Detection of four Plasmodium species in blood from humans by 18S rRNA gene subunit-based and specific real-time PCR assays

Résumé : Un nombre croissant de cas de malaria chez les voyageurs et migrants a été rapporté. Bien que l'analyse microscopique des frottis sanguins reste traditionnellement l'outil diagnostic de référence, sa fiabilité dépend considérablement de l'expertise de l'examinateur, pouvant elle-même faire défaut sous nos latitudes. Une PCR multiplex en temps réel a donc été développée en vue d'une standardisation du diagnostic. Un ensemble d'amorces génériques ciblant une région hautement conservée du gène d'ARN ribosomial 18S du genre Plasmodium a tout d'abord été conçu, dont le polymorphisme du produit d'amplification semblait suffisant pour créer quatre sondes spécifiques à l'espèce P. falciparum, P. malariae, P. vivax et P. ovale. Ces sondes utilisées en PCR en temps réel se sont révélées capables de détecter une seule copie de plasmide de P. falciparum, P. malariae, P. vivax et P. ovale spécifiquement. La même sensibilité a été obtenue avec une sonde de screening pouvant détecter les quatre espèces. Quatre-vingt-dix-sept échantillons de sang ont ensuite été testés, dont on a comparé la microscopie et la PCR en temps réel pour 66 (60 patients) d'entre eux. Ces deux méthodes ont montré une concordance globale de 86% pour la détection de plasmodia. Les résultats discordants ont été réévalués grâce à des données cliniques, une deuxième expertise microscopique et moléculaire (laboratoire de Genève et de l'Institut Suisse Tropical de Bâle), ainsi qu'à l'aide du séquençage. Cette nouvelle analyse s'est prononcé en faveur de la méthode moléculaire pour tous les neuf résultats discordants. Sur les 31 résultats positifs par les deux méthodes, la même réévaluation a pu donner raison 8 fois sur 9 à la PCR en temps réel sur le plan de l'identification de l'espèce plasmodiale. Les 31 autres échantillons ont été analysés pour le suivi de sept patients sous traitement antimalarique. Il a été observé une baisse rapide du nombre de parasites mesurée par la PCR en temps réel chez six des sept patients, baisse correspondant à la parasitémie déterminée microscopiquement. Ceci suggère ainsi le rôle potentiel de la PCR en temps réel dans le suivi thérapeutique des patients traités par antipaludéens. Abstract : There have been reports of increasing numbers of cases of malaria among migrants and travelers. Although microscopic examination of blood smears remains the "gold standard" in diagnosis, this method suffers from insufficient sensitivity and requires considerable expertise. To improve diagnosis, a multiplex real-time PCR was developed. One set of generic primers targeting a highly conserved region of the 18S rRNA gene of the genus Plasmodium was designed; the primer set was polymorphic enough internally to design four species-specific probes for P. falciparum, P. vivax, P. malarie, and P. ovale. Real-time PCR with species-specific probes detected one plasmid copy of P. falciparum, P. vivax, P. malariae, and P. ovale specifically. The same sensitivity was achieved for all species with real-time PCR with the 18S screening probe. Ninety-seven blood samples were investigated. For 66 of them (60 patients), microscopy and real-time PCR results were compared and had a crude agreement of 86% for the detection of plasmodia. Discordant results were reevaluated with clinical, molecular, and sequencing data to resolve them. All nine discordances between 18S screening PCR and microscopy were resolved in favor of the molecular method, as were eight of nine discordances at the species level for the species-specific PCR among the 31 samples positive by both methods. The other 31 blood samples were tested to monitor the antimalaria treatment in seven patients. The number of parasites measured by real-time PCR fell rapidly for six out of seven patients in parallel to parasitemia determined microscopically. This suggests a role of quantitative PCR for the monitoring of patients receiving antimalaria therapy.

How does the body-scale model affect back-calculated growth: the example of arctic charr Salvelinus alpinus (L.) of Lake Geneva (Switzerland)

When back-calculating fish length from scale measurements, the choice of the body-scale relationship is a fundamental step. Using data from the arctic charrSalvelinus alpinus (L.) of Lake Geneva (Switzerland) we show the need for a curvilinear model, on both statistical and biological grounds. From several 2-parameters models, the log-linear relationship appears to provide the best fit. A 3-parameters, Bertalanffy model did not improve the fit. We show moreover that using the proportional model would lead to important misinterpretations of the data.

Experiències amb interacció de cos sencer generades en temps real amb entorns d’aigua virtual

L’objectiu d’aquest projecte ha estat la realització de dues aplicacions independents entre elles la interacció de les quals és a temps real i a cos sencer. En una aplicació l’usuari se li dona la possibilitat de, mitjançant el seu propi cos, transformar gotes que cauen en forma de plujavirtual en diferents flocs de neu. En la segona aplicació l’usuari pot generar onades en una superfície “aquosa”.Les dues aplicacions s’han creat amb la finalitat que puguin ser instal·lades i exposades en el centre de visitants de l’Expo2008 a Saragossa. Aquest fet ha estat fonamental per la tria de la temàtica de les aplicacions.Per assolir aquest objectiu, des del punt de vista del projectista, diferents estudis s’han hagut de portar terme. S’han realitzat estudis per tal de saber el funcionament del sistema de càmeres i del seu calibratge i de com es capta la informació referent a l’usuari. Aquestes parts són fonamentals per tal de poder gestionar bé la interacció i, sense les quals, no seria possible eldesenvolupament de les experiències. Un altre tipus d’estudi és el de la interacció, que és el que condueix l’eix principal del projecte, i el de la generació d’efectes visuals gràfics.

SIIMTA, Sistema Integrat d’Interacció de cos sencer generada en temps real i MusicoTeràpia per a persones amb discapacitats com l’Autisme

L’objectiu d’aquest Projecte Final de Carrera és el poder desenvolupar una nova eina de treball, segons el paradigme de l’interacció a cos sencer, per als terapeutes de la Fundació Tekhnikós, per a poder afavorir nous processos d’expressió, comunicació i interactivitat entre elpacient i el terapeuta en les sessions de Musicoteràpia.El Projecte tracta d’un sistema d’interacció a cos sencer per a pacients amb diferentspatologies o síndromes de conducta. Mitjançant un sistema de captura que treballa en el camp dels infrarojos, el pacient és capaç d’interactuar amb els elements de l’aplicació a través de lagesticulació. El moviment d’aquestes partícules (vegis el Glossari de termes en l’Annexe)generarà diferents sons segons posició i direcció en l’espai de la pantalla motivant al pacient a explorar el comportament el sistema de diferents maneres. Aquests estímuls sonors estan basatsen l’experiència prèvia de la Fundació amb les seves sessions de Musitoràpia, basades enl’observació de les reaccions del pacient davant estímuls sonors. Aquesta eina permetràexperimentat tot un món nou d’estímuls per al pacient, tant sonors, com visuals gràcies a la possibilitat de configuració de molts dels paràmetres del sistema. Gràcies al sistema de visió artifical s’aconsegueix una interacció màquina / pacient a cos sencer no intrusiu, molt interessant tenint en compte que l’aplicació està destinada a un gran espectre de patologies o síndromes. A més tenint en compte la tipologia general del pacient s’haoptat per una estètica molt minimalista, evitant qualsevol referència icònica per tal de no sobre estimular al pacient abans de que pugui interactuar amb el sistema.

Early detection of microcirculatory perfusion changes with a high resolution, real time laser Doppler imaging camera--frostbite case study.

A 41-year-old male presented with severe frostbite that was monitored clinically and with a new laser Doppler imaging (LDI) camera that records arbitrary microcirculatory perfusion units (1-256 arbitrary perfusion units (APU's)). LDI monitoring detected perfusion differences in hand and foot not seen visually. On day 4-5 after injury, LDI showed that while fingers did not experience any significant perfusion change (average of 31±25 APUs on day 5), the patient's left big toe did (from 17±29 APUs day 4 to 103±55 APUs day 5). These changes in regional perfusion were not detectable by visual examination. On day 53 postinjury, all fingers with reduced perfusion by LDI were amputated, while the toe could be salvaged. This case clearly demonstrates that insufficient microcirculatory perfusion can be identified using LDI in ways which visual examination alone does not permit, allowing prognosis of clinical outcomes. Such information may also be used to develop improved treatment approaches.

Breathhold three-dimensional coronary magnetic resonance angiography using real-time navigator technology.

The acquisition duration of most three-dimensional (3D) coronary magnetic resonance angiography (MRA) techniques is considerably prolonged, thereby precluding breathholding as a mechanism to suppress respiratory motion artifacts. Splitting the acquired 3D volume into multiple subvolumes or slabs serves to shorten individual breathhold duration. Still, problems associated with misregistration due to inconsistent depths of expiration and diaphragmatic drift during sustained respiration remain to be resolved. We propose the combination of an ultrafast 3D coronary MRA imaging sequence with prospective real-time navigator technology, which allows correction of the measured volume position. 3D volume splitting using prospective real-time navigator technology, was successfully applied for 3D coronary MRA in five healthy individuals. An ultrafast 3D interleaved hybrid gradient-echoplanar imaging sequence, including T2Prep for contrast enhancement, was used with the navigator localized at the basal anterior wall of the left ventricle. A 9-cm-thick volume, with in-plane spatial resolution of 1.1 x 2.2 mm, was acquired during five breathholds of 15-sec duration each. Consistently, no evidence of misregistration was observed in the images. Extensive contiguous segments of the left anterior descending coronary artery (48 +/- 18 mm) and the right coronary artery (75 +/- 5 mm) could be visualized. This technique has the potential for screening for anomalous coronary arteries, making it well suited as part of a larger clinical MR examination. In addition, this technique may also be applied as a scout scan, which allows an accurate definition of imaging planes for subsequent high-resolution coronary MRA.

Uniform Manual Real Property Acquisition and Relocation Assistance Section II - Chapter 111 Uniform Rules and Procedures April, 2006

The purpose of this chapter is to implement Iowa Code chapter 316 and sections 6B.42, 6B.45, 6B.54 and 6B.55, as required by the Uniform Relocation Assistance and Real Property Acquisition Policies Act of 1970, Pub. L. 91-646, as amended by the Uniform Relocation Act Amendments of 1987, Title IV, Pub. L. No. 100-17 , Sec. 104, Pub. L. 105-117, and federal regulations adopted pursuant thereto.

Genometrics as an essential tool for the assembly of whole genome sequences: the example of the chromosome of Bifidobacterium longum NCC2705.

BACKGROUND: Analysis of the first reported complete genome sequence of Bifidobacterium longum NCC2705, an actinobacterium colonizing the gastrointestinal tract, uncovered its proteomic relatedness to Streptomyces coelicolor and Mycobacterium tuberculosis. However, a rapid scrutiny by genometric methods revealed a genome organization totally different from all so far sequenced high-GC Gram-positive chromosomes. RESULTS: Generally, the cumulative GC- and ORF orientation skew curves of prokaryotic genomes consist of two linear segments of opposite slope: the minimum and the maximum of the curves correspond to the origin and the terminus of chromosome replication, respectively. However, analyses of the B. longum NCC2705 chromosome yielded six, instead of two, linear segments, while its dnaA locus, usually associated with the origin of replication, was not located at the minimum of the curves. Furthermore, the coorientation of gene transcription with replication was very low. Comparison with closely related actinobacteria strongly suggested that the chromosome of B. longum was misassembled, and the identification of two pairs of relatively long homologous DNA sequences offers the possibility for an alternative genome assembly proposed here below. By genometric criteria, this configuration displays all of the characters common to bacteria, in particular to related high-GC Gram-positives. In addition, it is compatible with the partially sequenced genome of DJO10A B. longum strain. Recently, a corrected sequence of B. longum NCC2705, with a configuration similar to the one proposed here below, has been deposited in GenBank, confirming our predictions. CONCLUSION: Genometric analyses, in conjunction with standard bioinformatic tools and knowledge of bacterial chromosome architecture, represent fast and straightforward methods for the evaluation of chromosome assembly.

PEG-b-PPS diblock copolymer aggregates for hydrophobic drug solubilization and release: cyclosporin A as an example

Micelles formed from amphiphilic block copolymers have been explored in recent years as carriers for hydrophobic drugs. In an aqueous environment, the hydrophobic blocks form the core of the micelle, which can host lipophilic drugs, while the hydrophilic blocks form the corona or outer shell and stabilize the interface between the hydrophobic core and the external medium. In the present work, mesophase behavior and drug encapsulation were explored in the AB block copolymeric amphiphile composed of poly(ethylene glycol) (PEG) as a hydrophile and poly(propylene sulfide) PPS as a hydrophobe, using the immunosuppressive drug cyclosporin A (CsA) as an example of a highly hydrophobic drug. Block copolymers with a degree of polymerization of 44 on the PEG and of 10, 20 and 40 on the PPS respectively (abbreviated as PEG44-b-PPS10, PEG44-b-PPS20, PEG44-b-PPS40) were synthesized and characterized. Drug-loaded polymeric micelles were obtained by the cosolvent displacement method as well as the remarkably simple method of dispersing the warm polymer melt, with drug dissolved therein, in warm water. Effective drug solubility up to 2 mg/mL in aqueous media was facilitated by the PEG- b-PPS micelles, with loading levels up to 19% w/w being achieved. Release was burst-free and sustained over periods of 9-12 days. These micelles demonstrate interesting solubilization characteristics, due to the low glass transition temperature, highly hydrophobic nature, and good solvent properties of the PPS block

Reverse transcription quantitative real-time polymerase chain reaction reference genes in the spared nerve injury model of neuropathic pain: validation and literature search.

BACKGROUND: The reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is a widely used, highly sensitive laboratory technique to rapidly and easily detect, identify and quantify gene expression. Reliable RT-qPCR data necessitates accurate normalization with validated control genes (reference genes) whose expression is constant in all studied conditions. This stability has to be demonstrated.We performed a literature search for studies using quantitative or semi-quantitative PCR in the rat spared nerve injury (SNI) model of neuropathic pain to verify whether any reference genes had previously been validated. We then analyzed the stability over time of 7 commonly used reference genes in the nervous system - specifically in the spinal cord dorsal horn and the dorsal root ganglion (DRG). These were: Actin beta (Actb), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal proteins 18S (18S), L13a (RPL13a) and L29 (RPL29), hypoxanthine phosphoribosyltransferase 1 (HPRT1) and hydroxymethylbilane synthase (HMBS). We compared the candidate genes and established a stability ranking using the geNorm algorithm. Finally, we assessed the number of reference genes necessary for accurate normalization in this neuropathic pain model. RESULTS: We found GAPDH, HMBS, Actb, HPRT1 and 18S cited as reference genes in literature on studies using the SNI model. Only HPRT1 and 18S had been once previously demonstrated as stable in RT-qPCR arrays. All the genes tested in this study, using the geNorm algorithm, presented gene stability values (M-value) acceptable enough for them to qualify as potential reference genes in both DRG and spinal cord. Using the coefficient of variation, 18S failed the 50% cut-off with a value of 61% in the DRG. The two most stable genes in the dorsal horn were RPL29 and RPL13a; in the DRG they were HPRT1 and Actb. Using a 0.15 cut-off for pairwise variations we found that any pair of stable reference gene was sufficient for the normalization process. CONCLUSIONS: In the rat SNI model, we validated and ranked Actb, RPL29, RPL13a, HMBS, GAPDH, HPRT1 and 18S as good reference genes in the spinal cord. In the DRG, 18S did not fulfill stability criteria. The combination of any two stable reference genes was sufficient to provide an accurate normalization.

A Fazenda Real, campo de contradições entre a Coroa

A 4 de Maio de 1512, D. Manuel I, rei de Portugal, fez mercê do ofício de almoxarife da vila da Ribeira Grande a Álvaro Dias, escudeiro de Sua Casa e morador da ilha de Santiago. A 20 de Fevereiro de 1534, D. João III confirmou a dita carta. 2 Durante o período que medeia entre estas duas datas decorre a vivência administrativa de uma das personagens mais interessantes e representativas da ilha de Santiago na primeira metade do século XVI. Esta figura, que foi ao mesmo tempo oficial régio, armador e mercador, representou como almoxarife os interesses da Fazenda Real na vila da Ribeira Grande. Como morador de Santiago, que armava navios e comerciava com a costa da Guiné, pertencia à camada social dominante da ilha. Pelo facto de colocar os seus negócios acima dos deveres que tinha perante a Coroa, Álvaro Dias foi por várias vezes destituído do ofício. No entanto, aproveitando-se das fraquezas existentes nas estruturas da administração, conseguiu reter, até à sua morte, a carta de almoxarife.

A CONTABILIDADE E O REAL VALOR DAS EMPRESAS: FOCO NO CAPITAL INTELECTUAL

Este trabalho mostra que a contabilidade pode não estar a evidenciar o real valor do património das empresas (um dos seus objectivos), constatamos nos últimos tempos, que os relatórios fornecidos pela contabilidade financeira não retratam certas realidades das empresas, visto que o valor contabilístico se distancia cada vez mais do valor de mercado, principalmente nas empresas de alta tecnologia e serviços, assim sendo as demonstrações financeiras podem estar experimentando uma perda de relevância para a tomada de decisões de investimentos, de crédito e de gestão. Dentro deste contexto, ressaltamos a necessidade de a contabilidade evidenciar naquelas demonstrações tais informações para que possa divulgar aos vários utentes da informação o real valor da empresa. Visto que o Capital Intelectual é importante para as empresas, torna-se necessário relatar não só informações financeiras como também não financeiras. Este trabalho apresenta um estudo descritivo/quantitativo da empresa CVTelecom, com o objectivo de avaliar o grau de divulgação do Capital Intelectual na empresa. A principal constatação foi o seguinte: uma participação activa da empresa em divulgar o Capital Intelectual, uma forma não normalizada através da divulgação voluntária do Capital Intelectual cujo veículo de divulgação, é o Relatório de Gestão e o Balanço Social. This work shows that the Accounting cannot be evidencing the Real value of the patrimony of the enterprises (one of their objectives), we verified in the last times, that the reports supplied by the Financial Accounting don't portray certain realities of the enterprises, because the value Accounting of the enterprises go away more and more of his market value, mainly in the companies of high technology and services, soon the financial demonstrations are trying a loss of relevance for socket of decisions of investments, of credit and of administration. Inside of this context, we emphasized the need of the Accounting to evidence in the demonstrations such financial information so that it can publish to the several users of the information the Real value of the company. Because the Intellectual Capital is important for the enterprises, it becomes necessary to tell not only financial information as well as any financial. This work presents a descriptive-quantitative study of the enterprise CVtelecom, with the objective of evaluating the degree of popularization of the Intellectual Capital in the enterprise and the impact in the performance of this enterprise. The main verification was the following: A participation active of the company in publishing the Intellectual Capital in spite of being in a way no normalized but a voluntary popularization of the Intellectual Capital, whose popularization vehicle is the Report of Administration and the Social Swinging.

A multi-objective model for a multi-period distribution management problem

The problems arising in commercial distribution are complex and involve several players and decision levels. One important decision is relatedwith the design of the routes to distribute the products, in an efficient and inexpensive way.This article deals with a complex vehicle routing problem that can beseen as a new extension of the basic vehicle routing problem. The proposed model is a multi-objective combinatorial optimization problemthat considers three objectives and multiple periods, which models in a closer way the real distribution problems. The first objective is costminimization, the second is balancing work levels and the third is amarketing objective. An application of the model on a small example, with5 clients and 3 days, is presented. The results of the model show the complexity of solving multi-objective combinatorial optimization problems and the contradiction between the several distribution management objective.