Development of comprehensive liquid chromatography (LC x LC) approaches for the analysis of complex copolymer structures

Knowledge of the exact composition and purity of advanced copolymers is of utmost importance given their influence on the final physical properties. The development of analytical techniques to examine these impurities is therefore crucial with respect of large industrial productions. A comprehensive two-dimensional HPLC separation technique with a slow size exclusion (SEC) separation in the first dimension and a fast reversed phase liquid chromatography (RPLC) separation in the second dimension was therefore developed. For the SEC dimension, two 2 mm internal diameter columns were packed with a PLgel Mixed-D packing and in the RPLC dimension a Kinetex (C18) column packed with solid core/porous shell particles were applied. The optimized setup allowed for successful qualitative and semi-quantitative analysis of various types of in-house prepared copolymers, including block, graft and palm-tree types of structures

The orphan adapter protein SLY1 as a novel anti-apoptotic protein required for thymocyte development

<p>Abstract</p> <p>Background</p> <p>SH3 containing Lymphocyte Protein (SLY1) is a putative adapter protein exclusively expressed in lymphocytes which is involved in antigen receptor induced activation. We previously have generated SLY1^Δ/Δmice harbouring a partial deletion in the N-terminal region of SLY1 which revealed profound immunological defects in T and B cell functions.</p> <p>Results</p> <p>In this study, T cell development in SLY1^-/-and SLY1^Δ/Δmice was analysed <it>ex vivo </it>and upon cultivation with the bone marrow stromal cell line OP9. SLY1-deficient thymocytes were compromised in inducing nutrient receptor expression and ribosomal protein S6 phosphorylation, indicating a defect in mTOR complex activation. Furthermore, SLY1 was identified as a novel anti-apoptotic protein required for developmental progression of T cell precursors to the CD4⁺CD8⁺double-positive stage by protecting from premature programmed cell death initiation in developing CD4^-CD8^-double-negative thymocytes. In addition, SLY1 phosphorylation was differentially regulated upon Notch ligand-mediated stimulation and expression of the preTCR.</p> <p>Conclusion</p> <p>Thus, our results suggest a non-redundant role for SLY1 in integrating signals from both receptors in early T cell progenitors in the thymus.</p

Creation of functional viruses from non-functional cDNA clones obtained from an RNA virus population by the use of ancestral reconstruction

RNA viruses have the highest known mutation rates. Consequently it is likely that a high proportion of individual RNA virus genomes, isolated from an infected host, will contain lethal mutations and be non-functional. This is problematic if the aim is to clone and investigate high-fitness, functional cDNAs and may also pose problems for sequence-based analysis of viral evolution. To address these challenges we have performed a study of the evolution of classical swine fever virus (CSFV) using deep sequencing and analysis of 84 full-length cDNA clones, each representing individual genomes from a moderately virulent isolate. In addition to here being used as a model for RNA viruses generally, CSFV has high socioeconomic importance and remains a threat to animal welfare and pig production. We find that the majority of the investigated genomes are non-functional and only 12% produced infectious RNA transcripts. Full length sequencing of cDNA clones and deep sequencing of the parental population identified substitutions important for the observed phenotypes. The investigated cDNA clones were furthermore used as the basis for inferring the sequence of functional viruses. Since each unique clone must necessarily be the descendant of a functional ancestor, we hypothesized that it should be possible to produce functional clones by reconstructing ancestral sequences. To test this we used phylogenetic methods to infer two ancestral sequences, which were then reconstructed as cDNA clones. Viruses rescued from the reconstructed cDNAs were tested in cell culture and pigs. Both reconstructed ancestral genomes proved functional, and displayed distinct phenotypes in vitro and in vivo. We suggest that reconstruction of ancestral viruses is a useful tool for experimental and computational investigations of virulence and viral evolution. Importantly, ancestral reconstruction can be done even on the basis of a set of sequences that all correspond to non-functional variants

Ocular findings in children between 0 and 6 years of age, residing in the city of São Caetano do Sul, SP

PURPOSE: To establish the prevalence of refractive errors, strabismus, amblyopia and anisometropia, among children in a population, using two methods of screening (at school and at the National Polio Vaccination Day). METHODS: We examined 2,640 children, between 0 and 6 years of age. The children, residents of the city São Caetano do Sul, were divided into two groups (A and B). Group A: 476 children, with an age range of a few months to 6 years, had their eyes examined by 10 ophthalmologists and 11 ophthalmology technicians, during the National Polio Vaccination Day (June 20,1998). Group B: 2,164 children attending municipal schools (EMEIs and EMIs), between 4 and 6 years of age, who had their eyes examined by two ophthalmologists during the year of 1998. RESULTS: In group A, the prevalence of strabismus was 3.36% and that of anisometropia was 1.26%. The most common type of refractive error was hypermetropia £ 2D and 14.11% required optical prescription. In group B, the prevalence of strabismus was 1.43%. In the group who failed in the screening, the prevalence of amblyopia was 1.39% and that of anisometropia was 2.8%. The most common type of refractive error found was hypermetropic astigmatism and 56.48% required optical prescription. CONCLUSION: The prevalence of the ocular findings in children in São Caetano do Sul, although being a city with a privileged socioeconomic profile, is similar to that reported in the literature. Ophthalmic examination on National Polio Vaccination Day showed to be an efficient screening tool.OBJETIVO: Determinar a prevalência de erros refrativos, estrabismo, ambliopia e anisometropia nas crianças pertencentes à população em estudo, utilizando dois métodos de rastreamento (triagem na escola e triagem durante o dia da vacinação contra a poliomielite). MÉTODOS: Foram examinadas 2.640 crianças, residentes da cidade de São Caetano do Sul, entre zero e seis anos de idade, divididas em duas amostras (A e B). A amostra A consistiu de 476 crianças que foram examinadas por dez oftalmologistas e onze técnicos oftálmicos, durante o Dia Nacional de Vacinação (20 de junho de 1998) contra a Poliomielite. A amostra B consistiu de 2.164 crianças que freqüentavam escolas municipais, entre quatro e seis anos de idade e foram examinadas por dois oftalmologistas durante o ano de 1998. RESULTADOS: Na amostra A, a prevalência de estrabismo foi de 3,36% e de anisometropia foi de 1,26%. O principal tipo de erro refrativo encontrado foi a hipermetropia < 2D. Necessitaram de prescrição óptica 14,11% das crianças. Em relação à amostra B, a prevalência de estrabismo foi de 1,43%. Nas crianças que falharam na triagem, a prevalência de ambliopia foi de 1,39%, 2,8% de anisometropia e necessitaram de prescrição óptica, 56,48%. O principal tipo de erro refrativo encontrado foi o astigmatismo hipermetrópico composto. CONCLUSÃO: Embora em município com perfil socioeconômico privilegiado, a prevalência de estrabismo, anisometropia, ambliopia e erro refrativo não foi inferior à relatada na literatura. Com relação à metodologia de rastreamento, o Dia Nacional de Vacinação contra a Poliomielite mostrou-se eficiente ferramenta metodológica já que houve grande participação da população alvo.UNIFESP Departamento de Oftalmologia Setor de Cirurgia RefrativaUNIFESP Departamento de Oftalmologia Setor de CórneaUNIFESP Departamento de Oftalmologia Setor de GlaucomaUNIFESP, Depto. de Oftalmologia Setor de Cirurgia RefrativaUNIFESP, Depto. de Oftalmologia Setor de CórneaUNIFESP, Depto. de Oftalmologia Setor de GlaucomaSciEL

Metagenomics for broad and improved parasite detection: a proof-of-concept study using swine faecal samples

Efficient and reliable identification of emerging pathogens is crucial for the design and implementation of timely and proportionate control strategies. This is difficult if the pathogen is so far unknown or only distantly related with known pathogens. Diagnostic metagenomics – an undirected, broad and sensitive method for the efficient identification of pathogens – was frequently used for virus and bacteria detection, but seldom applied to parasite identification. Here, metagenomics datasets prepared from swine faeces using an unbiased sample processing approach with RNA serving as starting material were re-analysed with respect to parasite detection. The taxonomic identification tool RIEMS, used for initial detection, provided basic hints on potential pathogens contained in the datasets. The suspected parasites/intestinal protists (Blastocystis, Entamoeba, Iodamoeba, Neobalantidium, Tetratrichomonas) were verified using subsequently applied reference mapping analyses on the base of rRNA sequences. Nearly full-length gene sequences could be extracted from the RNA-derived datasets. In the case of Blastocystis, subtyping was possible with subtype (ST)15 discovered for the first known time in swine faeces. Using RIEMS, some of the suspected candidates turned out to be false-positives caused by the poor status of sequences in publicly available databases. Altogether, 11 different species/STs of parasites/intestinal protists were detected in 34 out of 41 datasets extracted from metagenomics data. The approach operates without any primer bias that typically hampers the analysis of amplicon-based approaches, and allows the detection and taxonomic classification including subtyping of protist and metazoan endobionts (parasites, commensals or mutualists) based on an abundant biomarker, the 18S rRNA. The generic nature of the approach also allows evaluation of interdependencies that induce mutualistic or pathogenic effects that are often not clear for many intestinal protists and perhaps other parasites. Thus, metagenomics has the potential for generic pathogen identification beyond the characterisation of viruses and bacteria when starting from RNA instead of DNA

The effect of superior pedicle breast reductions on breast sensation

The aim of this study was to assess nipple, areola and breast skin sensation after breast reductions with two different superior pedicle techniques: a short, vertical scar technique compared to a long, inverted-T scar technique. Thirty-six women with a vertical technique (group I) and ten women with an inverted-T technique (group II) with a resection weight of ≤500g per breast completed their 1-year follow-up. The four modalities used to evaluate sensation were pressure with Semmes-Weinstein filaments, vibration with a vibrometer, and temperature and pain perception on a qualitative basis. The evaluation revealed that 1-year after breast reduction, the sensation was either reduced, unaltered, or improved in both groups. In the nipple, the mean sensation was markedly reduced throughout all qualities in both groups with the exception of pain, which was enhanced. In the areola, the mean sensation was also reduced in all qualities in both groups. In the quadrants of the skin, mean sensation was improved in terms of pressure and vibration in group I (8.3% normal pressure values preoperatively vs. 70% normal pressure values postoperatively) but reduced in the lower quadrant of the skin in group II with the inverted-T scar. This reduction of pressure was also significant (p = 0.04) in comparison with group I. Apart from this difference between the two groups, this study showed that in breast reductions with a superior pedicle technique, the long-scar technique did not lead to a greater reduction of sensation in the nipple and areola than the short-scar techniqu