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Individuals with chronic kidney disease have elevated levels of oxidative stress and are at a significantly higher risk of skeletal fracture. Advanced glycation end products (AGEs), which accumulate in bone and compromise mechanical properties, are known to be driven in part by oxidative stress. The goal of this study was to study effects of N-acetylcysteine (NAC) on reducing oxidative stress and improving various bone parameters, most specifically mechanical properties, in an animal model of progressive CKD. Male Cy/+ (CKD) rats and unaffected littermates were untreated (controls) or treated with NAC (80 mg/kg, IP) from 30 to 35 weeks of age. Endpoint measures included serum biochemistries, assessments of systemic oxidative stress, bone morphology, and mechanical properties, and AGE levels in the bone. CKD rats had the expected phenotype that included low kidney function, elevated parathyroid hormone, higher cortical porosity, and compromised mechanical properties. NAC treatment had mixed effects on oxidative stress markers, significantly reducing TBARS (a measure of lipid peroxidation) while not affecting 8-OHdG (a marker of DNA oxidation) levels. AGE levels in the bone were elevated in CKD animals and were reduced with NAC although this did not translate to a benefit in bone mechanical properties. In conclusion, NAC failed to significantly improve bone architecture/geometry/mechanical properties in our rat model of progressive CKD.
BACKGROUND - Previous studies in our laboratory have demonstrated that a magnetic resonance imaging method called diffusion tensor imaging (DTI) can differentiate between crush and complete transection peripheral nerve injuries in a rat model ex vivo. DTI measures the directionally dependent effect of tissue barriers on the random diffusion of water molecules. In ordered tissues such as nerves, this information can be used to reconstruct the primary direction of diffusion along fiber tracts, which may provide information on fiber tract continuity after nerve injury and surgical repair.
METHODS - Sprague-Dawley rats were treated with different degrees of partial transection of the sciatic nerve followed by immediate repair and euthanized after 1 week of recovery. Nerves were then harvested, fixed, and scanned with a 7 Tesla magnetic resonance imaging to obtain DTIand fiber tractography in each sample. Additional behavioral (sciatic function index, foot fault asymmetry) and histological (Toluidine blue staining) assessments were performed for validation.
RESULTS - Tractography yielded a visual representation of the degree of injury that correlated with behavioral and histological evaluations.
CONCLUSIONS - DTI tractography is a noninvasive tool that can yield a visual representation of a partial nerve transection as early as 1 week after surgical repair.
© The Association of Military Surgeons of the United States. All rights reserved. For permissions, please e-mail: firstname.lastname@example.org.
Spinal cord injuries (SCIs) are a leading cause of disability and can severely impact the quality of life. However, to date, the processes of spontaneous repair of damaged spinal cord remain incompletely understood, partly due to a lack of appropriate longitudinal tracking methods. Noninvasive, multiparametric magnetic resonance imaging (MRI) provides potential biomarkers for the comprehensive evaluation of spontaneous repair after SCI. In this study in rats, a clinically relevant contusion injury was introduced at the lumbar level that impairs both hindlimb motor and sensory functions. Quantitative MRI measurements were acquired at baseline and serially post-SCI for up to 2 wk. The progressions of injury and spontaneous recovery in both white and gray matter were tracked longitudinally using pool-size ratio (PSR) measurements derived from quantitative magnetization transfer (qMT) methods, measurements of water diffusion parameters using diffusion tensor imaging (DTI) and intrasegment functional connectivity derived from resting state functional MRI. Changes in these quantitative imaging measurements were correlated with behavioral readouts. We found (a) a progressive decrease in PSR values within 2 wk post-SCI, indicating a progressive demyelination at the center of the injury that was validated with histological staining, (b) PSR correlated closely with fractional anisotropy and transverse relaxation of free water, but did not show significant correlations with behavioral recovery, and (c) preliminary evidence that SCI induced a decrease in functional connectivity between dorsal horns below the injury site at 24 h. Findings from this study not only confirm the value of qMT and DTI methods for assessing the myelination state of injured spinal cord but indicate that they may also have further implications on whether therapies targeted towards remyelination may be appropriate. Additionally, a better understanding of changes after SCI provides valuable information to guide and assess interventions.
© 2020 John Wiley & Sons, Ltd.
Exploring the interactions between the Ca binding protein calmodulin (CaM) and its target proteins remains a challenging task. Members of the Munc13 protein family play an essential role in short-term synaptic plasticity, modulated via the interaction with CaM at the presynaptic compartment. In this study, we focus on the bMunc13-2 isoform expressed in the brain, as strong changes in synaptic transmission were observed upon its mutagenesis or deletion. The CaM‒bMunc13-2 interaction was previously characterized at the molecular level using short bMunc13-2-derived peptides only, revealing a classical 1‒5‒10 CaM binding motif. Using larger protein constructs, we have now identified for the first time a novel and unique CaM binding site in bMunc13-2 that contains an -terminal extension of a classical 1‒5‒10 CaM binding motif. We characterize this motif using a range of biochemical and biophysical methods and highlight its importance for the CaM‒bMunc13-2 interaction.
Nerve regeneration after injury must occur in a timely fashion to restore function. Unfortunately, current methods (e.g., electrophysiology) provide limited information following trauma, resulting in delayed management and suboptimal outcomes. Herein, we evaluated the ability of diffusion MRI to monitor nerve regeneration after injury/repair. Sprague-Dawley rats were divided into three treatment groups (sham = 21, crush = 23, cut/repair = 19) and ex vivo diffusion tensor imaging (DTI) and diffusion kurtosis imaging (DKI) was performed 1-12 weeks post-surgery. Behavioral data showed a distinction between crush and cut/repair nerves at 4 weeks. This was consistent with DTI, which found that thresholds based on the ratio of radial and axial diffusivities (RD/AD = 0.40 ± 0.02) and fractional anisotropy (FA = 0.53 ± 0.01) differentiated crush from cut/repair injuries. By the 12 week, cut/repair nerves whose behavioral data indicated a partial recovery were below the RD/AD threshold (and above the FA threshold), while nerves that did not recover were on the opposite side of each threshold. Additional morphometric analysis indicated that DTI-derived normalized scalar indices report on axon density (RD/AD: r = -0.54, p < 1e-3; FA: r = 0.56, p < 1e-3). Interestingly, higher-order DKI analyses did not improve our ability classify recovery. These findings suggest that DTI may provide promising biomarkers for distinguishing successful/unsuccessful nerve repairs and potentially identify cases that require reoperation.
Human but not mouse islets transplanted into immunodeficient NSG mice effectively accumulate lipid droplets (LDs). Because chronic lipid exposure is associated with islet β-cell dysfunction, we investigated LD accumulation in the intact human and mouse pancreas over a range of ages and states of diabetes. Very few LDs were found in normal human juvenile pancreatic acinar and islet cells, with numbers subsequently increasing throughout adulthood. While accumulation appeared evenly distributed in postjuvenile acinar and islet cells in donors without diabetes, LDs were enriched in islet α- and β-cells from donors with type 2 diabetes (T2D). LDs were also found in the islet β-like cells produced from human embryonic cell-derived β-cell clusters. In contrast, LD accumulation was nearly undetectable in the adult rodent pancreas, even in hyperglycemic and hyperlipidemic models or 1.5-year-old mice. Taken together, there appear to be significant differences in pancreas islet cell lipid handling between species, and the human juvenile and adult cell populations. Moreover, our results suggest that LD enrichment could be impactful to T2D islet cell function.
© 2019 by the American Diabetes Association.
In the brain, AMPA-type glutamate receptors (AMPARs) form complexes with their auxiliary subunits and mediate the majority of fast excitatory neurotransmission. Signals transduced by these complexes are critical for synaptic plasticity, learning, and memory. The two major categories of AMPAR auxiliary subunits are transmembrane AMPAR regulatory proteins (TARPs) and cornichon homologs (CNIHs); these subunits share little homology and play distinct roles in controlling ion channel gating and trafficking of AMPAR. Here, I report high-resolution cryo-electron microscopy structures of AMPAR in complex with CNIH3. Contrary to its predicted membrane topology, CNIH3 lacks an extracellular domain and instead contains four membrane-spanning helices. The protein-protein interaction interface that dictates channel modulation and the lipids surrounding the complex are revealed. These structures provide insights into the molecular mechanism for ion channel modulation and assembly of AMPAR/CNIH3 complexes.
Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.
This Letter details our efforts to develop new M PAM scaffolds with improved pharmacological properties. This endeavor involved replacing the 3,4-dimethylpyridazine core with two novel cores: a 2,3-dimethyl-2H-indazole-5-carboxamide core or a 1-methyl-1H-benzo[d][1,2,3]triazole-6-carboxamide core. Due to shallow SAR, these cores were further evolved into two unique tricyclic cores: an 8,9-dimethyl-8H-pyrazolo[3,4-h]quinazoline core and an 1-methyl-1H-[1,2,3]triazolo[4,5-h]quinazoline core. Both tricyclic cores displayed low nanomolar potency against both human and rat M.
Copyright © 2019 Elsevier Ltd. All rights reserved.
Identifying early indicators of toxicant-induced organ damage is critical to provide effective treatment. To discover such indicators and the underlying mechanisms of toxicity, we used gentamicin as an exemplar kidney toxicant and performed systematic perturbation studies in Sprague Dawley rats. We obtained high-throughput data 7 and 13 h after administration of a single dose of gentamicin (0.5 g/kg) and identified global changes in genes in the liver and kidneys, metabolites in the plasma and urine, and absolute fluxes in central carbon metabolism. We used these measured changes in genes in the liver and kidney as constraints to a rat multitissue genome-scale metabolic network model to investigate the mechanism of gentamicin-induced kidney toxicity and identify metabolites associated with changes in tissue gene expression. Our experimental analysis revealed that gentamicin-induced metabolic perturbations could be detected as early as 7 h postexposure. Our integrated systems-level analyses suggest that changes in kidney gene expression drive most of the significant metabolite alterations in the urine. The analyses thus allowed us to identify several significantly enriched injury-specific pathways in the kidney underlying gentamicin-induced toxicity, as well as metabolites in these pathways that could serve as potential early indicators of kidney damage.
© The Author(s) 2020. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please e-mail: email@example.com.
Peptides and biologics provide unique opportunities to modulate intracellular targets not druggable by conventional small molecules. Most peptides and biologics are fused with cationic uptake moieties or formulated into nanoparticles to facilitate delivery, but these systems typically lack potency due to low uptake and/or entrapment and degradation in endolysosomal compartments. Because most delivery reagents comprise cationic lipids or polymers, there is a lack of reagents specifically optimized to deliver cationic cargo. Herein, we demonstrate the utility of the cytocompatible polymer poly(propylacrylic acid) (PPAA) to potentiate intracellular delivery of cationic biomacromolecules and nano-formulations. This approach demonstrates superior efficacy over all marketed peptide delivery reagents and enhances delivery of nucleic acids and gene editing ribonucleoproteins (RNPs) formulated with both commercially-available and our own custom-synthesized cationic polymer delivery reagents. These results demonstrate the broad potential of PPAA to serve as a platform reagent for the intracellular delivery of cationic cargo.