Nomadic enhancers: tissue-specific cis-regulatory elements of yellow have divergent genomic positions among Drosophila species

cis-regulatory DNA sequences known as enhancers control gene expression in space and time. They are central to metazoan development and are often responsible for changes in gene regulation that contribute to phenotypic evolution. Here, we examine the sequence, function, and genomic location of enhancers controlling tissue- and cell-type specific expression of the yellow gene in six Drosophila species. yellow is required for the production of dark pigment, and its expression has evolved largely in concert with divergent pigment patterns. Using Drosophila melanogaster as a transgenic host, we examined the expression of reporter genes in which either 5' intergenic or intronic sequences of yellow from each species controlled the expression of Green Fluorescent Protein. Surprisingly, we found that sequences controlling expression in the wing veins, as well as sequences controlling expression in epidermal cells of the abdomen, thorax, and wing, were located in different genomic regions in different species. By contrast, sequences controlling expression in bristle-associated cells were located in the intron of all species. Differences in the precise pattern of spatial expression within the developing epidermis of D. melanogaster transformants usually correlated with adult pigmentation in the species from which the cis-regulatory sequences were derived, which is consistent with cis-regulatory evolution affecting yellow expression playing a central role in Drosophila pigmentation divergence. Sequence comparisons among species favored a model in which sequential nucleotide substitutions were responsible for the observed changes in cis-regulatory architecture. Taken together, these data demonstrate frequent changes in yellow cis-regulatory architecture among Drosophila species. Similar analyses of other genes, combining in vivo functional tests of enhancer activity with in silico comparative genomics, are needed to determine whether the pattern of regulatory evolution we observed for yellow is characteristic of genes with rapidly evolving expression patterns.     

Molecularly imprinted cryogels for human interferon‐alpha purification from human gingival fibroblast culture

Interferons are important proteins for the immune system because of their antiviral, anti‐proliferating and immunomodulatory activities. Therapeutic value of these proteins against certain types of tumors caused interest and investigations aimed to obtain highly purified interferons. Molecular imprinting is an efficient method for purification with high selectivity, specificity and good reproducibility. In this study, we utilized advantages of molecular imprinting technique for the purification of interferon from human gingival fibroblast culture. For this purpose, interferon α‐2b imprinted poly(hydroxyethyl methacrylate) cryogel (hIFN‐α‐MIP) was prepared. Optimum adsorption conditions were determined, and maximum adsorption capacity of hIFN‐α‐MIP cryogel was found as 254.8 × 10⁴ IU/g from aqueous solution. All interferon measurements are expressed as International Unit (IU), which is a unit measurement used to quantify biologically active substances like interferon based on their biological activity or effect. Selectivity experiments were performed using competitive proteins and repeated adsorption–desorption studies showed that the adsorption capacity maintained almost at a constant value after ten cycles. For the purification of interferon from human gingival fibroblast culture, fast protein liquid chromatography was used and the specific activity of the purified interferon α‐2b on HeLa cell line was found between the values 3.45 × 10⁸ IU/mg and 3.75 × 10⁸ IU/mg. The results are promising, and the molecular imprinting technique is effective for the purification of interferon α‐2b. Copyright © 2013 John Wiley & Sons, Ltd.     

The Association of PTPN22 R620W Polymorphism Is Stronger with Late-Onset AChR-Myasthenia Gravis in Turkey

A functional single nucleotide polymorphism (SNP) of the PTPN22 gene encoding a protein tyrosine phosphatase has been associated with autoimmune disorders including myasthenia gravis (MG). As the PTPN22 R620W polymorphism has a wide variation of allele frequencies among different populations, this polymorphism was investigated in MG in Turkey. An emphasis is put on MG subgroups according to autoantibody (Abs) production and presence of thymoma. DNA samples from 416 patients with clinically diagnosed generalized MG (231 with Abs to acetylcholine receptor, AChR-MG), 53 with Abs to muscle-specific kinase (MuSK-MG), 55 patients with no detectable Abs (SN-MG), 77 patients with thymoma (TAMG) and 293 healthy controls (HC) were genotyped for the SNP (PTPN22 R620W, C1858T, rs2476601). The PTPN22 T allele was increased in AChR-MG patients (odds ratio [OR]: 2.5, 95%CI: 1.2–5.1). The association was stronger in late disease-onset AChR (LOMG, OR: 3.1, 95%CI: 1.2–8.2). MuSK-MG, SN-MG and TAMG groups did not carry the variant allele more frequently than the HC. In contrast to findings in other autoimmune diseases, the distribution of the PTPN22 polymorphism in this population provides a susceptibility marker for AChR-MG. The strongest association is detected in patients with LOMG.     

SIRT2 ablation has no effect on tubulin acetylation in brain, cholesterol biosynthesis or the progression of Huntington's disease phenotypes in vivo

Huntington's disease (HD) is a devastating neurodegenerative disorder for which there are no disease-modifying treatments. The molecular pathogenesis of HD is complex and many mechanisms and cellular processes have been proposed as potential sites of therapeutic intervention. However, prior to embarking on drug development initiatives, it is essential that therapeutic targets can be validated in mammalian models of HD. Previous studies in invertebrate and cell culture HD models have suggested that inhibition of SIRT2 could have beneficial consequences on disease progression. SIRT2 is a NAD(+)-dependent deacetylase that has been proposed to deacetylate α-tubulin, histone H4 K16 and to regulate cholesterol biogenesis - a pathway which is dysregulated in HD patients and HD mouse models. We have utilized mice in which SIRT2 has been reduced or ablated to further explore the function of SIRT2 and to assess whether SIRT2 loss has a beneficial impact on disease progression in the R6/2 mouse model of HD. Surprisingly we found that reduction or loss of SIRT2 had no effect on the acetylation of α-tubulin or H4K16 or on cholesterol biosynthesis in the brains of wild type mice. Equally, genetic reduction or ablation of SIRT2 had no effect on HD progression as assessed by a battery of physiological and behavioural tests. Furthermore, we observed no change in aggregate load or levels of soluble mutant huntingtin transprotein. Intriguingly, neither the constitutive genetic loss nor acute pharmacological inhibition of SIRT2 affected the expression of cholesterol biosynthesis enzymes in the context of HD. Therefore, we conclude that SIRT2 inhibition does not modify disease progression in the R6/2 mouse model of HD and SIRT2 inhibition should not be prioritised as a therapeutic option for HD.     

Fabrication of Sub-10-nm Plasmonic Gaps for Ultra-Sensitive Raman Spectroscopy

Plasmonics - The past two decades have witnessed the explosion of activities in the field of surface enhanced Raman spectroscopy (SERS). SERS platforms employ nano-structures that excite plasmonic...