Vernalization and photoperiod-related changes in the DNA methylation state in winter and spring rapeseed

Vernalization-induced flowering is an effect of the epigenetic regulation of gene expression through DNA methylation and histone modifications. Vernalization-mediated silencing of a floral repressor through histone modifications was shown in Arabidopsis thaliana. However, for Brassica napus L., the mechanism underlying vernalization is unclear, and the roles of DNA methylation and histone modifications have not been established. This study revealed the profiles of changes in the DNA methylation state during vernalization (after 14, 35, 56 days) and the subsequent growth in long- or short-day photoperiods (after 2, 7, 14 days) in the winter and spring rapeseed using TLC and MSAP techniques. TLC analysis showed a significant decrease in the amount of 5-methylcytosine (m⁵C) in genomic DNA in both cultivars at the beginning of vernalization, but upon its termination, the winter rape showed a reduced level of m⁵C contrary to a significantly increased level in the spring rape. MSAP analysis revealed that winter and spring rapeseed differed in the MSAP loci which were demethylated/methylated in the course of the experiment and presented diverse profiles of changes in the methylation state. The winter rape showed permanent demethylations at 69.2 % of MSAP loci in the course of vernalization that were mostly preserved upon its termination. The spring rape showed similar numbers of demethylations and methylations that were mainly transient. The study provides evidence of the role of DNA methylation in vernalization for rapeseed and for the significant prevalence of demethylations at the beginning of vernalization, which is necessary for the transition to reproductive growth.     

Structure-Function Analysis of Heterodimer Formation,Oligomerization, and Receptor Binding of the Staphylococcus aureus Bi-component Toxin LukGH

The bi-component leukocidins of Staphylococcus aureus are important virulence factors that lyse human phagocytic cells and contribute to immune evasion. The γ-hemolysins (HlgAB and HlgCB) and Panton-Valentine leukocidin (PVL or LukSF) were shown to assemble from soluble subunits into membrane-bound oligomers on the surface of target cells, creating barrel-like pore structures that lead to cell lysis. LukGH is the most distantly related member of this toxin family, sharing only 30–40% amino acid sequence identity with the others. We observed that, unlike other leukocidin subunits, recombinant LukH and LukG had low solubility and were unable to bind to target cells, unless both components were present. Using biolayer interferometry and intrinsic tryptophan fluorescence we detected binding of LukH to LukG in solution with an affinity in the low nanomolar range and dynamic light scattering measurements confirmed formation of a heterodimer. We elucidated the structure of LukGH by x-ray crystallography at 2.8-Å resolution. This revealed an octameric structure that strongly resembles that reported for HlgAB, but with important structural differences. Structure guided mutagenesis studies demonstrated that three salt bridges, not found in other bi-component leukocidins, are essential for dimer formation in solution and receptor binding. We detected weak binding of LukH, but not LukG, to the cellular receptor CD11b by biolayer interferometry, suggesting that in common with other members of this toxin family, the S-component has the primary contact role with the receptor. These new insights provide the basis for novel strategies to counteract this powerful toxin and Staphylococcus aureus pathogenesis.     

Extraintestinal gamogony of Aggregata octopiana in the reared common octopus (Octopus vulgaris) (Cephalopoda: Octopodidae)

Aggregata octopiana (Apicomplexa, Aggregatidae) is the most prevalent coccidian in the wild common octopus (Octopus vulgaris), whose heteroxenous life cycle includes gamogony and sporogony undergoing in the octopus digestive tract. In the infected reared octopi, we observed an unusual extraintestinal distribution of the coccidian, with both gamogony and sporogony ongoing in dermal and gill tissue. Oocysts and macrogamonts were embedded in the dermal connective tissue of octopian arms, demarcated by a thin cyst wall or multilayered dark membrane. In gill connective and epithelial tissue all developmental stages were observed, eliciting hemocytic infiltration. Sometimes a complete substitution of the tissue by cysts and developmental stages occurred, resulting in necrosis of gill tissue.     

Biosimilars--global issues, national solutions

Biotechnology derived medicinal products are presently the best characterized biologicals with considerable production and clinical experience, and have revolutionized the treatment of some of the most difficult-to-treat diseases, prolonging and improving the quality of life and patient care. They are also currently one of the fastest growing segments of the pharmaceutical industry market. The critical challenge that the biopharmaceutical industry is facing is the expiry of patents for the first generation of biopharmaceuticals, mainly recombinant DNA derived products, such as interferons, growth hormone and erythropoetin. The question that immediately arose was how should such copies of the originator products be licensed, bearing in mind that they are highly complex biological molecules produced by equally complex biological production processes with their inherent problem of biological variability. Copying biologicals is much more complex than copying small molecules and the critical issue was how to handle the licensing of products if relying in part on data from an innovator product. Since 2004 there has been considerable international consultation on how to deal with biosimilars and biological copy products. This has led to a better understanding of the challenges in the regulatory evaluation of the quality, safety and efficacy of "biosimilars", to the exchange of information between regulators, as well as to the identification of key issues. The aim of this article is to provide a brief overview of the scientific and regulatory challenges faced in developing and evaluating similar biotherapeutic products for global use. It is intended as an introduction to the series of articles in this special issue of Biologicals devoted to similar biotherapeutic products.     

Tissue transglutaminase in celiac disease: role of autoantibodies

In celiac disease (CD), gluten, the disease-inducing toxic component in wheat, induces the secretion of IgA-class autoantibodies which target tissue transglutaminase (tTG). These autoantibodies are produced in the small-intestinal mucosa, and, during gluten consumption, they can also be detected in patients' serum but disappear slowly from the circulation on a gluten-free diet. Interestingly, after adoption of a gluten-free diet the serum autoantibodies disappear from the circulation more rapidly than the small-intestinal mucosal autoantibody deposits. The finding of IgA deposits on extracellular tTG in the liver, kidney, lymph nodes and muscles of patients with CD indicates that tTG is accessible to the gut-derived autoantibodies. Although the specific autoantibody response directed against tTG is very characteristic in celiac patients, their role in the immunopathology of the celiac mucosal lesion is a matter of debate. Here we report a brief summary of anti-tTG antibody effects demonstrating that these antibodies are functional and not mere bystanders in the disease pathogenesis. In fact, they inhibit intestinal epithelial cell differentiation, induce intestinal epithelial cell proliferation, increase epithelial permeability and activate monocytes and disturb angiogenesis.     

Met receptor tyrosine kinase signaling induces secretion of the angiogenic chemokine interleukin-8/CXCL8 in pancreatic cancer

At diagnosis, the majority of pancreatic cancer patients present with advanced disease when curative resection is no longer feasible and current therapeutic treatments are largely ineffective. An improved understanding of molecular targets for effective intervention of pancreatic cancer is thus urgent. The Met receptor tyrosine kinase is one candidate implicated in pancreatic cancer. Notably, Met is over expressed in up to 80% of invasive pancreatic cancers but not in normal ductal cells correlating with poor overall patient survival and increased recurrence rates following surgical resection. However the functional role of Met signaling in pancreatic cancer remains poorly understood. Here we used RNA interference to directly examine the pathobiological importance of increased Met signaling for pancreatic cancer. We show that Met knockdown in pancreatic tumor cells results in decreased cell survival, cell invasion, and migration on collagen I in vitro. Using an orthotopic model for pancreatic cancer, we provide in vivo evidence that Met knockdown reduced tumor burden correlating with decreased cell survival and tumor angiogenesis, with minimal effect on cell growth. Notably, we report that Met signaling regulates the secretion of the pro-angiogenic chemokine interleukin-8/CXCL8. Our data showing that the interleukin-8 receptors CXCR1 and CXCR2 are not expressed on pancreatic tumor cells, suggests a paracrine mechanism by which Met signaling regulates interleukin-8 secretion to remodel the tumor microenvironment, a novel finding that could have important clinical implications for improving the effectiveness of treatments for pancreatic cancer.