Interaction of four monoterpenes contained in essential oils with model membranes: implications for their antibacterial activity

The present article reports the antimicrobial efficacy of four monoterpenes (thymol, carvacrol, p-cymene, and gamma-terpinene) against the Gram-positive bacterium Staphylococcus aureus and the Gram-negative bacterium Escherichia coli. For a better understanding of their mechanism of action, the damage caused by these four monoterpenes on biomembranes was evaluated by monitoring the release, following exposure to the compounds under study, of the water-soluble fluorescent marker carboxyfluorescein (CF) from large unilamellar vesicles (LUVs) with different lipidic composition (phosphatidylcholine, PC, phosphatidylcholine/phosphatidylserine, PC/PS, 9:1; phosphatidylcholine/stearylamine, PC/SA, 9:1). Furthermore, the interaction of these terpenes with dimyristoylphosphatidylcholine multilamellar vesicles as model membranes was monitored by means of differential scanning calorimetry (DSC) technique. Finally, the results were related also with the relative lipophilicity and water solubility of the compounds examined. We observed that thymol is considerably more toxic against S. aureus than the other three terpenes, while carvacrol and p-cymene are the most inhibitory against E. coli. Thymol and carvacrol, but not gamma-terpinene and p-cymene, caused a concentration-dependent CF leakage from all kinds of LUVs employed; in particular, thymol was more effective on PC and PC/SA LUVS than on PC/PS vesicles, while carvacrol challenge evoked a CF leakage from PC/PS LUVs similar to that induced from PC/SA LUVs, and lower than that measured with PC vesicles. Concerning DSC experiments, these four terpenes caused a decrease in Tm and (especially carvacrol and p-cymene) DeltaH values, very likely acting as substitutional impurities. Taken together, our findings lead us to speculate that the antimicrobial effect of thymol, carvacrol, p-cymene, and gamma-terpinene may result, partially at least, from a gross perturbation of the lipidic fraction of the plasmic membrane of the microorganism. In addition to being related to the physicochemical characteristics of the compounds (such as lipophilicity and water solubility), this effect seems to be dependent on the lipidic composition and net surface charge of the microbic membranes. Furthermore, the compounds might cross the cell membranes, thus penetrating into the interior of the cell and interacting with intracellular sites critical for antibacterial activity.     

Removal of Chromium, Copper, and Nickel from an Electroplating Effluent Using a Flocculent Brewer’s Yeast Strain of Saccharomyces cerevisiae

The release of heavy metals in aquatic systems due to the discharge of industrial wastewaters is a matter of environmental concern. Heat-inactivated cells of a flocculent strain of Saccharomyces cerevisiae were used in the bioremediation, in a batch mode, of a real electroplating effluent containing Cu, Ni, and Cr. In this approach, no previous reduction of Cr(VI) to Cr(III) was required. Cr(VI) was selectively removed (98%) by yeast biomass at pH 2.3. At this pH, Cr(VI) is mainly in the form of HCrO ₄ ^? and yeast surface is surrounded by H⁺ ions, which enhance the Cr(VI) interaction with biomass binding sites by electrostatic forces. Subsequently, pH of the effluent was raised up to 6.0; this pH maximizes the efficiency of cations removal since at this pH the main binding groups of yeast cells are totally or partially deprotonated. The passage of effluent through a series of sequential batches, at pH 6.0, allowed, after the third batch, the removal of Cu(II), Ni (II), Cr total, and Cr(VI) in the effluent to values below the legal limit of discharge. The strategy proposed in the present work can be used in plants for the treatment of heavy metals rich industrial effluents containing simultaneously Cr(VI) and Cr(III).     

Qualitative and quantitative polymerase chain reaction (PCR) for detection of Leishmania in spleen samples from naturally infected dogs

Because infected dogs are widely considered to be the main domestic reservoir for Leishmania infantum (syn Leishmania chagasi) parasites in Brazil, the diagnosis of canine visceral leishmaniasis (CVL) must be made both accurately and promptly. The present study attempted to standardize a conventional polymerase chain reaction (cPCR) protocol for the detection of L. infantum DNA in canine spleen samples. Quantitative PCR (qPCR) technique was used to confirm the presence of Leishmania DNA in the canine spleen fragments. A comparison was made between the efficacies of these molecular diagnostic techniques and conventional parasitological and serological methods. cPCR protocols for spleen samples were standardized using primers that amplify a 145 bp fragment, located at the parasite kinetoplast minicircle. The genus specificity of the cPCR protocol was assessed by its inability to amplify the DNA of other common canine pathogens, such as Ehrlichia canis, Babesia canis, Toxoplasma gondii and Trypanosoma cruzi. cPCR protocol sensitivity was tested by assessing the reaction detection limit, determined to be 10 fg of L. infantum reference strain DNA, which corresponds to a range of 0.03-0.1 parasites per fragment. Standardized cPCR protocol was used to detect the presence of Leishmania in 45 dog spleen samples. Our results showed that 40% of the spleen fragment cultures were positive for Leishmania parasites, 58% of the dog serum samples tested positive using ELISA, and parasite DNA was detected in 44% using qPCR, while 47% of the spleen samples using cPCR. Diagnostic methods performance was assessed and revealed a better degree of ascertainment for cPCR when compared to other diagnostic methods. The sensitivity of ELISA was 83.3%, qPCR was 83.3%, and cPCR was 88.9%; PPV for ELISA was 57.7%, qPCR was 75% and cPCR was 76.2%; the Kappa coefficients were found to be 0.40 (fair) for ELISA, 0.64 (substantial) for qPCR and 0.68 (substantial) for cPCR. In both oligosymptomatic and polysymptomatic dogs, cPCR revealed the better performance analysis when compared to other diagnostic methods. The findings presented herein establish cPCR as the most indicated test to detect Leishmania when compared to the other two diagnostic methods evaluated. Despite the fact that the qPCR protocol provides a highly accurate quantification of parasites when targeting the SSU rRNA gene, this technique does not significantly improve the diagnosis of CVL when compared with the performance of the cPCR protocol, which focused on the kinetoplast minicircle.     

Ion-trap tandem mass spectrometry for the analysis of polychlorinated dibenzo-p-dioxins, dibenzofurans, and dioxin-like polychlorinated biphenyls in food

This paper reports on the applicability of gas chromatography coupled to ion-trap tandem mass spectrometry (GC/ITMS/MS) for the analysis of polychlorinated dibenzo- p-dioxins (PCDDs), dibenzofurans (PCDFs), and dioxin-like polychlorinated biphenyls (dl-PCBs) in food. MS/MS parameters were selected to achieve the high sensitivity and selectivity required for food analysis. Good precision (RSD=5-18% for PCDD/Fs and 6-14% for dl-PCBs) and low limits of detection for PCDD/Fs (0.1-0.93 pg/g of fat) and dl-PCBs (0.1-0.89 pg/g of fat) were obtained. A comparative study of the congener-specific determination using both GC/ITMS/MS and GC-high resolution mass spectrometry (GC/HRMS) was performed by analyzing several matrices such as milk, fish oil, chicken, pork, fish, eggs, and a chicken compound feed, at low pg/g levels. The results using GC/ITMS/MS were in good agreement with those obtained by GC/HRMS. Consequently, GC/ITMS/MS is proposed for the analysis of PCDD/Fs and dl-PCBs in food and feed samples.     

Towards the identification of ecological management units: A multidisciplinary approach for the effective management of bottlenose dolphins in the southern Iberian Peninsula

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Biological Potential of Flaxseed Protein Hydrolysates Obtained by Different Proteases

Plant Foods for Human Nutrition - Flaxseed meal, a byproduct of flaxseed oil extraction, was treated as low-value agrowaste for a long time despite its high protein content. Flaxseed meal has...     

Chlorella vulgaris Extracts as Modulators of the Health Status and the Inflammatory Response of Gilthead Seabream Juveniles (Sparus aurata)

This study aimed to evaluate the effects of short-term supplementation, with 2% Chlorella vulgaris (C. vulgaris) biomass and two 0.1% C. vulgaris extracts, on the health status (experiment one) and on the inflammatory response (experiment two) of gilthead seabream (Sparus aurata). The trial comprised four isoproteic (50% crude protein) and isolipidic (17% crude fat) diets. A fishmeal-based (FM), practical diet was used as a control (CTR), whereas three experimental diets based on CTR were further supplemented with a 2% inclusion of C. vulgaris biomass (Diet D1); 0.1% inclusion of C. vulgaris peptide-enriched extract (Diet D2) and finally a 0.1% inclusion of C. vulgaris insoluble fraction (Diet D3). Diets were randomly assigned to quadruplicate groups of 97 fish/tank (IBW: 33.4 ± 4.1 g), fed to satiation three times a day in a recirculation seawater system. In experiment one, seabream juveniles were fed for 2 weeks and sampled for tissues at 1 week and at the end of the feeding period. Afterwards, randomly selected fish from each group were subjected to an inflammatory insult (experiment two) by intraperitoneal injection of inactivated gram-negative bacteria, following 24 and 48 h fish were sampled for tissues. Blood was withdrawn for haematological procedures, whereas plasma and gut tissue were sampled for immune and oxidative stress parameters. The anterior gut was also collected for gene expression measurements. After 1 and 2 weeks of feeding, fish fed D2 showed higher circulating neutrophils than seabream fed CTR. In contrast, dietary treatments induced mild effects on the innate immune and antioxidant functions of gilthead seabream juveniles fed for 2 weeks. In the inflammatory response following the inflammatory insult, mild effects could be attributed to C. vulgaris supplementation either in biomass form or extract. However, the C. vulgaris soluble peptide-enriched extract seems to confer a protective, anti-stress effect in the gut at the molecular level, which should be further explored in future studies.     

Selection for tetracycline-resistant Chlamydia suis in treated pigs

The aim of this study was to investigate Chlamydia suis in a pig farm with an outbreak of conjunctivitis and diarrhea. Eye swabs and pooled fecal samples were investigated for the presence of C. suis by real-time PCR and ArrayTube microarray. Samples positive for C. suis by ArrayTube microarray assay were further tested for the presence of the tet(C) resistance gene by PCR. In the first examination, C. suis was identified in 12 six-week-old pigs showing conjunctivitis. Of these, the tet(C) gene-coding region was amplified in one pooled fecal sample and one eye swab, respectively. After oral treatment with tetracycline, clinical symptoms disappeared. Subsequently, all eye swabs investigated from 10 healthy pigs were positive for C. suis and the tet(C) gene-coding region. The present study reports rapid selection for tetracycline-resistant C. suis after antibiotic treatment.     

Presence of the <Emphasis Type="Italic">Eucalyptus</Emphasis> gall wasp <Emphasis Type="Italic">Ophelimus maskelli</Emphasis> and its parasitoid <Emphasis Type="Italic">Closterocerus chamaeleon</Emphasis> in Portugal: First record,geographic distribution and host preference

The Eucalyptus gall wasp Ophelimus maskelli (Hymenoptera: Eulophidae) and its parasitoid Closterocerus chamaeleon (Hymenoptera: Eulophidae) were observed for the first time in Portugal, in 2006 and 2007, respectively. Data on the distribution of O. maskelli in Portugal, differences in the susceptibility of two host species, Eucalyptus globulus and Eucalyptus camaldulensis, and parasitism by C. chamaeleon are given.     

Partially defatted black soldier fly larva meal inclusion in piglet diets: effects on the growth performance,nutrient digestibility,blood profile,gut morphology and histological features

Background: The aim of this trial was to investigate the effects of different inclusion levels of a partial y defatted black soldier fly(BSF, Hermetia il ucens L.) larva meal on the growth performance, nutrient digestibility, blood profile, gut morphology and histological features of piglets. A total of 48 newly weaned piglets were individual y weighed(initial body weight(IBW): 6.1 ± 0.16 kg) and randomly al ocated to 3 dietary treatments(4 boxes as replicates/treatment and 4 animals/box). BSF larva meal was included at increasing levels(0% [BSF0], 5% [BSF5] and 10% [BSF10]) in isonitrogenous and isoenergetic diets formulated for two feeding phases: I(from d 1 to d 23) and I(from d 24 to d 61). The weight gain(WG), average daily gain(ADG), average daily feed intake(ADFI) and feed conversion ratio(FCR) were calculated for each feeding phase and for the whole trial. The haematochemical parameters and nutrient digestibility of the piglets were also evaluated. A total of 3 piglets per box were slaughtered on d 61 and the slaughtered piglets were submitted to morphometric investigations and histopathological examinations.Results: No overal significant differences were observed for growth performance(P 0.05), except for the ADFI of phase I, which showed a linear response to increasing BSF meal levels(P 0.05, maximum for the BSF10 group). Dietary BSF meal inclusion did not significantly influence the blood profile, except as far as monocytes and neutrophils are concerned,and these showed a linear and quadratic response, respectively, to increasing BSF meal levels(P 0.05, maximum for the BSF10 and BSF5 groups, respectively). On the other hand, the nutrient digestibility, gut morphology and histological features were not affected by dietary BSF meal inclusion(P 0.05).Conclusions: The obtained results show that a partial y defatted BSF larva meal can be used as a feed ingredient in diets for weaned piglets without negatively affecting their growth performance, nutrient digestibility, blood profile, gut morphology or histological features.