound.The operations were successful without any complications,which can provide reference for clinical treatment of mitral stenosis.

In industrial production,the expression level of drug proteins in Chinese hamster ovary cells(CHO)is influenced by many factors:the regulatory elements on transcription and translation,the ge-nomic integration sites,and the expression system.Transcription,as the first step of gene expression,largely affects protein expression,and the promoter plays a crucial role in the initiation of transcription.Most of the promoters were screened through transient transfection or random integration,but the presence of unclear copy number or random integration sites makes it difficult to accurately evaluate the promoter activity.To some extent,site-specific integration can reduce the impact of positional effects on exogenous genes and may potentially increase the expression level of exogenous genes.In the early stage of our re-search,multiple sites that can stably express exogenous proteins were identified and verified in the CHO cell genome.In this study,one of these sites(2c6)was selected for the evaluation of promoter activity.The CRISPR/Cas9 gene editing technique was used to site-specifically integrate the reporter gene(EG-FP)regulated by the simian virus early promoter(SV40),mouse elongation factor-1α(mEF-1α),chicken β-actin(cACTB)promoter,and human phosphoglycerate kinase promoter(hPGK)into the 2c6 site,respectively.The mean fluorescence intensity of the cells was analyzed by flow cytometry,and the mRNA level of EGFP was detected by qPCR to comprehensively evaluate the activity of the promoter.The results showed that the activities of the mEF-1α and mACTB promoters were better than those of SV40 and hPGK.The results of the secondary flow cytometry sorting showed that site-specific integration can more accurately evaluate the activity of the promoter in the CHO cell expression system.

OBJECTIVES: To explore the cytotoxicity of four wild mushrooms involved in a case of Yunnan sudden unexplained death (YNSUD), to provide the experimental basis for prevention and treatment of YNSUD. METHODS: Four kinds of wild mushrooms that were eaten by family members in this YNSUD incident were collected and identified by expert identification and gene sequencing. Raw extracts from four wild mushrooms were extracted by ultrasonic extraction to intervene HEK293 cells, and the mushrooms with obvious cytotoxicity were screened by Cell Counting Kit-8 (CCK-8). The selected wild mushrooms were prepared into three kinds of extracts, which were raw, boiled, and boiled followed by enzymolysis. HEK293 cells were intervened with these three extracts at different concentrations. The cytotoxicity was detected by CCK-8 combined with lactate dehydrogenase (LDH) Assay Kit, and the morphological changes of HEK293 cells were observed under an inverted phase contrast microscope. RESULTS: Species identification indicated that the four wild mushrooms were Butyriboletus roseoflavus, Boletus edulis, Russula virescens and Amanita manginiana. Cytotoxicity was found only in Amanita manginiana. The raw extracts showed cytotoxicity at the mass concentration of 0.1 mg/mL, while the boiled extracts and the boiled followed by enzymolysis extracts showed obvious cytotoxicity at the mass concentration of 0.4 mg/mL and 0.7 mg/mL, respectively. In addition to the obvious decrease in the number of HEK293 cells, the number of synapses increased and the refraction of HEK293 cells was poor after the intervention of Amanita manginiana extracts. CONCLUSIONS The extracts of Amanita manginiana involved in this YNSUD case has obvious cytotoxicity, and some of its toxicity can be reduced by boiled and enzymolysis, but cannot be completely detoxicated. Therefore, the consumption of Amanita manginiana is potentially dangerous, and it may be one of the causes of the YNSUD.
Humans ; HEK293 Cells ; Sincalide ; China ; Amanita ; Death, Sudden

BACKGROUND: Butylphthalide (NBP) and edaravone (EDV) injection are common acute ischemic stroke medications in China, but there is a lack of large real-world safety studies on them. This study aimed to determine the incidence of adverse events, detect relevant safety signals, and assess the risk factors associated with these medications in real-world populations. METHODS: In this study, data of acute ischemic stroke patients were extracted from the electronic medical record database of six tertiary hospitals between January 2019 and August 2021. Baseline confounders were eliminated using propensity score matching. The drugs' safety was estimated by comparing the results of 24 laboratory tests standards on liver function, kidney function, lipid level, and coagulation function. The drugs' relative risk was estimated by logistic regression. A third group with patients who did not receive NBP or EDV was constructed as a reference. Prescription sequence symmetry analysis was used to evaluate the associations between adverse events and NBP and EDV, respectively. RESULTS: 81,292 patients were included in this study. After propensity score matching, the NBP, EDV, and third groups with 727 patients in each group. Among the 15 test items, the incidence of adverse events was lower in the NBP group than in the EDV group, and the differences were statistically significant. The multivariate logistic regression equation revealed that NBP injection was not a promoting factor for abnormal laboratory test results, whereas EDV had statistically significant effects on aspartate transaminase, low-density lipoprotein cholesterol and total cholesterol. Prescription sequence symmetry analysis showed that NBP had a weak correlation with abnormal platelet count. EDV had a positive signal associated with abnormal results in gamma-glutamyl transferase, alanine aminotransferase, aspartate aminotransferase, prothrombin time, and platelet count. CONCLUSIONS In a large real-world population, NBP has a lower incidence of adverse events and a better safety profile than EDV or other usual medications.