Buprenorphine and norbuprenorphine quantification in human plasma by simple protein precipitation and ultra-high performance liquid chromatography tandem mass spectrometry.

A highly sensitive ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method was developed for the quantification of buprenorphine and its major metabolite norbuprenorphine in human plasma. In order to speed up the process and decrease costs, sample preparation was performed by simple protein precipitation with acetonitrile. To the best of our knowledge, this is the first application of this extraction technique for the quantification of buprenorphine in plasma. Matrix effects were strongly reduced and selectivity increased by using an efficient chromatographic separation on a sub-2μm column (Acquity UPLC BEH C18 1.7μm, 2.1×50mm) in 5min with a gradient of ammonium formate 20mM pH 3.05 and acetonitrile as mobile phase at a flow rate of 0.4ml/min. Detection was made using a tandem quadrupole mass spectrometer operating in positive electrospray ionization mode, using multiple reaction monitoring. The procedure was fully validated according to the latest Food and Drug Administration guidelines and the Société Française des Sciences et Techniques Pharmaceutiques. Very good results were obtained by using a stable isotope-labeled internal standard for each analyte, to compensate for the variability due to the extraction and ionization steps. The method was very sensitive with lower limits of quantification of 0.1ng/ml for buprenorphine and 0.25ng/ml for norbuprenorphine. The upper limit of quantification was 250ng/ml for both drugs. Trueness (98.4-113.7%), repeatability (1.9-7.7%), intermediate precision (2.6-7.9%) and internal standard-normalized matrix effects (94-101%) were in accordance with international recommendations. The procedure was successfully used to quantify plasma samples from patients included in a clinical pharmacogenetic study and can be transferred for routine therapeutic drug monitoring in clinical laboratories without further development.

Ultra-performance liquid chromatography mass spectrometry and sensitive bioassay methods for quantification of posaconazole plasma concentrations after oral dosing.

Posaconazole (POS) is a new antifungal agent for prevention and therapy of mycoses in immunocompromised patients. Variable POS pharmacokinetics after oral dosing may influence efficacy: a trough threshold of 0.5 ?g/ml has been recently proposed. Measurement of POS plasma concentrations by complex chromatographic techniques may thus contribute to optimize prevention and management of life-threatening infections. No microbiological analytical method is available. The objective of this study was to develop and validate a new simplified ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method and a sensitive bioassay for quantification of POS over the clinical plasma concentration range. The UPLC-MS/MS equipment consisted of a triple quadrupole mass spectrometer, an electrospray ionization (ESI) source, and a C(18) analytical column. The Candida albicans POS-hypersusceptible mutant (MIC of 0.002 ?g/ml) ?cdr1 ?cdr2 ?flu ?mdr1 ?can constructed by targeted deletion of multidrug efflux transporters and calcineurin genes was used for the bioassay. POS was extracted from plasma by protein precipitation with acetonitrile-methanol (75%/25%, vol/vol). Reproducible standard curves were obtained over the range 0.014 to 12 (UPLC-MS/MS) and 0.028 to 12 ?g/ml (bioassay). Intra- and interrun accuracy levels were 106% ± 2% and 103% ± 4% for UPLC-MS/MS and 102% ± 8% and 104% ± 1% for bioassay, respectively. The intra- and interrun coefficients of variation were 7% ± 4% and 7% ± 3% for UPLC-MS/MS and 5% ± 3% and 4% ± 2% for bioassay, respectively. An excellent correlation between POS plasma concentrations measured by UPLC-MS/MS and bioassay was found (concordance, 0.96). In 26 hemato-oncological patients receiving oral POS, 27/69 (39%) trough plasma concentrations were lower than 0.5 ?g/ml. The UPLC-MS/MS method and sensitive bioassay offer alternative tools for accurate and precise quantification of the plasma concentrations in patients receiving oral posaconazole.

Targeted sequence capture and Ultra High Throughput sequencing for gene discovery in inherited diseases

L'introduction des technologies de séquençage de nouvelle génération est en vue de révolutionner la médecine moderne. L'impact de ces nouveaux outils a déjà contribué à la découverte de nouveaux gènes et de voies cellulaires impliqués dans la pathologie de maladies génétiques rares ou communes. En revanche, l'énorme quantité de données générées par ces systèmes ainsi que la complexité des analyses bioinformatiques nécessaires, engendre un goulet d'étranglement pour résoudre les cas les plus difficiles. L'objectif de cette thèse a été d'identifier les causes génétiques de deux maladies héréditaires utilisant ces nouvelles techniques de séquençage, couplées à des technologies d'enrichissement de gènes. Dans ce cadre, nous avons développé notre propre méthode de travail (pipeline) pour l'alignement des fragments de séquence (reads). Suite à l'identification de gènes, nous avons réalisé une analyse fonctionnelle pour élucider leur rôle dans la maladie. Dans un premier temps, nous avons étudié et identifié des mutations impliquées dans une forme récessive de la rétinite pigmentaire qui est à ce jour la dégénérescence rétinienne héréditaire la plus fréquente. En particulier, nous avons constaté que des mutations faux-sens dans le gène FAM161A étaient la cause de la rétinite pigmentaire préalablement associé avec le locus RP28. De plus, nous avons démontré que ce gène avait des fonctions au niveau du cil du photorécepteur, complétant le large spectre des cilliopathies rétiniennes héréditaires. Dans un second temps, nous avons exploré la possibilité qu'un syndrome, relativement fréquent en pédiatrie de fièvre récurrente, appelé PFAPA (acronyme de fièvre périodique avec adénite stomatite, pharyngite et cervical aphteuse) puisse avoir une origine génétique. L'étiologie de cette maladie n'étant pas claire, nous avons tenté d'identifier le spectre génétique de patients PFAPA. Comme nous n'avons pas pu mettre à jour un nouveau gène unique muté et responsable de la maladie chez tous les individus dépistés, il semblerait qu'un modèle génétique plus complexe suggérant l'implication de plusieurs gènes dans la pathologie ait été identifié chez les patients touchés. Ces gènes seraient notamment impliqués dans des processus liés à l'inflammation ce qui élargirait l'impact de ces études à d'autres maladies auto-inflammatoires.

Transcranial Doppler for predicting delayed cerebral ischemia after subarachnoid hemorrhage.

OBJECTIVE: Transcranial Doppler (TCD) is widely used to monitor the temporal course of vasospasm after subarachnoid hemorrhage (SAH), but its ability to predict clinical deterioration or infarction from delayed cerebral ischemia (DCI) remains controversial. We sought to determine the prognostic utility of serial TCD examination after SAH. METHODS: We analyzed 1877 TCD examinations in 441 aneurysmal SAH patients within 14 days of onset. The highest mean blood flow velocity (mBFV) value in any vessel before DCI onset was recorded. DCI was defined as clinical deterioration or computed tomographic evidence of infarction caused by vasospasm, with adjudication by consensus of the study team. Logistic regression was used to calculate adjusted odds ratios for DCI risk after controlling for other risk factors. RESULTS: DCI occurred in 21% of patients (n = 92). Multivariate predictors of DCI included modified Fisher computed tomographic score (P = 0.001), poor clinical grade (P = 0.04), and female sex (P = 0.008). After controlling for these variables, all TCD mBFV thresholds between 120 and 180 cm/s added a modest degree of incremental predictive value for DCI at nearly all time points, with maximal sensitivity by SAH day 8. However, the sensitivity of any mBFV more than 120 cm/s for subsequent DCI was only 63%, with a positive predictive value of 22% among patients with Hunt and Hess grades I to III and 36% in patients with Hunt and Hess grades IV and V. Positive predictive value was only slightly higher if mBFV exceeded 180 cm/s. CONCLUSION: Increased TCD flow velocities imply only a mild incremental risk of DCI after SAH, with maximal sensitivity by day 8. Nearly 40% of patients with DCI never attained an mBFV more than 120 cm/s during the course of monitoring. Given the poor overall sensitivity of TCD, improved methods for identifying patients at high risk for DCI after SAH are needed.

Suitability of Ultra Wideband for Mobile Devices

Mobiililaitteet ovat kehittyneet merkittävästi viimeisen vuosikymmenen aikana. Ne eivät enää ole pelkästään puhelimia tai kämmentietokoneita, vaan multimediatietokoneita, jotka pystyvät tuottamaan korkealaatuisia elämyksiä käyttäjälleen.Sisällön laadun paraneminen johtaa väistämättä myös siirrettävän tietomäärän kasvamiseen. Siksi uusia, nopeampia tiedonsiirtomenetelmiä on otettava käyttöön. Ultra Wideband (UWB) on uusi radioaaltotekniikka, jokamahdollistaa korkeat tiedonsiirtonopeudet lyhyillä etäisyyksillä. Tekniikka operoi erittäin leveällä 3.1 - 10.6 gigahertsin taajuuskaistalla.UWB on suunnattu nopeaan, langattomaan tiedonsiirtoon personal area networkin (PAN) laitteiden välillä. Tämän diplomityön tarkoituksena on arvioida UWBn soveltuvuutta mobiililaitteisiin. Arviointi tehdään teoreettiselta pohjalta, sillä UWBtä ei vielä ole implementoitu kaupallisiin laitteisiin. Ensin esitellään UWBn tekniikka ja ominaisuudet, jonka jälkeen sen soveltuvuutta arvioidaan esimerkkikäyttötapausten valossa. Esimerkkikäyttötapauksiksi on valittu kolme tyypillistä mutta erilaista toimintoa, jotka mahdollisimman hyvin kuvaavat mobiililaitteiden käyttöä.

Mudanças na ultra-estrutura da parede celular de mangas 'Tommy Atkins' tratadas com cloreto de cálcio na pré-colheita

Mangas 'Tommy Atkins' produzidas na região de Ibirá, São Paulo, foram pulverizadas na pré-colheita com cloreto de cálcio, nas concentrações de 0,0%, 2,5% e 5,0%, em três épocas de seu desenvolvimento (40; 60 e 90 dias após a floração) a fim de verificar a influência do cálcio na estrutura da parede celular destes frutos através de microscopia eletrônica de transmissão, imediatamente após a colheita e depois de 35 dias de armazenamento. Para fixar o material da polpa, utilizou-se metodologia descrita por Jacob e Gowanlock (1995). Nas condições experimentais, verificou-se que os frutos do tratamento-controle (sem cloreto de cálcio), no dia da colheita, já apresentavam desestruturação da parede celular e dissolução da lamela média (LM). A degradação da parede celular ocorre inicialmente na LM, levando à formação de espaços vazios bastante distintos, apresentando uma dissolução ainda maior, com o armazenamento prolongado (35 dias). Os frutos tratados com cloreto de cálcio a 5,0% apresentaram uma LM bem definida e ausência de espaços vazios, mesmo após o armazenamento, mostrando ser uma concentração efetiva na preservação da lamela média.

Século XXI: nova cultivar de goiabeira de dupla finalidade

A goiabeira (Psidium guajava L.) é uma espécie que vem se tornando de grande importância em diversas regiões do Brasil, principalmente no Estado de São Paulo, maior produtor nacional. Desde 1985, a UNESP/FCAV, Câmpus de Jaboticabal, vem desenvolvendo um programa de melhoramento genético da goiabeira, com o objetivo de obter plantas com boas características agronômicas e com frutos que possam ser destinados tanto à industrialização quanto ao consumo na forma de fruta fresca. Partindo-se de 219 plantas, oriundas de diversos cruzamentos, e após dez anos de avaliação, chegou-se à cultivar Século XXI, cujas principais características são: planta muito produtiva com ciclo precoce (130 dias da floração à colheita), frutos grandes, com polpa espessa, róseo-avermelhada, ótimo sabor e com poucas e pequenas sementes.

Estabelecimento e multiplicação in vitro de Pyrus calleryana D-6 em sistema de cultura 'dupla-fase'

Este trabalho objetivou estabelecer e propagar in vitro o porta-enxerto de pereira Pyrus calleryana D-6. Os explantes foram retirados de plantas-matrizes e submetidos à desinfestação com imersão em álcool a 70% por dois minutos, seguido de solução de hipoclorito de sódio a 1,5% + Tween 20 por 20 minutos, e após inoculados em meio MS com BAP (4,4 µM), AG3 (0,3 µM) e ANA (0,05 µM). Na fase de multiplicação, as microestacas caulinares, contendo 2-3 gemas, foram transferidas para frascos com 50 mL de meios MS e MS1 modificado com (NH4NO3)/2 e (CaCl2.2H2O)*2, suplementados com sacarose (30 gL-1), ágar (7 gL-1) e diferentes concentrações de BAP (0 e 6,7 µM), AIB (0 e 0,5 µM), AG3 (0 e 0,3µM). Para o sistema 'dupla-fase' (líquido-geleificado), os tratamentos receberam, aos 30 dias de cultura in vitro, 10 mL de meio MS líquido, sem fitorreguladores, e foram avaliados após 60 dias de cultura. Os resultados mostraram que 23,5% das gemas introduzidas obtiveram proliferação, sendo que o maior problema no estabelecimento in vitro foi a oxidação que afetou 44,8% das gemas introduzidas. O sistema 'dupla-fase' demonstrou ser muito efetivo para multiplicação in vitro do porta-enxerto de pereira Pyrus calleryana D-6. A maior taxa de multiplicação (16,8 brotos/explante) e o maior comprimento dos brotos (33 mm) foram obtidos no meio MS contendo BAP (6,7µM) e AIB (0,5 µM).