Resazurin to determine the minimum inhibitory concentration on antifungal susceptibility assays for Fonsecaea sp. using a modified EUCAST protocol.

Chromoblastomycosis is a fungal chronic disease, which affects humans, especially in cutaneous and subcutaneous tissues. There is no standard treatment for Chromoblastomycosis, and it is a therapeutic challenge, due natural resistance of their causative agents, inadequate response of patients and common cases of relapse. Protocols for determination of antifungal drugs susceptibility are not standardized for chromoblastomycosis agents and endpoint definition is usually based on visual inspection, which depends on the analyst, making it sometimes inaccurate. We presented a colorimetric and quantitative methodology based on resazurin reduction to resofurin to determine the metabolic status of viable cells of Fonsecaea sp. Performing antifungal susceptibility assay by a modified EUCAST protocol allied to resazurin, we validated the method to identify the minimum inhibitory concentrations of itraconazole, fluconazole, amphotericin B, and terbinafine for eight Fonsecaea clinical isolates. According to our data, resazurin is a good indicator of metabolic status of viable cells, including those exposed to antifungal drugs. This work aimed to test resazurin as an indicator of the metabolic activity of Fonsecaea species in susceptibility assays to antifungal drugs. Species of this genus are the main causative agents of Chromoblastomycosis, which affects humans.

A model of metformin mitochondrial metabolism in metachromatic leukodystrophy: first description of human Schwann cells transfected with CRISPR-Cas9.

Metachromatic leukodystrophy is a neurological lysosomal deposit disease that affects public health despite its low incidence in the population. Currently, few reports are available on pathophysiological events related to enzyme deficiencies and subsequent sulfatide accumulation. This research aims to examine the use of metformin as an alternative treatment to counteract these effects. This was evaluated in human Schwann cells (HSCs) transfected or non-transfected with CRISPR-Cas9, and later treated with sulfatides and metformin. This resulted in transfected HSCs showing a significant increase in cell reactive oxygen species (ROS) production when exposed to 100 µM sulfatides (p = 0.0007), compared to non-transfected HSCs. Sulfatides at concentrations of 10 to 100 µM affected mitochondrial bioenergetics in transfected HSCs. Moreover, these analyses showed that transfected cells showed a decrease in basal and maximal respiration rates after exposure to 100 µM sulfatide. However, maximal and normal mitochondrial respiratory capacity decreased in cells treated with both sulfatide and metformin. This study has provided valuable insights into bioenergetic and mitochondrial effects of sulfatides in HSCs for the first time. Treatment with metformin (500 µM) restored the metabolic activity of these cells and decreased ROS production.

Biological removal of hexavalent chromium: evaluation of the metabolic activity of native and Cr(VI)-acclimated activated sludge using a respirometric method.

Hexavalent chromium becomes in one of the tops internationally concern environmental issues due to its wide usage in several industrial activities. There are two stable oxidation states of chromium in the environment which differ significantly on its toxicity; Cr(III) has lower solubility, mobility and lesser biological toxicity in comparison with Cr(VI). While Cr(VI) is a well-known carcinogen, Cr(III) is an essential dietary element. For this reason, most technologies focus attention on the reduction of Cr(VI) to Cr(III). On this context, the ability of microorganisms to reduce Cr(VI) to Cr(III) has gained attention. The objectives of the present work were to analyze the effect of Cr(VI) on the activated sludge community in a continuous reactor, and to evaluate the differences on the metabolic activity of native (NAS) and Cr(VI)-acclimated activated sludge (CrAAS) using a respirometric method. Results showed that the activated sludge community had the capability to acclimate to the presence of Cr(VI). On the other hand, the increase of the initial Cr(VI) concentration from 0 to 100 mgCr/L leads to a decrease in the specific exogenous respiration rate (qEx ) values, but this reduction was more noticeably in the case of NAS in comparison with CrAAS. The respirometric curves were well described by the proposed mathematical model. It was concluded that the CrAAS tolerated a Cr(VI) concentration about one order of magnitude higher than NAS, which was positively reflected in the respiration rate first-order decay constant (kd ), the specific maximum exogenous respiration rate (qExm ), and the observed oxidation coefficient (YO/S ) values.

Soymilk fermentation: effect of cooling protocol on cell viability during storage and in vitro gastrointestinal stress.

This work covers soymilk fermentation by starter and probiotic cultures and explores the influence of cooling protocol on cell viability, organic acid production, sugar consumption, fatty acid profile, and cell survival to in vitro gastrointestinal stress. After fermentation at 37 °C by mono- or co-cultures of Streptococcus thermophilus (St), Lactobacillus bulgaricus (Lb), and Lactobacillus paracasei (Lp), fermented soymilk was cooled directly at 4 °C for 28 days or cooled in two phases (TPC), i.e., by preceding that step by another at 25 °C for 8 h. Soybean milk fermentation by Lb alone lasted longer (15 h) than by StLb or StLbLp (9 h). In ternary culture, TPC increased Lp viability, linoleic, and lactic acid concentrations by 3.8, 22.6, and 96.2%, respectively, whereas the cooling protocol did not influence Lp and St counts after in vitro gastrointestinal stress. Graphical abstract.

Cannabidiol Prevents the Expression of the Locomotor Sensitization and the Metabolic Changes in the Nucleus Accumbens and Prefrontal Cortex Elicited by the Combined Administration of Cocaine and Caffeine in Rats.

In the last years, clinical and preclinical researchers have increased their interest in non-psychotomimetic cannabinoids, like cannabidiol (CBD), as a strategy for treating psychostimulant use disorders. However, there are discrepancies in the pharmacological effects and brain targets of CBD. We evaluated if CBD was able to prevent the locomotor sensitization elicited by cocaine and caffeine co-administration. The effect of CBD on putative alterations in the metabolic activity of the medial prefrontal cortex (mPFC) and nucleus accumbens (NAc), and its respective subregions (cingulated, prelimbic, and infralimbic cortices, and NAc core and shell) associated to the behavioral response, was also investigated. Rats were intraperitoneally and repeatedly treated with CBD (20 mg/kg) or its vehicle, followed by the combination of cocaine and caffeine (Coc+Caf; 5 mg/kg and 2.5 mg/kg, respectively) or saline for 3 days. After 5 days of withdrawal, all animals were challenged with Coc+Caf (day 9). Locomotor activity was automatically recorded and analyzed by a video-tracking software. The metabolic activity was determined by measuring cytochrome oxidase-I (CO-I) staining. Locomotion was significantly and similarly increased both in Veh-Coc+Caf- and CBD-Coc+Caf-treated animals during the pretreatment period (3 days); however, on day 9, the expression of the sensitization was blunted in CBD-treated animals. A hypoactive metabolic response and a hyperactive metabolic response in mPFC and NAc subregions respectively were observed after the behavioral sensitization. CBD prevented almost all these changes. Our findings substantially contribute to the understanding of the functional changes associated with cocaine- and caffeine-induced sensitization and the effect of CBD on this process.

Candidemia Candida albicans clusters have higher tendency to form biofilms than singleton genotypes†.

The capacity of Candida spp. to form biofilms allows them to attach either to living or inert surfaces, promoting their persistence in hospital environments. In a previous study, we reported strain-to-strain variations in Candida spp. biofilm development, suggesting that some genotypes may be greater biofilm formers than others. In this study, we hypothesize that isolates pertaining to clusters may be found more frequently in the environment due to their ability to form biofilms compared to singleton genotypes. Two hundred and thirty-nine Candida spp. isolates (78 clusters) from candidemia patients admitted to 16 hospitals located in different cities and countries-and the same number of singleton genotypes used as controls-were tested in terms of biofilm formation using the crystal violet and the XTT reduction assays. Candida albicans clusters showed higher biofilm formation in comparison to singleton genotypes (P < .01). The biofilms formed by intra-hospital C. albicans clusters showed higher metabolic activity (P < .05). Furthermore, marked variability was found among species and type of cluster. We observed that the higher the number of isolates, the higher the variability of biofilm production by isolates within the cluster, suggesting that the production of biofilm by isolates of the same genotype is quite diverse and does not depend on the type of cluster studied. In conclusion, candidemia Candida spp. clusters-particularly in the case of C. albicans-show significantly more biomass production and metabolic activity than singleton genotypes.

Effect of antiprotozoal molecules on hypnospores of Perkinsus spp. parasite.

Perkinsus protozoan parasites have been associated with high mortality of bivalves worldwide, including Brazil. The use of antiproliferative drugs to treat the Perkinsosis is an unusual prophylactic strategy. However, because of their environment impact it could be used to control parasite proliferation in closed system, such as hatchery. This study evaluated the anti-Perkinsus activity potential of synthesized and commercial compounds. Viability of hypnospores of Perkinsus spp. was assessed in vitro. Cells were incubated with three 2-amino-thiophene (6AMD, 6CN, 5CN) and one acylhydrazone derivatives (AMZ-DCL), at the concentrations of 31.25; 62.5; 125; 250 and 500 µM and one commercial chlorinated phenoxy phenol derivative, triclosan (2, 5, 10 and 20 µM), for 24-48 h. Two synthetic molecules (6CN and AMZ-DCL) caused a significant decline (38 and 39%, respectively) in hypnospores viability, at the highest concentration (500 µM), after 48 h. Triclosan was the most cytotoxic compound, causing 100% of mortality at 20 µM after 24 h and at 10 µM after 48 h. Cytotoxic effects of the compounds 6CN, AMZ-DCL, and triclosan were investigated by measuring parasite's zoosporulation, morphological changes and metabolic activities (esterase activity, production of reactive oxygen species and lipid content). Results showed that zoosporulation occurred in few cell. Triclosan caused changes in the morphology of hypnospores. The 6CN and AMZ-DCL did not alter the metabolic activities studied whilst Triclosan significantly increased the production of reactive oxygen species and changed the amount and distribution of lipids in the hypnospores. These results suggest that three compounds had potential to be used as antiprotozoal drugs, although further investigation of their mechanism of action must be enlightened.

Characterization of MXR activity in the sea anemone Bunodosoma cangicum exposed to copper.

Transmembrane proteins of the ABC family contribute to a multiple xenobiotic resistance (MXR) phenotype in cells, driving the extrusion of toxic substances. This phenotype promotes a high degree of protection against xenobiotics. The present study provides a better understanding of the MXR activity in the podal disk cells of Bunodosoma cangicum exposed to copper, and further establishes the relationship between protein activity (measured by accumulation of rhodamine-B) and bioaccumulation of copper in these cells. Sea anemone cells were exposed for 24h to copper (0, 7.8 and 15.6µg/L) in presence and absence of MXR blocker (verapamil 50µM). Results indicate that copper exposure increases intracellular metal content when ABC proteins were blocked, causing an increase in cellular death. The present study also verified the relationship between MXR activity, ATP depletion, and general metabolic activity (by MTT). MXR activity decreased in treatment groups exposed to copper concentrations of 15.6µg/L and 10mM energy depleting potassium cyanide. Metabolic activity increased in cells exposed to 7.8µgCu/L, but 15.6µgCu/L was similar to 0 and 7.8µg/L. The presence of copper decreased the ABC proteins expression. The present study improves the knowledge of MXR in anemone cells and shows that this activity is closely associated with copper extrusion. Also, the copper exposure is able to modify the metabolic state and to lead to cytotoxicity when cells cannot defend themselves.

Chronic stress effects in contralateral medial pterygoid muscle of rats with occlusion alteration.

Temporomandibular disorder (TMD) has a high prevalence in our society, characterized by a severe pain condition of the masticatory muscles and temporomandibular joint. Despite the indication of multiple factor initiators of TMD, there is still controversy about its etiology and its pathophysiology is poorly understood. Using rats as experimental animals we investigated the effect of unpredictable chronic stress with or without unilateral molar extraction on the contralateral medial pterygoid muscle. Our hypothesis is that these two factors induce changes in morphology, oxidative metabolism and oxidative stress of muscle fibers. Young adult male Wistar rats (±200g) were divided into four groups: a group with extraction and unpredictable chronic stress (E+US); with extraction and without stress (E+C); without extraction and with unpredictable chronic stress (NO+US); and a control group without either extraction or stress (NO+C). The animals were subjected to unilateral extraction of the upper left molars, under intraperitoneal anesthesia with 4% Xylazine (10mg/kg) and 10% Ketamine (80mg/kg) on day zero. The rats of groups E+US and NO+US were submitted to different protocols of stress, from the 14th day after the extraction. The protocols were different every day for five consecutive days, which were repeated from the 6th day for five days more. Contralateral medial pterygoid muscles were obtained on the 24th day after the start of the experiment for morphological, metabolic, capillary density, and oxidative stress analysis. The data from capillary density showed a decrease of capillaries in animals subjected to dental extraction, compared with those without extraction and an increase of laminin expression in the group submitted to the unpredictable chronic stress when compared to the unexposed to stress. SDH test revealed a decrease of light fibers in the group submitted to unilateral extraction of molars, compared with this area in the control group. In E+US and NO+US groups, the deeply stained fibers increased compared to NO+C.·The exodontia factor was able to increase the ROS activity in muscle, whereas the stress factor does not significantly alter ROS in this tissue. It was concluded that both unpredictable chronic stress and the extraction induce metabolic and density of capillary changes in the contralateral medial pterygoid muscle to extraction, suggesting that these factors for a longer period of this experiment could induce muscle damage related to TMD.

Effect of Gamma Radiation on Growth and Metabolic Activities of Arthrospira platensis

This work aimed to study the influence of gamma radiation on the growth and production of some active substances of Arthrospira platensis. Biomass production was significantly inhibited (p ≤ 0.05) by 21 and 34%, with respect to the control at 2.0 and 2.5 kGy, respectively. Chlorophyll-a content showed 11% reduction at 2.5 kGy compared to the control. As a result of growth and Chl-a inhibition, chlorophyll productivity recorded a continuous significant decrease below the control in the cells exposed to 1, 1.5, 2 and 2.5 kGy by 8, 12, 15 and 25%, respectively after 15 days of incubation. In addition, phycobillins productivity showed significant decrease by 10 and 36% below the control at 2 and 2.5 kGy of gamma radiation, respectively. Protein production decreased significantly by 24% at 1.5 kGy; low doses of gamma irradiation (0.5, 1.0 and 1.5 kGy) induced carbohydrate production by 106, 246 and 146%, respectively. Lipid content increased significantly over the control at 0.5 kGy of gamma irradiation by 22%, which was decreased at higher doses. Interestingly, carotenoid productivity showed significant increase at all used gamma doses up to 155% over the control.

Biotechnological potential of Clostridium butyricum bacteria.

In response to demand from industry for microorganisms with auspicious biotechnological potential, a worldwide interest has developed in bacteria and fungi isolation. Microorganisms of interesting metabolic properties include non-pathogenic bacteria of the genus Clostridium, particularly C. acetobutylicum, C. butyricum and C. pasteurianum. A well-known property of C. butyricum is their ability to produce butyric acid, as well as effectively convert glycerol to 1,3-propanediol (38.2 g/L). A conversion rate of 0.66 mol 1,3-propanediol/mol of glycerol has been obtained. Results of the studies described in the present paper broaden our knowledge of characteristic features of C. butyricum specific isolates in terms of their phylogenetic affiliation, fermentation capacity and antibacterial properties.

Biotechnological potential of Clostridium butyricum bacteria

In response to demand from industry for microorganisms with auspicious biotechnological potential, a worldwide interest has developed in bacteria and fungi isolation. Microorganisms of interesting metabolic properties include non-pathogenic bacteria of the genus Clostridium, particularly C. acetobutylicum, C. butyricum and C. pasteurianum. A well-known property of C. butyricum is their ability to produce butyric acid, as well as effectively convert glycerol to 1,3-propanediol (38.2 g/L). A conversion rate of 0.66 mol 1,3-propanediol/mol of glycerol has been obtained. Results of the studies described in the present paper broaden our knowledge of characteristic features of C. butyricum specific isolates in terms of their phylogenetic affiliation, fermentation capacity and antibacterial properties.

Biotechnological potential of Clostridium butyricum bacteria

In response to demand from industry for microorganisms with auspicious biotechnological potential, a worldwide interest has developed in bacteria and fungi isolation. Microorganisms of interesting metabolic properties include non-pathogenic bacteria of the genus Clostridium, particularly C. acetobutylicum, C. butyricum and C. pasteurianum. A well-known property of C. butyricum is their ability to produce butyric acid, as well as effectively convert glycerol to 1,3-propanediol (38.2 g/L). A conversion rate of 0.66 mol 1,3-propanediol/mol of glycerol has been obtained. Results of the studies described in the present paper broaden our knowledge of characteristic features of C. butyricum specific isolates in terms of their phylogenetic affiliation, fermentation capacity and antibacterial properties.

Microbial populations and activities of mangrove, restinga and Atlantic forest soils from Cardoso Island, Brazil.

AIM: Mangroves provide a distinctive ecological environment that differentiates them from other ecosystems. This study deal to evaluate the frequency of microbial groups and the metabolic activities of bacteria and fungi isolated from mangrove, restinga and Atlantic forest soils. METHODS AND RESULTS: Soil samples were collected during the summer and winter at depths of 0-2, 2-5 and 5-10 cm. Except for fungi, the counts of the total, sporulating, Gram-negative, actinomycetes, nitrifying and denitrifying bacteria decreased significantly in the following order: Atlantic forest >mangrove > restinga. The counts of micro-organisms decreased by 11 and 21% from the surface to the 2-5 and 5-10 cm layers, but denitrifying bacteria increased by 44 and 166%, respectively. A larger growth of micro-organisms was verified in the summer compared with the winter, except for actinomycetes and fungi. The average frequency of bacteria isolated from mangrove, restinga and Atlantic forest soils was 95, 77 and 78%, and 93, 90 and 95% for fungi, respectively. Bacteria were amylolytic (33%), producers of acid phosphatase (79%) and solubilizers (18%) of inorganic phosphate. The proportions of fungi were 19, 90 and 27%. CONCLUSION: The mangrove soil studied had higher chemical characteristics than the Atlantic forest, but the high salinity may have restricted the growth of microbial populations. SIGNIFICANCE AND IMPACT OF THE STUDY: Estimates of the microbial counts and activities were important to elucidate the differences of mangrove ecosystem from restinga and Atlantic forest.