Tribuna. Reorientar el diagnóstico

Las técnicas de diagnóstico por imagen han revolucionado muchas especialidades, como la oncología, donde su uso ha permitido reducir la morbi-mortalidad de lesiones detectadas precozmente. Sin embargo, ni todas las técnicas ni todos los tumores son iguales.

Single-step extraction of fluconazole from plasma by ultra-filtration for the measurement of its free concentration by high performance liquid chromatography.

High performance liquid chromatography (HPLC) is the reference method for measuring concentrations of antimicrobials in blood. This technique requires careful sample preparation. Protocols using organic solvents and/or solid extraction phases are time consuming and entail several manipulations, which can lead to partial loss of the determined compound and increased analytical variability. Moreover, to obtain sufficient material for analysis, at least 1 ml of plasma is required. This constraint makes it difficult to determine drug levels when blood sample volumes are limited. However, drugs with low plasma-protein binding can be reliably extracted from plasma by ultra-filtration with a minimal loss due to the protein-bound fraction. This study validated a single-step ultra-filtration method for extracting fluconazole (FLC), a first-line antifungal agent with a weak plasma-protein binding, from plasma to determine its concentration by HPLC. Spiked FLC standards and unknowns were prepared in human and rat plasma. Samples (240 microl) were transferred into disposable microtube filtration units containing cellulose or polysulfone filters with a 5 kDa cut-off. After centrifugation for 60 min at 15000g, FLC concentrations were measured by direct injection of the filtrate into the HPLC. Using cellulose filters, low molecular weight proteins were eluted early in the chromatogram and well separated from FLC that eluted at 8.40 min as a sharp single peak. In contrast, with polysulfone filters several additional peaks interfering with the FLC peak were observed. Moreover, the FLC recovery using cellulose filters compared to polysulfone filters was higher and had a better reproducibility. Cellulose filters were therefore used for the subsequent validation procedure. The quantification limit was 0.195 mgl(-1). Standard curves with a quadratic regression coefficient > or = 0.9999 were obtained in the concentration range of 0.195-100 mgl(-1). The inter and intra-run accuracies and precisions over the clinically relevant concentration range, 1.875-60 mgl(-1), fell well within the +/-15% variation recommended by the current guidelines for the validation of analytical methods. Furthermore, no analytical interference was observed with commonly used antibiotics, antifungals, antivirals and immunosuppressive agents. Ultra-filtration of plasma with cellulose filters permits the extraction of FLC from small volumes (240 microl). The determination of FLC concentrations by HPLC after this single-step procedure is selective, precise and accurate.

Ultra high throughput sequencing excludes MDH1 as candidate gene for RP28-linked retinitis pigmentosa.

PURPOSE: Mutations in IDH3B, an enzyme participating in the Krebs cycle, have recently been found to cause autosomal recessive retinitis pigmentosa (arRP). The MDH1 gene maps within the RP28 arRP linkage interval and encodes cytoplasmic malate dehydrogenase, an enzyme functionally related to IDH3B. As a proof of concept for candidate gene screening to be routinely performed by ultra high throughput sequencing (UHTs), we analyzed MDH1 in a patient from each of the two families described so far to show linkage between arRP and RP28. METHODS: With genomic long-range PCR, we amplified all introns and exons of the MDH1 gene (23.4 kb). PCR products were then sequenced by short-read UHTs with no further processing. Computer-based mapping of the reads and mutation detection were performed by three independent software packages. RESULTS: Despite the intrinsic complexity of human genome sequences, reads were easily mapped and analyzed, and all algorithms used provided the same results. The two patients were homozygous for all DNA variants identified in the region, which confirms previous linkage and homozygosity mapping results, but had different haplotypes, indicating genetic or allelic heterogeneity. None of the DNA changes detected could be associated with the disease. CONCLUSIONS: The MDH1 gene is not the cause of RP28-linked arRP. Our experimental strategy shows that long-range genomic PCR followed by UHTs provides an excellent system to perform a thorough screening of candidate genes for hereditary retinal degeneration.

Alterations in Postural Control during the World's Most Challenging Mountain Ultra-Marathon

We investigated postural control (PC) effects of a mountain ultra-marathon (MUM): a 330-km trail run with 24000 m of positive and negative change in elevation. PC was assessed prior to (PRE), during (MID) and after (POST) the MUM in experienced ultra-marathon runners (n = 18; finish time = 126+/-16 h) and in a control group (n = 8) with a similar level of sleep deprivation. Subjects were instructed to stand upright on a posturographic platform over a period of 51.2 seconds using a double-leg stance under two test conditions: eyes open (EO) and eyes closed (EC). Traditional measures of postural stability (center of pressure trajectory analysis) and stabilogram-diffusion analysis (SDA) parameters were analysed. For the SDA, a significantly greater short-term effective diffusion was found at POST compared with PRE in the medio-lateral (ML; Dxs) and antero-posterior (AP) directions (Dys) in runners (p<0.05) The critical time interval (Ctx) in the ML direction was significantly higher at MID (p<0.001) and POST (p<0.05) than at PRE in runners. At MID (p<0.001) and POST (p<0.05), there was a significant difference between the two groups. The critical displacement (Cdx) in the ML was significantly higher at MID and at POST (p<0.001) compared with PRE for runners. A significant difference in Cdx was observed between groups in EO at MID (p<0.05) and POST (p<0.005) in the ML direction and in EC at POST in the ML and AP directions (p<0.05). Our findings revealed significant effects of fatigue on PC in runners, including, a significant increase in Ctx (critical time in ML plan) in EO and EC conditions. Thus, runners take longer to stabilise their body at POST than at MID. It is likely that the mountainous characteristics of MUM (unstable ground, primarily uphill/downhill running, and altitude) increase this fatigue, leading to difficulty in maintaining balance.

Fast quantification of ten psychotropic drugs and metabolites in human plasma by ultra-high performance liquid chromatography tandem mass spectrometry for therapeutic drug monitoring.

A sensitive and selective ultra-high performance liquid chromatography (UHPLC) tandem mass spectrometry (MS/MS) method was developed for the fast quantification of ten psychotropic drugs and metabolites in human plasma for the needs of our laboratory (amisulpride, asenapine, desmethyl-mirtazapine, iloperidone, mirtazapine, norquetiapine, olanzapine, paliperidone, quetiapine and risperidone). Stable isotope-labeled internal standards were used for all analytes, to compensate for the global method variability, including extraction and ionization variations. Sample preparation was performed by generic protein precipitation with acetonitrile. Chromatographic separation was achieved in less than 3.0min on an Acquity UPLC BEH Shield RP18 column (2.1mm×50mm; 1.7μm), using a gradient elution of 10mM ammonium formate buffer pH 3.0 and acetonitrile at a flow rate of 0.4ml/min. The compounds were quantified on a tandem quadrupole mass spectrometer operating in positive electrospray ionization mode, using multiple reaction monitoring. The method was fully validated according to the latest recommendations of international guidelines. Eight point calibration curves were used to cover a large concentration range 0.5-200ng/ml for asenapine, desmethyl-mirtazapine, iloperidone, mirtazapine, olanzapine, paliperidone and risperidone, and 1-1500ng/ml for amisulpride, norquetiapine and quetiapine. Good quantitative performances were achieved in terms of trueness (93.1-111.2%), repeatability (1.3-8.6%) and intermediate precision (1.8-11.5%). Internal standard-normalized matrix effects ranged between 95 and 105%, with a variability never exceeding 6%. The accuracy profiles (total error) were included in the acceptance limits of ±30% for biological samples. This method is therefore suitable for both therapeutic drug monitoring and pharmacokinetic studies.

Diagnóstico diferencial del tobillo doloroso

El propósito de ésta comunicación es hacer un pequeño repaso de las diferentes patologías que pueden ocasionar dolor a nivel de la articulación del tobillo para poder establecer un diagnóstico diferencial de las mismas. Si bien las más frecuentes suelen producirse durante la práctica deportiva sobre todo en niños, adolescentes y adultos jóvenes, no hemos de olvidar las diversas patologías articulares que afectan dicha articulación y que en muchas ocasiones son procesos secundarios a enfermedades sistémicas, que se manifiestan con mayor frecuencia en la edad adulta. Se trata pues de una breve descripción de cada patología, la exploración clínica, pruebas complementarias y tratamientos más indicados, para cada una de ellas.

Competencias interculturales transversales: su diagnóstico en equipos de trabajo de baja qualificación

La competencia intercultural es la competencia profesional que permite un correcto desempeño y una adecuada integración en la empresa culturalmente diversa. El Modelo CIT de Competencias Interculturales Transversales describe el conjunto de conocimientos, comportamientos y actitudes a través de dos macro competencias como Diagnosticar Interculturalmente y Afrontar los requerimientos derivados de la interculturalidad. La presente investigación se plantea dos objetivos: a) Identificar las competencias interculturales de Diagnosticar y b) Identificar las competencias interculturales de Afrontar en 12 grupos de trabajo multiculturales de baja cualificación. El estudio de naturaleza cualitativa ha mostrado que la jerarquía y las normas organizativas son los elementos más diagnosticados como sensibles a la diferencia cultural. En relación a la competencia de Afrontar, se han identificado competencias de resolución de problemas, trabajo en equipo y comunicación intercultural; aplicados indistintamente tanto por los supervisores y responsables de los grupos como por parte de los operarios y personal de base. Las estrategias aplicadas por las personas participantes en el estudio, valoradas como competentes por sus empresas, han permitido la apreciación y estudio de la diferencia cultural y la adaptación del comportamiento a la nueva realidad organizacional.

Competencias interculturales transversales: su diagnóstico en equipos de trabajo de baja qualificación

La competencia intercultural es la competencia profesional que permite un correcto desempeño y una adecuada integración en la empresa culturalmente diversa. El Modelo CIT de Competencias Interculturales Transversales describe el conjunto de conocimientos, comportamientos y actitudes a través de dos macro competencias como Diagnosticar Interculturalmente y Afrontar los requerimientos derivados de la interculturalidad. La presente investigación se plantea dos objetivos: a) Identificar las competencias interculturales de Diagnosticar y b) Identificar las competencias interculturales de Afrontar en 12 grupos de trabajo multiculturales de baja cualificación. El estudio de naturaleza cualitativa ha mostrado que la jerarquía y las normas organizativas son los elementos más diagnosticados como sensibles a la diferencia cultural. En relación a la competencia de Afrontar, se han identificado competencias de resolución de problemas, trabajo en equipo y comunicación intercultural; aplicados indistintamente tanto por los supervisores y responsables de los grupos como por parte de los operarios y personal de base. Las estrategias aplicadas por las personas participantes en el estudio, valoradas como competentes por sus empresas, han permitido la apreciación y estudio de la diferencia cultural y la adaptación del comportamiento a la nueva realidad organizacional.

Diagnóstico rápido e dialogado em estudos de desenvolvimento rural sustentável

O principal objetivo deste trabalho foi verificar se a suposição de predominância da agricultura familiar na microbacia do Taquara Branca, Sumaré, SP, poderia ser sustentada com base em informações concretas. Foi possível a utilização de amostragem probabilística, pois estava disponível um mapa com os limites das 106 propriedades da microbacia, sendo sorteadas 33 delas para a aplicação de questionários do tipo cross-section. A técnica multivariada empregada foi a análise de agrupamentos, para a qual foi utilizado o procedimento PROC CLUSTER do SAS. Nessa análise só foram considerados os dados referentes aos 22 respondentes que exerciam atividade agrícola. Após a análise unidimensional, algumas perguntas foram selecionadas com base na variabilidade entre as respostas e, a partir destas, construíram-se as variáveis necessárias para se proceder à análise multidimensional. A análise de agrupamento permitiu identificar claramente três grupos naturais entre as 22 propriedades. De acordo com as características dos componentes de cada grupo, foi possível classificá-los em: grupo 1, doze propriedades de agricultura familiar; grupo 2, seis propriedades de agricultura não familiar em pequenas áreas; e grupo 3, quatro propriedades de agricultura não familiar em grandes áreas. Embora não seja predominante (apenas 36%), a agricultura familiar é o grupo mais freqüente na microbacia do Taquara Branca.

Peroxisome proliferator activated receptor BETA/DELTA as a central player in the skin response to ultra-violets radiation

Previous studies in the lab of Dr. Liliane Michalik, have shown thai the nuclear hormone receptor Peroxisome Proliferator Activated Receptor beta/delta (PPARß/ö) is an important regulator of skin homeostasis, being involved in the regulation of keratinocyte differentiation, inflammation, apoptosis, arid mouse skin wound healing. Studies of PPARß/ö knock out mice have suggested a possible role for this receptor in cancer. However, contradictory observations of the role for PPARß/ö on tumor growth have been published, depending on cellular contexts and biological models. Given the controversial role of PPARß/ö in skin carcinoma development, the main aim of this PhD work has been to further explore the implication of PPARß/ö in skin response to UV and skin tumor growth. This PhD dissertation is divided in four chapters. The first chapter describes the core part of the project, where I explored the changes in miRNA expression in the skin upon chronic UV irradiation of PPARß/ö wild type and knock-out mice. This analysis shed light on a miRNA- PPARß/ö signature and also predicted thai miR-21-3p (previously named miR-21*) is a key regulator of the PPARß/ö-dependent UV response in the pre-lesiona! skin. Using mice acutely UV-irradiated, ! further demonstrated that miR-21-3p is indirectly regulated by PPARß/ö through activation of Transforming Growth Factor (TGFß)-1 under UV exposure. I also show that miR-21-3p is deregulated in human cutaneous squamous celi carcinoma. In cultured keratinocytes, application of a miR-21 -3p mimic oligonucleotide sequence leads to the regulation of lipid metabolism-related pathway. In the second chapter, I demonstrate that the usage of an mRNA/miRNA combined bioinformatics analysis leads to the discovery of important pathways involved in the PPARß/ö-miRNA response of the skin to chronic UV irradiation, indeed, I validated angiogenesis and lipid metabolism as important functions regulated by PPARß/ö in this context. In the third chapter, we demonstrate that PPARß/5 knockout mice have decreased cutaneous squamous cell carcinomas incidence compared to wild type mice and that PPARß/5 directly activates the cSrc kinase gene. In the last chapter, we review novel insights into PPAR functions in keratinocytes and liver, with emphasis on PPARß/ö but also on PPARa. In summary, this PhD study shows that i) PPARß/5 is able to regulate biological function through regulation of miRNAs, and specifically through miR-21-3p, the passenger miRNA of the oncomiR miR-21, and that ii) the PPARß/5-dependent skin response to UV involves the regulation of angiogenesis and lipid metabolism. Furthermore, the bioinformatics study highlights the relevance of performing integrated mRNA and miRNA genome-wide studies in order to better screen mRNAs and/or miRNAs of interest in the biological context of diseases. - Des études préalables dans le laboratoire du Dr. Liliane Michalik ont démontré que le récepteur nucléaire PPARß/5 est un régulateur important de l'homéostasie de la peau, étant impliqué dans la régulation de la différenciation des keratinocytes, dans l'inflammation, dans l'apoptose et dans la cicatrisation de la peau chez !a souris. L'étude de souris knock-out pour le gène PPARß/5, ont suggérées un rôle possible de ce récepteur dans le cancer. Cependant, des observations opposées ont été publiées suggérant un rôle pro- ou anti- cancer selon le tissue impliqué et le type- cellulaire. En considérant cette controverse autour du rôle de PPARß/5 dans le développement des cancers de la peau, le but principal de mon projet de recherche aura été d'approfondir l'exploration du rôle de PPARß/5 dans la réponse de la peau aux UVs et dans le développement du cancer. Cette dissertation de thèse est divisée en quatre parties. Une première partie, représentant le coeur de mon travail de recherche, décrit la découverte de l'implication des microRNAs (rniRNAs) dans la réponse aux UVs de PPARß/ö et plus spécifiquement l'implication du miRNA miR- 21 -3p (précédemment nommé miR-21*). En étudiant un modèle de souris irradiées de manière aigüe aux UVs, nous montrons que ia régulation de miR-21-3p est PPARß/ö-däpenaante et que cette régulation à lieu par l'intermédiaire du facteur de transcription TGFß-1. Dans des cultures de keratinocytes Humains, la transfecticn d'une séquence oligonucléotidique similaire à celle de miR-21-3p (mimic), montre l'implication de rniR-21-3p dans des fonctions importantes pour le développement des cancers telles que le métabolisme des lipides. Dans un second chapitre, nous montrons que l'usage d'une méthode bioinformatique combinant l'expression des ARN messagers et des miRNAs permet de mettre en évidence des fonctions biologiques importantes lors de ia réponse de PPARß/ö à l'irradiation chronique. L'angiogenèse, le stress oxydatif et le métabolisme des lipides font partie de ces fonctions régulées par PPARß/5 dans la peau irradiée aux UVs. Nous mettons également en évidence la régulation du gène LpcatS par PPARß/5 dans la peau irradiée aux UV ainsi que dans des keratinocytes humains suggérant un rôle pour PPARß/5 dans le remodelage des lipides membranaires. Dans une troisième partie, nous établissons un lien entre la régulation de l'oncogène Src et l'activation de PPARß/5 dans les carcinomes spinocellulaires de la peau. Finalement dans un quatrième chapitre, nous faisons une revue des dernières recherches portées sur le rôle de PPARß/5 et de PPARa dans le foie et ia peau. En résumé ce projet de thèse représente un avancement pour la recherche sur rimplication de PPARß/5 dans la réponse aux UVs de la peau. Pour la première fois, un lien est établi entre ce facteur de transcription et la régulation de microRNAs dans le cadre du carcinome spinocellulare. Jusqu'alors resté dans l'ombre de rniR-21-5p, miR-21-3p est en fait fortement augmenté à la fois dans un modèle de souris d'irradiation aux UVs ainsi que dans ie carcinome spinocellulare chez i'humain. De nouvelles fonctions biologiques pour PPARß/5 ont été également mises en évidence dans ce travail, comme la régulation de l'angiogenèse ou du métabolisme des lipides dans Sa peau. De plus cette dissertation valorise l'intérêt d'une association entre le travail de laboratoire et celui de la bioinformatique.

Iowa Ultra Thin Whitetopping at Two Years of Age, HR-559, 1996

An 11.6 km research project was constructed in 1994 on a portion of Iowa Highway 21 in Iowa County, from U.S. 6 to Iowa Highway 212. This research is intended to evaluate the effect of four primary variables on long term performances of the PCC concrete overlay, commonly called whitetopping. The variables are thickness (50 mm, 100 mm, 150 mm, and 200 mm), joint spacing (0.6 m squares, 1.2 m squares, 1.8 m squares, and 4.6 m spacing), fiber use (concrete with and without polypropolene fibers) and surface preparation (patch only, scarifying the surface, and cold-in-place recycling). After two years, only two sections exhibit a small amount of debonding and distress cracking. Both sections are 50 mm thick. Within each of these two sections, only 2% of the area is affected. Two other 50 mm thick sections have a small number of cracks but no debonding has been found. No adverse effects of these cracks are evident. Three asphalt overlay sections were also constructed. In each asphalt section, transverse cracks have recently been found. At two years of age, the research sections are performing very well. An insignificant number of cracks and no distressed areas have been found in any research sections thicker than 50 mm.

Xerostomía: diagnóstico y tratamiento

La xerostomía o disminución de la cantidad de saliva en la cavidad bucal suele tener una serie de consecuencias: unas directas, tales como el aumento en la incidencia de caries y enfermedad periodontal, y otras indirectas, como la dificultad en la masticación o la mala adaptación de las prótesis. De ahí la importancia de una correcta lubrificación salival de la cavidad bucal. En el presente trabajo se realiza una revisión de los problemas ocasionados por la xerostomía, así como de su diagnóstico y tratamiento, aspectos éstos un tanto conflictivos, debido a la dificultad de objetivar la sintomatología y de conseguir resultados duraderos.

Mesiodens: concepto, evaluación clínica, diagnóstico y tratamiento

La presencia de dientes supernumerarios en la línea media maxilar, conocidos bajo el término de mesiodens, puede causar diferentes alteraciones tales como malposición de dientes vecinos, erupción anómala o retraso de erupción de los incisivos centrales superiores permanentes, diastema interincisal y formación de quistes, entre otros. En este artículo presentamos una revisión bibliográfica exhaustiva de este tipo de patología y evaluamos los aspectos clínico, etiológico, diagnóstico y terapéutico.

Diagnóstico radiológico de un cuerpo extraño en fosas nasales. A propósito de un caso con retraso de erupción de incisivos laterales.

Se presenta el caso de un paciente con un objeto inanimado en fosa nasal izquierda, que acudió a la Clínica Odontológica de Bellvitge, por la ausencia de los incisivos laterales permanentes en el maxilar superior. Al confirmar la presencia de los incisivos, a partir de una radiografía oclusal, descubrimos, de forma casual, un objeto radiopaco al que acompañaba una específica sintomatología. Después de la intervención del otorrinolaringólogo, dicha sintomatología desapareció. Referimos el manejo de los cuerpos extraños en fosas nasales y aprovechamos la ocasión para revisar las pautas generales de orientación diagnóstico-terapéuticas frente a esta patología.