Interaction of tau with the neural membrane cortex is regulated by phosphorylation at sites that are modified in paired helical filaments

The axonal microtubule-associated phosphoprotein tau interacts with neural plasma membrane (PM) components during neuronal development (Brandt, R., Léger, J., and Lee, G. (1995) J. Cell Biol. 131, 1327-1340). To analyze the mechanism and potential regulation of tau's PM association, a method was developed to isolate PM-associated tau using microsphere separation of surface-biotinylated cells. We show that tau's PM association requires an intact membrane cortex and that PM-associated tau and cytosolic tau are differentially phosphorylated at sites detected by several Alzheimer's disease (AD) diagnostic antibodies (Ser(199)/Ser(202), Thr(231), and Ser(396)/Ser(404)). In polar neurons, the association of endogenous tau phosphoisoforms with the membrane cortex correlates with an enrichment in the axonal compartment. To test for a direct effect of AD-specific tau modifications in determining tau's interactions, a phosphomutant that simulates an AD-like hyperphosphorylation of tau was produced by site-directed mutagenesis of Ser/Thr residues to negatively charged amino acids (Glu). These mutations completely abolish tau's association with the membrane cortex; however, the construct retains its capability to bind to microtubules. The data suggest that a loss of tau's association with the membrane cortex as a result of phosphorylation at sites that are modified during disease contributes to somatodendritic tau accumulation, axonal microtubule disintegration, and neuronal death characteristic for AD.     

Global expression profiling of theophylline response genes in macrophages: evidence of airway anti-inflammatory regulation

Background

Theophylline has been used widely as a bronchodilator for the treatment of bronchial asthma and has been suggested to modulate immune response. While the importance of macrophages in asthma has been reappraised and emphasized, their significance has not been well investigated. We conducted a genome-wide profiling of the gene expressions of macrophages in response to theophylline.

Methods

Microarray technology was used to profile the gene expression patterns of macrophages modulated by theophylline. Northern blot and real-time quantitative RT-PCR were also used to validate the microarray data, while Western blot and ELISA were used to measure the levels of IL-13 and LTC4.

Results

We identified dozens of genes in macrophages that were dose-dependently down- or up-regulated by theophylline. These included genes related to inflammation, cytokines, signaling transduction, cell adhesion and motility, cell cycle regulators, and metabolism. We observed that IL-13, a central mediator of airway inflammation, was dramatically suppressed by theophylline. Real-time quantitative RT-PCR and ELISA analyses also confirmed these results, without respect to PMA-treated THP-1 cells or isolated human alveolar macrophages. Theophylline, rolipram, etazolate, db-cAMP and forskolin suppressed both IL-13 mRNA expression (~25%, 2.73%, 8.12%, 5.28%, and 18.41%, respectively) and protein secretion (<10% production) in macrophages. These agents also effectively suppressed LTC4 expression.

Conclusion

Our results suggest that the suppression of IL-13 by theophylline may be through cAMP mediation and may decrease LTC4 production. This study supports the role of theophylline as a signal regulator of inflammation, and that down regulation of IL-13 by theophylline may have beneficial effects in inflammatory airway diseases.     

Clinical-grade manufacturing of DC from CD14+ precursors: experience from phase I clinical trials in CML and malignant melanoma

BACKGROUND: We evaluated a clinical-grade protocol for the manufacture of mature DC from CD14 + precursors derived from normal donors and patients suffering from CML and stage IV malignant melanoma. We manufactured six products for CML patients and five for melanoma patients and administered them as vaccines in phase I clinical trials. METHODS: We isolated CD 14+ cells from apheresis products by immunomagnetic separation and incubated them in X-VIVO 15' medium supplemented with human AB serum, GM-CSF and IL-4 for 7 days, and with additional tumor necrosis factor (TNF)-a, IL-lIf, IL-6 and prostaglandin E2 for 3 days. Some cells were electroporated and transfected with mRNA isolated from melanoma tissue. DC were characterized by flow cytometry for the expression of CD83, CD86 andCD14. RESULTS: CD14+ cells constituted 14.4+/-6.2% (mean + SD) of nucleated cells in apheresis products and 98.3+/- 3.6% of isolated cells. Normal DC and CML DC were 77.4+/-7.3% CD83+ and 93.5+/- 7.0% CD86+.Corresponding values for electroporated DC from melanoma patients were 66.1 + 7.2% and 94.1 + 7.8%. The yield of CD83+ DC from isolated CD14+ cells was 18.1 + 7.2% for normal and CML patients and 9.8 + 3.7% for melanoma patients. DC viability was 92.7 + 5.8%; after cryopreservation and thawing it was 77+/-13.5%. DISCUSSION: Our method yielded viable and mature DC free of bacteria and mycoplasma. This robust and reproducible method provides cells of consistent phenotype and viability. Cryopreservation in single-dose aliquots allows multiple DC vaccine doses to be manufactured from a single apheresis product.     

Effect of the Learning Climate of Residency Programs on Faculty’s Teaching Performance as Evaluated by Residents

Background

To understand teaching performance of individual faculty, the climate in which residents’ learning takes place, the learning climate, may be important. There is emerging evidence that specific climates do predict specific outcomes. Until now, the effect of learning climate on the performance of the individual faculty who actually do the teaching was unknown.

Objectives

This study: (i) tested the hypothesis that a positive learning climate was associated with better teaching performance of individual faculty as evaluated by residents, and (ii) explored which dimensions of learning climate were associated with faculty’s teaching performance.

Methods and Materials

We conducted two cross-sectional questionnaire surveys amongst residents from 45 residency training programs and multiple specialties in 17 hospitals in the Netherlands. Residents evaluated the teaching performance of individual faculty using the robust System for Evaluating Teaching Qualities (SETQ) and evaluated the learning climate of residency programs using the Dutch Residency Educational Climate Test (D-RECT). The validated D-RECT questionnaire consisted of 11 subscales of learning climate. Main outcome measure was faculty’s overall teaching (SETQ) score. We used multivariable adjusted linear mixed models to estimate the separate associations of overall learning climate and each of its subscales with faculty’s teaching performance.

Results

In total 451 residents completed 3569 SETQ evaluations of 502 faculty. Residents also evaluated the learning climate of 45 residency programs in 17 hospitals in the Netherlands. Overall learning climate was positively associated with faculty’s teaching performance (regression coefficient 0.54, 95% confidence interval: 0.37 to 0.71; P<0.001). Three out of 11 learning climate subscales were substantially associated with better teaching performance: ‘coaching and assessment’, ‘work is adapted to residents’ competence’, and ‘formal education’.

Conclusions

Individual faculty’s teaching performance evaluations are positively affected by better learning climate of residency programs.     

Modeling short-range stiffness of feline lower hindlimb muscles

The short-range stiffness (SRS) of skeletal muscles is a critical property for understanding muscle contributions to limb stability, since it represents a muscle's capacity to resist external perturbations before reflexes or voluntary actions can intervene. A number of studies have demonstrated that a simple model, consisting of a force-dependent active stiffness connected in series with a constant passive stiffness, is sufficient to characterize the SRS of individual muscles over the entire range of obtainable forces. The purpose of this study was to determine if such a model could be used to characterize the SRS-force relationship in a number of architecturally distinct muscles. Specifically, we hypothesized that the active and passive stiffness components for a specific muscle can be estimated from anatomical measurements, assuming uniform active and passive stiffness properties across all muscles. This hypothesis was evaluated in six feline lower hindlimb muscle types with different motor unit compositions and architectures. The SRS-force relationships for each muscle type were predicted based on anatomical measurements and compared to experimental data. The model predictions were accurate to within 30%, when uniform scaling properties were assumed across all muscles. Errors were the greatest for the extensor digitorum longus (EDL). When this muscle was removed from the analysis, prediction errors dropped to less than 8%. Subsequent analyses suggested that these errors might have resulted from differences in the tendon elastic modulus, as compared to the other muscles tested.     

Pilot-scale conversion of lime-treated wheat straw into bioethanol: quality assessment of bioethanol and valorization of side streams by anaerobic digestion and combustion

Introduction  

The limited availability of fossil fuel sources, worldwide rising energy demands and anticipated climate changes attributed to an increase of greenhouse gasses are important driving forces for finding alternative energy sources. One approach to meeting the increasing energy demands and reduction of greenhouse gas emissions is by large-scale substitution of petrochemically derived transport fuels by the use of carbon dioxide-neutral biofuels, such as ethanol derived from lignocellulosic material.