Functional proteomics,human genetics and cancer biology of GIPC family members

GIPC1, GIPC2 and GIPC3 consist of GIPC homology 1 (GH1) domain, PDZ domain and GH2 domain. The regions around the GH1 and GH2 domains of GIPC1 are involved in dimerization and interaction with myosin VI (MYO6), respectively. The PDZ domain of GIPC1 is involved in interactions with transmembrane proteins [IGF1R, NTRK1, ADRB1, DRD2, TGFβR3 (transforming growth factorβ receptor type III), SDC4, SEMA4C, LRP1, NRP1, GLUT1, integrin α5 and VANGL2], cytosolic signaling regulators (APPL1 and RGS19) and viral proteins (HBc and HPV-18 E6). GIPC1 is an adaptor protein with dimerizing ability that loads PDZ ligands as cargoes for MYO6-dependent endosomal trafficking. GIPC1 is required for cell-surface expression of IGF1R and TGFβR3. GIPC1 is also required for integrin recycling during cell migration, angiogenesis and cytokinesis. On early endosomes, GIPC1 assembles receptor tyrosine kinases (RTKs) and APPL1 for activation of PI3K–AKT signaling, and G protein-coupled receptors (GPCRs) and RGS19 for attenuation of inhibitory Gα signaling. GIPC1 upregulation in breast, ovarian and pancreatic cancers promotes tumor proliferation and invasion, whereas GIPC1 downregulation in cervical cancer with human papillomavirus type 18 infection leads to resistance to cytostatic transforming growth factorβ signaling. GIPC2 is downregulated in acute lymphocytic leukemia owing to epigenetic silencing, while Gipc2 is upregulated in estrogen-induced mammary tumors. Somatic mutations of GIPC2 occur in malignant melanoma, and colorectal and ovarian cancers. Germ-line mutations of the GIPC3 or MYO6 gene cause nonsyndromic hearing loss. As GIPC proteins are involved in trafficking, signaling and recycling of RTKs, GPCRs, integrins and other transmembrane proteins, dysregulation of GIPCs results in human pathologies, such as cancer and hereditary deafness.     

Modification of Gold Electrode via Electrografting of the in situ Generated 3‐Carboxy‐1,2,4‐triazoldiazonium Salt for Label‐free Determination of Carcinoembryonic Antigen

In present study electrografting of the in situ generated 3‐carboxy‐1,2,4‐triazoldiazonium chloride on the Au disk electrode have been studied. The electrode film thickness differences between electrodeposited due to the aryl radical structure 3‐carboxy‐1,2,4‐triazoldiazonium chloride and 4‐carboxyphenyldiazonium salt was shown. The mechanism of 3‐carboxy‐1,2,4‐triazoldiazonium chloride electrografting has been proposed. 4‐nitroanilin was used to investigate the carbodiimide crosslinking capacity. It was established that electrodeposited films are suitable for carbodiimide crosslinking but the reaction proceed only on the “external” electrografted layer. Under the chosen optimal parameters, the label‐free electrochemical immunosensor have been developed. 3‐carboxy‐1,2,4‐triazoldiazonium chloride electrografting provided improvement of analytical characteristics in respect to electrodeposited 4‐carboxyphenyldiazonium salt. The linear range for carcinoembryonic antigen (CEA) detection is 10–10⁴ ng ? ml^?1, the limit of detection estimated as 0.2 ng ? ml^?1. The developed immunosensor is stable during 30 day′s storage and selective against excess of bovine serum albumin as an interfering reagent.     

Critical Review of Existing MHC I Immunopeptidome Isolation Methods

Major histocompatibility complex class I (MHC I) plays a crucial role in the development of adaptive immune response in vertebrates. MHC molecules are cell surface protein complexes loaded with short peptides and recognized by the T-cell receptors (TCR). Peptides associated with MHC are named immunopeptidome. The MHC I immunopeptidome is produced by the proteasome degradation of intracellular proteins. The knowledge of the immunopeptidome repertoire facilitates the creation of personalized antitumor or antiviral vaccines. A huge number of publications on the immunopeptidome diversity of different human and mouse biological samples—plasma, peripheral blood mononuclear cells (PBMCs), and solid tissues, including tumors—appeared in the scientific journals in the last decade. Significant immunopeptidome identification efficiency was achieved by advances in technology: the immunoprecipitation of MHC and mass spectrometry-based approaches. Researchers optimized common strategies to isolate MHC-associated peptides for individual tasks. They published many protocols with differences in the amount and type of biological sample, amount of antibodies, type and amount of insoluble support, methods of post-fractionation and purification, and approaches to LC-MS/MS identification of immunopeptidome. These parameters have a large impact on the final repertoire of isolated immunopeptidome. In this review, we summarize and compare immunopeptidome isolation techniques with an emphasis on the results obtained.     

Signal amplification strategy for sensitive immunoassay of prostate specific antigen (PSA) based on ferrocene incorporated polystyrene spheres

A new kind of signal amplification strategy based on ferrocene (Fc) incorporated polystyrene spheres (PS-Fc) was proposed. The synthesized PS-Fc displayed narrow size distribution and good stability. PS-Fc was applied as label to develop immunosensors for prostate specific antigen (PSA) after the typical sandwich immunoreaction by linking anti-PSA antibody (Ab₂) onto PS-Fc. After the fabrication of the immunosensor, tetrahydrofuran (THF) was dropped to dissolve PS and release the contained Fc for the following stripping voltammetric detection. PS-Fc as a new electrochemical label prevented the leakage of Fc and greatly amplified the immunosensor signal. In addition, the good biocompatibility of PS could maintain the bioactivity of the antibodies. The response current was linear to the logarithm of PSA concentration in the range from 0.01 ng mL⁻¹ to 20 ng mL⁻¹ with a detection limit of 1 pg mL⁻¹. The immunosensor results were validated through the detection of PSA in serum samples with satisfactory results.     

Ultrasensitive electrochemical immunoassay for carcinoembryonic antigen based on three-dimensional macroporous gold nanoparticles/graphene composite platform and multienzyme functionalized nanoporous silver label

Three-dimensional macroporous gold nanoparticles/graphene composites (3D-AuNPs/GN) were synthesized through a simple two-step process, and were used to modify working electrode sensing platform, based on which a facile electrochemical immunoassay for sensitive detection of carcinoembryonic antigen (CEA) in human serum was developed. In the proposed 3D-AuNPs/GN, AuNPs were distributed not just on the surface, but also on the inside of graphene. And this distribution property increased the area of sensing surface, resulting in capturing more primary antibodies as well as improving the electronic transmission rate. In the presence of CEA, a sandwich-type immune composite was formed on the sensing platform, and the horseradish peroxidase-labeled anti-CEA antibody (HRP-Ab₂)/thionine/nanoporous silver (HRP-Ab₂/TH/NPS) signal label was captured. Under optimal conditions, the electrochemical immunosensor exhibited excellent analytical performance: the detection range of CEA is from 0.001 to 10 ng mL⁻¹ with low detection limit of 0.35 pg mL⁻¹ and low limit of quantitation (LOQ) of 0.85 pg mL⁻¹. The electrochemical immunosensor showed good precision, acceptable stability and reproducibility, and could be used for the detection of CEA in real samples. The proposed method provides a promising platform of clinical immunoassay for other biomolecules     

Improved porous silicon microarray based prostate specific antigen immunoassay by optimized surface density of the capture antibody

Enriching the surface density of immobilized capture antibodies enhances the detection signal of antibody sandwich microarrays. In this study, we improved the detection sensitivity of our previously developed P-Si (porous silicon) antibody microarray by optimizing concentrations of the capturing antibody. We investigated immunoassays using a P-Si microarray at three different capture antibody (PSA – prostate specific antigen) concentrations, analyzing the influence of the antibody density on the assay detection sensitivity. The LOD (limit of detection) for PSA was 2.5 ng mL⁻¹, 80 pg mL⁻¹, and 800 fg mL⁻¹ when arraying the PSA antibody, H117 at the concentration 15 μg mL⁻¹, 35 μg mL⁻¹, and 154 μg mL⁻¹, respectively. We further investigated PSA spiked into human female serum in the range of 800 fg mL⁻¹ to 500 ng mL⁻¹. The microarray showed a LOD of 800 fg mL⁻¹ and a dynamic range of 800 fg mL⁻¹ to 80 ng mL⁻¹ in serum spiked samples.     

铜蒸气激光照射对血管平滑肌细胞凋亡的诱导作用及对其增殖的影响

目的：研究510.6nm铜蒸气激光照射对体外培养的血管平滑肌细胞（VSMC）凋亡的诱导作用，以及对增殖细胞核抗原（PCNA）表达的影响，探讨铜蒸气激光照射在经皮冠状动脉成形术（PTCA）后再狭窄（RS）的防治作用。方法：贴块法培养兔VSMC，510.6nm铜蒸气激光照射后透射电镜观察凋亡细胞形态学改变，TUNEL法计数凋亡细胞，免疫组化染色法计数照光对PCNA阳性表达率的影响。结果：激光照射后，VSMC凋亡率较未照光组增加12.8倍，而PCNA表达阳性率降低27．9％倍；电镜下观察细胞呈典型的凋亡形态学改变。结论：铜蒸气激光照射可以诱导VSMC凋亡，而且抑制其增殖，在RS的防治中具有一定的作用。     

抗肿瘤双特异免疫导向治疗制剂CAtin的表达及活性分析

结合生物信息学方法与已知癌胚抗原(Carcinoembryonic antigen, CEA)特异性单链抗体(Single chain Fv fragment, scFv)核苷酸序列, 经分子设计和密码子优化后, 通过化学方法合成CEA二硫键稳定性单链抗体(Disulfide stabilized single chain Fv fragments, scdsFv)基因片段. 将凋亡素基因(Apoptin)通过一段柔性连接肽(Linker)连接在CEA scdsFv基因下游, 并克隆入大肠杆菌表达载体质粒pET28a, 转化BL21感受态菌后经异丙基-β-D-硫代半乳糖苷(Isopropyl β-D-1-thiogalactopyranoside, IPTG)诱导, 表达融合蛋白CAtin. SDS-PAGE和Western-Blot分析表明, 目的蛋白得到良好表达, 经条件优化后表达量最高可达44.1 mg/L. 融合蛋白经分步洗涤法和谷胱甘肽对表达的目的蛋白进行初步纯化和复性后, 利用人肝癌细胞(HCC)对所制备融合蛋白进行亲和力测定、细胞结合活性测定和特异性细胞杀伤活性分析. 结果显示, 所制备融合蛋白不仅能够有效地与上述肿瘤细胞结合, 并对其具有明显的杀伤活性, 表明成功制备了具有特异性识别和特异性杀伤活性的双特异抗肿瘤免疫导向制剂.     

Chemical and Synthetic Biology Approaches for Cancer Vaccine Development

Cancer vaccines have been considered promising therapeutic strategies and are often constructed from whole cells, attenuated pathogens, carbohydrates, peptides, nucleic acids, etc. However, the use of whole organisms or pathogens can elicit unwanted immune responses arising from unforeseen reactions to the vaccine components. On the other hand, synthetic vaccines, which contain antigens that are conjugated, often with carrier proteins, can overcome these issues. Therefore, in this review we have highlighted the synthetic approaches and discussed several bioconjugation strategies for developing antigen-based cancer vaccines. In addition, the major synthetic biology approaches that were used to develop genetically modified cancer vaccines and their progress in clinical research are summarized here. Furthermore, to boost the immune responses of any vaccines, the addition of suitable adjuvants and a proper delivery system are essential. Hence, this review also mentions the synthesis of adjuvants and utilization of biomaterial scaffolds, which may facilitate the design of future cancer vaccines.     

一种基于动态识别邻域的免疫网络分类算法及其性能分析

针对传统免疫网络分类算法在记忆细胞确定上缺乏有效的指导,该文提出一种基于动态识别邻域的免疫网络分类算法。算法采用核函数表示机制来描述抗体-抗原之间的亲和度;利用抗原对构造动态识别邻域来指导抗体群体的进化,并选择邻域中距离对偶抗原最近的抗体为记忆细胞。算法被应用于多分类问题及高维分类问题来进行算法性能分析,同时,算法被应用于多个标准数据集的分类来评估算法的整体性能。分类结果表明该算法对于标准测试数据集有良好的分类性能,这说明基于动态识别邻域的训练方法能够有效地指导记忆细胞的生成,显著地改善分类器的性能。