DNA barcode reveals occurrence of threatened species and hidden diversity on Teleost fish trade in the Coastal Amazon.

This study aimed to identify the teleost fish species sold in Bragança, a major fishing hub on the north coast of Brazil. The COI gene analysis was performed for the identification of fish species. The local market uses common names that are not accurate and do not reflect the diversity of the species. 204 sequences were obtained, with 119 haplotypes. 83 species were identified by comparing with public databases and constructing phylogenetic trees, with Carangidae being the most prevalent family. The study also found Haemulon atlanticus, Menticirrhus cuiaranensis and Hoplias misioneira, a newly described species from the Amazon basin, among the samples. Additionally, 73 commercial names were recorded, including 10 categories, and the illegal trade of Epinephelus itajara was detected. The DNA Barcode method proved to be effective for discriminating the species. The study highlights that common and commercial names are vague and underestimate the fish diversity, and that Brazil needs to revise its regulations for commercial and scientific names.

Genetic connectivity and population expansion inferred from multilocus analysis in Lutjanus alexandrei (Lutjanidae-Perciformes), an endemic snapper from Northeastern Brazilian coast.

Previous studies about the genetic diversity, connectivity and demographic history in Lutjanidae fishes have reported a common pattern of genetic homogeneity and expansion in populations from Western South Atlantic. In the present work, we inferred the population structure, the levels of genetic diversity and the demographic history of the Brazilian snapper Lutjanus alexandrei, a recently described and endemic species from Northeastern coast of Brazil. Five different fragments, including mitochondrial DNA (Control Region, Cyt b and ND4) and nuclear DNA (Myostatin and S7) regions were analyzed in 120 specimens of L. alexandrei from four localities in Northeastern Brazil, representing the first study of population genetics in this species. High levels of genetic diversity were observed following a panmictic pattern, probably related to the larval dispersal by the current tides along the Brazilian coast. In addition, both demographic history and neutrality tests indicated that L. alexandrei has undergone population expansion during Pleistocene. In this sense, the sea level variation from this period could have increased the available resources and suitable habitats for the Brazilian snapper.

Molecular characterization of juvenile fish from the Amazon estuary using DNA barcoding approach.

The efficiency of the DNA barcoding relies on sequencing fragment of the Cytochrome C Subunit I (COI) gene, which has been claimed as a tool to biodiversity identification from distinct groups. Accordingly, the goal of this study was to identify juvenile fish species along an estuary of Caeté River in the Brazilian Blue Amazon based on. For this purpose, we applied the DNA barcoding and discuss this approach as a tool for discrimination of species in early ontogenetic stages. A 500-bp fragment was obtained from 74 individuals, belonging to 23 species, 20 genera, 13 families and seven orders. About 70% of the 46 haplotypes revealed congruence between morphological and molecular species identification, while 8% of them failed in identification of taxa and 22% demonstrated morphological misidentification. These results proved that COI fragments were effective to diagnose fish species at early life stages, allowing identifying all samples to a species-specific status, except for some taxa whose COI sequences remain unavailable in public databases. Therefore, we recommend the incorporation of DNA barcoding to provide additional support to traditional identification, especially in morphologically controversial groups. In addition, periodic updates and comparative analyses in public COI datasets are encouraged.

Arboviruses in Free-Ranging Birds and Hematophagous Arthropods (Diptera, Nematocera) from Forest Remnants and Urbanized Areas of an Environmental Protection Area in the Amazon Biome.

The rapid and disorderly urbanization in the Amazon has resulted in the insertion of forest fragments into cities, causing the circulation of arboviruses, which can involve hematophagous arthropods and free-ranging birds in the transmission cycles in urban environments. This study aimed to evaluate the circulation of arboviruses in free-ranging birds and hematophagous arthropods captured in an Environmental Protection Area in the Belem metropolitan area, Brazil. Birds were captured using mist nets, and hematophagous arthropods were collected using a human protected attraction technique and light traps. The birds' sera were subjected to a hemagglutination inhibition test to detect antibodies against 29 arbovirus antigens. Arthropod macerates were inoculated into C6/36 and VERO cell cultures to attempt viral isolation and were tested using indirect immunofluorescence, subsequent genetic sequencing and submitted for phylogenetic analysis. Four bird sera were positive for arbovirus, and one batch of Psorophora ferox was positive for Flavivirus on viral isolation and indirect immunofluorescence. In addition, the Ilheus virus was detected in the sequencing and phylogenetic analysis. The presence of antibodies in sera from free-ranging birds and the isolation of Ilheus virus in Psorophora ferox indicate the circulation of arboviruses in forest remnants in the urban center of Belem.

Inhibition of Staphylococcus aureus TetK and MsrA efflux pumps by hydroxyamines derived from lapachol and norlachol.

The present study aimed to evaluate the in vitro efflux pump inhibitory capacity of hydroxyamines derived from lapachol and norlachol, where compounds 3, 4, and 5 were tested against the S. aureus strains: RN4220 carrying the pUL5054 plasmid; and IS-58, endowed with the PT181 plasmid. The substances were synthesized from 2-hydroxy-quinones, lapachol and nor-lapachol obtaining the corresponding 2-methoxylated derivatives via dimethyl sulfate alkylation in a basic medium, which then reacted chemoselectively with 2-ethanolamine and 3-propanolamine to form the corresponding amino alcohols. The antibacterial action of the substances was quantified by determining the Minimum Inhibitory Concentration (MIC), while a microdilution assay was carried out to ascertain efflux pump inhibition of Staphylococcus aureus strains carrying the MsrA macrolide and the TetK tetracycline efflux pumps with the substances at a sub-inhibitory concentration. The results were subjected to statistical analysis by an ANOVA test and Bonferroni post hoc test. The MIC from the substances exhibited a value ≥ 1024 µg/mL. However, a significant reduction (p < 0.0001) of the erythromycin, tetracycline and ethidium bromide MIC was demonstrated when these were in combination with the substances, with this effect being due to a supposed efflux pump inhibition. The tested substances demonstrated effectiveness at decreasing the MIC of erythromycin, tetracycline and ethidium bromide, potentially by inhibiting the MsrA macrolide and the TetK tetracycline efflux pumps present in the tested S. aureus strains.

Aquaculture Waste: Potential Synthesis of Polyhydroxyalkanoates.

Petroleum-based plastics commonly and widely used on a daily basis are a threat to ecological health as they do not degrade in an ecologically feasible time frame. A class of natural polymers known as polyhydroxyalkanoates (PHAs) represents an up-and-coming alternative to petroleum-based materials, as they share properties similar to those of commodity plastics, such as polyethylene, polystyrene, among others, with the advantage of being biodegradable. PHAs are naturally produced by microorganisms under stress, and various farming practices have been proposed to be used for the synergistic and sustainable production of PHA for commercial purposes. Aquaculture has demonstrated particular potential for the production of PHA; however, a large struggle in commercializing these polymers is in procuring necessary feedstocks for manufacture outside of the laboratory environment. Through the coupling of PHA production and biofloc technology in aquaculture, the impediments to commercial exploitation can be potentially surmounted, while also providing for higher production efficiency in aquafarms. This mini-review covers the basic aspects of biofloc technology applied to aquaculture for the commercial production of PHA in large scale and offers a brief perspective on the next steps associated with the research and implementation of PHA production with biofloc technology.

New insights about species delimitation in red snappers (Lutjanus purpureus and L. campechanus) using multilocus data.

Lutjanus campechanus and Lutjanus purpureus are two commercially important lutjanid fishes (snappers) with non-sympatric distribution throughout Western Atlantic. Even though both taxa have traditionally been regarded as valid species, their taxonomic status remains under debate. In the present study, we used phylogeographic approaches and molecular methods of species delimitation to elucidate the taxonomic issues between both species, based on 1478 base pairs from four genomic regions. We found haplotypes shared between the two species, particularly in relation to nuclear DNA (nuDNA) sequences. The molecular delimitation of species supported the discrimination of L. purpureus and L. campechanus as distinct evolutionary units. Nonetheless, a unidirectional gene flow was found from L. campechanus towards L. purpureus. Therefore, it seems plausible to infer that L. campechanus and L. purpureus are two evolutionary units in which the apparent sharing of haplotypes should be driven by introgression.

Adsorption of Vi Capsular Antigen of Salmonella Typhi in Chitosan-Poly (Methacrylic Acid) Nanoparticles.

The development of a nanoparticulate system for the carrier antigen is now an important tool in the vaccination process, since a smaller number of doses is necessary for effective immunization. Thus, in this work a nanoparticulate system using polymers of chitosan and poly (methacrylic acid) (CS-PMAA) to adsorb the Vi antigen of Salmonella Typhi was developed. CS-PMAA nanoparticles with different proportions of chitosan and poly (methacrylic acid) were obtained and reached sizes from 123.9 ± 2.48 to 234.9 ± 2.66 nm, and spherical shapes were seen in transmission microscopy. At pH 7.2, the nanoparticles had a cationic surface charge that contributed to the adsorption of the Vi antigen. Qualitative analyses of the isolated Vi antigen were performed using Fourier-transform infrared spectroscopy, which indicated the presence of all the characteristic bands of the capsular polysaccharide, and nuclear magnetic resonance, which showed signals for the five hydrogens and the N-acetyl and O-acetyl groups which are characteristic of the Vi antigen structure. In the adsorption kinetics study, the Vi capsular antigen, contained in a phosphate buffer solution of pH 7.2, experienced 55% adsorption on the 1-1% (CS-PMAA) nanoparticles. The adsorption kinetics results showed the ability of the nanoparticulate system to adsorb the Vi antigen.

Forensic analysis reveals fraud in fillets from the "Gurijuba" Sciades parkeri (Ariidae - Siluriformes): a vulnerable fish in Brazilian Coastal Amazon.

The utilization of molecular tools for the certification of fishery products has been increasing over the last years. In general, economically important species are replaced by less valuable species, characterizing a commercial fraud. We evaluated the authenticity of 107 frozen fillets tagged as Gurijuba (Sciades parkeri) and Uritinga (Sciades proops) from local markets in northern amazon coast by sequencing two mitochondrial genes: Cytochrome oxidase subunit I and cytochrome b (Cyt b). About 16% of fillets putatively related to S. parkeri were replaced by S. proops. The Gurijuba faces high fishing pressure, being currently listed by the International Union for Conservation of Nature as vulnerable. Forensic analysis with DNA markers, proved to be highly efficient in the discrimination of the processed seafood products, providing unequivocal identification of species, revealing commercial fraud in the fillets of the Gurijuba, and revealing the utility of Cytb sequences as barcode in fishes.

Comparative analyses of species delimitation methods with molecular data in snappers (Perciformes: Lutjaninae).

The integration of approaches that allow the incorporation of stochasticity of gene histories with phylogenetic methods resulted in new approaches for the old issue of species delimitation. Nevertheless, coalescent methods seem problematic for taxa with large effective population size and shallow temporal diversification (like marine fishes). Here, we investigate the performance of single-locus (cytochrome oxidase 1, commonly used in DNA barcoding initiatives) methods for molecular species delimitation in snappers of Lutjaninae from the Western Atlantic and Pacific Eastern. Our results show incongruences among methods. ABGD, PTP and mPTP trend towards a lower number of estimated species. Phylogenetic-coalescent methods with single threshold were majority congruent for a same number of lineages. On the other hand, algorithms with multiple thresholds tend to estimate a higher number of potential species. We do not endorse the use of single-locus for species delimitation, but we do reinforce that single-locus data is sufficient to flag many problems.

Molecular protocol for authentication of snappers (Lutjanidae-Perciformes) based on multiplex PCR.

Fraud involving fish products is regularly reported and investigated using genetic methods. However, no such approach has been applied to snappers, despite the commercial relevance of these fish. This study proposes an authentication protocol with multiplex PCR for three species of snappers (Lutjanus purpureus, L. synagris, and Ocyurus chrysurus). The protocol yielded a distinct triple-banding pattern for L. purpureus, whereas L. synagris and O. chrysurus showed a double banding pattern of different sizes, thereby allowing differentiation of the three species. The protocol was validated using fillets labeled as snapper or "Pargo" in Brazil, previously identified using DNA sequencing, amongst which substitution with Rhomboplites aurorubens was detected. When subjected to the new protocol, banding characteristic of L. purpureus were detected whilst R. aurorubens generated only the control band. Our study provides a practical tool for investigating substitutions and might assist in quality control and increase food safety for consumers.

Study of molecular interactions between Chitosan and Vi Antigen.

Chitosan has attracted much interest due to its special physical and chemical properties related to drug administration. Nanoparticles delivery systems from Vi Antigen are a promising approach in the struggle against typhoid fever. In this paper, we reported the obtainment and the characterization of Vi Antigen by Infrared spectroscopy as well as Molecular Modeling and Computational Chemistry studies of the Chitosan-Vi Antigen interaction through theoretical models. The results of the theoretical and experimental Infrared spectroscopy showed important bands related to N-Acetyl and O-Acetyl groups present in Vi Antigen. Important interactions related to its adsorption were observed through three-dimensional optimized structures. Two models were proposed for the Chitosan-Vi Antigen in adsorption system, one as a monomer and another as an optimized tetrasaccharide antigen. The Molecular Modeling studies presented the best conformation and binding site on the nanoparticle Chitosan-Vi Antigen in models proposed. Interactions were observed between O-Acetyl and N-Acetyl groups the Vi Antigen and hydroxy, amino and methyl groups the Chitosan.

Lack of Spatial Subdivision for the Snapper Lutjanus purpureus (Lutjanidae - Perciformes) from Southwest Atlantic Based on Multi-Locus Analyses.

The Caribbean snapper Lutjanus purpureus is a marine species fish commonly found associated with rocky seabeds and is widely distributed along of Western Atlantic. Data on stock delineation and stock recognition are essential for establishing conservation measures for commercially fished species. However, few studies have investigated the population genetic structure of this economically valuable species, and previous studies (based on only a portion of the mitochondrial DNA) provide an incomplete picture. The present study used a multi-locus approach (12 segments of mitochondrial and nuclear DNA) to elucidate the levels of genetic diversity and genetic connectivity of L. purpureus populations and their demographic history. L. purpureus has high levels of genetic diversity, which probably implies in high effective population sizes values for the species. The data show that this species is genetically homogeneous throughout the geographic region analyzed, most likely as a result of dispersal during larval phase. Regarding demographic history, a historical population growth event occurred, likely due to sea level changes during the Pleistocene.

High levels of genetic connectivity among populations of yellowtail snapper, Ocyurus chrysurus (Lutjanidae-Perciformes), in the western South Atlantic revealed through multilocus analysis.

In the present study, five loci (mitochondrial and nuclear) were sequenced to determine the genetic diversity, population structure, and demographic history of populations of the yellowtail snapper, Ocyurus chrysurus, found along the coast of the western South Atlantic. O. chrysurus is a lutjanid species that is commonly associated with coral reefs and exhibits an ample geographic distribution, and it can therefore be considered a good model for the investigation of phylogeographic patterns and genetic connectivity in marine environments. The results reflected a marked congruence between the mitochondrial and nuclear markers as well as intense gene flow among the analyzed populations, which represent a single genetic stock along the entire coast of Brazil between the states of Pará and Espírito Santo. Our data also showed high levels of genetic diversity in the species (mainly mtDNA), as well a major historic population expansion, which most likely coincided with the sea level oscillations at the end of the Pleistocene. In addition, this species is intensively exploited by commercial fisheries, and data on the genetic structure of its populations will be essential for the development of effective conservation and management plans.

Inadequate prenatal care utilization and associated factors in São Luís, Brazil.

BACKGROUND: Over the last decades there has been a reduction of social inequalities in Brazil, as well as a strong expansion of health services, including prenatal care. The objective of the present study was to estimate the rate of inadequate prenatal care utilization and its associated factors in São Luís, Brazil, in 2010 and to determine whether there was a reduction of inequity in prenatal care use by comparing the present data to those obtained from a previous cohort started in 1997/98. METHODS: Data from the BRISA (Brazilian birth cohort studies of Ribeirão Preto and São Luís) population-based cohort, which started in 2010 (5067 women), were used. The outcome variable was the inadequate utilization of prenatal care, classified according to the recommendations of the Brazilian Ministry of Health. The explanatory variables were organized into three hierarchical levels based on the Andersen's behavioral model of the use of health services: predisposing, enabling and need factors. RESULTS: Only 2.0% of the women did not attend at least one prenatal care visit. The rate of inadequate prenatal care utilization was 36.7%. Despite an improved adequacy of prenatal care use from 47.3% in 1997/98 to 58.2% in 2010, social inequality persisted: both low maternal schooling (prevalence ratio (PR) = 2.78; 95% confidence interval (95% CI) 2.23-3.47 for 0 to 4 years of study) and low family income, less than 0.5 monthly minimum wage per capita (PR = 1.37; 95% CI 1.22-1. 54), continued to be associated with higher rates of inadequate prenatal care utilization. Racial disparity regarding adequate utilization of prenatal services was detected, with black (PR = 1.19; 95% CI 1.04-1.36) and mulatto (PR = 1.14; 95% CI 1.02-1.26) women showing higher rates of inadequate use. On the other hand, women covered by the FHP - Family Health Program (PR = 0.92; 95% CI 0.85-0.98) showed a lower rate of inadequate prenatal care utilization. CONCLUSIONS: Despite strong expansion of health services and expressive improvements in adequate prenatal care use and social indicators, inequalities in prenatal care use still persist. The FHP seems to be effective in reducing inadequate prenatal care utilization.

Lysosome-associated protein 1 (LAMP-1) and lysosome-associated protein 2 (LAMP-2) in a larger family carrier of Fabry disease.

This study investigated the potential relationship between the expression levels of lysosome-associated membrane proteins (LAMP) 1 and 2 and responses to enzyme replacement therapy (ERT) in the members of a single family with Fabry disease (FD). LAMP levels were assessed by flow cytometry in leukocytes from 17 FD patients who received an eight-month course of ERT course and 101 healthy individuals. We found that phagocytic cells from the FD patients had higher expression levels of both LAMP-1 and LAMP-2, relative to the levels in phagocytes from the healthy controls (p=0.001). Furthermore, the LAMP-1 and LAMP-2 levels in phagocytes from the FD carriers continuously decreased with ERT administration to reach levels similar to those in healthy controls. We suggest that LAMP-1 and LAMP-2 could be used as additional markers with which to assess ERT effectiveness in FD.

[Access to and use of the services of the family health strategy from the perspective of managers, professionals and users]. / Acesso e utilização dos serviços na Estratégia Saúde da Família na perspectiva dos gestores, profissionais e usuários.

Questionnaires adapted to the Brazilian reality (Primary Care Assessment Tool) to evaluate access to and use of services were distributed to 30 managers, 80 professionals and a random sample of 882 Family Health Strategy (FHS) users in São Luís in the state of Maranhão. The differences between the evaluations of managers, professionals and users were identified by the Kruskal-Wallis test and Dunn's post test. Users faced difficulties in access to and use of the services, which were also considered unsatisfactory by managers and professionals. Access was the dimension with the worst evaluation, and non-functioning of units after 6 p.m. and at weekends were the main difficulties. The evaluations of the managers and professionals were almost always divergent from those of the users, with the evaluation of the managers being more favorable. The fact that the service was free of charge, the use of preventive services and the FHS services before the specialized care were well evaluated. The operation of units should attend users' needs, especially that of the working population. Strengthening social participation in local management of the FHS may assist in identifying these needs, thereby reducing the divergent opinions of the players involved.

Efficiency of utilization of nitrogen coated with urease inhibitor in maize.

This study aimed to evaluate under field conditions the efficiency in the use ofN coated with urease inhibitor in maize. The experiment was conducted in the year of 2007/2008. The experimental design was a randomized block design in a factorial 2 x 6, with five repetitions, constituted the N sources (common and coated with urease inhibitor) and levels (0, 40, 80, 120, 160 and 200 kg ha-1 of N) sidedressing nitrogen application in the growth stage V4. Based on the data obtained were determined recovery efficiencies, utilization, agronomic and physiological N applied. In all cases, the efficiency levels for maize were influenced by levels of sidedressing nitrogen application, in which increasing levels of N resulted in a decrease of the efficiencies, regardless of the source being common urea or coated with urease inhibitor.

Prevalencia de terceros molares incluidos en estudiantes de una escuela pública de São Luís, Maranhão, Brasil / Prevalence of third molars in students of a public school in São Luís, Maranhão, Brazil

Los terceros molares presentan características muy particulares referentes a la erupción, inclusión, y complicaciones que requieren una evaluación clínica y radiológica más detallada para definir mejor el diagnostico y tratamiento cuando necesario. El objetivo era investigar la prevalencia de los terceros molares incluidos en estudiantes de una escuela pública de la ciudad de São Luís - Maranhão. Fue realizado un estudio transversal descriptivo de las características clínicas y radiológicas de los terceros molares de 155 estudiantes desde los 18 a los 21 años de edad de ambos sexos de un Instituto Federal de Educación Tecnológica de Maranhão. Los molares presentes con rizogénesis completa fueron clasificados según su nivel de inclusión. Todos los datos fueron registrados en historias clínicas propias, tablas del EPI - INFO 2000 y sometidos al test chi-cuadrado para verificar la existencia de asociaciones entre las variables del estudio, con un nivel significativo del 5%. De una población de 155 estudiantes, se obtuvo 531 (85,6%) terceros molares presentes y 89 (14,4%) terceros molares ausentes. De aquellos presentes 462 (87%) terceros molares fueron evaluados en cuanto a su nivel de inclusión. De estos, 239 (51,7%) se encontraban en algún estadio de la fase de inclusión, siendo que 201 (84,1%) estaban parcialmente incluidos y 38 (15,9%) totalmente incluidos. Hubo asociación positiva entre dientes incluidos y terceros molares (p = 0.0015). Los molares incluidos fueron una condición frecuente en la muestra (51%), donde fueron observados terceros molares en distintas fases de inclusión

Third molars have characteristics that are very peculiar regarding the eruption, inclusion and complications. Accordingly, they often require particular clinical and radiological assessments, in order to better define diagnosis and treatment. The aim of this study was to investigate the prevalence of dental inclusions of the third molars within students from a public school in São Luís - Maranhão. We conducted a transverse, descriptive study assessing the clinical characteristics, as well as the radiographies of the third molars, from 155 students (ages from 18-21 years, both genders), from the Federal Institute of Technological Education, in Maranhão. Teeth with complete rhizogeneses were classified according to different inclusion state. Data were transferred to summary files, and then entered in a database (EPI-INFO 2000). Groups were compared using the Chi-Square test in order to assess associations among the study variables. Significance level was established in 5%. Among 155 students, 531 (85.6%) teeth were present, and 89 (14.4%) were not. Among the present, 462 (87%) third molars were assessed according to the inclusion pattern. From them, 239 (51.7%) were in any inclusion state, while (84.1%) had partial inclusion and 38 (15,9%) in total inclusion We found a positive association between dental inclusion and third molars (p=0.0015). Dental inclusion was frequent in our sample (51%), where we observed third molars in different patterns of inclusion