Objective To explore the construction of α-1,3-galactosyltransferase (GGTA1) gene-knockout (GTKO) Diannan miniature pigs and the kidney xenotransplantation from pigs to rhesus macaques, and to assess the effectiveness of GTKO pigs. Methods The GTKO Diannan miniature pigs were constructed using the CRISPR/Cas9 gene-editing system and somatic cell cloning technology. The phenotype of GTKO pigs was verified through polymerase chain reaction, Sanger sequencing and immunofluorescence staining. Flow cytometry was used to detect antigen-antibody (IgM) binding and complement-dependent cytotoxicity. Kidney xenotransplantation was performed from GTKO pigs to rhesus macaques. The humoral immunity, cellular immunity, coagulation and physiological indicators of the recipient monkeys were monitored. The function and pathological changes of the transplanted kidneys were analyzed using ultrasonography, hematoxylin-eosin staining, immunohistochemical staining and immunofluorescence staining. Results Single-guide RNA (sgRNA) targeting exon 4 of the GGTA1 gene in Diannan miniature pigs was designed. The pGL3-GGTA1-sgRNA1-GFP vector was transfected into fetal fibroblasts of Diannan miniature pigs. After puromycin selection, two cell clones, C59# and C89#, were identified as GGTA1 gene-knockout clones. These clones were expanded to form cell lines, which were used as donor cells for somatic cell nuclear transfer. The reconstructed embryos were transferred into the oviducts of trihybrid surrogate sows, resulting in 13 fetal pigs. Among them, fetuses F04 and F11 exhibited biallelic mutations in the GGTA1 gene, and F04 had a normal karyotype. Using this GTKO fetal pig for recloning and transferring the reconstructed embryos into the oviducts of trihybrid surrogate sows, seven surviving piglets were obtained, all of which did not express α-Gal epitope. The binding of IgM from the serum of rhesus monkey 20# to GTKO pig PBMC was reduced, and the survival rate of GTKO pig PBMC in the complement-dependent cytotoxicity assay was higher than that of wild-type pig. GTKO pig kidneys were harvested and perfused until completely white. After the left kidney of the recipient monkey was removed, the pig kidney was heterotopically transplanted. Following vascular anastomosis and blood flow restoration, the pig kidney rapidly turned pink without hyperacute rejection (HAR). Urine appeared in the ureter 6 minutes later, indicating successful kidney transplantation. The right kidney of the recipient was then removed. Seven days after transplantation, the transplanted kidney had good blood flow, the recipient monkey's serum creatinine level was stable, and serum potassium and cystatin C levels were effectively controlled, although they increased 10 days after transplantation. Seven days after transplantation, the levels of white blood cells, lymphocytes, monocytes and eosinophils in the recipient monkey increased, while platelet count and fibrinogen levels decreased. The activated partial thromboplastin time, thrombin time and prothrombin time remained relatively stable but later showed an upward trend. The recipient monkey survived for 10 days. At autopsy, the transplanted kidney was found to be congested, swollen and necrotic, with a small amount of IgG deposition in the renal tissue, and a large amount of IgM, complement C3c and C4d deposition, as well as CD68⁺ macrophage infiltration. Conclusions The kidneys of GTKO Diannan miniature pigs may maintain normal renal function for a certain period in rhesus macaques and effectively overcome HAR, confirming the effectiveness of GTKO pigs for xenotransplantation.

Voice biomarkers， as an emerging smart medical technology， are now being used in applications such as assisting in the diagnosis and treatment of diseases， facilitating accurate and personalized medical services for patients. However， it also raises many ethical issues， including informed consent， privacy protection， accuracy and reliability， data security， legal risks， and other issues. This paper systematically sorted out the ethical issues in the applications of voice biomarkers in the medical field， summarized these issues， such as informed consent， privacy protection， accuracy and reliability， data security， and legal risks， as well as explored the corresponding coping strategies. These countermeasures encompassed utilizing new media platforms to raise public awareness of voice biomarkers， strengthening supervision and management to promote the privacy protection of voice biomarkers， reducing algorithm biases to promote the general benefits of voice biomarkers to the public， establishing multidisciplinary teams to protect the data security of voice biomarkers， and encouraging medical professionals and researchers to participate in policy research， with a view to providing references for promoting and regulating the applications of voice biomarkers in the medical field.

ObjectiveTo explore the interaction between bruceoside B and gut microbiota and the inhibitory activity of its metabolites on human lung cancer A549 cells， and to explore the value of bruceoside B in the treatment of non-small cell lung cancer（NSCLC）. MethodBruceoside B was co-incubated with the human gut microbiota under anoxic conditions in vitro， and ultra high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry（UPLC-Q-TOF-MS） was used to analyze the metabolic transformation products. Cell counting kit-8（CCK-8） assay was performed to determine the effects of bruceoside B and its metabolites on the proliferation of human lung cancer A549 cells and the half inhibitory concentration（IC₅₀） was calculated. Five healthy male rats were gavaged with bruceoside B（2 mg·kg^-1） for 7 days after adaptive feeding. The feces of rats were collected before and after administration. 16S rRNA sequencing was used to assess gut microbiota. ResultBruceoside B was mainly metabolized to brusatol by human gut microbiota， the IC₅₀ of bruceoside B and the conversion product to A549 cells were 1 755.50， 19.57 μmol·L^-1， respectively， and the conversion product had a better activity at inhibiting A549 cells proliferation than bruceoside B. Additionally， The results of intestinal flora analysis showed no significant differences in α diversity and β diversity of gut microbiota after administration. In terms of species abundance， at the phylum level， bruceoside B decreased the relative abundance of Actinobacteriota and Proteobacteria， increased the relative abundance of Firmicutes， Patescibacteria and Cyanobacteria. At the genus level， bruceoside B decreased the relative abundance of Staphylococcus， Aerococcus and Psychrobacter， increased the relative abundance of Romboutsia， Lactobacillus， Clostridium sensu stricto 1， Norank-f-norank-o-Clostridia-UCG-014， Turicibacter， Allobaculum and Candidatus Saccharimonas. The results of functional prediction showed that the gut microbiota functional compositions were relatively stable. ConclusionBruceoside B can be deglycosylated by intestinal flora and converted into brusatol， with a significant increase in antitumor activity. The administration of bruceoside B will not cause significant changes in the structure and function of the intestinal flora， resulting in intestinal microecological balance disorders， and the administration appears to be beneficial to the intestinal flora of NSCLC patients.

AIM:To study whether glycyrrhizic acid(GL)can resist the ototoxicity of cisplatin(CDDP)in mice and its molecular mechanism.METHODS:Male C57BL/6J mice were divided into 5 groups:control group,DMSO(5%)group,CDDP(4 mg/kg)group,CDDP+low-dose(50 mg/kg)GL group,and CDDP+high-dose(100 mg/kg)GL group(n=14).Auditory brainstem response(ABR)was used to detect hearing changes of mice.HE staining was used to observe the morphological change of cochlear stria vascular in mice.Evans blue(EB)staining was used to observe the per-meability change of the blood-labyrinth barrier(BLB).Immunohistochemical technique was used to detect the expression and distribution of adhesion protein VE-cadherin and tight junction protein ZO-1 on the cochlear stria.ELISA assay and immunofluorescence technology were employed to detect the expression of tumor necrosis factor-α(TNF-α)and interleu-kin-1β(1L-1β).RESULTS:In CDDP group,ABR waveforms of all frequencies were disturbed,the hearing threshold was significantly increased,and I wave latency was prolonged(P＜0.05).In CDDP+GL group,ABR waveforms of various frequencies were well differentiated,the hearing threshold was significantly decreased,and the latency of I-wave was shortened(P＜0.01).The disordered morphology and more vacuoles in the stria vascularis were observed by HE staining in CDDP group.The GL alleviated CDDP-induced damage in the stria vascularis.In EB staining,CDDP caused an increase in per-meability of BLB(P＜0.01),which was improved by GL treatment(P＜0.01).Immunohistochemical results showed that the expression of VE-cadherin and ZO-1 in CDDP group were decreased(P＜0.01),which was restored in CDDP+GL group(P＜0.01).The ELISA and immunofluorescence results showed that the expression of IL-1β and TNF-α was in-creased after CDDP treatment(P＜0.01),which was restored in CDDP+GL group(P＜0.01).CONCLUSION:The GL alleviates CDDP-induced hearing loss in mice by inhibiting CDDP-induced inflammation and reducing the permeability of BLB.

BACKGROUND:A pathophysiological feature of septic organ failure is endothelial dysfunction in sepsis(EDS).The physiological and pathological mechanism of sepsis is considered to be vascular leakage caused by endothelial dysfunction.These pathological changes lead to systemic organ injury.However,an analysis using bibliometric methods has not yet been conducted in the field of EDS.This study was conducted to provide an overview of knowledge structure and research trends in the field of EDS. METHODS:Based on previous research,a literature search was performed in the Web of Science Core Collection(WoSCC)for publications associated with EDS published between the year 2003 and 2023.Various types of data from the publications,such as citation frequency,authorship,keywords and highly cited articles,were extracted.The"Create Citation Report"feature in the WoSCC was employed to calculate the Hirsch index(h-index)and average citations per item(ACI)of authors,institutions,and countries.To conduct bibliometric and visualization analyses,three bibliometric tools were used,including R-bibliometrix,CiteSpace(co-citation analysis of references),and VOSviewer(co-authorship analysis of institutions,co-authorship analysis of authors,co-occurrence analysis of keywords). RESULTS:After excluding invalid records,the study finaly included 4,536 publications with 135,386 citations.Most of these publications originated in the USA,China,Germany,Canada,and Japan.Harvard University emerged as the most prolific institution,while professor Jong-Sup Bae and his research team at Kyungpook National University emerged as authors with the greatest influence.The"protein C","tissue factor","thrombin","glycocalyx","acute kidney injury","syndecan-1"and"biomarker"were identified as prominent areas of research.Future research may focus on molecular mechanisms(such as as vascular endothelial[VE]-cadherin regulation)and therapeutic interventions to enhance endothelial repair and function. CONCLUSION:Our findings show a growing interest in EDS research.Key areas for future research include signaling pathways,molecular mechanisms,endothelial repair,and interactions between endothelial cells and other cell types in sepsis.

Background::Pathological scars are a disorder that can lead to various cosmetic, psychological, and functional problems, and no effective assessment methods are currently available. Assessment and treatment of pathological scars are based on cutaneous manifestations. A two-photon microscope (TPM) with the potential for real-time non-invasive assessment may help determine the under-surface pathophysiological conditions in vivo. This study used a portable handheld TPM to image epidermal cells and dermal collagen structures in pathological scars and normal skin in vivo to evaluate the effectiveness of treatment in scar patients. Methods::Fifteen patients with pathological scars and three healthy controls were recruited. Imaging was performed using a portable handheld TPM. Five indexes were extracted from two dimensional (2D) and three dimensional (3D) perspectives, including collagen depth, dermo-epidermal junction (DEJ) contour ratio, thickness, orientation, and occupation (proportion of collagen fibers in the field of view) of collagen. Two depth-dependent indexes were computed through the 3D second harmonic generation image and three morphology-related indexes from the 2D images. We assessed index differences between scar and normal skin and changes before and after treatment.Results::Pathological scars and normal skin differed markedly regarding the epidermal morphological structure and the spectral characteristics of collagen fibers. Five indexes were employed to distinguish between normal skin and scar tissue. Statistically significant differences were found in average depth ( t = 9.917, P <0.001), thickness ( t = 4.037, P <0.001), occupation ( t= 2.169, P <0.050), orientation of collagen ( t = 3.669, P <0.001), and the DEJ contour ratio ( t = 5.105, P <0.001). Conclusions::Use of portable handheld TPM can distinguish collagen from skin tissues; thus, it is more suitable for scar imaging than reflectance confocal microscopy. Thus, a TPM may be an auxiliary tool for scar treatment selection and assessing treatment efficacy.

AIM:This study aimed to explore the induction of ferroptosis in non-small-cell lung cancer(NSCLC)cells by tubeimoside II(TBMS II)and to elucidate the underlying molecular mechanisms.METHODS:H460 NSCLC cells were cultured in vitro.Cell survival rates were assessed by using MTT assays,and doses of TBMS II resulting in below 50%survival were selected for further experimentation.Cell migration was evaluated using Transwell assays and the effects of TBMS II on H460 cell proliferation were assessed by colony formation assays.Flow cytometry and fluores-cence microscopy were used to assess changes in lipid peroxidation(lipid ROS),and the levels of GSH,T-AOC,MDA,and Fe2+were measured using commercial kits.Protein levels of GPX4,SLC7A11,FTH1,NCOA4,P62,and LC3 were examined using Western blot.Changes in mitochondrial structure were detected by transmission electron microscopy,and immunofluorescence was used to assess LC3 co-localization of FTH1 and NCOA4,as well as co-localization of LC3 and NCOA4 with lysosomes.RESULTS:Compared with the control group,TBMS II dose-dependently reduced H460 cell via-bility,migration,and clone formation,accompanied by the appearance of vacuoles within the cells.TBMS II treatment al-so led to decreased GSH and T-AOC levels,while increasing the cellular contents of MDA,indicating oxidative stress.Ad-ditionally,there was a decrease in the expression of the antioxidant proteins SLC7A11 and GPX4 in the cells,while lipid ROS and Fe2+levels were increased in proportion to the TBMS II concentration.The ferroptosis inhibitor ferrostatin-1 re-versed cell death caused by TBMS II,suggesting ferroptosis induction.Furthermore,increasing the TBMS II concentra-tion resulted in an upregulation of the autophagy marker proteins LC3 II/LC3 I and P62,indicative of increased autopha-gy.TBMS II also affected mitochondrial morphology in the cells,as seen in reduced mitochondrial fluorescence intensity.Protein expression of NCOA4 increased with higher TBMS II concentrations,while that of FTH1 decreased.Co-localiza-tion of LC3 II with FTH1 and NCOA4,as well as the lysosomal association of LC3 II and FTH1,also increased in a dose-dependent manner.CONCLUSION:TBMS II induces ferritinophagy in H460 cells,leading to decreased cell viability and increased ferroptosis.

Objective:To investigate the protective effect of ulinastatin on sepsis-acute kidney injury (SA-AKI) by NF-κB signaling pathway.Methods:Total of 60 mice were randomly(random number) divided into sham group, cecal ligation puncture group (CLP group) and ulinastatin treatment group (CLP+UTI group). Ulinastatin treatment group was intraperitoneally injected with ulinastatin 50 000 U/kg once a day. 24 hours after operation, five mice were sacrificed, the kidney tissues were collected to observe renal histopathology by HE staining. The macrophage infiltration was observed by immunohistochemistry. The remaining mice in each group were used to calculate the survival rate of 7-day after operation. HK-2 cells were stimulated by LPS to obtain the SA-AKI model, and the cells were divided into control group, LPS group and LPS + UTI group. CCK-8 assay was used to detect cell viability, EdU assay was used to detect cell proliferation, and JC-1 assay was used to detect mitochondrial damage. The phosphorylation degree of NF-κB was detected by western blot. Inflammatory factors concentrations of cellular supernatant were detected by ELISA assay.Results:Compared with the sham group, the kidney tissue of mice in CLP group showed that kidney pathological obvious changed, the infiltration of macrophages increased, and the survival rate of mice decreased. CLP+ UTI group reduced the pathological changes and the infiltration of macrophages, improved the survival rate of mice. Compared with control group, LPS group obviously inhibited the cells activity and proliferation of HK-2 cells, damaged the mitochondrial membrane potential of HK-2 cells. Compared with LPS group, LPS+ UTI group attenuated the phosphorylation of NF-κB, decreased the secretion of inflammatory factors, rescued the activity and proliferation of HK-2 cells, and reduced the damage of HK-2 mitochondrial membrane potential.Conclusions:Ulinastatin can reduce mitochondrial damage, inhibit the secretion of inflammatory factors and improve the function of renal tubular epithelial cells through regulating NF-κB signaling pathway.

Objective:To construct the "Internet +" peripherally inserted central catheter (PICC) home nursing service quality evaluation index system, so as to provide objective basis for standardizing the "Internet +" PICC home nursing service quality evaluation.Methods:Based on the three-dimensional quality structure model of "structure-process-outcome", combined with literature research, semi-structured interviews, group discussions and Delphi method, 20 experts who were engaged in intravenous therapy nursing and had management or practical experience in "Internet + nursing services" were selected for two rounds of consultation to determine the "Internet?+" PICC home nursing service quality evaluation index system. The positive coefficient of experts was expressed by the effective response rate of the questionnaire and the rate of expert opinion submission. The degree of expert authority was expressed by the expert authority coefficient. The coordination degree of expert opinions was represented by the Kendall harmony coefficient. The concentration degree of expert opinions was usually expressed in terms of the importance assigned to the indicator, the coefficient of variation, and the full score rate (%) .Results:The effective response rates of the two rounds of consultation questionnaires were all 100%, with expert authority coefficients of 0.793 and 0.848 respectively. The Kendall coordination coefficients of expert opinions were 0.202-0.216 and 0.222-0.270 respectively ( P<0.05). After the second round of expert consultation, the mean importance assigned to all indicators was greater than 4.0, the coefficient of variation was less than 0.2, and the full score rate was greater than 20%. The final "Internet +"PICC home nursing service quality evaluation index system included 3 first-level indicators, 12 second-level indicators and 64 third-level indicators. Conclusions:The "Internet + " PICC home nursing service quality evaluation index system constructed is scientific, reliable and practical, and reflects the specialty characteristics. It can effectively evaluate the "Internet + " PICC home nursing service quality and provide guidance for continuous improvement.

BACKGROUND: Systemic lupus erythematosus (SLE) is a complex autoimmune disease, and the mechanism of SLE is yet to be fully elucidated. The aim of this study was to explore the role of two-pore segment channel 2 (TPCN2) in SLE pathogenesis. METHODS: Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to detect the expression of TPCN2 in SLE. We performed a loss-of-function assay by lentiviral construct in Jurkat and THP-1 cell. Knockdown of TPCN2 were confirmed at the RNA level by qRT-PCR and protein level by Western blotting. Cell Count Kit-8 and flow cytometry were used to analyze the cell proliferation, apoptosis, and cell cycle of TPCN2-deficient cells. In addition, gene expression profile of TPCN2-deficient cells was analyzed by RNA sequencing (RNA-seq). RESULTS: TPCN2 knockdown with short hairpin RNA (shRNA)-mediated lentiviruses inhibited cell proliferation, and induced apoptosis and cell-cycle arrest of G2/M phase in both Jurkat and THP-1 cells. We analyzed the transcriptome of knockdown-TPCN2-Jurkat cells, and screened the differential genes, which were enriched for the G2/M checkpoint, complement, and interleukin-6-Janus kinase-signal transducer and activator of transcription pathways, as well as changes in levels of forkhead box O, phosphatidylinositol 3-kinase/protein kinase B/mechanistic target of rapamycin, and T cell receptor pathways; moreover, TPCN2 significantly influenced cellular processes and biological regulation. CONCLUSION TPCN2 might be a potential protective factor against SLE.
Apoptosis/genetics* ; Cell Division ; Humans ; Jurkat Cells ; Lupus Erythematosus, Systemic/genetics* ; RNA, Small Interfering/genetics*