Researchers commonly use cyclization recombination enzyme/locus of X-over P1 (Cre/loxP) technology-based conditional gene knockouts of model mice to investigate the functional roles of genes of interest in Sertoli and Leydig cells within the testis. However, the shortcomings of these genetic tools include high costs, lengthy experimental periods, and limited accessibility for researchers. Therefore, exploring alternative gene silencing techniques is of great practical value. In this study, we employed adeno-associated virus (AAV) as a vector for gene silencing in Sertoli and Leydig cells. Our findings demonstrated that AAV serotypes 1, 8, and 9 exhibited high infection efficiency in both types of testis cells. Importantly, we discovered that all three AAV serotypes exhibited exquisite specificity in targeting Sertoli cells via tubular injection while demonstrating remarkable selectivity in targeting Leydig cells via interstitial injection. We achieved cell-specific knockouts of the steroidogenic acute regulatory ( Star ) and luteinizing hormone/human chorionic gonadotropin receptor (Lhcgr) genes in Leydig cells, but not in Sertoli cells, using AAV9-single guide RNA (sgRNA)-mediated gene editing in Rosa26-LSL-Cas9 mice. Knockdown of androgen receptor ( Ar ) gene expression in Sertoli cells of wild-type mice was achieved via tubular injection of AAV9-short hairpin RNA (shRNA)-mediated targeting. Our findings offer technical approaches for investigating gene function in Sertoli and Leydig cells through AAV9-mediated gene silencing.
Animals ; Male ; Leydig Cells/metabolism* ; Mice ; Dependovirus/genetics* ; Sertoli Cells/metabolism* ; Gene Silencing ; Genetic Vectors ; Testis/cytology*

OBJECTIVES: To evaluate the application value of genetic newborn screening (gNBS) in the Yunnan region. METHODS: A prospective study was conducted with a random selection of 3 001 newborns born in the Yunnan region from February to December 2021. Traditional newborn screening (tNBS) was used to test biochemical indicators, and targeted next-generation sequencing was employed to screen 159 genes related to 156 diseases. Positive-screened newborns underwent validation and confirmation tests, and confirmed cases received standardized treatment and long-term follow-up. RESULTS: Among the 3 001 newborns, 166 (5.53%) were initially positive for genetic screening, and 1 435 (47.82%) were genetic carriers. The top ten genes with the highest variation frequency were GJB2 (21.29%), DUOX2 (7.27%), HBA (6.14%), GALC (3.63%), SLC12A3 (3.33%), HBB (3.03%), G6PD (2.94%), SLC25A13 (2.90%), PAH (2.73%), and UNC13D (2.68%). Among the initially positive newborns from tNBS and gNBS, 33 (1.10%) and 47 (1.57%) cases were confirmed, respectively. A total of 48 (1.60%) cases were confirmed using gNBS+tNBS. The receiver operating characteristic curve analysis demonstrated that the areas under the curve for tNBS, gNBS, and gNBS+tNBS in diagnosing diseases were 0.866, 0.982, and 0.968, respectively (P<0.05). DeLong's test showed that the area under the curve for gNBS and gNBS+tNBS was higher than that for tNBS (P<0.05). CONCLUSIONS gNBS can expand the range of disease detection, and its combined use with tNBS can significantly shorten diagnosis time, enabling early intervention and treatment.
Humans ; Infant, Newborn ; Neonatal Screening ; Genetic Testing ; Female ; Male ; Follow-Up Studies ; Prospective Studies ; China

ObjectiveBased on the ultrastructure of enteric nervous system （ENS）-platelet-derived growth factor receptor α positive （PDGFRα⁺） cell - smooth muscle cell （SMC） network， the effect and mechanism of Shuwei Decoction （舒胃汤） for rats with functional dyspepsia （FD） of liver-depression and spleen-deficiency syndrome were explored. MethodsFifty SD rats were divided into blank group， model group， low-， medium- and high-dose Shuwei Decoction groups randomly， with 10 rats in each group. The blank group was not set up， and the other 4 groups were set up with the modified composite cause （chronic restraint stress + tail irritation + excessive fatigue + diet disorder） for 21 days， resulting in FD model rats with liver-depression and spleen-deficiency syndrome. After successful modelling， rats in the low-， medium- and high-dose groups were given 3.78， 7.56 and 15.12 g/（kg·d） of Shuwei Decoction by gavage， respectively， while rats in the blank group and the model group were given 10 ml/（kg·d） of saline by gavage； all of them were given gavage once a day for 14 days. Body mass increase were collected before and after gavage， and compared after the experiment； the elevated cross maze experiment was conducted to observe the percentage of staying time and the percentage of times entering the open arm of rats； the gastric emptying rate and small intestinal propulsion rate were measured by black semi-solid paste； HE staining was used to observe histopathology of the gastric antrum； the Western Blotting and RT-qPCR were used to detect the expression levels of PDGFRα， small conductance calcium activated potassium （SK3）， and purinergic G protein-coupled receptor P2Y1 （P2Y1 R）， and mRNA expression in gastric antrum； immunofluorescence double staining was used to detect the co-localization of PDGFRα with receptor tyrosine kinase （C-kit）， protein gene product 9.5 （PGP9.5）， Sk3， and smooth muscle cells （SMCs） in gastric sinusoidal tissues； and transmission electron microscopy was used to observe the ultrastructure of the ENS-PDGFRα⁺ cell-SMC network. ResultsHE staining results showed that the mucosal folds of gastric tissue were clear in all groups， no organic lesions such as bleeding， ulceration and swelling were observed， and the arrangement of gastric glands in the lamina propria of gastric sinusoidal tissues in model group and low-dose Shuwei Decoction group was disordered. The arrangement of gastric glands in lamina propria in medium- and high-dose Shuwei Decoction groups was more orderly， and the lesion degree was less than that in model group and low-dose Shuwei Decoction group. Compared with the blank group， the growth value of body mass， the percentage of open arm retention time， the percentage of times entering the open arm， the small intestine propulsion rate， and the gastric empty rate of rats in the model group significantly decreased after gavage； the expressions of PDGFRα， Sk3， P2Y1 protein and mRNA in gastric sinusoidal tissues decreased； the co-localization expression of PDGFRα with C-kit， Sk3， SMC and PGP9.5 significantly decreased （P＜0.05 or P＜0.01）； the ultrastructure of ENS-PDGFRα⁺ cell-SMC network was damaged without gap junction. Compared with model group and low-dose Shuwei Decoction group， the growth value of body mass， the percentage of open arm retention time， the percentage of times entering the open arm， the small intestine propulsion rate， and the gastric empting rate of rats in medium- and high-dose Shuwei Decoction groups increased after gavage； the expressions of PDGFRα， Sk3， P2Y1 protein and mRNA in gastric sinusoidal tissues increased； the co-localization expression of PDGFRα with C-kit， Sk3， SMC and PGP9.5 significantly increased （P＜0.05 or P＜0.01）； the ultrastructure of ENS-PDGFRα⁺ cell-SMC network was intact. Compared with medium-dose Shuwei Decoction group， the protein expressions of PDGFRα， Sk3 and P2Y1， the mRNA expressions of PDGFRα and Sk3， and the co-localization expressions of PDGFRα and C-kit， Sk3， SMC， and PGP9.5 in high-dose Shuwei Decoction group increased （P＜0.05 or P＜0.01）. ConclusionShuwei Decoction can improve the pathological lesion of the gastric antrum in the FD model rats with liver-depression and spleen-deficiency syndrome and increase the gastric emptying rate as well as the small intestinal propusion rate. The mechanism of Shuwei Decoction on FD rats with liver-depression and spleen-deficiency syndrome is related to the protection of the ultrastructural integrity of ENS-PDGFRα⁺ cell-SMC network.

Objective: To describe fertility and explore factors associated with it among pre-conception couples of childbearing age. Methods: Based on the pre-conceptional offspring trajectory study of the School of Public Health of Fudan University, couples of childbearing age who participated in the pre-conception physical examination in Shanghai Jiading District from 2016 to 2021 were recruited and followed up. Couples' time to pregnancy (TTP) was analyzed and Cox proportional hazards regression model was used to explore the factors associated with TTP. Kaplan-Meier was used to calculate each menstrual cycle's cumulative pregnancy rate. Results: A total of 1 095 preconception couples were included in the analysis, the M(Q₁,Q₃)of TTP was 4.33 (2.41, 9.78) menstrual cycles. Age of women (FR=0.90, 95%CI: 0.85-0.95, P<0.001), women who were overweight or obese before pregnancy (FR=0.36, 95%CI: 0.24-0.55, P<0.001), women who were exposed to second-hand smoking (FR=0.63, 95%CI: 0.44-0.92, P=0.016), women whose home or office had been renovated in the past 2 years and had a particular smell (FR=0.46, 95%CI: 0.26-0.81, P=0.008) were risk factors for impaired fertility. Regular menstrual cycles (FR=1.64, 95%CI: 1.16-2.31, P=0.005), females who often drank tea/coffee (FR=1.55, 95%CI: 1.11-2.17, P=0.011) and males who took folic acid before conception (FR=2.35, 95%CI: 1.38-4.23, P=0.002) were associated with better fertility. The cumulative pregnancy rate of 3, 6, and 12 menstrual cycles was 37.6%, 64.4%, and 78.4%, respectively. Conclusion: Older couples, overweight or obesity before pregnancy, irregular menstruation, exposure to secondhand smoke and decoration pollutants in females are associated with impaired fertility. Frequent tea/coffee drinking before pregnancy in females and taking folic acid before pregnancy in males are associated with shortened conception time.
Pregnancy ; Male ; Humans ; Female ; Cohort Studies ; Overweight/complications* ; Coffee ; Intention ; China/epidemiology* ; Fertility ; Obesity/complications* ; Tea

Rhei Rhizoma is commonly used as a traditional Chinese medicine with multiple botanical origins. Different botanical sources may have different pharmacological activities. The germplasm resources of commercial Rhei Rhizoma were determined based on the chloroplast gene matK, and the anthraquinone and free anthraquinone content was determined by UPLC to analyze quality of commercial Rhei Rhizoma. Eighty-nine commercial Rhei Rhizoma samples were collected from 40 cities in 27 provinces. DNA was extracted and the matK gene was amplified by PCR. Results indicated that the collected samples were from the same botanical origin, Rheum palmatum, and 8 genotypes were identified, including Rp1, Rp2, Rp3, Rp4, Rp5, Rp6, Rp10 and Rp12. Rp4 and Rp6, cultivated in Gansu, Sichuan and Yunnan provinces were the main circulating genotypes, representing 40.45% and 37.08% of the total samples, respectively. Phylogenetic tree analysis showed that the eight genotypes were mainly divided into two branches, of which the main genotypes Rp4 and Rp6 were in one branch. Genetic distance analysis indicated that the genetic separation of the eight genotypes was between 0.001 and 0.010. UPLC analysis indicated that 93.26% of the samples met the Pharmacopoeia standards. There were significant differences in the content of total anthraquinone and free anthraquinone among the samples, in which the difference in free anthraquinone was 1.01% and the difference in total anthraquinone content was 3.79%, indicating that the quality of commercial Rhei Rhizoma samples varies considerably. There was no significant difference in the content of total anthraquinone and free anthraquinone in commercial Rhei Rhizoma among different collection provinces and genotypes. This study will help guide the circulation of Rhei Rhizoma in the market and provides valuable insights for molecular identification and quality analysis of other traditional Chinese medicines.

Objective: To determine the characteristics and progress of the visual acuity and refractive state of schoolchildren in Huangzhong District, Xining City, Qinghai Province in China. Methods: Cohort study. Department of Ophthalmology, Beijing Children's Hospital carried out a cohort study by collecting the visual acuity and refractive state of Grade 1-5 schoolchildren among 16 primary schools in Huangzhong District, Xining City, Qinghai Province in September 2020 and July 2021. Cycloplegic retinoscopy with eye drop which contained tropicamide (0.5%) and phenylephrine hydrochloride (0.5%) was performed in children with low vision(<1.0). Myopia was defined as the spherical equivalent (SE) ≤-0.5 D after cycloplegic retinoscopy. Measurement data was analyzed by t-test and enumeration data was analyzed by χ² test. Multiple linear regression was used to analyze the influencing factors. Results: The 2 489 individuals with repeated tests in two years were included in the follow-up study, among whom the prevalence of myopia was 26.24%(653/2 489) in 2020, while 32.94% (820/2 489)respectively in 2021. The incidence of myopia in one school year from grades 1 to 5 was 11.19%(47/420), 5.44%(21/386), 6.39%(25/391), 11.52%(44/382) and 11.67%(30/257). The average SE of children in all grades in 2021 increased negatively from the previous year (Grade 1 to Grade 5 increased respectively: 0.40 D, 0.69 D, 0.62 D, 0.52 D and 0.37 D). Conclusions: The prevalence of myopia among schoolchildren in Huangzhong District, Xining City, Qinghai Province was relatively high. There were two peaks of myopia incidence in the first, fourth and fifth grades. Female, age, and the baseline of SE were the related influencing factors for myopia progression.
Child ; Cohort Studies ; Female ; Follow-Up Studies ; Humans ; Mydriatics ; Myopia/epidemiology* ; Ophthalmic Solutions ; Phenylephrine ; Prevalence ; Prospective Studies ; Tropicamide

Scutellaria baicalensis is a commonly used Chinese medicinal herb. In this study, we identified the germplasm resources of commercial S. baicalensis samples based on trnH-psbA, petA-psbJ, and ycf4-cemA sequences according to the available chloroplast genome sequencing results, and measured the content of baicalin by HPLC. Through the above means we determined the best DNA barcode that can be used to detect the germplasm resources and evaluate the quality of commercial S. baicalensis samples. A total of 104 samples were collected from 24 provinces, from which DNA was extracted for PCR amplification. The amplification efficiencies of trnH-psbA, petA-psbJ, and ycf4-cemA sequences were 100%, 59.62%, and 25.96%, respectively. The results of sequence analysis showed that 5, 4, and 2 haplotypes were identified based on trnH-psbA, petA-psbJ, and ycf4-cemA sequences, respectively. However, the sequences of haplotypes in commercial samples were different from that of the wild type, and the joint analysis of three fragments of S. baicalensis only identified 6 haplotypes. Furthermore, the phylogenetic analysis and genetic distance analysis indicated that trnH-psbA could be used to identify S. baicalensis from adulterants. The above analysis showed that trnH-psbA was the best fragment for identifying the germplasm resources of commercial S. baicalensis samples. We then analyzed the haplotypes(THap1-THap5) of commercial S. baicalensis samples based on trnH-psbA and found that THap2 was the main circulating haplotype of the commercial samples, accounting for 86.55% of the total samples, which indicated the scarce germplasm resources of commercial S. baicalensis samples. The content of baicalin in all the collected commercial S. baicalensis samples exceeded the standard in Chinese Pharmacopoeia and had significant differences(maximum of 12.21%) among samples, suggesting that the quality of commercial S. baicalensis samples varied considerably. However, there was no significant difference in baicalin content between different provinces or between different haplotypes. This study facilitates the establishment of the standard identification system for S. baicalensis, and can guide the commercial circulation and reasonable medication of S. baicalensis.
Chromatography, High Pressure Liquid ; DNA Barcoding, Taxonomic/methods* ; DNA, Plant/genetics* ; Phylogeny ; Scutellaria baicalensis/genetics*

Immune checkpoints (ICs) are immunosuppressive molecules expressed on immune cells, which can regulate immune cells' activation. Immune checkpoint inhibitors (ICIs) which can block the interaction of immune checkpoints and their ligands, improve the cytotoxic effect of the immune system on tumor cells. Immunotherapy such as employing ICIs has gradually become a conventional therapeutic strategy for cancer treatment. However, the low response rate and the emergence of drug resistance have seriously affected the clinical efficacy of ICIs. Reactive oxygen species (ROS) are electronic reduction products of active oxygen, as well as natural by-products of cell metabolism, which can be used as regulators of intercellular signals. Tumor microenvironment (TME) is often in the state of oxidative stress (OS), which is the imbalance between oxidative system and antioxidant system. ROS can affect the interaction with its ligands by regulating the expression and activity of immune checkpoints in TME, thus affecting the anti-tumor effect of immune cells. Accumulating studies have shown that ROS could regulate tumor immune checkpoints through several pathways. Due to different types and stages of tumor, it would be clinical beneficial to understand the mechanistic link of ROS on tumor immune checkpoint, and choose appropriate ROS regulators combined with immune checkpoint inhibitors to maximize anti-tumor effects. This article reviews the common metabolic sources and characteristics of ROS, the regulatory effect and mechanism of ROS on tumor immune checkpoints and its therapeutic application.

To explore the effect of tanshinone IIA (TanIIA) on the occurrence and development of breast cancer, we employed the mouse mammary tumor virus-polyomavirus middle T antigen (MMTV-PyMT) transgenic mice as a spontaneous breast cancer mouse model. Animal welfare and experimental procedures were in accordance with the regulations of the Animal Ethics Committee of Nanjing University of Chinese Medicine. The animals were divided into control group, low-dose TanIIA treatment group (30 mg·kg^-1·day^-1), and high-dose TanIIA treatment group (60 mg·kg^-1·day^-1). The treatment was administered orally and daily for 5 weeks. The mice were sacrificed after final treatment. Mammary gland and lung were collected for histopathology studies. We evaluated the chemoprophylaxis effect of TanIIA on breast cancer in mice according to the pathological characteristics of breast cancer at different stages of development. Immunofluorescence staining were employed for blood vessel analysis. The expression levels of E-cadherin, proliferating nuclear antigen (PCNA), and oncogene c-Myc were detected by immunohistochemistry. Flow cytometry was used to analyze cell cycle and Cytoscape was used to construct drug-disease protein-protein interaction (PPI) network. Our results showed that TanIIA inhibits breast tumor progression by delaying malignancy from adenoma to early carcinoma, and inhibits blood vessel formation during tumor development. TanIIA (60 mg·kg^-1·day^-1) inhibits the expression levels of PCNA and c-Myc, upregulates the expression of E-cadherin. In addition, cell cycle experiments showed that the cell cycle of PyMT primary mammary cells in the high-dose TanIIA group was arrested in the G0/G1 phase. Our study demonstrated that TanIIA can significantly inhibit breast tumor progression in MMTV-PyMT mouse model, which may be related to the inhibition of angiogenic switch and cell cycle arrest.

Objective:To understand the time for observation and related factors in the clinics after vaccination among children's parents.Methods:From December 2019 to January 2020, parents of children aged 0-3 years were recruited by multiple-stage sampling from 34 vaccination clinics in 12 districts and counties in 6 provinces (Shandong, Guangdong, Henan, Sichuan, Inner Mongolia, and Liaoning). A questionnaire survey on the time of observation after vaccination was conducted. A multivariate logistic regression model was used to analyze the related factors of parental observation time after vaccination.Results:A total of 3 292 parents of 0-3 year's old children were selected, and 3 178 parents were finally included in the analysis. 87.85%(2 792/3 178) of the parents reported that the observation time after vaccination at clinics was ≥30 minutes. Multivariate logistic regression analysis showed that, after adjusting for the regions, the main factors affecting the observation time at clinics after vaccination among parents appeared as observation time informed by physicians at the clinic appeared ≥30 minutes ( OR=31.622, 95% CI: 19.847-50.384), parents were medical personnel ( OR=2.779, 95% CI: 1.505-5.133), parents being volunteers working on vaccination-related publicity and education activities ( OR=1.986, 95% CI: 1.438-2.743), parents aged 35 years old or above ( OR=1.900, 95% CI: 1.215-2.971), being parents of the first child ( OR=1.663, 95% CI: 1.282-2.156), per capita annual income of the family as 8 000- Yuan ( OR=1.646, 95% CI: 1.168-2.319), children aged 0-12 months old ( OR=1.646, 95% CI: 1.203-2.252) or 13-24 months old ( OR=1.506, 95% CI: 1.064-2.133), obedient to physicians' advice at the clinic ( OR=1.481, 95% CI: 1.067-2.055). Conclusions:The proportions of parents observed for ≥30 minutes at the clinics of vaccination were high. When the information was from the physicians at the vaccination clinic, the observation time was the most critical factor for parents to observe at clinics as required.