Biotin-avidin sandwich elisa with specific human isotypes IgG1 and IgG4 for Culicidae mosquito blood meal identification from an epizootic yellow fever area in Brazil

With a view toward investigating the feeding behavior of Culicidae mosquitoes from an area of epizootic yellow fever transmission in the municipalities of Garruchos and Santo Antônio das Missões, Rio Grande do Sul State, Brazil, specimens were collected by aspiration from September 2005 to April 2007. The engorged females were submitted to blood meal identification by enzyme-linked immunosorbent assay (ELISA). A total of 142 blood-engorged samples were examined for human or monkey blood through species-specific IgG. Additional tests for specificity utilizing isotypes IgG1 and IgG4 of human monoclonal antibodies showed that only anti-human IgG1 was effective in recognizing blood meals of human origin. The results indicated a significant difference (p = 0.027) in detection patterns in samples of Haemagogus leucocelaenus recorded from human blood meals at Santo Antônio das Missões, which suggests some degree of exposure, since it was an area where epizootic outbreaks have been reported.

Biotin/avidin sandwich enzyme-linked immunosorbent assay for Culicidae mosquito blood meal identification

The knowledge of mosquitoes Culicidae host feeding patterns is basic to understand the roles of different species and to indicate their importance in the epidemiology of arthropod-borne diseases. A laboratory assay was developed aiming at standardizing the biotin-avidin sandwich enzyme-linked immunosorbent assay, which was unprecedented for mosquito blood meal identification. The enzyme-linked immunosorbent assay (ELISA) activity was evaluated by the detection of titers on each sample of the 28 blood-fed Culex quinquefasciatus. In light of the high sensitivity that the technique permits, by means of small quantities of specific antibodies commercially provided and phosphatase substrate which reinforces additional dilutions, human and rat blood meals were readily identified in all laboratory-raised Culex quinquefasciatus tested. The assay was effective to detect human blood meal dilutions up to 1:4,096, which enables the technique to be applied in field studies. Additionally, the present results indicate a significant difference between the detection patterns recorded from human blood meal which corroborate the results of host feeding patterns.

Biotin/Avidin sandwich enzyme-linked immunosorbent assay for Culicidae mosquito blood meal identification

The knowledge of mosquitoes Culicidae host feeding patterns is basic to understand the roles of different species and to indicate their importance in the epidemiology of arthropod-borne diseases. A laboratory assay was developed aiming at standardizing the biotin-avidin sandwich enzyme-linked immunosorbent assay, which was unprecedented for mosquito blood meal identification. The enzyme-linked immunosorbent assay (ELISA) activity was evaluated by the detection of titers on each sample of the 28 blood-fed Culex quinquefasciatus. In light of the high sensitivity that the technique permits, by means of small quantities of specific antibodies commercially provided and phosphatase substrate which reinforces additional dilutions, human and rat blood meals were readily identified in all laboratory-raised Culex quinquefasciatus tested. The assay was effective to detect human blood meal dilutions up to 1:4,096, which enables the technique to be applied in field studies. Additionally, the present results indicate a significant difference between the detection patterns recorded from human blood meal which corroborate the results of host feeding patterns

Gender identification of five genera of stingless bees (Apidae, Meliponini) based on wing morphology

Currently, the identification of pollinators is a critical necessity of conservation programs. After it was found that features extracted from patterns of wing venation are sufficient to discriminate among insect species, various studies have focused on this structure. We examined wing venation patterns of males and workers of five stingless bee species in order to determine if there are differences between sexes and if these differences are greater within than between species. Geometric morphometric analyses were made of the forewings of males and workers of Nannotrigona testaceicornis, Melipona quadrifasciata, Frieseomelitta varia, and Scaptotrigona aff. depilis and Plebeia remota. The patterns of males and workers from the same species were more similar than the patterns of individuals of the same sex from different species, and the patterns of both males and workers, when analyzed alone, were sufficiently different to distinguish among these five species. This demonstrates that we can use this kind of analysis for the identification of stingless bee species and that the sex of the individual does not impede identification. Computer-assisted morphometric analysis of bee wing images can be a useful tool for biodiversity studies and conservation programs.

The Effect of Spatial Scale on Predicting Time Series: A Study on Epidemiological System Identification

A susceptible-infective-recovered (SIR) epidemiological model based on probabilistic cellular automaton (PCA) is employed for simulating the temporal evolution of the registered cases of chickenpox in Arizona, USA, between 1994 and 2004. At each time step, every individual is in one of the states S, I, or R. The parameters of this model are the probabilities of each individual (each cell forming the PCA lattice ) passing from a state to another state. Here, the values of these probabilities are identified by using a genetic algorithm. If nonrealistic values are allowed to the parameters, the predictions present better agreement with the historical series than if they are forced to present realistic values. A discussion about how the size of the PCA lattice affects the quality of the model predictions is presented. Copyright (C) 2009 L. H. A. Monteiro et al.

System Identification and Prediction of Dengue Fever Incidence in Rio de Janeiro

Identification, prediction, and control of a system are engineering subjects, regardless of the nature of the system. Here, the temporal evolution of the number of individuals with dengue fever weekly recorded in the city of Rio de Janeiro, Brazil, during 2007, is used to identify SIS (susceptible-infective-susceptible) and SIR (susceptible-infective-removed) models formulated in terms of cellular automaton (CA). In the identification process, a genetic algorithm (GA) is utilized to find the probabilities of the state transition S -> I able of reproducing in the CA lattice the historical series of 2007. These probabilities depend on the number of infective neighbors. Time-varying and non-time-varying probabilities, three different sizes of lattices, and two kinds of coupling topology among the cells are taken into consideration. Then, these epidemiological models built by combining CA and GA are employed for predicting the cases of sick persons in 2008. Such models can be useful for forecasting and controlling the spreading of this infectious disease.

Genetic trend estimates of meat quality traits in a male broiler line

The present research was conducted to estimate the genetic trends for meat quality traits in a male broiler line. The traits analyzed were initial pH, pH at 6 h after slaughter, final pH, initial range of falling pH, final range of falling pH, lightness, redness, yellowness, weep loss, drip loss, shrink loss, and shear force. The number of observations varied between 618 and 2125 for each trait. Genetic values were obtained by restricted maximum likelihood, and the numerator relationship matrix had 107,154 animals. The genetic trends were estimated by regression of the broiler average genetic values with respect to unit of time (generations), and the average genetic trend was estimated by regression coefficients. Generally, for the traits analyzed, small genetic trends were obtained, except for drip loss and shear force, which were higher. The small magnitude of the trends found could be a consequence of the absence of selection for meat quality traits in the line analyzed. The estimates of genetic trends obtained were an indication of an improvement in the meat quality traits in the line analyzed, except for drip loss.

Identification of a Highly Antigenic Linear B Cell Epitope within Plasmodium vivax Apical Membrane Antigen 1 (AMA-1)

Apical membrane antigen 1 (AMA-1) is considered to be a major candidate antigen for a malaria vaccine. Previous immunoepidemiological studies of naturally acquired immunity to Plasmodium vivax AMA-1 (PvAMA-1) have shown a higher prevalence of specific antibodies to domain II (DII) of AMA-1. In the present study, we confirmed that specific antibody responses from naturally infected individuals were highly reactive to both full-length AMA-1 and DII. Also, we demonstrated a strong association between AMA-1 and DII IgG and IgG subclass responses. We analyzed the primary sequence of PvAMA-1 for B cell linear epitopes co-occurring with intrinsically unstructured/ disordered regions (IURs). The B cell epitope comprising the amino acid sequence 290-307 of PvAMA-1 (SASDQPTQYEEEMTDYQK), with the highest prediction scores, was identified in domain II and further selected for chemical synthesis and immunological testing. The antigenicity of the synthetic peptide was identified by serological analysis using sera from P. vivax-infected individuals who were knowingly reactive to the PvAMA-1 ectodomain only, domain II only, or reactive to both antigens. Although the synthetic peptide was recognized by all serum samples specific to domain II, serum with reactivity only to the full-length protein presented 58.3% positivity. Moreover, IgG reactivity against PvAMA-1 and domain II after depletion of specific synthetic peptide antibodies was reduced by 18% and 33% (P = 0.0001 for both), respectively. These results suggest that the linear epitope SASDQPTQYEEEMTDYQK is highly antigenic during natural human infections and is an important antigenic region of the domain II of PvAMA-1, suggesting its possible future use in pre-clinical studies.

Identification of possible targets of the Aspergillus fumigatus CRZ1 homologue, CrzA

Background: Calcineurin, a serine/threonine-specific protein phosphatase, plays an important role in the control of cell morphology and virulence in fungi. Calcineurin regulates localization and activity of a transcription factor called CRZ1. Recently, we characterize Aspergillus fumigatus CRZ1 homologue, AfCrzA. Here, we investigate which pathways are influenced by A. fumigatus AfCrzA during a short pulse of calcium by comparatively determining the transcriptional profile of A. fumigatus wild type and.AfcrzA mutant strains. Results: We were able to observe 3,622 genes modulated in at least one timepoint in the mutant when compared to the wild type strain (3,211 and 411 at 10 and 30 minutes, respectively). Decreased mRNA abundance in the Delta crzA was seen for genes encoding calcium transporters, transcription factors and genes that could be directly or indirectly involved in calcium metabolism. Increased mRNA accumulation was observed for some genes encoding proteins involved in stress response. AfCrzA overexpression in A. fumigatus increases the expression of several of these genes. The deleted strain of one of these genes, AfRcnA, belonging to a class of endogenous calcineurin regulators, calcipressins, had more calcineurin activity after exposure to calcium and was less sensitive to menadione 30 mu M, hydrogen peroxide 2.5 mM, EGTA 25 mM, and MnCl(2) 25 mM. We constructed deletion, overexpression, and GFP fusion protein for the closely related A. nidulans AnRcnA. GFP

Identification of Neoplasias of Breast Tissues Using a Powder Diffractometer

An investigation was carried out to study the potential use of the angular distribution of scattered photons by human breast samples for a rapid identification of neoplasias of breast tissues. This technique has possible applications as diagnostic aid for breast cancer. In this work, a commercial powder diffractometer was used to obtain the scattering profiles from breast tissues histopathologically classified as normal breast tissues, fibroadenomas (benign breast diseases) and carcinomas (malignant breast diseases), in the interval 0.02 angstrom(-1) < x < 0.62 angstrom(-1). The experimental methods and data corrections are discussed in detail, and they included background subtraction, polarization, self-attenuation and geometric effects. The validation of the experimental procedure was achieved through an analysis of water sample. The results showed that the scattering profile is a unique impression of each type of tissue, being correlated with their microscopic morphological features. Multivariate analysis was applied to these profiles in order to verify if the information carried by these scattering profiles allow the differentiation between normal, benign and malignant breast tissues. The statistical analysis results showed that a correct identification of 75% of the analyzed samples is accomplished. The values of sensibility and specificity of this method in correctly differentiating between normal and neoplastic samples were 95.6% and 82.3%, respectively, while the values for differentiation between benign and malignant neoplasias were 78.6% and 62.5%. These initial results indicate the feasible use of commercial powder diffractometer to provide a rapid diagnostic with a high sensitivity.

Development of Hepatitis C Virus Genotyping by Real-Time PCR Based on the NS5B Region

Background: Hepatitis C virus (HCV) genotyping is the most significant predictor of the response to antiviral therapy. The aim of this study was to develop and evaluate a novel real-time PCR method for HCV genotyping based on the NS5B region. Methodology/Principal Findings: Two triplex reaction sets were designed, one to detect genotypes 1a, 1b and 3a; and another to detect genotypes 2a, 2b, and 2c. This approach had an overall sensitivity of 97.0%, detecting 295 of the 304 tested samples. All samples genotyped by real-time PCR had the same type that was assigned using LiPA version 1 (Line in Probe Assay). Although LiPA v. 1 was not able to subtype 68 of the 295 samples (23.0%) and rendered different subtype results from those assigned by real-time PCR for 12/295 samples (4.0%), NS5B sequencing and real-time PCR results agreed in all 146 tested cases. Analytical sensitivity of the real-time PCR assay was determined by end-point dilution of the 5000 IU/ml member of the OptiQuant HCV RNA panel. The lower limit of detection was estimated to be 125 IU/ml for genotype 3a, 250 IU/ml for genotypes 1b and 2b, and 500 IU/ml for genotype 1a. Conclusions/Significance: The total time required for performing this assay was two hours, compared to four hours required for LiPA v. 1 after PCR-amplification. Furthermore, the estimated reaction cost was nine times lower than that of available commercial methods in Brazil. Thus, we have developed an efficient, feasible, and affordable method for HCV genotype identification.

Fluorescence and reflectance spectroscopy for identification of atherosclerosis in human carotid arteries using principal components analysis

Objectives: The aim of this work was to verify the differentiation between normal and pathological human carotid artery tissues by using fluorescence and reflectance spectroscopy in the 400- to 700-nm range and the spectral characterization by means of principal components analysis. Background Data: Atherosclerosis is the most common and serious pathology of the cardiovascular system. Principal components represent the main spectral characteristics that occur within the spectral data and could be used for tissue classification. Materials and Methods: Sixty postmortem carotid artery fragments (26 non-atherosclerotic and 34 atherosclerotic with non-calcified plaques) were studied. The excitation radiation consisted of a 488-nm argon laser. Two 600-mu m core optical fibers were used, one for excitation and one to collect the fluorescence radiation from the samples. The reflectance system was composed of a halogen lamp coupled to an excitation fiber positioned in one of the ports of an integrating sphere that delivered 5 mW to the sample. The photo-reflectance signal was coupled to a 1/4-m spectrograph via an optical fiber. Euclidean distance was then used to classify each principal component score into one of two classes, normal and atherosclerotic tissue, for both fluorescence and reflectance. Results: The principal components analysis allowed classification of the samples with 81% sensitivity and 88% specificity for fluorescence, and 81% sensitivity and 91% specificity for reflectance. Conclusions: Our results showed that principal components analysis could be applied to differentiate between normal and atherosclerotic tissue with high sensitivity and specificity.

Suppression subtractive hybridization profiles of radial growth phase and metastatic melanoma cell lines reveal novel potential targets

Background: Melanoma progression occurs through three major stages: radial growth phase (RGP), confined to the epidermis; vertical growth phase (VGP), when the tumor has invaded into the dermis; and metastasis. In this work, we used suppression subtractive hybridization (SSH) to investigate the molecular signature of melanoma progression, by comparing a group of metastatic cell lines with an RGP-like cell line showing characteristics of early neoplastic lesions including expression of the metastasis suppressor KISS1, lack of alpha v beta 3-integrin and low levels of RHOC. Methods: Two subtracted cDNA collections were obtained, one (RGP library) by subtracting the RGP cell line (WM1552C) cDNA from a cDNA pool from four metastatic cell lines (WM9, WM852, 1205Lu and WM1617), and the other (Met library) by the reverse subtraction. Clones were sequenced and annotated, and expression validation was done by Northern blot and RT-PCR. Gene Ontology annotation and searches in large-scale melanoma expression studies were done for the genes identified. Results: We identified 367 clones from the RGP library and 386 from the Met library, of which 351 and 368, respectively, match human mRNA sequences, representing 288 and 217 annotated genes. We confirmed the differential expression of all genes selected for validation. In the Met library, we found an enrichment of genes in the growth factors/receptor, adhesion and motility categories whereas in the RGP library, enriched categories were nucleotide biosynthesis, DNA packing/repair, and macromolecular/vesicular trafficking. Interestingly, 19% of the genes from the RGP library map to chromosome 1 against 4% of the ones from Met library. Conclusion: This study identifies two populations of genes differentially expressed between melanoma cell lines from two tumor stages and suggests that these sets of genes represent profiles of less aggressive versus metastatic melanomas. A search for expression profiles of melanoma in available expression study databases allowed us to point to a great potential of involvement in tumor progression for several of the genes identified here. A few sequences obtained here may also contribute to extend annotated mRNAs or to the identification of novel transcripts.

Genome Survey Sequence Analysis and Identification of Homologs of Major Surface Protease (gp63) Genes in Trypanosoma rangeli

In this study, 222 genome survey sequences were generated for Trypanosoma rangeli strain P07 isolated from an opossum (Didelphis albiventris) in Minas Gerais State, Brazil. T. rangeli sequences were compared by BLASTX (Basic Local Alignment Search Tool X) analysis with the assembled contigs of Leishmania braziliensis, Leishmania infantum, Leishmania major, Trypanosoma brucei, and Trypanosoma cruzi. Results revealed that 82% (182/222) of the sequences were associated with predicted proteins described, whereas 18% (40/222) of the sequences did not show significant identity with sequences deposited in databases, suggesting that they may represent T. rangeli-specific sequences. Among the 182 predicted sequences, 179 (80.6%) had the highest similarity with T. cruzi, 2 (0.9%) with T. brucei, and 1 (0.5%) with L. braziliensis. Computer analysis permitted the identification of members of various gene families described for trypanosomatids in the genome of T. rangeli, such as trans-sialidases, mucin-associated surface proteins, and major surface proteases (MSP or gp63). This is the first report identifying sequences of the MSP family in T. rangeli. Multiple sequence alignments showed that the predicted MSP of T. rangeli presented the typical characteristics of metalloproteases, such as the presence of the HEXXH motif, which corresponds to a region previously associated with the catalytic site of the enzyme, and various cysteine and proline residues, which are conserved among MSPs of different trypanosomatid species. Reverse transcriptase-polymerase chain reaction analysis revealed the presence of MSP transcripts in epimastigote forms of T. rangeli.