Secondary structure is required for 3' splice site recognition in yeast

Higher order RNA structures can mask splicing signals, loop out exons, or constitute riboswitches all of which contributes to the complexity of splicing regulation. We identified a G to A substitution between branch point (BP) and 3' splice site (3'ss) of Saccharomyces cerevisiae COF1 intron, which dramatically impaired its splicing. RNA structure prediction and in-line probing showed that this mutation disrupted a stem in the BP-3'ss region. Analyses of various COF1 intron modifications revealed that the secondary structure brought about the reduction of BP to 3'ss distance and masked potential 3'ss. We demonstrated the same structural requisite for the splicing of UBC13 intron. Moreover, RNAfold predicted stable structures for almost all distant BP introns in S. cerevisiae and for selected examples in several other Saccharomycotina species. The employment of intramolecular structure to localize 3'ss for the second splicing step suggests the existence of pre-mRNA structure-based mechanism of 3'ss recognition.     

Intravitreal Bevacizumab with or without triamcinolone for refractory diabetic macular oedema

We evaluate the effect of intravitreal injections of Bevacizumab (IVB) alone or combined with triamcinolone (IVT) in the first injection for treatment of refractory diabetic macular oedema (DME). Sixty eyes of sixty patients with refractory DME were included. Half of them received injections of IVB (1.25 mg/0.05 ml) or combined IVB and IVT (1.25 mg/0.05 ml and 2 mg/0.05 ml respectively). The primary outcome measure was change in central macular thickness (CMT). Secondary outcome measures were change in best-corrected logMAR visual acuity (BCVA) and incidence of potential adverse events. Central macular thickness was reduced significantly in both the IVB and IVB/IVT groups. At week 24, CMT change compared to the baseline was -93.7 microm (95% CI, -172.2 to -19.26) in the IVB group and -93.1 microm (95% CI, -154.4 to -29.7) in the IVB/IVT group. There was not a significant difference between the IVB and the IVB/IVT groups. Improvement of BCVA was initiated at weeks 6 and 12 in the IVB/IVT and IVB groups respectively. Anterior chamber reaction was noticed in six (20%) and six (20%) eyes respectively in the IVB and IVB/IVT groups the day after injection, and it resolved with no sequel. Elevation of intraocular pressure (IOP) occurred in two eyes (6%) in the IVB/IVT group. Intravitreal injections of Bevacizumab had a beneficial effect on refractory DME in terms of CMT reduction and BCVA improvement. Addition of triamcinolone in the injection seemed to induce earlier visual improvement; however, it did not show any significant additive effect later during follow-up.     

Competitive canalization of PIN-dependent auxin flow from axillary buds controls pea bud outgrowth

Shoot branching is one of the major determinants of plant architecture. Polar auxin transport in stems is necessary for the control of bud outgrowth by a dominant apex. Here, we show that following decapitation in pea (Pisum sativum L.), the axillary buds establish directional auxin export by subcellular polarization of PIN auxin transporters. Apical auxin application on the decapitated stem prevents this PIN polarization and canalization of laterally applied auxin. These results support a model in which the apical and lateral auxin sources compete for primary channels of auxin transport in the stem to control the outgrowth of axillary buds.     

Discovery of highly potent, nonsteroidal 17β-hydroxysteroid dehydrogenase type 1 inhibitors by virtual high-throughput screening

17β-Hydroxysteroid dehydrogenase type 1 (17β-HSD1) catalyzes the formation of the potent proliferation-stimulating hormone estradiol, and it is thus involved in the development of hormone-dependent breast cancer. Due to its high substrate specificity and the known relationships between its overexpression and disease incidence, 17β-HSD1 is considered an attractive target for drug development. Here, we have used structure-based virtual high-throughput screening to successfully identify potent nonsteroidal 17β-HSD1 inhibitors. Computational screening of a drug-like database containing 13 million compounds identified hits with a 2-benzylidenebenzofuran-3(2H)-one scaffold that we show to be highly potent 17β-HSD1 inhibitors. The most potent in the series, compound 1, showed an IC(50) of 45nM in our 17β-HSD1 inhibition assay, and also showed good selectivity for 17β-HSD1 over 17β-HSD2.     

Cell plate restricted association of DRP1A and PIN proteins is required for cell polarity establishment in Arabidopsis

The polarized transport of the phytohormone auxin [1], which is crucial for the regulation of different stages of plant development [2, 3], depends on the asymmetric plasma membrane distribution of the PIN-FORMED (PIN) auxin efflux carriers [4,?5]. The PIN polar localization results from clathrin-mediated endocytosis (CME) from the plasma membrane and subsequent polar recycling [6]. The Arabidopsis genome encodes two groups of dynamin-related proteins (DRPs) that show homology to mammalian dynamin-a protein required for fission of endocytic vesicles during CME [7, 8]. Here we show by coimmunoprecipitation (coIP), bimolecular fluorescence complementation (BiFC), and F?rster resonance energy transfer (FRET) that members of the DRP1 group closely associate with PIN proteins at the cell plate. Localization and phenotypic analysis of novel drp1 mutants revealed a requirement for DRP1 function in correct PIN distribution and in auxin-mediated development. We propose that rapid and specific internalization of PIN proteins mediated by the DRP1 proteins and the associated CME machinery from the cell plate membranes during cytokinesis is an important mechanism for proper polar PIN positioning in interphase cells.