Objective To establish a rapid and accurate droplet digital PCR(ddPCR)detection method for detecting Staphylococcus aureus(SA)in laboratory animals and the environment.Methods Using the heat-stable nuclease gene(nuc)of SA as the target gene,a pair of specific primers and probes are designed within its conserved region.Optimize the reaction conditions,test the dynamic range,and evaluate the specificity and stability of the method.Using the same template,test reactions were performed with both ddPCR and real-time quantitative PCR(qPCR)method to assess the interchangeability between the two approaches.Finally,the method is applied to the detection of various clinical samples.Results The kinetic range of the established SA ddPCR method is 100～15 000 copies/μL,with a detection limit of 2.5 copies and a quantification limit of 10 copies;The specificity of this method was tested,and only SA showed positive droplets,while no positive droplets were found for other pathogens;After measuring three parallel samples,the standard deviation and relative standard deviation were calculated.It was found that within the dynamic detection interval of ddPCR,as the target copy number gradually decreased,the relative standard deviation showed an upward trend,but remained below 25％.This result indicates that the detection method has good stability.Conclusions The established ddPCR method for detecting SA has the advantages of high sensitivity,strong specificity,good stability,and good reproducibility.This method can be applied for the detection of SA in laboratory animals.

The intravitreal injection of anti-vascular endothelial growth factor (VEGF) agents is currently the first-line therapy for retinopathy of prematurity (ROP) in clinical practice.This therapy has several advantages, including ease of operation, rapid onset of action, and minimal adverse reactions.However, the high reactivation rate observed in clinical applications limits its utility.Understanding reactivation characteristics is crucial for patient management and clinical trial design.The International Classification of Retinopathy of Prematurity (Third edition) updated the definition of reactivation and related concepts.This article summarizes the clinical manifestations and mechanisms of ROP reactivation after anti-VEGF therapy in recent studies and analyzes high-risk factors associated with reactivation, including those related to the infant, the disease, and the medication.The goal is to provide guidance for the early identification, prevention, and intervention in clinical practice.Nevertheless, significant clinical controversies remain regarding the prevention and retreatment of ROP reactivation.Future research should accumulate more evidence-based medical data and conduct large-scale prospective studies to further standardize diagnostic and treatment processes.

Objective We aimed to investigate the effects of different non-surgical embryo transfer devices,number of transferred embryos,embryo stage,and embryos obtained from different mouse strains on the efficiency of non-surgical embryo transfer in mice,and to compare the efficiencies of surgical and non-surgical embryo transfer,in order to establish a stable non-surgical embryo transfer technology system.Methods Mouse embryo transfer was carried out using non-surgical method.Results The pregnancy rates using two different non-surgical transfer devices were(75.00±0.00)％and(66.67±14.43)％,and the birth rates were(46.11±6.31)％and(18.89±0.96)％,respectively.Transfer of 10,15,and 20 embryos resulted in pregnancy rates of(66.67±11.55)％,(80.00±0.00)％,and(66.67±23.09)％,and birth rates of(29.33±4.16)％,(38.67±4.81)％,and(17.00±3.46)％,respectively.When blastocysts and morulae were transferred non-surgically,the resulting pregnancy rates were(80.00±0.00)％and(46.67±11.55)％and the birth rates were(38.67±4.81)％and(10.22±2.77)％,respectively.Four strains(C57BL/6J,ICR,genetically modified mice A,genetically modified mice B)were used as donors for non-surgical embryo transfer,with resulting pregnancy rates of(66.67±11.55)％,(80.00±0.00)％,(73.33±11.55)％,and(80.00±0.00)％,and birthrates of(26.67±2.67)％,(38.67±4.81)％,(32.00±3.53)％,and(29.34±2.31)％,respectively.Fifteen pseudo-pregnant mice were transplanted surgically and 15 were transplanted non-surgically,with pregnancy rates of(80.00±0.00)％and(86.67±11.55)％,and birth rates of(38.67±4.81)％and(36.00±5.82)％,respectively.Conclusions Transfer device A resulted in a higher birth rate in this study.The embryo transfer efficiency was higher when 15 embryos were transferred into unilateral uterine horns of pseudo-pregnant 2.5-day recipients.Blastocyst-stage embryo transfer was more efficient than morula-stage transfer.There was no significant difference in efficiency between surgical and non-surgical embryo transfer procedures.

As an important and fundamental resource of scientific research,laboratory animals have become essential tools for the continuous advancements in life sciences,medical research,drug development,and other fields.With the development of related laws and regulations,the welfare of laboratory animals is increasingly valued by the general public and international research communities alike.To ensure the welfare of laboratory animals,the ethical review and conduct of laboratory animal practitioners should be standardized,incorporating and adapting advanced international practices with those in China.This article primarily outlines the process for reviewing and approving animal use protocols,along with the standards for evaluation,with the aim of providing a reference for researchers writing animal use protocols and for International Animal Care and Use Committee members conducting ethical reviews of laboratory animal welfare.

The intravitreal injection of anti-vascular endothelial growth factor (VEGF) agents is currently the first-line therapy for retinopathy of prematurity (ROP) in clinical practice.This therapy has several advantages, including ease of operation, rapid onset of action, and minimal adverse reactions.However, the high reactivation rate observed in clinical applications limits its utility.Understanding reactivation characteristics is crucial for patient management and clinical trial design.The International Classification of Retinopathy of Prematurity (Third edition) updated the definition of reactivation and related concepts.This article summarizes the clinical manifestations and mechanisms of ROP reactivation after anti-VEGF therapy in recent studies and analyzes high-risk factors associated with reactivation, including those related to the infant, the disease, and the medication.The goal is to provide guidance for the early identification, prevention, and intervention in clinical practice.Nevertheless, significant clinical controversies remain regarding the prevention and retreatment of ROP reactivation.Future research should accumulate more evidence-based medical data and conduct large-scale prospective studies to further standardize diagnostic and treatment processes.

OBJECTIVE: To observe the effect of electroacupuncture at "Sishencong" (EX-HN1) and "Fengchi" (GB20) on hippocampal neuronal ferritinophagy mediated by the nuclear receptor coactivator 4 (NCOA4)/ferritin heavy chain 1 (FTH1) signaling pathway in vascular dementia (VD) rats, and to explore the potential mechanisms of electroacupuncture for VD. METHODS: A total of 60 male rats of SPF grade were randomly divided into a blank group (12 rats), a sham surgery group (12 rats) and a modeling group (36 rats). In the modeling group, the modified 4-vessel occlusion method was used to establish the VD model. The 24 successfully modeled rats were randomly divided into a model group and an electroacupuncture group, with 12 rats in each group. In the electroacupuncture group, electroacupuncture was applied at left and right "Sishencong" (EX-HN1), and bilateral "Fengchi" (GB20), with continuous wave, in frequency of 2 Hz and current intensity of 1 mA, 30 min a time, once daily for 21 consecutive days. The learning and memory abilities were assessed using the Morris water maze test before modeling, after modeling and after intervention, as well as the novel object recognition test after intervention. After intervention, the neuronal morphology in the hippocampus was observed by Nissl staining; the iron deposition was observed by Prussian blue staining; the reactive oxygen species (ROS) level was detected by dihydroethidium (DHE) fluorescence staining; the levels of iron, malondialdehyde (MDA) and superoxide dismutase (SOD) in the hippocampal tissue were measured by the colorimetric assay, TBA method, and WST-1 method, respectively; the positive expression of NCOA4, FTH1 and glutathione peroxidase 4 (GPX4) was detected by immunohistochemistry; the protein expression of NCOA4, FTH1, GPX4, and the ratio of microtubule-associated protein 1 light chain 3B (LC3B) Ⅱ/Ⅰ in the hippocampus were detected by Western blot. RESULTS: Compared with the sham surgery group, in the model group, the escape latency was prolonged, and the number of platform crossings reduced (P<0.01), the recognition index (RI) was decreased (P<0.01); the hippocampal neurons displayed a blurred laminar structure, disorganized cellular arrangement, and the number of Nissl bodies was decreased (P<0.01); the percentage of iron deposition area in the hippocampus was increased (P<0.01); in the hippocampus, the levels of ROS, iron, MDA, and the protein expression of NCOA4, as well as the LC3B Ⅱ/Ⅰ ratio were increased (P<0.01), the SOD level, and the protein expression of FTH1 and GPX4 were decreased (P<0.01). Compared with the model group, in the electroacupuncture group, the escape latency was shortened and the number of platform crossings was increased (P<0.01), the RI was increased (P<0.01); the hippocampal neurons exhibited more regular morphology, better-organized cellular structure, and the number of Nissl bodies was increased (P<0.05); the percentage of iron deposition area in the hippocampus reduced (P<0.01); in the hippocampus, the levels of ROS, iron, MDA, and the protein expression of NCOA4, as well as the LC3B Ⅱ/Ⅰ ratio were decreased (P<0.01, P<0.05), the SOD level, and the protein expression of FTH1 and GPX4 were increased (P<0.01). CONCLUSION Electroacupuncture at "Sishencong" (EX-HN1) and "Fengchi" (GB20) can improve learning and memory abilities in VD rats, and its mechanism may be associated with the regulation of the hippocampal NCOA4/FTH1 signaling pathway, inhibition of ferritinophagy, and alleviation of oxidative stress damage.
Animals ; Electroacupuncture ; Dementia, Vascular/genetics* ; Male ; Rats ; Signal Transduction ; Humans ; Memory ; Rats, Sprague-Dawley ; Nuclear Receptor Coactivators/genetics* ; Ferritins/genetics* ; Learning ; Hippocampus/metabolism* ; Acupuncture Points

BACKGROUND: The choice of unicompartmental knee arthroplasty (UKA) vs . total knee arthroplasty (TKA) in the surgical treatment of knee osteoarthritis (KOA) remains controversial. This study aimed to perform a systematic review and meta-analysis of randomized controlled trials (RCTs) to compare the clinical results of UKA and TKA for treating unicompartmental KOA. METHODS: PubMed, Embase, and the Cochrane Library were systematically searched for articles published up to January 2, 2023. The literature was rigorously screened to include only RCTs comparing UKA and TKA for unicompartmental KOA. A systematic review and meta-analysis were performed to calculate the mean difference (MD), relative risk (RR), and 95% confidence interval (CI) according to the Cochrane standards. RESULTS: Thirteen publications involving 683 UKAs and 683 TKAs were analyzed. Except for one study with a follow-up period of 15 years, all outcome measures reported were within 5 years of follow-up. Meta-analysis showed better knee recovery (MD: 1.23; 95% CI: 1.01-1.45; P  <0.001), greater knee function (MD: 1.78; 95% CI: 0.34-3.22; P  = 0.020), less pain (MD: 0.75; 95% CI: 0.43-1.06; P  <0.001), and better health status (MD: 3.75; 95% CI: 0.81-6.69; P  = 0.010) after UKA than TKA. However, considering the minimal clinically important difference values for these variables, the findings were not clinically relevant. Moreover, UKA patients had fewer complications (RR: 0.59; 95% CI: 0.45-0.78; P  <0.001) and shorter hospital stays (MD: -0.89; 95% CI: -1.57 to -0.22; P  = 0.009) than did TKA patients. There were no statistically significant differences in terms of postoperative range of movement, revision, failure, operation time, and patient satisfaction. CONCLUSIONS In terms of clinical efficacy, there was no obvious advantage of UKA over TKA in the surgical treatment of knee OA when considering the minimal clinically important difference. The main advantage of UKA over TKA is that it leads to fewer complications and a shorter length of hospital stay. It is ideal to perform prospective studies with longer follow-up periods to fully evaluate the long-term efficacy and safety of the two procedures in the future.
Humans ; Arthroplasty, Replacement, Knee/methods* ; Osteoarthritis, Knee/surgery* ; Randomized Controlled Trials as Topic ; Treatment Outcome

Objective To explore the value of ZOOMit intravoxel incoherent motion diffusion weighted imaging(IVIM-DWI)technology in assessing renal function in diabetic kidney disease(DKD)and non-diabetic kidney disease(non-DKD).Methods Based on the estimated glomerular filtration rate(eGFR),50 DKD patients and 50 non-DKD patients were enrolled,with each group contained 21 mild cases(mild group)and 29 moderate to severe cases(moderate to severe group).All subjects underwent ZOOMit IVIM-DWI examination and the parameter values of the renal cortex and medulla were measured.The differences of IVIM-DWI parameters in renal cortex and medulla between DKD and non-DKD patients,and the correlation with eGFR,and diagnostic efficacy were compared.Results In the DKD patients,the f value of the medulla,the D value of the cortex,and the D* value of the medulla showed statistically significant differences between mild group and moderate to severe group(t=7.836,3.337,2.515,P<0.05),and all were positively correlated with eGFR(r=0.750,0.375,0.404,P<0.05).In the non-DKD patients,the f value of the medulla and the D* value of the medulla showed statistically significant differences between mild group and moderate to severe group(t=2.619,2.453,P<0.05),and were positively correlated with eGFR(r=0.425,0.360,P<0.05).In both DKD and non-DKD patients,the f value of the medulla showed the best efficacy in distinguishing between mild and moderate to severe DKD,with the area under the curve(AUC)=0.961(DeLong test P<0.05).Conclusion ZOOMit IVIM-DWI technology can be used for renal function in DKD and non-DKD,with the f value of the medulla being more suitable for the assessment of renal function in DKD.

OBJECTIVE To investigate the effects and mechanism of total alkaloids of Corydalis Rhizoma （TAC） on the regulation of cuproptosis in rats with diabetic cardiomyopathy （DCM） based on silence information regulator 1（Sirt1）/tumor protein 53（P53）signaling pathway. METHODS DCM rat model was induced by high-fat and high-sugar diet and intraperitoneal injection of streptozotocin. Thirty-two model rats were randomly divided into model group， TAC low-dose， medium-dose and high-dose groups （7， 10.5， 14 mg/kg）， with 8 rats in each group. An additional 8 rats were assigned to normal control group. Related drugs or normal saline were administered intragastrically in each group， once a day， for 4 weeks. After the last medication， the fasting blood glucose （FBG） levels of the rats were measured. The levels of myocardial creatine kinase （CK）， creatine kinase isoenzyme （CK-MB）， and lactate dehydrogenase （LDH） in serum and myocardial tissue of rats were all detected. The pathological morphology， fibrosis degree， and Cu2+ deposition of myocardial tissue in rats were observed. The levels of Cu2+ and glutathione （GSH） in myocardial tissue， the expressions of Sirt1/P53 signaling pathway-related proteins [Sirt1， P53， solute carrier family 7 membrane 11 （SLC7A11）]， and iron-sulfur cluster-related proteins [ferredoxin 1 （FDX1）， lipoic acid synthetase （LIAS）， aconitase 2 （ACO2）， NADH-ubiquinone oxidoreductase core subunit S8 （NDUFS8）， dihydrolipoamide acetyltransferase （DLAT）， dihydrolipoamide succinyltransferase （DLST）]， and heat shock protein 70 （HSP70） were all determined. RESULTS Compared with normal control group， the model group exhibited significantly elevated levels of FBG， CK， CK-MB and LDH in both serum and myocardial tissue， as well as increased 2+ levels of Cu in myocardial tissue and the expression of P53 and HSP70 proteins （P＜0.05）； the level of GSH and the expression levels of Sirt1， SLC7A11， FDX1， LIAS， ACO2， NDUFS8， DLAT， and DLST proteins in myocardial tissue were all significantly decreased （P＜0.05）； the myocardial tissue exhibited severe pathological damage， with numerous inflammatory cell infiltrations and significant fibrosis， as well as increased deposition of Cu2+. Compared with model group， most of the above quantitative indicators in rats were significantly reversed in TAC groups （P＜0.05）； the pathological damage to the myocardial tissue was alleviated， with reduced fibrosis and Cu2+ deposition. CONCLUSIONS TAC can ameliorate DCM in rats， and its mechanism of action may be related to activating the activity of the Sirt1/P53 signaling pathway， promoting the chelation of GSH with Cu2+， and inhibiting cuproptosis of cardiomyocyte.

BACKGROUND:The capability of repairing articular cartilage damage is very limited,and tissue engineering technology provides new therapeutic options for repairing damaged cartilage,in which the interaction and induction between chondrocytes,bone marrow mesenchymal stem cells,and synovial mesenchymal stem cells is the basis of autologous healing of cartilage damage. OBJECTIVE:To construct the chondrocyte-bone marrow mesenchymal stem cell-synovial mesenchymal stem cell co-culture system to simulate the in vitro microenvironment of chondrocytes,and to explore the optimal cell inoculation ratio,meanwhile to observe the effects of icariin-containing serum on the proliferation of chondrocytes and the chondrogenic differentiation of stem cells in the system. METHODS:Rat knee chondrocytes,bone marrow mesenchymal stem cells and synovial mesenchymal stem cells were extracted,cultured and identified,and a chondrocyte-bone marrow mesenchymal stem cell-synovial mesenchymal stem cell non-contact co-culture system was constructed according to different cell inoculation ratios.After 72 hours of co-culturing,the chondrocyte proliferative activity and phenotypic ability were observed,and the co-culture system with the best overall effect was selected.New Zealand white rabbits were gavaged with icariin solution(0.25 mg/mL)to prepare icariin-containing serum,and cultured in conventional complete medium(high sugar DMEM culture medium containing 10%fetal bovine serum and 1%double antibody by volume)as the control group,while the experimental group was intervened by adding 10%icariin-containing serum by volume on the basis of above.The proliferative activity of chondrocytes and the expression of collagen type II were tested for the two groups after 24 and 48 hours.The differentiation of bone marrow mesenchymal stem cells and synovial mesenchymal stem cells into chondrocytes in the co-culture system was tested by immunofluorescence staining after 14 days. RESULTS AND CONCLUSION:(1)The three kinds of cells grew normally adherently to the wall in different ratios of co-culture,where chondrocytes showed the best proliferative activity and phenotypic ability in the co-culture system when chondrocytes:bone marrow mesenchymal stem cells:synovial mesenchymal stem cells=2:1:1.(2)Compared with the control group,the proliferative activity and type II collagen expression of chondrocytes in the experimental group were significantly increased after 24 hours(P<0.01),and the two groups still had difference after 48 hours(P<0.05).The two groups showed obvious chondrogenic differentiation of bone marrow mesenchymal stem cells and synovial mesenchymal stem cells after 14 days(P<0.01),and some of the cells appeared round or oval,and the cytoplasmic type II collagen immunofluorescence staining was positive.The fluorescence intensity of the experimental group was significantly higher than that of the control group(P<0.01).(3)The results showed that the chondrocyte-bone marrow mesenchymal stem cell-synovial mesenchymal stem cell co-culture system could be successfully established by the non-contact co-culture method,and the best chondrocyte proliferative activity and phenotypic ability could be obtained when the cell ratio was 2:1:1.Icariin-containing serum had the promoting effect on chondrocyte proliferation,and chondrogenic differentiation of bone marrow mesenchymal stem cells and synovial mesenchymal stem cells in the system.