Single-cell analysis of paralytic shellfish toxins in Alexandrium tamarense by HPLC with post-column fluorescent derivatization

We developed a methodology for analyzing the C-toxin (C2) content in single Alexandrium tamarense cells; this method was based on high performance liquid chromatography (HPLC). C2 is the main paralytic shellfish toxin (PST) detected in a clonal culture of A. tamarense, which is a common causative organism in cases of paralytic shellfish poisoning in Japan. This HPLC method employs post-column fluorescent derivatization (FL). Mobile phase, column size, flow rate, reagent concentrations, and lamp type for the fluorescent detector were all optimized for the detection of C2. With this improved methodology, we could measure 1 fmol of C2 with a signal to noise ratio (S/N) = 2. Clonal heterogeneity within the toxic strain, which was maintained for 13 years after re-isolation from the original clonal culture, ranged from <1 fmol to 700 fmol cell⁻¹. This report is the first to demonstrate definitively that PST content varies on a cell-by-cell basis in a clonal culture of a dinoflagellate that causes paralytic shellfish poisoning.     

一种酵母细胞生长现象的实时单细胞拉曼光谱观察

用拉曼镊子观察单个即发活性干酵母(Saccharomy cescerevisiae)细胞在2.0%葡萄糖溶液中的活化过程,收集其拉曼光谱。结果发现,在某一批次的产品中,酵母细胞的1364cm-1峰强度随着细胞的活化而显著增加,531cm-1、652cm-1、1053cm-1等源自葡萄糖或葡萄糖基的信号峰也会随细胞的生长而增强,随后增强的还有1432cm-1、1448cm-1、1561cm-1等源自脂类物质的峰,而源自蛋白质及脂类的1000cm-1、1445cm-1、1655cm-1等峰的信号强度基本不变,酵母细胞代谢活跃的标志峰1603cm-1也基本不变。该批次产品中,10次实验有7次观察到上述现象,而在别的批次产品中并没有观察到该现象。用单细胞拉曼光谱实时记录了这一特殊的生长现象。     

Reporter gene expression at single-cell level characterized with real-time RT-PCR, chemiluminescence, fluorescence, and electrochemical imaging

mRNA from single cells was quantified using real-time RT-PCR after recording the address and reporter protein activity with chemiluminescence, fluorescence, and electrochemical techniques, using luciferase, green fluorescent protein, and secreted alkaline phosphatase. mRNA copy number ranging from below 103 to 10⁷ in single cells showed a lognormal distribution for both externally introduced reporter genes and internally expressed genes. The fluctuation in the gene expression decreased with the increase of the number of cells picked but did not decrease with the increase of mRNA copy number per cell. We found that the correlation coefficients for mRNA and protein expression in logarithmic plot at single-cell level were much lower than 1.00.     

Protists’ microbiome: A fine-scale,snap-shot field study on the ciliate Euplotes

Host-microbiome relationships play a fundamental role in the evolution and ecology of any living being. As unicellular organisms, protists represent a unique eukaryotic model to investigate selection mechanisms of the prokaryotic microbiome at the cellular level. Field investigations are central to disentangle relative importance of selective drivers in nature. Here we performed an analysis on data from a snap-shot field study reported previously on bacterial microbiomes associated to natural populations of protist ciliates of the genus Euplotes to detect at a fine scale any influence of habitat and/or host identity in microbiome selection. Comparative analyses revealed environment at a relatively large scale (sampling area) as the main driving factor in shaping prokaryotic communities’ structures. No evidence of habitat as key-factor emerged when a smaller spatial scale was considered (pond/channel or site). When only microbiomes of ciliates from the same site were compared, a clear assessment on the influence of host identity at the species level was not achieved, probably due to the small and unbalanced number of individuals for the two considered host species. Starting from this point, wider sampling campaigns will contribute in the future to depict a general view of the drivers influencing the prokaryotic microbiomes of natural protist populations.     

单细胞转录组数据分析中的数学

近年来,高通量单细胞测序技术为生物学研究带来了深刻的新发现,单细胞 RNA 测序技术的快速发展使得人们能够以单个细胞的分辨率定量研究细胞全基因组的转录水平。如何从高维的单细胞 RNA 测序数据中获取关于细胞和基因组的有用信息,是单细胞 RNA 测序数据分析中备受关注的重要问题。为此,广大研究者对单细胞 RNA 测序数据分析问题进行了大量的研究,开发了许多计算方法和相应的软件包。本综述将总结单细胞 RNA 测序数据分析的常用方法及其所涉及的数学基础,主要内容包括对原始数据进行预处理、聚类分析、拷贝数变异分析和非负矩阵分解等。本文重点介绍相应数据处理方法背后的数学原理,揭示如何利用数学工具获取单细胞 RNA 测序数据的信息,以便于数学工作者进行单细胞数据分析工作和对现有方法进行深入探索与改进。     

Trace elemental analysis of cancer-afflicted intestine by PIXE technique

Trace elemental analysis was carried out in the biological samples of cancer-afflicted intestine using the particle-induced X-ray emission technique (PIXE). A 2-MeV proton beam was employed to excite the samples. From the present results, it can be seen that the concentration of the elements Cr, Fe, and Ni are higher in the cancerous tissue of the intestine than those observed in the normal tissue, whereas the concentration levels of the element Zn is slightly lower in the cancer tissue of intestine than that observed in the normal tissue. The concentrations of S, Cl, K, Ca, Ti, Mn, Co, and Cu in the cancer tissue of the intestine are in agreement with those observed in the normal tissues within standard deviations. The present results support the previous observations that Ni and Cr are carcinogenic agents. The observed slightly low levels of zinc in the cancer tissue of the intestine suggest that zinc could possibly inhibit the tumor growth and development of neoplastic transformation. For correctly assessing the role played by the trace elements in initiating, promoting, or inhibiting cancer in various organs, there is a need for the acquisition of more data by trace elemental analysis from several investigations of this type undertaken in different regions.