Extensive unfolding of the C-LytA choline-binding module by submicellar concentrations of sodium dodecyl sulphate

We have investigated the stability of the choline-binding module C-LytA against sodium dodecyl sulphate (SDS)-induced unfolding at pH 7.0 and 20 C. A major intermediate with an unfolded N-terminal region accumulates at around 0.75 mM SDS, whereas 2.0 mM SDS was sufficient for a complete unfolding. This might be the first report of a protein being extensively unfolded by submicellar concentrations of SDS, occurring through formation of detergent clusters on the protein surface. All transitions were reversible upon SDS complexation with b-cyclodextrin, allowing the calculation of thermodynamic parameters. A model for the unfolding of C-LytA by SDS is presented and compared to a previous denaturation scheme by guanidine hydrochloride

Novel Approaches To Fight Streptococcus pneumoniae

Streptococcus pneumoniae affects millions of people worldwide. It is responsible for a wide spectrum of serious illnesses such as pneumonia, meningitis and bacteraemia. The highest rate of pneumococcal disease (and the highest mortality) occurs in young children, as well as in the elderly and the immunocompromised patients. Identification of S. pneumoniae in diagnostic procedures may significantly improve thanks to the descripion of new PCR-derived techniques. Vaccination based on the polysaccharidic capsule, together with benzylpenicillin-derived drugs, constitute the current choices to tackle pneumococcal diseases. However, the wide serotype diversity of S. pneumoniae and the emergence of antibiotic-resistant strains is fostering the development of new methods to fight this microorganism. In this sense, patents documenting the use of novel antibiotics of the fluoroquinolone or tetracycline families have recently been described. Moreover, surface-associated proteins are receiving an increasingly special attention, as they are synthesized by most pneumococcal strains and play an important role in virulence. New patented protein-based vaccines take into consideration these polypeptides. In this article we present the main relevant characteristics of this pathogen and review the most recent methods that have been patented for the prevention, diagnostic and treatment of the pneumococcal diseases

Bogdanov-Takens resonance in time-delayed systems

We analyze the oscillatory dynamics of a time-delayed dynamical system subjected to a periodic external forcing. We show that, for certain values of the delay, the response can be greatly enhanced by a very small forcing amplitude. This phenomenon is related to the presence of a Bogdanov- Takens bifurcation and displays some analogies to other resonance phenomena, but also substantial differences.Comment: 14 pages, 8 figure

Inhibition of pneumococcal choline-binding proteins and cell growth by esters of bicyclic amines

Streptococcus pneumoniae is one of the major pathogens worldwide. The use of currently available antibiotics to treat pneumococcal diseases is hampered by increasing resistance levels; also, capsular polysaccharide-based vaccination is of limited efficacy. Therefore, it is desirable to find targets for the development of new antimicrobial drugs specifically designed to fight pneumococcal infections. Choline-binding proteins are a family of polypeptides, found in all S. pneumoniae strains, that take part in important physiologic processes of this bacterium. Among them are several murein hydrolases whose enzymatic activity is usually inhibited by an excess of choline. Using a simple chromatographic procedure, we have identified several choline analogs able to strongly interact with the choline-binding module (C-LytA) of the major autolysin of S. pneumoniae. Two of these compounds (atropine and ipratropium) display a higher binding affinity to C-LytA than choline, and also increase the stability of the protein. CD and fluorescence spectroscopy analyses revealed that the conformational changes of C-LytA upon binding of these alkaloids are different to those induced by choline, suggesting a different mode of binding. In vitro inhibition assays of three pneumococcal, choline-dependent cell wall lytic enzymes also demonstrated a greater inhibitory efficiency of those molecules. Moreover, atropine and ipratropium strongly inhibited in vitro pneumococcal growth, altering cell morphology and reducing cell viability, a very different response than that observed upon addition of an excess of choline. These results may open up the possibility of the development of bicyclic amines as new antimicrobials for use against pneumococcal pathologies

Estimation of comprehensive two-dimensional gas chromatographic response from one-dimensional gas chromatography data

Trabajo presentado a la XV Reunión Científica de la Sociedad Española de Cromatografía y Técnicas Afines (SECyTA) celebrada en Castellón de la Plana del 28 al 30 de octubre de 2015Optimization of a separation in comprehensive two-dimensional gas chromatography (GC×GC) requires the selection of an appropriate set column, and the optimisation of their respective dimensions, temperature programs and flow rates. Until not so long ago, a time consuming trial and error process was the only way to select the columns and chromatographic conditions for the two GC dimensions. During the last few years, several attempts have been proposed to estimate the chromatographic response in GC×GC [1-4]. Although most of these methods are helpful contributions, some of them focussed exclusively on the retention time estimation and do not consider the effect of peak width, also essential for a correct prediction of the chromatographic separation capability. Others are based on theoretical formulas not applicable to the extreme experimental conditions used in GC×GC, or are only valid for specific types of compounds or stationary phases: these models cannot be corrected for different separation problems. In this work, modelization of retention time and peak width has been carried out from experimental 1D GC data in order to estimate the response in GC×GC separation for different column sets operated under different flows and temperatures. Several theoretical and experimental models are proposed for estimation of 1D and 2D retention time and peak width; in experimental models, 1D GC data are required together with information on column characteristics and operation conditions. Models for hold-up time, retention factor and peak width were first validated in 1D GC from the fit between experimental and calculated data. Their validation in GC×GC was carried out by using n-alkanes and a mixture of disaccharides as their trimethylsilyl oxime derivatives. Comparison of experimental and estimated data showed good results for retention time in both first and second dimension. Peak widths presented, however, some errors, caused by the no consideration on estimation of the effect of the modulator or the rather unusual chromatographic conditions used. The developed programme is very versatile as it can be used for the calculation of the chromatographic response of compounds with different polarity and volatility, analysed under different pressures and temperature ramps, and with different sets of columns. [1] J.V. Seeley, E.M. Libby, K.A.H. Edwards, S.K. Seeley, J. Chromatogr. A 1216 (2009) 1650. [2] F.L. Dorman, P.D. Schettler, L.A. Vogt, J.W. Cochran, J. Chromatogr. A 1186 (2008) 196. [3] Y. Zhao, J. Zhang, B. Wang, S.H. Kim, A. Fang, B. Bogdanov, Z. Zhou, C. McClain, X. Zhang, J. Chromatogr. A 1218 (2011) 2577. [4] S. Zhu, S. He, D.R. Worton, A.H. Goldstein, J. Chromatogr. A 1233 (2012) 147. Acknowledgements: This work has been funded by Ministerio de Economía y Competitividad (project CTQ2012-32957), Comunidad Autónoma de Madrid (Spain) and European funding from FEDER program (AVANSECAL-CM S2013/ABI-3028) and Fundación Ramón Areces. ACS thanks Ministerio de Economía y Competitividad of Spain for a Ramón y Cajal contract.CTQ2012-32957Peer reviewe

Synthesis and characterization of NaNiF3·3H2O: An unusual ordered variant of the ReO3 type

et al.A new hydrated sodium nickel fluoride with nominal composition NaNiF3·3H2O was synthesized using an aqueous solution route. Its structure was solved by means of ab initio methods from powder X-ray diffraction and neutron diffraction data. NaNiF3·3H2O crystallizes in the cubic crystal system, space group Pn3̅ with a = 7.91968(4) Å. The framework, derived from the ReO3 structure type, is built from NaX6 and NiX6 (X = O, F) corner-shared octahedra, in which F and O atoms are randomly distributed on a single anion site. The 2a × 2a × 2a superstructure arises from the strict alternate three-dimensional linking of NaX6 and NiX6 octahedra together with the simultaneous tilts of the octahedra from the cube axis (φ = 31.1°), with a significant participation of hydrogen bonding. NaNiF3·3H2O corresponds to a fully cation-ordered variant of the In(OH)3 structure, easily recognizable when formulated as NaNi(XH)6 (X = O, F). It constitutes one of the rare examples for the a+a+a+ tilting scheme with 1:1 cation ordering in perovskite-related compounds. The Curie-like magnetic behavior well-reflects the isolated paramagnetic Ni2+ centers without worth mentioning interactions. While X-ray and neutron diffraction data evidence Na/Ni order in combination with O/F disorder as a main feature of this fluoride, results from Raman and magic-angle spinning NMR spectroscopies support the existence of specific anion arrangements in isolated square windows identified in structural refinements. In particular, formation of water molecules derives from unfavorable FH bond formation.We thank Ministerio de Ciencia e Innovación and Comunidad de Madrid for funding Project Nos. MAT2010-19837-C06 and P2009/PPQ-1626, respectively. Financial support from Universidad CEU San Pablo is also acknowledged.Peer reviewe

One-loop corrections to ALP couplings

The plethora of increasingly precise experiments which hunt for axion-like particles (ALPs), as well as their widely different energy reach, call for the theoretical understanding of ALP couplings at loop-level. We derive the one-loop contributions to ALP-SM effective couplings, including finite corrections. The complete leading-order — dimension five — effective linear Lagrangian is considered. The ALP is left off-shell, which is of particular impact on LHC and accelerator searches of ALP couplings to γγ, ZZ, Zγ, WW, gluons and fermions. All results are obtained in the covariant Rξ gauge. A few phenomenological consequences are also explored as illustration, with flavour diagonal channels in the case of fermions: in particular, we explore constraints on the coupling of the ALP to top quarks, that can be extracted from LHC data, from astrophysical sources and from Dark Matter direct detection experiments such as PandaX, LUX and XENON1T. Furthermore, we clarify the relation between alternative ALP bases, the role of gauge anomalous couplings and their interface with chirality-conserving and chirality-flip fermion interactions, and we briefly discuss renormalization group aspect

Caracterización estructural y espectroscópica de fibras cristalinas de Ce0.4Zr0.6O2 crecidas mediante el método de fusión zonal asistida por láser

[EN] A structural and spectroscopic characterization of crystalline rods of Ce0.4Zr0.6O2 grown by the laser floating zone (LFZ) method is presented. A precursor rod of Ce0.4Zr0.6O2 composition was sintered at 1500 ºC in air atmosphere and then processed by the LFZ technique with a CO2 laser. The processed material was characterized by XRD, SEM and Raman spectroscopy. In the as-grown, dark-color processed rod, the Raman spectrum evolves radially from a t’-like one, corresponding to Ce0.37Zr0.63O2 composition, at the edge of the rod, to a very broad and weak, cubic-like one, at the center. The degree of cerium reduction and oxygen non-stoichiometry were determined through measurements of the electronic Raman spectrum of Ce3+. A strong Ce3+ signal was found at the core of the rod, indicating strong reduction, whereas no Ce3+ signal was detected at the edge. To restore oxygen and Ce4+ content a section of the fiber was reoxidized at 620 ºC for 24 h. A very homogeneous spectrum was found in the reoxidized sample, that was assigned to a t’-phase of composition Ce0.42Zr0.58O2.[ES] Presentamos una caracterización estructural y espectroscópica de fibras cristalinas de Ce0.4Zr0.6O2 crecidas mediante fusión zonal asistida por láser (LFZ). Una barra del material precursor, de composición Ce0.4Zr0.6O2, fue sinterizada a 1500 ºC en atmósfera de aire y después procesada por LFZ con un láser de CO2. El material procesado fue caracterizado por difracción de rayos X, microscopía electrónica de barrido y espectroscopía Raman. En la fibra recién procesada, de color gris oscuro, el espectro Raman varía radialmente desde un espectro de tipo t’, correspondiente a una composición Ce0.37Zr0.63O2, en el borde de la fibra, hasta un espectro muy ensanchado y débil, de aspecto cúbico, en el centro. El grado de reducción del cerio y la no estequiometría del oxígeno se determinaron a través de las medidas del Raman electrónico del Ce3+. La señal de Ce3+ era muy intensa en el centro de la fibra, lo que indica una fuerte reducción, mientras que no se detectó en el borde. Para restablecer el contenido de oxígeno y de Ce4+ una parte de la fibra fue reoxidada a 620 ºC durante 24 h. El espectro de la muestra reoxidada, de color amarillo pálido y muy homogénea, fue asignado a una fase t’ de composición Ce0.42Zr0.58O2.Financial support from Spanish project MAT2004-03070-C05-03 is acknowledged.Peer reviewe

Rational stabilization of the C-LytA affinity tag by protein engineering

The C-LytA protein constitutes the choline-binding module of the LytA amidase from Streptococcus pneumoniae. Owing to its affinity for choline and analogs, it is regularly used as an affinity tag for the purification of proteins in a single chromatographic step. In an attempt to build a robust variant against thermal denaturation, we have engineered several salt bridges on the protein surface. All the stabilizing mutations were pooled in a single variant, C-LytAm7, which contained seven changes: Y25K, F27K, M33E, N51K, S52K, T85K and T108K. The mutant displays a 7 degrees C thermal stabilization compared with the wild-type form, together with a complete reversibility upon heating and a higher kinetic stability. Moreover, the accumulation of intermediates in the unfolding of C-LytA is virtually abolished for C-LytAm7. The differences in stability become more evident when the proteins are bound to a DEAE-cellulose affinity column, as most of wild-type C-LytA is denatured at approximately 65 degrees C, whereas C-LytAm7 may stand temperatures up to 90 degrees C. Finally, the change in the isoelectric point of C-LytAm7 enhances its solubility at acidic pHs. Therefore, C-LytAm7 behaves as an improved affinity tag and supports the engineering of surface salt bridges as an effective approach for protein stabilization

Drug Repositioning as a Therapeutic Strategy against Streptococcus pneumoniae: Cell Membrane as Potential Target

A collection of repurposing drugs (Prestwick Chemical Library) containing 1200 compounds was screened to investigate the drugs’ antimicrobial effects against planktonic cultures of the respiratory pathogen Streptococcus pneumoniae. After four discrimination rounds, a set of seven compounds was finally selected, namely (i) clofilium tosylate; (ii) vanoxerine; (iii) mitoxantrone dihydrochloride; (iv) amiodarone hydrochloride; (v) tamoxifen citrate; (vi) terfenadine; and (vii) clomiphene citrate (Z, E). These molecules arrested pneumococcal growth in a liquid medium and induced a decrease in bacterial viability between 90.0% and 99.9% at 25 µM concentration, with minimal inhibitory concentrations (MICs) also in the micromolar range. Moreover, all compounds but mitoxantrone caused a remarkable increase in the permeability of the bacterial membrane and share a common, minimal chemical structure consisting of an aliphatic amine linked to a phenyl moiety via a short carbon/oxygen linker. These results open new possibilities to tackle pneumococcal disease through drug repositioning and provide clues for the design of novel membrane-targeted antimicrobials with a related chemical structure.Depto. de Sanidad AnimalFac. de VeterinariaTRUEMCIN/AEI/ 10.13039/501100011033pu