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Seeds show important variation as plant regenerative units among species, but their evolutionary co-variations with other plant characteristics are still poorly understood. Whilst a positive association of seed mass with genome size (GS) and life forms has already been documented, a broad-scale quantification of their evolutionary correlation and adaptive selection has never been conducted. Here, we tested for correlated evolution of seed mass and GS towards distinct selective regimes related to life form in angiosperms. In particular, we tested the hypothesis that the selection toward lighter seeds and smaller genomes is stronger for annual plants, ensuring high regenerative potential. Using multivariate evolutionary models over a dataset containing 3242 species, we showed an overall positive correlated evolution of seed mass and GS deviating from a pure drift process. Instead, evolutionary changes in seed and genome sizes were driven by adaptive selection towards optimal values differing among life forms. Specifically, the evolutionary optima towards which the seed and genome sizes evolve show a covariation toward small values in annuals, intermediate values in perennial herbs and a trade-off between seed mass and GS in woody plants. Moreover, the evolutionary correlation between seed mass and GS is strongest in annuals as an adaption to complete their life cycle in a short time window, when environmental conditions are favourable for regeneration and development to maturity. The asymmetry in the correlated evolution acting on seed and genome sizes due to life form could explain how life-history traits interplay with functional traits and how plants have evolved diverse successful life-history strategies.

At present, nuclear condensation and fragmentation have been estimated also using Hoechst probes in fluorescence microscopy and flow cytometry. However, none of the methods used the Hoechst probes for quantitative spectrofluorometric assessment. Therefore, the aim of the present study was to develop a spectrofluorometric assay for detection of nuclear condensation and fragmentation in the intact cells. We used human hepatoma HepG2 and renal HK-2 cells cultured in 96-well plates treated with potent apoptotic inducers (i.e. cisplatin, staurosporine, camptothecin) for 6-48 h. Afterwards, the cells were incubated with Hoechst 33258 (2 mu g/mL) and the increase of fluorescence after binding of the dye to DNA was measured. The developed spectrofluorometric assay was capable to detect nuclear changes caused by all tested apoptotic inducers. Then, we compared the outcomes of the spectrofluorometric assay with other methods detecting cell impairment and apoptosis (i.e. WST-1 and glutathione tests, TUNEL, DNA ladder, caspase activity, PARP-1 and JNKs expressions). We found that our developed spectrofluorometric assay provided results of the same sensitivity as the TUNEL assay but with the advantages of being fast processing, low-cost and a high throughput. Because nuclear condensation and fragmentation can be typical markers of cell death, especially in apoptosis, we suppose that the spectrofluorometric assay could become a routinely used method for characterizing cell death processes. V současné době se k detekci jaderné kondenzace a fragmentace využívají fluorescenční sondy Hoechst. Žádná z metod však nepoužívá sondy Hoechst pro kvantitativní spektrofluorometrické hodnocení. Cílem této studie bylo vyvinout spektrofluorometrický test pro detekci jaderné kondenzace a fragmentace v intaktních buňkách. Použili jsme buňky lidského hepatomu HepG2 a renální HK-2 buňky kultivované v 96-jamkových inkubovanými s apoptotickými induktory (tj. cisplatinou, staurosporinem, kamptotecinem) po dobu 6-48 hodin. Poté byly buňky inkubovány s Hoechst 33258 (2 ug/ml) a bylo detekováno zvýšení intenzity fluorescence po navázání barviva na DNA. Vyvinutý spektrofluorometrický test byl schopen detekovat jaderné změny způsobené všemi testovanými apoptotickými induktory. Poté jsme výsledky spektrofluorometrického testu porovnali s dalšími metodami detekujícími poškození buněk a apoptózu (tj. testy WST-1 a glutathionu, TUNEL, DNA ladder, aktivita kaspázy, exprese PARP-1 a JNKs). Zjistili jsme, že náš vyvinutý spektrofluorometrický test poskytuje výsledky se stejnou citlivostí jako test TUNEL, ale s výhodami rychlého zpracování, nízkých nákladů a vysokého výkonu. Protože jaderná kondenzace a fragmentace mohou být typickými markery buněčné smrti, zejména při apoptóze, předpokládáme, že spektrofluorometrický test by se mohl stát rutinně používanou metodou pro charakterizaci procesů buněčné smrti.

Studies of telomeres and telomere biology often critically rely on the detection of telomeric DNA and measurements of the length of telomere repeats in either single cells or populations of cells. Several methods are available that provide this type of information and it is often not clear what method is most appropriate to address a specific research question. The major variables that need to be considered are the material that is or can be made available and the accuracy of measurements that is required. The goal of this review is to provide a comprehensive summary of the most commonly used methods and discuss the advantages and disadvantages of each. Methods that start with genomic DNA include telomere restriction fragment (TRF) length analysis, PCR amplification of telomere repeats relative to a single copy gene by Q-PCR or MMQPCR and single telomere length analysis (STELA), a PCR-based approach that accurately measures the full spectrum of telomere lengths from individual chromosomes. A different set of methods relies on fluorescent in situ hybridization (FISH) to detect telomere repeats in individual cells or chromosomes. By including essential calibration steps and appropriate controls these methods can be used to measure telomere repeat length or content in chromosomes and cells. Such methods include quantitative FISH (Q-FISH) and flow FISH which are based on digital microscopy and flow cytometry, respectively. Here the basic principles of various telomere length measurement methods are described and their strengths and weaknesses are highlighted. Some recent developments in telomere length analysis are also discussed. The information in this review should facilitate the selection of the most suitable method to address specific research question about telomeres in either model organisms or human subjects. (C) 2011 Elsevier B.V. All rights reserved.

Studies of telomeres and telomere biology often critically rely on the detection of telomeric DNA and measurements of the length of telomere repeats in either single cells or populations of cells. Several methods are available that provide this type of information and it is often not clear what method is most appropriate to address a specific research question. The major variables that need to be considered are the material that is or can be made available and the accuracy of measurements that is required. The goal of this review is to provide a comprehensive summary of the most commonly used methods and discuss the advantages and disadvantages of each. Methods that start with genomic DNA include telomere restriction fragment (TRF) length analysis, PCR amplification of telomere repeats relative to a single copy gene by Q-PCR or MMQPCR and single telomere length analysis (STELA), a PCR-based approach that accurately measures the full spectrum of telomere lengths from individual chromosomes. A different set of methods relies on fluorescent in situ hybridization (FISH) to detect telomere repeats in individual cells or chromosomes. By including essential calibration steps and appropriate controls these methods can be used to measure telomere repeat length or content in chromosomes and cells. Such methods include quantitative FISH (Q-FISH) and flow FISH which are based on digital microscopy and flow cytometry, respectively. Here the basic principles of various telomere length measurement methods are described and their strengths and weaknesses are highlighted. Some recent developments in telomere length analysis are also discussed. The information in this review should facilitate the selection of the most suitable method to address specific research question about telomeres in either model organisms or human subjects. (C) 2011 Elsevier B.V. All rights reserved.

Objectives: Brain single-photon emission computed tomography (SPECT) has been proposed as a potentially useful modality for the metabolic assessment of various brain tumors. Material and methods: In a 10-patient prospective pilot study we evaluated whether (99m)Tc-Tetrofosmin ((99m)Tc-TF) uptake correlates with glioma proliferative activity assessed by flow cytometric analysis. (99m)Tc-TF brain SPECT was performed shortly before surgical tumor excision. Results: Eight patients were diagnosed with glioblastoma multiform and 2 with anaplastic astrocytoma. All tumors were aneuploid. We found a significant positive linear correlation between 99mTc-TF uptake and percentage of tumor cells on the S-phase of the cell cycle (r=0.92, P=0.001). Conclusion: Initial evidence suggests that 99mTc-TF could provide a non-invasive indicator of glioma proliferative activity. (C) 2009 Elsevier B.V. All rights reserved. Clin Neurol Neurosurg

Phaeocystis pouchetii is an ecologically important colony-forming marine phytoplankton species in northern hemisphere cold waters. It plays a central role in the processing of biogeochemically important elements in the upper ocean during spring blooms. Here, we report highly significant quantitative relationships among colony size, cell number and particulate carbon and nitrogen contents of field populations of P. pouchetii, which provide the means to quantitatively convert its biological expression into units of biogeochemical significance. Populations were sampled from mesocosms incubated in situ in western Norway and either fertilized with nitrate and phosphate or left unamended. Phaeocystis colony blooms developed in both scenarios, but were much greater in fertilized treatments. Colonies from the latter treatments were larger, contained higher concentrations of cells and more particulate carbon and nitrogen than those in the unfertilized mesocosm. Considering all data, log cell number increased linearly with log colony volume with a slope of 0.54. Log carbon and nitrogen increased with log colony volume, with respective slopes of 0.92 and 1.22, indicating a significant component of non-cellular carbon and nitrogen within the colonies. Carbon and nitrogen contents of colonies were linearly related, and fertilized colonies contained more nitrogen relative to carbon than unfertilized colonies. These equations are particularly applicable to contemporary studies of P. pouchetii because they represent colonies growing in environments that mimic a continuum from natural to eutrophicated ecosystems.

We report on the construction of sex-specific linkage maps, the identification of sex-linked markers and the genome size estimation for the brine shrimp Artemia franciscana. Overall, from the analysis of 433 AFLP markers segregating in a 112 full-sib family we identified 21 male and 22 female linkage groups (2n = 42), covering 1,041 and 1,313 cM respectively. Fifteen putatively homologous linkage groups, including the sex linkage groups, were identified between the female and male linkage map. Eight sex-linked AFLP marker alleles were inherited from the female parent, supporting the hypothesis of a WZ–ZZ sex-determining system. The haploid Artemia genome size was estimated to 0.93 Gb by flow cytometry. The produced Artemia linkage maps provide the basis for further fine mapping and exploring of the sex-determining region and are a possible marker resource for mapping genomic loci underlying phenotypic differences among Artemia species.

Malaria parasites must undergo sexual and sporogonic development in mosquitoes before they can infect their vertebrate hosts. We report the discovery and characterization of MISFIT, the first protein with paternal effect on the development of the rodent malaria parasite Plasmodium berghei in Anopheles mosquitoes. MISFIT is expressed in male gametocytes and localizes to the nuclei of male gametocytes, zygotes and ookinetes. Gene disruption results in mutant ookinetes with reduced genome content, microneme defects and altered transcriptional profiles of putative cell cycle regulators, which yet successfully invade the mosquito midgut. However, developmental arrest ensues during the ookinete transformation to oocysts leading to malaria transmission blockade. Genetic crosses between misfit mutant parasites and parasites that are either male or female gamete deficient reveal a strict requirement for a male misfit allele. MISFIT belongs to the family of formin-like proteins, which are known regulators of the dynamic remodeling of actin and microtubule networks. Our data identify the ookinete-to-oocyst transition as a critical cell cycle checkpoint in Plasmodium development and lead us to hypothesize that MISFIT may be a regulator of cell cycle progression. This study offers a new perspective for understanding the male contribution to malaria parasite development in the mosquito vector., Author Summary The unicellular protozoan parasites that cause malaria must undergo sexual development and subsequent proliferation in mosquitoes before they can infect humans and cause malaria. We characterized the first protein with paternal effect on the development of malaria parasites in the mosquito. This protein, which we named MISFIT, is produced in the progenitor cells of male gametes and found in the nuclei of these cells as well as in the nuclei of zygotes and their invasive forms, termed ookinetes. Disruption of the gene that encodes MISFIT leads to ookinetes with reduced DNA content, a defective secretory machinery and altered expression of various regulators of DNA replication and cell division. These mutant parasites stop developing immediately after traversing the mosquito gut, leading to malaria transmission blockage. Our study offers a new perspective for understanding the sexual development of malaria parasites in the mosquito vector, which leads to transmission of one of the most devastating diseases of mankind.

The hyperthermophilic archaea Acidianus hospitalis , Aeropyrum pernix , Pyrobaculum aerophilum , Pyrobaculum calidifontis , and Sulfolobus tokodaii representing three different orders in the phylum Crenarchaeota were analyzed by flow cytometry and combined phase-contrast and epifluorescence microscopy. The overall organization of the cell cycle was found to be similar in all species, with a short prereplicative period and a dominant postreplicative period that accounted for 64 to 77% of the generation time. Thus, in all Crenarchaeota analyzed to date, cell division and initiation of chromosome replication occur in close succession, and a long time interval separates termination of replication from cell division. In Pyrobaculum , chromosome segregation overlapped with or closely followed DNA replication, and further genome separation appeared to occur concomitant with cellular growth. Cell division in P. aerophilum took place without visible constriction.

Phaeocystis pouchetii is an ecologically important colony-forming marine phytoplankton species in northern hemisphere cold waters. It plays a central role in the processing of biogeochemically important elements in the upper ocean during spring blooms. Here, we report highly significant quantitative relationships among colony size, cell number and particulate carbon and nitrogen contents of field populations of P. pouchetii, which provide the means to quantitatively convert its biological expression into units of biogeochemical significance. Populations were sampled from mesocosms incubated in situ in western Norway and either fertilized with nitrate and phosphate or left unamended. Phaeocystis colony blooms developed in both scenarios, but were much greater in fertilized treatments. Colonies from the latter treatments were larger, contained higher concentrations of cells and more particulate carbon and nitrogen than those in the unfertilized mesocosm. Considering all data, log cell number increased linearly with log colony volume with a slope of 0.54. Log carbon and nitrogen increased with log colony volume, with respective slopes of 0.92 and 1.22, indicating a significant component of non-cellular carbon and nitrogen within the colonies. Carbon and nitrogen contents of colonies were linearly related, and fertilized colonies contained more nitrogen relative to carbon than unfertilized colonies. These equations are particularly applicable to contemporary studies of P. pouchetii because they represent colonies growing in environments that mimic a continuum from natural to eutrophicated ecosystems.