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 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.

Molecular identification of food sources in triatomines in the Brazilian northeast: roles of goats and rodents in Chagas disease epidemiology

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Application of Cognitive Engineering Principles to the Redesign of a Dichotomous Identification Key for Parasitology

Dichotomous identification keys are used throughout biology for identification of plants, insects, and parasites. However, correct use of identification keys can be difficult as they are not usually intended for novice users who may not be familiar with the terminology used or with the morphology of the organism being identified. Therefore, we applied cognitive engineering principles to redesign a parasitology identification key for the Internet. We addressed issues of visual clutter and spatial distance by displaying a single question couplet at a time and by switching to the appropriate next couplet after the user made a choice. Our analysis of the original paper-based key versus the Web-based approach found that of 26 applicable cognitive engineering principles, the paper key did not meet 4 (15%) and partially met 11 (42%). In contrast, the redesigned key met 100% of 32 applicable cognitive engineering principles.

Identification of functional domains and evolution of Tc1-like transposable elements.

Tc1-like transposable elements from teleost fish have been phylogenetically examined to determine the mechanisms involved in their evolution and conserved domains of function. We identified two new functional domains in these elements. The first is a bipartite nuclear localization signal, indicating that transposons can take advantage of the transport machinery of host cells for nuclear uptake of their transposases. The second is a novel combination of a paired domain-related protein motif juxtaposed to a leucine zipper-like domain located in the putative DNA-binding regions of the transposases. This domain coexists with a special inverted repeat structure in certain transposons in such phylogenetically distant hosts as fish and insects. Our data indicate that reassortment of functional domains and horizontal transmission between species are involved in the formation and spread of new types of transposable elements.

The loss of female sex pheromone after mating in the corn earworm moth Helicoverpa zea: identification of a male pheromonostatic peptide.

Female moths often become depleted of sex pheromone after mating as the various components of virgin behavior are switched off. In examining a potential male contribution to these events in the corn earworm moth Helicoverpa zea, we have characterized a basic polypeptide from the tissues producing (accessory glands) and storing (duplex) the seminal fluids. The peptide evokes the depletion of sex pheromone when injected into virgin females. This pheromonostatic peptide (PSP) is 57 amino acids long and contains a single disulfide bridge. It is blocked at the N terminus with pyroglutamate and at the C terminus by amidation. As little as 23 ng of peptide evokes the near-complete depletion of pheromone in decapitated (neck-ligated) females that had been injected with pheromone biosynthesis-activating neuropeptide. Activity is approximately 15-fold less in intact virgins, showing that the head limits the expression of activity in these injected females. Females mated to surgically impaired males, capable of producing a spermatophore but not transferring spermatozoa or seminal fluids, are depleted of pheromone by injected peptide. Females whose abdominal nerve cords have been severed are not depleted of pheromone after mating. Thus, neural signals either descending or ascending via the nerve cord are required for the depletion of pheromone after mating. PSP, from the seminal fluids, may participate in this process by direct or indirect action on the glandular tissue; if so, it represents an unusual mechanism in insects for the regulation by seminal fluids of postmating reproductive behavior.

The Coccidae, tables for the identification of the subfamilies and some of the more important genera and species, together with discussions of their anatomy and life history,

Bibliography: p. 466-474.

Identification and molecular modeling of a novel, plant-like, human purple acid phosphatase

Purple acid phosphatases are a family of binuclear metallohydrolases that have been identified in plants, animals and fungi. Only one isoform of similar to 35 kDa has been isolated from animals, where it is associated with bone resorption and microbial killing through its phosphatase activity, and hydroxyl radical production, respectively. Using the sensitive PSI-BLAST search method, sequences representing new purple acid phosphatase-like proteins have been identified in mammals, insects and nematodes. These new putative isoforms are closely related to the similar to 55 kDa purple acid phosphatase characterized from plants. Secondary structure prediction of the new human isoform further confirms its similarity to a purple acid phosphatase from the red kidney bean. A structural model for the human enzyme was constructed based on the red kidney bean purple acid phosphatase structure. This model shows that the catalytic centre observed in other purple acid phosphatases is also present in this new isoform. These observations suggest that the sequences identified in this study represent a novel subfamily of plant-like purple acid phosphatases in animals and humans. (c) 2006 Elsevier B.V. All rights reserved.

Identification of Conserved Splicing Motifs in Mutually Exclusive Exons of 15 Insect Species

Background: During alternative splicing, the inclusion of an exon in the final mRNA molecule is determined by nuclear proteins that bind cis-regulatory sequences in a target pre-mRNA molecule. A recent study suggested that the regulatory codes of individual RNA-binding proteins may be nearly immutable between very diverse species such as mammals and insects. The model system Drosophila melanogaster therefore presents an excellent opportunity for the study of alternative splicing due to the availability of quality EST annotations in FlyBase. Methods: In this paper, we describe an in silico analysis pipeline to extract putative exonic splicing regulatory sequences from a multiple alignment of 15 species of insects. Our method, ESTs-to-ESRs (E2E), uses graph analysis of EST splicing graphs to identify mutually exclusive (ME) exons and combines phylogenetic measures, a sliding window approach along the multiple alignment and the Welch’s t statistic to extract conserved ESR motifs. Results: The most frequent 100% conserved word of length 5 bp in different insect exons was “ATGGA”. We identified 799 statistically significant “spike” hexamers, 218 motifs with either a left or right FDR corrected spike magnitude p-value < 0.05 and 83 with both left and right uncorrected p < 0.01. 11 genes were identified with highly significant motifs in one ME exon but not in the other, suggesting regulation of ME exon splicing through these highly conserved hexamers. The majority of these genes have been shown to have regulated spatiotemporal expression. 10 elements were found to match three mammalian splicing regulator databases. A putative ESR motif, GATGCAG, was identified in the ME-13b but not in the ME-13a of Drosophila N-Cadherin, a gene that has been shown to have a distinct spatiotemporal expression pattern of spliced isoforms in a recent study. Conclusions: Analysis of phylogenetic relationships and variability of sequence conservation as implemented in the E2E spikes method may lead to improved identification of ESRs. We found that approximately half of the putative ESRs in common between insects and mammals have a high statistical support (p < 0.01). Several Drosophila genes with spatiotemporal expression patterns were identified to contain putative ESRs located in one exon of the ME exon pairs but not in the other.

Identification and characterization of a glycosulfatase-encoding gene cluster in Bifidobacterium breve UCC2003

Bifidobacteria constitute a specific group of commensal bacteria, typically found in the gastrointestinal tract (GIT) of humans and other mammals. Bifidobacterium breve strains are numerically prevalent among the gut microbiota of many healthy breast-fed infants. In the current study, we investigated glycosulfatase activity in a bacterial nursling stool isolate, B. breve UCC2003. Two putative sulfatases were identified on the genome of B. breve UCC2003. The sulfated monosaccharide N-acetylglucosamine-6-sulfate (GlcNAc-6-S) was shown to support growth of B. breve UCC2003, while, N-acetylglucosamine-3-sulfate, N-acetylgalactosamine-3-sulfate and N-acetylgalactosamine-6-sulfate, did not support appreciable growth. Using a combination of transcriptomic and functional genomic approaches, a gene cluster, designated ats2, was shown to be specifically required for GlcNAc-6-S metabolism. Transcription of the ats2 cluster is regulated by a ROK-family transcriptional repressor. This study represents the first description of glycosulfatase activity within the Bifidobacterium genus. Bifidobacteria are saccharolytic organisms naturally found in the digestive tract of mammals and insects. Bifidobacterium breve strains utilize a variety of plant and host-derived carbohydrates which allow them to be present as prominent members of the infant gut microbiota as well as being present in the gastrointestinal tract of adults. In this study, we introduce a previously unexplored area of carbohydrate metabolism in bifidobacteria, namely the metabolism of sulfated carbohydrates. B. breve UCC2003 was shown to metabolize N-acetylglucosamine-6-sulfate (GlcNAc-6-S) through one of two sulfatase-encoding gene clusters identified on its genome. GlcNAc-6-S can be found in terminal or branched positions of mucin oligosaccharides, the glycoprotein component of the mucous layer that covers the digestive tract. The results of this study provide further evidence of this species' ability to utilize mucin-derived sugars, a trait which may provide a competitive advantage in both the infant and adult gut.

Identification de caractéristiques agronomiques pour le diagnostic et la prise de décision de régénération des vergers de cacaoyers en Côte d’Ivoire

The rehabilitation of the old cocoa ( Theobroma cacao L. ) farms is one of the major challenges for a sustainable cocoa production. A study was carried out to set up a guide which could be used as a decision making tool for a quick and efficient diagnosis of the old cocoa orchards and to choose the appropriate regeneration option (rehabilitation or replanting). A sample of 90 rehabilitated cocoa farms and of 75 replanted cocoa farms was surveyed in 12 regions representing the three main cocoa producing sectors in the country. Data were collected on the key agronomic characteristics of these cacao farms. These were cocoa variety, farm size, age, yield, planting density, number of shade trees and the level of damages caused by insects and diseases. The results showed that age, planting density and yield were the discriminating criteria of these farms. The average values of these criteria were 25 to 30 years for the age, 800 to 1 000 trees ha-1 for the planting density and 250 to 400 kg ha-1 an-1 for the yield. Based on these criteria and their average values, a decision making guide was designed for the diagnosis of cocoa farms and the choice of regeneration option. According to this guide, old cocoa farms (more than 25 years), degraded and unproductive should be replanted. However, younger farms having planting density and yield higher than the average values above should be rehabilitated.

Mucopolysaccharidosis Type Ii: Identification Of 30 Novel Mutations Among Latin American Patients.

In this study, 103 unrelated South-American patients with mucopolysaccharidosis type II (MPS II) were investigated aiming at the identification of iduronate-2-sulfatase (IDS) disease causing mutations and the possibility of some insights on the genotype-phenotype correlation The strategy used for genotyping involved the identification of the previously reported inversion/disruption of the IDS gene by PCR and screening for other mutations by PCR/SSCP. The exons with altered mobility on SSCP were sequenced, as well as all the exons of patients with no SSCP alteration. By using this strategy, we were able to find the pathogenic mutation in all patients. Alterations such as inversion/disruption and partial/total deletions of the IDS gene were found in 20/103 (19%) patients. Small insertions/deletions/indels (<22 bp) and point mutations were identified in 83/103 (88%) patients, including 30 novel mutations; except for a higher frequency of small duplications in relation to small deletions, the frequencies of major and minor alterations found in our sample are in accordance with those described in the literature.

Identification Of Target Genes Using Gene Expression Profile Of Granulocytes From Patients With Chronic Myeloid Leukemia Treated With Tyrosine Kinase Inhibitors.

Differential gene expression analysis by suppression subtractive hybridization with correlation to the metabolic pathways involved in chronic myeloid leukemia (CML) may provide a new insight into the pathogenesis of CML. Among the overexpressed genes found in CML at diagnosis are SEPT5, RUNX1, MIER1, KPNA6 and FLT3, while PAN3, TOB1 and ITCH were decreased when compared to healthy volunteers. Some genes were identified and involved in CML for the first time, including TOB1, which showed a low expression in patients with CML during tyrosine kinase inhibitor treatment with no complete cytogenetic response. In agreement, reduced expression of TOB1 was also observed in resistant patients with CML compared to responsive patients. This might be related to the deregulation of apoptosis and the signaling pathway leading to resistance. Most of the identified genes were related to the regulation of nuclear factor κB (NF-κB), AKT, interferon and interleukin-4 (IL-4) in healthy cells. The results of this study combined with literature data show specific gene pathways that might be explored as markers to assess the evolution and prognosis of CML as well as identify new therapeutic targets.

High-throughput Analysis By Sp-ldi-ms For Fast Identification Of Adulterations In Commercial Balsamic Vinegars.

Balsamic vinegar (BV) is a typical and valuable Italian product, worldwide appreciated thanks to its characteristic flavors and potential health benefits. Several studies have been conducted to assess physicochemical and microbial compositions of BV, as well as its beneficial properties. Due to highly-disseminated claims of antioxidant, antihypertensive and antiglycemic properties, BV is a known target for frauds and adulterations. For that matter, product authentication, certifying its origin (region or country) and thus the processing conditions, is becoming a growing concern. Striving for fraud reduction as well as quality and safety assurance, reliable analytical strategies to rapidly evaluate BV quality are very interesting, also from an economical point of view. This work employs silica plate laser desorption/ionization mass spectrometry (SP-LDI-MS) for fast chemical profiling of commercial BV samples with protected geographical indication (PGI) and identification of its adulterated samples with low-priced vinegars, namely apple, alcohol and red/white wines.

Metals And (metallo)proteins Identification In Vitreous Humor Focusing On Post-mortem Biochemistry.

This work describes the evaluation of metals and (metallo)proteins in vitreous humor samples and their correlations with some biological aspects in different post-mortem intervals (1-7 days), taking into account both decomposing and non-decomposing bodies. After qualitative evaluation of the samples involving 26 elements, representative metal ions (Fe, Mg and Mo) are determined by inductively coupled plasma mass spectrometry after using mini-vial decomposition system for sample preparation. A significant trend for Fe is found with post-mortem time for decomposing bodies because of a significant increase of iron concentration when comparing samples from bodies presenting 3 and 7 days post-mortem interval. An important clue to elucidate the role of metals is the coupling of liquid chromatography with inductively coupled plasma mass spectrometry for identification of metals linked to proteins, as well as mass spectrometry for the identification of those proteins involved in the post-mortem interval.