Cats, owing to their physiological and immunological similarities with humans, have become increasingly valuable as model animals in virology research, drug development, and vaccine evaluation. They are irreplaceable in studies of feline immunodeficiency virus, feline coronavirus, and other related pathogens. However, cats are temperamentally sensitive, exhibit strong stress responses, and possess well-developed nervous systems as well as sharp claws and teeth. Consequently, the biosafety risks associated with infectious experiments using cats in animal biosafety level 2 laboratory (ABSL-2) are significantly higher than those encountered with conventional rodents. Drawing on long-term ABSL-2 operational experience, this article systematically reviews the entire workflow of infectious experiments in laboratory cats — from animal selection, pre-entry preparation, reception and quarantine, housing management, to infectious experimental procedures and incident response — identifying and addressing critical risk points at each stage. For strain selection, SPF-grade shorthair cats with defined genetic backgrounds and docile temperaments are recommended; sex and age should be scientifically matched to experimental objectives. During pre-entry preparation, emphasis is placed on dual-credential personnel management, health surveillance, standardized disinfection of environments and cages, feed and water standards, and robust record-keeping. During reception and quarantine, standardized protocols are established for transport control, appearance inspection, isolation quarantine, pathogen exclusion, and positive-reinforcement training. During infectious experimentation, a "three-fixed" husbandry principle is clearly implemented: dedicated caretakers, fixed feeding/cleaning times, and fixed cage positions. Disinfectant selection, autoclaving of waste, and daily veterinary rounds are rigorously enforced. Operational risk control includes detailed measures for graded personal protection, animal anesthesia and restraint, zoned operation within biosafety cabinets, and disposal of experimental waste. Contingency plans are formulated to address animal death, escape, personnel exposure, and spills of infectious materials. This study provides a reproducible and scalable technical pathway and operational standard for conducting infectious experiments in laboratory cats in ABSL-2 laboratories, offering a reference for other facilities undertaking similar work.

ObjectiveTo prepare rabbit anti-porcine inhibin polypeptide-keyhole limpet hemocyanin(KLH) conjugated polyclonal antibody and evaluate its effect on superovulation in C57BL/6J mice. MethodsNew Zealand white rabbits were immunized with a synthesized porcine inhibin polypeptide conjugated with KLH to produce anti-inhibin serum (AIS, i.e., inhibin polyclonal antibody). Female C57BL/6J mice received intraperitoneal injections of purified AIS in combination with pregnant mare serum gonadotropin (PMSG), followed by human chorionic gonadotropin (hCG) after 48 hours to induce superovulation. Oocytes obtained from superovulation were collected and counted 15 hours post-hCG administration, and the number of 2-cell embryos was assessed 24 hours after in vitro fertilization. ResultsAIS prepared by immunizing New Zealand White rabbits with KLH-conjugated porcine inhibin polypeptide was subjected to titer determination by indirect ELISA, showing titers reaching 1∶ 512 000. SDS-polyacrylamide gel electrophoresis analysis of ammonium sulfate-purified AIS revealed distinct 50 kDa and 25 kDa bands corresponding to the theoretical molecular weights of IgG antibody heavy and light chains, confirming successful production of porcine inhibin polyclonal antibody. Compared with conventional superovulation methods, AIS diluted 10-fold combined with PMSG significantly increased the number of oocytes obtained from superovulation in mice (P<0.05) by approximately 1.5-fold. ConclusionPorcine inhibin polyclonal antibody, as an improved superovulation reagent, can improve superovulation efficiency in C57BL/6J mice, and shows promising prospects for future applications.

Objective To evaluate the efficacy and safety of recombinant staphylokinase in patients with acute ST-segment elevation myocardial infarction(STEMI)by a multi-center,randomized,position-controlled,parallel post-marketing clinical trial.Methods This study was a multi-center,randomized,positive drug parallel control,non-inferiority clinical trial.From July 2019 to June 2022,a total of 251 patients with STEMI were enrolled in 31 hospitals.Patients were randomly assigned to receive intravenous staphylokinase or alteplase in a ratio of 1∶1.Vascular recanalization was evaluated by clinical indicators 30 minutes,60 minutes and 120 minutes after the initiation of thrombolysis.Coronary angiography was performed 90 to 120 minutes after the initiation of thrombolysis.The proportion of infarct-related artery(IRA)with thrombolysis in myocardial infarction(TIMI)grade Ⅱ and Ⅲ,corrected TIMI frame count(CTFC)and TIMI myocardial perfusion grade(TMPG)were analyzed Major adverse cardiac events(MACE,including all-cause death,rehospitalization,reinfarction,urgent target vessel revascularization)and bleeding events were followed up at 30 days(±2 days)after thrombolysis.Results After excluding 7 subjects who did not use thrombolytic drugs,244 subjects were finally eligibled from 31 hospitals(117 in trial group and 127 in control group),and 232 subjects completed the follow-up(111 in trial group and 121 in control group).The vascular recanalization rate evaluated by clinical indicators at 120 minutes after thrombolysis was 85.6％ in trial group and 83.5％ in control group(P=0.657).The difference between the two groups was 2.11(95％CI-7.19-11.41).Given that the lower confidence limit of the 95％CI was greater than-12％,the non-inferiority of the vascular recanalization rate was established based on clinical judgment.Coronary angiography showed that the total patency rate of IRA(TIMIⅡ-Ⅲ)was 77.5％ in trial group and 77.7％ in control group(P=0.970).The difference between the two groups was-0.21(95％CI-10.95-10.54),with the lower bound of the 95％CI exceeding-12％.Therefore,the non-inferiority of the TIMI blood flow grade was confirmed,indicating that the total patency rate of IRA in the trial group was not inferior to that in the control group.The CTFC was(32.7±17.6)frames in trial group and(37.6±16.6)frames in control group,with no statistically significant difference between the two groups(P=0.054).The difference between the two groups was-4.9(95％CI-10.0-0.1).As the lower limit of the 95％CI exceeded-12％,the noninferiority of CTFC was successfully demonstrated.The proportions of TMPG 0-Ⅲ were 20.7％,6.3％,2.7％and 69.4％in trial group,and 22.3％,4.1％,6.6％ and 66.9％ in control group,respectively.There was no significant difference in TIMI myocardial perfusion grade between the two groups(P=0.086).The incidence of MACE was 7.7％ in trial group and 7.1％ in control group within 30 days after the initiation of thrombolysis,and there was no significant difference between the two groups(P=0.857).Further analysis showed that there was no significant difference in cardiovascular mortality(3.4％ vs.4.7％,P=0.751).All 244 subjects were included in the safety analysis set.There was no significant difference in the total incidence of bleeding events between the two groups(22.2％ vs.15.0％,P=0.144).There was no significant difference in the incidence of major bleeding(1.7％ vs.0.8％,P=0.609).Conclusions Recombinant staphylokinase is simple to use and has a rapid onset of action.The efficacy and safety of recombinant staphylokinase are not inferior to alteplase in the treatment of acute STEMI.

Aim To investigate the impact of G pro-tein-coupled estrogen receptor 1(GPER1),also known as GPR30 playing a significant role in the nerv-ous system,on neuronal damage and cognitive dysfunc-tion following epileptic seizures.Methods The pro-tein expression levels of GPER1 and the DNA damage marker γ-H2AX in epileptic rats were assessed using Western blot.The hippocampal neuronal damage and apoptosis in pilocarpine-induced epilepsy models were evaluated using Nissl and TUNEL staining techniques,compared with GPER1 knockdown(GPER1-KD)rats with wild-type(WT)controls.The behavioral activi-ties,including memory and spatial learning,were mo-nitored during the chronic phase of epilepsy using the IntelliCage system.Results Compared to the control group,GPER1 protein expression in the cerebral cortex and hippocampus significantly increased 24 hours post-epilepsy onset.In the GPER1-KD+EP group,hipp-ocampal neuronal damage was more severe,with a sig-nificant increase in apoptotic neurons compared to the WT+EP group.The IntelliCage data revealed that during free exploration,nose contact,position learn-ing,and reverse position learning stages in the GPER1-KD+EP group exhibited fewer visits and a higher error rate than in the WT+EP group.Conclu-sions Deficiency in GPER1 impairs memory and spa-tial learning abilities following epilepsy,potentially due to exacerbated neuronal injury,apoptosis,and inflam-mation.GPER1 represents a promising therapeutic tar-get for mitigating post-epileptic nerve damage and cog-nitive impairment.

Objective:To investigate the effect and mechanism of injectable photopolymerizable porous gelatin methacrylate anhydride (Porous GelMA)/methacrylated silk fibroin (SilMA) composite hydrogel (PSE) loaded with human umbilical cord mesenchymal stem cell-derived exosomes (hUCMSC-Exos) in promoting knee joint cartilage regeneration.Methods:The porous GelMA solution (60 g/L) was mixed with SilMA solution (200 g/L) at a volume ratio of 6∶1 . The mixture was ultraviolet-irradiated for 30 seconds to form a cured Porous GelMA/SilMA hydrogel (P/S6). The hUCMSC-Exos was isolated via differential centrifugation coupled with ultrafiltration and then was incorporated into the Porous GelMA/SilMA composite solution at 200 μg/ml, followed by ultraviolet irradiation for 30 seconds to generate Exos-loaded PSE. Primary rat chondrocytes (P1) were divided into control group, P/S6 group, and PSE group to characterize the porosity, compressive strength, and sustained exosome release kinetics of PSE hydrogel. Chondrocytes were allocated to control group, interleukin-1β (IL-1β) group, P/S6 group, and PSE group, among which the last three groups were preconditioned with 10 ng/ml IL-1β for 24 hours, and then cultured in complete medium, P/S6 extract and PSE extract for 3 days, respectively, to establish in vitro cartilage defect models, while the control group remained untreated. Western blot and qRT-PCR analysis were conducted to quantify the expression levels of antibody to aggrecan core protein (ACAN), sex-determining region Y-box transcription factor 9 (SOX9), matrix metalloproteinase-13 (MMP13) and collagen type II (COL II). Murine monocyte-macrophage leukemia cells (RAW264.7) were divided into control group, P/S6 group, and PSE group, which were then cultured in complete medium, PSE extract, and PSE extract medium for 3 days, respectively. qRT-PCR was employed to detect the expression levels of recombinant arginase-1 protein (ARG1), mannose receptor (CD206), and inducible nitric oxide synthase (iNOS). Transcriptomic sequencing was used to identify differentially expressed genes during PSE-mediated chondrocyte regeneration, followed by functional enrichment analysis of key signaling pathways. Twenty-four SD rats were selected to establish cartilage defect models and assigned to injury control group, P/S6 group, and PSE group according to the random number table (8 rats per group). The right knee joints of the rats were surgically exposed, and cylindrical osteochondral defects (a diameter of 2.0 mm× a depth of 1.0 mm) were surgically created in the center of the femoral trochlear groove using a drill bit. The injury control group received phosphate-buffered saline, while the P/S6 group and PSE group were injected with corresponding hydrogels followed by photo-crosslinking. Incisions then were closed in layers. At 6 and 10 weeks after injury, specimens were harvested for HE staining and safranin O-fast green staining to evaluate cartilage regeneration and immunohistochemistry staining to quantify the positive area fractions for COL II, MMP13, ARG1, and CD206 in the defect areas. Results:PSE hydrogel exhibited compressive strength matching native cartilage (0.41 MPa), high porosity (85%), and sustained exosome release capacity (cumulative release rate of approximately 85% over 14 days). In chondrocyte repair experiments, compared to the IL-1β group, the PSE group demonstrated significantly upregulated expression of anabolic markers of cartilage (COL II expression increased by 2.1-fold, ACAN by 1.8-fold, and SOX9 by 1.5-fold) ( P<0.01) as well as significantly suppressed expression of catabolic markers (MMP13 expression decreased by 52%) ( P<0.01). In macrophage polarization assays, the PSE group exhibited ARG1 expression increased by 68% when compared to the control group ( P<0.01), thus promoting M2 polarization of macrophages. Transcriptomic analysis revealed that PSE enhanced extracellular matrix (ECM) synthesis by activating the phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) pathway and ECM-receptor interaction pathway, as well as by suppressing inflammation-related gene expression. Histological evaluation in animal experiments revealed regeneration of hyaline cartilage with smooth, continuous surfaces in the defect areas in the PSE group. At 10 weeks after surgery, the neocartilage-positive area in the PSE group was (9.94±0.26)%, significantly larger than (1.67±0.11)% in the injury control group ( P<0.01). Besides, the CD206? M2 macrophage-positive area reached (14.44±0.23)% in the PSE group, significantly larger than (3.41±0.36)% in the injury control group ( P<0.01). Conclusions:The PSE hydrogel successfully engineered in the study can significantly promote regenerative repair of knee cartilage defects through a dual mechanism of enhanced ECM anabolism and remodeled inflammatory microenvironment. The core mechanisms involve specific activation of the PI3K/Akt pathway (boosting chondrocyte proliferation and survival) and ECM-receptor interaction pathway (driving ECM synthesis and assembly) by exosome-loaded PSE, while effectively polarizing macrophages toward an anti-inflammatory M2 phenotype so as to coordinately regulate cartilage ECM metabolism and suppress inflammatory responses.

Objective:To investigate the effect and mechanism of injectable photopolymerizable porous gelatin methacrylate anhydride (Porous GelMA)/methacrylated silk fibroin (SilMA) composite hydrogel (PSE) loaded with human umbilical cord mesenchymal stem cell-derived exosomes (hUCMSC-Exos) in promoting knee joint cartilage regeneration.Methods:The porous GelMA solution (60 g/L) was mixed with SilMA solution (200 g/L) at a volume ratio of 6∶1 . The mixture was ultraviolet-irradiated for 30 seconds to form a cured Porous GelMA/SilMA hydrogel (P/S6). The hUCMSC-Exos was isolated via differential centrifugation coupled with ultrafiltration and then was incorporated into the Porous GelMA/SilMA composite solution at 200 μg/ml, followed by ultraviolet irradiation for 30 seconds to generate Exos-loaded PSE. Primary rat chondrocytes (P1) were divided into control group, P/S6 group, and PSE group to characterize the porosity, compressive strength, and sustained exosome release kinetics of PSE hydrogel. Chondrocytes were allocated to control group, interleukin-1β (IL-1β) group, P/S6 group, and PSE group, among which the last three groups were preconditioned with 10 ng/ml IL-1β for 24 hours, and then cultured in complete medium, P/S6 extract and PSE extract for 3 days, respectively, to establish in vitro cartilage defect models, while the control group remained untreated. Western blot and qRT-PCR analysis were conducted to quantify the expression levels of antibody to aggrecan core protein (ACAN), sex-determining region Y-box transcription factor 9 (SOX9), matrix metalloproteinase-13 (MMP13) and collagen type II (COL II). Murine monocyte-macrophage leukemia cells (RAW264.7) were divided into control group, P/S6 group, and PSE group, which were then cultured in complete medium, PSE extract, and PSE extract medium for 3 days, respectively. qRT-PCR was employed to detect the expression levels of recombinant arginase-1 protein (ARG1), mannose receptor (CD206), and inducible nitric oxide synthase (iNOS). Transcriptomic sequencing was used to identify differentially expressed genes during PSE-mediated chondrocyte regeneration, followed by functional enrichment analysis of key signaling pathways. Twenty-four SD rats were selected to establish cartilage defect models and assigned to injury control group, P/S6 group, and PSE group according to the random number table (8 rats per group). The right knee joints of the rats were surgically exposed, and cylindrical osteochondral defects (a diameter of 2.0 mm× a depth of 1.0 mm) were surgically created in the center of the femoral trochlear groove using a drill bit. The injury control group received phosphate-buffered saline, while the P/S6 group and PSE group were injected with corresponding hydrogels followed by photo-crosslinking. Incisions then were closed in layers. At 6 and 10 weeks after injury, specimens were harvested for HE staining and safranin O-fast green staining to evaluate cartilage regeneration and immunohistochemistry staining to quantify the positive area fractions for COL II, MMP13, ARG1, and CD206 in the defect areas. Results:PSE hydrogel exhibited compressive strength matching native cartilage (0.41 MPa), high porosity (85%), and sustained exosome release capacity (cumulative release rate of approximately 85% over 14 days). In chondrocyte repair experiments, compared to the IL-1β group, the PSE group demonstrated significantly upregulated expression of anabolic markers of cartilage (COL II expression increased by 2.1-fold, ACAN by 1.8-fold, and SOX9 by 1.5-fold) ( P<0.01) as well as significantly suppressed expression of catabolic markers (MMP13 expression decreased by 52%) ( P<0.01). In macrophage polarization assays, the PSE group exhibited ARG1 expression increased by 68% when compared to the control group ( P<0.01), thus promoting M2 polarization of macrophages. Transcriptomic analysis revealed that PSE enhanced extracellular matrix (ECM) synthesis by activating the phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) pathway and ECM-receptor interaction pathway, as well as by suppressing inflammation-related gene expression. Histological evaluation in animal experiments revealed regeneration of hyaline cartilage with smooth, continuous surfaces in the defect areas in the PSE group. At 10 weeks after surgery, the neocartilage-positive area in the PSE group was (9.94±0.26)%, significantly larger than (1.67±0.11)% in the injury control group ( P<0.01). Besides, the CD206? M2 macrophage-positive area reached (14.44±0.23)% in the PSE group, significantly larger than (3.41±0.36)% in the injury control group ( P<0.01). Conclusions:The PSE hydrogel successfully engineered in the study can significantly promote regenerative repair of knee cartilage defects through a dual mechanism of enhanced ECM anabolism and remodeled inflammatory microenvironment. The core mechanisms involve specific activation of the PI3K/Akt pathway (boosting chondrocyte proliferation and survival) and ECM-receptor interaction pathway (driving ECM synthesis and assembly) by exosome-loaded PSE, while effectively polarizing macrophages toward an anti-inflammatory M2 phenotype so as to coordinately regulate cartilage ECM metabolism and suppress inflammatory responses.

Objective:To explore the effect of C1q like protein 4(C1ql4)on migration and invasion of breast cancer and its mechanism.Methods:Real-time fluorescence quantitative PCR was used to detect the expression level of C1ql4 mRNA in tumor tissues and paracancerous tissues from 50 cases of breast cancer,as well as the breast cells including T549 and MCF-7 breast cancer cells,and MCF-10A normal breast cells.The siRNA targeting C1ql4 gene(siC1ql4)was synthesized and transfected into BT549 breast cancer cells by liposome transfection,and the silencing efficiency was detected by Western blotting.MTT assay was used to detect the proliferative ability of the cells in each group,the invasive ability of the cells was detected by Transwell assay,and the migratory ability of the cells was detected by scratch healing assay.Western blotting was used to detect the proliferative ability of the cells in each group.The expression levels of Snail,Slug,nuclear factor-κB(NF-κB)and phospho-NF-κB(p-NF-κB)were detected.The immunofluorescence was used to localize the distribution of NF-κB.Real-time fluorescence PCR was used to detect the mRNA expression levels of the NF-κB target genes TNF-α and IL-1β.Results:The expression level of C1ql4 mRNA in breast cancer tissues was significantly higher than that in paracancerous tissues,and the expression level of C1ql4 mRNA in BT549 and MCF-7 breast cancer cells was significantly higher than that in MCF-10A normal breast cells(P<0.05).After silencing C1ql4 expression,the proliferation,invasion and migration abilities of BT549 cells were significantly decreased(all P<0.001);the mRNA and prorein expression levels of Snail and Slug were significantly down-regulated(both P<0.001),and the mRNA expression levels of TNFα and IL-1β were significantly reduced(both P<0.001).The amount of NF-κB entering the nucleus was reduced,and the ratio of p-NF-κB to NF-κB was reduced(both P<0.05).Conclusion:The C1ql4 gene may regulate migration and invasion of breast cancer cells and promote breast cancer progression through the NF-κB pathway.

AIM To study the chemical constituents from Euphorbia humifusa Willd.and their in vitro anti-hepatoma activity.METHODS Silica gel,D101 macroporous adsorption resin and semi-preparative RP-HPLC were used for isolated and purified,then the structures of obtained compounds were identified by physicochemical properties and spectral data.The anti-hepatocellular carcinoma activity was determined by MTT mothod.RESULTS Eighteen compounds were isolated and identified as 22-O-angeloyl-R1-barrigenol(1),dimethyl 3,3'-[oxybis(4,1-phenylene)](2E,2'E)-diacrylate(2),N-(3-methoxy-1,3-dioxopropyl)-D-tryptophan methyl ester(3),N-acetyltryptophan methyl ester(4),N-(methoxycarbonyl)-tryptophan methyl ester(5),(3β,5α,17β)-4,4,8,14-tetramethyl-18-norandrostane-3,17-diol(6),3β,18,19β-trihydroxylupane(7),pregnenolone(8),3-hydroxy-5,6-epoxy-7-megastigmen-9-one(9),dehydrovomifoliol(10),loliolide(11),2,2'-oxybis(1,4-di-tert-butylbenzene)(12),dibutyl phthalate(13),4-methoxycinnamic acid(14),3,4-dimethoxycinnamic acid(15),methyl 4-hydroxybenzoate(16),kaempferol(17),quercetin(18).The IC50 values of compounds 1,7 and 8 on HepG2 cells were(17.27±0.92),(19.11±2.14)and(7.53±1.09)μmol/L,respectively.CONCLUSION Compounds 1-16 are first isolated from this plant.Compounds 1,7 and 8 have anti-hepatoma activity.

Objective To study the spatial correlation characteristics of the number of large medical equipment alloca-tion in China's provinces and cities,with a view to providing a reference basis for the optimization of the allocation of large medical equipment.Methods It analyzed the spatial autocorrelation of China's large medical equipment alloca-tion and its influencing factors by using the Moran's I,grey correlation and other methods.Results The results of the spatial autocorrelation analysis showed that the allocation of large medical equipment in China was positively corre-lated spatially,and the spatial agglomeration was significant(Global Moran's I index value was 0.392,P＜0.05).The grey correlation of GDP,year-end resident population,geographic area,number of tertiary hospitals,number of beds in medical institutions,number of practicing(assistant)physicians,and annual discharges in each province and city were 0.913,0.919,0.745,0.883,0.907,0.929,0.880,respectively.Conclusion China's large-scale medi-cal equipment configuration of spatial autocorrelation properties,distributed in the eastern Beijing-Tianjin-Hebei,Yangtze River Delta region and some provinces and cities in the west of the large-scale medical equipment configura-tion level in the formation of high-value agglomeration and low-value agglomeration area respectively.At the same time,the equipment configuration has the characteristics of high-quality medical resources concentration,oriented to the needs of the masses,taking into account economic and social development,etc.It is recommended to fur-ther optimise the spatial structure of equipment allocation,incorporate the configuration of large-scale medical equip-ment into the consideration of regional health resource planning at all levels,and enhance the linkage of the equip-ment configuration planning between provinces and cities.

Objective:To investigate the effect of GBP3 on replication of adult T-lymphocytic leukemia virus type 1(HTLV-1).Methods:Expression of GBP3 in HTLV-1-infected HeLa cell and THP1 cell was detected by Western blot.The knock-down efficiency of siRNAs targeting GBP3 in HeLa and THP1 cells was evaluated by Western blot.Effects of GBP3 overexpression or knockdown on expression of HTLV-1 proviral transcripts Tax,px,HBZ,Gag,ENV,5'UTR,and viral proteins Tax,p19 were investigated by RT-qPCR and Western blot.GTPase-defective mutant of GBP3,GBP3K51A was constructed to explore whether the effects of GBP3 on HTLV-1 infection were dependent on its GTPase activity.Results:GBP3 expression was upregulated in HTLV-1 infected HeLa and THP1 cells.GBP3 overexpression decreased expression of HTLV-1 proviral transcripts and viral proteins,whereas the knockdown of GBP3 has the opposite effects.Overexpression of GBP3K51A increased expression of HTLV-1 proviral transcripts and viral proteins.Conclusion:HTLV-1 virus infection can induce expression of GBP3;overexpression of GBP3 inhibits virus replication and may depend on GTPase.