ObjectiveThe utilization of assisted reproductive technology to rescue genetically modified mouse strains with reproductive disorders provides a reference for improving techniques to preserve valuable experimental mouse strains. MethodsIn vitro fertilization-embryo transfer (IVF-ET) technology was performed on 28 strains of infertile male mice aged 9-18 months. Several indicators such as sperm density and sperm motility in infertile male mice were assessed to select the most viable sperm for IVF-ET experiments. Fertility rate, abnormal egg rate, and birth rate were recorded after the birth of the pups. An optimized ovarian transplantation procedure was applied to 12 strains of infertile female mice aged 8-18 months. 6-week-old female mice with the same genetic background were selected as recipients. One intact ovary was removed from each recipient mouse, and the contralateral oviduct was ligated. An ovary from a donor mouse was isolated and transplanted orthotopically into the side where the ovary had been removed in the recipient mouse. Twenty-one days post-surgery, recipient mice were co-housed with 8-week-old wild type male mice of the same genetic background for breeding. Data such as the pregnancy rate and live birth rate of the recipients were recorded after the birth of the pups. ResultsIVF-ET successfully rescued 28 mouse strains, with the oldest male mice being 18 months old. The success rate of the first round of IVF-ET experiments was 89.29% (25/28). The average fertility rate of IVF in infertile male mice was (51.01±14.97)%, the abnormal egg rate was (9.03±5.28)%, and the birth rate of offspring mice was (18.60±7.03)%. 39 out of 40 ovarian transplant recipient mice survived, with a pregnancy rate of 33.33% (13/39) for ovarian transplant recipients, and a live birth rate of 17.95% (7/39). Four mouse strains were successfully rescued using optimized ovarian transplantation technology, with the oldest female mice being 18 months old. 8 strains were not rescued as they failed to produce offspring that survived to sexual maturity. ConclusionIVF-ET is an effective approach for rescuing mice with reproductive disorders caused by different reasons, especially for those beyond the optimal breeding age. Ovarian transplantation technology can also be used as an alternative for aged female mice. But its success rate is relatively lower than that of IVF-ET, and carries a higher experimental risk.

BACKGROUND:Fibromyalgia syndrome,as a common rheumatic disease,is related to central sensitization and immune abnormalities.However,the specific mechanism has not been elucidated,and there is a lack of specific diagnostic markers.Exploring the possible pathogenesis of this disease has important clinical significance. OBJECTIVE:To screen the potential diagnostic marker genes of fibromyalgia syndrome and analyze the possible immune infiltration characteristics based on bioinformatics methods,such as weighted gene co-expression network analysis(WGCNA),and machine learning. METHODS:Gene expression profiles in peripheral serum of fibromyalgia syndrome patients and healthy controls were obtained from the gene expression omnibus(GEO)database.The differentially co-expressed genes were screened in the expression profile by differential analysis and WGCNA analysis.Least absolute shrinkage and selection operator(LASSO)and support vector machine-recursive feature elimination(SVM-RFE)machine learning algorithm were further used to identify hub biomarkers,and draw receiver operating characteristic curve(ROC)to evaluate the accuracy of diagnosing fibromyalgia syndrome.Finally,single sample gene set enrichment analysis(ssGSEA)and gene set enrichment analysis(GSEA)were used to evaluate the immune cell infiltration and pathway enrichment in patients with fibromyalgia syndrome. RESULTS AND CONCLUSION:Eight down-regulated differentially expressed genes(DEGs)were obtained after differential analysis of the GSE67311 dataset according to the conditions of log2|(FC)|>0 and P<0.05.After WGCNA analysis,497 genes were included in the module(MEdarkviolet)with the highest positive correlation(r=0.22,P=0.04),and 19 genes were included in the module(MEsalmon2)with the highest negative correlation(r=-0.41,P=6×10-5).After intersecting DEGs and the module genes of WGCNA,seven genes were obtained.Four genes were screened out by LASSO regression algorithm and five genes were screened out by SVM-RFE machine learning algorithm.After the intersection of the two,three core genes were identified,which were germinal center associated signaling and motility like,integrin beta-8,and carboxypeptidase A3.The areas under the ROC curve of the three core genes were 0.744,0.739,and 0.734,respectively,indicating that they have good diagnostic value and can be used as biomarkers for fibromyalgia syndrome.The results of immune infiltration analysis showed that memory B cells,CD56 bright NK cells,and mast cells were significantly down-regulated in patients with fibromyalgia syndrome compared with the control group(P<0.05),and were significantly positively correlated with the above three biomarkers(P<0.05).The enrichment analysis suggested that there were nine fibromyalgia syndrome enrichment pathways,mainly related to olfactory transduction pathway,neuroactive ligand-receptor interaction,and infection pathway.The above results showed that the occurrence and development of fibromyalgia syndrome are related to the involvement of multiple genes,abnormal immune regulation,and multiple pathways imbalance.However,the interactions between these genes and immune cells,as well as their relationships with various pathways need to be further investigated.

In order to provide a reference basis for the development of relevant compound preparations， this article takes a comprehensive analysis of the usage and dosage of famous classical formulas in Han dynasty from various perspectives， and gives corresponding countermeasures on this basis. Through the comprehensive analysis of the classification and statistics of Zhongjing's medication characteristics， decoction methods， administration and dosage， and combining conversion methods of weights and measures by ancient medical practitioners， along with the dosage and administration of the listed Han dynasty famous classical formulas， it was found that the "Jiangxi method" served as a general guideline for administration according to Zhongjing's original text. This method allowed for flexible dosing based on the conversion of the ancient measurements to modern equivalents［13.8 g per Liang（两）］， ensuring the safe and effective medication of these formulas. After combing， it is found that although the dosage of single medicine is large in famous classical formulas from Han dynasty， the administration is flexible. The crude drug amount per administration serves as the foundational dose， with the frequency of administration adjusted flexibly according to the condition. This dosing approach becomes the key for the rational development of compound formulations of famous classical formulas. Based on the conclusions of the study， it is recommended that when developing compound formulations of famous classical formulas in Han dynasty， the original administration method and dosage should be respected. The original crude drug amount per administration should be considered as the daily foundational dose， with the frequency of administration described within a range（1 to N times per day， where N is the maximum number of administrations as per the original text）. The specific frequency of administration can be adjusted flexibly by clinical practitioners based on the individual condition. This approach should also be adopted in toxicological studies， where the dosage per administration serves as the basis for toxicity research， and the toxicity profile at the maximum administration frequency should be observed， providing guidance on the clinical safety range. Corresponding drug labels should provide information within a range to indicate toxicological risk intervals.

The important role of liquid-liquid phase separation in a series of biological processes,including regulation of gene transcription and translation,stress response,autophagy and the establishment of synaptic structure,has been widely accepted.Abnormal phase separation is associated with many human diseases,including neurodevelopmental disorders and neurodegenerative diseases.Studies have shown that some proteins associated with epigenetic modifications are also subject to liquid-liquid phase separation,suggesting that epigenetic modifications regulate the development and disease of the nervous system by regulating phase separation.This review summarized the important roles of epigenetic modification and phase separation in neurodevelopment and neurodiseases,and focused on the important roles of proteins related to epigenetic modification with phase separation characteristics.Understanding the correlation between epigenetic modification and phase separation will help fully understand the underlying mechanisms of neurodevelopment and neurodiseases,and will further provide new targets and strategies for the treatment of related diseases.

Objective:To investigate the discomfort of chest wall approach area in patients undergoing endoscopic thyroidectomy by a gasless unilateral axillary approach (GUA), and to analyze its influencing factors. To provide a basis for the development of targeted improvement measures.Methods:A total of 153 patients with GUA from May. 2023 to Aug. 2023 in the Department of Thyroid Surgery, the First Affiliated Hospital of Zhengzhou University were selected as the study subjects. The general information questionnaire was collected one day before operation, the pain scales were assessed one day and three days after operation, and the pain and numbness scales were assessed one month after operation. The t test or χ2 test was used for comparison of baseline data between groups according to different types of variables. The patients were divided into two groups: less than moderate pain group (two postoperative average VAS scores<4) and more than moderate pain group (two postoperative average VAS scores ≥4). Mild numbness group (postoperative VAS score<4) ; Moderate and severe numbness group (postoperative VAS score ≥4). Multivariate binary Logistic regression was performed with pain discomfort and numbness discomfort as dependent variables to find possible influencing factors. Results:A total of 153 valid questionnaires were collected. There were 125 patients in the moderate pain group; There were 28 patients in the moderate and above pain group. There were 94 patients in the mild numbness group. There were 59 patients in the moderate to severe numbness group. Multivariate binary Logistic regression results showed that, exercise habits ( OR=0.07 95% CI=0.006, 0.409), operation duration ( OR=1.026 95% CI=1.001, 1.054), total drainage volume ( OR=1.122 95% CI=1.07, P<0.05), 1.198), and drainage tube indwelling time ( OR=0.012 95% CI=0.0, 0.187) had an impact on the discomfort of the chest wall approach area, and the difference was statistically significant ( P<0.05). Gender, BMI, marital status, education, occupation, handed-side surgery, handed-side axillary surgery, smokess and alcohol history, intraoperative blood loss, and length of hospital stay had no effect on the discomfort of chest wall approach area, and the difference was not statistically significant ( P>0.05) . Conclusion:Exercise habits, operation duration, total drainage volume, and drainage duration are independent predictors of discomfort in GUA patients.

ObjectiveTo explore the molecular mechanism implicated in the treatment of primary myelofibrosis （PMF） using Chinese medicinal herbs （CMH） by bioinformatics and molecular dynamics. MethodsData mining was performed to find the high-frequency CMH in treating PMF between the year of 1985 and 2024 by searching CNKI， Chinese Science and Technology Journal Database （CCD）， and China Academic Journal Database （CSPD）. TCMSP， SwissTargetPrediction and related reports were used to collect the main active ingredients of high-frequency CMH and their targets. The PMF datasets GSE44426 and GSE124281 were downloaded from GEO database， and R software was used for data normalization and differentially expressed genes （DEGs） screening. Key module hub genes were obtained by weighted gene co-expression network analysis （WGCNA） analysis. The common intersection genes of active ingredient targets， DEGs and key module hub genes of CMH were selected， and the target network was generated using Cytoscape 3.9.2 software. The core target network was generated by topological analysis， while key pathways were selected by GO and KEGG pathway enrichment analysis， and protein interaction relationships were obtained from the String database， so as to construct drug-ingredient-target network and protein interaction network （PPI） relationship diagrams. Discovery Studio 2020 software was used to perform molecular docking， and the GROMACS program was used to perform molecular dynamics simulation. ResultsA total of 21 prescriptions were collected involving 121 herbs. There were 9 herbs with a frequency ≥10 times， which were Danshen （Radix et Rhizoma Salviae Miltiorrhizae）， Huangqi （Radix Astragali）， Baizhu （Rhizoma Atractylodis Macrocephalae）， Danggui （Radix Angelicae Sinensis）， Dangshen （Radix Codonopsis）， Gancao （Radix et Rhizoma Glycyrrhizae）， Baishao （Radix Paeoniae Alba）， Fuling （Poria） and Shudihuang （Radix Rehmanniae Praeparata） from high- to low-frequency. A total of 98 active ingredients and 1125 potential targets were obtained from 9 high-frequency CMH. GSE44426 and GSE124281 data sets screened out 24 gene samples， including 14 of the healthy control group and 10 of the PMF group， and identified 319 DEGs between the two groups， including 122 up-regulated genes and 197 down-regulated genes. WGCNA screened out 24 co-expression module genes and found that the five modules closely related to the onset of PMF were MEpink， MEdarkred， MEblack， MEgrey， and MEturquoise， involving 7112 key module hub genes. The GO and KEGG enrichment analyses indicated that lipids and the atherosclerosis pathways were mainly involved in the mechanism of above high-frequency CMH in treating PMF， which included six hub protein targets： HSP90AA1， HSP90AB1， SRC， MAPK1， IL1B and IL10. From the drug-ingredient-target network， seven active ingredients of CMH targeting at these six hub targets were found， including verbascoside， verbascos isoflavone， kaempferol， luteolin， naringenin， quercetin and pachymic acid. The molecular docking and molecular dynamics analyses showed that the key CMH were Shudihuang， Huangqi， Baishao， Danshen， Gancao and Fuling， and among the seven active ingredients， calycosin had the highest binding affinity with HSP90AB1. ConclusionThe main CMH for the treatment of PMF may be Shudihuang， Huangqi， Baishao， Danshen， Gancao and Fuling， and the active ingredients include verbascoside， verbascos isoflavones， kaempferol， luteolin， naringenin， quercetin and pachymic acid. The relevant targets are HSP90AA1， HSP90AB1， SRC， MAPK1， IL-10， and IL-1β， and the most critical pathways are lipid and atherosclerosis pathways.

Objective: To investigate the differences in enolase and laminin levels in vaginal epithelial tissues between mice successfully infected withGardnerellaand mice not infected with Gardnerella, providing information for further exploration of the correlation between enolase and laminin levels and the incidence of bacterial vaginosis. Methods: Gardnerella strains isolated, purified, and identified from vaginal secretions of patients with bacterial vaginosis were used to infect the vagina of mice and establish a mouse model of bacterial vaginosis. Successful and failed mice was defined as successful and failed groups, respectively. Differential expression of enolase and laminin in the vaginal epithelial tissue of two groups of mice was detected by Western blot. Modeling success rate was statistically analyzed, and the expression differences of enolase and laminin was compared between two groups. Results: One strain of Gardnerella vaginalis infected 10 SPF grade KM mice, 7 mice met the diagnostic criteria for bacterial vaginosis, and 3 mice failed to model, with a success rate of 70%. Western blot was used to detect protein expression levels, and the levels of laminin and enolase in the successfully modeled mouse vaginal epithelial tissue were significantly higher than those in the failed modeling group, with statistical differences between the two groups(P<0.05). Conclusion Enolase and laminin may be involved in the occurrence of bacterial vaginosis, however, further research is needed to determine the mechanisms through which they trigger the occurrence and development of the disease.

Objective To compare the anti-inflammatory,antitumor and anti-bacterial effects of the single extract(in granules)and the prepared drug in pieces of Forsythia Suspense(Lianqiao,a traditional Chinese herbal medicine).Methods In zebrafish embryo models of CuSO4 exposure,tail transection and LPS microinjection-induced inflammation,the anti-inflammatory effects of 10 μg/mL DEX,single extract of Forsythia Suspense,and the water extract of the prepared drug(400,600,and 800 μg/mL)were evaluated by observing neutrophil counts,RT-qPCR,HE staining and survival analysis.Zebrafish embryo models bearing different human tumor cell xenografts were used to assess the anti-tumor effect of the drugs in different dosage forms by fluorescence staining and HE staining.The microbroth dilution method was used to evaluate the antibacterial efficacy of the drugs.Results In the zebrafish embryo models of inflammation,both of the two dosage forms of Forsythia Suspense significantly inhibited neutrophil aggregation,reduced the mRNA expressions of TNF-α,IL-6,P38,Jnk,Erk and P65,and increased the survival rate of zebrafish.They both showed obvious inhibitory effects against xenografts of different human cancer cells including colon cancer cells(HCT116),pancreas adenocarcinoma cells(PANC-1),lung cancer cells(A549),liver cancer cells(Hep3B)and cervical carcinoma cells(Hela)in zebrafish embryos,and exhibited strong anti-bacterial effects at the concentration of 15.63 mg/mL.Conclusion The two dosage forms of Forsythia Suspense have similar anti-inflammatory,antitumor and antibacterial effects,but their effects for inhibiting IL-6,P65,and Jnk mRNA expressions and HCT116 cell proliferation differ significantly at low doses in zebrafish.

Objective To construct myeloid-specific Spi1 gene knockout mice and analyze their genotypes,so as to provide animal model basis for the study of pathological mechanism of diseases and drug targets.Methods Ac-cording to the principle of CRISPR/Cas9 technology and Cre/LoxP system,sgRNA and Donor vectors were de-signed and constructed.The transcript of Exon 2(Exon 2)was used as the knockout region,and Loxp elements were placed on both sides of Exon 2.Cas9 protein,sgRNA and Donor vector were mixed and microinjected into the fertilized eggs of C57BL/6J mice,the fertilized eggs were transplanted into the uterus of C57BL/6J pregnant female mice,and F0 generation was obtained after 19～20 days.Positive F0 mice were mated with C57BL/6J mice to ob-tain stable F1 Spi1flox/+mice.Spi1flox/+mice of F1 generation were selfed to obtain Spi1flox/flox mice.Spi1flox/flox mated with Lyz2-Cre+mice to obtain Spi1flox/+/Lyz2-Cre+mice,and then mated with Spi1flox/flox,the Spi1flox/flox/Lyz2-Cre+mice were myeloid-specific Spi1 gene knockout(KO)mice.Spi1flox/flox/Lyz2-cre-mice were used as wild-type(WT)mice.DNA of WT and KO mice was extracted,and the genotypes were identified by agarose gel electro-phoresis after PCR amplification.Western blot was used to detect the expression of spleen focus forming virus provi-ral integration oncogene,Spi-1/purine rich box-1(PU.1)in immune cells of WT and KO mice.Results The results of PCR identification showed that the genotype of mice with only 220 bp amplified by flox primer was Spi1flox/flox homozygote,and the genotype of mice with 700 bp amplified by Lyz2-Cre primer was Lyz2-Cre+.Western blot showed that compared with WT group,the protein PU.1 was not expressed in bone marrow-derived macropha-ges(BMDMs)and peritoneal macrophages(PM)in KO group(P<0.01).There was no significant difference of statistics in the expression level of PU.1 in T cells between KO mice and WT mice.The results of PCR and West-ern blot showed that myeloid-specific Spi1 KO mice were successfully constructed.Conclusion The myeloid-spe-cific Spi1 gene KO mice are successfully constructed and identified,which provides animal model basis for further revealing the potential mechanism of PU.1 inimmune regulation.

Objective To breed and identify the T lymphocyte-conditional Spi1 knockout mice for the further in-vestgation of the specific role of Spi1-encoded protein PU.1.Methods The Lck-Cre mice were mated with Spi1flox/flox mice to obtain Lck-Cre×Spi1flox/flox mice(T lymphocyte-specific Spi1 knockout mice),and the genotype was determined by polymerase chain reaction(PCR)and agarose gel electrophoresis.Magnetic beads were used to sort out the splenic T lymphocytes,and the knockdown efficiency of PU.1 in T cells was detected by Western blot,quantitative real-time PCR(qPCR)and flow cytometry.Results The Lck-Cre×Spi1flox/flox mouse genotype was stably inherited.Compared with Spi1flox/flox mice,the expression level of PU.1 was significantly reduced in splenic T cells of Lck-Cre×Spi1flox/flox mice.Conclusion In this study,the T lymphocyte-specific Spi1 knockout mice was successfully constructed by applying Cre/LoxP system and CRISPR/Cas9 technology,which provided a reliable an-imal model for the subsequent experiments of the specific role of PU.1 in T cell-related diseases.