The cellular thermal shift assay (CETSA) is a biophysical technique allowing direct studies of ligand binding to proteins in cells and tissues. The proteome-wide implementation of CETSA with mass spectrometry detection (MS-CETSA) has now been successfully applied to discover targets for orphan clinical drugs and hits from phenotypic screens, to identify off-targets, and to explain poly-pharmacology and drug toxicity. Highly sensitive multidimensional MS-CETSA implementations can now also access binding of physiological ligands to proteins, such as metabolites, nucleic acids, and other proteins. MS-CETSA can thereby provide comprehensive information on modulations of protein interaction states in cellular processes, including downstream effects of drugs and transitions between different physiological cell states. Such horizontal information on ligandmodulation in cells is largely orthogonal to vertical information on the levels of different proteins and therefore opens novel opportunities to understand operational aspects of cellular proteomes.

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Considerable efforts are currently focused on the biology of DC in view of their possible clinical use as adjuvant for the generation of antigen-specific immunity and lifelong immunologic memory or for the treatment of tumors. We assessed the role of Nattectin a C-type lectin identified in the Thalassophryne nattereri fish venom in DC maturation. Nattectin induced a significant neutrophilic recruitment into peritoneal cavity of mice, followed by macrophages, with lipidic mediators and IL-12 p70 synthesis. Macrophages derived from 7day-Nattectin mice were CD11c+CD11b(low)Ly6(high)F4/80R(high) and express high levels of MHC class II and CD80 molecules. Culture of peritoneal exudates derived macrophages from 7day Nattectin-mice and immature BMDCs with Nattectin markedly increased the surface expression of CD40, CD80, CD86, and MHC class II in a dose-dependent manner, and the production of MMP-2 and MMP-9 distributed in nucleus and cytoplasm of cells, that was associated with strong activity in the culture supernatant. Nattectin treated DCs secreted IL-12 p70 and IL-10. The Nattectin-treated BMDC or macrophage-derived DCs were highly efficient at Ag capture. The specific immune response elicited by Nattectin was characterized by the production of specific antibodies IgG1 and mainly IgG2a with IL-10 and IFN-γ synthesis by splenic cells. These results enable us to address that Nattectin induces the recruitment of Ly6C(high) monocytes into the peritoneum, which exhibit a pro-inflammatory profile, where they differentiate into proliferating F4/80R(high) macrophages. Macrophage-derived DCs mature in the presence of the cytokine milieu generated against Nattectin, exhibiting T cell co-stimulatory molecule expression and induced a Th1 polarized response.

The use of taxanes has for decades been crucial for treatment of several cancers. A major limitation of these therapies is inherent or acquired drug resistance. A key to improved outcome of taxane-based therapies is to develop tools to predict and monitor drug efficacy and resistance in the clinical setting allowing for treatment and dose stratification for individual patients. To assess treatment efficacy up to the level of drug target engagement, we have established several formats of tubulin-specific Cellular Thermal Shift Assays (CETSAs). This technique was evaluated in breast and prostate cancer models and in a cohort of breast cancer patients. Here we show that taxanes induce significant CETSA shifts in cell lines as well as in animal models including patient-derived xenograft (PDX) models. Furthermore, isothermal dose response CETSA measurements allowed for drugs to be rapidly ranked according to their reported potency. Using multidrug resistant cancer cell lines and taxane-resistant PDX models we demonstrate that CETSA can identify taxane resistance up to the level of target engagement. An imaging-based CETSA format was also established, which in principle allows for taxane target engagement to be accessed in specific cell types in complex cell mixtures. Using a highly sensitive implementation of CETSA, we measured target engagement in fine needle aspirates from breast cancer patients, revealing a range of different sensitivities. Together, our data support that CETSA is a robust tool for assessing taxane target engagement in preclinical models and clinical material and therefore should be evaluated as a prognostic tool during taxane-based therapies.

Cathorops spixii is the most common venomous fish on the Brazilian coast. Apart from the involvement with defense against pathogens, the possible contribution of skin mucus components to the development of injuries caused by venomous fish species has not been investigated. Thus, the present study was conducted to gain a better understanding of the peptide and protein components of fish skin mucus and the sting venom from the catfish C. spixii. Our results show that sting venom and skin mucus have distinct constituents that distinguished them like structural proteins, chaperones, ion transport, carbohydrate metabolism, oxidoreductase, cell cycle and protein binding present in sting venom and like tropomyosin 3 isoform 2 and energy metabolim proteins in skin mucus. But in a group of common 13 proteins we identified and isolated a WAP65 protein. The peptide fractions caused more harmful effects, such as venular stasis, hemorrhage and changes in the arteriolar wall diameter, and the protein fractions produced a typical inflammatory process in post-capillary venules. And finally we showed for the first time the presence WAP65 in sting venom and skin mucus of C. spixii using LC/MS/MS and also we purified this protein in the sting venom. Wap65 shows inflammatory action, working at different doses inducing an increase in the number of leukocytes rolling and adhering to the endothelium.

BackgroundTemperature, humidity, vision, and particularly odor, are external cues that play essential roles to mosquito blood feeding and oviposition. Entomological and behavioral studies employ well-established methods to evaluate mosquito attraction or repellency and to identify the source of the blood meal. Despite the efficacy of such methods, the costs involved in the production or acquisition of all parts, components and the chemical reagents involved are unaffordable for most researchers from poor countries. Thus, a simple and relatively low-cost method capable of evaluating mosquito preferences and the blood volume ingested is desirable.Principal FindingsBy using Evans blue (EB) vital dye and few standard laboratory supplies, we developed and validated a system capable of evaluating mosquito’s choice between two different host sources of blood. EB-injected and PBS-injected mice submitted to a number of situations were placed side by side on the top of a rounded recipient covered with tulle fabric and containing Aedes aegypti mosquitoes. Homogenates from engorged mosquitoes clearly revealed the blood source (EB- or PBS-injected host), either visually or spectrometrically. This method was able to estimate the number of engorded mosquitoes, the volume of blood ingested, the efficacy of a commercial repellent and the attractant effects of black color and human sweat.SignificanceDespite the obvious limitations due to its simplicity and to the dependence of a live source of blood, the present method can be used to assess a number of host variables (diet, aging, immunity, etc) and optimized for several aspects of mosquito blood feeding and vector-host interactions. Thus, it is proposed as an alternative to field studies, and it could be used for initial screenings of chemical compound candidates for repellents or attractants, since it replicates natural conditions of exposure to mosquitoes in a laboratory environment.

Classical studies have shown that Aedes aegypti salivary secretion is responsible for the sensitization to mosquito bites and many of the components present in saliva are immunogenic and capable of inducing an intense immune response. Therefore, we have characterized a murine model of adjuvant-free systemic allergy induced by natural exposure to mosquito bites. BALB/c mice were sensitized by exposure to A. aegypti mosquito bites and intranasally challenged with phosphate-buffered saline only or the mosquito’s salivary gland extract (SGE). Blood, bronchoalveolar lavage (BAL) and lung were collected and evaluated for cellularity, histopathological analyses, cytokines and antibody determination. Respiratory pattern was analyzed by Penh measurements and tracheal segments were obtained to study in vitro reactivity to methacholine. BAL recovered from sensitized mice following challenge with SGE showed an increased number of eosinophils and Th2 cytokines such as IL-4, IL-5 and IL-13. Peribronchoalveolar eosinophil infiltration, mucus and collagen were also observed in lung parenchyma of sensitized mice, suggesting the development of a typical Th2 response. However, the antibody profile in serum of these mice evidenced a mixed-type response with presence of both, IgG1/IgE (Th2-related) and IgG2a (Th1-related) isotypes. In addition, changes in breathing pattern and tracheal reactivity to methacholine were not found. Taken together, our results show that A. aegypti bites trigger an atypical allergic reaction, with some classical cellular and soluble Th2 components in the lung, but also systemic Th1 and Th2 antibody isotypes and no change in either the respiratory pattern or the trachea responsiveness to agonist.

Combined proteomic and transcriptomic approaches to study the composition of the venom of Thalassophryne nattereri venomous fish revealed the primary structures of the major toxins as a family of proteases natterins, never described on venoms and a C-type lectin nattectin. To gain new insights into the mechanisms of venom pathogenesis and to further elucidate the role of its major toxins, the natterins and nattectin, we undertook in vitro investigations using these isolated toxins. Here we demonstrated the specific ability of the nattectin to bind types I and V collagen and natterins to bind and cleave type I collagen as well as type IV collagen, disrupting cell attachment and HeLa cells survival. Natterins have cytotoxic effect on both adherent cells or at in suspension, showing direct induction of necrosis that is followed by cell detachment. Nattectin improves integrin-mediated HeLa cell adhesion and resistance to apoptosis by its binding to RGD-dependent integrins, especially the β1 subunit. Based on our studies we now report that extracellular matrix (ECM) components as well as the integrin β1 subunit are targets for the natterins and nattectin. The ECM degradation or remodeling activities exerted by these toxins affect cell-cell and cell-ECM adhesion and survival and impair inflammatory cell migration into inflamed tissues.