Practical time-gated luminescence flow cytometry. I: concepts.

The method of time-gated detection of long-lifetime (1-2,000 micros) luminescence-labeled microorganisms following rapid excitation pulses has proved highly efficient in suppressing nontarget autofluorescence (<0.1 micros), scatterings, and other prompt stray light (Hemmila and Mukkala, Crit Rev Clin Lab Sci 2001;38:441-519). The application of such techniques to flow cytometry is highly attractive but there are significant challenges in implementing pulsed operation mode to rapid continuous flowing sample to achieve high cell analysis rates (Leif R, Vallarino L, Rare-earth chelates as fluorescent markers in cell separation and analysis, In: Cell Separation Science and Technology, ACS Symposium Series 464, American Chemical Society, 1991, pp 41-58; Condrau et al., Cytometry 1994;16:187-194; Condrau et al., Cytometry 1994;16:195-205; Shapiro HM, Improving signals from labels: Amplification and other techniques, In: Practical Flow Cytometry, 4th ed., Wiley, New York, 2002, p 345). We present here practical approaches for achieving high cell analysis rates at 100% detection efficiency, using time-gated luminescence (TGL) flow cytometry. In particular, we report that new-generation UV LEDs are practical sources in TGL flow cytometry.Spatial effects of long-lived luminescence from the target fluorophore in a fast-flowing sample stream have been investigated; excitation and detection requirements in TGL flow cytometry were theoretically analyzed; two practical approaches, a triggered model and a continuous flow-section model, were considered as a function of flow speed, sizes and relative positions of the excitation/detection spots, label lifetime, excitation pulse duration/intensity, and detection duration. A particular configuration using LED excitation to detect europium dye-labeled targets in such a system has been modeled in detail.In the triggered model, TGL mode is confined to a low repetition rate (<1 kHz) and engaged only while a target particle is present in the excitation zone. In the flow-section model, TGL mode is engaged continuously at high repetition rates to permit much higher cell arrival rates. The detection of 5.7-microm europium calibration beads in a UV LED-excited TGL flow cytometer has been shown to be feasible with a calculated signal-to-background ratio up to 11:1.     

Magnetoelastic transducers for monitoring coagulation, clot inhibition, and fibrinolysis

Magnetoelastic transduction has been used to detect and monitor the viscosity changes that occur during the biological reactions of coagulation and fibrinolysis. Magnetoelastic sensors can be used, because the characteristic resonance frequency of the magnetoelastic strip shifts in response to the changes in fluid viscosity. At a set frequency, the output signal can be obtained over time to develop a coagulation and/or dissolution profile, which display the change in viscosity of a plasma sample that has undergone either coagulation or fibrinolysis. For coagulation screening, an exogenous tissue factor is added to an anticoagulated plasma sample to initiate coagulation. Further studies were performed to investigate fibrinolysis through the addition of plasmin. Plasmin is used in two different ways-as a competitive inhibitor before the initiation of clotting and also as a protease to dissolve the previously formed clot. This method is a viable option for the monitoring of processes that are paramount to maintaining hemostasis.     

Vertical Distribution of Ammonia-Oxidizing Archaea and Bacteria in Sediments of a Eutrophic Lake

In order to characterize the vertical variation of abundance and community composition of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) in sediments of a eutrophic lake, Lake Taihu, molecular techniques including real-time PCR, clone library, and sequencing were carried out in this study. Abundances of archaeal amoA gene (ranged from 2.34 × 10⁶ to 4.43 × 10⁷ copies [g dry sediment]^?1) were higher than those of bacterial amoA gene (ranged from 5.02 × 10⁴ to 6.91 × 10⁶ copies [g dry sediment]^?1) for all samples and both of them exhibited negative correlations with the increased depths. Diversities of archaeal and bacterial amoA gene increased with the elevated depths. There were no significant variations of AOB community structures derived from different sediment depths, whereas obvious differences were observed for the AOA community compositions. The information acquired in this study would be useful to elucidate the roles of AOA and AOB in the nitrogen cycling of freshwater ecosystems.     

Heterologous expression of OsSIZ1, a rice SUMO E3 ligase,enhances broad abiotic stress tolerance in transgenic creeping bentgrass

Sumoylation is a posttranslational regulatory process in higher eukaryotes modifying substrate proteins through conjugation of small ubiquitin‐related modifiers (SUMOs). Sumoylation modulates protein stability, subcellular localization and activity; thus, it regulates most cellular functions including response to environmental stress in plants. To study the feasibility of manipulating SUMO E3 ligase, one of the important components in the sumoylation pathway in transgenic (TG) crop plants for improving overall plant performance under adverse environmental conditions, we have analysed TG creeping bentgrass (Agrostis stolonifera L.) plants constitutively expressing OsSIZ1, a rice SUMO E3 ligase. Overexpression of OsSIZ1 led to increased photosynthesis and overall plant growth. When subjected to water deficiency and heat stress, OsSIZ1 plants exhibited drastically enhanced performance associated with more robust root growth, higher water retention and cell membrane integrity than wild‐type (WT) controls. OsSIZ1 plants also displayed significantly better growth than WT controls under phosphate‐starvation conditions, which was associated with a higher uptake of phosphate (Pi) and other minerals, such as potassium and zinc. Further analysis revealed that overexpression of OsSIZ1 enhanced stress‐induced SUMO conjugation to substrate in TG plants, which was associated with modified expression of stress‐related genes. This strongly supports a role sumoylation plays in regulating multiple molecular pathways involved in plant stress response, establishing a direct link between sumoylation and plant response to environmental adversities. Our results demonstrate the great potential of genetic manipulation of sumoylation process in TG crop species for improved resistance to broad abiotic stresses.     

高邮湖大型底栖动物群落结构及影响因素

跨流域调水工程沿线调蓄湖泊生态状况直接决定工程能否顺利实施。基于2018年4和9月高邮湖底栖动物调查数据,对底栖动物区系组成、群落结构及主要影响因子进行了研究。调查期间共鉴定出底栖动物42种,隶属3门7纲22科;软铗小摇蚊（Microchironomus tener）、霍甫水丝蚓（Limnodrilus hoffmeisteri）、齿吻沙蚕（Nephtys sp.）、苏氏尾鳃蚓（Branchiura sowerbyi）、厚唇嫩丝蚓（Teneridrilus mastix）为调查期间优势物种,相对丰度分别为24.0%、17.7%、8.1%、7.2%和6.5%;两次调查期间底栖动物优势物种组成存在较大差异。密度、物种丰度、香浓指数在4月份显著高于9月份;非度量多维标度排序（NMDS）和多响应置换过程（MRPP）从群落层面证实了两次调查期间底栖动物群落结构的差异性。指示物种分析表明,软铗小摇蚊、霍甫水丝蚓和有栉管水蚓（Aulodrilus pectinatus）是造成两次调查底栖动物群落差异的关键物种。基于距离的冗余分析（dbRDA）表明,高邮湖底栖动物群落在4月份主要受硅酸盐和总磷影响;9月份主要受水深和叶绿素a影响。     

Wah Soon Chow,a teacher,a friend and a colleague

Photosynthesis Research - To finish this special issue, some friends, colleagues and students of Prof. Chow (Emeritus Professor, the Research School of Biology, the Australian National University)...     

WRKY12调控植物发育的分子机制

WRKY转录因子是高等植物中最大的转录因子家族之一,参与植物多个生长发育进程,其调控网络复杂。WRKY12是具有典型代表性的WRKY家族成员。文中对WRKY12在多个生长发育过程中的最新调控机制进行了综述,并且比较了WRKY12与WRKY13之间的功能差异。这为深入研究WRKY12调控植物发育机制提供参考,也为探索WRKY家族其他成员自我调控以及WRKY家族因子之间的协同机制提供较为清晰的研究思路和借鉴策略。     

功能雄性不育茄子差异基因及代谢途径分析

以茄子功能雄性不育系 (S13) 和可育系 (F142) 为材料,对花蕾不同发育时期的细胞学特征进行观察,发现S13花药中环形细胞簇的解体时期比F142延迟2 d,开花当天F142的裂口组织及内绒毡层的细胞解体,而S13中无此现象发生。转录组测序分析表明,F142和S13开花前8天、5天和开花当天的花药共有1 436个差异表达基因 (DEGs) (651个表达上调,785个表达下调)。GO显著性分析表明差异基因主要富集在生物学过程中涉及单细胞生物过程、代谢过程和细胞过程的单基因簇 (Unigene) 较多,分子功能中涉及较多的有催化活性和结合功能;KEGG注释发现,差异基因主要富集在次生代谢产物的生物合成、代谢途径、内质网蛋白质加工、氨基酸的生物合成、碳代谢和植物激素信号转导。选取16 465个基因进行加权基因共表达网络构建分析 (Weighted gene co-expression network analysis,WGCNA),共鉴定到15个基因共表达模块,其中3个为S13在3个花蕾发育时期高度相关特异性模块 (Plum2、Royalblue和Bisque4模块);KEGG富集表明,特异性模块可以富集到苯丙烷类生物合成、光合作用、卟啉与叶绿素代谢、α-亚麻酸代谢、多糖的生物合成和代谢、脂肪酸降解以及戊糖与葡萄糖醛酸的相互转化等相关通路,这些基因可能在S13花蕾发育过程中发挥重要作用。研究结果为进一步探索茄子花药开裂机制提供了一定的参考。