Altitudinal distribution and body condition of migratory fish juveniles Megaleporinus obtusidens (Valenciennes) and Salminus brasiliensis (Cuvier) in the main channel of the Uruguay River, Southern Brazil.

In this study, we analyzed the altitudinal distribution and body condition of the juvenile migratory fish Megaleporinus obtusidens and Salminus brasiliensis in the Uruguay River, a South American subtropical river. We used the presence/absence data and condition factor (K) of juveniles as indicators to try to characterize some recruitment patterns of migratory fish in the main channel of the Uruguay River. Gillnet sampling was conducted during two reproductive years, 2015-2016 and 2016-2017 (November and March of each reproductive year), in three Uruguay River stretches at different levels of altitude. The nets remained in the water for 10-12 hours per night or day for 10 days during each field trip (NOV-15, MAR-16, NOV-16, and MAR-17). The abundance of M. obtusidens juveniles were similar between the two reproductive years, whereas S. brasiliensis capture was higher during the 2015-2016 reproductive year. The probability of finding M. obtusidens juveniles in the Uruguay River was negatively correlated (p < 0.05) with the altitudinal gradient of the sampling points, as shown by the GLM model. For S. brasiliensis, the logit model indicated a non-significant probabilistic relationship (p > 0.05) between the presence of juveniles of this fish species and the altitude gradient. Regarding the body condition of juveniles, the water quality of the sampling points did not affect the juvenile condition factor for either species; however, a positive relationship (p < 0.05) was found with the water level for both species, even with an adjusted R2 of 0.13 for M. obtusidens and 0.48 to S. brasiliensis. Altitude gradient and water level were factors that may affect the juvenile distribution and body condition of these species, respectively, in the Uruguay River. Therefore, this information can be used in hydropower generation policies to conserve migratory fish populations in the Uruguay River Basin.

Antifungal activity and toxicological parameters of 8-hydroxyquinoline-5-sulfonamides using alternative animal models.

AIM: The purpose of this study was to evaluate the antifungal activity and toxicological parameters of 8-hydroxyquinoline derivatives PH151 and PH153 using alternative animal models, to understand their behaviour when subjected to in vivo experiments. METHODS AND RESULTS: We used Toll-deficient Drosophila melanogaster to test the protective effect of compounds against Candida albicans infection. Toxicological parameters were investigated in chicken and zebrafish embryos. PH151 and PH153 showed low toxicity and the treated flies with these compounds had a significantly higher survival rate than untreated flies after 7 days of infection. The compounds did not cause interruption of chicken embryogenesis. Zebrafish embryos exposed to compounds showed dose-dependent toxicity. CONCLUSIONS: The data supported the potential of PH151 and PH153 for the treatment of systemic candidiasis and demonstrated to be appropriate drug candidates for further studies using mammalian models. SIGNIFICANCE AND IMPACT OF THE STUDY: The increased incidence of Candida infections resistant to antifungals currently available requires acceleration of the discovery of new agents with properties of inhibiting this fungal pathogen. In this study, we have described the antifungal potential and toxicity of two 8-hydroxyquinoline derivatives using in vivo alternative models, and the results confirm their potential to be developed as new drug candidates.

Acute embryonic exposure of zebrafish to permethrin induces behavioral changes related to anxiety and aggressiveness in adulthood.

Permethrin (PM) is one of the most used synthetic pyrethroid worldwide. Exposure to this compound during pregnancy and early childhood has been indicated as a risk factor for neurodevelopmental disorders. We evaluated the long-term effects of embryonic PM exposure in different stages of zebrafish development. Briefly, embryos (3 hpf) were exposed to sub-lethal concentrations of PM (25 and 50 µg.L-1) during 24 h and then behavioral parameters were evaluated during embryonic (28 hpf), eleutheroembryonic (3 dpf), larval (7 dpf), and adult stages (90 dpf). PM exposure decreased spontaneous movement at 28 hpf and decreased thigmotaxis in eleutheroembryos. The long-term effects of PM include changes in non-motor behaviors such as fear and anxiety in larva and adults. Adults embryonically exposed to PM also showed a significant increase in aggressiveness parameters. These results demonstrated that embryonic exposure to PM induces persistent neurotoxic effects in adulthood, which can impair the cognitive and behavioral fitness of non-target species contributing to a rise in neurodevelopmental disorders.

Oxidative stress markers in fish (Astyanax sp. and Danio rerio) exposed to urban and agricultural effluents in the Brazilian Pampa biome.

Aquatic ecosystems are under constant risk due to industrial, agricultural, and urban activities, compromising water quality and preservation of aquatic biota. The assessment of toxicological impacts caused by pollutants to aquatic environment using biomarker measurements in fish can provide reliable data to estimate sublethal effects posed by chemicals in contaminated areas. In this study, fish (Astyanax sp. and Danio rerio) exposed to agricultural and urban effluents at the Vacacaí River, Brazil, were tested for potential signs of aquatic contamination. This river comprehends one of the main watercourses of the Brazilian Pampa, a biome with a large biodiversity that has been neglected in terms of environmental and social-economic development. Sites S1 and S2 were chosen by their proximity to crops and wastewater discharge points, while reference site was located upstream of S1 and S2, in an apparently non-degraded area. Fish muscle and brain tissues were processed for determination of acetylcholinesterase as well as oxidative stress-related biomarkers. The results showed signs of environmental contamination, hallmarked by significant changes in cholinesterase activity, expression of metallothionein, antioxidant enzymes, glutathione levels, and activation of antioxidant/cell stress response signaling pathways in fish exposed to contaminated sites when compared to reference. Based on these results, it is evidenced that urban and agricultural activities are posing risk to the environmental quality of water resources at the studied area. It is also demonstrated that cell stress biomarkers may serve as important tools for biomonitoring and development of risk assessment protocols in the Pampa biome.

A Study on the Quality and Identity of Brazilian Pampa Biome Honey: Evidences for Its Beneficial Effects against Oxidative Stress and Hyperglycemia.

We characterized, for the first time, the quality and identity of Brazilian Pampa biome honey and its antioxidant properties in vitro (FRAP, DDPH and ABTS). The potential protective effect of honey against oxidative stress induced by iron (Fe) and paraquat, (PQ) in a Drosophila melanogaster model (in vivo) was also tested. The results indicated that all honey samples tested showed antioxidant activity in vitro. Flies treated with honey showed increased lifespan and were protected against oxidative stress induced by Fe and PQ. Despite the high concentration of sugars in honey (approximately 70-80%), our results demonstrate a hypoglycemic-like effect of honey in Drosophila. Thus, this study demonstrates the high quality of Brazilian Pampa biome honey as well as its significant antioxidant activity in vitro and in vivo, pointing to the potential use of this natural product as an alternative in the therapy of oxidative stress-associated diseases.

Is the lobster cockroach Nauphoeta cinerea a valuable model for evaluating mercury induced oxidative stress?

Organic and inorganic forms of mercury are highly neurotoxic environmental contaminants. The exact mechanisms involved in mercury neurotoxicity are still unclear. Oxidative stress appears to play central role in this process. In this study, we aimed to validate an insect-based model for the investigation of oxidative stress during mercury poisoning of lobster cockroach Nauphoeta cinerea. The advantages of using insects in basic toxicological studies include the easier handling, rapid proliferation/growing and absence of ethical issues, comparing to rodent-based models. Insects received solutions of HgCl2 (10, 20 and 40mgL(-1) in drinking water) for 7d. 24h after mercury exposure, animals were euthanized and head tissue samples were prepared for oxidative stress related biochemical determinations. Mercury exposure caused a concentration dependent decrease in survival rate. Cholinesterase activity was unchanged. Catalase activity was substantially impaired after mercury treatment 40mgL(-1). Likewise, GST had a significant decrease, comparing to control. Peroxidase and thioredoxin reductase activity was inhibited at concentrations of 20mgL(-1) and 40mgL(-1) comparing to control. These results were accompanied by decreased GSH levels and increased hydroperoxide and TBARS formation. In conclusion, our results show that mercuric compounds are able to induce oxidative stress signs in insect by modulating survival rate as well as inducing impairments on important antioxidant systems. In addition, our data demonstrates for the first time that Nauphoeta cinerea represents an interesting animal model to investigate mercury toxicity and indicates that the GSH and thioredoxin antioxidant systems plays central role in Hg induced toxicity in insects.

A new mechanism of dominance in hypophosphatasia: the mutated protein can disturb the cell localization of the wild-type protein.

The dominant negative effect of mutations is rare in metabolic diseases and its mechanism has not been studied much. Hypophosphatasia, a bone inherited metabolic disorder, is a good model because the disease can be dominantly transmitted. The gene product activity depends on a homodimeric configuration and many mutations have been reported in the ALPL gene responsible for the disease. Using CFP/YFP-tagged-TNSALP plasmids, transfections in COS cells and confocal fluorescence analyses, we studied the point mutation G232V (c.746G>T). We showed that the G232V protein sequestrates some of the wild-type protein into the cells and prevents it from reaching the membrane where it plays its physiological role.

A molecular approach to dominance in hypophosphatasia.

Hypophosphatasia is an inherited disorder characterized by defective bone mineralization and a deficiency of tissue-nonspecific alkaline phosphatase (TNSALP) activity. The disease is highly variable in its clinical expression, because of various mutations in the TNSALP gene. In approximately 14% of the patients tested in our laboratory, only one TNSALP gene mutation was found, despite exhaustive sequencing of the gene, suggesting that missing mutations are harbored in intron or regulatory sequences or that the disease is dominantly transmitted. The distinction between these two situations is of importance, especially in terms of genetic counseling, but dominance is sometimes difficult to conclusively determine by using familial analysis since expression of the disease may be highly variable, with parents of even severely affected children showing no or extremely mild symptoms of the disease. We report here the study of eight point mutations (G46 V, A99T, S164L, R167 W, R206 W, G232 V, N461I, I473F) found in patients with no other detectable mutation. Three of these mutations, G46 V, S164L, and I473F, have not previously been described. Pedigree and/or serum alkaline phosphatase data suggested possible dominant transmission in families with A99T, R167 W, and G232 V. By means of site-directed mutagenesis, transfections in COS-1 cells, and three-dimensional (3D) modeling, we evaluated the possible dominant effect of these eight mutations. The results showed that four of these mutations (G46 V, A99T, R167 W, and N461I) exhibited a negative dominant effect by inhibiting the enzymatic activity of the heterodimer, whereas the four others did not show such inhibition. Strong inhibition resulted in severe hypophosphatasia, whereas partial inhibition resulted in milder forms of the disease. Analysis of the 3D model of the enzyme showed that mutations exhibiting a dominant effect were clustered in two regions, viz., the active site and an area probably interacting with a region having a particular biological function such as dimerization, tetramerization, or membrane anchoring.

Pathogenesis of ectrodactyly in the Dactylaplasia mouse: aberrant cell death of the apical ectodermal ridge.

Dactylaplasia, or Dac, was recently mapped to the distal portion of mouse chromosome 19 and shown to be inherited as an autosomal semi-dominant trait characterized by missing central digital rays. The most common locus for human split hand split foot malformation, also typically characterized by missing central digital rays, is 10q25, a region of synteny to the Dac locus. The Dac mouse appears to be an ideal genotypic and phenotypic model for this human malformation syndrome. Several genes lie in this region of synteny, however, only Fibroblast Growth Factor 8, or Fgf-8, has been implicated to have a role in limb development. We demonstrate that the developmental mechanism underlying loss of central rays in Dac limbs is dramatic cell death of the apical ectodermal ridge, or AER. This cell death pattern is apparent in E10.5-11.5 Dac limb buds stained with the supravital dye Nile Blue Sulfate. We demonstrate that Fgf8 expression in wild type limbs colocalizes spatially and temporally with AER cell death in Dac limbs. Furthermore, in our mapping panel, there is an absence of recombinants between Fgf-8 and the Dac locus in 133 backcross progeny with a median linkage estimate of approximately 0.5 cM. Thus, our results demonstrate that cell death of the AER in Dac limbs silences the role of the AER as key regulator of limb outgrowth, and that Fgf-8 is a strong candidate for the cause of the Dac phenotype.

A split hand-split foot (SHFM3) gene is located at 10q24-->25.

The split hand-split foot (SHSF) malformation affects the central rays of the upper and lower limbs. It presents either as an isolated defect or in association with other skeletal or non-skeletal abnormalities. An autosomal SHSF locus (SHFM1) was previously mapped to 7q22.1. We report the mapping of a second autosomal SHSF locus to 10q24-->25. A panel of families was tested with 17 marker loci mapped to the 10q24-->25 region. Maximum lod scores of 3.73, 4.33 and 4.33 at a recombination fraction of zero were obtained for the loci D10S198, PAX2 and D10S1239, respectively. An 19 cM critical region could be defined by haplotype analysis and several genes with a potential role in limb morphogenesis are located in this region. Heterogeneity testing indicates the existence of at least one additional autosomal SHSF locus.

A second autosomal split hand/split foot locus maps to chromosome 10q24-q25.

Ectrodactyly (split hand/split foot malformation, SHSF) is a human limb malformation characterized by absent central digital rays, deep median cleft, and syndactyly of remaining digits. The disorder is genetically heterogeneous, with at least two loci thus far determined: an autosomal locus at 7q21 designated SHFM1 and an X-linked locus at Xq26 designated SHFM2. Cytogenetic analysis of sporadic SHSF patients and linkage studies in extended pedigrees both suggest more than one autosomal locus exists. We report a novel SHSF locus suggested by a stillborn infant with ectrodactyly and other malformations who inherited an unbalanced translocation resulting in monosomy 4p15.1-4pter and trisomy for 10q25.2-qter. To investigate 10q25 as a possible split hand/split foot locus, microsatellite markers spanning 52 cM of 10q were utilized for linkage analysis of a large autosomal dominant SHSF pedigree in which the region encompassing SHFM1 previously was excluded as containing the causative mutation. The marker D10S583 was fully informative in the family, giving a maximum LOD score of 4.21 at recombination theta = 0.00. Recombination haplotypes define the 9 cM region between D10S541 and D10S574 as inclusive for this second autosomal SHSF locus, for which we propose the designation SHFM3.

A contiguous gene deletion syndrome at 7q21-q22 and implications for a relationship between isolated ectrodactyly and syndromic ectrodactyly.

Ectrodactyly is a genetically heterogeneous human limb developmental malformation with an autosomal locus at 7q21 designated SHFD1. We report a patient with ectrodactyly and a small interstitial deletion of 7q21, detected by high resolution karyotype, which provides additional evidence for the existence of an ectrodactyly gene in this region and further restricts the size of the critical region containing SHFD1. A review of previously published patients with ectrodactyly and similar deletions suggests a contiguous gene deletion syndrome at 7q21-q22 which consists of ectrodactyly, growth retardation, developmental delay, hypertelorism, ear malformation, components of the Robin sequence, and GU abnormalities. Moreover, the clinical, genetic, and cytogenetic similarities of a number of multiple congenital anomaly syndromes which include ectrodactyly as just one component lead us to propose that a common genetic basis may underlie many of these syndromes. Thus, evaluation for related physical anomalies and high resolution cytogenetic studies are indicated in patients with ectrodactyly.

c-myc expression in the thyroid. I: Normal, adenomatous, and cancerous thyroid tissue.

Recent investigations have suggested that myc oncogene expression may be important in the development or progression of thyroid tumors. The purposes of the present study were to assess cellular (c)-myc expression in thyroid adenomas (n = 5), as well as in thyroid cancer (n = 4) and in normal thyrocytes (n = 7). Total RNA was prepared by extraction with guanididium thiocyanate and ultracentrifugation through a CsCl2 cushion. 30 micrograms total RNA was size fractionated on a 1% (w/v) agarose/formaldehyde gel and transferred to nylon membranes. These membranes were hybridized to a 32P-labelled third exon c-myc DNA. Following hybridization, blots were washed under high stringency and subjected to autoradiography; radioautographic bands were assessed visually or were quantitated by scanning densitometry. Nodular tissue had approximately the same degree of expression of the 2.4 Kb c-myc message as the surrounding normal tissue from the same gland (0.66 +/- 0.09 vs. 1.0 +/- 0.26, respectively); normal thyrocytes were capable in every instance of expressing the 2.4 Kb c-myc message. Thyroid cancer tissue expressed this message (0.91 +/- 0.17) but only at a level comparable to normal tissue. No other bands of hybridization were detected in any samples. We conclude that c-myc oncogene expression is comparable in normal thyrocytes and in thyroid nodules or thyroid cancer samples. These findings support a role for c-myc in both normal and neoplastic thyrocyte growth.

c-myc expression in the thyroid. II: Thyrocytes and peripheral and intrathyroidal lymphocytes from patients with autoimmune thyroid disease.

Enhanced oncogene expression observed in lymphocytes from patients with systemic lupus erythematosus has suggested the importance of studying oncogene expression and regulation in the cellular events of autoimmune thyroid diseases (AITD). The present study examines oncogene expression in peripheral and intrathyroidal lymphocytes from patients with Hashimoto's disease (HD) and Graves' disease (GD). Intrathyroidal lymphocytes from a patient with primary thyroid lymphoma were also examined. Lymphocytes were isolated by Ficoll-Hypaque gradients, and total RNA was prepared by extraction with guanididium thiocyanate and ultracentrifugation through a cesium chloride cushion. RNA concentrations were determined by O.D. readings at 260/280 nm and each sample subjected to gel electrophoresis with ethidium bromide staining to assure the integrity of the RNA. 30 micrograms total RNA was size fractionated on a 1% (w/v) agarose/formaldehyde gel and transferred to nylon membranes. These membranes were hybridized with nick-translated 32P labelled c-myc DNA (exon III), washed at high stringency and subjected to autoradiography. Specific bands were quantitated by scanning densitometry. Five RNA samples from GD thyroids had 2.4 Kb bands corresponding to c-myc with a mean O.D. (+/- SD) of 0.76 +/- 0.23, whereas 7 from normal thyroid glands had mean O.D. of 1.0 +/- 0.26. Peripheral lymphocytes from 7 GD patients had a mean O.D. of 1.41 +/- 0.25, 4 HD patients had a mean O.D. of 1.05 +/- 0.10 and 2 normal patients had a mean O.D. of 1.4 +/- 0.14. The readings for a sample obtained from intrathyroidal lymphocytes of a patient with HD and thyroid lymphoma were 1.0 and 1.4, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)