Integrating experiment, theory and simulation to determine the structure and dynamics of mammalian chromosomes.

Eukaryotic chromosomes are complex polymers, which largely exceed in size most biomolecules that are usually modelled in computational studies and whose molecular interactions are to a large extent unknown. Since the folding of the chromatin fiber in the cell nucleus is tightly linked to biological function and gene expression in particular, characterizing the conformational and dynamical properties of chromosomes has become crucial in order to better understand how genes are regulated. In parallel with the development of experimental techniques allowing to measure physical contacts within chromosomes inside the cell nucleus, a large variety of physical models to study the structure and mechanisms of chromosome folding have recently emerged. Such models can be roughly divided into two classes, based on whether they adopt specific hypotheses on the interaction mechanism within chromosomes, or learn those interactions on the available experimental data using the principle of maximum entropy. All of them have played a key role in interpreting experimental data and advancing our understanding the folding principles of the chromatin fiber.

Modelling genome-wide topological associating domains in mouse embryonic stem cells.

Chromosome conformation capture (3C)-based techniques such as chromosome conformation capture carbon copy (5C) and Hi-C revealed that the folding of mammalian chromosomes is highly hierarchical. A fundamental structural unit in the hierarchy is represented by topologically associating domains (TADs), sub-megabase regions of the genome within which the chromatin fibre preferentially interacts. 3C-based methods provide the mean contact probabilities between chromosomal loci, averaged over a large number of cells, and do not give immediate access to the single-cell conformations of the chromatin fibre. However, coarse-grained polymer models based on 5C data can be used to extract the single-cell conformations of single TADs. Here, we extend this approach to analyse around 2500 TADs in murine embryonic stem cells based on high-resolution Hi-C data. This allowed to predict the cell-to-cell variability in single contacts within genome-wide TADs and correlations between them. Based on these results, we predict that TADs are more similar to ideal chains than to globules in terms of their physical size and three-dimensional shape distribution. Furthermore, we show that their physical size and the degree of structural anisotropy of single TADs are correlated with the level of transcriptional activity of the genes that it harbours. Finally, we show that a large number of multiplets of genomic loci co-localize more often than expected by random, and these loci are particularly enriched in promoters, enhancers and CTCF-bound sites. These results provide the first genome-wide structural reconstruction of TADs using polymeric models obeying the laws of thermodynamics and reveal important universal trends in the correlation between chromosome structure and transcription.

Physiological and biochemical responses of sunflower (Helianthus annuus L.) exposed to nano-CeO2 and excess boron: Modulation of boron phytotoxicity.

Little is known about the interaction of nanoparticles (NPs) with soil constituents and their effects in plants. Boron (B), an essential micronutrient that reduces crop production at both deficiency and excess, has not been investigated with respect to its interaction with cerium oxide NPs (nano-CeO2). Considering conflicting results on the nano-CeO2 toxicity and protective role as antioxidant, their possible modulation on B toxicity in sunflower (Helianthus annuus L.) was investigated. Sunflower was cultivated for 30 days in garden pots containing original or B-spiked soil amended with nano-CeO2 at 0-800 mg kg-1. At harvest, Ce and B concentrations in tissues, biomass, and activities of stress enzymes in leaves were determined. Results showed that in the original soil, Ce accumulated mainly in roots, with little translocation to stems and leaves, while reduced root Ce was observed in plants from B-spiked soil. In the original soil, higher levels of nano-CeO2 reduced plant B concentration. Although morphological effects were not visible, changes in biomass and oxidative stress response were observed. Sunflower leaves from B-spiked soil showed visible symptoms of B toxicity, such as necrosis and chlorosis in old leaves, as well as an increase of superoxide dismutase (SOD) activity. However, at high nano-CeO2 level, SOD activity decreased reaching values similar to that of the control. This study has shown that nano-CeO2 reduced both the B nutritional status of sunflower in original soil and the B phytotoxicity in B-spiked soil.

Looping probability of random heteropolymers helps to understand the scaling properties of biopolymers.

Random heteropolymers are a minimal description of biopolymers and can provide a theoretical framework to the investigate the formation of loops in biophysical experiments. The looping probability as a function of polymer length was observed to display in some biopolymers, like chromosomes in cell nuclei or long RNA chains, anomalous scaling exponents. Combining a two-state model with self-adjusting simulated-tempering calculations, we calculate numerically the looping properties of several realizations of the random interactions within the chain. We find a continuous set of exponents upon varying the temperature, which arises from finite-size effects and is amplified by the disorder of the interactions. We suggest that this could provide a simple explanation for the anomalous scaling exponents found in experiments. In addition, our results have important implications notably for the study of chromosome folding as they show that scaling exponents cannot be the sole criteria for testing hypothesis-driven models of chromosome architecture.

Impact of TiO2 nanoparticles on Vicia narbonensis L.: potential toxicity effects.

This work was aimed to provide further information about toxicology of TiO2 nanoparticles (NPs) on Vicia narbonensis L., considering different endpoints. After exposure to TiO2 nanoparticle suspension (mixture of rutile and anatase, size <100 nm) at four different concentrations (0.2, 1.0, 2.0 and 4.0 ‰), the seeds of V. narbonensis were let to germinate in controlled environmental conditions. After 72 h, the extent of the success of the whole process (seed germination plus root elongation) was recorded as the vigour index, an indicator of possible phytotoxicity. After the characterisation of the hydric state of different materials, oxidative stress and enzymatic and nonenzymatic antioxidant responses were considered as indicators of possible cytotoxicity and to assess if damage induced by TiO2 NPs was oxidative stress-dependent. Cytohistochemical detection of in situ DNA fragmentation as genotoxicity endpoint was monitored by TUNEL reaction. The treatments with TiO2 NPs in our system induced phytotoxic effects, ROS production and DNA fragmentation. The nonenzymatic and enzymatic antioxidant responses were gradually and differentially activated and were able to maintain the oxidative damage to levels not significantly different from the control. On the other hand, the results of DNA fragmentation suggested that the mechanisms of DNA repair were not effective enough to eliminate early genotoxicity effects.

Cytological and molecular characterization of Vicia barbazitae Ten. & Guss.

Vicia barbazitae, a taxon belonging to section Vicia of subgenus Vicia, was recovered and analysed by cytological, karyological and molecular methods with the aim of both proposing a general characterisation of this species and studying the relationships among the species of section Vicia . Phylogenetic relationships among the species of the section Vicia and those of the sections Microcarinae, Wiggersia and Atossa were also analysed. Automated karyotype analysis has been determined after Feulgen's reaction; chromosome banding was performed by sequence-specific fluorochrome staining. Fluorescent chromosome banding showed CMA(+)/DAPI(-) NOR-associated heterochromatin in the satellite pair. Karyomorphological parameters, based on symmetry indices, the dendrogram of linkage distance constructed on 37 chromosome parameters, as well as the molecular data based on internal transcribed spacer sequences provided information about phylogenetic position of this species inside the section Vicia and among the species belonging to the sections Microcarinae, Wiggersia, Atossa and Vicia. From our karyological and molecular results, it emerges that V. barbazitae can be considered a natural member of section Vicia.

Exogenous cytokinin treatments of an Ni hyper-accumulator, Alyssum murale, grown in a serpentine soil: implications for phytoextraction.

Application of exogenous plant growth regulators was examined as a viable technique to increase the efficiency of plant metal extraction from contaminated soils. The aim of this study was to investigate the alteration of Ni phytoextraction by Alyssum murale, a Ni hyperaccumulator, following the application of cytokinins. The following parameters were investigated: Ni accumulation, plant growth, gas exchange, stomata behavior and the concentration of nonprotein thiols (glutathione, y-Glu-Cys, and phytochelatins). In a pot experiment, A. murale plants grown in a serpentine soil were treated with a mix of naturally occurring cytokinins. Results showed that Ni accumulation in plants ranged from 4000 to 7000 mg kg(-1) confirming the hyper-accumulation ability from the soil used. Cytokinin treatments produced a significant increase in plant biomass and transpiration rate whereas no significant variation in Ni accumulation or the concentration of non-protein thiols was observed. The results suggest that A. murale is a plant species sensitive to cytokinin treatment and that cytokinin treatment is potentially useful in increasing the phytoextraction capability by increasing biomass. Moreover, for first time, evidence was obtained that the Ni hyperaccumulation mechanism is independent of water flux and transpiration rate.

Desenvolvimento de formulações de comprimidos tamponados mastigáveis de didanosina com perfil de dissolução e capacidade neutralizante ácida otimizados / Development of didanosine chewable buffered tablets with optimized dissolution profile and acid neutralizing capacity.

Neste estudo buscou-se desenvolver formulações de comprimidos tamponados mastigáveis (CTM) de didanosina com eficiência de dissolução (ED%) e capacidade neutralizante ácida (CNA) otimizados, tendo como base o medicamento referência e especialidades farmacêuticas disponíveis no mercado nacional. Cinco formulações de CTM foram produzidas e avaliadas quanto a ED% e CNA, por meio de ensaio de dissolução e titulação ácido-base, respectivamente. Os resultados iniciais de CNA foram próximos aos encontrados para as especialidades farmacêuticas, aproximadamente 12 mEq HCl, porém distantes do medicamento referência (especialidade A, CNA = 17,93 mEq HCl). Já as formulações derivadas de CTM-4 conduziram à obtenção de comprimidos tamponados com CNA otimizada de aproximadamente 17,5 mEq HCl, o mesmo ocorrendo para ED%, (61,33% e 62,00%, CTM-4-2-1 e3, respectivamente). Esse resultado mostra-se próximo ao valor de 59,33% da especialidade A, quando utilizado o mesmo método de dissolução, indicando haver equivalência entre estas formulações e o medicamento referência para estes parâmetros.

The aim in this study was to develop chewable buffered tablets (CBT) of didanosine with optimized dissolution efficiency (DE) and acid-neutralizing capacity (ANC), using the reference medicine and other pharmaceutical didanosine products available in Brazil as models. Five CBT formulations were prepared and assessed for DE and ANC, through the dissolution test and acid-base titration, respectively. The initial ANC results fell short of those for the reference medicine (product A, ANC= 17.93 mEq HCl), but were close to those obtained for other pharmaceutical products (approximately 12 mEq HCl). The formulations derived from CBT-4 resulted in buffered tablets with an optimized ANC of 17.5 mEq HCl, approximately. The same was found for DE (61.33% and 62.00%, CBT-4-2-1 and CBT 3, respectively). This result proved to be close to that of product A (59.33%), when the same method was used for the dissolution test, indicating that both formulations and the reference medicine were equivalent with respect to these properties.

Expression of HtKNOT1, a class I KNOX gene, overlaps cell layers and development compartments of differentiating cells in stems and flowers of Helianthus tuberosus.

In plant, post-embryonic development relies on the activities of indeterminate cell populations termed meristems, spatially clustered cell lineages, wherein a subset divides indeterminately. For correct growth, the plant must maintain a constant flow of cells through the meristem, where the input of dividing pluripotent cells offsets the output of differentiating cells. KNOTTED1-like homeobox (KNOX) genes are expressed in specific patterns in the plant meristems and play important roles in maintaining meristematic cell identity. We have analyzed the expression pattern of HtKNOT1, a class I KNOX gene of Helianthus tuberosus, in stems, inflorescence meristems, floral meristems and floral organs. HtKNOT1 is expressed in cambial cells, phloem cells and xylematic parenchyma within apical stem internodes, while in basal internodes HtKNOT1 expression was restricted to the presumptive initials and recently derived phloem cells. In the reproductive phase, HtKNOT1 mRNAs were detected in both the inflorescence and floral meristems as well within lateral organ primordia (i.e. floral bracts, petals, stamens and carpels). In more differentiated flowers, the expression of HtKNOT1 was restricted to developing ovules and pollen mother cells. HtKNOT1 may play a dual role being required to maintain the meristem initials as well as initiating differentiation and/or conferring new cell identity. In particular, it is possible that HtKNOT1 cooperates at floral level with additional factors that more specifically control floral organs and pollen development in H. tuberosus.

Proliferation-dependent pattern of expression of a dihydrofolate reductase-thymidylate synthase gene from Daucus carota.

The pattern of expression of a carrot dhfr-ts gene was evaluated in different plant organs, in somatic embryos, and in hypocotyl explants induced to dedifferentiate in vitro by the addition of the synthetic auxin 2,4 dichorophenoxyacetic acid. The promoter of this gene was also placed upstream of a uidA (GUS) reporter gene and, using biolistic and protoplasts transient expression assays, was shown to drive a particularly high level of expression in actively growing suspension cells. The results from these expression analyses combined with the presence of putative cell cycle-related cis-acting elements in the dhfr-ts promoter, strongly point to a cell division-dependent expression of this gene.

The antifungal Dm-AMP1 protein from Dahlia merckii expressed in Solanum melongena is released in root exudates and differentially affects pathogenic fungi and mycorrhizal symbiosis.

• Transformed aubergine plants constitutively expressing the Dm-AMP1 antimicrobial defensin (from Dahlia merckii) were generated and characterized. • Transgenic plants were selected on kanamycin and screened by polymerase chain reaction analysis. The expression of Dm-AMP1 in plant tissues and its release in root exudates were detected by Western blot analyses. Dm-AMP1 localization was performed by immunohistochemical experiments. • Dm-AMP1 expression ranged from 0.2% to 0.48% of total soluble proteins in primary transformants and from 0.16% to 0.66% in F2 plants. Transformed clones showed resistance to the pathogenic fungus Botrytis cinerea, whose development on leaves was reduced by 36-100%, with respect to controls. The protein was released in root exudates of the transformed plants and was active in reducing the growth of the co-cultured pathogenic fungus Verticillium albo-atrum, whereas it did not interfere with recognition responses and symbiosis establishment by the arbuscular mycorrhizal fungus Glomus mosseae. • Dm-AMP1 transformants may represent a useful model to study the interactions between genetically modified plants and pathogenic fungi or beneficial nontarget microorganisms.

Floral genes expressed in tomato hypocotyl explants in liquid culture.

This paper confirms, at molecular level, previous data showing that small explants of many plants do form a floral meristem and express specific floral genes after only few days in culture. After 15-20 days of culture, small tomato hypocotyl explants develop differentiated structures often resembling primitive ancestral reproductive organs. Other specific reproductive functions such as chromosomal segregation (somatic meiosis) were also present and demonstrated by means of a cytological and histological analysis. By reverse transcriptase-PCR and in situ hybridization it was found that these structures are indeed able to express flower-specific genes. The TM8 gene, a tomato gene that is expressed very early during floral development, is detectable on the proliferating hypocotyl explants during the first week of culture. The MON9612 gene, which in vivo is expressed only by tomato pistils and ovules, is detectable on the ovulelike structures developed after 20 days of culture. The construction of transgenic tomato plants expressing the GUS gene under the control of the MON9612 promoter allowed us to follow the induction and the expression of this gene during explant proliferation and development of the flowerlike structures. These data confirm the hypothesis that a floral reprogramming can be induced in plant explants as a consequence of wounding and growth factors action. It appears to be an effort to survive stress by means of an unscheduled reproductive program.

Genome plasticity during the acquisition of embryogenic competence

Hypocotyl explants from carrot and other species experience concomitant segregation events and differentiation of homeotic structures during the first 20 days of culture on 2,4-dichlorophenoxyacetic acid (2,4-D). In addition to these cyto-morphological changes, significant amounts of nuclear DNA are lost, the molecular details of which we investigate in this paper. We have developed a slot-blot analysis assay to study the DNA content of a series of carrot samples; besides the leaves, this survey ranged over different culture timepoints: hypocotyls, cell lines, and somatic embryo stages. We carried on to study the relationship between this DNA loss and sequence complexity modulation. Results from probing sequences that correspond to different degrees of complexity, such as medium repetitive and unique sequences as well as sequences belonging to both classes (ribosomal cistrons, ubiquitin, actin, and chalcone synthase), consistently manifested a reduction in DNA levels during the acquisition of embryogenic competence. In some cases, the cultured cells would contain only 10% of the gene copies observed in the reference tissues. Modulation trends also showed that DNA levels of most sequences recover at the torpedo-plantlet stage, which again correlates DNA modulation and the acquisition of embryogenic competence. These results suggest that similar DNA variations may occur in plants in vivo during meiosis, possibly so that meiotic division may be properly completed.

Carrot DNA-methyltransferase is encoded by two classes of genes with differing patterns of expression.

In the present study, the isolation and characterization of two distinct cDNAs that code for carrot DNA (cytosine-5)-methyltransferase (DNA-METase) are reported. The screening of a cDNA library with a carrot genomic DNA fragment, previously obtained by PCR using degenerate primers, has led to the isolation of clones that belong to two distinct classes of genes (Met1 and Met2) which differ in sequence and size. Met1-5 and Met2-21 derived amino acid sequences are more than 85% identical for most of the polypeptide and completely diverge at the N-terminus. The larger size of the Met2-21 cDNA is due to the presence of nearly perfect fivefold repeat of a 171 bp sequence present only once in the Met1-5 cDNA. Northern and in situ hybridization analyses with young carrot plants and somatic embryos indicate that both genes are maximally expressed in proliferating cells (suspension cells, meristems and leaf primordia), but differ quantitatively and spatially in their mode of expression. Polyclonal antibodies were raised in rabbit using fusion proteins corresponding to the regulatory and catalytic regions of the most highly expressed gene (Met1-5). In nuclear carrot extracts, both antibodies were found to recognize a band of about 200 kDa along with some additional bands of lower size. These results provide the first direct demonstration that DNA-METases of a higher eukaryote are encoded by a gene family.

Regulation of a carotenoid biosynthesis gene promoter during plant development.

Carotenoids are terpenoid pigments which are accumulated in the chloroplasts of leaves and in the chromoplasts of many flowers and fruits. Phytoene desaturase (Pds), the second dedicated enzyme in carotenoid biosynthesis, is encoded in tomato by a single copy gene. A 2 kb fragment from the tomato Pds gene, comprising 1.5 kb from the promoter and 0.5 kb from the 5' non-translated region, is able to drive developmentally regulated expression of the GUS reporter gene in transgenic tomato and tobacco plants. In tomato, high levels of Pds/GUS expression are found in organs and at stages of development where chromoplasts are formed: petals, anthers and ripening fruits. Tobacco petals and fruits, which do not contain chromoplasts, show instead low levels of Pds/GUS expression. Transgenic tobacco seedlings were subjected to treatment with a range of inhibitors of carotenoid and chlorophyll biosynthesis. The results indicate that, in green tissues, carotenoid and chlorophyll levels are tightly co-regulated and that a chemically induced arrest in pigment biosynthesis results in activation of the Pds promoter. The promoter is also induced in etiolated seedlings, which contain much lower carotenoid levels than light-grown seedlings. These data suggest that in green tissues Pds gene transcription may respond to end-product regulation.

On the occurrence of somatic meiosis in embryogenic carrot cell cultures.

During the establishment of an embryogenic cell line from a carrot hypocotyl explant, processes closely resembling meiotic divisions are seen. A microdensitometric analysis revealed that the amount of cellular DNA diminished in the majority of cells to the haploid level. However, the diploid level was re-established in a matter of a few days. The genetic consequences of this segregation were studied by analyzing restriction fragment length polymorphisms (RFLP) and randomly amplified polymorphic DNAs (RAPD). The results showed that the great majority of embryos regenerated from segregants and that different segregants had different genetic constitutions.

Influence of oral contraceptives on fasting gallbladder volume.

A total of 615 healthy fertile women (518 pill users and 97 non-users) were examined by real-time ultrasonography for fasting gallbladder volume, gallstones and biliary dysmorphism. None of the six examined combinations of oral contraceptives appeared to influence fasting gallbladder volume significantly. When fasting gallbladder volumes were reanalyzed according to the presence or absence of recognized biliary risk factors, significant modifications were detected, in both pill users and non-users. These changes related only to age and parity. Relative risks of cholelithiasis and biliary dysmorphism were not affected by contraceptive treatment.

PIP: In Italy, researchers used real-time ultrasonography for fasting (no solid food for 24 hours) gallbladder volume to compare data on 518 women using low-dose, combined oral contraceptives (OCs) for 12-120 months with data on 97 women who had never used OCs to examine the effect of OCs on the gallbladder. The 615 healthy, fertile 16-52 year old women were clients of Contraceptive Counseling Services of the Department of Obstetrics and Gynaecology at the University of Ferrara and the Local Sanitary Unit. No significant difference in fasting gallbladder volumes existed between cases and controls. OC use was not associated with fasting gallbladder volume in relation to duration of treatment, family cholelithiasis, smoking, or age. As parity increased so did fasting gallbladder volume in both groups (p = 0.04 for controls and p = 0.01 for cases). Fasting gallbladder volume increased with age in both groups but not significantly so. Biliary dysmorphism existed in 17.8% of OC users and 24.7% of controls. Cholelithiasis was present in 2.9% of OC users and 3.1% of controls. OC use did not affect the relative risk of biliary dysmorphism and cholelithiasis (0.66 and 0.95, respectively). In conclusion, low-dose OCs do not affect gallbladder motility.