The CRISPR/dCas9 system is a gene editing tool that has proven to be highly efficient and precise. By utilizing transcriptional activators, such as VP64, p65, and Rta, the system can effectively and stably activate target genes. Sox17, a transcription factor belonging to the SOX family, plays a crucial role in the differentiation of the germ layers and the determination of cell fates during the early stages of embryonic development. Sheep embryonic stem cells (sESCs) are characterized by their capacity for self-renewal and multidirectional differentiation, serving as a significant in vitro model for studying the mechanisms of cell differentiation during early embryonic development. However, the importing of exogenous genes into sESCs is challenging due to their unique growth characteristics. The objective of this study was to investigate the conditions necessary for successfully activating Sox17 in sESCs. To this end, we employed the CRISPR/dCas9 system along with liposome transfection, lentivirus invasion, and electroporation to activate Sox17 in sESCs. The expression of Sox17 was then determined by fluorescence quantitative PCR, on the basis of which the performance of different transfection methods was compared. The results indicated that the electroporation group had the best transfection effect and the highest Sox17 expression among the three transfection methods. The efficient and stable gene activation protocol will provide a reference for embryonic stem cell research in other species, especially livestock animals, and lay the foundation for the subsequent study of gene function and realization of precise cell fate regulation by regulating gene expression in sheep embryonic stem cells.
Animals ; CRISPR-Cas Systems/genetics* ; Sheep ; SOXF Transcription Factors/genetics* ; Embryonic Stem Cells/cytology* ; Genetic Vectors/genetics* ; Cell Differentiation/genetics* ; Transfection ; Gene Editing/methods*

Objective In order to shorten the transparency time of clear,unobstructed brain imaging cocktails and computational analysis(CUBIC),improve the transparency efficiency,and explore the possibility of applying hydrophilic tissue transparency technique,this study was conducted to optimize the perfusion of CUBIC technique and compare it with four hydrophilic tissue clearing method in terms of tissue transparency effect,transparency time,area change,volume change and adeno-associated virus(AAV)fluorescence retention.Methods Brain,liver,spleen and kidney of 6 adult Institute of Cancer Research(ICR)mice were subjected to clearing treatment by SeeDB,FRUIT,ScaleS and CUBIC method,respectively.The area and gray value of the samples were measured by Image J 1.8.0,and the volume before and after transparency was measured by drainage method to compare the transparency effect,time and size deformation of each group.Perfusion optimization of the CUBIC was performed by improving the perfusion rate with the optimal perfusion dose,each group of the experimental sample size was 6.Fluorescence preservation by different techniques was evaluated by injecting AAV in the motor cortex of 16 adult mice and taking the cervical spinal segments for transparency treatment after four weeks,and the fluorescence photographs were measured by Image J 1.8.0 to measure the mean fluorescent intensity.Results The optimal perfusion rate and dose of CUBIC was 15 ml/min and 200 ml respectively.For transparency ability and speed,the perfusion CUBIC had the lowest mean gray value and took the shortest time,while CUBIC consumed the longest time,and SeeDB,FRUIT,and ScaleS did not show good transparency ability.In terms of area and volume changes,several techniques showed different degrees of expansion after transparency of tissues or organs.In terms of fluorescence retention,perfusion CUBIC showed the best retention of green fluorescent protein(GFP)fluorescence signal,followed by CUBIC,ScaleS,FRUIT,and SeeDB.Conclusion Perfusion CUBIC technique shows the best tissue transparency,the shortest transparency time,and the most AAV fluorescence retention compared with other techniques.

The characteristic features of dissociative conversion disorder entail a partial or complete loss of normal integration in memory,identity awareness,immediate sensation,and motor control.This article documents a case of the disorder presenting with an onset of depressive mood,diminished interest,and facial tics,subsequently accompanied by intermittent fainting spells with limb convulsions.Despite multiple consultations in both general hospitals and psychiatric settings,and despite treatment with various antidepressants and antiepileptic medications,the patient's symptoms showed no significant improvement.The report of this case deepens the understanding of a complex and commonly misdiagnosed condition,offering valuable insights for the diagnosis and treatment of patients exhibiting similar symptoms.

Objective To investigate the MRI manifestations of acquired immunodeficiency syndrome complicated with subtentorial progressive multifocal leukoencephalopathy(PML).Methods Sixteen patients with acquired immunodeficiency syndrome complicated with subtentorial PML confirmed by surgery,pathology,or clinical diagnosis were selected.The MRI features of plain scan and enhanced scan were analyzed and summarized,retrospectively.Results Among the 16 acquired immunodeficiency syndrome patients complicated with subtentorial PML,in terms of lesion distribution,the left cerebellar hemisphere was involved in 10 cases(62.5％),and the right cerebellar hemisphere was involved in 10 cases(62.5％).The left pontine arm was involved in 11 cases(68.8％),the right pontine arm was involved in 10 cases(62.5％),the medulla oblongata was involved in 6 cases(37.5％),the pontine region was involved in 11 cases(68.8％),the midbrain was involved in 7 cases(43.8％),and the supratentorial lesions were involved in 5 cases(31.3％).Subtentorial lesions were found to be asymmetrically distributed in all patients.In terms of MRI imaging findings,acquired immunodeficiency syndrome patients complicated with subtentorial PML lesions exhibited low signal on T,WI and high signal on T2 WI/T2-fluid attenuated inversion recovery(FLAIR)images.The"shrimp sign"and"crescent cerebellar lesion"was respectively observed in 3 and 7 cases on T2 WI.There were no diffusion limitations on diffusion weighted imaging(DWI),no significant enhancement after contrast-enhanced scanning,and no mass effect in all the 16 cases.Conclusion The MRI imaging findings of acquired immunodeficiency syndrome complicated with subtentorial PML exhibit similar features to those observed in PML affecting the cerebral hemispheres.A characteristic"shrimp sign"is often observed on T2WI.The presence of typical MRI imaging findings,along with the patient's clinical history and the detection of JC virus deoxyribonucleic acid(DNA)in the cerebrospinal fluid(CSF),can aid in the accurate diagnosis of PML.

This study constructed a LHCGR-CRE-luc-HEK293 transgenic cell line according to the activation of the cAMP signaling pathway after recombinant human chorionic gonadotropin binding to the receptor. The biological activity of recombinant human chorionic gonadotropin was assayed using a luciferase assay system. The relative potency of the samples was calculated using four-parameter model. And the method conditions were optimized to validate the specificity, relative accuracy, precision and linearity of the method. The results showed that there was a quantitative potency relationship of human chorinonic gonadotropin (hCG) in the method and it was in accordance with the four-parameter curve. After optimization, the conditions were determined as hCG dilution concentration of 2.5 μg·mL^-1, dilution ratio of 1∶4, cell number of 10 000-15 000 cells/well, and induction time of 6 h. The method had good specificity, relative accuracy with relative bias ranging from -8.9% to 3.4%, linear regression equation correlation coefficient of 0.996, intermediate precision geometric coefficient of variation ranging from 3.3% to 15.0%, and linearity range of 50% to 200%. This study successfully established and validated a reporter gene method to detect hCG biological activity, which can be used for hCG biological activity assay and quality control.

Objective To develop and validate a deep learning model for automatic identification of liver CT contrast-enhanced phases.Methods A total of 766 patients with liver CT contrast-enhanced images were retrospectively collected.A three-phase classification model and an arterial phase(AP)classification model were developed,so as to automatically identify liver CT contrast-enhanced phases as early arterial phase(EAP)or late arterial phase(LAP),portal venous phase(PVP),and equilibrium phase(EP).In addition,221 patients with liver CT contrast-enhanced images in 5 different hospitals were used for external validation.The annotation results of radiologists were used as a reference standard to evaluate the model performances.Results In the external validation datasets,the accuracy in identifying each enhanced phase reached to 90.50%-99.70%.Conclusion The automatic identification model of liver CT contrast-enhanced phases based on residual network may provide an efficient,objective,and unified image quality control tool.

Establishing a new paradigm for high-quality development in public hospitals,embracing a comprehensive,systematic,and innovative strategic management philosophy,and instituting a modern hospital management system hold significant practical importance.It investigates the pathways through which the Guangzhou Women and Children's Medical Center has fulfilled its strategic commitments over its 17-year history.It delves into the meaning and value of constructing hospital strategic commitments,explores pathways for realization,analyzes effects.It aims to understand the normative role of strategic commitments in shaping the development paths of specialized women's and children's hospitals and to provide a reference for the high-quality development of similar hospitals across China.

Objective To explore the potential mechanism of action of paeoniflorin in diabetes mellitus,the related targets and pathways were preliminarily discussed,based on the network pharmacology and molecular docking technology.Methods Analyze the potential targets of paeoniflorin using the Swiss Target Prediction database.Genecards and OMIM databases yielded the genes of diabetes-related illnesses.After taking the intersection of the two,protein-protein interaction network(PPI)was established using STRING and Cytoscape programs to search for key genes with strong correlation and complete gene ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis.Use AutoDockTools and Pymol programs to complete protein molecule docking validation.Results The pharmacologically-related study revealed 63 targets associated with paeoniflorin,4 758 genes related to diabetes,and 50 intersection targets.15 key genes including vascular endothelial growth factor A(VEGFA),epidermal growth factor receptor(EGFR),V-Ha-ras harvey(HRAS),V-src sarcoma(SRC)and heat shock protein hs 90-alpha(HSP90AA1)were screened.RAs-associated protein 1,Ras,calcium and other signaling pathways were obtained by KEGG pathway analysis.Molecular docking results showed that paeoniflorin had good binding ability with key genes.Conclusion Paeoniflorin can treat diabetes through multiple targets and pathways,and this mechanism can provide a basis for the application of paeoniflorin in anti diabetes and drug research and development.

Objective To observe clinical efficacy and safety of adalimumab(ADA)injection combined with methotrexate(MTX)tablets in the treatment of patients rheumatoid arthritis(RA)and its influences on bone metabolism.Methods According to cohort method,the RA patients were divided into treatment group and control group.The control group was treated with oral MTX tablets(10 mg/once,once/week),while treatment group was given subcutaneous injection of ADA ejection(40 mg/once,once/2 weeks)on basis of control group.Both groups were treated for 24 weeks.The clinical curative effect,symptoms improvement,joint function and serum bone metabolism indexes[bone alkaline phosphatase(B-ALP),bone glaprotein(BGP),carboxyterminal propeptide of type Ⅰ procollagen(PICP)]in the two groups were compared,and safety was evaluated.Results There were 37 cases in control group and 43 cases in treatment group.After treatment,total response rates of treatment group and control group were 93.02％(40 cases/43 cases)and 75.68％(28 cases/37 cases),the difference was statistically significant(P＜0.05).After treatment,morning stiffness time in treatment group and control group were(29.27±7.05)and(48.63±12.25)min,the number of joint swelling were 2.64±0.66 and 4.04±1.09,number of joint tenderness were 4.51±1.12 and 6.23±2.07,levels of serum B-ALP were(20.76±2.82)and(26.41±3.05)U·L-1;levels of serum BGP were(27.45±3.41)and(23.86±3.17)ng·mL-1;levels of serum PICP were(47.39±5.63)and(41.68±5.24)ng·mL-1,the differences were statistically significant(P＜0.05).The adverse drug reactions in treatment group were mainly on headache,pruritus at the injection site,diarrhea and nausea,while which in control group were mainly on diarrhea,anorexia and nausea.The difference in incidence of adverse reactions between treatment group and control group was not statistically significant(11.63％vs 8.11％,P＞0.05).Conclusion Clinical curative effect of ADA injection combined with MTX tablets is significant in RA patients,which can significantly relieve clinical symptoms,promote recovery of joint function,reduce levels of serum rheumatoid factor,relieve inflammatory response and improve bone metabolism.

Establishing a new paradigm for high-quality development in public hospitals,embracing a comprehensive,systematic,and innovative strategic management philosophy,and instituting a modern hospital management system hold significant practical importance.It investigates the pathways through which the Guangzhou Women and Children's Medical Center has fulfilled its strategic commitments over its 17-year history.It delves into the meaning and value of constructing hospital strategic commitments,explores pathways for realization,analyzes effects.It aims to understand the normative role of strategic commitments in shaping the development paths of specialized women's and children's hospitals and to provide a reference for the high-quality development of similar hospitals across China.