Evaluation of bacterial growth inhibition by mercaptopropionic acid in metallo-β-lactamase detection on multidrug-resistant Acinetobacter baumannii

INTRODUCTION: Metallo-β-lactamase (MBL) has been reported all over the world. METHODS: The inhibitory effect of mercaptopropionic acid (MPA) on bacterial growth was evaluated by comparison between disk diffusion and broth dilution methodology with determination of the minimum inhibitory concentration (MIC) for multidrug-resistant Acinetobacter baumanni strains. RESULTS: MPA significantly inhibited growth of the strains. CONCLUSIONS: The use of MPA can affect the results in phenotypic methods of MBL detection.

The influence of menopausal status on bone turnover and disease control in breast cancer patients with metastatic bone disease treated with chemotherapy plus zoledronic acid : an exploratory retrospective cohort study

RESUMO: Introdução: Uma meta-análise recente demonstrou que uso adjuvante de ácido zoledrónico (AZ) em mulheres pós-menopáusicas com cancro da mama precoce (CM) conduz a redução do risco de morte por CM em 17%. Investigámos o efeito do estado hormonal (pré [PrM] vs pós-menopausa tardia [PoM]) na remodelação óssea e controlo de doença em mulheres com CM e metástases ósseas (MO) tratadas com AZ e quimioterapia (QT). Métodos: Neste estudo de coorte retrospetivo, colhemos variáveis clinico-patológicas e quantificámos o telopéptido N-terminal (NTX) urinário e marcadores tumorais (MT) séricos em mulheres com CM e MO tratadas com QT e AZ. As doentes foram divididas em PrM (<45 anos) e PoM (>60 anos). Endpoints do estudo: variação do NTX, CA15.3 e CEA nos meses 3, 6 e 9, tempo até falência de QT de primeira-linha e sobrevivência. Quando apropriado foram usados os testes de Wilcoxon rank-sum, modelo de efeitos lineares mistos, teste log-rank e modelo de Cox. Resultados: Quarenta doentes foram elegíveis para análise (8 PrM e 32 PoM). Depois da introdução de AZ e QT, os níveis de NTX e MT caíram no coorte global. O perfil de resposta não diferiu entre grupos no mês 3 ou em tempos posteriores (valor-p para interação tempo-estado hormonal no mês 3=0.957). Ademais, o perfil de resposta dos MT também não diferiu entre grupos. O tempo mediano até falência de primeira-linha de QT em PrM e PoM foi de 15.2 e 17.4 meses, respetivamente. Não foi identificada diferença significativa entre grupos, quer em análise univariada quer após controlo para envolvimento visceral (p=0.399 e 0.469, respetivamente). Igualmente, não houve diferenças em termos de sobrevivência. Conclusões: Neste coorte, não foram identificadas diferenças no controlo de NTX ou MT em função do estado menopausico. Igualmente, não foi identificada diferença no tempo até falência de primeira-linha de CT ou sobrevivência.----------- ABSTRACT: Background: A recent meta-analysis showed that the adjuvant use of zoledronic acid (ZA) in postmenopausal women with early breast cancer (BC) leads to a reduction in the risk of breast cancer death by 17%. We investigated the effect of the hormonal status (pre [PrM] vs late post menopause [PoM]) on bone turnover and disease control among women with BC and boné metastases (BM) treated with ZA and chemotherapy (CT). Methods: In this retrospective cohort study, we collected clinicopathologic variables, urinary Nterminal telopeptide (NTX) and serum tumor marker levels from women with BC and BM treated with CT and ZA. Patients were divided in PrM (<45 years) and PoM (>60 years). Study endpoints were NTX, CA15.3 and CEA variation at 3, 6 and 9 months, and time to first-line CT failure and survival. We performed multilevel mixed-effects linear regression models to assess the variation of repeated measures and cox regression models for time to event outcomes. Results: Forty patients were eligible for analysis (8 PrM and 32 PoM). After introduction of ZA and CT, NTX and tumor markers declined in the overall cohort. Response profile was similar between menopausal groups at month 3 and at later time points (p-value for time-hormonal status interaction at month 3=0.957). Furthermore, tumor markers response profile was also equal between groups. Median time to first-line CT failure in PrM and PoM women was 15.2 and 17.4 months, respectively. No significant difference between groups was found, either using a univariate analysis or after controlling for visceral disease involvement (p=0.399 and 0.469, respectively). Likewise, no differences in survival were found. Conclusions: In this cohort, no differences were found in terms of NTX or tumor markers control according to menopausal status. Similarly, no difference in time to first-line CT failure or survival was found.

Larvicidal activity of crude extracts from Larrea cuneifolia (Zygophyllaceae) and of its metabolite nordihydroguaiaretic acid against the vector Culex quinquefasciatus (Diptera: Culicidae)

INTRODUCTION: The aim of the present study was to analyze the larvicidal activity of different crude extracts of Larrea cuneifolia and its most abundant lignan, nordihydroguaiaretic acid (NDGA), against Culex quinquefasciatus. METHODS: Chloroform, methanol, and aqueous extracts from L. cuneifolia and NDGA were tested against larvae of Cx. quinquefasciatus under laboratory conditions. RESULTS: The chloroform extract showed the highest larvicidal effect, with an estimated LC50 of 0.062 mg/ml. NDGA also demonstrated significant larvicidal activity with an estimated LC50 of 0.092 mg/ml. CONCLUSIONS: These results indicate that the chloroform extract of L. cuneifolia and NDGA are promising insecticides of botanical origin that could be useful for controlling Cx. quinquefasciatus.

Unravelling the role of alpha 2,6 sialic acid on mouse dentritic cells functions

RESUMO: As células dendríticas (CDs) são fundamentais na imunomodulação e iniciação de respostas imunes adaptativas, enquanto os ácidos siálicos (Sias) são potenciais imunomoduladores. Estas células expressam níveis elevados da sialiltransferase ST6Gal-1, que transfere Sias para a posição terminal de oligossacáridos. De facto, a maturação de CDs está associada a uma diminuição da sialilação na sua superfície celular. Apesar de ter função biológica desconhecida, a forma solúvel, extracelular de ST6Gal-1 aumenta em cancros e inflamação. Ainda assim, esta foi recentemente identificada como moduladora da hematopoiese. Considerando o importante papel das CDs na iniciação de respostas anticancerígenas, uma ligação entre a sialilação extrínseca induzida por ST6Gal-1 extracelular e o seu papel na modulação de CDs deve ser identificada. Neste trabalho hipotetizou-se que a sialilação α2,6 extrínseca de CDs diminui o seu perfil de maturação mediante ativação por lipopolissacarídeo (LPS). O objetivo principal foi sialilar extrinsecamente em α2,6 CDs da medula óssea de murganhos, avaliando os seus perfis de maturação e de libertação de citocinas, após estimulação com LPS (por Citometria de Fluxo e ELISA, respetivamente). Ao contrário da hipótese, o perfil celular não foi modulado, usando várias abordagens. Por outro lado, a consequência da falta de α2,6 Sias na maturação de CDs foi avaliada analisando: 1) CDs da medula óssea de murganhos tratadas com sialidase, 2) CDs da medula óssea e 3) CDs das vias aéreas, ambas de murganhos deficientes em ST6Gal-1, comparando com a estirpe selvagem. Estes resultados sugerem que a perta total de α2,6 Sias se relaciona com o aumento da expressão do complexo de histocompatibilidade principal de classe II. Apesar de controverso, é provável existirem mecanismos inerentes à ativação por LPS, reduzindo a eficácia de ST6Gal-1 extracelular. Por outro lado, a modificação no perfil de CDs de murganhos deficientes em ST6Gal-1 poderá relacionar-se com uma predisposição para um estado inflamatório severo. Com isto, o trabalho desenvolvido abriu futuras linhas de investigação, nomeadamente explorar outros fatores envolvidos na (de)sialilação α2,6 de CDs, podendo ter impacto em imunoterapia com uso de CDs.--------------------------ABSTRACT: Dendritic cells (DCs) are vital for immunomodulation and the initiation of adaptive immune responses, whereas sialic acids (Sias) are potential immunomodulators. These cells express high levels of sialyltransferase ST6Gal-1, responsible for transferring Sias to the terminal position of oligosaccharide chains. Indeed, DCs’ maturation is associated with decreased cell surface sialylation. Although its biological significance is unknown, the soluble, extracellular form of ST6Gal-1 increases in cancers and inflammation. However, extracellular ST6Gal-1 was recently identified as modulator of hematopoiesis. Considering that DCs play a crucial role in the initiation of a productive anti-cancer immune response, a link between extrinsic sialylation by the extracellular ST6Gal-1 on DC function needs to be investigated. We hypothesize that extrinsic α2,6 sialylation of DCs diminishes their maturation features upon lipopolysaccharide (LPS) stimulation. The main goal was to extrinsically α2,6 sialylate mice bone marrow derived DCs (BMDCs) and to evaluate their maturation and cytokine profiles upon LPS stimulation (by Flow Cytometry and ELISA, respectively). Unlike the hypothesis, we observed that BMDCs’ profile is not modulated, even using several approaches. In contrast, the consequence of lacking cell surface α2,6 Sias in DC maturation was assessed by analysing: 1) sialidase treated BMDCs, 2) BMDCs from mice lacking ST6Gal-1 and 3) DCs from mice airways, comparing wild type with ST6Gal-1 knockout mice. These results suggest that overall lack in α2,6 Sias is related with increased expression of major histocompatibility class II (MHC-II). Although appearing to be controversial findings, other intracellular mechanisms might be occurring upon LPS-induced BMDC activation, probably reducing extracellular ST6Gal-1 effect. In opposite, the modification observed in DC profile of ST6Gal-1 knockout mice might be related to its predisposition to a more severe inflammatory status. With this, the developed work opened future lines of investigation, namely exploring other factors involved in α2,6 (de)sialylation of DC, which might have influence in immunotherapy using DCs.

Evaluation of three enzyme immunoassays and a nucleic acid amplification test for the diagnosis of Clostridium difficile-associated diarrhea at a university hospital in Brazil

Introduction Despite the known importance of Clostridium difficile as a nosocomial pathogen, few studies regarding Clostridium difficile infection (CDI) in Brazil have been conducted. To date, the diagnostic tests that are available on the Brazilian market for the diagnosis of CDI have not been evaluated. The aim of this study was to compare the performances of four commercial methods for the diagnosis of CDI in patients from a university hospital in Brazil. Methods Three enzyme immunoassays (EIAs) and one nucleic acid amplification test (NAAT) were evaluated against a cytotoxicity assay (CTA) and toxigenic culture (TC). Stool samples from 92 patients with suspected CDI were used in this study. Results Twenty-five (27.2%) of 92 samples were positive according to the CTA, and 23 (25%) were positive according to the TC. All EIAs and the NAAT test demonstrated sensitivities between 59 and 68% and specificities greater than 91%. Conclusions All four methods exhibited low sensitivities for the diagnosis of CDI, which could lead to a large number of false-negative results, an increased risk of cross-infection to other patients, and overtreatment with empirical antibiotics.

Bioproduction of succinic acid from renewable feedstocks by Actinobacillus succinogenes 130Z

Succinic acid (SA) is a highly versatile building block that is used in a wide range of industrial applications. The biological production of succinic acid has emerged in the last years as an efficient alternative to the chemical production based on fossil fuels. However, in order to fully replace the competing petro-based chemical process from which it has been produced so far, some challenges remain to be surpassed. In particular, one main obstacle would be to reduce its production costs, mostly associated to the use of refined sugars. The present work is focused on the development of a sustainable and cost-e↵ective microbial production process based on cheap and renewable resources, such as agroindustrial wastes. Hence, glycerol and carob pods were identified as promising feedstocks and used as inexpensive carbon sources for the bioproduction of succinic acid by Actinobacillus succinogenes 130Z, one of the best naturally producing strains. Even though glycerol is a highly available carbon source, as by-product of biodiesel production, its consumption by A. succinogenes is impaired due to a redox imbalance during cell growth. However, the use of an external electron acceptor such as dimethylsulfoxide (DMSO) may improve glycerol metabolism and succinic acid production by this strain. As such, DMSO was tested as a co-substrate for glycerol consumption and concentrations of DMSO between 1 and 4% (v/v) greatly promoted glycerol consumption and SA production by this biocatalyst. Aiming at obtaining higher succinic acid yield and production rate, batch and fed-batch experiments were performed under controlled cultivation conditions. Batch experiments resulted in a succinic acid yield on glycerol of 0.95 g SA/g GLY and a production rate of 2.13 g/L.h, with residual production of acetic and formic acids. In fed-batch experiment, the SA production rate reached 2.31 g/L.h, the highest value reported in the literature for A. succinogenes using glycerol as carbon source. DMSO dramatically improved the conversion of glycerol by A. succinogenes and may be used as a co-substrate, opening new perspectives for the use of glycerol by this biocatalyst. Carob pods, highly available in Portugal as a residue from the locust bean gum industry, contain a significant amount of fermentable sugars such as sucrose, glucose and fructose and were also used as substrate for succinic acid production. Sugar extraction from raw and roasted carobs was optimized varying solid/water ratio and extraction time, maximizing sugar recovery while minimizing the extraction of polyphenols. Kinetic studies of glucose, fructose and sucrose consumption by A. succinogenes as individual carbon sources till 30 g/L were first determined to assess possible metabolic diferences. Results showed no significant diferences related to sugar consumption and SA production between the diferent sugars. Carob pods water extracts were then used as carbon source during controlled batch cultivations. (...)

Ursodeoxycholic acid does not interfere with in vivo Helicobacter pylori colonization

A low frequency of Helicobacter pylori in the gastric mucosa of patients with alkaline gastritis has been reported. At the same time, it can be noted that the growth of bacteria can be inhibited by bile acids. We studied 40 patients with chronic gastritis related to Helicobacter pylori in order to determine the effect of ursodeoxycholic acid on this infection. Diagnoses of the infection and the inflammatory process were obtained by histologic study of gastric biopsies collected during endoscopy. Two groups were studied: group I received ursodeoxycholic acid - 300 mg/day, and group II received the placebo, twice a day, both for 28 days. The colonization by Helicobacter pylori and the intensity of the mononuclear and polymorphonuclear inflammatory infiltrate were determined before (time 1) and after (time 2) treatment. Ursodeoxycholic acid had no effect on the Helicobacter pylori infection. A significant reduction in the intensity of the mononuclear inflammatory infiltrate of the gastric antrum mucosa was observed in patients from group I, when we compared not only times 1 and 2 but also groups I and II. However, this was not the case with the body mucosa. We concluded that ursodeoxycholic acid had no action on the colonization by Helicobacter pylori or on the polymorphonuclear inflammatory infiltrate, but it caused a significant reduction in the intensity of the mononuclear inflammatory infiltrate of the gastric antrum.

The effect of intravenous zoledronic acid on glucocorticoid-induced multiple vertebral fractures in juvenile systemic lupus erythematosus

Glucocorticoids are widely used in the treatment of lupus patients, and adverse effects, which include osteoporosis and associated fractures, are frequent. Treatment of osteoporosis of young patients should be effective and not harmful to bone growth and remodeling. Bisphosphonates are drugs that decrease the incidence of bone fractures, but their use in juvenile patients is still controversial because of their possible side effects on the growing skeleton. However, recently published studies showed that linear growth continued normally after treatment with these drugs, and there was no excessive suppression of bone remodeling or mineralization defects. Zoledronic acid is a new intravenous bisphosphonate that has been approved by the US FDA for use with hypercalcemia of malignancies and might be an effective treatment for postmenopausal osteoporosis. The authors report a case of a young girl with systemic lupus who developed multiple vertebral collapses due to glucocorticoid therapy, and zoledronic acid was used producing significant clinical and densitometric improvement.

Evaluation of antibacterial activity of caffeic acid encapsulated by -cyclodextrins

Context: Caffeic acid is described as antibacterial, but this bioactive molecule has some issues regarding solubility and stability to environmental stress. Thus, encapsulation devices are required. Objective: The aim of this work was to study the effect of the caffeic acid encapsulation by cyclodextrins on its antibacterial activity. Materials and methods: The interactions between the caffeic acid and three cyclodextrins (-cyclodextrin (CD), 2-hydroxypropyl--cyclodextrin (HPCD) and methyl--cyclodextrin were study. Results and discussion: The formation of an aqueous soluble inclusion complex was confirmed for CD and HPCD with a 1:1 stoichiometry. The CD/caffeic acid complex showed higher stability than HPCD/caffeic acid. Caffeic acid antibacterial activity was similar at pH 3 and pH 5 against the three bacteria (K. pneumoniae, S. epidermidis and S. aureus). Conclusions: The antibacterial activity of the inclusion complexes was described here for the first time and it was shown that the caffeic acid activity was remarkably enhanced by the cyclodextrins encapsulation.

Engineering of fatty acid production and secretion in Saccharomyces cerevisiae

Tese de Doutoramento em Ciências - Especialidade em Biologia

Thermal characterization of polyhydroxyalkanoates and poly(lactic acid) blends obtained by injection molding

In this work we present the thermal characterization of the full scope of polyhydroxyalcanoate and poly(lactic acid) blends obtain by injection molding. Blends of polyhydroxyalcanoate and poly(lactic acid) (PHA/PLA) were prepared in different compositions ranging from 0–100% in steps of 10%. The blends were injection molded and then characterized by differential scanning calorimetry (DSC), scanning electron microscopy (SEM) and wide angle X-ray diffraction (WAXD). The increment of PHA fraction increased the degree of crystallinity of the blend and the miscibility of the base polymers as verified by the Fox model. The WAXD analysis indicates that the presence of PHA hindered the PLA crystallization. The crystallization evolution trough PHA weight fraction (wf) shows a phase inversion around 50-60%. SEM analyses confirmed that the miscibility of PHA/PLA blends increased with the incorporation of PHA and became total for values of PHA higher that 50%.

Aminobenzocoumarinylmehtyl esters as photoactive precursors for the release of butyric acid

The evaluation of the photorelease of a carboxylic acid drug, using butyric acid as a representative model, was carried out by using 7-amino-4-chloromethyl-2-oxo-2Hnaphtho[1,2-b] pyran, an aminobenzocoumarin, and its mono- and di-methylated or ethylated derivatives. This study was intended to improve the release of butyric acid from benzocoumarins by the addition of an amino group to the heterocycle by applying the knowledge of second-generation coumarinylmethyl-based photoremovable protecting groups. Photolysis studies were performed on the resultant ester cages by irradiation in a photochemical reactor at 254, 300, 350 and 419 nm, using methanol/HEPES buffer 80:20 solutions as solvent. The data obtained showed that these new fluorescent aminobenzocoumarins are superior to all the previously tested benzocoumarins with the same or different ring fusions. As well as the photolysis, the photophysics of the compounds were characterised by both steady state and time-resolved methods.

Microencapsulation of ellagic acid from pomegranate husk and karaya gum by spray drying

Objective: The aim of this study was to obtain and characterize microcapsules with Ellagic Acid (EA) from pomegranate as core material and Karaya Gum (KG) as wall material. Methods: EA was obtained from dry pomegranate peel powder via methanolysis and quantified by HPLC. Microcapsules were obtained preparing a dispersion containing KG and EA in phosphate buffer pH 8. The dispersion was processed in a spray dryer under specific conditions (inlet temperature at 150 °C, feed flow at 30% and aspirator at 100 %) for obtaining of microcapsules. Fourier transform infrared (FTIR) spectroscopy, differential scanning calorimetry (DSC) and scanning electron microscopy (SEM) were used for characterization. Results: Obtained material contains 98.03±2.82 mg EA/g of pomegranate peel. FTIR showed that there were changes in the molecular structure of microcapsules referred to raw materials. SEM confirmed that particles obtained had micron-size (1-5 µm). DSC analysis showed that raw materials had glass transition temperatures of 79.58 and 83.41 °C and for microcapsules the value was67.25 °C. Conclusion: Methanolysis is a viable technique for the obtaining of EA from the peel of pomegranate. KG shows good potential for be used as wall material for EA microencapsulation.

Photoactivation of butyric acid from 6-aminobenzocoumarin cages

A new benzocoumarin bearing an amino group is proposed as a photocleavable protecting group for carboxylic acids. The novel heterocycle, 6-amino-4-chloromethyl-2-oxo-2H-naphtho[1,2-b]pyran was used in the preparation of ester conjugates of butyric acid, and of the corresponding mono- and di-methylated or ethylated derivatives. The photolability of the ester conjugates was studied by irradiation at selected wavelengths in methanol/HEPES buffer (80:20) solutions, and the release of butyric acid was followed with HPLC/UV and 1H NMR monitoring. Release of the carboxylic acid was faster for the monoalkylated derivatives (approximately within 20 min), at the longer wavelengths of irradiation (350 and 419 nm). The photophysics of the heterocyclic conjugates was also evaluated by both steady state and time-resolved methods.