Acute toxicity and regenerative dose finding of an extract of Miconia ferruginata DC. in a mouse model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a severe disease with no cure caused by a genetic abnormality, promoting progressive muscle degeneration. Corticosteroids are used drugs in treatment associated with adverse effects. The extract of Miconia ferruginata (Melastomataceae) (MF) has demonstrated potent antioxidant and anti-inflammatory potential in vitro. This study used a DMD model (mdx) to determine the toxic dose of this plant and found a possible non-toxic dose with therapeutic effects. The mdx groups received an intraperitoneal injection of 0 (control group), 50, 100, 200, 300, and 2000 mg kg-1 of the aqueous leaf extract following a single-dose acute toxicity protocol and were observed for 14 days. The range of toxicity of the extract and LD50 were determined. Histopathological analysis, the quantification of fibrosis, and immunohistochemical analysis of the tissues were performed. The results demonstrated that 2000 mg kg-1 was highly toxic, inducing histopathological changes in the tissues evaluated, with 100% mortality in 48 hours. The other doses caused no behavioral changes or signs of toxicity. The MF extract led reduction in histopathological changes, fibrosis, and inflammation, a reduction in HSP70 and an increase in MCL-1 proteins. Doses of 50-200 mg kg-1 demonstrated regenerative tissue and anti-inflammatory potential.

Label-free electrochemical impedance immunosensor based on modified screen-printed gold electrodes for the diagnosis of canine visceral leishmaniasis.

Leishmaniasis is a disease with high impact on public health in many countries. Visceral leishmaniasis (VL) is a vectorial zoonosis, with dogs as primary reservoirs in the domestic environment. VL presents high morbidity, mortality and importance in epidemiology in the American continent. In the present study, the first label-free electrochemical impedance immunosensor using screen-printed electrodes (SPEs) for the detection of anti-Leishmania infantum antibodies was developed. The soluble antigens of L. infantum were immobilized on an SPE by a 3-mercaptopropionic acid monolayer. Electrochemical impedance spectroscopy (EIS) was used for detecting bimolecular interactions occurring at the electrode surface. The addition of real samples consisting of canine and human sera positive and negative for VL presented high sensitivity and selectivity through EIS. Based on the results, a sensitive, specific, rapid and simple immunosensor was developed successfully with potential application for the serological diagnosis of leishmaniasis disease.

Blood analytes of oceanic-juvenile loggerhead sea turtles (Caretta caretta) from Azorean waters: reference intervals, size-relevant correlations and comparisons to neritic loggerheads from western Atlantic coastal waters.

Blood analyte reference intervals are scarce for immature life stages of the loggerhead sea turtle (Caretta caretta). The objectives of this study were to (1) document reference intervals of packed cell volume (PCV) and 20 plasma chemistry analytes from wild oceanic-juvenile stage loggerhead turtles from Azorean waters, (2) investigate correlations with body size (minimum straight carapace length: SCLmin) and (3) compare plasma chemistry data to those from older, larger neritic juveniles (<80 cm SCLmin) and adult loggerheads (≥80 cm SCLmin) that have recruited to the West Atlantic in waters around Cape Canaveral, Florida. Twenty-eight Azorean loggerhead turtles with SCLmin of 17.6-60.0 cm (mean 34.9 ± 12.1 cm) were captured, sampled and immediately released. Reference intervals are reported. There were several biologically relevant correlations of blood analytes with SCLmin: positive correlations of PCV, proteins and triglycerides with SCLmin indicated somatic growth, increasing diving activity and/or diet; negative correlations of tissue enzymes with SCLmin suggested faster growth at smaller turtle size, while negative correlations of electrolytes with SCLmin indicated differences in diet, environmental conditions and/or osmoregulation unique to the geographic location. Comparisons of loggerhead turtles from the Azores (i.e. oceanic) and Cape Canaveral (i.e. neritic) identified significant differences regarding diet, somatic growth, and/or environment: in Azorean turtles, albumin, triglycerides and bilirubin increased with SCLmin, while alkaline phosphatase, lactate dehydrogenase and sodium decreased. In larger neritic Cape Canaveral turtles, aspartate aminotransferase increased with SCLmin, while the albumin:globulin ratio, phosphorus and cholesterol decreased. These differences suggest unique physiological disparities between life stage development and migration, reflecting biological and habitat differences between the two populations. This information presents biologically important data that is applicable to stranded individual turtles and to the population level, a tool for the development of conservation strategies, and a baseline for future temporal and spatial investigations of the Azorean loggerhead sea turtle population.

Glycotriazole-peptides derived from the peptide HSP1: synergistic effect of triazole and saccharide rings on the antifungal activity.

This work proposes a strategy that uses solid-phase peptide synthesis associated with copper(I)-catalyzed azide alkyne cycloaddition reaction to promote the glycosylation of an antimicrobial peptide (HSP1) containing a carboxyamidated C-terminus (HSP1-NH2). Two glycotriazole-peptides, namely [p-Glc-trz-G1]HSP1-NH2 and [p-GlcNAc-trz-G1]HSP1-NH2, were prepared using per-O-acetylated azide derivatives of glucose and N-acetylglucosamine in the presence of copper(II) sulfate pentahydrate (CuSO4·5H2O) and sodium ascorbate as a reducing agent. In order to investigate the synergistic action of the carbohydrate motif linked to the triazole-peptide structure, a triazole derivative [trz-G1]HSP1-NH2 was also prepared. A set of biophysical approaches such as DLS, Zeta Potential, SPR and carboxyfluorescein leakage from phospholipid vesicles confirmed higher membrane disruption and lytic activities as well as stronger peptide-LUVs interactions for the glycotriazole-peptides when compared to HSP1-NH2 and to its triazole derivative, which is in accordance with the performed biological assays: whereas HSP1-NH2 presents relatively low and [trz-G1]HSP1-NH2 just moderate fungicidal activity, the glycotriazole-peptides are significantly more effective antifungal agents. In addition, the glycotriazole-peptides and the triazole derivative present strong inhibition effects on ergosterol biosynthesis in Candida albicans, when compared to HSP1-NH2 alone. In conclusion, the increased fungicidal activity of the glycotriazole-peptides seems to be the result of (A) more pronounced membrane-disruptive properties, which is related to the presence of a saccharide ring, together with (B) the inhibition of ergosterol biosynthesis, which seems to be related to the presence of both the monosaccharide and the triazole rings.

Plastic ingestion in oceanic-stage loggerhead sea turtles (Caretta caretta) off the North Atlantic subtropical gyre.

Juvenile oceanic-stage sea turtles are particularly vulnerable to the increasing quantity of plastic coming into the oceans. In this study, we analysed the gastrointestinal tracts of 24 juvenile oceanic-stage loggerheads (Caretta caretta) collected off the North Atlantic subtropical gyre, in the Azores region, a key feeding ground for juvenile loggerheads. Twenty individuals were found to have ingested marine debris (83%), composed exclusively of plastic items (primarily polyethylene and polypropylene) identified by µ-Fourier Transform Infrared Spectroscopy. Large microplastics (1-5mm) represented 25% of the total number of debris and were found in 58% of the individuals sampled. Average number of items was 15.83±6.09 (±SE) per individual, corresponding to a mean dry mass of 1.07±0.41g. The results of this study demonstrate that plastic pollution acts as another stressor for this critical life stage of loggerhead turtles in the North Atlantic.

Exploitation promotes earlier sex change in a protandrous patellid limpet, Patella aspera Röding, 1798.

Exploitation of organisms can prompt the reduction in the number and size of target populations consequently affecting reproductive output and replenishment. Here, we investigated the effects of exploitation on the population structure of a protandrous patellid limpet, Patella aspera, an overexploited Macaronesian endemic. Timed dives were used to collect animals across eleven islands of Macaronesia. Individuals were inspected for sex, size, and gonad stage. Using catch effort (time per person) per island coastal perimeter as a surrogate for exploitation intensity, we found that limpet abundance (CPUE) and mean size tended to decrease with exploitation intensity. When considering the sex of animals separately, the size of the largest male, but not females, decreased with exploitation. In contrast, the size of the smallest male remained relatively consistent, whereas the size of the smallest female decreased significantly with exploitation. As exploitation is mostly targeting larger individuals, results suggest that males are compensating the removal of larger females, by undergoing sex change at smaller and presumably earlier sizes. These results have wider implications for the conservation of P. aspera, as a reduction in female size will likely affect the numbers of oocytes produced, hence fecundity. Regulations promoting the protection of the larger-sized animals should be enforced to safeguard the replenishment of the population.

Applicability of a novel immunoassay based on surface plasmon resonance for the diagnosis of Chagas disease.

BACKGROUND: We defined the methodological criteria for the interpretation of the results provided by a novel immunoassay based on surface plasmon resonance (SPR) to detect antibodies anti-Trypanosoma cruzi in human sera (SPRCruzi). Then, we evaluated its applicability as a diagnostic tool for Chagas disease. METHODS: To define the cut-off point and serum dilution factor, 57 samples were analyzed at SPRCruzi and the obtained values of SPR angle displacement (ΔθSPR) were submitted to statistical analysis. Adopting the indicated criteria, its performance was evaluated into a wide panel of samples, being 99 Chagas disease patients, 30 non-infected subjects and 42 with other parasitic/infectious diseases. In parallel, these samples were also analyzed by ELISA. RESULTS: Our data demonstrated that 1:320 dilution and cut-off point at ∆θSPR=17.2 m° provided the best results. Global performance analysis demonstrated satisfactory sensitivity (100%), specificity (97.2%), positive predictive value (98%), negative predictive value (100%) and global accuracy (99.6%). ELISA and SPRCruzi showed almost perfect agreement, mainly between chagasic and non-infected individuals. However, the new immunoassay was better in discriminate Chagas disease from other diseases. CONCLUSION: This work demonstrated the applicability of SPRCruzi as a feasible, real time, label free, sensible and specific methodology for the diagnosis of Chagas disease.

Development of a label-free immunosensor based on surface plasmon resonance technique for the detection of anti-Leishmania infantum antibodies in canine serum.

In this work, a surface plasmon resonance (SPR) immunosensor was developed using an 11-mercaptoundecanoic acid (11-MUA) modified gold SPR sensor chip for the detection of anti-Leishmania infantum antibodies. The soluble antigens of L. infantum were securely immobilized on an SPR gold disk by an 11-MUA self-assembled monolayer. Cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and scanning electrochemical microscopy (SECM) techniques were employed in the characterization of the antigen immobilization. After the immunosensor construction, canine serum positive for visceral leishmaniasis was added to its surface and showed significant variation in the SPR angle, indicating excellent sensitivity of the technique for antigen-antibody interaction detection. Moreover, the addition of negative serum was accompanied by a smaller response, demonstrating that the immunosensor shows good specificity against anti-L. infantum antibodies. Therefore, this work demonstrates the successful development of an SPR sensor for anti-L. infantum antibodies detection in short time, showing a great perspective as a sensing system of visceral leishmaniasis in endemic regions.

Clinical and laboratory status of patients with chronic Chagas disease living in a vector-controlled area in Minas Gerais, Brazil, before and nine years after aetiological treatment

Twenty-eight Chagas disease patients (CD), 22 with the indeterminate clinical form (IND) and six with the cardiac or digestive form (CARD/DIG), were treated with benznidazole and underwent clinical and laboratorial analysis before (IND and CARD/DIG) and nine years after [patients after treatment (CDt), patients with the indeterminate clinical form at treatment onset (INDt) and with the cardiac or digestive form at treatment onset (CARD/DIGt)] treatment. The data demonstrate that 82.1 percent of CDt patients (23/28) remained clinically stable and 95.4 percent of the INDt (21/22) and 33.3 percent of the CARD/DIGt (2/6) patients showed unaltered physical and laboratorial examinations. The clinical evolution rate was 2 percent/year and was especially low in INDt patients (0.5 percent/year) relative to CARD/DIGt patients (7.4 percent/year). Positive haemoculture in treated patients was observed in 7.1 percent of the cases. None of the INDt (0/21) and 33.3 percent of the CARD/DIGt (2/6) patients displayed positive cultures. The PCR presented a positive rate significantly higher (85.2 percent, 23/27) than haemoculture and two samples from the same patient revealed the same result 57.7 percent of the patients. Conventional serology-ELISA on 16 paired samples remained positive in all individuals. Semi-quantitative ELISA highlighted significant decreases in reactivity, particularly in INDt relative to IND. Non-conventional serology-FC-ALTA-IgG, after treatment, showed positive results in all sera and 22 paired samples examined at seven and nine years after treatment, demonstrated significantly lower reactivity, particularly in INDt patients. This study was retrospective in nature, had a low number of samples and lacked an intrinsic control group, but the data corroborate other results found in the literature. The data also demonstrate that, even though a cure has not been detected in the none-treated patients, the benefits for clinical evolution ...

Clinical and laboratory status of patients with chronic Chagas disease living in a vector-controlled area in Minas Gerais, Brazil, before and nine years after aetiological treatment.

Twenty-eight Chagas disease patients (CD), 22 with the indeterminate clinical form (IND) and six with the cardiac or digestive form (CARD/DIG), were treated with benznidazole and underwent clinical and laboratorial analysis before (IND and CARD/DIG) and nine years after [patients after treatment (CDt), patients with the indeterminate clinical form at treatment onset (INDt) and with the cardiac or digestive form at treatment onset (CARD/DIGt)] treatment. The data demonstrate that 82.1% of CDt patients (23/28) remained clinically stable and 95.4% of the INDt (21/22) and 33.3% of the CARD/DIGt (2/6) patients showed unaltered physical and laboratorial examinations. The clinical evolution rate was 2%/year and was especially low in INDt patients (0.5%/year) relative to CARD/DIGt patients (7.4%/year). Positive haemoculture in treated patients was observed in 7.1% of the cases. None of the INDt (0/21) and 33.3% of the CARD/DIGt (2/6) patients displayed positive cultures. The PCR presented a positive rate significantly higher (85.2%, 23/27) than haemoculture and two samples from the same patient revealed the same result 57.7% of the patients. Conventional serology-ELISA on 16 paired samples remained positive in all individuals. Semi-quantitative ELISA highlighted significant decreases in reactivity, particularly in INDt relative to IND. Non-conventional serology-FC-ALTA-IgG, after treatment, showed positive results in all sera and 22 paired samples examined at seven and nine years after treatment, demonstrated significantly lower reactivity, particularly in INDt patients. This study was retrospective in nature, had a low number of samples and lacked an intrinsic control group, but the data corroborate other results found in the literature. The data also demonstrate that, even though a cure has not been detected in the none-treated patients, the benefits for clinical evolution were selectively observed in the group of INDt patients and did not occur for CARD/DIGt patients.

Trypanosoma cruzi: blood parasitism kinetics and their correlation with heart parasitism intensity during long-term infection of Beagle dogs.

The goals of the present study were to evaluate the kinetics of blood parasitism by examination of fresh blood, blood culture (BC) and PCR assays and their correlation with heart parasitism during two years of infection in Beagle dogs inoculated with the Be-78, Y and ABC Trypanosoma cruzi strains. Our results showed that the parasite or its kDNA is easily detected during the acute phase in all infected animals. On the other hand, a reduced number of positive tests were verified during the chronic phase of the infection. The frequency of positive tests was correlated with T. cruzi strain. The percentage of positive BC and blood PCR performed in samples from animals inoculated with Be-78 and ABC strains were similar and significantly larger in relation to animals infected with the Y strain.Comparison of the positivity of PCR tests performed using blood and heart tissue samples obtained two years after infection showed two different patterns associated with the inoculated T. cruzi strain: (1) high PCR positivity for both blood and tissue was observed in animals infected with Be-78 or ABC strains; (2) lower and higher PCR positivity for the blood and tissue, respectively, was detected in animals infected with Y strains. These data suggest that the sensitivity of BC and blood PCR was T. cruzi strain dependent and, in contrast, the heart tissue PCR revealed higher sensitivity regardless of the parasite stock.

Trypanosoma cruzi: blood parasitism kinetics and their correlation with heart parasitism intensity during long-term infection of Beagle dogs

The goals of the present study were to evaluate the kinetics of blood parasitism by examination of fresh blood, blood culture (BC) and PCR assays and their correlation with heart parasitism during two years of infection in Beagle dogs inoculated with the Be-78, Y and ABC Trypanosoma cruzi strains. Our results showed that the parasite or its kDNA is easily detected during the acute phase in all infected animals. On the other hand, a reduced number of positive tests were verified during the chronic phase of the infection. The frequency of positive tests was correlated with T. cruzi strain. The percentage of positive BC and blood PCR performed in samples from animals inoculated with Be-78 and ABC strains were similar and significantly larger in relation to animals infected with the Y strain.Comparison of the positivity of PCR tests performed using blood and heart tissue samples obtained two years after infection showed two different patterns associated with the inoculated T. cruzi strain: (1) high PCR positivity for both blood and tissue was observed in animals infected with Be-78 or ABC strains; (2) lower and higher PCR positivity for the blood and tissue, respectively, was detected in animals infected with Y strains. These data suggest that the sensitivity of BC and blood PCR was T. cruzi strain dependent and, in contrast, the heart tissue PCR revealed higher sensitivity regardless of the parasite stock.