呼吸道合胞病毒IgM抗体AlphaLISA快速检测方法的建立

呼吸道合胞病毒(Respiratory syncytial virus,RSV)属于副粘液病毒科,是一种下呼吸道感染最主要的RNA病毒.呼吸道合胞病毒感染是引起全球婴幼儿高致死率的呼吸道感染病原,仅次于疟疾,但用于检测RSV感染的选择相对较少.本文通过抗原抗体结合原理,建立呼吸道合胞病毒(RSV)IgM抗体AlphaLISA快速检测方法.小鼠抗人IgM单克隆抗体偶联的受体微球与临床血清样品中RSV特异性IgM抗体结合,RSV特异性IgM抗体再与生物素标记的RSV抗原结合,生物素连接偶联链霉亲和素的供体微球;受体微球和供体微球的距离被拉近小于200nm,680nm荧光激发供体微球生成单线态氧,扩散给受体微球,发射波长为520～620nm的荧光,荧光强度与血清中RSV特异性IgM抗体呈正比.结果显示,该方法批内变异系数与批间变异系数均小于10％,不与其他呼吸道病原体发生交叉反应,与间接免疫荧光法具有较好的一致性,总符合率达83.33％.该方法具有微量检测、快速省时、操作简便等优势,可快速检测RSV的IgM抗体,为早期确诊RSV感染提供高效可行方案.     

SHED aggregate exosomes shuttled miR‐26a promote angiogenesis in pulp regeneration via TGF‐β/SMAD2/3 signalling

ObjectivesPulp regeneration brings big challenges for clinicians, and vascularization is considered as its determining factor. We previously accomplished pulp regeneration with autologous stem cells from deciduous teeth (SHED) aggregates implantation in teenager patients, however, the underlying mechanism needs to be clarified for regenerating pulp in adults. Serving as an important effector of mesenchymal stem cells (MSCs), exosomes have been reported to promote angiogenesis and tissue regeneration effectively. Here, we aimed to investigate the role of SHED aggregate‐derived exosomes (SA‐Exo) in the angiogenesis of pulp regeneration.Materials and MethodsWe extracted exosomes from SHED aggregates and utilized them in the pulp regeneration animal model. The pro‐angiogenetic effects of SA‐Exo on SHED and human umbilical vein endothelial cells (HUVECs) were evaluated. The related mechanisms were further investigated.ResultsWe firstly found that SA‐Exo significantly improved pulp tissue regeneration and angiogenesis in vivo. Next, we found that SA‐Exo promoted SHED endothelial differentiation and enhanced the angiogenic ability of HUVECs, as indicated by the in vitro tube formation assay. Mechanistically, miR‐26a, which is enriched in SA‐Exo, improved angiogenesis both in SHED and HUVECs via regulating TGF‐β/SMAD2/3 signalling.ConclusionsIn summary, these data reveal that SA‐Exo shuttled miR‐26a promotes angiogenesis via TGF‐β/SMAD2/3 signalling contributing to SHED aggregate‐based pulp tissue regeneration. These novel insights into SA‐Exo may facilitate the development of new strategies for pulp regeneration.     

Extensive profiling of the expressions of tRNAs and tRNA-derived fragments (tRFs) reveals the complexities of tRNA and tRF populations in plants

Evidence is emerging that t RNA-derived fragments(t RFs) are regulatory molecules. Studies of t RFs in plants have been based on conventional small RNA sequencing, and focused on profiling of t RF-5 and t RF-3 species. A more comprehensive and quantitative analysis of the entire t RF population is highly necessary. Here, we employ t RNA-seq and YAMAT-seq, and develop a bioinformatics tool to comprehensively profile the expressions of t RNAs and t RFs in plants. We show that in Arabidopsis,approximately half of t RNA genes are extremely weakly expressed, accounting for only 1% of total t RNA abundance, while～12% of t RNA genes contribute to ～80% of t RNA abundance. Our t RNA sequencings in various plants reveal that t RNA expression profiles exhibit a cross-species conserved pattern. By characterizing the composition of a highly heterogeneous t RF population, we show that t RNA halves and previously unnoticed 10–16-nt tiny t RFs represent substantial portions. The highly accumulated 13-nt and 16-nt tiny t RFs in Arabidopsis indicate that tiny t RFs are not random t RNA degradation products. Finally,we provide a user-friendly database for displaying the dynamic spatiotemporal expressions of t RNAs and t RFs in the model plants Arabidopsis and rice.     

不稳定EGFP细胞模型的构建及其在基因编辑体系评价中的应用*

目的:为了更好地评价基因编辑效率,满足高通量筛选应用中快速、高效的检测要求,在细胞上建立一个原位检测方法具有重要的意义。通过检测荧光蛋白信号强度的变化可以评价CRISPR系统在细胞中的基因编辑情况,然而这一方法的效率受限于荧光蛋白较长的半衰期。方法:将鸟氨酸脱羧酶降解结构域(含PEST序列)与EGFP融合,通过慢病毒系统感染HEK-293T细胞,获得了表达单拷贝、EGFP-PEST报告基因的稳转细胞系。结果:与EGFP相比,EGFP-PEST在细胞内的降解速度明显加快,荧光水平在4 h内显著降低。利用该模型比较了3种商品化脂质体介导的CRISPR/Cas9基因编辑效率,能够在2~4 d实现定性和定量评价。结论:这一模型能够快速、灵敏地指示基因编辑效果,可以用于不同CRISPR系统或新递送工具的高通量筛选和评价。     

环磷酰胺辅助化疗对不同年龄乳腺癌患者卵巢功能的损伤情况及机制研究

目的:以月经变化情况及血清激素水平为观察指标,探讨环磷酰胺辅助化疗方案对绝经前不同年龄段乳腺癌患者卵巢功能的损伤情况及可能的机制研究。方法:收集2017年1月至2018年12月期间在我院接受环磷酰胺辅助化疗的绝经前乳腺癌患者77例。患者年龄范围为26-48岁,并按年龄分为两组:≤35岁组为16例;>35岁组为61例。调查研究两组患者化疗前后的月经情况,以及血清激素水平(E、FSH、AMH)。同时进行动物实验,采用单剂量环磷酰胺(200 mg/kg)处理不同月龄组(3月龄和6月龄)雌性C57BL/6J小鼠,每组小鼠12例。在环磷酰胺处理前及处理后一周,检测小鼠血清AMH水平,计数小鼠卵巢卵泡数量。结果:环磷酰胺化疗后,>35岁组与≤35岁组间比较,>35岁组闭经率更高,复经率更低,血清AMH和E2水平下降幅度更大(P<0.05),FSH水平升高幅度也大于≤35岁组,但差异无明显统计学意义(P>0.05)。不同年龄组内比较,≤35岁组患者化疗后的血清AMH和E2下降水平无明显差异(P>0.05);血清FSH水平明显升高(P<0.05)。而>35岁组的患者化疗后,血清AMH和E2水平明显下降,FSH水平明显升高,差异均有统计学意义(P<0.05)。不同月龄组小鼠环磷酰胺处理前后,血清AMH水平明显下降,卵巢原始卵泡和生长卵泡数明显较少,差异均有统计学意义(P<0.05),且6月龄组小鼠下降程度更明显(P<0.05)。结论:环磷酰胺辅助化疗方案明显损伤患者卵巢功能,且对年龄越大者损伤程度越严重,更易导致不可逆性卵巢损伤。     

Comprehensive landscape and interference of clonal haematopoiesis mutations for liquid biopsy: A Chinese pan-cancer cohort

Tumour-derived DNA found in the plasma of cancer patients provides the probability to detect somatic mutations from circulating cell-free DNA (cfDNA) in plasma samples. However, clonal hematopoiesis (CH) mutations affect the accuracy of liquid biopsy for cancer diagnosis and treatment. Here, we integrated landscape of CH mutations in 11,725 pan-cancer patients of Chinese and explored effects of CH on liquid biopsies in real-world. We first identified 5933 CHs based on panel sequencing of matched DNA of white blood cell and cfDNA on 301 genes for 5100 patients, in which CH number of patients had positive correlation with their diagnosis age. We observed that canonical genes related to CH, including DNMT3A, TET2, ASXL1, TP53, ATM, CHEK2 and SF3B1, were dominant in the Chinese cohort and 13.29% of CH mutations only appeared in the Chinese cohort compared with the Western cohort. Analysis of CH gene distribution bias indicated that CH tended to appear in genes with functions of tyrosine kinase regulation, PI3K-Akt signalling and TP53 activity, suggesting unfavourable effects of CH mutations in cancer patients. We further confirmed effect of driver genes carried by CH on somatic mutations in liquid biopsy of cancer patients. Forty-eight actionable somatic mutations in 17 driver genes were considered CH genes in 92 patients (1.80%) of the Chinese cohort, implying potential impacts of CH on clinical decision-making. Taken together, this study exhibits strong evidence that gene mutations from CH interfere accuracy of liquid biopsies using cfDNA in cancer diagnosis and treatment in real-world.     

Microscale grooves regulate maturation development of hPSC-CMs by the transient receptor potential channels (TRP channels)

The use of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) is limited in drug discovery and cardiac disease mechanism studies due to cell immaturity. Micro-scaled grooves can promote the maturation of cardiomyocytes by aligning them in order, but the mechanism of cardiomyocytes alignment has not been studied. From the level of calcium activity, gene expression and cell morphology, we verified that the W20H5 grooves can effectively promote the maturation of cardiomyocytes. The transient receptor potential channels (TRP channels) also play an important role in the maturation and development of cardiomyocytes. These findings support the engineered hPSC-CMs as a powerful model to study cardiac disease mechanism and partly mimic the myocardial morphological development. The important role of the TRP channels in the maturation and development of myocardium is first revealed.     

Loss of longitudinal superiority marks the microarchitecture deterioration of osteoporotic cancellous bones

Biomechanics and Modeling in Mechanobiology - Osteoporosis (OP), a skeletal disease making bone mechanically deteriorate and easily fracture, is a global public health issue due to its high...