Whole-cell detection of live lactobacillus acidophilus on aptamer-decorated porous silicon biosensors

This work describes the design of optical aptamer-based porous silicon (PSi) biosensors for the direct capture of Lactobacillus acidophilus. Aptamers are oligonucleotides (single-stranded DNA or RNA) that can bind their targets with high affinity and specificity, making them excellent recognition elements for biosensing applications. Herein, aptamer Hemag1P, which specifically targets the important probiotic L. acidophilus, was utilized for direct bacteria capture onto oxidized PSi Fabry-Perot thin films. Monitoring changes in the reflectivity spectrum (using reflective interferometric Fourier transform spectroscopy) allows for bacteria detection in a label-free, simple and rapid manner. The performance of the biosensor was optimized by tuning the PSi nanostructure, its optical properties, as well as the immobilization density of the aptamer. We demonstrate the high selectivity and specificity of this simple "direct-capture" biosensing scheme and show its ability to distinguish between live and dead bacteria. The resulting biosensor presents a robust and rapid method for the specific detection of live L. acidophilus at concentrations relevant for probiotic products and as low as 10(6) cells per mL. Rapid monitoring of probiotic bacteria is crucial for quality, purity and safety control as the use of probiotics in functional foods and pharmaceuticals is becoming increasingly popular.DFG/CHE 279/32-

Phenotype-oriented NGS panels for mucopolysaccharidoses : validation and potential use in the diagnostic flowchart

Mucopolysaccharidosis (MPS) are a group of rare genetic disorders caused by deficiency in the activity of specific lysosomal enzymes required for the degradation of glycosaminoglycans (GAGs). A defect in the activity of these enzymes will result in the abnormal accumulation of GAGs inside the lysosomes of most cells, inducing progressive cellular damage and multiple organ failure. DNA samples from 70 patients with biochemical diagnosis of different MPSs genotypes confirmed by Sanger sequencing were used to evaluate a Next Generation Sequencing (NGS) protocol. Eleven genes related to MPSs were divided into three different panels according to the clinical phenotype. This strategy led to the identification of several pathogenic mutations distributed across all exons of MPSs-related genes. We were able to identify 96% of all gene variants previously identified by Sanger sequencing, showing high sensitivity in detecting different types of mutations. Furthermore, new variants were not identified, representing 100% specificity of the NGS protocol. The use of this NGS approach for genotype identification in MPSs is an attractive option for diagnosis of patients. In addition, the MPS diagnosis workflow could be divided in a two-tier approach: NGS as a first-tier followed by biochemical confirmation as a second-tier

Evaluar para enseñar ciencias naturales

Una de las preocupaciones centrales de las gestiones en educación en los últimos años han sido los resultados de las pruebas de evaluación estandarizadas que se aplican en la región desde hace dos décadas. En una operación de reducción conceptual, solemos asociar los términos calidad educativa a lo que estas pruebas revelan sobre el rendimiento de los estudiantes. En este análisis, quienes señalan los magros resultados de los países parecen poner bajo sospecha la inversión realizada en el sector educativo, los programas que proponen mejoras para el trabajo en el aula y, al fin de cuentas, el trabajo de los docentes en las escuelas. Lejos de sumar argumentos a un diagnóstico que reitera la gravedad y avanza poco en proponer otras estrategias de trabajo en las aulas, este material propone un conjunto de preguntas en torno a las prácticas de evaluación. Es resultado del trabajo realizado por el programa Ciencia y Tecnología con Creatividad (CTC) durante tres años en escuelas primarias de las provincias de Buenos Aires y de Tucumán para promover mejoras en la enseñanza de las Ciencias Naturales.Fil: Attias, Ayelén. No especifíca;Fil: González, Delia. No especifíca;Fil: Dussel, Inés. No especifíca;Fil: Furman, Melina Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de San Andrés. Escuela de Educación; ArgentinaFil: Montes, Nancy. No especifíca;Fil: Segal, Analia Edit. No especifíca

Dynamics of physiological and biochemical changes\ud during somatic embryogenesis of Acca sellowiana

Feijoa (Acca sellowiana [O. Berg] Burret [Myrtaceae]) is a native fruit species of southern Brazil and northern Uruguay. This species is amenable to somatic embryogenesis and therefore suitable as a model system for comparative studies of zygotic and somatic embryo development. In seed plants, embryogenesis involves three main steps, which are regulated by many factors, such as hormones, proteins, polyamines, and transcription factors. In the present work, the dynamics of protein, sugar, starch, amino acid, and polyamine accumulation were assayed during somatic embryogenesis of A. sellowiana. Protein, starch, amino acid, and polyamine levels accumulated unevenly during the induction phase of somatic embryogenesis, while the sugar content remained stable. Throughout the different developmental stages of somatic embryogenesis, synthesis and accumulation of proteins and amino acids showed patterns similar to those reported previously during the development of zygotic embryos of this same species. Differential patterns of polyamine accumulation were observed. This is important because these compounds affect the synthesis of other endogenous growth regulators, such as auxinindole-3-acetic acid that is mainly involved in the establishment of embryo polarity. Taken together, the present work brings new insights to the physiological and biochemical dynamics that occur during somatic embryogenesis of A. sellowiana

Immunochip analysis identifies multiple susceptibility loci for systemic sclerosis

In this study, 1,833 systemic sclerosis (SSc) cases and 3,466 controls were genotyped with the Immunochip array. Classical alleles, amino acid residues, and SNPs across the human leukocyte antigen (HLA) region were imputed and tested. These analyses resulted in a model composed of six polymorphic amino acid positions and seven SNPs that explained the observed significant associations in the region. In addition, a replication step comprising 4,017 SSc cases and 5,935 controls was carried out for several selected non-HLA variants, reaching a total of 5,850 cases and 9,401 controls of European ancestry. Following this strategy, we identified and validated three SSc risk loci, including DNASE1L3 at 3p14, the SCHIP1-IL12A locus at 3q25, and ATG5 at 6q21, as well as a suggested association of the TREH-DDX6 locus at 11q23. The associations of several previously reported SSc risk loci were validated and further refined, and the observed peak of association in PXK was related to DNASE1L3. Our study has increased the number of known genetic associations with SSc, provided further insight into the pleiotropic effects of shared autoimmune risk factors, and highlighted the power of dense mapping for detecting previously overlooked susceptibility loci

Underlying mechanisms of effective adoptive T cell therapy against a B cell lymphoma

© 2014 Dr. Gabriela Segal WassermanThe immune system protects the body from potential dangers and mediates the destruction of infected or tumoural cells. The elimination of these target cells is predominantly executed by cytotoxic CD8 + T cells (CTLs) that destroy any cell presenting their cognate antigen, namely infected or tumoural cells. CTLs have characteristics that make them attractive for adoptive T cell therapy (ACT). This type of therapy infuses tumour-specific CTLs into cancer patients and mediates tumour regression. So far, the results obtained with ACT in clinical trials have been promising, but this therapy is still not the standard of care and many factors that affect the outcome remain unknown. To study the factors that affect ACT, our laboratory has previously established a tumour model based on a B-cell lymphoma, the Eμ-myc tumour. This tumour has been transfected with GFP or with GFP-OVA. Utilizing these tumours together with transgenic T cells against OVA (OT-I) for ACT, we established that one of the key factors for the success of ACT is the tumour burden. We have proven that a high amount of tumour cells in the host triggers a quick deletion and inactivation of the transferred OT-I CTLs, a process that has shown to be antigen specific and directly mediated by the tumour cells. In this thesis, we studied several factors that determine the outcome of ACT to deepen the understanding of the characteristics and mechanisms of the T cell inactivation occurring after ACT. In chapter three, it was established that using CTLs with a low affinity against the target antigen OVA (OT-3 cells) as ACT, resulted in tumour elimination when the tumour load was low. In addition, we show the importance of the level of antigen expression within the tumour, as high levels are required for successful ACT outcomes. Finally, we study the role of the avidity of the tumour: T cell interaction for the inactivation process, and demonstrate that this is an irrelevant factor for the inactivation to occur. One of the complications associated with ACT is autoimmunity triggered by the destruction of healthy tissue. To study the relationship between ACT and autoimmunity in chapter four, we utilize ACT in a system where the tumour antigen is shared with healthy tissue. We determine that it is possible to eliminate a tumour expressing a shared antigen when targeting utilizing ACT, but successful ACT includes healthy tissue destruction in this setting. One of the factors limiting the outcomes of ACT in our model is the deletion of the T cells once in the tumour-bearing host. To further investigate this we image ACT in situ and visualize the deletion of the T cells in organs like spleen and LN in chapter five. We establish a system utilizing two-photon microscopy (TPM) and fluorescently tagged cells to visualize the motility of the transferred T cells in the LN and determine a correlation between lower motility and dysfunction of the T cells. Tumours hijack tolerance mechanisms to avoid anti-tumour attack; a strategy that can compromise the results obtained with ACT and may be the limiting factor for the success in our model. To examine the mechanisms that drive CTL inactivation, we characterize the transcriptome of inactivated T cells in chapter six, and analyse their gene expression pattern. We establish that the inactivated T cells display a gene-expression profile reminiscent of a combination of tolerance-associated pathways, suggesting a novel mechanism of tolerance regulating inactivation in these T cells. In this thesis, we provide a model to study ACT in detail and to gain insight into the factors affecting the outcome of ACT. The work undertaken gives us a greater understanding of the inactivation process occurring in T cells after ACT