985 resultados para high-resolution comparative genomic hybridization


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Amino acid sequencing by recombinant DNA technology, although dramatically useful, is subject to base reading errors, is indirect, and is insensitive to posttranslational processing. Mass spectrometry techniques can provide molecular weight data from even relatively large proteins for such cDNA sequences and can serve as a check of an enzyme's purity and sequence integrity. Multiply-charged ions from electrospray ionization can be dissociated to yield structural information by tandem mass spectrometry, providing a second method for gaining additional confidence in primary sequence confirmation. Here, accurate (+/- 1 Da) molecular weight and molecular ion dissociation information for human muscle and brain creatine kinases has been obtained by electrospray ionization coupled with Fourier-transform mass spectrometry to help distinguish which of several published amino acid sequences for both enzymes are correct. The results herein are consistent with one published sequence for each isozyme, and the heterogeneity indicated by isoelectric focusing due to 1-Da deamidation changes. This approach appears generally useful for detailed sequence verification of recombinant proteins.

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Este estudo teve como objetivos (a) identificar mecanismos pelos quais rearranjos cromossômicos citogeneticamente equilibrados possam estar associados de maneira causal a determinados quadros clínicos e (b) contribuir para a compreensão dos mecanismos de formação desses rearranjos. Para isso, foram estudados 45 rearranjos cromossômicos citogeneticamente equilibrados (29 translocações, 10 inversões e seis rearranjos complexos), detectados em pacientes que apresentavam malformações congênitas, comprometimento do desenvolvimento neuropsicomotor ou déficit intelectual. Foram 31 rearranjos cromossômicos esporádicos, três familiais que segregavam com o quadro clínico e mais 11 rearranjos cromossômicos herdados de genitores fenotipicamente normais. Inicialmente os pontos de quebra desses rearranjos foram mapeados por hibridação in situ fluorescente (FISH). A busca por microdeleções e duplicações genômicas foi realizada por a-CGH. A investigação dos pontos de quebra prosseguiu com a aplicação da técnica de Mate-Pair Sequencing (MPS), que permite localizar as quebras em segmentos de 100 pb - 1 kb, na maioria dos casos. Para obter os segmentos de junção das quebras no nível de pares de bases, os segmentos delimitados por MPS foram sequenciados pelo método de Sanger. A análise por aCGH revelou microdeleções ou microduplicações localizadas nos cromossomos rearranjados, em 12 dos 45 pacientes investigados (27%). A análise de 27 rearranjos por MPS permitiu a caracterização dos pontos de junção das quebras. MPS expandiu o número de pontos de quebra, detectados por análise do cariótipo ou aCGH, de 114 para 156 (em resolução < 2kb, na maioria dos casos). O número de pontos de quebra/rearranjo variou de 2 a 20. Os 156 pontos de quebra resultaram em 86 variantes estruturais equilibradas e outras 32 variantes não equilibradas. Perdas e ganhos de segmentos submiscroscópicos nos cromossomos rearranjados constituíram a principal causa ou, provavelmente, contribuíram para o quadro clínico de 12 dos 45 pacientes. Em cinco desses 12 rearranjos foram detectadas por MPS a interrupção de genes já relacionados à doença, ou provável alteração de sua região reguladora, contribundo para o quadro clínico. Em quatro dos 33 rearranjos não associados a perdas ou ganhos de segmentos, a análise por MPS revelou a interrupção de genes que já foram anteriormente relacionados a doenças, explicando-se, assim, as características clínicas dos portadores; outro rearranjo pode ter levando alteração da expressão gênica de gene sensível a dosagem e ao quadro clínico. Um rearranjo cromossômico familial, identificado na análise após bandamento G como uma translocação equilibrada, t(2;22)(p14;q12), segregava com quadro de atraso do desenvolvimento neuropsicomotor e dificuldade de aprendizado associados a dismorfismos. A combinação das análises por FISH, aCGH e MPS revelou que se tratava, na verdade, de rearranjo complexo entre os cromossomos 2, 5 e 22, incluindo 10 quebras. A segregação de diferentes desequilíbrios submicroscópicos em indivíduos afetados e clinicamente normais permitiu a compreensão da variabilidade clínica observada na família. Rearranjos equilibrados detectados em indivíduos afetados, mas herdados de genitores clinicamente normais, são, em geral, considerados como não tendo relação com o quadro clínico, apesar da possibilidade de desequilíbrios cromossômicos gerados por permuta desigual na meiose do genitor portador do rearranjo. Neste trabalho, a investigação de 11 desses rearranjos por aCGH não revelou perdas ou ganhos de segmentos nos cromossomos rearranjados. No entanto, a análise por aCGH da portadora de um desses rearranjos - inv(12)mat - revelou deleção de 8,7 Mb no cromossomo 8, como causa de seu fenótipo clínico. Essa deleção estava relacionada com outro rearranjo equilibrado também presente em sua mãe, independente da inversão. Para compreender os mecanismos de formação de rearranjos citogeneticamente equilibrados, investigamos os segmentos de junção no nível de pares de base. A análise por MPS que levou, na maioria dos casos, ao mapeamento dos pontos de quebras em segmentos <1kb permitiu o sequenciamento pelo método de Sanger de 51 segmentos de junções de 17 rearranjos. A ocorrência de blunt fusions ou inserções e deleções <10 pb, e a ausência de homologia ou a presença de micro homologia de 2 pb a 4 pb de extensão indicaram o mecanismo de junção de extremidades não homólogas (non-homologous end joinging; NHEJ), na maioria das 51 junções caracterizadas. As características de três dos quatro rearranjos mais complexos, com 17-20 quebras, indicaram sua formação pelo mecanismo de chromothripsis. Este estudo mostra a importância da análise genômica de variações de número de cópias por microarray, juntamente com o mapeamento dos pontos de quebra por MPS, para determinar a estrutura de rearranjos cromossômicos citogeneticamente equilibrados e seu impacto clínico. O mapeamento dos segmentos de junção por MPS, permitindo o sequenciamento pelo método de Sanger, foi essencial para a compreensão de mecanismos de formação desses rearranjos

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We present the most recent results of our ongoing long-term high resolution spectroscopic study of nearby (d ≤ 25 pc) FGK stars which aim is to characterize the local properties of the Galaxy, in particular the star-formation history. A through analysis has been carried out for 253 cool stars in the solar neighborhood. This includes radial and rotational velocities determinations, chromospheric activity levels inference, kinematic analysis, and age estimates. This study does not only shed new light on the issue of stellar formation history but also contributes to any present or future mission aiming to detect extra-solar planets. Exo-planets are likely to be found orbiting around nearby cool stars and their detection and characterization is highly dependent on the precise determination of fundamental stellar parameters such as age, activity levels. Therefore, our study is of paramount importance to ensure the success of any such mission.

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We present a library of Utrecht echelle spectrograph (UES) observations of a sample of F, G, K and M field dwarf stars covering the spectral range from 4800 Å to 10600 Å with a resolution of 55000. These spectra include some of the spectral lines most widely used as optical and near-infrared indicators of chromospheric activity such as Hβ, Mg I b triplet, Na I D_1, D_2, He I D_3, Hα, and Ca II IRT lines, as well as a large number of photospheric lines which can also be affected by chromospheric activity. The spectra have been compiled with the aim of providing a set of standards observed at high-resolution to be used in the application of the spectral subtraction technique to obtain the active-chromosphere contribution to these lines in chromospherically active single and binary stars. This library can also be used for spectral classification purposes. A digital version with all the spectra is available via ftp and the World Wide Web (WWW) in both ASCII and FITS formats.

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Accurate characterization of the microporous structure in porous solids is of paramount importance for several applications such as energy and gas storage, nanoconfinement reactions, and so on. Among the different techniques for precise textural characterization, high-precision gas adsorption measurement of probe molecules at cryogenic temperatures (e.g., N2 at 77.4 K and Ar at 87.3 K) is the most widely used, after appropriate calibration of the sample holder with a probe gas, which does not experience physisorption processes. Although traditionally helium has been considered not to be adsorbed in porous solids at cryogenic temperatures, here we show that even at 77.4 K (high above its boiling temperature, 4 K) the use of He in the calibration step can give rise to erroneous interpretations when narrow micropores/constrictions are present.

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Background: The pupillary light reflex characterizes the direct and consensual response of the eye to the perceived brightness of a stimulus. It has been used as indicator of both neurological and optic nerve pathologies. As with other eye reflexes, this reflex constitutes an almost instantaneous movement and is linked to activation of the same midbrain area. The latency of the pupillary light reflex is around 200 ms, although the literature also indicates that the fastest eye reflexes last 20 ms. Therefore, a system with sufficiently high spatial and temporal resolutions is required for accurate assessment. In this study, we analyzed the pupillary light reflex to determine whether any small discrepancy exists between the direct and consensual responses, and to ascertain whether any other eye reflex occurs before the pupillary light reflex. Methods: We constructed a binocular video-oculography system two high-speed cameras that simultaneously focused on both eyes. This was then employed to assess the direct and consensual responses of each eye using our own algorithm based on Circular Hough Transform to detect and track the pupil. Time parameters describing the pupillary light reflex were obtained from the radius time-variation. Eight healthy subjects (4 women, 4 men, aged 24–45) participated in this experiment. Results: Our system, which has a resolution of 15 microns and 4 ms, obtained time parameters describing the pupillary light reflex that were similar to those reported in previous studies, with no significant differences between direct and consensual reflexes. Moreover, it revealed an incomplete reflex blink and an upward eye movement at around 100 ms that may correspond to Bell’s phenomenon. Conclusions: Direct and consensual pupillary responses do not any significant temporal differences. The system and method described here could prove useful for further assessment of pupillary and blink reflexes. The resolution obtained revealed the existence reported here of an early incomplete blink and an upward eye movement.

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Context. The ongoing Gaia-ESO Public Spectroscopic Survey is using FLAMES at the VLT to obtain high-quality medium-resolution Giraffe spectra for about 105 stars and high-resolution UVES spectra for about 5000 stars. With UVES, the Survey has already observed 1447 FGK-type stars. Aims. These UVES spectra are analyzed in parallel by several state-of-the-art methodologies. Our aim is to present how these analyses were implemented, to discuss their results, and to describe how a final recommended parameter scale is defined. We also discuss the precision (method-to-method dispersion) and accuracy (biases with respect to the reference values) of the final parameters. These results are part of the Gaia-ESO second internal release and will be part of its first public release of advanced data products. Methods. The final parameter scale is tied to the scale defined by the Gaia benchmark stars, a set of stars with fundamental atmospheric parameters. In addition, a set of open and globular clusters is used to evaluate the physical soundness of the results. Each of the implemented methodologies is judged against the benchmark stars to define weights in three different regions of the parameter space. The final recommended results are the weighted medians of those from the individual methods. Results. The recommended results successfully reproduce the atmospheric parameters of the benchmark stars and the expected Teff-log  g relation of the calibrating clusters. Atmospheric parameters and abundances have been determined for 1301 FGK-type stars observed with UVES. The median of the method-to-method dispersion of the atmospheric parameters is 55 K for Teff, 0.13 dex for log  g and 0.07 dex for [Fe/H]. Systematic biases are estimated to be between 50−100 K for Teff, 0.10−0.25 dex for log  g and 0.05−0.10 dex for [Fe/H]. Abundances for 24 elements were derived: C, N, O, Na, Mg, Al, Si, Ca, Sc, Ti, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Y, Zr, Mo, Ba, Nd, and Eu. The typical method-to-method dispersion of the abundances varies between 0.10 and 0.20 dex. Conclusions. The Gaia-ESO sample of high-resolution spectra of FGK-type stars will be among the largest of its kind analyzed in a homogeneous way. The extensive list of elemental abundances derived in these stars will enable significant advances in the areas of stellar evolution and Milky Way formation and evolution.

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PURPOSE: To evaluate in a pilot study the visual, refractive, corneal topographic, and aberrometric changes after wavefront-guided LASIK or photorefractive keratectomy (PRK) using a high-resolution aberrometer to calculate the treatment for aberrated eyes. METHODS: Twenty aberrated eyes of 18 patients undergoing wavefront-guided LASIK or PRK using the VISX STARS4IR excimer laser and the iDesign aberrometer (Abbott Medical Optics, Inc., Santa Ana, CA) were enrolled in this prospective study. Three groups were differentiated: keratoconus post-CXL group including 11 keratoconic eyes (10 patients), post-LASIK group including 5 eyes (5 patients) with previous decentered LASIK treatments, and post-RK group including 4 eyes (3 patients) with previous radial keratotomy. Visual, refractive, contrast sensitivity, corneal topographic, and ocular aberrometric changes were evaluated during a 6-month follow-up. RESULTS: An improvement in uncorrected (UDVA) and corrected visual acuity (CDVA) associated with a reduction in the spherical equivalent was observed in the three groups, but was only statistically significant in the keratoconus post-CXL and post-LASIK groups (P ≤ .04). All eyes gained one or more lines of CDVA after surgery. Improvements in contrast sensitivity were observed in the three groups, but they were only statistically significant in the keratoconus post-CXL and post-LASIK groups (P ≤ .04). Regarding aberrations, a reduction was observed in trefoil aberrations in the keratoconus post-CXL group (P = .05) and significant reductions in higher-order and primary coma aberrations in the post-LASIK group (P = .04). CONCLUSIONS: Wavefront-guided laser enhancements using the evaluated platform seem to be safe and effective to restore the visual function in aberrated eyes.

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Resistance to antibiotics used against Neisseria gonorrhoeae infections is a major public health concern. Antimicrobial resistance (AMR) testing relies on time-consuming culture-based methods. Development of rapid molecular tests for detecting AMR determinants could provide valuable tools for surveillance, epidemiological studies and to inform individual case management. We developed a fast (<1.5 hrs) SYBR-green based real-time PCR method with high resolution melting (HRM) analysis. One triplex and three duplex reactions included two sequences for N. gonorrhoeae identification and seven determinants of resistance to extended-spectrum cephalosporins (ESCs), azithromycin, ciprofloxacin, and spectinomycin. The method was validated by testing 39 previously fully-characterized N. gonorrhoeae strains, 19 commensal Neisseria spp., and an additional panel of 193 gonococcal isolates. Results were compared with culture-based AMR determination. The assay correctly identified N. gonorrhoeae and the presence or absence of the seven AMR determinants. There was some cross-reactivity with non-gonococcal Neisseria species and the detection limit was 10(3)-10(4) gDNA copies/reaction. Overall, the platform accurately detected resistance to ciprofloxacin (sensitivity and specificity, 100%), ceftriaxone (sensitivity 100%, specificity 90%), cefixime (sensitivity 92%, specificity 94%), azithromycin and spectinomycin (both sensitivity and specificity, 100%). In conclusion, our methodology accurately detects mutations generating resistance to antibiotics used to treat gonorrhea. Low assay sensitivity prevents direct diagnostic testing of clinical specimens but this method can be used to screen collections of gonococcal isolates for AMR more quickly than with current culture-based AMR testing.

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Higher resolution pore-water samples were recovered at intervals of 0.3 to 3 m from selected cores during Leg 119 in order to identify zones where active geochemical reactions were occurring. In addition to shipboard measurements, solid- and dissolved-phase samples were analyzed at my shore-based laboratory. Solid-phase samples were analyzed for redox conditions, carbon, total metals, and leachable metals. Pore-water samples were analyzed for ammonia, silica, sulfate, and major cations. Data are presented in tables for 400 samples from Site 739 in Prydz Bay, East Antarctica, and Sites 736, 737, 738, 744, 745, and 746 at the Kerguelen Ridge, South Indian Ocean.