Objective To establish a flow cytometric assay for detecting heat shock protein A2(HSPA2)in sperm and explore the role of HSPA2 expression levels in predicting low fertilization rates in in vitro fertilization(IVF).Methods The principle of in-direct immunofluorescence(IIF)was used to fluorescently stain sperm HSPA2.After the sperm sample was permeabilized and sealed,rabbit anti-human HSPA2 antibody(primary antibody)and fluorescein isothiocyanate(FITC)labeled goat anti-rabbit IgG antibody(secondary antibody)were sequentially added as detection tubes.At the same time,a sample without primary anti-body was set up as a control tube,and the positive rates of the two tubes were measured by flow cytometer.The ratio of the posi-tive rate of the detection tube to the control tube(positive rate ratio)was calculated.The optimal number of sperm for detection and the optimal working dilutions of primary and secondary antibodies were explored using the chessboard method.Under the optimal conditions,the repeatability,linear range and reference range of the method were evaluated separately,in order to estab-lish a preliminary method for detecting sperm HSPA2 expression levels using flow cytometry.After the establishment of the method,preliminary testing was conducted on a total of 85 sperm samples from couples who underwent IVF at the Reproductive Medicine Center of Jiangmen Central Hospital in 2023.The ratio of HSPA2 positivity rates between the group with IVF success-ful(n=63)and the group with low fertilization rate(n=22)was compared,and the receiver operating characteristic(ROC)curve was used to analyze the threshold.Results The positive rate of HSPA2 in the control tube was relatively low,showing a low background signal,while the fluorescence signal of the detection tube was significantly enhanced,indicating that this method can effectively detect HSPA2.The optimal number of sperm samples for detection determined by the chessboard method was 2×106,and the optimal working dilutions for primary and secondary antibodies were 1∶300 and 1∶400,respectively.Evaluation of repeatability and linear range showed good methodological performance.Comparative analysis between the group with IVF success-ful and the group with low fertilization rate showed that the ratio of sperm HSPA2 positivity rate in the group with low fertilization rate(6.19±4.07)was lower than successful fertilization group(10.69±8.26),the difference was statistically significant(t=2.446,P<0.05).The ROC curve and Youden index showed that the best predictive power was achieved when the cutoffvalue for the ratio of positivity rate was 5.5067,with a sensitivity and a specificity of 71.4%,55.5%,respectively.Conclusion A flow cytometric method for detecting HSPA2 in sperm is successfully established.The expression level of sperm HSPA2 detected by this method suggests its predictive value for low fertilization rate in IVF,providing a basis for future clinical scientific selection of fertilization methods.

Objective To establish a flow cytometric assay for detecting heat shock protein A2(HSPA2)in sperm and explore the role of HSPA2 expression levels in predicting low fertilization rates in in vitro fertilization(IVF).Methods The principle of in-direct immunofluorescence(IIF)was used to fluorescently stain sperm HSPA2.After the sperm sample was permeabilized and sealed,rabbit anti-human HSPA2 antibody(primary antibody)and fluorescein isothiocyanate(FITC)labeled goat anti-rabbit IgG antibody(secondary antibody)were sequentially added as detection tubes.At the same time,a sample without primary anti-body was set up as a control tube,and the positive rates of the two tubes were measured by flow cytometer.The ratio of the posi-tive rate of the detection tube to the control tube(positive rate ratio)was calculated.The optimal number of sperm for detection and the optimal working dilutions of primary and secondary antibodies were explored using the chessboard method.Under the optimal conditions,the repeatability,linear range and reference range of the method were evaluated separately,in order to estab-lish a preliminary method for detecting sperm HSPA2 expression levels using flow cytometry.After the establishment of the method,preliminary testing was conducted on a total of 85 sperm samples from couples who underwent IVF at the Reproductive Medicine Center of Jiangmen Central Hospital in 2023.The ratio of HSPA2 positivity rates between the group with IVF success-ful(n=63)and the group with low fertilization rate(n=22)was compared,and the receiver operating characteristic(ROC)curve was used to analyze the threshold.Results The positive rate of HSPA2 in the control tube was relatively low,showing a low background signal,while the fluorescence signal of the detection tube was significantly enhanced,indicating that this method can effectively detect HSPA2.The optimal number of sperm samples for detection determined by the chessboard method was 2×106,and the optimal working dilutions for primary and secondary antibodies were 1∶300 and 1∶400,respectively.Evaluation of repeatability and linear range showed good methodological performance.Comparative analysis between the group with IVF success-ful and the group with low fertilization rate showed that the ratio of sperm HSPA2 positivity rate in the group with low fertilization rate(6.19±4.07)was lower than successful fertilization group(10.69±8.26),the difference was statistically significant(t=2.446,P<0.05).The ROC curve and Youden index showed that the best predictive power was achieved when the cutoffvalue for the ratio of positivity rate was 5.5067,with a sensitivity and a specificity of 71.4%,55.5%,respectively.Conclusion A flow cytometric method for detecting HSPA2 in sperm is successfully established.The expression level of sperm HSPA2 detected by this method suggests its predictive value for low fertilization rate in IVF,providing a basis for future clinical scientific selection of fertilization methods.

A 50-year-old male patient was scheduled to undergo epiglottic mass resection under general anesthesia due to an epiglottic cyst. Before anesthesia induction, the patient received dexamethasone, methylprednisolone, midazolam, and ondansetron by intravenous injectionin sequence. After 2 minutes, the patient complained of palpitations, abdominal spasmodic pain, cyanosis of the lips, and patchy changes in the skin on the chest and body. The electrocardiogram monitor showed a heart rate of 175 beats per minute, but his cuff blood pressure cannot be measured. His blood oxygen saturation was 0.76, and he did not respond to the call afterwards. Oxygen through a face mask and pressure ventilation, intravenous injection of 20 mg of esmolol twice were given immediately. The patient′s consciousness recovered, the heart rate gradually decreased to 60 beats per minute (sinus rhythm), and the blood pressure increased to 74/50 mmHg. Continuous IV pumping of norepinephrine 8 μg/min was given. After 25 minutes, the patient′s bedside electrocardiogram showed atrial fibrillation with ventricular differential conduction, myocardial injury or acute myocardial infarction, and QT interval prolongation. Then intravenous injection of furosemide 40 mg was given, his above symptoms were improved,his blood pressure recovered to 110-120/70 mmHg, blood oxygen saturation was 1.00, the skin spots on his chest and body disappeared, and his abdominal pain was alleviated. Anesthesiologists and pharmacists evaluated the patient′s adverse reactions and considered that there was a high possibility of type I Kounis syndrome caused by the combination of glucocorticoids, midazolam, and ondansetron.

A 50-year-old male patient was scheduled to undergo epiglottic mass resection under general anesthesia due to an epiglottic cyst. Before anesthesia induction, the patient received dexamethasone, methylprednisolone, midazolam, and ondansetron by intravenous injectionin sequence. After 2 minutes, the patient complained of palpitations, abdominal spasmodic pain, cyanosis of the lips, and patchy changes in the skin on the chest and body. The electrocardiogram monitor showed a heart rate of 175 beats per minute, but his cuff blood pressure cannot be measured. His blood oxygen saturation was 0.76, and he did not respond to the call afterwards. Oxygen through a face mask and pressure ventilation, intravenous injection of 20 mg of esmolol twice were given immediately. The patient′s consciousness recovered, the heart rate gradually decreased to 60 beats per minute (sinus rhythm), and the blood pressure increased to 74/50 mmHg. Continuous IV pumping of norepinephrine 8 μg/min was given. After 25 minutes, the patient′s bedside electrocardiogram showed atrial fibrillation with ventricular differential conduction, myocardial injury or acute myocardial infarction, and QT interval prolongation. Then intravenous injection of furosemide 40 mg was given, his above symptoms were improved,his blood pressure recovered to 110-120/70 mmHg, blood oxygen saturation was 1.00, the skin spots on his chest and body disappeared, and his abdominal pain was alleviated. Anesthesiologists and pharmacists evaluated the patient′s adverse reactions and considered that there was a high possibility of type I Kounis syndrome caused by the combination of glucocorticoids, midazolam, and ondansetron.

Objective:To explore the related risk factors for systemic embolism (SE) in patients aged≥75 years with non-valvular atrial fibrillation (NVAF).Methods:A case-control study. NVAF patients aged≥75 years who were hospitalized at the First Affiliated Hospital of Xinjiang Medical University from October 2018 to October 2020 were divided into no SE ( n=1 127) and SE ( n=433) groups according to the occurrence of SE after NVAF. Multivariate logistic regression was used to analyze SE-related factors in patients with NVAF without anticoagulation treatment. Results:In the multivariate model, the following factors were associated with an increased risk of SE in patients with NVAF: history of AF≥5 years [odds ratio ( OR)=2.75, 95% confidence interval ( CI) 1.98-3.82, P<0.01], lipoprotein(a)>300 g/L ( OR=2.07, 95% CI 1.50-2.84, P<0.01), apolipoprotein (Apo)B>1.2 g/L ( OR=1.91, 95% CI 1.25-2.93, P=0.003), left ventricular ejection fraction (LVEF) of 30%-49% ( OR=2.45, 95% CI 1.63-3.69, P<0.01), left atrial diameter>40 mm ( OR=1.54, 95% CI 1.16-2.07, P=0.003), and CHA 2DS 2-VASc score≥3 ( OR=15.14, 95% CI 2.05-112.13, P=0.01). ApoAI>1.6 g/L was negatively correlated with the occurrence of SE ( OR=0.28, 95% CI 0.15-0.51, P<0.01). Conclusions:History of AF≥5 years, lipoprotein(a)>300 g/L, elevated ApoB, left atrial diameter>40 mm, LVEF of 30%-49%, and CHA 2DS 2-VASC score≥3 are independent risk factors for SE whereas ApoAI>1.6 g/L is a protective factor against SE in patients with NVAF.

Objective:To investigate the effect of miRNA-1-3p (miR-1-3p) on expression of myocyte enhancer factor 2A (MEF2A) and the biological function of osteosarcoma cells.Methods:The tumor tissues and adjacent normal tissues of 20 patients with osteosarcoma who were clinically diagnosed in Shanxi Provincial Cancer Hospital from January 2019 to January 2020 were collected, and the expression of miR-1-3p in the samples was detected by real-time fluorescent quantitative polymerase chain reaction (qRT-PCR). The expression of miR-1-3p in osteosarcoma cell lines U2-OS, SAOS-2, MG63, SW1353 and human normal osteoblast cell line hFOB1.19 was detected by qRT-PCR, then the cell line with the lowest expression of miR-1-3p was selected for follow-up experiments. An overexpression miR-1-3p vector was constructed (miR-1-3p mimcs). The miR-1-3p overexpression group was transfected with miR-1-3p mimcs, and the control group was transfected with empty vector (miR-1-3p nc). CCK-8 method was used to detect the proliferation activity of cells; flow cytometry was used to detect the changes of cell apoptosis and cell cycle. miRwalk database was used to predict the miR-1-3p target gene, and the target gene was verified by dual-luciferase reporter gene assay; Western blot was used to detect the expression of MEF2A protein in cells of each group.Results:Compared with adjacent tissues, the expression of miR-1-3p in osteosarcoma tissues was down-regulated (0.31±0.14 vs. 0.62±0.21), and the difference was statistically significant ( t = 5.31, P<0.01). The expression of miR-1-3p in U2-OS cells was the lowest; compared with the control group, the proliferation activity of U2-OS cells was inhibited in miR-1-3p overexpression group (48 h absorbance value 0.56±0.01 vs. 0.77±0.03, t = 2.77, P<0.01; 72 h absorbance value 0.87±0.02 vs. 1.40±0.03, t = 2.93, P<0.01); G 1/S cell cycle arrest increased [G 1 phase (38.24±0.55)% vs. (32.11±0.80)%, t = 9.27, P = 0.01; S phase (61.24±0.90)% vs. (67.78±0.83)%, t = 7.52, P = 0.02]; early apoptotic rate increased [(11.20±0.12)% vs. (1.50±0.12)%, t = 2.91, P<0.05], miRwalk database predicted that the miR-1-3p target gene was MEF2A. The result of dual-luciferase reporter gene assay showed that miR-1-3p bound to MEF2A 3'UTR, and the luciferase activity of U2-OS cells in miR-1-3p overexpression group was lower than that in the control group (renilla luciferase/firefly luciferase activity ratio 0.53±0.06 vs. 1.00±0.04, t = 4.04, P < 0.05). Western blot showed that the expression of MEF2A protein in U2-OS cells of miR-1-3p overexpression group was lower than that of the control group (protein relative expression 0.41±0.14 vs. 0.77±0.12, t = 3.93, P < 0.05). Conclusions:The low expression of miR-1-3p may be associated with the proliferation, apoptosis and cycle changes of osteosarcoma cells. miR-1-3p can negatively regulate the expression of MEF2A protein and regulate the occurrence and development of osteosarcoma.

Background::Leukocyte telomere length shortening is a characteristic of premature senescence, a process that can be accelerated by oxidative stress. In general, patients with end-stage renal disease undergoing regular hemodialysis (HD) are repeatedly exposed to oxidative stress. Patients undergoing HD tend to have cardiovascular diseases associated with oxidative stress and inflammation. Therefore, we assumed that telomere length is associated with HD vintage and the degree of vascular calcification.Methods::A total of 144 patients undergoing regular HD before kidney transplantation and 62 patients on hemodialysis, but not undergoing kidney transplantation, were enrolled. We measured common laboratory values, such as calcium, phosphate, and hemoglobin levels, and assessed the degree of vascular calcification in the patients. The leukocyte telomere length was measured using reverse transcription polymerase chain reaction, and Spearman correlation was used for correlation analysis.Results::The leukocyte telomere length was negatively associated with age (rho = -0.306, P <0.01); it was shorter in middle-aged patients than in young patients (13.48 ± 4.80 vs. 15.86 ± 4.51, P < 0.01). The telomere length was significantly different among patients aged 52-74 years in groups with different HD vintages. Additionally, the telomere length was positively associated with serum hemoglobin (Hb) levels in all patients (rho = 0.290, P < 0.01). There was a significant difference among patients divided into three groups according to the degree of anemia (17.09 ± 5.64 vs. 14.40 ± 4.07 vs. 13.99 ± 3.95, P < 0.01). Further, a significant difference was observed in the telomere length among patients with different degrees of vascular calcification (16.79 ± 4.91 vs. 13.61 ± 2.82 vs. 14.62 ± 3.63 vs. 10.71 ± 3.74, P < 0.01). The telomere length was shorter in the patients on hemodialysis who did not receive a kidney transplant than in the surgical patients (8.12 ± 1.83 vs. 14.33 ± 4.63, P < 0.01). Conclusion::This study demonstrated that the telomere length was significantly correlated with HD vintage in patients of a certain age group. The telomere length was shorter in patients on hemodialysis who matched for age and dialysis vintage with kidney transplant patients. It was also associated with vascular calcification and serum Hb levels in all patients undergoing HD.

BK polyomavirus-associated nephropathy (BKPyVAN) is a common cause of allograft failure. However, differentiation between BKPyVAN and type I T cell-mediated rejection (TCMR) is challenging when simian virus 40 (SV40) staining is negative, because of the similarities in histopathology. This study investigated whether donor-derived cell-free DNA (ddcfDNA) can be used to differentiate BKPyVAN. Target region capture sequencing was applied to detect the ddcfDNAs of 12 recipients with stable graft function, 22 with type I TCMR, 21 with proven BKPyVAN, and 5 with possible PyVAN. We found that urinary ddcfDNA levels were upregulated in recipients with graft injury, whereas plasma ddcfDNA levels were comparable for all groups. The median urinary concentrations and fractions of ddcfDNA in proven BKPyVAN recipients were significantly higher than those in type I TCMR recipients (10.4 vs. 6.1 ng/mL,

Background::Leukocyte telomere length shortening is a characteristic of premature senescence, a process that can be accelerated by oxidative stress. In general, patients with end-stage renal disease undergoing regular hemodialysis (HD) are repeatedly exposed to oxidative stress. Patients undergoing HD tend to have cardiovascular diseases associated with oxidative stress and inflammation. Therefore, we assumed that telomere length is associated with HD vintage and the degree of vascular calcification.Methods::A total of 144 patients undergoing regular HD before kidney transplantation and 62 patients on hemodialysis, but not undergoing kidney transplantation, were enrolled. We measured common laboratory values, such as calcium, phosphate, and hemoglobin levels, and assessed the degree of vascular calcification in the patients. The leukocyte telomere length was measured using reverse transcription polymerase chain reaction, and Spearman correlation was used for correlation analysis.Results::The leukocyte telomere length was negatively associated with age (rho = -0.306, P <0.01); it was shorter in middle-aged patients than in young patients (13.48 ± 4.80 vs. 15.86 ± 4.51, P < 0.01). The telomere length was significantly different among patients aged 52-74 years in groups with different HD vintages. Additionally, the telomere length was positively associated with serum hemoglobin (Hb) levels in all patients (rho = 0.290, P < 0.01). There was a significant difference among patients divided into three groups according to the degree of anemia (17.09 ± 5.64 vs. 14.40 ± 4.07 vs. 13.99 ± 3.95, P < 0.01). Further, a significant difference was observed in the telomere length among patients with different degrees of vascular calcification (16.79 ± 4.91 vs. 13.61 ± 2.82 vs. 14.62 ± 3.63 vs. 10.71 ± 3.74, P < 0.01). The telomere length was shorter in the patients on hemodialysis who did not receive a kidney transplant than in the surgical patients (8.12 ± 1.83 vs. 14.33 ± 4.63, P < 0.01). Conclusion::This study demonstrated that the telomere length was significantly correlated with HD vintage in patients of a certain age group. The telomere length was shorter in patients on hemodialysis who matched for age and dialysis vintage with kidney transplant patients. It was also associated with vascular calcification and serum Hb levels in all patients undergoing HD.

Objective To study the correlation between serum miRNA-21 (miR-21) level and chemosensitivity and prognosis of osteosarcoma patients. Methods A total of 68 osteosarcoma patients who were treated in Shanxi Provincial Cancer Hospital from August 2016 to August 2017 were selected. All patients received routine chemotherapy after admission. They were followed up for one year. According to the effectiveness of chemotherapy, they were divided into effective group (52 cases) and ineffective group (16 cases). The general clinicopathological characteristics of the two groups were recorded. Univariate logistic regression model was used to analyze the serum miR-21 level and chemosensitivity in osteosarcoma patients. The prognostic efficacy of serum miR-21 was evaluated by receiver operating characteristic (ROC) curve. Results The serum miR-21 expression in the effective group was higher than that in the ineffective group (6.6 ±0.7 vs. 5.2 ±0.7), and the tumor metastasis rate in the effective group was lower than that in the ineffective group [33.9％ (18/52) vs. 37.5％ (6/16)], and the differences between the two groups were statistically significant (both P< 0.05). There were no significant differences in gender, age, body mass index (BMI), tumor location, size of tumors and TNM stage between the two groups (all P>0.05). Univariate logistic regression model showed that serum miR-21 and metastasis were the factors affecting chemosensitivity (both P<0.05). ROC curve showed that the area under the curve (AUC) of using serum miR-21 to predict the prognosis of osteosarcoma patients was 0.774. Youden index indicated that the best cut-off points of serum miR-21 and chemosensitivity for predicting the prognosis of osteosarcoma patients was 6.25. Conclusion Serum miR-21 level in osteosarcoma patients is significantly correlated with chemosensitivity and prognosis, and it can be used as an effective index to predict the effect of chemotherapy and prognosis of patients with osteosarcoma.