Tumor-Localized Administration of α-GalCer to Recruit Invariant Natural Killer T Cells and Enhance Their Antitumor Activity against Solid Tumors

Invariant natural killer T (iNKT) cells have the capacity to mount potent anti-tumor reactivity and have therefore become a focus in the development of cell-based immunotherapy. iNKT cells attack tumor cells using multiple mechanisms with a high efficacy; however, their clinical application has been limited because of their low numbers in cancer patients and difficulties in infiltrating solid tumors. In this study, we aimed to overcome these critical limitations by using α-GalCer, a synthetic glycolipid ligand specifically activating iNKT cells, to recruit iNKT to solid tumors. By adoptively transferring human iNKT cells into tumor-bearing humanized NSG mice and administering a single dose of tumor-localized α-GalCer, we demonstrated the rapid recruitment of human iNKT cells into solid tumors in as little as one day and a significantly enhanced tumor killing ability. Using firefly luciferase-labeled iNKT cells, we monitored the tissue biodistribution and pharmacokinetics/pharmacodynamics (PK/PD) of human iNKT cells in tumor-bearing NSG mice. Collectively, these preclinical studies demonstrate the promise of an αGC-driven iNKT cell-based immunotherapy to target solid tumors with higher efficacy and precision.     

Technical Validation and Clinical Implications of Ultrasensitive PCR Approaches for EGFR-Thr790Met Mutation Detection in Pretreatment FFPE Samples and in Liquid Biopsies from Non-Small Cell Lung Cancer Patients

In pretreatment tumor samples of EGFR-mutated non-small cell lung cancer (NSCLC) patients, EGFR-Thr790Met mutation has been detected in a variable prevalence by different ultrasensitive assays with controversial prognostic value. Furthermore, its detection in liquid biopsy (LB) samples remains challenging, being hampered by the shortage of circulating tumor DNA (ctDNA). Here, we describe the technical validation and clinical implications of a real-time PCR with peptide nucleic acid (PNA-Clamp) and digital droplet PCR (ddPCR) for EGFR-Thr790Met detection in diagnosis FFPE samples and in LB. Limit of blank (LOB) and limit of detection (LOD) were established by analyzing negative and low variant allele frequency (VAF) FFPE and LB specimens. In a cohort of 78 FFPE samples, both techniques showed an overall agreement (OA) of 94.20%. EGFR-Thr790Met was detected in 26.47% of cases and was associated with better progression-free survival (PFS) (16.83 ± 7.76 vs. 11.47 ± 1.83 months; p = 0.047). In LB, ddPCR was implemented in routine diagnostics under UNE-EN ISO 15189:2013 accreditation, increasing the detection rate of 32.43% by conventional methods up to 45.95%. During follow-up, ddPCR detected EGFR-Thr790Met up to 7 months before radiological progression. Extensively validated ultrasensitive assays might decipher the utility of pretreatment EGFR-Thr790Met and improve its detection rate in LB studies, even anticipating radiological progression.     

Single-Circulating Tumor Cell Whole Genome Amplification to Unravel Cancer Heterogeneity and Actionable Biomarkers

The field of single-cell analysis has advanced rapidly in the last decade and is providing new insights into the characterization of intercellular genetic heterogeneity and complexity, especially in human cancer. In this regard, analyzing single circulating tumor cells (CTCs) is becoming particularly attractive due to the easy access to CTCs from simple blood samples called “liquid biopsies”. Analysis of multiple single CTCs has the potential to allow the identification and characterization of cancer heterogeneity to guide best therapy and predict therapeutic response. However, single-CTC analysis is restricted by the low amounts of DNA in a single cell genome. Whole genome amplification (WGA) techniques have emerged as a key step, enabling single-cell downstream molecular analysis. Here, we provide an overview of recent advances in WGA and their applications in the genetic analysis of single CTCs, along with prospective views towards clinical applications. First, we focus on the technical challenges of isolating and recovering single CTCs and then explore different WGA methodologies and recent developments which have been utilized to amplify single cell genomes for further downstream analysis. Lastly, we list a portfolio of CTC studies which employ WGA and single-cell analysis for genetic heterogeneity and biomarker detection.     

Facile Microwave Assisted Synthesis of Silver Nanostars for Ultrasensitive Detection of Biological Analytes by SERS

We report a very simple, rapid and reproducible method for the fabrication of anisotropic silver nanostars (AgNS) that can be successfully used as highly efficient SERS substrates for different bioanalytes, even in the case of a near-infra-red (NIR) excitation laser. The nanostars have been synthesized using the chemical reduction of Ag⁺ ions by trisodium citrate. This is the first research reporting the synthesis of AgNS using only trisodium citrate as a reducing and stabilizing agent. The key elements of this original synthesis procedure are rapid hydrothermal synthesis of silver nanostars followed by a cooling down procedure by immersion in a water bath. The synthesis was performed in a sealed bottom flask homogenously heated and brought to a boil in a microwave oven. After 60 s, the colloidal solution was cooled down to room temperature by immersion in a water bath at 35 °C. The as-synthesized AgNS were washed by centrifugation and used for SERS analysis of test molecules (methylene blue) as well as biological analytes: pharmaceutical compounds with various Raman cross sections (doxorubicin, atenolol & metoprolol), cell lysates and amino acids (methionine & cysteine). UV-Vis absorption spectroscopy, (Scanning) Transmission Electron Microscopy ((S)TEM) and Atomic Force Microscopy (AFM) have been employed for investigating nanostars’ physical properties.     

Induced Zinc Loss Produces Heterogenous Biological Responses in Melanoma Cells

Zinc levels in serum and/or tissue are reported to be altered in melanoma with unknown effects on melanoma development and biology. The purpose of this study was to examine the effects of acute chelation of free intracellular zinc pools in melanoma cell lines Bowes and A375, as well as selected melanoma tissue explants with high or low intracellular free zinc. Zinc chelating agent TPEN at the concentration of 25 µM was employed during 48 h, which significantly reduced intracellular free zinc while decreasing melanoma cell proliferation, inducing G1/S arrest and cell damage leading to mitochondrial, caspase-dependent apoptosis. Chelation of free zinc was also associated with increased generation of superoxide in cell lines but not marked lysosomal membrane damage. Conversely, melanoma explant cultures mostly displayed time-dependent loss of lysosomal membrane integrity in the presence of slowly growing superoxide levels. Loss of free zinc-dependent p53 activity was similarly disparate in individual melanoma models. Surviving melanoma cells were arrested in the cell cycle, and varying proportions of them exhibited features characteristic of premature senescence, which increased in time despite zinc reloading. The present results show that melanoma cells with varying free zinc levels respond to its acute loss in a number of individual ways, reflecting activated mechanisms including oxidative stress, lysosomal damage, and p53 activity leading to heterogenous outcomes including cell death, transient, and/or permanent cell cycle arrest and premature senescence.     

基于ST7单片机的太阳能直流供电系统控制器设计

设计一种用ST7单片机构成的全数字化太阳能直流供电系统控制器。系统在ST7单片机的控制下,能将太阳能电池转换的直流电进行有效存储和合理使用。系统充分利用了ST7单片机的软件资源,最大程度地简化了硬件电路,使电源具有较高的性价比和可靠性。     

基于模糊穴─穴映射的多变量模糊系统分析

在简要介绍多变量模糊系统的模糊穴－穴映射（ＦＣＭ－Ｉ）模型的基础上，提出了一种对多变量模糊系统进行分解的方法。此法的核心在于认为任何复杂系统的动态都是由其隐含的稳定子动态和不稳定于动态聚合而成，而系统特性则主要取决于决定性子动态的性质。并介绍了确定系统决定性子动态和基于其上的多变量模糊系统渐近分析方法。     

多功能Pt-Ce6纳米平台作为过氧化氢纳米酶和NIR-Ⅱ光热剂用于增强PDT/PTT肿瘤治疗

实体肿瘤的缺氧严重影响着基于氧气的光动力疗法(PDT)的效果.另外,单一治疗模式通常难以达到满意的治疗效果.为此,我们设计合成了一种多功能纳米复合材料Pt-Ce6用于克服肿瘤乏氧,实现PDT/PTT协同治疗.在该体系中,我们使用多孔Pt纳米粒子作为过氧化氢纳米酶、近红外二区(NIR-Ⅱ)光热转换剂和光敏剂二氢卟吩e6(...     

UG-75/5.3-M_3型锅炉冷态流化特性的试验研究

对UG－75／5．3－M3型循环流化床锅炉在点火底料不同厚度下的料层阻力进行了测试，并获得了该型锅炉在颗粒为0～5mm时料层阻力特性曲线，进而得出了料层阻力与料层厚度关系的计算公式和临界流化风量，为该种锅炉点火时风量的控制及运行中料层厚度的调节提供了依据。