Comparative in vitro toxicity of a graphene oxide-silver nanocomposite and the pristine counterparts toward macrophages

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Graphene oxide (GO) is a highly oxidized graphene form with oxygen functional groups on its surface. GO is an excellent platform to support and stabilize silver nanoparticles (AgNP), which gives rise to the graphene oxide-silver nanoparticle (GOAg) nanocomposite. Understanding how this nanocomposite interacts with cells is a toxicological challenge of great importance for future biomedical applications, and macrophage cells can provide information concerning the biocompatibility of these nanomaterials. The cytotoxicity of the GOAg nanocomposite, pristine GO, and pristine AgNP was compared toward two representative murine macrophages: a tumoral lineage (J774) and peritoneal macrophages collected from Balb/c mouse. The production of reactive oxygen species (ROS) by J774 macrophages was also monitored. We investigated the internalization of nanomaterials by transmission electron microscopy (TEM). The quantification of internalized silver was carried out by inductively coupled plasma mass spectrometry (ICP-MS). Nanomaterial stability in the cell media was investigated overtime by visual observation, inductively coupled plasma optical emission spectrometry (ICP OES), and dynamic light scattering (DLS). Results: The GOAg nanocomposite was more toxic than pristine GO and pristine AgNP for both macrophages, and it significantly induced more ROS production compared to pristine AgNP. TEM analysis showed that GOAg was internalized by tumoral J774 macrophages. However, macrophages internalized approximately 60 % less GOAg than did pristine AgNP. The images also showed the degradation of nanocomposite inside cells. Conclusions: Although the GOAg nanocomposite was less internalized by the macrophage cells, it was more toxic than the pristine counterparts and induced remarkable oxidative stress. Our findings strongly reveal a synergistic toxicity effect of the GOAg nanocomposite. The toxicity and fate of nanocomposites in cells are some of the major concerns in the development of novel biocompatible materials and must be carefully evaluated.Graphene oxide (GO) is a highly oxidized graphene form with oxygen functional groups on its surface. GO is an excellent platform to support and stabilize silver nanoparticles (AgNP), which gives rise to the graphene oxide-silver nanoparticle (GOAg) nanoco14CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)140560/2014-9The authors thank the National Council for Technological and Scientific Development (CNPq) for the PhD student scholarship (140560/2014-9) and the financial support. The authors also acknowledge Dr. Daniel Ruiz Abanádes for suggestions, Renata Magueta fo

Leucine-Rich Diet Modulates the Metabolomic and Proteomic Profile of Skeletal Muscle during Cancer Cachexia

Background: Cancer-cachexia induces a variety of metabolic disorders, including skeletal muscle imbalance. Alternative therapy, as nutritional supplementation with leucine, shows a modulatory effect over tumour damage in vivo and in vitro. Method: Adult rats distributed into Control (C), Walker tumour-bearing (W), control fed a leucine-rich diet (L), and tumour-bearing fed a leucine-rich diet (WL) groups had the gastrocnemius muscle metabolomic and proteomic assays performed in parallel to in vitro assays. Results: W group presented an affected muscle metabolomic and proteomic profile mainly related to energy generation and carbohydrates catabolic processes, but leucine-supplemented group (WL) recovered the energy production. In vitro assay showed that cell proliferation, mitochondria number and oxygen consumption were higher under leucine effect than the tumour influence. Muscle proteomics results showed that the main affected cell component was mitochondria, leading to an impacted energy generation, including impairment in proteins of the tricarboxylic cycle and carbohydrates catabolic processes, which were modulated and improved by leucine treatment. Conclusion: In summary, we showed a beneficial effect of leucine upon mitochondria, providing information about the muscle glycolytic pathways used by this amino acid, where it can be associated with the preservation of morphometric parameters and consequent protection against the effects of cachexia

Butyrate Protects Mice from Clostridium difficile-Induced Colitis through an HIF-1-Dependent Mechanism

Antibiotic-induced dysbiosis is a key factor predisposing intestinal infection by Clostridium difficile. Here, we show that interventions that restore butyrate intestinal levels mitigate clinical and pathological features of C. difficile-induced colitis. Butyrate has no effect on C. difficile colonization or toxin production. However, it attenuates intestinal inflammation and improves intestinal barrier function in infected mice, as shown by reduced intestinal epithelial permeability and bacterial translocation, effects associated with the increased expression of components of intestinal epithelial cell tight junctions. Activation of the transcription factor HIF-1 in intestinal epithelial cells exerts a protective effect in C. difficile-induced colitis, and it is required for butyrate effects. We conclude that butyrate protects intestinal epithelial cells from damage caused by C. difficile toxins via the stabilization of HIF-1, mitigating local inflammatory response and systemic consequences of the infection

Morphological, biochemical and molecular evaluation of the elastogenesis in the adult tissues of the mouse pubic symphysis during and after pregnancy

Orientadores: Paulo Pinto Joazeiro, Cláudio Chrysostomo WerneckDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: A organização das fibras elásticas envolve a síntese e a deposição de moléculas em uma sequência altamente regulada para assegurar as características elásticas nos estágios iniciais do desenvolvimento. Durante e prenhez, os tecidos pélvicos ricos em fibras elásticas se alteram para permitir um parto seguro e essa remodelação é essencial para o parto normal. A sínfise púbica de camundongos também remodela em um processo controlado por hormônios. Este fenômeno compreende a "transformação" da fibrocartilagem em um ligamento interpúbico (LIp) seguido por seu relaxamento antes do parto. Após o primeiro parto, o processo de retorno ocorre e assegura a homeostase dos tecidos pélvicos. Ainda, alterações no suporte dos órgãos pélvicos foram descritas em animais geneticamente modificados para proteínas envolvidas na elastogênese como a lysyl oxidase-like 1 (LOXL1), fibulina-3 e 5. Como ligamentos são as principais estruturas de suporte dos órgãos pélvicos, o objetivo deste estudo foi avaliar a elastogênse no desenvolvimento do LIp durante a prenhez de camundongos. Assim, camundongos selvagens C57Bl/06 e deficientes em fibrilina-1 virgens, prênhes e no pós-parto foram estudados usando microscopia de luz convencional, microscopia confocal a laser, microscopia eletrônica de transmissão, western blotting e real-time PCR. Ambos os animais selvagens e deficientes em fibrilina-1 apresentaram classicamente a separação dos ossos púbicos, a formação e relaxamento do LIp e a involução deste no pós-parto. Esses processos sugeriram um padrão no qual as células controlam a remodelação da matriz extracelular sob sinalização hormonal e molecular. A ultra-estrutura dos tecidos fibrocartilaginosos apresentou delgadas microfibrilas aleatoriamente distribuídas entre os fibrocondrócitos. Na formação do LIp, foram observadas fibras elásticas com conglomerados de material amorfo distribuídos entre as microfibrilas. O LIp mostrou fibras elásticas e todos os componentes teciduais alinhados na direção da abertura da articulação interpúbica antes do parto. O estudo imuno-histoquímico e de expressão gênica relativa quantitativa indicou que durante o desenvolvimento do LIp em camundongos selvagens, elastina/tropoelastina, fibrilina-1 e 2, LOXL1, fibulina-5 e TGF-? foram regulados espacial e temporalmente, e estas moléculas poderiam contribuir para a síntese de novas fibras elásticas que asseguram a elasticidade necessária para a cintura pélvica durante o preparo para o parto e também no fechamento da articulação no pós-parto. Entretanto, se comparados com o animal selvagem, a análise indicou alteração na expressão gênica relativa da tropoelastina, fibrilina-1, LOXL1, fibulina-5 e TGF-?, diferentemente da morfologia muito similar observada em camundongos selvagens. Neste estudo, o camundongo deficiente em fibrilina-1 não apresentou prolapso de órgãos pélvicos após o primeiro parto como o deficiente em LOXL1 (Liu et al., 2004), nem modificações morfológicas que poderiam ser relacionadas ao enfraquecimento dos tecidos pélvicos. No entanto, este é o primeiro estudo que relata disfunções pélvicas nos camundongos deficientes em fibrilina-1 multíparos, usados como matrizes reprodutivas. Em conclusão, a formação das fibras elásticas que ocorreu na sínfise púbica de camundongos durante a vida adulta possui características únicas de um modelo que pode ser usado para compreensão dos processos normais e patológicos, principalmente aqueles relacionados aos animais geneticamente modificados para proteínas envolvidas na elastogênese. Assim, este trabalho traz à luz as evidências das profundas modificações que a sínfise púbica de camundongos passa durante a prenhez com a síntese de novas fibras elásticas, o que pode contribuir na compreensão dos mecanismos biológicos para formação das fibras elásticasAbstract: The organization of elastic fibers involves the synthesis and the deposition of molecules in a high regulated sequence to ensure the elastic characteristics in the early stages of development. During pregnancy, elastic fibers-enriched pelvic tissues change to allow safe delivery and this remodeling is essential to the vaginal delivery. The mouse pubic symphysis articulation also remodels in a controlled hormonal process. This phenomenon comprises the "transformation" of the fibrocartilage into an interpubic ligament (IpL) followed by its relaxation before parturition. After the first parturition, recovery process occurs to ensure the pelvic tissue homeostasis. Adding to that, pelvic organ support impairment had been described in genetically modified mouse for the proteins involved in the elastogenesis such as lysyl oxidase-like 1 (LOXL-1), fibulin-3 and -5. Since, ligaments are the main supportive structures of pelvic organs, the aim of this study was to evaluate the elastogenesis in the IpL development during mouse pregnancy. Thus virgin, pregnant and postpartum C57Bl/06 wild-type and fibrillin-1mg?/+ female mice were studied using light, confocal, transmission electron microscopy, western blotting and real-time PCR. Both, wild-type and fibrillin-1mg?/+ female mice showed classically the separation of the pubic bones, the formation and relaxation of the IpL and the recovery at postpartum. These processes suggested a pattern which cells control the extracellular matrix remodeling under hormonal and molecular signaling. The ultra-structure of the fibrocartilaginous tissue had slender bundles of microfibrils randomly distributed among the fibrochondrocytes. By the time IpL is formed, there were seen elastic fibers, which consist of small conglomerates of amorphous material, distributed among the bundles of microfibrils. The IpL showed elastic fibers and all tissue compounds aligned to the opening axis of the articulation before parturition. The immunohistochemical study and quantitative gene expression indicated that during IpL development in wild-type mice, tropoelastin/elastin, fibrillin-1, fibrillin-2, LOXL-1, fibulin-5 and TGF-? were spatial and temporal regulated, and these molecules might contribute to the synthesis of new elastic fibers that assure the elasticity that is needed to the pelvic girdle during preparation for parturition and also the recovery at postpartum. However, compared to wild-type mice, alterations were found in the quantitative gene expression of elastin, fibrillin-1, LOXL-1, fibulin-5 and TGF- ?, different from the morphology that was very similar to the one that was observed in wild-type mice. In this study, the fibrillinmg?/+ mice did not show pelvic organ prolapse after the first parturition as LOXL1-/- did (Liu et al., 2004), neither morphological modifications that could be related to the weakness of pelvic tissue. However, this is the first work about pelvic dysfunctions in multiparous fibrillin-1mg?/+ mice used as reproductive matrices. In conclusion, the elastic fiber assembly that occurred in the mouse pubic symphysis during the adult life has characteristics of a model that could be used to understand normal and pathological processes, mainly those related to genetically modified mice for the proteins involved in the elastogenesis. Then, this work may bring readers up-to-date with accumulating evidence that the mouse pubic symphysis undergoes remarkable modifications during pregnancy with new synthesized elastic fibers and may contribute to our understanding of the biological mechanisms about elastic fiber assemblyMestradoHistologiaMestre em Biologia Celular e Estrutura

Localization and function of focal adhesion kinase and Calsarcin-1 in rat cardiomyocytes : using of molecular probes and lentivirus

Orientador: Kleber Gomes FranchiniTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: Há um crescente avanço no desenvolvimento das tecnologias que permitam a localização de proteínas em células por microscopias de luz e eletrônica combinadas, com o uso das sondas moleculares miniSOG e Apex2, por exemplo. Adicionalmente busca-se compreender como proteínas são responsáveis pelas funções celulares e teciduais. A Quinase de Adesão Focal (FAK), proteína da cascata de sinalização das integrinas é considerada uma mediadora em potencial do estresse mecânico nos cardiomiócitos. Sabe-se que em cardiomiócitos submetidos a estímulos hipertróficos, ocorre rápida ativação da FAK e sua redistribuição subcelular, contudo são pouco conhecidos os mecanismos envolvidos nesses processos. Outra proteína de grande importância no coração é a Calsarcina-1 (CS1), regulador negativo da via de Calcineurina, crucial no desenvolvimento da hipertrofia cardíaca. Entretanto os mecanismos envolvidos nessa regulação negativa, assim como a distribuição subcelular de CS1 são pouco conhecidos. Visando à interação entre essas áreas para estudo da distribuição espaço-temporal dos componentes celulares e de proteínas, bem como a importância da FAK e CS1 no sarcômero e seu papel na sinalização hipertrófica sob estímulo mecânico, o objetivo geral desse trabalho foi explorar a capacidade das proteínas FAK e CS1 para incorporar geneticamente sondas moleculares que permitissem monitorar por meio de imagens o comportamento dessas moléculas-chave na biologia do disco Z em MVRNs submetida ao estiramento mecânico. Para isso, foram realizados ensaios de localização subcelular com uso de sondas moleculares aplicadas à microscopia correlativa, bem como ensaios bioquímicos, moleculares e de atividade enzimática. Os dados confirmaram a FAK associada às fibras de actina e adesões focais em células H9c2 e foi demonstrado à microscopia de luz que FAK wild-type (wt) translocou-se parcialmente para o compartimento nuclear após estimulação do agonista fenilefrina, enquanto que FAK Y397F (forma mutante inativa) não apresentou mesmo fenótipo. Por outro lado, apesar da padronização e expressão das construções com FAK e miniSOG ou Apex2 em células HEK 293T e H9c2, não foi conclusiva a localização subcelular da FAK, por meio do uso de microscopia eletrônica em MVRN. Provavelmente devido à distribuição difusa da maior parte das moléculas de FAK, não foi identificada uma região elétron-densa conclusiva à microscopia eletrônica de transmissão. No tocante à importância da CS1, observou-se que o estiramento cíclico não induziu o aumento na expressão proteica ou gênica relativa de CS1 e CnA, assim como não houve alteração na atividade fosfatase de CnA. No entanto houve redução da interação de CS1 e CnA, bem como alteração na localização de CS1 em MVRN sob estímulo mecânico. Dados de superexpressão e silenciamento de CS1 corroboram a regulação negativa de CS1 à CnA em MVRN sob estímulo mecânico. Baseando-se em dados estruturais, especulou-se que como o sítio de ligação de NFAT e CS1 à CnA são muito próximos e ao mesmo tempo distante do sítio ativo da fosfatase, é possível que o papel de CS1 na regulação negativa de CnA ocorra por impedimento espacial ao fator de transcrição NFAT. Portanto, esses resultados podem contribuir para uma possível inferência farmacológica, visto que a via de Calcineurina-NFAT é uma das principais mediadoras de hipertrofia em cardiomiócitos, mediante estímulos patológicosAbstract: There is an increasing move towards the development of technologies that allow the localization of proteins in cells by combined electron and light microscopy, with the use of molecular probes such as miniSOG and APEX2. Additionally we seek to understand how proteins are responsible for the cellular and tissue functions. The Focal Adhesion Kinase (FAK) is protein of integrin signaling cascade considered as a potential mediator of mechanical stress in cardiomyocytes. It is known that in cardiomyocytes subjected to hypertrophic stimuli by rapid activation of FAK and its subcellular redistribution, however the mechanisms involved in these processes are poorly understood. Another very important protein in the heart is Calsarcin-1 (CS1), a negative regulator of the Calcineurin pathway which is crucial in the development of cardiac hypertrophy. However the mechanisms involved in the negative regulation as well as the subcellular distribution CS1 are poorly understood. Aiming at the interaction between these areas to study the spatial-temporal distribution of cellular and protein components, and the importance of FAK and CS1 in the sarcomere and its role in hypertrophic signaling under mechanical stimulation, the aim of this study was to explore the ability of FAK and CS1 to incorporate genetically molecular probes that allow monitoring through images the behavior of these key molecules in the Z disc biology in MVRNs subjected to mechanical stretch. To this end, we performed subcellular localization assays using molecular probes applied to the correlative microscopy, biochemical and molecular assays and enzymatic activity. These data confirm the FAK associated with actin and focal adhesions fibers in H9c2 cells and has been shown by light microscopy that FAK wild-type (wt) is partially translocated to the nuclear compartment after stimulation of the agonist phenylephrine, while FAK Y397F (inactive mutant form) did not show the same phenotype. Moreover, despite standardization and expression of FAK and miniSOG or APEX2 in HEK 293T cells and H9c2, it was inconclusive subcellular localization of FAK, through the use of electron microscopy, in MVRN. Probably due to the diffuse distribution of most FAK molecules, it has no conclusive electron-dense region in transmission electron microscopy. Regarding the importance of CS1, it was observed that the cyclic stretch did not induce an increase in protein expression or gene relative CS1 and CnA, as there was no change in the phosphatase activity of CnA. However there was less interaction CS1 and CnA and change in CS1 location in MVRN under mechanical stimulation. CS1 overexpression and silencing corroborate the negative regulation of the CS1 over CnA in MVRN under mechanical stimulation. Based on structural data, it has been speculated that as the NFAT and CS1 binding sites are very close in CnA and at the same time distant from the active site of the phosphatase, it is possible that the role of CS1 in the negative regulation of CnA occur by steric hindrance to the NFAT transcription factor. Therefore, these results may contribute to a possible pharmacological inference, whereas Calcineurin-NFAT pathway is a major mediator of hypertrophy in cardiac myocytes by pathologic stimuliDoutoradoBiologia TecidualDoutor em Biologia Celular e Estrutura

Colorectal Cancer Cell-Derived Small Extracellular Vesicles Educate Human Fibroblasts to Stimulate Migratory Capacity

Colorectal cancer (CRC) is in the top 10 cancers most prevalent worldwide, affecting equally men and women. Current research on tumor-derived extracellular vesicles (EVs) suggests that these small extracellular vesicles (sEVs) play an important role in mediating cell-to-cell communication and thus potentially affecting cancer progression via multiple pathways. In the present study, we hypothesized that sEVs derived from different CRC cell lines differ in their ability to reprogram normal human fibroblasts through a process called tumor education. The sEVs derived from CRC cell lines (HT29 and HCT116) were isolated by a combination of ultrafiltration and polymeric precipitation, followed by characterization based on morphology, size, and the presence or absence of EV and non-EV markers.

Histomorphometric evaluation of bone-guided regeneration in maxillary sinus floor augmentation using nano-hydroxyapatite/beta-tricalcium phosphate composite biomaterial: a case report

Background: The development of techniques in biomaterials design and production added to advanced surgical procedures which enabled better and more predictable clinical out-comes. Maxillary sinus floor augmentation (MSFA) is among the more studied bone-guided regeneration procedure in the literature. The MSFA could be considered the gold standard procedure for bone-guided regeneration as it provides suitable functional and aesthetic solutions to alveolar ridge atrophy due to tooth loss. Purpose: This study aimed to conduct a detailed histomorphometric evaluation of collagen production in SFAs bone-guided regeneration, using nano-hydroxyapatite/beta-tricalcium phos-phate (nano-HA/beta-TCP) composite. Patients and Methods: A 52-year-old female had the left upper second premolar con-demned due to periodontal disease, then a tooth implant replacement was planned. Due to maxillary sinus pneumatization, the MSFA had to be done before implant placement. Nano-HA/beta-TCP composite (2g) was used in the MSFA procedure. After nine months of the healing process, during the Cone Morse implant installation process, bone samples were collected for histologic analysis (sirius red, hematoxylin/eosin, polarized microscopy). Six months after implant installation, a ceramic crown was installed according to the patient's request. Results: Proper masticatory function and aesthetics were re-established. The histomorpho-metric evaluation indicated that nano-HA/beta-TCP composite did not show any area devoid of cellular activity in sirius red or hematoxylin/eosin staining and the percentage (%) of new bone collagen fibers was achieved using polarization technique evaluation. Conclusion: According to these results, nano-HA/beta-TCP composite presented clinical and histomorphometric properties suit to be used as bone-guided regeneration biomaterial in MSFA. Furthermore, nano-HA/beta-TCP composite provided a favorable nano-environment to bone cells, enhancing bone matrix production.info:eu-repo/semantics/publishedVersio