Quantitative live cell imaging of a tauopathy model enables the identification of a polypharmacological drug candidate that restores physiological microtubule interaction

Tauopathies such as Alzheimer's disease are characterized by aggregation and increased phosphorylation of the microtubule-associated protein tau. Tau's pathological changes are closely linked to neurodegeneration, making tau a prime candidate for intervention. We developed an approach to monitor pathological changes of aggregation-prone human tau in living neurons. We identified 2-phenyloxazole (PHOX) derivatives as putative polypharmacological small molecules that interact with tau and modulate tau kinases. We found that PHOX15 inhibits tau aggregation, restores tau's physiological microtubule interaction, and reduces tau phosphorylation at disease-relevant sites. Molecular dynamics simulations highlight cryptic channel-like pockets crossing tau protofilaments and suggest that PHOX15 binding reduces the protofilament's ability to adopt a PHF-like conformation by modifying a key glycine triad. Our data demonstrate that live-cell imaging of a tauopathy model enables screening of compounds that modulate tau-microtubule interaction and allows identification of a promising polypharmacological drug candidate that simultaneously inhibits tau aggregation and reduces tau phosphorylation.</p

Gentamicin supplemented polyvinylidenfluoride mesh materials enhance tissue integration due to a transcriptionally reduced MMP-2 protein expression

<p>Abstract</p> <p>Background</p> <p>A beneficial effect of gentamicin supplemented mesh material on tissue integration is known. To further elucidate the interaction of collagen and MMP-2 in chronic foreign body reaction and to determine the significance of the MMP-2-specific regulatory element (RE-1) that is known to mediate 80% of the MMP-2 promoter activity, the spatial and temporal transcriptional regulation of the MMP-2 gene was analyzed at the cellular level.</p> <p>Methods</p> <p>A PVDF mesh material was surface modified by plasma-induced graft polymerization of acrylic acid (PVDF+PAAc). Three different gentamicin concentrations were bound to the provided active sites of the grafted mesh surfaces (2, 5 and 8 μg/mg). 75 male transgenic MMP-2/LacZ mice harbouring the LacZ reporter gene under control of MMP-2 regulatory sequence -1241/+423, excluding the RE-1 were randomized to five groups. Bilateral of the abdominal midline one of the five different meshes was implanted subcutaneously in each animal. MMP-2 gene transcription (anti-ß-galactosidase staining) and MMP-2 protein expression (anti-MMP-2 staining) were analyzed semiquantitatively by immunohistochemistry 7, 21 and 90 days after mesh implantation. The collagen type I/III ratio was analyzed by cross polarization microscopy to determine the quality of mesh integration.</p> <p>Results</p> <p>The perifilamentary ß-galactosidase expression as well as the collagen type I/III ratio increased up to the 90^thday for all mesh modifications, whereas no significant changes could be observed for MMP-2 protein expression between days 21 and 90. Both the 5 and 8 μg/mg gentamicin group showed significantly reduced levels of ß-galactosidase expression and MMP-2 positive stained cells when compared to the PVDF group on day 7, 21 and 90 respectively (5 μg/mg: p < 0.05 each; 8 μg/mg: p < 0.05 each). Though the type I/III collagen ratio increased over time for all mesh modifications significant differences to the PVDF mesh were only detected for the 8 μg/mg group at all 3 time points (p < 0.05 each).</p> <p>Conclusions</p> <p>Our current data indicate that lack of RE-1 is correlated with increased mesh induced MMP-2-gene expression for coated as well as for non-coated mesh materials. Gentamicin coating reduced MMP-2 transcription and protein expression. For the 8 μg/mg group this effect is associated with an increased type I/III collagen ratio. These findings suggest that gentamicin is beneficial for tissue integration after mesh implantation, which possibly is mediated via RE-1.</p

Cytokine Plasma Levels: Reliable Predictors for Radiation Pneumonitis?

BACKGROUND: Radiotherapy (RT) is the primary treatment modality for inoperable, locally advanced non-small-cell lung cancer (NSCLC), but even with highly conformal treatment planning, radiation pneumonitis (RP) remains the most serious, dose-limiting complication. Previous clinical reports proposed that cytokine plasma levels measured during RT allow to estimate the individual risk of patients to develop RP. The identification of such cytokine risk profiles would facilitate tailoring radiotherapy to maximize treatment efficacy and to minimize radiation toxicity. However, cytokines are produced not only in normal lung tissue after irradiation, but are also over-expressed in tumour cells of NSCLC specimens. This tumour-derived cytokine production may influence circulating plasma levels in NSCLC patients. The aim of the present study was to investigate the prognostic value of TNF-alpha, IL-1beta, IL-6 and TGF-beta1 plasma levels to predict radiation pneumonitis and to evaluate the impact of tumour-derived cytokine production on circulating plasma levels in patients irradiated for NSCLC. METHODOLOGY/PRINCIPAL FINDINGS: In 52 NSCLC patients (stage I-III) cytokine plasma levels were investigated by ELISA before and weekly during RT, during follow-up (1/3/6/9 months after RT), and at the onset of RP. Tumour biopsies were immunohistochemically stained for IL-6 and TGF-beta1, and immunoreactivity was quantified (grade 1-4). RP was evaluated according to LENT-SOMA scale. Tumour response was assessed according to RECIST criteria by chest-CT during follow-up. In our clinical study 21 out of 52 patients developed RP (grade I/II/III/IV: 11/3/6/1 patients). Unexpectedly, cytokine plasma levels measured before and during RT did not correlate with RP incidence. In most patients IL-6 and TGF-beta1 plasma levels were already elevated before RT and correlated significantly with the IL-6 and TGF-beta1 production in corresponding tumour biopsies. Moreover, IL-6 and TGF-beta1 plasma levels measured during follow-up were significantly associated with the individual tumour responses of these patients. CONCLUSIONS/SIGNIFICANCE: The results of this study did not confirm that cytokine plasma levels, neither their absolute nor any relative values, may identify patients at risk for RP. In contrast, the clear correlations of IL-6 and TGF-beta1 plasma levels with the cytokine production in corresponding tumour biopsies and with the individual tumour responses suggest that the tumour is the major source of circulating cytokines in patients receiving RT for advanced NSCLC

Imaging of Tau and Microtubules to Study Mechanisms of Tau Pathologies and Neurodegeneration

The nervous system undergoes constant remodeling and adapting of its structural organization to fulfill its function, the propagation and processing of information. The structural backbone of neurons are arrays of microtubules (MTs) maintaining their specialized morphologies and serving as railway system for the transport of cargoes and information. The functional regulation of this complex system is based on many different factors, while microtubule-associated proteins (MAPs) like tau being one of them. The tau protein is of particular interest as it plays a central role in numerous neurodegenerative diseases, called tauopathies, where Alzheimer’s Disease (AD) is the most common and prominent example. Hallmark of tauopathies such as AD is the aggregation and increased phosphorylation of tau, while also many other post-translational modifications (PTMs) of the protein are described and linked to neurodegeneration. Scope of this thesis are investigations on how the neuronal MTs, MT-dynamics and MT-dependent transport are affected by post-translational changes of the tau protein. A new type of toxic gain of function of tau is described, as it impairs axonal transport when cleaved at a diseases relevant site and microtubule-targeting drugs are introduced as pharmacological modifiers of MAP-microtubule interaction. The effect of MT-stabilization by Epothilone D on the MT-cytoskeleton as well as the effect of a tau knockout is further investigated by cutting-edge microscopy (DNA-PAINT SMLM) and algorithm-based quantification of the MT array. Changes of the MT organization and structure on the level of individual MTs upon stabilization and tau knockout are described. Accumulating evidence suggests that pathologically modified monomeric and soluble oligomeric forms of tau should be considered as harmful tau species. Hence, a cell-based model allowing investigations of disease-like modified tau, its oligomerization, and MT-interaction is implemented enabling the screening for modulators of early-stage aggregation and phosphorylation of tau. Potential drug candidates with polypharmacological activity are presented

Tendon response to pharmaco-mechanical stimulation of the chronically retracted rotator cuff in sheep

PURPOSE: Chronic tearing of tendons is associated with molecular and structural alterations causing biomechanical changes, which compromise musculotendinous function and become limiting factors for tendon repair. This study investigated the histological response of chronically retracted sheep rotator cuff tendons to mechanical and pharmacological stimulation in view of tendon repair. METHODS: Sixteen weeks after experimental release of the infraspinatus tendon in 20 sheep, the retracted musculotendinous unit was subjected to continuous traction either with [anabolic steroids (nandrolone) group/insulin-like growth factor (IGF) group] or without (control group) additional pharmacological treatment during 6 weeks. A new degeneration score for tendinous tissues (DSTT), based on established knowledge on histological changes associated with tendon degeneration, was used for histological analysis at the time of tendon release, at the beginning of continuous re-lengthening and at repair in all animals. RESULTS: The DSTT score (inter-observer correlation: r = 0.83), quantifiably representing tendon degeneration, improved from 15.5 (SD 1.3) points before to 9.8 (SD 3.8) points after re-lengthening. It improved in a qualitatively and quantitatively similar fashion if pharmacological stimulation was added. The nandrolone group improved from 13.7 (SD 1.6) to 9.8 (SD 1.9) and the IGF group from 13.3 (SD 3.6) to 8.8 (SD 1.8) points. CONCLUSION: Mechanical stimulation significantly reduced tissue degeneration. However, the addition of a pharmacological stimulation with anabolic steroids or IGF had neither a measurable positive nor negative effect on the degenerative process. Therefore, this investigation does neither support the additional pharmacological use of the anabolic steroid nandrolone or of IGF decanoate for restoration of tendon degeneration, nor otherwise provide evidence for additional tendon damage, if those substances are used to alter the muscular metabolism

Relationship intention as a mediator between relational benefits and customer loyalty in the tour operator industry

Relationship and customer loyalty management have been an important field of research in marketing for decades. However, the focus for many years was mainly on the benefits of relationship marketing for companies. This article contributes to a deeper understanding of the benefits of relationship marketing for customers in the travel industry. It investigates the effects between relational benefits, relationship intention and intentional loyalty using the tour operator industry as an example. Relationship intention will be introduced as a variable influencing intentional loyalty. The role of relationship intention in the customer benefit-intentional loyalty context will be analyzed by testing different path models. The measurement model is estimated, based on a confirmatory factor analysis using LISREL with a sample of 1,702 tour operator customers. The structural model as well as the estimate path coefficients were analyzed applying a partial least square approach (PLS) using SmartPLS. This article not only contributes to the further development of models explaining intentional loyalty, but also to the development of practical insights about the influence of different measures and tools on intentional loyalty from a customer benefit perspective. It can be concluded that the intention of a customer to invest in a relationship is dependent on the perceived level of relationship benefits. Further, the results show, that the buying behavior of customers is influenced by the perceived relational benefits. With the management of customer relations, a provider can influence the behavior of the customer to exploit the earning potential efficiently. Therefore, a provider has to be successful in creating new relational benefits for his customers through measures of customer relation management