Application of self-quenched JH consensus primers for real-time quantitative PCR of IGH gene to minimal residual disease evaluation in multiple myeloma.

Monitoring multiple myeloma patients for relapse requires sensitive methods to measure minimal residual disease and to establish a more precise prognosis. The present study aimed to standardize a real-time quantitative polymerase chain reaction (PCR) test for the IgH gene with a JH consensus self-quenched fluorescence reverse primer and a VDJH or DJH allele-specific sense primer (self-quenched PCR). This method was compared with allele-specific real-time quantitative PCR test for the IgH gene using a TaqMan probe and a JH consensus primer (TaqMan PCR). We studied nine multiple myeloma patients from the Spanish group treated with the MM2000 therapeutic protocol. Self-quenched PCR demonstrated sensitivity of >or=10(-4) or 16 genomes in most cases, efficiency was 1.71 to 2.14, and intra-assay and interassay reproducibilities were 1.18 and 0.75%, respectively. Sensitivity, efficiency, and residual disease detection were similar with both PCR methods. TaqMan PCR failed in one case because of a mutation in the JH primer binding site, and self-quenched PCR worked well in this case. In conclusion, self-quenched PCR is a sensitive and reproducible method for quantifying residual disease in multiple myeloma patients; it yields similar results to TaqMan PCR and may be more effective than the latter when somatic mutations are present in the JH intronic primer binding site.

Incomplete DJH rearrangements of the IgH gene are frequent in multiple myeloma patients: immunobiological characteristics and clinical implications.

DH-JH rearrangements of the Ig heavy-chain gene (IGH) occur early during B-cell development. Consequently, they are detected in precursor-B-cell acute lymphoblastic leukemias both at diagnosis and relapse. Incomplete DJH rearrangements have also been occasionally reported in mature B-cell lymphoproliferative disorders, but their frequency and immunobiological characteristics have not been studied in detail. We have investigated the frequency and characteristics of incomplete DJH as well as complete VDJH rearrangements in a series of 84 untreated multiple myeloma (MM) patients. The overall detection rate of clonality by amplifying VDJH and DJH rearrangements using family-specific primers was 94%. Interestingly, we found a high frequency (60%) of DJH rearrangements in this group. As expected from an immunological point of view, the vast majority of DJH rearrangements (88%) were unmutated. To the best of our knowledge, this is the first systematic study describing the incidence of incomplete DJH rearrangements in a series of unselected MM patients. These results strongly support the use of DJH rearrangements as PCR targets for clonality studies and, particularly, for quantification of minimal residual disease by real-time quantitative PCR using consensus JH probes in MM patients. The finding of hypermutation in a small proportion of incomplete DJH rearrangements (six out of 50) suggests important biological implications concerning the process of somatic hypermutation. Moreover, our data offer a new insight in the regulatory development model of IGH rearrangements.

De novo methylation of tumor suppressor gene p16/INK4a is a frequent finding in multiple myeloma patients at diagnosis.

The p16 gene competes with cyclin D for binding to CDK4/CDK6 and therefore inhibits CDK4/6 complex kinase activity, resulting in dephosphorylation of pRb and related G1 growth arrest. Inactivation of this gene has been involved in a variety of tumors by different mechanisms: homozygous/hemyzygous deletions, point mutations and methylation of a 5' CpG island into exon E1alpha of the p16 gene. Homozygous deletions have been rarely found in multiple myeloma (MM) and no point mutations have been reported. Two recent studies have reported a high prevalence of methylation in the exon E1alpha of the p16 gene, but included only a small number of cases. We have analyzed the methylation pattern of exon E1alpha of the p16 gene in 101 untreated MM and five primary plasma cell leukemias (PCL). A PCR assay, relying on the inability of some restriction enzymes to digest methylated sequences, was used to analyze the methylation status. Southern blot analysis was used to confirm these results. Forty-one of 101 MM patients (40.5%) as well as four of the five (80%) primary PCL patients had shown methylation of the exon E1alpha. Our study confirms that hypermethylation of the p16 gene is a frequent event in MM. Leukemia (2000) 14, 183-187.

Rice-Infecting Pseudomonas Genomes Are Highly Accessorized and Harbor Multiple Putative Virulence Mechanisms to Cause Sheath Brown Rot

Sheath rot complex and seed discoloration in rice involve a number of pathogenic bacteria that cannot be associated with distinctive symptoms. These pathogens can easily travel on asymptomatic seeds and therefore represent a threat to rice cropping systems. Among the rice-infecting Pseudomonas, P. fuscovaginae has been associated with sheath brown rot disease in several rice growing areas around the world. The appearance of a similar Pseudomonas population, which here we named P. fuscovaginae-like, represents a perfect opportunity to understand common genomic features that can explain the infection mechanism in rice. We showed that the novel population is indeed closely related to P. fuscovaginae. A comparative genomics approach on eight rice-infecting Pseudomonas revealed heterogeneous genomes and a high number of strain-specific genes. The genomes of P. fuscovaginae-like harbor four secretion systems (Type I, II, III, and VI) and other important pathogenicity machinery that could probably facilitate rice colonization. We identified 123 core secreted proteins, most of which have strong signatures of positive selection suggesting functional adaptation. Transcript accumulation of putative pathogenicity-related genes during rice colonization revealed a concerted virulence mechanism. The study suggests that rice-infecting Pseudomonas causing sheath brown rot are intrinsically diverse and maintain a variable set of metabolic capabilities as a potential strategy to occupy a range of environments.

High-resolution image reconstruction from multiple low-resolution images.

In this paper, we demonstrate a digital signal processing (DSP) algorithm for improving spatial resolution of images captured by CMOS cameras. The basic approach is to reconstruct a high resolution (HR) image from a shift-related low resolution (LR) image sequence. The aliasing relationship of Fourier transforms between discrete and continuous images in the frequency domain is used for mapping LR images to a HR image. The method of projection onto convex sets (POCS) is applied to trace the best estimate of pixel matching from the LR images to the reconstructed HR image. Computer simulations and preliminary experimental results have shown that the algorithm works effectively on the application of post-image-captured processing for CMOS cameras. It can also be applied to HR digital image reconstruction, where shift information of the LR image sequence is known.

Bayesian alignment of continuous molecular shapes using random fields

Statistical methodology is proposed for comparing molecular shapes. In order to account for the continuous nature of molecules, classical shape analysis methods are combined with techniques used for predicting random fields in spatial statistics. Applying a modification of Procrustes analysis, Bayesian inference is carried out using Markov chain Monte Carlo methods for the pairwise alignment of the resulting molecular fields. Superimposing entire fields rather than the configuration matrices of nuclear positions thereby solves the problem that there is usually no clear one--to--one correspondence between the atoms of the two molecules under consideration. Using a similar concept, we also propose an adaptation of the generalised Procrustes analysis algorithm for the simultaneous alignment of multiple molecular fields. The methodology is applied to a dataset of 31 steroid molecules.

Contributions to gait recognition using multiple views

Esta tesis se centra en la identificación de personas a través de la forma de caminar. El problema del reconocimiento del paso ha sido tratado mediante diferentes enfoques, en los dominios 2D y 3D, y usando una o varias vistas. Sin embargo, la dependencia con respecto al punto de vista, y por tanto de la trayectoria del sujeto al caminar sigue siendo aún un problema abierto. Se propone hacer frente al problema de la dependencia con respecto a la trayectoria por medio de reconstrucciones 3D de sujetos caminando. El uso de reconstrucciones varias ventajas que cabe destacar. En primer lugar, permite explotar una mayor cantidad de información en contraste con los métodos que extraen los descriptores de la marcha a partir de imágenes, en el dominio 2D. En segundo lugar, las reconstrucciones 3D pueden ser alineadas a lo largo de la marcha como si el sujeto hubiera caminado en una cinta andadora, proporcionando así una forma de analizar el paso independientemente de la trayectoria seguida. Este trabajo propone tres enfoques para resolver el problema de la dependencia a la vista: 1. Mediante la utilización de reconstrucciones volumétricas alineadas. 2. Mediante el uso de reconstrucciones volumétricas no alineadas. 3. Sin usar reconstrucciones. Se proponen además tres tipos de descriptores. El primero se centra en describir el paso mediante análisis morfológico de los volúmenes 3D alineados. El segundo hace uso del concepto de entropa de la información para describir la dinámica del paso humano. El tercero persigue capturar la dinámica de una forma invariante a rotación, lo cual lo hace especialmente interesante para ser aplicado tanto en trayectorias curvas como rectas, incluyendo cambios de dirección. Estos enfoques han sido probados sobre dos bases de datos públicas. Ambas están especialmente diseñadas para tratar el problema de la dependencia con respecto al punto de vista, y por tanto de la dependencia con respecto a la trayectoria. Los resultados experimentales muestran que para el enfoque basado en reconstrucciones volumétricas alineadas, el descriptor del paso basado en entropa consigue los mejores resultados, en comparación con métodos estrechamente relacionados del Estado del Arte actual. No obstante, el descriptor invariante a rotación consigue una tasa de reconocimiento que supera a los métodos actuales sin requerir la etapa previa de alineamiento de las reconstrucciones 3D.

Complete genome sequence of Deltapapillomavirus 4 (bovine papillomavirus 2) from a bovine papillomavirus lesion in Amazon Region, Brazil

The complete genome sequence of bovine papillomavirus 2 (BPV2) from Brazilian Amazon Region was determined using multiple-primed rolling circle amplification followed by Illumina sequencing. The genome is 7,947 bp long, with 45.9% GC content. It encodes seven early (E1, E2, E4, E5, E6, E7, and E8) and two late (L1 and L2) genes. The complete genome of a BPV2 can help in future studies since this BPV type is highly reported worldwide although the lack of complete genome sequences available.

Trajectory design of a multiple flyby mission to asteroids

In designing the trajectory for a multiple flyby mission to asteroids the choice of the targets is the most challenging problem. This dissertation faces this problem in the framework of the recently issued medium-size mission call (M5) from ESA: CASTAway. Starting from the preliminary work done in [6], this thesis develops a methodology for sequencing the potential targets in a multiple flyby mission. In order to reduce the computational time, the complete database of known small bodies is firstly pruned on the base of heuristic considerations. Using the assumption of small manoeuvres, a chief orbit concept could be used. Thus, two heuristic thresholds are defined in order to exclude non-promising targets given a chief orbit. The sequencing process takes chief orbit and promising targets as inputs and gives a set of candidate sequences. The results of such a process are analysed in the CASTAway framework and the best feasible sequence studied in details.

Characterization of DNA sequence properties through network and statistical approaches

In this thesis we will see that the DNA sequence is constantly shaped by the interactions with its environment at multiple levels, showing footprints of DNA methylation, of its 3D organization and, in the case of bacteria, of the interaction with the host organisms. In the first chapter, we will see that analyzing the distribution of distances between consecutive dinucleotides of the same type along the sequence, we can detect epigenetic and structural footprints. In particular, we will see that CG distance distribution allows to distinguish among organisms of different biological complexity, depending on how much CG sites are involved in DNA methylation. Moreover, we will see that CG and TA can be described by the same fitting function, suggesting a relationship between the two. We will also provide an interpretation of the observed trend, simulating a positioning process guided by the presence and absence of memory. In the end, we will focus on TA distance distribution, characterizing deviations from the trend predicted by the best fitting function, and identifying specific patterns that might be related to peculiar mechanical properties of the DNA and also to epigenetic and structural processes. In the second chapter, we will see how we can map the 3D structure of the DNA onto its sequence. In particular, we devised a network-based algorithm that produces a genome assembly starting from its 3D configuration, using as inputs Hi-C contact maps. Specifically, we will see how we can identify the different chromosomes and reconstruct their sequences by exploiting the spectral properties of the Laplacian operator of a network. In the third chapter, we will see a novel method for source clustering and source attribution, based on a network approach, that allows to identify host-bacteria interaction starting from the detection of Single-Nucleotide Polymorphisms along the sequence of bacterial genomes.

The Clinical Impact Of Cerebellar Grey Matter Pathology In Multiple Sclerosis.

The cerebellum is an important site for cortical demyelination in multiple sclerosis, but the functional significance of this finding is not fully understood. To evaluate the clinical and cognitive impact of cerebellar grey-matter pathology in multiple sclerosis patients. Forty-two relapsing-remitting multiple sclerosis patients and 30 controls underwent clinical assessment including the Multiple Sclerosis Functional Composite, Expanded Disability Status Scale (EDSS) and cerebellar functional system (FS) score, and cognitive evaluation, including the Paced Auditory Serial Addition Test (PASAT) and the Symbol-Digit Modalities Test (SDMT). Magnetic resonance imaging was performed with a 3T scanner and variables of interest were: brain white-matter and cortical lesion load, cerebellar intracortical and leukocortical lesion volumes, and brain cortical and cerebellar white-matter and grey-matter volumes. After multivariate analysis high burden of cerebellar intracortical lesions was the only predictor for the EDSS (p<0.001), cerebellar FS (p = 0.002), arm function (p = 0.049), and for leg function (p<0.001). Patients with high burden of cerebellar leukocortical lesions had lower PASAT scores (p = 0.013), while patients with greater volumes of cerebellar intracortical lesions had worse SDMT scores (p = 0.015). Cerebellar grey-matter pathology is widely present and contributes to clinical dysfunction in relapsing-remitting multiple sclerosis patients, independently of brain grey-matter damage.

Multiple Desmoid Tumors In A Patient With Gardner's Syndrome - Report Of A Case.

Desmoid tumor (DT) is a common manifestation of Gardner's Syndrome (GS), although it is a rare condition in the general population. DT in patients with GS is usually located in the abdominal wall and/or intra-abdominal cavity. We report a case of a 32 years-old female patient with familial adenomatous polyposis (FAP), who was already submitted to total colectomy and developed multiple DT, located in the abdominal wall and in the left breast. The patient underwent several surgical procedures, with a multidisciplinary team of surgeons. Wide surgical resections of the left breast and the abdominal wall tumors were performed in separate steps. Polypropylene mesh reconstruction and muscle flaps were needed to cover the defects of the thoracic and abdominal walls. After partial necrosis of the adipose-cutaneous flap in the abdomen that required a new skin graft, she had a satisfactory outcome with complete healing of the surgical incisions. DT is frequent in GS, however, breast localization is very rare, with few cases reported in the literature. Recurrence of DT is not negligible, even after a wide surgical resection. GS patients must be followed up closely, and clinical examination, associated with imaging studies, should be performed to detect any signs of tumor. DT represents one of the most significant causes of the morbidity and mortality that affects FAP patients following colectomy. In general, the surgical procedures to excise DT are highly complex, requiring a multidisciplinary team.

The Effect Of Pelvic Floor Muscle Training Alone Or In Combination With Electrostimulation In The Treatment Of Sexual Dysfunction In Women With Multiple Sclerosis.

Sexual dysfunction (SD) affects up to 80% of multiple sclerosis (MS) patients and pelvic floor muscles (PFMs) play an important role in the sexual function of these patients. The objective of this paper is to evaluate the impact of a rehabilitation program to treat lower urinary tract symptoms on SD of women with MS. Thirty MS women were randomly allocated to one of three groups: pelvic floor muscle training (PFMT) with electromyographic (EMG) biofeedback and sham neuromuscular electrostimulation (NMES) (Group I), PFMT with EMG biofeedback and intravaginal NMES (Group II), and PFMT with EMG biofeedback and transcutaneous tibial nerve stimulation (TTNS) (Group III). Assessments, before and after the treatment, included: PFM function, PFM tone, flexibility of the vaginal opening and ability to relax the PFMs, and the Female Sexual Function Index (FSFI) questionnaire. After treatment, all groups showed improvements in all domains of the PERFECT scheme. PFM tone and flexibility of the vaginal opening was lower after the intervention only for Group II. All groups improved in arousal, lubrication, satisfaction and total score domains of the FSFI questionnaire. This study indicates that PFMT alone or in combination with intravaginal NMES or TTNS contributes to the improvement of SD.

Genome Sequence Of Streptomyces Wadayamensis Strain A23, An Endophytic Actinobacterium From Citrus Reticulata.

The actinobacterium Streptomyces wadayamensis A23 is an endophyte of Citrus reticulata that produces the antimycin and mannopeptimycin antibiotics, among others. The strain has the capability to inhibit Xylella fastidiosa growth. The draft genome of S. wadayamensis A23 has ~7.0 Mb and 6,006 protein-coding sequences, with a 73.5% G+C content.

Genome Sequence Of Bacillus Safensis Cfa06, Isolated From Biodegraded Petroleum In Brazil.

Bacillus safensis is a microorganism recognized for its biotechnological and industrial potential due to its interesting enzymatic portfolio. Here, as a means of gathering information about the importance of this species in oil biodegradation, we report a draft genome sequence of a strain isolated from petroleum.