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Short interfering RNA (siRNA) showed to be a viable alternative to a better prognosis in cancer therapy. Nevertheless, the successful application of this strategy still depends on the development of nanocarriers for the safe delivery of siRNA into the diseased tissue, which mostly occurs by passive accumulation. When an external magnetic field is applied, magnetic nanoparticles biodistribution is partially modulated to favor accumulation in a target tissue. In this work we designed a novel magnetic responsive siRNA nanocarrier. The new delivery system is composed of superparamagnetic iron oxide nanoparticles (SPIONs) coated with calcium phosphate (CaP) and PEG-polyanion block copolymers, which are known to be biocompatible. The nanoparticles presented rounded shape with small size and narrow distribution suitable for biomedical applications. TEM images showed dark spheres in the core surrounded by a lower electron density material in the corona. The X-ray photoelectron spectra (XPS) confirmed CaP-polymer coating of the magnetic core. In addition, the coating procedure did not affect the superparamagnetic property as showed using a vibrating sample magnetometer (VSM). With a high loading efficiency (80%), the nanoparticles enhanced vascular endothelium growth factor (VEGF) silencing in breast cancer cells in vitro, at gene and protein levels (~60% and 40%, respectively), without associated toxicity. Iron and siRNA quantification showed that the novel nanoparticles move towards a magnetic source carrying siRNA molecules. Therefore, these novel nanoparticles are a promising tool for cancer therapy based on RNAi effect, added by a magnetic capability to further modulate siRNA accumulation in the target tissue.

The development of new treatments for malignant melanoma, which has the worst prognosis among skin neoplasms, remains a challenge. The tumor microenvironment aids tumor cells to grow and resist to chemotherapeutic treatment. One way to mimic and study the tumor microenvironment is by using three-dimensional (3D) co-culture models (spheroids). In this study, a melanoma heterospheroid model composed of cancer cells, fibroblasts, and macrophages was produced by liquid-overlay technique using the agarose gel. The size, growth, viability, morphology, cancer stem-like cells population and inflammatory profile of tumor heterospheroids and monospheroids were analyzed to evaluate the influence of stromal cells on these parameters. Furthermore, dacarbazine cytotoxicity was evaluated using spheroids and two-dimensional (2D) melanoma model. After finishing the experiments, it was observed the M2 macrophages induced an anti-inflammatory microenvironment in heterospheroids; fibroblasts cells support the formation of the extracellular matrix, and a higher percentage of melanoma CD271 was observed in this model. Additionally, melanoma spheroids responded differently to the dacarbazine than the 2D melanoma culture as a result of their cellular heterogeneity and 3D structure. The 3D model was shown to be a fast and reliable tool for drug screening, which can mimic the in vivo tumor microenvironment regarding interactions and complexity. GRAPHIC ABSTRACT: [Image: see text]

We have studied the influence of the organic molecule DL-Dithiothreitol on the properties of isotropic conductive adhesives for application in microelectronics. The Ag fillers were prepared from commercial micro flakes or submicron particles from waste jewelry. The results show that coating of the Ag recycled fillers with thiol avoided the agglomeration of the particles improving the dispersion and the electrical and mechanical properties. By comparing the samples, the better properties were obtained from adhesives with recycled Ag particles relative to the ones formulated with commercial Ag flakes and improvement can be attributed to the absence of the lubricant layer and smaller size of the particles produced from recycled silver.

Chlorogenic acids (CGA) are the most abundant phenolic compounds in green coffee beans and in the human diet and have been suggested to mitigate several cardiometabolic risk factors. Here, we aimed to evaluate the effect of a water-based standardized green coffee extract (GCE) on cardiometabolic parameters in ApoE-/- mice and to explore the potential underlying mechanisms. Mice were fed an atherogenic diet without (vehicle) or with GCE by gavage (equivalent to 220 mg/kg of CGA) for 14 weeks. We assessed several metabolic, pathological, and inflammatory parameters and inferred gut microbiota composition, diversity, and functional potential. Although GCE did not reduce atherosclerotic lesion progression or plasma lipid levels, it induced important favorable changes. Specifically, improved metabolic parameters, including fasting glucose, insulin resistance, serum leptin, urinary catecholamines, and liver triglycerides, were observed. These changes were accompanied by reduced weight gain, decreased adiposity, lower inflammatory infiltrate in adipose tissue, and protection against liver damage. Interestingly, GCE also modulated hepatic IL-6 and total serum IgM and induced shifts in gut microbiota. Altogether, our results reveal the cooccurrence of these beneficial cardiometabolic effects in response to GCE in the same experimental model and suggest potential mediators and pathways involved.

Breast cancer is a major cause of death among women worldwide. Resistance to conventional therapies has been observed in HER2-positive breast cancer patients, indicating the need for more effective treatments. Small interfering RNA (siRNA) therapy is an attractive strategy against HER2-positive tumors, but its success depends largely on the efficient delivery of agents to target tissues. In this study, we prepared a magnetic hybrid nanostructure composed of iron oxide nanoparticles coated with caffeic acid and stabilized by layers of calcium phosphate and PEG-polyanion block copolymer for incorporation of siRNA. Transmission electron microscopy images showed monodisperse, neutrally charged compact spheres sized100 nm. Dynamic light scattering and nanoparticle tracking analysis revealed that the nanostructure had an average hydrodynamic diameter of 130 nm. Nanoparticle suspensions remained stable over 42 days of storage at 4 and 25 °C. Unloaded caffeic acid-magnetic calcium phosphate (Caf-MCaP) nanoparticles were not cytotoxic, and loaded nanoparticles were successfully taken up by the HER2-positive breast cancer cell line HCC1954, even more so under magnetic guidance. Nanoparticles escaped endosomal degradation and delivered siRNA into the cytoplasm, inducing HER2 gene silencing.

Fiber fermentation by gut microbiota yields short-chain fatty acids (SCFAs) that are either absorbed by the gut or excreted in feces. Studies are conflicting as to whether SCFAs are beneficial or detrimental to cardiometabolic health, and how gut microbiota associated with SCFAs is unclear. In this study of 441 community-dwelling adults, we examined associations of fecal SCFAs, gut microbiota diversity and composition, gut permeability, and cardiometabolic outcomes, including obesity and hypertension. We assessed fecal microbiota by 16S rRNA gene sequencing, and SCFA concentrations by gas chromatography/mass spectrometry. Fecal SCFA concentrations were inversely associated with microbiota diversity, and 70 unique microbial taxa were differentially associated with at least one SCFA (acetate, butyrate or propionate). Higher SCFA concentrations were associated with a measure of gut permeability, markers of metabolic dysregulation, obesity and hypertension. Microbial diversity showed association with these outcomes in the opposite direction. Associations were significant after adjusting for measured confounders. In conclusion, higher SCFA excretion was associated with evidence of gut dysbiosis, gut permeability, excess adiposity, and cardiometabolic risk factors. Studies assessing both fecal and circulating SCFAs are needed to test the hypothesis that the association of higher fecal SCFAs with obesity and cardiometabolic dysregulation is due to less efficient SCFA absorption.

Synthesis of nanoparticles free from toxic chemicals and solvents is highly seen for large-scale production processes, particularly for use in biomedical/biotechnological applications. So far, although several methods for synthesis of metal nanoparticles using citrus extracts have been described, none of them clarify which compounds are responsible for both reduction and stabilization of NPs. Here we report the role of citrus flavonoids, hesperidin, hesperetin, rutin, naringenin, quercetin and diosmin, in the synthesis of gold nanoparticles (AuNP) at room temperature. Only in the presence of the citrus flavonoids, diosmin (Dm), and hesperetin (Ht), the reduction of HAuCl4 in concentrations as high as 7 mM under alkaline conditions yielded concentrated and self-stabilized suspensions of uniform spherical nanoparticles with a narrow size distribution. We went further and focused on Ht, the most abundant flavonoid aglycone from citrus fruits known for its medicinal properties. HtAuNPs were characterized using high-resolution transmission electron microscopy, dynamic light scattering, X-ray photoelectron spectrometry and UV-Vis spectrophotometry. The NPs remained stable for months without significant changes in their shape and optical properties. Theoretical calculations using density functional theory were used to identify the functional groups involved in the electron transfer from the Ht molecules to gold, which seems to be the consequence of an initial complexation, leading to the reduction of Au3+ ions into Au0. Besides, this procedure provides a one-pot method, showing potential for large-scale.

Maleic anhydride (MA) grafted poly(lactic acid) (PLA) (PLA-g-MA) was synthesized from PLA. Proton nuclear magnetic resonance confirmed the grafting of the MA. PLA-g-MA and PLA were used to prepare polymeric nanoparticles. Particle size distributions were measured by dynamic light scattering, and colloidal stability was determined by (zeta) ζ-potential. The ζ-potential becomes more negative for PLA-g-MA than PLA nanoparticle dispersions, due to the presence of deprotonated carboxylic acid groups on the backbone of the PLA and confirms the MA grafting results. Maleic anhydride grafted on PLA backbone improves the covalent conjugation with ovalbumin (OVA) compared to OVA physically adsorbed on the particles. The chemical conjugation was carried out via amide linkages between the carboxylic groups of the nanoparticles, activated with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and the amino groups of the protein. The amount of protein conjugated was measured by using the bicinchoninic acid method and is threefold higher compared to the adsorbed OVA. Moreover, the PLA-g-MA nanoparticles increased the amount of conjugated OVA by 36 wt% compared to PLA nanoparticles. OVA adsorption and OVA conjugation provided colloidal dispersions with excellent stability.

Microsporidia are a group of intracellular pathogens causing self-limited and severe diseases in immunocompetent and immunocompromised individuals, respectively. A cellular type 1 adaptive response, mediated by IL-12, IFNg, CD4+ and CD8+ T cells has been shown to be essential for host resistance, and dendritic cells (DC) play a key role at eliciting anti-microsporidial immunity. We investigated the in vitro response of DC and DC precursors/progenitors to infection with Encephalitozoon intestinalis (Ei), a common agent of human microsporidosis. Ei-exposed DC cultures up-regulated the surface expression of MHC class II and the costimulatory molecules CD86 and CD40, only when high loads of spores were used. A vigorous secretion of IL-6 but not of IL-1b or IL-12p70 was also observed in these cultures. Ei-exposed DC cultures consisted of immature infected and mature bystander DC, as assessed by MHC class II and costimulatory molecules expression, suggesting that intracellular Ei spores deliver inhibitory signals in DC. Moreover, Ei selectively inhibited the secretion of IL-12p70 in LPS-stimulated DC. Whereas Ei-exposed DC promoted allogeneic naive T cell proliferation and IL-2 and IFNg secretion in DC-CD4+ T cell co-cultures, separated co-cultures with bystander or infected DCs showed stimulation or inhibition of IFNg secretion, respectively. When DC precursors/progenitors were exposed to Ei spores, a significant inhibition of DC differentiation was observed without shifting the development towards cells phenotypically or functionally compatible with myeloid-derived suppressor cells. Neutralization experiments demonstrated that this inhibitory effect is IL-6-dependent. Altogether this investigation reveals a novel potential mechanism of immune escape of microsporidian parasites through the modulation of DC differentiation and maturation.

Many studies have demonstrated that the flavonoid quercetin protects against cardiovascular disease (CVD) and related risk factors. Atherosclerosis, the underlying cause of CVD, is also attenuated by oral quercetin administration in animal models. Although macrophages are key players during fatty streak formation and plaque progression and aggravation, little is known about the effects of quercetin on atherogenic macrophages. Here, we report that primary bone marrow-derived macrophages internalized less oxidized low-density lipoprotein (oxLDL) and accumulated less intracellular cholesterol in the presence of quercetin. This reduction of foam cell formation correlated with reduced surface expression of the oxLDL receptor CD36. Quercetin also targeted the lipopolysaccharide-dependent, oxLDL-independent pathway of lipid droplet formation in macrophages. In oxLDL-stimulated macrophages, quercetin inhibited reactive oxygen species production and interleukin (IL)-6 secretion. In a system that evaluated cholesterol crystal-induced IL-1β secretion via nucleotide-binding domain and leucine-rich repeat containing protein 3 inflammasome activation, quercetin also exhibited an inhibitory effect. Dyslipidemic apolipoprotein E-deficient mice chronically treated with intraperitoneal quercetin injections had smaller atheromatous lesions, reduced lipid deposition, and less macrophage and T cell inflammatory infiltrate in the aortic roots than vehicle-treated animals. Serum levels of total cholesterol and the lipid peroxidation product malondialdehyde were also reduced in these mice. Our results demonstrate that quercetin interferes with both key proatherogenic activities of macrophages, namely foam cell formation and pro-oxidant/proinflammatory responses, and these effects may explain the atheroprotective properties of this common flavonoid.

Several podophyllotoxin derivatives modified in the E-ring were prepared and evaluated for their cytotoxicity on four neoplastic cell lines (P-388, A-549, HT-29 and MEL-28) and for their antiherpetic activity against Herpes simplex virus type II. The trimethoxyphenyl moiety was oxidized to ortho-quinone and further condensed with diamines and enamines to form different heterocycles. Most of the compounds maintained their cytotoxicity at the muM level and some of them showed antiherpetic activity.

MicroRNA-21 (miR-21) has been described as upregulated in tissue and serum samples from patients with tumor diseases such as hepatocellular carcinoma. In addition, circulating miR-21 has been reported as a marker of necroinflammatory activity in patients with hepatitis C virus (HCV) and HCV-induced hepatocellular carcinoma (HCC). Thus, the expression of miR-21 by reverse transcription quantitative real-time PCR (RT-qPCR) in serum samples (n = 216) from patients with hepatic cirrhosis (n = 187) compared with control group (n = 29) was evaluated. Initially, peripheral blood was collected in tubes containing separating gel and clot activator and centrifuged at 820 × g for 10 min at room temperature. Then, the supernatants were transferred to new microtubes and the samples were centrifuged at 16,000 × g for 10 min to precipitate cell debris. Next, total serum RNA was isolated from 500 μL of serum from each sample and eluted in 50 μL of RNase-free water using a mirVanaTM PARISTM kit (AM1556, Ambion, Carlsbad, CA), according to the manufacturer's instructions. After that, total RNA quality obtained was evaluated by NanoVueTM Plus spectrophotometer (GE Healthcare, Buckinghamshire, UK) and the RT-qPCR was done using Taqman microRNA assays (Applied Biosystems, Foster City, CA). In the present work, the expression of miR-16 was not differentially expressed between healthy (Cq = 24.7 ± 3.0) and cirrhosis groups (Cq = 25.3 ± 3.0; P = 0.2741). Therefore, miR-16 was used as internal control to evaluate the expression of miR-21. The relative expression of miR-21 found was not statistically significant considering the control (1.0 ± 0.6) and cirrhosis groups (2.0 ± 3.5; P = 0.1478). The low expression of miR-21 indicates the patients did not yet develop a malignant disease or necroinflammatory activity. To better evaluate hepatic function, miR-34a, miR-122, and miR-885-5p were also analyzed, because these miRNAs have already been described as potential biomarkers for detection and assessment of liver diseases. The relative expressions found were significantly different for miR-34a (1.0 ± 1.5 vs 3.5 ± 8.1, P = 0.0077), miR-122 (1.0 ± 10.1 vs 1.2 ± 33.6, P < 0.0001) and miR-885-5p (1.0 ± 4.8 vs 1.9 ± 12.8, P = 0.0001). These miRNAs were found to be upregulated with fold-change for miR-34a, miR-122, and miR-885-5p of 4.1, 7.1, and 3.2, respectively. Due to the fact that these miRNAs were previously studied separately in independent studies, we applied a logistic regression model to obtain a miRNA panel not yet described. The logit model, logit(p = Cirrhosis) = 0.59 + 1.57 x miR-34a + 0.50 x miR-122 - 0.14 x miR-885-5p, was used to construct the ROC curve and, thus, to evaluate the diagnostic accuracy for the established miRNA panel. The area under the ROC curve (AUC) for the miRNA panel was 0.859 (95% confidence interval, 0.792-0.912; sensitivity: 84.8%; specificity: 73.7%; P < 0.0001), providing the high diagnostic accuracy for cirrhosis. A serum microRNA panel having considerable clinical value in cirrhosis diagnosis, chronic hepatitis viral, and hepatocellular carcinoma was found, thus allowing patients not to lose the window of treatment being benefited with a more appropriate therapy. Citation Format: Alex Evangelista Amaral, Júlia Cisilotto, Michele Patrícia Rode, Evelyn Winter, Adny Henrique Silva, Jelver Sierra-Restrepo, Leonardo de Lucca Schiavon, Tânia Beatriz Creczynski-Pasa. Serum microRNA panel to diagnose important liver diseases [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr B78.

Pentacyclic triterpenes are a large family of plant metabolites that exhibit a wide array of biological activities. The genus Cecropia, which encompasses many plant species, has been used as traditional medicine for the treatment of inflammatory diseases and is known to produce many active pentacyclic triterpenes. In this study we investigated the chemical composition of a pentacyclic triterpene fraction from the roots of Cecropia telenitida Cuatrec., Urticaceae. A novel compound, which we termed yarumic acid, and four known molecules (serjanic acid, spergulagenic acid A, 20-hydroxy-ursolic acid and goreishic acid I) were isolated and characterised. In a dendritic cell (DC)-based assay, we demonstrated that non-toxic doses of these pentacyclic triterpenes inhibited the secretion of at least one of the proinflammatory cytokines tested (IL-1β, IL-12p40, IL-12p70, TNF-α). Spergulagenic acid A also inhibited nitric oxide production in lipopolysaccharide-stimulated dendritic cell. Serjanic acid and spergulagenic acid A, which were the most potent abundant compounds in the pentacyclic triterpene fraction, showed the most activity in the dendritic cell-based assay. These results show that all pentacyclic triterpenes might contribute to the anti-inflammatory activities of C. telenitida. Moreover, yarumic acid as well as the four known pentacyclic triterpenes, can be exploited as potential immunomodulatory/anti-inflammatory agents. Keywords: Cecropia telenitida, Pentacyclic triterpene, Natural product, Cytokine modulation, Anti-inflammatory activity, Dendritic cells

Clementines are especially appreciated for their delicious flavor, and recent years have seen a great increase in the consumption of clementine juice. In previous decades, antioxidant compounds have received particular attention because of widely demonstrated beneficial health effects. In this work, the organoleptic, volatile flavor, and antioxidant quality of clementine juice were studied with regard to the influence on them by different juice extraction systems: plug inside fruit and rotating cylinders. The results showed that juice extracted by the former method presented higher yields and hesperidin content, which was related to higher antioxidant activity, demonstrated by ORAC and LDL assays. The organoleptic quality was not affected by the processing technique, whereas there were significant differences in the chemical flavor profile. There are important differences in chemical and functional quality between juice extraction techniques, which must be taken into account when employing processing systems to produce high-quality products.

Based on two lead cytotoxic spongiane diterpenes, a new series of C7-oxygenated derivatives were synthesized and evaluated for their antitumor activity in vitro against the cancer cell lines HeLa and HEp-2. In general, introduction of either hydroxyl or acetoxy groups at C-7 did not improve the resultant cytotoxicity, while the presence of a butyrate ester led to more active compounds (CC(50)=4.0-9.5 microM).