Current status of whitening agents and enzymes in Dentistry

Abstract This study reviews the knowledge on the use of conventional dental whitening and the use of enzymes as a new approach in bleaching. A review of the literature was based on academic articles and on patents related to the use of enzymes in dental bleaching. Tooth whitening techniques used nowadays are well reported in the literature, and its mechanism of action consists of an oxidoreduction reaction with the release of free radicals. The great instability of radicals, when in contact with the tissues, promotes oxidation and reduction in the size of the pigment chains incorporated into them. These pigments are eventually broken down into smaller and smaller molecular chains and end up being diffused from the dental structure. In turn, the use of enzymes aimed at tooth whitening can be a less harmful alternative to the tooth because their specificity regarding the substrate makes them of great interest to perform specific reactions, reducing collateral effects. The use of proteolytic enzymes and oxidoreductases paired with the application of peroxides, can be a promising alternative for obtaining even better results in the dental bleaching process.

Reusable Enzymatic Strip for Detection of Arsenic

HIGHLIGHTS Arsenic is considered as one of the highly hazardous elements in the environment and a serious carcinogen for the human health. An enzymatic method has been described by using arsenite oxidase for arsenic detection. Residual activity of the immobilized enzyme was 43% of the initial activity after being recycled 10 times.

Abstract Arsenic is considered as one of the highly hazardous elements in the environment and a serious carcinogen for the human health. More attention has taken towards the arsenic due to its presence in ground water in India, China, Bangladesh, Inner Mongolia and several other regions of the world. It's been a challenge to remove arsenic due to the lack of its efficient detection approach in the complicated environmental matrix. The proposed method describes an enzymatic method for arsenic determination using arsenite oxidase, which catalyzes the oxidation of arsenite to arsenate. Hence, a colorimetric PVC strip with immobilized arsenite oxidase has been developed to detect the arsenic concentration and also having potential for the field-testing. The influence of the optimal conditions i.e. pH, temperature, storage stability, and reusability of free and immobilized enzyme were evaluated and compared. The results have shown that the stabilities were significantly enhanced compared with free counterpart. Residual activity of the immobilized enzyme was 43% of the initial activity after being recycled 10 times. We approve that this novel low cost immobilized carrier presents a new approach in large scale applications and expected to act as a model for establishment of indigenous arsenic sensor in miniature form.

Exploring the five-paced viper (Deinagkistrodon acutus) venom proteome by integrating a combinatorial peptide ligand library approach with shotgun LC-MS/MS

Snake venoms are complex mixtures of toxic proteins or peptides encoded by various gene families that function synergistically to incapacitate prey. In the present study, in order to unravel the proteomic repertoire of Deinagkistrodon acutus venom, some trace abundance components were analyzed. Methods Shotgun proteomic approach combined with shotgun nano-LC-ESI-MS/MS were employed to characterize the medically important D. acutus venom, after collected samples were enriched with the combinatorial peptide ligand library (CPLL). Results This avenue helped us find some trace components, undetected before, in D. acutus venom. The results indicated that D. acutus venom comprised 84 distinct proteins from 10 toxin families and 12 other proteins. These results are more than twice the number of venom components obtained from previous studies, which were only 29 distinct proteins obtained through RP-HPLC for the venom of the same species. The present results indicated that in D. acutus venom, the most abundant components (66.9%) included metalloproteinases, serine proteinases, and C-type lectin proteins; the medium abundant components (13%) comprised phospholipases A2 (PLA2) and 5'-nucleotidases and nucleases; whereas least abundant components (6%) were aminopeptidases, L-amino acid oxidases (LAAO), neurotoxins and disintegrins; and the trace components. The last were undetected before the use of conventional shotgun proteomics combined with shotgun nano-LC-ESI-MS/MS, such as cysteine-rich secretory proteins Da-CRPa, phospholipases B-like 1, phospholipases B (PLB), nerve growth factors (NGF), glutaminyl-peptide cyclortransferases (QC), and vascular non-inflammatory molecules 2 (VNN2). Conclusion These findings demonstrated that the CPLL enrichment method worked well in finding the trace toxin proteins in D. acutus venom, in contrast with the previous venomic characterization of D. acutus by conventional LC-MS/MS. In conclusion, this approach combined with the CPLL enrichment was effective for allowing us to explore the hidden D. acutus venomic profile and extended the list of potential venom toxins.(AU)

P-I metalloproteinases and L-amino acid oxidases from Bothrops species inhibit angiogenesis

Snake venoms are composed of pharmacologically active proteins that are evolutionarily diverse, stable and specific to targets. Hence, venoms have been explored as a source of bioactive molecules in treating numerous diseases. Recent evidences suggest that snake venom proteins may affect the formation of new blood vessels. Excessive angiogenesis has been implicated in several pathologies including tumours, diabetic retinopathy, arthritis, inter alia. In the present study, we have examined the effects of P-I metalloproteinases isolated from Bothrops moojeni (BmMP-1) and Bothrops atrox (BaMP-1) and L-amino acid oxidases (LAAO) isolated from B. moojeni (BmLAAO) and B. atrox (BaLAAO) on biochemical and functional aspects of angiogenesis. Methods: P-I metalloproteinases and LAAO were purified from venom by molecular size exclusion and ion-exchange chromatography and subsequently confirmed using mass spectrometry. The P-I metalloproteinases were characterized by azocaseinolytic, fibrinogenolytic and gelatinase activity and LAAO activity was assessed by enzyme activity on L-amino acids. Influence of these proteins on apoptosis and cell cycle in endothelial cells was analysed by flow cytometry. The angiogenic activity was determined by in vitro 3D spheroid assay, Matrigel tube forming assay, and in vivo agarose plug transformation in mice. Results: P-I metalloproteinases exhibited azocaseinolytic activity, cleaved α and partially β chain of fibrinogen, and displayed catalytic activity on gelatin. LAAO showed differential activity on L-amino acids. Flow cytometry analysis indicated that both P-I metalloproteinases and LAAO arrested the cells in G0/G1 phase and further induced both necrosis and apoptosis in endothelial cells. In vitro, P-I metalloproteinases and LAAO exhibited significant anti-angiogenic properties in 3D spheroid and Matrigel models by reducing sprout outgrowth and tube formation. Using agarose plug transplants in mice harbouring P-I metalloproteinases and LAAO we demonstrated a marked disruption of vasculature at the periphery. Conclusion: Our research suggests that P-I metalloproteinases and LAAO exhibit anti-angiogenic properties in vitro and in vivo.(AU)

Cardioprotective effect of eicosapentaenoic acid during ischemia and reperfusion / Efeito cardioprotetor do ácido eicosapentaenóico durante a isquemia e reperfusão

Objective: In this work, rats isolated hearts were infused EPA before the ischemia period and during reperfusion for available get well in parameter relatives to redox reactions. Methods: The effect of EPA was tested on isolated hearts induced to ischemia and reperfusion, treatment occurred at different times (ischemia or reperfusion). Antioxidant capacity against peroxyl radicals, glutathione cysteine ligase activity, glutathione concentration, lactate dehydrogenase, and creatine kinase concentration was analyzed. Results: Hearts treated with eicosapentaenoic acid had the minor generation of species reactive oxygen and lipid damage after reperfusion. The GSH concentration was higher when the hearts were treated with eicosapentaenoic acid in the period of reperfusion. Conclusion: In conclusion, this study demonstrates that the dose of EPA (20µM) used before ischemia can act as a cardioprotective antioxidant molecule, prevented damage heart from ischemic and reperfusion injury

Objetivo: Neste trabalho, corações isolados de ratos foram infundidos com EPA antes do período de isquemia e durante a reperfusão para obtenção de melhora em parâmetros relativos às reações redox. Métodos: O efeito do EPA foi testado em corações isolados induzidos a isquemia e reperfusão, o tratamento ocorreu em diferentes momentos (isquemia ou reperfusão). A capacidade antioxidante contra os radicais peroxil, atividade da glutationa cisteína ligase, concentração de glutationa, lactato desidrogenase e concentração de creatina quinase foi analisada. Resultados: Corações tratados com ácido eicosapentaenóico tiveram a menor geração de espécies reativas de oxigênio e danos lipídicos após a reperfusão. A concentração de GSH foi maior quando os corações foram tratados com ácido eicosapentaenóico no período de reperfusão. Conclusão: Em conclusão, este estudo demonstra que a dose de EPA (20µM) utilizada antes da isquemia pode atuar como uma molécula antioxidante cardioprotetora, prevenindo danos ao coração por isquemia e lesão de reperfusão.

Venomics and antivenomics of the poorly studied Brazil's lancehead, Bothrops brazili (Hoge, 1954), from the Brazilian State of Pará

The Brazil's lancehead, Bothrops brazili, is a poorly studied pit viper distributed in lowlands of the equatorial rainforests of southern Colombia, northeastern Peru, eastern Ecuador, southern and southeastern Venezuela, Guyana, Suriname, French Guiana, Brazil, and northern Bolivia. Few studies have been reported on toxins isolated from venom of Ecuadorian and Brazilian B. brazili. The aim of the present study was to elucidate the qualitative and quantitative protein composition of B. brazili venom from Pará (Brazil), and to carry out a comparative antivenomics assessment of the immunoreactivity of the Brazilian antibothropic pentavalent antivenom [soro antibotrópico (SAB) in Portuguese] against the venoms of B. brazili and reference species, B. jararaca. Methods: We have applied a quantitative snake venomics approach, including reverse-phase and two-dimensional electrophoretic decomplexation of the venom toxin arsenal, LC-ESI-MS mass profiling and peptide-centric MS/MS proteomic analysis, to unveil the overall protein composition of B. brazili venom from Pará (Brazil). Using third-generation antivenomics, the specific and paraspecific immunoreactivity of the Brazilian SAB against homologous (B. jararaca) and heterologous (B. brazili) venoms was investigated. Results: The venom proteome of the Brazil's lancehead (Pará) is predominantly composed of two major and three minor acidic (19%) and two major and five minor basic (14%) phospholipase A2 molecules; 7-11 snake venom metalloproteinases of classes PI (21%) and PIII (6%); 10-12 serine proteinases (14%), and 1-2 L-amino acid oxidases (6%). Other toxins, including two cysteine-rich secretory proteins, one C-type lectin-like molecule, one nerve growth factor, one 5'-nucleotidase, one phosphodiesterase, one phospholipase B, and one glutaminyl cyclase molecule, represent together less than 2.7% of the venom proteome. Third generation antivenomics profile of the Brazilian pentabothropic antivenom showed paraspecific immunoreactivity against all the toxin classes of B. brazili venom, with maximal binding capacity of 132.2 mg venom/g antivenom. This figure indicates that 19% of antivenom's F(ab')2 antibodies bind B. brazili venom toxins. Conclusion: The proteomics outcome contribute to a deeper insight into the spectrum of toxins present in the venom of the Brazil's lancehead, and rationalize the pathophysiology underlying this snake bite envenomings. The comparative qualitative and quantitative immunorecognition profile of the Brazilian pentabothropic antivenom toward the venom toxins of B. brazili and B. jararaca (the reference venom for assessing the bothropic antivenom's potency in Brazil), provides clues about the proper use of the Brazilian antibothropic polyvalent antivenom in the treatment of bites by the Brazil's lancehead.(AU)

Germination temperatures affect the physiological quality of seeds of lettuce cultivars / Qualidade fisiológica de sementes de alface sob diferentes temperaturas na germinação

The objective of this study was to evaluate the physiological quality allied to biochemical quality of lettuce seeds by germination and enzymes expression at 20, 25, 30, 35, 40 and 42ºC. Germination speed index and percentage of germination were estimated. Isoenzyme expressions were assessed by alcohol dehydrogenase (ADH), malate dehydrogenase (MDH), catalase (CAT), esterase (EST), pyruvate decarboxylase (PDC) and glutamate oxaloacetate transferase (GOT). The experiment consisted of a completely randomized design in a factorial scheme 4x6, with four cultivars and six different temperatures, with four replications. The highest germination and vigor were observed for cv. 'Everglades' at 35°C, which proved that this cultivar is thermotolerant. Catalase can be considered a genetic marker for the identification ofthermotolerant lettuce cultivars. Cultivar 'Everglades' has potential to be used in lettuce breeding programs aimed at cultivars tolerant to high temperatures during germination.

O objetivo deste estudo foi avaliar a qualidade fisiológica e bioquímica de sementes de alface por meio da germinação e expressão de enzimas a 20, 25, 30, 35, 40 e 42ºC. As variáveis velocidade de germinação e o índice de velocidade de germinação foram estimadas. As expressões das enzimas alcool desidrogenase (ADH), malato desidrogenase (MDH), catalase (CAT), esterase (EST), piruvate descarboxilase (PDC) e glutamato oxaloacetato transferase (GOT) foram avaliadas. Para análise dos genótipos foi empregado o delineamento inteiramente casualizado em esquema fatorial 4x6, testando quatro cultivares e seis diferentes temperaturas, com quatro repetições. A maior germinação e vigor foram observadas para a cv. 'Everglades' a 35°C, o que prova que esta cultivar é termotolerante. A catalase pode ser considerada um marcador para a identificação de cultivares de alface termotolerantes. A cultivar 'Everglades' tem potential para uso em programas de melhoramento visando tolerância à alta temperatura durante a germinação.

Efecto de la procedencia y el almacenamiento en la calidad del café / Effect of provenance and storage on coffee quality

Resumen Antecedentes. La calidad del grano de café ha sido relacionada con su procedencia, su manejo agronómico y sus condiciones de almacenamiento. Objetivo. Determinar la actividad de la polifenil oxidasa, el contenido de lípidos, el color y las características organolépticas de cafés provenientes de 3 subestaciones experimentales. Materiales y métodos. Se siguió un diseño completamente aleatorio en arreglo factorial factorial 3x6 (lugares de procedencia del café y tiempo de almacenamiento respectivamente). Resultados. La actividad de la polifenil oxidasa es mayor en el café fresco-para las tres procedencias. El café procedente de Naranjal presentó actividades enzimáticas más altas que los cafés provenientes de las subestaciones Supía y la Catalina. El análisis de varianza mostró el efecto de la procedencia sobre la variable actividad enzimática. La actividad de la polifenil oxidasa en los cafés estudiados decrece con el tiempo de almacenamiento. El contenido de lípidos es menor a menor en la subestación de la Catalina. Todos los cafés fueron caracterizados de buena calidad en el tiempo cero de almacenamiento. Las características de aroma, intensidad del aroma y cuerpo presentaron altibajos en los diferentes meses de almacenamiento. El café de Naranjal, obtuvo en promedio una calificación aceptable a lo largo de los seis meses de almacenamiento. Conclusiones. Se encontraron diferencias significativas para las variables estudiadas por efecto de la procedencia y el almacenamiento. La actividad enzimática de la PFO presentó etapas de activación/inhibición, durante los seis meses de almacenamiento.

Abstract Background. The quality of the coffee bean has been related to its origin, the agronomic management and the storage conditions. Objective. To determine the activity of the polyphenyl oxidase, the lipid content, the color and the organoleptic characteristics of coffees from 3 experimental substations. Materials and methods. A completely randomized design was followed in a 3x6 factorial arrangement (places of coffee origin and storage time respectively). Results. The activity of polyphenyl oxidase is greater in fresh coffee-for the three provenances. The coffee from Naranjal presented higher enzymatic activities than the coffees from the Supía and the Catalina substations. The analysis of variance showed the effect of provenance on the enzyme activity variable. The activity of the polyphenyl oxidase in the coffees studied decreases with storage time. The lipid content is lower at a lower height in the Catalina. All coffees were characterized as good quality at zero storage time; but the characteristics of aroma, intensity of aroma and body presented ups and downs in the different months of storage. Naranjal coffee, on average, obtained an acceptable rating throughout the six months of storage. Conclusions. Significant differences were found for the variables studied due to the effect of provenance and storage. The enzymatic activity of the PFO showed activation / inhibition stages, during the six months of storage.

Phenotypic Characterization and Synthesis of Extracellular Catecholase from a Newly Isolated Bacterium Pseudomonas sp. BSC-6

Abstract Catecholase (EC 1.10.3.1), an oxidoreductase enzyme is a key member of polyphenol oxidase family which catalyze the degradation of catechol. This enzyme possesses vast applications in diverse areas and is found in bacteria, fungi, mushrooms, higher plants, arthropods, amphibians and mammals. Catechol, a phenolic compound, is used as a starting material in the synthesis of various industrial compounds such as inhibitors, antioxidants, pesticides etc. The release of this phenolic compound in the environment causes toxicity to both flora and fauna. In the present studies, emphasis has been laid on isolation, screening and characterization of catechol degrading bacterium coupled with synthesis of catecholase enzyme. Further, the selected isolated strain was phenotypically characterized and was found to be member of genus Pseudomonas. Among all the isolates, BSC-6 was found as best isolate with maximum extracellular catecholase activity of 152.32 IU/L obtained after scale up studies. The herein synthesized bacterial catecholase may be employed for wide applications particularly in bioremediation of phenol enriched polluted sites.

Rectal application of argan oil improves healing of colorectal anastomosis in rats

Abstract Purpose: To investigate the possible effects of argan oil on the healing of colorectal anastomoses. Methods: I n Group 1 (sham), laparotomy was performed and the colon was mobilized. In the control (Group 2) and argan oil (Group 3) groups, colonic resection and anastomosis were applied. To the control and sham groups, 2 mL of 0.9% NaCl was administred rectally, and in the argan oil group, 2 mL/day argan oil was applied rectally for 7 days. Results: The mean bursting pressures of the argan oil and sham groups were significantly higher than the values in the control group. A significant difference was determined between the tissue hydroxyproline and prolidase levels of control group and other groups. Histopathologically, argan oil showed significant beneficial effects on colonic wound healing. In the argan oil and sham groups, the tissue malondialdehyde and fluorescent oxidation product levels were found to be lower and total sulfhydryl levels were higher than the control group. Conclusions: The rectally administered argan oil was observed to have significantly ameliorated wound healing parameters and exerted a significant antioxidant effect. This is the first study in the literature about the beneficial effects of argan oil on colorectal anastomoses.

Genetic engineering of Theileria parva lactate dehydrogenase gene: a new anti-theilerial target

Theileria parva is the causative agent of East Coast Fever (ECF), a tick borne disease, which results in major economic losses in cattle. Major problems in dealing with this illness are the high cost of drugs, development of resistance, and absence of effective vaccines. Thus, exploiting new targets for cost effective and higher therapeutic value drugs are imperative. Glycolysis is the main pathway for generation of ATP in T. parva, given its development inside erythrocytes. Thus, the enzymes of this pathway may prove potential targets for designing new-generation anti-theilerials. Lactate dehydrogenase of T. parva (TpLDH) has the highest activity of all glycolytic enzymes and thus we selected this enzyme as the potential therapeutic target. Our study is the first to report the isolation, removal of introns through directed mutagenesis, and cloning of TpLDH and showing that amino acid insertions or deletions most notably corresponded to a 5-amino acid sequence (Asn-91A, Glu-91B, Glu-91C, Trp-91D, Asn-91E) between Ser-91 ve Arg-92 of the enzyme. This region is also present in other apicomplexan such as Babesia bovis, a pathogen of cattle and Plasmodium falciparum, a human pathogen. Providing as the attachment site for the enzyme inhibitors and not being present in LDH of respective hosts, we propose this site as an attractive drug target. The work here is expected to lead new studies on detailed structural and kinetic aspects of apicomplexan LDHs and development of new inhibitors.(AU)

Kinetic investigations and stability studies of two Bothrops L-amino acid oxidases

L-amino acid oxidases isolated from snake venoms (SV-LAAOs) are enzymes that have great therapeutic potential and are currently being investigated as tools for developing new strategies to treat various diseases, including cancer and bacterial infections. The main objective of this study was to make a brief evaluation of the enzymatic stability of two Bothrops LAAOs, one isolated from Bothrops jararacussu (BjussuLAAO-II) and the other from Bothrops moojeni (BmooLAAO-I) venoms. Methods and results: The enzymatic activity and stability of both LAAOs were evaluated by microplate colorimetric assays, for which BjussuLAAO-II and BmooLAAO-I were incubated with different L-amino acid substrates, in the presence of different ions, and at different pH ranges and temperatures. BjussuLAAO-II and BmooLAAO-I demonstrated higher affinity for hydrophobic amino acids, such as Phe and Leu. The two enzymes showed high enzymatic activity in a wide temperature range, from 25 to 75 °C, and presented optimum pH around 7.0. Additionally, Zn2+, Al3+, Cu2+ and Ni2+ ions negatively modulated the enzymatic activity of both LAAOs. As to stability, BjussuLAAO-II and BmooLAAO-I showed high enzymatic activity for 42 days stored at 4°C in neutral pH solution. Moreover, the glycan portions of both LAAOs were analyzed by capillary electrophoresis, which revealed that BjussuLAAO-II presented two main glycan portions with relative masses of 7.78 and 8.13 CGU, while BmooLAAO-I showed three portions of 7.58, 7.94 and 8.37 CGU. Conclusions: Our results showed that, when stored properly, BjussuLAAO-II and BmooLAAO-I present enzymatic stability over a long time period, which is very important to allow the use of these enzymes in pharmacological studies of great impact in the medical field.(AU)

Evaluation of the commercial kit SIRE nitratase for detecting resistant mycobacterium tuberculosis in Brazil

Abstract INTRODUCTION: This study aimed to evaluate a new commercial kit, Kit SIRE Nitratase-PlastLabor, for testing the drug susceptibility of clinical Mycobacterium tuberculosis isolates. METHODS: The accuracy of the Kit SIRE Nitratase was evaluated by examining the susceptibility (streptomycin, isoniazid, rifampicin, and ethambutol) of 40 M. tuberculosis isolates, using the proportion method with Lowenstein-Jensen medium or the BACTEC MGIT 960 system. RESULTS: The detection accuracy for streptomycin, isoniazid, rifampicin, and ethambutol was 95%, 97.5%, 100%, and 80%, respectively. CONCLUSIONS: The exceptional accuracy demonstrated by Kit SIRE Nitratase for isoniazid and rifampicin makes the kit an attractive option for screening M. tuberculosis strain resistance.

Over-expression of dehydroascorbate reductase enhances oxidative stress tolerance in tobacco

Background: Ascorbic acid (Asc) is one of the most abundant antioxidants and it serves as a major contributor to protect plants against oxidative damage. Plants use two enzymes that participate in the metabolic recycling of Asc. One of these two enzymes is dehydroascorbate reductase (DHAR). It directly regenerates Asc from its oxidized state and thus prevents Asc from being irreversibly hydrolyzed to 2, 3-diketogulonic acid. This study aimed to examine whether over-expression of DHAR leads to an enhanced oxidative stress tolerance in tobacco plants. Results: In this study, we functionally characterized a novel JcDHAR gene from Jatropha curcas and found via quantitative RT-PCR analysis that JcDHAR can be induced with H2O2, salt and PEG stresses. The DHAR activities of transgenic tobacco plants increased from 2.0 to 5.3 fold compared to wild-type plants. As a result, the transgenic plants displayed enhanced tolerance to oxidative stress. Conclusions: Our results indicate that JcDHAR expression can effectively enhance the tolerance to oxidative stress in plants.

Taxonomic status of two morphotypes of Coryphaena hippurus (Perciformes: Coryphaenidae)

Two Coryphaena hippurus morphotypes (dourado and palombeta) are found along the Brazilian coast and are considered by Rio de Janeiro’s fisherman and fishmongers as two different species. Furthermore, these morphotypes are commercialized under different values and suffer different fishing pressure. Therefore, a definition of their taxonomic status is an important economic and biological matter. In order to investigate this problem, allozyme electrophoresis method was undertaken for seventeen loci on 117 individuals of C. hippurus sampled at Cabo Frio/RJ (Brazil). The data indicate homogeneity between the morphotypes gene pools. Nevertheless, differences were found for genetic variation among dourado and palombeta, especially due to alcohol dehydrogenase locus. Natural selection hypothesis is discussed in explaining these findings.(AU)

Dois morfotipos de Coryphaena hippurus (dourado e palombeta) encontrados ao longo da costa brasileira são considerados espécies diferentes por pescadores e mercadores das regiões de desembarque do estado do Rio de Janeiro. Além disso, esses morfotipos são comercializados por valores diferentes e sofrem diferentes pressões de pesca. Desta forma, a definição do status taxonômico desses morfotipos é importante, tanto em termos econômicos quanto biológicos. A fim de investigar esse problema foi utilizado o método de eletroforese de aloenzimas com a amostragem de dezessete loci para 117 indivíduos dos dois morfotipos de C. hippurus obtidos em desembarques pesqueiros na região de Cabo Frio/RJ (Brasil). Os dados indicaram uma homogeneidade entre os conjuntos gênicos dos morfotipos. A despeito disso, diferenças entre os conjuntos gênicos de dourado e palombeta foram encontradas, devido, especialmente, ao locus álcool desidrogenase. A hipótese de seleção natural é discutida como possível explicação para esses resultados.(AU)

Over-expression of Mycobacterium neoaurum 3-ketosteroid-∆ 1-dehydrogenase in Corynebacterium crenatum for efficient bioconversion of 4-androstene-3, 17-dione to androst-1, 4-diene-3, 17-dione

Background: 3-Ketosteroid-∆¹-dehydrogenase (KSDD), a flavoprotein enzyme, catalyzes the bioconversion of 4-androstene-3,17-dione (AD) to androst-1,4-diene-3,17-dione (ADD). To date, there has been no report about characterization of KSDD from Mycobacterium neoaurum strains, which were usually employed to produce AD or ADD by fermentation. Results: In this work, Corynebacterium crenatum was chosen asa new host for heterologous expression of KSDD from M. neoaurum JC-12 after codon optimization of the KSDD gene. SDS-PAGE and western blotting results indicated that the recombinant C. crenatum harboring the optimized ksdd (ksdd n) gene showed significantly improved ability to express KSDD. The expression level of KSDD was about 1.6-fold increased C. crenatum after codon optimization. After purification of the protein, we first characterized KSDD from M. neoaurum JC-12, and the results showed that the optimum temperature and pH for KSDD activity were 30°C and pH 7.0, respectively. The Km and Vmax values of purified KSDD were 8.91 µM and 6.43 mM/min. In this work, C. crenatum as a novel whole-cell catalyst was also employed and validated for bioconversion of AD to ADD. The highest transformation rate of AD to ADD by recombinant C. crenatum was about 83.87% after 10 h reaction time, which was more efficient than M. neoaurum JC-12 (only 3.56% at 10 h). Conclusions: In this work, basing on the codon optimization, overexpression, purification and characterization of KSDD, we constructed a novel system, the recombinant C. crenatum SYPA 5-5 expressing KSDD, to accumulate ADDfromADefficiently. This work provided new insights into strengthening sterol catabolism by overexpressing the key enzyme KSDD, for efficient ADD production.

Planejamento de fármacos anti-T. cruzi: Síntese de compostos nitrofurânicos, avaliação da atividade biológica in vitro e estudos de relações estrutura-atividade / Design of anti-T. cruzi compounds: Synthesis of 5-nitrofuran derivatives, in vitro biological activity and study of structure-activity relationships

A doença de Chagas afeta cerca de 6 a 7 millhões de pessoas no mundo, principalmente América Latina. A busca de alternativas terapêuticas para esta enfermidade tem grande relevância para a sociedade, já que as opções atuais são limitadas, sendo disponível apenas o benznidazol (BZD) e nifurtimox. Os derivados nitroheterocíclicos são considerados compostos bioativos com número crescente de estudos na comunidade científica contra seu agente etiológico, o Trypanosoma cruzi. Neste sentido, o presente trabalho tem por objetivo a identificação de derivados do 5-nitrofurano com atividade frente a diferentes cepas do T. cruzi, assim como estudar possíveis modo de ação desta classe de compostos. Esta investigação envolve estudos computacionais com o propósito de construir modelos quantitativos de relações estrutura-atividade (QSAR multivariado) que possam auxiliar na previsão de novas estruturas com perfil farmacológico otimizado. No presente trabalho foram realizadas as etapas de planejamento, síntese e identificação de 36 compostos com resultados satisfatórios quanto à identificação estrutural, pureza e rendimento, que foi da ordem de 70%. A determinação da atividade anti-T. cruzi in vitro dos compostos obtidos foi realizada frente às cepas Silvio X10 cl1, Y, Bug 2149 cl10 e Colombiana na forma epimastigota do parasito. A maioria dos compostos analisados apresentou maior capacidade de inibição de crescimento do parasito, comparado ao BZD: Silvio X10 cl1 - IC50 = 29,16 ±2,90 µM, Y - IC50 = 40,40 ±3,37µM, Bug 2149 cl10 - IC50 = 30,63 ±3,21 µM, Colombiana - IC50 = 47,91 ±4,96 µM. O composto mais ativo (BSF-35) apresentou os seguintes valores: Silvio X10 cl1 - IC50 = 3,17 ±0,32 µM, Y - IC50 = 1,17 ±0,12 µM, Bug 2149 cl10 - IC50 = 1,81 ±0,18 µM e Colombiana - IC50 = 3,06 ±0,23 µM. Foram realizados cálculos de propriedades moleculares das estruturas tridimensionais dos compostos, seguido pela análise exploratória de dados por análise de agrupamentos hierárquicos (HCA) e análise de componentes principais (PCA), possibilitando o reconhecimento de padrões do conjunto. Considerando esta análise prévia, foram obtidos modelos QSAR com abordagem multivariada, aplicando algorítmo OPS e método de regressão por quadrados mínimos parciais, PLS. Os melhores modelos gerados foram obtidos considerando os compostos benzenos substituídos para as quatro cepas estudadas. Os descritores que mais influenciaram na análise foram o ClogP (coeficiente de partição) e cargas CHELPG. Considerando as informações obtidas, foram planejados e sintetizados quatro novos compostos com objetivo de obter compostos mais ativos e validar os modelos QSAR. Estes compostos apresentaram alta atividade frente a forma epimastigota das quatro cepas estudadas. Os compostos mais ativos foram avaliados quanto a citotoxicidade frente células LLC-MK2 e apresentaram seletividade até 25 vezes superior ao BZD. Estudos in vitro frente a forma amastigota da cepa Y em células U2OS foram realizados com metodologia fenotípica de análise de alto conteúdo (HCA') e os compostos apresentaram atividade até 64 vezes superior ao BZD e com seletividade de até 50 vezes superior a este fármaco. Quanto à determinação da atividade dos compostos frente às enzimas tripanotiona redutase (TcTR) e glutationa redutase (GR), os compostos analisados não apresentaram atividade relevante, indicando não ser este o mecanismo desta classe de compostos. Com finalidade de explorar outro possível mecanismo de ação dos compostos 5-nitrofurânicos, foi realizada a análise de potencial de redução da membrana mitocondrial, porém a morte parasitária não foi atribuída à despolarização da membrana em estudos simultâneos com iodeto de propídio

Chagas disease affects approximately 6-7 millions people worldwide, especially Latin America. The search for therapeutic alternatives for this disease is of great relevance to society, as current options are limited and there are only two available drugs: benznidazole (BZD) and nifurtimox. The nitroheterocyclic derivatives are considered bioactive compounds with increasing number of studies in the scientific community against its etiologic agent, Trypanosoma cruzi. In this sense, this work aims to identify derivatives of 5-nitrofuran with activity against different strains of T. cruzi, and to study possible mode of action of this compounds. This research involves computational studies to obtain models of quantitative structure-activity relationships (QSAR multivariate) that can help predict new structures with optimized pharmacological profile. In this work were carried out the design, synthesis and identification of 36 compounds with satisfactory results regarding the structural identification, purity and yield (approximately 70%). The determination of anti-T. cruzi activity in vitro of the compounds obtained was carried out with Silvio X10 cl1, Y, Bug 2149 CL10 and Colombiana strains of epimastigote form of the parasite. Most of the compounds examined showed greater capacity of growth inhibition of the parasite compared to the BZD (Silvio X10 CL1 - IC 50 = 29.16 ± 2.90 µM, Y - IC50 = 40.40 ± 3,37µM, 2149 CL10 Bug - IC 50 = 30.63 ± 3.21 µM, Colombiana - IC 50 = 47.91 ± 4.96 µM). The most active compound (BSF-35) showed the following values: Silvio X10 cl1 - IC 50 = 3.17 ± 0.32 uM, Y - IC 50 = 1.17 ± 0.12 µM, Bug 2149 CL10 - IC50 = 1, 81 ± 0.18 µM and Colombiana - IC 50 = 3.06 ± 0.23 µM. Calculations were performed for the molecular properties of three-dimensional structures of the compounds, followed by exploratory data analysis by hierarchical cluster analysis (HCA) and principal component analysis (PCA), allowing the recognition of the set. Considering this preliminary analysis were obtained QSAR models with multivariate approach, using OPS algorithm and regression method of partial least squares, PLS. The best generated models were obtained considering the benzyl substituted compounds for the four strains. The descriptors that most influenced the analysis were ClogP (partition coefficient) and CHELPG charges. Considering the information obtained, four new compounds were designed and synthesized to obtain more active compounds and validate QSAR models. These compounds showed high activity against epimastigote form of the four strains studied. The most active compounds were evaluated for cytotoxicity against LLC-MK2 cells and the compounds selectivity values were up to 25 times higher than BZD. In vitro studies against amastigote form of the Y strain in U2OS cells were performed with phenotypic method of high content analysis (HCA') and the compounds showed activity to 64 times higher than BZD and selectivity of up to 50 times. The activity of the compounds against trypanothione reductase enzymes (TcTR) and glutathione reductase (GR) showed no significant activity, indicating that this is not the mechanism of this class of compounds. In order to exploit another possible mechanism of action of 5-nitrofuran derivatives, analysis reduction of mitochondrial membrane potential was held, however the cell death was not attributed to membrane depolarization in simultaneous studies with propidium iodide

Seudoquiste pancreático infectado por Shewanella putrefaciens: reporte de caso / Pancreatic pseudocyst infected by Shewanella putrefaciens: A case report

Shewanella putrefaciens es un bacilo gramnegativo, no fermentador, oxidasa positivo, productor de sulfato de hidrógeno, que de manera inusual causa cuadros infecciosos en humanos. Este germen oportunista produce infecciones en pacientes inmunosuprimidos y/o con antecedente de exposición a comida o agua de mar contaminados con el germen. A continuación presentamos un reporte de caso de un paciente con una infección del páncreas por este germen.

Shewanella putrefaciens is a rare Gram-negative bacillus, non-fermenting, oxidasepositive, and a hydrogen sulfide producer, which infrequently causes human infections. This opportunist germ causes infections in immunosuppressed people and in those with a history of contact with seawater or food contaminated with the microbe. We present a case of a patient with a pancreatic infection caused by this microbe.

GenoType MTBDR plus 1.0 ® para la detección de resistencia cruzada entre isoniacida y etionamida en aislamientos de Mycobacterium tuberculosis multirresistentes / GenoType MTBDR plus 1.0 ® for the detection of cross-resistance between isoniazide and ethionamide in isolates of multidrug-resistant Mycobacterium tuberculosis

Introducción. Una parte de los aislamientos de Mycobacterium tuberculosis multirresistente también presenta resistencia a la etionamida. Es importante determinar si la resistencia a la isoniacida es independiente o se cruza con la resistencia a la etionamida, ya que si sucede lo segundo habría que reevaluar el tratamiento antituberculoso de segunda línea. La prueba molecular GenoType MTBDR plus ® detecta las mutaciones asociadas con la resistencia a isoniacida y podría detectar la resistencia cruzada a la etionamida. Objetivo. Evaluar la prueba GenoType MTBDR plus ® y comparar su desempeño con el de la secuenciación, en la detección de mutaciones en el gen katG y en el promotor inhA en aislamientos clínicos de M. tuberculosis multirresistente. Materiales y métodos. Se utilizaron el estuche comercial GenoType MTBDR plus 1.0 ® y la secuenciación para evaluar mutaciones en el gen katG y en el promotor inhA en 30 aislamientos de M. tuberculosis multirresistente con resistencia a la etionamida. La cepa de laboratorio H37Rv y tres aislamientos sensibles a los medicamentos de primera línea, sirvieron de control. Resultados. Al comparar los resultados de la secuenciación y de la prueba GenoType MTBDR plus ® , el índice kappa fue de 1. Todos los aislamientos resistentes a la isoniacida y la etionamida tenían las mutaciones detectadas con la prueba GenoTypeMTBDR plus ® en el gen katG, y 40 % de ellos, las detectadas en el promotor inhA. Mediante secuenciación se encontraron, además, mutaciones en katG en posiciones diferentes a las detectadas por la prueba GenoType MTBDR plus ® . Conclusión. La prueba GenoTypeMTBDR plus ® tiene la capacidad de detectar rápidamente la resistencia a isoniacida. Además, los resultados del estudio sugieren que también podría utilizarse como prueba de tamización para detectar la resistencia cruzada a etionamida.

Introduction: A variable proportion of isolates of multidrug-resistant Mycobacterium tuberculosis also presents resistance to ethionamide. It is important to determine whether resistance to isoniazid is independent or crossed with resistance to ethionamide, given that this could lead to the re-evaluation of second-line anti-tuberculosis treatment. The GenoType MTBDR plus ® molecular test is used for the detection of MDR-MTB, as it identifies mutations associated with resistance to isoniazide and could detect cross-resistance with ethionamide. Objective: To evaluate the performance of GenoType MTBDR plus ® in comparison with sequencing in the detection of mutations in gene katG and promotor inhA in clinical isolates of multidrug-resistant M. tuberculosis . Materials and methods: The GenoType MTBDR plus 1.0 ® commercial kit and sequencing were used to evaluate mutations in gene katG and promotor inhA in 30 multidrug-resistant M. tuberculosis isolates that were resistant to ethionamide. The laboratory strain H37Rv and three pan-sensitive isolates acted as controls. Results: The kappa index for the comparison between the results of sequencing and GenoType MTBDR plus ® was 1. All the isolates resistant to isoniazid and ethionamide had the mutations detected by GenoTypeMTBDR plus ® in the katG gene and 40% of them in promotor inhA. Sequencing also revealed katG mutations in positions different to those detected by GenoType MTBDR plus ® . Conclusion: GenoType MTBDR plus ® is able to detect resistance to isoniazid rapidly. Our results suggest that it could also be used to screen for cross-resistance with ethionamide.