RESUMO
Triggering receptor expressed on myeloid cells 2 (TREM2) is a single transmembrane molecule uniquely expressed in microglia. TREM2 mutations are genetically linked to Nasu-Hakola disease and associated with multiple neurodegenerative disorders, including Alzheimer's disease. TREM2 may regulate microglial inflammation and phagocytosis through coupling to the adaptor protein TYRO protein-tyrosine kinase-binding protein (TYROBP). However, there is no functional system for monitoring this protein-protein interaction. We developed a luciferase-based modality for real-time monitoring of TREM2-TYROBP coupling in live cells that utilizes split-luciferase complementation technology based on TREM2 and TYROBP fusion to the C- or N-terminal portion of the Renilla luciferase gene. Transient transfection of human embryonic kidney 293 cells with this reporter vector increased luciferase activity upon stimulation with an anti-TREM2 antibody, which induces their homodimerization. This was confirmed by ELISA-based analysis of the TREM2-TYROBP interaction. Antibody-mediated TREM2 stimulation enhanced spleen tyrosine kinase (SYK) activity and uptake of Staphylococcus aureus in microglial cell line BV-2 in a kinase-dependent manner. Interestingly, the TREM2 T66M mutation significantly enhanced luciferase activity without stimulation, indicating constitutive coupling to TYROBP. Finally, flow cytometry analyses indicated significantly lower surface expression of T66M TREM2 variant than wild type or other TREM2 variants. These results demonstrate that our TREM2 reporter vector is a novel tool for monitoring the TREM2-TYROBP interaction in real time.
Assuntos
Citometria de Fluxo/métodos , Teste de Complementação Genética/métodos , Luciferases de Renilla/metabolismo , Glicoproteínas de Membrana/metabolismo , Microglia/metabolismo , Receptores Imunológicos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Animais , Linhagem Celular , Humanos , Lipodistrofia/genética , Lipodistrofia/metabolismo , Glicoproteínas de Membrana/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Osteocondrodisplasias/genética , Osteocondrodisplasias/metabolismo , Receptores Imunológicos/genética , Panencefalite Esclerosante Subaguda/genética , Panencefalite Esclerosante Subaguda/metabolismo , Quinase Syk/genética , Quinase Syk/metabolismoRESUMO
Tau-tubulin kinase-1 (TTBK1) is a central nervous system (CNS)-specific protein kinase implicated in the pathological phosphorylation of tau. TTBK1-transgenic mice show enhanced neuroinflammation in the CNS. Double-transgenic mice expressing TTBK1 and frontotemporal dementia with parkinsonism-17-linked P301L (JNPL3) tau mutant (TTBK1/JNPL3) show increased accumulation of oligomeric tau protein in the CNS and enhanced loss of motor neurons in the ventral horn of the lumbar spinal cord. To determine the role of TTBK1-induced neuroinflammation in tauopathy-related neuropathogenesis, age-matched TTBK1/JNPL3, JNPL3, TTBK1, and non-transgenic littermates were systematically characterized. There was a striking switch in the activation phenotype and population of mononuclear phagocytes (resident microglia and infiltrating macrophages) in the affected spinal cord region: JNPL3 mice showed accumulation of alternatively activated microglia, whereas TTBK1 and TTBK1/JNPL3 mice showed accumulation of classically activated infiltrating peripheral monocytes. In addition, expression of chemokine ligand 2, a chemokine important for the recruitment of peripheral monocytes, was enhanced in TTBK1 and TTBK1/JNPL3 but not in other groups in the spinal cord. Furthermore, primary cultured mouse motor neurons showed axonal degeneration after transient expression of the TTBK1 gene or treatment with conditioned media derived from lipopolysaccharide-stimulated microglia; this was partially blocked by silencing of the endogenous TTBK1 gene in neurons. These data suggest that TTBK1 accelerates motor neuron neurodegeneration by recruiting proinflammatory monocytes and enhancing sensitivity to neurotoxicity in inflammatory conditions.
Assuntos
Demência Frontotemporal/genética , Degeneração Neural/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas tau/genética , Animais , Modelos Animais de Doenças , Demência Frontotemporal/metabolismo , Demência Frontotemporal/patologia , Camundongos , Camundongos Transgênicos , Mutação , Degeneração Neural/metabolismo , Degeneração Neural/patologia , Neurônios/metabolismo , Neurônios/patologia , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Medula Espinal/patologia , Tauopatias/genética , Tauopatias/metabolismo , Tauopatias/patologia , Proteínas tau/metabolismoRESUMO
OBJECTIVES: Students often express uncertainty regarding changing their answers on multiple choice tests despite multiple studies quantitatively showing the benefits of changing answers. METHODS: Data was collected from 86 first-year podiatric medical students over one semester for the course of Biochemistry, as shown in electronic testing data collected via ExamSoft's® Snapshot Viewer. Quantitative analysis was performed comparing frequency of changing answers and whether students changed their answers from incorrect-to-correct, correct-to-incorrect, or incorrect-to-incorrect. A correlation analysis was performed to assess the relationship between the frequency of each type of answer change and class rank. Independent-sample t-tests were used to assess differences in the pattern of changing answers amongst the top and bottom performing students in the class. RESULTS: The correlation between total changes made from correct-to-incorrect per total answer changes and class rank yielded a positive correlation of r = 0.218 (P = .048). There was also a positive correlation of r = 0.502 (P < .000) observed in the number of incorrect-to-incorrect answer changes per total changes made compared to class rank. A negative correlation of r = -0.382 (P < .000) was observed when comparing class rank and the number of changed answers from incorrect-to-correct. While most of the class benefited from changing answers, a significant positive correlation of r = 0.467 (P < .000) for percent ultimately incorrect (regardless of number of changes) and class rank was observed. CONCLUSION: Analysis revealed that class rank correlated to likelihood of a positive gain from changing answers. Higher ranking students were more likely to gain points from changing their answer compared to lower ranking. Top students changed answers less frequently and changed answers to an ultimately correct answer more often, while bottom students changed answers from an incorrect answer to another incorrect answer more frequently than top students.
RESUMO
Cluster EC ClearAsMud and cluster EA4 Kauala are lytic Siphoviridae bacteriophages that were isolated from soil in southern California using Microbacterium foliorum NRRL B-24224 as the host. The ClearAsMud and Kauala genomes are 52,987 bp and 39,378 bp, respectively, and contain 92 and 56 predicted protein-coding genes, respectively.