262,205 research outputs found
Set up and optimization of a tandem mass spectrometry procedure for enzymatic assays of lysosomal storage disorders and feasibility evaluations for a newborn screening program
No full textLSDs are a cluster of about 50 different inheritable pathologies mainly due to the deficit of lysosomal enzymes, which lead to severe and progressive multiorgan dysfunction. Biochemically well defined, they are characterized by an ubiquitous accumulation of undegraded macromolecules and, although individually rare, they present an overall incidence around 1 in 1500-7000 newborns.
Main therapies for these pathologies are Enzyme Replacement and Hematopoietic Stem Cell Transplantation, both needing an early application to maximize their benefits; up to now, laboratory tests are carried out only following a particular clinical suspect and they allow a correct diagnosis when symptoms (sometimes irreversible) are already clearly developed.
Other medical approaches, as enzyme enhancement therapy, substrate deprivation/reduction therapy and gene therapy, are on trial.
Moreover, still very little is known on the individual determinants of therapeutic efficacy and, at the moment, the same therapeutic scheme is applied to all patients, although it is becoming clear that much better results could be obtained for each single patient if personalized therapeutic protocols could be defined.
Both of these questions drive to the search of new diagnostic approaches able to detect patients in the population at the earliest possible time, in order to allow a very precocious application of therapies, and able to rapidly test enzymes bio-availability in blood, to facilitate the development of personalized therapeutic protocols, to reduce unnecessary treatments and side effects and to lead to a more cost-effective healthcare.
Finally, recent studies on high number of samples, have reported an unexpected high incidence of these disorders, underlined how LSDs might have been since now underestimated.
For all these reasons in the last years a new easy, cheap, and reliable diagnostic test, using Tandem Mass Spectrometry, was developed allowing to measure enzymatic activities in Dried Blood Spots (DBS).
This work was addressed to the set up and optimization of this method for the detection of the enzymatic activities for six lysosomal storage disorders; the choice of the diseases to take into consideration in this study was made according to the availability of a therapy and of reagents for MS/MS analysis.
The first part of the work was conducted at the Centogene Laboratory of Vienna; after the Gaucher and Niemann Pick A/B assays set up, they were multiplexed in a single test together with Pompe, Fabry and Krabbe assays. Then, the reliability and sensitivity of the method were investigated through a cross comparison work between two laboratories (the Vienna Centogene Laboratory and a research group of the Wadsworth Center of NY). An evaluation of the most critical steps of the protocol that needed to be optimized was made.
Finally, a pilot newborn screening project was started in collaboration with the Hospital of Szeged. Ten thousand Hungarian anonymous samples were analyzed singly, then samples presenting low activities for one of the enzymes were re-tested in duplicate. Confirmed positive samples were sent to a third laboratory in Rostock for molecular analysis, so the detection of mutation for pathological samples and an evaluation of the false positive rate was possible.
These studies confirm, not only the reliability of using DBS as biological specimens and the feasibility of the analysis by tandem mass spectrometry, but also the easy application of the method for screening intent.
The second part of the work was carry out in Padova, in collaboration with the Tandem Mass Spectrometry Laboratory of the Department of Pediatrics.
Here the multiplex assay was reproduced and the test for Mucopolysaccharidosis type I was added.
Considering the preliminary results obtained in Vienna the protocol has been modified, by reducing the sample manipulation, through the introduction of an Ultra Performance Liquid Chromatography connected in line with the MS/MS spectrometer, and by optimizing the analytical parameters.
The aim of the work was to maximize MS/MS sensitivity, precision and accuracy, to allow all enzyme activities to show an unambiguous difference between DBS obtained from healthy controls and LSD affected patients.
The new defined protocol for enzyme activity detection described in this thesis will be potentially applied for several different purpose: it can be used both for the screening of wide populations, with the possibility to add new enzymatic tests for those disorders that will have a therapy available, or for the enzyme bioavailability determination in single patientsLe malattie da accumulo lisosomiale (LSD) rappresentano un gruppo di circa 50 disordini genetici, dovuti principalmente al deficit di specifiche idrolasi con conseguente accumulo di substrati non degradati nei lisosomi di organi e tessuti. Le LSD sono malattie molto complesse, gravemente debilitanti o letali, caratterizzate da un’estrema variabilità per quanto riguarda età di esordio, sintomatologia e rapidità di decorso clinico.
Pur essendo singolarmente rare, esse presentano un’incidenza complessiva stimata di 1:1500-7000 nati vivi.
Le principali terapie a disposizione, oltre alle cure palliative, sono la terapia enzimatica sostitutiva (ERT) e il trapianto di cellule staminali ematopoietiche; entrambe presentano massima efficacia se iniziate precocemente, prima cioè dell’insorgenza delle principali manifestazioni cliniche. Tuttavia, spesso una diagnosi precoce non è possibile poiché i test di laboratorio vengono eseguiti solo in seguito ad un evidente sospetto clinico. Altre strategie terapeutiche in fase di trial comprendono l’enzyme enhancement therapy, la terapia da inibizione/riduzione del substrato e la terapia genica.
Ad oggi inoltre, i soggetti in trattamento vengono tutti sottoposti allo stesso protocollo terapeutico; sarebbe invece opportuno riuscire a sviluppare protocolli terapeutici personalizzati andando a valutare rapidamente la biodisponibilità dell’enzima somministrato, durante il follow up dei pazienti.
Entrambe queste problematiche hanno portato, negli ultimi anni, alla ricerca di nuove tecniche diagnostiche, sia adatte per l’analisi di un elevato numero di campioni, che rispondenti alla necessità di permettere una veloce stima dell’attività degli enzimi di interesse in singoli soggetti.
Il dosaggio enzimatico su Dried Blood Spot (DBS) mediante spettrometria in tandem massa si è rivelato un metodo ideale per la diagnosi delle malattie lisosomiali, poiché esso si distingue per affidabilità, sensibilità, rapidità e accuratezza e consente l’analisi enzimatica multipla in un elevato numero di campioni contemporaneamente.
Infine, studi recenti di screening per alcune LSD condotti sia su ampie popolazioni sia su specifici gruppi a rischio (pazienti con infarto criptogenico o con insufficienza renale) hanno riportato un’incidenza complessiva sorprendentemente elevata, oltre a diversi casi, in particolare della malattia di Fabry, di mancato riconoscimento della patologia; ciò prova come questi disordini siano stati fino ad oggi sottovalutati, e sottolinea l’importanza di una corretta e tempestiva diagnosi.
Questo lavoro è dedicato alla messa a punto e all’ottimizzazione di questa nuova metodica per lo studio dell’attività enzimatica per sei malattie da accumulo lisosomiale (Pompe, Fabry, Niemann Pick A/B, Gaucher, Krabbe e Mucopolisaccaridosi di tipo I). La scelta delle patologie è stata determinata sia, per ragioni etiche, dalla disponibilità di una terapia possibile, sia dalla disponibilità dei reagenti per la MSMS da parte del CDC di Atlanta.
La prima parte del lavoro è stata svolta presso il laboratorio Centogene di Vienna, e ha previsto la messa a punto dei saggi per la malattia di Gaucher e Niemann Pick A/B, che sono stati successivamente unificati in un unico test insieme ai saggi per le malattie di Pompe, Fabry e Krabbe.
In seguito, uno studio incrociato tra due laboratori (il laboratorio Centogene di Vienna e un gruppo di ricerca del Wadsworth Center di NY), su più di 400 campioni ha permesso di valutare l’affidabilità e la sensibilità della tecnica utilizzata, oltre ad individuare i passaggi critici del protocollo che sarebbero stati in seguito ottimizzati.
Infine, nel corso di questo progetto è stato iniziato uno studio pilota di screening neonatale in collaborazione con l’Ospedale di Szeged (Ungheria). Diecimila campioni anonimi sono stati analizzati singolarmente; i campioni che presentavano una bassa attività per uno degli enzimi di interesse sono stati ri-analizzati in duplicato e nel caso di conferma, inviati ad un terzo laboratorio (Centogene di Rostock) per l’analisi molecolare. In questo modo è stata possibile sia un’identificazione delle mutazioni nei campioni patologici, sia una valutazione della percentuale di falsi positivi.
Questi lavori hanno confermato non solo l’affidabilità nell’utilizzo dei DBS come campione biologico e la sensibilità della spettrometria in tandem massa per l’analisi, ma anche la facile organizzazione e applicazione di tale metodica per progetti di screening.
La seconda parte del progetto è stata svolta a Padova in collaborazione con la sezione di spettrometria di massa del Dipartimento di Pediatria.
Qui è stato riprodotto il saggio multiplo per i 5 enzimi sopra citati ed è stato aggiunto inoltre il test per la Mucopolisaccaridosi di tipo I.
Tenendo conto dei risultati preliminari ottenuti a Vienna il protocollo è stato così modificato: è stata ridotta al minimo la parte preparativa dei campioni, sostituita da una cromatografia liquida collegata online con lo spettrometro di massa; inoltre, tutti i parametri analitici sono stati minuziosamente ottimizzati. Lo scopo è stato quello si massimizzare la sensibilità e precisione di rilevamento degli analiti per garantire una inconfutabile discriminazione tra soggetti sani e patologici.
Questa nuova metodica può essere applicata per scopi diversi: sia per lo screening di ampie popolazioni, con la possibilità di aggiungere nuovi test al pannello delle malattie screenate via via che sarà per esse disponibile una terapia, sia per una rapida valutazione della bio-disponibilità dell’enzima in singoli soggett
Eric Legnini réunit Kellylee Evans et Sandra Nkaké en hommage à Ray Charles
No full textL'année 1959 n'en finit pas d'être célébrée. Après Monk au Town Hall de New York, le festival de jazz à la Villette a consacré un week-end l'année dernière à une série de reprises de « Kind Of Blue » de Miles Davis, « Giant Steps » de John Coltrane, « Mingus Ah Um » de Charles Mingus ou encore « Time Out » de Dave Brubeck. Éric Legnini avait choisi quant à lui Ray Charles, et "What'd I Say". "C'est le moment, m'explique-t-il, où Ray Charles popularise le Gospel, tel qu'il était chanté à l'ég..
Eric Legnini réunit Kellylee Evans et Sandra Nkaké en hommage à Ray Charles
No full textL'année 1959 n'en finit pas d'être célébrée. Après Monk au Town Hall de New York, le festival de jazz à la Villette a consacré un week-end l'année dernière à une série de reprises de « Kind Of Blue » de Miles Davis, « Giant Steps » de John Coltrane, « Mingus Ah Um » de Charles Mingus ou encore « Time Out » de Dave Brubeck. Éric Legnini avait choisi quant à lui Ray Charles, et "What'd I Say". "C'est le moment, m'explique-t-il, où Ray Charles popularise le Gospel, tel qu'il était chanté à l'ég..
Going Beyond Counting First Authors in Author Co-citation Analysis
No full textThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
The interplay between the HUR protein and the long non-coding RNA linc-MD1 drives the progression from early to late muscle differentiation stages
No full textThe lack of the Celf2a splicing factor converts a Duchenne genotype into a Becker phenotype
No full textSubstitutions, deletions and duplications in the dystrophin gene lead to either the severe Duchenne muscular dystrophy (DMD) or mild Becker muscular dystrophy depending on whether out-of-frame or in-frame transcripts are produced. We identified a DMD case (GSΔ44) where the correlation between genotype and phenotype is not respected, even if carrying a typical Duchenne mutation (exon 44 deletion) a Becker-like phenotype was observed. Here we report that in this patient, partial restoration of an in-frame transcript occurs by natural skipping of exon 45 and that this is due to the lack of Celf2a, a splicing factor that interacts with exon 45 in the dystrophin pre-mRNA. Several experiments are presented that demonstrate the central role of Celf2a in controlling exon 45 splicing; our data point to this factor as a potential target for the improvement of those DMD therapeutic treatments, which requires exon 45 skipping
A Long Noncoding RNA Controls Muscle Differentiation by Functioning as a Competing Endogenous RNA
No full textRecently, a new regulatory circuitry has been identified in which RNAs can crosstalk with each other by competing for shared microRNAs. Such competing endogenous RNAs (ceRNAs) regulate the distribution of miRNA molecules on their targets and thereby impose an additional level of post-transcriptional regulation. Here we identify a muscle-specific long noncoding RNA, linc-MD1, which governs the time of muscle differentiation by acting as a ceRNA in mouse and human myoblasts. Downregulation or overexpression of linc-MD1 correlate with retardation or anticipation of the muscle differentiation program, respectively. We show that linc-MD1 "sponges" miR-133 and miR-135 to regulate the expression of MAML1 and MEF2C, transcription factors that activate muscle-specific gene expression. Finally, we demonstrate that linc-MD1 exerts the same control over differentiation timing in human myoblasts, and that its levels are strongly reduced in Duchenne muscle cells. We conclude that the ceRNA network plays an important role in muscle differentiation
Protecting Animals 36: Author Witi Ihimaera
No full textIn this very special episode of Knowing Animals I am joined by beloved New Zealand author Witi Ihimaera. Witi has written many books featuring nonhuman animals. He offers us a non-colonial lens through which to think about the human/nonhuman relationship
Author Under Sail The Imagination of Jack London, 1893-1902
No full textIn Author Under Sail, Jay Williams offers the first complete literary biography of Jack London as a professional writer engaged in the labor of writing. It examines the authorial imagination in London's work, the use of imagination in both his fiction and nonfiction, and the ways he defined imagination in the creative process in his business dealings with his publishers, editors, and agents. In this first volume of a two-volume biography, Williams traverses the years 1893 to 1902, from London's "Story of a Typhoon" to The People of the Abyss. The Jack London who emerges in the pages of Author Under Sail is a writer whose partnership with publishers, most notably his productive alliance with George Brett of Macmillan, was one of the most formative in American literary history. London pioneered many author models during the heyday of realism and naturalism, blurring the boundaries of these popular genres by focusing on absorption and theatricality and the representation of the seen and unseen. London created an impassioned, sincere, and extremely personal realism unlike that of other American writers of the time. Author Under Sail is a literary tour de force that reveals the full range of London as writer, creative citizen, and entrepreneur at the same time it sheds light on the maverick side of machine-age literature.Intro -- Title Page -- Copyright Page -- Dedication -- Contents -- Acknowledgments -- Introduction -- 1. Spirit Truth -- 2. From Absorption to Theatricality and Back Again -- 3. "I Will Build a New Present" -- 4. Sons as Authors -- 5. Fathers as Publishers -- 6. The Daughter as Author -- 7. Lovers as Authors -- 8. At Sea with the Family -- 9. Yellow News, Yellow Stories -- 10. The Return Home -- Notes -- Bibliography -- Index -- About Jay WilliamsIn Author Under Sail, Jay Williams offers the first complete literary biography of Jack London as a professional writer engaged in the labor of writing. It examines the authorial imagination in London's work, the use of imagination in both his fiction and nonfiction, and the ways he defined imagination in the creative process in his business dealings with his publishers, editors, and agents. In this first volume of a two-volume biography, Williams traverses the years 1893 to 1902, from London's "Story of a Typhoon" to The People of the Abyss. The Jack London who emerges in the pages of Author Under Sail is a writer whose partnership with publishers, most notably his productive alliance with George Brett of Macmillan, was one of the most formative in American literary history. London pioneered many author models during the heyday of realism and naturalism, blurring the boundaries of these popular genres by focusing on absorption and theatricality and the representation of the seen and unseen. London created an impassioned, sincere, and extremely personal realism unlike that of other American writers of the time. Author Under Sail is a literary tour de force that reveals the full range of London as writer, creative citizen, and entrepreneur at the same time it sheds light on the maverick side of machine-age literature.Description based on publisher supplied metadata and other sources.Electronic reproduction. Ann Arbor, Michigan : ProQuest Ebook Central, YYYY. Available via World Wide Web. Access may be limited to ProQuest Ebook Central affiliated libraries
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