An epigenetic view of plant cells cultured in vitro: somaclonal variation and beyond

Epigenetic mechanisms are highly dynamic events that modulate gene expression. As more accurate and powerful tools for epigenetic analysis become available for application in a broader range of plant species, analysis of the epigenetic landscape of plant cell cultures may turn out to be crucial for understanding variant phenotypes. In vitro plant cell and tissue culture methodologies are important for many ongoing plant propagation and breeding programmes as well as for cutting-edge research in several plant model species. Although it has long been known that in vitro conditions induce variation at several levels, most studies using such conditions rely on the assumption that in vitro cultured plant cells/tissues mostly conform genotypically and phenotypically. However, when large-scale clonal propagation is the aim, there has been a concern in confirming true-to-typeness using molecular markers for evaluating stability. While in most reports genetic variation has been found to occur at relatively modest frequencies, variation in DNA methylation patterns seems to be much more frequent and in some cases it has been directly implicated in phenotypic variation. Recent advances in the field of epigenetics have uncovered highly dynamic mechanisms of chromatin remodelling occurring during cell dedifferentiation and differentiation processes on which in vitro adventitious plant regeneration systems are based. Here, an overview of recent findings related to developmental switches occurring during in vitro culture is presented. Additionally, an update on the detection of epigenetic variation in plant cell cultures will be provided and discussed in the light of recent progress in the plant epigenetics field.     

<Emphasis Type="Italic">Fusarium verticillioides</Emphasis> and fumonisin contamination in <Emphasis Type="Italic">Bt</Emphasis> and non-<Emphasis Type="Italic">Bt</Emphasis> maize cultivated in Brazil

Fusarium verticillioides is one of the main pathogens of maize, causing ear and stalk rots. This fungus is also able to produce high levels of fumonisins, which have been linked to various illnesses in humans and animals. Previous studies have shown that maize hybrids genetically modified with the cry genes from the bacterium Bacillus thuringiensis (Bt) presented lower incidence of F. verticillioides and fumonisin levels, presumably through the reduction of insects, which could act as vectors of fungi. The aim of this study was to assess the incidence of F. verticillioides and the concentration of fumonisins in Bt and isogenic non-Bt hybrids (2B710Hx, 30F35YG, 2B710, and 30F35, respectively). The samples of 2B710Hx and 30F35YG presented lower F. verticillioides frequency than 2B710 and 30F35 samples. However, there was no statistical difference between fumonisin contamination when Bt and non-Bt samples were compared (P > 0.05). The results suggest that other environmental parameters could possibly trigger fumonisin production during plant development in the field; consequently, other management strategies should be applied to aid controlling fumonisin contamination in maize.     

Development of a Teat Bio-sealant and Evaluation of its Technological and Functional Properties

A teat bio-sealant was developed using Weissella cibaria, and the bio-sealant’s technological and functional properties were assessed. The development included four experimental phases that were analyzed using independent experimental designs. Initially, sterilized or pasteurized Aloe vera gels were used, and the effect of heat treatment was investigated. In the second phase, the effects of time, storage temperature, and addition of cryopreservatives on the viability of the probiotic were observed. The third phase consisted of evaluating the synergistic effects of the cryopreservatives. The fourth phase involved selecting a material that would provide viscosity to the teat sealant. Technological and functional properties were measured in terms of viability of W. cibaria, and antimicrobial activity against Staphylococcus aureus and Streptococcus agalactiae was also analyzed. A mixture of milk powder and glycerol preserved this antimicrobial activity. Pullulan provided greater viscosity and maintained the technological and functional properties of the bio-sealant for 29 days. This teat bio-sealant can be used as an alternative for the prevention of bovine mastitis.     

Cytologic features of pilocytic astrocytoma in cerebrospinal fluid specimens

OBJECTIVE: To illustrate the cytomorphologic features of pilocytic astrocytoma (PA) in cerebrospinal fluid (CSF) samples. STUDY DESIGN: A search of records from 1965 to 2001 was performed to identify all patients with a diagnosis of PA in whom CSF samples were examined. Slides from CSF samples originally reported as atypical, suspicious or positive were reviewed and the cytomorphologic features assessed. RESULTS: Two hundred ninety-three patients with a diagnosis of PA were identified. Of these, 44 had a total of 65 cytologic preparations of CSF. In 34 patients (77.2%) the CSF cytology was negative, in 5 (11.4%) either atypical or suspicious, and in 5 (11.4%) positive for neoplastic cells. The tumors in the 5 positive cases arose in the cerebellar hemispheres (2), cerebellar vermis (1), thalamus (1) and tectum with extension into the fourth ventricle (1). All positive samples were hypercellular, with an average of 5 cell clusters per case (range, 3-11). The clusters were composed of cohesive epithelioid cells with a mean of 8 cells per cluster. In addition, some cases had scattered, isolated, single cells. These single neoplastic cells had prominent, hairlike cytoplasmic processes. The cells in clusters appeared epithelioid, with oval nuclei, mild nuclear pleomorphism, finely or slightly coarsely granular chromatin and cobweblike cytoplasm. CONCLUSION: The cytomorphologic features of PAs recapitulate their histologic characteristics. The tumor cells are recognizable in CSF samples and readily distinguishable from histiocytes and ependymal cells.     

Functional and structural characterization of synthetic cardosin B-derived rennet

The potential of using a synthetic cardosin-based rennet in cheese manufacturing was recently demonstrated with the development and optimization of production of a recombinant form of cardosin B in Kluyveromyces lactis. With the goal of providing a more detailed characterization of this rennet, we herein evaluate the impact of the plant-specific insert (PSI) on cardosin B secretion in this yeast, and provide a thorough analysis of the specificity requirements as well as the biochemical and structural properties of the isolated recombinant protease. We demonstrate that the PSI domain can be substituted by different linker sequences without substantially affecting protein secretion and milk clotting activity. However, the presence of small portions of the PSI results in dramatic reductions of secretion yields in this heterologous system. Kinetic characterization and specificity profiling results clearly suggest that synthetic cardosin B displays lower catalytic efficiency and is more sequence selective than native cardosin B. Elucidation of the structure of synthetic cardosin B confirms the canonical fold of an aspartic protease with the presence of two high mannose-type, N-linked glycan structures; however, there are some differences in the conformation of the flap region when compared to cardosin A. These subtle variations in catalytic properties and the more stringent substrate specificity of synthetic cardosin B help to explain the observed suitability of this rennet for cheese production.     

Histone hyperacetylation in mitosis prevents sister chromatid separation and produces chromosome segregation defects

Posttranslational modifications of core histones contribute to driving changes in chromatin conformation and compaction. Herein, we investigated the role of histone deacetylation on the mitotic process by inhibiting histone deacetylases shortly before mitosis in human primary fibroblasts. Cells entering mitosis with hyperacetylated histones displayed altered chromatin conformation associated with decreased reactivity to the anti-Ser 10 phospho H3 antibody, increased recruitment of protein phosphatase 1-delta on mitotic chromosomes, and depletion of heterochromatin protein 1 from the centromeric heterochromatin. Inhibition of histone deacetylation before mitosis produced defective chromosome condensation and impaired mitotic progression in living cells, suggesting that improper chromosome condensation may induce mitotic checkpoint activation. In situ hybridization analysis on anaphase cells demonstrated the presence of chromatin bridges, which were caused by persisting cohesion along sister chromatid arms after centromere separation. Thus, the presence of hyperacetylated chromatin during mitosis impairs proper chromosome condensation during the pre-anaphase stages, resulting in poor sister chromatid resolution. Lagging chromosomes consisting of single or paired sisters were also induced by the presence of hyperacetylated histones, indicating that the less constrained centromeric organization associated with heterochromatin protein 1 depletion may promote the attachment of kinetochores to microtubules coming from both poles.     

The empowerment of translational research: lessons from laminopathies

The need for a collaborative approach to complex inherited diseases collectively referred to as laminopathies, encouraged Italian researchers, geneticists, physicians and patients to join in the Italian Network for Laminopathies, in 2009. Here, we highlight the advantages and added value of such a multidisciplinary effort to understand pathogenesis, clinical aspects and try to find a cure for Emery-Dreifuss muscular dystrophy, Mandibuloacral dysplasia, Hutchinson-Gilford Progeria and forms of lamin-linked cardiomyopathy, neuropathy and lipodystrophy.     

Polymorphisms in the TNFA and IL6 Genes Represent Risk Factors for Autoimmune Thyroid Disease

Background

Autoimmune thyroid disease (AITD) comprises diseases including Hashimoto''s thyroiditis and Graves'' disease, both characterized by reactivity to autoantigens causing, respectively, inflammatory destruction and autoimmune stimulation of the thyroid-stimulating hormone receptor. AITD is the most common thyroid disease and the leading form of autoimmune disease in women. Cytokines are key regulators of the immune and inflammatory responses; therefore, genetic variants at cytokine-encoding genes are potential risk factors for AITD.

Methods

Polymorphisms in the IL6-174 G/C (rs1800795), TNFA-308 G/A (rs1800629), IL1B-511 C/T (rs16944), and IFNGR1-56 T/C (rs2234711) genes were assessed in a case-control study comprising 420 Hashimoto''s thyroiditis patients, 111 Graves'' disease patients and 735 unrelated controls from Portugal. Genetic variants were discriminated by real-time PCR using TaqMan SNP genotyping assays.

Results

A significant association was found between the allele A in TNFA-308 G/A and Hashimoto''s thyroiditis, both in the dominant (OR = 1.82, CI = 1.37–2.43, p-value = 4.4×10⁻⁵) and log-additive (OR = 1.64, CI = 1.28–2.10, p-value = 8.2×10⁻⁵) models. The allele C in IL6-174 G/C is also associated with Hashimoto''s thyroiditis, however, only retained significance after multiple testing correction in the log-additive model (OR = 1.28, CI = 1.06–1.54, p-value = 8.9×10⁻³). The group with Graves'' disease also registered a higher frequency of the allele A in TNFA-308 G/A compared with controls both in the dominant (OR = 1.85, CI = 1.19–2.87, p-value = 7.0×10⁻³) and log-additive (OR = 1.69, CI = 1.17–2.44, p-value = 6.6×10⁻³) models. The risk for Hashimoto''s thyroiditis and Graves'' disease increases with the number of risk alleles (OR for two risk alleles is, respectively, 2.27 and 2.59).

Conclusions

This study reports significant associations of genetic variants in TNFA and IL6 with the risk for AITD, highlighting the relevance of polymorphisms in inflammation-related genes in the etiopathogenesis of AITD.