Chronic PKA phosphorylation of ryanodine receptor 2 (RyR2) has been shown to increase diastolic sarcoplasmic reticulum (SR) Ca2+ leakage and lead to cardiac dysfunction. We hypothesize that intracellular gene delivery of an RyR2-targeting phosphorylation site-specific nanobody could preserve the contractility of the failing myocardium. In the present study, we acquired RyR2-specific nanobodies from a phage display library that were variable domains of Camelidae heavy chain-only antibodies. One of the nanobodies, AR185, inhibited RyR2 phosphorylation in vitro and was chosen for further investigation. We investigated the potential of adeno-associated virus (AAV)9-mediated cardiac expression of AR185 to combat postischemic heart failure (HF). AAV gene delivery elevated the intracellular expression of the AR185 protein in a rat model of ischemic HF, and this treatment normalized the systolic and diastolic dysfunction of the failing myocardium in vivo by reversing myocardial Ca2+ handling. Furthermore, AR185 gene transfer to failing cardiomyocytes reduced the frequency of SR calcium leaks, thereby restoring the attenuated intracellular calcium transients and SR calcium load. Moreover, AR185 gene transfer inhibited the PKA-mediated phosphorylation of RyR2 in failing cardiomyocytes. Our results provide preclinical experimental evidence that the cardiac expression of RyR2 nanobodies with AAV9 vectors is a promising therapeutic strategy for HF.-Li, T., Shen, Y., Lin, F., Fu, W., Liu, S., Wang, C., Liang, J., Fan, X., Ye, X., Tang, Y., Ding, M., Yang, Y., Lei, C., Hu, S. Targeting RyR2 with a phosphorylation site-specific nanobody reverses dysfunction of failing cardiomyocytes in rats.

Chronic PKA phosphorylation of ryanodine receptor 2 (RyR2) has been shown to increase diastolic sarcoplasmic reticulum (SR) Ca2+ leakage and lead to cardiac dysfunction. We hypothesize that intracellular gene delivery of an RyR2-targeting phosphorylation site-specific nanobody could preserve the contractility of the failing myocardium. In the present study, we acquired RyR2-specific nanobodies from a phage display library that were variable domains of Camelidae heavy chain-only antibodies. One of the nanobodies, AR185, inhibited RyR2 phosphorylation in vitro and was chosen for further investigation. We investigated the potential of adeno-associated virus (AAV)9-mediated cardiac expression of AR185 to combat postischemic heart failure (HF). AAV gene delivery elevated the intracellular expression of the AR185 protein in a rat model of ischemic HF, and this treatment normalized the systolic and diastolic dysfunction of the failing myocardium in vivo by reversing myocardial Ca2+ handling. Furthermore, AR185 gene transfer to failing cardiomyocytes reduced the frequency of SR calcium leaks, thereby restoring the attenuated intracellular calcium transients and SR calcium load. Moreover, AR185 gene transfer inhibited the PKA-mediated phosphorylation of RyR2 in failing cardiomyocytes. Our results provide preclinical experimental evidence that the cardiac expression of RyR2 nanobodies with AAV9 vectors is a promising therapeutic strategy for HF.-Li, T., Shen, Y., Lin, F., Fu, W., Liu, S., Wang, C., Liang, J., Fan, X., Ye, X., Tang, Y., Ding, M., Yang, Y., Lei, C., Hu, S. Targeting RyR2 with a phosphorylation site-specific nanobody reverses dysfunction of failing cardiomyocytes in rats.

Abstract Purpose: Both EGFR and PI3K-Akt signaling pathways have been used as therapeutically actionable targets, but resistance is frequently reported. In this report, we show that enrichment of the cancer stem cell (CSC) subsets and dysregulation of Notch signaling underlie the challenges to therapy and describe the development of bispecific antibodies targeting both HER and Notch signaling. Experimental Design: We utilized cell-based models to study Notch signaling in drug-induced CSC expansion. Both cancer cell line models and patient-derived xenograft tumors were used to evaluate the antitumor effects of bispecific antibodies. Cell assays, flow cytometry, qPCR, and in vivo serial transplantation assays were employed to investigate the mechanisms of action and pharmacodynamic readouts. Results: We found that EGFR/Notch targeting bispecific antibodies exhibited a notable antistem cell effect in both in vitro and in vivo assays. Bispecific antibodies delayed the occurrence of acquired resistance to EGFR inhibitors in triple-negative breast cancer cell line–based models and showed efficacy in patient-derived xenografts. Moreover, the EGFR/Notch bispecific antibody PTG12 in combination with GDC-0941 exerted a stronger antitumor effect than the combined therapy of PI3K inhibitor with EGFR inhibitors or tarextumab in a broad spectrum of epithelial tumors. Mechanistically, bispecific antibody treatment inhibits the stem cell–like subpopulation, reduces tumor-initiating cell frequency, and downregulates the mesenchymal gene expression. Conclusions: These findings suggest that the coblockade of EGFR and Notch signaling has the potential to increase the response to PI3K inhibition, and PTG12 may gain clinical efficacy when combined with PI3K blockage in cancer treatment.

Genetically engineered T cells expressing a chimeric antigen receptor (CAR) are rapidly emerging a promising new treatment for haematological and non-haematological malignancies. CAR-T therapy can induce rapid and durable clinical responses but is associated with unique acute toxicities. Moreover, CAR-T cells are vulnerable to immunosuppressive mechanisms. Here, we report that CAR-T cells release extracellular vesicles, mostly in the form of exosomes that carry CAR on their surface. The CAR-containing exosomes express a high level of cytotoxic molecules and inhibit tumour growth. Compared with CAR-T cells, CAR exosomes do not express Programmed cell Death protein 1 (PD1), and their antitumour effect cannot be weakened by recombinant PD-L1 treatment. In a preclinical in vivo model of cytokine release syndrome, the administration of CAR exosomes is relatively safe compared with CAR-T therapy. This study supports the use of exosomes as biomimetic nanovesicles that may be useful in future therapeutic approaches against tumours.

Post-ischaemic heart failure is a major cause of death worldwide. Reperfusion of infarcted heart tissue after myocardial infarction has been an important medical intervention to improve outcomes. However, disturbances in Ca2+ and redox homeostasis at the cellular level caused by ischaemia/reperfusion remain major clinical challenges. In this study, we investigated the potential of adeno-associated virus (AAV)-9-mediated cardiac expression of a Type-2 ryanodine receptor (RyR2) degradation-associated gene, Presenilin 1 (PSEN1), to combat post-ischaemic heart failure. Adeno-associated viral PSEN1 gene delivery elevated PSEN1 protein expression in a post-infarction rat heart failure model, and this administration normalised the contractile dysfunction of the failing myocardium in vivo and in vitro by reversing myocardial Ca2+ handling and function. Moreover, PSEN1 gene transfer to failing cardiomyocytes reduced sarcoplasmic reticulum (SR) Ca2+ leak, thereby restoring the diminished intracellular Ca2+ transients and SR Ca2+ load. Moreover, PSEN1 gene transfer reversed the phosphorylation of RyR2 in failing cardiomyocytes. However, selective autophagy inhibition did not prevent the PSEN1-induced blockade of RyR2 degradation, making the participation of autophagy in PSEN1-associated RyR2 degradation unlikely. Our results established a role of the cardiac expression of PSEN1 with AAV9 vectors as a promising therapeutic approach for post-ischaemic heart failure.

Transmission electron microscope (TEM) image plays an important role in the researches of biological tissue and cell morphology.High quality ultrathin slice staining is the key factor in TEM sample preparation.Aiming at the problems in ultrathin section batch staining work,a new type of grids fixing equipment depicted in this paper is developed to overcome the inconvenience of the traditional instrument.It can improve the quality and efficiency of batch staining.

Aiming at solving the management problems in electron microscopy(EM) analysis work,including separation of sample information and relevant electron microscope image,the traceable EM image management and retrieval information system depicted in this paper is developed to overcome the inconvenience of getting results and making image transferring and tracing.It can realize the comprehensive management and traceable management of Electron microscopy images for experimental information,sample data and electron microscopy images,real-time transmission of result images,and traceability of electron microscopy image by content-based image retrieval algorithm.

Epidermal growth factor receptor (EGFR) blockade and radiation are efficacious in the treatment of cancer, but resistance is commonly reported. Studies have suggested that dysregulation of Notch signaling and enrichment of the cancer stem cell population underlie these treatment challenges. Our data show that dual targeting of EGFR and Notch2/3 receptors with antibody CT16 not only inhibited signaling mediated by these receptors but also showed a strong anti-stem cell effect both in vitro and in vivo. Treatment with CT16 prevented acquired resistance to EGFR inhibitors and radiation in non-small cell lung cancer (NSCLC) cell line models and patient-derived xenograft tumors. CT16 also had a superior radiosensitizing impact compared with EGFR inhibitors. CT16 in combination with radiation had a larger antitumor effect than the combination of radiation with EGFR inhibitors or tarextumab. Mechanistically, CT16 treatment inhibits the stem cell-like subpopulation, which has a high mesenchymal gene expression and DNA repair activity, and reduces tumor-initiating cell frequency. This finding highlights the capacity of a combined blockade of EGFR and Notch signaling to augment the response to radiation and suggests that CT16 may achieve clinical efficacy when combined with radiation in NSCLC treatment.

Although gold nanorods ( AuNRs) have been widely used in biomedicine field due to their many special properties, their potential biological risks are still controversial. In this study, the author investigated the molecular mechanism of autophagy induced by AuNRs in A549 cells using laser scanning confocal microscopy, western blotting and other molecular biological methods from the perspective of cellular oxidative stress. The results showed that A549 cells treated with 4μg·mL-1 of AuNRs for 6 hours could increase the expression of autophagy marker protein LC3 - II and induce LC3 protein transferred from the nucleus to the cytoplasm which promote the formation of autophagy vesicles. Further studies showed that AuNRs can reduce the mitochondrial membrane potential, ATP content, UCP2 protein expression level, cell antioxidant capacity and increase ROS accumulation in A549 cells, which maybe eventually lead to autophagy. 10 mmol·L-1 antioxidant NAC could reverse the changes of mitochondria, cell function and inhibit the occurrence of autophagy. This study provided a strong experimental evidence for the further understanding of their biological risks and possible mechanisms.

生物医学透射电镜已越来越广泛地应用到各个医学及科研领域，透射电镜技术实验课教学也显得尤为重要，为了能够培养出更多实验能力较强、操作技能扎实的学生，实验教学的改革是必然的。

The human epidermal growth factor receptor (EGFR) targeting chimeric monoclonal antibody, cetuximab (Erbitux®), is a widely used drug in the treatment of metastatic colorectal cancer. However, the activation of the extensive crosstalk among the EGFR family receptors as well as other tyrosine kinase receptors (RTKs) impairs the efficacy of the drug by fueling acquired resistance. To identify the responsible potential activation pathway underlying cetuximab resistance and generate novel treatment strategies, cetuximab-resistant colorectal cancer cell lines were generated and validated and a functional RNAi screen targeting human RTKs was used to identify extensive receptor tyrosine kinase signaling networks established in resistant cancer cells. MET, Axl, and IGF-1R were identified as contributors to the acquired resistance to cetuximab. Targeting vectored immunoprophylaxis (VIPs) to different RTKs were generated and characterized. Different VIP approaches were evaluated in vivo with parental and cetuximab-resistance xenografts and the RTKs in resistant cancer xenografts were inhibited with VIPs via re-sensitization to cetuximab treatment. Combination of VIPs was more broadly efficacious, mechanistically, due to co-blocking the EGFR/Axl/MET signaling pathway, which was cross-activated in the resistant cell lines. Moreover, a VIP-based procedural treatment strategy not only eliminated the tumor but also afforded long-lasting protection from tumor recurrence and resistance. Overall, EGFR-related RTK pathway-network activation represents a novel mechanism underlying cetuximab resistance. A broad VIP combination strategy and VIP-based procedural treatment strategy may be a recommended addition to cetuximab-based targeted therapy. Our results establish a new principle to achieve combined RTK inhibition and reverse drug resistance using a VIP approach.

目的:观察破壁灵芝孢子粉对人肺癌细胞(A549细胞)超微结构的影响及对生长的抑制作用.方法:选取A549细胞并采用离体实验研究破壁灵芝孢子粉对肺癌细胞的作用.利用透射电镜研究破壁灵芝孢子粉对A549细胞超微结构的影响;利用CCK-8研究破壁灵芝孢子粉对A549细胞生长的抑制作用;利用乳酸脱氢酶(LDH)研究破壁灵芝孢子粉对细胞膜通透性的影响.结果:透射电镜观察显示破壁灵芝孢子粉可进入A549细胞,细胞中线粒体出现肿胀等现象;10 mg/ml破壁灵芝孢子粉可导致大量A549细胞肿胀、崩解.CCK-8检测结果显示破壁灵芝孢子粉导致A549细胞的存活率明显下降.LDH检测显示破壁灵芝孢子粉可使A549细胞的LDH释放量明显增加,且存在浓度依赖关系.结论:破壁灵芝孢子粉对人肺癌细胞具有显著抑制作用.

Gold nanoparticles, which have unique physicochemical characteristics, are being used for an increasingly wide range of applications in biomedical research. In this study, gold nanorods (width of 25 nm, length of 52 nm) were found to be internalized by A549 cells and were primarily localized in the lysosomes and membranous vesicles. The integrity of the membranes of A549 cells exposed to gold nanorods for 4h was damaged, as indicated by laser scanning confocal microscopy (LSCM). Increased lactate dehydrogenase (LDH) leakage and decreased cell viability further indicated the concentration-dependent cytotoxicity of the gold nanorods to the A549 cells. Reactive oxygen species (ROS) production was induced in the A549 cells by the gold nanorods, and this effect was positively correlated with the concentration of the gold nanorods. The results of this study indicated that exposure to gold nanorods caused dose-dependent cytotoxicity in A549 cells and that oxidative stress may be the main factor causing cytotoxicity.

To explore the mechanism of sarcoplasmic reticulum calcium uptake in heart failure myocytes. Methods: Adult rat ventricular myocytes were isolated by enzymatic digestion from the rat model of chronic heart failure, which was produced by ligation of the left anterior descending coronary artery. Sarcoplasmic reticulum calcium content was measured by laser scanning confocal microscopy;western blot method was used to analyze the differences in expression of calcium handling proteins in sham and heart failure. Results:The fluorescence intensity of ratsmyocytesin heart failure group was much lower than the sham group, and the caffeine induced calcium transients in heart failure group was lower than that of the sham group. The expression of PLN phosphorylation level decreased, but the level of overall PLN did not change. The expression of SERCA2a decreased and the level of PP1 increased. Conclusion:The main reasons for reducing sarcoplasmic reticulum calcium uptake were the reduction of SERCA2a expression and the decrease of SERCA2a activity.

透射电镜生物样品的取材是电镜样品制备过程中的关键步骤之一，并由实验者亲自动手操作完成。作者根据多年电镜实验课的教学经验，就指导学生取材的正确方法及培养学生的动手能力，谈谈实验教学中的一些方法和体会。

Objective To investigate the influence of Fe3O4 magnetic nanoparticles on human keratinocyte cells(HaCaT cells).Methods HaCaT cells were incubated with different concentrations of Fe3O4 magnetic nanoparticles for 4 h at 37℃ with 5% CO2.Then transmission electron microscopy(TEM) was used to observe the way nanoparticles entering HaCaT cells and the ultrastructure of HaCaT cells.Results The mean diameter of Fe3O4 magnetic nanoparticles was 12 nm.The nanoparticles of different concentrations could enter the HaCaT cells by phagocytosis.After entering the cells the particles were released from phagocytic vesicles and exerted influence on the nearby mitochondria,leading to mitochondria swelling and cristae dissolving.Conclusion Fe3O4 magnetic nanoparticles can damage the ultrastructure of mitochondria near the particles in HaCaT cells,and the effect is in a concentration-dependent manner.

Objective: Taking the second class as the carrier,we expand the depth and breadth of the students’ knowledge of biophysics,mobilize the enthusiasm of students’ learning,and develop their practical ability and scientific ideas.Method: In the second semester of medical undergraduates,students in the form of interest groups,carry out the derived electron microscopy,sample preparation,electron microscopy experimental activities.Result: Students’ practical ability is trained,and their interest in learning is improved significantly.Conclusion: Lab platform can help teachers and students complete the second classroom activities,so that the process and content of university education has been improved,but how to combine students’ knowledge,ability and quality still need to constant exploration in teaching practice.

随着电镜技术应用的普及,利用电镜观察肾小球超微结构进行临床诊断的样品日益增多.传统的超薄切片染色方法不能1次进行大批量染色操作(费时、费工,且易造成污染).为此,笔者对传统的电镜染色法作了探索和改进,最终做出了制作简便、染色量大、染色效果稳定的电镜超薄切片染色装置.

生物医学电镜技术实验课过去是一门重要的选修课,现在由于分子生物学技术的兴起,传统的形态学方法越来越被边缘化,学时减少,特别是实验部分的简化.面对教学目标的转变与改革,培养学生们的创新能力与实践操作能力是现在教学的重要内容.因此,在生物技术专业本科生与研究生中开展电镜技术实验课程教学显得十分重要,可使学生们从抽象的理论形态学知识过渡到感性的实验实践之中.在生物医学领域,电子显微技术与生物医学研究、医学诊断等多学科的结合变得越来越重要,已成为相关科研人员和医学工作者不可或缺的科学研究手段和临床诊断工作中的重要工具,同时生物电子显微镜技术是实践性极强的一门技术.