Chloride intracellular channel 3: a secreted pro-invasive oxidoreductase

No abstract available

Tumor stiffness extends its grip on the metastatic microenvironment

The increased stiffness of a tumor triggers a multitude of responses that aid cancer cell dissemination. Stiffness-induced expression of CCN1 mediates autocrine signaling in the endothelium to upregulate N-Cadherin levels. This permits more stable interactions with cancer cells and increases their ability to spread into the circulation

Proteome-wide analysis of cysteine oxidation reveals metabolic sensitivity to redox stress

Reactive oxygen species (ROS) are increasingly recognised as important signalling molecules through oxidation of protein cysteine residues. Comprehensive identification of redox-regulated proteins and pathways is crucial to understand ROS-mediated events. Here, we present stable isotope cysteine labelling with iodoacetamide (SICyLIA), a mass spectrometry-based workflow to assess proteome-scale cysteine oxidation. SICyLIA does not require enrichment steps and achieves unbiased proteome-wide sensitivity. Applying SICyLIA to diverse cellular models and primary tissues provides detailed insights into thiol oxidation proteomes. Our results demonstrate that acute and chronic oxidative stress causes oxidation of distinct metabolic proteins, indicating that cysteine oxidation plays a key role in the metabolic adaptation to redox stress. Analysis of mouse kidneys identifies oxidation of proteins circulating in biofluids, through which cellular redox stress can affect whole-body physiology. Obtaining accurate peptide oxidation profiles from complex organs using SICyLIA holds promise for future analysis of patient-derived samples to study human pathologies

Mass spectrometry-based methods for identifying oxidized proteins in disease:advances and challenges

Many inflammatory diseases have an oxidative aetiology, which leads to oxidative damage to biomolecules, including proteins. It is now increasingly recognized that oxidative post-translational modifications (oxPTMs) of proteins affect cell signalling and behaviour, and can contribute to pathology. Moreover, oxidized proteins have potential as biomarkers for inflammatory diseases. Although many assays for generic protein oxidation and breakdown products of protein oxidation are available, only advanced tandem mass spectrometry approaches have the power to localize specific oxPTMs in identified proteins. While much work has been carried out using untargeted or discovery mass spectrometry approaches, identification of oxPTMs in disease has benefitted from the development of sophisticated targeted or semi-targeted scanning routines, combined with chemical labeling and enrichment approaches. Nevertheless, many potential pitfalls exist which can result in incorrect identifications. This review explains the limitations, advantages and challenges of all of these approaches to detecting oxidatively modified proteins, and provides an update on recent literature in which they have been used to detect and quantify protein oxidation in disease

In-depth proteomics identifies a role for autophagy in controlling reactive oxygen species mediated endothelial permeability

Endothelial cells (ECs) form the inner layer of blood vessels and physically separate the blood from the surrounding tissue. To support tissues with nutrients and oxygen, the endothelial monolayer is semipermeable. When EC permeability is altered, blood vessels are not functional, and this is associated with disease. A comprehensive knowledge of the mechanisms regulating EC permeability is key in developing strategies to target this mechanism in pathologies. Here we have used an in vitro model of human umbilical vein endothelial cells mimicking the formation of a physiologically permeable vessel and performed time-resolved in-depth molecular profiling using stable isotope labeling by amino acids in cell culture mass spectrometry (MS)-proteomics. Autophagy is induced when ECs are assembled into a physiologically permeable monolayer. By using siRNA and drug treatment to block autophagy in combination with functional assays and MS proteomics, we show that ECs require autophagy flux to maintain intracellular reactive oxygen species levels, and this is required to maintain the physiological permeability of the cells

Robô Solucionador de Sudoku

TCC (graduação) - Universidade Federal de Santa Catarina. Campus Araranguá. Engenharia da Computação.Os avanços tecnológicos dos dispositivos de aquisição de imagens, aliados ao crescente aprimoramento do hardware computacional, estão impulsionando o desenvolvimento dos sistemas de visão de máquina. Estes sistemas são atualmente empregados para solucionar problemas de naturezas diversas, em áreas como medicina, biologia e, em especial, na indústria. Este trabalho apresenta o projeto de um robô, construído exclusivamente com peças de LEGO, capaz de solucionar jogos de Sudoku escritos em papel. Trata-se de um plotador cartesiano controlado por um sistema de visão de máquina. O projeto visa introduzir princípios, modelos e aplicações dos sistemas de visão. O processo executado pelo robô inicia-se com a captura de uma imagem do seu espaço de trabalho, utilizando-se de uma webcam. A imagem capturada é então analisada pelo sistema de visão, que identifica o jogo, interpreta-o e o soluciona em memória. Finalmente, a solução é escrita pelo robô por meio de uma caneta acoplada ao seu elemento terminal.Technological advances in image acquisition devices, combined with the increasingly improvements in computer hardware, are boosting the development of machine vision systems. These systems are currently employed to solve a lot of problems, in areas such as medicine, biology and particularly in the industry. This paper presents the design of a Sudoku solver robot that was built exclusively with LEGO bricks. It consists of a plotter printer, controlled by a machine vision system. The project aims to introduce principles, models and applications of vision systems. The process executed by the robot starts capturing an image of its workspace with a webcam. The captured image is then analyzed by the vision system, which identifies the game, interprets and solves it. Finally, the solution is written by the robot with a pen that is attached to its terminal element

Avaliação da corrente de soldagem TIG empregada na montagem de conectores em dutos flexíveis

Devido à exploração de petróleo em águas cada vez mais profundas, foi necessário desenvolver novas tecnologias, dentre elas estão inseridos os dutos flexíveis e conectores. Os dutos flexíveis são estruturas submarinas que fazem o transporte de óleo entre o poço de produção e a plataforma petrolífera, enquanto que os conectores são montados nas extremidades dos dutos flexíveis e tem a função de garantir o estanque e conexão entre dutos e plataforma ou poço. Dentro da montagem de conectores existe uma etapa de solda para ancoragem da Armadura de Pressão que é uma das camadas do duto flexível, está etapa é crítica, pois abaixo desta camada existe uma camada polimérica que pode ser danificada caso o calor gerado durante a soldagem seja excessivo. Corpos de prova foram soldados através do processo Tungsten Inert Gás (TIG) em uma Armadura de Pressão, o processo ocorreu com variações de corrente de soldagem (60, 70 e 80 A), para posteriormente avaliar a qualidade das soldas feitas nestes intervalos. Essa avaliação consistiu em realizar ensaios de temperatura de soldagem na face oposta da solda (face de contato com polímeros), inspeção visual das amostras, inspeção pelo método de Partícula Magnéticas, Macrografia e Metalografia da seção transversal dos cordões de solda e microdureza. Foi observado que apresentaram mais defeitos as amostras soldadas com corrente de soldagem de 60 A, enquanto que melhor corrente de soldagem foi a de 70 A, pois apresentou maior qualidade dos cordões de solda, cordões livres de defeitos e menor temperatura na face oposta da solda.Due to the exploration of oil in increasingly deeper waters, it was necessary to develop new technologies, including flexible pipes and End Fittings. Flexible Pipes are underwater structures that transport oil between the production well and the oil platform, while the End Fittings are assembly at the ends of the flexible pipes and have the function of guaranteeing the watertight connection between the pipes and the platform or well. Inside the assembly of connectors there is a welding step for anchoring the Pressure Armor, which is one of the layers of the flexible pipe, this step is critical, because below this layer there is a polymeric layer that can be damaged if the heat generated in the welding is excessive. Specimens were welded using the Tungsten Inert Gas (TIG) process in a Pressure Armor, the process occurred with variations in welding current (60, 70 and 80 A), to later assess the quality of the welds made in these intervals. This evaluation consisted of performing welding temperature tests on the opposite face of the weld (contact face with polymers), visual inspection of samples, inspection by the Magnetic Particle method, Macrography and Metallography of the cross section of the welds and microhardness. It was observed that the samples welded with a welding current of 60 A had more defects, while the best welding current was that of 70 A, as it presented higher quality of the weld beads, bead free of defects and lower temperature on the opposite side of the weld

The CDC42 effector protein MRCKß autophosphorylates on Threonine 1108

The CDC42 small GTPase is a major influence on actin-myosin cytoskeleton organization and dynamics, signalling via effector proteins including the Myotonic dystrophy related CDC42-binding protein kinases (MRCK) α and β. We previously identified Serine 1003 of MRCKα as a site of autophosphorylation, and showed that a phosphorylation-sensitive antibody raised against this site could be used as a surrogate indicator of kinase activity. In this study, a kinase-dead version of MRCKβ was established by mutation of the conserved Lysine 105 to Methionine (K105M), which was then used for mass spectrometry analysis to identify phosphorylation events that occurred in catalytically-competent MRCKβ but not in the kinase-dead form. A total of ten phosphorylations were identified on wild-type MRCKβ, of which the previously undescribed Threonine 1108 (Thr1108) was not found on kinase-dead MRCKβ K105M, consistent with this being due to autophosphorylation. Mutation of Thr1108 to non-phosphorylatable Alanine (T1108A) or phosphomimetic Glutamate (T1108E) did not affect the ability of MRCKβ to phosphorylate recombinant myosin light chain in vitro, or observably alter the subcellular localization of green fluorescent protein (GFP)-tagged MRCKβ expressed in MDA MB 231 human breast cancer cells. Although phosphorylation of Thr1108 did not appear to contribute to MRCKβ function or regulation, the identification of this phosphorylation does make it possible to characterize whether this site could be used as a surrogate biomarker of kinase activity and inhibitor efficacy as we previously demonstrated for Ser 1003 in MRCKα

A proteomic approach to identify endosomal cargoes controlling cancer invasiveness

We have previously shown that Rab17 - a small GTPase associated with epithelial polarity - is specifically suppressed by ERK2 signalling to promote an invasive phenotype. However, the mechanisms through which Rab17 loss permits invasiveness, and the endosomal cargoes that are responsible for mediating this are not known. Using quantitative mass spectrometry-based proteomics, we have found that knockdown of Rab17 leads to highly selective reduction in the cellular levels of a v-SNARE (Vamp8). Moreover, proteomics and immunofluorescence indicate that Vamp-8 is associated with Rab17 at late endosomes. Reduced levels of Vamp8 promote transition between ductal carcinoma in situ (DCIS) and a more invasive phenotype. We developed an unbiased proteomic approach to elucidate the complement of receptors that redistributes between endosomes and the plasma membrane, and have pin-pointed neuropilin-2 (NRP2) as a key pro-invasive cargo of Rab17/Vamp8-regulated trafficking. Indeed, reduced Rab17 or Vamp8 levels lead to increased mobilisation of NRP2-containing late endosomes and upregulated cell surface expression of NRP2. Finally, we show that NRP2 is required for the basement membrane disruption which accompanies transition between DCIS and a more invasive phenotype

Re-design de munição: equipamento não letal para uso expresso de forças policiais em emprego individual

Visando facilitar o trabalho dos agentes da Polícia Militar, o GP5-AR foi criado com o intuito de servir e agir essencialmente como equipamento/armamento não letal, sendo empregado em casos específicos de modo individual. O processo metodológico envolveu a interação com o público alvo, coletar dados, conhecer os equipamentos e armamentos, além do mercado e seguir e aplicar a metodologia projetual passo a passo de Bernd Löbach (2001), a qual é voltada para a escala industrial, contendo fases de: preparação; geração; avaliação e realização, as quais foram realizadas conforme instruções do autor. Ao decorrer do projeto foram analisadas as alternativas criadas e dentre elas somente uma foi escolhida, a qual recebeu melhorias, desenvolvimento de imagens em 3D e protótipo em escala real. O equipamento foi criado visando sempre a segurança, facilidade e eficiência para o agente aplicador, de modo que tenha mais êxito em suas ocorrências, evitando o contato aproximado do indivíduo