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Peptides have fantastic functions like defensins that make up the natural defense in different species from vertebrates to bacteria. Another fantastic example are prions that cause transmissible diseases devoid of nucleic acid. So, because of their high specificity, peptides can be used for different therapeutic goals. In this work we describe in vitro action of a peptide named L18R this sequence is part of a group of four distinct sequences that were previously tested and showed activity against fungi and tumors, which motivated us to investigate this activity against murine B16F10 melanoma. Thus, has been demonstrated that L18R had activity against tumor, for interfering in cell viability, migration and in cell cycle. To conclude, the present work suggests a functional molecule against tumor.

The parasitism of cellular organisms is a topic of high relevance to human health and is under constant investigation. The process by which the flagellate protozoan Trypanosoma cruzi invades the host cells, thus causing the Chagas disease, is still not fully understood. In this work we study the role of AFAP-1L1, galectin-3 and WASP proteins on host cell invasion and characterize the biological functions of the P21 recombinant form, called P21-His6. The AFAP-1L1, galectin-3 and WASP proteins participate in numerous biological activities on the host cell, while the P21 protein, a recently characterized protein present in the membrane of T. cruzi, is presented in all developmental stages of the parasite. We assessed the involvement of galectin-3 in the intracellular traffic of T. cruzi by immunofluorescence tracking, and the role of the other host proteins by knockdown cell lines. The biological activities of P21-His6 were studied by selection of specific bacteriophages ligands with Biopanning technique, with implantation of sponges in Balb/c animals, treating or nor with P21-His6, and evaluating chemotaxis response when treating C57Bl/6 animals. We observed that galectin-3 is present in approximately 20% of cellular invasion events, forming structures containing polarized galectin-3 (G3CS) that fades over time. We also observed that AFAP-1L1 seems to have some importance in controlling early multiplications, and that reduction of WASP protein expression seems to facilitate parasite proliferation in parallel to the reduction of invasion. Thus, we conclude that these proteins are important for establishing T. cruzi infection. We observed that the P21-His6 has anti-angiogenic activity and capacity for cellular recruitment probably through interaction with the CXCR4 receptor especially of activated macrophages and neutrophils. We also obtained mimetic CXCR4 bacteriophages capable of specifically binding to P21-His6 and reducing its biological effects in vitro. Thus we conclude that P21-His6 has high chemotactic capacity that can be inhibited by specific ligands, and may have therapeutic anti-angiogenic activity. Together these results may open the door to novel therapeutic interventions. O parasitismo de organismos celulares é alvo de constantes estudos e o processo pelo qual o protozoário flagelado Trypanosoma cruzi invade a célula do hospedeiro ainda tem lacunas a serem preenchidas. As proteínas AFAP-1L1, galectina-3 e WASP são proteínas importantes para a célula do hospedeiro, participando de inúmeras atividades biológicas. Já a proteína P21, secretada pelo T. cruzi, foi recentemente caracterizada, se mostrando presente em todas as formas evolutivas do parasita. Nesse trabalho, estudamos a importância das proteínas AFAP-1L1, galectina-3 e WASP do hospedeiro durante a invasão e multiplicação do T. cruzi e também buscamos caracterizar funções biológicas da forma recombinante da P21, chamada P21-His6. A participação da galectina-3 durante o tráfego intracelular de T. cruzi foi acompanhada por imunofluorescência, e a importância das outras proteínas do hospedeiro foram analisadas através de linhagens celulares knockdown, com reduzida expressão, para as proteínas de interesse. Os estudos das atividades biológicas da P21-His6 se deram pela seleção de bacteriófagos ligantes específicos com a técnica de Biopanning, com o implante de esponjas interescapulares e a avaliação do processo inflamatório de corpo estranho e também pelo seu tratamento in vivo em animais Balb/c. Observamos que a proteína galectina-3 está presente em aproximadamente 20% dos eventos de invasão celular, formando estruturas contendo galectina-3 polarizada que vão desaparecendo ao longo do tempo, também observamos que a proteína AFAP-1L1 parece ter alguma importância no controle da multiplicação em tempos iniciais, assim como a redução da expressão proteína WASP parece facilitar a multiplicação do parasita, paralelamente ao fato de reduzir a invasão do mesmo. Observamos que a P21-His6 tem capacidade de recrutamento celular, provavelmente pela sua interação com o receptor CXCR4, especialmente de macrófagos e neutrófilos ativados, e que também possui atividade anti-angiogênica. Obtivemos também bacteriófagos miméticos do CXCR4, capaz de se ligar especificamente à P21-His6 e reduzir seus efeitos biológicos in vitro. Sendo assim concluímos a importância das proteínas do hospedeiro Galectina-3, AFAP-1L1 e WASP durante o estabelecimento da infecção por T. cruzi e concluímos que a P21-His6 pode ter função terapêutica pela sua atividade anti-angiogência, que possui alta capacidade quimiotática e que pode ser inibida por ligantes específicos. Mestre em Imunologia e Parasitologia Aplicadas

Electrochemical procedures have emerged as a powerful tool for the synthesis of complex organic molecules including transformation of natural products. In this study, the electrosynthesis of biseugenol in MeOH afforded one new oxidized derivative, which was characterized as methoxyloxo-biseugenol (MOB) by nuclear magnetic resonance (NMR) and electrospray ionization-high resolution mass spectrometry (ESI-HRMS) analysis. Since biseugenol was previously described as a cytotoxic natural product, MOB was also tested against tumoral cells B16F10-Nex2 (murine metastatic melanoma) and SKMEL-25 (human metastatic melanoma) as well as against nontumoral cells MØ Raw 264.7 (murine macrophage immortalized) and HUVEC (human umbilical endothelium). As results, MOB showed inhibitory concentration that affects 50% of cells (IC50)values of 9.5 ± 1.6 mg mL-1 (B16F10Nex2), 13.2 ± 2.5 mg mL-1 (SKMEL-25), 19.9 ± 3.5 mg mL-1 (MØ Raw 264.7) and 36.3 ± 7.4 mg mL-1 (HUVEC). Therefore, selectivity index (SI) values of MOB to tumoral cells B16F10Nex2 and SKMEL-25 were calculated as 2.1 and 2.8, respectively, higher than biseugenol (1.4 and 1.7, respectively). Based on these results, the superior cytotoxic activity of MOB in comparison to biseugenol could be associated, at least in part, to the presence of a non-aromatic ring and a conjugated carbonyl system instead of a phenol moiety.

Trypanosomatids regulate gene expression mainly at the post-transcriptional level through processing, exporting and stabilising mRNA and control of translation. In most eukaryotes, protein synthesis is regulated by phosphorylation of eukaryotic initiation factor 2 (eIF2) at serine 51. Phosphorylation halts overall translation by decreasing availability of initiator tRNAmet to form translating ribosomes. In trypanosomatids, the N-terminus of eIF2α is extended with threonine 169 the homologous phosphorylated residue. Here, we evaluated whether eIF2α phosphorylation varies during the Trypanosoma cruzi life cycle, the etiological agent of Chagas' disease. Total levels of eIF2α are diminished in infective and non-replicative trypomastigotes compared with proliferative forms from the intestine of the insect vector or amastigotes from mammalian cells, consistent with decreased protein synthesis reported in infective forms. eIF2α phosphorylation increases in proliferative intracellular forms prior to differentiation into trypomastigotes. Parasites overexpressing eIF2αT169A or with an endogenous CRISPR/Cas9-generated eIF2αT169A mutation were created and analysis revealed alterations to the proteome, largely unrelated to the presence of μORF in epimastigotes. eIF2αT169A mutant parasites produced fewer trypomastigotes with lower infectivity than wild type, with increased levels of sialylated mucins and oligomannose glycoproteins, and decreased galactofuranose epitopes and the surface protease GP63 on the cell surface. We conclude that eIF2α expression and phosphorylation levels affect proteins relevant for intracellular progression of T. cruzi.

Host actin cytoskeleton polymerization has been shown to play an important role during Trypanosoma cruzi internalization into mammalian cell. The structure and dynamics of the actin cytoskeleton in cells are regulated by a vast number of actin-binding proteins. Here we aimed to verify the impact of AFAP-1L1, during invasion and multiplication of T. cruzi. Knocking-down AFAP-1L1 increased parasite cell invasion and intracellular multiplication. Thus, we have shown that the integrity of the machinery formed by AFAP-1L1 in actin cytoskeleton polymerization is important to hinder parasite infection.

Trypanosoma cruzi has high biological and biochemical diversity and variable tissue tropism. Here we aimed to verify the kinetics of cytokine and chemokine in situ secretion in animals infected with two distinct T. cruzi strains after oral inoculation. Also, we investigated parasite migration, residence and pathological damage in stomach, heart and spleen. Our results showed that host immune response against T. cruzi infection is an intricate phenomenon that depends on the parasite strain, on the infected organ and on the time point of the infection. We believe that a wide comprehension of host immune response will potentially provide basis for the development of immunotherapeutic strategies in order to clear parasitism and minimize tissue injury. In this context, we find that KC poses as a possible tool to be used.

Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) CNPq Fundacao de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ) NIH Some 1,3-diarylureas and 1-((1,4-trans)-4-aryloxycyclohexyl)-3-arylureas (cHAUs) activate heme-regulated kinase causing protein synthesis inhibition via phosphorylation of the eukaryotic translation initiation factor 2 (eIF2) in mammalian cancer cells. To evaluate if these agents have potential to inhibit trypanosome multiplication by also affecting the phosphorylation of eIF2 alpha subunit (eIF2 alpha), we tested 25 analogs of 1,3-diarylureas and cHAUs against Trypanosoma cruzi, the agent of Chagas disease. One of them (I-17) presented selectivity close to 10-fold against the insect replicative forms and also inhibited the multiplication of T. cruzi inside mammalian cells with an EC50 of 1-3 mu M and a selectivity of 17-fold. I-17 also prevented replication of African trypanosomes (Trypanosoma brucei bloodstream and procyclic forms) at similar doses. It caused changes in the T. cruzi morphology, arrested parasite cell cycle in G1 phase, and promoted phosphorylation of eIF2 alpha with a robust decrease in ribosome association with mRNA. The activity against T. brucei also implicates eIF2 alpha phosphorylation, as replacement of WT-eIF2 alpha with a non-phosphorylatable eIF2 alpha, or knocking down eIF2 protein kinase-3 by RNAi increased resistance to I-17. Therefore, we demonstrate that eIF2 alpha phosphorylation can be engaged to develop trypanosome-static agents in general, and particularly by interfering with activity of eIF2 kinases. Univ Fed Sao Paulo, Escola Paulista Med, Dept Microbiol Imunol & Parasitol, BR-04039032 Sao Paulo, SP, Brazil Univ Fed Rio de Janeiro, Inst Biofis Carlos Chagas Filho, Lab Parasitol Mol, Rio De Janeiro, RJ, Brazil Inst Butantan, Sao Paulo, SP, Brazil Univ Sao Paulo, Inst Ciencias Biomed, Dept Microbiol, Sao Paulo, SP, Brazil Brigham & Womens Hosp, Dept Med, Hematol Lab Translat Res, 75 Francis St, Boston, MA 02115 USA Harvard Med Sch, 75 Francis St, Boston, MA 02115 USA Univ Fed Sao Paulo, Escola Paulista Med, Dept Microbiol Imunol & Parasitol, BR-04039032 Sao Paulo, SP, Brazil FAPESP: 2015/20031-0 FAPESP: 2014/01577-2 CNPq: 445655/2014-3 NIH: R01 CA152312 Web of Science

The seasonal chemical composition of essential oils from Inga laurina was determined by GC/MS. In the stem bark’s essential oil extracted during the dry season, the presence of terpenoids (30.05%) stood out, and phytol (9.76%) was the major compound identified. For the stem bark oil obtained during the rainy season, in addition to terpenoids (26.63%), a large amount of fatty acids (46.84%) were identified, in particular palmitic acid (25.40%). Regarding the leaves’ essential oil obtained in the dry season, esters (42.35%) were the main components. The main ester present was (Z)-hex-3-enyl benzoate (10.15%) and the major compound of this oil was (Z)-hex-3-en-1-ol (14.23%). Terpenoids (33.84%), long-chain alkanes (27.04%) and fatty acids (21.72%) were the main components of the essential oil from leaves in the rainy season. Phytol (33.21%), nonacosane (21.95%) and palmitic acid (15.20%) were the major compounds identified. The antimicrobial activity against aerobic and anaerobic oral bacteria was evaluated by the microdilution broth method and cytotoxic activity was carried out with Vero cells. The essential oils from the rainy season showed a better inhibition of the bacterial growth with Minimal Inhibitory Concentrations (MIC) values of 25 or 50 µg·mL−1 for aerobic bacteria, and high selectivity against bacteria was observed. The large amount of fatty acids in rainy season oils may be related to the better inhibitory effects observed.

The chemical composition of the essential oils from leaves, bark and wood of Cassia bakeriana Craib. was determined by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS). Alcohols, aldehydes and fatty acids were the major components in leaf and bark oil, while wood essential oil was rich in fatty acids. Terpenes such as linalool, (E)-nerolidol and phytol were present in low concentrations. The antimicrobial activity against aerobic and anaerobic oral bacteria was evaluated using the microdilution method, as was the cell viability test carried out with Vero cells. The oils from leaves and bark showed high antimicrobial activity, with minimum inhibitory concentrations between 62.5 and 125 µg·mL⁻¹ for most of the tested bacteria, including Streptococcus mutans, the main etiological agent of dental caries. Leaves oil displayed the lowest cytotoxic effect (EC₅₀ of 153 µg·mL⁻¹), while wood oil exhibited the highest toxicity to Vero cells. C. bakeriana oils are thus a source of biologically active compounds against aerobic and anaerobic oral microorganisms. This study is the first report on the chemical composition, antimicrobial activity and cytotoxicity of C. bakeriana.

Histones are well-conserved proteins that form the basic structure of chromatin in eukaryotes and undergo several post-translational modifications, which are important for the control of transcription, replication, DNA damage repair, and chromosome condensation. In early branched organisms, histones are less conserved and appear to contain alternative sites for modifications, which could reveal evolutionary unique functions of histone modifications in gene expression and other chromatin-based processes. Here, by using high-resolution mass spectrometry, we identified and quantified histone post-translational modifications in two life cycle stages of Trypanosoma cruzi, the protozoan parasite that causes Chagas disease. We detected 44 new modifications, namely: 18 acetylations, seven monomethylations, seven dimethylations, seven trimethylations, and four phosphorylations. We found that replicative (epimastigote stage) contains more histone modifications than nonreplicative and infective parasites (trypomastigote stage). Acetylations of lysines at the C-terminus of histone H2A and methylations of lysine 23 of histone H3 were found to be enriched in trypomastigotes. In contrast, phosphorylation in serine 23 of H2B and methylations of lysine 76 of histone H3 predominates in proliferative states. The presence of one or two methylations in the lysine 76 was found in cells undergoing mitosis and cytokinesis, typical of proliferating parasites. Our findings provide new insights into the role of histone modifications related to the control of gene expression and cell-cycle regulation in an early divergent organism.

Structural studies of proteins normally require large quantities of pure material that can only be obtained through heterologous expression systems and recombinant technique. In these procedures, large amounts of expressed protein are often found in the insoluble fraction, making protein purification from the soluble fraction inefficient, laborious, and costly. Usually, protein refolding is avoided due to a lack of experimental assays that can validate correct folding and that can compare the conformational population to that of the soluble fraction. Herein, we propose a validation method using simple and rapid 1D (1)H nuclear magnetic resonance (NMR) spectra that can efficiently compare protein samples, including individual information of the environment of each proton in the structure.

The invasion of host cells by the intracellular protozoan Trypanosoma cruzi requires interactions with host cell molecules, and the replication of the parasite requires escape from a parasitophorous vacuole into the host cell cytosol. Galectin-3, a member of β-galactosidase-binding lectin family, has numerous extracellular and intracellular functions. In this study, we investigated the role of galectin-3 during the invasion and intracellular trafficking of T. cruzi extracellular amastigotes (EAs). Endogenous galectin-3 from mouse peritoneal macrophages accumulated around the pathogen during cell invasion by EAs. In addition, galectin-3 accumulated around parasites after their escape from the parasitophorous vacuole. Thus, galectin-3 behaved as a novel marker of phagolysosome lysis during the infection of host cells by T. cruzi.

BACKGROUND: P21 is a secreted protein expressed in all developmental stages of Trypanosoma cruzi. The aim of this study was to determine the effect of the recombinant protein based on P21 (P21-His(6)) on inflammatory macrophages during phagocytosis. FINDINGS: Our results showed that P21-His(6) acts as a phagocytosis inducer by binding to CXCR4 chemokine receptor and activating actin polymerization in a way dependent onthe PI3-kinase signaling pathway. CONCLUSIONS: Thus, our results shed light on the notion that native P21 is a component related to T. cruzi evasion from the immune response and that CXCR4 may be involved in phagocytosis. P21-His(6) represents an important experimental control tool to study phagocytosis signaling pathways of different intracellular parasites and particles.