Objective To establish a highly sensitive and specific method for detecting human DNA based on real time quantitative PCR(qPCR)technique for the rapid detection of potential DNA con-tamination sources in DNA laboratories.Methods Primers and probes were designed with Primer Ex-pressTM software using the reference sequence of human 18S rRNA gene as a template,and the opti-mal prime-probe combination was screened by matrix method.The PCR products of the target se-quence of human 18S rRNA gene were used to construct the plasmid,and a plasmid standard was used to draw the standard curve of the qPCR system.According to the Minimum Information for Pub-lication of Quantitative Real-time PCR Experiments(MIQE)guidelines,the specificity,sensitivity,re-peatability and application effect of the qPCR system were evaluated.Results The sensitivity of the qPCR system established in this study was 5.3×10-5 ng/μL,which showed good specificity for human DNA samples.The correlation coefficient of the qPCR system was-0.999,and amplification efficiency was 100％.Both the intra-batch and inter-batch variation coefficients were less than 2％.Conclusion The established human DNA detection method based on qPCR technique has good specificity,high sen-sitivity,and robust stability.It can be used for rapid detection of DNA contamination and daily moni-toring of the accumulated human DNA in the laboratory environment.

Objective To investigate the role and molecular mechanism of SOX4 in Helicobacter pylori(H.pylori)-mediated gastric mucosal epithelial dysplasia.Methods The expression of SOX4 in gastric tissues and cells was analyzed with reverse transcription-polymerase chain reaction(RT-PCR),Western blotting,and immunohistochemical staining.The effects of SOX4 on gastric epithelial cell proliferation and colony formation were determined with CCK-8 and colony formation assays.A PCR array was used to screen downstream target genes involved in H.pylori-induced dysplasia mediated by SOX4.The transcriptional regulation and binding sites of the target gene MLH3 by SOX4 were elucidated with luciferase reporter assay,promoter truncation assay,and chromatin immunoprecipitation(ChIP).Results SOX4 expression was significantly increased in H.pylori-infected gastric tissues(P<0.05).Overexpression of SOX4 markedly enhanced the proliferation and colony formation abilities of normal gastric epithelial cells(P<0.05).Elevated SOX4 led to the dysregulation of MLH3 and other DNA damage repair-related molecules after H.pylori infection in gastric epithelial cells(|logFC|>1,P<0.05).H.pylori promoted MLH3 expression in gastric epithelial cells through SOX4.SOX4 transcriptionally activated MLH3 expression by binding to the 5th site of the MLH3 promoter.The increased expression of SOX4 and MLH3 is associated with poor prognosis of gastric cancer patients.Conclusion SOX4 is closely associated with H.pylori-induced dysplasia in gastric epithelial cells.Upregulation of SOX4 promotes H.pylori-related dysplasia by transcriptionally activating MLH3,leading to the imbalance of proliferation and colony formation in gastric epithelial cells.

Objective:To investigate the efficacy of rigid ureteroscopic laser lithotripsy combined with N-trap(RULL+N-trap),which is scoop-shaped,in the treatment of ureteral stones with a maximum diameter ≥1.5 cm.Methods:This retrospective cohort study included patients diagnosed with ureteral calculi who underwent rigid ureteroscopic lithotripsy(RULL)combined with N-Trap stone entrapment system at the Department of Urology,Peking University Third Hospital,by the same surgical team between June 2021 and September 2024.A total of 364 patients were initially enrolled.After excluding 21 patients due to missing critical outcome variables,two distinct cohorts were established:38 patients with ureteral stones measuring ≥1.5 cm in maximum diameter,and 305 patients with stones＜1.5 cm in maximum diameter.To minimize selection bias and control for confounding variables,propensity score matching(PSM)was employed.This resulted in two well-balanced groups:31 patients with stones 1.5 cm in maximum diameter and 31 patients with stones＜1.5 cm in maximum diameter,matched on baseline demographic and clinical characteristics.The primary outcomes assessed between the two groups included stone clearance.Secondary outcomes included changes in renal function indicators,specifically serum creatinine(SCr)and estimated glomerular filtration rate(GFR),and other factors like postopera-tive hospital stay and operative time.Results:In the matched cohort,the patients with stones ≥1.5 cm in maximum diameter had significantly longer operative time compared with those with smaller stones:(85.8±28.8)min vs.(62.4±24.6)min(P＜0.05).Postoperative length of hospital stay showed no significant difference:(2.26±1.79)d vs.(2.03±0.80)d(P＞0.05).The stone clearance on postoperative day one was 90.3％in the study group vs.100.0％in the control group(P＞0.05).One month postoperatively,the stone clearance was 93.5％vs.100.0％,respectively(P＞0.05).Changes in SCr were(-6.58±16.10)μmol/L vs.(-13.70±12.50)μmol/L,and changes in GFR were(5.92±14.90)mL/(min·1.73 m2)vs.(7.47±11.20)m L/(min·1.73 m2),with no statistically significant differences observed between the two groups for either renal function marker(P＞0.05).Conclusion:Ureteroscopic lithotripsy combined with N-trap is an optional method for treating ureteral stones with a maximum diameter ≥1.5 cm.The overall therapeutic efficacy is comparable,with the added benefit of significantly reducing the economic burden on patients.

Objective:To establish a complete system for the isolation, purification, identification, and culture of Kaposiform hemangioendothelioma-derived endothelial cells (KHE-ECs), to analyze the phenotype of KHE-ECs, and to explore the possibility of establishing a KHE-EC bank.Methods:A novel digestion solution for KHE tumors (patent number: CN202410500224.2) was formulated using collection fluid, Liberase TM and dispase stock solutions, and was used to process tumor tissues to obtain cells. High-purity KHE-ECs were purified using CD31 + immunomagnetic beads. The EGM-2 complete medium containing 10% fetal bovine serum and 2% penicillin-streptomycin solution was employed for cell culture. To verify the characteristics of KHE-ECs, immunofluorescence assay was conducted to determine the expression of endothelial cell-specific markers CD31 and CD34, KHE disease markers podoplanin (D2-40), prospero-related homeobox 1 (Prox-1), and lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1), as well as an infantile hemangioma-specific diagnostic marker glucose transporter 1 (GLUT-1). Human umbilical vein endothelial cells (HUVECs) served as controls for the phenotype analysis of KHE-ECs, including cell viability, cytoskeleton, proliferation, migration, invasion, tube formation, and sprouting ability. Results:Primary cells were successfully isolated from KHE tumor tissues, and high-purity KHE-ECs were obtained by using CD31 + immunomagnetic beads. The cells exhibited typical spindle-shaped morphology and an adherent growth pattern. Immunofluorescence assay showed that KHE-ECs expressed CD31, CD34, D2-40, Prox-1, and LYVE1, but did not express GLUT-1. There were significant differences in cell morphology, cell viability, and cytoskeletal structures between KHE-ECs and HUVECs. Additionally, the KHE-EC group showed significantly increased percentages of proliferative cells (29.1% ± 2.5%), numbers of migratory cells (114.3 ± 9.4) and invasive cells (110.0 ± 6.1), tube length (32 121.0 ± 892.0 μm), and number of sprouting cells (25.0 ± 3.6) compared with the HUVEC group (13.0% ± 2.2%, 38.0 ± 3.6, 35.3 ± 2.3, 25 345.0 ± 448.1 μm, 5.0 ± 1.0, respectively, all P ≤ 0.001) . Conclusion:An innovative digestion solution specifically for KHE tumors was formulated for the first time, and high-purity and well-growing KHE-EC strains were successfully isolated and purified by using the novel digestion solution in combination with CD31 + immunomagnetic beads, providing a stable and reliable cell source for subsequent experimental studies on KHE and laying the foundation for establishing a KHE-EC bank.

Ischemic stroke (IS) is a globally life-threatening disease. Presently, few therapeutic medicines are available for treating IS, and rt-PA is the only drug approved by the US Food and Drug Administration (FDA) in the US. In fact, many agents showing excellent neuroprotection but no blood flow-improving activity in animals have not achieved ideal clinical efficacy, while thrombolytic drugs only improving blood flow without neuroprotection have limited their wider application. To address these challenges and meet the huge unmet clinical need, we have designed and identified a novel compound AAPB with dual effects of neuroprotection and cerebral blood flow improvement. AAPB significantly reduced cerebral infarction and neural function deficit in tMCAO rats, pMCAO rats, and IS rhesus monkeys, as well as displayed exceptional safety profiles and excellent pharmacokinetic properties in rats and dogs. AAPB has now entered phase I of clinical trials fighting IS in China.

Objective:To investigate the optimal timing of oral propranolol treatment for proliferative infantile hemangiomas (IH) .Methods:A bidirectional cohort study was conducted. Infants with proliferative IH receiving oral propranolol treatment were collected from the Department of Pediatric Surgery, West China Hospital, Sichuan University between June 2015 and May 2019, and their general information and IH-related clinical data were analyzed. The primary outcome was the satisfactory regression rate of IH during 6-12 months of continuous oral propranolol treatment; secondary outcomes included the time to achieve satisfactory regression, incidence of adverse reactions, incidence of IH ulceration, and IH recurrence rate. Multivariate logistic regression was performed to identify factors influencing the satisfactory regression of IH after propranolol treatment, and a receiver operating characteristic (ROC) curve was employed to determine the optimal age for initiating propranolol therapy.Results:A total of 122 IH infants were enrolled in the study, including 32 males (26.2%) and 90 females (73.8%), with ages ( M[ Q1, Q3]) of 8.6 [6.3, 12.3] weeks. IH was located on the head and face in 56 cases (45.9%). There were 57 cases (46.7%) of localized IH, 53 (43.4%) of segmental IH, and 86 (70.5%) of mixed-type IH. Ulceration occurred in 17 cases (13.9%). After 6 months of propranolol treatment, 8 patients (6.6%) experienced treatment failure, and 12 (9.8%) experienced relapse within 6 months after discontinuation of propranolol. During 6 months of oral propranolol treatment, 56 infants (45.9%) experienced mild to moderate adverse reactions, with no drug-related deaths observed. Multivariate logistic regression analysis revealed that the age at initiation of propranolol treatment was an independent factor influencing satisfactory regression of IH ( OR = 0.879, 95% CI: 0.808 - 0.957). ROC curve analysis revealed that the optimal age for starting propranolol therapy was 9.9 weeks, with a sensitivity of 75.7% and a specificity of 61.5%. Infants aged ≤ 9.9 weeks (73 cases) had a significantly higher satisfactory regression rate (72.6% [53/73]) compared with those aged > 9.9 weeks (49 cases, 34.7% [17/49]; χ2 = 17.23, P < 0.001) ; the time to achieve satisfactory regression of IH was significantly shorter in the infants aged ≤ 9.9 weeks ( M[ Q1, Q3]: 46.0 [38.5, 48.0] weeks) than in those aged > 9.9 weeks (57.0 [40.0, 73.5] weeks; Z = -2.01, P = 0.045) . Conclusion:For IH infants requiring systemic therapy, initiation of oral propranolol before the age of 10 weeks appeared to improve the satisfactory regression rate of IH.

OBJECTIVE: To observe the clinical efficacy of warming acupuncture combined with western medication for oligoasthenoteratozoospermia of kidney-yang insufficiency and its effects on the levels of interleukin (IL)-6 and IL-10 in seminal plasma. METHODS: A total of 60 patients with oligoasthenoteratozoospermia of kidney-yang insufficiency were randomly divided into a combination group and a medication group, with 30 cases in each group. The medication group was treated with levocarnitine oral solution orally, 10 mL once, 3 times a day. On the basis of the treatment in the medication group, warming acupuncture was applied at Baihui (GV20), Guanyuan (CV4) and Mingmen (GV4) in the combination group, once every other day, 3 times a week. Both groups were treated for 12 weeks. Before and after treatment, the TCM syndrome score was observed, the semen routine indexes (the sperm concentration, progressive [PR] sperm motility, PR + non-progressive [NP] sperm motility and sperm malformation rate), the serum sex hormones indexes (follicle-stimulating hormone [FSH], luteinizing hormone [LH], testosterone [T] and estradiol [E₂]), as well as the IL-6 and IL-10 levels in seminal plasma were detected, and the clinical efficacy was evaluated after treatment in the two groups. RESULTS: After treatment, except for the hyposexuality score in the medication group, the each item scores and total scores of TCM syndrome were decreased compared with those before treatment (P<0.01, P<0.05), the sperm malformation rates, serum FSH and LH levels, IL-6 levels in the seminal plasma were decreased compared with those before treatment (P<0.01, P<0.05), the PR sperm motility, PR + NP sperm motility, serum T levels, IL-10 levels in the seminal plasma were increased compared with those before treatment (P<0.01, P<0.05) in the two groups; the sperm concentration was increased compared with that before treatment in the combination group (P<0.01). After treatment, compared with the medication group, except for the hyposexuality and frequent nocturia scores, the each item scores and total score of TCM syndrome were lower (P<0.01, P<0.05); the sperm concentration, PR sperm motility and PR + NP sperm motility, serum T level, IL-10 level in the seminal plasma were higher (P<0.01, P<0.05); sperm malformation rate, serum FSH and LH levels, IL-6 level in the seminal plasma were lower (P<0.01, P<0.05) in the combination group. The total effective rate was 83.8% (25/30) in the combination group, which was superior to 60.0% (18/30) in the medication group (P<0.05). CONCLUSION Warming acupuncture combined with western medication can effectively treat oligoasthenoteratozoospermia of kidney-yang insufficiency, regulate the levels of sex hormones, and its mechanism may be related to the down-regulation of IL-6 level and the up-regulation of IL-10 level in seminal plasma.
Humans ; Male ; Interleukin-10/genetics* ; Interleukin-6/genetics* ; Adult ; Semen/metabolism* ; Acupuncture Therapy ; Oligospermia/drug therapy* ; Yang Deficiency/physiopathology* ; Kidney/physiopathology* ; Young Adult ; Asthenozoospermia/drug therapy* ; Combined Modality Therapy ; Treatment Outcome

Jiuwei Xifeng Granules have become a Chinese patent medicine in the market. Because the formula contains Os Draconis, a top-level protected fossil of ancient organisms, the formula was to be improved by replacing Os Draconis with Ostreae Concha. To evaluate whether the improved formula has the same effectiveness and safety as the original formula, a randomized, double-blind, parallel-controlled, equivalence clinical trial was conducted. This study enrolled 288 tic disorder(TD) of children and assigned them into two groups in 1∶1. The treatment group and control group took the modified formula and original formula, respectively. The treatment lasted for 6 weeks, and follow-up visits were conducted at weeks 2, 4, and 6. The primary efficacy endpoint was the difference in Yale global tic severity scale(YGTSS)-total tic severity(TTS) score from baseline after 6 weeks of treatment. The results showed that after 6 weeks of treatment, the declines in YGTSS-TSS score showed no statistically significant difference between the two groups. The difference in YGTSS-TSS score(treatment group-control group) and the 95%CI of the full analysis set(FAS) were-0.17[-1.42, 1.08] and those of per-protocol set(PPS) were 0.29[-0.97, 1.56], which were within the equivalence boundary [-3, 3]. The equivalence test was therefore concluded. The two groups showed no significant differences in the secondary efficacy endpoints of effective rate for TD, total score and factor scores of YGTSS, clinical global impressions-severity(CGI-S) score, traditional Chinese medicine(TCM) response rate, or symptom disappearance rate, and thus a complete evidence chain with the primary outcome was formed. A total of 6 adverse reactions were reported, including 4(2.82%) cases in the treatment group and 2(1.41%) cases in the control group, which showed no statistically significant difference between the two groups. No serious suspected unexpected adverse reactions were reported, and no laboratory test results indicated serious clinically significant abnormalities. The results support the replacement of Os Draconis by Ostreae Concha in the original formula, and the efficacy and safety of the modified formula are consistent with those of the original formula.
Adolescent ; Child ; Child, Preschool ; Female ; Humans ; Male ; Double-Blind Method ; Drugs, Chinese Herbal/therapeutic use* ; Tic Disorders/drug therapy* ; Treatment Outcome