RESUMO
For use in a broad spectrum of cell culture applications, we have devised a novel method, termed High-Throughput Metabolic Screening (HTMS), with which to more rapidly screen the overall activity of major metabolic pathways of mammalian cells. This current protocol uses adaptations of theoretical and experimental techniques from metabolic and cell culture engineering. First, HTMS makes use of a simplified metabolic network for metabolic flux analysis. Despite its simplicity, the network is capable of generating flux distributions and ATP production rates that are comparable to a more detailed network. Second, HTMS makes use of microtiter well-plate technology and adaptations of well-known enzymatic assays to increase precision and throughput for cell culture experiments. Multireplicate, multiparallel cultures in the sub-milliliter scale yield very precise metabolic rates using common laboratory equipment and at a fraction of the cost and time of traditional experiments with T-flasks, spinner flasks, or bioreactor systems. The simplicity of the network and the well-plate assays synergistically comprise a new, extremely useful, broadly applicable, and relatively inexpensive way to probe cell cultures for metabolic effects, screen drugs and toxins, optimize media, and support the development of bioprocesses. The simplified network and cell culture and analytical assays are also useful for undergraduate, graduate, and professional training.
Assuntos
Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Técnicas de Química Combinatória/métodos , Metabolismo/fisiologia , Modelos Biológicos , Animais , Células Cultivadas , Técnicas de Química Combinatória/instrumentação , Simulação por Computador , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos/instrumentação , Avaliação Pré-Clínica de Medicamentos/métodos , Metabolismo Energético/fisiologia , Humanos , Hibridomas/efeitos dos fármacos , Hibridomas/metabolismo , Hibridomas/fisiologia , Mamíferos , Complexos Multienzimáticos/fisiologia , Controle de Qualidade , Sirolimo/farmacologiaRESUMO
The purpose was to assess the feasibility of a care transition intervention for kidney transplant recipients (KTRs) with diabetes. Results document improved quality indicators and reduced resource utilization. These findings imply that a care transition intervention for KTRs with diabetes is feasible and associated with improved patient outcomes.
Assuntos
Assistência Ambulatorial/métodos , Doenças Cardiovasculares/epidemiologia , Diabetes Mellitus/terapia , Transplante de Rim , Ambulatório Hospitalar , Farmacêuticos , Comportamento de Redução do Risco , Idoso , Diabetes Mellitus/sangue , Estudos de Viabilidade , Feminino , Grupos Focais , Seguimentos , Hemoglobinas Glicadas/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Avaliação de Resultados em Cuidados de Saúde , Avaliação de Resultados da Assistência ao Paciente , Qualidade da Assistência à Saúde , TransplantadosRESUMO
Microalbuminuria in humans with Type 1 diabetes (T1D) is associated with increased urinary excretion of megalin, as well as many megalin ligands, including vitamin-D-binding protein (VDBP). We examined the DBA/2J diabetic mouse, nephropathy prone model, to determine if megalin and VDBP excretion coincide with the development of diabetic nephropathy. Megalin, VDBP, and 25-hydroxy-vitamin D (25-OHD) were measured in urine, and genes involved in vitamin D metabolism were assessed in renal tissues from diabetic and control mice at 10, 15, and 18 weeks following the onset of diabetes. Megalin, VDBP, and 25-OHD were increased in the urine of diabetic mice. 1-α hydroxylase (CYP27B1) mRNA in the kidney was persistently increased in diabetic mice, as were several vitamin D-target genes. These studies show that intrarenal vitamin D handling is altered in the diabetic kidney, and they suggest that in T1D, urinary losses of VDBP may portend risk for intrarenal and extrarenal vitamin D deficiencies.
Assuntos
Diabetes Mellitus Tipo 1/complicações , Diabetes Mellitus Tipo 1/metabolismo , Nefropatias Diabéticas/etiologia , Nefropatias Diabéticas/metabolismo , Endocitose , Vitamina D/metabolismo , Animais , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/induzido quimicamente , Diabetes Mellitus Tipo 1/genética , Nefropatias Diabéticas/patologia , Modelos Animais de Doenças , Suscetibilidade a Doenças , Endocitose/fisiologia , Rim/metabolismo , Rim/patologia , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Camundongos , Camundongos Endogâmicos DBA , Transdução de Sinais/fisiologia , EstreptozocinaRESUMO
Skeletal changes accompanying aging are associated with both increased risk of fractures and impaired fracture healing, which, in turn, is due to compromised bone regeneration potential. These changes are associated with increased serum levels of selected proinflammatory cytokines, e.g., tumor necrosis factor alpha (TNF-alpha). We have used a unique model of bone regeneration to demonstrate (1) that aged-related deficits in direct bone formation can be restored to young mice by treatment with TNF blockers and (2) that the cyclin-dependent kinase inhibitor p21 is a candidate for mediation of the osteoinhibitory effects of TNF. It has been hypothesized recently that TNF antagonists may represent novel anabolic agents, and we believe that the data presented here represent a successful test of this hypothesis.