RESUMO
Vaccinia-related kinase 1 (VRK1) and the δ and ε isoforms of casein kinase 1 (CK1) are linked to various disease-relevant pathways. However, the lack of tool compounds for these kinases has significantly hampered our understanding of their cellular functions and therapeutic potential. Here, we describe the structure-based development of potent inhibitors of VRK1, a kinase highly expressed in various tumor types and crucial for cell proliferation and genome integrity. Kinome-wide profiling revealed that our compounds also inhibit CK1δ and CK1ε. We demonstrate that dihydropteridinones 35 and 36 mimic the cellular outcomes of VRK1 depletion. Complementary studies with existing CK1δ and CK1ε inhibitors suggest that these kinases may play overlapping roles in cell proliferation and genome instability. Together, our findings highlight the potential of VRK1 inhibition in treating p53-deficient tumors and possibly enhancing the efficacy of existing cancer therapies that target DNA stability or cell division.
Assuntos
Inibidores de Proteínas Quinases , Proteínas Serina-Treonina Quinases , Pteridinas , Humanos , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/síntese química , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Pteridinas/farmacologia , Pteridinas/química , Pteridinas/síntese química , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proliferação de Células/efeitos dos fármacos , Relação Estrutura-Atividade , Caseína Quinase Idelta/antagonistas & inibidores , Caseína Quinase Idelta/metabolismo , Caseína Quinase 1 épsilon/antagonistas & inibidores , Caseína Quinase 1 épsilon/metabolismo , Linhagem Celular TumoralRESUMO
Endolysins are bacteriophage (or phage)-encoded enzymes that catalyse the peptidoglycan breakdown in the bacterial cell wall. The exogenous action of recombinant phage endolysins against Gram-positive organisms has been extensively studied. However, the outer membrane acts as a physical barrier when considering the use of recombinant endolysins to combat Gram-negative bacteria. This study aimed to evaluate the antimicrobial activity of the SAR-endolysin LysKpV475 against Gram-negative bacteria as single or combined therapies, using an outer membrane permeabilizer (polymyxin B) and a phage, free or immobilized in a pullulan matrix. In the first step, the endolysin LysKpV475 in solution, alone and combined with polymyxin B, was tested in vitro and in vivo against ten Gram-negative bacteria, including highly virulent strains and multidrug-resistant isolates. In the second step, the lyophilized LysKpV475 endolysin was combined with the phage phSE-5 and investigated, free or immobilized in a pullulan matrix, against Salmonella enterica subsp. enterica serovar Typhimurium ATCC 13311. The bacteriostatic action of purified LysKpV475 varied between 8.125 µgâ¯ml-1 against Pseudomonas aeruginosa ATCC 27853, 16.25 µgâ¯ml-1 against S. enterica Typhimurium ATCC 13311, and 32.50 µgâ¯ml-1 against Klebsiella pneumoniae ATCC BAA-2146 and Enterobacter cloacae P2224. LysKpV475 showed bactericidal activity only for P. aeruginosa ATCC 27853 (32.50 µgâ¯ml-1) and P. aeruginosa P2307 (65.00 µgâ¯ml-1) at the tested concentrations. The effect of the LysKpV475 combined with polymyxin B increased against K. pneumoniae ATCC BAA-2146 [fractional inhibitory concentration index (FICI) 0.34; a value lower than 1.0 indicates an additive/combined effect] and S. enterica Typhimurium ATCC 13311 (FICI 0.93). A synergistic effect against S. enterica Typhimurium was also observed when the lyophilized LysKpV475 at â MIC was combined with the phage phSE-5 (m.o.i. of 100). The lyophilized LysKpV475 immobilized in a pullulan matrix maintained a significant Salmonella reduction of 2 logs after 6 h of treatment. These results demonstrate the potential of SAR-endolysins, alone or in combination with other treatments, in the free form or immobilized in solid matrices, which paves the way for their application in different areas, such as in biocontrol at the food processing stage, biosanitation of food contact surfaces and biopreservation of processed food in active food packing.
Assuntos
Antibacterianos , Endopeptidases , Glucanos , Polimixina B , Fagos de Salmonella , Endopeptidases/farmacologia , Endopeptidases/química , Endopeptidases/metabolismo , Polimixina B/farmacologia , Antibacterianos/farmacologia , Antibacterianos/química , Fagos de Salmonella/genética , Fagos de Salmonella/fisiologia , Fagos de Salmonella/química , Glucanos/química , Glucanos/farmacologia , Animais , Testes de Sensibilidade Microbiana , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Negativas/virologia , Camundongos , Salmonella typhimurium/virologia , Salmonella typhimurium/efeitos dos fármacos , Bacteriófagos/fisiologia , Bacteriófagos/genética , Proteínas Virais/genética , Proteínas Virais/metabolismo , Proteínas Virais/farmacologia , Proteínas Virais/químicaRESUMO
The dual-specificity protein kinase MKK3 has been implicated in tumor cell proliferation and survival, yet its precise role in cancer remains inconclusive. A critical step in elucidating the kinase's involvement in disease biology is the identification of potent, cell-permeable kinase inhibitors. Presently, MKK3 lacks a dedicated tool compound for these purposes, along with validated methods for the facile screening, identification, and optimization of inhibitors. In this study, we have developed a TR-FRET-based enzymatic assay for the detection of MKK3 activity in vitro and a BRET-based assay to assess ligand binding to this enzyme within intact human cells. These assays were instrumental in identifying hit compounds against MKK3 that share a common chemical scaffold, sourced from a library of bioactive kinase inhibitors. Initial hits were subsequently expanded through the synthesis of novel analogs. The resulting structure-activity relationship (SAR) was rationalized using molecular dynamics simulations against a homology model of MKK3. We expect our findings to expedite the development of novel, potent, selective, and bioactive inhibitors, thus facilitating investigations into MKK3's role in various cancers.
Assuntos
Neoplasias , Pirimidinas , Humanos , MAP Quinase Quinase 3 , Pirimidinas/química , Relação Estrutura-Atividade , Fosforilação , Proliferação de Células , Inibidores de Proteínas Quinases/químicaRESUMO
New antibiotics are urgently needed to counter the emergence of antimicrobial-resistant pathogenic bacteria. A major challenge in antibiotic drug discovery is to turn potent biochemical inhibitors of essential bacterial components into effective antimicrobials. This difficulty is underpinned by a lack of methods to investigate the physicochemical properties needed for candidate antibiotics to permeate the bacterial cell envelope and avoid clearance by the action of bacterial efflux pumps. To address these issues, here we used a target engagement assay to measure the equilibrium and kinetic binding parameters of antibiotics targeting dihydrofolate reductase (DHFR) in live bacteria. We also used this assay to identify novel DHFR ligands having antimicrobial activity. We validated this approach using the Gram-negative bacteria Escherichia coli and the emerging human pathogen Mycobacterium abscessus. We expect the use of target engagement assays in bacteria to expedite the discovery and progression of novel, cell-permeable antibiotics with on-target activity.
Assuntos
Antibacterianos , Anti-Infecciosos , Antibacterianos/química , Anti-Infecciosos/farmacologia , Escherichia coli/metabolismo , Bactérias Gram-Negativas , Humanos , Tetra-Hidrofolato Desidrogenase/químicaRESUMO
The inappropriate disposal of toxic compounds generated by industrial activity has considerably impacted the environment. Microbial communities inhabiting contaminated sites may represent ecological alternatives for the decontamination of environments. The present work aimed to search the potential of fungi isolated from wastewater treatment plant of a textile industry for bioremediation processes. Twenty-three fungi previously isolated from textile effluent were evaluated for their abilities to degrade pollutants using heavy metal and hydrocarbon tolerance assays. One isolate was subjected to pyrene degradation due its ability to tolerate hydrocarbon. The majority of isolates were resistant to at least two metals tested, i.e. chrome, copper, lead and aluminum. Isolates Penicillium sp. ITF 2, Penicillium rubens ITF 4, Penicillium sp. ITF 12 and ITF 20 (not identified) showed tolerance to tested heavy metals in all concentrations. ITF 12 and ITF 20 were able to tolerate benzene, toluene and hexane, separately. ITF 12 was able to degrade 24.9% of pyrene after 5 days of cultivation. The results encourage future studies to optimize the tolerance and degradation assay using the isolates that showed the best results, as well as studies on the treatment of environments contaminated with heavy metals and hydrocarbons, including industrial textile effluents.
Assuntos
Fungos , Metais Pesados , Biodegradação Ambiental , Metais Pesados/análise , Pirenos/metabolismo , TêxteisRESUMO
The inappropriate disposal of toxic compounds generated by industrial activity has considerably impacted the environment. Microbial communities inhabiting contaminated sites may represent ecological alternatives for the decontamination of environments. The present work aimed to search the potential of fungi isolated from wastewater treatment plant of a textile industry for bioremediation processes. Twenty-three fungi previously isolated from textile effluent were evaluated for their abilities to degrade pollutants using heavy metal and hydrocarbon tolerance assays. One isolate was subjected to pyrene degradation due its ability to tolerate hydrocarbon. The majority of isolates were resistant to at least two metals tested, i.e. chrome, copper, lead and aluminum. Isolates Penicillium sp. ITF 2, Penicillium rubens ITF 4, Penicillium sp. ITF 12 and ITF 20 (not identified) showed tolerance to tested heavy metals in all concentrations. ITF 12 and ITF 20 were able to tolerate benzene, toluene and hexane, separately. ITF 12 was able to degrade 24.9% of pyrene after 5 days of cultivation. The results encourage future studies to optimize the tolerance and degradation assay using the isolates that showed the best results, as well as studies on the treatment of environments contaminated with heavy metals and hydrocarbons, including industrial textile effluents.
Assuntos
Fungos , Metais Pesados , Biodegradação Ambiental , Metais Pesados/análise , Pirenos/metabolismo , TêxteisRESUMO
The discovery of potent and selective inhibitors for understudied kinases can provide relevant pharmacological tools to illuminate their biological functions. DYRK1A and DYRK1B are protein kinases linked to chronic human diseases. Current DYRK1A/DYRK1B inhibitors also antagonize the function of related protein kinases, such as CDC2-like kinases (CLK1, CLK2, CLK4) and DYRK2. Here, we reveal narrow spectrum dual inhibitors of DYRK1A and DYRK1B based on a benzothiophene scaffold. Compound optimization exploited structural differences in the ATP-binding sites of the DYRK1 kinases and resulted in the discovery of 3n, a potent and cell-permeable DYRK1A/DYRK1B inhibitor. This compound has a different scaffold and a narrower off-target profile compared to current DYRK1A/DYRK1B inhibitors. We expect the benzothiophene derivatives described here to aid establishing DYRK1A/DYRK1B cellular functions and their role in human pathologies.
Assuntos
Proteínas Serina-Treonina Quinases , Proteínas Tirosina Quinases , Humanos , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases , Proteínas Tirosina Quinases/metabolismo , TiofenosRESUMO
ABSTRACT The present study aims to establish the fatty acid profile of a variety of traditional foods consumed regularly in Paraguay. Thirty-two high-lipid content foods were evaluated by gas chromatography coupled with a flame ionization detector (GC-FID). Meat and dairy products were found to present high levels of saturated fatty acids, and this led to an increase in the atherogenicity and thrombogenicity indices of these products. Among the monounsaturated acids evaluated, oleic acid (18:1n-9c) was mainly found in olives (71.7%) and chipa (62.7%), which had been industrially prepared with the addition of oleic acid. Milk-based ice cream (5.1%) and chipa guazú (3.8%) presented the highest content of trans fatty acids. The n-6/n-3 fatty acids ratio obtained for the traditional sopa Paraguaya and empanada (fried pasty) was higher than 37:1; this can be attributed to the addition of soybean and corn oils in the preparation of these foods. The data evaluated and presented in this study can contribute toward the improvement of eating habits among the Paraguayan population, as well as help combat the risk factors commonly associated with bad eating habits and chronic health issues.
RESUMEN El presente estudio tuvo como objetivo establecer los perfiles de ácidos grasos de una variedad de alimentos tradicionales que se consumen regularmente en Paraguay. Se evaluaron treinta y dos alimentos con alto contenido de lípidos mediante cromatografía de gases acoplada a detector de ionización de llama (GC-FID). Los productos cárnicos y lácteos presentaron un elevado nivel de ácidos grasos saturados, aumentando los índices de aterogenicidad y trombogenicidad. Entre los monoinsaturados, el ácido oleico (18: 1n-9c) se encuentró principalmente en aceitunas (71,7%) y chipa (62,7%), este último preparado industrialmente por adición de ácido oleico. En cuanto al contenido de ácidos grasos trans, los valores más altos se encontraron en los helados a base de leche (5,1%) y chipa guazú (3,8%). Los valores de la relación n-6 / n-3 fueron superiores a 37:1 en sopa paraguaya y empanada (frita) debido a la adición de aceite de soja y maíz utilizado en la preparación de alimentos. Los datos evaluados y presentados en este estudio pueden contribuir a la mejora de los hábitos alimentarios de la población paraguaya, así como ayudar a combatir los factores de riesgo comúnmente asociados con los malos hábitos alimentarios y los problemas crónicos de salud.
RESUMO
Los marcadores son herramientas para ayudar a obtener información que mejore la producción animal, especialmente en nutrición. El objetivo de este estudio fue evaluar las dosis de óxido de hierro como marcador fecal a través de sus efectos sobre la coloración fecal de las ovejas. El diseño experimental fue un cuadrado latino 4 x 4, que comprende cuatro dosis de adición de óxido de hierro (Fe2O3) de 0, 5, 10 y 20% del concentrado, equivalente a 0, 7,5, 15, 30 g de animal-1, respectivamente. Cuatro ovejas macho no castradas fueron mantenidas en jaulas metabólicas adaptadas, alimentadas con una dieta con proporción de forraje: concentrado de 84:16, heno Tifton 85 triturado y el suplemento energético a base de maíz molido. El período experimental duró 47 días, contando 15 días para la adaptación, con cada subperíodo compuesto por 7 días más un descanso. Los animales recibieron tratamientos con dosis de Fe2O3 mezcladas con el concentrado el primer día de cada subperíodo experimental y las colecciones de heces totales fueron seguidas diariamente directamente desde la jaula. No hubo efectos de los tratamientos sobre el consumo voluntario de materia seca, comportamiento y heces totales. El color de las heces de los animales se observó en todos los tratamientos después de 22 h y permaneció bajo diferentes intensidades hasta 130 h, y las dosis de 10 y 20% persistieron hasta 154 h después...(AU)
Markers are tools to support getting information that improves animal production, especially in nutrition. The aim of this study was to evaluate doses of iron oxide as a fecal marker through its effects on fecal coloring of sheep. The experimental design was a 4 x 4 Latin square, comprising four doses of iron oxide (Fe2O3) addition of 0, 5, 10 and 20% of the concentrate, equivalent to 0, 7.5, 15, 30 g animal-1, respectively. Four non-castrated male sheep were kept in adapted metabolic cages, fed a diet with proportion of forage: concentrate of 84: 16, with crushed Tifton 85 hay and the energy supplement based on ground corn. The experimental period lasted 47 days, counting 15 days for adaptation, with each subperiod comprising 7 days plus one rest. The animals received treatments with Fe2O3 doses mixed with the concentrate on the first day of each experimental subperiod and total feces collections were followed daily directly from the cage. There were no effects of treatments on voluntary consumption of dry matter, behavior and total feces. The color of the animals' feces was observed in all treatments after 22 h and remained under different intensities until 130 h, and the doses of 10 and 20% persisted until 154 h after supply. The Fe2O3 has the potential to be used as a fecal marker, becoming a tool to support in animal experimentation through its...(AU)
Os marcadores são ferramentas que auxiliam a obtenção de informações que aprimoram a produção animal, principalmente em nutrição. Objetivou-se avaliar doses de óxido de ferro como marcador fecal e seus efeitos na coloração das fezes de ovinos. O delineamento experimental foi em quadrado latino 4 x 4, compreendendo quatro doses de adição de óxido de ferro (Fe2O3) de 0, 5, 10 e 20% do concentrado, equivalentes a 0, 7,5, 15, 30 g animal-1, respectivamente. Foram utilizados quatro ovinos machos não castrados mantidos em gaiolas metabólicas adaptadas, alimentados com dieta com relação volumoso: concentrado de 84: 16, sendo feno de Tifton 85 triturado e o suplemento energético a base de milho moído. O período experimental teve duração de 47 dias, contabilizando 15 dias para adaptação, sendo que cada subperíodo compreendeu 7 dias mais um de descanso. Os animais receberam os tratamentos com as doses de Fe2O3 misturadas ao concentrado no primeiro dia de cada subperíodo experimental e seguia-se as coletas de fezes total diariamente diretamente da gaiola. Não foram verificados efeitos dos tratamentos no consumo voluntário de matéria seca, comportamento ingestivo e total de fezes. A coloração das fezes dos animais foi observada em todos os tratamentos a partir das 22 h e permaneceu sob diferentes intensidades até 130 h, sendo que as doses de 10 e 20% persistiram até 154 h após o...(AU)
Assuntos
Animais , Ovinos , Biomarcadores/análise , Compostos Ferrosos/análise , Fezes , Ração Animal , PigmentaçãoRESUMO
PURPOSE: To determine the prevalence of probable sleep bruxism (SB) in preschoolers and associated factors. METHODS: A cross-sectional, population-based study was carried out with 862 dyads of parents/preschoolers aged 5 from Teresina, Brazil. Data collection was performed through a questionnaire answered by parents/guardians about socioeconomic, demographic, and health condition data. Each child was examined for diagnosis of probable SB based on the presence of dental wear associated or not with the report of teeth grinding by parents/guardians. The analysis of independent variables was stratified into four levels of determinants: demographic characteristics of the child (distal), characteristics of the family, environmental factors (intermediate), and health conditions (proximal). Descriptive analysis and bivariate and multivariate Poisson's regression were performed using a hierarchical approach (p < 0.05). RESULTS: The prevalence of probable SB was 36%. In the final adjusted multivariate hierarchical model, a preschooler who was the only child (PR = 1.25; 95%CI = 1.02-1.51), with breathing problems (PR = 1.43; 95%CI = 1.19-1.73), and having parents/guardians with possible sleep bruxism (PR = 1.65; 95%CI = 1.32-2.07) had a higher prevalence of probable SB. CONCLUSION: The prevalence of probable SB in preschoolers was high and associated with the condition of being an only child, the presence of breathing problems, and having parents/guardians with possible sleep bruxism.
Assuntos
Filho Único/estatística & dados numéricos , Pais , Transtornos Respiratórios/epidemiologia , Bruxismo do Sono/epidemiologia , Brasil/epidemiologia , Pré-Escolar , Estudos Transversais , Feminino , Humanos , Masculino , Prevalência , Fatores de Risco , Bruxismo do Sono/genética , Inquéritos e QuestionáriosRESUMO
Los marcadores son herramientas para ayudar a obtener información que mejore la producción animal, especialmente en nutrición. El objetivo de este estudio fue evaluar las dosis de óxido de hierro como marcador fecal a través de sus efectos sobre la coloración fecal de las ovejas. El diseño experimental fue un cuadrado latino 4 x 4, que comprende cuatro dosis de adición de óxido de hierro (Fe2O3) de 0, 5, 10 y 20% del concentrado, equivalente a 0, 7,5, 15, 30 g de animal-1, respectivamente. Cuatro ovejas macho no castradas fueron mantenidas en jaulas metabólicas adaptadas, alimentadas con una dieta con proporción de forraje: concentrado de 84:16, heno Tifton 85 triturado y el suplemento energético a base de maíz molido. El período experimental duró 47 días, contando 15 días para la adaptación, con cada subperíodo compuesto por 7 días más un descanso. Los animales recibieron tratamientos con dosis de Fe2O3 mezcladas con el concentrado el primer día de cada subperíodo experimental y las colecciones de heces totales fueron seguidas diariamente directamente desde la jaula. No hubo efectos de los tratamientos sobre el consumo voluntario de materia seca, comportamiento y heces totales. El color de las heces de los animales se observó en todos los tratamientos después de 22 h y permaneció bajo diferentes intensidades hasta 130 h, y las dosis de 10 y 20% persistieron hasta 154 h después...
Markers are tools to support getting information that improves animal production, especially in nutrition. The aim of this study was to evaluate doses of iron oxide as a fecal marker through its effects on fecal coloring of sheep. The experimental design was a 4 x 4 Latin square, comprising four doses of iron oxide (Fe2O3) addition of 0, 5, 10 and 20% of the concentrate, equivalent to 0, 7.5, 15, 30 g animal-1, respectively. Four non-castrated male sheep were kept in adapted metabolic cages, fed a diet with proportion of forage: concentrate of 84: 16, with crushed Tifton 85 hay and the energy supplement based on ground corn. The experimental period lasted 47 days, counting 15 days for adaptation, with each subperiod comprising 7 days plus one rest. The animals received treatments with Fe2O3 doses mixed with the concentrate on the first day of each experimental subperiod and total feces collections were followed daily directly from the cage. There were no effects of treatments on voluntary consumption of dry matter, behavior and total feces. The color of the animals' feces was observed in all treatments after 22 h and remained under different intensities until 130 h, and the doses of 10 and 20% persisted until 154 h after supply. The Fe2O3 has the potential to be used as a fecal marker, becoming a tool to support in animal experimentation through its...
Os marcadores são ferramentas que auxiliam a obtenção de informações que aprimoram a produção animal, principalmente em nutrição. Objetivou-se avaliar doses de óxido de ferro como marcador fecal e seus efeitos na coloração das fezes de ovinos. O delineamento experimental foi em quadrado latino 4 x 4, compreendendo quatro doses de adição de óxido de ferro (Fe2O3) de 0, 5, 10 e 20% do concentrado, equivalentes a 0, 7,5, 15, 30 g animal-1, respectivamente. Foram utilizados quatro ovinos machos não castrados mantidos em gaiolas metabólicas adaptadas, alimentados com dieta com relação volumoso: concentrado de 84: 16, sendo feno de Tifton 85 triturado e o suplemento energético a base de milho moído. O período experimental teve duração de 47 dias, contabilizando 15 dias para adaptação, sendo que cada subperíodo compreendeu 7 dias mais um de descanso. Os animais receberam os tratamentos com as doses de Fe2O3 misturadas ao concentrado no primeiro dia de cada subperíodo experimental e seguia-se as coletas de fezes total diariamente diretamente da gaiola. Não foram verificados efeitos dos tratamentos no consumo voluntário de matéria seca, comportamento ingestivo e total de fezes. A coloração das fezes dos animais foi observada em todos os tratamentos a partir das 22 h e permaneceu sob diferentes intensidades até 130 h, sendo que as doses de 10 e 20% persistiram até 154 h após o...
Assuntos
Animais , Biomarcadores/análise , Compostos Ferrosos/análise , Fezes , Ovinos , Pigmentação , Ração AnimalRESUMO
Deoxyhypusine synthase (DHS) catalyzes the first step of the post-translational modification of eukaryotic translation factor 5A (eIF5A), which is the only known protein containing the amino acid hypusine. Both proteins are essential for eukaryotic cell viability, and DHS has been suggested as a good candidate target for small molecule-based therapies against eukaryotic pathogens. In this work, we focused on the DHS enzymes from Brugia malayi and Leishmania major, the causative agents of lymphatic filariasis and cutaneous leishmaniasis, respectively. To enable B. malayi (Bm)DHS for future target-based drug discovery programs, we determined its crystal structure bound to cofactor NAD+. We also reported an in vitro biochemical assay for this enzyme that is amenable to a high-throughput screening format. The L. major genome encodes two DHS paralogs, and attempts to produce them recombinantly in bacterial cells were not successful. Nevertheless, we showed that ectopic expression of both LmDHS paralogs can rescue yeast cells lacking the endogenous DHS-encoding gene (dys1). Thus, functionally complemented dys1Δ yeast mutants can be used to screen for new inhibitors of the L. major enzyme. We used the known human DHS inhibitor GC7 to validate both in vitro and yeast-based DHS assays. Our results show that BmDHS is a homotetrameric enzyme that shares many features with its human homologue, whereas LmDHS paralogs are likely to form a heterotetrameric complex and have a distinct regulatory mechanism. We expect our work to facilitate the identification and development of new DHS inhibitors that can be used to validate these enzymes as vulnerable targets for therapeutic interventions against B. malayi and L. major infections.
Assuntos
Anti-Helmínticos/farmacologia , Antiprotozoários/farmacologia , Brugia Malayi/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Proteínas de Helminto/antagonistas & inibidores , Leishmania major/efeitos dos fármacos , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/antagonistas & inibidores , Proteínas de Protozoários/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Anti-Helmínticos/química , Antiprotozoários/química , Brugia Malayi/enzimologia , Brugia Malayi/genética , Brugia Malayi/crescimento & desenvolvimento , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/química , Proteínas de Helminto/química , Proteínas de Helminto/genética , Proteínas de Helminto/metabolismo , Ensaios de Triagem em Larga Escala , Leishmania major/enzimologia , Leishmania major/genética , Leishmania major/crescimento & desenvolvimento , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/química , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Alinhamento de SequênciaRESUMO
RESUMO O objetivo deste estudo consiste em compreender o cuidado à saúde bucal de pessoas com fenda orofacial e suas expressões. Esta pesquisa teve a história de vida como recurso metodológico. Analisaram-se as narrativas de quatro mulheres com idades entre 19 e 28 anos, moradoras da zona urbana de um município do Nordeste brasileiro. Foram estruturados quatro encontros, gravados e transcritos, em que vivenciou-se a construção de uma identidade e formação do vínculo, resgate das redes sociais e comunitárias de apoio, caminhos percorridos nos serviços de saúde e a percepção sobre os serviços. A análise dos dados foi inspirada na abordagem hermenêutica de P. Ricoeur. Identificaram-se quatro unidades de sentido: Ser-fissurado?; Rede de cuidados; Fluxos assistenciais em saúde bucal para pessoas com fenda orofacial e suas expressões; e Percepção sobre os serviços de saúde. Concluiu-se que há uma rede fragmentada quanto ao cuidado à saúde bucal, com falhas na comunicação, articulação e integração dos serviços de saúde, pouco envolvimento e apropriação dos casos pelas equipes de atenção básica, com importantes repercussões dos estigmas sociais sobre o paciente. Assim, estratégias para o aprimoramento das relações, bem como articulação e integração entre os serviços devem ser desenvolvidas na tentativa de superar tais problemas.
ABSTRACT The objective of this study was to understand the oral health care of people with orofacial cleft and their expressions. This research had the history of life as a methodological resource. It was analyzed the narratives of four women aged between 19 and 28 years, living in the urban area of a Brazilian northeast municipality. Four meetings were structured, recorded and transcribed, in which we experienced the construction of an identity and formation of a bond, rescue of social and community support networks, paths taken in health services and perception on services. The analysis of the data was inspired by the hermeneutic approach of P. Ricoeur. Four sense units were identified: Be a person with orofacial cleft?; Health care network; Oral health care flows for people with orofacial clefts and their expressions; and, Perception about health services. It was concluded that there is a fragmented oral health care network, with poor communication, articulation and integration of health services, low involvement and appropriation of cases by primary care teams, with important repercussions of social stigmas on the patient. Thus, strategies for improving relations, as well as articulation and integration among services should be developed in an attempt to overcome such problems.
RESUMO
Calcium/Calmodulin-dependent Protein Kinase Kinase 2 (CAMKK2) acts as a signaling hub, receiving signals from various regulatory pathways and decoding them via phosphorylation of downstream protein kinases - such as AMPK (AMP-activated protein kinase) and CAMK types I and IV. CAMKK2 relevance is highlighted by its constitutive activity being implicated in several human pathologies. However, at present, there are no selective small-molecule inhibitors available for this protein kinase. Moreover, CAMKK2 and its closest human homolog, CAMKK1, are thought to have overlapping biological roles. Here we present six new co-structures of potent ligands bound to CAMKK2 identified from a library of commercially-available kinase inhibitors. Enzyme assays confirmed that most of these compounds are equipotent inhibitors of both human CAMKKs and isothermal titration calorimetry (ITC) revealed that binding to some of these molecules to CAMKK2 is enthalpy driven. We expect our results to advance current efforts to discover small molecule kinase inhibitors selective to each human CAMKK.
Assuntos
Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/química , Inibidores de Proteínas Quinases/química , Animais , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Descoberta de Drogas , Humanos , Ligantes , Conformação Molecular , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ligação Proteica , Inibidores de Proteínas Quinases/farmacologia , Proteínas Recombinantes , Relação Estrutura-AtividadeRESUMO
Among gliomas types, glioblastoma is considered the most malignant and the worst form of primary brain tumor. It is characterized by high infiltration rate and great angiogenic capacity. The presence of an inflammatory microenvironment contributes to chemo/radioresistance, resulting in poor prognosis for patients. Recent data show that thiazolidinones have a wide range of pharmacological properties, including anti-inflammatory and antiglioma activities. Nanocapsules of biodegradable polymers become an alternative to cancer treatment since they provide targeted drug delivery and could overcome blood-brain barrier. Therefore, here we investigated the in vitro antiglioma activity and the potential in vivo toxicity of 2- (2-methoxyphenyl) -3- ((piperidin-1-yl) ethyl) thiazolidin-4-one-loaded polymeric nanocapsules (4L-N). Nanocapsules were prepared and characterized in terms of particle size, polydispersity index, zeta potential, pH, molecule content and encapsulation efficiency. Treatment with 4L-N selectively decreased human U138MG and rat C6 cell lines viability and proliferation, being even more efficient than the free-form molecule (4L). In addition, 4L-N did not promote toxicity to primary astrocytes. We further demonstrated that the treatment with sub-therapeutic dose of 4L-N did not alter weight, neither resulted in mortality, toxicity or peripheral damage to Wistar rats. Finally, 4L as well as 4L-N did not alter makers of oxidative damage, such as TBARS levels and total sulfhydryl content, and did not change antioxidant enzymes SOD and CAT activity in liver and brain of treated rats. Taken together, these data indicate that the nanoencapsulation of 4L has potentiated its antiglioma effect and does not cause in vivo toxicity.
Assuntos
Neoplasias Encefálicas/tratamento farmacológico , Glioma/tratamento farmacológico , Nanocápsulas/química , Piperidinas/toxicidade , Piperidinas/uso terapêutico , Polímeros/química , Tiazolidinas/toxicidade , Tiazolidinas/uso terapêutico , Animais , Apoptose/efeitos dos fármacos , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Astrócitos/patologia , Biomarcadores Tumorais/sangue , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Neoplasias Encefálicas/sangue , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Liberação Controlada de Fármacos , Glioma/sangue , Glioma/patologia , Humanos , Luz , Fígado/efeitos dos fármacos , Fígado/patologia , Masculino , Estresse Oxidativo/efeitos dos fármacos , Piperidinas/síntese química , Piperidinas/química , Polímeros/síntese química , Ratos Wistar , Tiazolidinas/síntese química , Tiazolidinas/química , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismo , Testes de Toxicidade , Redução de Peso/efeitos dos fármacosRESUMO
INTRODUCTION: according to the nutriment addition scheme from the current Mexican legislation, there is no data about overdose or adverse effects caused by a nutriment, or any information showing the risk for the population in Mexico. This work is classified as descriptive and observational. AIM: to assess the risk of consuming fortified food products (FFP) in Mexico. METHODS: the study was done in three phases: a) selection of the FFP and acquisition of the information from the nutritional facts label; b) elaboration of six diets according to the socioeconomic status, both in rural and urban areas, based on the ENIGH and ENSANUT surveys; and c) comparison of these diets with regimes containing FFP, calculated for an adult-equivalent (2,828 kcal). RESULTS: the FFP represent 10% of all the products in the market, being milk, corn and wheat flour, and their byproducts the most abundant. The six diets containing FFP were deficient in calcium, ascorbic acid and vitamins D and E. However, vitamins from the B complex were over the recommendation values. In general, any added nutriment was over the tolerable upper intake levels (UL). CONCLUSIONS: we demonstrated that the nutriment concentrations in the FFP do not reach the UL values and are not a risk for the Mexican population; however, they improve the nutritional contribution of the FFP.
INTRODUCCIÓN: en México no existen datos de sobredosis o reacciones adversas causadas por algún nutrimento o dato alguno que indique riesgo a la población de acuerdo al esquema de adición de nutrimentos de la legislación mexicana vigente. Este trabajo se clasifica como descriptivo y observacional. OBJETIVO: valorar el riesgo por consumo de productos alimenticios adicionados (PAA) en México. MÉTODOS: se realizó en tres fases: a) selección de PAA y obtención de la información nutrimental de las etiquetas; b) elaboración de seis dietas de acuerdo al estrato socioeconómico, tanto en el ámbito rural como en el urbano con base a las encuestas ENIGH y ENSANUT; y c) comparación de estas dietas con dietas que incluyen PAA, calculadas para un adulto equivalente (2.828 kcal). RESULTADOS: los PAA representan el 10% del total de productos presentes en el mercado. Los más frecuentes son: leche, harinas de maíz y trigo y sus derivados. Las seis dietas con PAA presentaron deficiencias en calcio, ácido ascórbico, vitamina D y E. Sin embargo, las vitaminas del complejo B superaron la recomendación. En general, ningún nutrimento adicionado se encontró por arriba del nivel de ingestión tolerable superior (UL). CONCLUSIONES: se demostró que las concentraciones de los nutrimentos en los PAA no alcanzan los UL y no representan un riesgo para la población mexicana, sin embargo, mejoran su aporte nutrimental.
Assuntos
Dieta , Alimentos Fortificados/efeitos adversos , Alimentos Fortificados/análise , Adulto , Animais , Ácido Ascórbico , Cálcio da Dieta , Farinha , Rotulagem de Alimentos , Humanos , México , Leite , Recomendações Nutricionais , Fatores de Risco , Fatores Socioeconômicos , Vitaminas/análise , Zea maysRESUMO
Glioblastoma is the most devastating primary brain tumor. Current treatment is palliative, making necessary the development of new therapeutic strategies to offer alternatives to patients. Therefore, endophytes represent an interesting source of natural metabolites with anticancer potential. These microorganisms reside in tissues of living plants and act to improve their growth. Evidence revealed that several medicinal plants are colonized by endophytic fungi producer of antitumor metabolites. Achyrocline satureioides is a Brazilian medicinal plant characterized by its properties against gastrointestinal disturbances, anticancer and antioxidant effects. However, there are no reports describing the endophytic composition of A. satureioides. The present study proposes the isolation of endophytic fungus from A. satureioides, extract preparation, phytochemical characterization and evaluation of its antiglioma potential. Our data showed that crude extracts of endophyte decreased glioma viability with IC50 values of 1.60-1.63 µg/mL to eDCM (dichloromethane extract) and 37.30-55.12 µg/mL to eEtAc (ethyl acetate extract), respectively. Crude extracts induced cell death by apoptosis with modulation of redox status. In order to bioprospect anticancer metabolites, endophytic fungus extracts were subjected to guided fractionation and purification yielded five fractions of each extract. Six of ten fractions showed selective antiproliferative activity against glioma cells, with IC50 values ranged from 0.95 to 131.3 µg/mL. F3DCM (from eDCM) and F3EtAc (from eEtAc) fractions promoted C6 glioma toxicity with IC50 of 1.0 and 27.05 µg/mL, respectively. F3EtAc fraction induced late apoptosis and arrest in G2/M stage, while F3DCM promoted apoptosis with arrest in Sub-G1 phase. Moreover, F3DCM increased antioxidant defense and decreased ROS production. Additionally, F3DCM showed no cytotoxic activity against astrocytes, revealing selective effect. Based on promising potential of F3DCM, we identified the production of Sch-642305, a lactone, which showed antiproliferative properties with IC50 values of 1.1 and 7.6 µg/mL to C6 and U138MG gliomas, respectively. Sch-642305 promoted arrest on cell cycle in G2/M inducing apoptosis. Furthermore, this lactone decreased glioma cell migration and modulated redox status, increasing superoxide dismutase and catalase activities and enhancing sulfhydryl content, consequently suppressing reactive species of oxygen generation. Taken together, these results indicate that metabolites produced by endophytic fungus isolated from A. satureioides have therapeutic potential as antiglioma agent.
RESUMO
The calcium/calmodulin-dependent protein kinases (CAMKKs) are upstream activators of CAMK1 and CAMK4 signalling and have important functions in neural development, maintenance and signalling, as well as in other aspects of biology such as Ca2+ signalling in the cardiovascular system. To support the development of specific inhibitors of CAMKKs we have determined the crystal structure of CAMKK1 with two ATP-competitive inhibitors. The structures reveal small but exploitable differences between CAMKK1 and CAMKK2, despite the high sequence identity, which could be used in the generation of specific inhibitors. Screening of a kinase inhibitor library revealed molecules that bind potently to CAMKK1. Isothermal titration calorimetry revealed that the most potent inhibitors had binding energies largely dependent on favourable enthalpy. Together, the data provide a foundation for future inhibitor development activities.
Assuntos
Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/química , Desenho de Fármacos , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/metabolismo , Calorimetria , Domínio Catalítico , Avaliação Pré-Clínica de Medicamentos , Hesperidina/química , Hesperidina/farmacologia , Humanos , Estrutura Secundária de ProteínaRESUMO
The PR-1 proteins (pathogenesis-related protein 1) are involved in plant defense mechanisms against various pathogens. The genome of cacao (Theobroma cacao) encodes 14 PR-1 proteins, named TcPR-1a to TcPR-1n. Two of them, TcPR-1f and TcPR-1g, have a C-terminal expansion with high similarity to protein kinase domains, suggesting a receptor-like kinase (RLK) protein architecture. Moreover, TcPR-1g is highly expressed during cacao response to Witches' Broom Disease, caused by the fungus Moniliopthora perniciosa. Here we describe a structural genomics approach to clone, express and purify the kinase domains of TcPR-1f and TcPR-1g. Escherichia coli BL21(DE3)-R3 cells were used for protein expression and co-expression of Lambda Protein Phosphatase was critical for successfully obtaining soluble recombinant protein. We expect that the ability to express and purify the kinase domains of TcPR-1f and TcPR-1g will further our understanding of the role these proteins play during cacao defense response.