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ABSTRACT The CONSORT 2010 statement provides minimum guidelines for reporting randomized trials. Its widespread use has been instrumental in ensuring transparency in the evaluation of new interventions. More recently, there has been a growing recognition that interventions involving artificial intelligence (AI) need to undergo rigorous, prospective evaluation to demonstrate impact on health outcomes. The CONSORT-AI (Consolidated Standards of Reporting Trials-Artificial Intelligence) extension is a new reporting guideline for clinical trials evaluating interventions with an AI component. It was developed in parallel with its companion statement for clinical trial protocols: SPIRIT-AI (Standard Protocol Items: Recommendations for Interventional Trials-Artificial Intelligence). Both guidelines were developed through a staged consensus process involving literature review and expert consultation to generate 29 candidate items, which were assessed by an international multi-stakeholder group in a two-stage Delphi survey (103 stakeholders), agreed upon in a two-day consensus meeting (31 stakeholders) and refined through a checklist pilot (34 participants). The CONSORT-AI extension includes 14 new items that were considered sufficiently important for AI interventions that they should be routinely reported in addition to the core CONSORT 2010 items. CONSORT-AI recommends that investigators provide clear descriptions of the AI intervention, including instructions and skills required for use, the setting in which the AI intervention is integrated, the handling of inputs and outputs of the AI intervention, the human-AI interaction and provision of an analysis of error cases. CONSORT-AI will help promote transparency and completeness in reporting clinical trials for AI interventions. It will assist editors and peer reviewers, as well as the general readership, to understand, interpret and critically appraise the quality of clinical trial design and risk of bias in the reported outcomes.
RESUMO A declaração CONSORT 2010 apresenta diretrizes mínimas para relatórios de ensaios clínicos randomizados. Seu uso generalizado tem sido fundamental para garantir a transparência na avaliação de novas intervenções. Recentemente, tem-se reconhecido cada vez mais que intervenções que incluem inteligência artificial (IA) precisam ser submetidas a uma avaliação rigorosa e prospectiva para demonstrar seus impactos sobre os resultados de saúde. A extensão CONSORT-AI (Consolidated Standards of Reporting Trials - Artificial Intelligence) é uma nova diretriz para relatórios de ensaios clínicos que avaliam intervenções com um componente de IA. Ela foi desenvolvida em paralelo à sua declaração complementar para protocolos de ensaios clínicos, a SPIRIT-AI (Standard Protocol Items: Recommendations for Interventional Trials - Artificial Intelligence). Ambas as diretrizes foram desenvolvidas por meio de um processo de consenso em etapas que incluiu revisão da literatura e consultas a especialistas para gerar 29 itens candidatos. Foram feitas consultas sobre esses itens a um grupo internacional composto por 103 interessados diretos, que participaram de uma pesquisa Delphi em duas etapas. Chegou-se a um acordo sobre os itens em uma reunião de consenso que incluiu 31 interessados diretos, e os itens foram refinados por meio de uma lista de verificação piloto que envolveu 34 participantes. A extensão CONSORT-AI inclui 14 itens novos que, devido à sua importância para as intervenções de IA, devem ser informados rotineiramente juntamente com os itens básicos da CONSORT 2010. A CONSORT-AI preconiza que os pesquisadores descrevam claramente a intervenção de IA, incluindo instruções e as habilidades necessárias para seu uso, o contexto no qual a intervenção de IA está inserida, considerações sobre o manuseio dos dados de entrada e saída da intervenção de IA, a interação humano-IA e uma análise dos casos de erro. A CONSORT-AI ajudará a promover a transparência e a integralidade nos relatórios de ensaios clínicos com intervenções que utilizam IA. Seu uso ajudará editores e revisores, bem como leitores em geral, a entender, interpretar e avaliar criticamente a qualidade do desenho do ensaio clínico e o risco de viés nos resultados relatados.
RESUMEN
resumen está disponible en el texto completo
ABSTRACT The SPIRIT 2013 statement aims to improve the completeness of clinical trial protocol reporting by providing evidence-based recommendations for the minimum set of items to be addressed. This guidance has been instrumental in promoting transparent evaluation of new interventions. More recently, there has been a growing recognition that interventions involving artificial intelligence (AI) need to undergo rigorous, prospective evaluation to demonstrate their impact on health outcomes. The SPIRIT-AI (Standard Protocol Items: Recommendations for Interventional Trials-Artificial Intelligence) extension is a new reporting guideline for clinical trial protocols evaluating interventions with an AI component. It was developed in parallel with its companion statement for trial reports: CONSORT-AI (Consolidated Standards of Reporting Trials-Artificial Intelligence). Both guidelines were developed through a staged consensus process involving literature review and expert consultation to generate 26 candidate items, which were consulted upon by an international multi-stakeholder group in a two-stage Delphi survey (103 stakeholders), agreed upon in a consensus meeting (31 stakeholders) and refined through a checklist pilot (34 participants). The SPIRIT-AI extension includes 15 new items that were considered sufficiently important for clinical trial protocols of AI interventions. These new items should be routinely reported in addition to the core SPIRIT 2013 items. SPIRIT-AI recommends that investigators provide clear descriptions of the AI intervention, including instructions and skills required for use, the setting in which the AI intervention will be integrated, considerations for the handling of input and output data, the human-AI interaction and analysis of error cases. SPIRIT-AI will help promote transparency and completeness for clinical trial protocols for AI interventions. Its use will assist editors and peer reviewers, as well as the general readership, to understand, interpret and critically appraise the design and risk of bias for a planned clinical trial.
RESUMO A declaração SPIRIT 2013 tem como objetivo melhorar a integralidade dos relatórios dos protocolos de ensaios clínicos, fornecendo recomendações baseadas em evidências para o conjunto mínimo de itens que devem ser abordados. Essas orientações têm sido fundamentais para promover uma avaliação transparente de novas intervenções. Recentemente, tem-se reconhecido cada vez mais que intervenções que incluem inteligência artificial (IA) precisam ser submetidas a uma avaliação rigorosa e prospectiva para demonstrar seus impactos sobre os resultados de saúde. A extensão SPIRIT-AI (Standard Protocol Items: Recommendations for Interventional Trials - Artificial Intelligence) é uma nova diretriz de relatório para protocolos de ensaios clínicos que avaliam intervenções com um componente de IA. Essa diretriz foi desenvolvida em paralelo à sua declaração complementar para relatórios de ensaios clínicos, CONSORT-AI (Consolidated Standards of Reporting Trials - Artificial Intelligence). Ambas as diretrizes foram desenvolvidas por meio de um processo de consenso em etapas que incluiu revisão da literatura e consultas a especialistas para gerar 26 itens candidatos. Foram feitas consultas sobre esses itens a um grupo internacional composto por 103 interessados diretos, que participaram de uma pesquisa Delphi em duas etapas. Chegou-se a um acordo sobre os itens em uma reunião de consenso que incluiu 31 interessados diretos, e os itens foram refinados por meio de uma lista de verificação piloto que envolveu 34 participantes. A extensão SPIRIT-AI inclui 15 itens novos que foram considerados suficientemente importantes para os protocolos de ensaios clínicos com intervenções que utilizam IA. Esses itens novos devem constar dos relatórios de rotina, juntamente com os itens básicos da SPIRIT 2013. A SPIRIT-AI preconiza que os pesquisadores descrevam claramente a intervenção de IA, incluindo instruções e as habilidades necessárias para seu uso, o contexto no qual a intervenção de IA será integrada, considerações sobre o manuseio dos dados de entrada e saída, a interação humano-IA e a análise de casos de erro. A SPIRIT-AI ajudará a promover a transparência e a integralidade nos protocolos de ensaios clínicos com intervenções que utilizam IA. Seu uso ajudará editores e revisores, bem como leitores em geral, a entender, interpretar e avaliar criticamente o delineamento e o risco de viés de um futuro estudo clínico.
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The development of biomaterials to improve wound healing is a critical clinical challenge and an active field of research. As it is well described that oxygen plays a critical role in almost each step of the wound healing process, in this work, an oxygen producing photosynthetic biomaterial was generated, characterized, and further modified to additionally release other bioactive molecules. Here, alginate hydrogels were loaded with the photosynthetic microalgae Chlamydomonas reinhardtii, showing high integration as well as immediate oxygen release upon illumination. Moreover, the photosynthetic hydrogel showed high biocompatibility in vitro and in vivo, and the capacity to sustain the metabolic oxygen requirements of zebrafish larvae and skin explants. In addition, the photosynthetic dressings were evaluated in 20 healthy human volunteers following the ISO-10993-10-2010 showing no skin irritation, mechanical stability of the dressings, and survival of the photosynthetic microalgae. Finally, hydrogels were also loaded with genetically engineered microalgae to release human VEGF, or pre-loaded with antibiotics, showing sustained release of both bioactive molecules. Overall, this work shows that photosynthetic hydrogels represent a feasible approach for the local delivery of oxygen and other bioactive molecules to promote wound healing. STATEMENT OF SIGNIFICANCE: As oxygen plays a key role in almost every step of the tissue regeneration process, the development of oxygen delivering therapies represents an active field of research, where photosynthetic biomaterials have risen as a promising approach for wound healing. Therefore, in this work a photosynthetic alginate hydrogel-based wound dressing containing C. reinhardtii microalgae was developed and validated in healthy skin of human volunteers. Moreover, hydrogels were modified to additionally release other bioactive molecules such as recombinant VEGF or antibiotics. The present study provides key scientific data to support the use of photosynthetic hydrogels as customizable dressings to promote wound healing.
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Hidrogeles , Oxígeno , Animales , Humanos , Hidrogeles/farmacología , Oxígeno/farmacología , Factor A de Crecimiento Endotelial Vascular , Pez Cebra , Vendajes , Materiales Biocompatibles , Antibacterianos , Alginatos/farmacologíaRESUMEN
resumen está disponible en el texto completo
ABSTRACT The SPIRIT 2013 statement aims to improve the completeness of clinical trial protocol reporting by providing evidence-based recommendations for the minimum set of items to be addressed. This guidance has been instrumental in promoting transparent evaluation of new interventions. More recently, there has been a growing recognition that interventions involving artificial intelligence (AI) need to undergo rigorous, prospective evaluation to demonstrate their impact on health outcomes. The SPIRIT-AI (Standard Protocol Items: Recommendations for Interventional Trials-Artificial Intelligence) extension is a new reporting guideline for clinical trial protocols evaluating interventions with an AI component. It was developed in parallel with its companion statement for trial reports: CONSORT-AI (Consolidated Standards of Reporting Trials-Artificial Intelligence). Both guidelines were developed through a staged consensus process involving literature review and expert consultation to generate 26 candidate items, which were consulted upon by an international multi-stakeholder group in a two-stage Delphi survey (103 stakeholders), agreed upon in a consensus meeting (31 stakeholders) and refined through a checklist pilot (34 participants). The SPIRIT-AI extension includes 15 new items that were considered sufficiently important for clinical trial protocols of AI interventions. These new items should be routinely reported in addition to the core SPIRIT 2013 items. SPIRIT-AI recommends that investigators provide clear descriptions of the AI intervention, including instructions and skills required for use, the setting in which the AI intervention will be integrated, considerations for the handling of input and output data, the human-AI interaction and analysis of error cases. SPIRIT-AI will help promote transparency and completeness for clinical trial protocols for AI interventions. Its use will assist editors and peer reviewers, as well as the general readership, to understand, interpret and critically appraise the design and risk of bias for a planned clinical trial.
RESUMO A declaração SPIRIT 2013 tem como objetivo melhorar a integralidade dos relatórios dos protocolos de ensaios clínicos, fornecendo recomendações baseadas em evidências para o conjunto mínimo de itens que devem ser abordados. Essas orientações têm sido fundamentais para promover uma avaliação transparente de novas intervenções. Recentemente, tem-se reconhecido cada vez mais que intervenções que incluem inteligência artificial (IA) precisam ser submetidas a uma avaliação rigorosa e prospectiva para demonstrar seus impactos sobre os resultados de saúde. A extensão SPIRIT-AI (Standard Protocol Items: Recommendations for Interventional Trials - Artificial Intelligence) é uma nova diretriz de relatório para protocolos de ensaios clínicos que avaliam intervenções com um componente de IA. Essa diretriz foi desenvolvida em paralelo à sua declaração complementar para relatórios de ensaios clínicos, CONSORT-AI (Consolidated Standards of Reporting Trials - Artificial Intelligence). Ambas as diretrizes foram desenvolvidas por meio de um processo de consenso em etapas que incluiu revisão da literatura e consultas a especialistas para gerar 26 itens candidatos. Foram feitas consultas sobre esses itens a um grupo internacional composto por 103 interessados diretos, que participaram de uma pesquisa Delphi em duas etapas. Chegou-se a um acordo sobre os itens em uma reunião de consenso que incluiu 31 interessados diretos, e os itens foram refinados por meio de uma lista de verificação piloto que envolveu 34 participantes. A extensão SPIRIT-AI inclui 15 itens novos que foram considerados suficientemente importantes para os protocolos de ensaios clínicos com intervenções que utilizam IA. Esses itens novos devem constar dos relatórios de rotina, juntamente com os itens básicos da SPIRIT 2013. A SPIRIT-AI preconiza que os pesquisadores descrevam claramente a intervenção de IA, incluindo instruções e as habilidades necessárias para seu uso, o contexto no qual a intervenção de IA será integrada, considerações sobre o manuseio dos dados de entrada e saída, a interação humano-IA e a análise de casos de erro. A SPIRIT-AI ajudará a promover a transparência e a integralidade nos protocolos de ensaios clínicos com intervenções que utilizam IA. Seu uso ajudará editores e revisores, bem como leitores em geral, a entender, interpretar e avaliar criticamente o delineamento e o risco de viés de um futuro estudo clínico.
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As hypoxic tumors show resistance to several clinical treatments, photosynthetic microorganisms have been recently suggested as a promising safe alternative for oxygenating the tumor microenvironment. The relationship between organisms and the effect microalgae have on tumors is still largely unknown, evidencing the need for a simple yet representative model for studying photosynthetic tumor oxygenation in a reproducible manner. Here, we present a 3D photosynthetic tumor model composed of human melanoma cells and the microalgae Chlamydomonas reinhardtii, both seeded into a collagen scaffold, which allows for the simultaneous study of both cell types. This work focuses on the biocompatibility and cellular interactions of the two cell types, as well as the study of photosynthetic oxygenation of the tumor cells. It is shown that both cell types are biocompatible with one another at cell culture conditions and that a 10:1 ratio of microalgae to cells meets the metabolic requirement of the tumor cells, producing over twice the required amount of oxygen. This 3D tumor model provides an easy-to-use in vitro resource for analyzing the effects of photosynthetically produced oxygen on a tumor microenvironment, thus opening various potential research avenues.
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Chlamydomonas reinhardtii , Microalgas , Neoplasias , Humanos , Microalgas/metabolismo , Fotosíntesis , Chlamydomonas reinhardtii/metabolismo , Oxígeno/metabolismo , Comunicación Celular , Microambiente TumoralRESUMEN
We performed a retrospective screening of 428 serum samples for anti-SARS-CoV-2 immunoglobulin during a period of low prevalence. Employing two different serological tests yielded discrepant results for 10 samples; highlighting an increased risk of potential false positive results and the need for further confirmatory testing before publication of data.
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COVID-19 , SARS-CoV-2 , Brasil , Reacciones Falso Positivas , Humanos , Inmunoglobulinas , Prevalencia , Estudios Retrospectivos , Sensibilidad y EspecificidadRESUMEN
Surgical sutures represent the gold standard for wound closure, however, their main purpose is still limited to a mechanical function rather than playing a bioactive role. Since oxygen and pro-regenerative growth factors have been broadly described as key players for the healing process, in this study we evaluated the feasibility of generating photosynthetic sutures that, in addition to mechanical fixation, could locally and stably release oxygen and recombinant human growth factors (VEGF, PDGF-BB, or SDF-1α) at the wound site. Here, photosynthetic genetically modified microalgae were seeded in commercially available sutures and their distribution and proliferation capacity was evaluated. Additionally, the mechanical properties of seeded sutures were compared to unseeded controls that showed no significant differences. Oxygen production, as well as recombinant growth factor release was quantified in vitro over time, and confirmed that photosynthetic sutures are indeed a feasible approach for the local delivery of bioactive molecules. Finally, photosynthetic sutures were tested in order to evaluate their resistance to mechanical stress and freezing. Significant stability was observed in both conditions, and the feasibility of their use in the clinical practice was therefore confirmed. Our results suggest that photosynthetic gene therapy could be used to produce a new generation of bioactive sutures with improved healing capacities. STATEMENT OF SIGNIFICANCE: Disruption of the vascular network is intrinsic to trauma and surgery, and consequently, wound healing is characterized by diminished levels of blood perfusion. Among all the blood components, oxygen and pro-regenerative growth factors have been broadly described as key players for the healing process. Therefore, in this study we evaluated the feasibility of generating photosynthetic sutures that, in addition to mechanical fixation, could locally and stably release oxygen and recombinant human growth factors at the wound site. This novel concept has never been explored before for this type of material and represents the first attempt to create a new generation of bioactive sutures with improved regenerative capabilities.
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Portadores de Fármacos , Péptidos y Proteínas de Señalización Intercelular , Oxígeno , Suturas , Heridas y Lesiones , Células 3T3 , Animales , Pared Celular/química , Chlamydomonas reinhardtii/química , Portadores de Fármacos/química , Portadores de Fármacos/farmacocinética , Portadores de Fármacos/farmacología , Células Endoteliales de la Vena Umbilical Humana , Humanos , Péptidos y Proteínas de Señalización Intercelular/química , Péptidos y Proteínas de Señalización Intercelular/farmacocinética , Péptidos y Proteínas de Señalización Intercelular/farmacología , Ratones , Microalgas/química , Oxígeno/química , Oxígeno/farmacocinética , Oxígeno/farmacología , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacocinética , Proteínas Recombinantes/farmacología , Heridas y Lesiones/metabolismo , Heridas y Lesiones/patología , Heridas y Lesiones/terapiaRESUMEN
Epithelial cells can acquire invasive and tumorigenic capabilities through epithelial-mesenchymal-transition (EMT). The glycan-binding protein galectin-8 (Gal-8) activates selective ß1-integrins involved in EMT and is overexpressed by certain carcinomas. Here we show that Gal-8 overexpression or exogenous addition promotes proliferation, migration, and invasion in nontumoral Madin-Darby canine kidney (MDCK) cells, involving focal-adhesion kinase (FAK)-mediated transactivation of the epidermal growth factor receptor (EGFR), likely triggered by α5ß1integrin binding. Under subconfluent conditions, Gal-8-overexpressing MDCK cells (MDCK-Gal-8H) display hallmarks of EMT, including decreased E-cadherin and up-regulated expression of vimentin, fibronectin, and Snail, as well as increased ß-catenin activity. Changes related to migration/invasion included higher expression of α5ß1 integrin, extracellular matrix-degrading MMP13 and urokinase plasminogen activator/urokinase plasminogen activator receptor (uPA/uPAR) protease systems. Gal-8-stimulated FAK/EGFR pathway leads to proteasome overactivity characteristic of cancer cells. Yet MDCK-Gal-8H cells still develop apical/basolateral polarity reverting EMT markers and proteasome activity under confluence. This is due to the opposite segregation of Gal-8 secretion (apical) and ß1-integrins distribution (basolateral). Strikingly, MDCK-Gal-8H cells acquired tumorigenic potential, as reflected in anchorage-independent growth in soft agar and tumor generation in immunodeficient NSG mice. Therefore, Gal-8 can promote oncogenic-like transformation of epithelial cells through partial and reversible EMT, accompanied by higher proliferation, migration/invasion, and tumorigenic properties.
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Transición Epitelial-Mesenquimal , Receptores ErbB/metabolismo , Galectinas/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Transducción de Señal , Animales , Cadherinas/metabolismo , Carcinogénesis , Perros , Quinasa 1 de Adhesión Focal/metabolismo , Humanos , Integrina beta1/metabolismo , Células de Riñón Canino Madin Darby , Masculino , Ratones , Neoplasias Experimentales , Proteínas Recombinantes/metabolismo , Transfección , Regulación hacia Arriba , Activador de Plasminógeno de Tipo Uroquinasa/metabolismoRESUMEN
Galectin-8 (Gal-8) is a member of a glycan-binding protein family that regulates the immune system, among other functions, and is a target of antibodies in autoimmune disorders. However, its role in multiple sclerosis (MS), an autoimmune inflammatory disease of the central nervous system (CNS), remains unknown. We study the consequences of Gal-8 silencing on lymphocyte subpopulations and the development of experimental autoimmune encephalitis (EAE), to then assess the presence and clinical meaning of anti-Gal-8 antibodies in MS patients. Lgals8/Lac-Z knock-in mice lacking Gal-8 expression have higher polarization toward Th17 cells accompanied with decreased CCR6+ and higher CXCR3+ regulatory T cells (Tregs) frequency. These conditions result in exacerbated MOG35-55 peptide-induced EAE. Gal-8 eliminates activated Th17 but not Th1 cells by apoptosis and ameliorates EAE in C57BL/6 wild-type mice. ß-gal histochemistry reflecting the activity of the Gal-8 promoter revealed Gal-8 expression in a wide range of CNS regions, including high expression in the choroid-plexus. Accordingly, we detected Gal-8 in human cerebrospinal fluid, suggesting a role in the CNS immune-surveillance circuit. In addition, we show that MS patients generate function-blocking anti-Gal-8 antibodies with pathogenic potential. Such antibodies block cell adhesion and Gal-8-induced Th17 apoptosis. Furthermore, circulating anti-Gal-8 antibodies associate with relapsing-remitting MS (RRMS), and not with progressive MS phenotypes, predicting clinical disability at diagnosis within the first year of follow-up. Our results reveal that Gal-8 has an immunosuppressive protective role against autoimmune CNS inflammation, modulating the balance of Th17 and Th1 polarization and their respective Tregs. Such a role can be counteracted during RRMS by anti-Gal-8 antibodies, worsening disease prognosis. Even though anti-Gal-8 antibodies are not specific for MS, our results suggest that they could be a potential early severity biomarker in RRMS.
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Autoanticuerpos/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Galectinas/inmunología , Esclerosis Múltiple/inmunología , Linfocitos T Reguladores/inmunología , Células Th17/inmunología , Animales , Apoptosis/fisiología , Encéfalo/inmunología , Encéfalo/metabolismo , Adhesión Celular/fisiología , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/metabolismo , Femenino , Galectinas/genética , Galectinas/metabolismo , Silenciador del Gen , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Esclerosis Múltiple/genética , Esclerosis Múltiple/metabolismo , Pronóstico , Linfocitos T Reguladores/metabolismo , Células Th17/metabolismoRESUMEN
BACKGROUND: Glioblastoma is one of the most aggressive cancers of the brain. Malignant traits of glioblastoma cells include elevated migration, proliferation and survival capabilities. Galectins are unconventionally secreted glycan-binding proteins that modulate processes of cell adhesion, migration, proliferation and apoptosis by interacting with beta-galactosides of cell surface glycoproteins and the extracellular matrix. Galectin-8 is one of the galectins highly expressed in glioblastoma cells. It has a unique selectivity for terminally sialylated glycans recently found enhanced in these highly malignant cells. A previous study in glioblastoma cell lines reported that Gal-8 coating a plastic surface stimulates two-dimensional motility. Because in other cells Gal-8 arrests proliferation and induces apoptosis, here we extend its study by analyzing all of these processes in a U87 glioblastoma cell model. METHODS: We used immunoblot and RT-PCR for Gal-8 expression analysis, recombinant Gal-8 produced in a bacteria system for Gal-8 treatment of the cells, and shRNA in lentivirus transduction for Gal-8 silencing. Cell migration as assessed in transwell filters. Cell proliferation, cell cycle and apoptosis were analyzed by FACS. RESULTS: Gal-8 as a soluble stimulus triggered chemotactic migration of U87 cells across the polycarbonate filter of transwell chambers, almost as intensively as fetal bovine serum. Unexpectedly, Gal-8 also enhanced U87 cell growth. Co-incubation of Gal-8 with lactose, which blocks galectin-glycan interactions, abrogated both effects. Immunoblot showed Gal-8 in conditioned media reflecting its secretion. U87 cells transduced with silencing shRNA in a lentiviral vector expressed and secreted 30-40 % of their normal Gal-8 levels. These cells maintained their migratory capabilities, but decreased their proliferation rate and underwent higher levels of apoptosis, as revealed by flow cytometry analysis of cell cycle, CFSE and activated caspase-3 staining. Proliferation seemed to be more sensitive than migration to Gal-8 expression levels. CONCLUSIONS: Gal-8, either secreted or exogenously enriched in the media, and acting through extracellular glycan interactions, constitutes a strong stimulus of directional migration in glioblastoma U87 cells and for the first time emerges as a factor that promotes proliferation and prevents apoptosis in cancerous cells. These properties could potentially contribute to the exaggerated malignancy of glioblastoma cells.
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Neoplasias Encefálicas/patología , Galectinas/fisiología , Glioblastoma/patología , Animales , Apoptosis/fisiología , Neoplasias Encefálicas/genética , Bovinos , Línea Celular Tumoral , Movimiento Celular/fisiología , Proliferación Celular/fisiología , Citometría de Flujo/métodos , Galectina 1/análisis , Galectina 1/fisiología , Galectina 3/análisis , Galectina 3/fisiología , Galectinas/análisis , Galectinas/farmacología , Glioblastoma/genética , Humanos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Tiempo , Células Tumorales CultivadasRESUMEN
BACKGROUND: Glioblastoma is one of the most aggressive cancers of the brain. Malignant traits of glioblastoma cells include elevated migration, proliferation and survival capabilities. Galectins are unconventionally secreted glycan-binding proteins that modulate processes of cell adhesion, migration, proliferation and apoptosis by interacting with beta-galactosides of cell surface glycoproteins and the extracellular matrix. Galectin-8 is one of the galectins highly expressed in glioblastoma cells. It has a unique selectivity for terminally sialylated glycans recently found enhanced in these highly malignant cells. A previous study in glioblastoma cell lines reported that Gal-8 coating a plastic surface stimulates two-dimensional motility. Because in other cells Gal-8 arrests proliferation and induces apoptosis, here we extend its study by analyzing all of these processes in a U87 glioblastoma cell mode.l METHODS: We used immunoblot and RT-PCR for Gal-8 expression analysis, recombinant Gal-8 produced in a bacteria system for Gal-8 treatment of the cells, and shRNA in lentivirus transduction for Gal-8 silencing. Cell migration as assessed in transwell filters. Cell proliferation, cell cycle and apoptosis were analyzed by FACS. RESULTS: Gal-8 as a soluble stimulus triggered chemotactic migration of U87 cells across the polycarbonate filter of transwell chambers, almost as intensively as fetal bovine serum. Unexpectedly, Gal-8 also enhanced U87 cell growth. Co-incubation of Gal-8 with lactose, which blocks galectin-glycan interactions, abrogated both effects. Immunoblot showed Gal-8 in conditioned media reflecting its secretion. U87 cells transduced with silencing shRNA in a lentiviral vector expressed and secreted 30-40 % of their normal Gal-8 levels. These cells maintained their migratory capabilities, but decreased their proliferation rate and underwent higher levels of apoptosis, as revealed by flow cytometry analysis of cell cycle, CFSE and activated caspase-3 staining. Proliferation seemed to be more sensitive than migration to Gal-8 expression levels. CONCLUSIONS: Gal-8, either secreted or exogenously enriched in the media, and acting through extracellular glycan interactions, constitutes a strong stimulus of directional migration in glioblastoma U87 cells and for the first time emerges as a factor that promotes proliferation and prevents apoptosis in cancerous cells. These properties could potentially contribute to the exaggerated malignancy of glioblastoma cells.
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Humanos , Animales , Bovinos , Neoplasias Encefálicas/patología , Glioblastoma/patología , Galectinas/fisiología , Factores de Tiempo , Neoplasias Encefálicas/genética , Células Tumorales Cultivadas , Movimiento Celular/fisiología , Apoptosis/fisiología , Glioblastoma/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Galectinas/análisis , Galectinas/farmacología , Galectina 1/análisis , Galectina 1/fisiología , Galectina 3/análisis , Galectina 3/fisiología , Línea Celular Tumoral , Proliferación Celular/fisiología , Citometría de Flujo/métodosRESUMEN
Gulf of Mexico (Gulf) fisheries account for 41% of the U.S. marine recreational fish catch and 16% of the nation's marine commercial fish landings. Mercury (Hg) concentrations are elevated in some fish species in the Gulf, including king mackerel, sharks, and tilefish. All five Gulf states have fish consumption advisories based on Hg. Per-capita fish consumption in the Gulf region is elevated compared to the U.S. national average, and recreational fishers in the region have a potential for greater MeHg exposure due to higher levels of fish consumption. Atmospheric wet Hg deposition is estimated to be higher in the Gulf region compared to most other areas in the U.S., but the largest source of Hg to the Gulf as a whole is the Atlantic Ocean (>90%) via large flows associated with the Loop Current. Redistribution of atmospheric, Atlantic and terrestrial Hg inputs to the Gulf occurs via large scale water circulation patterns, and further work is needed to refine estimates of the relative importance of these Hg sources in terms of contributing to fish Hg levels in different regions of the Gulf. Measurements are needed to better quantify external loads, in-situ concentrations, and fluxes of total Hg and methylmercury in the water column, sediments, and food web.
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Mercurio/química , Agua de Mar/química , Contaminantes Químicos del Agua/química , Contaminantes Atmosféricos/química , Animales , Exposición a Riesgos Ambientales , Cadena Alimentaria , Sedimentos Geológicos/química , Humanos , Mercurio/metabolismo , Contaminantes Químicos del Agua/metabolismoRESUMEN
Anthropogenic activities influence the biogeochemical cycles of mercury, both qualitatively and quantitatively, on a global scale from sources to sinks. Anthropogenic processes that alter the temporal and spatial patterns of sources and cycling processes are changing the impacts of mercury contamination on aquatic biota and humans. Human exposure to mercury is dominated by the consumption of fish and products from aquaculture operations. The risk to society and to ecosystems from mercury contamination is growing, and it is important to monitor these expanding risks. However, the extent and manner to which anthropogenic activities will alter mercury sources and biogeochemical cycling in tropical and sub-tropical coastal environments is poorly understood. Factors as (1) lack of reliable local/regional data; (2) rapidly changing environmental conditions; (3) governmental priorities and; (4) technical actions from supra-national institutions, are some of the obstacles to overcome in mercury cycling research and policy formulation. In the tropics and sub-tropics, research on mercury in the environment is moving from an exploratory "inventory" phase towards more process-oriented studies. Addressing biodiversity conservation and human health issues related to mercury contamination of river basins and tropical coastal environments are an integral part of paragraph 221 of the United Nations document "The Future We Want" issued in Rio de Janeiro in June 2012.