CroniCare: Platform for the Design and Implementation of Follow-Up, Control and Self-Management Interventions for Chronic and Multimorbidity Patients Based on Mobile Technologies.

The project proposes to facilitate the design and evaluation of interventions based on mobile technologies and information systems in order to improve the capacity for self-management, empowerment and control of chronic and multimorbidity patients. The system allows to create customizable apps according to the needs of primary care and specialized care. The project includes an evaluation of the impact of the care model, as well as the effectiveness and efficiency of the intervention through a study with 124 multimorbidity patients.

Nondisjunction in favor of a chromosome: the mechanism of rye B chromosome drive during pollen mitosis.

B chromosomes (Bs) are supernumerary components of the genome and do not confer any advantages on the organisms that harbor them. The maintenance of Bs in natural populations is possible by their transmission at higher than Mendelian frequencies. Although drive is the key for understanding B chromosomes, the mechanism is largely unknown. We provide direct insights into the cellular mechanism of B chromosome drive in the male gametophyte of rye (Secale cereale). We found that nondisjunction of Bs is accompanied by centromere activity and is likely caused by extended cohesion of the B sister chromatids. The B centromere originated from an A centromere, which accumulated B-specific repeats and rearrangements. Because of unequal spindle formation at the first pollen mitosis, nondisjoined B chromatids preferentially become located toward the generative pole. The failure to resolve pericentromeric cohesion is under the control of the B-specific nondisjunction control region. Hence, a combination of nondisjunction and unequal spindle formation at first pollen mitosis results in the accumulation of Bs in the generative nucleus and therefore ensures their transmission at a higher than expected rate to the next generation.

Painting the rye genome with genome-specific sequences.

We used rye-specific repetitive DNA sequences in fluorescence in situ hybridization (FISH) to paint the rye genome and to identify rye DNA in a wheat background. A 592 bp fragment from the rye-specific dispersed repetitive family R173 (named UCM600) was cloned and used as a FISH probe. UCM600 is dispersed over the seven rye chromosomes, being absent from the pericentromeric and subtelomeric regions. A similar pattern of distribution was also observed on the rye B chromosomes, but with weaker signals. The FISH hybridization patterns using UCM600 as probe were comparable with those obtained with the genomic in situ hybridization (GISH) procedure. There were, however, sharper signals and less background with FISH. UCM600 was combined with the rye-specific sequences Bilby and pSc200 to obtain a more complete painting. With these probes, the rye chromosomes were labeled with distinctive patterns; thus, allowing the rye cultivar 'Imperial' to be karyotyped. It was also possible to distinguish rye chromosomes in triticale and alien rye chromatin in wheat-rye addition and translocation lines. The distribution of UCM600 was similar in cultivated rye and in the wild Secale species Secale vavilovii Grossh., Secale sylvestre Host, and Secale africanum Stapf. Thus, UCM600 can be used to detect Secale DNA introgressed from wild species in a wheat background.

Agrobacterium-mediated transformation of maize (Zea mays) with Cre-lox site specific recombination cassettes in BIBAC vectors.

The Cre/loxP site-specific recombination system has been applied in various plant species including maize (Zea mays) for marker gene removal, gene targeting, and functional genomics. A BIBAC vector system was adapted for maize transformation with a large fragment of genetic material including a herbicide resistance marker gene, a 30 kb yeast genomic fragment as a marker for fluorescence in situ hybridization (FISH), and a 35S-lox-cre recombination cassette. Seventy-five transgenic lines were generated from Agrobacterium-mediated transformation of a maize Hi II line with multiple B chromosomes. Eighty-four inserts have been localized among all 10 A chromosome pairs by FISH using the yeast DNA probe together with a karyotyping cocktail. No inserts were found on the B chromosomes; thus a bias against the B chromosomes by the Agrobacterium-mediated transformation was revealed. The expression of a cre gene was confirmed in 68 of the 75 transgenic lines by a reporter construct for cre/lox mediated recombination. The placement of the cre/lox site-specific recombination system in many locations in the maize genome will be valuable materials for gene targeting and chromosome engineering.

Improvement of Agrobacterium-mediated transformation in Hi-II maize (Zea mays) using standard binary vectors.

High-frequency transformation of maize (Zea mays L.) using standard binary vectors is advantageous for functional genomics and other genetic engineering studies. Recent advances in Agrobacterium tumefaciens-mediated transformation of maize have made it possible for the public to transform maize using standard binary vectors without a need of the superbinary vector. While maize Hi-II has been a preferred maize genotype to use in various maize transformation efforts, there is still potential and need in further improving its transformation frequency. Here we report the enhanced Agrobacterium-mediated transformation of immature zygotic embryos of maize Hi-II using standard binary vectors. This improved transformation process employs low-salt media in combined use with antioxidant L-cysteine alone or L-cysteine and dithiothreitol (DTT) during the Agrobacterium infection stage. Three levels of N6 medium salts, 10, 50, and 100%, were tested. Both 10 and 50% salts were found to enhance the T-DNA transfer in Hi-II. Addition of DTT to the cocultivation medium also improves the T-DNA transformation. About 12% overall and the highest average of 18% transformation frequencies were achieved from a large number of experiments using immature embryos grown in various seasons. The enhanced transformation protocol established here will be advantageous for maize genetic engineering studies including transformation-based functional genomics.

Construction and behavior of engineered minichromosomes in maize.

Engineered minichromosomes were constructed in maize by modifying natural A and supernumerary B chromosomes. By using telomere-mediated chromosomal truncation, it was demonstrated that such an approach is feasible for the generation of minichromosomes of normal A chromosomes by selection of spontaneous polyploid events that compensate for the deficiencies produced. B chromosomes are readily fractionated by biolistic transformation of truncating plasmids. Foreign genes were faithfully expressed from integrations into normal B chromosomes and from truncated miniB chromosomes. Site-specific recombination between the terminal transgene on a miniA chromosome and a terminal site on a normal chromosome was demonstrated. It was also found that the miniA chromosome did not pair with its progenitor chromosomes during meiosis, indicating a useful property for such constructs. The miniB chromosomes are faithfully transmitted from one generation to the next but can be changed in dosage in the presence of normal B chromosomes. This approach for construction of engineered chromosomes can be easily extended to other plant species because it does not rely on cloned centromere sequences, which are species-specific. These platforms will provide avenues for studies on plant chromosome structure and function and for future developments in biotechnology and agriculture.

Molecular and functional dissection of the maize B chromosome centromere.

The centromere of the maize (Zea mays) B chromosome contains several megabases of a B-specific repeat (ZmBs), a 156-bp satellite repeat (CentC), and centromere-specific retrotransposons (CRM elements). Here, we demonstrate that only a small fraction of the ZmBs repeats interacts with CENH3, the histone H3 variant specific to centromeres. CentC, which marks the CENH3-associated chromatin in maize A centromeres, is restricted to an approximately 700-kb domain within the larger context of the ZmBs repeats. The breakpoints of five B centromere misdivision derivatives are mapped within this domain. In addition, the fraction of this domain remaining after misdivision correlates well with the quantity of CENH3 on the centromere. Thus, the functional boundaries of the B centromere are mapped to a relatively small CentC- and CRM-rich region that is embedded within multimegabase arrays of the ZmBs repeat. Our results demonstrate that the amount of CENH3 at the B centromere can be varied, but with decreasing amounts, the function of the centromere becomes impaired.

Advances in plant chromosome identification and cytogenetic techniques.

Recent developments that improve our ability to distinguish slightly diverged genomes from each other, as well as to distinguish each of the nonhomologous chromosomes within a genome, add a new dimension to the study of plant genomics. Differences in repetitive sequences among different species have been used to develop multicolor fluorescent in situ hybridization techniques that can define the components of allopolyploids in detail and reveal introgression between species. Bacterial artificial chromosome probes and repetitive sequence arrays have been used to distinguish each of the nonhomologous somatic chromosomes within a species. Such karyotype analysis opens new avenues for the study of chromosomal variation and behavior, as well as for the localization of individual genes and transgenes to genomic position.

Characterization of the constriction with neocentric activity of 5RL chromosome in wheat.

In wheat-5RL monotelosomic and ditelosomic addition lines, a proximal constriction located on the long arm of rye chromosome 5R shows neocentric activity at metaphase I of meiosis. In some pollen mother cells this region is unusually stretched, acquires kinetic activity and co-orients with the true centromeres. In the work described here we characterized the putative neocentric constriction of 5RL using various approaches. Fluorescence in situ hybridization (FISH) revealed that the rye subtelomeric repetitive DNA sequence pSc119.2 is a constituent of the 5RL constriction. This FISH site corresponds with a heterochromatic C-band in normal rye. Other subtelomeric (pSc34, pSc74, pSc200), centromeric (CCS1, Bilby) and Arabidopsis-type telomeric sequences produce no detectable hybridization signal on the constriction. Immunolocalization with anti-alpha-tubulin antibodies showed that microtubules are bound to the constriction in a similar way to their binding to true centromeres. Silver staining demonstrated that proteins are accumulated at the constriction, the signal being more prominent than that observed at the centromere and telomeres of 5RL. The frequency of neocentric activity in different plants varied dramatically in different generations and in siblings grown in different years, suggesting that activation of the neocentric site is dependent on internal features and environmental conditions.