In situ vaccination and gene-mediated PD-L1 blockade for enhanced tumor immunotherapy

Despite of the promising achievements of immune checkpoints blockade therapy (ICB) in the clinic, which was often limited by low objective responses and severe side effects. Herein, we explored a synergistic strategy to combine in situ vaccination and gene-mediated anti-PD therapy, which was generated by unmethylated cytosine-phosphate-guanine (CpG) and pshPD-L1 gene co-delivery. PEI worked as the delivery carrier to co-deliver the CpG and pshPD-L1 genes, the formed PDC (PEI/DNA/CpG) nanoparticles were further shielded by aldehyde modified polyethylene glycol (OHC-PEG-CHO) via pH responsive Schiff base reaction for OHC-PEG-CHO-PEI/DNA/CpG nanoparticles (P(PDC) NPs) preparation. All steps could be finished within 30 min. Such simple nanoparticles achieved the synergistic antitumor efficacy in B16F10 tumor-bearing mice, and the amplified T cell responses, together with enhanced NK cells infiltration were observed after the combined treatments. In addition, the pH responsive delivery system reduced the side effects triggered by anti-PD therapy. The facile and effective combination strategy we presented here might provide a novel treatment for tumor inhibition.     

Toll-like receptor 9-mediated inhibition of apoptosis occurs through suppression of FoxO3a activity and induction of FLIP expression

Synthetic oligodeoxynucleotides (ODN) with a CpG-motif are recognized by Toll-like receptor 9 (TLR9) and pleiotropic immune responses are elicited. Stimulation of macrophages with TLR9 agonist prevented apoptosis induced by serum deprivation through increased expression of FLICE-like inhibitory protein (FLIP). CpG ODN-mediated anti-apoptosis depended on the TLR9-Akt-FoxO3a signaling pathway. Inhibition of TLR9 by small interfering (si) RNA or an inhibitor suppressed CpG ODN-mediated anti-apoptosis. Analysis of signaling pathways revealed that the anti-apoptotic effect of CpG ODN required phosphorylation of FoxO3a and its translocation from the nucleus to the cytosol. Overexpression of FoxO3a increased apoptosis induced by serum deprivation and CpG ODN blocked these effects through FLIP expression. In contrast, siRNA knock-down of FoxO3a decreased apoptosis by serum deprivation. In addition, Akt activation was involved in CpG ODN-induced phosphorylation of FoxO3a, expression of FLIP, and anti-apoptosis. Taken together, these results demonstrate the involvement of Akt-FoxO3a in TLR9-mediated anti-apoptosis and indicate that FoxO3a is a distinct regulator for FLIP expression.     

焦测序技术及其在遗传分析中的应用

焦测序技术是一种新的实时DNA测序技术。它在DNA聚合酶、三磷酸腺苷硫酸化酶、荧光素酶和三磷酸腺苷双磷酸酶4种酶的协同作用下,使引物延伸聚合脱氧核糖核酸(dNTP)释放焦磷酸盐(PP i)、PP i转换三磷酸腺苷(ATP)、ATP产生荧光信号与dNTP和ATP的降解等化学反应偶联起来,检测结果准确可靠。本文综述了焦测序技术的基本原理、操作步骤和它在单核苷酸多态性(SNP)研究、微生物的分型鉴定和基因甲基化检测等遗传分析中的应用,并对焦测序技术的发展作了展望。     

胃癌细胞MGC803中LTF基因的表达及其启动子甲基化相关性研究

目的 检测胃癌细胞株MGC803内乳铁蛋白（LTF）基因的甲基化和表达情况,探讨LTF的DNA 启动子区甲基化异常 与其在胃癌内表达的相关性。方法 用亚硫酸氢盐测序PCR 方法检测MGC803 启动子区甲基化状态,使用real-time PCR 和Western blot 分别检测LTF 在MGC803内的mRNA 和蛋白表达水平。同时使用去甲基化剂5-aza-CdR 处理胃癌细胞株,观察给药前后的DNA 甲基化状态和mRNA 表达情况。结果 MGC803 启动子甲基化率达59.8%,使用5-aza-CdR 处理的两组（2滋mol/L组和10滋mol/L组）和空白对照组甲基化率差异、LTF 的mRNA表达差异均有统计学意义（均P＜0.05）,并且随着MGC803启动子甲基化程度的下降,其mRNA 和蛋白表达增加;而不同浓度药物处理组间差异均无统计学意义（均P＞0.05）。结论 LTF 基因在胃癌细胞株MGC803 内表现为较高甲基化状态,而且LTF的表达和其启动子区甲基化情况相关,使用去甲基化剂能逆转DNA 的甲基化情况从而使其mRNA 重新表达。     

Voltammetric Label‐free Detection of DNA Hypermethylation Using Polypyrrole‐modified Microelectrode Array

An alternative strategy for the label‐free electrochemical detection of DNA hypermethylation using a microelectrode array as an oligodinucleotide (ODN) detector is presented. It relies on the oligonucleotide dependent electrostatic affinity interaction firstly with unmethylated ODN and then follow‐up with methylated DNA. The methylated cytosine status is quantified by monitoring the relative change in the exchange current at the ODN‐detector before and after the bisulfite treated DNA samples. This novel aproach displays unique advantages such as small working volumes of the analytes, low damage to DNA samples, easy integration of oligonucleides on the detector and signal evaluation.     

Separation‐free single‐base extension assay with fluorescence resonance energy transfer for rapid and convenient determination of DNA methylation status at specific cytosine and guanine dinucleotide sites

A separation‐free single‐base extension (SBE) assay utilizing fluorescence resonance energy transfer (FRET) was developed for rapid and convenient interrogation of DNA methylation status at specific cytosine and guanine dinucleotide sites. In this assay, the SBE was performed in a tube using an allele‐specific oligonucleotide primer (i.e., extension primer) labeled with Cy3 as a FRET donor fluorophore at the 5′‐end, a nucleotide terminator (dideoxynucleotide triphosphate) labeled with Cy5 as a FRET acceptor, a PCR amplicon derived from bisulfite‐converted genomic DNA, and a DNA polymerase. A single base‐extended primer (i.e., SBE product) that was 5′‐Cy3‐ and 3′‐Cy5‐tagged was formed by incorporation of the Cy5‐labeled terminator into the 3′‐end of the extension primer, but only if the terminator added was complementary to the target nucleotide. The resulting SBE product brought the Cy3 donor and the Cy5 acceptor into close proximity. Illumination of the Cy3 donor resulted in successful FRET and excitation of the Cy5 acceptor, generating fluorescence emission from the acceptor. The capacity of the developed assay to discriminate as low as 10% methylation from a mixture of methylated and unmethylated DNA was demonstrated at multiple cytosine and guanine dinucleotide sites.     

AMS-8-NH2的合成及其对ODN的负载释放

以阴离子表面活性剂肉豆蔻酰基谷氨酸钠(C₁₄GluA-Na)为模板剂, 3-氨丙基三甲氧基硅烷(APS)为辅助结构导向剂, 合成了介孔材料AMS-8-NH₂, 并结合XRD分析、N₂吸附/脱附技术及²⁹Si魔角旋转核磁共振(MAS NMR)等技术手段对纳米孔洞材料AMS-8-NH₂进行了表征. 结果表明, AMS-8-NH₂是高度有序的纳米孔洞材料, 其孔径和比表面积分别为3.2 nm和706 m²/g; 有机胺基团通过化学键连接在材料内表面, 这些基团的覆盖度达到25%. 以AMS-8-NH₂为载体, 寡核苷酸(ODN)为模型生物分子研究在不同条件下AMS-8-NH₂对ODN的吸附和释放性能, 实验结果表明, 当ODN浓度一定时, 溶液的pH值越低, 对ODN的吸附量越大, pH=4.7时对ODN的最大负载量可达214 mg/g; 在释放过程中, 材料对ODN具有较高的缓释能力, 随环境pH值增加, 释放量增加.     

Mechanistic study on DNA mutation of the cytosine methylation reaction at C5 position

DNA methylation is a crucial epigenetic mark connected to conventionally changing the DNA bases, typically by adding methyl groups into DNA bases. Methylation of cytosine at the C5 position (5-methylcytosine) occurs mostly in the context of cytosine-phosphate-guanine dinucleotides, the methylation of which has important impacts on gene regulation and expression. However, the mechanistic details of this reaction are still debatable concerning the concertedness of the key reaction steps and the roles played by the base that abstracts the proton in the β-elimination and water molecules at the active site. To gain a deeper insight into the formation of 5-mehtylcytosine, an extensive density functional theory (DFT) study was performed with the B3LYP functional in conjunction with different basis sets. Our study has clearly established the mechanistic details of this methylation approach, based on which the roles of conserved active site residues, such as glutamic acid and waters, are well understood. Our results show that the reaction of 5-methylcytosine follows a concerted mechanism in which water molecules are critically involved. Moreover, arginine and alanine give more significant catalytic effects than glutamic acid on the 5-methylcytosine process. Considering the effect of Alanine, Arginine, and one water bridging molecule, the activation energy is 31 kJ mol^?1 calculated at B3LYP/6-31G(d) level of theory.