Two exopolyphosphatases with distinct molecular architectures and substrate specificities from the thermophilic green-sulfur bacterium Chlorobium tepidum TLS

The genome of the thermophilic green-sulfur bacterium Chlorobium tepidumTLS possesses two genes encoding putative exopolyphosphatases (PPX; EC 3.6.1.11), namely CT0099 (ppx1, 993 bp) and CT1713 (ppx2, 1557 bp). The predicted polypeptides of 330 and 518 aa residues are Ppx-GppA phosphatases of different domain architectures - the largest one has an extra C-terminal HD domain - which may represent ancient paralogues. Both ppxgenes were cloned and overexpressed in Escherichia coli BL21(DE3). While CtPPX1 was validated as a monomeric enzyme, CtPPX2 was found to be a homodimer. Both PPX homologues were functional, K+-stimulated phosphohydrolases, with an absolute requirement for divalent metal cations and a marked preference for Mg2+. Nevertheless, they exhibited remarkably different catalytic specificities with regard to substrate classes and chain lengths. Even though both enzymes were able to hydrolyse the medium-size polyphosphate (polyP) P13-18 (polyP mix with mean chain length of 13-18 phosphate residues), CtPPX1 clearly reached its highest catalytic efficiency with tripolyphosphate and showed substantial nucleoside triphosphatase (NTPase) activity, while CtPPX2 preferred long-chain polyPs (>300 Pi residues) and did not show any detectable NTPase activity. These catalytic features, taken together with the distinct domain architectures and molecular phylogenies, indicate that the two PPX homologues of Chl. tepidum belong to different Ppx-GppA phosphatase subfamilies that should play specific biochemical roles in nucleotide and polyP metabolisms. In addition, these results provide an example of the remarkable functional plasticity of the Ppx-GppA phosphatases, a family of proteins with relatively simple structures that are widely distributed in the microbial world. © 2014 The Authors.España, Gobierno BFU2004-00843, BFU2007- 61887 and BFU2010-15622Junta de AndalucÍa BIO118

Inorganic Polyphosphate in the Microbial World. Emerging Roles for a Multifaceted Biopolymer

inorganic polyphosphates (polyP) are linear polymers of tens to hundreds orthophosphate residues linked by phosphoanhydride bonds. These fairly abundant biopolymers occur in all extant forms of life, from prokaryotes to mammals, and could have played a relevant role in prebiotic evolution. Since the first identification of polyP deposits as metachromatic or volutin granules in yeasts in the nineteenth century, an increasing number of varied physiological functions have been reported. Due to their "high energy" bonds analogous to those in ATP and their properties as polyanions, polyP serve as microbial phosphagens for a variety of biochemical reactions, as a buffer against alkalis, as a storage of Ca(2+) and as a metal-chelating agent. In addition, recent studies have revealed polyP importance in signaling and regulatory processes, cell viability and proliferation, pathogen virulence, as a structural component and chemical chaperone, and as modulator of microbial stress response. This review summarizes the current status of knowledge and future perspectives of polyP functions and their related enzymes in the microbial world.España, MINECO BFU2004-00843, BFU2007-61887, BFU2010-15622España, Junta de Andalucía BIO-26

Purification of Starch Granules from Arabidopsis Leaves and Determination of Granule-Bound Starch Synthase Activity

Starch constitutes the most important carbon reserve in plants and is composed of branched amylopectin and linear amylose. The latter is synthesized exclusively by the Granule-Bound Starch Synthase (GBSS, EC 2.4.1.21). Here we report a readily reproducible, specific and highly sensitive protocol, which includes the isolation of intact starch granules from Arabidopsis thaliana leaves and the subsequent determination of GBSS activity. We have applied this method to study GBSS activity in diurnal cycles in vegetative growth and during the photoperiodic transition to flowering in Arabidopsis (Tenorio et al., 2003; Ortiz-Marchena et al., 2014).España,MINECO CSD2007-00057, BIO2008-02292, and BIO2011-28847-C02-00España, Junta de Andalucía P06-CVI-01450 and P08-AGR-0358

Characterization of the sucrose phosphate phosphatase (SPP) isoforms from Arabidopsis thaliana and role of the S6PPc domain in dimerization

Sucrose-phosphate phosphatase (SPP) catalyses the final step in the sucrose biosynthesis pathway. Arabidopsis thaliana genome codifies four SPP isoforms. In this study, the four Arabidopsis thaliana genes coding for SPP isoforms have been cloned, expressed in Escherichia coli and the kinetic and regulatory properties of the purified enzymes analysed. SPP2 is the isoform showing the highest activity, with SPP3b and SPP3a showing lower activity levels. No activity was detected for SPP1. We propose that this lack of activity is probably due to the absence of an essential amino acid participating in catalysis and/or in the binding of the substrate, sucrose-6-phosphate (Suc6P). The expression patterns of Arabidopsis SPP genes indicate that SPP2 and SPP3b are the main isoforms expressed in different tissues and organs, although the non-catalytic SPP1 is the main isoform expressed in roots. Thus, SPP1 could have acquired new unknown functions. We also show that the three catalytically active SPPs from Arabidopsis are dimers. By generating a chimeric SPP composed of the monomeric cyanobacterial SPP fused to the higher plant non-catalytic S6PPc domain (from SPP2), we show that the S6PPc domain is responsible for SPP dimerization. This is the first experimental study on the functionality and gene expression pattern of all the SPPs from a single plant species.Ministerio de Economía y Competitividad TRANSPLANTA Consolider 28317Junta de Andalucía P08-AGR-03582 y CVI-28

Effect of intravenous pulses of methylprednisolone 250 mg versus dexamethasone 6 mg in hospitalised adults with severe COVID ‐19 pneumonia: An open‐label randomised trial

Producción CientíficaBackground: The efficacy and safety of high versus medium doses of glucocorticoids for the treatment of patients with COVID-19 has shown mixed outcomes in controlled trials and observational studies. We aimed to evaluate the effectiveness of methylprednisolone 250 mg bolus versus dexamethasone 6 mg in patients with severe COVID-19. Methods: A randomised, open-label, controlled trial was conducted between February and August 2021 at four hospitals in Spain. The trial was suspended after the first interim analysis since the investigators considered that continuing the trial would be futile. Patients were randomly assigned in a 1:1 ratio to receive dexamethasone 6 mg once daily for up to 10 days or methylprednisolone 250 mg once daily for 3 days. Results: Of the 128 randomised patients, 125 were analysed (mean age 60 ± 17 years; 82 males [66%]). Mortality at 28 days was 4.8% in the 250 mg methylprednisolone group versus 4.8% in the 6 mg dexamethasone group (absolute risk difference, 0.1% [95% CI, −8.8 to 9.1%]; p = 0.98). None of the secondary outcomes (admission to the intensive care unit, non-invasive respiratory or high-flow oxygen support, additional immunosuppressive drugs, or length of stay), or prespecified sensitivity analyses were statistically significant. Hyperglycaemia was more frequent in the methylprednisolone group at 27.0 versus 8.1% (absolute risk difference, −18.9% [95% CI, −31.8 to - 5.6%]; p = 0.007). Conclusions: Among severe but not critical patients with COVID-19, 250 mg/d for 3 days of methylprednisolone compared with 6 mg/d for 10 days of dexamethasone did not result in a decrease in mortality or intubation

Modelos de riesgo para la predicción de mortalidad hospitalaria en ancianos con neumonía por COVID-19

Producción CientíficaObjetivo. Los objetivos son comparar la utilidad pronóstica de tres escalas de gravedad (Pneumonia Severity Index: PSI; CURB-65 scale; Severity Community Acquired Pneumonia Score: SCAP) y diseñar un nuevo modelo predictivo de mortalidad hospitalaria en pacientes mayores de 75 años ingresados por neumonía por COVID-19. Método. Estudio retrospectivo de pacientes mayores de 75 años ingresados por neumonía por COVID-19 desde el servicio de urgencias entre el 12 de marzo y el 27 de abril de 2020. Se recogieron variables demográficas (edad, sexo, institucionalización), clínicas (síntomas, comorbilidades, índice de Charlson) y analíticas (bioquímica en suero, gasometría, hematimetría, hemostasia). Se derivó un modelo de riesgo y se compararon las escalas de gravedad PSI, CURB-65 y SCAP para predecir la mortalidad intrahospitalaria por cualquier causa. Resultados. Se incluyeron 186 pacientes, con una mediana de edad de 85 años (RIC 80-89), un 44,1% varones. La mortalidad fue del 47,3%. Las escalas PSI, CURB-65 y SCAP tuvieron un área bajo la curva (ABC) de 0,74 (IC 95% 0,64-0,82), 0,71 (IC 95% 0,62-0,79) y 0,72 (IC 95% 0,63-0,81), respectivamente. El modelo predictivo compuesto por la ausencia o presencia de síntomas (tos y disnea), comorbilidad (índice de Charlson) y datos analíticos (aspartato-aminotransferasa, potasio, urea y lactato-deshidrogenasa) tuvo un ABC de 0,81 (IC 95% 0,73-0,88). Conclusión. Este estudio muestra que la escala PSI tiene una capacidad predictiva de mortalidad moderada, notablemente mejor que las escalas CURB-65 y SCAP. Se propone un nuevo modelo predictivo de mortalidad que mejora significativamente el rendimiento de estas escalas, siendo necesario verificar su validez externa.Junta de Castilla y León (project GRS COVID 09/A/2020)Ministerio de Ciencia e Innovación – Agencia Estatal de Investigación, cofinanciada por el Fondo Social Europeo (grant RYC2019-028566-I)Gerencia Regional de Salud de Castilla y León (grant INT/M/15/20

Procedimiento para obtener compuestos de alto valor añadido a partir de hoja de olivo

Fecha de presentación internacional: 08.02.2005. - Titular: Consejo Superior de Investigaciones Científicas (CSIC). - Universidad Autónoma de Madrid[EN] The invention relates to a method of obtaining high-value-added compounds from olive leaves. The inventive method comprises the following steps, namely: a first step in which the leaves are subjected to solid-liquid extraction with organic solvents, preferably hexane or ethanol, and the crude extract thus obtained is vacuum concentrated; and a second step in which the crude extract is fractionated by means of supercritical CO2 countercurrent column extraction and separation into two cells with different fixed pressure and/or temperature conditions which alter the dissolving power of the CO2, thereby precipitating different compounds. The invention can be used to extract natural products of interest from olive leaves for the food, pharmaceutical and cosmetic industries, such as waxes, squalene, beta -carotene, alpha -tocopherol, oleuropein, hydroxytyrosol and other phenolic compounds, beta-sitosterol, alpha - and beta-amyrin, erythrodiol, uvaol and other terpenic alcohols, oleanolic acid, ursolic acid and maslinic acid, among others.[ES] Mediante el procedimiento de la invención se obtienen compuestos de alto valor añadido a partir de hoja de olivo. En una primera etapa se someten las hojas a extracción sólido-líquido con disolventes orgánicos, preferentemente hexano o etanol, y se concentra a vacío el extracto bruto obtenido. En una segunda etapa se realiza un fraccionamiento de ese extracto bruto por extracción en contracorriente en columna con COz supercrítico y separación en dos celdas en que se fijan diferentes condiciones de presión y/o temperatura que cambian el poder disolvente del CO2, precipitando diferentes compuestos. El procedimiento de la invención permite extraer de la hoja de olivo productos naturales de interés para las industrias alimentaria, farmacéutica y cosmética, como ceras, escualeno, beta- caroteno, alfa-tocoferol, oleuropeina, hidroxitirosol y otros compuestos fenólicos, beta-sitosterol, a y beta-amirina, eritrodiol, uvaol y otros alcoholes terpénicos, ácido oleanólico, ácido ursólico y ácido maslínico, entre otros.Peer reviewe