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Shoukri M.M.,National Biotechnology Center | El-Dali A.,KFSHRC
Contemporary Clinical Trials | Year: 2013

A crucial step in designing a new study is to estimate the required sample size. For a design involving cluster sampling, the appropriate sample size depends on the so-called design effect, which is a function of the average cluster size and the intracluster correlation coefficient (ICC). It is well-known that under the framework of hierarchical and generalized linear models, a reduction in residual error may be achieved by including risk factors as covariates. In this paper we show that the covariate design, indicating whether the covariates are measured at the cluster level or at the within-cluster subject level affects the estimation of the ICC, and hence the design effect. Therefore, the distinction between these two types of covariates should be made at the design stage. In this paper we use the nested-bootstrap method to assess the accuracy of the estimated ICC for continuous and binary response variables under different covariate structures. The codes of two SAS macros are made available by the authors for interested readers to facilitate the construction of confidence intervals for the ICC. Moreover, using Monte Carlo simulations we evaluate the relative efficiency of the estimators and evaluate the accuracy of the coverage probabilities of a 95% confidence interval on the population ICC. The methodology is illustrated using a published data set of blood pressure measurements taken on family members. © 2013 The Authors.


Gonzalez-Martin A.,Scripps Research Institute | Adams B.D.,Yale University | Lai M.,Scripps Research Institute | Shepherd J.,Scripps Research Institute | And 5 more authors.
Nature Immunology | Year: 2016

Autoreactive B cells have critical roles in a large diversity of autoimmune diseases, but the molecular pathways that control these cells remain poorly understood. We performed an in vivo functional screen of a lymphocyte-expressed microRNA library and identified miR-148a as a potent regulator of B cell tolerance. Elevated miR-148a expression impaired B cell tolerance by promoting the survival of immature B cells after engagement of the B cell antigen receptor by suppressing the expression of the autoimmune suppressor Gadd45α, the tumor suppressor PTEN and the pro-apoptotic protein Bim. Furthermore, increased expression of miR-148a, which occurs frequently in patients with lupus and lupus-prone mice, facilitated the development of lethal autoimmune disease in a mouse model of lupus. Our studies demonstrate a function for miR-148a as a regulator of B cell tolerance and autoimmunity. © 2016 Nature America, Inc.


Valverde J.,National Biotechnology Center | Orozco A.,Autonomous University of Madrid
2016 IEEE 36th Central American and Panama Convention, CONCAPAN 2016 | Year: 2017

The advance of Molecular Nanobiotechnology grows at a fast pace and a quantity of data and biological information emerges vertiginously at a smaller scale each time. This multilateral effect, necessarily appeals to the rational design of new bioinformatics and bio computing systems, along with the structured and non-structured systematization (NoSQL) at the organization, management, processing and storage of atomic-molecular originated data, applied in new forms of scientific consumption, research and technology such as Big Data and Cloud communities through mobile device consulting (Apps) and Computational Biology System. Also, on the molecular nanobiotechnology applied within the medical molecular context, it is necessary to provide a clear work oriented link inside the functionality of the platform's genomic devices and integrated infrastructures as a service (SaaS, Paas, IaaS). Therefore, Bioinformatics can help and find techniques, methods and combined computing algorithms, with the object to resolve several problems and emerging actions in a hybrid integration of scientific fields. Therefore, the technological revolution and evolution on the Omics Science has had considerable advances at a parallel front through Ultra-Sequencing techniques (NGS) and high speed provisions of complete genome readings in short time and with low cost, deepening the metabolic inter-relations and unstable biological circuits in several primary attention diseases in an easier way, including the example development on this research support and through DNA nanotechnology (molecular estructure of insuline), with the object to provide immediate traslational bioinformatics services to diagnose and provide prognosis of solutions for several primary attention requirements on molecular medicine. © 2016 IEEE.


Almarcegui R.J.,University of Chile | Navarro C.A.,University of Chile | Paradela A.,National Biotechnology Center | Albar J.P.,National Biotechnology Center | And 2 more authors.
Research in Microbiology | Year: 2014

The response of Acidithiobacillus ferrooxidans ATCC 23270 to copper was analyzed in sulfur-grown cells by using quantitative proteomics. Fortyseven proteins showed altered levels in cells grown in the presence of 50mM copper sulfate. Of these proteins, 24 were up-regulated and 23 down-regulated. As seen before in ferrous iron-grown cells, there was a notorious up-regulation of RND-type Cus systems and different RND-type efflux pumps, indicating that these proteins are very important in copper resistance. Copper also triggered the down-regulation of the major outer membrane porin of A. ferrooxidans in sulfur-grown bacteria, suggesting they respond to the metal by decreasing the influx of cations into the cell. On the contrary, copper in sulfur-grown cells caused an overexpression of putative TadA and TadB proteins known to be essential for biofilm formation in bacteria. Surprisingly, sulfur-grown microorganisms showed increased levels of proteins related with energy generation (rus and petII operons) in the presence of copper. Although rus operon is overexpressed mainly in cells grown in ferrous iron, the up-regulation of rusticyanin in sulfur indicates a possible role for this protein in copper resistance as well. Finally, copper response in A. ferrooxidans appears to be influenced by the substrate being oxidized by the microorganism. © 2014 Institut Pasteur.


Almarcegui R.J.,University of Chile | Navarro C.A.,University of Chile | Paradela A.,National Biotechnology Center | Albar J.P.,National Biotechnology Center | And 2 more authors.
Journal of Proteome Research | Year: 2014

Acidithiobacillus ferrooxidans is an extremophilic bacterium used in biomining processes to recover metals. The presence in A. ferrooxidans ATCC 23270 of canonical copper resistance determinants does not entirely explain the extremely high copper concentrations this microorganism is able to stand, suggesting the existence of other efficient copper resistance mechanisms. New possible copper resistance determinants were searched by using 2D-PAGE, real time PCR (qRT-PCR) and quantitative proteomics with isotope-coded protein labeling (ICPL). A total of 594 proteins were identified of which 120 had altered levels in cells grown in the presence of copper. Of this group of proteins, 76 were up-regulated and 44 down-regulated. The up-regulation of RND-type Cus systems and different RND-type efflux pumps was observed in response to copper, suggesting that these proteins may be involved in copper resistance. An overexpression of most of the genes involved in histidine synthesis and several of those annotated as encoding for cysteine production was observed in the presence of copper, suggesting a possible direct role for these metal-binding amino acids in detoxification. Furthermore, the up-regulation of putative periplasmic disulfide isomerases was also seen in the presence of copper, suggesting that they restore copper-damaged disulfide bonds to allow cell survival. Finally, the down-regulation of the major outer membrane porin and some ionic transporters was seen in A. ferrooxidans grown in the presence of copper, indicating a general decrease in the influx of the metal and other cations into the cell. Thus, A. ferrooxidans most likely uses additional copper resistance strategies in which cell envelope proteins are key components. This knowledge will not only help to understand the mechanism of copper resistance in this extreme acidophile but may help also to select the best fit members of the biomining community to attain more efficient industrial metal leaching processes. © 2013 American Chemical Society.


PubMed | National Biotechnology Center, University of Maryland University College, Indian Institute of Technology Madras and Tata Institute of Fundamental Research
Type: Journal Article | Journal: Journal of the American Chemical Society | Year: 2016

Thermosensing is critical for the expression of virulence genes in pathogenic bacteria that infect warm-blooded hosts. Proteins of the Hha-family, conserved among enterobacteriaceae, have been implicated in dynamically regulating the expression of a large number of genes upon temperature shifts. However, there is little mechanistic evidence at the molecular level as to how changes in temperature are transduced into structural changes and hence the functional outcome. In this study, we delineate the conformational behavior of Cnu, a putative molecular thermosensor, employing diverse spectroscopic, calorimetric and hydrodynamic measurements. We find that Cnu displays probe-dependent unfolding in equilibrium, graded increase in structural fluctuations and temperature-dependent swelling of the dimensions of its native ensemble within the physiological range of temperatures, features that are indicative of a highly malleable native ensemble. Site-specific fluorescence and NMR experiments in combination with multiple computational approaches-statistical mechanical model, coarse-grained and all-atom MD simulations-reveal that the fourth helix of Cnu acts as a unique thermosensing module displaying varying degrees of order and orientation in response to temperature modulations while undergoing a continuous unfolding transition. Our combined experimental-computational study unravels the folding-functional landscape of a natural thermosensor protein, the molecular origins of its unfolding complexity, highlights the role of functional constraints in determining folding-mechanistic behaviors, and the design principles orchestrating the signal transduction roles of the Hha protein family.


Schonfelder J.,National Biotechnology Center | Schonfelder J.,IMDEA Madrid Institute for Advanced Studies | Schonfelder J.,CIC Nanogune | Perez-Jimenez R.,CIC Nanogune | And 4 more authors.
Nature Communications | Year: 2016

A major drive in protein folding has been to develop experimental technologies to resolve the myriads of microscopic pathways and complex mechanisms that purportedly underlie simple two-state folding behaviour. This is key for cross-validating predictions from theory and modern computer simulations. Detecting such complexity experimentally has remained elusive even using methods with improved time, structural or single-molecule resolution. Here, we investigate the mechanical unfolding of cold shock protein B (Csp), a showcase two-state folder, using single-molecule force-spectroscopy. Under controlled-moderate pulling forces, the unfolding of Csp emerges as highly heterogeneous with trajectories ranging from single sweeps to different combinations of multiple long-lived mechanical intermediates that also vary in order of appearance. Steered molecular dynamics simulations closely reproduce the experimental observations, thus matching unfolding patterns with structural events. Our results provide a direct glimpse at the nanoscale complexity underlying two-state folding, and postulate these combined methods as unique tools for dissecting the mechanical unfolding mechanisms of such proteins.


Munoz V.,National Biotechnology Center | Munoz V.,IMDEA Madrid Institute for Advanced Studies | Munoz V.,University of California at Merced | Campos L.A.,National Biotechnology Center | And 2 more authors.
Current Opinion in Structural Biology | Year: 2016

Theory and simulations predict that the structural concert of protein folding reactions is relatively low. Experimentally, folding cooperativity has been difficult to study, but in recent years we have witnessed major advances. New analytical procedures in terms of conformational ensembles rather than discrete states, experimental techniques with improved time, structural, or single-molecule resolution, and combined thermodynamic and kinetic analysis of fast folding have contributed to demonstrate a general scenario of limited cooperativity in folding. Gradual structural disorder is already apparent on the unfolded and native states of slow, two-state folding proteins, and it greatly increases in magnitude for fast folding domains. These results demonstrate a direct link between how fast a single-domain protein folds and unfolds, and how cooperative (or structurally diverse) is its equilibrium unfolding process. Reducing cooperativity also destabilizes the native structure because it affects unfolding more than folding. We can thus define a continuous cooperativity scale that goes from the 'pliable' two-state character of slow folders to the gradual unfolding of one-state downhill, and eventually to intrinsically disordered proteins. The connection between gradual unfolding and intrinsic disorder is appealing because it suggests a conformational rheostat mechanism to explain the allosteric effects of folding coupled to binding. © 2015 Elsevier Ltd.


PubMed | National Biotechnology Center, Yale University and Scripps Research Institute
Type: Journal Article | Journal: Nature immunology | Year: 2016

Autoreactive B cells have critical roles in a large diversity of autoimmune diseases, but the molecular pathways that control these cells remain poorly understood. We performed an in vivo functional screen of a lymphocyte-expressed microRNA library and identified miR-148a as a potent regulator of B cell tolerance. Elevated miR-148a expression impaired B cell tolerance by promoting the survival of immature B cells after engagement of the B cell antigen receptor by suppressing the expression of the autoimmune suppressor Gadd45, the tumor suppressor PTEN and the pro-apoptotic protein Bim. Furthermore, increased expression of miR-148a, which occurs frequently in patients with lupus and lupus-prone mice, facilitated the development of lethal autoimmune disease in a mouse model of lupus. Our studies demonstrate a function for miR-148a as a regulator of B cell tolerance and autoimmunity.


PubMed | National Biotechnology Center and CIC Nanogune
Type: | Journal: Nature communications | Year: 2016

A major drive in protein folding has been to develop experimental technologies to resolve the myriads of microscopic pathways and complex mechanisms that purportedly underlie simple two-state folding behaviour. This is key for cross-validating predictions from theory and modern computer simulations. Detecting such complexity experimentally has remained elusive even using methods with improved time, structural or single-molecule resolution. Here, we investigate the mechanical unfolding of cold shock protein B (Csp), a showcase two-state folder, using single-molecule force-spectroscopy. Under controlled-moderate pulling forces, the unfolding of Csp emerges as highly heterogeneous with trajectories ranging from single sweeps to different combinations of multiple long-lived mechanical intermediates that also vary in order of appearance. Steered molecular dynamics simulations closely reproduce the experimental observations, thus matching unfolding patterns with structural events. Our results provide a direct glimpse at the nanoscale complexity underlying two-state folding, and postulate these combined methods as unique tools for dissecting the mechanical unfolding mechanisms of such proteins.

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