Objective:To perform RHD gene detection on a blood sample with serological weak D phenotype.Methods:A specimen received by the People's Hospital of Zhijin County was serologically identified by the microcolumn gel method and saline method.RHD gene detection was conducted by the PCR-SSP method,and the full sequence determination of the 10 exons amplified was performed.The sequencing results were compared with the ISBT database to determine the genotype.Bioinformatics tool was used to predict the functional damage of mutant proteins,and Alphafold-3 was used for tertiary structural modeling of wild-type and mutant RhD proteins,and the structures of the two proteins were compared and analyzed to explore the reasons why mutations lead to weak serological manifestations.Results:The patient's genotype was identified as RHD*DV.5/RHD*01N.01 heterozygote,with the complete deletion of RHD genes on one chromosome,unable to express the D antigen.On the other chromosome,a G＞A mutation occurred at the 697th base of the 5th exon,resulting in a partial D phenotype.This mutation causes internal hydrogen bond changes at the 233 position of RhD protein,resulting in a change in the conformation of the protein,affecting binding to the corresponding antibody.Conclusion:The patient is a heterozygous mutant individual with RHD*DV.5/RHD*01N.01,exhibiting a partial D phenotype serologically.This variation is extremely rare and has been scarcely reported globally.

Objective:To investigate the effects of different BMI classifications on rehabilitation outcomes and complications after total knee arthroplasty(TKA).Methods:Clinical data of 88 patients who underwent TKA in our hospital from January 2022 to June 2024 were retrospectively analyzed.According to the World Health Organization(WHO)BMI standards,patients were divided into normal weight group(18.5 ≤BMI＜24.9 kg/m2),overweight group(25.0≤BMI＜29.9 kg/m2),mild obesity group(30.0≤BMI＜34.9 kg/m2),and severe obesity group(BMI≥35.0 kg/m2).Baseline data,operation time,intraoperative blood loss,hospital stay,postoperative knee function scores,pain scores,range of motion(ROM),and incidence of complications were compared among groups.Results:With the increase of BMI,operation time was prolonged,intraoperative blood loss increased,and hospital stay extended,with statistically significant differences(P＜0.05).At 3-month and 6-month follow-ups,Knee Society Score(KSS)and Western Ontario and McMaster Universities Osteoarthritis Index(WOMAC)showed that functional recovery decreased with increasing BMI(P＜0.05).Regarding postoperative complications,the incidence of poor incision healing,infection,and deep vein thrombosis(DVT)in the obese groups was significantly higher than in the normal weight and overweight groups(P＜0.05).Conclusion:BMI is an important factor affecting rehabilitation outcomes and complications after TKA.Patients with high BMI have slower functional recovery and higher complication rates.For patients with high BMI,preoperative weight loss and individualized rehabilitation programs should be considered to improve TKA prognosis.

BACKGROUND:The cryopreservation technology enables tissues/cells to be stored for a long time in a low-temperature environment while maintaining the integrity of their activity and function,which is of great significance for the construction of cell therapy,tissue engineering and biological sample banks.Cryoprotective agents often contain dimethyl sulfoxide and serum.To avoid the toxic side effects of dimethyl sulfoxide,the complexity of serum components and immune responses,although some finished cryoprotective agents have been marketed,they are faced with many difficulties such as high cost and limited application.Therefore,there is an urgent need to develop a cryoprotective agent with clear components and the ability to solve the above problems.OBJECTIVE:To evaluate the effects of a novel cryoprotectant on cryopreservation efficiency of different tissue and cell sources.METHODS:By applying the novel cryoprotectant as an experimental group with the commercially available and widely used cryoprotectant(control group)to umbilical cord Wharton's jelly tissue,umbilical cord mesenchymal stem cells,umbilical cord blood/peripheral blood mononuclear cells,NK and CIK cells,comparative analyses were conducted in terms of cell morphology,number,viability,surface markers,differentiation potential,and cell-killing toxicity assay before cryopreservation and after resuscitation thawing.We confirmed the cryopreservation effect of the new cryoprotectant and its potential application value.RESULTS AND CONCLUSION:(1)The novel cryoprotectant facilitated the normal growth of cryopreserved Wharton's jelly tissue upon recovery,exhibiting mesenchymal stem cell morphology.No significant differences were observed between the experimental and control groups in terms of cell recovery rate,surface markers,and differentiation potential.(2)There was no significant difference in the number and viability of cells between the experimental group and the control group after cryopreservation of cord blood/peripheral blood mononuclear cells,and the cryo-resuscitated cell numbers and viability of derived NK cells/CIK cells did not show significant difference between the experimental and control groups.(3)For NK cells derived and differentiated from cord blood/peripheral blood mononuclear cells,there was no significant difference in the proportion of CD56+CD16+cell subpopulations between the experimental group and the control group.For CIK cells derived and differentiated from cord blood/peripheral blood mononuclear cells,there was no significant difference in the proportions of CD3+CD8+and CD3+CD56+cell subpopulations between the experimental group and the control group.(4)In terms of cytotoxicity testing,when the effective-target ratio of immune cells and melanoma cell line Mel624 was 20:1,whether it was NK cells/CIK cells derived from cord blood or peripheral blood mononuclear cells,there was no significant difference in the tumoricidal activity of cells between the experimental group and the control group.These findings suggest that the novel cryoprotectant can replace existing commercially available and widely used cryoprotectants,and is applicable to Wharton's jelly tissue,umbilical cord mesenchymal stem cells,umbilical cord blood/peripheral blood mononuclear cells,as well as NK and CIK cells,providing a solid technical foundation for the scaling,standardization,and commercialization of universal cryoprotectants.

Hydrogel is a type of polymer material that has been increasingly studied in the field of medical research due to its excellent biocompatibility, tunable mechanical properties, and drug delivery capabilities. In recent years, hydrogels have shown a wide range of application potentials in the treatment of urological surgical diseases. The research on hydrogels is gradually advancing in areas such as drug delivery within the bladder, treatment of incontinence and bladder vesicoureteral reflux, diagnostic and therapeutic applications for urological tumors, male reproduction, hemostasis in urological surgery, treatment of urolithiasis, tissue engineering for urethral defects and strictures, construction and coating applications of ureteral stent. At the same time, there are also some challenges that need to be addressed. This paper introduces the basic characteristics of hydrogel materials, and discussed the research progress of these materials in the diagnosis and treatment of urological diseases.

An assay was established for detection of toxigenic Clostridioides difficile by combining microfluidic chip analysis with immunochromatography,and its performance was evaluated and compared with those of the Xpert C.difficile/Epi and VIDAS CD AB tests.Primer pairs were designed according to the tcdB and tpi genes in C.difficile.The specificity,limit of detection,reproducibility,and stability were evaluated.A total of 215 stool samples from patients with diarrhea were collected and tested in parallel with the Xpert C.difficile/Epi,VIDAS CDAB,and our assay.C.difficile was isolated from samples,and the tcdB gene was identified when discrepant results were obtained from the three above assays.Our assay showed no cross-reaction with other diarrhea-associated pathogens.Its reproducibility was 100％in testing of two standard plasmids containing tcdB and tpi genes at two concentrations(105 and 102 copies/μL).Two standard plasmids were detected after the PCR and immunochromatography reagents had been stored for 3,6,9,and 12 months,and all the results were posi-tive.The limit of detection was 10 copies/μL for toxigenic C.difficile.Testing of 33 samples positive for C.difficile with our assay(33/215,15.3％)yielded findings statistically coherent with those of the Xpert C.difficile/Epi test(kappa value=0.965).The sensitivity,specificity,positive predictive value,and negative predictive value of our assay,with respect to Xpert C.difficile/Epi as the standard,were 94.3％,100.0％,100.0％,and 98.9％;these values were significantly higher than those of VIDAS CDAB(60.0％,98.9％,91.3％,and 92.7％)(Kappa=0.714,OR=157.50,95％CI:62.03-847.28,P=0.013).In conclusion,our newly developed assay is specific,stable,and reproducible,and may be used for rapid and accu-rate detection of toxigenic C.difficile.The assay could be used for C.difficile infection screening in outpatient and emergen-cy,community medical service center,and epidemiological settings.

Objective:To investigate the changes and effects of the Wnt/β-catenin pathway during hypoxia-reoxygenation in human renal tubular epithelial(HK-2)cells.Methods:HK-2 cells were randomly divided into control group(Control),model group(Model),model+interference empty vector group(Model+NC),and model+interference group(Model+Si-β-catenin).Cells in the control group were cultured routinely.Cells in the Model group were intervened with hypoxia for 6 hours and reoxygenation for 2 hours to construct a hypoxia-reoxygenation model.HK-2 cells in the Model+NC group and the Model+Si-β-catenin group were transfected with β-catenin interference empty vector and β-catenin interference vector respectively,and then a hypoxia-reoxygenation model was constructed.After corresponding interventions,cells were collected.The apoptosis of cells in each group was detected by flow cytometry.The expression of matrix metalloproteinase-7(MMP-7)and α-smooth muscle actin(α-SMA)was detected by immunofluorescence.The expression level of Wnt pathway-related proteins was detected by Western blotting.Results:Compared with that in the control group,the apoptosis rate of cells in the Model group was significantly up-regulated,and there was no significant difference in cell apoptosis between the Model group,the Model+NC group and the Model+Si-β-catenin group.The results of WB detection showed that there was no significant difference in the expression of GSK-3β protein in the cells among the control group,the model group,the model+NC group and the model+Si-β-catenin group.Compared with that in the control group,the expression of p-GSK-3β protein in the Model group was significantly up-regulated,while there was no significant difference in the expression of p-GSK-3βprotein between the Model group,the Model+NC group and the Model+Si-β-catenin group.The results of immunofluorescence detection showed that MMP-7 and α-SMA in the Model group were significantly increased compared with that in the control group,while there was no significant difference between the Model group and the Model+NC group.Compared with the Model+NC group,MMP-7 and α-SMA in the Model+Si-β-catenin group were significantly down-regulated.Conclusion:The Wnt/β-catenin pathway is activated in HK-2 cells during hypoxia-reoxygenation and promotes the expression of fibrosis-related proteins.Inhibiting β-catenin can improve its fibrosis induction level.

Objective:To investigate the effects of different BMI classifications on rehabilitation outcomes and complications after total knee arthroplasty(TKA).Methods:Clinical data of 88 patients who underwent TKA in our hospital from January 2022 to June 2024 were retrospectively analyzed.According to the World Health Organization(WHO)BMI standards,patients were divided into normal weight group(18.5 ≤BMI＜24.9 kg/m2),overweight group(25.0≤BMI＜29.9 kg/m2),mild obesity group(30.0≤BMI＜34.9 kg/m2),and severe obesity group(BMI≥35.0 kg/m2).Baseline data,operation time,intraoperative blood loss,hospital stay,postoperative knee function scores,pain scores,range of motion(ROM),and incidence of complications were compared among groups.Results:With the increase of BMI,operation time was prolonged,intraoperative blood loss increased,and hospital stay extended,with statistically significant differences(P＜0.05).At 3-month and 6-month follow-ups,Knee Society Score(KSS)and Western Ontario and McMaster Universities Osteoarthritis Index(WOMAC)showed that functional recovery decreased with increasing BMI(P＜0.05).Regarding postoperative complications,the incidence of poor incision healing,infection,and deep vein thrombosis(DVT)in the obese groups was significantly higher than in the normal weight and overweight groups(P＜0.05).Conclusion:BMI is an important factor affecting rehabilitation outcomes and complications after TKA.Patients with high BMI have slower functional recovery and higher complication rates.For patients with high BMI,preoperative weight loss and individualized rehabilitation programs should be considered to improve TKA prognosis.

An assay was established for detection of toxigenic Clostridioides difficile by combining microfluidic chip analysis with immunochromatography,and its performance was evaluated and compared with those of the Xpert C.difficile/Epi and VIDAS CD AB tests.Primer pairs were designed according to the tcdB and tpi genes in C.difficile.The specificity,limit of detection,reproducibility,and stability were evaluated.A total of 215 stool samples from patients with diarrhea were collected and tested in parallel with the Xpert C.difficile/Epi,VIDAS CDAB,and our assay.C.difficile was isolated from samples,and the tcdB gene was identified when discrepant results were obtained from the three above assays.Our assay showed no cross-reaction with other diarrhea-associated pathogens.Its reproducibility was 100％in testing of two standard plasmids containing tcdB and tpi genes at two concentrations(105 and 102 copies/μL).Two standard plasmids were detected after the PCR and immunochromatography reagents had been stored for 3,6,9,and 12 months,and all the results were posi-tive.The limit of detection was 10 copies/μL for toxigenic C.difficile.Testing of 33 samples positive for C.difficile with our assay(33/215,15.3％)yielded findings statistically coherent with those of the Xpert C.difficile/Epi test(kappa value=0.965).The sensitivity,specificity,positive predictive value,and negative predictive value of our assay,with respect to Xpert C.difficile/Epi as the standard,were 94.3％,100.0％,100.0％,and 98.9％;these values were significantly higher than those of VIDAS CDAB(60.0％,98.9％,91.3％,and 92.7％)(Kappa=0.714,OR=157.50,95％CI:62.03-847.28,P=0.013).In conclusion,our newly developed assay is specific,stable,and reproducible,and may be used for rapid and accu-rate detection of toxigenic C.difficile.The assay could be used for C.difficile infection screening in outpatient and emergen-cy,community medical service center,and epidemiological settings.

Objective:To investigate the changes and effects of the Wnt/β-catenin pathway during hypoxia-reoxygenation in human renal tubular epithelial(HK-2)cells.Methods:HK-2 cells were randomly divided into control group(Control),model group(Model),model+interference empty vector group(Model+NC),and model+interference group(Model+Si-β-catenin).Cells in the control group were cultured routinely.Cells in the Model group were intervened with hypoxia for 6 hours and reoxygenation for 2 hours to construct a hypoxia-reoxygenation model.HK-2 cells in the Model+NC group and the Model+Si-β-catenin group were transfected with β-catenin interference empty vector and β-catenin interference vector respectively,and then a hypoxia-reoxygenation model was constructed.After corresponding interventions,cells were collected.The apoptosis of cells in each group was detected by flow cytometry.The expression of matrix metalloproteinase-7(MMP-7)and α-smooth muscle actin(α-SMA)was detected by immunofluorescence.The expression level of Wnt pathway-related proteins was detected by Western blotting.Results:Compared with that in the control group,the apoptosis rate of cells in the Model group was significantly up-regulated,and there was no significant difference in cell apoptosis between the Model group,the Model+NC group and the Model+Si-β-catenin group.The results of WB detection showed that there was no significant difference in the expression of GSK-3β protein in the cells among the control group,the model group,the model+NC group and the model+Si-β-catenin group.Compared with that in the control group,the expression of p-GSK-3β protein in the Model group was significantly up-regulated,while there was no significant difference in the expression of p-GSK-3βprotein between the Model group,the Model+NC group and the Model+Si-β-catenin group.The results of immunofluorescence detection showed that MMP-7 and α-SMA in the Model group were significantly increased compared with that in the control group,while there was no significant difference between the Model group and the Model+NC group.Compared with the Model+NC group,MMP-7 and α-SMA in the Model+Si-β-catenin group were significantly down-regulated.Conclusion:The Wnt/β-catenin pathway is activated in HK-2 cells during hypoxia-reoxygenation and promotes the expression of fibrosis-related proteins.Inhibiting β-catenin can improve its fibrosis induction level.

In 2015,the International Ascites Club proposed a new definition of hepatorenal syndrome-acute kidney injury based on the progression of hepatorenal syndrome,and studies are still being conducted to explore the exact pathogenesis of hepatorenal syndrome-acute kidney injury.Intestinal barrier plays an important bridging role in liver-kidney connection,and intestinal flora disturbance,bacterial translocation,and endotoxins entering the blood cause damage to the kidneys by releasing proinflammatory cytokines and activating immune-related cells.The entrance of bile acid into the circulation system also directly or indirectly lead to the development and progression of hepatorenal syndrome-acute kidney injury.This article reviews the crosstalk mechanism of hepatorenal syndrome-acute kidney injury from the perspective of the intestinal barrier and further clarifies the key role of the liver-gut-kidney axis in the pathogenesis of this disease,in order to provide new treatment ideas.