Increased stem cell proliferation in atherosclerosis accelerates clonal hematopoiesis

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Analytical cytometric approaches to heterogeneous cell populations in solid tumors: a review

The problems encountered in studying the heterogeneity of cells in solid tumors is reviewed with emphasis on the role of various analytical cytometric assays for studying both the biology and the dynamics and proliferating, quiescent and dead malignant cells in vitro and in vivo. Due to advances in cytometric technology, many interesting in vitro studies on tumor cells heterogeneity have been and will be conducted over the next several years. For example, the acidic acridine orange staining of HeLa cells in suspension culture does readily discriminate between proliferating and quiescent cells. Some of these assays have been and others will be extended to in vivo studies. However, it is obvious that either the current analytical cytometric techniques must be modified and refined to permit better resolution for the complex situation in vivo or other new analytical cytometric techniques will have to be developed before many interesting studies on tumor cell heterogeneity in vivo can be addressed with reasonable efficiency.     

Limits to adaptation in asexual populations

In asexual populations, the rate of adaptation is basically limited by the frequency and properties of spontaneous beneficial mutations. Hence, knowledge of these mutational properties and how they are affected by particular evolutionary conditions is a precondition for understanding the process of adaptation. Here, we address how the rate of adaptation of asexual populations is limited by its population size and mutation rate, as well as by two factors affecting the fraction of mutations that confer a benefit, i.e. the initial adaptedness of the population and the variability of the environment. These factors both influence which mutations are likely to occur, as well as the probability that they will ultimately contribute to adaptation. We attempt to separate the consequences of these basic population features in terms of their effect on the rate of adaptation by using results from evolution experiments with microorganisms.     

Biomass partitioning in response to resources availability: A comparison between native and invaded ranges in the clonal invader Carpobrotus edulis

Identifying the mechanism underlying plant invasiveness is a fast-moving research topic in current ecology. Phenotypic plasticity has been pointed out as a trait that can contribute to plant invasiveness. This experiment examines the presence of rapid adaptive evolution favoring plastic biomass partitioning during the invasion process. With that aim, we tested differences in patterns of biomass allocation between populations of Carpobrotus edulis from South Africa (native area) and the Iberian Peninsula (invaded area) growing under different nutrient, water and light availabilities in a common garden experiment. Here we demonstrate that biomass partitioning in response to nutrient availability in C. edulis differs between populations from native and invaded ranges, indicating that this trait could be under selection during the invasion process. Thus, nutrient shortage significantly increased the proportional production of roots in populations from the invaded range, but not in populations from the native area. This plastic root-foraging response may contribute to the optimization of nutrient uptake by plants, and therefore could be considered as an adaptive strategy. Understanding the ecological implications of rapid evolution for plastic biomass partitioning is important in determining processes of plant adaptation to new environments, and contributes to disentangling the mechanisms underlying plant invasiveness.     

克隆整合对遮荫白夹竹分株根际土壤细菌生物特征的影响

克隆整合被认为是克隆植物维持生态优势的重要手段,其通过分株间生理整合缓解资源异质性带来的压力。文章以根状茎克隆植物白夹竹(Phyllostachys bissetii)为研究对象,探讨异质性光照下克隆整合对白夹竹分株根际土壤细菌生物特征的影响。白夹竹克隆片段包含一个近端分株(proximal ramet)和一个远端分株(distal ramet),近端分株或远端分株分别置于80%遮荫环境,另一分株置于全光照环境;同时,分株间根状茎保持连接或割断处理。研究结果表明,不论白夹竹克隆片段近端分株遮荫还是远端分株遮荫,克隆整合均显著促进了遮荫分株根际土壤溶解性有机碳(DOC)含量和微生物生物量碳、氮含量(MBC,MBN);根状茎连接条件下遮荫分株根际土壤胞外N-乙酰基-β-氨基葡萄糖苷酶(NAGase)、脲酶(Urease)活性显著高于根状茎割断处理的遮荫分株;对遮荫分株根际土壤基因组DNA的16S rDNA V3、V4可变区的测序结果表明,可操作分类单元(OTUs)归于13个门,415个属。与根际土壤氮素转化相关的菌群Nitrosomonadaceae(uncultured),Nitrospira,Nitrospinaceae(uncultured),Xanthobacteraceae(uncultured),Bradyrhizobium相对丰度较小(最高值为11.8%)。基于OTUs的主成分分析(PCA)表明,根状茎割断处理并没有对遮荫分株根际土壤细菌生物群落结构产生显著性影响。克隆整合显著促进了遮荫分株根际土壤C有效性,进而刺激了微生物调控的土壤有机质(SOM)周转过程。克隆整合对异质生境下克隆植物土壤细菌生物特征的影响可能部分解释了克隆植物的生态优势。     

It's time to regulate: Coping with product‐induced nongenetic clonal instability in CHO cell lines via regulated protein expression

Clonal instability and titer loss during Chinese hamster ovary (CHO) cell line development (CLD) has several underlying causes, the most prominent of which are DNA copy number loss and DNA silencing. However, in some cases, clonal instability is due to the toxicity of the therapeutic protein(s) that clones express. Unlike DNA copy number loss, which may occur in some clones or DNA silencing that is prevalent in certain regions of the genome, the hallmark of product induced clonal instability is its manifestation in all the selected clones. To circumvent such product induced clonal instability, we have developed a vector construct that utilizes a regulated protein expression system in which the constitutive expression of the target protein(s) is prevented unless doxycycline is added to the culture. We have then successfully used this system to express, at high titers, an antibody for which constitutive expression results in clonal instability perhaps due to intracellular accumulation of the antibody. Our data shows that unlike the constitutively expressed or continuously induced clones, uninduced clones do not display instability. Furthermore, maintaining the uninduced clones in culture for months or subjecting them to freeze‐thaws did not have any effects on their titers. All together, our findings suggest that a regulated expression system could be suitable for production of difficult proteins that trigger instability. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1432–1440, 2014     

Genome-wide analysis of single nucleotide variants allows for robust and accurate assessment of clonal derivation in cell lines used to produce biologics

A clonally derived (or “monoclonal”) cell line is a cell population derived from a single progenitor cell. Clonally derived cell lines are required for many biotechnological applications. For instance, recombinant mammalian cells used to produce therapeutic proteins are expected by regulatory authorities to be clonally derived. Assurance of clonal derivation (or “clonality”) is usually obtained from the characterization of the procedure used for cell cloning, for instance by assessing the success rate of single-cell sorting but not by assessing the cell line itself. We have developed a method to assess clonal derivation directly from the genetic makeup of cells. The genomic test of clonality is based on whole-genome sequencing and statistical analysis of single nucleotide variants. This approach quantifies the clonal fractions present in nonclonal samples and it provides a measure of the probability that a cell line is derived from a single cell. Upon experimental validation of the test, we show that it is highly accurate and that it can robustly detect minor clonal fractions of as little as 1% of the cell population. Moreover, we find that it is applicable to various cell line development protocols. This approach can simplify development protocols and shorten timelines while ensuring clonal derivation with high confidence.     

Recent mathematical approaches to reconstruct phylogenies: A chemosystematist's and botanist's view

Some basic problems of mathematical phylogenetics are discussed. While algorithms regularly depend on the principle of parsimony, some features of phylogenesis interfere with that principle. Nonrandomness of the distribution of mutations as well as the inconstancy of the molecular clock in time and within a given sequence can bias the calculated relationships of closely related taxa. True comparability of sequences is difficult to establish, since this requires defining of homology of positions and of functions of amino acids as well. Parallelism and convergence can give rise to errors in establishing homology. Furthermore, they are difficult to be integrated into a consistent mathematical approach. The asymmetry of identity versus difference matrices is discussed. Common mathematical procedures implicate the monophyletic hypothesis as well as strict dichotomy of the final tree. Real phylogeny demands to introduce more flexible approaches with respect to monophyly and dichotomy. Graph theory offers the possibility to prove the minimality of a given tree and to develop more flexible approaches. The consequences of altering the procedures to compare sequences can be tested. This is demonstrated by calculations with a set of 15 cytochromes from various taxa. To interpret a calculated tree requires a sharp distinction between taxonomy and phyletics. This is exemplified for the case of directly linked taxa. The limits of resolving uncertain relationships by accumulating more data are mentioned.This paper was partly presented at the Symposium Theory and practice of botanical classification, during the 14th International Botanical Congress, Berlin, July 1987.     

Expedited approaches to whole cell electron tomography and organelle mark-up in situ in high-pressure frozen pancreatic islets

We have developed a simplified, efficient approach for the 3D reconstruction and analysis of mammalian cells in toto by electron microscope tomography (ET), to provide quantitative information regarding ‘global’ cellular organization at 15–20 nm resolution. Two insulin-secreting beta cells—deemed ‘functionally equivalent’ by virtue of their location at the periphery of the same pancreatic islet—were reconstructed in their entirety in 3D after fast-freezing/freeze-substitution/plastic embedment in situ within a glucose-stimulated islet of Langerhans isolated intact from mouse pancreata. These cellular reconstructions have afforded several unique insights into fundamental structure–function relationships among key organelles involved in the biosynthesis and release of the crucial metabolic hormone, insulin, that could not be provided by other methods. The Golgi ribbon, mitochondria and insulin secretory granules in each cell were segmented for comparative analysis. We propose that relative differences between the two cells in terms of the number, dimensions and spatial distribution (and for mitochondria, also the extent of branching) of these organelles per cubic micron of cellular volume reflects differences in the two cells’ individual capacity (and/or readiness) to respond to secretagogue stimulation, reflected by an apparent inverse relationship between the number/size of insulin secretory granules versus the number/size of mitochondria and the Golgi ribbon. We discuss the advantages of this approach for quantitative cellular ET of mammalian cells, briefly discuss its application relevant to other complementary techniques, and summarize future strategies for overcoming some of its current limitations.