Objective:To establishment an assay for HIV-1 intact proviral DNA assay through droplet digital PCR (ddPCR).Methods:DNA was extracted by culturing 8E5 cells, a Tlymphocyte cell line containing a single copy of integrated HIV-1 provirus. Serial diluting DNA were prepared by amplified 1-fold, 5-fold, 25-fold, 625-fold, 3 125-fold, and 15 625-fold across the HIV-1 Ψ region, env region, and eukaryotic chromosome 10 RPP30 regions, and the linear relationship was calculated and the minimum detection concentration. DNA solution of 5 μl, 3.1 μl, 2.5 μl was added to the ddPCR mixture respectively, with each dilution undergoing two batches of detection, and each was repeated four times. The intra-batch variation coefficient was detected, while the inter-batch variation coefficient was detected by the same DNA amount and different DNA amounts to determine the stability; 8E5 cell was used to detect the intact proviral content in cells.Results:The linear fitting goodness of Ψ region, env region and RPP30 region are R2≥0.999, R2≥0.993, R2≥0.996 in 6 dilutions of DNA, respectively. At a 3 125-fold dilution, the lowest positive droplets were detected in the Ψ region, env region and RPP30 region were 3, 2 and 2, respectively, the detected concentrations were 2.37 copies/μl, 1.21 copies/μl and 1.58 copies/μl. The ddPCR repeatability experimental detecting DNA showed that the Ψ region of the intra-batch variation coefficients ranged from 0.66% to 3.43%, with the inter-batch variation coefficients of the same DNA at 3.19%, 4.3% and 3.45% respectively, and the inter-batch variation coefficients of the different DNA at only 4.35%. The env region of the intra-batch variation coefficients ranged from 0.7% to 3.20%, with the inter-batch variation coefficients of the same DNA at 3.18%, 4.52% and 3.4% respectively, and the inter-batch variation coefficients of the different DNA at only 4.02%. The RPP30 region of the intra-batch variation coefficients ranged from 0.91% to 2.91%, with the inter-batch variation coefficients of the same DNA at 3%, 4.55% and 3.37% respectively, and the inter-batch variation coefficients of the different DNA at only 3.98%. The proportion of 8E5 cells containing defective provirus and the proportion of intact provirus were calculated to be approximately 90% and 45%, respectively. Conclusions:Droplet digital PCR used to detect HIV-1 intact proviral DNA, showed strong stability and provided a technical means for HIV-1 infection reservoir detection.

Objective:To analyze the clinical characteristics and management strategy of carotid web (CW)-related stroke and improve the understanding of CW.Methods:The clinical data of 6 patients with CW-related stroke treated at Xuanwu Hospital, Capital Medical University, from January 2018 to September 2022 were retrospectively analyzed. Relevant data included clinical manifestations, bronchial magnetic resonance imaging (MRI), CT angiography (CTA)/digital subtraction angiography (DSA), carotid ultrasonography, and transcranial color-coded Doppler sonography (TCCD). Treatments and follow-ups were also reported.Results:All 6 patients were middle-aged or young, without traditional risk factors for cerebrovascular disease. The male-to-female ratio was 2∶4. All patients had recurrent strokes, with a score of 2-4 on the National Institutes of Health Stroke Scale at the time of onset. Head MRI showed 5 patients with cerebral infarction in the middle cerebral artery supply area; 1 patient had no intracranial infarction. No significant stenosis of the vessels at the site of the CW was observed in any of the 6 patients. Four patients had ipsilateral stenosis or occlusion of the middle cerebral artery, 1 patient had microembolic signals in the middle cerebral artery, and 1 patient had no significant abnormalities in the intracranial artery. All 6 patients were initially diagnosed by ultrasound. Diagnoses were confirmed by CTA/DSA in 5 cases; the other patient did not show any significant abnormalities. All 6 patients underwent a carotid endarterectomy after a pathological examination to confirm the diagnosis. Postoperatively, regular antiplatelet aggregation and statin lipid-lowering therapies were administered. A follow-up of 0.5 to 5.2 years showed no stroke recurrence in any patient.Conclusions:CW-related stroke is a rare cause of cryptogenic stroke and has a high recurrence rate. For stroke patients who do not have traditional risk factors for cerebrovascular disease while repeatedly experiencing embolic events of the internal carotid artery system, attention should be paid to the combination of the carotid and cerebral arteries, and comprehensive hemodynamic characteristics should be checked for the presence of a CW. Carotid artery ultrasound and TCCD combined with other imaging methods can improve the detection rate of a CW. Simple antiplatelet aggregation therapy cannot effectively prevent stroke recurrence. For patients with confirmed CW-related cerebral infarction, surgical resection can be performed, and the treatment effect can be evaluated using ultrasonography.

Objective:To observe the effect of Tuina(Chinese therapeutic massage)combined with functional training in improving clinical symptoms and balance function in patients with meniscus injury. Methods:A total of 93 patients with grade Ⅰ-Ⅱ meniscus injury were randomly divided into a functional training group,a Tuina group,and a combination group,with 31 cases in each group.The Tuina group received Tuina manipulation.The functional training group underwent functional training under the supervision of physicians.The combination group received Tuina manipulation in addition to the functional training.The treatment course of all three groups was 8 weeks.The clinical symptoms were observed before the intervention and after 4 and 8 weeks of interventions,and the changes in knee injury and osteoarthritis outcome score(KOOS)and Y-balance test(YBT)were compared. Results:There was no statistically significant difference in the total response rate among the three groups after treatment(P>0.05).The total and subscale KOOS in the three groups were significantly higher than those before treatment after 4 and 8 weeks of interventions(P<0.05).There was a statistically significant difference in the total KOOS between the combination group and the functional training group after 4 and 8 weeks of interventions(P<0.05);there was a statistically significant difference in the score of daily living ability and sports and recreation function between the two groups after 8 weeks of interventions(P<0.05).The YBT balance coefficients of the affected and non-affected knees of the three groups were significantly improved after 4 and 8 weeks of interventions(P<0.05),and there was no statistically significant difference among the groups(P>0.05). Conclusion:Tuina,functional training,and their combination can improve clinical symptoms and balance function in patients with grade Ⅰ-Ⅱ meniscus injury.Tuina combined with functional training is significantly effective in improving daily living ability and sports and recreation function.

Objective:To observe the effect of Yi Jin Jing(Sinew-transforming Qigong Exercises)intervention on anxiety in asymptomatic patients with Corona Virus Disease 2019(COVID-19)infection during quarantine.Methods:A total of 160 asymptomatic patients with COVID-19 infection were stratified by gender and divided into an observation group and a control group by the stratified randomization method,with 80 cases in each group.The control group was given basic nursing in the cabin,and the observation group was given additional Yi Jin Jing exercises once a day,20 min each time,and trained continuously until 1 d before leaving the cabin.The Hamilton anxiety scale(HAMA)score was compared between the two groups.Results:A total of 154 cases were included for data analysis in this study,including 74 cases in the observation group and 80 cases in the control group.After intervention,the HAMA scores in both groups increased(P<0.05),while the score in the observation group was lower than that in the control group(P<0.05).In the control group,the HAMA score of females after intervention was higher than that of males.The scores of mental anxiety and somatic anxiety in both groups were higher than those before intervention,while the score of mental anxiety in the observation group was significantly lower than that in the control(P<0.05).However,there was no statistical difference in the somatic score between the two groups(P>0.05).Conclusion:Patients with COVID-19 infection will get anxious during quarantine.Yi Jin Jing exercise can effectively reduce the aggravation of anxiety in asymptomatic patients with COVID-19 infection during concentrated quarantine.

Since microdroplets are able to be generated rapidly in large amount and each droplet can be well controlled as an independent micro-cultivator, droplet microfluidic technology can be potentially used in the culture of microorganisms, and provide the microbial culture with high throughput manner. But its application mostly stays in the laboratory-level building and using for scientific research, and the wide use of droplet microfluidics in microbial technology has been limited by the key problems that the operation for microdroplets needs high technical requirements with wide affecting factors and the difficulties in integration of automatic microdroplet instrumentation. In this study, by realizing and integrating the complicated operations of droplet generation, cultivation, detection, splitting, fusion and sorting, we design a miniaturized, fully automated and high-throughput microbial microdroplet culture system (MMC). The MMC can be widely used in microbial growth curve test, laboratory adaptive evolution, single factor and multi-level analysis of microbial culture, metabolite detection and so on, and provide a powerful instrument platform for customized microbial evolution and screening aiming at efficient strain engineering.
Industrial Development ; Microfluidics

[Abstract] Objective: To investigate the effect of HMGB1 gene on the growth of human epithelial ovarian cancer xenografts in nude mice, and to lay a foundation for finding new targets for the treatment of ovarian cancer. Methods: Human epithelial ovarian cancer SKOV3 cells in logarithmic growth phase were selected to establish a human epithelial ovarian cancer xenograft model in nude mice. Nude mice with successful model establishment were randomly divided into control group and HMGB1-siRNA group. On the 7th, 9th, 11th, 14th, and 16th days after cell inoculation, the same amount of saline and HMGB1-siRNA were respectively injected into two groups of mice under the armpit.After 3 weeks, the nude mice were sacrificed by cervical dislocation, the tumor tissues were separated, and the volume of the tumor was measured. The apoptosis of transplanted tumor cells was detected by Tunnel staining. The expressions of HMGB1, STAT3 and p-STAT3 were detected by Western blotting. The expression of vascular endothelial growth factorA(VEGF-A) and microvascularization were detected by immunohistochemistry. Results: Compared with the control group, the growth of tumor volume slowed down in HMGB1 siRNA group, and on the 21st day, the tumor volume of HMGB1-siRNA group was significantly smaller than that of the control group (P<0.05). HMGB1-siRNA successfully knocked down the expression of HMGB1 mRNA in transplanted tumor tissue. The apoptosis rate of tissue cells in HMGB1-siRNA group was significantly increased ([34±8]% vs [6±2]%, P=0.04), and the expressions of HMGB1 and p-STAT3 were significantly reduced (P<0.05). The expression of VEGF-Aand the number of microvessels were significantly lower than those of the control group (both P<0.05). Conclusion: Knockdown of HMGB1 gene reduces the expression of VEGF-A and microvessel formation possibly by inhibiting the HMGB1/STAT3 signaling pathway, thereby promoting the apoptosis of tumor tissues and slowing the growth of xenografts.

Objective: To investigate the correlation between ultrasonographic features of papillary thyroid carcinoma and central cervical lymph node metastasis. Methods: We retrospectively analyzed 486 patients with papillary thyroid carcinoma(PTC), pathologically confirmed after surgery in Tianjin Medical University Cancer Institute & Hospital. All patients were divided into central cervical lymph node metastasis group and non-metastasis group. No lateral cervical lymph node metastasis was found in preoperative ultrasonography and postoperative pathology. The characteristics of the ultrasound was observed and analyzed. Results: 297 out of 486 patients with papillary thyroid carcinomahad central metastasis, and the other 189 cases did not. Take pathology results as a standard, the sensitivity, specificity, positive predictive value, negative predictive value and accuracy rate of preoperative ultrasound diagnosis in PTC patients with central cervical lymph node metastasis were 35.3%, 88.6%, 83.2%, 47.4%, 56.6%, respectively. Univariate analysis showed that multi-focus, taller-than-wide, diameter>1 cm, located in the lower pole, ill-defined margin, hypoechogenicity, micro-calcification, capsule invasion more than 1/4 perimeter of papillary thyroid carcinoma were significantly associated with central cervical lymph node metastasis (all P<0.05). Multivariate analysis showed that diameter>1 cm, micro-calcification, capsule invasion more than 1/4 perimeter of papillary thyroid carcinoma became independent risk factors of central cervical neck lymph node metastasis (all P<0.05). Conclusions Preoperative description of ultrasonographical features has important value to assess central cervical lymph node metastasis in patients with papillary thyroid carcinoma. More information could be provided for clinical treatment. When the papillary thyroid carcinoma presented as diameter>1 cm, micro-calcification, and capsule invasion more than 1/4 perimeter of, there will be a greater risk of central cervical lymph node metastasis, and we shall suggest prophylactic central lymph cervical node dissection.

Symptomatic venous thromboembolism (VTE) has a six-to seven-fold risk of occurring in cancer patients compared with non-cancer patients. VTE is the second most common cause of death among patients with cancer, and cancer-associated VTE is be-coming increasingly prevalent. Therefore, early diagnosis and treatment of cancer-associated VTE is particularly important. This study presents a pancreatic cancer-associated deep vein thrombosis (DVT) patient who engaged in a multidisciplinary comprehensive discus-sion in the Interventional Therapy Department, Tianjin Medical University Cancer Hospital to enhance concern, interdisciplinary com-munication, and cooperation in terms of cancer-associated VTE diagnosis and treatment strategies.

Objective To investigate the effect of β-asarone on hippocampal neurons of experimental depression rats. Methods Eighty male adult SD rats were randomly divided into 4 groups, namely normal group, model group, fluoxetine(1.2 mg/kg) group and β-asarone (25 mg/kg) group. Depression rat model was established. The medication groups were given corresponding agents respectively. After treatment for 21 days, the number of apoptotic cells and the histological features in the hippocampus of rats were detected by flow cytometry and TUNEL, respectively. Results Compared with the normal group, typical apoptotic cells were found, apoptotic cell count and apoptotic rate were increased in the hippocampal CA3 and DG area of the model group (P<0.01). Compared with the model group, apoptotic cell count and apoptotic rate were decreased in the hippocampal area ofβ-asarone group and fluoxetine group (P<0.01). Conclusion The protective mechanism of β-asarone for the hippocampal cells of depression rats is probably associated with the reduction of the apoptosis of hippocampal neurons.

Objective To investigate the effect of the polymorphisms of multidrug resistance 1 (MDR1) C3435T and G2677T on Tacrolimus (Tac) individualized treatment and prognosis of grafts in the renal transplantation recipients (RTRs).Methods One hundred and twenty-seven RTRs who treated with Tac regimen and had a stable graft function were enrolled,and were divided into adjuvant treatment group and non-adjuvant treatment group according to whether given adjuvant drugs to raise Tac trough concentrations. MDR1 C3435T and G2677T SNPs were detected by using sequence specific primers PCR.Tac trough concentrations of whole blood were measured by using enzymelabeled immunosorbent assay.Tac concentration-to-dose ratio (C/D) standardized by body weight was compared according to the various genotypes and haplotypes of MDR1 C3435T and G2677TA SNPs.Results Adjuvant treatment group including 36 recipients had a higher frequency of C genotype of C3435T than un-adjuvant treatment group (68.05％ vs 48.35％,P ＜ 0.01 ). The frequency of G2677TA polymorphisms was of no significant difference between the two group recipients (P＞ 0.05).As to non-adjuvant treatment recipients,the mean Tac DD required and C/D were not significantly different among various polymorphisms of MDR1 G2677T/A and C3435T or various haplotypes (P＞0.05).During A follow-up period of 4 years,13 recipients suffered graft dysfunction in which 84.6％ (11/13) carried 3435C genotype (P＞0.05).Conclusion The frequency of MDR1 C3435T polymorphisms in RTRs is high in the recipients given adjuvant treatment to raise Tac concentrations.Recipients with 3435C genotype were prone to graft dysfunction.