Leishmaniases comprise a group of infectious parasitic diseases caused by various species of Leishmania and are considered a significant public health problem worldwide. Only a few medications, ...including miltefosine, amphotericin B, and meglumine antimonate, are used in current therapy. These medications are associated with severe side effects, low efficacy, high cost, and the need for hospital support. Additionally, there have been occurrences of drug resistance. Additionally, only a limited number of drugs, such as meglumine antimonate, amphotericin B, and miltefosine, are available, all of which are associated with severe side effects. In this context, the need for new effective drugs with fewer adverse effects is evident. Therefore, this study investigated the anti-Leishmania activity of a dichloromethane fraction (DCMF) extracted from Arrabidaea brachypoda roots. This fraction inhibited the viability of L. infantum, L. braziliensis, and L. Mexicana promastigotes, with IC50 values of 10.13, 11.44, and 11.16 µg/mL, respectively, and against L. infantum amastigotes (IC50 = 4.81 µg/mL). Moreover, the DCMF exhibited moderate cytotoxicity (CC50 = 25.15) towards RAW264.7 macrophages, with a selectivity index (SI) of 5.2. Notably, the DCMF caused damage to the macrophage genome only at 40 µg/mL, which is greater than the IC50 found for all Leishmania species. The results suggest that DCMF demonstrates similar antileishmanial effectiveness to isolated brachydin B, without causing genotoxic effects on mammalian cells. This finding is crucial because the isolation of the compounds relies on several steps and is very costly while obtaining the DCMF fraction is a simple and cost-effective process. Furthermore, In addition, the potential mechanisms of action of brachydins were also investigated. The computational analysis indicates that brachydin compounds bind to the Triosephosphate isomerase (TIM) enzyme via two main mechanisms: destabilizing the interface between the homodimers and interacting with catalytic residues situated at the site of binding. Based on all the results, DCMF exhibits promise as a therapeutic agent for leishmaniasis due to its significantly reduced toxicity in comparison to the adverse effects associated with current reference treatments.
•DCMF showed antileishmanial activity for promastigotes and amastigotes of L. infantum.•DCMF proved to be more selective than the antileishmanial reference drugs.•DCMF significantly decreased of infected macrophages and internalized amastigotes.•Suggest that DCMF act by disrupting cell membranes rather than causing DNA damage.•Molecular dynamics showed two binding modes for brachydins on the TIM enzyme.
•A detailed protocol for culturing adult human brain slices is presented.•Cell viability and neurotransmitter release are only slightly affected in vitro.•Cultured slices are sensitive to Alzheimer’s ...disease-associated Aβ oligomers.•These cultures may represent a preferred model to study neurodegenerative diseases.
Slice cultures have been prepared from several organs. With respect to the brain, advantages of slice cultures over dissociated cell cultures include maintenance of the cytoarchitecture and neuronal connectivity. Slice cultures from adult human brain have been reported and constitute a promising method to study neurological diseases. Despite this potential, few studies have characterized in detail cell survival and function along time in short-term, free-floating cultures.
We used tissue from adult human brain cortex from patients undergoing temporal lobectomy to prepare 200 μm-thick slices. Along the period in culture, we evaluated neuronal survival, histological modifications, and neurotransmitter release. The toxicity of Alzheimer’s-associated Aβ oligomers (AβOs) to cultured slices was also analyzed.
Neurons in human brain slices remain viable and neurochemically active for at least four days in vitro, which allowed detection of binding of AβOs. We further found that slices exposed to AβOs presented elevated levels of hyperphosphorylated Tau, a hallmark of Alzheimer’s disease.
Although slice cultures from adult human brain have been previously prepared, this is the first report to analyze cell viability and neuronal activity in short-term free-floating cultures as a function of days in vitro.
Once surgical tissue is available, the current protocol is easy to perform and produces functional slices from adult human brain. These slice cultures may represent a preferred model for translational studies of neurodegenerative disorders when long term culturing in not required, as in investigations on AβO neurotoxicity.
PsaA (pneumococcal surface antigen A) is a S. pneumoniae virulence factor that belongs to the metal transport system. The Manganese PsaA binding has been associated with oxidative stress resistance ...becoming a pivotal element in the bacteria virulence. It has been shown that Zinc inhibits the Manganese acquisition and promotes bacteria toxicity. We have performed a PsaA conformational analysis both in the presence (Zn-rPsaA) and in the absence of Zinc (free-rPsaA). We performed experiments in the presence of different Zinc concentrations to determine the metal minimum concentration which induced a conformational change. The protein in free and Zn-binding condition was also studied in pH ranging 2.6-8.0 and in temperature ranging 25oC-85oC. pH experiments showed a decrease of fluorescence intensity only in acidic medium. Analysis of the heat-induced denaturation demonstrated that Zinc-binding promoted an increase in melting temperature from 55oC (free-rPsaA) to 78.8oC (Zn-rPsaA) according to fluorescence measurements. In addition, the rPsaA stabilization by Zinc was verified through analysis of urea and guanidine hydrochloride denaturation. Data showed that Zinc promoted an increase in the rPsaA stability and its removal by EDTA can lead to a PsaA intermediate conformation. These findings can be considered in the development of vaccines containing PsaA as antigen.
In this work, the construction and performance evaluation of PVC membranes ion‐selective electrodes for the determination of tetracycline type antibiotics is described. Electrodes with the best ...responses were based on plastic membranes containing 31% (w/w) PVC, 68% (w/w) of dibutylphthalate as plasticizer and 1% (w/w) of β‐cyclodextrin as ionophore. The electrodes were responsive over 6 months to tetracycline, oxytetracycline, doxycycline and chlortetracycline in glycine buffer solution (pH 2), in the dynamic range 2×10−5–10−2 mol L−1 with a constant slope of about 55 mV/dec To enable large scale analysis reducing wastes as associated costs tubular shape electrodes were coupled to a sequential‐injection analysis system and its performance evaluated using pharmaceutical samples and waste waters from a treatment facility. The sample throughput of 51 samples h−1 was enabled by the system as well as results that favorably agree with those provided by chromatographic analysis.
Rocio virus (ROCV) is a highly neuropathogenic mosquito-transmitted flavivirus responsible for an unprecedented outbreak of human encephalitis during 1975-1976 in Sao Paulo State, Brazil. Previous ...studies have shown an increased number of inflammatory macrophages in the central nervous system (CNS) of ROCV-infected mice, implying a role for macrophages in the pathogenesis of ROCV. Here, we show that ROCV infection results in increased expression of CCL2 in the blood and in infiltration of macrophages into the brain. Moreover, we show, using CCR2 knockout mice, that CCR2 expression is essential for macrophage infiltration in the brain during ROCV infection and that the lack of CCR2 results in increased disease severity and mortality. Thus, our findings show the protective role of CCR2-mediated infiltration of macrophages in the brain during ROCV infection.
Phospholipases A2 (PLA2s) comprise a superfamily of glycerophospholipids hydrolyzing enzymes present in many organisms in nature, whose catalytic activity was majorly unveiled by analysis of snake ...venoms. The latter have pharmaceutical and biotechnological interests and can be divided into different functional sub-classes. Our goal was to identify important residues and their relation to the functional and class-specific characteristics in the PLA2s family with special emphasis on snake venom PLA2s (svPLA2s). We identified such residues by conservation analysis and decomposition of residue coevolution networks (DRCN), annotated the results based on the available literature on PLA2s, structural analysis and molecular dynamics simulations, and related the results to the phylogenetic distribution of these proteins. A filtered alignment of PLA2s revealed 14 highly conserved positions and 3 sets of coevolved residues, which were annotated according to their structural or functional role. These residues are mostly involved in ligand binding and catalysis, calcium-binding, the formation of disulfide bridges and a hydrophobic cluster close to the binding site. An independent validation of the inference of structure-function relationships from our co-evolution analysis on the svPLA2s family was obtained by the analysis of the pattern of selection acting on the Viperidae and Elapidae lineages. Additionally, a molecular dynamics simulation on the Lys49 PLA2 from Agkistrodon contortrix laticinctus was carried out to further investigate the correlation of the Lys49-Glu69 pair. Our results suggest this configuration can result in a novel conformation where the binding cavity collapses due to the approximation of two loops caused by a strong salt bridge between Glu69 and Arg34. Finally, phylogenetic analysis indicated a correlation between the presence of residues in the coevolved sets found in this analysis and the clade localization. The results provide a guide for important positions in the family of PLA2s, and potential new objects of investigation.
•Our results serve as a guide to structurally and functionally relevant residues.•14 highly conserved positions and 3 sets of coevolved residues are disclosed.•Phylogenetic analysis reveals class-specific features and divergent group of PLA2s.
Dry forests in the Caatinga biome of Brazil are seasonal ecosystems where diversity is driven by water availability. Understanding how the distribution of communities is driven by temporal climate ...changes has intrigued researchers for decades. However, temporal diversity patterns should be more evident in dry environments, since seasonality is characterized by being highly limiting to insect activities. Cerambycid beetles are considered good ecological indicators because they respond well to impacts of environmental changes. Thus, we asked two questions: (i) How do climatic changes affect the diversity of these insects across seasons? (ii) Are diversity components correlated with increasing air humidity, rainfall, and temperature? Our results showed a marked seasonality of cerambycid beetles, with higher abundance and richness in the wet season. The mean temperature and relative humidity were predictors of the composition of beetle assemblages. However, the variation of cerambycid assemblages between seasons is related mainly to species turnover. Our study demonstrates that the combined effect of temperature and humidity drives the temporal distribution of the cerambycids in dry forests. Although thermal sensitivity was low, the decrease in air moisture during the dry season was the limiting factor for these insects. Species turnover increased continuously with air moisture and temperature rise, creating temporal segregation among cerambycid species and maintaining the stability of the assemblage. Thus, our results are consistent with mechanisms invoking activity patterns, desiccation resistance, and physiologic constraints that predict a decrease in richness and abundance of the cerambycids from warmer and moister to colder and drier conditions.
Background: Low-frequency electrical stimulation (LFES) is an adjuvant method for heart failure (HF) patients with restrictions to start an exercise. However, the impact on molecular changes in ...circulating is unknown. We investigated the effects of 10 weeks of home-based LFES on plasma cytokines profile, redox biomarkers, metalloproteinases (MMPs) activity, and exercise performance in HF patients. Methods: Twenty-four HF patients (52.45 ± 9.15 years) with reduced ejection fraction (HFrEF) (EF < 40%), were randomly assigned to a home-based LFES or sham protocol. Plasma cytokines profile was assessed through interleukins, interferon-gamma, and tumor necrosis factor levels. Oxidative stress was evaluated through ferric reducing antioxidant power, thiobarbituric acid-reactive substances, and inducible nitric oxide synthase. The MMPs activity were analyzed by zymography. Cardiorespiratory capacity and muscle strength were evaluated by cardiopulmonary test and isokinetic. Results: LFES was able to increase the active-MMP2 activity post compared to pre-training (0.057 to 0.163, p = 0.0001), while it decreased the active-MMP9 (0.135 to 0.093, p = 0.02). However, it did not elicit changes in cytokines, redox biomarkers, or exercise performance (p > 0.05). Conclusion: LFES protocol is a promising intervention to modulate MMPs activity in HFrEF patients, although with limited functional effects. These preliminary responses may help the muscle to adapt to future mechanical demands dynamically.
In the present study, a nested-PCR system, targeting the TbD1 region, involving the performance of conventional PCR followed by real-time PCR, was developed to detect Mycobacterium bovis in ...bovine/bubaline tissue homogenates. The sensitivity and specificity of the reactions were assessed with DNA samples extracted from tuberculous and non-tuberculous mycobacteria, as well as other actinomycetales species and DNA samples extracted directly from bovine and bubaline tissue homogenates. In terms of analytical sensitivity, the DNA of M. bovis AN5 was detected up to 1.56 ng with conventional PCR, 97.6 pg with real-time PCR, and 1.53 pg with nested-PCR in the reaction mixture. The nested-PCR exhibited 100% analytical specificity for M. bovis when tested with the DNA of reference strains of environmental mycobacteria and closely-related Actinomycetales. A clinical sensitivity value of 76.0% was detected with tissue samples from animals that exhibited positive results in the comparative intradermal tuberculin test (CITT), as well as from those with lesions compatible with tuberculosis (LCT) that rendered positive cultures. A clinical specificity value of 100% was detected with tissue samples from animals with CITT- results, with no visible lesions (NVL) and negative cultures. No significant differences were found between the nested-PCR and culture in terms of detecting CITT+ animals with LCT or with NVL. No significant differences were recorded in the detection of CITT- animals with NVL. However, nested-PCR detected a significantly higher number of positive animals than the culture in the group of animals exhibiting LCT with no previous records of CITT. The use of the nested-PCR assay to detect M. bovis in tissue homogenates provided a rapid diagnosis of bovine and bubaline tuberculosis.
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