A general definition and nomenclature for alternative splicing events

Understanding the molecular mechanisms responsible for the regulation of the transcriptome present in eukaryotic cells isone of the most challenging tasks in the postgenomic era. In this regard, alternative splicing (AS) is a key phenomenoncontributing to the production of different mature transcripts from the same primary RNA sequence. As a plethora ofdifferent transcript forms is available in databases, a first step to uncover the biology that drives AS is to identify thedifferent types of reflected splicing variation. In this work, we present a general definition of the AS event along with anotation system that involves the relative positions of the splice sites. This nomenclature univocally and dynamically assignsa specific ‘‘AS code’’ to every possible pattern of splicing variation. On the basis of this definition and the correspondingcodes, we have developed a computational tool (AStalavista) that automatically characterizes the complete landscape of ASevents in a given transcript annotation of a genome, thus providing a platform to investigate the transcriptome diversityacross genes, chromosomes, and species. Our analysis reveals that a substantial part—in human more than a quarter—ofthe observed splicing variations are ignored in common classification pipelines. We have used AStalavista to investigate andto compare the AS landscape of different reference annotation sets in human and in other metazoan species and found thatproportions of AS events change substantially depending on the annotation protocol, species-specific attributes, andcoding constraints acting on the transcripts. The AStalavista system therefore provides a general framework to conductspecific studies investigating the occurrence, impact, and regulation of AS.

Multiple non-collinear TF-map alignments of promoter regions

Background: The analysis of the promoter sequence of genes with similar expression patterns isa basic tool to annotate common regulatory elements. Multiple sequence alignments are on thebasis of most comparative approaches. The characterization of regulatory regions from coexpressedgenes at the sequence level, however, does not yield satisfactory results in manyoccasions as promoter regions of genes sharing similar expression programs often do not shownucleotide sequence conservation.Results: In a recent approach to circumvent this limitation, we proposed to align the maps ofpredicted transcription factors (referred as TF-maps) instead of the nucleotide sequence of tworelated promoters, taking into account the label of the corresponding factor and the position in theprimary sequence. We have now extended the basic algorithm to permit multiple promotercomparisons using the progressive alignment paradigm. In addition, non-collinear conservationblocks might now be identified in the resulting alignments. We have optimized the parameters ofthe algorithm in a small, but well-characterized collection of human-mouse-chicken-zebrafishorthologous gene promoters.Conclusion: Results in this dataset indicate that TF-map alignments are able to detect high-levelregulatory conservation at the promoter and the 3'UTR gene regions, which cannot be detectedby the typical sequence alignments. Three particular examples are introduced here to illustrate thepower of the multiple TF-map alignments to characterize conserved regulatory elements inabsence of sequence similarity. We consider this kind of approach can be extremely useful in thefuture to annotate potential transcription factor binding sites on sets of co-regulated genes fromhigh-throughput expression experiments.

Differentiated evolutionary rates in alternative exons and the implications for splicing regulation

Background: Alternatively spliced exons play an important role in the diversification of gene function in most metazoans and are highly regulated by conserved motifs in exons and introns. Two contradicting properties have been associated to evolutionary conserved alternative exons: higher sequence conservation and higher rate of non-synonymous substitutions, relative to constitutive exons. In order to clarify this issue, we have performed an analysis of the evolution of alternative and constitutive exons, using a large set of protein coding exons conserved between human and mouse and taking into account the conservation of the transcript exonic structure. Further, we have also defined a measure of the variation of the arrangement of exonic splicing enhancers (ESE-conservation score) to study the evolution of splicing regulatory sequences. We have used this measure to correlate the changes in the arrangement of ESEs with the divergence of exon and intron sequences. Results: We find evidence for a relation between the lack of conservation of the exonic structure and the weakening of the sequence evolutionary constraints in alternative and constitutive exons. Exons in transcripts with non-conserved exonic structures have higher synonymous (dS) and non-synonymous (dN) substitution rates than exons in conserved structures. Moreover, alternative exons in transcripts with non-conserved exonic structure are the least constrained in sequence evolution, and at high EST-inclusion levels they are found to be very similar to constitutive exons, whereas alternative exons in transcripts with conserved exonic structure have a dS significantly lower than average at all EST-inclusion levels. We also find higher conservation in the arrangement of ESEs in constitutive exons compared to alternative ones. Additionally, the sequence conservation at flanking introns remains constant for constitutive exons at all ESE-conservation values, but increases for alternative exons at high ESE-conservation values. Conclusion: We conclude that most of the differences in dN observed between alternative and constitutive exons can be explained by the conservation of the transcript exonic structure. Low dS values are more characteristic of alternative exons with conserved exonic structure, but not of those with non-conserved exonic structure. Additionally, constitutive exons are characterized by a higher conservation in the arrangement of ESEs, and alternative exons with an ESE-conservation similar to that of constitutive exons are characterized by a conservation of the flanking intron sequences higher than average, indicating the presence of more intronic regulatory signals.

A non-interventional phase IV Belgian survey to assess the antiviral effectiveness of pegylated interferon-alpha-2b and ribavirin treatment according to the stage of liver fibrosis in previously untreated patients with genotype 1/4/5/6 chronic hepatitis C (PRACTICE).

BACKGROUND AND STUDY AIMS: This was an observational, non-interventional, multicenter, phase IV study, in patients with genotype 1/4/5/6 chronic hepatitis C (CHC). The primary objectives were to evaluate SVR in patients with no or minimal fibrosis (METAVIR F0-F1) versus well established fibrosis (F2-F4), and to estimate response on Weeks 12, 24 and 48 on treatment in previously untreated patients with genotypes 1/4/5/6 CHC. PATIENTS AND METHODS: 538 patients treated with pegylated interferon alfa 2b 1.5 mcg/kg in combination with ribavirin 800-1200 mg/day were enrolled in 55 sites in Belgium and Luxembourg, 505 being considered for the analysis. 40% of the patients were female and 60% male, the average age was 47.5 years, 10.5% were 65 or older. RESULTS: SVR was observed in 35% of the patients, EVR in 68%, of which pEVR in 33% and cEVR in 35%. SVR was observed in 43% of the low fibrosis group (F0, F1) and 30% of the high fibrosis group (F2, F3, F4) (p = 0.005). SVR rates were 34% for genotype 1, 37% for genotype 4, and 47% for genotype 5 (NS). Multivariate analysis showed that EVR and baseline METAVIR score are independent prognostic factors for SVR. CONCLUSIONS: This trial confirms that fibrosis stage and early viral response are the most important key-factors to predict sustained response, suggesting that the earlier patients are treated, the better the outcome. Non-invasive techniques enable us to closely monitor progression of fibrosis, allowing a better selection of patients for antiviral treatment in the DAA-era.

Longevity and fecundity of Dichroplus maculipennis (Orthoptera, Acrididae) at non-outbreaking and outbreaking situations

Dichroplus maculipennis is one of the most characteristic and damaging grasshopper species of Argentina, mainly in areas of the Pampas and Patagonia regions. We estimated and compared the longevity and fecundity of adult female D. maculipennis under controlled conditions (30ºC, 14L:10D, 40% RH) from individuals collected as last instar nymphs (VI) in the field and with a known recent history of low and high density conditions. Densities of D. maculipennis at the collecting sites were 0.95 individuals per m² in 2006 and 46 ind/m² in 2009, representing non-outbreaking and outbreaking situations, respectively. Adult female longevity in 2006 (67.96 ± 3.2 days) was significantly higher (p < 0.05) than in 2009 (37.44 ± 1.98 days). The number of egg-pods per female was 3.32 ± 0.44 for 2006 and 1.62 ± 0.26 for 2009. The average fecundity in 2006 (89.29 ± 11.9 eggs/female) was significantly greater (p < 0.05) than that in 2009 (36.27 ± 5.82 eggs/female). While it was observed that the oviposition rate was higher in 2006, this difference was not significant (p > 0.05). The fecundity curves showed that the highest values were at weeks 11 and 13 for the 2006 females, and at week 6 for those of 2009. Since the daily oviposition rate at low and high densities was not significantly different, the diminished fecundity rate at high density is attributable to their reduced longevity.

Identification of novel functional TBP-binding sites and general factor repertoires.

Our current knowledge of the general factor requirement in transcription by the three mammalian RNA polymerases is based on a small number of model promoters. Here, we present a comprehensive chromatin immunoprecipitation (ChIP)-on-chip analysis for 28 transcription factors on a large set of known and novel TATA-binding protein (TBP)-binding sites experimentally identified via ChIP cloning. A large fraction of identified TBP-binding sites is located in introns or lacks a gene/mRNA annotation and is found to direct transcription. Integrated analysis of the ChIP-on-chip data and functional studies revealed that TAF12 hitherto regarded as RNA polymerase II (RNAP II)-specific was found to be also involved in RNAP I transcription. Distinct profiles for general transcription factors and TAF-containing complexes were uncovered for RNAP II promoters located in CpG and non-CpG islands suggesting distinct transcription initiation pathways. Our study broadens the spectrum of general transcription factor function and uncovers a plethora of novel, functional TBP-binding sites in the human genome.

Evaluation of the immune response to infection with metastatic and non-metastatic "Leishmania guyanensis" parasites

ABSTRACT : Les infections par le parasite Leishmania guyanensis se caractérisent par une dissémination depuis le site initial d'infection jusqu'aux tissus naso-pharyngés, responsable de la Leishmaniose à lésions secondaires muco-cutanées (LMC). Les lésions des patients atteints de LMC montrent une massive infiltration de cellules immunitaires, une réponse immunitaire élevée et la présence de parasites (bien qu'en très faible quantité). La LMC engendre une augmentation de l'expression de TNFa ainsi qu'un défaut dans le contrôle de la réponse immunitaire caractérisé par une absence de réponse à l'IL 10. La réponse immunitaire de l'hôte ainsi que la virulence du parasite sont deux facteurs reconnus pour le contrôle de l'infection. Le mécanisme de la pathogenèse de la LMC restent grandement incompris, surtout le mécanisme de dissémination de l'infection du site d'inoculation jusqu'aux sites secondaires d'infection (métastases) ainsi que les détails de la réponse de l'hôte contre le pathogène. Dans un modèle d'infection d' hamsters avec des parasites du Nouveau Monde, la classification des parasites Leishmania se fait en fonction de leur capacité à développer des métastases. Ce modéle d'infection a permis de caractériser différentes souches de parasites selon la classification de l'Organisation Mondiale de la Sante (OMS) tel que la souche de référence W>É-II/BR/78/M5313 qui est reconnue comme hautement métastatique alors que ces clones dérivés de M5313 montrent de grandes variations quand a leur capacité à créer des métastases. Les clones 13 et 21 sont métastatiques (M+) alors que les clones 3 et 17 sont nonmétastatiques (NI-). Les objectifs de cette thèse ont été d'étudier le rôle de la réponse immunitaire innée des macrophages après infection in vitro avec différents clones métastatiques et non-métastatiques du parasite L. guyanensis, ainsi que d'étudier la réponse immunitaire générée suite à une infection in vivo par les clones M+ et M- de L. guyanensis dans un modèle marin. L'analyse de la .réponse immunitaire des macrophages in vitro montrent qu'il y aune augmentation significative de leur statut d'activation après infection par des parasites M+ indiquée par la modulation des marqueurs d'activation de surface CD80, CD86 et CD40, ainsi que une augmentation significative de CXCL 10, CCLS, IL6 et TNFa au niveau transcription de l'ARNm et au niveau de la protéine. Cette phénomène d'activation a été observée chez les deux souches de souris C57BL/6 et BALB/c. L'utilisation d'un inhibiteur d'entrée des parasites (Cytochalsin D) ou d'un inhibiteur des fonctions endosomales (Chloroquine) diminue de manière significative la réponse des macrophages aux parasites M+. L'utilisation de macrophages déficients en TLR, MyD88, et TRIF a démontré que la réponse générée après infection par les parasites M+ était dépendante de la voie de signalisation de TRIF et TLR3. Lors d'infection in vivo par des parasites M5313, au moins 50% des souris BALB/c présentent un phénotype sensible caractérisé par des lésions non-nécrotiques qui ne guérissent pas, persistent plus de 13 semaines après infection et contiennent un nombre considérable de parasites. Ces souris développent une réponse immunitaire de type T helper 2 (Th2) avec un niveau élevé d'IL-4 et d'IL-10. Les autres souris ont un phénotype non-sensible, les souris développant peu ou pas de lésion, avec peu de parasites et une réponse immunitaire diminuée, caractérisée par un niveau faible d'IFNy, d'IL4 et d'IL10. De plus, les souris BALB/c infectées par un parasite L. guyanensis isolé à partir des lésions muco-cutanées d'un patient humain atteint de LMC ont démontrés un phénotype similaire aux souris infectées par la souche M5313 avec 50% des souris développant des lésions persistantes, alors qu'un parasite dérivé des lésions cutanées humains n'a montré qu'une faible sensibilité avec une lésion transitoire qui finit par guérir. Nous avons montré que la sensibilité de ces souris BALB/c dépend de l'IL-4 et de l'IL-10 car les souris IL-10-/sur fond génétique BALB/c ainsi que les souris BALB/c traitée avec de l'anti-IL4 étaient capables de contrôler l'infection par M5313. Les souris C57BL/6 sont résistantes à l'infection par le parasite M5313. Elles développent une lésion transitoire qui guérit 9 semaines après infection. Ces souris résistantes ont un très faible taux de parasites au site d'infection et développent une réponse immunitaire de type Thl avec un niveau élevé d'IFNr et peu d'IL4 et d'IL10. Les infections in vivo de souris déficientes en MyD88, TRIF, TLR3 ou TLR9 (sur fond génétique C57BL/6) ont indiqué que MyD88 et TLR9 étaient impliqués dans la résistance à l'infection par L. guyanensi, et que TRIF et TLR3 avaient un rôle important dans la sensibilité. Ce travail met en évidence le fait que la réponse immunitaire de l'hôte est modulée par le parasite selon leur caractérisation d'être soit M+ ou M-. Nous avons démontré également que plusieurs gènes et voies de signalisations étaient impliqués dans cette réponse favorisant le développement d'une LMC. ABSTRACT : Leishmania guyanensis parasites are able to disseminate from the initial site of cutaneous skin infection to the nasopharyngeal tissues resulting in destructive secondary lesions and the disease Mucocutaneous Leishmaniasis (MCL). The secondary lesions in patients have intense immune cell infiltration, elevated immune responses and the presence (albeit at low levels) of parasites. More specifically, MCL patients produce higher levels of TNFa and display impairment in their ability to control the immune response due to a defect in their ability to respond to IL10. Little is known about the pathogenesis of MCL, especially about the dissemination of the infection from the site of inoculation to secondary sites (metastasis) and the response of the host to the pathogen. The hamster model of L. guyanensis infection has previously characterized the WHO reference strain, L. guyanensis WHI/BR/78/M5313, as being highly metastatic. Clones of parasites derived from this reference strain show a differential ability to metastasize. This thesis studied the differential immune response generated by macrophages in vitro, or by mice in vivo, following infection with L. guyanensis parasites. A significant increase in the activation status of macrophages derived from C57BL/6 or BALB/c mice was observed after in vitro infection with L. guyanensis parasites when compared to non-metastatic parasites. This change in status was evidenced by the increased expression of surface activation markers, together with the chemokines, CXCL 10, CCLS, and cytokines, IL6 and TNFa. Furthermore, in vitro infection of macrophages isolated from mice deficient in either a specific Toll Like Receptor (TLR) or the adaptor molecules MyD88 or TRIF, indicated that the immune response generated following L. guyanensis metastatic parasite infection was reliant on the TRIF dependent TLR3 signalling pathway. In vivo footpad infection of BALB/c mice with the L. guyanensis M5313 parasites showed a reproducible susceptible phenotype, whereby at least 50% of infected mice developed non-healing, nonnecrosing lesions with high parasitemia that persisted over 13 weeks post infection. This phenotype was characterized by a Th2 type cytokine immune response with increased levels of IL4 and IL10 detected in the draining lymph nodes. IL 10 deficient mice on a BALB/c background, or BALB/c mice treated with anti-IL4 were able to control infection with L. guyanensis M5313 parasites, thereby proving that these cytokines were indeed implicated in the susceptibility to infection. Moreover, infection of BALB/c mice with patient isolated L. guyanensis parasites confirmed that MCL derived parasites were able to induce a susceptibility phenotype similar to that of L. guyanensis M5313. C57BL/6 mice, on the other hand, were highly resistant to infection with L. guyanensis M5313 parasites and produced transient footpad swelling that healed by week 9 post infection, together with low degrees of footpad parasitemia and a Thl polarized immune response. Infection of mice deficient in MyD88, TRIF, TLR3, and TLR9 (on a C57BL/6 background), indicated that MyD88 and TLR9 were involved in the resistance of these mice to infection, and that TRIF and TLR3 were involved in the susceptibility. This study has shown that the host response can be differentially modulated depending on the infecting parasite with several genes and pathways being identified that could be involved in promoting the development of MCL.

Qualités des sites internet anglophones traitant du trouble obsessionnel complusif

Enjeux et contexte : Dans les questions de santé mentale, internet constitue de plus en plus une source d'information pour les personnes souffrant de troubles psychiatriques ainsi que de leurs proches. D'autre part, le trouble obsessionnel compulsif revêt une importance grandissante et bénéficie d'un intérêt croissant en raison de sa fréquence et de la charge qu'il représente pour le patient et pour la société. Les patients souffrant de trouble obsessionnel compulsif, qu'ils soient diagnostiqués ou non, ainsi que leurs proches peuvent être amenés à rechercher une information de bonne qualité sur net sur le sujet. Cette étude vise à évaluer la qualité de l'information issue de l'internet concernant les sites anglophones traitant du trouble obsessionnel compulsif et de comparer les résultats des requêtes en utilisant un moteur de recherche général (Google) à celles obtenues avec un moteur de recherche spécialisé (Omni Medical Search). Des mots-clés relatifs au trouble obsessionnel compulsif ont été introduits dans Google et Omni Medical Search. Les sites retenus ont été évalués selon leur responsabilité, interactivité, lisibilité et la qualité de leur contenu. Le Label HON et la version brève de l'échelle DISCERN ont été utilisés comme indicateurs possibles de la qualité du contenu. Sur les 235 adresses retrouvées, 53 sites retenus ont été analysés. Résultats : La qualité du contenu des sites examinés est relativement bonne. L'utilisation d'un moteur de recherche spécialisé ne constitue pas un avantage par comparaison au moteur général utilisé par la grande majorité des internautes. Un score > 16 de la version brève du DISCERN est associé à une meilleure qualité du contenu. En conclusion : cette étude montre que le contenu des sites web concernant le trouble obsessionnel compulsif est acceptable. L'utilisation d'un moteur de recherche spécialisé n'offre pas d'avantage par rapport à Google. Comme implications pratiques : internet renferme des sites de haute qualité sur le trouble obsessionnel compulsif. L'accès à ces sites ne nécessite pas l'utilisation d'un moteur de recherche spécialisé. En revanche, une discussion entre le patient et le soignant à propos de l'information disponible sur internet demeure indispensable Perspectives : en dépit des limitations de notre étude, on peut dire que l'information contenue dans le web concernant le trouble obsessionnel compulsif est acceptable. Le contenu et la présentation de cette information pourraient être améliorés. Quant à l'internaute qui cherche une information de qualité, il pourrait être guidé par deux éléments : le HON et la version brève du DISCERN.

Nuclear and cytoplasmic triiodothyronine-binding sites in primary sensory neurons and Schwann cells: radioautographic study during development.

The effects of the thyroid hormones on target cells are mediated through nuclear T3 receptors. In the peripheral nervous system, nuclear T3 receptors were previously detected with the monoclonal antibody 2B3 mAb in all the primary sensory neurons throughout neuronal life and in peripheral glia at the perinatal period only (Eur. J. Neurosci. 5, 319, 1993). To determine whether these nuclear T3 receptors correspond to functional ones able to bind T3, cryostat sections and in vitro cell cultures of dorsal root ganglion (DRG) or sciatic nerve were incubated with 0.1 nM [125I]-labeled T3, either alone to visualize the total T3-binding sites or added with a 10(3) fold excess of unlabeled T3 to estimate the part due to the non-specific T3-binding. After glutaraldehyde fixation, radioautography showed that the specific T3-binding sites were largely prevalent. The T3-binding capacity of peripheral glia in DRG and sciatic nerve was restricted to the perinatal period in vivo and to Schwann cells cultured in vitro. In all the primary sensory neurons, specific T3-binding sites were disclosed in foetal as well as adult rats. The detection of the T3-binding sites in the nucleus indicated that the nuclear T3 receptors are functional. Moreover the concomitant presence of both T3-binding sites and T3 receptors alpha isoforms in the perikaryon of DRG neurons infers that: 1) [125I]-labeled T3 can be retained on the T3-binding 'E' domain of nascent alpha 1 isoform molecules newly-synthesized on the perikaryal ribosomes; 2) the alpha isoforms translocated to the nucleus are modified by posttranslational changes and finally recognized by 2B3 mAb as nuclear T3 receptor. In conclusion, the radioautographic visualization of the T3-binding sites in peripheral neurons and glia confirms that the nuclear T3 receptors are functional and contributes to clarify the discordant intracellular localization provided by the immunocytochemical detection of nuclear T3 receptors and T3 receptor alpha isoforms.

PEASANT AND SCIENTIFIC KNOWLEDGE ON PLANOSOLS AS A SOURCE OF MATERIALS IN THE MAKING OF NON-INDUSTRIAL POTTERY

Ethnopedological studies have mainly focused on agricultural land uses and associated practices. Nevertheless, peasant and indigenous populations use soil and land resources for a number of additional purposes, including pottery. In the present study, we describe and analyze folk knowledge related to the use of soils in non-industrial pottery making by peasant potters, in the municipality of Altinho, Pernambuco State, semiarid region at Brazil. Ethnoscientific techniques were used to record local knowledge, with an emphasis on describing the soil materials recognized by the potters, the properties they used to identify those soil materials, and the criteria employed by them to differentiate and relate such materials. The potters recognized three categories of soil materials: “terra” (earth), “barro” (clay) and, “piçarro” (soft rock). The multi-layered arrangement of these materials within the soil profiles was similar to the arrangement of the soil horizon described by formal pedologists. “Barro vermelho” (red clay) was considered by potters as the principal ceramic resource. The potters followed morphological and utilitarian criteria in distinguishing the different soil materials. Soils from all of these sites were sodium-affected Alfisols and correspond to Typic Albaqualf and Typic Natraqualf in the Soil Taxonomy (Soil Survey Staff, 2010).

High-throughput SELEX SAGE method for quantitative modeling of transcription-factor binding sites.

The ability to determine the location and relative strength of all transcription-factor binding sites in a genome is important both for a comprehensive understanding of gene regulation and for effective promoter engineering in biotechnological applications. Here we present a bioinformatically driven experimental method to accurately define the DNA-binding sequence specificity of transcription factors. A generalized profile was used as a predictive quantitative model for binding sites, and its parameters were estimated from in vitro-selected ligands using standard hidden Markov model training algorithms. Computer simulations showed that several thousand low- to medium-affinity sequences are required to generate a profile of desired accuracy. To produce data on this scale, we applied high-throughput genomics methods to the biochemical problem addressed here. A method combining systematic evolution of ligands by exponential enrichment (SELEX) and serial analysis of gene expression (SAGE) protocols was coupled to an automated quality-controlled sequence extraction procedure based on Phred quality scores. This allowed the sequencing of a database of more than 10,000 potential DNA ligands for the CTF/NFI transcription factor. The resulting binding-site model defines the sequence specificity of this protein with a high degree of accuracy not achieved earlier and thereby makes it possible to identify previously unknown regulatory sequences in genomic DNA. A covariance analysis of the selected sites revealed non-independent base preferences at different nucleotide positions, providing insight into the binding mechanism.

Enhanced pulse propagation in non-linear arrays of oscillators

The propagation of a pulse in a nonlinear array of oscillators is influenced by the nature of the array and by its coupling to a thermal environment. For example, in some arrays a pulse can be speeded up while in others a pulse can be slowed down by raising the temperature. We begin by showing that an energy pulse (one dimension) or energy front (two dimensions) travels more rapidly and remains more localized over greater distances in an isolated array (microcanonical) of hard springs than in a harmonic array or in a soft-springed array. Increasing the pulse amplitude causes it to speed up in a hard chain, leaves the pulse speed unchanged in a harmonic system, and slows down the pulse in a soft chain. Connection of each site to a thermal environment (canonical) affects these results very differently in each type of array. In a hard chain the dissipative forces slow down the pulse while raising the temperature speeds it up. In a soft chain the opposite occurs: the dissipative forces actually speed up the pulse, while raising the temperature slows it down. In a harmonic chain neither dissipation nor temperature changes affect the pulse speed. These and other results are explained on the basis of the frequency vs energy relations in the various arrays

Which anatomical sites should be sampled for screening of methicillin-resistant Staphylococcus aureus carriage by culture or by rapid PCR test?

The nose is the anatomical site usually recommended for methicillin-resistant Staphylococcus aureus (MRSA) screening. Other sites are also recommended, but are more controversial. We showed that the sensitivities of MRSA detection from nasal swabs alone were 48% and 62% by culture or by rapid PCR test, respectively. These percentages increased to 79% and 92% with the addition of groin swabs, and to 96% and 99% with the addition of groin and throat swabs. In conclusion, neither by culture nor by rapid PCR test is nose sampling alone sufficient for MRSA detection. Additional anatomical sites should include at least the groin and throat.

Feasibility of RNA studies on illegitimate transcription for molecular characterization of splicing mutations in the ATP7B gene: a case report.

Approximately 520 Wilson disease-causing mutations in the ATP7B gene have been described to date. In this study we report DNA and RNA analyses carried out for molecular characterization of a consensus sequence splicing mutation found in homozygosity in a Swiss Wilson disease patient. RNA analysis of 1946 +6 T→C in both the peripheral lymphoblasts and liver resulted in the production in the propositus of only an alternative transcript lacking exons 6, 7, and 8 resulting most likely in alterations of cell biochemistry and disease. The patient presents an early form of severe hepatic disease characterized by hepatosplenomegaly, reduced hepatic function, anemia and thrombocytopenia indicating that 1946 +6 T→C is a severe mutation. Since identical results were obtained from both peripheral lymphoblasts and liver they also suggest that RNA studies of illegitimate transcripts can be safely used for molecular characterization of ATP7B splicing mutations, thus improving genetic counseling and diagnosis of Wilson disease. Moreover these studies, contribute to reveal the exact molecular mechanisms producing Wilson disease.

High-magnification vascular imaging to reject false-positive sites in situ during Hexvix® fluorescence cystoscopy

Fluorescence imaging for detection of non-muscle-invasive bladder cancer is based on the selective production and accumulation of fluorescing porphyrins-mainly, protoporphyrin IX-in cancerous tissues after the instillation of Hexvix®. Although the sensitivity of this procedure is very good, its specificity is somewhat limited due to fluorescence false-positive sites. Consequently, magnification cystoscopy has been investigated in order to discriminate false from true fluorescence positive findings. Both white-light and fluorescence modes are possible with the magnification cystoscope, allowing observation of the bladder wall with magnification ranging between 30× for standard observation and 650×. The optical zooming setup allows adjusting the magnification continuously in situ. In the high-magnification (HM) regime, the smallest diameter of the field of view is 600 microns and the resolution is 2.5 microns when in contact with the bladder wall. With this cystoscope, we characterized the superficial vascularization of the fluorescing sites in order to discriminate cancerous from noncancerous tissues. This procedure allowed us to establish a classification based on observed vascular patterns. Seventy-two patients subject to Hexvix® fluorescence cystoscopy were included in the study. Comparison of HM cystoscopy classification with histopathology results confirmed 32/33 (97%) cancerous biopsies and rejected 17/20 (85%) noncancerous lesions.