Optimization and evaluation of surface-enhanced laser-desorption/ionization time-of-flight mass spectrometry for protein profiling of cerebrospinal fluid

Cerebrospinal fluid (CSF) potentially carries an archive of peptides and small proteins relevant to pathological processes in the central nervous system (CNS) and surrounding brain tissue. Proteomics is especially well suited for the discovery of biomarkers of diagnostic potential in CSF for early diagnosis and discrimination of several neurodegenerative diseases. ProteinChip surface-enhanced laser-desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) is one such approach which offers a unique platform for high throughput profiling of peptides and small proteins in CSF. In this study, we evaluated methodologies for the retention of CSF proteins < 20 kDa in size, and identify a strategy for screening small proteins and peptides in CSF. ProteinChip array types, along with sample and binding buffer conditions, and matrices were investigated. By coupling the processing of arrays to a liquid handler reproducible and reliable profiles, with mean peak coefficients of variation < 20%, were achieved for intra- and inter-assays under selected conditions. Based on peak m/z we found a high degree of overlap between the tested array surfaces. The combination of CM10 and IMAC30 arrays was sufficient to represent between 80–90% of all assigned peaks when using either sinapinic acid or α-Cyano-4-hydroxycinnamic acid as the energy absorbing matrices. Moreover, arrays processed with SPA consistently showed better peak resolution and higher peak number across all surfaces within the measured mass range. We intend to use CM10 and IMAC30 arrays prepared in sinapinic acid as a fast and cost-effective approach to drive decisions on sample selection prior to more in-depth discovery of diagnostic biomarkers in CSF using alternative but complementary proteomic strategies

Calidad de vida del paciente con insuficiencia renal crónica en hemodiálisis, clínica Nefrología LTDA, Santa Marta septiembre-noviembre 2006.

La presente investigación tiene por objetivo, describir la calidad de vida del paciente con insuficiencia renal crónica en tratamiento de hemodiálisis, que asiste a la Clínica Nefrología Ltda. Se trata de un estudio descriptivo, cuantitativo de corte transversal; la muestra está conformada por 84 pacientes, quienes cumplieron con los criterios de inclusión, entre los meses de septiembre - noviembre 2006. En esta investigación se tuvieron en cuenta los aspectos éticos, se obtuvo el consentimiento informado de forma verbal, lo cual permitió la libre decisión de participar o no en este estudio. El análisis se basó en responder la pregunta, ¿cómo es la calidad de vida de los pacientes con insuficiencia renal crónica en tratamiento con hemodiálisis? Se aplicó el instrumento propuesto por Betty Ferrell y colaboradores: “escala sobre calidad de vida paciente con enfermedad crónica”. El análisis de los datos se llevó a cabo a través del paquete estadístico SPSS versión 13. La edad media de los participantes fue de 36 a 59 años, el 65% fueron del sexo masculino, el 67 % de estrato socioeconómico bajo y el 57% con más de 37 meses de tratamiento. Se encontró problema en el bienestar psicológico en aspectos como contener/lidiar con su vida, cambios en su apariencia y autoconcepto, temor al diagnóstico inicial, ansiedad/desesperación y al retroceso de la enfermedad, y en el bienestar social en la aflicción que la familia siente por la enfermedad, sentir insuficiente apoyo y carga económica. Cabe resaltar que en el bienestar físico no se halló problema con respecto a la fatiga/agotamiento, dolor, y nauseas que padece el paciente al enfrentarse a la enfermedad y en el bienestar espiritual la creencia en Dios fortalece al paciente y lo ayuda a renovar fuerzas para enfrentar la situación. Los hallazgos permitieron evidenciar como las acciones encaminadas al mantenimiento de la salud, por parte de los pacientes tienen impacto nocivo en su calidad de vida, representada en el bienestar físico, psicológico, social y espiritual

Flow-Excited Turbine Rotor Instability

The problem of steam whirl is one of the technological limits that now prohibit the development of power-generating turbomachinery substantially above GW. Due to steam flow, self-excited vibrations develop at high loads in the form of stable limit cycles that, at even higher loads, deteriorate to chaotic vibration of high amplitude

Stability Analysis for Autonomous Dynamical Switched Systems through Nonconventional Lyapunov Functions

The stability of autonomous dynamical switched systems is analyzed by means of multiple Lyapunov functions. The stability theorems given in this paper have finite number of conditions to check. It is shown that linear functions can be used as Lyapunov functions. An example of an exponentially asymptotically stable switched system formed by four unstable systems is also given

The challenges of clinical trials in fragile X syndrome

RATIONALE: Advances in understanding the underlying mechanisms of conditions such as fragile X syndrome (FXS) and autism spectrum disorders have revealed heterogeneous populations. Recent trials of novel FXS therapies have highlighted several challenges including subpopulations with possibly differential therapeutic responses, the lack of specific outcome measures capturing the full range of improvements of patients with FXS, and a lack of biomarkers that can track whether a specific mechanism is responsive to a new drug and whether the response correlates with clinical improvement. OBJECTIVES: We review the phenotypic heterogeneity of FXS and the implications for clinical research in FXS and other neurodevelopmental disorders. RESULTS: Residual levels of fragile X mental retardation protein (FMRP) expression explain in part the heterogeneity in the FXS phenotype; studies indicate a correlation with both cognitive and behavioral deficits. However, this does not fully explain the extent of phenotypic variance observed or the variability of drug response. Post hoc analyses of studies involving the selective mGluR5 antagonist mavoglurant and the GABAB agonist arbaclofen have uncovered significant therapeutic responses following patient stratification according to FMR1 promoter methylation patterns or baseline severity of social withdrawal, respectively. Future studies designed to quantify disease modification will need to develop new strategies to track changes effectively over time and in multiple symptom domains. CONCLUSION: Appropriate selection of patients and outcome measures is central to optimizing future clinical investigations of these complex disorders

Reciprocal changes in DNA methylation and hydroxymethylation and a broad repressive epigenetic switch characterize FMR1 transcriptional silencing in fragile X syndrome.

BACKGROUND: Fragile X syndrome (FXS) is the most common form of inherited intellectual disability, resulting from the loss of function of the fragile X mental retardation 1 (FMR1) gene. The molecular pathways associated with FMR1 epigenetic silencing are still elusive, and their characterization may enhance the discovery of novel therapeutic targets as well as the development of novel clinical biomarkers for disease status. RESULTS: We have deployed customized epigenomic profiling assays to comprehensively map the FMR1 locus chromatin landscape in peripheral mononuclear blood cells (PBMCs) from eight FXS patients and in fibroblast cell lines derived from three FXS patient. Deoxyribonucleic acid (DNA) methylation (5-methylcytosine (5mC)) and hydroxymethylation (5-hydroxymethylcytosine (5hmC)) profiling using methylated DNA immunoprecipitation (MeDIP) combined with a custom FMR1 microarray identifies novel regions of DNA (hydroxy)methylation changes within the FMR1 gene body as well as in proximal flanking regions. At the region surrounding the FMR1 transcriptional start sites, increased levels of 5mC were associated to reciprocal changes in 5hmC, representing a novel molecular feature of FXS disease. Locus-specific validation of FMR1 5mC and 5hmC changes highlighted inter-individual differences that may account for the expected DNA methylation mosaicism observed at the FMR1 locus in FXS patients. Chromatin immunoprecipitation (ChIP) profiling of FMR1 histone modifications, together with 5mC/5hmC and gene expression analyses, support a functional relationship between 5hmC levels and FMR1 transcriptional activation and reveal cell-type specific differences in FMR1 epigenetic regulation. Furthermore, whilst 5mC FMR1 levels positively correlated with FXS disease severity (clinical scores of aberrant behavior), our data reveal for the first time an inverse correlation between 5hmC FMR1 levels and FXS disease severity. CONCLUSIONS: We identify novel, cell-type specific, regions of FMR1 epigenetic changes in FXS patient cells, providing new insights into the molecular mechanisms of FXS. We propose that the combined measurement of 5mC and 5hmC at selected regions of the FMR1 locus may significantly enhance FXS clinical diagnostics and patient stratification

CGG Repeat-Induced FMR1 Silencing Depends on the Expansion Size in Human iPSCs and Neurons Carrying Unmethylated Full Mutations

In fragile X syndrome (FXS), CGG repeat expansion greater than 200 triplets is believed to trigger FMR1 gene silencing and disease etiology. However, FXS siblings have been identified with more than 200 CGGs, termed unmethylated full mutation (UFM) carriers, without gene silencing and disease symptoms. Here, we show that hypomethylation of the FMR1 promoter is maintained in induced pluripotent stem cells (iPSCs) derived from two UFM individuals. However, a subset of iPSC clones with large CGG expansions carries silenced FMR1. Furthermore, we demonstrate de novo silencing upon expansion of the CGG repeat size. FMR1 does not undergo silencing during neuronal differentiation of UFM iPSCs, and expression of large unmethylated CGG repeats has phenotypic consequences resulting in&nbsp;neurodegenerative features. Our data suggest that UFM individuals do not lack the cell-intrinsic ability to silence FMR1 and that inter-individual variability in the CGG repeat size required for silencing exists in the FXS population

Protein synthesis levels are increased in a subset of individuals with fragile X syndrome.

Fragile X syndrome (FXS) is a monogenic form of intellectual disability and autism spectrum disorder caused by the absence of the fragile X mental retardation protein (FMRP). In biological models for the disease, this leads to upregulated mRNA translation and as a consequence, deficits in synaptic architecture and plasticity. Preclinical studies revealed that pharmacological interventions restore those deficits, which are thought to mediate the FXS cognitive and behavioral symptoms. Here, we characterized the de novo rate of protein synthesis in patients with FXS and their relationship with clinical severity. We measured the rate of protein synthesis in fibroblasts derived from 32 individuals with FXS and from 17 controls as well as in fibroblasts and primary neurons of 27 Fmr1 KO mice and 20 controls. Here, we show that levels of protein synthesis are increased in fibroblasts of individuals with FXS and Fmr1 KO mice. However, this cellular phenotype displays a broad distribution and a proportion of fragile X individuals and Fmr1 KO mice do not show increased levels of protein synthesis, having measures in the normal range. Because the same Fmr1 KO animal measures in fibroblasts predict those in neurons we suggest the validity of this peripheral biomarker. Our study offers a potential explanation for the comprehensive drug development program undertaken thus far yielding negative results and suggests that a significant proportion, but not all individuals with FXS, may benefit from the reduction of excessive levels of protein synthesis