New tools provide a second look at HDV ribozyme structure, dynamics and cleavage.

The hepatitis delta virus (HDV) ribozyme is a self-cleaving RNA enzyme essential for processing viral transcripts during rolling circle viral replication. The first crystal structure of the cleaved ribozyme was solved in 1998, followed by structures of uncleaved, mutant-inhibited and ion-complexed forms. Recently, methods have been developed that make the task of modeling RNA structure and dynamics significantly easier and more reliable. We have used ERRASER and PHENIX to rebuild and re-refine the cleaved and cis-acting C75U-inhibited structures of the HDV ribozyme. The results correct local conformations and identify alternates for RNA residues, many in functionally important regions, leading to improved R values and model validation statistics for both structures. We compare the rebuilt structures to a higher resolution, trans-acting deoxy-inhibited structure of the ribozyme, and conclude that although both inhibited structures are consistent with the currently accepted hammerhead-like mechanism of cleavage, they do not add direct structural evidence to the biochemical and modeling data. However, the rebuilt structures (PDBs: 4PR6, 4PRF) provide a more robust starting point for research on the dynamics and catalytic mechanism of the HDV ribozyme and demonstrate the power of new techniques to make significant improvements in RNA structures that impact biologically relevant conclusions.

NgBR is essential for endothelial cell glycosylation and vascular development.

NgBR is a transmembrane protein identified as a Nogo-B-interacting protein and recently has been shown to be a subunit required for cis-prenyltransferase (cisPTase) activity. To investigate the integrated role of NgBR in vascular development, we have characterized endothelial-specific NgBR knockout embryos. Here, we show that endothelial-specific NgBR knockout results in embryonic lethality due to vascular development defects in yolk sac and embryo proper. Loss of NgBR in endothelial cells reduces proliferation and promotes apoptosis of the cells largely through defects in the glycosylation of key endothelial proteins including VEGFR2, VE-cadherin, and CD31, and defective glycosylation can be rescued by treatment with the end product of cisPTase activity, dolichol phosphate. Moreover, NgBR functions in endothelial cells during embryogenesis are Nogo-B independent. These data uniquely show the importance of NgBR and protein glycosylation during vascular development.

Punctuated evolution and transitional hybrid network in an ancestral cell cycle of fungi.

Although cell cycle control is an ancient, conserved, and essential process, some core animal and fungal cell cycle regulators share no more sequence identity than non-homologous proteins. Here, we show that evolution along the fungal lineage was punctuated by the early acquisition and entrainment of the SBF transcription factor through horizontal gene transfer. Cell cycle evolution in the fungal ancestor then proceeded through a hybrid network containing both SBF and its ancestral animal counterpart E2F, which is still maintained in many basal fungi. We hypothesize that a virally-derived SBF may have initially hijacked cell cycle control by activating transcription via the cis-regulatory elements targeted by the ancestral cell cycle regulator E2F, much like extant viral oncogenes. Consistent with this hypothesis, we show that SBF can regulate promoters with E2F binding sites in budding yeast.

A vibrational spectroscopic investigation of rhodanine and its derivatives in the solid state

Raman and infrared spectra are reported for rhodanine, 3-aminorhodanine and 3-methylrhodanine in the solid state. Comparisons of the spectra of non-deuterated/deuterated species facilitate discrimination of the bands associated with N-H, NH2, CH2 and CH3 vibrations. DFT calculations of structures and vibrational spectra of isolated gas-phase molecules, at the B3-LYP/cc-pVTZ and B3-PW91/cc-pVTZ level, enable normal coordinate analyses in terms of potential energy distributions for each vibrational normal mode. The cis amide I mode of rhodanine is associated with bands at ~ 1713 and 1779 cm-1, whereas a Raman and IR band at ~ 1457 cm-1 is assigned to the amide II mode. The thioamide II and III modes of rhodanine, 3-aminorhodanine and 3-methylrhodanine are observed at 1176 and 1066/1078; 1158 and 1044; 1107 and 984 cm-1 in the Raman and at 1187 and 1083; 1179 and 1074; 1116 and 983 cm-1 in the IR spectra, respectively.

Vibrational spectroscopy and DFT calculations of di-amino acid cyclic peptides. Part I: cyclo(Gly-Gly), cyclo(L-Ala-L-Ala) and cyclo(L-Ala-Gly) in the solid state and in aqueous solution

Investigations of the vibrational spectra of cyclo(Gly-Gly), cyclo(L-Ala-L-Ala) and cyclo(t-Ala-Gly) are reported. Raman scattering and Fourier transform infrared (FTIR) spectra of solid-state and aqueous protonated samples, as well as their corresponding N-deuterated isotopomers, have been examined. In addition, density functional theory (DFT) (B3-LYP/cc-pVDZ) calculations of molecular structures and their associated vibrational modes were carried out. In each case, the calculated structures of lowest energy for the isolated gas-phase molecules have boat conformations. Assignments have been made for the observed Raman and FTIR vibrational bands of the cyclic di-amino acid peptides (CDAPs) examined. Raman polarization studies of aqueous phase samples are consistent with C-2 and C-1 symmetries for the six-membered rings of cyclo(L-Ala-L-Ala) and cydo(L-Ala-Gly), respectively. There is a good correlation between experimental and calculated vibrational bands for the three CDAPs. These data are in keeping with boat conformations for cydo(L-Ala-L-Ala) and cyclo(L-Ala-Gly) molecules, predicted by the ab initio calculations, in both the solid and aqueous solution states. However, Raman spectroscopic results might infer that cyclo(L-AlaGly) deviates only slightly from planarity in the solid state. The potential energy distributions of the amide I and II modes of a cis-peptide linkage are shown to be significantly different from those of the trans-peptides. For example, deuterium shifts have shown that the cis-amide I vibrations found in cyclo(Gly-Gly), cyclo(L-Ala-L-Ala), and cyclo(L-Ala-Gly) have larger N-H contributions compared to their trans-amide counterparts. Compared to trans-amide II vibrations, cis-amide II vibrations show a considerable decrease in N-H character.

Vibrational spectroscopic studies of the structure of di-amino acid peptides. Part II: cyclo(L-Asp-L-Asp) in the solid state and in aqueous solution

Solid-state protonated and N,O-deuterated Fourier transform infrared (IR) and Raman scattering spectra together with the protonated and deuterated Raman spectra in aqueous solution of the cyclic di-amino acid peptide cyclo(L-Asp-L-Asp) are reported. Vibrational band assignments have been made on the basis of comparisons with previously cited literature values for diketopiperazine (DKP) derivatives and normal coordinate analyses for both the protonated and deuterated species based upon DFT calculations at the B3-LYP/cc-pVDZ level of the isolated molecule in the gas phase. The calculated minimum energy structure for cyclo(L-Asp-L-Asp), assuming C-2 symmetry, predicts a boat conformation for the DKP ring with both the two L-aspartyl side chains being folded slightly above the ring. The C=O stretching vibrations have been assigned for the side-chain carboxylic acid group (e.g. at 1693 and 1670 cm(-1) in the Raman spectrum) and the cis amide I bands (e.g. at 1660 cm(-1) in the Raman spectrum). The presence of two bands for the carboxylic acid C=O stretching modes in the solid-state Raman spectrum can be accounted for by factor group splitting of the two nonequivalent molecules in a crystallographic unit cell. The cis amide II band is observed at 1489 cm(-1) in the solid-state Raman spectrum, which is in agreement with results for cyclic di-amino acid peptide molecules examined previously in the solid state, where the DKP ring adopts a boat conformation. Additionally, it also appears that as the molecular mass of the substituent on the C-alpha atom is increased, the amide II band wavenumber decreases to below 1500 cm(-1); this may be a consequence of increased strain on the DKP ring. The cis amide II Raman band is characterized by its relatively small deuterium shift (29 cm(-1)), which indicates that this band has a smaller N-H bending contribution than the trans amide II vibrational band observed for linear peptides.

IR/Raman spectroscopy and DFT calculations of cyclic di-amino acid peptides. Part III: comparison of solid state and solution structures of cyclo(L-Ser-L-Ser)

B3-LYP/cc-pVDZ calculations of the gas-phase structure and vibrational spectra of the isolated molecule cyclo(L-Ser-L-Ser), a cyclic di-amino acid peptide (CDAP), were carried out by assuming C-2 symmetry. It is predicted that the minimum-energy structure is a boat conformation for the diketopiperazine (DKP) ring with both L-Beryl side chains being folded slightly above the ring. An additional structure of higher energy (15.16 kJ mol(-1)) has been calculated for a DKP ring with a planar geometry, although in this case two fundamental vibrations have been calculated with imaginary wavenumbers. The reported X-ray crystallographic structure of cyclo(L-Ser-L-Ser), shows that the DKP ring displays a near-planar conformation, with both the two L-Beryl side chains being folded above the ring. It is hypothesized that the crystal packing forces constrain the DKP ring in a planar conformation and it is probable that the lower energy boat conformation may prevail in the aqueous environment. Raman scattering and Fourier-transform infrared (FT-IR) spectra of solid state and aqueous solution samples of cyclo(L-Ser-L-Ser) are reported and discussed. Vibrational band assignments have been made on the basis of comparisons with the calculated vibrational spectra and band wavenumber shifts upon deuteration of labile protons. The experimental Raman and IR results for solid-state samples show characteristic amide I vibrations which are split (Raman:1661 and 1687 cm(-1), IR:1666 and 1680 cm(-1)), possibly due to interactions between molecules in a crystallographic unit cell. The cis amide I band is differentiated by its deuterium shift of ~ 30 cm(-1), which is larger than that previously reported for trans amide I deuterium shifts. A cis amide II mode has been assigned to a Raman band located at 1520 cm(-1). The occurrence of this cis amide II mode at a wavenumber above 1500 cm(-1) concurs with results of previously examined CDAP molecules with low molecular weight substituents on the C-alpha atoms, and is also indicative of a relatively unstrained DKP ring.

Vibrational spectra and crystal structure of the di-amino acid peptide cyclo(L-Met-L-Met): Comparison of experimental data and DFT calculations

Experimental Raman and FT-IR spectra of solid-state non-deuterated and N-deuterated samples of cyclo(L-Met-L-Met) are reported and discussed. The Raman and FT-IR results show characteristic amide I vibrations (Raman: 1649 cm-1, infrared: 1675 cm-1) for molecules exhibiting a cis amide conformation. A Raman band, assigned to the cis amide II vibrational mode, is observed at sim1493 cm-1 but no IR band is observed in this region. Cyclo(L-Met-L-Met) crystallises in the triclinic space group P1 with one molecule per unit cell. The overall shape of the diketopiperazine (DKP) ring displays a (slightly distorted) boat conformation. The crystal packing employs two strong hydrogen bonds, which traverse the entire crystal via translational repeats. B3-LYP/cc-pVDZ calculations of the structure of the molecule predict a boat conformation for the DKP ring, in agreement with the experimentally determined X-ray structure. Copyright © 2009 John Wiley & Sons, Ltd.

Synthesis and properties of polyelectrolyte microgel particles

A series of cationic poly(N-isopropylacrylamide/4-vinylpyridine) [poly(NIPAM/4-VP)] polyelectrolyte co-polymer microgels have been prepared by surfactant free emulsion polymerization (SFEP) with varying compositions of 4-VP and NIPAM. The compositions of 4-VP were 15, 25, 35, 45, 55 wt.% relative to NIPAM. The temperature and pH responsive swelling–deswelling properties of these microgels have been investigated using dynamic light scattering (DLS) and electrophoretic mobility measurements. DLS results have shown that the particle diameter of the poly(NIPAM/4-VP) microgels decreases with increasing concentration (wt.%) of 4-VP over the 20–60 °C temperature range due to the increased amount of hydrophobic group. The particle size of all poly(NIPAM/4-VP) microgel series increases with decreasing pH, as the 4-VP units become more protonated at low pH below the pKa (5.39) of the monomer 4-VP. Electrophoretic mobility results have shown that electrophoretic mobility increases as the temperature/pH increases at a constant background ionic strength (1 × 10− 4 mol dm− 3 NaCl). These results are in good agreement with DLS results. The temperature/pH sensitivity of these microgels depends on the ratio of NIPAM/4-VP concentration in the co-polymer microgel systems. The combined temperature/pH responsiveness of these polyelectrolyte microgels can be used in applications where changes in particle size with small change in pH or temperature is of great consequence.

Sign language in Spanish television. Study on reception

The present article analyses the preferences of the deaf who use sign language and are users of the TV interpretation service to sign language, as well as the characteristics with which TV channels provide that service in television in Spain. The objective is to establish whether the way in which the aforementioned accessibility service is provided matches the preferences of users or differ from them. The analysis presents the opinion on this service of the deaf that use the Spanish sign language as their first language for communication. A study has also been conducted on the programmes broadcast with sign language during week 10-16/03/2014. The main data collected reveal that the deaf are dissatisfied with broadcasting times. They ask for news programmes with sign language, they would rather have the interpretation carried out by deaf people who use sign language and they prefer that the interpreter is the main image on screen. Concerning the analysis of the programmes broadcast, the study shows that the majority of programmes with sign language are broadcast at night, they are entertainment programmes, the interpretation is carried out by hearing people who use sign language and that their image is displayed in a corner of the screen.

Sports programs of the Spanish radio in the social network Facebook: compared study (2010-2015)

Social networks have increasingly become a showcase where the media can be promoted. Like many other media, radio stations have made use of social networks to promote themselves in a better way and, sometimes, to keep more feedback with their listeners. But not all programs make the same use and not all of them have managed to reach in the same way his followers. This article discusses the consolidation in the social networks of the major radio sports programs in Spain. Through a comparative analysis between 2010 and 2015, throughout the text, the authors have tried to observe the evolution of the programs and, at the same time, to establish comparisons between the followers that these programs have on social networks and the number of listeners as EGM.

Valor social de la fotografía de guerra: Robert Capa en la Guerra civil española

A partir del análisis de las relaciones entre sociología, fotografía y documentación se realiza un recorrido por algunas de las principales fotografías realizadas por Robert Capa durante la Guerra civil española.

Polystyrene and asphaltene micelles within blends with a bitumen of an SBS block copolymer and styrene and butadiene homopolymers

Using fluorescence microscopy, DSC and DMTA we have explored blends of a bitumen with a styrene-butadiene-styrene (SBS) block copolymer, and with blends of the bitumen with SBS and one or two homopolymers - a polystyrene and a poly(cis-butadiene). The SBS polymer was progressively replaced with quantities of the homopolymers both together in the proportions found in the block copolymer and then by each homopolymer separately. At low temperatures the blends are all softer than the bitumen itself, so the polymers plasticise the bitumen-rich phase, and above 50°C the blends' stiffness (E') falls below a plateau only when a critical proportion of the block copolymer has been replaced with the two homopolymers: this supports the idea of an extensive network created by the polystyrene-rich spherical microphases that is effective even when the polystyrene microphases have melted. In one polymer blend the stiffness rose as the temperature was raised above 100°C, suggesting the development of a mesophase based upon polybutadiene plus asphaltenes, in another E' was enhanced and E" remained constant as the temperature rose above 70°C, perhaps for a similar reason; in some loss process appeared and the stiffness fell as temperature rose; but in others a good part of the SBS was replaced by either polystyrene or polybutadiene without changing the appearance of a rubbery plateau, that is, without a diminution of the mechanical properties of the soft matter.

Tandem enzyme-catalysed oxidations of alkyl phenyl sulfides and alkyl benzenes: enantiocomplementary routes to chiral phenols.

Dioxygenase-catalysed trioxygenation of alkyl phenyl sulfides and alkyl benzenes yields enantiopure cis-dihydrodiol sulfoxides and triols respectively; naphthalene cis-dihydrodiol dehydrogenase-catalysed aromatisation of these diastereoisomers gives enantiopure catechols of either configuration.

Tyrosine phosphorylated SOCS-3 inhibits STAT activation but binds p120-RasGAP and activates Ras

Suppressors of cytokine signalling (SOCS, also known as CIS and SSI) are encoded by immediate early genes that act in a feedback loop to inhibit cytokine responses and activation of 'signal transducer and activator of transcription' (STAT). Here we show that SOCS-3 is strongly tyrosine-phosphorylated in response to many growth factors, including interleukin-2 (IL-2), erythropoietin (EPO), epidermal growth factor (EGF) and platelet-derived growth factor (PDGF). The principal phosphorylation sites on SOCS-3 are residues 204 and 221 at the carboxy terminus, and upon phosphorylation tyrosine 221 interacts with the Ras inhibitor p120 RasGAP. After IL-2 stimulation, phosphorylated SOCS-3 strongly inhibits STAT5 activation but, by binding to RasGAP, maintains activation of extracellular-signal-regulated kinase (ERK). A tyrosine mutant of SOCS-3 still blocks STAT phosphorylation, but also strongly inhibits IL-2-dependent activation of ERK and cell proliferation. Moreover, it also inhibits EPO- and PDGF-induced proliferation and ERK activation. Therefore, although SOCS proteins inhibit growth-factor responses, tyrosine phosphorylation of SOCS-3 can ensure cell survival and proliferation through the Ras pathway.