Optimizing the capture of protein interaction partners in the biacore T100 for mapping signaling pathways with low cell amounts

Comunicaciones a congreso

Production of a biodiesel-like biofuel without glycerol generation, by using Novozym 435, an immobilized Candida antarctica lipase

Background: Novozym 435, a commercial lipase from Candida antarctica, recombinant, expressed in Aspergillus niger, immobilized on macroporous acrylic resin, has been already described in the obtention of biodiesel. It is here evaluated in the production of a new biofuel that integrates the glycerol as monoglyceride (MG) together with two fatty acid ethyl esters (FAEE) molecules by the application of 1,3-selective lipases in the ethanolysis reaction of sunflower oil. Results: Response surface methodology (RSM) is employed to estimate the effects of main reaction. Optimum conditions for the viscosity, selectivity, and conversion were determined using a multifactorial design of experiments with three factors run by the software Stat Graphics version XV.I. The selected experimental parameters were reaction temperature, oil/ethanol ratio and alkaline environment. On the basis of RSM analysis, the optimum conditions for synthesis were 1/6 oil/EtOH molar ratio, 30°C, and 12.5 μl of NaOH 10 N aqueous solutions, higher stirring than 300 rpm, for 2 h and 0.5 g of biocatalyst. Conclusions: These obtained results have proven a very good efficiency of the biocatalyst in the studied selective process. Furthermore, it was allowed sixteen times the successive reuse of the biocatalyst with good performanc

Biocatalytic behaviour of immobilized Rhizopus oryzae lipase in the 1,3-selective ethanolysis of sunflower oil to obtain a biofuel similar to biodiesel

A new biofuel similar to biodiesel was obtained in the 1,3-selective transesterification reaction of sunflower oil with ethanol using as biocatalyst a Rhizopus oryzae lipase (ROL) immobilized on Sepiolite, an inorganic support. The studied lipase was a low cost powdered enzyme preparation, Biolipase-R, from Biocon-Spain, a multipurpose additive used in food industry. In this respect, it is developed a study to optimize the immobilization procedure of these lipases on Sepiolite. Covalent immobilization was achieved by the development of an inorganic-organic hybrid linker formed by a functionalized hydrocarbon chain with a pendant benzaldehyde, bonded to the AlPO4 support surface. Thus, the covalent immobilization of lipases on amorphous AlPO 4/sepiolite (20/80 wt %) support was evaluated by using two different linkers (p-hydroxybenzaldehyde and benzylamine-terephthalic aldehyde, respectively). Besides, the catalytic behavior of lipases after physical adsorption on the demineralized sepiolite was also evaluated. Obtained results indicated that covalent immobilization with the p-hydroxybenzaldehyde linker gave the best biocatalytic behavior. Thus, this covalently immobilized lipase showed a remarkable stability as well as an excellent capacity of reutilization (more than five successive reuses) without a significant loss of its initial catalytic activity. This could allow a more efficient fabrication of biodiesel minimizing the glycerol waste production. © 2014 by the authors.Grants from the Spanish Ministry of Economy and Competitiveness (Project ENE 2011-27017), Spanish Ministry of Education and Science (Projects CTQ2010-18126 and CTQ2011-28954-C02-02), FEDER funds and Junta de Andalucía FQM 0191, PO8-RMN-03515 and P11-TEP-7723 are gratefully acknowledged by the authors. We are also gratefully to Biocon®-Spain, for the kindly supply of the BIOLIPASE-R®. We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI)Peer Reviewe

Effect of easily biodegradable amendments on heavy metal solubilization and accumulation in technical crops ‐ a field trial

Heavy‐metal soil pollution is a common environmental problem all over the world. In the induced phytoextraction process plants can transport and translocate to their above‐ground tissues significant concentration of contaminants. Since some heavy metals are mostly presented in non‐soluble state, the application of chelating agents is needed. In this study we introduced into the rhizosphere of maize (var. Kneja 530) and sunflower (var. Favorit) plants 1 and 5 mmol l−1 of EDDS and NTA, and 5 g kg−1 of nutrients to enhance the accumulated concentrations of cadmium, lead and zinc. The fraction of CaCl2‐extractable metals increased incrementing the concentration of chelates. This was clearly pronounced for all the metals in the treatments with NTA and for Pb in those with EDDS. Generally, under all the experimental conditions the accumulated heavy‐metal concentrations were found to be significantly higher than in the control. Thus, in the case of higher concentration of NTA and EDDS, the accumulated Cd in the leaves of sunflower was found to be threefold higher. The highest accumulation of Pb in the leaves of maize was observed when nutrients were added (62 mg kg−1). On the other hand, negative effects of the used chelating agents on soil bacteria and fungi at plant harvest were not observed. Moreover, when nutrients were added, the number of studied microorganisms significantly increased. Lengvai biodegradavimą pagerinančių pataisymų priemonių poveikis sunkiųjų metalų tirpumui ir akumuliacijai techniniuose pasėliuose – lauko bandymai Santrauka Dirvožemio užterštumas sunkiaisiais metalais yra dažna aplinkos problema visame pasaulyje. Inicijuotas fitoekstrakcijos procesas augaluose gali perkelti į jų antžeminius audinius dideles teršalų koncentracijas. Kadangi kai kurie sunkieji metalai dažniausiai esti netirpios būsenos, reikia pritaikyti chelatų agentus. Į kukurūzus buvo įterpta rizosfera (var. Kneja 530) ir saulėgrąžos (var. Favorit) EDDS bei NTA 1 ir 5 mmol–1 ir 5 g kg–1 mineralinių medžiagų, padidinant kadmio, švino ir cinko akumuliuotas koncentracijas. Ekstrahuotos CaCl2 metalo frakcijos padidino chelatų koncentracijos didėjimą. Tai pasireiškė visiems metalams, apdorotiems su NTA, ir švinui, paveiktam su EDDS. Visuose eksperimentuose akumuliuoto sunkiojo metalo koncentracijos buvo daug didesnės nei kontroliniame bandinyje. Vadinasi, didesnės NTA ir EDDS koncentracijos atveju akumuliuotas Cd saulėgrąžos lapuose buvo 3 kartus didesnis. Didžiausia Pb akumuliacija miežių lapuose išmatuota, pridėjus mineralinių medžiagų (62 mg kg–1). Kita vertus, neigiamas chelatų agentų naudojimo poveikis dirvožemio bakterijoms ir grybeliams augalų derliuje nebuvo nustatytas. Be to, kai buvo pridėta mineralinių medžiagų, padaugėjo nagrinėjamų mikroorganizmų. Reikšminiai žodžiai: sunkieji metalai, fitoekstrakcija, kviečiai, saulėgrąžos, EDDS, NTA, lauko bandymai, rizosferos mikroorganizmai. Полевые испытания влияния мер, способствующих деградации тяжелых металлов, их разложению и накапливанию в технических посевах Резюме Загрязнение почв тяжелыми металлами – наиболее часто отмечаемая проблема во всем мире. Инициированный процесс фитоэкстракции в растениях может способствовать переносу значительных концентраций загрязнителей в их поверхностные ткани. В связи с тем, что некоторые тяжелые металлы находятся в нерастворенном состоянии, необходимо применение агентов хелатов. В настоящем исследовании в кукурузу была введена ризосфера (вар. Kneja 530) и подсолнечник (вар. favorit) EDDS и NTA 1 и 5 ммоль–1 ir 5 г кг–1 минеральных веществ для увеличения аккумулированных концентраций кадмия, свинца и цинка. Экстрагированные фракции металла CaCl2 способствовали увеличению концентрации хелатов. Это подтверждено исследованием всех металлов, подвергшихся воздействию NTA, и свинца, обработанного с помощью EDDS. Во всех экспериментах концентрации аккумулированного тяжелого металла были значительно больше, чем в контрольном образце. Следовательно, в случае большей концентрации NTA и EDDS содержание аккумулированного в листьях подсолнечника Cd было в три раза больше. Наибольшая аккумуляция Cd в листьях ячменя зафиксирована в случае добавления минеральных веществ (62 мг кг–1). Не было обнаружено и отрицательного воздействия от применения агентов хелатов на бактерии почвы и грибки в урожае растений. Кроме того, в результате добавления минеральных веществ количество исследуемых микроорганизмов увеличилось. Ключевые слова: тяжелые металлы, фитоэкстракция, ячмень, подсолнечник, NTA, EDDS полевые испытания, микроорганизмы ризосферы. First Published Online: 14 Oct 201

Diagnostic value of quantitative SPECT/CT in assessing active sacroiliitis in patients with axial spondylarthritis and/or inflammatory low back pain

Background. The diagnostic accuracy of bone scintigraphy (BS) increases with SPECT/CT imaging. It would therefore be appropriate to reassess the diagnostic utility of scintigraphy in sacroiliitis with axial spondyloarthritis (SpA). The aim of this study was to compare the diagnostic performance of MRI, SPECT/CT and a combination of both techniques in sacroiliitis, and to evaluate the correlation between quantitative SPECT/CT indices and quantitative MRI inflammatory lesion scores. Methods. Thirty-one patients with active SpA and 22 patients with inflammatory low back pain underwent MRI and SPECT/CT of the sacroiliac joints. The diagnostic accuracy of both techniques was calculated using clinical diagnosis as the gold standard. The correlation between MRI and SPECT/CT was calculated by comparing the SPECT/CT activity indices and the Berlin/SPARCC scoring systems for MRI. Results. The sensitivity and specificity values in quantitative SPECT/CT, taking the sacroiliac/promontory ratio of >1.36 as the cut-off value, were close to those from MRI published in the literature. The combination of both techniques increased sensitivity while maintaining high specificity. There was a moderate correlation between SPECT/CT and MRI total scores. This correlation was improved by using solely the MRI inflammation scores. Conclusion. Quantitative SPECT/CT showed better diagnostic accuracy than planar scintigraphy and showed a moderate correlation with MRI scores in active sacroiliitis. The combination of both tests increased the diagnostic accuracy. Quantitative SPECT/CT could play a relevant role in the diagnosis of active sacroiliitis in patients with high a suspicion of SpA and a negative/inconclusive MRI test or in patients with whom MRI studies cannot be carried out.Fundamento. La precisión diagnóstica de la gammagrafía ósea (GO) aumenta con las imágenes SPECT/TAC haciendo conveniente reevaluar su utilidad diagnóstica en la sacroilitis de la espondiloartritis axial (EA). El objetivo fue comparar el rendimiento diagnóstico de la resonancia magnética (RM), la SPECT/TC y ambas pruebas combinadas, y evaluar la correlación entre los índices cuantitativos de ambas técnicas. Métodos. A 31 pacientes con EA activa y 22 con lumbalgia inflamatoria se les realizó una RM y una SPECT/TC de las articulaciones sacroilíacas y se calculó la precisión diagnóstica de ambas técnicas respecto al diagnóstico clínico. La correlación entre ambas pruebas se calculó comparando los índices de actividad del SPECT/TC con los sistemas de puntuación Berlín y SPARCC de RM. Resultados. Los valores de sensibilidad y especificidad de la SPECT/TC cuantitativa, tomando como punto de corte el cociente sacroilíaca/promontorio >1,36, fueron similares a los publicados para la RM. La combinación de ambas técnicas aumentó la sensibilidad manteniendo una alta especificidad. La correlación entre las escalas totales de RM y SPECT/TC fue moderada y mejoraba al utilizar únicamente las escalas de inflamación. Conclusiones. La SPECT/TC cuantitativa muestra una mejor precisión diagnóstica que la GO planar en la sacroilitis activa y una correlación moderada con las puntuaciones de RM. La combinación de ambas técnicas aumenta la precisión diagnóstica. Por tanto, la SPECT/TAC cuantitativa podría tener un papel relevante en el diagnóstico de sacroilitis en pacientes con alta sospecha de EA y RM negativa/no concluyente o en aquellos pacientes en los que no se pueda realizar una RM

Accurate Treatment of Large Supramolecular Complexes by Double-Hybrid Density Functionals Coupled with Nonlocal van der Waals Corrections

In this work, we present a thorough assessment of the performance of some representative double-hybrid density functionals (revPBE0-DH-NL and B2PLYP-NL) as well as their parent hybrid and GGA counterparts, in combination with the most modern version of the nonlocal (NL) van der Waals correction to describe very large weakly interacting molecular systems dominated by noncovalent interactions. Prior to the assessment, an accurate and homogeneous set of reference interaction energies was computed for the supramolecular complexes constituting the L7 and S12L data sets by using the novel, precise, and efficient DLPNO-CCSD(T) method at the complete basis set limit (CBS). The correction of the basis set superposition error and the inclusion of the deformation energies (for the S12L set) have been crucial for obtaining precise DLPNO-CCSD(T)/CBS interaction energies. Among the density functionals evaluated, the double-hybrid revPBE0-DH-NL and B2PLYP-NL with the three-body dispersion correction provide remarkably accurate association energies very close to the chemical accuracy. Overall, the NL van der Waals approach combined with proper density functionals can be seen as an accurate and affordable computational tool for the modeling of large weakly bonded supramolecular systems.Financial support by the “Ministerio de Economía y Competitividad” (MINECO) of Spain and European FEDER funds through projects CTQ2011-27253 and CTQ2012-31914 is acknowledged. The support of the Generalitat Valenciana (Prometeo/2012/053) is also acknowledged. J.A. thanks the EU for the FP7-PEOPLE-2012-IEF-329513 grant. J.C. acknowledges the “Ministerio de Educación, Cultura y Deporte” (MECD) of Spain for a predoctoral FPU grant

Evaluation of 12 GWAS-drawn SNPs as biomarkers of rheumatoid arthritis response to TNF inhibitors. A potential SNP association with response to etanercept

Research in rheumatoid arthritis (RA) is increasingly focused on the discovery of biomarkers that could enable personalized treatments. The genetic biomarkers associated with the response to TNF inhibitors (TNFi) are among the most studied. They include 12 SNPs exhibiting promising results in the three largest genome-wide association studies (GWAS). However, they still require further validation. With this aim, we assessed their association with response to TNFi in a replication study, and a meta-analysis summarizing all nonredundant data. The replication involved 755 patients with RA that were treated for the first time with a biologic drug, which was either infliximab (n = 397), etanercept (n = 155) or adalimumab (n = 203). Their DNA samples were successfully genotyped with a single-base extension multiplex method. Lamentably, none of the 12 SNPs was associated with response to the TNFi in the replication study (p > 0.05). However, a drug-stratified exploratory analysis revealed a significant association of the NUBPL rs2378945 SNP with a poor response to etanercept (B = -0.50, 95% CI = -0.82, -0.17, p = 0.003). In addition, the metaanalysis reinforced the previous association of three SNPs: rs2378945, rs12142623, and rs4651370. In contrast, five of the remaining SNPs were less associated than before, and the other four SNPs were no longer associated with the response to treatment. In summary, our results highlight the complexity of the pharmacogenetics of TNFi in RA showing that it could involve a drug-specific component and clarifying the status of the 12 GWAS-drawn SNPsThis work was supported by the Instituto de Salud Carlos III (ISCIII, Spain) through grants PI14/01651, PI17/01606 and RD16/0012/0014 to AG and PI12/01909 to JJG-R. These grants are partially financed by the European Regional Development Fund of the EU (FEDER

Evaluation of 12 GWAS-drawn SNPs as biomarkers of rheumatoid arthritis response to TNF inhibitors. A potential SNP association with response to etanercept

Research in rheumatoid arthritis (RA) is increasingly focused on the discovery of biomarkers that could enable personalized treatments. The genetic biomarkers associated with the response to TNF inhibitors (TNFi) are among the most studied. They include 12 SNPs exhibiting promising results in the three largest genome-wide association studies (GWAS). However, they still require further validation. With this aim, we assessed their association with response to TNFi in a replication study, and a meta-analysis summarizing all non-redundant data. The replication involved 755 patients with RA that were treated for the first time with a biologic drug, which was either infliximab (n = 397), etanercept (n = 155) or adalimumab (n = 203). Their DNA samples were successfully genotyped with a single-base extension multiplex method. Lamentably, none of the 12 SNPs was associated with response to the TNFi in the replication study (p > 0.05). However, a drug-stratified exploratory analysis revealed a significant association of the NUBPL rs2378945 SNP with a poor response to etanercept (B = -0.50, 95% CI = -0.82, -0.17, p = 0.003). In addition, the meta-analysis reinforced the previous association of three SNPs: rs2378945, rs12142623, and rs4651370. In contrast, five of the remaining SNPs were less associated than before, and the other four SNPs were no longer associated with the response to treatment. In summary, our results highlight the complexity of the pharmacogenetics of TNFi in RA showing that it could involve a drug-specific component and clarifying the status of the 12 GWAS-drawn SNP

Validation Study Of Genetic Biomarkers Of Response To Tnf Inhibitors In Rheumatoid Arthritis

Genetic biomarkers are sought to personalize treatment of patients with rheumatoid arthritis (RA), given their variable response to TNF inhibitors (TNFi). However, no genetic biomaker is yet sufficiently validated. Here, we report a validation study of 18 previously reported genetic biomarkers, including 11 from GWAS of response to TNFi. The validation was attempted in 581 patients with RA that had not been treated with biologic antirheumatic drugs previously. Their response to TNFi was evaluated at 3, 6 and 12 months in two ways: change in the DAS28 measure of disease activity, and according to the EULAR criteria for response to antirheumatic drugs. Association of these parameters with the genotypes, obtained by PCR amplification followed by single-base extension, was tested with regression analysis. These analyses were adjusted for baseline DAS28, sex, and the specific TNFi. However, none of the proposed biomarkers was validated, as none showed association with response to TNFi in our study, even at the time of assessment and with the outcome that showed the most significant result in previous studies. These negative results are notable because this was the first independent validation study for 12 of the biomarkers, and because they indicate that prudence is needed in the interpretation of the proposed biomarkers of response to TNFi even when they are supported by very low p values. The results also emphasize the requirement of independent replication for validation, and the need to search protocols that could increase reproducibility of the biomarkers of response to TNFi

Lack of validation of genetic variants associated with anti-tumor necrosis factor therapy response in rheumatoid arthritis: a genome-wide association study replication and meta-analysis

Introduction: In this study, our aim was to elucidate the role of four polymorphisms identified in a prior large genome-wide association study (GWAS) in which the investigators analyzed the responses of patients with rheumatoid arthritis (RA) to treatment with tumor necrosis factor inhibitors (TNFi). The authors of that study reported that the four genetic variants were significantly associated. However, none of the associations reached GWAS significance, and two subsequent studies failed to replicate these associations. Methods: The four polymorphisms (rs12081765, rs1532269, rs17301249 and rs7305646) were genotyped in a total of 634 TNFi-treated RA patients of Spanish Caucasian origin. Four outcomes were evaluated: changes in the Disease Activity Score in 28 joints (DAS28) after 6 and 12 months of treatment and classification according to the European League Against Rheumatism (EULAR) response criteria at the same time points. Association with DAS28 changes was assessed by linear regression using an additive genetic model. Contingency tables of genotype and allele frequencies between EULAR responder and nonresponder patients were compared. In addition, we combined our data with those of previously reported studies in a meta-analysis including 2,998 RA patients. Results: None of the four genetic variants showed an association with response to TNFi in any of the four outcomes analyzed in our Spanish patients. In addition, only rs1532269 yielded a suggestive association (P = 0.0033) with the response to TNFi when available data from previous studies were combined in the meta-analysis. Conclusion: Our data suggest that the rs12081765, rs1532269, rs17301249 and rs7305646 genetic variants do not have a role as genetic predictors of TNFi treatment outcomes