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Glioblastoma is the most life‐threatening tumour of the central nervous system. Temozolomide (TMZ) is the first‐choice oral drug for the treatment of glioblastoma, although it shows low efficacy. Silver nanoparticles (AgNPs) have been shown to exhibit biocidal activity in a variety of microorganisms, including some pathogenic microorganisms. Herein, the antiproliferative effect of AgCl‐NPs on glioblastoma cell lines (GBM02 and GBM11) and on astrocytes was evaluated through automated quantitative image‐based analysis (HCA) of the cells. The cells were treated with 0.1‐5.0 μg/ml AgCl‐NPs or with 9.7‐48.5 μg/ml TMZ. Cells that received combined treatment were also analysed. At a maximum tested concentration of AgCl‐NPs, GBM02 and GBM11, the growth decreased by 93% and 40%, respectively, following 72 h of treatment. TMZ treatment decreased the proliferation of GBM02 and GBM11 cells by 58% and 34%, respectively. Combinations of AgCl‐NPs and TMZ showed intermediate antiproliferative effects; the lowest concentrations caused an inhibition similar to that obtained with TMZ, and the highest concentrations caused inhibition similar to that obtained with AgCl‐NPs alone. No significant changes in astrocyte proliferation were observed. The authors’ findings showed that HCA is a fast and reliable approach that can be used to evaluate the antiproliferative effect of the nanoparticles at the single‐cell level and that AgCl‐NPs are promising agents for glioblastoma treatment.

The urgency for new materials in oncology is immediate. In this study we have developed the g-C

Ewing’s sarcoma is the most aggressive connective tissue tumor, mainly affecting children and adolescents; the 5 year survival rate is only 50%. Current treatments have poor effectiveness, and more efficient treatments are being sought. Silver-based nanoparticles, such as silver chloride nanoparticles (AgCl-NPs) and silver/silver chloride (Ag/AgCl-NPs) nanoparticles, can be biologically produced and can release Ag+ ions into solution; however, their antitumor activity has been minimally investigated. The aim of this study was to evaluate the antitumor potential of AgCl-NPs and Ag/AgCl-NPs against Ewing’s sarcoma cells. A673 cells (Ewing’s sarcoma) were treated for 72 h with 0–12.5 μg ml−1 of Ag/AgCl-NPs or 0–40 μg ml−1 of AgCl-NPs. Human cells from the RPE-1 cell line (pigmented retinal epithelium) were used as a model of nontumor cells. The RPE-1 cells were less affected by the administration of AgCl-NPs or Ag/AgCl-NPs, with small reductions in the number of cells and viability and a small increase in apoptosis rates, while lysosomal damage, changes in reactive oxygen species (ROS) production, loss of mitochondrial membrane potential and alterations in microfilaments or cell areas were not observed. A673 tumor cells had significantly reduced number and viability levels when treated with AgCl-NPs, with reductions of 65.05% and 99.17%, respectively, whereas with Ag/AgCl-NP treatment, reductions of 65.53% and 92.51% were observed, respectively. When treated with silver-based nanoparticles, A673 cells also showed a significant increase in ROS production and loss of mitochondrial membrane potential, which culminated in an increase in the percentage of apoptosis among the population. Lysosomal damage was also observed when A673 cells were treated with the highest concentration of AgCl-NPs. In conclusion, the results showed that both AgCl-NPs and Ag/AgCl-NPs had some antitumor activity with minimal effects against healthy cells, which demonstrated the possibility of their use in cancer therapy.

This study aimed to evaluate the effects of ohmic heating (OH) on probiotic inactivation, cell viability and morphology of the probiotic strains Lactobacillus acidophilus LA 05 (LA), Lacticaseibacillus casei 01 (LC), and Bifidobacterium animalis Bb 12 (BA) to develop paraprobiotics. OH at different electric field magnitudes (4, 8, and 12 V/cm at 60 Hz) and conventional heat treatment (CONV) were performed to determine the most adequate condition for the obtainment of paraprobiotics. Analysis of culturability, flow cytometry (FC), and Scanning electron microscope (SEM) was carried out. The complete inactivation by CONV was achieved only in the following conditions: LA - 95 °C/5 min, LC and BA - 95 °C/7 min. The same temperature profile was used in OH treatments to study the OH electrical effects. The OH treatment (8 V/cm) caused lower damage to the cell membrane integrity compared to the CONV treatment (p 0.05). The OH showed to be adequate technology for the efficient production of paraprobiotics.

The effect of different types of sugar (sucrose, demerara, brown, fructose, coconut sugar, and honey) on sheep milk kefir was evaluated. Microbial counts (Lactobacillus, Lactococcus, Leuconostoc, yeast), antagonistic activity against foodborne pathogens, microstructure (scanning electron microscopy), and antiproliferative activity of cancer cells were evaluated. Furthermore, the antioxidant activity (DPPH), inhibitory activity of angiotensin-converting enzyme (ACE), α-amylase, and α-glucosidase, lactose content, lactic and acetic acids and ethanol, fatty acid profile and volatile organic compounds were determined. The addition of sugars increased the Lactobacillus population (up to 2.24 log CFU/mL), metabolites concentration, antagonistic activity against pathogens, antioxidant activity (11.1 to 24.1%), ACE inhibitory activity (27.5 to 37.6%), α-amylase inhibition (18 to 37.4%), and anti-proliferative activity. Furthermore, it improved the fatty acid profile and volatile compounds. The results suggest that the replacement of sucrose with different types of sugar constitute an interesting option to be used in sheep milk kefir formulations.

Lignocellulosic biomass is one of the most abundant raw materials in the world, and it is mainly composed of carbohydrate polymers (cellulose and hemicellulose) and lignin. Its applications vary from the production of pulp and paper, to the most recent plant-based bioethanol production, which has challenge due to low hydrolysis conversion rates by the inherit recalcitrance of biomass. The biomass is naturally resistant due the high complexity in the component organization and interaction in the cell wall. The application of pretreatment technologies is one of the most used strategies to overcome biomass recalcitrance. These techniques often require a catalyst to modify the lignocellulosic structure which can be acids, alkaline compounds, ionic solutions, organic solvents, and even pressurized steam among others. The type of catalyst dictates the name of the pretreatment involved. This work presents an overview of these strategies, along with some recent contributions from the scientific community to improve biomass conversion technologies. The discussion is focused on the key factors related to the recalcitrance and conversion process, as well as the composition and physicochemical properties.

Glioblastomas (GBM) are aggressive brain tumors with very poor prognosis. While silver nanoparticles represent a potential new strategy for anticancer therapy, the silver/silver chloride nanoparticles (Ag/AgCl-NPs) have microbicidal activity, but had not been tested against tumor cells. Here, we analyzed the effect of biogenically produced Ag/AgCl-NPs (from yeast cultures) on the proliferation of GBM02 glioblastoma cells (and of human astrocytes) by automated, image-based high-content analysis (HCA). We compared the effect of 0.1–5.0 µg mL(−1) Ag/AgCl-NPs with that of 9.7–48.5 µg mL(−1) temozolomide (TMZ, chemotherapy drug currently used to treat glioblastomas), alone or in combination. At higher concentrations, Ag/AgCl-NPs inhibited GBM02 proliferation more effectively than TMZ (up to 82 and 62% inhibition, respectively), while the opposite occurred at lower concentrations (up to 23 and 53% inhibition, for Ag/AgCl-NPs and TMZ, respectively). The combined treatment (Ag/AgCl-NPs + TMZ) inhibited GBM02 proliferation by 54–83%. Ag/AgCl-NPs had a reduced effect on astrocyte proliferation compared with TMZ, and Ag/AgCl-NPs + TMZ inhibited astrocyte proliferation by 5–42%. The growth rate and population doubling time analyses confirmed that treatment with Ag/AgCl-NPs was more effective against GBM02 cells than TMZ (~ 67-fold), and less aggressive to astrocytes, while Ag/AgCl-NP + TMZ treatment was no more effective against GBM02 cells than Ag/AgCl-NPs monotherapy. Taken together, our data indicate that 2.5 µg mL(−1) Ag/AgCl-NPs represents the safest dose tested here, which affects GBM02 proliferation, with limited effect on astrocytes. Our findings show that HCA is a useful approach to evaluate the antiproliferative effect of nanoparticles against tumor cells. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s10616-018-0253-1) contains supplementary material, which is available to authorized users.

Made available in DSpace on 2018-12-11T17:17:57Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-05-01 Lignocellulosic biomasses such as banana pseudostem are attractive cellulose sources for bioenergy production, and for the use in biorefinery processes. However, pretreatment of lignocellulosic material is required to remove hemicellulose and lignin, while increasing cellulose accessibility to enzymatic hydrolysis (i.e., decreasing biomass recalcitrance). The effect of different concentrations of acid (H2SO4), alkaline (NaOH) and peroxide (H2O2) pretreatments on the chemical composition, cellulose accessibility, and enzymatic digestibility of banana pseudostem were studied. The water insoluble solids (WIS) recovery was low (∼30%) for the severe pretreatment conditions applied, indicating high material solubilization. Acid pretreatment completely removed the hemicellulose content, whereas alkaline and peroxide pretreatments reduced its amount to 4.38 and 8.68%, respectively. In contrast, the lignin content increased (from 17.26 to 39.99%) after severe acid pretreatment, while alkaline and peroxide pretreatments reduced the lignin content to 7.65% and 7.17%, respectively. In line with hemicellulose and lignin removal, the cellulose content increased from 60.84 to 75.48 and 74.37%, respectively for alkaline and peroxide pretreatments, with no alteration for acid. Dye adsorption assays showed that alkaline and acid pretreatments resulted in high internal and external specific surface areas – indicative of high cellulose accessibility – when compared with peroxide pretreatments. Overall, alkaline and acid pretreatments resulted in the highest glucose yields from enzymatic hydrolysis of banana pseudostem, compared with peroxide pretreatment. In conclusion, concentrations of each pretreatment that led to the highest glucose yields was identified, confirming that the banana pseudostem is a great source of fermentable sugars, with high potential for biofuel production. Bioenergy Research Institute (IPBEN) Universidade Estadual Paulista (UNESP) Laboratory of Microscopy Applied to Life Science – Lamav National Institute of Metrology Quality and Technology – Inmetro Center for the Study of Social Insects (CEIS) Universidade Estadual Paulista (UNESP) Laboratory of Cellular Ultrastructure Hertha Meyer Federal University of Rio de Janeiro – UFRJ Bioenergy Research Institute (IPBEN) Universidade Estadual Paulista (UNESP) Center for the Study of Social Insects (CEIS) Universidade Estadual Paulista (UNESP)

In a previous paper (RSC Adv., 2015, 5, 66886–66893), we showed that the combination of silver nanoparticles (NanoAg) with doxycycline (DO) culminated in an increased bactericidal activity towards E. coli. Herein we further investigated the metabolic changes that occurred on Staphylococcus aureus upon exposure to NanoAg with the help of attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) coupled with multivariate data analysis. It has been discovered that the combination of DO with NanoAg produced metabolic changes in S. aureus that were not simply the overlap of the treatments with DO and NanoAg separately. Our results suggest that DO and NanoAg act synergistically to impede protein synthesis by the bacteria.

The urgency for new materials in oncology is immediate. In this study we have developed the g-C3N4, a graphitic-like structure formed by periodically linked tris-s-triazine units. The g-C3N4 has been synthesized by a simple and fast thermal process. XRD has shown the formation of the crystalline sheet with a compacted structure. The graphite-like structure and the functional groups have been shown by Raman and FTIR spectroscopy. TEM image and AFM revealed the porous composed of five or six C-N layers stacked. DRS and Photoluminescence analyses confirmed the structure with band gap of 2.87 eV and emission band at 448 nm in different wavelengths excitation conditions. The biological results showed inhibitory effect on: cancer cell lines and non-toxic effect in normal cell lines. To the best of our knowledge, this is the first work demonstrating the cytotoxic effects of 2D g-C3N4 in a cancer cell line, without any external or synergistic influence. The biodistribution/tissue accumulation showed that g-C3N4 present a tendency to accumulation on the lung in the first 2 hours, but after 24 hours the profile of the biodistribution change and it is found mainly in the liver. Thus, 2D-g-C3N4 showed great potential for the treatment of several cancer types.

This study aimed to evaluate the impact in nutritional and rheological parameters on sheep milk ice cream by fat replacement for different prebiotic fibers (inulin, fructo-oligosaccharide, galacto-oligosaccharide, short-chain fructo-oligosaccharide, resistant starch, soluble corn fiber, and polydextrose), also consumer perception (Pivot Profile). Low caloric content, around 98 kcal/100 g, were observed due to prebiotic fibers supplementation; without loss on moisture, carbohydrates or protein values. Sheep milk ice creams presented a pseudoplastic behavior when submitted to flow curve analysis. Moreover, inulin and fructo-oligosaccharide have proven to be a promising alternative as fat substitutes in sheep milk ice cream formulation, due to the similar rheological properties, such as hardness, viscoelasticity and consistency. Sheep milk ice creams containing inulin or fructo-oligosaccharide were also perceived creamier and brighter than control full-fat ice sheep milk ice cream. In addition, most prebiotic ice creams were cited as sweeter than control, meaning those fibers could act as sweeteners. In conclusion, the replacement of sheep milk fat by prebiotics to manufacture sheep milk ice cream can be an effective alternative to improve the nutritional and physiological aspects due to low caloric value and functionality provided by prebiotics.

Gold nanoparticles (GNPs) are regarded as potential platforms for drug delivery. However, their interaction with live organisms must be understood prior to their utilization as drug carriers. The present study reports the anti-inflammatory, analgesic and anti-tumor effects of GNPs. The biodistribution of GNPs and their effect on various tissues have also been studied. GNPs were synthesized through an environmentally friendly route and characterized with TEM and UV–vis. After HT-29 cells had been exposed to GNPs, apoptosis was assessed with Annexin V and propidium iodide staining and caspase-3 activity determined with a confocal laser scanning microscope. GNPs were administrated to male and female Swiss mice for posterior assessment of their anti-inflammatory and analgesic properties. The biodistribution of GNPs and their impact on tissues were studied with UV–vis and histopathological analysis, respectively. Cell apoptosis was observed in a dose-dependent manner for GNPs concentrations ranging from 40 μg/mL to 80 μg/mL (p < 0.05). The best anti-inflammatory activity was observed at the dose of 1500 μg/kg, which caused a reduction of 49.3% in leukocyte migration. GNPs showed peripheral analgesia at the dose of 1500 μg/kg and have been found in liver, spleen, kidney and lungs. Histopathological examination revealed extravasation of red blood cells in lungs. The study draws attention to gold nanoparticles as a resource for technological innovation in the anti-inflammatory, analgesic and anti-tumor fields. GNPs have biological effects that deserve investigation to assess their full interaction with organic systems.

Made available in DSpace on 2021-06-25T10:14:12Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-01-01 Lignocellulosic materials are complex, recalcitrant, and difficult to release fermentable carbohydrates, such as xylose. Xylose is the feedstock for the development of products with high added value, such as ethanol, butanol, xylitol, and furfural. A high xylose yield was obtained from epidermis-free stem and external fraction (containing epidermis) of sugarcane with minimal formation of degradation products using dilute acid pretreatment. The influences of variables such as sulfuric acid concentration (% m/m or % m/v), reaction time (min), and solid/liquid ratio (% m/v) on acid pretreatment were investigated through a central composite design. In study region 1, stem and external fraction had xylose yields of 58.69 and 55.33%, as well as 1.79 and 2.16% of furfural. The optimal condition to obtain high xylose yield was observed with the use of 50% m/m or 1% m/v of sulfuric acid. The highest xylose extraction was achieved in the epidermis-free stem, leading to a less recalcitrant fraction. Xylose has a key role in obtaining products with high added value. For this reason, this study obtained high xylose yield applying optimal conditions of dilute acid pretreatment, with minimum degradation products. Laboratory of Microscopy Applied to Life Science – Lamav National Institute of Metrology Quality and Technology – Inmetro Institute for Research in Bioenergy IPBEN University of São Paulo State (UNESP) Institute for Research in Bioenergy IPBEN University of São Paulo State (UNESP)

Made available in DSpace on 2020-12-12T02:48:42Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-01-01 The sugarcane bagasse is a heterogeneous material and needs a pretreatment to breakdown its complex structure to make cellulose accessible to enzyme action. This study aimed to evaluate pseudo-lignin formation, enzymatic hydrolysis of sugarcane fractions (leaf, external fraction, internode, and node), and bagasse after partial delignification and acid pretreatment. The leaf and external fraction presented the highest content of lignin, and external fraction was the most recalcitrant material resulting in lower glucose release. Pretreatment with diluted sulfuric acid (20% m/m or 2% m/v) generated 5 g/L of acetic acid and 2.07 g/L of 5-hydroxymethylfurfural (external fraction in natura and leaf extractive-free, respectively). Furfural ranged between 0.11 g/L (node delignified) and 0.06 g/L (leaf, external fraction, and node in natura). A decrease was observed in pseudo-lignin formed with extractive-free and delignified biomasses, with different structure compared with non-delignified samples. The biomass partial delignification and subsequent pretreatment with dilute acid generate a material with fragmented anatomical structure, with improved cellulose accessibility, favoring enzymatic hydrolysis achieving more than 90% of glucose yield (up to 12 g/L). This study has shown strategies to decrease material heterogeneity and avoid pseudo-lignin formation as it results in lower recalcitrance and better efficiency of the enzymatic hydrolysis. Institute for Research in Bioenergy (IPBEN) São Paulo State University (UNESP) Laboratory of Microscopy Applied to Life Science(Lamav) National Institute of Metrology Quality and Technology (Inmetro) Institute for Research in Bioenergy (IPBEN) São Paulo State University (UNESP)

We show that sodium 9,10-epoxy-12-hydroxytetradecanoate (SEAR), an epoxidized derivative of ricinoleic acid, simultaneously functioned as reducing and stabilizing agents in the synthesis of silver nanoparticles in alkaline aqueous medium. The advantage of using SEAR is its biodegradability and nontoxicity, which are important characteristics for mitigation of environmental impact upon discharge of nanoparticles into terrestrial and aquatic ecosystems. The SEAR concentration was found to impact considerably the size distribution of silver nanoparticles (AgNPs). A concentration below the SEAR critical micelle concentration (CMC) generated 23 nm sized AgNPs with 10 nm standard deviation, while 50 nm sized AgNPs (σ=21 nm) were obtained at a concentration above the SEAR CMC. FTIR analysis revealed that the carboxylate that constitutes the SEAR hydrophilic head binds directly to the AgNPs surface promoting stabilization in solution. Finally, AgNPs turned into Ag2S upon contact with wastewater samples from Wastewater Treatment Plant at Federal University of Rio Grande do Norte (UFRN), Brazil, which is an interesting result, since Ag2S is more environmentally friendly than pure AgNPs.

SUMMARY Biodiesel is a renewable fuel produced mostly from edible and non-edible vegetables, by transesterification of neutral lipids (triacylglycerols). However, vegetable oil-based biodiesel production competes with food crops for arable land, increasing food prices and leading to biodiversity loss. The production of biodiesel from oleaginous microorganisms – particularly microalgae – has attracted attention due to the higher lipid productivity of these organisms, when compared with vegetables. Several environmental factors – including light, temperature, pH and the presence of nutrients (particularly nitrogen, phosphorus and iron) – influence directly the ability of microalgae to produce and store triacylglycerols and other lipids, and also modulate microalgal growth. Although some environmental factors affect several species in a similar manner, differential responses between species are frequent, highlighting the importance of identifying optimal cultivation conditions for each species, to balance growth and lipid productivity for biodiesel production. Here, we reviewed the particular influence of the physicochemical and nutritional factors on the growth and lipid productivity of different green oleaginous microalgae species.

Made available in DSpace on 2018-12-11T16:41:04Z (GMT). No. of bitstreams: 0 Previous issue date: 2016-06-01 Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) Sugarcane culm has different types of tissue organization, and its heterogeneity can influence the bagasse quality generated in the sugar/ethanol industry. The heterogeneity of the sugarcane bagasse contributes to the intrinsic recalcitrance of the biomass, impairing its conversion to ethanol. To determine the effects of sugarcane culm heterogeneity, the internode and node fractions were pretreated with acid, alkali, and peroxide. The investigation focused on the change in the fraction morphology, crystallinity, chemical composition, and enzymatic digestibility. Scanning electron microscopy (SEM) revealed different anatomical traits between the node and the internode. Vascular bundles appeared in larger number and diameter in the periphery of both the node and the internode. The internode and node responded differently to the pretreatments, with slight differences in the amount of hemicellulose and lignin removal. Furthermore, the internode was more susceptible to the acid and alkaline pretreatments than the node, generating a material with better digestibility. The fractions pretreated with peroxide showed similar enzymatic digestibility. In SEM images, the internode showed more structural damage after pretreatments than the node fibers. These results provide insight that pretreated sugarcane bagasse has fractions with different enzymatic digestibility related to their anatomy, and that conversion of the entire biomass is not feasible. Laboratory of Biotechnology (Labio) Metrology Applied to Life Science Division-National Institute of Metrology Quality and Technology (Inmetro) Laboratory of Biomass Characterization Bioenergy Research Institute (IPBEN) São Paulo State University (UNESP) Laboratory of Biocatalysis National Institute of Technology (INT) Federal University of Rio de Janeiro Chemistry Institute Department of Biochemistry Laboratory of Cell Structure Institute of Biophysics Carlos Chagas Filho Rio de Janeiro Federal University National Science and Technology Institute in Structural Biology and Bioimaging The National Institute of Science and Technology Structural Biology and Bioimaging Federal University of Rio de Janeiro-UFRJ Laboratory of Biomass Characterization Bioenergy Research Institute (IPBEN) São Paulo State University (UNESP)

Biodiesel from oleaginous microorganisms is a viable substitute for a fossil fuel. Current methods for microorganism lipid productivity evaluation do not analyze lipid dynamics in single cells. Here, we described a high-content image analysis (HCA) as a promising strategy for screening oleaginous microorganisms for biodiesel production, while generating single-cell lipid dynamics data in large cell density. Rhodotorula slooffiae yeast were grown in standard (CTL) or lipid trigger medium (LTM), and lipid droplet (LD) accumulation was analyzed in deconvolved confocal microscopy images of cells stained with the lipophilic fluorescent Nile red (NR) dye using automated cell and LD segmentation. The 'vesicle segmentation' method yielded valid morphometric results for limited lipid accumulation in smaller LDs (CTL samples) and for high lipid accumulation in larger LDs (LTM samples), and detected LD localization changes. Thus, HCA can be used to analyze the lipid accumulation patterns likely to be encountered in screens for biodiesel production.

Quinoxaline derivatives have attracted considerable attention due to their vast range of applications that includes electroluminescence and biomedicine. Concerning the latter, the literature has shown that compounds with a quinoxaline motif bind quite efficiently to phosphatidylinositol-4,5-bisphosphate 3-kinases (PI3Ks), which are enzymes found to be overexpressed in some types of neoplasms. In the present study, gold nanoparticles (AuNPs) were easily functionalized with 2,3-diethanolminoquinoxaline (DEQX) and 2-(2,3-dihydro-[1,4]oxazino[2,3-b]quinoxalin-4-yl)ethanol (OAQX). We made use of glycerol in alkaline media as reducing agent and the quinoxalines served as capping ligands to stabilize the AuNPs. This is the first report on the modification of a nanostructure with quinoxalines. Functionalization confers nanoparticles the required specificity to target only cancer cells, which opens possibilities for phototherapy since the modified AuNPs would concentrate in the tumor tissue as a consequence of PI3Kα overexpression. Molecular dynamics simulations have shown that DEQX and OAQX are potential inhibitors of PI3Kα since they bind to the active site of the enzyme in a way similar to other known inhibitors.

Stem cell tissue constructs are likely to come into contact with silver-based nanoparticles—such as silver chloride nanoparticles (AgCl–NPs)—used as microbicidals at the implant site or in cosmetics. However, the effect of silver-based nanoparticles on 3D cell cultures with potential for tissue engineering has received little attention. Here, we examined the effect of sub-lethal doses (5, 10 and 25 µg/mL, for 1, 7 and 21 days) of AgCl–NPs produced by ‘green’ bacterial-based synthesis on spheroid 3D cultures of human adipose tissue stem cells (ASCs). Light microscopy analysis revealed that the shape and diameter of ASC spheroids remained largely unchanged after AgCl–NP treatment. Flow cytometry analysis with 7-AAD and 2′,7′-dichlorofluorescein diacetate revealed no statistically significant differences in cell death but showed an increase of ROS levels for the untreated group and significant differences for the groups treated with 5 and 10 µg/mL at day 7 (p = 0.0395, p = 0.0266, respectively). Electron microscopy analysis showed limited cell damage in the periphery of AgCl–NP-treated spheroids. However, treatment with AgCl–NP had statistically significant effects on the secretion of IL-6, IL-8, IL-1β and IL-10 by spheroids, at specific treatment periods and concentrations, and particularly for IL-6, IL-8 and IL-1β. TGF-β1 and -β2 secretion also changed significantly throughout the treatment period. Our results indicate that, despite having little effect on cell viability and morphology, sub-lethal AgCL–NP doses modulate ROS production at day 7 for the groups treated with 5 and 10 µg/mL and also modulate the secretory profile of ASC spheroids. Thus, the use of skin implants or products containing Ag-NPs may promote long-term disturbances in subcutaneous adipose tissue homeostasis.

CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior FAPESP - Fundação de Amparo à Pesquisa do Estado de São Paulo Spray drying is a widely used method for producing milk powder. This process is not aimed to cause microbial inactivation, thus sporeforming bacteria may be abundant in the microbiota of milk powder. The first aim of this study was to determine the inactivation kinetics parameters in capillary tubes of three Bacillus cereus strains (436, B63, 540) in three menstrua (whole milk, phosphate buffer, and talc suspension) at 90, 100, and 110 C. D-values for B. cereus in the three menstrua were not significantly different at the highest tested temperature (p > 0.05). Thus, talc was chosen as a carrier agent to allow the recovery of B. cereus from spray dried materials given its low interference on inactivation kinetics. B. cereus spores were also inoculated in whole milk and skim milk following spray drying at 95, 105, and 110 C (outlet temperature). After the spray drying runs, B. cereus spores were counted and the number of decimal reductions (g) calculated. A correlation between the small diameter of the particles with the survival of spores of three B. cereus strains was found, and B. cereus 436 presented consistently the lowest g no matter temperature and a carrier agent. The highest g was found when talc powder was used, which suggest that this carrier agent does not protect B. cereus spores during spray drying. Spray drying of milk can lead to up to 4 g (strain 540) of B. cereus spores but depending on the strain less than one g (strain 436) could be observed. This study contributes to the knowledge on the microbiology of low water activity foods by providing novel findings regarding the fate of three B. cereus strains to different spray drying conditions. Acknowledging the variability of inactivation of B. cereus during spray drying is key in the current context of food safety in which the quantification of effects of unit operations must be known for the validation of processes and development of more robust formulations.

In a previous paper (

Cryptococcus neoformans is a fungal pathogen that causes life-threatening infections in immunocompromised individuals. Its main virulence factor is an extracellular polysaccharide capsule whose structure, assembly and dynamics remain poorly understood. In this study, we apply improved protocols for sample preparation and recently-developed scanning microscopy techniques to visualize the ultrastructure of the C. neoformans capsule at high-resolution (up to 1 nm) and improved structural preservation. Although most capsule structures in nature consist of linear polymers, we show here that the C. neoformans capsule is a 'microgel-like' structure composed of branched polysaccharides. Moreover, we imaged the capsule-to-cell wall link, which is formed by thin fibers that branch out of thicker capsule filaments, and have one end firmly embedded in the cell wall structure. Together, our findings provide compelling ultrastructural evidence for a branched and complex capsule conformation, which may have important implications for the biological activity of the capsule as a virulence factor.

Here, we provide the first evidence of yeast strains assisted Ag/AgCl-NPs production in vitro. The formed nanoparticles were characterized by spectroscopic and electron microscopy approaches. UV-vis supported the biosynthesis. TEM analysis evidenced that the nanoparticles mainly presented a circular shape and their diameters varied mostly being in the range 2 to 10 nm. XRD analysis showed a crystalline structure, with diffraction peaks corresponding to metallic silver and silver chloride nanoparticles, and when analyzed by high-resolution transmission electron microscopy (HRTEM), instead of being round, (111) (octahedral) and (200) (cubic) symmetry facets appeared systematically in one side of the nanoparticles. Analysis of ultra-thin sections by TEM indicated that the domain of the synthesis of Ag/AgCl-NPs was mainly between the cell wall and the plasma membrane. By using 3D reconstruction obtained from focused ion beam scanning electron microscopy (FIB/SEM) the spatial distribution of the domains of nanoparticle synthesis was mapped and nanoaggregates of Ag/AgCl-NPs up 35 nm in diameter were observed. Extracellular synthesis also occurred; in accordance with the fact that conditioned media from yeast isolates were as efficient at producing Ag/AgCl-NPs as live-cell cultures. Exposure of Gram-positive Staphylococcus aureus and Gram-negative Klebsiella pneumoniae cultures to Ag/AgCl-NPs led to a strong growth inhibition as shown by optical density measurements. The Ag/AgCl-NPs described here have characteristics compatible with a strong potential for use in the biotechnology industry, particularly for biomedical applications.

Gamma aminobutyric acid (GABA) is widely known as a neurotransmitter and signal transduction molecule found in vertebrates, plants, and some protozoan organisms. However, the presence of GABA and its role in trypanosomatids is unknown. Here, we report the presence of intracellular GABA and the biochemical characterization of its uptake in Trypanosoma cruzi, the etiological agent of Chagas' disease. Kinetic parameters indicated that GABA is taken up by a single transport system in pathogenic and nonpathogenic forms. Temperature dependence assays showed a profile similar to glutamate transport, but the effect of extracellular cations Na(+) , K(+) , and H(+) on GABA uptake differed, suggesting a different uptake mechanism. In contrast to reports for other amino acid transporters in T. cruzi, GABA uptake was Na(+) dependent and increased with pH, with a maximum activity at pH 8.5. The sensitivity to oligomycin showed that GABA uptake is dependent on ATP synthesis. These data point to a secondary active Na(+) /GABA symporter energized by Na(+) -exporting ATPase. Finally, we show that GABA occurs in the parasite's cytoplasm under normal culture conditions, indicating that it is regularly taken up from the culture medium or synthesized through an still undescribed metabolic pathway.

Borrelia burgdorferisensu lato, the causative agent of Lyme disease, is transmitted to humans through the bite of infectedIxodesspp. ticks. Successful infection of vertebrate hosts necessitates sophisticated means of the pathogen to escape the vertebrates’ immune system. One strategy employed by Lyme disease spirochetes to evade adaptive immunity involves a highly coordinated regulation of the expression of outer surface proteins that is vital for infection, dissemination, and persistence. Here we characterized the expression pattern of bacterial surface antigens using different microscopy techniques, from fluorescent wide field to super-resolution and immunogold-scanning electron microscopy. A fluorescent strain ofB. burgdorferispirochetes was labeled with monoclonal antibodies directed against various bacterial surface antigens. Our results indicate that OspA is more evenly distributed over the surface than OspB and OspC that were present as punctate areas.

The effect of the Lactobacillus casei 01 and inulin addition on sheep milk ice cream during storage (-18 °C, 150 days) was investigated. Control, probiotic and synbiotic ice cream (10% w/w sheep milk cream; 10% w/w sheep milk cream, L. casei 01, 6 log CFU/mL; 10% w/w inulin, L. casei 01, 6 log CFU/mL, respectively) were manufactured. Microbiological counts (probiotic count, survival after in vitro gastrointestinal resistance, Caco-2 cell adhesion), bioactivity and microstructure were analysed. Physical and textural characteristics, colour parameters, thermal analysis and organic acids/volatile compounds were also evaluated. All formulations supported L. casei 01 viability and maintained above the minimum therapeutic level (>6 log CFU/mL) during storage. Inulin did not affect L. casei 01 survival after the passage through simulated gastrointestinal tract and adhesion to Caco-2 cells while improved the ACE-inhibitory and antioxidant activity. L. casei 01 addition produced several volatile compounds, such as carboxylic acids, alcohols, aldehydes and ketones. Also, scanning electron microscopy showed an interaction between probiotic bacteria and inulin fibre on synbiotic ice cream and the adhesion of L. casei to Caco-2 cells was observed.

The emergence of multi-resistant pathogens has necessitated the investigation of new strategies to cope with this ever-increasing threat to public health. In this context, we combined silver nanoparticles (AgNPs) with doxycycline (DO), an antibiotic from the class of tetracyclines, to evaluate the potentiality of this hybrid as a bactericidal agent against E. coli. Polyvinylpyrrolidone (PVP) was used as a stabilizer to prevent the excessive growth and agglomeration of AgNPs. Interestingly, DO bound directly to PVP and had its concentration increased around the particle as a consequence of this interaction. As a result, the AgNPs/DO conjugates presented enhanced bactericidal properties compared to the individual components. Stabilizing agents are generally unwanted on the surfaces of nanoparticles because of their potential to block adsorption surface sites. However, we have shown that PVP played a paramount role in concentrating DO around the particle, which culminated in an increased bactericidal activity towards E. coli.

The present work describes a microcontrolled photometer based on light-emitting-diodes (LEDs) for detection of Pb2+ using gold nanoparticles (AuNPs). The photometer makes use of a single LED as a light source, a sensor TCS230 (TAOS, USA) and an Arduino electronic card as an acquisition system. On the sensor, the light from the three closely adjoined red, green, and blue LEDs composing the “white” light source LED is contact-coupled to the map-illumination pointed toward the detection cell. To maintain a constant light intensity, a common white-color LED (emitting a 450–620 nm continuous spectrum) was employed as a controllable light source. Software was written in C++ to control the photometer through a USB interface and for data acquisition. Pb2+ measurements are based on the color change of AuNPs due to their aggregation provoked by Pb2+. The method showed excellent selectivity for Pb2+ compared to other 19 metal ions (Ag+, Al3+, Ba2+, Ca2+, Cd2+, Co2+, Cr3+, Cu2+, Fe2+, Hg+, K+, Li+, Mg2+, Mn2+, Na+, Ni2+, Sn2+, Sr2+, and Zn2+). Pb2+ was detected with the photometer and also monitored via UV-Vis. Solutions containing Pb2+ in the concentration range from 0.6 to 10 mmol L−1 were employed to construct the analytical curves, proving a limit of detection (LOD) of 0.89 mmol L−1. The sensitivity was compared to that obtained with a UV-Vis spectrophotometer at 520 nm. A repeatability of 4.11% (expressed as the relative standard deviation of 10 measurements) was obtained. The proposed method was successfully applied to detect Pb2+ in spiked water samples.

The lignocellulosic biomass of plant cell wall is a source of sugar that can be used for second generation of bioethanol. The technology used for converting the fermentable sugar in the cell wall into bioethanol involves a pretreatment step that helps to decrease the recalcitrance of biomass to deconstruction. Atomic force microscopy (AFM), due to its ability to generate high resolution images with negligible sample preparation, is a powerful tool for studying biological samples on a nanometer scale. In this work, we elucidated the microfibrillar arrangement of the sugarcane cell wall and analyzed the effect of a thermochemical pretreatment at the nanoscale level using AFM images. Initially, we isolated the sugarcane cell wall to improve the AFM analysis. The cellulose microfibrils distribution in the sugarcane cell wall was clarified by AFM. Cellulose microfibrils were organized in a polylaminated structure with a distinct orientation. The measured filaments were approximately 20 nm thick, which corresponds to the size of cellulose microfibrils in other crops. Additionally, we investigated the effect of the thermochemical pretreatment on the cell wall structure. The thermochemical-pretreated cell wall had undergone loss of filaments and formation of globular structures, presumably containing lignin. The surface roughness was also dramatically changed. Our results provide insights that may help to understand the sugarcane cell wall organization and the mechanism by which thermochemical pretreatments modify the cell wall structure to improve its digestibility.

Chlorophyll is a commercially important natural green pigment responsible for the absorption of light energy and its conversion into chemical energy via photosynthesis in plants and algae. This bioactive compound is widely used in the food, cosmetic, and pharmaceutical industries. Chlorophyll has been consumed for health benefits as a nutraceutical agent with antioxidant, anti-inflammatory, antimutagenic, and antimicrobial properties. Microalgae are photosynthesizing microorganisms which can be extracted for several high-value bioproducts in the biotechnology industry. These microorganisms are highly efficient at adapting to physicochemical variations in the local environment. This allows optimization of culture conditions for inducing microalgal growth and biomass production as well as for changing their biochemical composition. The modulation of microalgal culture under controlled conditions has been proposed to maximize chlorophyll accumulation. Strategies reported in the literature to promote the chlorophyll content in microalgae include variation in light intensity, culture agitation, and changes in temperature and nutrient availability. These factors affect chlorophyll concentration in a species-specific manner; therefore, optimization of culture conditions has become an essential requirement. This paper provides an overview of the current knowledge on the effects of key environmental factors on microalgal chlorophyll accumulation, focusing on small-scale laboratory experiments.

Silver nanoparticles are powerful antimicrobial agents. Here, the synthesis of silver chloride nanoparticles (AgCl-NPs) was consistently evidenced from a commercially valuable microalgae species, Chlorella vulgaris. Incubation of C. vulgaris conditioned medium with AgNO3 resulted in a medium color change to yellow/brown (with UV-vis absorbance at 415nm), indicative of silver nanoparticle formation. Energy-dispersive X-ray spectroscopy (EDS) of purified nanoparticles confirmed the presence of both silver and chlorine atoms, and X-ray diffraction (XRD) showed the typical pattern of cubic crystalline AgCl-NPs. Transmission electron microscopy (TEM) showed that most particles (65%) were spherical, with average diameter of 9.8±5.7nm. Fourier transform infrared spectroscopy (FTIR) of purified nanoparticle fractions suggested that proteins are the main molecular entities involved in AgCl-NP formation and stabilization. AgCl-NPs (from 10μg/mL) decreased by 98% the growth of Gram-positive Staphylococcus aureus and Gram-negative Klebsiella pneumoniae bacterial pathogens, and had a dose-dependent effect on cell viability, which was measured by automated image-based high content screening (HCS). Ultrastructural analysis of treated bacteria by TEM revealed the abnormal arrangement of the chromosomal DNA. Our findings strongly indicated that the AgCl-NPs from C. vulgaris conditioned medium is a promising 'green' alternative for biomedical application as antimicrobials.

The protist parasite Trypanosoma cruzi has evolved the ability to transit between completely different hosts and to replicate in adverse environments. In particular, the epimastigote form, the replicative stage inside the vector, is subjected to nutritional and osmotic stresses during its development. In this work, we describe the biochemical and global gene expression changes of epimastigotes under hyperosmotic conditions. Hyperosmotic stress resulted in cell shrinking within a few minutes. Depending on the medium osmolarity, this was followed by lack of volume recovery for at least 2 h or by slow recovery. Experiments with inhibitors, or with cells in which an aquaporin gene (TcAQP1) was knocked down or overexpressed, revealed its importance for the cellular response to hyperosmotic stress. Furthermore, the adaptation to this new environment was shown to involve the regulation of the polyphosphate polymerization state as well as changes in amino acid catabolism to generate compatible osmolytes. A genome-wide transcriptional analysis of stressed parasites revealed down-regulation of genes belonging to diverse functional categories and up-regulation of genes encoding trans-sialidase-like and ribosomal proteins. Several of these changes were confirmed by Northern blot analyses. Sequence analysis of the 3'UTRs of up- and down-regulated genes allowed the identification of conserved structural RNA motifs enriched in each group, suggesting that specific ribonucleoprotein complexes could be of great importance in the adaptation of this parasite to different environments through regulation of transcript abundance.

Acidocalcisomes are acidic calcium and polyphosphate storage organelles found in a diverse range of organisms. Here we present evidence that the biogenesis of acidocalcisomes in Trypanosoma brucei is linked to the expression of adaptor protein-3 (AP-3) complex. Localization studies in cell lines expressing β3 and δ subunits of AP-3 fused to epitope tags revealed their partial co-localization with the vacuolar proton pyrophosphatase, a marker of acidocalcisomes, with the Golgi marker Golgi reassembly and stacking protein, and with antibodies against the small GTPase Rab11. Ablation of the β3 subunit by RNA interference (RNAi) resulted in disappearance of acidocalcisomes from both procyclic and bloodstream form trypanosomes, as revealed by immmunofluorescence and electron microscopy assays, with no alterations in trafficking of different markers to lysosomes. Knockdown of the β3 subunit resulted in lower acidic calcium, pyrophosphate, and polyphosphate content as well as defects in growth in culture, resistance to osmotic stress, and virulence in mice. Similar results were obtained by knocking down the expression of the δ subunit of AP-3. These results indicate that AP-3 is essential for the biogenesis of acidocalcisomes and for growth and virulence of T. brucei.

Endocytic activity is particularly intense in Trypanosoma cruzi epimastigotes, while in amastigotes and trypo- mastigotes it is untraceable. Cargo molecules enters through the cytostome or flagellar pocket at the parasite anterior re- gion, goes along a branched early endosomal network of tubules and vesicles spread from nuclear periphery to the pos- terior pole, until delivery to reservosomes, the final compartment. Reservosomes are acid compartments that store protein and lipid cargo and also accumulate lysosomal hydrolases, modulating digestive activity. Although T. cruzi infective forms are unable to uptake molecules, its lysosome related organelles represent a potential targets for anti-parasitic chemotherapy.

Reservosomes are late endosomes present only in members of the Schizotrypanum subgenus of the Trypanosoma genus and are defined as the site of storage of endocytosed macromolecules and lysosomal enzymes. They have been extensively described in Trypanosoma cruzi epimastigote: are bounded by a membrane unit, present an electron-dense protein matrix with electron-lucent lipid inclusions, being devoid of inner membranes. Here we performed a detailed ultrastructural analysis of these organelles using a variety of electron microscopy techniques, including ultrathin sectioning, uranyl acetate stained preparations, and freeze fracture, either in intact epimastigotes or in isolated reservosomes. New informations were obtained. First, both isolated and in situ reservosomes presented small profiles of inner membranes that are morphologically similar to the membrane surrounding the organelle. In uranyl acetate stained preparations, internal membrane profiles turned out to be longer than they appeared in ultrathin section images and traversed the organelle diameter. Internal vesicles were also found. Second, endocytosed cargo are not associated with internal vesicles and reach reservosomes on board of vesicles that fuse with the boundary membrane, delivering cargo directly into reservosome lumen. Third, electron-lucent bodies with saturated lipid core surrounded by a membrane monolayer and with unusual rectangular shape were also observed. Fourth, it was possible to demonstrate the presence of intramembranous particles on the E face of both internal vesicles and the surrounding membrane. Collectively, these results indicate that reservosomes have a complex internal structure, which may correlate with their multiple functions.

SUMMARYThe actin cytoskeleton controls pivotal cellular processes such as motility and cytokinesis, as well as cell-cell and cell-substrate interactions. Assembly and spatial organization of actin filaments are dynamic events regulated by a large repertoire of actin-binding proteins. This report presents the first detailed characterization of theTrypanosoma cruziactin (TcActin). Protein sequence analysis and homology modelling revealed that the overall structure ofT. cruziactin is conserved and that the majority of amino-acid changes are concentrated on the monomer surface. Immunofluorescence assays using specific polyclonal antibody against TcActin revealed numerous rounded and punctated structures spread all over the parasitic body. No pattern differences could be found between epimastigotes and trypomastigotes or amastigotes. Moreover, in detergent extracts, TcActin was localized only in the soluble fraction, indicating its presence in the G-actin form or in short filaments dissociated from the microtubule cytoskeleton. The trypanosomatid genome was prospected to identify actin-binding and actin-related conserved proteins. The main proteins responsible for actin nucleation and treadmilling in higher eukaryotes are conserved inT. cruzi.

Autophagy is the major mechanism used by eukaryotic cells to degrade and recycle proteins and organelles. Bioinformatics analysis of the genome of the protozoan parasite Trypanosoma cruzi revealed the presence of all components of the Atg8 conjugation system, whereas Atg12, Atg5, and Atg10 as the major components of the Atg12 pathway could not be identified. The two TcATG4 (autophagin) homologs present in the genome were found to correctly process the two ATG8 homologs after the conserved Gly residue. Functional studies revealed that both ATG4 homologues but only one T. cruzi ATG8 homolog (TcATG8.1) complemented yeast deletion strains. During starvation of the parasite, TcAtg8.1, but not TcAtg8.2, was found by immunofluorescence to be located in autophagosome-like vesicles. This confirms its function as an Atg8/LC3 homolog and its potential to be used as an autophagosomal marker. Most importantly, autophagy is involved in differentiation between developmental stages of T. cruzi, a process that is essential for parasite maintenance and survival. These findings suggest that the autophagy pathway could represent a target for a novel chemotherapeutic strategy against Chagas disease.

Epimastigotes multiplies in the insect midgut by taking up nutrients present in the blood meal including heme bound to hemoglobin of red blood cell. During blood meal digestion by vector proteases in the posterior midgut, hemoglobin is clipped off into amino acids, peptides, and free heme. In this paper, we compared the heme and hemoglobin uptake kinetics and followed their intracellular trafficking. Addition of heme to culture medium increased epimastigote proliferation in a dose-dependent manner, while medium supplemented with hemoglobin enhanced growth after 3-day lag phase. Medium supplemented with globin-derived peptides stimulated cell proliferation in a dose-independent way. Using Palladium mesoporphyrin IX (Pd-mP) as a fluorescent heme-analog, we observed that heme internalization proceeded much faster than that observed by hemoglobin-rhodamine. Binding experiments showed that parasites accumulated the Pd-mP into the posterior region of the cell whereas hemoglobin-rhodamine stained the anterior region. Finally, using different specific inhibitors of ABC transporters we conclude that a P-glycoprotein homologue transporter is probably involved in heme transport through the plasma membrane.