Construction and Characterization of a CTLA-4-Targeted scFv–Melittin Fusion Protein as a Potential Immunosuppressive Agent for Organ Transplant

Immunotoxins with selective cytotoxicity are frequently used as therapeutic immunosuppressive agents in solid-organ transplantation because of their efficiency and high specificity. In this study, we present a new recombinant immunotoxin termed anti-CTLA-4-scFv–melittin prepared from Escherichia coli aimed at clearing activated T cells at the same time avoiding all-round decline in systematic immunity. This fusion protein is composed of anti-CTLA-4-scFv unit and melittin analog unit with properties of low immunogenicity and selective cytotoxicity to CTLA-4-positive T cells. In preliminary biological activity assays, our results confirmed the feasibility of activated T cell clearance strategy and there were significant differences in cell survival rates between CTLA-4-positive group and control group at all experimental concentrations of the immunotoxin. The selective cytotoxicity, low immunogenicity, and low production cost make it an attractive alternate to traditional immunosuppressants.     

Prenatal diagnosis of Pallister-Killian syndrome and literature review

Pallister-Killian syndrome (PKS) is a rare sporadic genetic disorder usually caused by mosaicism of an extra isochromosome of 12p (i(12p)). This retrospective study analysed the prenatal ultrasound manifestations and molecular and cytogenetic results of five PKS foetuses. Samples of amniotic fluid and/or cord blood, skin biopsy and placenta were collected. Conventional karyotyping and single nucleotide polymorphism array (SNP array) were performed on all the amniotic fluid or cord blood samples. Copy number variants sequencing (CNV-seq) and fluorescence in situ hybridization (FISH) were also used for the validation for one foetus. All the five foetuses were from pregnancies with advanced parental age. Two foetuses involved structural abnormalities and one foetus had only soft markers, all of which included increased nuchal translucency. The rest two foetuses had normal ultrasounds in the second trimester, which has rarely been reported before. The karyotype revealed typical i(12p) in four cases and a small supernumerary marker chromosome consisting of 12p and 20p in the remaining one case. The proportion of cells with i(12p) ranged from 0 to 100% in cultural cells, while SNP array results suggested 2−4 copies of 12p. For one foetus, metaphase FISH showed normal results, but the interphase FISH suggested cell lines with two, three and four copies of 12p in the amniotic fluid. Advanced parental age may be an important risk factor for PKS, and there were no typical ultrasound manifestations related to PKS. A combination of karyotype analysis and molecular diagnosis is an effective method for the diagnosis of PKS.     

采伐剩余物管理对杉木人工林土壤nosZ型反硝化细菌群落多样性的影响

反硝化细菌是土壤氧化亚氮(N₂O)排放的关键因子。以杉木人工林为研究对象,设置4种采伐剩余物处理方式(RF:对照;RB:火烧;MT:粉碎;NR:移除),采用高通量测序技术,以nosZ为标记基因,测定了自2018年9月—2020年9月,2年期间土壤nosZ型反硝化细菌群落的组成和丰度。研究结果显示,4种采伐剩余物处理中的土壤nosZ型反硝化细菌90%以上来自变形菌门,优势菌属包括固氮螺菌属、中慢生根瘤菌属、动胶菌属、伯克霍尔德菌属、嗜酸菌属、慢生根瘤菌属、假单胞菌属、固氮弧菌属以及无色杆菌属;样本间差异物种的显著性分析表明,在处理完成半年时,火烧相较于对照于β-变形菌纲水平显著增加了nosZ基因丰度;在处理完成一年时,火烧分别于红螺菌目、红螺菌科、固氮螺菌属水平显著高于粉碎;粉碎相较于移除在处理完成一年时,于γ-变形菌纲和产碱菌科水平显著增加了nosZ基因丰度;在处理完成两年时,粉碎处理的nosZ基因丰度在变形菌门水平显著高于对照和火烧。α多样性数据显示,处理完成一年时,粉碎处理相较于对照和移除显著增加了Shannon和Simpson指数;处理完成两年时,粉碎和火烧...     

High‐fat diet‐induced dysbiosis mediates MCP‐1/CCR2 axis‐dependent M2 macrophage polarization and promotes intestinal adenoma‐adenocarcinoma sequence

High‐fat diet (HFD) is a well‐known risk factor for gut microbiota dysbiosis and colorectal cancer (CRC). However, evidence relating HFD, gut microbiota and carcinogenesis is limited. Our study aimed to demonstrate that HFD‐induced gut dysbiosis promoted intestinal adenoma‐adenocarcinoma sequence. In clinical study, we found that HFD increased the incidence of advanced colorectal neoplasia (AN). The expression of monocyte chemoattractant protein 1 (MCP‐1), CC chemokine receptor 2 (CCR2) and CD163 in CRC patients with HFD was significantly higher than that in CRC patients with normal diet. When it comes to the Apc^min/+ mice, HFD consumption could induce gut dysbiosis and promote intestinal carcinogenesis, accompanying with activation of MCP‐1/CCR2 axis that recruited and polarized M2 tumour‐associated macrophages. Interestingly, transfer of faecal microbiota from HFD‐fed mice to another batch of Apc^min/+ mice in the absence of HFD could also enhance carcinogenesis without significant body weight gain and induced MCP‐1/CCR2 axis activation. HFD‐induced dysbiosis could also be transmitted. Meanwhile, antibiotics cocktail treatment was sufficient to inhibit HFD‐induced carcinogenesis, indicating the vital role of dysbiosis in cancer development. Conclusively, these data indicated that HFD‐induced dysbiosis accelerated intestinal adenoma‐adenocarcinoma sequence through activation of MCP‐1/CCR2 axis, which would provide new insight into better understanding of the mechanisms and prevention for HFD‐related CRC.     

Absent in melanoma 2 enhances anti‐tumour effects of CAIX promotor controlled conditionally replicative adenovirus in renal cancer

Conditionally replicative adenoviruses (CRAds) were promising approach for solid tumour treatment, but its oncolytic efficiency and toxicity are still not satisfactory for further clinical application. Here, we developed the CAIX promotor (CAIX^promotor)‐controlled CRAd armed with a tumour suppressor absent in melanoma 2 (AIM2) to enhance its oncolytic potency. The CAIX^promotor‐AIM2 adenoviruses (Ad‐CAIX^promotor‐AIM2) could efficiently express E1A and AIM2 in renal cancer cells. Compared with Ad‐CAIX^promotor, Ad‐CAIX^promotor‐AIM2 significantly inhibited cell proliferation and enhanced cell apoptosis and cell killing, thus resulting in the oncolytic efficiency in 786‐O cells or OSRC‐2 cells. To explore the therapeutic effect, various Ads were intratumourally injected into OSRC‐2‐xenograft mice. The tumour growth was remarkably inhibited in Ad‐CAIX^promotor‐AIM2‐treated group as demonstrated by reduced tumour volume and weight with a low toxicity. The inflammasome inhibitor YVAD‐CMK resulted in the reduction of anti‐tumour activity by Ad‐CAIX^promotor‐AIM2 in vitro or in vivo, suggesting that inflammasome activation response was required for the enhanced therapeutic efficiency. Furthermore, lung metastasis of renal cancer mice was also suppressed by Ad‐CAIX^promotor‐AIM2 treatment accompanied by the decreased tumour fossil in lung tissues. These results indicated that the tumour‐specific Ad‐CAIX^promotor‐AIM2 could be applied for human renal cancer therapy. The therapeutic strategy of AIM2‐based CRAds could be a potential and promising approach for the therapy of primary solid or metastasis tumours.     

河北泥河湾盆地下卜庄遗址端刮器功能的实验分析

端刮器常见于旧石器时代晚期的细石器工业中,形制比较固定,在探讨人类行为、生计以及环境适应等方面具有重要的研究价值。国外的研究涉及制作工艺、使用方式与功能等多个方面,而国内的研究多为类型学分析,对其功能的研究相对薄弱。河北泥河湾盆地下卜庄遗址出土了一定数量的端刮器遗存,本文通过模拟打制与使用实验,结合微痕分析方法,对它们的功能进行了探索,结果表明：1）下卜庄遗址的端刮器可能主要用来加工兽皮,且加工湿皮的现象较多;2）端刮器也可能直接被用来加工木材;3）部分端刮器微痕之间的关系表明,一些端刮器在使用过程中存在对刃缘的重新修理,当它们不再适合处理兽皮时会被用来加工木材,而后废弃。     

模拟降水量变化对荒漠草原土壤酶活性的影响及其相关因素分析

为明确荒漠草原土壤酶活性对降水格局改变的响应机制, 该研究基于宁夏荒漠草原降水量不同梯度变化(减少50%、减少30%、自然降水、增加30%和增加50%)的野外试验(2014年开始试验), 于2016年5-7月采样, 测定分析不同降水梯度2年后对土壤酶活性的影响, 并分析酶活性与植物生物量、微生物生物量C∶N∶P生态化学计量特征以及土壤理化性质的关系。结果表明: (1)与自然降水量相比, 减少30%降水量对3种土壤酶活性均无显著影响, 减少50%降水量显著降低了土壤蔗糖酶活性(^P < 0.05); 增加降水量显著提高了土壤蔗糖酶和磷酸酶活性(^P < 0.05), 但对脲酶活性无显著影响。(2)减少降水量对植物生物量影响较小(尤其减少30%降水量), 但不同程度地降低了微生物生物量C、N、P, 提高了微生物生物量C∶N和C∶P; 增加降水量则不同程度提高了植物生物量及微生物生物量C、N、P。(3)土壤蔗糖酶和磷酸酶活性随植物及微生物生物量增加而增加; 对土壤酶活性影响显著的土壤因子包括: 含水量、NO₃^- N、NH₄⁺ N、C∶P、有机C、全N、C∶N和pH (P < 0.05)。研究认为, 减少降水量(尤其是减少30%降水量)对土壤酶活性影响较小, 增加降水量促进了植物的生长、刺激微生物活性, 进而提高了土壤酶活性, 但随着植物生物量增加, 土壤有机C输入增多, 磷酸酶活性相应增强并促进了有机P的矿化, 导致土壤微生物P限制增加。     

屯昌猪MYLPF基因序列分析及其组织表达量检测

为获得屯昌猪MYLPF基因的CDS区序列并分析其分子结构特征,研究屯昌猪、杜洛克猪以及其杂交F1代猪不同组织中的MYLPF mRNA表达水平,本研究以GenBank上公布的猪MYLPF基因序列(登录号:NM_001006592)为参考设计引物,通过RT-PCR扩增、测序获得屯昌猪MYLPF基因CDS区.结果显示:该基因CDS区长度510 bp,编码169个氨基酸,在CDS区存在33 G＞T的同义突变;该基因编码产物既没有跨膜结构,也不存在信号肽,属于非分泌型蛋白,主要在细胞质中发挥作用,预测存在1个潜在的糖基化位点和9个磷酸化位点,α螺旋区域在预测的二级结构中占比最大.荧光定量PCR检测结果显示MYLPF基因在屯昌猪背最长肌中表达量最高,屯昌猪背最长肌中MYLPF mRNA的表达量均显著高于杜洛克猪及其杂交F1代猪.本研究为探明MYLPF基因对猪肌内脂肪的影响提供了分子理论依据.     

Extrapolating the effect of deleterious nsSNPs in the binding adaptability of flavopiridol with CDK7 protein: a molecular dynamics approach

Background

Recent reports suggest the role of nonsynonymous single nucleotide polymorphisms (nsSNPs) in cyclin-dependent kinase 7 (CDK7) gene associated with defect in the DNA repair mechanism that may contribute to cancer risk. Among the various inhibitors developed so far, flavopiridol proved to be a potential antitumor drug in the phase-III clinical trial for chronic lymphocytic leukemia. Here, we described a theoretical assessment for the discovery of new drugs or drug targets in CDK7 protein owing to the changes caused by deleterious nsSNPs.

Methods

Three nsSNPs (I63R, H135R, and T285M) were predicted to have functional impact on protein function by SIFT, PolyPhen2, I-Mutant3, PANTHER, SNPs&GO, PhD-SNP, and screening for non-acceptable polymorphisms (SNAP). Furthermore, we analyzed the native and proposed mutant models in atomic level 10 ns simulation using the molecular dynamics (MD) approach. Finally, with the aid of Autodock 4.0 and PatchDock, we analyzed the binding efficacy of flavopiridol with CDK7 protein with respect to the deleterious mutations.

Results

By comparing the results of all seven prediction tools, three nsSNPs (I63R, H135R, and T285M) were predicted to have functional impact on the protein function. The results of protein stability analysis inferred that I63R and H135R exhibited less deviation in root mean square deviation in comparison with the native and T285M protein. The flexibility of all the three mutant models of CDK7 protein is diverse in comparison with the native protein. Following to that, docking study revealed the change in the active site residues and decrease in the binding affinity of flavopiridol with mutant proteins.

Conclusion

This theoretical approach is entirely based on computational methods, which has the ability to identify the disease-related SNPs in complex disorders by contrasting their costs and capabilities with those of the experimental methods. The identification of disease related SNPs by computational methods has the potential to create personalized tools for the diagnosis, prognosis, and treatment of diseases.

Lay abstract

Cell cycle regulatory protein, CDK7, is linked with DNA repair mechanism which can contribute to cancer risk. The main aim of this study is to extrapolate the relationship between the nsSNPs and their effects in drug-binding capability. In this work, we propose a new methodology which (1) efficiently identified the deleterious nsSNPs that tend to have functional effect on protein function upon mutation by computational tools, (2) analyze d the native protein and proposed mutant models in atomic level using MD approach, and (3) investigated the protein-ligand interactions to analyze the binding ability by docking analysis. This theoretical approach is entirely based on computational methods, which has the ability to identify the disease-related SNPs in complex disorders by contrasting their costs and capabilities with those of the experimental methods. Overall, this approach has the potential to create personalized tools for the diagnosis, prognosis, and treatment of diseases.