A simplified minimal residual disease polymerase chain reaction method at early treatment points can stratify children with acute lymphoblastic leukemia into good and poor outcome groups

Background Minimal residual disease is an important independent prognostic factor in childhood acute lymphoblastic leukemia. The classical detection methods such as multiparameter flow cytometry and real-time quantitative polymerase chain reaction analysis are expensive, time-consuming and complex, and require considerable technical expertise. Design and Methods We analyzed 229 consecutive children with acute lymphoblastic leukemia treated according to the GBTLI-99 protocol at three different Brazilian centers. Minimal residual disease was analyzed in bone marrow samples at diagnosis and on days 14 and 28 by conventional homo/heteroduplex polymerase chain reaction using a simplified approach with consensus primers for IG and TCR gene rearrangements. Results At least one marker was detected by polymerase chain reaction in 96.4%, of the patients. By combining the minimal residual disease results obtained on days 14 and 28, three different prognostic groups were identified: minimal residual disease negative on days 14 and 28, positive on day 14/negative on day 28, and positive on both. Five-year event-free survival rates were 85%, 75.6%,, and 27.8%, respectively (p<0.0001). The same pattern of stratification held true for the group of intensively treated children. When analyzed in other subgroups of patients such as those at standard and high risk at diagnosis, those with positive B-derived CD10, patients positive for the TEL/AML1 transcript, and patients in morphological remission on a day 28 marrow, the event-free survival rate was found to be significantly lower in patients with positive minimal residual disease on day 28. Multivariate analysis demonstrated that the detection of minimal residual disease on day 28 is the most significant prognostic factor. Conclusions This simplified strategy for detection of minimal residual disease was feasible, reproducible, cheaper and simpler when compared with other methods, and allowed powerful discrimination between children with acute lymphoblastic leukemia with a good and poor outcome.

In Vitro Identification of Novel Plasminogen-Binding Receptors of the Pathogen Leptospira interrogans

Background: Leptospirosis is a multisystem disease caused by pathogenic strains of the genus Leptospira. We have reported that Leptospira are able to bind plasminogen (PLG), to generate active plasmin in the presence of activator, and to degrade purified extracellular matrix fibronectin. Methodology/Principal Findings: We have now cloned, expressed and purified 14 leptospiral recombinant proteins. The proteins were confirmed to be surface exposed by immunofluorescence microscopy and were evaluated for their ability to bind plasminogen (PLG). We identified eight as PLG-binding proteins, including the major outer membrane protein LipL32, the previously published rLIC12730, rLIC10494, Lp29, Lp49, LipL40 and MPL36, and one novel leptospiral protein, rLIC12238. Bound PLG could be converted to plasmin by the addition of urokinase-type PLG activator (uPA), showing specific proteolytic activity, as assessed by its reaction with the chromogenic plasmin substrate, D-Val-Leu-Lys 4-nitroanilide dihydrochloride. The addition of the lysine analog 6-aminocaproic acid (ACA) inhibited the protein-PLG interaction, thus strongly suggesting the involvement of lysine residues in plasminogen binding. The binding of leptospiral surface proteins to PLG was specific, dose-dependent and saturable. PLG and collagen type IV competed with LipL32 protein for the same binding site, whereas separate binding sites were observed for plasma fibronectin. Conclusions/Significance: PLG-binding/activation through the proteins/receptors on the surface of Leptospira could help the bacteria to specifically overcome tissue barriers, facilitating its spread throughout the host.

Identification of three distinguishable phenotypes in golden retriever muscular dystrophy

Duchenne muscular dystrophy (DMD) is a human disease characterized by progressive and irreversible skeletal muscle degeneration caused by mutations in genes coding for important muscle proteins. Unfortunately, there is no efficient treatment for this disease; it causes progressive loss of motor and muscular ability until death. The canine model (golden retriever muscular dystrophy) is similar to DMD, showing similar clinical signs. Fifteen dogs were followed from birth and closely observed for clinical signs. Dogs had their disease status confirmed by polymerase chain reaction analysis and genotyping. Clinical observations of musculoskeletal, morphological, gastrointestinal, respiratory, cardiovascular, and renal features allowed us to identify three distinguishable phenotypes in dystrophic dogs: mild (grade I), moderate (grade II) and severe (grade III). These three groups showed no difference in dystrophic alterations of muscle morphology and creatine kinase levels. This information will be useful for therapeutic trials, because DMD also shows significant, inter- and intra-familiar clinical variability. Additionally, being aware of phenotypic differences in this animal model is essential for correct interpretation and understanding of results obtained in pre-clinical trials.

Additive genetic relationship of longevity with fertility and production traits in Nellore cattle based on bivariate models

Survival or longevity is an economically important trait in beef cattle. The main inconvenience for its inclusion in selection criteria is delayed recording of phenotypic data and the high computational demand for including survival in proportional hazard models. Thus, identification of a longevity-correlated trait that could be recorded early in life would be very useful for selection purposes. We estimated the genetic relationship of survival with productive and reproductive traits in Nellore cattle, including weaning weight (WW), post-weaning growth (PWG), muscularity (MUSC), scrotal circumference at 18 months (SC18), and heifer pregnancy (HP). Survival was measured in discrete time intervals and modeled through a sequential threshold model. Five independent bivariate Bayesian analyses were performed, accounting for cow survival and the five productive and reproductive traits. Posterior mean estimates for heritability (standard deviation in parentheses) were 0.55 (0.01) for WW, 0.25 (0.01) for PWG, 0.23 (0.01) for MUSC, and 0.48 (0.01) for SC18. The posterior mean estimates (95% confidence interval in parentheses) for the genetic correlation with survival were 0.16 (0.13-0.19), 0.30 (0.25-0.34), 0.31 (0.25-0.36), 0.07 (0.02-0.12), and 0.82 (0.78-0.86) for WW, PWG, MUSC, SC18, and HP, respectively. Based on the high genetic correlation and heritability (0.54) posterior mean estimates for HP, the expected progeny difference for HP can be used to select bulls for longevity, as well as for post-weaning gain and muscle score.

A frequency approach to identifying asteroid families II. Families interacting with nonlinear secular resonances and low-order mean-motion resonances

Aims. In an earlier paper we introduced a new method for determining asteroid families where families were identified in the proper frequency domain (n, g, g + s) ( where n is the mean-motion, and g and s are the secular frequencies of the longitude of pericenter and nodes, respectively), rather than in the proper element domain (a, e, sin(i)) (semi-major axis, eccentricity, and inclination). Here we improve our techniques for reliably identifying members of families that interact with nonlinear secular resonances of argument other than g or g + s and for asteroids near or in mean-motion resonant configurations. Methods. We introduce several new distance metrics in the frequency space optimal for determining the diffusion in secular resonances of argument 2g - s, 3g - s, g - s, s, and 2s. We also regularize the dependence of the g frequency as a function of the n frequency (Vesta family) or of the eccentricity e (Hansa family). Results. Our new approaches allow us to recognize as family members objects that were lost with previous methods, while keeping the advantages of the Carruba & Michtchenko (2007, A& A, 475, 1145) approach. More important, an analysis in the frequency domain permits a deeper understanding of the dynamical evolution of asteroid families not always obtainable with an analysis in the proper element domain.

Optical identification of the transient supersoft X-ray source RX J0527.8-6954, in the LMC

Context. Close binary supersoft X-ray sources (CBSS) are binary systems that contain a white dwarf with stable nuclear burning on its surface. These sources, first discovered in the Magellanic Clouds, have high accretion rates and near-Eddington luminosities (10(37)-10(38) erg s(-1)) with high temperatures (T = 2-7 x 10(5) K). Aims. The total number of known objects in the MC is still small and, in our galaxy, even smaller. We observed the field of the unidentified transient supersoft X-ray source RX J0527.8-6954 in order to identify its optical counterpart. Methods. The observation was made with the IFU-GMOS on the Gemini South telescope with the purpose of identifying stars with possible He II or Balmer emission or else of observing nebular extended jets or ionization cones, features that may be expected in CBSS. Results. The X-ray source is identified with a B5e V star that is associated with subarcsecond extended H alpha emission, possibly bipolar. Conclusions. If the primary star is a white dwarf, as suggested by the supersoft X-ray spectrum, the expected orbital period exceeds 21 h; therefore, we believe that the 9.4 h period found so far is not associated to this system.

A SEARCH FOR UNRECOGNIZED CARBON-ENHANCED METAL-POOR STARS IN THE GALAXY

We have developed a new procedure to search for carbon-enhanced metal-poor (CEMP) stars from the Hamburg/ESO (HES) prism-survey plates. This method employs an extended line index for the CH G band, which we demonstrate to have superior performance when compared to the narrower G-band index formerly employed to estimate G-band strengths for these spectra. Although CEMP stars have been found previously among candidate metal-poor stars selected from the HES, the selection on metallicity undersamples the population of intermediate-metallicity CEMP stars (-2.5 <= [Fe/H] <= -1.0); such stars are of importance for constraining the onset of the s-process in metal-deficient asymptotic giant branch stars (thought to be associated with the origin of carbon for roughly 80% of CEMP stars). The new candidates also include substantial numbers of warmer carbon-enhanced stars, which were missed in previous HES searches for carbon stars due to selection criteria that emphasized cooler stars. A first subsample, biased toward brighter stars (B < 15.5), has been extracted from the scanned HES plates. After visual inspection (to eliminate spectra compromised by plate defects, overlapping spectra, etc., and to carry out rough spectral classifications), a list of 669 previously unidentified candidate CEMP stars was compiled. Follow-up spectroscopy for a pilot sample of 132 candidates was obtained with the Goodman spectrograph on the SOAR 4.1 m telescope. Our results show that most of the observed stars lie in the targeted metallicity range, and possess prominent carbon absorption features at 4300 angstrom. The success rate for the identification of new CEMP stars is 43% (13 out of 30) for [Fe/H] < -2.0. For stars with [Fe/H] < -2.5, the ratio increases to 80% (four out of five objects), including one star with [Fe/H] < -3.0.

The B0.5 IVe CoRoT target HD 49330 II. Spectroscopic ground-based observations

Context. We present spectroscopic ground-based observations of the early Be star HD 49330 obtained simultaneously with the CoRoT-LRA1 run just before the burst observed in the CoRoT data. Aims. Ground-based spectroscopic observations of the early Be star HD 49330 obtained during the precursor phase and just before the start of an outburst allow us to disantangle stellar and circumstellar contributions and identify modes of stellar pulsations in this rapidly rotating star. Methods. Time series analysis (TSA) is performed on photospheric line profiles of He I and Si III by means of the least squares method. Results. We find two main frequencies f1 = 11.86 c d(-1) and f2 = 16.89 c d(-1) which can be associated with high order p-mode pulsations. We also detect a frequency f3 = 1.51 c d(-1) which can be associated with a low order g-mode. Moreover we show that the stellar line profile variability changed over the spectroscopic run. These results are in agreement with the results of the CoRoT data analysis, as shown in Huat et al. (2009). Conclusions. Our study of mid-and short-term spectroscopic variability allows the identification of p-and g-modes in HD 49330. It also allows us to display changes in the line profile variability before the start of an outburst. This brings new constraints for the seimic modelling of this star.

Automated supervised classification of variable stars in the CoRoT programme Method and application to the first four exoplanet fields

Aims. In this work, we describe the pipeline for the fast supervised classification of light curves observed by the CoRoT exoplanet CCDs. We present the classification results obtained for the first four measured fields, which represent a one-year in-orbit operation. Methods. The basis of the adopted supervised classification methodology has been described in detail in a previous paper, as is its application to the OGLE database. Here, we present the modifications of the algorithms and of the training set to optimize the performance when applied to the CoRoT data. Results. Classification results are presented for the observed fields IRa01, SRc01, LRc01, and LRa01 of the CoRoT mission. Statistics on the number of variables and the number of objects per class are given and typical light curves of high-probability candidates are shown. We also report on new stellar variability types discovered in the CoRoT data. The full classification results are publicly available.

PFLEIDERER 2: IDENTIFICATION OF A NEW GLOBULAR CLUSTER IN THE GALAXY

We provide evidence that indicates the star cluster Pfleiderer 2, which is projected in a rich field, as a newly identified Galactic globular cluster. Since it is located in a crowded field, core extraction and decontamination tools were applied to reveal the cluster sequences in B, V, and I color-magnitude diagrams (CMDs). The main CMD features of Pfleiderer 2 are a tilted red giant branch and a red horizontal branch, indicating a high metallicity around solar. The reddening is E(B - V) = 1.01. The globular cluster is located at a distance of d(circle dot) = 16 +/- 2 kpc from the Sun. The cluster is located 2.7 kpc above the Galactic plane and at a distance of R(GC) = 9.7 kpc from the Galactic center, which is unusual for a metal-rich globular cluster.

Heteroduplex formation and S1 digestion for mapping alternative splicing sites

The identification of alternatively spliced transcripts has contributed to a better comprehension of developmental mechanisms, tissue-specific physiological processes and human diseases. Polymerase chain reaction amplification of alternatively spliced variants commonly leads to the formation of heteroduplexes as a result of base pairing involving exons common between the two variants. S1 nuclease cleaves single-stranded loops of heteroduplexes and also nicks the opposite DNA strand. In order to establish a strategy for mapping alternative splice-prone sites in the whole transcriptome, we developed a method combining the formation of heteroduplexes between 2 distinct splicing variants and S1 nuclease digestion. For 20 consensuses identified here using this methodology, 5 revealed a conserved splice site after inspection of the cDNA alignment against the human genome (exact splice sites). For 8 other consensuses, conserved splice sites were mapped at 2 to 30 bp from the border, called proximal splice sites; for the other 7 consensuses, conserved splice sites were mapped at 40 to 800 bp, called distal splice sites. These latter cases showed a nonspecific activity of S1 nuclease in digesting double-strand DNA. From the 20 consensuses identified here, 5 were selected for reverse transcription-polymerase chain reaction validation, confirming the splice sites. These data showed the potential of the strategy in mapping splice sites. However, the lack of specificity of the S1 nuclease enzyme is a significant obstacle that impedes the use of this strategy in large-scale studies.

Alternative splicing enriched cDNA libraries identify breast cancer-associated transcripts

Background: Alternative splicing (AS) is a central mechanism in the generation of genomic complexity and is a major contributor to transcriptome and proteome diversity. Alterations of the splicing process can lead to deregulation of crucial cellular processes and have been associated with a large spectrum of human diseases. Cancer-associated transcripts are potential molecular markers and may contribute to the development of more accurate diagnostic and prognostic methods and also serve as therapeutic targets. Alternative splicing-enriched cDNA libraries have been used to explore the variability generated by alternative splicing. In this study, by combining the use of trapping heteroduplexes and RNA amplification, we developed a powerful approach that enables transcriptome-wide exploration of the AS repertoire for identifying AS variants associated with breast tumor cells modulated by ERBB2 (HER-2/neu) oncogene expression. Results: The human breast cell line (C5.2) and a pool of 5 ERBB2 over-expressing breast tumor samples were used independently for the construction of two AS-enriched libraries. In total, 2,048 partial cDNA sequences were obtained, revealing 214 alternative splicing sequence-enriched tags (ASSETs). A subset with 79 multiple exon ASSETs was compared to public databases and reported 138 different AS events. A high success rate of RT-PCR validation (94.5%) was obtained, and 2 novel AS events were identified. The influence of ERBB2-mediated expression on AS regulation was evaluated by capillary electrophoresis and probe-ligation approaches in two mammary cell lines (Hb4a and C5.2) expressing different levels of ERBB2. The relative expression balance between AS variants from 3 genes was differentially modulated by ERBB2 in this model system. Conclusions: In this study, we presented a method for exploring AS from any RNA source in a transcriptome-wide format, which can be directly easily adapted to next generation sequencers. We identified AS transcripts that were differently modulated by ERBB2-mediated expression and that can be tested as molecular markers for breast cancer. Such a methodology will be useful for completely deciphering the cancer cell transcriptome diversity resulting from AS and for finding more precise molecular markers.

Identification and Phylogenetic Analysis of Heme Synthesis Genes in Trypanosomatids and Their Bacterial Endosymbionts

It has been known for decades that some insect-infecting trypanosomatids can survive in culture without heme supplementation while others cannot, and that this capability is associated with the presence of a betaproteobacterial endosymbiont in the flagellate's cytoplasm. However, the specific mechanisms involved in this process remained obscure. In this work, we sequence and phylogenetically analyze the heme pathway genes from the symbionts and from their hosts, as well as from a number of heme synthesis-deficient Kinetoplastida. Our results show that the enzymes responsible for synthesis of heme are encoded on the symbiont genomes and produced in close cooperation with the flagellate host. Our evidence suggests that this synergistic relationship is the end result of a history of extensive gene loss and multiple lateral gene transfer events in different branches of the phylogeny of the Trypanosomatidae.

Identification and Characterization of Ixodes scapularis Antigens That Elicit Tick Immunity Using Yeast Surface Display

Repeated exposure of rabbits and other animals to ticks results in acquired resistance or immunity to subsequent tick bites and is partially elicited by antibodies directed against tick antigens. In this study we demonstrate the utility of a yeast surface display approach to identify tick salivary antigens that react with tick-immune serum. We constructed an Ixodes scapularis nymphal salivary gland yeast surface display library and screened the library with nymph-immune rabbit sera and identified five salivary antigens. Four of these proteins, designated P8, P19, P23 and P32, had a predicted signal sequence. We generated recombinant (r) P8, P19 and P23 in a Drosophila expression system for functional and immunization studies. rP8 showed anti-complement activity and rP23 demonstrated anti-coagulant activity. Ixodes scapularis feeding was significantly impaired when nymphs were fed on rabbits immunized with a cocktail of rP8, rP19 and rP23, a hall mark of tick-immunity. These studies also suggest that these antigens may serve as potential vaccine candidates to thwart tick feeding.

Antimicrobial activities of Garcinia kola on oral Fusobacterium nucleatum and biofilm

The extracts from the root, bark and seed of Garcinia kola are currently used in traditional medicine in Nigeria. The aim of this study was to evaluate the inhibitory activity of crude extracts of G. kola on Fusobacterium nucleatum isolated from the oral cavity. Methanol and aqueous extracts were prepared from the seed and the minimal inhibitory concentration was evaluated by the agar dilution method, using a Wilkins-Chalgren agar supplemented with horse blood (5%), hemin (5 mu g/ml) and menadione (1 mu g/ml). Antimicrobial activity of plant extracts on microbial biofilms was determined in microtiter plates. The seed of G. kola demonstrated significant inhibitory action on F. nucleatum isolates at a concentration of 1.25 and 12.5 mg/ml for amoxicillin resistant strain. It was able to inhibit the microbial biofilm formed by the association of F. nucleatum with Porphyromonas gingivalis ATCC 33277, Aggregatibacter actinomycetemcomitans ATCC 33384 and Prevotella intermedia ATCC 2564 at a concentration of 25 mg/ml. The in-vitro inhibitory effect of G. kola on F. nucleatum population suggests a potential role for its use in oral hygiene.