Identification of Potential Multitarget Compounds against Alzheimer's Disease through Pharmacophore-Based Virtual Screening.

Alzheimer's disease (AD) is a neurodegenerative disease characterized by progressive loss of cognitive functions, and it is the most prevalent type of dementia worldwide, accounting for 60 to 70% of cases. The pathogenesis of AD seems to involve three main factors: deficiency in cholinergic transmission, formation of extracellular deposits of ß-amyloid peptide, and accumulation of deposits of a phosphorylated form of the TAU protein. The currently available drugs are prescribed for symptomatic treatment and present adverse effects such as hepatotoxicity, hypertension, and weight loss. There is urgency in finding new drugs capable of preventing the progress of the disease, controlling the symptoms, and increasing the survival of patients with AD. This study aims to present new multipurpose compounds capable of simultaneously inhibiting acetylcholinesterase (AChE), butyrylcholinesterase (BChE)-responsible for recycling acetylcholine in the synaptic cleft-and beta-secretase 1 (BACE-1)-responsible for the generation of amyloid-ß plaques. AChE, BChE, and BACE-1 are currently considered the best targets for the treatment of patients with AD. Virtual hierarchical screening based on a pharmacophoric model for BACE-1 inhibitors and a dual pharmacophoric model for AChE and BChE inhibitors were used to filter 214,446 molecules by QFITBACE > 0 and QFITDUAL > 56.34. The molecules selected in this first round were subjected to molecular docking studies with the three targets and further evaluated for their physicochemical and toxicological properties. Three structures: ZINC45068352, ZINC03873986, and ZINC71787288 were selected as good fits for the pharmacophore models, with ZINC03873986 being ultimately prioritized for validation through activity testing and synthesis of derivatives for SAR studies.

Penicillium Ochrochloron RLS11 Secretome Containing Carbohydrate-Active Enzymes Improves Commercial Enzyme Mixtures During Sugarcane Straw Saccharification.

Filamentous fungi are prolific producers of carbohydrate-active enzymes (CAZymes) and important agents that carry out plant cell wall degradation in natural environments. The number of fungal species is frequently reported in the millions range, with a huge diversity and genetic variability, reflecting on a vast repertoire of CAZymes that these organisms can produce. In this study, we evaluated the ability of previously selected ascomycete and basidiomycete fungi to produce plant cell wall-degrading enzyme (PCWDE) activities and the potential of the culture supernatants to increase the efficiency of the Cellic® CTec2/HTec2 for steam-exploded sugarcane straw saccharification. The culture supernatant of Penicillium ochrochloron RLS11 showed a promising supplementation effect on Cellic® CTec2/HTec2, and we conducted the whole-genome sequencing and proteomic analysis for this fungus. The size of the assembled genome was 38.06 Mbp, and a total of 12,015 protein-coding genes were identified. The repertoire of PCWDE-coding genes was comparatively high among Penicillium spp. and showed an expansion in important cellulases and xylanases families, such as GH3, GH6, GH7, and GH11. The proteomic analysis indicated cellulases that probably enhanced the biomass saccharification performance of the Cellic® CTec2/HTec2, which included enzymes from GH3, GH6, and GH7 families.

A Chrysoporthe cubensis enzyme cocktail produced from a low-cost carbon source with high biomass hydrolysis efficiency.

Low cost and high efficiency cellulolytic cocktails can consolidate lignocellulosic ethanol technologies. Sugarcane bagasse (SCB) is a low cost agro-industrial residue, and its use as a carbon source can reduce the costs of fungi cultivation for enzyme production. Chrysoporthe cubensis grown under solid state fermentation (SSF) with wheat bran has potential to produce efficient enzymatic extracts for SCB saccharification. This fungus was grown under submersed fermentation (SmF) and SSF with in natura SCB, pretreated with acid or alkali and with others carbon sources. In natura SCB induced the highest carboxymethylcellulase (CMCase), xylanase, ß-xylosidase, α-galactosidase and mannanase activities by C. cubensis under SSF. In natura and washed SCB, inducers of enzyme production under SSF, did not induce high cellulases and hemicellulases production by C. cubensis in SmF. The C. cubensis enzymatic extract produced under SSF with in natura SCB as a carbon source was more efficient for lignocelulolic biomass hydrolysis than extracts produced under SSF with wheat bran and commercial cellulolytic extract. Chrysoporthe cubensis showed high potential for cellulases and hemicellulases production, especially when grown under SSF with in natura SCB as carbon source.

Purification and Characterization of Xylanases from the Fungus Chrysoporthe cubensis for Production of Xylooligosaccharides and Fermentable Sugars.

Xylanases from the pathogen fungus Chrysoporthe cubensis were produced under solid state fermentation (SSF) using wheat bran as carbon source. The enzymatic extracts were submitted to ion exchange (Q Sepharose) and gel filtration chromatography methods (Sephadex S-200) for purification. The xylanases were divided into three groups: P1 showed better performance at 60 °C and pH 4.0, P2 at 55 °C and pH 3.0, and P3 at 80 °C and pH 3.0. Oat spelt xylan was the best substrate hydrolyzed by P1 and P3, while beechwood xylan was better degraded by P2. Carboxymethyl cellulose (CMC) and p-nitrophenyl-ß-D-xylopyranoside (p-NPßXyl) were not hydrolyzed by any of the xylanases. The K M' or K M values, using oat spelt xylan as substrate, were 2.65 mg/mL for P1, 1.81 mg/mL for P2, and 1.18 mg/mL for P3. Xylobiose and xylotriose were the main xylooligosaccharides of oat spelt xylan degradation, indicating that the xylanases act as endo-ß-1,4-xylanases. Xylanases also proved to be efficient for hydrolysis of sugarcane bagasse when used as supplement of a commercial cocktail due to the increase of the reducing sugar release.

Production and application of an enzyme blend from Chrysoporthe cubensis and Penicillium pinophilum with potential for hydrolysis of sugarcane bagasse.

Blending of the enzyme extracts produced by different fungi can result in favorable synergetic enhancement of the enzyme blend with regards to the main cellulase activities, as well as the inclusion of accessory enzymes that may not be as abundant in enzyme extracts produced by predominantly cellulase producing fungi. The Chrysoporthe cubensis:Penicillium pinophilum 50:50 (v/v) blend produced herein presented good synergy, especially for FPase and endoglucanase activities which were 76% and 48% greater than theoretical, respectively. This enzyme blend was applied to sugarcane bagasse previously submitted to a simple alkali pretreatment. Glucan hydrolysis efficiency reached an excess of 60% and xylan conversion exceeded 90%. Increasing the hydrolysis temperature from 45 to 50°C also resulted in a 16-20% increase in conversion of both glucan and xylan fractions. The blended enzyme extract obtained therefore showed great potential for application in the lignocellulose hydrolysis process.

Chrysoporthe cubensis: a new source of cellulases and hemicellulases to application in biomass saccharification processes.

The plant pathogenic fungus Chrysoporthe cubensis was cultivated under solid state employing different substrates and the highest endoglucanase (33.84Ug(-1)), FPase (2.52Ug(-1)), ß-glucosidase (21.55Ug(-1)) and xylanase (362.38Ug(-1)) activities were obtained using wheat bran as carbon source. Cellulases and xylanase produced by C. cubensis showed maximal hydrolysis rate at pH 4.0 and in a temperature range of 50-60°C. All enzymatic activities were highly stable at 40 and 50°C through 48h of pre-incubation. Saccharification of alkaline pretreated sugarcane bagasse by crude enzyme extract from C. cubensis resulted in release of 320.8mg/g and 288.7mg/g of glucose and xylose, respectively. On another hand, a similar assay employing commercial cellulase preparation resulted in release of 250.6mg/g and 62.1mg/g of glucose and xylose, respectively. Cellulolytic extract from C. cubensis showed a great potential to be used in biomass saccharification processes.

Characterization of cellulolytic extract from Pycnoporus sanguineus PF-2 and its application in biomass saccharification.

The aim of this work was to evaluate the biochemical features of the white-rot fungi Pycnoporus sanguineus cellulolytic complex and its utilization to sugarcane bagasse hydrolysis. When cultivated under submerged fermentation using corn cobs as carbon source, P. sanguineus produced high FPase, endoglucanase, ß-glucosidase, xylanase, mannanase, α-galactosidase, α-arabinofuranosidase, and polygalacturonase activities. Cellulase activities were characterized in relation to pH and temperature. ß-Glucosidase and FPase activities were higher at 55 °C, pH 4.5, and endoglucanase activity was higher at 60 °C, in a pH range of 3.5-4.0. All cellulase activities were highly stable at 40 and 50 °C through 48 h of pre-incubation. Crude enzymatic extract from P. sanguineus was applied in a saccharification experiment using acid-treated and alkali-treated sugarcane bagasse as substrate, and the hydrolysis yields were compared to that obtained by a commercial cellulase preparation. Reducing sugar yields of 60.4% and 64.0% were reached when alkali-treated bagasse was hydrolyzed by P. sanguineus extract and commercial cellulase, respectively. Considering the glucose production, it was observed that P. sanguineus extract and commercial cellulase ensured yields of 22.6% and 36.5%, respectively. The saccharification of acid-treated bagasse was lower than that of alkali-treated bagasse regardless of the cellulolytic extract. The present work showed that P. sanguineus has a great potential as an enzyme producer for biomass saccharification.

Treinamento em natação atenua a disfução contrátil de cardiomiócitos de ratos diabéticos / Swimming training attenuates contractile dysfunction in diabetic rat cardiomyocytes / Entrenamiento en natación atenúa la disfunción contráctil de cardiomiocitos de ratones diabéticos

FUNDAMENTO: O diabete experimental promove disfunção contrátil em cardiomiócitos, mas os efeitos do treinamento em natação nesta disfunção não são conhecidos. OBJETIVO: Testar os efeitos de um programa de treino em natação (PTN) sobre a disfunção contrátil de cardiomiócitos de ratos com diabete experimental. MÉTODOS: Ratos Wistar (idade: 30 dias; peso corporal médio: 84,19 g) com diabete induzida por estreptozotocina (60 mg/kg de peso corporal; glicemia > 300 mg/dl) foram alocados em diabéticos sedentários (DS, n = 10) e diabéticos exercitados (DE, n = 13). Animais da mesma idade e peso serviram de controles sedentários (CS, n = 10) e controles exercitados (CE, n = 06). Os animais DE e CE foram submetidos a um PTN (05 dias/semana, 90 min/dia), por 08 semanas. Os miócitos do ventrículo esquerdo (VE) foram isolados e estimulados eletricamente a 3,0 Hz em temperatura ambiente (∼ 25º C). RESULTADOS: O diabete reduziu a função contrátil nos cardiomiócitos dos animais em relação aos controles (i.e., menor amplitude de contração, maior tempo de contração e relaxamento). O PTN atenuou a redução na amplitude de contração (CS, 11 ± 0,2 por cento vs DE, 11,6 ± 0,2 por cento), o tempo para o pico de contração (CS, 319 ± 5,8 ms vs DE, 333 ± 4,8 ms) e o tempo para 50 por cento de relaxamento (CS, 619 ± 22,2 ms vs DE, 698 ± 18,6 ms) dos cardiomiócitos dos animais diabéticos. O diabete reduziu as dimensões dos cardiomiócitos, porém, o PTN minimizou a redução da largura e volume celular, sem alterar o comprimento. CONCLUSÃO: O programa de treino em natação atenuou a disfunção contrátil dos miócitos do VE de ratos com diabete experimental.

BACKGROUND: Experimental diabetes promotes contractile dysfunction in cardiomyocytes, but the effects of swimming in this disorder are not known. OBJECTIVE: To test the effects of a swimming training program (STP) on cardiomyocyte contractile dysfunction in rats with experimental diabetes. METHODS: Wistar rats (age: 30 days; mean body weight: 84.19 g) with diabetes induced by streptozotocin (60 mg/kg body weight; glucose > 300 mg/dl) were divided into sedentary diabetic rats (SD, n = 10) and exercised diabetic rats (ED, n = 13). Animals of same age and weight served as sedentary controls (SC, n = 10) and exercised controls (EC, n = 06). Animals and ED and EC underwent a STP (05 days/week, 90 min/day) for 08 weeks. Left ventricular (LV) myocytes were isolated and electrically stimulated at 3.0 Hz at room temperature (∼ 25º C). RESULTS: Diabetes reduced contractile function in cardiomyocytes of animals compared to controls (i.e., lower amplitude of contraction, longer duration of contraction and relaxation). The STP attenuated the reduced amplitude of contraction (SC, 11 ± 0.2 percent vs ED, 11.6 ± 0.2 percent), time to peak contraction (SC, 319 ± 5.8 ms vs ED, 333 ± 4.8 ms) and time to 50.0 percent of relaxation (SC, 619 ± 22.2 ms vs ED 698 ± 18.6 ms) of cardiomyocytes of diabetic rats. Diabetes reduced the size of cardiomyocytes, however, the STP minimized the reduction of cell volume and width, without changing length. CONCLUSION: The swimming training program attenuated the contractile dysfunction of the LV myocytes of rats with experimental diabetes.

FUNDAMENTO: La diabetes experimental promueve el trastorno contráctil de los cardiomiocitos, pero los efectos del entrenamiento en natación en este trastorno no se conocen. OBJETIVO: Probar los efectos de un programa de capacitación en natación (PTN) sobre el trastorno contráctil de cardiomiocitos de ratas con diabetes experimental. MÉTODOS: Ratas Wistar (edad: 30 días, peso corporal medio: 84,19 g) con diabetes inducida por estreptozotocina (60 mg/kg de peso corporal, glucosa > 300 mg/dl) fueron divididos en diabéticos sedentarios (DS, n = 10) y diabéticos ejercitados (DE, n = 13). Animales de la misma edad y peso sirvieron de controles sedentarios (CS, n = 10) y controles ejercitados (CE, n = 06). Los animales DE y CE se sometieron a un PTN (05 días/semana, 90 min/día), por 08 semanas. Los miocitos del ventrículo izquierdo (VI) fueron aislados y estimulados eléctricamente a 3,0 Hz en temperatura ambiente (~ 25º C). RESULTADOS: La diabetes disminuyó la función contráctil de los cardiomiocitos de los animales en comparación con los controles (es decir, menor amplitud de la contracción, la duración de la contracción y relajación). El PTN atenuó la reducción de la amplitud de la contracción (CS, 11 ± 0,2 por ciento frente a la DE, 11,6 ± 0,2 por ciento), el tiempo para la contracción máxima (CS, 319 ± 5,8 ms vs DE, 333 ± 4,8 ms) y el tiempo para el 50 por ciento de relajación (CS, 619 ± 22,2 ms vs DE, 698 ± 18,6 ms) de los cardiomiocitos de los animales diabéticos. La diabetes redujo las dimensiones de los cardiomiocitos, sin embargo, el PTN minimizó la reducción de la anchura y volumen celular, sin cambiar la longitud. CONCLUSIÓN: El programa de entrenamiento de natación atenuó la disfunción contráctil de los miocitos del VI de las ratas con diabetes experimental.

Cellulases and hemicellulases from endophytic Acremonium species and its application on sugarcane bagasse hydrolysis.

The aim of this work was to have cellulase activity and hemicellulase activity screenings of endophyte Acremonium species (Acremonium zeae EA0802 and Acremonium sp. EA0810). Both fungi were cultivated in submerged culture (SC) containing L -arabinose, D -xylose, oat spelt xylan, sugarcane bagasse, or corn straw as carbon source. In solid-state fermentation, it was tested as carbon source sugarcane bagasse or corn straw. The highest FPase, endoglucanase, and xylanase activities were produced by Acremonium sp. EA0810 cultivated in SC containing sugarcane bagasse as a carbon source. The highest ß-glucosidase activity was produced by Acremonium sp. EA0810 cultivated in SC using D -xylose as carbon source. A. zeae EA0802 has highest α-arabinofuranosidase and α-galactosidase activities in SC using xylan as a carbon source. FPase, endoglucanase, ß-glucosidase, and xylanase from Acremonium sp. EA0810 has optimum pH and temperatures of 6.0, 55 °C; 5.0, 70 °C; 4.5, 60 °C; and 6.5, 50 °C, respectively. α-Arabinofuranosidase and α-galactosidase from A. zeae EA0802 has optimum pH and temperatures of 5.0, 60 °C and 4.5, 45 °C, respectively. It was analyzed the application of Acremonium sp. EA0810 to hydrolyze sugarcane bagasse, and it was achieved 63% of conversion into reducing sugar and 42% of conversion into glucose.

Swimming training attenuates contractile dysfunction in diabetic rat cardiomyocytes.

BACKGROUND: Experimental diabetes promotes contractile dysfunction in cardiomyocytes, but the effects of swimming in this disorder are not known. OBJECTIVE: To test the effects of a swimming training program (STP) on cardiomyocyte contractile dysfunction in rats with experimental diabetes. METHODS: Wistar rats (age: 30 days; mean body weight: 84.19 g) with diabetes induced by streptozotocin (60 mg/kg body weight; glucose > 300 mg/dl) were divided into sedentary diabetic rats (SD, n = 10) and exercised diabetic rats (ED, n = 13). Animals of same age and weight served as sedentary controls (SC, n = 10) and exercised controls (EC, n = 06). Animals and ED and EC underwent a STP (05 days/week, 90 min/day) for 08 weeks. Left ventricular (LV) myocytes were isolated and electrically stimulated at 3.0 Hz at room temperature (∼ 25º C). RESULTS: Diabetes reduced contractile function in cardiomyocytes of animals compared to controls (i.e., lower amplitude of contraction, longer duration of contraction and relaxation). The STP attenuated the reduced amplitude of contraction (SC, 11 ± 0.2% vs ED, 11.6 ± 0.2%), time to peak contraction (SC, 319 ± 5.8 ms vs ED, 333 ± 4.8 ms) and time to 50.0% of relaxation (SC, 619 ± 22.2 ms vs ED 698 ± 18.6 ms) of cardiomyocytes of diabetic rats. Diabetes reduced the size of cardiomyocytes, however, the STP minimized the reduction of cell volume and width, without changing length. CONCLUSION: The swimming training program attenuated the contractile dysfunction of the LV myocytes of rats with experimental diabetes.

Purification and characterization of Aspergillus terreus α-galactosidases and their use for hydrolysis of soymilk oligosaccharides.

α-Galactosidases has the potential to hydrolyze α-1-6 linkages in raffinose family oligosaccharides (RFO). Aspergillus terreus cells cultivated on wheat bran produced three extracellular forms of α-galactosidases (E1, E2, and E3). E1 and E2 α-galactosidases presented maximal activities at pH 5, while E3 α-galactosidase was more active at pH 5.5. The E1 and E2 enzymes showed stability for 6 h at pH 4-7. Maximal activities were determined at 60, 55, and 50 °C, for E1, E2, and E3 α-galactosidase, respectively. E2 α-galactosidase retained 90% of its initial activity after 70 h at 50 °C. The enzymes hydrolyzed ρNPGal, melibiose, raffinose and stachyose, and E1 and E2 enzymes were able to hydrolyze guar gum and locust bean gum substrates. E1 and E3 α-galactosidases were completely inhibited by Hg²âº, Ag⁺, and Cu²âº. The treatment of RFO present in soy milk with the enzymes showed that E1 α-galactosidase reduced the stachyose content to zero after 12 h of reaction, while E2 promoted total hydrolysis of raffinose. The complete removal of the oligosaccharides in soy milk could be reached by synergistic action of both enzymes.

Propriedades enzimáticas da enzima ALS de Cyperus difformis e mecanismo de resistência da espécie ao herbicida pyrazosulfuron-ethyl / Enzymatic properties of Cyperus difformis ALS enzyme and mechanism resistance of the species to pyrazosulfuron-ethyl herbicide

Cyperus difformis L. é uma planta daninha ocorrente em lavouras de arroz irrigado, que tem apresentado dificuldade de controle devido à resistência a herbicidas inibidores da enzima ALS. Os objetivos deste trabalho foram investigar características cinéticas da enzima ALS de biótipos de C. difformis e determinar as bases bioquímicas da resistência da espécie ao herbicida pyrazosulfuron-ethyl. Para isso, foram conduzidos experimentos em laboratório do BIOAGRO/UFV. O método utilizado baseou-se na metologia utilizada por CAREY et al. (1997) e adaptada por VARGAS et al. (1999), com algumas modificações. Foram avaliadas a concentração de substrato (piruvato) que fornece velocidade inicial igual à metade da velocidade máxima de reação (K M) e velocidade máxima de reação (Vmáx), bem como a atividade da enzima ALS na presença do inibidor (pyrazosulfuron-ethyl). Diante dos resultados, pode-se observar que a resistência de C. difformis a pyrazosulfuron-ethyl é decorrente da insensibilidade da enzima ALS ao herbicida, não acarretando, porém, prejuízo aos parâmetros cinéticos K M e Vmáx da enzima ALS.

Cyperus difformis L. is a weed that occurs in flooded rice, which has presented difficulty in controlling due to the resistance to ALS inhibiting herbicides. The objectives of this research were to investigate kinetic characteristics of ALS enzyme from C. difformis biotypes and to determine the biochemical bases of resistance from the species to pyrazosulfuron-ethyl herbicide. For that, experiments were conducted at the BIOAGRO/UFV laboratory. The method used was based on the methodology used by CAREY et al. (1997) and adapted by VARGAS et al. (1999), with some modifications. It was evaluated substratum concentration (pyruvate) that provides initial velocity equal to half the speed reaction (K M) and maximum velocity of reaction (Vmáx), as well the activity of the ALS enzyme in the presence of the inhibitor (pyrazosulfuron-ethyl). According to the results, it is possible to observe that C. difformis resistance to pyrazosulfuron-ethyl is due to the insensibility of the ALS enzyme to the herbicide, however without penalty to K M and Vmáx kinetic parameters of the ALS enzyme.

Hydrolysis of galacto-oligosaccharides in soy molasses by α -galactosidases and invertase from Aspergillus terreus

Two α -galactosidase (P1 and P2) and one invertase present in the culture of Aspergillus terreus grown on wheat straw for 168 h at 28ºC were partially purified by gel filtration and hydrophobic interaction chromatographies. Optimum pH and temperatures for P1, P2 and invertase preparations were 4.5-5.0, 5.5 and 4.0 and 60, 55 and 65ºC, respectively. The K M app for ρ -nitrophenyl-α -D-galactopyranoside were 1.32 mM and 0.72 mM for P1 and P2, respectively, while the K M app value for invertase, using sacarose as a substrate was 15.66 mM. Enzyme preparations P1 and P2 maintained their activities after pre-incubation for 3 h at 50ºC and invertase maintained about 90 percent after 6 h at 55 ºC. P1 and P2 presented different inhibition sensitivities by Ag+, D-galactose, and SDS. All enzyme preparations hydrolyzed galacto-ologosaccharides present in soymolasses.

Duas α-galactosidases (P1 e P2) e uma invertase produzidas no sobrenadante da cultura do fungo Aspergillus terreus quando crescido por 168 h a 28ºC com farelo de trigo como fonte de carbono foram parcialmente purificadas por cromatografias de gel filtração e interação hidrofóbica. O pH e temperatura ótimos para as preparações P1, P2 e invertase foram entre 4,5-5,0, 5,5 e 4,0 e 60, 55 e 65ºC, respectivamente. O K M app para ρ-nitrofenil-α-D-galactopiranosideo foi 1.32 mM e 0.72 mM para P1 e P2, respectivamente. O valor de K M app para invertase usando sacarose como substrato foi de 15,66 mM. As preparações enzimáticas P1 e P2 mantiveram suas atividades após 3 h de pré-incubação a 50 ºC e a invertase manteve cerca de 90 por cento após 6 h a 55 ºC. P1 e P2 foram diferentemente sensíveis à inibição por Ag+, D-galactose e SDS. As preparações enzimáticas hidrolisaram os galactooligossacarídeos presentes em melaço de soja.

Covalent immobilization of alpha-galactosidase from Penicillium griseoroseum and its application in oligosaccharides hydrolysis.

Partially purified alpha-Galactosidase from Penicillium griseoroseum was immobilized onto modified silica using glutaraldehyde linkages. The effective activity of immobilized enzyme was 33%. Free and immobilized alpha-galactosidase showed optimal activity at 45 degrees C and pH values of 5 and 4, respectively. Immobilized alpha-galactosidase was more stable at higher temperatures and pH values. Immobilized alpha-galactosidase from P. griseoroseum maintained 100% activity after 24 h of incubation at 40 degrees C, while free enzyme showed only 32% activity under the same incubation conditions. Defatted soybean flour was treated with free and immobilized alpha-galactosidase in batch reactors. After 8 h of incubation, stachyose was completely hydrolyzed in both treatments. After 8 h of incubation, 39% and 70% of raffinose was hydrolyzed with free and immobilized alpha-galactosidase respectively. Immobilized alpha-galactosidase was reutilized eight times without any decrease in its activity.