Correction of defective CFTR/ENaC function and tightness of cystic fibrosis airway epithelium by amniotic mesenchymal stromal (stem) cells

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, with most of the mortality given by the lung disease. Human amniotic mesenchymal stromal (stem) cells (hAMSCs) hold great promise for regenerative medicine in the field of lung disease; however, their potential as therapeutics for CF lung disease has not been fully explored. In the present study, hAMSCs were analysed in co‐cultures on Transwell filters with CF immortalized airway epithelial cells (CFBE41o‐ line) at different ratios to exploit their potency to resume basic defects associated with CF. The results show that F‐actin content was increased in co‐cultures as compared with CF cells and actin was reorganized to form stress fibres. Confocal microscopy studies revealed that co‐cultures had a tendency of increased expression of occludin and ZO‐1 at the intercellular borders, paralleled by a decrease in dextran permeability, suggestive of more organized tight junctions (TJs). Spectrofluorometric analysis of CFTR function demonstrated that hAMSC‐CFBE co‐cultures resumed chloride transport, in line with the appearance of the mature Band C of CFTR protein by Western blotting. Moreover, hAMSC‐CFBE co‐cultures, at a 1:5 ratio, showed a decrease in fluid absorption, as opposed to CFBE cell monolayers that displayed a great rate of fluid resorption from the apical side. Our data show that human amniotic MSCs can be used in co‐culture with CF respiratory epithelial cells to model their engraftment into the airways and have the potential to resume a tight epithelium with partial correction of the CF phenotype.     

From Cassyrane to Cashmeran – The Molecular Parameters of Odorants

This review, including some new experimental results, is the summary of a talk at the ‘flavors & fragrances 2013’ conference in Leipzig, organized jointly by the GDCh, the Liebig‐Vereinigung, and the EuCheMS. After times of searching for natural odor principles and serendipitous discoveries by chemical inspiration, directed odorant design today offers the highest hit rates for the discovery of new odorants, although serendipity still plays a role. Keeping intact the electronic shape required for a certain olfactophore‐binding geometry, one can add or subtract structural elements, rigidify molecular structures, or introduce more structural flexibility. To find out which structural features are critical, the ‘seco‐approach’, in which different fragments are removed by cutting strategic bonds, is the most analytical. Following this approach, such ingredients as Serenolide, Sylkolide, and Pomarose were designed. Transferring this design principle from the family of damascones to that of the theaspiranes led to the discovery of Cassyrane, though completely different structural features turned out to be relevant. Application of the seco‐concept to a 3,7a‐substituted 2,6,7,7a‐tetrahydro‐1H‐inden‐5‐yl musk lead structure derived from carotol resulted in the discovery of a new family of dienone musks with novel structure? odor correlations. However, cutting the C(2)? O bond of Cassyrane and oxidizing the resulting seco‐structure to the 1,2,5,1″‐tetradehydro derivative links the family of dienone musks with that of blackcurrant odorants, but the resulting target structures turned out to be potent orris odorants. (3E,5E)‐5‐(tert‐Butyl)octadeca‐3,5‐dien‐2‐one even possesses the lowest odor threshold in the whole ionone family (0.036 ng/l air), which could be rationalized by a superposition analysis on (?)‐cis‐γ‐irone. In the course of the synthesis of these high‐impact orris odorants, we discovered that, depending on the reaction conditions, the dehydration step of the intermediate 5‐hydroxyalk‐3‐yn‐2‐ones was accompanied by a carbenium‐ion rearrangement. Depending on the substitution pattern, these rearrangement products and their derivatives possessed interesting musky‐woody olfactory properties reminiscent of Cashmeran, demonstrating that the same structural elements can code for completely different odors, i.e., cassis, musk, orris, violet, and Cashmeran‐type, depending only on their spatial arrangement.     

Subtractive and differential hybridization molecular analyses of <Emphasis Type="Italic">Ceratitis capitata</Emphasis> XX/XY versus XX embryos to search for male-specific early transcribed genes

The agricultural pest Ceratitis capitata, also known as the Mediterranean fruit fly or Medfly, is a fruit crop pest of very high economic relevance in different continents. The strategy to separate Ceratitis males from females (sexing) in mass rearing facilities is a useful step before the sterilization and release of male-only flies in Sterile Insect Technique control programs (SIT). The identification of genes having early embryonic male-specific expression, including Y-linked genes, such as the Maleness factor, could help to design novel and improved methods of sexing in combination with transgenesis, aiming to confer conditional female-specific lethality or female-to-male sexual reversal.We used a combination of Suppression Subtractive Hybrydization (SSH), Mirror Orientation Selection (MOS) and differential screening hybridization (DSH) techniques to approach the problem of isolating corresponding mRNAs expressed in XX/XY embryos versus XX-only embryos during a narrow developmental window (8-10 hours after egg laying, AEL ). Here we describe a novel strategy we have conceived to obtain relatively large amounts of XX-only embryos staged at 8-10 h AEL and so to extract few micrograms of polyA+ required to apply the complex technical procedure. The combination of these 3 techniques led to the identification of a Y-linked putative gene, CcGm2, sharing high sequence identity to a paralogous gene, CcGm1, localized either on an autosome or on the X chromosome.We propose that CcGm2 is a first interesting putative Y-linked gene which could play a role in sex determination. The function exterted by this gene should be investigated by novel genetic tools, such as CRISPR-CAS9, which will permit to target only the Y-linked paralogue, avoiding to interfere with the autosomal or X-linked paralogue function.     

Complete sequencing of Novosphingobium sp. PP1Y reveals a biotechnologically meaningful metabolic pattern

Background

Novosphingobium sp. strain PP1Y is a marine α-proteobacterium adapted to grow at the water/fuel oil interface. It exploits the aromatic fraction of fuel oils as a carbon and energy source. PP1Y is able to grow on a wide range of mono-, poly- and heterocyclic aromatic hydrocarbons. Here, we report the complete functional annotation of the whole Novosphingobium genome.

Results

PP1Y genome analysis and its comparison with other Sphingomonadal genomes has yielded novel insights into the molecular basis of PP1Y’s phenotypic traits, such as its peculiar ability to encapsulate and degrade the aromatic fraction of fuel oils. In particular, we have identified and dissected several highly specialized metabolic pathways involved in: (i) aromatic hydrocarbon degradation; (ii) resistance to toxic compounds; and (iii) the quorum sensing mechanism.

Conclusions

In summary, the unraveling of the entire PP1Y genome sequence has provided important insight into PP1Y metabolism and, most importantly, has opened new perspectives about the possibility of its manipulation for bioremediation purposes.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-384) contains supplementary material, which is available to authorized users.     

Exceptional ancient DNA preservation and fibre remains of a Sasanian saltmine sheep mummy in Chehrābād,Iran

Mummified remains have long attracted interest as a potential source of ancient DNA. However, mummification is a rare process that requires an anhydrous environment to rapidly dehydrate and preserve tissue before complete decomposition occurs. We present the whole-genome sequences (3.94 X) of an approximately 1600-year-old naturally mummified sheep recovered from Chehrābād, a salt mine in northwestern Iran. Comparative analyses of published ancient sequences revealed the remarkable DNA integrity of this mummy. Hallmarks of postmortem damage, fragmentation and hydrolytic deamination are substantially reduced, likely owing to the high salinity of this taphonomic environment. Metagenomic analyses reflect the profound influence of high-salt content on decomposition; its microbial profile is predominated by halophilic archaea and bacteria, possibly contributing to the remarkable preservation of the sample. Applying population genomic analyses, we find clustering of this sheep with Southwest Asian modern breeds, suggesting ancestry continuity. Genotyping of a locus influencing the woolly phenotype showed the presence of an ancestral ‘hairy’ allele, consistent with hair fibre imaging. This, along with derived alleles associated with the fat-tail phenotype, provides genetic evidence that Sasanian-period Iranians maintained specialized sheep flocks for different uses, with the ‘hairy’, ‘fat-tailed’-genotyped sheep likely kept by the rural community of Chehrābād''s miners.     

The solution structure of the first PHD finger of autoimmune regulator in complex with non-modified histone H3 tail reveals the antagonistic role of H3R2 methylation

Plant homeodomain (PHD) fingers are often present in chromatin-binding proteins and have been shown to bind histone H3 N-terminal tails. Mutations in the autoimmune regulator (AIRE) protein, which harbours two PHD fingers, cause a rare monogenic disease, autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED). AIRE activates the expression of tissue-specific antigens by directly binding through its first PHD finger (AIRE-PHD1) to histone H3 tails non-methylated at K4 (H3K4me0). Here, we present the solution structure of AIRE-PHD1 in complex with H3K4me0 peptide and show that AIRE-PHD1 is a highly specialized non-modified histone H3 tail reader, as post-translational modifications of the first 10 histone H3 residues reduce binding affinity. In particular, H3R2 dimethylation abrogates AIRE-PHD1 binding in vitro and reduces the in vivo activation of AIRE target genes in HEK293 cells. The observed antagonism by R2 methylation on AIRE-PHD1 binding is unique among the H3K4me0 histone readers and represents the first case of epigenetic negative cross-talk between non-methylated H3K4 and methylated H3R2. Collectively, our results point to a very specific histone code responsible for non-modified H3 tail recognition by AIRE-PHD1 and describe at atomic level one crucial step in the molecular mechanism responsible for antigen expression in the thymus.     

TIMP-1-GPI in combination with hyperthermic treatment of melanoma increases sensitivity to FAS-mediated apoptosis

Resistance to apoptosis is a prominent feature of malignant melanoma. Hyperthermic therapy can be an effective adjuvant treatment for some tumors including melanoma. We developed a fusion protein based on the tissue inhibitor of matrix metalloproteinase-1 linked to a glycosylphosphatidylinositol anchor (TIMP-1-GPI). The TIMP-1-GPI-fusion protein shows unique properties. Exogenous administration of TIMP-1-GPI can result in transient morphological changes to treated cells including modulation of proliferation and decreased resistance to apoptosis. The effect of TIMP-1-GPI on the biology of melanoma in the context of a defined hyperthermic dose was evaluated in vitro. Clonogenic assays were used to measure cell survival. Gelatinase zymography determined secretion of MMP-2 and MMP-9. Monoclonal antibody against FAS/CD95 was applied to induce apoptosis. The expression of pro- and anti-apoptotic proteins and the secretion of immunoregulatory cytokines were then evaluated using Western blot and ELISA. TIMP-1-GPI combined with a sub-lethal hyperthermic treatment (41.8°C for 2 h) suppressed tumor cell growth capacity as measured by clonogenic assay. The co-treatment also significantly suppressed tumor cell proliferation, enhanced FAS receptor surface expression increased tumor cell susceptibility to FAS-mediated killing. The increased sensitivity to FAS-induced apoptosis was linked to alterations in the apoptotic mediators Bcl-2, Bax, Bcl-XL and Apaf-1. The agent works in concert with sub-lethal hyperthermic treatment to render melanoma cells sensitive to FAS killing. The targeted delivery of TIMP-1-GPI to tumor environments in the context of regional hyperthermic therapy could be optimized through the use of thermosensitive liposomes. Elfriede Noessner, Peter J. Nelson are equal contributors.