Isolation and monoculture of functional primary astrocytes from the adult mouse spinal cord

Astrocytes are a widely heterogenic cell population that play major roles in central nervous system (CNS) homeostasis and neurotransmission, as well as in various neuropathologies, including spinal cord injury (SCI), traumatic brain injury, and neurodegenerative diseases, such as amyotrophic lateral sclerosis. Spinal cord astrocytes have distinct differences from those in the brain and accurate modeling of disease states is necessary for understanding disease progression and developing therapeutic interventions. Several limitations to modeling spinal cord astrocytes in vitro exist, including lack of commercially available adult-derived cells, lack of purchasable astrocytes with different genotypes, as well as time-consuming and costly in-house primary cell isolations that often result in low yield due to small tissue volume. To address these issues, we developed an efficient adult mouse spinal cord astrocyte isolation method that utilizes enzymatic digestion, debris filtration, and multiple ACSA-2 magnetic microbead purification cycles to achieve an astrocyte monoculture purity of ≅93–98%, based on all markers assessed. Importantly, the isolated cells contain active mitochondria and express key astrocyte markers including ACSA-1, ACSA-2, EAAT2, and GFAP. Furthermore, this isolation method can be applied to the spinal cord of male and female mice, mice subjected to SCI, and genetically modified mice. We present a primary adult mouse spinal cord astrocyte isolation protocol focused on purity, viability, and length of isolation that can be applied to a multitude of models and aid in targeted research on spinal-cord related CNS processes and pathologies

Absolutely continuous invariant measures for random non-uniformly expanding maps

We prove existence of (at most denumerable many) absolutely continuous invariant probability measures for random one-dimensional dynamical systems with asymptotic expansion. If the rate of expansion (Lyapunov exponents) is bounded away from zero, we obtain finitely many ergodic absolutely continuous invariant probability measures, describing the asymptotics of almost every point. We also prove a similar result for higher-dimensional random non-uniformly expanding dynamical systems. The results are consequences of the construction of such measures for skew-products with essentially arbitrary base dynamics and asymptotic expansion along the fibers. In both cases our method deals with either critical or singular points for the random maps.Comment: 30 pages; 2 figures. Keywords: non-uniform expansion, random dynamics, slow recurrence, singular and critical set, absolutely continuous invariant measures, skew-product. To appear in Math Z, 201

Chronic high glucose downregulates mitochondrial calpain 10 and contributes to renal cell death and diabetes-induced renal injury

Whereas most calpains are cytosolic proteases, calpain 10 is resident in mitochondria and is important in mitochondrial homeostasis. Because calpain 10 has been implicated in type 2 diabetes, we studied its possible role in diabetes-induced renal dysfunction. We treated renal proximal tubular cells with high glucose (17mmol/l) and found decreased mitochondrial calpain 10 mRNA and protein at 96h compared with cells incubated with 0 or 5mmol/l glucose or 17mmol/l D-mannitol. High glucose increased mitochondrial calpain 10 substrates (NDUFB8 and ATP synthase β), decreased basal and uncoupled respiration, and initiated cell apoptosis as indicated by cleaved caspase 3 and nuclear condensation. Renal calpain 10 protein and mRNA were specifically decreased in streptozotocin-induced diabetic rats with kidney dysfunction, and in diabetic ob/ob mice. In agreement with our in vitro data, the kidneys of streptozotocin-induced diabetic rats had elevated calpain 10 substrates and cleaved caspase 3. Finally, specific siRNA-induced knockdown of calpain 10 in the proximal tubules of control rats resulted in decreased renal function as evidenced by increased serum creatinine, and increased caspase 3 cleavage compared with rats receiving scrambled siRNA. Thus, the glucose-induced loss of calpain 10 in vivo results in renal cell apoptosis and organ failure through accumulation of mitochondrial calpain 10 substrates and mitochondrial dysfunction. Whether this is a major cause of the decreased renal function in diabetic nephropathy will require further studies

Deploying gas power with CCS: The role of operational flexibility, merit order and the future energy system

Combined cycle gas turbine (CCGT) power plants are an important part of many electricity systems. By fitting them with carbon capture their CO2 emissions could be virtually eliminated. We evaluate CCGT plants with different variations of post combustion capture using amine solvents, covering a range of options, including solvent storage, partial capture and shifting the energy penalty in time. The analysis is based on the UK electricity system in 2025. The behaviour of individual CCGT plants is governed by the plant’s place in the merit order and to a lesser extent by CO2 reduction targets for the electricity system. In the UK, CCGT plants built from 2016 onwards will emit ~90% of the CO2 emissions of the whole CCGT fleet in 2025. The typical ‘base case’ CCGT plant with capture is designed to capture 90% of the CO2 emissions and to operate dynamically with the power plant. Downsizing the capture facility could be attractive for low-merit plants, i.e. plants with high short-run marginal costs. Solvent storage enables electricity generation to be decoupled in time from the energy penalty associated with carbon capture. Beyond a few minutes of solvent storage, substantial tanks would be needed. If solvent storage is to play an important role, it will require definitions of ‘capture ready’ to be expanded to ensure sufficient land is available

Loss of calpain 10 causes mitochondrial dysfunction during chronic hyperglycemia

We showed that renal calpain 10, a mitochondrial and cytosolic Ca(2+)-regulated cysteine protease, is specifically decreased in kidneys of diabetic rats and mice, and is associated with diabetic nephropathy. The goals of this study were to examine renal calpain 10 and mitochondrial dysfunction in streptozotocin-induced hyperglycemic rats and determine the effects of siRNA-mediated knock down of renal calpain 10 on mitochondrial function. Four weeks after streptozotocin injection, calpain 10 protein and mRNA were decreased and calpain 10 substrates accumulated. We detected increased state 2 respiration in isolated renal mitochondria and increased markers of mitochondrial fission and mitophagy. All changes were prevented by daily insulin injection. Compared to scrambled siRNA, calpain 10 siRNA resulted in a marked decrease in renal calpain 10 at 2, 5 and 7 days. In concert with the loss of renal calpain 10, calpain 10 substrates accumulated, mitochondrial fusion decreased, mitochondrial fission and mitophagy increased. In summary, insulin-sensitive hyperglycemia induced loss of renal calpain 10 is correlated with renal mitochondrial dysfunction, fission and mitophagy, and specific depletion of renal calpain 10 produces similar mitochondrial defects. These results provide evidence that diabetes-induced renal mitochondrial dysfunction and renal injury may directly result from the loss of renal calpain 10

Design, Development, Physicochemical Characterization, and In Vitro Drug Release of Formoterol PEGylated PLGA Polymeric Nanoparticles

Polymeric nanoparticles’ drug delivery systems represent a promising platform for targeted controlled release since they are capable of improving the bioavailability and tissue localization of drugs compared to traditional means of administration. Investigation of key parameters of nanoparticle preparation and their impact on performance, such as size, drug loading, and sustained release, is critical to understanding the synthesis parameters surrounding a given nanoparticle formulation. This comprehensive and systematic study reports for the first time and focuses on the development and characterization of formoterol polymeric nanoparticles that have potential application in a variety of acute and chronic diseases. Nanoparticles were prepared by a variety of solvent emulsion methods with varying modifications to the polymer and emulsion system with the aim of increasing drug loading and tuning particle size for renal localization and drug delivery. Maximal drug loading was achieved by amine modification of polyethylene glycol (PEG) conjugated to the poly(lactic-co-glycolic acid) (PLGA) backbone. The resulting formoterol PEGylated PLGA polymeric nanoparticles were successfully lyophilized without compromising size distribution by using either sucrose or trehalose as cryoprotectants. The physicochemical characteristics of the nanoparticles were examined comprehensively, including surface morphology, solid-state transitions, crystallinity, and residual water content. In vitro formoterol drug release characteristics from the PEGylated PLGA polymeric nanoparticles were also investigated as a function of both polymer and emulsion parameter selection, and release kinetics modeling was successfully applied. © 2022 by the authors. Licensee MDPI, Basel, Switzerland.Open access journalThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]