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Cyclotides are mini-proteins with potent bioactivities and outstanding potential for agricultural and pharmaceutical applications. More than 450 different plant cyclotides have been isolated from six angiosperm families. In Brazil, studies involving this class of natural products are still scarce, despite its rich floristic diversity. Herein were investigated the cyclotides from

Made available in DSpace on 2021-06-25T10:59:01Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-01-01 Antimicrobial peptides (AMPs) are part of the innate immune system of many species. AMPs are short sequences rich in charged and non-polar residues. They act on the lipid phase of the plasma membrane without requiring membrane receptors. Polybia-MP1 (MP1), extracted from a native wasp, is a broad-spectrum bactericide, an inhibitor of cancer cell proliferation being non-hemolytic and non-cytotoxic. MP1 mechanism of action and its adsorption mode is not yet completely known. Its adsorption to lipid bilayer and lytic activity is most likely dependent on the ionization state of its two acidic and three basic residues and consequently on the bulk pH. Here we investigated the effect of bulk acidic (pH 5.5) and neutral pH (7.4) solution on the adsorption, insertion, and lytic activity of MP1 and its analog H-MP1 to anionic (7POPC:3POPG) model membrane. H-MP1 is a synthetic analog of MP1 with lysines replaced by histidines. Bulk pH changes could modulate this peptide efficiency. The combination of different experimental techniques and molecular dynamics (MD) simulations showed that the adsorption, insertion, and lytic activity of H-MP1 are highly sensitive to bulk pH in opposition to MP1. The atomistic details, provided by MD simulations, showed peptides contact their N-termini to the bilayer before the insertion and then lay parallel to the bilayer. Their hydrophobic faces inserted into the acyl chain phase disturb the lipid-packing. Department of Physics IBILCE UNESP-São Paulo State University, Cristóvão Colombo, 2265-Jardim Nazareth Department of Basic and Applied Biology Institute of Biosciences UNESP-São Paulo State University Department of Physics IBILCE UNESP-São Paulo State University, Cristóvão Colombo, 2265-Jardim Nazareth Department of Basic and Applied Biology Institute of Biosciences UNESP-São Paulo State University

The present review aims the discussion of the impact of the bioprospection initiative developed by the projects associated to BIOprospecTA, a subprogram of the program BIOTA, supported by FAPESP. This review brings a summary of the main results produced by the projects investigating natural products (NPs) from non-plants organisms, as examples of the success of this initiative, focusing on the progresses achieved by the projects related to NPs from macroalgae, marine invertebrates, arthropods and associated microorganisms. Macroalgae are one of the most studied groups in Brazil with the isolation of many bioactive compounds including lipids, carotenoids, phycocolloids, lectins, mycosporine-like amino acids and halogenated compounds. Marine invertebrates and associated microorganisms have been more systematically studied in the last thirty years, revealing unique compounds, with potent biological activities. The venoms of Hymenopteran insects were also extensively studied, resulting in the identification of hundreds of peptides, which were used to create a chemical library that contributed for the identification of leader models for the development of antifungal, antiparasitic, and anticancer compounds. The built knowledge of Hymenopteran venoms permitted the development of an equine hyperimmune serum anti honeybee venom. Amongst the microorganisms associated with insects the bioprospecting strategy was to understand the molecular basis of intra- and interspecies interactions (Chemical Ecology), translating this knowledge to possible biotechnological applications. The results discussed here reinforce the importance of BIOprospecTA program on the development of research with highly innovative potential in Brazil. Resumo A presente revisão discute o impacto das iniciativas de bioprospecção desenvolvidas pelos projetos associados ao BIOprospecTA, subprograma do programa BIOTA, apoiado pela FAPESP. Esta revisão traz um resumo dos principais resultados produzidos pelos projetos de investigação de produtos naturais (PNs) de organismos não vegetais, como exemplos do sucesso desta iniciativa, com foco nos avanços alcançados pelos projetos relacionados a PNs de macroalgas, invertebrados marinhos, artrópodes e microrganismos associados. As macroalgas são um dos grupos mais estudados no Brasil com o isolamento de muitas substâncias bioativas, incluindo lipídios, carotenóides, ficocolóides, lectinas, aminoácidos do tipo micosporina e substâncias halogenadas. Invertebrados marinhos e microrganismos associados têm sido estudados de forma mais sistemática nos últimos trinta anos, revelando substâncias únicas, com potentes atividades biológicas. Os venenos de insetos himenópteros também foram amplamente estudados, resultando na identificação de centenas de peptídeos, que foram utilizados para criar uma biblioteca química que contribuiu para a identificação de modelos para o desenvolvimento de substâncias antifúngicas, antiparasitárias e anticancerígenas. O conhecimento construído dos venenos de himenópteros permitiu o desenvolvimento de um soro equino anti-peçonha de abelha. Dentre os microrganismos associados a insetos, a estratégia de bioprospecção foi compreender as bases moleculares das interações intra e interespécies (Ecologia Química), traduzindo esse conhecimento para possíveis aplicações biotecnológicas. Os resultados aqui discutidos reforçam a importância do programa BIOprospecTA no desenvolvimento de pesquisas com alto potencial inovador no Brasil.

The fibroin-based silk fibers of weaver ants are an alternative biomaterial to be investigated and explored for potential biomedical applications. In this context, the silk fibers from the nest of the weaver ant Camponotus textor was solubilized and fractionated by gel permeation. The different fractions were collected, pooled and submitted to analysis with a series of biochemical methods, nuclear magnetic resonance (NMR) spectroscopy, analytical proteomic strategies, and data treatment with bioinformatic tools to perform the structural characterization of the fibroin-based silk fibers produced by the ant. Our data demonstrated the identification of one fibroin proteoform in the ant silk fibers. The protein chracterized as a glycoprotein with MW around 40 kDa and presenting 66% (w/w) of total sugars attached to it through O-linked carbohydrates. The 3D of protein was modeled, revealing a structure predominantly constituted of coiled-coil secondary units in the whole model, featuring at least four superhelices (arrangement with multiple α-helices). The scientific outcomes reported herein may be relevant for the development of novel approaches for the synthetic or recombinant production of novel silk-based polymers for biomedical applications. BIOLOGICAL SIGNIFICANCE: The present investigation significantly expanded knowledge regarding to the fibroin-based silk fibers from weaver ants, contributing to improvements in our understanding of the properties and characteristics of these silk fibers. For example, as reported here, carbohydrates were detected in the ants' silk for the first time presenting the fibroin as a glycoprotein. Moreover, the 3D structure provided new insights into the secondary structures considering the whole model of the protein.

Made available in DSpace on 2022-04-29T08:36:37Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-12-01 Cryptococcosis is associated with high rates of morbidity and mortality, especially in AIDS patients. Its treatment is carried out by combining amphotericin B and azoles or flucytosine, which causes unavoidable toxicity issues in the host. Thus, the urgency in obtaining new antifungals drives the search for antimicrobial peptides (AMPs). This study aimed to extend the understanding of the mechanism of action of an AMP analog from wasp peptide toxins, MK58911-NH2, on Cryptococcus neoformans. We also evaluated if MK58911-NH2 can act on cryptococcal cells in macrophages, biofilms, and an immersion zebrafish model of infection. Finally, we investigated the structure-antifungal action and the toxicity relationship of MK58911-NH2 fragments and a derivative of this peptide (MH58911-NH2). The results demonstrated that MK58911-NH2 did not alter the fluorescence intensity of the cell wall-binding dye calcofluor white or the capsule-binding dye 18b7 antibody-fluorescein isothiocyanate (FITC) in C. neoformans but rather reduced the number and size of fungal cells. This activity reduced the fungal burden of C. neoformans in both macrophages and zebrafish embryos as well as within biofilms. Three fragments of the MK58911-NH2 peptide showed no activity against Cryptococcus and not toxicity in lung cells. The derivative peptide MH58911-NH2, in which the lysine residues of MK58911-NH2 were replaced by histidines, reduced the activity against extracellular and intracellular C. neoformans. On the other hand, it was active against biofilms and showed reduced toxicity. In summary, these results showed that peptide MK58911-NH2 could be a promising agent against cryptococcosis. This work also opens a perspective for the verification of the antifungal activity of other derivatives. Department of Clinical Analysis School of Pharmaceutical Sciences São Paulo State University-UNESP Department of Basic and Applied Biology Institute of Biosciences São Paulo State University-UNESP Department of Clinical Analysis School of Pharmaceutical Sciences São Paulo State University-UNESP Department of Basic and Applied Biology Institute of Biosciences São Paulo State University-UNESP

Aim: In this work, mastoparan analog peptides from wasp venom were tested against Candida albicans and safety assays were performed using cell culture and model zebrafish. Materials & methods: Minimal inhibitory concentration was determined and toxicity was performed using human skin keratinocyte and embryo zebrafish. Also, permeation of peptides through embryo chorion was performed. Results: The peptides demonstrated anti- C. albicans activity, with low cytotoxicity and nonteratogenicity in Danio rerio. The compounds had different permeation through chorion, suggesting that this occurs due to modifications in their amino acid sequence. Conclusion: The results showed that the studied peptides can be used as structural study models for novel potential antifungal agents.

Insect venom phospholipases have been identified in nearly all clinically relevant social Hymenoptera, including bees, wasps and ants. Among other biological roles, during the envenoming process these enzymes cause the disruption of cellular membranes and induce hypersensitive reactions, including life threatening anaphylaxis. While phospholipase A2 (PLA2) is a predominant component of bee venoms, phospholipase A1 (PLA1) is highly abundant in wasps and ants. The pronounced prevalence of IgE-mediated reactivity to these allergens in sensitized patients emphasizes their important role as major elicitors of Hymenoptera venom allergy (HVA). PLA1 and -A2 represent valuable marker allergens for differentiation of genuine sensitizations to bee and/or wasp venoms from cross-reactivity. Moreover, in massive attacks, insect venom phospholipases often cause several pathologies that can lead to fatalities. This review summarizes the available data related to structure, model of enzymatic activity and pathophysiological roles during envenoming process of insect venom phospholipases A1 and -A2.

Made available in DSpace on 2021-06-25T10:59:22Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-05-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Anionic lipid membrane electrostatic potential and solution pH can influence cationic peptide adsorption to these bilayers, especially those containing simultaneously acid and basic residues. Here, we investigate the effects of the pH solution on MP1 (IDWKKLLDAAKQIL-NH2) adsorption to anionic (7POPC:3POPG) lipid vesicles in comparison to its analog H-MP1, with histidines substituting lysines. We used the association of adsorption isotherms and constant pH molecular dynamic simulations (CpHMD) to explore the effects of membrane potential and pH on peptides’ adsorption on this lipid membrane. We analyzed the fluorescence and zeta potential adsorption isotherms using the Gouy–Chapman theory. In CpHMD simulations for the peptides in solution and adsorbed on the lipid bilayer, we used the conformations obtained by conventional MD simulations at a µs timescale. Non-equilibrium Monte Carlo simulations provided the protonation states of acidic and basic residues. CpHMD showed average pKa shifts of two to three units, resulting in a higher net charge for the analog than for MP1, strongly modulating the peptide adsorption. The fractions of the protonation of acidic and basic residues and the peptides’ net charges obtained from the analysis of the adsorption isotherms were in reasonable agreement with those from CpHMD. MP1 adsorption was almost insensitive to solution pH. H-MP1 was much more sensitive to partitioning, at acidic pH, with an affinity ten times higher than in neutral ones. IBILCE Department of Physics UNESP—São Paulo State University Institute of Biosciences Department of Basic and Applied Biology UNESP—São Paulo State University IBILCE Department of Physics UNESP—São Paulo State University Institute of Biosciences Department of Basic and Applied Biology UNESP—São Paulo State University FAPESP: 2010/18169-3 FAPESP: 2015/25619-9 FAPESP: 2016/16212-5 FAPESP: 2018/01841-2

Made available in DSpace on 2021-06-25T11:10:07Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-02-01. Added 1 bitstream(s) on 2021-07-15T15:05:39Z : No. of bitstreams: 1 S0103-50532021000200237.pdf: 4679375 bytes, checksum: 70081961e9c71ddc7039a07f706743c0 (MD5) Maytenus ilicifolia (Celastraceae) is a medicinal plant that is native to southern Brazil and is popularly known as espinheira-santa. From a biosynthesis perspective, this species accumulates quinonemethide triterpenes and sesquiterpene pyridine alkaloids as major secondary metabolites that exhibit interesting biological properties, with antitumoral and antiprotozoal activities, respectively, being the most frequently reported. Additionally, the restricted accumulation of such compounds in the roots raises questions about the expression of proteins involved in such compartmentalization and their possible biological and/or ecological role in M. ilicifolia. Thus, this article describes the use of shotgun proteomics and histochemical studies for the characterization of the main biosynthetic pathways involved in the regulation of the metabolism in M. ilicifolia roots. This combined approaches also resulted in the identification of a series of proteins involved in the quinonemethide triterpenes and sesquiterpene pyridine alkaloids, providing evidences of their differential compartmentalization Instituto de Química Universidade Estadual Paulista (Unesp) Scientific and Technological Bioresource Nucleus BIOREN-UFRO Universidad de La Frontera (UFRO) Laboratório de Botânica Faculdade de Ciências Farmacêuticas de Ribeirão Preto Universidade de São Paulo (USP) Departamento de Biologia Geral e Aplicada Instituto de Biociências Universidade Estadual Paulista (Unesp) Instituto de Química Universidade Estadual Paulista (Unesp) Departamento de Biologia Geral e Aplicada Instituto de Biociências Universidade Estadual Paulista (Unesp)

Made available in DSpace on 2022-04-29T08:35:27Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-11-19 Several molecular mechanisms are involved in the genetic code interpretation during translation, as codon degeneration for the incorporation of rare amino acids. One mechanism that stands out is selenocysteine (Sec), which requires a specific biosynthesis and incorporation pathway. In Bacteria, the Sec biosynthesis pathway has unique features compared with the eukaryote pathway as Ser to Sec conversion mechanism is accomplished by a homodecameric enzyme (selenocysteine synthase, SelA) followed by the action of an elongation factor (SelB) responsible for delivering the mature Sec-tRNASec into the ribosome by the interaction with the Selenocysteine Insertion Sequence (SECIS). Besides this mechanism being already described, the sequential events for Sec-tRNASec and SECIS specific recognition remain unclear. In this study, we determined the order of events of the interactions between the proteins and RNAs involved in Sec incorporation. Dissociation constants between SelB and the native as well as unacylated-tRNASec variants demonstrated that the acceptor stem and variable arm are essential for SelB recognition. Moreover, our data support the sequence of molecular events where GTP-activated SelB strongly interacts with SelA.tRNASec. Subsequently, SelB.GTP.tRNASec recognizes the mRNA SECIS to deliver the tRNASec to the ribosome. SelB in complex with its specific RNAs were examined using Hydrogen/Deuterium exchange mapping that allowed the determination of the molecular envelopes and its secondary structural variations during the complex assembly. Our results demonstrate the ordering of events in Sec incorporation and contribute to the full comprehension of the tRNASec role in the Sec amino acid biosynthesis, as well as extending the knowledge of synthetic biology and the expansion of the genetic code. Physics Institute of Sao Carlos University of Sao Paulo, Trabalhador Sao Carlense Av., 400 Department of Chemistry and Biochemistry University California – Santa Cruz, 1156 High St. Physical Sciences Laboratory State University of Northern Rio de Janeiro Darcy Ribeiro – UENF, Av. Alberto Lamego, 2000 Faculty of Science Philosophy and Letters University of Sao Paulo Department of Biochemistry and Molecular Biology University of British Columbia, 2350 Health Science Mall Department of Physical Chemistry Chemistry Institute of the São Paulo State University – UNESP Physics Department Institute of Biosciences Letters and Exact Sciences (IBILCE) São Paulo State University – UNESP Department of General and Applied Biology Institute of Biosciences of Rio Claro São Paulo State University – UNESP Bioscience Institute of Universidade Estadual Paulista, Rubião Jr. Department of Genetics and Evolution Federal University of São Carlos – UFSCar Department of Physical Chemistry Chemistry Institute of the São Paulo State University – UNESP Physics Department Institute of Biosciences Letters and Exact Sciences (IBILCE) São Paulo State University – UNESP Department of General and Applied Biology Institute of Biosciences of Rio Claro São Paulo State University – UNESP Bioscience Institute of Universidade Estadual Paulista, Rubião Jr.

Made available in DSpace on 2021-06-25T10:59:40Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-01-01 Salivary glands are omnipresent in termites and occur in all developmental stages and castes. They function to produce, store, and secrete compounds, ranging from a feeding function to defensive mechanisms. Here, we provide a complete morphological overview of the salivary glands in the soldierless species Ruptitermes reconditus and R. xanthochiton, and the first proteomic profile of the salivary glands in a Neotropical Apicotermitinae representative, R. reconditus. Salivary glands from both species were composed of several acini, roughly spherical structures composed of two types of central cells (type I and II) and peripheral parietal cells, as well as transporting ducts and two salivary reservoirs. Central cells were richly supplied with electron-lucent secretory vesicles and rough endoplasmic reticulum, a feature of protein-secreting cells. Parietal cells of Ruptitermes spp. had conspicuous characteristics such as electron-lucent secretory vesicles surrounded by mitochondria and well-developed microvilli. Moreover, different individuals showed variation in the secretory cycle of salivary acini, which may be related to polyethism. Ultrastructural analysis evidenced a high synthesis of secretion and also the occurrence of lysosomes and autophagic structures in central cells. Proteomic analysis of the salivary glands revealed 483 proteins divided into functional groups, highlighting toxins/defensins and compounds related to alarm communication and colony asepsis. Soldierless termites are quite successful, especially due to morphological adaptations of the workers, including unknown modifications of exocrine glands. Thus, according to our morphological and proteomic findings, we discuss the potential roles of the salivary gland secretion in different social aspects of the sampled species. Laboratório de Cupins Departamento de Biologia Geral e Aplicada Instituto de Biociências Univ Estadual Paulista UNESP Campus Rio Claro, Avenida 24A, 1515, Bela Vista Centro de Estudos de Insetos Sociais-CEIS Instituto de Biociências Univ Estadual Paulista UNESP Campus Rio Claro, Avenida 24A, 1515, Bela Vista Laboratório de Cupins Departamento de Biologia Geral e Aplicada Instituto de Biociências Univ Estadual Paulista UNESP Campus Rio Claro, Avenida 24A, 1515, Bela Vista Centro de Estudos de Insetos Sociais-CEIS Instituto de Biociências Univ Estadual Paulista UNESP Campus Rio Claro, Avenida 24A, 1515, Bela Vista

Made available in DSpace on 2021-06-25T10:44:24Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-01-01 In order to evaluate the use of matrix-assisted laser desorption ionization (MALDI) Mass Spectral Imaging (MSI) to Glioblastoma (GBM) studies, some sections of formalin fixed paraffin embedded samples of GBM tumors were submitted to classical immunoassays, to define the profile of distribution of some classical and well recognized molecular markers of GBM grade IV (Ki-67, S100, Glial GFAP, CD31 and CD34), while other sections of the same samples were submitted to in-tissue proteomic analysis by MALDI MSI, and both results compared to each other. The overlapping of the MALDI spectra obtained for the tryptic peptides with the immunohistochemical reactions of each marker protein were used to build a distribution map of the marker proteins all over the GBM tissue section. The results revealed a high correlation between both methods, indicating that MALDI MSI has enough sensitivity to be compared to the immunohistochemical methods, as well is sufficiently reliable to be used in biomarkers identification. Institute of Biosciences Department of Basic and Applied Biology Center of the Study of Social Insects Sao Paulo State University (UNESP) Center for the Study of Venoms and Venomous Animals (CEVAP) Sao Paulo State University (UNESP) Botucatu Medical School (FMB) Sao Paulo State University (UNESP) Institute of Biosciences Department of Basic and Applied Biology Center of the Study of Social Insects Sao Paulo State University (UNESP) Center for the Study of Venoms and Venomous Animals (CEVAP) Sao Paulo State University (UNESP) Botucatu Medical School (FMB) Sao Paulo State University (UNESP)

Made available in DSpace on 2020-12-12T02:19:36Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-08-07 Orb-weaving spiders use a highly strong, sticky and elastic web to catch their prey. These web properties alone would be enough for the entrapment of prey; however, these spiders may be hiding venomous secrets in the web, which current research is revealing. Here, we provide strong proteotranscriptomic evidence for the presence of toxin/neurotoxin-like proteins, defensins, and proteolytic enzymes on the web silk from Nephila clavipes spider. The results from quantitative-based transcriptomic and proteomic approaches showed that silk-producing glands produce an extensive repertoire of toxin/neurotoxin-like proteins, similar to those already reported in spider venoms. Meanwhile, the insect toxicity results demonstrated that these toxic components can be lethal and/or paralytic chemical weapons used for prey capture on the web, and the presence of fatty acids in the web may be a responsible mechanism opening the way to the web toxins for accessing the interior of prey's body, as shown here. Comparative phylogenomic-level evolutionary analyses revealed orthologous genes among two spider groups, Araneomorphae and Mygalomorphae, and the findings showed protein sequences similar to toxins found in the taxa Scorpiones and Hymenoptera in addition to Araneae. Overall, these data represent a valuable resource to further investigate other spider web toxin systems and also suggest that N. clavipes web is not a passive mechanical trap for prey capture, but it exerts an active role in prey paralysis/killing using a series of neurotoxins. Center of the Study of Social Insects Department of General and Applied Biology Institute of Biosciences of Rio Claro University of Saõ Paulo State (UNESP) Department of Pharmaceutical Chemistry University of Vienna Division of Computational System Biology Department of Microbiology and Ecosystem Science University of Vienna Department of Laboratory Medicine and Core Facility Genomics Medical University of Vienna Paracelsus Medical University Center of the Study of Social Insects Department of General and Applied Biology Institute of Biosciences of Rio Claro University of Saõ Paulo State (UNESP)

Made available in DSpace on 2020-12-12T02:04:03Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-05-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Phospholipase A1 (PLA1) is one of the three major allergens identified in the venom of P. paulista (Hymenoptera: Vespidae), a clinically relevant wasp from southeastern Brazil. The recombinant form of this allergen (rPoly p 1) could be used for the development of molecular diagnostic of venom allergy. Early attempts to produce rPoly p 1 using Escherichia coli BL21 (DE3) cells rendered high yields of the insoluble rPoly p 1 but with low levels of solubilized protein recovery (12%). Here, we aimed to improve the production of rPoly p 1 in E. coli by testing different conditions of expression, solubilization of the inclusion bodies and protein purification. The results showed that the expression at 16 °C and 0.1 mM of IPTG increased the production of rPoly p 1, still in the insoluble form, but with high solubilized protein yields after incubation with citrate–phosphate buffer with 0.15 M NaCl, 6 M urea, pH 2.6 at 25 ºC for 2 h. The venom allergen was also cloned in pPICZαA vector for soluble expression as a secreted protein in Pichia pastoris X-33 cells, rendering almost undetectable levels (nanograms) in the culture supernatant. In contrast, a sevenfold increase of the solubilized and purified rPoly p 1 yields (1.5 g/L of fermentation broth) was obtained after improved production in E. coli. The identity of the protein was confirmed with an anti-His antibody and MS spectra. Allergen-specific IgE (sIgE)-mediated recognition was evaluated in immunoblotting with sera of allergic patients (n = 40). Moreover, rPoly p 1 showed high levels of diagnostic sensitivity (95%). The optimized strategy for rPoly p 1 production described here, will provide the amounts of allergen necessary for the subsequent protein refolding, immunological characterization steps, and ultimately, to the development of molecular diagnostic for P. paulista venom allergy. Center for the Study of Social Insects Department of General and Applied Biology University of Sao Paulo State (UNESP/RC) Center for Genetic Engineering and Biotechnology. Biomedical Research Division System Biology Department, Ave. 31, e/ 158 and 190, Cubanacan, Playa, P.O. Box 6162 Laboratory of Translational Immunology School of Medical Sciences University of Campinas (UNICAMP), Campinas Arthropod Molecular Biology Laboratory-LBMA-IB University of Sao Paulo State (UNESP/RC), Ave. 24-A, 1515- Bela Vista Venoms and Venomous Animal Studies Center-CEVAP University of São Paulo State (UNESP) Lageado Experimental Farm, José Barbosa de Barros Street, 1780 Center for the Study of Social Insects Department of General and Applied Biology University of Sao Paulo State (UNESP/RC) Arthropod Molecular Biology Laboratory-LBMA-IB University of Sao Paulo State (UNESP/RC), Ave. 24-A, 1515- Bela Vista Venoms and Venomous Animal Studies Center-CEVAP University of São Paulo State (UNESP) Lageado Experimental Farm, José Barbosa de Barros Street, 1780 FAPESP: # 2014/13936-7

Made available in DSpace on 2020-12-12T02:05:15Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-04-15 Celiac disease (CD) is a chronic illness characterized by an inflammatory process triggered by gluten protein intake. Recent evidence has suggested that the lower relative abundance of bifidobacteria in the intestinal lumen may be associated with CD. Herein, we assessed the effect of the Bifidobacterium species Bifidobacterium bifidum, Bifidobacterium longum, Bembidion breve, Bifidobacterium animalis alone, and also a Bifidobacterium consortium on the digestion of intact gluten proteins (gliadins and glutenins) and the associated immunomodulatory responses elicited by the resulting peptides. The cytotoxicity and proinflammatory responses were evaluated through the activation of NF-kB p65 and the expression of cytokines TNF-α and IL-1β in Caco-2 cell cultures exposed to gluten-derived peptides. The peptides induced a clear reduction in cytotoxic responses and proinflammatory marker levels compared to the gluten fragments generated during noninoculated gastrointestinal digestion. These results highlight the possible use of probiotics based on bifidobacteria as a prospective treatment for CD. Department of Chemistry Federal University of São Carlos (UFSCar) Center of Study of Social Insects Department of Biology Institute of Biosciences of Rio Claro São Paulo State University (UNESP) Department of Genetics and Evolution Biotechnology Graduate Program (PPGBiotec) Federal University of São Carlos Center of Study of Social Insects Department of Biology Institute of Biosciences of Rio Claro São Paulo State University (UNESP)

Celiac disease (CD) is a chronic illness characterized by an inflammatory process triggered by gluten protein intake. Recent evidence has suggested that the lower relative abundance of bifidobacteria in the intestinal lumen may be associated with CD. Herein, we assessed the effect of the

Made available in DSpace on 2020-12-12T02:07:06Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-05-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Increasing resistance in antibiotic and chemotherapeutic treatments has been pushing studies of design and evaluation of bioactive peptides. Designing relies on different approaches from minimalist sequences and endogenous peptides modifications to computational libraries. Evaluation relies on microbiological tests. Aiming a deeper understanding, we chose the octapeptide Jelleine-I (JI) for its selective and low toxicity profile, designed small modifications combining the substitutions of Phe by Trp and Lys/His by Arg and tested the antimicrobial and anticancer activity on melanoma cells. Biophysical methods identified environment-dependent modulation of aggregation, but critical aggregation concentrations of JI and analogs in buffer show that peptides start membrane interactions as monomers. The presence of model membranes increases or reduces the partial aggregation of peptides. Compared to JI, analog JIF2WR shows the lowest tendency to aggregation on bacterial model membranes. JI and analogs are lytic to model membranes. Their composition-dependent performance indicates preference for the higher charged anionic bilayers in line with their superior performance toward Staphylococcus aureus and Streptococcus pneumoniae. JIF2WR presented the higher partitioning, higher lytic activity and lower aggregated contents. Despite these increased membranolytic activities, JIF2WR exhibited comparable antimicrobial activity in relation to JI at the expenses of some loss in selectivity. We found that the substitution Phe/Trp (JIF2W) tends to decrease antimicrobial but to increase anticancer activity and aggregation on model membranes and the toxicity toward human cells. However, the concomitant substitution Lys/His by Arg (JIF2WR) modulates some of these tendencies, increasing both the antimicrobial and the anticancer activity while decreasing the aggregation tendency. Departamento de Física Universidade Estadual Paulista (Unesp) Instituto de Biociências Letras e Ciências Exatas (Ibilce), R. Cristóvão Colombo, 2265 Departamento de Química e Ciências Ambientais Universidade Estadual Paulista (Unesp) Instituto de Biociências Letras e Ciências Exatas (Ibilce), R. Cristóvão Colombo, 2265 Faceres Medical School Laboratório de Neurobiologia Estrutural e Funcional (LaNEF) Departamento de Biofísica Universidade Federal de São Paulo R. Botucatu, 862, Edifício ECB, 7º andar Centro de Estudos de Insetos Sociais Universidade Estadual Paulista (Unesp), Câmpus Rio Claro, Av. 24-A, 1515 Departamento de Doenças Dermatológicas Infecciosas e Parasitárias Faculdade de Medicina de São José do Rio Preto Departamento de Física Universidade Estadual Paulista (Unesp) Instituto de Biociências Letras e Ciências Exatas (Ibilce), R. Cristóvão Colombo, 2265 Departamento de Química e Ciências Ambientais Universidade Estadual Paulista (Unesp) Instituto de Biociências Letras e Ciências Exatas (Ibilce), R. Cristóvão Colombo, 2265 Centro de Estudos de Insetos Sociais Universidade Estadual Paulista (Unesp), Câmpus Rio Claro, Av. 24-A, 1515 FAPESP: 2012/24259-0 FAPESP: 2014/08372-7 FAPESP: 2016/13368-4 FAPESP: 2016/16212-5 FAPESP: 2016/17951-6

Made available in DSpace on 2020-12-12T01:55:12Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-02-07 The proboscis extension response (PER) reflex may be used to condition the pairing of an odor with sucrose, which is applied to the antennae, in experiments to induce learning, where the odor represents a conditioned stimulus, while sucrose represents an unconditioned stimulus. A series of studies have been conducted on honeybees, relating learning and memory acquisition/retrieval using the PER as a strategy for accessing their ability to exhibit an unconditioned stimulus; however, the major metabolic processes involved in the PER are not well known. Thus, the aim of this investigation is profiling the metabolome of the honeybee brain involved in the PER. In this study, a semiquantitative approach of matrix-assisted laser desorption ionization (MALDI) mass spectral imaging (MSI) was used to profile the most abundant metabolites of the honeybee brain that support the PER. It was reported that execution of the PER requires the metabolic transformations of arginine, ornithine, and lysine as substrates for the production of putrescine, cadaverine, spermine, spermidine, 1,3-diaminopropane, and γ-aminobutyric acid (GABA). Considering the global metabolome of the brain of honeybee workers, the PER requires the consumption of large amounts of cadaverine and 1,3-diaminopropane, in parallel with the biosynthesis of high amounts of spermine, spermidine, and ornithine. To exhibit the PER, the brain of honeybee workers processes the conversion of l-arginine and l-lysine through the polyamine pathway, with different regional metabolomic profiles at the individual neuropil level. The outcomes of this study using this metabolic route as a reference are indicating that the antennal lobes and the calices (medial and lateral) were the most active brain regions for supporting the PER. Department of Biology Center of the Study of Social Insects Institute of Biosciences University of São Paulo State (UNESP) Department of Biology Center of the Study of Social Insects Institute of Biosciences University of São Paulo State (UNESP)

Phospholipase A1 (PLA1) is one of the three major allergens identified in the venom of P. paulista (Hymenoptera: Vespidae), a clinically relevant wasp from southeastern Brazil. The recombinant form of this allergen (rPoly p 1) could be used for the development of molecular diagnostic of venom allergy. Early attempts to produce rPoly p 1 using Escherichia coli BL21 (DE3) cells rendered high yields of the insoluble rPoly p 1 but with low levels of solubilized protein recovery (12%). Here, we aimed to improve the production of rPoly p 1 in E. coli by testing different conditions of expression, solubilization of the inclusion bodies and protein purification. The results showed that the expression at 16 °C and 0.1 mM of IPTG increased the production of rPoly p 1, still in the insoluble form, but with high solubilized protein yields after incubation with citrate–phosphate buffer with 0.15 M NaCl, 6 M urea, pH 2.6 at 25 ºC for 2 h. The venom allergen was also cloned in pPICZαA vector for soluble expression as a secreted protein in Pichia pastoris X-33 cells, rendering almost undetectable levels (nanograms) in the culture supernatant. In contrast, a sevenfold increase of the solubilized and purified rPoly p 1 yields (1.5 g/L of fermentation broth) was obtained after improved production in E. coli. The identity of the protein was confirmed with an anti-His antibody and MS spectra. Allergen-specific IgE (sIgE)-mediated recognition was evaluated in immunoblotting with sera of allergic patients (n = 40). Moreover, rPoly p 1 showed high levels of diagnostic sensitivity (95%). The optimized strategy for rPoly p 1 production described here, will provide the amounts of allergen necessary for the subsequent protein refolding, immunological characterization steps, and ultimately, to the development of molecular diagnostic for P. paulista venom allergy. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s13205-020-02202-8) contains supplementary material, which is available to authorized users.

Background Toxicological studies evaluating the possible harmful effects of pesticides on bees are important and allow the emergence of protection and pollinator conservation strategies. This study aimed to evaluate the effects of exposure to a sublethal concentration of imidacloprid (LC50/100 : 0.014651 ng imidacloprid µL-1 diet) on the distribution of certain proteins identified in the brain of Apis mellifera worker bees using a MALDI-imaging approach. This technique enables proteomic analysis of tissues in situ by monitoring the spatiotemporal dynamics of the biochemical processes occurring at a specific time in specific brain neuropils. For this purpose, foraging bees were exposed to an 8-day diet containing a sublethal concentration of imidacloprid corresponding to the LC50/100 . Bees were collected on day 8 of exposure, and their brains analyzed using protein density maps. Results The results showed that exposure to imidacloprid led to a series of biochemical changes, including alterations in synapse regulation, apoptosis regulation and oxidative stress, which may adversely impair the physiology of these colony bees. Conclusion Worker bee contact with even tiny amounts of imidacloprid had potent effects leading to the overexpression of a series of proteins related to important cellular processes that were possibly damaged by the insecticide. © 2018 Society of Chemical Industry.

Made available in DSpace on 2020-12-12T01:51:42Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-12-06 Invasive fungal infections, such as cryptococcosis and paracoccidioidomycosis are associated with significant rates of morbidity and mortality. Cryptococcosis, caused by Cryptococcus neoformans, is distributed worldwide and has received much attention as a common complication in patients with HIV. Invasive fungal infections are usually treated with a combination of amphotericin B and azoles. In addition, 5-fluorocytosine (5-FC) is applied in cryptococcosis, specifically to treat central nervous system infection. However, host toxicity, high cost, emerging number of resistant strains, and difficulty in developing new selective antifungals pose challenges. The need for new antifungals has therefore prompted a screen for inhibitory peptides, which have multiple mechanisms of action. The honeycomb moth Galleria mellonella has been widely used as a model system for evaluating efficacy of antifungal agents. In this study, a peptide analog from the mastoparan class of wasps (MK58911) was tested against Cryptococcus spp. and Paracoccidioides spp. In addition, peptide toxicity tests on lung fibroblasts (MRC5) and glioblastoma cells (U87) were performed. Subsequent tests related to drug interaction and mechanism of action were also performed, and efficacy and toxicity of the peptide were evaluated in vivo using the G. mellonella model. Our results reveal promising activity of the peptide, with an MIC in the range of 7.8–31.2 μg/mL, and low toxicity in MRC and U87 cells (IC50 > 500 μg/mL). Taken together, these results demonstrate that MK58911 is highly toxic in fungal cells, but not mammalian cells (SI > 16). The mechanism of toxicity involved disruption of the plasma membrane, leading to death of the fungus mainly by necrosis. In addition, no interaction with the drugs amphotericin B and fluconazole was found either in vitro or in vivo. Finally, the peptide showed no toxic effects on G. mellonella, and significantly enhanced survival rates of larvae infected with C. neoformans. Although not statistically significant, treatment of larvae with all doses of MK58911 showed a similar trend in decreasing the fungal burden of larvae. These effects were independent of any immunomodulatory activity. Overall, these results present a peptide with potential for use as a new antifungal drug to treat systemic mycoses. Department of Clinical Analysis School of Pharmaceutical Sciences São Paulo State University-UNESP Department of Biology Center for the Study of Social Insects Institute of Biosciences São Paulo State University-UNESP Department of Clinical Analysis School of Pharmaceutical Sciences São Paulo State University-UNESP Department of Biology Center for the Study of Social Insects Institute of Biosciences São Paulo State University-UNESP

Polybia paulista (Hymenoptera: Vespidae) is a neotropical social wasp from southeast Brazil. As most social Hymenoptera, venom from P. paulista comprises a complex mixture of bioactive toxins ranging from low molecular weight compounds to peptides and proteins. Several efforts have been made to elucidate the molecular composition of the P. paulista venom. Data derived from proteomic, peptidomic and allergomic analyses has enhanced our understanding of the whole envenoming process caused by the insect sting. The combined use of bioinformatics, -omics- and molecular biology tools have allowed the identification, characterization, in vitro synthesis and recombinant expression of several wasp venom toxins. Some of these P. paulista - derived bioactive compounds have been evaluated for the rational design of antivenoms and the improvement of allergy specific diagnosis and immunotherapy. Molecular characterization of crude venom extract has enabled the description and isolation of novel toxins with potential biotechnological applications. Here, we review the different approaches that have been used to unravel the venom composition of P. paulista. We also describe the main groups of P. paulista - venom toxins currently identified and analyze their potential in the development of component-resolved diagnosis of allergy, and in the rational design of antivenoms and novel bioactive drugs.

Bees are recognized worldwide for their social, economic, and environmental value. In recent decades they have been seriously threatened by diseases and high levels of pesticide use. The susceptibility of bees to insecticides makes them an important terrestrial model for assessing environmental quality, and various biomarkers have been developed for such assessments. The present study aimed to evaluate the activity of the enzymes acetylcholinesterase (AChE), carboxylesterase (CaE), and glutathione-S-transferase (GST) in Africanized honeybees exposed to fipronil. The results showed that fipronil at a sublethal dose (0.01 ng/bee) modulates the activity of CaE in all isoforms analyzed (CaE-1, CaE-2, and CaE-3) in both newly emerged and aged bees, and does not affect the activity of AChE or GST. The recovery of the bees after fipronil exposure was also investigated, and these results demonstrated that even the cessation of fipronil ingestion might not lead to complete recovery of individual bees. Even at low doses, fipronil was shown to cause changes in the activity of key enzymes in bees. The possible consequences of these changes are discussed. Environ Toxicol Chem 2017;36:1652-1660. © 2016 SETAC.

Made available in DSpace on 2018-12-11T17:00:34Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-01-16 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Dufour's gland is associated with the venom apparatuses of social wasps and bees. This location and its evolutionary adaptations indicate that it could be involved in the production of alarm pheromones in the social wasp Polybia paulista. To investigate this hypothesis, the volatile composition of this gland was analyzed and compared to that in the venom. Eighteen compounds were identified as secreted by Dufour's gland, and 16 of these compounds were also identified in the venom, suggesting that the compounds produced by the gland are secreted and mixed with venom in the venom reservoir of this wasp. These compounds were subjected to a field bioassay to investigate their potential action as alarm pheromones. Alcohols and aldehydes elicited the alert behavior in workers, luring them outside the nest, whereas acids attracted the workers in the direction of the source; fatty acid methyl esters elicited aggression. These results suggest that Dufour's gland produces alarm pheromones. To corroborate this hypothesis the proteomic complement of this gland was assigned using a shot-gun strategy; 59 proteins were identified, and the results indicate specialization of Dufour's gland for the metabolism of fatty acids (elongation, esterification unsaturation, reduction, and decarboxylation) in the biosynthesis of alarm pheromones. Biological significance The present knowledge about the role of Dufour's gland among aculeate Hymenoptera insects suggests that it may have many different roles related to the biosynthesis and secretion of chemical markers for different biological functions, though none are related to the elicitation of alarm behaviors for coordinating a mass attack of the colony against intruders. The present study combined the analysis of secreted volatile compounds (as metabolites) with proteome assignments and a field bioassay with synthetic compounds to clearly demonstrate that Dufour's gland does in fact biosynthesize alarm pheromones in social wasps. This strategy may be reproduced in other investigations related to pheromone production in other insects. Center of Study of Social Insects Department of Biology Institute of Biosciences of Rio Claro São Paulo State University (UNESP) Center of Study of Social Insects Department of Biology Institute of Biosciences of Rio Claro São Paulo State University (UNESP) FAPESP: 2011/51684-1

Made available in DSpace on 2020-12-12T01:51:19Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-02-20 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Termite soldiers constitute the defensive frontline of the colonies, despite workers also perform such tasks, especially within the Neotropical Apicotermitinae, in which all species are soldierless. Workers of the genus Ruptitermes display an extreme form of defense, characterized by body rupture and release of a sticky secretion. Previous observations suggested that such behavior may be advantageous against enemies, but the chemical composition of this secretion has been neglected. Here we firstly provide the proteomic profile of the defensive secretion of Ruptitermes reconditus and Ruptitermes pitan workers. Additionally, the mechanisms of action of this behavior was evaluated through different bioassays. A total of 446 proteins were identified in R. reconditus and 391 proteins in R. pitan, which were classified into: toxins, defensins and proteolytic enzymes; sticky components/ alarm communication; proteins related to detoxification processes; proteins involved in folding/conformation and post-translational modifications; housekeeping proteins; and uncharacterized/hypothetical proteins. According to the bioassays, the self-sacrifice is triggered by a physical stimulus, and the defensive secretion may cause immobility and death of the opponents. Assuming that termites are abundant in the tropics and therefore exposed to predators, suicidal behaviors seem to be advantageous, since the loss of an individual benefit the whole colony. Significance: Although recent studies have reported the biochemical composition of different weapons in soldiered species of termites, such efforts had not been applied to sordierless taxa up until now. Thus, this is the first report of the defensive mechanisms in soldierless termite species based on proteomic analysis. The diversity of compounds, which included toxin-like and mucin-like proteins, reflect the mechanisms of action of the defensive secretion released by termite workers, which may cause immobility and death of the opponents. Our findings may contribute to the knowledge regarding the development of defensive strategies in termites, especially in groups which lost the soldier caste during the evolution. Laboratório de Cupins Departamento de Biologia Instituto de Biociências Univ Estadual Paulista UNESP, Campus Rio Claro, Avenida 24A, 1515, Bela Vista Center for the Study of Social Insects Department of Biology Institute of Biosciences of Rio Claro Univ Estadual Paulista UNESP, Rio Claro Laboratório de Cupins Departamento de Biologia Instituto de Biociências Univ Estadual Paulista UNESP, Campus Rio Claro, Avenida 24A, 1515, Bela Vista Center for the Study of Social Insects Department of Biology Institute of Biosciences of Rio Claro Univ Estadual Paulista UNESP, Rio Claro CNPq: 150699/2017-4 FAPESP: 2013/26451-9 FAPESP: 2016/16212-5 FAPESP: 2017/10373-0 CAPES: 206/2018 CNPq: 301656/2013-4 CNPq: 305539/2014-0

Made available in DSpace on 2019-10-06T16:45:28Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-01-01 Termite societies are abundant in the tropics, and are therefore exposed to multiple enemies and predators, especially during foraging activity. Soldiers constitute a specialized defensive caste, although workers also participate in this process, and even display suicidal behavior, which is the case with the species Neocapritermes braziliensis. Here we describe the morphology, mechanisms of action, and proteomics of the salivary weapon in workers of this species, which due to the autothysis of the salivary glands causes their body rupture, in turn releasing a defensive secretion, observed during aggressiveness bioassays. Salivary glands are paired, composed of two translucent reservoirs, ducts and a set of multicellular acini. Histological and ultrastructural techniques showed that acini are composed of two types of central cells, and small parietal cells located in the acinar periphery. Type I central cells were abundant and filled with a large amount of secretion, while type II central cells were scarce and presented smaller secretion. Parietal cells were often paired and devoid of secretion. The gel-free proteomic approach (shotgun) followed by mass spectrometry revealed 235 proteins in the defensive secretion, which were classified into functional groups: (i) toxins and defensins, (ii) folding/conformation and post-translational modifications, (iii) salivary gland detoxification, (iv) housekeeping proteins and (v) uncharacterized and hypothetical proteins. We highlight the occurrence of neurotoxins previously identified in arachnid venoms, which are novelties for termite biology, and contribute to the knowledge regarding the defense strategies developed by termite species from the Neotropical region. Laboratório de Cupins Departamento de Biologia Instituto de Biociências Univ Estadual Paulista UNESP, Campus Rio Claro, Avenida 24A, 1515, Bela Vista Centro de Estudos de Insetos Sociais-CEIS Instituto de Biociências Univ Estadual Paulista UNESP, Campus Rio Claro, Avenida 24A, 1515, Bela Vista Laboratório de Cupins Departamento de Biologia Instituto de Biociências Univ Estadual Paulista UNESP, Campus Rio Claro, Avenida 24A, 1515, Bela Vista Centro de Estudos de Insetos Sociais-CEIS Instituto de Biociências Univ Estadual Paulista UNESP, Campus Rio Claro, Avenida 24A, 1515, Bela Vista

The olfactory conditioning of the bee proboscis extension reflex (PER) is extensively used as a paradigm in associative learning of invertebrates but with limited molecular investigations. To investigate which protein changes are linked to olfactory conditioning, a non-sophisticated conditioning model is applied using the PER in the honeybee (Apis mellifera). Foraging honeybees are assigned into three groups based on the reflex behavior and training: conditioned using 2-octanone (PER-conditioned), and sucrose and water controls. Thereafter, the brain synaptosomal proteins are isolated and analyzed by quantitative proteomics using stable isotope labeling (TMT). Additionally, the complex proteome dataset of the bee brain is generated with a total number of 5411 protein groups, including key players in neurotransmitter signaling. The most significant categories affected during olfactory conditioning are associated with "SNARE interactions in vesicular transport" (BET1 and VAMP7), ABC transporters, and fatty acid degradation pathways.

Made available in DSpace on 2019-10-04T12:37:47Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-05-30 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) The partial proteome of Polybia paulista wasp venom was previously reported elsewhere using a gel-dependent approach and resulted in the identification of a limited number of venom toxins. Here, we reinvestigated the P. paulista venom using a gel-free shotgun proteomic approach; the highly dynamic range of this approach facilitated the detection and identification of 1673 proteins, of which 23 venom proteins presented N-linked glycosylation as a posttranslational modification. Three different molecular forms of PLAT were identified as allergenic proteins, and two of these forms were modified by N-linked glycosylation. This study reveals an extensive repertoire of hitherto undescribed proteins that were classified into the following six different functional groups: (i) typical venom proteins; (ii) proteins related to the folding/conformation and PTMs of toxins; (iii) proteins that protect toxins from oxidative stress; (iv) proteins involved in chemical communication; (v) housekeeping proteins; and (vi) uncharacterized proteins. It was possible to identify venom toxin-like proteins that are commonly reported in other animal venoms, including arthropods such as spiders and scorpions. Thus, the findings reported here may contribute to improving our understanding of the composition of P. paulista venom, its envenoming mechanism and the pathologies experienced by the victim after the wasp stinging accident. Biological significance: The present study significantly expanded the number of proteins identified in P. paulista venom, contributing to improvements in our understanding of the envenoming mechanism produced by sting accidents caused by this wasp. For example, novel wasp venom neurotoxins have been identified, but no studies have assessed the presence of this type of toxin in social wasp venoms. In addition, 23 N-linked glycosylated venom proteins were identified in the P. paulista venom proteome, and some of these proteins might be relevant allergens that are immunoreactive to human IgE. Sao Paulo State Univ, Inst Biosci Rio Claro, Dept Biol, Ctr Study Social Insects, BR-13500 Rio Claro, SP, Brazil Univ Campinas UNICAMP, Fac Med, Lab Translat Immunol, Cidade Univ Zeferino Vaz, BR-13083887 Campinas, SP, Brazil Sao Paulo State Univ, Inst Biosci Rio Claro, Dept Biol, Ctr Study Social Insects, BR-13500 Rio Claro, SP, Brazil FAPESP: 2013/26451-9 FAPESP: 2016/16212-5 FAPESP: 2017/04680-7 FAPESP: 2017/10373-0 FAPESP: 2017/22405-3 CNPq: 301656/2013-4 CNPq: 150699/2017-4

Orb-weaving spiders can produce different silk fibers, which constitute outstanding materials characterized by their high strength and elasticity. Researchers have tried to reproduce the fibers of these proteins synthetically and/or by using recombinant DNA technology, but only a few of the natural physicochemical and biophysical properties have been obtained to date. Female orb-web-spiders present seven silk-glands, which synthesize the spidroins and a series of other proteins, which interact with the spidroins, resulting in silk fibers with notable physicochemical properties. Despite the recognized importance of the silk-glands for understanding how the fibers are produced and processed, the investigation of these glands is at a nascent stage. In the current study we present the assembled transcriptome of silk-producing glands from the orb-weaving spider Nephila clavipes, as well as develop a large-scale proteomic approach for in-depth analyses of silk-producing glands. The present investigation revealed an extensive repertoire of hitherto undescribed proteins involved in silk secretion and processing, such as prevention of degradation during the silk spinning process, transportation, protection against proteolytic autolysis and against oxidative stress, molecular folding and stabilization, and post-translational modifications. Comparative phylogenomic-level evolutionary analyses revealed orthologous genes among three groups of silk-producing organisms - (i) Araneomorphae spiders, (ii) Mygalomorphae spiders, and (iii) silk-producing insects. A common orthologous gene, which was annotated as silk gland factor-3 is present among all species analysed. This protein belongs to a transcription factor family, that is important and related to the development of the silk apparatus synthesis in the silk glands of silk-producing arthropods.

Aim: This study aims to understand which Candida orthopsilosis protein aids fungus adaptation upon its switching from planktonic growth to biofilm. Materials & methods: Ion mobility separation within mass spectrometry analysis combination were used. Results: Proteins mapped for different biosynthetic pathways showed that selective ribosome autophagy might occur in biofilms. Glucose, used as a carbon source in the glycolytic flux, changed to glycogen and trehalose. Conclusion: Candida orthopsilosis expresses proteins that combine a variety of mechanisms to provide yeasts with the means to adjust the catalytic properties of enzymes. Adjustment of the enzymes helps modulate the biosynthesis/degradation rates of the available nutrients, in order to control and coordinate the metabolic pathways that enable cells to express an adequate response to nutrient availability.

Made available in DSpace on 2018-11-26T16:56:30Z (GMT). No. of bitstreams: 0 Previous issue date: 2016-10-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Gert Lubec Proteomics Laboratory at the University of Vienna Major ampullate spidroin-2 (MaSp2) is one of the most important spider silk protein, but up to now no information is available regarding the post-translational modifications (PTMs) of this protein. A gel-based mass spectrometry strategy using collision-induced dissociation (CID) and electron-transfer dissociation (ETD) fragmentation methods was used to sequence Nephila clavipes MaSp2 (including the N- and C-terminal non repetitive domains, and the great part of the central core), and to assign a series of post-translational modifications (PTMs) on to the MaSp2 sequence. Two forms of this protein were identified, with different levels of phosphorylation along their sequences. These findings provide a basis for understanding mechanoelastic properties and can support the future design of recombinant spider silk proteins for biotechnological applications. (C) 2016 Elsevier B.V. All rights reserved. Sao Paulo State Univ, Inst Biosci Rio Claro, Dept Biol, Ctr Study Social Insects, BR-13500 Rio Claro, SP, Brazil Univ Vienna, Dept Pharmaceut Chem, Althanstr 14, A-1090 Vienna, Austria Sao Paulo State Univ, Inst Biosci Rio Claro, Dept Biol, Ctr Study Social Insects, BR-13500 Rio Claro, SP, Brazil FAPESP: 2010/19051-6 FAPESP: 2011/51684-1 FAPESP: 2013/26451-9

Endostatin (ES) is an antiangiogenic protein that exhibits antitumor activity in animal models. However, the activity observed in animals was not observed in human clinical trials. ES-BAX is a fusion protein composed of two functional domains: ES, which presents specificity and is internalized by activated endothelial cells and the proapoptotic BH3 domain of the protein BAX, a peptide inductor of cellular death when internalized. We have previously shown (Chura-Chambi et al., Cell Death Dis, 5, e1371, 2014) that ES-BAX presents improved antitumor activity in relation to wild-type ES. Secondary and tertiary structures of ES-BAX are similar to ES, as indicated by homology-modeling studies and molecular dynamics simulations. Tryptophan intrinsic fluorescence and circular dichroism spectroscopy corroborate these data. 15 N HSQC NMR indicates that ES-BAX is structured, but some ES residues have suffered chemical shift perturbations, suggesting that the BH3 peptide interacts with some parts of the ES protein. ES and ES-BAX present similar stability to thermal denaturation. The production of stable hybrid proteins can be a new approach to the development of therapeutic agents presenting specificity for tumoral endothelium and improved antitumor effect.

The social Hymenoptera increasingly arouse the attention of researchers due to the great economic and ecological importance to the species belonging to this order. The processes of pollination performed by these insects, the biological control, the structures of nests and colonies, the social organization, the accidents arising from their stings and the pharmacological potential of venoms synthesized by them, represent some of the features that make these individuals, targets of important studies. The sting apparatus and venom represent fundamental defense mechanisms of these insects. In order to characterize the convoluted and the venom gland of these species and gain an understanding of the relationship between the components, this study aimed to analyze through morphology and histochemical techniques the venom glands of the wasps, Polistes versicolor , Agelaia palipes palipes and Polybia paulista , and also the convoluted gland - a structure found in the reservoir, establishing the defining characteristics of these structures. In addition to the morphological features described about these structures, our results showed the presence of secretion produced by the convoluted gland cells which indicates that this structure contributes to the final composition of the venom produced and stored in the reservoir.

Made available in DSpace on 2019-10-04T12:30:55Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-10-02 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Gert Lubec Proteomics Laboratory The capture spiral of web from N. clavipes spider consists of a single type of spidroin - the flagelliform silk protein, a natural material representing a combination of strength and high elasticity. Flagelliform spider silk is the most extensible silk fibre produced by orb weaver spiders and the structure of this remarkable material is still largely unknown. In the present study we used a proteomic approach to elucidate the complete sequence and the post-translational modifications of flagelliform silk proteins. The long sequence of flagelliform silk protein presents 45 hydroxylated proline residues, which may contribute to explain the mechanoelastic property of these fibres, since they are located in the GPGGX motif. The 3D-structure of the protein was modelled considering the three domains together, i.e., the N- and C-terminal non-repetitive domains, and the central repetitive domain. In the resulting molecular model there is a predominance of random structures in the solid fibres of the silk protein. The N-terminal domain is composed of three alpha-helices and the C-terminal domain is composed of one small helical section. Proteomic data reported herein may be relevant for the development of novel approaches for the synthetic or recombinant production of novel silk-based spider polymers. Sao Paulo State Univ, Inst Biosci Rio Claro, Dept Biol, Ctr Study Social Insects, BR-13500 Rio Claro, SP, Brazil Paracelsus Med Univ, A-5020 Salzburg, Austria Sao Paulo State Univ, Inst Biosci Rio Claro, Dept Biol, Ctr Study Social Insects, BR-13500 Rio Claro, SP, Brazil FAPESP: 2010/19051-6 FAPESP: 2011/51684-1 FAPESP: 2013/26451-9 FAPESP: 2015/14220-8 FAPESP: 2016/16212-5 CNPq: 301656/2013-4 CNPq: 150699/2017-4

Aggressiveness in honeybees seems to be regulated by multiple genes, under the influence of different factors, such as polyethism of workers, environmental factors, and response to alarm pheromones, creating a series of behavioral responses. It is suspected that neuropeptides seem to be involved with the regulation of the aggressive behavior. The role of allatostatin and tachykinin-related neuropeptides in honeybee brain during the aggressive behavior is unknown, and thus worker honeybees were stimulated to attack and to sting leather targets hung in front of the colonies. The aggressive individuals were collected and immediately frozen in liquid nitrogen; the heads were removed and sliced at sagittal plan. The brain slices were submitted to MALDI spectral imaging analysis, and the results of the present study reported the processing of the precursors proteins into mature forms of the neuropeptides AmAST A (59-76) (AYTYVSEYKRLPVYNFGL-NH2), AmAST A (69-76) (LPVYNFGL-NH2), AmTRP (88-96) (APMGFQGMR-NH2), and AmTRP (254-262) (ARMGFHGMR-NH2), which apparently acted in different neuropils of the honeybee brain during the aggressive behavior, possibly taking part in the neuromodulation of different aspects of this complex behavior. These results were biologically validated by performing aggressiveness-related behavioral assays using young honeybee workers that received 1 ng of AmAST A (69-76) or AmTRP (88-96) via hemocele. The young workers that were not expected to be aggressive individuals presented a complete series of aggressive behaviors in the presence of the neuropeptides, corroborating the hypothesis that correlates the presence of mature AmASTs A and AmTRPs in the honeybee brain with the aggressiveness of this insect.

Made available in DSpace on 2018-11-26T17:52:01Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-06-15 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Among venomous animals, Hymenoptera have been suggested as a rich source of natural toxins. Due to their broad ecological diversity, venom from Hymenoptera insects (bees, wasps and ants) have evolved differentially thus widening the types and biological functions of their components. To date, insect toxinology analysis have scarcely uncovered the complex composition of bee, wasp and ant venoms which include low molecular weight compounds, highly abundant peptides and proteins, including several allergens. In Hymenoptera, these complex mixtures of toxins represent a potent arsenal of biological weapons that are used for self-defense, to repel intruders and to capture prey. Consequently, Hymenoptera venom components have a broad range of pharmacological targets and have been extensively studied, as promising sources of new drugs and biopesticides. In addition, the identification and molecular characterization of Hymenoptera venom allergens have allowed for the rational design of component-resolved diagnosis of allergy, finally improving the outcome of venom immunotherapy (VIT). Until recently, a limited number of Hymenoptera venoms had been unveiled due to the technical limitations of the approaches used to date. Nevertheless, the application of novel techniques with high dynamic range has significantly increased the number of identified peptidic and proteinaceous toxins. Considering this, the present review summarizes the current knowledge about the most representative Hymenoptera venom peptides and proteins which are under study for a better understanding of the insect-caused envenoming process and the development of new drugs and biopesticides. (C) 2018 Elsevier Ltd. All rights reserved. Sao Paulo State Univ, Biosci Inst Rio Claro, Biol Dept, Social Insect Study Ctr, BR-13500 Rio Claro, SP, Brazil Ctr Genet Engn & Biotechnol, Biomed Res Div, Syst Biol Dept, Ave 31,E-158 & 190,POB 6162, Havana 10600, Cuba Sao Paulo State Univ, Biosci Inst Rio Claro, Biol Dept, Social Insect Study Ctr, BR-13500 Rio Claro, SP, Brazil FAPESP: 2016/16212-5 CNPq: 301656/2013-4 FAPESP: 2017/22405-3 CNPq: 150699/2017-4

Made available in DSpace on 2018-12-11T17:16:09Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-01-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Molecular cross-reactivity caused by allergen homology or cross-reactive carbohydrate determinants (CCDs) is a major challenge for diagnosis and immunotherapy of insect venom allergy. Venom phospholipases A1 (PLA1s) are classical, mostly non-glycosylated wasp and ant allergens that provide diagnostic benefit for differentiation of genuine sensitizations from cross-reactivity. As CCD-free molecules, venom PLA1s are not causative for CCD-based cross-reactivity. Little is known however about the protein-based cross-reactivity of PLA1 within vespid species. Here, we address PLA1-based cross-reactivity among ten clinically relevant Hymenoptera venoms from Neotropical and temperate regions including Polybia paulista (paulistinha) venom and Vespula vulgaris (yellow jacket) venom. In order to evaluate cross-reactivity, sera of mice sensitized with recombinant PLA1 (rPoly p 1) from P. paulista wasp venom were used. Pronounced IgE and IgG based cross-reactivity was detected for wasp venoms regardless the geographical region of origin. The cross-reactivity correlated well with the identity of the primary sequence and 3-D models of PLA1 proteins. In contrast, these mice sera showed no reaction with honeybee (HBV) and fire ant venom. Furthermore, sera from patients monosensitized to HBV and fire ants did not recognize the rPoly p 1 in immunoblotting. Our findings reveal the presence of conserved epitopes in the PLA1s from several clinically relevant wasps as major cause of PLA1-based in vitro cross-reactivity. These findings emphasize the limitations but also the potential of PLA1-based HVA diagnostics. Laboratório de Biologia Molecular de Artrópodes-LBMA-IBRC-UNESP (Univ Estadual Paulista), Av. 24-A, n_1515, Bela Vista Laboratório de Imunologia Translacional Faculdade de Ciências Médicas FCM Universidade Estadual de Campinas-UNICAMP, Rua Vital Brasil, n_ 300, Cidade Universitária “Zeferino Vaz” Center for Genetic Engineering and Biotechnology Biomedical Research Division System Biology Department, Ave. 31, e/158 and 190, P.O. Box 6162, Cubanacan, Playa Centro de Estudos de Insetos Sociais-CEIS-IBRC-UNESP (Univ Estadual Paulista), Av. 24-A, n° 1515, Bela Vista Immunological Engineering Department of Engineering Aarhus University, Gustav Wieds Vej 10 Euroimmun AG, Seekamp 21 Centro de Estudos de Venenos e Animais Peçonhentos-CEVAP (Univ Estadual Paulista), Rua José Barbosa de Barros, 1780, Fazenda Experimental Lageado Laboratório de Biologia Molecular de Artrópodes-LBMA-IBRC-UNESP (Univ Estadual Paulista), Av. 24-A, n_1515, Bela Vista Centro de Estudos de Insetos Sociais-CEIS-IBRC-UNESP (Univ Estadual Paulista), Av. 24-A, n° 1515, Bela Vista Centro de Estudos de Venenos e Animais Peçonhentos-CEVAP (Univ Estadual Paulista), Rua José Barbosa de Barros, 1780, Fazenda Experimental Lageado FAPESP: 2013/26451-9

In this study, a series of mastoparan analogs were engineered based on the strategies of Ala and Lys scanning in relation to the sequences of classical mastoparans. Ten analog mastoparans, presenting from zero to six Lys residues in their sequences were synthesized and assayed for some typical biological activities for this group of peptide: mast cell degranulation, hemolysis, and antibiosis. In relation to mast cell degranulation, the apparent structural requirement to optimize this activity was the existence of one or two Lys residues at positions 8 and/or 9. In relation to hemolysis, one structural feature that strongly correlated with the potency of this activity was the number of amino acid residues from the C-terminus of each peptide continuously embedded into the zwitterionic membrane of erythrocytes-mimicking liposomes, probably due to the contribution of this structural feature to the membrane perturbation. The antibiotic activity of mastoparan analogs was directly dependent on the apparent extension of their hydrophilic surface, i.e., their molecules must have from four to six Lys residues between positions 4 and 11 of the peptide chain to achieve activities comparable to or higher than the reference antibiotic compounds. The optimization of the antibacterial activity of the mastoparans must consider Lys residues at the positions 4, 5, 7, 8, 9, and 11 of the tetradecapeptide chain, with the other positions occupied by hydrophobic residues, and with the C-terminal residue in the amidated form. These requirements resulted in highly active AMPs with greatly reduced (or no) hemolytic and mast cell degranulating activities.

Most reports about the 3-D structure of spidroin-1 have been proposed for the protein in solid state or for individual domains of these proteins. A gel-based mass spectrometry strategy using collision-induced dissociation (CID) and electron-transfer dissociation (ETD) fragmentation methods was used to completely sequence spidroins-1A and -1B and to assign a series of post-translational modifications (PTMs) on to the spidroin sequences. A total of 15 and 16 phosphorylation sites were detected on spidroin-1A and -1B, respectively. In this work, we present the nearly complete amino acid sequence of spidroin-1A and -1B, including the nonrepetitive N- and C-terminal domains and a highly repetitive central core. We also described a fatty acid layer surrounding the protein fibers and PTMs in the sequences of spidroin-1A and -1B, including phosphorylation. Thus, molecular models for phosphorylated spidroins were proposed in the presence of a mixture fatty acids/water (1:1) and submitted to molecular dynamics simulation. The resulting models presented high content of coils, a higher percentage of α-helix, and an almost neglected content of 310-helix than the previous models. Knowledge of the complete structure of spidroins-1A and -1B would help to explain the mechanical features of silk fibers. The results of the current investigation provide a foundation for biophysical studies of the mechanoelastic properties of web-silk proteins.