Topographical studies of the polypeptide subunits of the thylakoid cytochrome b6-f complex

The orientation of specific polypeptides of the cytochrome b6-f complex with respect to the chloroplast stromal phase has been studied using trinitrobenzenesulfonate (TNBS) and pronase E as impermeant modifying reagents. Of the four polypeptides of the complex (33,23,20 and 17 kDa), only cytochrome f was labeled by 14C-TNBS in unfractionated membranes. However, to a varying degree, all of the constituent polypeptides were sensitive to pronase digestion and, in the case of cytochrome f, it was possible, by immunoblotting techniques to identify several degradation products. These results are discussed in relation to the organization of the cytochrome complex in thylakoid membranes and argue for an exposure to the stromal phase of all of the polypeptides, while functional considerations indicate that at least cytochrome f and the Rieske iron-sulfur protein have a possible transmembrane organization.     

The Filament Sensor for Near Real-Time Detection of Cytoskeletal Fiber Structures

A reliable extraction of filament data from microscopic images is of high interest in the analysis of acto-myosin structures as early morphological markers in mechanically guided differentiation of human mesenchymal stem cells and the understanding of the underlying fiber arrangement processes. In this paper, we propose the filament sensor (FS), a fast and robust processing sequence which detects and records location, orientation, length, and width for each single filament of an image, and thus allows for the above described analysis. The extraction of these features has previously not been possible with existing methods. We evaluate the performance of the proposed FS in terms of accuracy and speed in comparison to three existing methods with respect to their limited output. Further, we provide a benchmark dataset of real cell images along with filaments manually marked by a human expert as well as simulated benchmark images. The FS clearly outperforms existing methods in terms of computational runtime and filament extraction accuracy. The implementation of the FS and the benchmark database are available as open source.     

Generation of Aggregates of Mouse Embryonic Stem Cells that Show Symmetry Breaking,Polarization and Emergent Collective Behaviour In Vitro

We have developed a protocol improving current Embryoid Body (EB) culture which allows the study of self-organization, symmetry breaking, axial elongation and cell fate specification using aggregates of mouse embryonic stem cells (mESCs) in suspension culture. Small numbers of mESCs are aggregated in basal medium for 48 hr in non-tissue-culture-treated, U-bottomed 96-well plates, after which they are competent to respond to experimental signals. Following treatment, these aggregates begin to show signs of polarized gene expression and gradually alter their morphology from a spherical mass of cells to an elongated, well organized structure in the absence of external asymmetry cues. These structures are not only able to display markers of the three germ layers, but actively display gastrulation-like movements, evidenced by a directional dislodgement of individual cells from the aggregate, which crucially occurs at one region of the elongated structure. This protocol provides a detailed method for the reproducible formation of these aggregates, their stimulation with signals such as Wnt/β-Catenin activation and BMP inhibition and their analysis by single time-point or time-lapse fluorescent microscopy. In addition, we describe modifications to current whole-mount mouse embryo staining procedures for immunocytochemical analysis of specific markers within fixed aggregates. The changes in morphology, gene expression and length of the aggregates can be quantitatively measured, providing information on how signals can alter axial fates. It is envisaged that this system can be applied both to the study of early developmental events such as axial development and organization, and more broadly, the processes of self-organization and cellular decision-making. It may also provide a suitable niche for the generation of cell types present in the embryo that are unobtainable from conventional adherent culture such as spinal cord and motor neurones.     

A Short Guide to the Climatic Variables of the Last Glacial Maximum for Biogeographers

Ecological niche models are widely used for mapping the distribution of species during the last glacial maximum (LGM). Although the selection of the variables and General Circulation Models (GCMs) used for constructing those maps determine the model predictions, we still lack a discussion about which variables and which GCM should be included in the analysis and why. Here, we analyzed the climatic predictions for the LGM of 9 different GCMs in order to help biogeographers to select their GCMs and climatic layers for mapping the species ranges in the LGM. We 1) map the discrepancies between the climatic predictions of the nine GCMs available for the LGM, 2) analyze the similarities and differences between the GCMs and group them to help researchers choose the appropriate GCMs for calibrating and projecting their ecological niche models (ENM) during the LGM, and 3) quantify the agreement of the predictions for each bioclimatic variable to help researchers avoid the environmental variables with a poor consensus between models. Our results indicate that, in absolute values, GCMs have a strong disagreement in their temperature predictions for temperate areas, while the uncertainties for the precipitation variables are in the tropics. In spite of the discrepancies between model predictions, temperature variables (BIO1-BIO11) are highly correlated between models. Precipitation variables (BIO12- BIO19) show no correlation between models, and specifically, BIO14 (precipitation of the driest month) and BIO15 (Precipitation Seasonality (Coefficient of Variation)) show the highest level of discrepancy between GCMs. Following our results, we strongly recommend the use of different GCMs for constructing or projecting ENMs, particularly when predicting the distribution of species that inhabit the tropics and the temperate areas of the Northern and Southern Hemispheres, because climatic predictions for those areas vary greatly among GCMs. We also recommend the exclusion of BIO14 and BIO15 from ENMs because those variables show a high level of discrepancy between GCMs. Thus, by excluding them, we decrease the level of uncertainty of our predictions. All the climatic layers produced for this paper are freely available in http://ecoclimate.org/.     

Making a living while starving in the dark: metagenomic insights into the energy dynamics of a carbonate cave

Carbonate caves represent subterranean ecosystems that are largely devoid of phototrophic primary production. In semiarid and arid regions, allochthonous organic carbon inputs entering caves with vadose-zone drip water are minimal, creating highly oligotrophic conditions; however, past research indicates that carbonate speleothem surfaces in these caves support diverse, predominantly heterotrophic prokaryotic communities. The current study applied a metagenomic approach to elucidate the community structure and potential energy dynamics of microbial communities, colonizing speleothem surfaces in Kartchner Caverns, a carbonate cave in semiarid, southeastern Arizona, USA. Manual inspection of a speleothem metagenome revealed a community genetically adapted to low-nutrient conditions with indications that a nitrogen-based primary production strategy is probable, including contributions from both Archaea and Bacteria. Genes for all six known CO₂-fixation pathways were detected in the metagenome and RuBisCo genes representative of the Calvin–Benson–Bassham cycle were over-represented in Kartchner speleothem metagenomes relative to bulk soil, rhizosphere soil and deep-ocean communities. Intriguingly, quantitative PCR found Archaea to be significantly more abundant in the cave communities than in soils above the cave. MEtaGenome ANalyzer (MEGAN) analysis of speleothem metagenome sequence reads found Thaumarchaeota to be the third most abundant phylum in the community, and identified taxonomic associations to this phylum for indicator genes representative of multiple CO₂-fixation pathways. The results revealed that this oligotrophic subterranean environment supports a unique chemoautotrophic microbial community with potentially novel nutrient cycling strategies. These strategies may provide key insights into other ecosystems dominated by oligotrophy, including aphotic subsurface soils or aquifers and photic systems such as arid deserts.     

Global Leukocyte DNA Methylation is Similar in African American and Caucasian Women Under Conditions of Controlled Folate Intake

DNA methylation is an epigenetic feature that may modify disease risk, and can be influenced by folate status as well as by methylenetetrahydrofolate reductase (MTHFR) C677T genotype. The aim of this study was to investigate the influence of ethnicity/race on global leukocyte DNA methylation under conditions of controlled folate intake. Caucasian (n=14) and African American (n-14) women (18-45y) possessing the MTHFR 677CC genotype consumed a folate restricted diet (135 μg/d as dietary folate equivalents, DFE) for 7 week followed by folate treatment with 400 or 800 μg DFE/d for 7 week. Global leukocyte DNA methylation was assessed via the cytosine extension assay at baseline (wk 0), after folate restriction (wk 7) and after folate treatment (wk 14). Ethnicity/race was not a determinant of global leukocyte DNA methylation. No differences (P>0.05) were detected in DNA methylation between African American and Caucasian women at baseline or any other study time point. In addition, folate intake did not modify global leukocyte DNA methylation. These data suggest that global leukocyte DNA methylation does not differ between Caucasian and African American women and that short-term folate restriction is not sufficient to modify methylation content in young women with the MTHFR 677CC genotype.     

Chloroplast Genome Diversity in Portuguese Grapevine (Vitis vinifera L.) Cultivars

Grapevine chloroplast (cp) DNA diversity was analysed for the first time through amplification and digestion of fragments of the large single copy (LSC) region by polymerase chain reaction–restriction fragment length polymorphism methodology and also by amplification of three microsatellite loci, previously described as polymorphic in grapevine. Thirty-eight grapevine cultivars collected mainly in the North of Portugal, including some neglected cultivars, four international cultivars (Chasselas, Muscat of Alexandria, Muscat of Hamburg and Pinot) and Vitis riparia and Vitis rupestris, were used in this study with the main goal of finding out their cpDNA diversity and compare the obtained results with previously published data on cultivars from other regions to ascertain their possible origin. Two different alleles were found in each of the three cpSSR loci. Allele variants of the three loci combined in a total of three different haplotypes (A, B and D). The most frequent haplotype, A, was previously reported as the most frequent in Iberian Peninsula and Occidental Europe. Haplotype B was unique to Rabigato, Muscat of Alexandria, V. riparia and V. rupestris. This haplotype was previously proposed to be an ancestral haplotype. Twenty-seven fragments of the LSC region of Vitis vinifera cpDNA were amplified and then digested with HinfI and TaqI restriction enzymes. Polymorphisms were found in the trnT-psbC (TC) and orf184-petA (OA) fragments. In the TC fragment, the polymorphism corresponds to a point mutation in a restriction site of TaqI and is only present in all cultivars with cpSSR haplotype D. In the OA fragment, a short deletion exclusive to the Rabigato cultivar was found. In this case, one sequence tagged site-based marker was developed and will be very useful in future phylogenetic and fingerprinting studies in a broader number of cultivars and in wild grapevine populations. Inference about the progenitors of the Touriga Franca cultivar is done. The present work supports and completes its origin as a descendent of the female and male parents, Marufo and Touriga Nacional.