Some solutions to obtain very efficient separations in isocratic and gradient modes using small particles size and ultra-high pressure.

The UHPLC strategy which combines sub-2 microm porous particles and ultra-high pressure (>1000 bar) was investigated considering very high resolution criteria in both isocratic and gradient modes, with mobile phase temperatures between 30 and 90 degrees C. In isocratic mode, experimental conditions to reach the maximal efficiency were determined using the kinetic plot representation for DeltaP(max)=1000 bar. It has been first confirmed that the molecular weight of the compounds (MW) was a critical parameter which should be considered in the construction of such curves. With a MW around 1000 g mol(-1), efficiencies as high as 300,000 plates could be theoretically attained using UHPLC at 30 degrees C. By limiting the column length to 450 mm, the maximal plate count was around 100,000. In gradient mode, the longest column does not provide the maximal peak capacity for a given analysis time in UHPLC. This was attributed to the fact that peak capacity is not only related to the plate number but also to column dead time. Therefore, a compromise should be found and a 150 mm column should be preferentially selected for gradient lengths up to 60 min at 30 degrees C, while the columns coupled in series (3x 150 mm) were attractive only for t(grad)>250 min. Compared to 30 degrees C, peak capacities were increased by about 20-30% for a constant gradient length at 90 degrees C and gradient time decreased by 2-fold for an identical peak capacity.

Isolation of genetic mutation leading to abnormal phenotype in human through ultra-high-throughput sequencing (UHTS)

The introduction of Next Generation Sequencing (NGS) facilitated the task of localizing DNA variation and identifying the genetic cause of yet unsolved Mendelian disorders. Using Whole Exome Capture method and NGS, we identified the causative genetic aberration responsible for a number of monogenic disorders previously undetermined. Due to the novelty of the NGS method we benchmarked different algorithms to assess their merits and defects. This allowed us to establish a pipeline that we successfully used to pinpoint genes responsible for a form of West's syndrome, a Complex Intellectual Disability syndrome associated with patellar dislocation and celiac disease, and correcting some erroneous molecular diagnosis of Alport's syndrome in a Saudi Arabian family.

Clinical pharmacogenomic testing of KRAS, BRAF and EGFR mutations by high resolution melting analysis and ultra-deep pyrosequencing

BACKGROUND: Epidermal growth factor receptor (EGFR) and its downstream factors KRAS and BRAF are mutated in several types of cancer, affecting the clinical response to EGFR inhibitors. Mutations in the EGFR kinase domain predict sensitivity to the tyrosine kinase inhibitors gefitinib and erlotinib in lung adenocarcinoma, while activating point mutations in KRAS and BRAF confer resistance to the anti-EGFR monoclonal antibody cetuximab in colorectal cancer. The development of new generation methods for systematic mutation screening of these genes will allow more appropriate therapeutic choices. METHODS: We describe a high resolution melting (HRM) assay for mutation detection in EGFR exons 19-21, KRAS codon 12/13 and BRAF V600 using formalin-fixed paraffin-embedded samples. Somatic variation of KRAS exon 2 was also analysed by massively parallel pyrosequencing of amplicons with the GS Junior 454 platform. RESULTS: We tested 120 routine diagnostic specimens from patients with colorectal or lung cancer. Mutations in KRAS, BRAF and EGFR were observed in 41.9%, 13.0% and 11.1% of the overall samples, respectively, being mutually exclusive. For KRAS, six types of substitutions were detected (17 G12D, 9 G13D, 7 G12C, 2 G12A, 2 G12V, 2 G12S), while V600E accounted for all the BRAF activating mutations. Regarding EGFR, two cases showed exon 19 deletions (delE746-A750 and delE746-T751insA) and another two substitutions in exon 21 (one showed L858R with the resistance mutation T590M in exon 20, and the other had P848L mutation). Consistent with earlier reports, our results show that KRAS and BRAF mutation frequencies in colorectal cancer were 44.3% and 13.0%, respectively, while EGFR mutations were detected in 11.1% of the lung cancer specimens. Ultra-deep amplicon pyrosequencing successfully validated the HRM results and allowed detection and quantitation of KRAS somatic mutations. CONCLUSIONS: HRM is a rapid and sensitive method for moderate-throughput cost-effective screening of oncogene mutations in clinical samples. Rather than Sanger sequence validation, next-generation sequencing technology results in more accurate quantitative results in somatic variation and can be achieved at a higher throughput scale.

Laser-ultraviolet-A induced ultra weak photon emission in human skin cells: A biophotonic comparison between keratinocytes and fibroblasts.

Photons participate in many atomic and molecular interactions and processes. Recent biophysical research has discovered an ultraweak radiation in biological tissues. It is now recognized that plants, animal and human cells emit this very weak biophotonic emission which can be readily measured with a sensitive photomultiplier system. UVA laser induced biophotonic emission of cultured cells was used in this report with the intention to detect biophysical changes between young and adult fibroblasts as well as between fibroblasts and keratinocytes. With suspension densities ranging from 1-8 x 106 cells/ml, it was evident that an increase of the UVA-laser-light induced photon emission intensity could be observed in young as well as adult fibroblastic cells. By the use of this method to determine ultraweak light emission, photons in cell suspensions in low volumes (100 microl) could be detected, in contrast to previous procedures using quantities up to 10 ml. Moreover, the analysis has been further refined by turning off the photomultiplier system electronically during irradiation leading to the first measurements of induced light emission in the cells after less than 10 micros instead of more than 100 milliseconds. These significant changes lead to an improvement factor up to 106 in comparison to classical detection procedures. In addition, different skin cells as fibroblasts and keratinocytes stemming from the same donor were measured using this new highly sensitive method in order to find new biophysical insight of light pathways. This is important in view to develop new strategies in biophotonics especially for use in alternative therapies.

Ultra high throughput sequencing in human DNA variation detection: a comparative study on the NDUFA3-PRPF31 region.

BACKGROUND: Ultra high throughput sequencing (UHTS) technologies find an important application in targeted resequencing of candidate genes or of genomic intervals from genetic association studies. Despite the extraordinary power of these new methods, they are still rarely used in routine analysis of human genomic variants, in part because of the absence of specific standard procedures. The aim of this work is to provide human molecular geneticists with a tool to evaluate the best UHTS methodology for efficiently detecting DNA changes, from common SNPs to rare mutations. METHODOLOGY/PRINCIPAL FINDINGS: We tested the three most widespread UHTS platforms (Roche/454 GS FLX Titanium, Illumina/Solexa Genome Analyzer II and Applied Biosystems/SOLiD System 3) on a well-studied region of the human genome containing many polymorphisms and a very rare heterozygous mutation located within an intronic repetitive DNA element. We identify the qualities and the limitations of each platform and describe some peculiarities of UHTS in resequencing projects. CONCLUSIONS/SIGNIFICANCE: When appropriate filtering and mapping procedures are applied UHTS technology can be safely and efficiently used as a tool for targeted human DNA variations detection. Unless particular and platform-dependent characteristics are needed for specific projects, the most relevant parameter to consider in mainstream human genome resequencing procedures is the cost per sequenced base-pair associated to each machine.

Molecular genetics of rare hereditary diseases via genome-wide and integrated approaches

ABSTRACTThe Online Mendelian Inheritance in Man database (OMIM) reports about 3000 Mendelian diseases of known causal gene and about 2000 that remain to be mapped. These cases are often difficult to solve because of the rareness of the disease, the structure of the family (too big or too small) or the heterogeneity of the phenotype. The goal of this thesis is to explore the current genetic tools, before the advent of ultra high throughput sequencing, and integrate them in the attempt to map the genes behind the four studied cases. In this framework we have studied a small family with a recessive disease, a modifier gene for the penetrance of a dominant mutation, a large extended family with a cardiac phenotype and clinical and/or allelic heterogeneity and we have molecularly analyzed a balanced chromosomal translocation.RESUMELa base de données des maladies à transmission mendélienne, Online Mendelian Inheritance in Man (OMIM), contient environ 3000 affections à caractère mendélien pour lesquelles le gène responsable est connu et environ 2000 qui restent à élucider.Les cas restant à résoudre sont souvent difficiles soit par le caractère intrinsèquement rare de ces maladies soit à cause de difficultés structurelles (famille trop petite ou trop étendue) ou hétérogénéité du phénotype ou génétique. Cette thèse s'inscrit avant l'arrivée des nouveaux outils de séquençage à haut débit. Son but est d'explorer les outils génétiques actuels, et de les intégrer pour trouver les gènes impliqués dans quatre cas représentant chacun une situation génétique différente : nous avons étudié une famille de quatre individus avec une transmission récessive, recherché un gène modificateur de la pénétrance de mutations dominantes, étudié une famille étendue présentant un phénotype cardiaque cliniquement et/ou allèliquement hétérogène et nous avons fait l'analyse moléculaire d'une translocation chromosomique balancée.

Isolamento e Caracterização Fenotípica e Molecular de Parasitos Leishmania sp. de Pacientes com Leishmaniose Tegumentar Americana (LTA) Atendidos no Hospital Universitário Lauro Wanderley da Universidade Federal da Paraíba

As leishmanioses são um complexo de doenças infecto-parasitárias endêmicas em 88 países que constituem um grave problema de saúde pública. Diversas espécies do gênero Leishmania são os agentes causadores da doença, dentre elas a espécie L. (V.) braziliensis associada a diferentes quadros clínicos da Leishmaniose Tegumentar Americana. No presente trabalho foram obtidos nove isolados de Leishmania sp. oriundos de LTA, sendo sete isolados (87%) oriundos de pacientes residentes no município de Pilões, um (13%) da cidade de João Pessoa, ambas regiões, do estado da Paraíba. Todos os nove isolados foram identificados como sendo da espécie L. (V.) braziliensis através de PCR específica. As análises de caracterização molecular pela técnica de RAPD-PCR mostraram que esses isolados compartilharam 62,63% revelando diferenças genotípicas entre elas. Contudo, os isolados AF e JSL, ambos provenientes de lesões disseminadas, tiveram o maior percentual de bandas compartilhadas dentre os isolados. Outra técnica molecular utilizada foi o SSR-PCR com o iniciador K7 que também foi capaz de demontrar polimorfismo entre os isolados. Nas análises de PCR-RLFP das regiões ITS1, foram encontradas perfis diferentes entre os isolados, onde MRSS, JMTS, AF, JCTS, JRL apresentaram o mesmo perfil de bandas e os isolados JSL, JCNS, MFTS e JAS tiveram, cada um, perfis de bandas distintos. Na análise de PCR-RLFP da região do gene hsp70, JSL, AF, JCTS, JRL, MFTS apresentaram o mesmo perfil de bandas e os isolados, MRSS, JMTS, JCNS e JAS perfis de bandas distintos. Adicionalmente foram também observadas diferenças fenotípicas entre estes isolados, visto que em dado momento de cultivo, todos apresentaram comportamento diferenciado, além de demonstrarem diferenças quanto à sensibilidade às drogas utilizadas na terapêutica das leishmanioses. Portanto estes estudos revelam que os isolados de L. (V.) braziliensis obtidos no estado da Paraíba demonstraram um significativo polimorfismo genético, revelando um alto nível de variação intraespecífica.

Costos metabólicos de Engraulis ringens y Sardinops sagax en relación al peso, temperatura y el nivel de actividad

El metabolismo estándar de la anchoveta es más alto que el de la sardina; en .las larvas por un factor promedio de 2.3 y por 2.1 en juveniles y adultos en el rango de temperaturas de 14-20ºC. Lo opuesto sucede en el metabolismo activo: para una velocidad de natación de un cuerpo por segundo( =metabolismo de rutina), la anchoveta adulta con un peso mayor de 20 g a 20ºC y mayor de 50 g a 14ºC gasta menos energía que la sardina de tamaños similares; incrementándose la velocidad a 3 c.p .seg a 17°C.todos los tamaños mayores de 1.5 g de Sardina necesitan más energía que las anchovetas de pesos similares (una sardina de 5 g 1.5 veces y una de 25 g 3.8 veces más). Un cambio de temperatura de 6ºc (de 14° C a 20°c) afecta a ambas especies en una forma diferente : asumiendo una velocidad de natación de 1 cuerpo por segundo, una larva de anchoveta( 0.1 g) tiene que incrementar sus gastos metabólicos dos veces más que la larva de sardina, pero una anchoveta adulta( 40 g)necesita solo un 60% de lo que requiere una sardina del mismo tamaño .

Efecto de la suplementación con ácidos grasos de cadena media sobre la evolución de peso en lechones con poco peso al nacimiento

La evolución de peso en los períodos de lactación y transición de 583 lechones fue estudiada mediante un análisis estadístico, evaluando el efecto de la suplementación con ácidos grasos de cadena media (AGCM) en lechones con poco peso al nacimiento. 188 de los 375 lechones que nacieron con un peso al nacimiento (PN) &1250 g recibieron 3mL de AGCM cada 24 h durante los primeros 3 días de vida; su peso medio al destete (día 28) fue inferior respecto al grupo control (lechones no suplementados) (-114,17 g). No obstante, 106 de los 180 lechones nacidos con un PN &1000 g fueron suplementados, y su peso medio al destete y a finales de transición (día 63) fue superior respecto al grupo control (destete: +315,16 g; día 63: +775,47 g). Finalmente, los lechones suplementados con PN&800 g tuvieron los peores resultados: su diferencia de peso medio al destete fue de -177,58 g respecto al grupo control. Por lo tanto, en esta prueba fueron estudiados los lechones con un PN entre 800 y 999 g porque el grupo suplementado al destete tuvo una diferencia de peso medio considerable respecto al grupo control:+511,58 g. Asimismo, considerando una probabilidad de error inferior a 0,05, no hubieron diferencias significativas en las diferentes categorías de PN analizadas. De todas maneras, es importante destacar el alto grado de significación en la suplementación con AGCM en lechones con PN entre 800 y 999g (P=0,059). Por otra parte, el PN del grupo suplementado con PN&1000 g fue inferior que el del grupo no suplementado con PN&1000 g; esta diferencia de PN fue significativa (P=0,004) y como consecuencia el grado de significación en la suplementación con AGCM en lechones con PN entre 800 y 999 g fue inferior al esperado. Además, en esta prueba se incluyeron algunos resultados generales y también un análisis simple de supervivencia, aunque no era el objetivo principal

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.

Influência do estádio de maturação e da embalagem de polietileno na frigoconservação de ameixa

Um experimento visando verificar a influência do estádio de maturação e da embalagem de polietileno, durante o armazenamento de ameixa (Prunus salicina Lindl.) cultivar Amarelinha, foi realizado na Embrapa - Centro de Pesquisa Agropecuária de Clima Temperado (CPACT), em Pelotas, RS. Foram utilizadas frutas em três estádios de maturação: verde, semimaduro e maduro, embaladas ou não em sacos de polietileno. O armazenamento refrigerado realizou-se a 0ºC e 90-95% UR, por 14, 28 e 42 dias, seguido da comercialização simulada por três dias a 25-26ºC. As frutas semimaduras foram as que perderam menos peso ao longo da frigoconservação e comercialização simulada. A embalagem de polietileno reduziu as perdas de peso para menos de 1% ao longo do armazenamento. As frutas não embaladas perderam até 7% em peso, apresentando sintomas de murchamento a partir dos 28 dias, principalmente as do estádio maduro. A firmeza de polpa e a acidez total titulável decresceram ao longo do experimento nos três estádios de maturação, e a perda elevada de acidez resultou em sobrematuração das frutas, principalmente aos 42 dias de armazenamento. Não ocorreu desintegração interna nas frutas e a incidência de podridões aumentou aos 42 dias. Conclui-se que o armazenamento refrigerado deve ser feito até os 28 dias.

Herança do peso de grãos primários e secundários de aveia

Aliar boa qualidade de grãos às variedades com alto rendimento, ciclo curto e adaptação é um dos objetivos dos programas de melhoramento de aveia no sul do Brasil. O peso dos grãos tem sido um dos critérios utilizados para determinar essa qualidade; porém, para melhor manipular o peso dos grãos nos programas de melhoramento, é necessário o estudo de suas bases genéticas em germoplasma adaptado de aveia. Este trabalho foi realizado com o objetivo de estudar a herança dos caracteres peso de grãos primários e secundários da aveia. Para isso, foram pesadas amostras de 20 grãos primários e secundários de um número variável de plantas nas gerações P1, P2, F1, F2 e F3 de cinco cruzamentos de aveia. Com esses dados, foram obtidas as distribuições de freqüência, variâncias, os efeitos gênicos predominantes, e estimados os valores de herdabilidade no sentido amplo, e usando regressão genitor-progênie. Os resultados mostraram que existe variabilidade em progênies precoces no peso de grãos primários e secundários, e que aditividade e dominância são os efeitos gênicos atuantes. Não foi detectada a presença de genes maiores para a expressão dessas características. Além disso, variações contínuas em F2 e herdabilidades baixas a moderadas indicam que seleção amena pode ser aplicada em gerações iniciais.

Incidência de ascite em frangos de corte alimentados com rações comerciais de alto nível energético

O trabalho objetivou avaliar os efeitos de diferentes rações comerciais com alto nível energético na incidência de ascite em frangos de corte. Foram utilizadas 1.200 aves de uma mesma linhagem comercial (Hubbard), distribuídas em 12 boxes, segundo um delineamento inteiramente ao acaso, com quatro tratamentos e três repetições de 100 aves cada. Os tratamentos foram constituídos por três diferentes rações comerciais trituradas (B, C e D) comparadas com o controle, uma ração farelada inicial (A), do primeiro ao 39º dia de idade. Não houve diferenças entre os tratamentos quanto ao consumo, peso e ganho de peso das aves. Em relação à conversão alimentar, o tratamento C apresentou resultado significativamente melhor; entretanto, foi observada neste mesmo tratamento, a maior taxa de mortalidade. O maior motivo dos óbitos registrados foi a síndrome ascítica. Conclui-se que existe um favorecimento de surto de ascite pelas rações com melhor conversão alimentar nas aves.

Laser-Ultraviolet-A induced ultra weak photon emission in human skin cells: A biophotonic comparison between keratinocytes and fibroblasts

Photons participate in many atomic and molecular interactions and processes. Recent biophysical research has discovered an ultraweak radiation in biological tissues. It is now recognized that plants, animal and human cells emit this very weak biophotonic emission which can be readily measured with a sensitive photomultiplier system. UVA laser induced biophotonic emission of cultured cells was used in this report with the intention to detect biophysical changes between young and adult fibroblasts as well as between fibroblasts and keratinocytes. With suspension densities ranging from 1-8x106 cells/ml, it was evident that an increase of the UVA-laser-light induced photon emission intensity could be observed in young as well as adult fibroblastic cells. By the use of this method to determine ultraweak light emission, photons in cell suspensions in low volumes (100 mu l) could be detected, in contrast to previous procedures using quantities up to 10 ml. Moreover, the analysis has been further refined by turning off the photomultiplier system electronically during irradiation leading to the first measurements of induced light emission in the cells after less than 10 mu s instead of more than 100 milliseconds. These significant changes lead to an improvement factor up to 106 in comparison to classical detection procedures. In addition, different skin cells as fibroblasts and keratinocytes stemining from the same donor were measured using this new highly sensitive method in order to find new biophysical insight of light pathways. This is important in view to develop new strategies in biophotonics especially for use in alternative therapies.