一种HBV DNA定量检测试剂的准确性分析

目的评价一种新型HBV DNA定量检测试剂的准确性。方法将第3代HBV DNA WHO国际标准品稀释为6种不同梯度浓度样本,8.5×10~5、8.5×10~4、8.5×10~3、8.5×10~2、85及42.5 IU/ml,用待评价试剂care HBV PCR Assay V3.0检测,并对实测值和理论值进行相关性分析;以Cobas Taqman HBV V2.0为参比试剂,用care HBV PCR Assay V3.0检测93份HBV感染者标本和30份健康者标本HBV DNA含量,并对2种试剂定量检测结果进行相关性和一致性分析;采用巢式PCR扩增漏检标本的HBV S区,直接测序法测定其基因型。结果 care HBV PCR Assay V3.0检测WHO国际标准品的实测值和理论值具有线性相关;2种定量试剂检测93份HBV感染者标本结果均阳性共68份,检测结果呈线性相关,差异无统计学意义(P>0.05),但care HBV PCR Assay V3.0检测值相对偏低;care HBV PCR Assay V3.0检测有14份标本漏检,其中13份标本Cobas Taqman HBV V2.0检测结果位于30~2 000 IU/ml间;对1份漏检标本的基因型检测结果为C型。结论 care HBV PCR Assay V3.0与Cobas Taqman HBV V2.0试剂检测结果有较好的相关性,但对基因型C型的标本可能存在漏检现象。

脐带间充质干细胞增强无精子症模型睾丸特异性基因表达

在生殖男科领域，探索人脐带间充质干细胞（HUCMSCs）对无精子症的治疗作用。HUCMSCs具有潜在的免疫抑制功能，并能够分泌多种细胞因子和生长因子，因此具有潜在的临床应用价值。作为探索，我们移植HUCMSCs进入无精子症小鼠睾丸间质，检测是否能够促进精子发生过程。从不同来源的脐带中分离HUCMSCs，移植进入白消安处理的小鼠睾丸间质中，采用注射生理盐水和HEK293细胞作为对照，对侧睾丸不注射。三周之后，RT-PCR检测10个生殖细胞特异性表达的基因，并检N3个特异性蛋白的表达。结果表明，注射人脐带间充质干细胞之后的表达明显高于对照组，证实生殖细胞特异性基因的表达上调，从而表明HUCMSCs对睾丸生精功能的恢复具有促进作用，为治疗无精子症探索一条新的途径。

NF-E2:a Novel Regulator of Alpha-hemoglobin Stabilizing Protein Gene Expression

Objective To investigate whether α-hemoglobin stabilizing protein (AHSP), the α-globin-specific molecular chaperone, is regulated by erythroid transcription factor NF-E2. Methods We established the stable cell line with NF-E2p45 (the larger subunit of NF-E2) short hairpin RNA to silence its expression. Western blot, real-time polymerase chain reaction, and chromatin immunoprecipitation (ChIP) analysis were performed to detect the expression of AHSP, the histone modifications at AHSP gene locus, and the binding of GATA-1 at the AHSP promoter with NF-E2p45 deficiency. ChIP was also carried out in dimethyl sulfoxide (DMSO)-induced DS19 cells and estrogen-induced G1E-ER4 cells to examine NF-E2 binding to the AHSP gene locus and its changes during cell erythroid differentiation. Finally, luciferase assay was applied in HeLa cells transfected with AHSP promoter fragments to examine AHSP promoter activity in the presence of exogenous NF-E2p45. Results We found that AHSP expression was highly dependent on NF-E2p45. NF-E2 bound to the regions across AHSP gene locus in vivo, and the transcription of AHSP was transactivated by exogenous NF-E2p45. In addition, we observed the decrease of H3K4 trimethylation and GATA-1 occupancy at the AHSP gene locus in NF-E2p45-deficient cells. Restoration of GATA-1 in G1E-ER4 cells in turn led to increased DNA binding of NF-E2p45. Conclusion NF-E2 may play an important role in AHSP gene regulation, providing new insights into the molecular mechanisms underlying the erythroid-specific expression of AHSP as well as new possibilities for β-thalassemia treatment.

Lysine-specific Demethylase 1 Represses THP-1 Monocyte-to-macrophage Differentiation

Objective To investigate the role of lysine-specific demethylase 1 (LSD1) in the process of THP-1 monocyte-to-macrophage differentiation.Methods Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blotting were performed to analyze the expression of LSD1 and interleukin-6 (IL-6) in THP-1 monocytes and THP-1-derived macrophages.Chromatin immunoprecipitation (ChIP) assay was applied to detect the occupancy of LSD1 and H3K4 methylation at IL-6 promoter during THP-1 monocyte-to-macrophage differentiation.IL-6 mRNA level and H3K4 methylation at IL-6 promoter were analyzed using qRT-PCR and ChIP assay in LSD1-knockdown THP-1 cells treated with 12-O-tetradecanoylphorbol-13-acetate (TPA) for 0,4,8,12,and 24 hours.Fluorescence activated flow cytometry was performed to reveal the percentage of macrophages differentiated from THP-1 monocytes.Results The expression of LSD1 reduced during THP-1 monocyte-to-macrophage differentiation (P<0.01).LSD1 occupancy decreased and H3K4 methylation increased at IL-6 promoter during the differentiation.With knockdown of LSD1,H3K4 methylation at IL-6 promoter was found increased after TPA treatment at different times points (all P<0.05,except 24 hours).The percentage of macrophages increased significantly in the THP-1 cells with LSD1 knockdown (P<0.05).Conclusions LSD1 is repressed during the monocyte-to-macrophage differentiation of THP-1 cells.Suppression of LSD1-mediated H3K4 demethylation may be required for THP-1 monocyte-to-macrophage differentiation.

An Ideal Hallmark Closest to Complete Cure of Chronic Hepatitis B Patients:High-sensitivity Quantitative HBsAg Loss

In the era of antiviral therapy,the main goal of treatment has shifted from the persistent inhibition of hepatitis B virus(HBV)replication to the pursuit of serological clearance of HBs surface antigen(HBsAg).Based on the life cycle of HBV,HBsAg originates from covalently closed circular DNA(cccDNA)and integrated HBV DNA,thus reflecting their transcriptional activity.Complete HBsAg loss may mean elimination or persistent inactivity of the HBV genome including cccDNA and integrated HBV DNA.HBsAg loss improves the recovery of abnormal immune function,which in turn,may further promote the clearance of residual viruses.Combined with functional cure and the great improvement of clinical outcomes,the continuous seroclearance of high-sensitivity quantitative HBsAg may represent the complete cure of chronic hepatitis B(CHB).For many other risk factors besides HBV itself,patients with HBsAg loss still need regular monitoring.In this review,we summarized the evolution of CHB treatment,the origin of serum HBsAg,the pattern of HBsAg seroclearance,and the effect of HBsAg loss on immune function and disease outcomes.In addition,we discuss the significance of high-sensitivity HBsAg detection and its possibility as a surrogate of complete cure.

Postoperative subacute static progressive stretch does not increase the risk of distal lower limb venous thromboembolism

Purpose:Static progressive stretch(SPS)can be applied to treat chronic joint stiffness.However,the impacts of subacute application of SPS to the distal lower limbs,where deep vein thrombosis(DVT)is common,on venous thromboembolism remain unclear.This study aims to explore the risk of venous thromboembolism events following subacute application of SPS.Methods:A retrospective cohort study was conducted on patients diagnosed with DVT following a lower extremity orthopedic surgery before being transferred to the rehabilitation ward from May 2017 to May 2022.Patients with unilateral lower limb comminuted para-articular fractures,transferred to rehabilitation ward for further treatment within 3 weeks after operation,followed up more than 12 weeks since initial manual physiotherapy,and diagnosed DVT by ultrasound before rehabilitation course were included in the study.Patients with polytrauma,without evidence of previous peripheral vascular disease or incompetence,had medication for thrombosis treatment or prophylaxis before the operation,detected with paralysis due to nervous system impairment,infected after operation during the regime,or with acute progression of DVT were excluded.The included patients were randomized to the standard physiotherapy and the SPS integrated groups for observation.Associated DVT and pulmonary embolism data were collected during the physiotherapy course to compare the groups.SSPS 28.0 and GraphPad Prism 9 were used for data processing.Ap<0.05 was set significant difference.Results:In total of 154 patients with DVT participating in this study,75 of them were treated with additional SPS for postoperative rehabilitation.The participants in the SPS group showed improved range of motion(12.3°±6.7°).However,in the SPS group,there was no difference in thrombosis volume between the start and termination(p=0.106,p=0.787,respectively),although difference was seen intra-therapy(p<0.001).Contingency analysis revealed the pulmonary embolism incidence(OR=0.703)in the SPS group compared to the mean physiotherapy.Conclusion:The SPS technique is a safe and reliable option to prevent potential joint stiffness without aggravating the risk of distal DVT for postoperative patients suffering from relevant trauma.

Impact of hepatitis C virus genotype 3 on liver disease progression in a Chinese national cohort

Background:Hepatitis C virus(HCV)genotype 3,particularly subtype 3b,is increasing in prevalence and distribution in China.This study evaluated the prevalence,regional distribution,clinical characteristics,host factors,treatment outcomes,and disease progression of patients with HCV genotype 3 in China.Methods:A 5-year follow-up was preceded by a cross-sectional study.Treatment choices were at the discretion of treating physicians.Estimated infection time to overall-disease-progression(defined by≥1 of:newly diagnosed cirrhosis;cirrhosis at baseline,Child-Turcotte-Pugh score increased 2 points or more;progression from compensated cirrhosis to decompensated cirrhosis;hepatocellular carcinoma;liver transplantation;or death)was calculated using the Kaplan-Meier method.Cox regression analyses were conducted to evaluate the risk factors for disease progression.Results:The cross-sectional study enrolled 997 patients,including 91 with HCV genotype 3 infection.Among them,subtype 3b(57.1%)was more dominant than subtype 3a(38.5%).Five hundred and twelve patients were included into the follow-up phase.Among patients analyzed for estimated infection time to overall-disease-progression,52/304(17.1%)patients with HCV genotype 1 and 4/41(9.8%)with HCV genotype 3(4/26 with genotype 3b,0/13 with genotype 3a,and 0/2 with undefined subtype of genotype 3)experienced overall-disease-progression.Patients with HCV genotype 3 were younger than those with genotype 1(mean age:39.5±8.7 vs.46.9±13.6 years)and demonstrated more rapid disease progression(mean estimated infection time to overall-disease-progression 27.1 vs.35.6 years).Conclusions:HCV genotype 3,specifically subtype 3b,is associated with more rapid progression of liver disease.Further analysis to compare HCV subtype 3a and 3b is needed in high prevalence regions.

HBcrAg Identifies Patients Failing to Achieve HBeAg Seroconversion Treated with Pegylated Interferon Alfa-2b

Background： We aimed to evaluate the usefulness of serum hepatitis B virus core-related antigens （HBcrAg） for predicting hepatitis B e antigen （HBeAg） seroconversion in HBeAg-positive chronic hepatitis B patients treated with conventional interferon （IFN） alfh-2b or pegylated IFN. Methods： Fifty-eight patients were enrolled： 29 for the training group and 29 for the validating group. HBcrAg was measured at baseline, week 12, end of the treatment, and 12- and 24-week follow-ups. Sixteen patients in the training group were enrolled in the long-term follow-up （LTFU）, during which time the dynamics of the HBcrAg was monitored. Results： The serum HBcrAg level gradually declined during treatment among the HBeAg seroconversion patients of the training group （from baseline, week 12, end of the treatment, 12-week follow-up to 24-week follow-up were II0,245 kU/ml, 3760 kU/ ml, 7410 kU/ml, 715 kU/ml, 200 kU/ml, respectively）. HBcrAg 〈19,565 kU/ml at week 24, HBcrAg 〈34,225 kU/ml at 12-week follow-up, and HBcrAg decrease 〉0.565 log10 kU/ml from the baseline to the end of treatment （EOT） had negative predictive values （NPVs） of 100% for HBeAg seroconversion at the end of follow-up, whereas the positive predictive values （PPVs） were 30.77%, 26.67%, and 25.00%, respectively. The patients with HBeAg seroconversion at the end of 24-week follow-up remained in seroconversion during the LTFU, during which time their serum HBcrAg levels steadily declined or even became undetectable, ranging from 0 to 2.1 kU/ml. Conclusions： Effective antiviral treatment can decrease HBcrAg levels in the serum. The NPVs of HBcrAg for predicting HBeAg seroconversion at 24-week follow-up was 100%, but the PPVs were not satisfactory （all 〈31%）. The serum HBcrAg levels of the patients with HBeAg seroconversion at the end of the 24-week follow-up steadily declined or even became undetectable during the LTFU.