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.

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.

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.

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)

Prodromal metabolic phenotype in MCI cybrids: implications for Alzheimer's disease.

Mild cognitive impairment (MCI) is considered a nosological entity or a translational state between normal aging and sporadic Alzheimer's disease (AD). From brain tissue to peripheral blood samples, it is evident that the early markers of metabolic dysfunction observed in AD have also been found in MCI subjects. These observations obtained from MCI and AD subjects leave open the possibility that mitochondrial dysfunction-induced oxidative damage happening a priori of symptom onset, may trigger other pathological hallmarks, namely Aß oligomerization. In this study, we used a citoplasmic hybrid (cybrid) model created by the repopulation of human teratocarcinoma (NT2) cells depleted of endogenous mitochondrial DNA (mtDNA) with platelets from age-matched controls, MCI and AD subjects. We found mitochondrial deficits in MCI and AD cybrids as compared with controls, such as a decrease in cytochrome c oxidase (COX) activity, a decrease in mitochondrial membrane potential and in mitochondrial cytochrome c content. Consequently, we analyzed parameters of oxidative damage and found that AD and MCI cybrids exhibit an increase in lipid peroxides, higher production of superoxide radicals, and higher content in protein carbonyls. Since our data clearly show alterations in mitochondrial-mediated oxidative damage in MCI cybrids we propose that mitochondrial dysfunction is an early event in idiopathic AD. Moreover, we found that mitochondrial Aß oligomeric content increases in AD, which may exacerbate initial mitochondrial damage. Altogether, our data strongly supports a key role for mitochondria/ mtDNA in aged-driven AD pathology.

The anti-adherence effect of Lippia sidoides Cham: extract against microorganisms of dental biofilm / Efeito anti-aderente de Lippia sidoides: extrato contra microorganismos do biofilme dental

Most illnesses affecting the oral cavity are proven to have infectious origin. Several categories of chemical agents have been used in the chemical control of dental biofilm through strategies that aim at reducing bacterial adhesion and inhibiting the growth and the proliferation of microorganisms on the tooth surface. The use of plants in folk medicine and in Dentistry, as well as the spread of successful cases, has led to scientific exploration, resulting in chemical-pharmacological knowledge of thousands of plants. The present study aimed to evaluate the anti-adherence activity of Lippia sidoides Cham., comparing the results with those of 0.12% chlorhexidine by means of an in vitro simulation of dental biofilm. The studied bacterial strains were Streptococcus mutans, Streptococcus sanguinis and Lactobacillus casei, main responsible for the biofilm adherence. The studied extract was effective in inhibiting the adherence of Streptococcus mutans up to a concentration of 1:16, compared to Chlorhexidine. Lippia sidoides Cham extract showed anti-adherence effect on the major microorganisms responsible for dental biofilm consolidation.

É fato comprovado que a maior parte das doenças que acometem a cavidade bucal são de origem infecciosa. Várias categorias de agentes químicos têm sido utilizadas no controle químico do biofilme dental através de estratégias que visam a redução da adesão bacteriana, a inibição do crescimento, e a proliferação dos microrganismos na superfície do dente. A utilização das plantas pela medicina popular, seu uso na Odontologia e a divulgação dos êxitos, conduziram a exploração científica resultando no conhecimento químico-farmacológico de milhares de plantas. A presente pesquisa objetivou avaliar a atividade antiaderente da folha da Lippia sidoides Cham. comparando seus resultados com a Clorexidina 0,12%, através de uma simulação, in vitro, do biofilme dental. As linhagens bacterianas utilizadas na pesquisa foram o Streptococcus mutans, Streptococcus sanguinis, e o Lactobacillus casaram, principais responsáveis pela aderência do biofilme. O extrato estudado mostrou-se efetivo na inibição de aderência das bactérias ensaiadas até uma concentração de 1:16, sobre o Streptococcus mutans, sendo comparável à Clorexidina. O extrato da Lippia sidoides Cham. demonstrou efeito antiederente, sobre os principais microrganismos responsáveis pela consolidação do biofilme dental.

Design of a microfluidic platform for monoclonal antibody extraction using an aqueous two-phase system.

The use of monoclonal antibodies (mAbs) in medical treatments and in laboratory techniques has a very important impact in the battle against many diseases, namely in the treatment of cancer, autoimmune diseases and neural disorders. Thus these biopharmaceuticals have become increasingly important, reinforcing the demand for efficient, scalable and cost-effective techniques for providing pure antibodies. Aqueous two-phase systems (ATPS) have shown potential for downstream processing of mAbs. In this work, an ATPS in a microfluidic platform was designed and tested for mAbs extraction. The system demonstrated the potential to be an effective tool to accelerate bioprocess design and optimization. The partition of immunoglobulin G (IgG) tagged with fluorescein isothiocyanate (FITC) in an ATPS of polyethylene-glycol (PEG)/phosphate buffer with NaCl was investigated using a PDMS microfluidic device fabricated using soft lithography techniques. Different structures were tested with different values of microchannel length (3.14-16.8 cm) and flow rates of the salt (1-2 µL/min) and PEG-rich phases (0.2-0.5 µL/min). A stable interphase between the phases was obtained and the phenomena of diffusion and of partition of the IgG from the salt-rich phase to the PEG-rich phase were measured by fluorescence microscopy. Process simulation allowed the modeling of the IgG diffusion and partitioning behavior observed in the microstructure. The reduction to the microscale does not greatly affect the antibody extraction yield when compared with macroscale results, but it does reduce the operation time, demonstrating the potentiality of this approach to process optimization.

Genes involved in protein metabolism of the probiotic lactic acid bacterium Lactobacillus delbrueckii UFV H2b20.

A basic requirement for the prediction of the potential use of lactic acid bacteria (LAB) in the dairy industry is the identification of specific genes involved in flavour-forming pathways. The probiotic Lactobacillus delbrueckii UFV H2b20 was submitted to a genetic characterisation and phylogenetic analysis of genes involved in protein catabolism. Eight genes belonging to this system were identified, which possess a closely phylogenetic relationship to NCFM strains representative, as it was demonstrated for oppC and oppBII, encoding oligopeptide transport system components. PepC, PepN, and PepX might be essential for growth of LAB, probiotic or not, since the correspondent genes are always present, including in L. delbrueckii UFV H2b20 genome. For pepX gene, a probable link between carbohydrate catabolism and PepX expression may exists, where it is regulated by PepR1/CcpA-like, a common feature between Lactobacillus strains and also in L. delbrueckii UFV H2b20. The well conserved evolutionary history of the ilvE gene is evidence that the pathways leading to branched-chain amino acid degradation, such as isoleucine and valine, are similar among L. delbrueckii subsp. bulgaricus strains and L. delbrueckii UFV H2b20. Thus, the involvement of succinate in flavour formation can be attributed to IlvE activity. The presence of aminopeptidase G in L. delbrueckii UFV H2b20 genome, which is absent in several strains, might improve the proteolytic activity and effectiveness. The nucleotide sequence encoding PepG revealed that it is a cysteine endopeptidase, belonging to Peptidase C1 superfamily; sequence analysis showed 99% identity with L. delbrueckii subsp. bulgaricus ATCC 11842 pepG, whereas protein sequence analysis revealed 100% similarity with PepG from the same organism. The present study proposes a schematic model to explain how the proteolytic system of the probiotic L. delbrueckii UFV H2b20 works, based on the components identified so far.

Substituição parcial de farelo de soja por ureia protegida na produção e composição do leite / Effects of partial replacement of soybean meal by protected urea on milk yield and composition

Avaliaram-se os efeitos da ureia protegida na produção e composição do leite, utilizando-se 34 vacas da raça Holandesa, com 128±60 dias em leite e produção de 41,6±6,7kg/dia no início do experimento, distribuídas em blocos por produção de leite, ordem de lactação e dias em lactação. As variáveis produção de leite, porcentagens e produções de gordura, proteína, lactose e sólidos totais do leite, contagem de células somáticas e nitrogênio ureico no leite foram mensuradas aos 30 e 60 dias do experimento. Os dados foram analisados por procedimento de modelos mistos com o modelo contendo como covariável as observações obtidas antes da adoção das dietas experimentais e os efeitos fixos de período, de tratamento e a interação de período versus tratamento. Os tratamentos (T) foram compostos por dietas isoenergéticas e isonitrogenadas com 1,66 EL L e 18,35 por cento de PB, definidas como: T1= 11,4 por cento farelo de soja e T2= 0,4 por cento ureia encapsulada + 9,0 por cento farelo de soja. Não foram observadas diferenças (P>0,05) nas produções diárias de leite e de sólidos, de nitrogênio ureico no leite e da contagem de células somáticas. A dieta com ureia protegida reduziu (P<0,05) as porcentagens de gordura e de sólidos totais do leite. A substituição parcial do farelo de soja por ureia protegida não reduziu o desempenho produtivo das vacas em lactação.

The effects of protected urea on milk yield and composition were evaluated. Thirty-four lactating Holstein cows, with 128±60 days in milk and yield of 41.6±6.7 kg/day at the beginning of the trial, were blocked by milk production, lactation number, and days in milk. Milk yield; yields and contents of fat, protein, lactose, total solids; somatic cell count; and milk urea nitrogen were measured on days 30 and 60 of the comparison period. Variables measured before the application of treatments were used as covariate in the statistical model. Data were analyzed with the mixed procedure of SAS with a model containing the continuous effect of the covariate and the fixed effects of period, treatment, and the interaction of period and treatment. Treatments (T) were isoenergetic and isonitrogenous diets with 1.66 EL L and 18.35 percent of CP, described as: T1= 11.4 percent soybean meal and T2= 0.4 percent encapsulated urea + 9.0 percent soybean meal. There was no treatment effect (P>0.05) on the daily production of milk and solids, milk urea nitrogen, and somatic cell count. The slow release urea diet reduced (P<0.05) milk fat and total solids contents. The partial replacement of soybean meal by protected urea did not decrease the performance of lactating cows.

Access to tuberculosis diagnosis in Itaboraí City, Rio de Janeiro, Brazil: the patient's point of view.

SETTING: Itaboraí Municipality in Rio de Janeiro, Brazil. OBJECTIVE: To evaluate access to tuberculosis (TB) diagnosis for users of the Family Health Program (FHP) and Reference Ambulatory Units (RAUs). DESIGN: A cross-sectional study was conducted in Itaboraí City, Rio de Janeiro, Brazil. Between July and October 2007, a sample of 100 TB patients registered consecutively with the TB Control Program was interviewed using the primary care assessment tool. The two highest scores, describing 'almost always' and 'always', or 'good' and 'very good', were used as a cut-off point to define high quality access to diagnosis. RESULTS: FHP patients were older and had less education than RAU interviewees. Sex and overcrowding did not differ in the two groups. Patient groups did not differ with regard to the number of times care was sought at a unit, transport problems, cost of attending units and availability of consultation within 24 h. Adequate access to diagnosis was identified by 62% of the FHP patients and 53% of the RAU patients (P = 0.01). CONCLUSION: In Itaboraí, Rio de Janeiro, TB patients believe that the FHP units provide greater access to TB diagnosis than RAUs. These findings will be used by the Department of Health to improve access to diagnosis in Itaboraí.

Efeitos cardiovasculares dos inibidores da acetilcolinesterase: relato de caso

Objetivos: demonstrar efeito cardiovascular provocado por inibidor da acetilcolinesterase (IAChE), potencializado pelo uso associado de beta bloqueador adrenérgico. Materiais e métodos: paciente M.R., 80 anos, sexo masculino, internado com quadro de rebaixamento do nível de consciência. Apresentava-se ictérico, hipotenso (PA: 80x50 mmHg) e bradicárdico (FC: 36bpm). Ausculta cardíaca com bulhas rítmicas, em 2 tempos, sem sopro. Pulmão e abdome sem alterações. Sem déficit focal e sem sinais meníngeos. Antecedentes de etilismo e tabagismo. Portador de hepatopatia crônica, hipotiroidismo, hipertensão arterial sistêmica, insuficiência renal crônica e síndrome demencial tipo Alzheimer. Fazia uso de Inibidor de ECA, Levotiroxina, Ácido fólico, Omeprazol, Diuréticos, Tiamina, Beta bloqueador e um, IAChE, ECG: bradicardia sinusal, FC: 36 bpm, Hemograma: discreta anemia macrocítica; leucograma sem alterações e plaquetopenia: 72000, Creatinina: 2,4mg/dl, Uréia: 125 mg/dl, Sódio: 134mmol/L, Potássio: 5,3 mmol/L . Glicemia: 97 mg/dl, Enzimas cardíacas normais. TGO: 97 U/L, TGP: 75 U/L, DHL: 249 U/L, Bilirrubina total: 1,45 mg/dl, Bilirrubina direta: 0,61 mg/dl , Bilirrubina indireta: 0,84 mg/dl, TAP: 16,1 seg, AP: 67%, INR:1,34, Proteínas totais: 5,9 g/dl, Albumina 2,3 g/dl, TSH: 3,4 mUl/ml. O beta bloqueador foi suspenso no dia da internação, porém o paciente manteve-se bradicárdico. Dois dias após, o IAChE foi suspenso. Conclusão: a freqüência de efeitos cardiovasculares associados aos IAChEs são: vertigem e síncope (1 a 10%), bradicardia, arritmias atriais, infarto do miocárdio, angina, convulsões (0,01 a 1%), bloqueio não atrial e atrioventricular (0,001 a 0,1%). Revisões revelam que a incidência de efeitos colaterais cardiovasculares são raros com o uso dos IAChEs

Use of immunoblotting assay improves the sensitivity of paracoccidioidomycosis diagnosis

The purpose of this work was to evaluate two serological assays: double immunodiffusion (DI) and immunoblotting (IB) in immunodiagnosis of paracoccidioidomycosis (PCM). We evaluated by IB assay 23 sera samples from patients with clinical confirmation of PCM, all of them with negative DI results against culture filtrate from Paracoccidioides brasiliensis isolate 113. For IB, as well as for comparative DI assay, we employed soluble components of the cell wall outer surface (SCCWOS) from P. brasiliensis isolate 113 cultivated at 36°C in Fava-Neto's agar medium for 5 and 10 days. Among the 20 sera samples analyzed by DI, 13 (65 percent) were negative and 7 (35 percent) were positive against SCCWOS obtained on the 5th and 10th days. By IB assay, 95.4 percent and 100 percent of sera reacted against gp43 and gp70 present in SCCWOS from the 5th day and 95.6 percent recognized these fractions when evaluated against SCCWOS from the 10th day. Our results demonstrated that the use of an immunoenzymatic assay significantly improves the sensitivity of PCM immunodiagnosis and also suggests that at least two serological tests for antibody detection should be adopted in cases of questionable diagnosis.

Flavonóides glicosilados de Herissantia tiubae (K. Schum) Brizicky (Malvaceae) e testes farmacológicos preliminares do canferol 3,7-di-O-a-L-ramnopiranosídeo / Glycosyl flavonoids from Herissantia tiubae (K. Schum) Brizicky (Malvaceae) and preliminary tests of kaempferol 3,7-di-O-a-L-rhamnopyranoside

Das partes aéreas de Herissantia tiubae (K. Schum.) Brizicky foram isolados, através de métodos cromatográficos, dois flavonóides glicosilados, canferol 3,7-di-O-a-L-ramnopiranosídeo e canferol 3-b-O-D-(6’’-E-p-cumaroil) glicosídeo. As estruturas foram identificadas com o uso de técnicas espectroscópicas de IV, RMN ¹H e 13C incluindo métodos bidimensionais, além de comparações com dados da literatura. O canferol 3,7-di-O-a-L-ramnopiranosídeo foi submetido a testes farmacológicos preliminares com a finalidade de avaliar o seu efeito sobre o sistema cardiovascular.

From the aerial parts of Herissantia tiubae (K. Schum.) Brizicky two flavonol glycosides were isolated by means of chromatographic methods, kaempferol 3,7-di-O-a-L-rhamnopyranoside and kaempferol 3-O-b-D-(6’’-E-p-coumaroyl) glucoside. Their structural identification was made by IV, ¹H and 13C NMR spectroscopy, including two dimensional techniques, together with comparison with literatura data. Preliminary tests were carried out with kaempferol 3,7-di-O-a-L-rhamnopyranoside in order to study its possible cardiovascular effect.

Calcium antagonism and the vasorelaxation of the rat aorta induced by rotundifolone

The vasorelaxing activity of rotundifolone (ROT), a major constituent (63.5 percent) of the essential oil of Mentha x villosa, was tested in male Wistar rats (300-350 g). In isolated rat aortic rings, increasing ROT concentrations (0.3, 1, 10, 100, 300, and 500 æg/ml) inhibited the contractile effects of 1 æM phenylephrine and of 80 or 30 mM KCl (IC50 values, reported as means ± SEM = 184 ± 6, 185 ± 3 and 188 ± 19 æg/ml, N = 6, respectively). In aortic rings pre-contracted with 1 æM phenylephrine, the smooth muscle-relaxant activity of ROT was inhibited by removal of the vascular endothelium (IC50 value = 235 ± 7 æg/ml, N = 6). Furthermore, ROT inhibited (pD2 = 6.04, N = 6) the CaCl2-induced contraction in depolarizing medium in a concentration-dependent manner. In Ca2+-free solution, ROT inhibited 1 æM phenylephrine-induced contraction in a concentration-dependent manner and did not modify the phasic contractile response evoked by caffeine (20 mM). In conclusion, in the present study we have shown that ROT produces an endothelium-independent vasorelaxing effect in the rat aorta. The results further indicated that in the rat aorta ROT is able to induce vasorelaxation, at least in part, by inhibiting both: a) voltage-dependent Ca² channels, and b) intracellular Ca2+ release selectively due to inositol 1,4,5-triphosphate activation. Additional studies are required to elucidate the mechanisms underlying ROT-induced relaxation.

Endothelium-dependent hypotensive and vasorelaxant effects of the essential oil from aerial parts of Mentha x villosa in rats.

Cardiovascular effects of an essential oil from the aerial parts of Mentha x villosa (OEMV) were tested in rats using a combined in vivo and in vitro approach. In non-anesthetized normotensive rats, OEMV (1, 5, 10, 20, 30 mg kg(-1) body wt., i.v.) induced a significant and dose-dependent hypotension (-3 +/- 1.8%; -6 +/- 0.7%; -40 +/- 6.7%; -58 +/- 3.8%; -57 +/- 2.1%, respectively) associated with decreases in heart rate (-1 +/- 0.3%; -9 +/- 0.9%; -17 +/- 3.2%; -72 +/- 3.1%; -82 +/- 1.4%, respectively). The hypotensive and bradycardic responses evoked by OEMV were attenuated and blocke by pre-treatment of the animals with atropine (2 mg kg(-1) body wt., i.v.). In isolated rat atrial preparations, OEMV (10, 100, 300, 500 microg ml(-1)) produced concentration-related negative chronotropic and inotropic effects (IC50 value = 229 +/- 17 and 120 +/- 13 microg ml(-1), respectively). In isolated rat aortic rings, increasing concentrations of OEM (10, 100, 300, 500 microg ml(-1)) were able to antagonize the effects of phenylephrine (1 microM), prostaglandin F2alpha (10 microM) and KCl (80 mM)-induced contractions (IC50 value = 255 +/- 9, 174 +/- 4 and 165 +/- 14 microg ml(-1), respectively). The vasorelaxant activity induced by OEMV was attenuated significantly by either endothelium removal (IC50 value = 304 +/- 9 microg ml(-1)), NG-nitro L-arginine methyl ester (L-NAME) 100 microM (IC50 value=359 +/- 18 microg ml(-1)), L-NAME 300 microM (IC50 value = 488 +/- 20 microg ml(-1)) or indomethacin 10 microM (IC50 value = 334 +/- 18 microg ml(-1)). However, it was not affected by atropine 1 microM (IC50 value = 247 +/- 12 microg ml(-1)). Furthermore, the hypotensive response induced by OEMV was attenuated significantly after nitric oxide (NO) synthase blockade (L-NAME, 20 mg kg(-1) body wt., i.v.), while bradycardia was not altered. The results suggest that the hypotensive effect induced by OEMV is probably due to its direct cardiodepressant action and peripheral vasodilation, which can be attributed to both endothelium-dependent (via EDRFs, at least NO and prostacyclin) and endothelium-independent mechanisms (such as Ca2+ channel blockade).

Agenesis and lipoma of corpus callosum. Case report.

The agenesis and lipoma of the corpus callosum is a very rare association. We report the case of a 18-years old woman with rare epileptic seizures since the age of 6 years, normal neurological examination, as well as normal electroencephalogram. The brain computed tomography scanning and the magnetic resonance showed the lipoma and the agenesis of the corpus callosum.