Neuroinflammation is a hallmark of various neurological disorders including autoimmune-, neurodegenerative and neuropsychiatric diseases. In neuroinflammation, activated microglia and astrocytes ...release soluble mediators such as cytokines, glutamate, and reactive oxygen species that negatively affect neuronal function and viability, and thus contribute to neurodegeneration during disease progression. Therefore, the development of neuroprotective strategies might be important in addition to treating inflammation in these diseases. Mitochondria are promising cellular targets for neuroprotective interventions: They are among the first structures affected in many neuroinflammatory diseases, with mitochondrial impairment ranging from impaired respiratory activity and reduced mitochondrial membrane potential to mitochondrial oxidation and fragmentation. Therefore, we developed a cell culture model that resembles an early state of inflammation-induced neuronal mitochondrial dysfunction preceding neuronal cell death, and can be used to test mito- and neuroprotective strategies. Rat primary cortical neurons were challenged with conditioned medium from mixed primary cultures of rat microglia and astrocytes that had been activated with lipopolysaccharide and ATP. When sublethal amounts of glia-conditioned medium were added to neurons for 24 h, mitochondrial membrane potential and ATP levels were decreased, whereas mitochondrial redox state remained unaffected. Effects on mitochondrial membrane potential and ATP levels were ameliorated by knock-down of the mitochondrial calcium uniporter in neurons. This study suggests that neuronal bioenergetic failure is an early event during neuroinflammation and it identifies the mitochondrial calcium uniporter as a candidate target for neuroprotection in this context.
•Neuronal mitochondrial membrane potential is particularly sensitive to inflammation.•Neuronal bioenergetic impairment occurs prior to cell death in neuroinflammation.•Inflammation-induced functional impairment of neuronal mitochondria involves Mcu.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Misfolded proteins in the endoplasmic reticulum (ER) activate the unfolded protein response (UPR), which enhances protein folding to restore homeostasis. Additional pathways respond to ER stress, but ...how they help counteract protein misfolding is incompletely understood. Here, we develop a titratable system for the induction of ER stress in yeast to enable a genetic screen for factors that augment stress resistance independently of the UPR. We identify the proteasome biogenesis regulator Rpn4 and show that it cooperates with the UPR. Rpn4 abundance increases during ER stress, first by a post-transcriptional, then by a transcriptional mechanism. Induction of
transcription is triggered by cytosolic mislocalization of secretory proteins, is mediated by multiple signaling pathways and accelerates clearance of misfolded proteins from the cytosol. Thus, Rpn4 and the UPR are complementary elements of a modular cross-compartment response to ER stress.
The inter-critically reheated grain coarsened heat affected zone (IC GC HAZ) has been reported as one of the most brittle section of high-strength low-alloy (HSLA) steels welds. The presence of ...micro-alloying elements in HSLA steels induces the formation of microstructural constituents, capable to improve the mechanical performance of welded joints. Following double welding thermal cycle, with second peak temperature in the range between Ac1 and Ac3, the IC GC HAZ undergoes a strong loss of toughness and fatigue resistance, mainly caused by the formation of residual austenite (RA). The present study aims to investigate the behavior of IC GC HAZ of a S355 steel grade, with the addition of different vanadium contents. The influence of vanadium micro-alloying on the microstructural variation, RA fraction formation and precipitation state of samples subjected to thermal cycles experienced during double-pass welding was reported. Double-pass welding thermal cycles were reproduced by heat treatment using a dilatometer at five different maximum temperatures of the secondary peak in the inter-critical area, from 720 °C to 790 °C. Although after the heat treatment it appears that the addition of V favors the formation of residual austenite, the amount of residual austenite formed is not significant for inducing detrimental effects (from the EBSD analysis the values are always less than 0.6%). Moreover, the precipitation state for the variant with 0.1 wt.% of V (high content) showed the presence of vanadium rich precipitates with size smaller than 60 nm of which, more than 50% are smaller than 15 nm.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Cell metabolism reprogramming to sustain energy production, while reducing oxygen and energy consuming processes is crucially important for the adaptation to hypoxia/ischemia. Adaptive metabolic ...rewiring is controlled by hypoxia-inducible factors (HIFs). Accumulating experimental evidence indicates that timely activation of HIF in brain-resident cells improves the outcome from acute ischemic stroke. However, the underlying molecular mechanisms are still incompletely understood. Thus, we investigated whether HIF-dependent metabolic reprogramming affects the vulnerability of brain-resident cells towards ischemic stress.
We used genetic and pharmacological approaches to activate HIF in the murine brain
and in primary neurons and astrocytes
. Numerous metabolomic approaches and molecular biological techniques were applied to elucidate potential HIF-dependent effects on the central carbon metabolism of brain cells. In animal and cell models of ischemic stroke, we analysed whether HIF-dependent metabolic reprogramming influences the susceptibility to ischemic injury.
Neuron-specific gene ablation of prolyl-4-hydroxylase domain 2 (PHD2) protein, negatively regulating the protein stability of HIF-α in an oxygen dependent manner, reduced brain injury and functional impairment of mice after acute stroke in a HIF-dependent manner. Accordingly, PHD2 deficient neurons showed an improved tolerance towards ischemic stress
, which was accompanied by enhanced HIF-1-mediated glycolytic lactate production through pyruvate dehydrogenase kinase-mediated inhibition of the pyruvate dehydrogenase. Systemic treatment of mice with roxadustat, a low-molecular weight pan-PHD inhibitor, not only increased the abundance of numerous metabolites of the central carbon and amino acid metabolism in murine brain, but also ameliorated cerebral tissue damage and sensorimotor dysfunction after acute ischemic stroke. In neurons and astrocytes roxadustat provoked a HIF-1-dependent glucose metabolism reprogramming including elevation of glucose uptake, glycogen synthesis, glycolytic capacity, lactate production and lactate release, which enhanced the ischemic tolerance of astrocytes, but not neurons. We found that strong activation of HIF-1 in neurons by non-selective inhibition of all PHD isoenzymes caused a HIF-1-dependent upregulation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 redirecting glucose-6-phosphate from pentose phosphate pathway (PPP) to the glycolysis pathway. This was accompanied by a reduction of NADPH production in the PPP, which further decreased the low intrinsic antioxidant reserve of neurons, making them more susceptible to ischemic stress. Nonetheless, in organotypic hippocampal cultures with preserved neuronal-glial interactions roxadustat decreased the neuronal susceptibility to ischemic stress, which was largely prevented by restricting glycolytic energy production through lactate transport blockade.
Collectively, our results indicate that HIF-1-mediated metabolic reprogramming alleviates the intrinsic vulnerability of brain-resident cells to ischemic stress.
When faced with proteotoxic stress, cells mount adaptive responses to eliminate aberrant proteins. Adaptive responses increase the expression of protein folding and degradation factors to enhance the ...cellular quality control machinery. However, it is unclear whether and how this augmented machinery acquires new activities during stress. Here, we uncover a regulatory cascade in budding yeast that consists of the hydrophilin protein Roq1/Yjl144w, the HtrA-type protease Ynm3/Nma111, and the ubiquitin ligase Ubr1. Various stresses stimulate ROQ1 transcription. The Roq1 protein is cleaved by Ynm3. Cleaved Roq1 interacts with Ubr1, transforming its substrate specificity. Altered substrate recognition by Ubr1 accelerates proteasomal degradation of misfolded as well as native proteins at the endoplasmic reticulum membrane and in the cytosol. We term this pathway stress-induced homeostatically regulated protein degradation (SHRED) and propose that it promotes physiological adaptation by reprogramming a key component of the quality control machinery.
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•Stress induces the synthesis of Roq1, which undergoes cleavage by the protease Ynm3•Cleaved Roq1 modulates substrate specificity of the N-end rule ubiquitin ligase Ubr1•Roq1-bound Ubr1 promotes selective proteasomal degradation of misfolded proteins•This pathway, termed SHRED, adapts protein quality control to changing cellular needs
Szoradi et al. uncover SHRED, a stress response pathway that hinges on the previously uncharacterized regulatory protein Roq1. Proteolytically cleaved Roq1 binds to the N-end rule ubiquitin ligase Ubr1 as a pseudosubstrate and directs Ubr1 toward misfolded proteins, thereby promoting the degradation of aberrant proteins.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The antibacterial activity of a calixarene derivative, p-tert-butylcalix6arene (Calix6), was assessed and was shown not to inhibit the growth of E. coli, S. aureus and B. subtilis bacteria. With the ...aim of gaining more insights into the absence of antibacterial activity of Calix6, the interaction of this derivative with DPPG, a bacterial cell membrane lipid, was studied. Langmuir monolayers were used as the model membrane. Pure DPPG and pure Calix6 monolayers, as well as binary DPPG:Calix6 mixtures were studied using surface pressure measurements, compressional modulus, Brewster angle and fluorescence microscopies, ellipsometry, polarization-modulation infrared reflection absorption spectroscopy and molecular dynamics simulations. Thermodynamic properties of the mixed monolayers were additionally calculated using thermodynamic parameters. The analysis of isotherms showed that Calix6 significantly affects the DPPG monolayers, modifying the isotherm profile and increasing the molecular area, in agreement with the molecular dynamics simulations. The presence of Calix6 in the mixed monolayers decreased the interfacial elasticity, indicating that calixarene disrupts the strong intermolecular interactions of DPPG hindering its organization into a compact arrangement. At low molar ratios of Calix6, the DPPG:Calix6 interactions are preferentially attractive, due to the interactions between the hydrophobic tails of DPPG and the tert-butyl groups of Calix6. Increasing the proportion of calixarene generates repulsive interactions. Calix6 significantly affects the hydrophobic tail organization, which was confirmed by PM-IRRAS measurements. Calix6 appears to be expelled from the mixed films at a biologically relevant surface pressure, π = 30 mN m-1, indicating a low interaction with the cell membrane model related to the absence of antibacterial activity.
Abstract Background context The publication of several recent studies showing minimal benefit for radiofrequency (RF) lumbar zygapophysial (l-z) joint denervation have led many investigators to ...reevaluate selection criteria. One controversial explanation for these findings is that the most commonly used cutoff value for selecting patients for l-z (facet) joint RF denervation, greater than 50% pain relief after diagnostic blocks, is too low and hence responsible for the high failure rate. Purpose To compare l-z joint RF denervation success rates between the conventional greater than or equal to 50% pain relief threshold and the more stringently proposed greater than or equal to 80% cutoff for diagnostic medial branch blocks (MBB). Study design/setting Multicenter, retrospective clinical data analysis. Patient sample Two hundred and sixty-two patients with chronic low back pain who underwent l-z RF denervation at three pain clinics. Outcome measures Outcome measures were greater than 50% pain relief based on visual analog scale or numerical pain rating score after RF denervation persisting at least 6 months postprocedure, and global perceived effect (GPE), which considered pain relief, satisfaction and functional improvement. Methods Data were garnered at three centers on 262 patients who underwent l-z RF denervation after obtaining greater than or equal to 50% pain relief after diagnostic MBB. Subjects were separated into those who received partial (greater than or equal to 50% but less than 80%) and near-complete (greater than or equal to 80%) pain relief from the MBB. Outcomes between groups were compared with multivariate analysis after controlling for 14 demographic and clinical variables. Results One hundred and forty-five patients obtained greater than or equal to 50% but less than 80% pain relief after diagnostic MBB, and 117 patients obtained greater than or equal to 80% relief. In the greater than or equal to 50% group, success rates were 52% and 67% based on pain relief and GPE, respectively. Among patients who experienced greater than 80% relief from diagnostic blocks, 56% obtained greater than or equal to 50% relief from RF denervation and 66% had a positive GPE. Conclusions Using more stringent pain relief criteria when selecting patients for l-z joint RF denervation is unlikely to improve success rates, and may lead to misdiagnosis and withholding a potentially valuable treatment from good candidates.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
A finite-element analysis is used to explore the impact of elastic material properties, boundary conditions, and geometry, including coiling, on the spatial characteristics of the compliance of the ...unloaded basilar membrane (BM). It is assumed that the arcuate zone is isotropic and the pectinate zone orthotropic, and that the radial component of the effective Young's modulus in the pectinate zone decreases exponentially with distance from base to apex. The results concur with tonotopic characteristics of compliance and neural data. Moreover, whereas the maximum compliance in a radial profile is located close to the boundary between the two zones in the basal region, it shifts to the midpoint of the pectinate zone for the apical BM; the width of the profile also expands. This shift begins near the 1 kHz characteristic place for guinea pig and the 2.4 kHz place for gerbil. Shift and expansion are not observed for linear rather than exponential decrease of the radial component of Young's modulus. This spatial change of the compliance profile leads to the prediction that mechanical excitation in the apical region of the organ of Corti is different to that in the basal region.
Thanks to the progress made in instruments and techniques, the methods in physical chemistry have developed rapidly over the past few decades, making them increasingly valuable for scientists of many ...disciplines. These two must-have volumes meet the needs of the scientific community for a thorough overview of all the important methods currently used. As such, this work bridges the gap between standard textbooks and review articles, covering a large number of methods, as well as the motivation behind their use. A uniform approach is adopted throughout both volumes, while the critical comparison of the advantages and disadvantages of each method makes this a valuable reference for physical chemists and other scientists working with these techniques.
The modeling of the mechanical process of hearing requires an accurate geometrical model of the inner ear (cochlea). The purpose of this study was the creation of a 3-D model of the fluid chambers of ...Guinea pig cochlea, which could serve as a basis for further mechanical modeling. Micro computer tomography used in this study is a noninvasive method to visualize bony structures. The visualization of the membranous labyrinth was achieved by additional staining of the specimen with OsO
4. The resulting stack of images has been transformed into a cylindrical coordinate system. To suppress noise on tomography images, a nonlinear smoothing method, anisotropic diffusion, were applied. A new approach has been proposed to estimate algorithm parameters automatically. Then, a segmentation using active contours (snakes) was performed. In this study, a new energy linking the contours on adjacent slices has been added to the standard approach. This compensates the inconsistencies between adjacent contours. The images segmented in this way were used as a basis for a 3-D reconstruction of the hearing organ.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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