Objective To prepare A29L protein, A29L neutralization epitope tandem protein(Neu6) and A29L-mRNA-lipid nanoparticles(LNPs) vaccine based on A29L antigen, a key neutralizing target of monkeypox virus(MPXV), and evaluate their immunogenicity, so as to provide experimental basis for the development of MPXV vaccine and antibody drugs.Methods The A29L and Neu6 proteins were expressed by E.coli prokaryotic system and purified. Female BALB/c mice were immunized at five sites by subcutaneous injection in the extremities and intraperitoneal injection with five mice in each group. After three times of immunization, blood samples were collected from posterior orbital venous plexus and serum was separated. The template sequence of A29L-mRNA was cloned into plasmid pBluescript Ⅱ SK(+), and the template plasmid pBlueScriptⅡSK(+)-A29L-mRNA was constructed. A29L-mRNA was synthesized by transcription in vitro and purified by capping. A29L-mRNA were encapsulated by INano~(TM)L nano-drug preparation system to prepare A29L-mRNA-LNPs, which wereinjectedintramuscularlyintothebackoffivefemale BALB/c mice. After three times of immunization, blood samples were collected from orbital venous plexus and serum was separated. The serum IgG titers were detected by indirect ELISA and the serum neutralizing antibody titers were detected by plaque reduction neutralization test(PRNT).Results A29L and Neu6proteins were obtained successfully, with the purity of more than 90%, which were expressed mainly in inclusion bodies and showed specific binding to mouse anti-A29L monoclonal antibodies. The particle size of A29L-mRNA-LNPs was uniform(71. 35 nm), the polymer dispersity index(PDI) was 0. 097, and the encapsulation efficiency was 93%. HEK-293T cells transfected with A29L-mRNA-LNPs in vitro effectively expressed A29L protein. The serum IgG antibody titers of mice in A29L and Neu6 groups were significantly higher than that of mRNA group(t = 5. 692 and 5. 774, respectively, each P < 0. 001),while the serum neutralizing antibody titer of Neu6 group was significantly higher than those of A29L and mRNA groups(t =5. 202 and 4. 475, respectively, each P < 0. 01).Conclusion The immunogenicity of A29L-mRNA-LNPs is weak, suggesting that the effect of single antigen mRNA vaccine is limited. A29L and Neu6 proteins expressed in prokaryotic cells have good immunogenicity, and Neu6 can significantly improve the neutralizing antibody level, indicating that multi-antigen combination strategy should be explored in the future, and Neu6 protein is expected to become a highly effective candidate immunogen for MPXV vaccine and neutralizing antibody development.

Cough variant asthma （CVA） is a chronic respiratory disease with cough as its main symptom. The occurrence of CVA is closely related to non-specific airway inflammation， and its pathogenesis involves environmental， genetic， immune， and other factors. In recent years， the advantages of traditional Chinese medicine （TCM） in the treatment of CVA have attracted the attention of experts and scholars in China and abroad， especially its prominent role in regulating immune balance， relieving cough symptoms in CVA patients， and reducing recurrence. T Helper cells 1 （Th1）， T helper cells 2 （Th2）， T helper cells 17 （Th17）， and regulatory T cells （Treg） are derived from CD4⁺ T cells. Immune imbalance of Th1/Th2 and Th17/Treg is a new hotspot in the pathogenesis of CVA and a potential key target in the treatment of CVA by TCM. Th cell subsets are in dynamic balance under physiological conditions， maintaining respiratory immune homeostasis in which pro-inflammatory cytokines and anti-inflammatory cytokines are balanced. Immature helper T cells （Th0） can be differentiated into Th1， Th2， Th17， Treg， and other cell subsets due to cytokine types in the microenvironment in the stage of CVA maturation. The proliferation of Th2 cells leads to eosinophilic airway inflammation. Excessive differentiation of Th17 cells induces neutrophil airway inflammation. Th1/Th2 and Th17/Treg cells are mutually restricted in number and function， and the immune imbalance of Th1/Th2 and Th17/Treg is easy to aggravate the generation of inflammatory response. Restoring immune balance is particularly important for the airway anti-inflammatory therapy of CVA. In this paper， the imbalance of Th1/Th2 and Th17/Treg and the pathogenesis of CVA were systematically expounded. Meanwhile， the latest research on the regulation of immune imbalance by TCM compound， single TCM， and its effective ingredients in the treatment of CVA was reviewed. It provides ideas and references for revealing the scientific connotation of TCM regulating immune balance therapy of CVA， as well as the development of clinical treatment and basic research of CVA.

Objective To establish a stable rat model of non-obese polycystic ovary syndrome(PCOS)with clinical characteristics.Methods Dehydroepiandrosterone(DHEA)was used to establish a PCOS rat model by subcutaneous injection.Three-week-old female SD rats were divided into a normal group,6 mg/kg DHEA model group,and 60 mg/kg DHEA model group.The model groups were subcutaneously injected with the corresponding dose of DHEA daily,while the normal group was subcutaneously injected with glycerol daily for 21 consecutive days.The model was evaluated with ovarian histopathology as the gold standard to determine the optimal dosage of DHEA to induce a PCOS rat model.On this basis,the optimal DHEA modeling dose was selected,and stop and continue modeling groups were set up to observe the model for 28 days and evaluate its maintenance.The stop modeling group was no longer given DHEA,and the continued modeling group was subcutaneously injected with 60 mg/kg DHEA every 48 h.The evaluation indicators included body mass,estrous cycle,fasting blood glucose,serum insulin,histopathologic morphology of the ovaries,and serum sex hormone levels.Results(1)Compared with the normal group,the 6 mg/kg and 60 mg/kg DHEA model groups showed no significant difference in body mass,and their estrous cycles were irregular.There were more cystically dilated large follicles in the ovaries;fewer mature follicles;reduced layers of granulosa cells,which were arranged in a sparse and disorganized manner;and fewer lutea in the 6 mg/kg and 60 mg/kg DHEA model groups than the normal group.Furthermore,serum T and E2 levels were significantly higher in the 60 mg/kg DHEA model group(P＜0.05)than the normal group.(2)The stop modeling group(A2 group)resumed regular estrous cycles after 2 weeks,various growth follicles and corpora lutea were observed in the ovarian tissues,the number of cystic follicles was reduced,the number of granulosa cell layers increased,mature follicles were visible,oocyte morphology was locally intact,and the levels of E2 and AMH were reduced compared with the normal group(A1 group)(P＜0.05).(3)The continue model group(B2 group)was in the late stage of estrous cycle for a long period,and there were more large follicles with cystic dilatation,fewer mature follicles,fewer layers of granulosa cells with a sparse and disordered arrangement,and significantly fewer corpus lutea in the ovaries compared with the normal group(B1 group).The levels of serum LH,LH/FSH,and T were elevated(P＜0.05).Conclusions Subcutaneous injection of 60 mg/kg DHEA for 21 consecutive days can be used to successfully construct a non-obese PCOS rat model that possesses clinical characteristics.Subcutaneous injection of 60 mg/kg DHEA every 48 hours maintains the stability of the model.

During the coronavirus disease 2019(COVID-19)pandemic season, cosmetic procedures have been hit hard. Cosmetic techniques must now change its emphasis from infection prevention to safe restart in response to patient desire generally. A key component of the approach is comprehending how COVID-19 affects invasive cosmetic techniques. This paper reviewed the relationship between COVID-19 and invasive cosmetic techniques and proposed coping strategies during the recovery period to increase awareness of COVID-19 among practitioners and patients, and to reduce the incidence of adverse events.

Objective:To investigate the influence of maternal autoimmune diseases and anticoagulants, including low-molecular-weight heparin (LMWH) and aspirin, on the fetal fraction of maternal plasma cell-free DNA of non-invasive prenatal testing (NIPT).Methods:A prospective cohort study was conducted on women with singleton pregnancies receiving NIPT in the Nanjing Drum Tower Hospital from March 2021 to July 2022. NIPT was carried out using a polymerase chain reaction (PCR)-free amplification platform. In this study, four types of maternal autoimmune diseases, which were antiphospholipid syndrome, undifferentiated connective tissue disease, Sj?gren's syndrome, and systemic lupus erythematosus (SLE), and two anticoagulants, LMWH and aspirin, were studied. Univariate and multivariate linear regression models were used to analyze the factors influencing fetal fraction of maternal plasma cell-free DNA.Results:A total of 4 102 singleton pregnant women were enrolled in the prospective cohort, and 3 948 were finally included after excluding the cases with unclear dosing time of LMWH or aspirin, other autoimmune diseases, conceiving through ovulation induction alone, and having true positive or failed NIPT result. There were 96 cases with antiphospholipid syndrome, 35 with undifferentiated connective tissue disease, 34 with Sj?gren's syndrome, and 18 with SLE. A total of 108 patients only received LMWH treatment, 121 only received aspirin treatment, and 113 received both LMWH and aspirin treatment. Univariate linear regression analysis showed that maternal body mass index at blood collection ( B=－0.423), conceived by assisted reproductive technology ( B=－0.803), male fetus ( B=－0.458), undifferentiated connective tissue disease ( B=1.774), and SLE ( B=3.467) had influence on the fetal fraction (all P<0.05). Multivariate linear regression analysis showed that maternal body mass index at blood collection ( B=－0.415), conceived by assisted reproductive technology ( B=－0.585), male fetus ( B=－0.322), SLE ( B=3.347) and undifferentiated connective tissue disease ( B=1.336) were factors influencing fetal fraction (all P<0.05). Conclusions:Maternal use of LMWH or aspirin does not affect fetal fraction when performing NIPT on a PCR-free amplification platform, but undifferentiated connective tissue disease and SLE are the influencing factors. Therefore, pregnant women should be informed before the NIPT that the fetal fraction of maternal plasma cell-free DNA may be affected by maternal autoimmune diseases.

During the coronavirus disease 2019(COVID-19)pandemic season, cosmetic procedures have been hit hard. Cosmetic techniques must now change its emphasis from infection prevention to safe restart in response to patient desire generally. A key component of the approach is comprehending how COVID-19 affects invasive cosmetic techniques. This paper reviewed the relationship between COVID-19 and invasive cosmetic techniques and proposed coping strategies during the recovery period to increase awareness of COVID-19 among practitioners and patients, and to reduce the incidence of adverse events.

ObjectiveTo systematically review the clinical experience of four sessions of Masters of Traditional Chinese Medicine and two sessions of National Famous Chinese Medicine Practitioners in treating ulcerative colitis （UC）. Data mining and analysis were conducted to clarify the diagnosis and treatment ideas and characteristics of prescription used by these famous doctors in treating UC. MethodsRelevant literature on the treatment of UC by renowned doctors was retrieved from the establishment of the database until March 31， 2023. The literature was collected from databases such as China National Knowledge Infrastructure （CNKI）， Wanfang Data Knowledge Service Platform， Chinese Science and Technology Periodicals Database， and China Biomedical Literature Database. The data mining techniques including frequency analysis， association rules， and cluster analysis were conducted using the Ancient and Modern Medical Case Cloud Platform V2.3.5. ResultsA total of 157 literatures were included in this study， including 115 clinical case data. The study found that UC can be categorized into 14 types of syndrome patterns for treatment， including large intestine dampness-heat syndrome （75，65.22%）， syndrome of dampness stagnancy due to spleen deficiency （23， 20.00%）， spleen-kidney yang deficiency syndrome （21， 18.26%）. The main affected organs were the spleen （85， 73.91%） and large intestine （75， 65.22%）， and they were closely related to liver （24， 20.87%） and the kidney （21， 18.26%）. The predominant pathogenic factors were dampness （83， 72.17%） ， heat （80， 69.57%） and qi deficiency （65， 56.52%）. The treatment involved 30 kinds of treatment methods， including heat-clearing and dampness-draining method （75， 65.22%）， pleen-tonifying and qi-boosting method （25，21.74%） and spleen-invigorating and dampness-transforming method （23， 20.00%）. The medication involved 187 ingredients， with the most commonly used being heat-clearing herbs （37， 19.79%） and tonifying herbs （27， 14.44%）. The tastes of the herbs were mostly sweet （85， 45.45%） ， bitter （80， 42.78%） ， and pungent （71， 37.97%）. The association rules revealed 16 high-frequency combinations mainly composed of Huanglian （黄连）， Baishao （白芍） and Gancao （甘草） along with Baizhu （白术）， Fuling （茯苓）， Muxiang （木香） and Danggui （当归）. ConclusionFamous doctors are skilled in diagnosing and treating UC based on the differentiation of the zang-fu organs and qi-blood. The key pathological mechanism is “spleen deficiency as the root， and large intestine damp-heat as the manifestation”. The core treatment approach is “heat-clearing， spleen-tonifying， and dampness-draining”， with the inclusion of “regulating qi and blood， and balancing cold and heat”.

OBJECTIVE: To detect potential variants in eight Chinese pedigrees affected with autosomal dominant polycystic kidney disease (ADPKD) and provide prenatal diagnosis for two of them. METHODS: Whole exome sequencing and high-throughput sequencing were carried out to detect variants of PKD1 and PKD2 genes in the probands. Sanger sequencing was used to validate the variants, and their pathogenicity was predicted by searching the ADPKD and protein variation databases. RESULTS: Eight PKD1 variants were detected, which have included five nonsense mutations and three missense mutations. Among these, four nonsense variants (PKD1: c.7555C>T, c.7288C>T, c.4957C>T, c.11423G>A) were known to be pathogenic, whilst one missense variant (PKD1: c.2180T>G) was classified as likely pathogenic. Three novel variants were detected, which included c.6781G>T (p.Glu2261*), c.109T>G (p.Cys37Gly) and c.8495A>G (p.Asn2832Ser). Prenatal testing showed that the fetus of one family has carried the same mutation as the proband, while the fetus of another family did not. CONCLUSION PKD1 variants, including three novel variants, have been identified in the eight pedigrees affected with ADPKD. Based on these results, prenatal diagnosis and genetic counseling have been provided.
DNA Mutational Analysis/methods* ; Female ; Humans ; Mutation ; Pedigree ; Polycystic Kidney, Autosomal Dominant/genetics* ; Pregnancy ; Prenatal Diagnosis ; TRPP Cation Channels/genetics*

Objective: Nonalcoholic fatty liver disease (NAFLD) has become a common chronic liver disease that is harmful to human health. Moreover, there is currently no FDA-approved first-line drug for the treatment of nonalcoholic steatohepatitis (NASH) or NAFLD. Traditional Chinese medicine (TCM) is widely used to ameliorate liver diseases, such as the traditional ancient recipe called Three Flower Tea (TFT), which consists of double rose (Rosa rugosa), white chrysanthemum (Chrysanthemum morifolium), and Daidaihua (Citrus aurantium). However, the mechanisms of the action of TFT are not clear. Therefore, this study aimed to elucidate the mechanisms of TFT against NAFLD in high-fat diet (HFD)-induced rats. Methods: This study utilized bioinformatics and network pharmacology to establish the active and potential ingredient-target networks of TFT. Furthermore, a protein–protein interaction (PPI) network was constructed, and enrichment analysis was performed to determine the key targets of TFT against NAFLD. Furthermore, an animal experiment was conducted to evaluate the therapeutic effect and confirm the key targets of TFT against NAFLD. Results: A total of 576 NAFLD-related genes were searched in GeneCards, and under the screening criteria of oral bioavailability (OB) ≥30% and drug-likeness (DL) ≥0.18, a total of 19 active ingredients and 210 targets were identified in TFT. Network pharmacology analysis suggested that 55 matching targets in PPIs were closely associated with roles for NAFLD treatment. Through the evaluation of network topology parameters, four key central genes, PPARγ, SREBP, AKT, and RELA, were identified. Furthermore, animal experiments indicated that TFT could reduce plasma lipid profiles, hepatic lipid profiles and hepatic fat accumulation, improve liver function, suppress inflammatory factors, and reduce oxidative stress. Through immunoblotting and immunofluorescence analysis, PPARγ, SREBP, AKT, and RELA were confirmed as targets of TFT in HFD-induced rats. Conclusion: In summary, our results indicate that TFT can prevent and treat NAFLD via multiple targets, including lipid accumulation, antioxidation, insulin sensitivity, and inflammation.

Objective:To investigate the abnormal results of chromosomal microarray analysis (CMA) in the subsequent pregnancy of women with adverse pregnancy history, and explore the applicability of CMA in women with different genetic etiology.Methods:Out of 5 563 pregnant women who received CMA test in Nanjing Drum Tower Hospital during June 2014 and July 2020, 169 cases that underwent prenatal diagnosis due to isolated adverse pregnancy history were retrospectively collected in this study. All the participants were divided into three groups based on the etiology type of probands, genetic origin and expected CMA outcome: high-risk group ( n=19, including 11 cases with inherited pathogenic copy number variations and eight cases with inherited chromosomal abnormalities), low-risk group ( n=113, including six cases with negative whole exome sequencing and/or CMA findings, 31 cases with confirmed monogenic disease, 47 cases with de novo pathogenic copy number variations and 29 cases with de novo chromosomal abnormalities), and unknown risk group ( n=40, none of the cases underwent genetic testing). Descriptive statistical analysis was used to summarize the abnormal detection of each group. Results:There were 169 mothers with 172 fetuses finally enrolled, including two twins and one woman with two singleton pregnancies. A total of nine cases of abnormal fetuses were detected by CMA, accounting for 5.2% (9/172). Among them, eight were in the high-risk group, which were all caused by parental abnormalities, and one case in the low-risk group was detected with a de novo 22q11.22q11.23 microduplication, which was arr[GRCh37]22q11.22q11.23(22,997,928-25,002,659)×3. No abnormality was detected in the 40 patients of unknown risk group. Conclusions:Clarifying the etiology of isolated adverse pregnancy history is crucial to the rational application of CMA. Monogenic disease, unknown cause or negative finding of CMA in probands may not be an indication for prenatal diagnosis of CMA.