Redox proteomics in melanoma cells: An optimized protocol.

Cancer development and progression are intimately related with post-translational protein modifications, e.g., highly reactive thiol moiety of cysteines enables structural rearrangements resulting in redox biological switches. In this context, redox proteomics techniques, such as 2D redox DIGE, biotin switch assay and OxIcat are fundamental tools to identify and quantify redox-sensitive proteins and to understand redox mechanisms behind thiol modifications. Given the great variability in redox proteomics protocols, problems including decreased resolution of peptides and low protein amounts even after enrichment steps may occur. Considering the biological importance of thiol's oxidation in melanoma, we adapted the biotin-switch assay technique for melanoma cells in order to overcome the limitations and improve coverage of detected proteins.

Automated, High-Throughput Screening of Hybrid Elastin-like Polypeptide/Polysaccharide Multilayer Film Deposition.

The self-assembled layer-by-layer technique has attracted a great deal of attention as a method for engineering bio-functional surfaces under mild chemical conditions. The production of multilayer films, starting from newly designed building blocks, may be laborious, considering the inherent limitations for anticipating how minimal changes in the macromolecular composition may impact both film deposition and performance. This paper presents an automated, high-throughput approach to depositing polyelectrolyte multilayers (PEMs) in multiwell plates, enabling the screening of nearly 100 film formulations in the same process. This high-throughput layer-by-layer (HT-LbL) method runs in an affordable, fully commercial platform using Python-coded routines that can be easily adapted for the materials science lab settings. The HT-LbL system was validated by investigating the deposition of polysaccharide-based films in multiwell plates, probing the absorbance signal of ionically stained polyelectrolyte multilayers (PEMs) prepared in one single batch. The HT-LbL method was also used to investigate the deposition of PEMs with a small library of genetically engineered elastin-like polypeptides (ELPs) with different levels of ionizable and hydrophobic amino acid residues. The deposition of ELP/chitosan films was assessed based on the signal of fluorescently labeled species (chitosan or ELP-mCherry), demonstrating that both electrostatic and hydrophobic residues are essential for film buildup. The growth and surface properties of ELP-mCherry/chitosan films also seemed susceptible to the assembly pH, forming a higher film growth and a rougher and more hydrophobic surface for both polyelectrolytes deposited under a low ionization degree. Overall, this study illustrates the challenge of predicting the growth and properties of multilayer films and how the HT-LbL can accelerate the development of multilayer films that demand high levels of testing and optimization.

Interplay of the Assembly Conditions on Drug Transport Mechanisms in Polyelectrolyte Multilayer Films.

The layer-by-layer film deposition is a suitable strategy for the design and functionalization of drug carriers with superior performance, which still lacks information describing the influence of assembly conditions on the mechanisms governing the drug release process. Herein, traditional poly(acrylic acid)/poly(allylamine) polyelectrolyte multilayers (PEM) were explored as a platform to study the influence of the assembly conditions such as pH, drug loading method, and capping layer deposition on the mechanisms that control the release of calcein, the chosen model drug, from PEM. Films with 20-40 bilayers were assembled at pH 4.5 or 8.8, and the drug loading process was carried out during- or post-film assembly. Release data were fitted to three release models, namely, Higuchi, Ritger-Peppas, and Berens-Hopfenberg, to investigate the mechanism governing the drug transport, such as the apparent diffusion and the relaxation time. The postassembly drug loading method leads to a higher drug loading capacity than the during-assembly method, attributed to the washing out of calcein during film assembly steps in the latter method. Higuchi's and Ritger-Peppas' model analyses indicate that the release kinetic constant increased with the number of bilayers for the postassembly method. The opposite trend is observed for the during-assembly method. The Berens-Hopfenberg release model enabled the decoupling of each drug transport mechanism's contribution, indicating the increase of the diffusion contribution with the number of bilayers for the postassembly method at pH 4.5 and the increase of the polymer relaxation contribution for the during-assembly method at pH 8.8. Deborah's number, which represents the ratio of the polymer relaxation time to the diffusion time, follows the trends observed for the relaxation contribution for the conditions investigated. The deposition of the capping phospholipid layer over the payload also favored the polymer relaxation contribution in the drug release, featuring new strategies to investigate the drug release in PEM.

Tracking Sulfonated Polystyrene Diffusion in a Chitosan/Carboxymethyl Cellulose Layer-by-Layer Film: Exploring the Internal Architecture of Nanocoatings.

Since chitosan presents the ability to interact with a wide range of molecules, it has been one of the most popular natural polymers for the construction of layer-by-layer thin films. In this study, depth-profiling X-ray photoelectron spectroscopy (XPS) was employed to track the diffusion of sulfonated polystyrene (SPS) in carboxymethyl cellulose/chitosan (CMC/Chi) multilayers. Our findings suggest that the CMC/Chi film does not constitute an electrostatic barrier sufficient to block diffusion of SPS, and that diffusion can be controlled by adjusting the diffusion time and the molecular weight of the polymers that compose the CMC/Chi system. In addition to monitoring the diffusion, it was also possible to observe a process of preferential interaction between Chi and SPS. Thus, the nitrogen N 1s peak, due to functional groups found exclusively in chitosan chains, was the key factor to identifying the molecular interactions involving chitosan and the different polyanions. Accordingly, the presence of a strong polyanion such as SPS shifts the N 1s peak to a higher level of binding energy. Such results highlight that understanding the fundamentals of polymer interactions is a major step to fine-tuning the internal architecture of LbL structures for specific applications (e.g., drug release).

Repurposing strategies for Chagas disease therapy: the effect of imatinib and derivatives against Trypanosoma cruzi.

Chagas disease (CD) is a neglected parasitic condition endemic in the Americas caused by Trypanosoma cruzi. Patients present an acute phase that may or not be symptomatic, followed by lifelong chronic stage, mostly indeterminate, or with cardiac and/or digestive progressive lesions. Benznidazole (BZ) and nifurtimox are the only drugs approved for treatment but not effective in the late chronic phase and many strains of the parasite are naturally resistant. New alternative therapy is required to address this serious public health issue. Repositioning and combination represent faster, and cheaper trial strategies encouraged for neglected diseases. The effect of imatinib (IMB), a tyrosine kinase inhibitor designed for use in neoplasias, was assessed in vitro on T. cruzi and mammalian host cells. In comparison with BZ, IMB was moderately active against different strains and forms of the parasite. The combination IMB + BZ in fixed-ratio proportions was additive. Novel 14 derivatives of IMB were screened and a 3,2-difluoro-2-phenylacetamide (3e) was as potent as BZ on T. cruzi but had low selectivity index. The results demonstrate the importance of phenotypic assays, encourage the improvement of IMB derivatives to reach selectivity and testify to the use of repurposing and combination in drug screening for CD.

Repurposing Strategy of Atorvastatin against Trypanosoma cruzi: In Vitro Monotherapy and Combined Therapy with Benznidazole Exhibit Synergistic Trypanocidal Activity.

Statins are inhibitors of cholesterol synthesis, but other biological properties, such as antimicrobial effects, have also been assigned to them, leading to their designation as pleiotropic agents. Our goal was to investigate the activity and selectivity of atorvastatin (AVA) against Trypanosoma cruzi by using in vitro models, aiming for more effective and safer therapeutic options through drug repurposing proposals for monotherapy and therapy in combination with benznidazole (BZ). Phenotypic screening was performed with different strains (Tulahuen [discrete typing unit {DTU} VI] and Y [DTU II]) and forms (intracellular forms, bloodstream trypomastigotes, and tissue-derived trypomastigotes) of the parasite. On assay of the Tulahuen strain, AVA was more active against intracellular amastigotes (selectivity index [SI] = 3). Also, against a parasite of another DTU (Y strain), this statin was more active (2.1-fold) and selective (2.4-fold) against bloodstream trypomastigotes (SI = 51) than against the intracellular forms (SI = 20). A cytomorphological approach using phalloidin-rhodamine permitted us to verify that AVA did not induced cell density reduction and that cardiac cells (CC) maintained their typical cytoarchitecture. Combinatory approaches using fixed-ratio methods showed that AVA and BZ gave synergistic interactions against both trypomastigotes and intracellular forms (mean sums of fractional inhibitory concentration indexes [∑FICIs] of 0.46 ± 0.12 and 0.48 ± 0.03, respectively). Thus, the repurposing strategy for AVA, especially in combination with BZ, which leads to a synergistic effect, is encouraging for future studies to identify novel therapeutic protocols for Chagas disease treatment.

In Vitro and In Vivo Studies of the Trypanocidal Effect of Novel Quinolines.

Therapies for human African trypanosomiasis and Chagas disease, caused by Trypanosoma brucei and Trypanosoma cruzi, respectively, are limited, providing minimal therapeutic options for the millions of individuals living in very poor communities. Here the effects of 10 novel quinolines are evaluated in silico and by phenotypic studies using in vitro and in vivo models. Absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties revealed that most molecules did not infringe on Lipinski's rules, which is a prediction of good oral absorption. These quinolines showed high probabilities of Caco2 permeability and human intestinal absorption and low probabilities of mutagenicity and of hERG1 inhibition. In vitro screens against bloodstream forms of T. cruzi demonstrated that all quinolines were more active than the reference drug (benznidazole [Bz]), except for DB2171 and DB2192, with five (DB2187, DB2131, DB2186, DB2191, and DB2217) displaying 50% effective concentrations (EC50s) of <3 µM (4-fold lower than that of Bz). Nine quinolines were more effective than Bz (2.7 µM) against amastigotes, showing EC50s ranging from 0.6 to 0.1 µM. All quinolines were also highly active in vitro against African trypanosomes, showing EC50s of ≤0.25 µM. The most potent and highly selective candidates for each parasite species were tested in in vivo models. Results for DB2186 were promising in mice with T. cruzi and T. brucei infections, reaching a 70% reduction of the parasitemia load for T. cruzi, and it cured 2 out of 4 mice infected with T. brucei DB2217 was also active in vivo and cured all 4 mice (100% cure rate) with T. brucei infection.

Prevalence of human papillomavirus types associated with cervical lesions in Sergipe state, Northeastern Brazil: high frequency of a possibly carcinogenic type.

Persistent infection with high-risk human papillomavirus (HPV) is the main cause of cervical cancer and the prevalence of HPV types varies depending on the geographic region. Therefore, this study assessed the prevalence of HPV types in women with cervical lesions from Sergipe state, Northeastern Brazil. A cross-sectional study was conducted in women with cervical lesions from March to December 2014. These lesions were investigated by PCR and HPV types were identified by DNA sequencing. 432 patients were included, of which 337 patients tested positive for HPV. Eighteen different HPV types were detected, and high-risk HPV types were detected in 69.2%. HPV 16 (63.4%) was the most prevalent HPV type found, followed by HPV 66 (4.6%), HPV 18 (1.6%) and HPV 45 (1.4%). These results highlight the importance of the high prevalence of HPV 66, which is a possibly carcinogenic virus type not covered by the available vaccines. The prevalence of HPV 16 was high in the studied population, reaffirming the importance of young vaccination. However, the high prevalence of HPV 66 found in this study shows the importance of monitoring the diversity of HPV types in different populations and geographic regions to better understand the impacts of current HPV vaccines.

Antitrypanosomal Activity of Sterol 14α-Demethylase (CYP51) Inhibitors VNI and VFV in the Swiss Mouse Models of Chagas Disease Induced by the Trypanosoma cruzi Y Strain.

Chagas disease is a life-threatening infection caused by a variety of genetically diverse strains of the protozoan parasite Trypanosoma cruzi The current treatment (benznidazole and nifurtimox) is unsatisfactory, and potential alternatives include inhibitors of sterol 14α-demethylase (CYP51), the cytochrome P450 enzyme essential for the biosynthesis of sterols in eukaryotes and the major target of clinical and agricultural antifungals. Here we performed a comparative investigation of two protozoon-specific CYP51 inhibitors, VNI and its CYP51 structure-based derivative VFV, in the murine models of infection caused by the Y strain of T. cruzi The effects of different treatment regimens and drug delivery vehicles were evaluated in animals of both genders, with benznidazole serving as the reference drug. Regardless of the treatment scheme or delivery vehicle, VFV was more potent in both genders, causing a >99.7% peak parasitemia reduction, while the VNI values varied from 91 to 100%. Treatments with VNI and VFV resulted in 100% animal survival and 0% natural relapse after the end of therapy, though, except for the 120-day treatment schemes with VFV, relapses after three cycles of immunosuppression were observed in each animal group, and quantitative PCR analysis revealed a very light parasite load in the blood samples (sometimes below or near the detection limit, which was 1.5 parasite equivalents/ml). Our studies support further investigations of this class of compounds, including their testing against other T. cruzi strains and in combination with other drugs.

Phenotypic Screening In Vitro of Novel Aromatic Amidines against Trypanosoma cruzi.

The current treatment of Chagas disease (CD), based on nifurtimox and benznidazole (Bz), is unsatisfactory. In this context, we performed the phenotypic in vitro screening of novel mono- and diamidines and drug interaction assays with selected compounds. Ten novel amidines were tested for their activities against bloodstream trypomastigote (BT) and amastigote forms of Trypanosoma cruzi (Y and Tulahuen strains) and their toxicities for mammalian host cells (L929 cells and cardiac cells). Seven of 10 molecules were more active than Bz against BT, with the most active compound being the diamidine DB2267 (50% effective concentration [EC50] = 0.23 µM; selectivity index = 417), which was 28-fold more active and about 3 times more selective than the standard drug. Five of the six monoamidines were also more active than Bz. The combination of DB2267 and DB2236 in fixed-ratio proportions showed an additive effect (sum of fractional inhibitory concentrations < 4) on BT. Interestingly, when intracellular forms were exposed to DB2267, its activity was dependent on the parasite strain, being effective (EC50 = 0.87 ± 0.05 µM) against a discrete typing unit (DTU) II strain (strain Y) but not against a representative DTU VI strain (strain Tulahuen) even when different vehicles (ß-cyclodextrin and dimethyl sulfoxide) were used. The intrinsic fluorescence of several diamidines allowed their uptake to be studied. Testing of the uptake of DB2236 (inactive) and DB2267 (active) by amastigotes of the Y strain showed that the two compounds were localized intracellularly in different compartments: DB2236 in the cytoplasm and DB2267 in the nucleus. Our present data encourage further studies regarding the activities of amidines and provide information which will help with the identification of novel agents for the treatment of CD.

Influence of different anaesthetic protocols over the sperm quality on the fresh, chilled (4°C) and frozen-thawed epididymal sperm samples in domestic dogs.

This study assessed the influence of three different anaesthetic protocols on semen quality obtained from the epididymis. Sixty male dogs undergoing to routine sterilization were assigned to three anaesthetic protocols: thiopental group (TG, n = 20), propofol group (PG, n = 20) and ketamine-dexmedetomidine group (KDG, n = 20). Immediately after orchidectomy, the cauda epididymides and vas deferent ducts were isolated and then a retrograde flushing was performed to collect spermatozoa. In experiment 1, after the initial evaluation of the semen (sperm concentration, sperm motility and the percentages of live spermatozoa, abnormal spermatozoa and acrosome membrane integrity), semen samples were diluted in Tris-glucose-egg yolk extender and chilled for 48 hr, and the sperm motility was assessed at 6, 24 and 48 hr. In experiment 2, semen samples were diluted in Tris-glucose-egg yolk extender and chilled for 24 hr, and then samples were frozen in two extenders with different glycerol concentrations, to reach a final concentration of 50-100 × 10(6) spermatozoa ml(-1) , 20% egg yolk, 0.5% Equex and 4% and 5% glycerol, respectively. Mean values of total sperm concentration, sperm viability and the percentages of intact acrosome and abnormal spermatozoa were not significantly different between experimental groups, and therefore, the anaesthetic protocols assessed did not affect sperm parameters mentioned above. However, our study confirmed a detrimental effect of the use of thiopental (TG) over the total sperm motility (p < 0.05) and progressive sperm motility (p < 0.05) of the fresh and chilled epididymal sperm samples. The anaesthetic protocols including the application of propofol or ketamine-dexmedetomidine can be used to recover sperm in domestic canids without significant changes in sperm quality compared when semen is collected routinely and these techniques could be applicable to endangered wild canids.

Dynamics of Notch signalling in the mouse oviduct and uterus during the oestrous cycle.

The oviduct and uterus undergo extensive cellular remodelling during the oestrous cycle, requiring finely tuned intercellular communication. Notch is an evolutionarily conserved cell signalling pathway implicated in cell fate decisions in several tissues. In the present study we evaluated the quantitative real-time polymerase chain reaction (real-time qPCR) and expression (immunohistochemistry) patterns of Notch components (Notch1-4, Delta-like 1 (Dll1), Delta-like 4 (Dll4), Jagged1-2) and effector (hairy/enhancer of split (Hes) 1-2, Hes5 and Notch-Regulated Ankyrin Repeat-Containing Protein (Nrarp)) genes in the mouse oviduct and uterus throughout the oestrous cycle. Notch genes are differentially transcribed and expressed in the mouse oviduct and uterus throughout the oestrous cycle. The correlated transcription levels of Notch components and effector genes, and the nuclear detection of Notch effector proteins, indicate that Notch signalling is active. The correlation between transcription levels of Notch genes and progesterone concentrations, and the association between expression of Notch proteins and progesterone receptor (PR) activation, indicate direct progesterone regulation of Notch signalling. The expression patterns of Notch proteins are spatially and temporally specific, resulting in unique expression combinations of Notch receptor, ligand and effector genes in the oviduct luminal epithelium, uterus luminal and glandular epithelia and uterine stroma throughout the oestrous cycle. Together, the results of the present study imply a regulatory role for Notch signalling in oviduct and uterine cellular remodelling occurring throughout the oestrous cycle.

Differential expression of Notch component and effector genes during ovarian follicle and corpus luteum development during the oestrous cycle.

Ovarian dynamics throughout the female oestrous cycle (EC) are characterised by cyclical follicle and corpus luteum (CL) development. These events are tightly regulated, involving extensive cell-to-cell communication. Notch is an evolutionarily well conserved cell-signalling pathway implicated in cell-fate decisions in several tissues. Here, we evaluated the extra-vascular expression patterns of Notch component and effector genes during follicle and CL development throughout the EC. Five mature CD1 female mice were killed at each EC stage. Blood samples were collected for progesterone measurement, ovaries were processed for immunohistochemistry and expression patterns of Notch components (Notch1, 2 and 3, Jagged1 and Delta-like1 and 4) and effectors (Hes1, Hes2 and Hes5) were characterised. Nuclear detection of Notch effectors indicates that Notch signalling is active in the ovary. Notch components and effectors are differentially expressed during follicle and CL development throughout the EC. The spatial and temporal specific expression patterns are associated with follicle growth, selection and ovulation or atresia and CL development and regression.

Frequencies of allele groups HLA-A, HLA-B and HLA-DRB1 in a population from the northwestern region of São Paulo State, Brazil.

The aim of this study was to estimate the HLA-A, HLA-B and HLA-DRB1 allele groups frequencies in a population of 1559 volunteer bone marrow donors from the northwestern region of São Paulo State grouped according to ethnicity. An additional objective was to compare the allele frequencies of the current study with data published for other Brazilian populations. The allele groups were characterized by the PCR-rSSO method using Luminex(®) technology. Twenty HLA-A, 32 HLA-B and 13 HLA-DRB1 allele groups were identified. The most common allele groups in European descent and mixed African and European descent samples were HLA-A*02, HLA-B*35 and HLA-DRB1*13, while HLA-A*02, HLA-B*35 and HLA-DRB1*11 were more common in African descent samples. The HLA-A*23, HLA-A*36, HLA-B*58 and HLA-B*81 allele groups were more common in sample from African descent than European descent, and the HLA-DRB1*08 was more common in mixed African and European descent than in European descent. Allele group frequencies were compared with samples from other Brazilian regions. The HLA-A*30 and HLA-A*23 were more common in this study than in the populations of Rio Grande do Sul and Paraná; and the HLA-A*01, HLA-B*18, HLA-B*57 and HLA-DRB1*11 were more common in this study than in the population of Piauí. The least frequent allele groups were HLA-A*31, HLA-B*15, HLA-B*40 and HLA-DRB1*08 for the population of Piauí, HLA-A*01 and HLA-A*11 for Parana, HLA-A*02 and -A*03 for Rio Grande do Sul and HLA-DRB1*04 for Paraná, Rio Grande do Sul and Piauí. These data provide an overview on the knowledge on HLA diversity in the population of the northwestern region of São Paulo State and show that the genes of this system are useful to distinguish different ethnic groups.

Effects of oocyte donor age and embryonic stage of development on transcription of genes coding for enzymes of the prostaglandins and progesterone synthesis pathways in bovine in vitro produced embryos.

The ability of early bovine embryos to produce prostaglandins (PGs) and progesterone (P4), and the role of these mediators in embryonic development and survival are poorly understood. In this study we tested the hypothesis that day 7 bovine embryos are able to transcribe genes coding for enzymes of the PGs (PTGS2, PGES, PGFS) and P4 (StAR, P450scc, 3ß-HSD) synthesis pathways, and that transcription levels of these genes are associated with developmental progression and heifer age-related [pre-pubertal (PP) versus post-pubertal cyclic (C)] oocyte competence. Compared with C heifer oocytes, PP heifer oocytes showed a lower (P < 0.0001) in vitro blastocyst rate, but in embryos developing until day 7, heifer age had no effect on quality grade. Day 7 quality grade 1-2 embryos were selected for RNA extraction and gene transcription analysis by qRT-PCR, in a 2 × 2 factorial design [age (PP or C) × embryonic stage (compact morulae and early blastocysts, CM + EBL, or blastocysts and expanded blastocysts, BL + BEX); 15 embryos/group]. Transcription levels of PTGS2, PGES, PGFS, P450scc and 3ß-HSD were not affected by heifer age but were higher (P < 0.01) in BL + BEX than in CM + EBL. In conclusion, the main limiting factor for embryo production from PP heifers is oocyte competence. Day 7 bovine embryos evidence transcription of genes coding for enzymes of PGs and P4 synthesis pathways, and transcription levels are associated with blastocyst differentiation. This prompts for an autocrine/paracrine action of PGs and P4 in early bovine embryonic development.

Molecular typing of Salmonella from Sergipe, Northeastern Brazil, showing the epidemiological relationship between poultry and human infection.

Randomly amplified polymorphic DNA (RAPD) has been widely used for epidemiological and phylogenetic purposes ow-ing to its rapidity and efficiency. The aim of this study was to perform genome typing of Salmonella samples isolated from different sources by RAPD profiling. Thirty-three Salmonella samples from the bacterial collection of the Laboratório de Virologia Comparada, Departamento de Morfologia, Universidade Federal de Sergipe, Brazil, and two standard samples were used. RAPD profiling was conducted using six primers of the Ready-To-Go RAPD system. The amplified products were electro-phoresed on 5% polyacrylamide gel and silver-stained. RAPD analysis resulted in reproducible and stable banding patterns and showed high genetic diversity among the isolated strains. The Primer P1-generated dendrogram showed an epidemiologic relationship between the human and poultry isolated samples, highlighting the usefulness of RAPD for molecular typing and epidemiological studies.

In vitro susceptibility of equine-obtained isolates of Corynebacterium pseudotuberculosis to gallium maltolate and 20 other antimicrobial agents.

This study's objective was to determine the in vitro antimicrobial activities of gallium maltolate (GaM) and 20 other antimicrobial agents against clinical equine isolates of Corynebacterium pseudotuberculosis. The growth of cultured isolates was not inhibited by any concentration of GaM. MIC data revealed susceptibility to commonly used antimicrobials.

In vitro investigation of the efficacy of novel diamidines against Trypanosoma cruzi.

Chagas' disease is a neglected tropical disease caused by Trypanosoma cruzi and constitutes a serious public health problem for Latin America. Its unsatisfactory chemotherapy stimulates the search for novel antiparasitic compounds. Amidines and related compounds exhibit well-known activity towards different microbes including T. cruzi. In this vein, our present aim was to evaluate the biological effect of 10 novel structurally related amidines in vitro against bloodstream and intracellular forms of the parasite as well as their potential toxicity on cardiac cell cultures. Our results show that although active against the extracellular forms, with some of them like DB2247 being 6-fold more effective than benznidazole and displaying very low toxicity (>96 µm), none presented superior trypanocidal effect against intracellular forms as compared with the reference drug. These results may be due to differences in susceptibility profiles related to distinct uptake/extrusion mechanisms and cellular targets between bloodstream and amastigote forms. The present study adds to the knowledge base for the future design of novel amidines that may provide promising activity against T. cruzi.