Dentine biomodification by sulphonamides pre-treatment: bond strength, proteolytic inhibition, and antimicrobial activity.

We evaluated the effects of dentine biomodification after pre-treatment with two sulphonamide carbonic anhydrase inhibitors (CAIs) of the N-[4-sulphamoylphenethylcarbamoyl]benzenesulphonamide type, investigating matrix metalloproteases activity, resin-dentine micro tensile bond strength, dentine surface wettability, and antimicrobial activities. Ninety-five sound-extracted human molars were selected for the study. Inhibitory effects were evaluated by gelatinase and collagenase activity tests and collagen degradation FT-IR spectroscopic analysis. Pre-treatment with the two CAIs kept the micro tensile values after 12 months of storage (32.23 ± 5.95) and cariogenic challenge (34.13 ± 2.71) similar to the initial, pre-treatment values (33.56 ± 4.34). A decreased Streptococcus mutans biofilm formation on dentine surfaces and antibacterial activity against planktonic bacteria were observed after CAI treatment. Dentine pre-treatment with sulphonamide CAIs maintained adhesion strength stability, allowed better dentine wettability, maintained matrix collagen, and showed anti-S. mutans activity.

Chagas Disease: Perspectives on the Past and Present and Challenges in Drug Discovery.

Chagas disease still has no effective treatment option for all of its phases despite being discovered more than 100 years ago. The development of commercial drugs has been stagnating since the 1960s, a fact that sheds light on the question of how drug discovery research has progressed and taken advantage of technological advances. Could it be that technological advances have not yet been sufficient to resolve this issue or is there a lack of protocol, validation and standardization of the data generated by different research teams? This work presents an overview of commercial drugs and those that have been evaluated in studies and clinical trials so far. A brief review is made of recent target-based and phenotypic studies based on the search for molecules with anti-Trypanosoma cruzi action. It also discusses how proteochemometric (PCM) modeling and microcrystal electron diffraction (MicroED) can help in the case of the lack of a 3D protein structure; more specifically, Trypanosoma cruzi carbonic anhydrase.

Evaluation of Kluyveromyces marxianus endo-polygalacturonase activity through ATR-FTIR.

The endo-polygalacturonase enzyme (endoPG: EC 3.2.1.15) plays an important role in the fruit juice and wine industries, so the development of new tools for the quantitative and qualitative analysis of its enzymatic action is necessary. In this work, we report the development of a simple, fast and practical method that did not use any chemical reagent to identify and evaluate the action of the endoPG enzyme, produced by the yeast Kluyveromyces marxianus CCT3172, using attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy combined with principal component analysis-linear discriminant analysis (PCA-LDA). This method evaluated the action of the endoPG enzyme on the polygalacturonic acid (PGA) substrate at 5 different times (0, 10, 15, 20 and 30 minutes), and at each time interval the samples were analyzed by ATR-FTIR. It was demonstrated that there was clear segregation between the samples that were and that were not subjected to the action of the endoPG enzyme, and it was also possible to distinguish the samples that were subjected to different incubation times with the enzyme. Through PCA-LDA it was possible to obtain wavelengths that are biomarkers for this enzymatic reaction and the observed changes as a function of hydrolysis duration were found to be in agreement with the breakdown of the glycosidic chain (1011 cm-1-CH-O- CH stretching) of PGA and release of oligosaccharides (1078 cm-1 C-OH elongation). The activity of the endoPG enzyme and the release of galacturonic acid were verified by the dinitrosalicylic acid (DNS) method in all samples. The efficacy of an automatic classifier using a principal component analysis-linear discriminant classifier (PCA-LDC) was evaluated to diagnose the action of the endoPG enzyme. The results showed an accuracy of 100% for the identification of the endoPG enzyme action and from 91.67% to 100% for classification according to the hydrolysis duration in which PGA was exposed to endoPG. The present study indicates that this methodology may be a new approach for the qualitative evaluation of the endoPG enzyme with the potential to be used in laboratories and industries.

Nanovesicle-based formulations for photoprotection: a safety and efficacy approach.

Vesicular nanosystems are versatile and they are able to encapsulate actives with different solubilities, such as lipophilic and hydrophilic compounds. The most well-known vesicular nanosystems are liposomes and niosomes, the last one is formed by non-ionic surfactants. In the present work, we developed photoprotective niosomes containing sunscreens (octyl methoxycinnamate, diethylamino hydroxybenzoyl hexyl benzoate and phenylbenzimidazole sulfonic acid), non-ionic surfactants, cholesterol and stearylamine (positive-charged lipid). Studies based on dynamic light scattering techniques, entrapment efficiency and morphology by transmission electron microscopy were performed to characterize the niosomes. In addition, rheology, pH, in vitro sun protection factor (SPF) efficacy and toxicity and in vivo and in vitro safety were determined for the niosome formulations F-N1 and F-N2. The mean sizes of N1 and N2 were 168 ± 5 nm and 192 ± 8 nm, respectively, and their morphologies were spherical, unilamellar and with an entrapment efficiency of more than 45% for each sunscreen. Both formulations, F-N1 and F-N2 presented characteristics of pseudoplastic non-Newtonian fluids, showing declining viscosity with increasing shear rate applied. SPF values were considered satisfactory, 34 ± 8 for formulation F-N1 and 34 ± 5 for F-N2. The formulations did not present toxicity when tested in macrophages and the pH was compatible with skin, which minimizes allergies. The in vitro safety assay showed lipophilic sunscreens greater affinity for the epidermis, since this layer contains natural lipids. In vivo safety assay suggests that the increased skin retention of N2 is directly correlated with the positive charge of stearylamine. Stable photoprotective niosomes were obtained and were shown to be promising nanostructures to be used against solar radiation.

Nanoemulsions of sulfonamide carbonic anhydrase inhibitors strongly inhibit the growth of Trypanosoma cruzi.

Sulfonamide carbonic anhydrase (CA, EC 4.2.1.1) inhibitors targeting the α-class enzyme from the protozoan pathogen Trypanosoma cruzi, responsible of Chagas disease, were recently reported. Although many such derivatives showed low nanomolar activity in vitro, they were inefficient anti-T. cruzi agents in vivo. Here, we show that by formulating such sulfonamides as nanoemulsions in clove (Eugenia caryophyllus) oil, highly efficient anti-protozoan effects are observed against two different strains of T. cruzi. These effects are probably due to an enhanced permeation of the enzyme inhibitor through the nanoemulsion formulation, interfering in this way with the life cycle of the pathogen either by inhibiting pH regulation or carboxylating reactions in which bicarbonate/CO2 are involved. This type of formulation of sulfonamides with T. cruzi CA inhibitory effects may lead to novel therapeutic approaches against this orphan disease.

Antileishmanial activity of sulphonamide nanoemulsions targeting the ß-carbonic anhydrase from Leishmania species.

The ß-carbonic anhydrase (CA, EC 4.2.1.1) from Leishmania spp. (LdcCA) is effectively inhibited by aromatic/heterocyclic sulphonamides, in the low nanomolar range, but no in vitro antileishmanial activity was detected for such compounds. We formulated some of these sulphonamides as nanoemulsions (NEs) in clove oil, and tested them in vitro against Leishmania infantum MHOM/BR/1974/PP75 and Leishmania amazonensis IFLA/BR/1967/PH8 strains. Interesting inhibitory concentrations IC50 were observed for some of the sulphonamides NEs, with IC50 as low as 3.90 µM (NE-3F) and 2.24 µM (NE-5B) for L. amazonensis and 3.47 µM (NE-5B) for L. infantum. Some of the investigated NEs displayed toxicity for macrophages beyond the parasites. For the same nonoemulsions, a selective index (SI) greater than for Amphotericin B. Haemolytic assay using human red blood cells indicate that the NEs were less cytotoxic than amphotericin B, a widely used antifungal agent. NEs demonstrated to be an excellent strategy for increasing the penetration of these hydrophilic drugs through membranes, with a huge increase of efficacy over the sulphonamide CA inhibitor (CAI) alone.

Development and evaluation of zinc phthalocyanine nanoemulsions for use in photodynamic therapy for Leishmania spp.

Photodynamic therapy (PDT) combines light with photosensitizers (PS) for production of reactive oxygen species (ROS) that can kill infectious microorganisms such as bacteria, fungi and protozoa. The application of nanotechnology has enabled the advancement of PDT because many PS are insoluble in water, necessitating a nanocarrier as a physiologically acceptable carrier. Nanoemulsions are efficient nanocarriers for solubilizing liposoluble drugs, like the PS, in water. Cutaneous (CL) and mucocutaneous leishmaniasis (ML) are caused by different species of the genus Leishmania, transmitted to humans by sandfly bites. Parasites are hosted in skin macrophages producing ulcerative lesions. Thus, a topical treatment, effective and inexpensive, for CL and ML is preferable to systemic interventions. There are topical treatments like paromomycin and amphotericin B, but they have many local side effects or a very high cost, limiting their use. This work aimed to develop a zinc phthalocyanine (photosensitizer) oil-in-water nanoemulsion, essential clove oil and polymeric surfactant (Pluronic® F127) for the formulation of a topical delivery system for use in PDT against Leishmania amazonensis and Leishmania infantum. The nanoemulsion was produced by a high-energy method and characterized by size, polydispersity, morphology, pH, content and stability studies. The toxicity in the dark and the photobiological activity of the formulations were evaluated in vitro for Leishmania and macrophages. The formulation presented was pH compatible with topical use, approximately 30 nm in size, with a polydispersity index ≤0.1 and remained stable at room and refrigerator temperature during the stability study (60 days). The zinc phthalocyanine nanoemulsion is effective in PDT against Leishmania spp.; use against skin infections can be a future application of this topical formulation, avoiding the use of oral or injectable medications, decreasing systemic adverse effects.

Hydroxamic acid derivatives: a promising scaffold for rational compound optimization in Chagas disease.

This work describes the antitrypanocidal activity of two hydroxamic acid derivatives containing o-ethoxy (HAD1) and p-ethoxy (HAD2) as substituent in the aromatic ring linked to the isoxazoline ring. HAD1 and HAD2 induced a significant reduction in the number of intracellular parasites and consequently showed activity on the multiplication of the parasite. Treatment of cardiomyocytes and macrophages with the compounds revealed no significant loss in cell viability. Ultrastructural alterations after treatment of cardiomyocytes or macrophages infected by Trypanosoma cruzi with the IC50 value of HAD1 revealed alterations to amastigotes, showing initial damage seen as swelling of the kinetoplast. This gave a good indication of the ability of the drug to permeate through the host cell membrane as well as its selectivity to the parasite target. Both compounds HAD1 and 2 were able to reduce the cysteine peptidases and decrease the activity of metallopeptidases.

A new class of quinazoline-sulfonamides acting as efficient inhibitors against the α-carbonic anhydrase from Trypanosoma cruzi.

The protozoan parasite Trypanosoma cruzi is the agent responsible for trypanosomiasis (Chagas disease) in humans and other animals. It has been recently reported that this pathogen encodes for an α-class carbonic anhydrase (CA, EC 4.2.1.1), denominated TcCA, which was shown to be crucial for its life cycle. Inhibition studies of a class of 4-oxoquinazoline containing a benzensulfonamide moiety and their 4-thioxo bioisosteres against the protozoan enzyme TcCA are described here. Most of 4-oxoquinazoline sulfonamides showed nanomolar TcCA inhibition activity with K(I)s in the same order of magnitude of acetazolamide (AAZ), whereas their thioxo bioisosters showed moderate anti-Trypanosoma CA potency with K(I)s in the micromolar range. The discovery of compounds incorporating a 4-oxoquinazoline ring as a low-nanomolar TcCA inhibitor is quite promising and it may be useful for developing anti-Trypanosoma agents with a novel mechanism of action compared to the clinically used drugs (such as benznidazole, nifurtimox) for which significant resistance and serious adverse effects due to their high-toxicity appeared.

Marine extremophiles: a source of hydrolases for biotechnological applications.

The marine environment covers almost three quarters of the planet and is where evolution took its first steps. Extremophile microorganisms are found in several extreme marine environments, such as hydrothermal vents, hot springs, salty lakes and deep-sea floors. The ability of these microorganisms to support extremes of temperature, salinity and pressure demonstrates their great potential for biotechnological processes. Hydrolases including amylases, cellulases, peptidases and lipases from hyperthermophiles, psychrophiles, halophiles and piezophiles have been investigated for these reasons. Extremozymes are adapted to work in harsh physical-chemical conditions and their use in various industrial applications such as the biofuel, pharmaceutical, fine chemicals and food industries has increased. The understanding of the specific factors that confer the ability to withstand extreme habitats on such enzymes has become a priority for their biotechnological use. The most studied marine extremophiles are prokaryotes and in this review, we present the most studied archaea and bacteria extremophiles and their hydrolases, and discuss their use for industrial applications.

Inhibitory effect of linalool-rich essential oil from Lippia alba on the peptidase and keratinase activities of dermatophytes.

Abstract Lippia alba (Miller) N.E. Brown is an aromatic plant known locally as "Erva-cidreira-do-campo" that has great importance in Brazilian folk medicine. The aim of our study was to evaluate the antidermatophytic potential of linalool-rich essential oil (EO) from L. alba and analyze the ability of this EO to inhibit peptidase and keratinase activities, which are important virulence factors in dermatophytes. The minimum inhibitory concentrations (MICs) of L. alba EO were 39, 156 and 312 µg/mL against Trichophyton rubrum, Epidermophyton floccosum and Microsporum gypseum, respectively. To evaluate the influence of L. alba EO on the proteolytic and keratinolytic activities of these dermatophytes, specific inhibitory assays were performed. The results indicated that linalool-rich EO from L. alba inhibited the activity of proteases and keratinases secreted from dermatophytes, and this inhibition could be a possible mechanism of action against dermatophytes. Due to the effective antidermatophytic activity of L. alba EO, further experiments should be performed to explore the potential of this linalool-rich EO as an alternative antifungal therapy.

Feather keratin hydrolysates obtained from microbial keratinases: effect on hair fiber.

BACKGROUND: Hair is composed mainly of keratin protein and a small amount of lipid. Protein hydrolysates, in particular those with low molecular weight distribution have been known to protect hair against chemical and environmental damage. Many types of protein hydrolysates from plants and animals have been used in hair and personal care such as keratin hydrolysates obtained from nails, horns and wool. Most of these hydrolysates are obtained by chemical hydrolysis and hydrothermal methods, but recently hydrolyzed hair keratin, feather keratin peptides, and feather meal peptides have been obtained by enzymatic hydrolysis using Bacillus spp in submerged fermentation. RESULTS: Keratin peptides were obtained by enzymatic hydrolysis of keratinases using Bacillus subtilis AMR. The microorganism was grown on a feather medium, pH 8.0 (1% feathers) and supplemented with 0.01% of yeast extract, for 5 days, at 28°C with agitation. The supernatant containing the hydrolysates was colleted by centrifugation and ultra filtered in an AMICON system using nano-membranes (Millipore - YC05). The Proteins and peptides were analyzed using HPTLC and MALDI-TOF-MS. Commercial preparations of keratin hydrolysates were used as a comparative standard. After five days the feather had been degraded (90-95%) by the peptidases and keratinases of the microorganism. MALDI-TOF mass spectrometry showed multiple peaks that correspond to peptides in the range of 800 to 1079 Daltons and the commercial hydrolysate was in the range of 900 to 1400 Da. HPTLC showed lower molecular mass peptides and amino acids in the enzymatic hydrolysate when compared with the commercial hydrolysate . A mild shampoo and a rinse off conditioner were formulated with the enzymatic hydrolysate and applied to hair fibers to evaluate the hydration, with and without heat, using a Corneometer® CM 825. The hydration was more efficient with heat, suggesting a more complete incorporation of hydrolysates into the fibers. Scanning Electron Microscopy showed deposits of organic matter in the junction of the cuticles that probably collaborates to the sealing of the cuticles, increasing the brightness and softness. CONCLUSIONS: These results show that the enzymatic method to produce keratin peptides for hair care products is an attractive and eco- friendly method with a great potential in the cosmetic industry.

Anion inhibition studies of the α-carbonic anhydrase from the protozoan pathogen Trypanosoma cruzi, the causative agent of Chagas disease.

The protozoan pathogen Trypanosoma cruzi, the causative agent of Chagas disease, encodes an α-class carbonic anhydrase (CA, EC 4.2.1.1), TcCA, which was recently shown to be crucial for its life cycle. Thiols, a class of strong TcCA inhibitors, were also shown to block the growth of the pathogen in vitro. Here we report the inhibition of TcCA by inorganic and complex anions and other molecules interacting with zinc proteins, such as sulfamide, sulfamic acid, phenylboronic/arsonic acids. TcCA was inhibited in the low micromolar range by iodide, cyanate, thiocyanate, hydrogensulfide and trithiocarbonate (KIs in the range of 44-93 µM), but the best inhibitor was diethyldithiocarbamate (KI=5 µM). Sulfamide showed an inhibition constant of 120 µM, but sulfamic acid was much less effective (KI of 10.6 mM). The discovery of diethyldithiocarbamate as a low micromolar TcCA inhibitor may be useful to detect leads for developing anti-Trypanosoma agents with a diverse mechanism of action compared to clinically used drugs (benznidazole, nifurtimox) for which significant resistance emerged.

The Potential of Allelochemicals from Microalgae for Biopesticides.

Improvements in agricultural productivity are required to meet the demand of a growing world population. Phytopathogens, weeds, and insects are challenges to agricultural production. The toxicity and widespread application of persistent synthetic pesticides poses a major threat to human and ecosystem health. Therefore, sustainable strategies to control pests are essential for agricultural systems to enhance productivity within a green paradigm. Allelochemicals are a less persistent, safer, and friendly alternative to efficient pest management, as they tend to be less toxic to non-target organisms and more easily degradable. Microalgae produce a great variety of allelopathic substances whose biocontrol potential against weeds, insects, and phytopathogenic fungi and bacteria has received much attention. This review provides up-to-date information and a critical perspective on allelochemicals from microalgae and their potential as biopesticides.

Therapeutic Potential of Bioactive Compounds from Brugmansia suaveolens Bercht. & J. Presl.

Brugmansia suaveolens Bercht. & J. Presl has been widely used due to the presence of different bioactive compounds. This review summarizes the latest advances and perspectives of the B. suaveolens plant species; it is a systematic literature review on aspects of botany, traditional uses, phytochemistry, pharmacology, and toxicology as therapeutic potential. In addition, 120 compounds are described, including alkaloids, flavonoids, terpenoids, steroids, amino acids, aromatics, and aliphatics. As for the therapeutic potential, it is described in extracts and compounds in the antitumor, anti-inflammatory, antioxidant, antimicrobial, antispasmodic, anticoagulant, and analgesic aspects, as well as the effects on the central nervous system. The toxicity of the genus stands out, especially the potential for organ toxicity. Therefore, this review evidenced the knowledge related to the traditional use based on the scientific research of Brugmansia suaveolens, highlighting an overview of bioactive compounds and biological and toxicological activities in order to provide a scientific basis for future studies on the value of this species for the development of new natural products.

Microbial Pigments: Major Groups and Industrial Applications.

Microbial pigments have many structures and functions with excellent characteristics, such as being biodegradable, non-toxic, and ecologically friendly, constituting an important source of pigments. Industrial production presents a bottleneck in production cost that restricts large-scale commercialization. However, microbial pigments are progressively gaining popularity because of their health advantages. The development of metabolic engineering and cost reduction of the bioprocess using industry by-products opened possibilities for cost and quality improvements in all production phases. We are thus addressing several points related to microbial pigments, including the major classes and structures found, the advantages of use, the biotechnological applications in different industrial sectors, their characteristics, and their impacts on the environment and society.

RdJ detection tests to identify a unique MRSA clone of ST105-SCCmecII lineage and its variants disseminated in the metropolitan region of Rio de Janeiro.

Hospital bloodstream infection (BSI) caused by methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of morbidity and mortality and is frequently related to invasive procedures and medically complex patients. An important feature of MRSA is the clonal structure of its population. Specific MRSA clones may differ in their pathogenic, epidemiological, and antimicrobial resistance profiles. Whole-genome sequencing is currently the most robust and discriminatory technique for tracking hypervirulent/well-adapted MRSA clones. However, it remains an expensive and time-consuming technique that requires specialized personnel. In this work, we describe a pangenome protocol, based on binary matrix (1,0) of open reading frames (ORFs), that can be used to quickly find diagnostic, apomorphic sequence mutations that can serve as biomarkers. We use this technique to create a diagnostic screen for MRSA isolates circulating in the Rio de Janeiro metropolitan area, the RdJ clone, which is prevalent in BSI. The method described here has 100% specificity and sensitivity, eliminating the need to use genomic sequencing for clonal identification. The protocol used is relatively simple and all the steps, formulas and commands used are described in this work, such that this strategy can also be used to identify other MRSA clones and even clones from other bacterial species.

Keratinases and sulfide from Bacillus subtilis SLC to recycle feather waste.

The aim of this study is to investigate the culture conditions of chicken feather degradation and keratinolytic enzyme production by the recently isolated Bacillus subtilis SLC and to evaluate the potential of the SLC strain to recycle feather waste discarded by the poultry industry. The SLC strain was isolated from the agroindustrial waste of a poultry farm in Brazil and was confirmed to belong to Bacillus subtilis by rDNA gene analysis. There was high keratinase production when the medium was at pH 8 (280 U ml(-1)). Activity was higher using the inoculum propagated for 72 h on 1% whole feathers supplemented with 0.1% yeast extract. In the enzymatic extract, the keratinases were active in the pH range from 2.0 to 12.0 with a maximum activity at pH 10.0 and temperature 60°C. For gelatinase the best pH was 5.0 and the best temperature was 37°C. All keratinases are serine peptidases. The crude enzymatic extract degraded keratin, gelatin, casein, and hemoglobin. Scanning electron microscopy showed Bacillus cells adhered onto feather surfaces after 98 h of culture and degraded feather filaments were observed. MALDI-TOF mass spectrometric analysis showed multiple peaks from 522 to 892 m/z indicating feather degradation. The presence of sulfide was detected on extracellular medium probably participating in the breakdown of sulfide bridges of the feather keratin. External addition of sulfide increased feather degradation.

Metabology: Analysis of metabolomics data using community ecology tools.

Several areas such as microbiology, botany, and medicine use genetic information and computational tools to organize, classify and analyze data. However, only recently has it been possible to obtain the chemical ontology of metabolites computationally. The systematic classification of metabolites into classes opens the way for adapting methods that previously used genetic taxonomy to now accept chemical ontology. Community ecology tools are ideal for this adaptation as they have mature methods and enable exploratory data analysis with established statistical tools. This study introduces the Metabology approach, which transforms metabolites into an ecosystem where the metabolites (species) are related by chemical ontology. In the present work, we demonstrate the applicability of this new approach using publicly available data from a metabolomics study of human plasma that searched for prognostic markers of COVID-19, and in an untargeted metabolomics study carried out by our laboratory using Lasiodiplodia theobromae fungal pathogen supernatants.

Development of Microalgae Biodiesel: Current Status and Perspectives.

Microalgae are regarded as a promising source of biodiesel. In contrast with conventional crops currently used to produce commercial biodiesel, microalgae can be cultivated on non-arable land, besides having a higher growth rate and productivity. However, microalgal biodiesel is not yet regarded as economically competitive, compared to fossil fuels and crop-based biodiesel; therefore, it is not commercially produced. This review provides an overall perspective on technologies with the potential to increase efficiency and reduce the general costs of biodiesel production from microalgae. Opportunities and challenges for large-scale production are discussed. We present the current scenario of Brazilian research in the field and show a successful case in the research and development of microalgal biodiesel in open ponds by Petrobras. This publicly held Brazilian corporation has been investing in research in this sector for over a decade.