Molecular identification of Sporothrix species involved in the first familial outbreak of sporotrichosis in the state of Espírito Santo, southeastern Brazil

Sporotrichosis is a widespread subcutaneous mycosis caused by the dimorphic fungi now known as the Sporothrix schenckii complex. This complex is comprised of at least six species, including Sporothrix albicans, Sporothrix brasiliensis, Sporothrix globosa, Sporothrix luriei, Sporothrix mexicana and S. schenckii. Cases of sporotrichosis have significantly increased in Brazil over the past decade, especially in the state of Rio de Janeiro (RJ), where an epidemic among cat owners has been observed. The zoonotic transmission from cats to humans suggests a common source of infection and indicates that animals can act as vectors. We performed a molecular characterisation of samples collected during the first outbreak of familial sporotrichosis caused by S. brasiliensis in the state of Espírito Santo, Brazil. These results represent the first description of such an outbreak outside the endemic area of zoonotic sporotrichosis in RJ.

COI barcode versus morphological identification of Culex ( Culex ) (Diptera: Culicidae) species: a case study using samples from Argentina and Brazil

Sequences of the cytochrome c oxidase subunit I (COI) mitochondrial gene from adults of 22 Culex ( Culex ) species from Argentina and Brazil were employed to assess species identification and to test the usefulness of COI for barcoding using the best close match (BCM) algorithm. A pairwise Kimura two-parameter distance matrix including the mean intra and interspecific distances for 71 COI barcode sequences was constructed. Of the 12 COI lineages recovered in the Neighbour-joining topology, five confirmed recognised morphological species ( Cx. acharistus , Cx. chidesteri , Cx. dolosus , Cx. lygrus and Cx. saltanensis ) with intraspecific divergences lower than 1.75%. Cx. bilineatus is formally resurrected from the synonymy of Cx. dolosus . Cx. maxi , Cx. surinamensis and the Coronator group species included were clustered into an unresolved lineage. The intraspecific distance of Cx. pipiens (3%) was almost twice the interspecific between it and Cx. quinquefasciatus (1.6%). Regarding the BCM criteria, the COI barcode successfully identified 69% of all species. The rest of the sequences, approximately 10%, 18% and 3%, remained as ambiguously, mis and unidentified, respectively. The COI barcode does not contain enough information to distinguish Culex ( Cux. ) species.

Identification of pharmaceutical tablets by Raman spectroscopy and chemometrics

Raman spectroscopy has become an attractive tool for the analysis of pharmaceutical solid dosage forms. In the present study it is used to ensure the identity of tablets. The two main applications of this method are release of final products in quality control and detection of counterfeits. Twenty-five product families of tablets have been included in the spectral library and a non-linear classification method, the Support Vector Machines (SVMs), has been employed. Two calibrations have been developed in cascade: the first one identifies the product family while the second one specifies the formulation. A product family comprises different formulations that have the same active pharmaceutical ingredient (API) but in a different amount. Once the tablets have been classified by the SVM model, API peaks detection and correlation are applied in order to have a specific method for the identification and allow in the future to discriminate counterfeits from genuine products. This calibration strategy enables the identification of 25 product families without error and in the absence of prior information about the sample. Raman spectroscopy coupled with chemometrics is therefore a fast and accurate tool for the identification of pharmaceutical tablets.

Mycobacteria mobility shift assay: a method for the rapid identification of Mycobacterium tuberculosis and nontuberculous mycobacteria

The identification of mycobacteria is essential because tuberculosis (TB) and mycobacteriosis are clinically indistinguishable and require different therapeutic regimens. The traditional phenotypic method is time consuming and may last up to 60 days. Indeed, rapid, affordable, specific and easy-to-perform identification methods are needed. We have previously described a polymerase chain reaction-based method called a mycobacteria mobility shift assay (MMSA) that was designed for Mycobacterium tuberculosis complex (MTC) and nontuberculous mycobacteria (NTM) species identification. The aim of this study was to assess the MMSA for the identification of MTC and NTM clinical isolates and to compare its performance with that of the PRA-hsp65 method. A total of 204 clinical isolates (102 NTM and 102 MTC) were identified by the MMSA and PRA-hsp65. For isolates for which these methods gave discordant results, definitive species identification was obtained by sequencing fragments of the 16S rRNA and hsp65 genes. Both methods correctly identified all MTC isolates. Among the NTM isolates, the MMSA alone assigned 94 (92.2%) to a complex or species, whereas the PRA-hsp65 method assigned 100% to a species. A 91.5% agreement was observed for the 94 NTM isolates identified by both methods. The MMSA provided correct identification for 96.8% of the NTM isolates compared with 94.7% for PRA-hsp65. The MMSA is a suitable auxiliary method for routine use for the rapid identification of mycobacteria.

Identification of blood meal sources of Lutzomyia longipalpis using polymerase chain reaction-restriction fragment length polymorphism analysis of the cytochrome B gene

An analysis of the dietary content of haematophagous insects can provide important information about the transmission networks of certain zoonoses. The present study evaluated the potential of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the mitochondrial cytochrome B (cytb) gene to differentiate between vertebrate species that were identified as possible sources of sandfly meals. The complete cytb gene sequences of 11 vertebrate species available in the National Center for Biotechnology Information database were digested with Aci I, Alu I, Hae III and Rsa I restriction enzymes in silico using Restriction Mapper software. The cytb gene fragment (358 bp) was amplified from tissue samples of vertebrate species and the dietary contents of sandflies and digested with restriction enzymes. Vertebrate species presented a restriction fragment profile that differed from that of other species, with the exception of Canis familiaris and Cerdocyon thous. The 358 bp fragment was identified in 76 sandflies. Of these, 10 were evaluated using the restriction enzymes and the food sources were predicted for four: Homo sapiens (1), Bos taurus (1) and Equus caballus (2). Thus, the PCR-RFLP technique could be a potential method for identifying the food sources of arthropods. However, some points must be clarified regarding the applicability of the method, such as the extent of DNA degradation through intestinal digestion, the potential for multiple sources of blood meals and the need for greater knowledge regarding intraspecific variations in mtDNA.

Identification of the SPLUNC1 ENaC-inhibitory domain yields novel strategies to treat sodium hyperabsorption in cystic fibrosis airway epithelial cultures.

The epithelial sodium channel (ENaC) is responsible for Na(+) and fluid absorption across colon, kidney, and airway epithelia. Short palate lung and nasal epithelial clone 1 (SPLUNC1) is a secreted, innate defense protein and an autocrine inhibitor of ENaC that is highly expressed in airway epithelia. While SPLUNC1 has a bactericidal permeability-increasing protein (BPI)-type structure, its NH2-terminal region lacks structure. Here we found that an 18 amino acid peptide, S18, which corresponded to residues G22-A39 of the SPLUNC1 NH2 terminus inhibited ENaC activity to a similar degree as full-length SPLUNC1 (∼2.5 fold), while SPLUNC1 protein lacking this region was without effect. S18 did not inhibit the structurally related acid-sensing ion channels, indicating specificity for ENaC. However, S18 preferentially bound to the βENaC subunit in a glycosylation-dependent manner. ENaC hyperactivity is contributory to cystic fibrosis (CF) lung disease. Unlike control, CF human bronchial epithelial cultures (HBECs) where airway surface liquid (ASL) height was abnormally low (4.2 ± 0.6 μm), addition of S18 prevented ENaC-led ASL hyperabsorption and maintained CF ASL height at 7.9 ± 0.6 μm, even in the presence of neutrophil elastase, which is comparable to heights seen in normal HBECs. Our data also indicate that the ENaC inhibitory domain of SPLUNC1 may be cleaved away from the main molecule by neutrophil elastase, suggesting that it may still be active during inflammation or neutrophilia. Furthermore, the robust inhibition of ENaC by the S18 peptide suggests that this peptide may be suitable for treating CF lung disease.

Limits of a rapid identification of common Mediterranean sandflies using polymerase chain reaction-restriction fragment length polymorphism

A total of 131 phlebotomine Algerian sandflies have been processed in the present study. They belong to the species Phlebotomus bergeroti, Phlebotomus alexandri, Phlebotomus sergenti, Phlebotomus chabaudi, Phlebotomus riouxi, Phlebotomus perniciosus, Phlebotomus longicuspis, Phlebotomus perfiliewi, Phlebotomus ariasi, Phlebotomus chadlii, Sergentomyia fallax, Sergentomyia minuta, Sergentomyia antennata, Sergentomyia schwetzi, Sergentomyia clydei, Sergentomyia christophersi and Grassomyia dreyfussi. They have been characterised by sequencing of a part of the cytochrome b (cyt b), t RNA serine and NADH1 on the one hand and of the cytochrome C oxidase I of the mitochondrial DNA (mtDNA) on the other hand. Our study highlights two sympatric populations within P. sergenti in the area of its type-locality and new haplotypes of P. perniciosus and P. longicuspis without recording the specimens called lcx previously found in North Africa. We tried to use a polymerase chain reaction-restriction fragment length polymorphism method based on a combined double digestion of each marker. These method is not interesting to identify sandflies all over the Mediterranean Basin.

Knockout confirmation for Hurries: rapid genotype identification of Trypanosoma cruzi transfectants by polymerase chain reaction directly from liquid culture

Gene knockout is a widely used approach to evaluate loss-of-function phenotypes and it can be facilitated by the incorporation of a DNA cassette having a drug-selectable marker. Confirmation of the correct knockout cassette insertion is an important step in gene removal validation and has generally been performed by polymerase chain reaction (PCR) assays following a time-consuming DNA extraction step. Here, we show a rapid procedure for the identification of Trypanosoma cruzi transfectants by PCR directly from liquid culture - without prior DNA extraction. This simple approach enabled us to generate PCR amplifications from different cultures varying from 106-108 cells/mL. We also show that it is possible to combine different primer pairs in a multiplex detection reaction and even to achieve knockout confirmation with an extremely simple interpretation of a real-time PCR result. Using the “culture PCR” approach, we show for the first time that we can assess different DNA sequence combinations by PCR directly from liquid culture, saving time in several tasks for T. cruzi genotype interrogation.

Identification of a novel splice-site mutation in the CYP1A2 gene.

AIMS: To identify the molecular basis for a low CYP1A2 metabolic status, as determined by a caffeine phenotyping test, in a 71-year-old, nonsmoking, Caucasian woman who presented with very high clozapine concentrations despite being administered a standard dose of the drug. METHODS: The nucleotide sequence of the 7 exons, exon-intron boundaries and 5'-flanking region of the CYP1A2 gene was analysed by direct sequencing. RESULTS: Only one heterozygous point mutation was identified in the donor splice site of intron 6 (3534G > A) of CYP1A2. This mutation could cause abnormal RNA splicing and therefore lead to a truncated nonfunctional enzyme. No other carrier of this mutation was identified in a population of 100 unrelated healthy Caucasians. CONCLUSIONS: This is the first report of a splice-site mutation affecting the CYP1A2 gene. This polymorphism is a likely explanation for the low CYP1A2 activity associated with high clozapine concentrations in this patient.

Discrepancies between Aedes aegypti identification in the field and in the laboratory after collection with a sticky trap

Currently, sticky traps are regularly employed to assist in the surveillance of Aedes aegypti infestation. We tested two alternative procedures for specimen identification performed by local health agents: directly in the field, as recommended by certain manufacturers, or after transportation to the laboratory. A total of 384 sticky traps (MosquiTRAP) were monitored monthly during one year in four geographically representative Brazilian municipalities. When the same samples were inspected in the field and in the laboratory, large differences were noted in the total number of mosquitoes recorded and in the number of specimens identified as Ae. aegypti by both procedures. Although field identification has the potential to speed vector surveillance, these results point to uncertainties in the evaluated protocol.

Identification of structural variation in mouse genomes.

Structural variation is variation in structure of DNA regions affecting DNA sequence length and/or orientation. It generally includes deletions, insertions, copy-number gains, inversions, and transposable elements. Traditionally, the identification of structural variation in genomes has been challenging. However, with the recent advances in high-throughput DNA sequencing and paired-end mapping (PEM) methods, the ability to identify structural variation and their respective association to human diseases has improved considerably. In this review, we describe our current knowledge of structural variation in the mouse, one of the prime model systems for studying human diseases and mammalian biology. We further present the evolutionary implications of structural variation on transposable elements. We conclude with future directions on the study of structural variation in mouse genomes that will increase our understanding of molecular architecture and functional consequences of structural variation.

Identification and in vitro reactivity of celiac immunoactive peptides in an apparent gluten-free beer.

Gluten content from barley, rye, wheat and in certain oat varieties, must be avoid in individuals with celiac disease. In most of the Western countries, the level of gluten content in food to be considered as gluten-free products is below 20 parts per million measured by ELISA based on specific anti-gluten peptide antibody. However, in beverages or food suffering complex hydrolytic processes as beers, the relative proportion of reactive peptides for celiac patients and the analytical techniques may differ, because of the diversity of the resulting peptide populations after fermentations. A beer below 20 parts per million of gluten but yet detectable levels of gluten peptides by anti-gliadin 33-mer antibodies (G12 and A1) was analyzed. We identified and characterized the relevant peptides for either antibody recognition or immunoactivity in celiac patients. The beer was fractionated by HPLC. The relative reactivity of the different HPLC fractions to the G12/A1 antibodies correlated to the reactivity of peripheral blood mononuclear cells isolated from 14 celiac individuals. Peptides from representative fractions classified according to the relative reactivity to G12/A1 antibodies were identified by mass spectrometry. The beer peptides containing sequences with similarity to those of previously described G12 and A1 epitopes were synthesized and confirmed significant reactivity for the antibodies. The most reactive peptides for G12/A1 also confirmed the highest immunogenicity by peripheral blood mononuclear cell activation and interferon γ production from celiac patients. We concluded that preparative HPLC combined with anti-gliadin 33-mer G12/A1 antibodies were very sensitive and specific methods to analyze the relevant immunogenic peptides in hydrolyzed gluten.

Bioactive endophytic fungi isolated from Caesalpinia echinata Lam. (Brazilwood) and identification of beauvericin as a trypanocidal metabolite from Fusarium sp.

Aiming to identify new sources of bioactive secondary metabolites, we isolated 82 endophytic fungi from stems and barks of the native Brazilian tree Caesalpinia echinata Lam. (Fabaceae). We tested their ethyl acetate extracts in several in vitro assays. The organic extracts from three isolates showed antibacterial activity against Staphylococcus aureus and Escherichia coli [minimal inhibitory concentration (MIC) 32-64 μg/mL]. One isolate inhibited the growth of Salmonella typhimurium (MIC 64 μg/mL) and two isolates inhibited the growth of Klebsiella oxytoca (MIC 64 μg/mL), Candida albicans and Candida tropicalis (MIC 64-128 μg/mL). Fourteen extracts at a concentration of 20 μg/mL showed antitumour activities against human breast cancer and human renal cancer cells, while two isolates showed anti-tumour activities against human melanoma cancer cells. Six extracts were able to reduce the proliferation of human peripheral blood mononuclear cells, indicating some degree of selective toxicity. Four isolates were able to inhibit Leishmania (Leishmania) amazonensis and one isolate inhibited Trypanosoma cruzi by at least 40% at 20 μg/mL. The trypanocidal extract obtained from Fusarium sp. [KF611679] culture was subjected to bioguided fractionation, which revealed beauvericin as the compound responsible for the observed toxicity of Fusarium sp. to T. cruzi. This depsipeptide showed a half maximal inhibitory concentration of 1.9 μg/mL (2.43 μM) in a T. cruzi cellular culture assay.

Identification of factors associated with diagnostic error in primary care.

BACKGROUND Missed, delayed or incorrect diagnoses are considered to be diagnostic errors. The aim of this paper is to describe the methodology of a study to analyse cognitive aspects of the process by which primary care (PC) physicians diagnose dyspnoea. It examines the possible links between the use of heuristics, suboptimal cognitive acts and diagnostic errors, using Reason's taxonomy of human error (slips, lapses, mistakes and violations). The influence of situational factors (professional experience, perceived overwork and fatigue) is also analysed. METHODS Cohort study of new episodes of dyspnoea in patients receiving care from family physicians and residents at PC centres in Granada (Spain). With an initial expected diagnostic error rate of 20%, and a sampling error of 3%, 384 episodes of dyspnoea are calculated to be required. In addition to filling out the electronic medical record of the patients attended, each physician fills out 2 specially designed questionnaires about the diagnostic process performed in each case of dyspnoea. The first questionnaire includes questions on the physician's initial diagnostic impression, the 3 most likely diagnoses (in order of likelihood), and the diagnosis reached after the initial medical history and physical examination. It also includes items on the physicians' perceived overwork and fatigue during patient care. The second questionnaire records the confirmed diagnosis once it is reached. The complete diagnostic process is peer-reviewed to identify and classify the diagnostic errors. The possible use of heuristics of representativeness, availability, and anchoring and adjustment in each diagnostic process is also analysed. Each audit is reviewed with the physician responsible for the diagnostic process. Finally, logistic regression models are used to determine if there are differences in the diagnostic error variables based on the heuristics identified. DISCUSSION This work sets out a new approach to studying the diagnostic decision-making process in PC, taking advantage of new technologies which allow immediate recording of the decision-making process.

Functional and molecular evolutionary analysis of the "Brevix Radix" gene family in mono-and dicotyledonous plants

ABSTRACTIn contrast to animals, plants cannot move from their place of birth and, therefore, need to adapt to their particular habitat in order to survive. Thus, plant development is remarkably plastic, making plants an ideal system for the isolation of genes that account for intraspecific natural variation and possibly environmental adaptation. However, to date, this approach mostly identified null alleles and missed mutations with subtle effects. For instance, BREVIS RADIX (BRX) has been isolated as a key regulator of root growth through a naturally occurring loss-of-function allele in the Arabidopsis thaliana accession Uk-1 and is the founding member of a highly-conserved plant-specific gene family.In this work, we show that a strong selective pressure is acting on the BRX gene family and dates back before the monocot-dicot divergence. However, functional diversification is observed mainly in dicotyledon BRX family genes and is correlated with acceleration in the evolutionary rates in the N-terminal regions. Population genetic data revealed that BRX is highly conserved across Arabidopsis accessions and presents signatures of adaptation. Interestingly, a seven amino acid deletion polymorphism in BRX sequence was found in a few accessions, which seems to be responsible for their enhanced primary root growth. Nevertheless, BRX might not only be active in the root, as suggested by its expression in the shoot. Indeed, leaves and cotyledons of brx mutants are significantly smaller than wild- type. This phenotype is a direct consequence of the absence of BRX function in the shoot rather than an indirect effect of an altered root system growth. Interestingly, cotyledons of brx plants reflect the same physiological defects as the root. Moreover, phenotypes in BRX gain-of-function plants, such as epinastic leaves and increased epidermal cell size, could be associated with an increase in leaf brassinosteroid content.Collectively, these results indicate that BRX contributes to local adaptation by ubiquitously regulating plant growth, probably through the modulation of brassinosteroid biosynthesis.RÉSUMÉContrairement à la plupart des animaux, les plantes ne peuvent se mouvoir et doivent ainsi s'adapter à leur environnement pour survivre. Pour cette raison, elles représentent un système idéal pour l'identification de gènes contribuant à la variation naturelle intra- spécifique, ainsi qu'à l'adaptation. Cependant, cette approche a, jusqu'à présent, surtout permis d'isoler des allèles nuls et non des mutations conférant des effets plus subtiles. C'est le cas du gène Β REVIS RADIX (BRX), un régulateur clé de la croissance racinaire, qui a été identifié grâce à un allèle non-fonctionnel présent dans l'accession naturelle d'Arabidopsis thaliana Uk-1. BRX et ses homologues des plantes mono- et dicotylédones forment une famille très conservée et spécifique aux plantes.Dans ce travail, nous démontrons que la famille de gènes BRX est soumise à une forte pression de sélection qui remonte avant la divergence entre mono- et dicotylédones. Cependant, une diversification fonctionnelle a été observée chez les gènes des dicotylédones et corrèle avec une accélération de la vitesse d'évolution dans leur région N- terminale. Une analyse génétique de différentes accessions naturelles d'Arabidopsis a révélé que BRX est hautement conservé et présente des signatures d'adaptation. Remarquablement, un polymorphisme de délétion de sept acides aminés a été détecté dans quelques accessions et a pour conséquence une plus forte croissance de la racine primaire. Néanmoins, il semble que le rôle de BRX ne se limite pas qu'à la racine, comme indiqué par son expression dans les parties aériennes de la plante. En effet, les mutants brx présentent des cotylédons et des feuilles significativement plus petits que le type sauvage, une conséquence directe de l'absence d'activité de BRX dans ces organes. Nous avons aussi noté que les cotylédons des mutants brx, à l'instar des racines, ont une perception altérée de l'auxine et peuvent être complémentés par l'application exogène de brassinostéroïdes. De plus, dans des plantes présentant un gain de fonction BRX, les feuilles sont épinastiques et les cellules de leur épiderme plus grandes. Ces phénotypes sont accompagnés d'une augmentation de la concentration de brassinostéroïdes dans les feuilles. Conjointement, ces résultats démontrent que BRX contribue à une adaptation locale de la plante par la régulation générale de sa croissance, probablement en modulant la biosynthèse des brassinostéroïdes.