16S rRNA基因在微生物生态学中的应用

16S rRNA(Small subunit ribosomal RNA)基因是对原核微生物进行系统进化分类研究时最常用的分子标志物(Biomarker),广泛应用于微生物生态学研究中。近些年来随着高通量测序技术及数据分析方法等的不断进步,大量基于16S rRNA基因的研究使得微生物生态学得到了快速发展,然而使用16S rRNA基因作为分子标志物时也存在诸多问题,比如水平基因转移、多拷贝的异质性、基因扩增效率的差异、数据分析方法的选择等,这些问题影响了微生物群落组成和多样性分析时的准确性。对当前使用16S rRNA基因分析微生物群落组成和多样性的进展情况做一总结,重点讨论当前存在的主要问题以及各种分析方法的发展,尤其是与高通量测序技术有关的实验和数据处理问题。     

Preservation of cell-based immunotherapies for clinical trials

In the unique supply chain of cellular therapies, preservation is important to keep the cell product viable. Many factors in cryopreservation affect the outcome of a cell therapy: (i) formulation and introduction of a freezing medium, (ii) cooling rate, (iii) storage conditions, (iv) thawing conditions and (v) post-thaw processing. This article surveys clinical trials of cellular immunotherapy that used cryopreserved regulatory, chimeric antigen receptor or gamma delta T cells, dendritic cells or natural killer (NK) cells. Several observations are summarized from the given information. The aforementioned cell types have been similarly frozen in media containing 5–10% dimethyl sulfoxide (DMSO) with plasma, serum or human serum albumin. Two common freezing methods are an insulated freezing container such as Nalgene Mr. Frosty and a controlled-rate freezer at a cooling rate of -1°C/min. Water baths at approximately 37°C have been commonly used for thawing. Post-thaw processing of cryopreserved cells varied greatly: some studies infused the cells immediately upon thawing; some diluted the cells in a carrier solution of varying formulation before infusion; some washed cells to remove cryoprotective agents; and others re-cultured cells to recover cell viability or functionality lost due to cryopreservation. Emerging approaches to preserving cellular immunotherapies are also described. DMSO-free formulations of the freezing media have demonstrated improved preservation of cell viability in T lymphocytes and of cytotoxic function in natural killer cells. Saccharides are a common type of molecule used as an alternative cryoprotective agent to DMSO. Improving methods of preservation will be critical to growth in the clinical use of cellular immunotherapies.     

拟南芥DNA去甲基化基因RLL4的定位与功能分析

该研究以携带2×35S:LUC报告基因的转基因拟南芥Col-LUC为亲本系,将其种子进行甲基磺酸乙酯(EMS)诱变,在M2代筛选出1株低荧光的候选突变体,命名为rll4(reduced LUC luminescence 4)。遗传学分析表明,rll4突变位点包含1个核基因隐性突变。图位克隆技术定位结果显示,突变基因的位点位于4号染色体2个分子标记CL417-B10M1和CL418-B2M2之间,这2个分子标记分别位于F20D10和F20M13BAC(bacterial artificial chromosome)克隆。酶切PCR(Chop-PCR)结果显示,rll4突变体中基因组DNA的部分位点甲基化显著升高。反转录PCR(RT-PCR)结果显示,rll4突变体中ROS1(REPRESSOR OF SILENCING 1)的表达量并没有明显变化,而一些RNA介导的DNA甲基化(RdDM)过程靶位点的基因表达量有明显下降。研究表明,RLL4位点很可能参与了拟南芥DNA去甲基化过程。     

我国棉花主产区棉蚜对吡虫啉的抗性监测及抗性机理

【目的】由于生长周期短、繁殖率高,棉蚜Aphis gossypii容易对杀虫剂产生抗药性。本研究旨在明确我国棉花主产区棉蚜对吡虫啉的抗性水平及抗性机理。【方法】采用浸叶法测定了北京海淀,河北廊坊和邯郸,山东德州,河南许昌,以及新疆奎屯和阿克苏地区棉蚜对吡虫啉的抗性水平;测定了不同种群棉蚜3种解毒酶（多功能氧化酶、羧酸酯酶、谷胱甘肽S-转移酶）及乙酰胆碱酯酶的活性;并对靶标基因烟碱型乙酰胆碱受体（nAChR）β1亚基基因进行了突变检测。【结果】北京海淀、河南许昌和河北邯郸的棉蚜对吡虫啉敏感;河北廊坊、新疆阿克苏、山东德州及新疆奎屯地区的棉蚜对吡虫啉的抗性倍数(resistance ratio, RR)分别为22.6, 26.3,53.5和61.1倍,为中等水平抗性。酶活力对比研究发现,阿克苏和奎屯地区的棉蚜多功能氧化酶的比活力分别是敏感种群(北京种群)的1.7和1.8倍,羧酸酯酶的比活力分别是敏感种群的1.6和1.7倍,谷胱甘肽S-转移酶的比活力均是敏感种群的1.5倍,但是乙酰胆碱酯酶比活力在棉蚜种群间差异不显著。靶标基因突变检测表明,河北廊坊、新疆阿克苏、山东德州及新疆奎屯棉蚜种群nAChR β1亚基均存在与吡虫啉抗性相关的精氨酸到苏氨酸（R81T）突变。【结论】结果提示,多功能氧化酶、羧酸酯酶和谷胱甘肽S-转移酶活力升高以及nAChR β1亚基R81T突变与棉蚜对吡虫啉的抗性形成相关。     

小麦叶片暗诱导衰老期间内肽酶的特性

The characterization of senescence-associated endopeptidase (EP) isoenzymes in wheat (Triticum aestivum L. var. Yangmai 158) leaves during dark-induced senescence was performed. It was found that there was much higher endoproteolytic activity in dark-induced wheat leaves than in control. Six endopeptidase isoenzymes (EP1-EP6) were identified by natural gradient杙olyacrylamide gel electrophoresis (PAGE) co-polymerized gelatin in the gel, five of which (EP1, EP2, EP4, EP5 and EP6) were only detected in senescing leaves. Treatment with 6-benzylaminopurine (6-BA) delayed the expression of these EP isoenzymes and abscisic acid (ABA) accelerated it. The activity of EP3 could be detected at a wider range of pH and temperature levels while EP4, EP5 and EP 6 could be only detected at pH 4-5 and 30 45 ℃, EP1 and EP2 at pH 3 5 and 30 ℃ 45 ℃. All of the EP isoenzymes showed high thermal stability, especially EP3, EP5 and EP6 which still had activities even by incubation at 55 ℃ for 1 h. By using different class-specific inhibitors, EP1 and EP2 were characterized as metal-dependent cysteine-proteases, EP4 as a serine-protease.     

浙江中南部近岸海域春季主要鱼类空间生态位

基于2016年4、5月(春季)在浙江中南部近岸海域进行的2个航次渔业资源调查数据,计算浙江中南部近岸海域主要鱼类的相对重要性指数(IRI)、平均拥挤度、生态位宽度及生态位重叠值(Q_ik)。结果表明: 浙江中南部近岸海域主要鱼类(IRI>100)共24种,生态位宽度值差异较大,相对重要性指数与生态位宽度值呈极显著正相关。春季鱼类的生态位重叠值极低,4月Q_ik>0.6的种对有6对,占总对数的3.9%;5月Q_ik>0.6的种对有5对,占总对数的4.8%。冗余分析表明,温度、盐度、深度和溶解氧是影响浙江中南部近岸海域主要鱼类分布的重要因素,主要鱼类在这些环境因子上存在生态分化现象。     

阿帕替尼联合多西他赛对多线治疗晚期非小细胞肺癌患者血清肿瘤标志物、免疫功能及生活质量的影响

目的:分析阿帕替尼联合多西他赛用于多线治疗晚期非小细胞肺癌患者的临床效果。方法:选择我院2017年1月到2019年8月共86例晚期非小细胞肺癌患者为研究对象,按照随机抽签法分为对照组、观察组各43例,地塞米松预处理后,对照组仅应用多西他赛治疗,观察组联合应用阿帕替尼与多西他赛治疗,3周为1个治疗周期,均治疗4个周期。比较两组治疗前、治疗4周期后血清肿瘤标志物水平、免疫功能及生活质量及不良反应发生情况。结果:观察组治疗4周期后总缓解率为44.19%(19/43),高于对照组总缓解率23.26%(10/43)(P<0.05);治疗4周期后两组患者细胞角蛋白19片段抗原21-1(CYFRA21-1)、癌胚抗原(CEA)、糖蛋白抗原125(CA125)、鳞状上皮细胞癌抗原(SCC)水平、CD8⁺、生命质量测定量表EORTC QLQ-C30(QLQ-C30)中的功能及症状总得分均较治疗前降低,且观察组低于对照组(P<0.05);CD3⁺、CD4⁺、CD4⁺/CD8⁺水平及QLQ-C30的总体健康状况及生活质量总得分均高于治疗前,且观察组高于对照组(P<0.05),两组患者治疗期间的不良反应发生率无差异(P>0.05)。结论:阿帕替尼联合多西他赛用于多线治疗晚期非小细胞肺癌患者,可在一定程度上提高缓解率,其改善血清肿瘤标志物水平的效果更优,有助于提高患者机体免疫功能和生活质量,且短期内不会增加不良反应。     

Evolutionary history of an alpine shrub Hippophae tibetana (Elaeagnaceae): allopatric divergence and regional expansion

Increasing evidence suggests that geological or climatic events in the past promoted allopatric speciation of alpine plants in the Qinghai‐Tibetan Plateau and adjacent region. However, few studies have been undertaken to examine whether such allopatric divergences also occurred within a morphologically uniform species. In the present study, we report the evolutionary history of an alpine shrub species, Hippophae tibetana, based on examining chloroplast DNA (cpDNA) and nuclear ribosomal internal transcribed spacer (ITS) DNA variations. We sequenced two cpDNA fragments (trnL‐F and trnS‐G) and the nuclear ITS region in 183 individuals collected from 21 natural populations. Ten chlorotypes and 17 ITS types were identified. Phylogenetic analyses of both chlorotypes and ITS sequence variations suggested two distinct lineages distributed in the eastern and western region, respectively. On the basis of the fast and low plant substitution rates, these two lineages were estimated to have diverged from each other between 1 and 4 million years ago, during the period of the major glaciations and orogenic processes. In addition, ITS has undergone the accelerated evolution in two populations in the southern Himalaya isolated by the high mountains with a surprising accumulation of the private variations. The east–west split was also supported by an analysis of molecular variance, which partitioned around 91% of the total cpDNA variance between these two groups of populations. A single chlorotype was found for most populations in eastern or western region, suggesting a recent postglacial expansion within each region. Star‐phylogeny and mismatch analyses of all chlorotypes within the eastern group of populations suggested an earlier regional expansion before the Last Glacial Maximum (LGM). The local fixture of the different chlorotypes in multiple populations suggested more than one refugia remained for eastern or western region. Coalescent tests rejected the hypothesis that all current populations originated from a single refugium during the LGM. Instead, they supported hypothesis that two lineages diverged before the late Pleistocene. These findings, when taken together, suggested that this species had experienced long allopatric divergence and recent regional range expansions in response to orogenic processes and the climate changes. The evolutionary history of this shrub species highlights importance of geographical isolations to the intraspecific divergence of alpine plants occurring in the world's ruff. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 102 , 37–50.     

建立“人工面神经技术”恢复周围性面瘫兔的闭眼功能

目的:建立"人工面神经技术"以恢复单侧周围性面瘫兔的眼轮匝肌闭眼功能,为周围性面瘫提供一种新的治疗手段。方法:制备单侧周围性面瘫兔,建立健侧眼轮匝肌肌电信号采集、中枢信号处理模式识别、患侧电流刺激眼轮匝肌系统。当健侧眼轮匝肌采集的肌电信号经信号识别、提取以及电脑分析判断,符合其闭眼刺激阈值时,即对人工电刺激器发出指令,由刺激电极直接作用于患侧眼轮匝肌,引起眼睑完全闭合结果:以最小电流刺激患侧眼轮匝肌,引起眼睑完全闭合刺激方式为正负矩形波,电流强度为0.30～0.50mA。结论:利用可植入式微机电技术,在单侧周围性面瘫模型建立"人工面神经反射弧",恢复患侧眼轮匝肌闭眼收缩,可维持双侧闭眼功能的对称性和同步性。     

异养硝化细菌的筛选、鉴定及其氨氮转化特性的研究

从巢湖湖底泥床中分离筛选出一株具有氨氮转化活性的异养型硝化菌株X5.经生理生化分析、16S rDNA序列测定及系统发育学比较,菌株X5为枯草芽孢杆菌属(Bacillus sp.).菌株X5对氨氮的转化能力受温度、pH值以及接种量等条件的影响.实验结果表明,菌株X5的最适氨氮转化条件为:25～35℃,16 h,pH值7.0～8.0以及6%的接种量.将菌株X5扩大培养后接种于含蓝藻的发酵培养基中,分别测定总氮(TN)、氨氮(NH4+-N)及硝态氮(NO-3-N)的含量.结果显示,菌株X5对蓝藻中的氮素具有一定的转化作用.