Mechanisms of Adsorption and Surface-Mediated Aggregation of Intrinsically Disordered Protein Tau at Model Surfaces

The adsorption and aggregation of an intrinsically disordered soluble protein, tau, into insoluble filaments is a defining hallmark of many neurodegenerative diseases, commonly referred to as tauopathies. In its native state, the protein tau’s function is to promote the assembly, and aid in the stabilization of microtubules. The microtubules allow for material transport through the axon, to and from the neuron. While the presence of aggregated tau protein fibrils are hypothesized to accelerate neuronal degradation, possibly by destabilizing microtubules, or disrupting cell membranes, more recent research has established the presence of soluble oligomeric species as being cytotoxic. These results necessitate a complete fundamental understanding of the governing principles that modulate the initial steps in the mechanisms of tau protein aggregation. The macromolecular environment, including the presence of surfaces such as the cell membrane, and the presence of macromolecules in a crowded environment, has been implicated in the aggregation of tau protein. However, the exact role of surfaces in modulating Tau protein aggregation has not been explored in detail. We hypothesize that Tau protein aggregation at model surfaces is modulated by two factors, the physicochemical properties of the surfaces, as well as the biochemistry of the protein molecules. The work presented in this thesis project employs a combination of biophysical techniques to study the adsorption and aggregation of a wild type and several mutations of tau protein at model surfaces. A Quartz Crystal Microbalance with Dissipation (QCM-D) was used to monitor the adsorption of different tau species at nanomolar concentrations, mimicking the in vivo situation, to surfaces with different surface charge, wettability and softness, while Atomic Force Microscopy (AFM) was utilized to obtain direct visualization of the proteins at these different surfaces. Our results indicate that the hydrophobic amino acid sequence in the microtubule binding region was the leading force driving the adsorption of tau proteins to different surfaces. Further, AFM images provided direct evidence of the presence of oligomeric tau species at the interfaces, establishing that the solid surface did in fact provide a template for the tau protein to form aggregates. Adsorption of different tau protein mutations to phospholipid covered surfaces of different fluidity indicated that tau protein oligomers can also cause destabilization or disintegration of lipid bilayers. Such disintegration may well be the cause of observed cell death in several tauopathies. In summary, this thesis establishes that both protein biochemistry and the physicochemical properties of the surface modulate surface mediated aggregation. The work described in this thesis also provides a foundation for further research focused on the role of surfaces as templates that mediate tau aggregation pathway in vivo. A complete understanding of the mechanisms of tau aggregation will ultimately lead to strategies for therapeutic solutions for neurodegenerative diseases

Detection of recombinant human lactoferrin and lysozyme produced in a bitransgenic cow

Lactoferrin and lysozyme are 2 glycoproteins with great antimicrobial activity, being part of the nonspecific defensive system of human milk, though their use in commercial products is difficult because human milk is a limited source. Therefore, many investigations have been carried out to produce those proteins in biological systems, such as bacteria, yeasts, or plants. Mammals seem to be more suitable as expression systems for human proteins, however, especially for those that are glycosylated. In the present study, we developed a bicistronic commercial vector containing a goat β-casein promoter and an internal ribosome entry site fragment between the human lactoferrin and human lysozyme genes to allow the introduction of both genes into bovine adult fibroblasts in a single transfection. Embryos were obtained by somatic cell nuclear transfer, and, after 6 transferences to recipients, 3 pregnancies and 1 viable bitransgenic calf were obtained. The presence of the vector was confirmed by fluorescent in situ hybridization of skin cells. At 13 mo of life and after artificial induction of lactation, both recombinant proteins were found in the colostrum and milk of the bitransgenic calf. Human lactoferrin concentration in the colostrum was 0.0098 mg/mL and that in milk was 0.011 mg/mL; human lysozyme concentration in the colostrum was 0.0022 mg/mL and that in milk was 0.0024 mg/mL. The molar concentration of both human proteins revealed no differences in protein production of the internal ribosome entry site upstream and downstream protein. The enzymatic activity of lysozyme in the transgenic milk was comparable to that of human milk, being 6 and 10 times higher than that of bovine lysozyme presentin milk. This work represents an important step to obtain multiple proteins or enhance single protein production by using animal pharming and fewer regulatory and antibiotic-resistant foreign sequences, allowing the design of humanized milk with added biological value for newborn nutrition and development. Transgenic animals can offer a unique opportunity to the dairy industry, providing starting materials suitable to develop specific products with high added value. Key words: bitransgenic cow, human lactoferrin, ELISA, human lysozyme.Fil: Kaiser, German Gustavo. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce. Grupo de Biotecnología de la Reproducción; ArgentinaFil: Mucci, Nicolas Crescencio. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce. Grupo de Biotecnología de la Reproducción; ArgentinaFil: Gonzalez, Vega. Universidad de Zaragoza. Facultad de Veterinaria. Tecnología de los Alimentos; EspañaFil: Sánchez, Lourdes. Universidad de Zaragoza. Facultad de Veterinaria. Tecnología de los Alimentos; EspañaFil: Parrón, José Antonio. Universidad de Zaragoza. Facultad de Veterinaria. Tecnología de los Alimentos; EspañaFil: Pérez, María Dolores. Universidad de Zaragoza. Facultad de Veterinaria. Tecnología de los Alimentos; EspañaFil: Calvo, Miguel. Universidad de Zaragoza. Facultad de Veterinaria. Tecnología de los Alimentos; EspañaFil: Aller Atucha, Juan Florencio. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce. Grupo de Biotecnología de la Reproducción; ArgentinaFil: Hozbor, Federico Andres. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce. Grupo de Biotecnología de la Reproducción; ArgentinaFil: Mutto, Adrián Angel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentin

Intense exercise for survival among men with metastatic castrate-resistant prostate cancer (INTERVAL-GAP4): A multicentre, randomized, controlled phase III study protocol

Introduction: Preliminary evidence supports the beneficial role of physical activity on prostate cancer outcomes. This phase III randomised controlled trial (RCT) is designed to determine if supervised high-intensity aerobic and resistance exercise increases overall survival (OS) in patients with metastatic castrate-resistant prostate cancer (mCRPC). Methods and analysis: Participants (n=866) must have histologically documented metastatic prostate cancer with evidence of progressive disease on androgen deprivation therapy (defined as mCRPC). Patients can be treatmentnaive for mCRPC or on first-line androgen receptor-targeted therapy for mCRPC (ie, abiraterone or enzalutamide) without evidence of progression at enrolment, and with no prior chemotherapy for mCRPC. Patients will receive psychosocial support and will be randomly assigned (1:1) to either supervised exercise (high-intensity aerobic and resistance training) or self-directed exercise (provision of guidelines), stratified by treatment status and site. Exercise prescriptions will be tailored to each participant’s fitness and morbidities. The primary endpoint is OS. Secondary endpoints include time to disease progression, occurrence of a skeletal-related event or progression of pain, and degree of pain, opiate use, physical and emotional quality of life, and changes in metabolic biomarkers. An assessment of whether immune function, inflammation, dysregulation of insulin and energy metabolism, and androgen biomarkers are associated with OS will be performed, and whether they mediate the primary association between exercise and OS will also be investigated. This study will also establish a biobank for future biomarker discovery or validation. Ethics and dissemination: Validation of exercise as medicine and its mechanisms of action will create evidence to change clinical practice. Accordingly, outcomes of this RCT will be published in international, peer-reviewed journals, and presented at national and international conferences. Ethics approval was first obtained at Edith Cowan University (ID: 13236 NEWTON), with a further 10 investigator sites since receiving ethics approval, prior to activation. Trial registration number: NCT02730338

Phospholipid Composition Modulates Carbon Nanodiamond-Induced Alterations in Phospholipid Domain Formation

This document is the Accepted Manuscript version of a Published Work that appeared in final form in Langmuir, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see http://doi.org/10.1021/la504923j.The focus of this work is to elucidate how phospholipid composition can modulate lipid nanoparticle interactions in phospholipid monolayer systems. We report on alterations in lipid domain formation induced by anionically engineered carbon nanodiamonds (ECNs) as a function of lipid headgroup charge and alkyl chain saturation. Using surface pressure vs area isotherms, monolayer compressibility, and fluorescence microscopy, we found that anionic ECNs induced domain shape alterations in zwitterionic phosphatidylcholine lipids, irrespective of the lipid alkyl chain saturation, even when the surface pressure vs area isotherms did not show any significant changes. Bean-shaped structures characteristic of dipalmitoylphosphatidylcholine (DPPC) were converted to multilobed, fractal, or spiral domains as a result of exposure to ECNs, indicating that ECNs lower the line tension between domains in the case of zwitterionic lipids. For membrane systems containing anionic phospholipids, ECN-induced changes in domain packing were related to the electrostatic interactions between the anionic ECNs and the anionic lipid headgroups, even when zwitterionic lipids are present in excess. By comparing the measured size distributions with our recently developed theory derived by minimizing the free energy associated with the domain energy and mixing entropy, we found that the change in line tension induced by anionic ECNs is dominated by the charge in the condensed lipid domains. Atomic force microscopy images of the transferred anionic films confirm that the location of the anionic ECNs in the lipid monolayers is also modulated by the charge on the condensed lipid domains. Because biological membranes such as lung surfactants contain both saturated and unsaturated phospholipids with different lipid headgroup charges, our results suggest that when studying potential adverse effects of nanoparticles on biological systems the role of lipid compositions cannot be neglected

Effets d'un entraînement combiné (cyclisme et course) sur la performance aérobie chez des athlètes de fond

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Paraspinal muscle oxygenation and mechanical efficiency are reduced in individuals with chronic low back pain

Abstract This study aimed to compare the systemic and local metabolic responses during a 5-min trunk extension exercise in individuals with chronic low back pain (CLBP) and in healthy individuals. Thirteen active participants with CLBP paired with 13 healthy participants performed a standardised 5-min trunk extension exercise on an isokinetic dynamometer set in continuous passive motion mode. During exercise, we used near-infrared spectroscopy to measure tissue oxygenation (TOI) and total haemoglobin-myoglobin (THb). We used a gas exchange analyser to measure breath-by-breath oxygen consumption (V̇O2) and carbon dioxide produced (V̇CO2). We also calculated mechanical efficiency. We assessed the intensity of low back pain sensation before and after exercise by using a visual analogue scale. In participants with CLBP, low back pain increased following exercise (+ 1.5 units; p < 0.001) and THb decreased during exercise (− 4.0 units; p = 0.043). Paraspinal muscle oxygenation (65.0 and 71.0%, respectively; p = 0.009) and mechanical efficiency (4.7 and 5.3%, respectively; p = 0.034) were both lower in participants with CLBP compared with healthy participants. The increase in pain sensation was related to the decrease in tissue oxygenation (R2 = − 0.420; p = 0.036). Decreases in total haemoglobin-myoglobin and mechanical efficiency could involve fatigability in exercise-soliciting paraspinal muscles and, therefore, exacerbate inabilities in daily life. Given the positive correlation between tissue oxygenation and exercise-induced pain exacerbation, muscle oxygenation may be related to persisting and crippling low back pain

Benefits of a four-week functional restoration program in chronic low back pain patients and three-month follow-up: focus on paraspinal muscle aerobic metabolism responses to exercise

International audienceBackground: Chronic low back pain (CLBP) is a major health concern characterized by paraspinal muscle fatigability. This can be improved following a functional restoration program. Muscle fatigability can be related to impairment in aerobic metabolism responses. In this study, we investigated paraspinal muscles aerobic metabolism in CLBP patients before and after a functional restoration program, in order to determine if the enhancement in patients' condition following the program is associated to changes in metabolism responses.Methods: Twenty-two CLBP patients (11 women, 11 men; 41.6±1.8 years; 73.7±3.1 kg; 1.74±0.02 m) were evaluated before and after a 4-week functional restoration program, with exercise therapy as the main component. Three months later, 12 patients were seen for a follow-up visit. During each testing session, patients performed a five-minute isokinetic trunk extension exercise in measuring pulmonary gas exchanges and paraspinal muscle oxygenation. Mechanical efficiency and onset V̇O2 kinetics were also calculated, in addition to usual questionnaires and exercises designed to evaluate psychosocial and physical factors.Results: At the end of the program, paraspinal muscle oxygenation, mechanical efficiency, and the V̇O2 onset kinetics were improved (P<0.05). All measures remained stable during the three-month follow-up except for paraspinal muscle oxygenation, which deteriorated (P<0.05). Return-to-work was associated with the level of workday physical activities and to a decrease in fear-avoidance beliefs.Conclusions: At the end of the program, aerobic metabolism responses were improved in paraspinal muscles in patients. These improvements were not associated with return-to-work, which was primarily influenced by socio-psychological factor

Evidence of exercise-induced O2 arterial desaturation in non-elite sportsmen and sportswomen following high-intensity interval-training

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Heterogeneity of muscle deoxygenation kinetics during two bouts of repeated heavy exercises

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