Efeitos de combinações entre o ácido anacárdico derivado da casca da castanha do caju (Anacardium occidentale) e o óleo de açaí (Euterpe oleracea Mart.), livres ou nanoestruturados, no tratamento de células de câncer de pele não melanoma, in vitro

Dissertação (mestrado)—Universidade de Brasília, Instituto de Ciências Biológicas, Pós-Graduação em Nanociência e Nanobiotecnologia, 2017.O Câncer de Pele Não Melanoma (CNPM) o tipo de câncer que possui maior incidência no Brasil e no mundo. O ácido anacárdico (AA) é um composto proveniente da casca da castanha do caju (Anacardium occidentale) que vem atraindo grande interesse nos últimos anos devido ás suas propriedades antitumorais, antibióticas, gastroprotetoras e antioxidantes. O açaí (Euterpe oleracea Mart.) também vem atraindo a atenção de pesquisadores, por ser rico em polifenóis com atividades como supressão tumoral, antiproliferativo e pró-apoptótica. Grande parte desses fitoquímicos que possuem atividades terapêuticas são pouco solúveis em soluções aquosas, o que dificulta sua administração e absorção no organismo. Desta forma, a encapsulação desses compostos em nanoestruturas se torna uma alternativa plausível para potencializar seus efeitos biológicos. Diante do exposto, o presente projeto de pesquisa tem como objetivo avaliar os efeitos de combinações entre o ácido anacárdico (AA) derivado da casca da castanha do caju (Anacardium occidentale) e o óleo de açaí (Euterpe oleracea Mart.), livres ou nanoestruturados, no tratamento de câncer de pele não melanoma in vitro. Os testes de estabilidade mostraram que a nanoemulsão à base de óleo de açaí (AçNE) apresentaram gotículas com diâmetro hidrodinâmico de ± 140 nm, com índice de polidespersão de 0,229, potencial de superfície de ± 17,6 mV e pH 7 por 120 dias. Foi possível modificar a superfície das AçNE adicionando polímeros de quitosana (CH), polietileno glicol (PEG) e fosfolipídios catiônicos DOTAP (1,2-Dioleoiloxi-3-(trimetilamónio) propano). Tais formulações não apresentaram efeito citotóxico nas linhagens A431 e HaCaT, independentemente do tipo de superfície. Os tratamentos AçNE associado ao AA provocaram uma significativa redução na viabilidade das células A431, porém não foi observado efeito de sinergismo entre os mesmos. Em contrapartida, quando ambos compostos foram adicionados na forma não-nanoestruturada, observou-se redução de 90% da viabilidade de células A431 em 24 horas. Dados de citometria de fluxo indicam que a combinação dos compostos livres resulta em morte celular por apoptose e bloqueio do ciclo celular. O presente estudo sugere que a combinação de óleo de açaí e AA é uma promissora alternativa terapêutica antitumoral a ser mais explorada em estudos futuros.Non-Melanoma Skin Cancer (CNPM) is the type of cancer that has the highest incidence in Brazil and worldwide. Anacardic acid (AA) is a compound derived from cashew nuts (Anacardium occidentale) that has attracted great interest in recent years due to its antitumor, antibiotic, gastroprotective and antioxidant properties. Açaí (Euterpe oleracea Mart.) has also attracted the attention of researchers, because it is rich in polyphenols which shows great activity as a tumor suppressor, antiproliferative and pro-apoptotic. Most of these phytochemicals that have therapeutic activities are poorly soluble in aqueous solutions, which hinders their administration and absorption in the body. In this way, the encapsulation of these compounds in nanostructures becomes a plausible alternative to enhance their biological effects. Thus, the present research project has the objective of evaluating the effects of anacardic acid (AA) derived from cashew nut shell (Anacardium occidentale) and açaí oil (Euterpe oleracea Mart.), free or nanostructured, in the treatment of non-melanoma skin cancer in vitro. The stability tests showed that the açaí oil-based nanoemulsion (AçNE) showed droplets with a hydrodynamic diameter of ± 140 nm, with a polydispersion index of 0.229, surface potential of ± 17.6 mV and pH 7 for 120 days. It was possible to modify the surface of the AçNE by adding polymers of chitosan (CH), polyethylene glycol (PEG) and cationic phospholipids DOTAP (1,2-Dioleoyloxy-3- (trimethylammonium) propane). Such formulations showed no cytotoxic effect on the A431 and HaCaT cell lines, regardless of surface type. The AçNE treatments associated with AA caused a significant reduction in the viability of A431 cells, but no synergism was observed between them. On the other hand, when both compounds were added in the non-nanostructured form, a 90% reduction in the viability of A431 cells was observed in 24 hours. Flow cytometry data indicate that the combination of the free compounds results in cell death by apoptosis and cell cycle block. The present study suggests that the combination of acai oil and AA is a promising alternative antitumor therapy to be further explored in future studies

Crystal Structure of the Full-Length Japanese Encephalitis Virus NS5 Reveals a Conserved Methyltransferase-Polymerase Interface

<div><p>The flavivirus NS5 harbors a methyltransferase (MTase) in its N-terminal ≈265 residues and an RNA-dependent RNA polymerase (RdRP) within the C-terminal part. One of the major interests and challenges in NS5 is to understand the interplay between RdRP and MTase as a unique natural fusion protein in viral genome replication and cap formation. Here, we report the first crystal structure of the full-length flavivirus NS5 from Japanese encephalitis virus. The structure completes the vision for polymerase motifs F and G, and depicts defined intra-molecular interactions between RdRP and MTase. Key hydrophobic residues in the RdRP-MTase interface are highly conserved in flaviviruses, indicating the biological relevance of the observed conformation. Our work paves the way for further dissection of the inter-regulations of the essential enzymatic activities of NS5 and exploration of possible other conformations of NS5 under different circumstances.</p></div

Global views of JEV NS5 structure.

<p>Structure of NS5 shown in orientations A) looking into the RdRP front channel (putative dsRNA channel); B) along the NTP entry channel; C) viewing from the top of RdRP. MTase is in cyan, RdRP palm in gray, thumb in blue, fingers in light red, N-terminal extension in pink, priming loop in purple, and signature sequence SGDD in magenta. Zinc ions are shown as brown spheres. The numbers defining the residue ranges of MTase and RdRP are shown in panel C. The three missing residues (271–273) in the linker are indicated by dashed lines in panel C. For clarity purposes, protein structures in all figures are shown in thin ribbon style with only the key β-sheet structures shown in regular cartoon style in most of the figure panels. “N-ext.” is used as abbreviation for “N-terminal extension”.</p

The RdRP region of flavivirus NS5.

<p>A) Stereo-pair images of NS5 viewing down into the RdRP active site (same view as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003549#ppat-1003549-g001" target="_blank">Fig. 1C</a>) with a color-coded bar defining structural elements underneath. Coloring scheme is as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003549#ppat-1003549-g001" target="_blank">Figure 1</a>, except that the index (green), middle (orange), ring (yellow), and pinky (light red) fingers are individually color-coded. Side chains of key residues in the MTase-RdRP interface are shown in sticks. The strand numbers of the 5-stranded β-sheet are indicated. B–C) Structural comparison of important elements in the core polymerase (B), the N-terminal extension and the priming loop (C). The JEV model is shown as thick ribbons and colored as in panel A. Three highly conserved charged residues in motif F (K459, E461, R474) and two structurally conserved motif G residues (A410, L411) interacting with the +1/+2 junction of the template strand are shown as spheres. The RNA duplex in the PV elongation complex (EC) model is shown in the motif G subpanel. The template strand in the priming loop subpanel is modeled using the PV EC model. Side chains of key priming loop residues W800, R797 and the invariant D536, D668 are shown in sticks. The putative priming NTP site (“p”) and the elongating NTP site (“e”) are indicated. D) Structure-based sequence alignment of RdRP motifs A–G, and other important elements. Three viruses from flavivirus genus, HCV and BVDV representing the other two genera of the <i>Flaviviridae</i> family, and PV representing viruses using primer-dependent strategy in genome replication are included in the alignment. Conserved active site residues (red text), MTase interacting residues (blue text and triangle), priming loop residues (purple text) are highlighted, the two invariant catalytic Asp residues are highlighted by asterisks, and residues in lower case letters either deviate from the consensus structure conformations or are not resolved in the crystal structures and are therefore included based only on sequence homology. The structurally conserved residues interacting with the template +1/+2 junction is highlighted by a red box, Colors at top of the alignment correspond to coloring of the structural elements in panel A. FLAV and PICO are used as abbreviations for <i>Flaviviridae</i> and <i>Picornaviridae</i> Families, respectively.</p

The MTase-RdRP interface.

<p>Stereo-pair images of 3,500 K composite SA-omit electron density map (contoured at 1.5σ) overlaid onto structural models centered at the MTase-RdRP interface. Coloring scheme is as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003549#ppat-1003549-g002" target="_blank">Figure 2A</a>. The GTR pivot is shown as thick ribbon. Side chains of six key hydrophobic residues (thick) that form the interface core and other residues (thin) at the peripheral of the interface are shown in sticks.</p

The uniqueness of the MTase-RdRP interface.

<p>Structures of JEV NS5 (middle) comparing to PV 3CD (left) and SARS-CoV nsp16/nsp10 complex (right). Viewing angle and coloring scheme are consistent with <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003549#ppat-1003549-g002" target="_blank">Figure 2A</a>. The RdRP portion of PV 3CD is color-coded according to NS5 RdRP and protease is colored red. The SARS-CoV nsp16 MTase is shown in cyan and nsp16 stimulatory factor is shown in blue. The methyl donor SAM bound in nsp16 is shown as sticks.</p

Data collection and refinement statistics.

a<p>One crystal was used for this structure.</p>b<p>Values in parentheses are for highest-resolution shell.</p>c<p>Ligand/ion include four Zinc ions, two bound to each NS5 molecule, and two SAH molecules, one bound to each NS5 molecule.</p

Different Environmental Drivers of Highly Pathogenic Avian Influenza H5N1 Outbreaks in Poultry and Wild Birds

<div><p>A large number of highly pathogenic avian influenza (HPAI) H5N1 outbreaks in poultry and wild birds have been reported in Europe since 2005. Distinct spatial patterns in poultry and wild birds suggest that different environmental drivers and potentially different spread mechanisms are operating. However, previous studies found no difference between these two outbreak types when only the effect of physical environmental factors was analysed. The influence of physical and anthropogenic environmental variables and interactions between the two has only been investigated for wild bird outbreaks. We therefore tested the effect of these environmental factors on HPAI H5N1 outbreaks in poultry, and the potential spread mechanism, and discussed how these differ from those observed in wild birds. Logistic regression analyses were used to quantify the relationship between HPAI H5N1 outbreaks in poultry and environmental factors. Poultry outbreaks increased with an increasing human population density combined with close proximity to lakes or wetlands, increased temperatures and reduced precipitation during the cold season. A risk map was generated based on the identified key factors. In wild birds, outbreaks were strongly associated with an increased Normalized Difference Vegetation Index (NDVI) and lower elevation, though they were similarly affected by climatic conditions as poultry outbreaks. This is the first study that analyses the differences in environmental drivers and spread mechanisms between poultry and wild bird outbreaks. Outbreaks in poultry mostly occurred in areas where the location of farms or trade areas overlapped with habitats for wild birds, whereas outbreaks in wild birds were mainly found in areas where food and shelters are available. The different environmental drivers suggest that different spread mechanisms might be involved: HPAI H5N1 spread to poultry via both poultry and wild birds, whereas contact with wild birds alone seems to drive the outbreaks in wild birds.</p></div

Environmental variables kept after the process of stepwise selection using 1000 bootstrapping training datasets. Italics indicate quadratic effects.

<p>Environmental variables kept after the process of stepwise selection using 1000 bootstrapping training datasets. Italics indicate quadratic effects.</p

Summary of the anthropogenic, physical environmental variables and interaction variables used in the analysis.

<p>Summary of the anthropogenic, physical environmental variables and interaction variables used in the analysis.</p