The circadian clock time tunes axonal regeneration.

Nerve injuries cause permanent neurological disability due to limited axonal regeneration. Injury-dependent and -independent mechanisms have provided important insight into neuronal regeneration, however, common denominators underpinning regeneration remain elusive. A comparative analysis of transcriptomic datasets associated with neuronal regenerative ability revealed circadian rhythms as the most significantly enriched pathway. Subsequently, we demonstrated that sensory neurons possess an endogenous clock and that their regenerative ability displays diurnal oscillations in a murine model of sciatic nerve injury. Consistently, transcriptomic analysis showed a time-of-day-dependent enrichment for processes associated with axonal regeneration and the circadian clock. Conditional deletion experiments demonstrated that Bmal1 is required for neuronal intrinsic circadian regeneration and target re-innervation. Lastly, lithium enhanced nerve regeneration in wild-type but not in clock-deficient mice. Together, these findings demonstrate that the molecular clock fine-tunes the regenerative ability of sensory neurons and propose compounds affecting clock pathways as a novel approach to nerve repair.

Improved peridynamic model and its application to crack propagation in rocks.

The conventional bond-based peridynamics (BB-PD) model is suitable for simulating the failure mode of homogeneous elastic-brittle materials. However, the strain hardening and subsequent strain softening characteristics of rocks under loading cannot be reflected. In addition, the fracture mechanisms of rock materials under tension and compression are completely different. To solve these problems, this paper proposes an improved BB-PD model using different fracture criteria in the tensile and compression stages of the bond based on previous improved models, and a critical failure condition obeying the Weibull distribution is introduced to reflect the heterogeneity of the rock. The crack propagation processes of an intact rock specimen, rock specimen with a single pre-existing flaw and rock specimens with two and three pre-existing flaws under compressive loading are simulated using the model, and its feasibility is verified by comparing with the results of previous laboratory tests. Next, the effects of the inclination angle and length on the wing crack propagation length are studied. Finally, the changes in the crack aggregation modes under different rock bridge inclination angles are simulated. Eight types of crack aggregation modes are found, and the conditions under which they may occur are analysed. The improved model proposed can effectively simulate the crack propagation and coalescing processes and has a wide application prospect for rock fracture simulations.

CBP/p300 activation promotes axon growth, sprouting, and synaptic plasticity in chronic experimental spinal cord injury with severe disability.

The interruption of spinal circuitry following spinal cord injury (SCI) disrupts neural activity and is followed by a failure to mount an effective regenerative response resulting in permanent neurological disability. Functional recovery requires the enhancement of axonal and synaptic plasticity of spared as well as injured fibres, which need to sprout and/or regenerate to form new connections. Here, we have investigated whether the epigenetic stimulation of the regenerative gene expression program can overcome the current inability to promote neurological recovery in chronic SCI with severe disability. We delivered the CBP/p300 activator CSP-TTK21 or vehicle CSP weekly between week 12 and 22 following a transection model of SCI in mice housed in an enriched environment. Data analysis showed that CSP-TTK21 enhanced classical regenerative signalling in dorsal root ganglia sensory but not cortical motor neurons, stimulated motor and sensory axon growth, sprouting, and synaptic plasticity, but failed to promote neurological sensorimotor recovery. This work provides direct evidence that clinically suitable pharmacological CBP/p300 activation can promote the expression of regeneration-associated genes and axonal growth in a chronic SCI with severe neurological disability.

Simulations of Fractures of Heterogeneous Orthotropic Fiber-Reinforced Concrete with Pre-Existing Flaws Using an Improved Peridynamic Model.

The propagation and coalescence of cracks in fiber-reinforced concretes (FRCs) is the direct cause of instability in many engineering structures. To predict the crack propagation path and failure mode of FRCs, an orthotropic-bond-based peridynamic (PD) model was established in this study. A kernel function reflecting long-range force was introduced, and the fiber bond was used to describe the macroanisotropy of the FRC. The crack propagation process of the FRC plate with flaws was simulated under uniaxial tensile loading. The results showed that under homogeneous conditions, the cracks formed along the centerline of the isotropic concrete propagate in a direction perpendicular to the load. Under anisotropic conditions, the cracks propagate strictly in the direction of the fiber bond. The failure degree of the FRC increases with the increase in heterogeneity. When the shape parameter is 10 and the fiber bond is 0°, the failure mode changes from tensile to shear failure. When the fiber bond is 45°, the FRC changes from a state where outer cracks penetrate the entire specimen to a state where cracks coalesce at the middle. It was found that the improved model can effectively simulate the crack propagation processes of orthotropic FRC materials.

The gut metabolite indole-3 propionate promotes nerve regeneration and repair.

The regenerative potential of mammalian peripheral nervous system neurons after injury is critically limited by their slow axonal regenerative rate1. Regenerative ability is influenced by both injury-dependent and injury-independent mechanisms2. Among the latter, environmental factors such as exercise and environmental enrichment have been shown to affect signalling pathways that promote axonal regeneration3. Several of these pathways, including modifications in gene transcription and protein synthesis, mitochondrial metabolism and the release of neurotrophins, can be activated by intermittent fasting (IF)4,5. However, whether IF influences the axonal regenerative ability remains to be investigated. Here we show that IF promotes axonal regeneration after sciatic nerve crush in mice through an unexpected mechanism that relies on the gram-positive gut microbiome and an increase in the gut bacteria-derived metabolite indole-3-propionic acid (IPA) in the serum. IPA production by Clostridium sporogenes is required for efficient axonal regeneration, and delivery of IPA after sciatic injury significantly enhances axonal regeneration, accelerating the recovery of sensory function. Mechanistically, RNA sequencing analysis from sciatic dorsal root ganglia suggested a role for neutrophil chemotaxis in the IPA-dependent regenerative phenotype, which was confirmed by inhibition of neutrophil chemotaxis. Our results demonstrate the ability of a microbiome-derived metabolite, such as IPA, to facilitate regeneration and functional recovery of sensory axons through an immune-mediated mechanism.

Reversible CD8 T cell-neuron cross-talk causes aging-dependent neuronal regenerative decline.

Aging is associated with increased prevalence of axonal injuries characterized by poor regeneration and disability. However, the underlying mechanisms remain unclear. In our experiments, RNA sequencing of sciatic dorsal root ganglia (DRG) revealed significant aging-dependent enrichment in T cell signaling both before and after sciatic nerve injury (SNI) in mice. Lymphotoxin activated the transcription factor NF-κB, which induced expression of the chemokine CXCL13 by neurons. This in turn recruited CXCR5+CD8+ T cells to injured DRG neurons overexpressing major histocompatibility complex class I. CD8+ T cells repressed the axonal regeneration of DRG neurons via caspase 3 activation. CXCL13 neutralization prevented CXCR5+CD8+ T cell recruitment to the DRG and reversed aging-dependent regenerative decline, thereby promoting neurological recovery after SNI. Thus, axonal regeneration can be facilitated by antagonizing cross-talk between immune cells and neurons.

Purification of mouse axoplasmic proteins from dorsal root ganglia nerves for proteomics analysis.

The study of neuronal signaling ex vivo requires the identification of the proteins that are represented within the neuronal axoplasm. Here, we describe a detailed protocol to isolate the axoplasm of peripheral and central axonal branches of sciatic dorsal root ganglia neurons in mice. The axoplasm is separated by 2D gel and digestion followed by proteomics analysis with MS/MS-LC. This protocol can be applied to dissect the axoplasmic protein expression signatures before and after a sciatic nerve or a spinal cord injury. For complete details on the use and execution of this protocol, please refer to Kong et al. (2020).

Evolution of PtCu tripod nanocrystals to dendritic triangular nanocrystals and study of the electrochemical performance to alcohol electrooxidation.

In the field of catalysis, the design and construction of nanomaterials is an efficient way to optimize the catalytic activity of catalysts. This study presents the synthesis of PtCu tripod nanocrystals with branching structures and high purity prepared using a simple hydrothermal method. The dendritic PtCu triangular nanocrystals were successfully synthesized by regulating the amount of I- ions to achieve different degrees of branching on PtCu nanocrystals, and the process was systematically studied and analyzed. Meanwhile, dumbbell nanocrystals of PtCu were successfully synthesized through slight adjustments to synthesis conditions. In electrochemical tests, the obtained dendritic PtCu triangular nanocrystals exhibited prominent electrocatalytic activity and long-term stability for ethylene glycol, methanol, and ethanol oxidation reactions due to the unique nanostructures as well as alloyed virtue, and were much better than commercial Pt/C. In addition, this study provides a general strategy for designing novel branched Pt-based nanomaterials with high electrocatalytic performance.

Enriched conditioning expands the regenerative ability of sensory neurons after spinal cord injury via neuronal intrinsic redox signaling.

Overcoming the restricted axonal regenerative ability that limits functional repair following a central nervous system injury remains a challenge. Here we report a regenerative paradigm that we call enriched conditioning, which combines environmental enrichment (EE) followed by a conditioning sciatic nerve axotomy that precedes a spinal cord injury (SCI). Enriched conditioning significantly increases the regenerative ability of dorsal root ganglia (DRG) sensory neurons compared to EE or a conditioning injury alone, propelling axon growth well beyond the spinal injury site. Mechanistically, we established that enriched conditioning relies on the unique neuronal intrinsic signaling axis PKC-STAT3-NADPH oxidase 2 (NOX2), enhancing redox signaling as shown by redox proteomics in DRG. Finally, NOX2 conditional deletion or overexpression respectively blocked or phenocopied enriched conditioning-dependent axon regeneration after SCI leading to improved functional recovery. These studies provide a paradigm that drives the regenerative ability of sensory neurons offering a potential redox-dependent regenerative model for mechanistic and therapeutic discoveries.

Cyclic Stretch of Either PNS or CNS Located Nerves Can Stimulate Neurite Outgrowth.

The central nervous system (CNS) does not recover from traumatic axonal injury, but the peripheral nervous system (PNS) does. We hypothesize that this fundamental difference in regenerative capacity may be based upon the absence of stimulatory mechanical forces in the CNS due to the protective rigidity of the vertebral column and skull. We developed a bioreactor to apply low-strain cyclic axonal stretch to adult rat dorsal root ganglia (DRG) connected to either the peripheral or central nerves in an explant model for inducing axonal growth. In response, larger diameter DRG neurons, mechanoreceptors and proprioceptors showed enhanced neurite outgrowth as well as increased Activating Transcription Factor 3 (ATF3).

AMPK controls the axonal regenerative ability of dorsal root ganglia sensory neurons after spinal cord injury.

Regeneration after injury occurs in axons that lie in the peripheral nervous system but fails in the central nervous system, thereby limiting functional recovery. Differences in axonal signalling in response to injury that might underpin this differential regenerative ability are poorly characterized. Combining axoplasmic proteomics from peripheral sciatic or central projecting dorsal root ganglion (DRG) axons with cell body RNA-seq, we uncover injury-dependent signalling pathways that are uniquely represented in peripheral versus central projecting sciatic DRG axons. We identify AMPK as a crucial regulator of axonal regenerative signalling that is specifically downregulated in injured peripheral, but not central, axons. We find that AMPK in DRG interacts with the 26S proteasome and its CaMKIIα-dependent regulatory subunit PSMC5 to promote AMPKα proteasomal degradation following sciatic axotomy. Conditional deletion of AMPKα1 promotes multiple regenerative signalling pathways after central axonal injury and stimulates robust axonal growth across the spinal cord injury site, suggesting inhibition of AMPK as a therapeutic strategy to enhance regeneration following spinal cord injury.

Shear Testing of the Interfacial Friction Between an HDPE Geomembrane and Solid Waste.

High-density polyethylene (HDPE) geomembrane is often used as an anti-seepage material in domestic and industrial solid waste landfills. To study the interfacial shear strength between the HDPE anti-seepage geomembrane and various solid wastes, we performed direct shear tests on the contact interface between nine types of industrial solid waste or soil (desulfurization gypsum, fly ash, red mud, mercury slag, lead-zinc slag, manganese slag, silica fume, clay and sand) and a geomembrane with a smooth or rough surface in Guizhou Province, China. Friction strength parameters like the interfacial friction angle and the apparent cohesion between the HDPE geomembrane and various solid wastes were measured to analyze the shear strength of the interface between a geomembrane with either a smooth or a rough surface and various solid wastes. The interfacial shear stress between the HDPE geomembrane and the industrial solid waste increased with shear displacement and the slope of the stress-displacement curve decreased gradually. When shear displacement increased to a certain range, the shear stress at the interface remained unchanged. The interfacial shear strength between the geomembrane with a rough surface and the solid waste was higher than for the geomembrane with a smooth surface. Consequentially, the interfacial friction angle for the geomembrane with a rough surface was larger. The geomembrane with a rough surface had a better shear resistance and the shear characteristics fully developed when it was in full contact with the solid waste.

Epigenomic signatures underpin the axonal regenerative ability of dorsal root ganglia sensory neurons.

Axonal injury results in regenerative success or failure, depending on whether the axon lies in the peripheral or the CNS, respectively. The present study addresses whether epigenetic signatures in dorsal root ganglia discriminate between regenerative and non-regenerative axonal injury. Chromatin immunoprecipitation for the histone 3 (H3) post-translational modifications H3K9ac, H3K27ac and H3K27me3; an assay for transposase-accessible chromatin; and RNA sequencing were performed in dorsal root ganglia after sciatic nerve or dorsal column axotomy. Distinct histone acetylation and chromatin accessibility signatures correlated with gene expression after peripheral, but not central, axonal injury. DNA-footprinting analyses revealed new transcriptional regulators associated with regenerative ability. Machine-learning algorithms inferred the direction of most of the gene expression changes. Neuronal conditional deletion of the chromatin remodeler CCCTC-binding factor impaired nerve regeneration, implicating chromatin organization in the regenerative competence. Altogether, the present study offers the first epigenomic map providing insight into the transcriptional response to injury and the differential regenerative ability of sensory neurons.

Facile Synthesis of IrCu Microspheres Based on Polyol Method and Study on Their Electro-Catalytic Performances to Oxygen Evolution Reaction.

The development of Ir-based catalyst with high efficiency for oxygen evolution reaction (OER) in acidic conditions is of great significance to the development of clean energy, but it still remains a significant challenge for shape controlled synthesis of Ir-based catalysts. This article presented a facile one-pot synthesis method that is based on polyol method for preparing IrCu microspheres. In the process of synthesis, formaldehyde solution and ethylene glycol were used as reducing agent and solvent, respectively, while poly(vinylpyrrolidone) was used as surfactant and dispersant, and all of them played important roles in the successful synthesis of Ir-Cu microspheres. The Ir-Cu microspheres, as synthesized, showed well sphere shape and smooth surface, while their alloy features were quite clear and the composition could be adjusted. Benefitting from the synergistic electronic effect between the Iridium and Cupric atoms from the alloy, the IrCu0.77 microspheres exhibited excellent electrocatalytic activity towards OER in 0.1 M HClO4 electrolyte, and to achieve 10 mA cm-2, IrCu0.77 microspheres only required the overpotential of 282 mV, which was much lower than that of commercial Ir/C catalysts.

PP4-dependent HDAC3 dephosphorylation discriminates between axonal regeneration and regenerative failure.

The molecular mechanisms discriminating between regenerative failure and success remain elusive. While a regeneration-competent peripheral nerve injury mounts a regenerative gene expression response in bipolar dorsal root ganglia (DRG) sensory neurons, a regeneration-incompetent central spinal cord injury does not. This dichotomic response offers a unique opportunity to investigate the fundamental biological mechanisms underpinning regenerative ability. Following a pharmacological screen with small-molecule inhibitors targeting key epigenetic enzymes in DRG neurons, we identified HDAC3 signalling as a novel candidate brake to axonal regenerative growth. In vivo, we determined that only a regenerative peripheral but not a central spinal injury induces an increase in calcium, which activates protein phosphatase 4 that in turn dephosphorylates HDAC3, thus impairing its activity and enhancing histone acetylation. Bioinformatics analysis of ex vivo H3K9ac ChIPseq and RNAseq from DRG followed by promoter acetylation and protein expression studies implicated HDAC3 in the regulation of multiple regenerative pathways. Finally, genetic or pharmacological HDAC3 inhibition overcame regenerative failure of sensory axons following spinal cord injury. Together, these data indicate that PP4-dependent HDAC3 dephosphorylation discriminates between axonal regeneration and regenerative failure.

SIP/CacyBP promotes autophagy by regulating levels of BRUCE/Apollon, which stimulates LC3-I degradation.

BRUCE/Apollon is a membrane-associated inhibitor of apoptosis protein that is essential for viability and has ubiquitin-conjugating activity. On initiation of apoptosis, the ubiquitin ligase Nrdp1/RNF41 promotes proteasomal degradation of BRUCE. Here we demonstrate that BRUCE together with the proteasome activator PA28γ causes proteasomal degradation of LC3-I and thus inhibits autophagy. LC3-I on the phagophore membrane is conjugated to phosphatidylethanolamine to form LC3-II, which is required for the formation of autophagosomes and selective recruitment of substrates. SIP/CacyBP is a ubiquitination-related protein that is highly expressed in neurons and various tumors. Under normal conditions, SIP inhibits the ubiquitination and degradation of BRUCE, probably by blocking the binding of Nrdp1 to BRUCE. On DNA damage by topoisomerase inhibitors, Nrdp1 causes monoubiquitination of SIP and thus promotes apoptosis. However, on starvation, SIP together with Rab8 enhances the translocation of BRUCE into the recycling endosome, formation of autophagosomes, and degradation of BRUCE by optineurin-mediated autophagy. Accordingly, deletion of SIP in cultured cells reduces the autophagic degradation of damaged mitochondria and cytosolic protein aggregates. Thus, by stimulating proteasomal degradation of LC3-I, BRUCE also inhibits autophagy. Conversely, SIP promotes autophagy by blocking BRUCE-dependent degradation of LC3-I and by enhancing autophagosome formation and autophagic destruction of BRUCE. These actions of BRUCE and SIP represent mechanisms that link the regulation of autophagy and apoptosis under different conditions.

Cbp-dependent histone acetylation mediates axon regeneration induced by environmental enrichment in rodent spinal cord injury models.

After a spinal cord injury, axons fail to regenerate in the adult mammalian central nervous system, leading to permanent deficits in sensory and motor functions. Increasing neuronal activity after an injury using electrical stimulation or rehabilitation can enhance neuronal plasticity and result in some degree of recovery; however, the underlying mechanisms remain poorly understood. We found that placing mice in an enriched environment before an injury enhanced the activity of proprioceptive dorsal root ganglion neurons, leading to a lasting increase in their regenerative potential. This effect was dependent on Creb-binding protein (Cbp)-mediated histone acetylation, which increased the expression of genes associated with the regenerative program. Intraperitoneal delivery of a small-molecule activator of Cbp at clinically relevant times promoted regeneration and sprouting of sensory and motor axons, as well as recovery of sensory and motor functions in both the mouse and rat model of spinal cord injury. Our findings showed that the increased regenerative capacity induced by enhancing neuronal activity is mediated by epigenetic reprogramming in rodent models of spinal cord injury. Understanding the mechanisms underlying activity-dependent neuronal plasticity led to the identification of potential molecular targets for improving recovery after spinal cord injury.

Facile Synthesis of Pd Nanocubes with Assistant of Iodide and Investigation of Their Electrocatalytic Performances Towards Formic Acid Oxidation.

This article presents a facile, one-pot method using the aqueous phase for the synthesis of high-quality Pd nanocubes. In this study, Pd chloride was used as the precursor, sodium iodide as capping agent, and poly(vinylpyrrolidone) as surfactant and reducing agent. The effects of different halogens on the morphology of Pd nanocrystals were investigated. The results showed that, in this synthesis system, the selection and proper amount of sodium iodide was essential to the preparation of high-quality Pd nanocubes. When iodide was replaced by other halogens (such as bromide and chloride), Pd nanocrystals with cubic morphology could not be obtained. In addition, we have found that NaBH4 can be used to efficiently remove inorganic covers, such as iodide, from the surface of Pd nanoparticles as synthesized. The Pd nanoparticles obtained were employed as electro-catalysts for formic acid oxidation, and they exhibited excellent catalytic activity and good stability towards this reaction.

Proteasomal deubiquitinase UCH37 inhibits degradation of ß-catenin and promotes cell proliferation and motility.

The ubiquitin-proteasome system degrades most cellular proteins in eukaryotes. UCH37, also known as UCH-L5, is a deubiquitinase binding to Rpn13, a receptor for ubiquitinated substrates in the 26 S proteasome. But, it remains unclear how UCH37 influences the proteasomal degradation of the ubiquitinated substrates. Because deletion of UCH37 is embryonically lethal in mice, this study aims to investigate the role of UCH37 in proteasomal degradation by constructing the UCH37-deficient cell lines using CRISPR/Cas9 technology. Our results demonstrated that deletion of UCH37 decreased the levels of proteasomal Rpn13, implying that UCH37 might facilitate incorporation of Rpn13 into the proteasome. Meanwhile, deletion of UCH37 decreased the levels of ß-catenin and the early endosomal protein Rab8. ß-Catenin interacts with TCF/LEF to control transcription, and is involved in development, tissue homeostasis and tumorigenesis. We further found that deletion of UCH37 increased the levels of the ubiquitinated ß-catenin and accelerated the hydrogen peroxide-stimulated degradation of ß-catenin. Deletion of UCH37 also down-regulated the transcription of c-Myc, a downstream effector of ß-catenin, and inhibited cell proliferation and motility. These results raise the possibility that UCH37 maintains the homeostasis of proteasomal degradation reciprocally by assisting the recruitment of the ubiquitin receptor Rpn13 into the proteasome and by reversing ubiquitination of certain critical substrates of the 26 S proteasome.

A "Culture" Shift: Broad Bacterial Detection, Identification, and Antimicrobial Susceptibility Testing Directly from Whole Blood.

BACKGROUND: The time required for bloodstream pathogen detection, identification (ID), and antimicrobial susceptibility testing (AST) does not satisfy the acute needs of disease management. Conventional methods take up to 3 days for ID and AST. Molecular diagnostics have reduced times for ID, but their promise to supplant culture is unmet because AST times remain slow. We developed a combined quantitative PCR (qPCR)-based ID+AST assay with sequential detection, ID, and AST of leading nosocomial bacterial pathogens. METHODS: ID+AST was performed on whole blood samples by (a) removing blood cells, (b) brief bacterial enrichment, (c) bacterial detection and ID, and (d) species-specific antimicrobial treatment. Broad-spectrum qPCR of the internal transcribed spacer between the 16S and 23S was amplified for detection. High-resolution melting identified the species with a curve classifier. AST was enabled by Ct differences between treated and untreated samples. RESULTS: A detection limit of 1 CFU/mL was achieved for Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, and Staphylococcus aureus. All species were accurately identified by unique melting curves. Antimicrobial minimum inhibitory concentrations were identified with Ct differences of ≥1 cycle. Using an RNA target allowed reduction of AST incubation time from 60 min to 5 min. Rapid-cycle amplification reduced qPCR times by 83% to 30 min. CONCLUSIONS: Combined, sequential ID+AST protocols allow rapid and reliable detection, ID, and AST for the diagnosis of bloodstream infections, enabling conversion of empiric to targeted therapy by the second dose of antimicrobials.