Computational identification of conserved microRNAs and their putative targets in the Hypericum perforatum L. flower transcriptome

MicroRNAs (miRNAs) have recently emerged as important regulators of gene expression in plants. Many miRNA families and their targets have been extensively studied in model species and major crops. We have characterized mature miRNAs along with their precursors and potential targets in Hypericum to generate a comprehensive list of conserved miRNA families and to investigate the regulatory role of selected miRNAs in biological processes that occur in the flower. St. John’s wort (Hypericum perforatum L., 2n = 4x = 32), a medicinal plant that produces pharmaceutically important metabolites with therapeutic activities, was chosen because it is regarded as an attractive model system for the study of apomixis. A computational in silico prediction of structure, in combination with an in vitro validation, allowed us to identify 7 pre-miRNAs, including miR156, miR166, miR390, miR394, miR396, and miR414. We demonstrated that H. perforatum flowers share highly conserved miRNAs and that these miRNAs potentially target dozens of genes with a wide range of molecular functions, including metabolism, response to stress, flower development, and plant reproduction. Our analysis paves the way toward identifying flower-specific miRNAs that may differentiate the sexual and apomictic reproductive pathways.     

Transient Inhibition of FGFR2b-Ligands Signaling Leads to Irreversible Loss of Cellular β-Catenin Organization and Signaling in AER during Mouse Limb Development

The vertebrate limbs develop through coordinated series of inductive, growth and patterning events. Fibroblast Growth Factor receptor 2b (FGFR2b) signaling controls the induction of the Apical Ectodermal Ridge (AER) but its putative roles in limb outgrowth and patterning, as well as in AER morphology and cell behavior have remained unclear. We have investigated these roles through graded and reversible expression of soluble dominant-negative FGFR2b molecules at various times during mouse limb development, using a doxycycline/transactivator/tet(O)-responsive system. Transient attenuation (≤24 hours) of FGFR2b-ligands signaling at E8.5, prior to limb bud induction, leads mostly to the loss or truncation of proximal skeletal elements with less severe impact on distal elements. Attenuation from E9.5 onwards, however, has an irreversible effect on the stability of the AER, resulting in a progressive loss of distal limb skeletal elements. The primary consequences of FGFR2b-ligands attenuation is a transient loss of cell adhesion and down-regulation of P63, β1-integrin and E-cadherin, and a permanent loss of cellular β-catenin organization and WNT signaling within the AER. Combined, these effects lead to the progressive transformation of the AER cells from pluristratified to squamous epithelial-like cells within 24 hours of doxycycline administration. These findings show that FGFR2b-ligands signaling has critical stage-specific roles in maintaining the AER during limb development.     

Genetic analysis of polynucleotide phosphorylase structure and functions

Polynucleotide phosphorylase (PNPase) is a phosphate-dependent 3' to 5' exonuclease widely diffused among bacteria and eukaryotes. The enzyme, a homotrimer, can also be found associated with the endonuclease RNase E and other proteins in a heteromultimeric complex, the RNA degradosome. PNPase negatively controls its own gene (pnp) expression by destabilizing pnp mRNA. A current model of autoregulation maintains that PNPase and a short duplex at the 5'-end of pnp mRNA are the only determinants of mRNA stability. During the cold acclimation phase autoregulation is transiently relieved and cellular pnp mRNA abundance increases significantly. Although PNPase has been extensively studied and widely employed in molecular biology for about 50 years, several aspects of structure-function relationships of such a complex protein are still elusive. In this work, we performed a systematic PCR mutagenesis of discrete pnp regions and screened the mutants for diverse phenotypic traits affected by PNPase. Overall our results support previous proposals that both first and second core domains are involved in the catalysis of the phosphorolytic reaction, and that both phosphorolytic activity and RNA binding are required for autogenous regulation and growth in the cold, and give new insights on PNPase structure-function relationships by implicating the alpha-helical domain in PNPase enzymatic activity.     

The Golgi-associated protein GRASP is required for unconventional protein secretion during development

During Dictyostelium development, prespore cells secrete acyl-CoA binding protein (AcbA). Upon release, AcbA is processed to generate a peptide called spore differentiation factor-2 (SDF-2), which triggers terminal differentiation of spore cells. We have found that cells lacking Golgi reassembly stacking protein (GRASP), a protein attached peripherally to the cytoplasmic surface of Golgi membranes, fail to secrete AcbA and, thus, produce inviable spores. Surprisingly, AcbA lacks a signal sequence and is not secreted via the conventional secretory pathway (endoplasmic reticulum-Golgi-cell surface). GRASP is not required for conventional protein secretion, growth, and the viability of vegetative cells. Our findings reveal a physiological role of GRASP and provide a means to understand unconventional secretion and its role in development.     

1H and 13C NMR study of the complex formed by copper(II) with the nucleoside antibiotic sinefungin

Sinefungin (SFG) is an antifungal and antiparasitic nucleoside antibiotic composed by ornithine and adenosine moieties both having the potential to bind copper(II). NMR studies performed at physiological pH have shown that the alpha-amino and the carboxylate groups in the ornithine unit are the preferred donor sites for Cu(II) binding. On the contrary, at acidic pH, Cu(II) complexation starts from adenosine nitrogen being the alpha-amino group still protonated and not available for metal binding. The proton paramagnetic relaxation enhancements measured at neutral pH allowed to obtain the 3D structure of the 1:2 Cu(II)-SFG complex. Molecular dynamics calculations were revealing for the existence of secondary Cu(II) interaction with the purine nitrogens of the adenosine moiety.     

Limited plasticity of mesenchymal stem cells cocultured with adult cardiomyocytes

In order to assess, in a controlled in vitro model, the differentiation potential of adult bone marrow derived stem cells we have developed a coculture procedure using adult rat cardiomyocytes and mesenchymal stem cells (MSCs) from transgenic GFP positive rats. We investigated in the cocultured MSCs the time course of cellular processes that are difficult to monitor in in vivo experiments. Adult rat cardiomyocytes and adult rat MSCs were cocultured for up to 7 days and analyzed by confocal microscopy. Several markers were studied by immunofluorescence technique. The fluorescent ST-BODIPY-Dihydropyridine was used to label calcium channels in living cells. Intracellular calcium was monitored with the fluorescent probe X-Rhod-1. Immunofluorescence experiments showed the presence of connexin-43 between cardiomyocytes and MSCs and between MSCs, while no sarcomeric structures were observed at any time of the coculture. We looked at the expression of calcium channels and development of voltage-dependent calcium signaling in cocultured MSCs. MSCs showed a time-dependent increase of labeling of ST-BODIPY-Dihydropyridine, reaching a relatively strong level after 72 h of coculture. The treatment with a non-fluorescent DHP, Nifedipine, completely abolished ST-BODIPY labeling. We investigated whether depolarization could modulate intracellular calcium. Depolarization-induced calcium transients increased in MSCs in relation to the coculture time. We conclude that MSCs cocultured with adult cardiomyocytes present preliminary evidence of voltage-dependent calcium modulation uncoupled with the development of nascent or adult myofibrils, thus showing a limited lineage specification and a low plasticity to differentiate in a full cardiomyocyte-like phenotype.     

GOLPH3 Is Essential for Contractile Ring Formation and Rab11 Localization to the Cleavage Site during Cytokinesis in Drosophila melanogaster

The highly conserved Golgi phosphoprotein 3 (GOLPH3) protein has been described as a Phosphatidylinositol 4-phosphate [PI(4)P] effector at the Golgi. GOLPH3 is also known as a potent oncogene, commonly amplified in several human tumors. However, the molecular pathways through which the oncoprotein GOLPH3 acts in malignant transformation are largely unknown. GOLPH3 has never been involved in cytokinesis. Here, we characterize the Drosophila melanogaster homologue of human GOLPH3 during cell division. We show that GOLPH3 accumulates at the cleavage furrow and is required for successful cytokinesis in Drosophila spermatocytes and larval neuroblasts. In premeiotic spermatocytes GOLPH3 protein is required for maintaining the organization of Golgi stacks. In dividing spermatocytes GOLPH3 is essential for both contractile ring and central spindle formation during cytokinesis. Wild type function of GOLPH3 enables maintenance of centralspindlin and Rho1 at cell equator and stabilization of Myosin II and Septin rings. We demonstrate that the molecular mechanism underlying GOLPH3 function in cytokinesis is strictly dependent on the ability of this protein to interact with PI(4)P. Mutations that abolish PI(4)P binding impair recruitment of GOLPH3 to both the Golgi and the cleavage furrow. Moreover telophase cells from mutants with defective GOLPH3-PI(4)P interaction fail to accumulate PI(4)P-and Rab11-associated secretory organelles at the cleavage site. Finally, we show that GOLPH3 protein interacts with components of both cytokinesis and membrane trafficking machineries in Drosophila cells. Based on these results we propose that GOLPH3 acts as a key molecule to coordinate phosphoinositide signaling with actomyosin dynamics and vesicle trafficking during cytokinesis. Because cytokinesis failures have been associated with premalignant disease and cancer, our studies suggest novel insight into molecular circuits involving the oncogene GOLPH3 in cytokinesis.     

A portable device for measuring donor corneal transparency in eye banks

To develop a portable device for measuring the donor corneal transparency and validate its efficacy for corneal evaluation in the eye-banks and for research. The transparency device (TD) has a light source, a detachable system for corneal insertion and a base for light transmission. The probe detects the transmitted light which is measured by a lux-meter. A contact lens was set as ‘control’ to reduce the light scattering concern, an empty petri-plate as ‘blank’ and the cornea as ‘sample’. Two experts and non-experts (masked) observed the corneas for subjective analysis which was then compared using the TD. The parameters observed were scars, foreign-body, stromal-deformities, folds, thickness and opacity which were then converted to a relative overall percentage by the observer. Twenty corneas were evaluated for correlation, five tissues to obtain standard-deviation and twenty-four pairs for a comparative study. Experts mimicked the eye-banks with long-term experience while non-experts mimicked the emerging eye-banks. Subjective values by the experts closely resembled the measurements by TD. The average correlation between the experts and the non-experts to TD was 0.985 and 0.960 respectively. TD showed higher reproducibility than experts followed by the non-experts. The comparative study showed that increase in thickness reduces the transparency. TD is portable, easy, efficient, maintains sterility and less expensive hence the emerging eye-banks and researchers can use to raise their standards and evaluate the transparency for in vitro tests and comparative studies. The suitable transparency for the cornea deemed for clinical applications was found to be >75 %.