ObjectiveTo evaluate pharmacological effects of Shengmai injection（SMI）on cerebral ischemia and study its neuroprotective mechanism. MethodsMale specific pathogen-free （SPF） Sprague-Dawley （SD） rats were randomly divided into a sham group， a model group， a low-dose SMI group（3 mL·kg^-1）， a middle-dose SMI group（6 mL·kg^-1）， a high-dose SMI group（12 mL·kg^-1）， and a Ginaton group（4 mL·kg^-1）according to the random number table method， with 12 rats in each group. The rat model of cerebral ischemia-reperfusion（MCAO/R）was prepared via the suture method. The administration groups were intraperitoneally injected with corresponding concentrations of SMI or Ginaton injection after reperfusion， which was conducted for 3 consecutive days. The sham group and model group were administered the equivalent volume of physiological saline. The pharmacological effects of SMI on brain injury in MCAO/R rats were evaluated by neurological function scores， cerebral infarction area， hematoxylin-eosin （HE） staining， Nissl staining， terminal deoxynucleotidyl transferase dUTP nick-end labeling （TUNEL） staining， and Western blot. The dominant link and key protein of SMI treating cerebral injury were explored using proteomic analysis. The related mechanisms of SMI were further validated using enzyme-linked immunosorbent assay （ELISA）， Western blot， and chloride ion fluorescence probe with oxygen-glucose deprivation/reoxygenation（OGD/R）-treated PC12 cells and MCAO/R rats. ResultsCompared with the sham group， the model group showed significantly increased neurological function scores， cerebral infarction area， neuronal apoptosis rate， and expression levels of apoptosis related proteins （P<0.05， P<0.01）and significantly decreased density of Nissl bodies and neurons（P<0.01）. Compared with the model group， the SMI groups exhibited significantly decreased neurological function scores， cerebral infarction area， neuronal apoptosis rate， and expression levels of apoptosis related proteins （P<0.05， P<0.01）and significantly increased density of Nissl bodies and neurons （P<0.05）. The proteomic analysis results showed that oxidative stress and inflammatory response were important processes of SMI intervening in MCAO/R injury， and the chloride intracellular channel protein 1 （CLIC1） was one of key proteins in its action network. The levels of representative indicators of oxidative stress and inflammatory response in the MCAO/R rats of the SMI groups were significantly reduced， compared with those in the model group（P<0.05， P<0.01）， and the expression levels of CLIC1 and downstream NOD-like receptor protein 3 （NLRP3） decreased （P<0.01）. In addition， the experimental results based on the OGD/R PC12 cells showed that SMI significantly increased the cell survival rate（P<0.01） and significantly decreased the intracellular chloride ion concentration（P<0.05）. ConclusionSMI has neuroprotective effects. Oxidative stress and inflammatory response are key processes of SMI intervening in MCAO/R injury. The potential mechanism is closely related to the regulation of CLIC1.

The long-term presence of non-steroidal anti-inflammatory drugs (NSAIDs) in the environmental water samples not only affects the life safety of aquatic organisms and disturbs the ecoenvironment, but also poses a serious threat to human health. In this study, amino-functionalized Fe3O4 nanoparticles (Fe3O4-NH2) were firstly prepared by solvothermal method. Subsequently, polyethyleneimine (PEI) with a branched chain structure was successfully grafted onto Fe3O4 nanoparticles by Schiff base reaction in aqueous solution at room temperature using glutaraldehyde as a cross-linking agent, and a recyclable PEI-grafted magnetic nano-sorbent (Fe3O4@PEI) was synthesized and applied for the detection of NSAIDs in the environmental water samples. The compositional properties of Fe3O4@PEI were investigated by different characterization methods and the parameters affecting the extraction of NSAIDs were optimized. Due to high adsorption of Fe3O4@PEI for NSAIDs, the quantitative analysis of four NSAIDs in the environmental water samples, ketoprofen, naproxen, diclofenac and tolfenamic acid, was performed in combination with high-performance liquid chromatography. A good linear relationship between the chromatographic peak area and concentration was observed in the range of 1−500 µg/mL. The recoveries of the samples at three different spiked levels ranged from 85.6% to 107.8%; the intra-day precision was less than 7.8% (n=6); and the inter-day precision was less than 9.5% (n=3). The method is simple, rapid, accurate and reliable, and can be used for the analysis of NSAIDs in the environmental water samples．

Clinicians need to focus on various points in the diagnosis and treatment of insomnia.This article prescribed the treatment protocol based on the unique features,such as insomnia in the elderly,women experiencing specific physiologi-cal periods,children insomnia,insomnia in sleep-breathing disorder patients,insomnia in patients with chronic liver and kidney dysfunction.It pro-vides some reference for clinicians while they make decision on diagnosis,differentiation and treat-ment methods.

ObjectiveTo explore the quality differences between steamed products and raw products of Citri Reticulatae Pericarpium（CRP）. MethodThe color of steamed products and raw products of CRP was determined from the perspective of appearance by electronic eye technique， and the quality differences between them was objectively characterized by the luminous value（L^*）， yellow-blue value（b^*）， red-green value（a^*） and total chromatic value（E^*_ab）. Based on this， ultra-high performance liquid chromatography（UPLC） was used to establish a fingerprint evaluation method with the mobile phase of acetonitrile（A）-0.1% formic acid aqueous solution（B） for gradient elution（0-5 min， 5%A； 5-30 min， 5%-20%A； 30-60 min， 20%-52%A）， detection wavelength at 270 nm， flow rate of 0.3 mL·min^-1 and column temperature of 30 ℃. The quality differences between steamed products and raw products of CRP were compared from the perspective of chemical composition， and correlation analysis was used to reveal the correlation between the difference in appearance color and the difference in internal chemical composition. ResultAfter being steamed， L^*， b^* and E^*_ab of CRP showed an overall decreasing trend， indicating that the color of the steamed products darkened and deepened from yellow to blue but still tended to be yellow， while a^* showed an overall increasing trend， indicating that the color of the steamed products tended to red. A total of 24 peaks were identified in the fingerprint profiles of raw products and steamed products of CRP， and 13 of the main peaks were identified. The precision， stability and repeatability studies showed that compared with the reference peak （peak 14， hesperidin）， the relative standard deviations（RSDs） of the relative peak area and relative retention time of the remaining peaks were<3.0%.The results of chemometric statistical analysis showed that there were some differences between raw products and steamed products of CRP， and 7 main differential components were identified， among which 5-hydroxymaltol（peak 1） and 5-hydroxymethylfurfural（peak 2） were the characteristic components of steamed products. The correlation analysis results showed that， in addition to the above two characteristic components， four components of peak 4， peak 10 （vicenin-2）， peak 23 （tangeretin） and peak 24 （5-demethylnobiletin） also correlated significantly with the color change （E^*_ab） of the samples （P<0.05， P<0.01）. ConclusionBefore and after steaming， not only the chemical composition changes， but also the color. Comparing the characteristic peaks of chemical composition difference and color difference before and after steaming of CRP， it is found that 5-hydroxymaltol， 5-hydroxymethylfurfural and peak 4 are common characteristic difference components， which can provide a reference for establishing the characteristic quality control method of steamed products， and quickly evaluating the quality difference between raw products and steamed products of CRP.

Objective:To investigate the application value of fiberoptic endoscopic evaluation of swallowing (FEES) in patients with dysphagia after stroke.Methods:A total of 200 patients with dysphagia who received treatment in Wenzhou Hospital of Traditional Chinese Medicine from January 2019 to December 2020 were included in this study. All patients underwent videofluoroscopic swallowing study (VFFS) and FEES. Examination results and the incidence of adverse reactions were compared between VFFS and FEES.Results:VFFS results revealed that oral food residue occurred in 122 patients, food leakage in 168 patients, food aspiration in 170 patients, epiglottis valley residue in 179 patients, pyriform sinus residue in 184 patients, cricopharyngeus muscle dysfunction in 61 patients, and abnormal esophageal peristalsis in 58 patients. FEES results revealed that food leakage occurred in 173 patients, food penetration in 151 patients, food aspiration in 180 patients, food retention in 166 patients, and vocal neoplasm in 21 patients. The incidence of adverse reactions generated in FEES was lower than that in VFFS (5.5% vs. 12.0%, χ2 = 5.29, P < 0.05). Conclusion:FEES has high sensitivity and safety in the detection of dysphagia after stroke, and it has important guiding significance for the treatment of dysphagia.

Gonadal somatic cells are the main players in gonad development and are important for sex determination and germ cell development.Here,using a time-series single-cell RNA sequencing(scRNA-seq)strategy,we analyzed fetal germ cells(FGCs)and gonadal somatic cells in human embryos and fetuses.Clustering analysis of testes and ovaries revealed several novel cell subsets,including POU5F1+SPARC+FGCs and KRT19+somatic cells.Furthermore,our data indicated that the bone morphogenetic protein(BMP)signaling pathway plays cell type-specific and develop-mental stage-specific roles in testis development and promotes the gonocyte-to-spermatogonium transition(GST)in late-stage testicular mitotic arrest FGCs.Intriguingly,testosterone synthesis function transitioned from fetal Sertoli cells to adult Leydig cells in a stepwise manner.In our study,potential interactions between gonadal somatic cells were systematically explored and we identified cell type-specific developmental defects in both FGCs and gonadal somatic cells in a Turner syndrome embryo(45,XO).Our work provides a blueprint of the complex yet highly ordered devel-opment of and the interactions among human FGCs and gonadal somatic cells.

Pyroptosis is a form of programmed cell death, and recently described as a new molecular mechanism of chemotherapy drugs in the treatment of tumors. Miltirone, a derivative of phenanthrene-quinone isolated from the root of Bunge, has been shown to possess anti-cancer activities. Here, we found that miltirone inhibited the cell viability of either HepG2 or Hepa1-6 cells, and induced the proteolytic cleavage of gasdermin E (GSDME) in each hepatocellular carcinoma (HCC) cell line, with concomitant cleavage of caspase 3. Knocking out switched miltirone-induced cell death from pyroptosis to apoptosis. Additionally, the induction effects of miltirone on GSDME-dependent pyroptosis were attenuated by siRNA-mediated caspase three silencing and the specific caspase three inhibitor Z-DEVD-FMK, respectively. Miltirone effectively elicited intracellular accumulation of reactive oxygen species (ROS), and suppressed phosphorylation of mitogen-activated and extracellular signal-regulated kinase (MEK) and extracellular regulated protein kinases 1/2 (ERK1/2) for pyroptosis induction. Moreover, miltirone significantly inhibited tumor growth and induced pyroptosis in the Hepa1-6 mouse HCC syngeneic model. These results provide a new insight that miltirone is a potential therapeutic agent for the treatment of HCC GSDME-dependent pyroptosis.

OBJECTIVE： To screen potential eEF2K inhibitor molecules， and to provide reference for the design and R&D of eEF2K inhibitor. METHODS： The eEF2K crystal structure model was constructed by homology modeling technique. The model was optimized by Loop optimization and molecular dynamics. With the help of SAVES online server， the above models were evaluated from three aspects such as Verify_3D， EERAT and Laplace diagram. Totally 55 eEF2K inhibitor molecules were collected. Hypogen pharmacophore model with activity prediction ability was constructed based on 28 of them （odd number， as training set） by Insight Ⅱ software and validated by other 27 （even number， as test set）. The optimal pharmacophore model was screened by fitting the predicted and experimental values of activity [i.e. negative logarithm of half inhibitory concentration （pIC50）] and using Ligand profiler thermogram. The virtual screening of small molecules of eEF2K inhibitors was carried out by combining the above pharmacophore model， Lipinski’s five rules and molecular docking method. RESULTS & CONCLUSIONS： The overall quality factor score of the crystal structure model of eEF2K protein was 93.697. Among them， 83.33％ of the amino acid Verify_3D score was more than or equal to 0.2， and 1.7％ of the total amino acids were located in the non-permissible region. The amino acid conformation and skeleton structure of the model were reasonable and the reliability of the model was high. Totally 9 Hypogen pharmacophore models （No. 02-10） with active predictive function were constructed， among which No. 03 pharmacophore model included 2 hydrogen bond receptors and 2 conjugated aromatic rings， which could better distinguish active and inactive molecules. The predicted value of pIC50 fitted the experimental value best （the correlation coefficient was 0.665 3）， and it had good predictive ability and high reliability. Finally， 9 potential eEF2K inhibitor molecules were obtained through virtual screening （pIC50 ranged from 1.074 to 1.185， and Dcoking-score of protein-molecule interaction ranged from －9.730 to －7.467）. Pro268， Asp267， Gln171， Phe121 and Glu212 may be the key amino acids for the interaction between eEF2K inhibitors and target proteins， including hydrogen bonds， salt bridges and hydrophobicity. These 9 molecules are expected to be the lead compounds for the development of eEF2K inhibitors.

Objective: To explore the feasibility of gender assignment in 46,XY disorders of sex development (DSD) with severe undermasculinisation mainly based on molecular diagnosis. Methods: A retrospective study of 45 patients of 46, XY DSD with severe undermasculinisation were admitted between November 2015 and October 2018 at Children′s Hospital, Zhejiang University School of Medicine. The initial social gender were all female, of whom the external genital manifestations were Prader 0 to 2; the degree of masculinity was scored using external masculinisation score (EMS); the position and development of the gonads were examined by ultrasound, cystoscopy and laparoscopy, also including assessing the development of the Wolffian tube and the Müllerian tube. The level and ratio of testosterone to dihydrotestosterone before and after hCG stimulation were evaluated for the function of Leydig cell and 5α-reductase-2. Gender role scales and sandbox games were used to assess gender role behavior. Genital sensitivity to androgen stimulation was assessed; A panel including 163 genes related to gender development were determined by second-generation sequencing in all 45 patients. Finally, a multidisciplinary team (MDT) makes a gender assignment after a comprehensive analysis mainly based on the molecular etiological diagnosis. Results: Thirty-nine out of 45 patients (87%) had an identifiable genetic etiology, and the remaining 6 (13%) were negative for genetic testing. Forty-five patients had EMS less than or equal to 3 points. Sexual psychological assessment was performed in 39 patients, with male dominance in 24 (62%) and female dominance in 15 (38%). The gender assignment was 23 cases (51%) for male and 19 cases (42%) for female, and 3 cases (7%) were not completely determined. Conclusions Molecular diagnosis provides a strong basis for appropriate gender assignment of 46, XY DSD children with severe undermasculinisation. Based on molecular diagnosis, each DSD should be analyzed by professional MDT to analyze the clinical symptoms/signs, gonadal development, gonad tumor risk, external genital morphology, sexual psychological assessment, potential fertility opportunities, parental views, Social and cultural factors, etc. make appropriate gender assignment.

Objective: To investigate the efficacy and safety of rapamycin in children with tuberous sclerosis complex (TSC) associated renal disease. Methods: A prospective self-control study was conducted. The clinical data of 92 children diagnosed with tuberous sclerosis complex associated kidney disease at the People′s Liberation Army General Hospital from January 2011 to January 2019 were collected. The long-term rapamycin treatment for all patients initiated at 1 mg/(m²·d), which was gradually adjusted to reach a blood concentration of 5-10 μg/L. The changes of the maximum diameter of renal lesions in children after rapamycin treatment were observed and analyzed with Wilcoxon test. Results: Ninety-two children, including 52 males and 40 females, who met the criteria were analyzed. Sixty patients had only renal angiomyolipoma(RAML), while 24 patients had only multiple renal cysts(MRC), and 8 patients had both lesions. The age of TSC diagnosis was 16.0 (7.0, 42.0) months, and the age of initial treatment with rapamycin was 63.5 (21.0, 103.0) months. The follow-up lasted for 12.0 (4.0, 23.0) months. Sequencing of TSC1 and TSC2 genes was performed in 54 children with TSC, including 3 patients (6%) with mutations in TSC1 gene and 51 patients (94%) with mutations in TSC2 gene. The maximum RAML diameter before treatment was 7.0 (4.0, 9.0) mm. The best effect reached at 3 months of treatment, with the diameter of 4.0 (0,7.0) mm. The maximum diameters at 6 months, 1 year and 1-2 years were 5.0 (0,9.8) mm, 5.0 (1.5, 8.5) mm, 5.5 (3.0, 9.0) mm, respectively, and were significantly different from the baseline (Z=-2.404,-2.350,-2.750,P=0.016,0.019,0.006, respectively). The maximum diameter after 2-3 years, and ≥3 years were 5.0 (3.9,7.0) mm and 6.0 (1.0, 11.0) mm, without significant difference from the baseline (Z=-0.856,-0.102,P=0.393,0.919, respectively).The maximum diameters of MRC after 3 months, 6 months, 1 year,1-2 years, 2-3 years, and ≥3 years were 11.0 (5.0, 14.0) mm,3.0 (0.0,11.0) mm,5.0 (0,21.0) mm,0 (0,14.0) mm,0 (0,10.0) mm, and 0 (0,18.3) mm, respectively, but were not significantly different rom the baseline (7.0 (5.0, 15.7) mm)(Z=-0.944,-1.214,-1.035,-1.896,-1.603,-1.214,P=0.345,0.225,0.301,0.058,0.109,0.225, respectively).Twenty-nine patients (32%) had oral ulcers during the entire treatment period, and no serious adverse reactions were observed. Conclusions Rapamycin could decrease the diameter of TSC-related RAML, but could not inhibit the growth of cysts. It is well tolerated in the treatment of renal diseases associated with tuberous sclerosis complex.