Antifungal activity of 2-chloro-N-phenylacetamide: a new molecule with fungicidal and antibiofilm activity against fluconazole-resistant Candida spp / Atividade antifúngica de 2-cloro-N-fenilacetamida: uma nova molécula com atividade fungicida e anti-biofilme contra Candida spp. resistentes a fluconazol

In the current context of emerging drug-resistant fungal pathogens such as Candida albicans and Candida parapsilosis, discovery of new antifungal agents is an urgent matter. This research aimed to evaluate the antifungal potential of 2-chloro-N-phenylacetamide against fluconazole-resistant clinical strains of C. albicans and C. parapsilosis. The antifungal activity of 2-chloro-N-phenylacetamide was evaluated in vitro by the determination of the minimum inhibitory concentration (MIC), minimum fungicidal concentration (MFC), inhibition of biofilm formation and its rupture, sorbitol and ergosterol assays, and association between this molecule and common antifungal drugs, amphotericin B and fluconazole. The test product inhibited all strains of C. albicans and C. parapsilosis, with a MIC ranging from 128 to 256 µg.mL-1, and a MFC of 512-1,024 µg.mL-1. It also inhibited up to 92% of biofilm formation and rupture of up to 87% of preformed biofilm. 2-chloro-N-phenylacetamide did not promote antifungal activity through binding to cellular membrane ergosterol nor it damages the fungal cell wall. Antagonism was observed when combining this substance with amphotericin B and fluconazole. The substance exhibited significant antifungal activity by inhibiting both planktonic cells and biofilm of fluconazole-resistant strains. Its combination with other antifungals should be avoided and its mechanism of action remains to be established.

No atual contexto de patógenos fúngicos resistentes emergentes tais como Candida albicans e Candida parapsilosis, a descoberta de novos agentes antifúngicos é uma questão urgente. Esta pesquisa teve como objetivo avaliar o potencial antifúngico da 2-cloro-N-fenilacetamida contra cepas clínicas de C. albicans e C. parapsilosis resistentes a fluconazol. A atividade antifúngica da substância foi avaliada in vitro através da determinação da concentração inibitória mínima (CIM), concentração fungicida mínima (CFM), ruptura e inibição da formação de biofilme, ensaios de sorbitol e ergosterol, e associação entre esta molécula e antifúngicos comuns, anfotericina B e fluconazol. O produto teste inibiu todas as cepas de C. albicans e C. parapsilosis, com uma CIM variando de 128 a 256 µg.mL-1, e uma CFM de 512-1,024 µg.mL-1. Também inibiu até 92% da formação de biofilme e causou a ruptura de até 87% de biofilme pré-formado. A 2-cloro-N-fenilacetamida não promoveu atividade antifúngica pela ligação ao ergosterol da membrana celular fúngica, tampouco danificou a parede celular. Antagonismo foi observado ao combinar esta substância com anfotericina B e fluconazol. A substância exibiu atividade antifúngica significativa ao inibir tanto as células planctônicas quanto o biofilme das cepas resistentes ao fluconazol. Sua combinação com outros antifúngicos deve ser evitada e seu mecanismo de ação deve ser estabelecido.

Antifungal activity of 2-chloro-N-phenylacetamide: a new molecule with fungicidal and antibiofilm activity against fluconazole-resistant Candida spp

Abstract In the current context of emerging drug-resistant fungal pathogens such as Candida albicans and Candida parapsilosis, discovery of new antifungal agents is an urgent matter. This research aimed to evaluate the antifungal potential of 2-chloro-N-phenylacetamide against fluconazole-resistant clinical strains of C. albicans and C. parapsilosis. The antifungal activity of 2-chloro-N-phenylacetamide was evaluated in vitro by the determination of the minimum inhibitory concentration (MIC), minimum fungicidal concentration (MFC), inhibition of biofilm formation and its rupture, sorbitol and ergosterol assays, and association between this molecule and common antifungal drugs, amphotericin B and fluconazole. The test product inhibited all strains of C. albicans and C. parapsilosis, with a MIC ranging from 128 to 256 µg.mL-1, and a MFC of 512-1,024 µg.mL-1. It also inhibited up to 92% of biofilm formation and rupture of up to 87% of preformed biofilm. 2-chloro-N-phenylacetamide did not promote antifungal activity through binding to cellular membrane ergosterol nor it damages the fungal cell wall. Antagonism was observed when combining this substance with amphotericin B and fluconazole. The substance exhibited significant antifungal activity by inhibiting both planktonic cells and biofilm of fluconazole-resistant strains. Its combination with other antifungals should be avoided and its mechanism of action remains to be established.

Resumo No atual contexto de patógenos fúngicos resistentes emergentes tais como Candida albicans e Candida parapsilosis, a descoberta de novos agentes antifúngicos é uma questão urgente. Esta pesquisa teve como objetivo avaliar o potencial antifúngico da 2-cloro-N-fenilacetamida contra cepas clínicas de C. albicans e C. parapsilosis resistentes a fluconazol. A atividade antifúngica da substância foi avaliada in vitro através da determinação da concentração inibitória mínima (CIM), concentração fungicida mínima (CFM), ruptura e inibição da formação de biofilme, ensaios de sorbitol e ergosterol, e associação entre esta molécula e antifúngicos comuns, anfotericina B e fluconazol. O produto teste inibiu todas as cepas de C. albicans e C. parapsilosis, com uma CIM variando de 128 a 256 µg.mL-1, e uma CFM de 512-1,024 µg.mL-1. Também inibiu até 92% da formação de biofilme e causou a ruptura de até 87% de biofilme pré-formado. A 2-cloro-N-fenilacetamida não promoveu atividade antifúngica pela ligação ao ergosterol da membrana celular fúngica, tampouco danificou a parede celular. Antagonismo foi observado ao combinar esta substância com anfotericina B e fluconazol. A substância exibiu atividade antifúngica significativa ao inibir tanto as células planctônicas quanto o biofilme das cepas resistentes ao fluconazol. Sua combinação com outros antifúngicos deve ser evitada e seu mecanismo de ação deve ser estabelecido.

Association study between ceftriaxone and a synthetic amide against strains of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a non-lactose fermenting Gram-negative bacteria responsible for causing numerous nosocomial infections. The present research aimed to analyze the anti-Pseudomonas aeruginosa potential of 2-Chloro-N-(4-fluoro-3-nitrophenyl)acetamide (A8). The antibacterial potential of A8 was evaluated from the Minimum Inhibitory Concentration (MIC), Minimum Bactericidal Concentration (MBC) and Association using the checkerboard method. MIC and MBC values were 512 µg/mL for all P. aeruginosa strains evaluated, demonstrating predominantly bactericidal activity. Furthermore, when A8 was associated with the drug ceftriaxone, pharmacological additivity and indifference were evidenced. In this sense, the synthetic amide was interesting, since it demonstrates the potential to become a possible candidate for an antimicrobial drug.

Genomic and phenotypic features of Acinetobacter baumannii isolated from oil reservoirs reveal a novel subspecies specialized in degrading hazardous hydrocarbons.

The genus Acinetobacter encompasses biotechnologically relevant species and nosocomial pathogens. In this study, nine isolates recovered from different oil reservoir samples showed the ability to grow with petroleum as the only carbon source and possessed the ability to emulsify kerosene. The whole genomes of the nine strains were sequenced and analyzed. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values of all strains were compared to the reference strains, and the results were below the reference values (<97.88 and 82, respectively), suggesting that the isolates belong to a new subspecies of Acinetobacter baumannii. The name Acinetobacter baumannii oleum ficedula is proposed. A comparison of the whole genome repertoire of 290 Acinetobacter species indicated that the strains in this study resemble non-pathogenic Acinetobacter strains. However, the new isolates resemble A. baumannii when comparing virulence factors. The isolates in this study carry many genes involved in hydrocarbon degradation, indicating the potential to degrade most toxic compounds listed by environmental regulatory agencies such as ATSDR, EPA, and CONAMA. In addition, despite the absence of known biosurfactant or bioemulsifier genes, the strains showed emulsifying activity, suggesting the presence of new pathways or genes related to this process. This study investigated the genomic, phenotypic, and biochemical features of the novel environmental subspecies A. baumannii oleum ficedula, revealing their potential to degrade hydrocarbons and to produce biosurfactants or bioemulsifiers. Applying these environmental subspecies in bioaugmentation strategies sheds light on future approaches to bioremediation. The study shows the importance of genomic analysis of environmental strains and their inclusion in metabolic pathways databases, highlighting unique enzymes/alternative pathways for consuming hazardous hydrocarbons.

Antifungal activity of 2-chloro-N-phenylacetamide: a new molecule with fungicidal and antibiofilm activity against fluconazole-resistant Candida spp.

In the current context of emerging drug-resistant fungal pathogens such as Candida albicans and Candida parapsilosis, discovery of new antifungal agents is an urgent matter. This research aimed to evaluate the antifungal potential of 2-chloro-N-phenylacetamide against fluconazole-resistant clinical strains of C. albicans and C. parapsilosis. The antifungal activity of 2-chloro-N-phenylacetamide was evaluated in vitro by the determination of the minimum inhibitory concentration (MIC), minimum fungicidal concentration (MFC), inhibition of biofilm formation and its rupture, sorbitol and ergosterol assays, and association between this molecule and common antifungal drugs, amphotericin B and fluconazole. The test product inhibited all strains of C. albicans and C. parapsilosis, with a MIC ranging from 128 to 256 µg.mL-1, and a MFC of 512-1,024 µg.mL-1. It also inhibited up to 92% of biofilm formation and rupture of up to 87% of preformed biofilm. 2-chloro-N-phenylacetamide did not promote antifungal activity through binding to cellular membrane ergosterol nor it damages the fungal cell wall. Antagonism was observed when combining this substance with amphotericin B and fluconazole. The substance exhibited significant antifungal activity by inhibiting both planktonic cells and biofilm of fluconazole-resistant strains. Its combination with other antifungals should be avoided and its mechanism of action remains to be established.

Intravaginal progesterone device (1. 9g) and estradiol benzoate for follicular control in the mare during spring and summer / [Implante intravaginal de progesterona (1, 9g) e benzoato de estradiol no controle folicular de éguas durante a primavera e o verão]

The objective of this study was to evaluate follicular growth and ovulatory rates in mares treated with an intravaginal progesterone device (P4) during the 10-day period, associated with the use of estradiol benzoate (EB). The results were compared during the transition period (ET) in the spring and the breeding season in the summer (ER). The variables were submitted to ANOVA (Tukey's test), considering P<0.05. No ovulation occurred during the permanence of the P4 implant in both experimental periods. The ovulatory rate in the ER was 100% (n = 8) and in the ET 62.5% (n = 5; P = 0.0547). Significant differences were observed (<0.001), in both periods, comparing follicular growth rates during the permanence of P4 device (ER: 1.33 ± 0.89mm/d; ET: 1.00 ± 0.81mm/d) to the period without P4 (ER: 3.63 ± 1.33 mm/d; ET: 3.31 ± 1.66 mm/d). The present study demonstrated applicability and efficiency of a hormonal protocol using P4 intravaginal device and EB for follicular control in mares, both during ET and ER.

O objetivo deste trabalho foi avaliar a taxa de crescimento folicular e a taxa ovulatória em éguas tratadas com dispositivo intravaginal de progesterona (P4) durante o período de 10 dias, associado à utilização de benzoato de estradiol (BE). Os resultados foram comparados durante o período de transição (ET) da primavera com a época de reprodução no verão (ER). As variáveis foram submetidas à ANOVA (teste de Tukey), considerando-se P<0,05. Nenhuma ovulação ocorreu durante a permanência do dispositivo de P4 em ambos os períodos experimentais. A taxa ovulatória na ER foi de 100% (n = 8) e na ET, de 62,5% (n=5; P=0,0547). Diferença significativas (<0,001) foram observadas, em ambos os períodos experimentais, comparando as taxas de crescimento folicular durante a permanência da P4 (ER: 1,33 ± 0,89mm/d; ET: 1,00 ± 0,81mm/d) com o período sem P4 (ER: 3,63 ± 1,33mm/d; ET: 3,31 ± 1,66mm/d). O presente estudo demonstrou aplicabilidade e eficiência do protocolo hormonal utilizando dispositivo intravaginal de P4 e BE para controle folicular de éguas, tanto na ET quanto na ER.

The importance of updated criteria in breeding soundness examination of commercial Nellore bulls for the herd reproductive improvement / [Importância de critérios atualizados na avaliação andrológica de touros Nelore a campo para a evolução reprodutiva do rebanho]

The aim of this work was to compare results of breeding soundness examination (BSE) of Nellore bulls (n=1257) according to evaluation criteria from two different classification tables (traditional-Table1 used since 1997 and an updated-Table2-proposed in 2020). Data were separated into 3 categories: questionable animals in Table1 and Table2 (Q1Q2), animals approved in Table1 and questionable in Table2 (A1Q2) and animals approved in Table1 and Table2 (A1A2). BSE parameters were submitted to ANOVA (P<005), according to age groups. Higher (P<0.0001) scrotal perimeter (PE) were observed in A1A2 category (18-24m=33.4±2.4cm; 24-36m=34.5±2.2cm; 36-48m=36.6±1.7cm; >48m=38.6±1.7cm) compared to A1Q2 (18-24m=29.05±0.98cm; 24-36m=30.3±0.6cm; 36-48m=32.9±1.0cm; >48m=34.8±1.0cm) and to Q1Q2 (24-36m=26.8±2.0cm; 36-48m=30.0±0.1cm; >48m=31.3±1.1cm), for all age groups. At the age of 36-48months (Q1Q2=2.7±0.3; A1Q2=3.2±0.3; A1A2=3.3±0.6) and >48months (Q1Q2=3.0±0.4; A1Q2=3.3±0.5; A1A2=3.4±0.5), animals with better andrological classifications presented higher (P<0.05) body condition score (BCS). Additionally, at age >48m, higher sperm Motility (P=0.0250) and Vigor (P=0.0335) were observed in animals A1Q2 (Mot=55.5±14.7%; V=3.21±0.82) and A1A2 (Mot=55.8±12.2%; V=3.23±0.81) compared to Q1Q2 (Mot=50.2±17.4%; V=2.77±0.82). It was concluded that bulls approved using strict selection criteria demonstrated higher PE and BCS, regardless of the age. The utilization of updated classification tables is highly recommended for further reproductive potential development of Nellore bulls in the field.(AU)

O objetivo deste estudo foi comparar os resultados obtidos no exame andrológico a campo de touros Nelore (n=1257) de acordo com os critérios de avaliação de duas tabelas de classificação (uma tabela tradicional - tabela 1 - proposta em 1997 e uma nova tabela atualizada - tabela 2 - proposta em 2020). Os dados foram separados em três categorias: animais questionáveis nas tabelas 1 e 2 (Q1Q2), animais aprovados na tabela 1 e questionáveis na tabela 2 (A1Q2) e animais aprovados nas tabelas 1 e 2 (A1A2). Os parâmetros foram submetidos à análise de variância (P<0,05), por faixa etária. Observou-se maior (P<0,0001) PE no grupo A1A2 (18-24m=33,4±2,4cm; 24-36m=34,5±2,2cm; 36-48m=36,6±1,7cm; >48m=38,6±1,7cm) em comparação ao grupo A1Q2 (18-24m=29,05±0,98cm; 24-36m=30,3±0,6cm; 36-48m=32,9±1,0cm; >48m=34,8±1,0cm) e este maior (P<0,0001) que Q1Q2 (24-36m=26,8±2,0cm; 36-48m=30,0±0,1cm; >48m=31,3±1,1cm) em todas as idades. Nas faixas etárias 36-48m (Q1Q2=2,7±0,3; A1Q2=3,2±0,3; A1A2=3,3±0,6) e >48m (Q1Q2=3,0±0,4;A1Q2=3,3±0,5; A1A2=3,4±0,5), animais com melhor classificação andrológica apresentaram melhor (P<0,05) escore de condição corporal (ECC). Adicionalmente, na idade >48m, maiores motilidade (P=0,0250) e vigor (P=0,0335) foram observados nos animais A1Q2 (Mot=55,5±14,7%; V=3,21±0,82) e A1A2 (Mot=55,8±12,2%; V=3,23±0,81) comparados aos animais Q1Q2 (Mot=50,2±17,4%; V=2,77±0,82). Concluiu-se que touros aprovados na tabela com critérios mais rigorosos de classificação (tabela 2) apresentaram maior PE e ECC, independentemente da idade. Assim, a utilização de tabelas classificatórias atualizadas é fundamental para maior desenvolvimento do potencial reprodutivo de touros Nelore a campo.(AU)

Braylin induces a potent vasorelaxation, involving distinct mechanisms in superior mesenteric and iliac arteries of rats.

Arterial hypertension is a risk factor for various cardiovascular and renal diseases, representing a major public health challenge. Although a wide range of treatment options are available for blood pressure control, many hypertensive individuals remain with uncontrolled hypertension. Thus, the search for new substances with antihypertensive potential becomes necessary. Coumarins, a group of polyphenolic compounds derived from plants, have attracted intense interest due to their diverse pharmacological properties, like potent antihypertensive activities. Braylin (6-methoxyseselin) is a coumarin identified in the Zanthoxylum tingoassuiba species, described as a phosphodiesterase-4 (PDE4) inhibitor. Although different coumarin compounds have been described as potent antihypertensive agents, the activity of braylin on the cardiovascular system has yet to be investigated. To investigate the vasorelaxation properties of braylin and its possible mechanisms of action, we performed in vitro studies using superior mesenteric arteries and the iliac arteries isolated from rats. In this study, we demonstrated, for the first time, that braylin induces potent vasorelaxation, involving distinct mechanisms from two different arteries, isolated from rats. A possible inhibition of phosphodiesterase, altering the cyclic adenosine monophosphate (cAMP)/cAMP-dependent protein kinase (PKA) pathway, may be correlated with the biological action of braylin in the mesenteric vessel, while in the iliac artery, the biological action of braylin may be correlated with increase of cyclic guanosine monophosphate (cGMP), followed by BKCa, Kir, and Kv channel activation. Together, these results provide evidence that braylin can represent a potential therapeutic use in preventing and treating cardiovascular diseases.

Intussuscepção uterina em uma cadela sem raça definida - relato de caso / Uterine intussusception in a crossbred female dog - case report

O presente relato descreve uma condição rara de intussuscepção uterina em uma cadela sem raça definida, quatro anos de idade, diagnosticada por meio de celiotomia exploratória e análise anatomopatológica. Foi realizada ovariossalpingo-histerectomia (OSH) como tratamento. Essa patologia geralmente requer manejo cirúrgico porque o diagnóstico inicial pode ser desafiador.(AU)

The present study describes a rare condition of uterine intussusception in a 4 year old crossbred female dog diagnosed by exploratory celiotomy and anatomopathological analysis. As treatment, a ovariohysterectomy (OH) was performed. This pathology usually requires surgical management because the initial diagnosis can be challenging.(AU)

Efeito da adição de plasma seminal oriundo de animais de alta e baixa fertilidade na criopreservação de espermatozoides da cauda do epidídimo e do ejaculado de garanhões subférteis / Effect of seminal plasma addition from high and low fertility animals on the cryopreservation of epididymal tail and ejaculated sperm from subfertile stallions

O objetivo do presente estudo foi avaliar o efeito da adição de plasma seminal de garanhões de alta e baixa fertilidade sobre a congelabilidade e a viabilidade de espermatozoides do ejaculado (EJ) e do epidídimo (EP) de garanhões subférteis. Foram utilizados seis garanhões com histórico de subfertilidade. Após coleta, espermatozoides do ejaculado foram divididos em três alíquotas: BotuSêmen® (EJ-CT); plasma seminal de alta qualidade espermática (EJ-PS1); e plasma seminal de baixa qualidade espermática (EJ-PS2). O mesmo protocolo foi realizado com espermatozoides da cauda do epidídimo após orquiectomia (EP-CT; EP-PS1; EP-PS2). Foram realizadas avaliações da cinética espermática pelo CASA e análises de integridade de membrana, acrossoma, fragmentação de DNA, capacitação espermática e peroxidação espermática por citometria de fluxo. Não foram observadas diferenças na cinética espermática entre EJ e EP, logo após a descongelação. Porém, foi observada maior (P<0,05) porcentagem de células com membranas plasmática e acrossomal íntegras nos grupos EP (EP-CT:31,7±7,5b; EP-PS1:35,2±7,0b; EP-PS2:33,9±7,2b) em comparação aos grupos EJ (EJ-CT:15,1±4,9a; EJ-PS1:11,7±4,5a; EJ-PS2:13,1±5,2a). Adicionalmente, foram observadas diferenças no índice de fragmentação de DNA (EJ-CT:2,6±0,6a; EJ-PS1:2,4±0,8a; EJ-PS2:3,0±0,8a; EP-CT:1,4±0,4b; EP-PS1:1,2±0,3b; EP-PS2:1,3±0,2b). Concluiu-se que a adição de 20% de plasma seminal, oriundo de animais férteis ou subférteis, previamente à congelação de espermatozoides epidídimários de animais subférteis não interfere na qualidade espermática.(AU)

The aim of this study was to compare the effect of the addition of seminal plasma from high and low fertility stallions on sperm viability of frozen-thawed sperm cells from ejaculate and from epididymal tail of subfertile stallions. Six stallions with a history of subfertility were used. After collection, ejaculate spermatozoa were divided into three aliquots: Botu-Semen® (EJ-CT); High-quality seminal plasma (EJ-PS1); Low-quality seminal plasma (EJ-PS2). The same was done with sperm cells from epididymis tail after orchiectomy (EP-CT; EP-PS1; EP-PS2). Evaluations of sperm kinetics were assessed by CASA and membrane and acrosome integrity, DNA fragmentation, sperm capacitation and sperm peroxidation were assessed by flow cytometry. After thawing, no differences were observed between ejaculated sperm (EJ) and epididymal sperm (EP) in any CASA evaluations. However, higher (P< 0.05) percentage of cells with intact plasma and acrossomal membranes was observed in EP groups (EP-CT:31.7±7.5b; EP-PS1:35.2±7.0b; EP-PS2:33.9±7.2b) compared to EJ groups (EJ-CT:15.1±4.9a, EJ-PS1:11.7±4.5a, EJ-PS2:13.1±5,2a). In addition, differences in DNA fragmentation index were observed (EJ-CT:2.6±0.6a; EJ-PS1:2.4±0.8a; EJ-PS2:3.0±0.8a; CT:1.4±0.4b; EP-PS1:1.2±0.3b; EP-PS2:1.3±0.2b). It was concluded that the addition of 20% seminal plasma from fertile or subfertile animals prior to the freezing of epididymal spermatozoa from subfertile animals does not interfere in sperm quality.(AU)

Acetylation as a major determinant to microtubule-dependent autophagy: Relevance to Alzheimer's and Parkinson disease pathology.

Protein post-translational modifications (PTMs) that potentiate protein aggregation have been implicated in several neurological disorders, including Alzheimer's (AD) and Parkinson's disease (PD). In fact, Tau and alpha-synuclein (ASYN) undergo several PTMs potentiating their aggregation and neurotoxicity. Recent data posits a role for acetylation in Tau and ASYN aggregation. Herein we aimed to clarify the role of Sirtuin-2 (SIRT2) and HDAC6 tubulin deacetylases as well as p300 acetyltransferase in AD and PD neurodegeneration. We used transmitochondrial cybrids that recapitulate pathogenic alterations observed in sporadic PD and AD patient brains and ASYN and Tau cellular models. We confirmed that Tau protein and ASYN are microtubules (MTs)-associated proteins (MAPs). Moreover, our results suggest that α-tubulin acetylation induced by SIRT2 inhibition is functionally associated with the improvement of MT dynamic determined by decreased Tau phosphorylation and by increased Tau/tubulin and ASYN/tubulin binding. Our data provide a strong evidence for a functional role of tubulin and MAPs acetylation on autophagic vesicular traffic and cargo clearance. Additionally, we showed that an accumulation of ASYN oligomers imbalance mitochondrial dynamics, which further compromise autophagy. We also demonstrated that an increase in Tau acetylation is associated with Tau phosphorylation. We found that p300, HDAC6 and SIRT2 influences Tau phosphorylation and autophagic flux in AD. In addition, we demonstrated that p300 and HDAC6 modulate Tau and Tubulin acetylation. Overall, our data disclose the role of Tau and ASYN modifications through acetylation in AD and PD pathology, respectively. Moreover, this study indicates that MTs can be a promising therapeutic target in the field of neurodegenerative disorders in which intracellular transport is altered.

Recycle of Immobilized Endocellulases in Different Conditions for Cellulose Hydrolysis.

The immobilization of cellulases could be an economical alternative for cost reduction of enzyme application. The derivatives obtained in the immobilization derivatives were evaluated in recycles of paper filter hydrolysis. The immobilization process showed that the enzyme recycles were influenced by the shape (drop or sheet) and type of the mixture. The enzyme was recycled 28 times for sheets E' and 13 times for drops B'. The derivative E' showed the highest stability in the recycle obtaining 0.05 FPU/g, RA of 10%, and FPU Yield of 1.64 times, higher than FPU spent or Net FPU Yield of 5.3 times, saving more active enzymes. The derivative B showed stability in recycles reaching 0.15 FPU/g of derivative, yield of Recovered Activity (RA) of 25%, and FPU Yield of 1.57 times, higher than FPU spent on immobilization or Net PFU Yield of 2.81 times. The latex increased stability and resistance of the drops but did not improve the FPU/gram of derivative.

NO production and potassium channels activation induced by Crotalus durissus cascavella underlie mesenteric artery relaxation.

Animal toxins are natural resources for pharmacological studies. The venom of Crotalus durissus cascavella (C.d. cascavella) may be a source in the bio-prospecting of new anti-hypertensive agents. The aim of this study was to investigate vascular effects of the venom of C.d. cascavella in normotensive rats. Studies were performed using isolated mesenteric artery segments and aortic endothelial cells. The cumulative administration of the venom of C.d. cascavella (0.001-30 µg/mL) on phenylephrine (Phe; 10 µM) pre-contracted rings induced a concentration-dependent vasorelaxation in the presence of vascular endothelium (Emax = 47.9 ± 5.0% n = 8), and its effect was almost abolished in the absence of endothelium (Emax = 5.8± 2.4% n = 5 (∗∗∗p < 0.001)). Tissue viability was maintained as there was no difference in the contractile capacity of rings before and after the administration of venom. The vasorelaxant effect of the venom was also abolished when arteries were pre-contracted with potassium chloride (KCl; 80 mM) (Emax = 6.4± 0.9% n = 5, ∗∗∗p < 0.001). When assessing the participation of endothelium-derived relaxing factors, it was noted that non-selective COX inhibition with indomethacin (10 µM) caused a significant reduction in the vasorelaxant effect of C.d. cascavella (*p < 0.05). When investigating the participation of NO released by endothelium, there was a significant reduction of the vasorelaxant effect of venom in rings treated with L-NAME (100 µM; Emax = 17.5± 2.2% n = 6; **p < 0.01). Similar results were noted in the presence of ODQ (10 µM), an inhibitor of soluble guanylyl cyclase (Emax = 11.2± 3.5%, n = 6) and PTIO (100 µM), a stable radical scavenger for nitric oxide (Emax = 10.77± 3.6%, n = 6). Moreover, the venom induced the release of NO by isolated aortic endothelial cells through amperometric studies. When assessing the participation of K+ channels on the vasodilatory response of the venom, tyrode solution with 20 mM of KCl caused a significant reduction in the relaxation response (p < 0.001) (Emax = 21.3 ± 8%, n = 7), as did inhibitor of delayed rectifier K+ channels (4-amynopiridine 1 mM; Emax = 9.5 ± 1.3, %, n = 5, ***p < 0.001), and vasorelaxation was almost abolished in the presence of Iberiotoxin (IbTx 100 nM). Therefore, these results suggest that the venom of C.d. cascavella induces vasorelaxation in superior mesenteric artery rings of normotensive rats in an endothelium-dependent manner. Specifically, the venom stimulates the generation of endothelium-derived relaxing factors, especially NO, and activates vascular smooth muscle hyperpolarization through K+ channels. These data illustrate that C.d. cascavella is a source of bioactive molecules and therefore has therapeutic potential in the treatment of cardiovascular diseases such as hypertension.

Determination of partition coefficients of biomolecules in a microfluidic aqueous two phase system platform using fluorescence microscopy.

Aqueous two phase systems (ATPS) offer great potential for selective separation of a wide range of biomolecules by exploring differences in molecular solubility in each of the two immiscible phases. However, ATPS use has been limited due to the difficulty in predicting the behavior of a given biomolecule in the partition environment together with the empirical and time-consuming techniques that are used for the determination of partition and extraction parameters. In this work, a fast and novel technique based on a microfluidic platform and using fluorescence microscopy was developed to determine the partition coefficients of biomolecules in different ATPS. This method consists of using a microfluidic device with a single microchannel and three inlets. In two of the inlets, solutions containing the ATPS forming components were loaded while the third inlet was fed with the FITC tagged biomolecule of interest prepared in milli-Q water. Using fluorescence microscopy, it was possible to follow the location of the FITC-tagged biomolecule and, by simply varying the pumping rates of the solutions, to quickly test a wide variety of ATPS compositions. The ATPS system is allowed 4min for stabilization and fluorescence micrographs are used to determine the partition coefficient.The partition coefficients obtained were shown to be consistent with results from macroscale ATPS partition. This process allows for faster screening of partition coefficients using only a few microliters of material for each ATPS composition and is amenable to automation. The partitioning behavior of several biomolecules with molecular weights (MW) ranging from 5.8 to 150kDa, and isoelectric points (pI) ranging from 4.7 to 6.4 was investigated, as well as the effect of the molecular weight of the polymer ATPS component.

Mitochondrial Metabolism Power SIRT2-Dependent Deficient Traffic Causing Alzheimer's-Disease Related Pathology.

Multiple lines of evidence state a major role for mitochondrial dysfunction in sporadic Alzheimer's disease (AD) etiopathogenesis. However, the molecular mechanism(s) triggered by mitochondrial deficits that lead to neurodegeneration remain elusive. Herein, we propose a new mechanism by which mitochondrial loss of potential leads to a dysfunction in autophagy/mitophagy due to the overactivation of SIRT2, a tubulin deacetylase that regulates microtubule network acetylation, and provide insights into the association between metabolism, phosphorylation, and Aß aggregation. We observed an increase in SIRT2 levels and a decrease in the acetylation of lys40 of tubulin in AD cells containing patient mtDNA as well as in AD brains. SIRT2 loss of function either with AK1 (a specific SIRT2 inhibitor) or by SIRT2 knockout recovers microtubule stabilization and improves autophagy, favoring cell survival through the elimination of toxic Aß oligomers. Our data provide strong evidence for a functional role of tubulin acetylation on autophagic vesicle traffic and mitochondria degradation. We propose that SIRT2 inhibition may improve microtubule assembly thus representing a valid approach as disease-modifying therapy for AD.

Evidences of antihypertensive potential of extract from Solanum capsicoides All. in spontaneously hypertensive rats.

BACKGROUND: Solanum capsicoides All. is morphologically similar to Solanum sisymbriifolium Lam. which is used in folk medicine in South America for antihypertensive and diuretics purposes. This similarity has led to species identification errors, which therefore may result in errors by patients. PURPOSE: To evaluate the antihypertensive and diuretics potential of the methanol extract from Solanum capsicoides All. (MeOH-Sc), in vitro and in vivo, in spontaneously hypertensive rats (SHR). METHODS: Initial experiments were performed in rat mesenteric artery to evaluate the in vitro vascular effect of MeOH-Sc and its fractions, in addition to the mechanisms involved during the observed effect. Mean arterial pressure (MAP) and heart rate (HR) were recorded in non-anesthetised hypertensive and normotensive rats. In another set of experiments, MeOH-Sc was administered for 21 consecutive days. Daily body weight measurements were conducted and MAP, HR and urinary volume were measured every 5 days. The mesenteric artery from treated animals was tested for phenylephrine and sodium nitroprussiate (SNP) sensitivity. RESULTS: Initially, MeOH-Sc and fractions relaxed phenylephrine-induced contractions in mesenteric artery rings. The vasorelaxant effect was not changed in the presence of a blocker of eNOS (L-NAME) in rings with an intact endothelium. In denuded-endothelium rings, the vasorelaxant response was significantly reduced in the presence of a cAMP inhibitor (SQ 22536 10 µM) in SHR but not in Wistar Kyoto rats (WKY). However, in the presence of a cGMP inhibitor (ODQ 10 µM), a curve shift to the right was observed in WKY animals, but not in SHR. Intravenous bolus injections of MeOH-Sc into non-anesthetised SHR and WKY, induced hypotension that was associated with an increase in HR. A significant antihypertensive effect was observed in animals that received MeOH-Sc orally for 21 days, which also prevented the development of cardiac hypertrophy. Urine volume from animals treated with MeOH-Sc significantly increased. Finally, MeOH-Sc induced beneficial changes in vascular responsiveness. CONCLUSION: MeOH-Sc has a potential antihypertensive effect in SHR.

Characterization of mesoporous calcium phosphates from calcareous marine sediments containing Si, Sr and Zn for bone tissue engineering.

Calcium phosphates (CAPs) can be produced from either biologically sourced materials or mineral deposits. The raw materials impart unique properties to the CAPs due to innate trace amounts of elements that affect the crystal structure, morphology and stoichiometry. Using calcium carbonate (CaCO3) precursors derived from fossilized calcareous marine sediments (FCMSs), we have synthesized a novel class of CAP biomaterials, termed fm-CaPs, with defined Ca/P molar ratios of 1.4 and 1.7 using a wet synthesis method. Compared with commercially available CAP biomaterials, such as hydroxyapatite (HA) and beta-tricalcium phosphate (ß-TCP), fm-CaP1.7 had a biphasic composition consisting of an HA phase (in a hexagonal system) and a ß-TCP phase (in a rhombohedral crystalline system), which is desirable for the current design of bone substitutes, whereas fm-CaP1.4 consisted of an HA phase and a beta-dicalcium pyrophosphate phase (in a tetragonal system). These bioceramics exhibited a fringe structure of regular crystallographic orientation with well-ordered mesoporous channels. The FCMS raw material imparted trace amounts of silicon (Si), strontium (Sr) and zinc (Zn) to fm-CaPs; these are elements that are important for bone formation. The cyto-compatibility of these biomaterials and their effects on cellular activity were evaluated using osteoblast cells. Cell proliferation assays revealed no signs of cytotoxicity, whereas cells growth was equal to or better than HA and ß-TCP controls. The SEM analysis of the cell and material interactions showed good cell spreading on the fm-CaP materials that was comparable to ß-TCP and in vitro assays suggested robust osteogenic differentiation, as seen by increased mineralization (alizarin red) and upregulation of osteogenic gene expression. Our results indicate that fm-CaP1.7, in particular, has chemical, physical and morphological properties that make this material suitable for applications that promote bone tissue regeneration.

Involvement of the ornithine decarboxylase gene in acid stress response in probiotic Lactobacillus delbrueckii UFV H2b20.

Amino acid decarboxylation is important for the maintenance of intracellular pH under acid stress. This study aims to carry out phylogenetic and expression analysis by real-time PCR of two genes that encode proteins involved in ornithine decarboxylation in Lactobacillus delbrueckii UFV H2b20 exposed to acid stress. Sequencing and phylogeny analysis of genes encoding ornithine decarboxylase and amino acid permease in L. delbrueckii UFV H2b20 showed their high sequence identity (99%) and grouping with those of L. delbrueckii subsp. bulgaricus ATCC 11842. Exposure of L. delbrueckii UFV H2b20 cells in MRS pH 3.5 for 30 and 60 min caused a significant increase in expression of the gene encoding ornithine decarboxylase (up to 8.1 times higher when compared to the control treatment). Increased expression of the ornithine decarboxylase gene demonstrates its involvement in acid stress response in L. delbrueckii UFV H2b20, evidencing that the protein encoded by that gene could be involved in intracellular pH regulation. The results obtained show ornithine decarboxylation as a possible mechanism of adaptation to an acidic environmental condition, a desirable and necessary characteristic for probiotic cultures and certainly important to the survival and persistence of the L. delbrueckii UFV H2b20 in the human gastrointestinal tract.

Máxima velocidade aeróbia calculada pelo custo da frequência cardíaca: relação com a performance / Velocidad aeróbica máxima calculada por el costo del ritmo cardíaco: relación con el rendimiento / Maximal aerobic speed calculated by heart rate cost: relationship with performance

Objetivos. Comparar a máxima velocidade aeróbia (MVA) calculada pelo custo de oxigênio (vVO2max) e o custo da frequência cardíaca (vFCmax) com a medida direta da MVA (Vpico) e verificar a relação entre a vFCmax e a performance em provas de 10 e 15 km de corredores recreacionais de meia idade. Método. Participaram 21 corredores (idade: 30‐49 anos), subdivididos em 2 grupos a partir da idade (G1 e G2). Os participantes foram submetidos a um teste incremental contínuo máximo em laboratório para determinação do consumo máximo de oxigênio. A MVA foi determinada a partir das propostas apresentadas na literatura com base no vVO2max e no vFCmax. Além disso, foram realizadas 2 performances em pista de atletismo (10 e 15 km). Resultados. A menor diferença entre as médias observada para a Vpico foi em relação à vFCmax de Lacour et al. (0,0 km h–1; p > 0,05). A maior diferença foi em relação à vFCmax de Di Prampero (1,55 km h–1; p < 0,05). O mesmo padrão de diferenças foi observado quando analisado o G1 e G2. A vFCmax determinada a partir de 2 diferentes métodos propostos na literatura se correlacionou com as provas de 10 e 15 km (0,55 ≤ r ≤ 0,82; p < 0,05). Conclusões. A vFCmax em corredores recreacionais de meia idade tem elevada relação com as performances de 10 e 15 km e não foi diferente da Vpico (para vFCmax de Lacour et al.), apresentando resultados semelhantes aos observados pelos métodos baseados no custo de oxigênio (AU)

Objetivo: Comparar la velocidad aeróbica máxima (VAM), calculada a través del costo de oxígeno (vVO2max) y del costo de la frecuencia cardíaca (vFCmáx), con la medida directa de la VAM (Vpico) y verificar la relación entre la vFCmax y la performance de 10 e 15 km de corredores recreativos de mediana edad. Método: Participaron 21 corredores recreativos (edades: 30-49 años) subdivididos en 2 grupos por edad (G1 y G2). Los participantes se sometieron a un test incremental continuo máximo en laboratorio para la determinación del consumo máximo de oxígeno. La MVA fue determinada a través de las propuestas presentadas en la literatura basada en el vVO2max y el vFCmáx. Además, se realizaron 2 pruebas en pista de atletismo (10 e 15 km). Resultados: La menor diferencia entre las medias observadas para la Vpico fue en relación con la vFCmax de Lacour et al. (0,0 km h-1; p > 0,05). La mayor diferencia fue en relación con la vFCmax de Di Prampero (1,55 km h-1; p < 0,05). El mismo patrón de diferencias fue observado cuando se analizaron el G1 y G2. La vFCmáx determinada a través de 2 distintos métodos propuestos en la literatura se correlacionó con las pruebas de 10 y 15 km (0,55 ≤ r ≤ 0,82; p < 0,05). Conclusiones: La vFCmáx, en corredores recreativos de mediana edad, tiene alta correlación con las pruebas de 10 y 15 km y no fue diferente de la Vpico (para vFCmáx de Lacour et al.), presentando resultados similares a los observados por los métodos basados en el costo de oxígeno

Objectives. To compare maximal aerobic speed (MAS) calculated by oxygen cost (vVO2max) and heart rate cost (vHRmax) with the direct measurement of MAS (Vpeak) and to verify the relationship between vHRmax and 10‐ and 15‐km performance in middle‐age recreationally runners. Method. Twenty one recreationally runners participated in this study (age: 30 to 49 years), allocated in two groups according to age (G1 and G2). Participants were submitted to an incremental continuous test of maximal effort in laboratory to determine maximal oxygen uptake. MAS was determined according to proposes presented in literature based on vVO2max and vHRmax. Besides, it was performed two performances in field track (10 and 15 km). Results. The lowest difference between the mean values observed and Vpeak was in relation to vHRmax from Lacour et al. (0.0 km h–1; p > 0.05). The highest was in relation to vHRmax from di Prampero (1.55 km h–1; p < 0.05). The same pattern of differences was observed when G1 and G2 were analyzed. The vHRmax determined according to two different methods presented in literature showed to be correlated with 10 and 15 km performances (0.55 ≤ r ≤ 0.82; p < 0.05). Conclusions. The vHRmax in middle‐aged recreational runners has elevated correlation with 10 and 15 km performances and was not different from Vpeak (to vHRmax from Lacour et al.) showing similar results than the method based on oxygen cost (AU)