Revival of light signalling in the postmortem mouse and human retina.

Death is defined as the irreversible cessation of circulatory, respiratory or brain activity. Many peripheral human organs can be transplanted from deceased donors using protocols to optimize viability. However, tissues from the central nervous system rapidly lose viability after circulation ceases1,2, impeding their potential for transplantation. The time course and mechanisms causing neuronal death and the potential for revival remain poorly defined. Here, using the retina as a model of the central nervous system, we systemically examine the kinetics of death and neuronal revival. We demonstrate the swift decline of neuronal signalling and identify conditions for reviving synchronous in vivo-like trans-synaptic transmission in postmortem mouse and human retina. We measure light-evoked responses in human macular photoreceptors in eyes removed up to 5 h after death and identify modifiable factors that drive reversible and irreversible loss of light signalling after death. Finally, we quantify the rate-limiting deactivation reaction of phototransduction, a model G protein signalling cascade, in peripheral and macular human and macaque retina. Our approach will have broad applications and impact by enabling transformative studies in the human central nervous system, raising questions about the irreversibility of neuronal cell death, and providing new avenues for visual rehabilitation.

NUDC is critical for rod photoreceptor function, maintenance, and survival.

NUDC (nuclear distribution protein C) is a mitotic protein involved in nuclear migration and cytokinesis across species. Considered a cytoplasmic dynein (henceforth dynein) cofactor, NUDC was shown to associate with the dynein motor complex during neuronal migration. NUDC is also expressed in postmitotic vertebrate rod photoreceptors where its function is unknown. Here, we examined the role of NUDC in postmitotic rod photoreceptors by studying the consequences of a conditional NUDC knockout in mouse rods (rNudC-/- ). Loss of NUDC in rods led to complete photoreceptor cell death at 6 weeks of age. By 3 weeks of age, rNudC-/- function was diminished, and rhodopsin and mitochondria were mislocalized, consistent with dynein inhibition. Levels of outer segment proteins were reduced, but LIS1 (lissencephaly protein 1), a well-characterized dynein cofactor, was unaffected. Transmission electron microscopy revealed ultrastructural defects within the rods of rNudC-/- by 3 weeks of age. We investigated whether NUDC interacts with the actin modulator cofilin 1 (CFL1) and found that in rods, CFL1 is localized in close proximity to NUDC. In addition to its potential role in dynein trafficking within rods, loss of NUDC also resulted in increased levels of phosphorylated CFL1 (pCFL1), which would purportedly prevent depolymerization of actin. The absence of NUDC also induced an inflammatory response in Müller glia and microglia across the neural retina by 3 weeks of age. Taken together, our data illustrate the critical role of NUDC in actin cytoskeletal maintenance and dynein-mediated protein trafficking in a postmitotic rod photoreceptor.

Evolution of the clinical-stage hyperactive TcBuster transposase as a platform for robust non-viral production of adoptive cellular therapies.

Cellular therapies for the treatment of human diseases, such as chimeric antigen receptor (CAR) T and natural killer (NK) cells have shown remarkable clinical efficacy in treating hematological malignancies; however, current methods mainly utilize viral vectors that are limited by their cargo size capacities, high cost, and long timelines for production of clinical reagent. Delivery of genetic cargo via DNA transposon engineering is a more timely and cost-effective approach, yet has been held back by less efficient integration rates. Here, we report the development of a novel hyperactive TcBuster (TcB-M) transposase engineered through structure-guided and in vitro evolution approaches that achieves high-efficiency integration of large, multicistronic CAR-expression cassettes in primary human cells. Our proof-of-principle TcB-M engineering of CAR-NK and CAR-T cells shows low integrated vector copy number, a safe insertion site profile, robust in vitro function, and improves survival in a Burkitt lymphoma xenograft model in vivo. Overall, TcB-M is a versatile, safe, efficient and open-source option for the rapid manufacture and preclinical testing of primary human immune cell therapies through delivery of multicistronic large cargo via transposition.

Optic cup morphogenesis requires neural crest-mediated basement membrane assembly.

Organogenesis requires precise interactions between a developing tissue and its environment. In vertebrates, the developing eye is surrounded by a complex extracellular matrix as well as multiple mesenchymal cell populations. Disruptions to either the matrix or periocular mesenchyme can cause defects in early eye development, yet in many cases the underlying mechanism is unknown. Here, using multidimensional imaging and computational analyses in zebrafish, we establish that cell movements in the developing optic cup require neural crest. Ultrastructural analysis reveals that basement membrane formation around the developing eye is also dependent on neural crest, but only specifically around the retinal pigment epithelium. Neural crest cells produce the extracellular matrix protein nidogen: impairing nidogen function disrupts eye development, and, strikingly, expression of nidogen in the absence of neural crest partially restores optic cup morphogenesis. These results demonstrate that eye formation is regulated in part by extrinsic control of extracellular matrix assembly.This article has an associated 'The people behind the papers' interview.

Metabolic changes and retinal remodeling in Heterozygous CRX mutant cats (CRXRDY/+).

CRX is a transcription factor essential for normal photoreceptor development and survival. The CRXRdy cat has a naturally occurring truncating mutation in CRX and is a large animal model for dominant Leber congenital amaurosis. This study investigated retinal remodeling that occurs as photoreceptors degenerate. CRXRdy/+ cats from 6 weeks to 10 years of age were investigated. In vivo structural changes of retinas were analyzed by fundus examination, confocal scanning laser ophthalmoscopy and spectral domain optical coherence tomography. Histologic analyses included immunohistochemistry for computational molecular phenotyping with macromolecules and small molecules. Affected cats had a cone-led photoreceptor degeneration starting in the area centralis. Initially there was preservation of inner retinal cells such as bipolar, amacrine and horizontal cells but with time migration of the deafferented neurons occurred. Early in the process of degeneration glial activation occurs ultimately resulting in formation of a glial seal. With progression the macula-equivalent area centralis developed severe atrophy including loss of retinal pigmentary epithelium. Microneuroma formation occured in advanced stages as more marked retinal remodeling occurred. This study indicates that retinal degeneration in the CrxRdy/+ cat retina follows the progressive, phased revision of retina that have been previously described for retinal remodeling. These findings suggest that therapy dependent on targeting inner retinal cells may be useful in young adults with preserved inner retinas prior to advanced stages of retinal remodeling and neuronal cell loss.

Retinal Pathoconnectomics: A Window into Neurodegeneration.

Over the past decade, the field of retinal connectomics has made huge strides in describing the precise topologies underlying retinal visual processing. The same techniques that allowed these advancements are also applicable to understanding the progression of rewiring in retinal remodeling: retinal pathoconnectomics. Pathoconnectomics is unique in its unbiased approach to understanding the impacts of deafferentation on the remaining network components and identifying aberrant connectivities leading to visual processing defects. Pathoconnectomics also paves the way for identifying underlying rules of rewiring that may be recapitulated throughout the nervous system in other neurodegenerative diseases.

Elastin Layer in Bruch's Membrane as a Target for Immunization or Tolerization to Modulate Pathology in the Mouse Model of Smoke-Induced Ocular Injury.

Age-related macular degeneration (AMD) is associated with an overactive complement system and an increase in circulating antibodies. Our search for potential neoantigens that can trigger complement activation in disease has led us to investigate elastin. A loss of the elastin layer (EL) of Bruch's membrane (BrM) has been reported in aging and AMD together with an increase of serum elastin-derived peptides and α-elastin antibodies. In the mouse model of cigarette smoke exposure (CSE), damage in BrM, loss of the EL, and vision loss are dependent on complement activation. We have examined the hypothesis that CSE generates immunogenic elastin neoepitopes that trigger an increase in α-elastin IgG and IgM antibodies, which can then bind to the neoepitopes in the target cells or membranes, triggering complement activation. Specifically, we showed that immunization with elastin peptide oxidatively modified by cigarette smoke (ox-elastin) exacerbated ocular pathology and vision loss in CSE mice. In contrast, mice receiving peptide immunotherapy (PIT) with ox-elastin did not lose vision over the smoking period and exhibited a more preserved BrM. Immunization and PIT correlated with humoral immunity and complement activation and IgG/IgM deposition in the RPE/BrM/choroid. Finally, PIT modulated immune markers IFNγ and IL-4. The data further support the hypothesis that complement activation, triggered by immune complex formation in target tissues, plays a role in ocular damage in the CSE model. As PIT with ox-elastin peptides reduces damage, we discuss the possibility that AMD progression might be preventable.

An anti-ANGPTL3/8 antibody decreases circulating triglycerides by binding to a LPL-inhibitory leucine zipper-like motif.

Triglycerides (TG) are required for fatty acid transport and storage and are essential for human health. Angiopoietin-like-protein 8 (ANGPTL8) has previously been shown to form a complex with ANGPTL3 that increases circulating TG by potently inhibiting LPL. We also recently showed that the TG-lowering apolipoprotein A5 (ApoA5) decreases TG levels by suppressing ANGPTL3/8-mediated LPL inhibition. To understand how LPL binds ANGPTL3/8 and ApoA5 blocks this interaction, we used hydrogen-deuterium exchange mass-spectrometry and molecular modeling to map binding sites of LPL and ApoA5 on ANGPTL3/8. Remarkably, we found that LPL and ApoA5 both bound a unique ANGPTL3/8 epitope consisting of N-terminal regions of ANGPTL3 and ANGPTL8 that are unmasked upon formation of the ANGPTL3/8 complex. We further used ANGPTL3/8 as an immunogen to develop an antibody targeting this same epitope. After refocusing on antibodies that bound ANGPTL3/8, as opposed to ANGPTL3 or ANGPTL8 alone, we utilized bio-layer interferometry to select an antibody exhibiting high-affinity binding to the desired epitope. We revealed an ANGPTL3/8 leucine zipper-like motif within the anti-ANGPTL3/8 epitope, the LPL-inhibitory region, and the ApoA5-interacting region, suggesting the mechanism by which ApoA5 lowers TG is via competition with LPL for the same ANGPTL3/8-binding site. Supporting this hypothesis, we demonstrate that the anti-ANGPTL3/8 antibody potently blocked ANGPTL3/8-mediated LPL inhibition in vitro and dramatically lowered TG levels in vivo. Together, these data show that an anti-ANGPTL3/8 antibody targeting the same leucine zipper-containing epitope recognized by LPL and ApoA5 markedly decreases TG by suppressing ANGPTL3/8-mediated LPL inhibition.

Hypoxia tolerance in the Norrin-deficient retina and the chronically hypoxic brain studied at single-cell resolution.

The mammalian CNS is capable of tolerating chronic hypoxia, but cell type-specific responses to this stress have not been systematically characterized. In the Norrin KO (NdpKO ) mouse, a model of familial exudative vitreoretinopathy (FEVR), developmental hypovascularization of the retina produces chronic hypoxia of inner nuclear-layer (INL) neurons and Muller glia. We used single-cell RNA sequencing, untargeted metabolomics, and metabolite labeling from 13C-glucose to compare WT and NdpKO retinas. In NdpKO retinas, we observe gene expression responses consistent with hypoxia in Muller glia and retinal neurons, and we find a metabolic shift that combines reduced flux through the TCA cycle with increased synthesis of serine, glycine, and glutathione. We also used single-cell RNA sequencing to compare the responses of individual cell types in NdpKO retinas with those in the hypoxic cerebral cortex of mice that were housed for 1 week in a reduced oxygen environment (7.5% oxygen). In the hypoxic cerebral cortex, glial transcriptome responses most closely resemble the response of Muller glia in the NdpKO retina. In both retina and brain, vascular endothelial cells activate a previously dormant tip cell gene expression program, which likely underlies the adaptive neoangiogenic response to chronic hypoxia. These analyses of retina and brain transcriptomes at single-cell resolution reveal both shared and cell type-specific changes in gene expression in response to chronic hypoxia, implying both shared and distinct cell type-specific physiologic responses.

Two-Experiment Examination of Habitual and Manipulated Foot Placement Angles on the Kinetics, Kinematics, and Muscle Forces of the Barbell Back Squat in Male Lifters.

This two-experiment study aimed to examine the effects of different habitual foot placement angles and also the effects of manipulating the foot placement angle on the kinetics, three-dimensional kinematics and muscle forces of the squat. In experiment 1, seventy lifters completed squats at 70% of their one repetition maximum using a self-preferred placement angle. They were separated based on their habitual foot angle into three groups HIGH, MEDIUM and LOW. In experiment 2, twenty lifters performed squats using the same relative mass in four different foot placement angle conditions (0°, 21°, 42° and control). Three-dimensional kinematics were measured using an eight-camera motion analysis system, ground reaction forces (GRF) using a force platform, and muscle forces using musculoskeletal modelling techniques. In experiment 1, the impulse of the medial GRF, in the descent and ascent phases, was significantly greater in the HIGH group compared to LOW, and in experiment 2 statistically greater in the 42° compared to the 21°, 0° and control conditions. Experiment 2 showed that the control condition statistically increased quadriceps muscle forces in relation to 0°, whereas the 0° condition significantly enhanced gluteus maximus, gastrocnemius and soleus forces compared to control. In experiment 1, patellofemoral joint stress was significantly greater in the HIGH group compared to LOW, and in experiment 2, patellar and patellofemoral loading were statistically greater in the control compared to the 42°, 21°, 0° and control conditions. Owing to the greater medial GRF's, increased foot placement angles may improve physical preparedness for sprint performance and rapid changes of direction. Reducing the foot angle may attenuate the biomechanical mechanisms linked to the aetiology of knee pathologies and to promote gluteus maximus, gastrocnemius and soleus muscular development. As such, though there does not appear to be an optimal foot placement angle, the observations from this study can be utilised by both strength and conditioning and sports therapy practitioners seeking to maximise training and rehabilitative adaptations.

Network Architecture of Gap Junctional Coupling among Parallel Processing Channels in the Mammalian Retina.

Gap junctions are ubiquitous throughout the nervous system, mediating critical signal transmission and integration, as well as emergent network properties. In mammalian retina, gap junctions within the Aii amacrine cell-ON cone bipolar cell (CBC) network are essential for night vision, modulation of day vision, and contribute to visual impairment in retinal degenerations, yet neither the extended network topology nor its conservation is well established. Here, we map the network contribution of gap junctions using a high-resolution connectomics dataset of an adult female rabbit retina. Gap junctions are prominent synaptic components of ON CBC classes, constituting 5%-25% of all axonal synaptic contacts. Many of these mediate canonical transfer of rod signals from Aii cells to ON CBCs for night vision, and we find that the uneven distribution of Aii signals to ON CBCs is conserved in rabbit, including one class entirely lacking direct Aii coupling. However, the majority of gap junctions formed by ON CBCs unexpectedly occur between ON CBCs, rather than with Aii cells. Such coupling is extensive, creating an interconnected network with numerous lateral paths both within, and particularly across, these parallel processing streams. Coupling patterns are precise with ON CBCs accepting and rejecting unique combinations of partnerships according to robust rulesets. Coupling specificity extends to both size and spatial topologies, thereby rivaling the synaptic specificity of chemical synapses. These ON CBC coupling motifs dramatically extend the coupled Aii-ON CBC network, with implications for signal flow in both scotopic and photopic retinal networks during visual processing and disease.SIGNIFICANCE STATEMENT Electrical synapses mediated by gap junctions are fundamental components of neural networks. In retina, coupling within the Aii-ON CBC network shapes visual processing in both the scotopic and photopic networks. In retinal degenerations, these same gap junctions mediate oscillatory activity that contributes to visual impairment. Here, we use high-resolution connectomics strategies to identify gap junctions and cellular partnerships. We describe novel, pervasive motifs both within and across classes of ON CBCs that dramatically extend the Aii-ON CBC network. These motifs are highly specific with implications for both signal processing within the retina and therapeutic interventions for blinding conditions. These findings highlight the underappreciated contribution of coupling motifs in retinal circuitry and the necessity of their detection in connectomics studies.

Peptide-based immunotherapy against oxidized elastin ameliorates pathology in mouse model of smoke-induced ocular injury.

PURPOSE: Age-related macular degeneration (AMD), the leading cause of blindness in western populations, is associated with an overactive complement system, and an increase in circulating antibodies against certain epitopes, including elastin. As loss of the elastin layer of Bruch's membrane (BrM) has been reported in aging and AMD, we previously showed that immunization with elastin peptide oxidatively modified by cigarette smoke (ox-elastin), exacerbated ocular pathology in the smoke-induced ocular pathology (SIOP) model. Here we asked whether ox-elastin peptide-based immunotherapy (PIT) ameliorates damage. METHODS: C57BL/6J mice were injected with ox-elastin peptide at two doses via weekly subcutaneous administration, while exposed to cigarette smoke for 6 months. FcγR-/- and uninjected C57BL/6J mice served as controls. Retinal morphology was assessed by electron microscopy, and complement activation, antibody deposition and mechanisms of immunological tolerance were assessed by Western blotting and ELISA. RESULTS: Elimination of Fcγ receptors, preventing antigen/antibody-dependent cytotoxicity, protected against SIOP. Mice receiving PIT with low dose ox-elastin (LD-PIT) exhibited reduced humoral immunity, reduced complement activation and IgG/IgM deposition in the RPE/choroid, and largely a preserved BrM. While there is no direct evidence of ox-elastin pathogenicity, LD-PIT reduced IFNγ and increased IL-4 within RPE/choroid. High dose PIT was not protective. CONCLUSIONS: These data further support ox-elastin role in ocular damage in part via elastin-specific antibodies, and support the corollary that PIT with ox-elastin attenuates ocular pathology. Overall, damage is associated with complement activation, antibody-dependent cell-mediated cytotoxicity, and altered cytokine signature.

Model-based comparison of current flow in rod bipolar cells of healthy and early-stage degenerated retina.

Retinal degenerative diseases, such as retinitis pigmentosa, are generally thought to initiate with the loss of photoreceptors, though recent work suggests that plasticity and remodeling occurs prior to photoreceptor cell loss. This degeneration subsequently leads to death of other retinal neurons, creating functional alterations and extensive remodeling of retinal networks. Retinal prosthetic devices stimulate the surviving retinal cells by applying external current using implanted electrodes. Although these devices restore partial vision, the quality of restored vision is limited. Further knowledge about the precise changes in degenerated retina as the disease progresses is essential to understand how current flows in retinas undergoing degenerative disease and to improve the performance of retinal prostheses. We developed computational models that describe current flow from rod photoreceptors to rod bipolar cells (RodBCs) in the healthy and early-stage degenerated retina. Morphologically accurate models of retinal cells with their synapses are constructed based on retinal connectome datasets, created using serial section transmission electron microscopy (TEM) images of 70 nm-thick slices of either healthy (RC1) or early-stage degenerated (RPC1) rabbit retina. The passive membrane and active ion currents of each cell are implemented using conductance-based models in the Neuron simulation environment. In response to photocurrent input at rod photoreceptors, the simulated membrane potential at RodBCs in early degenerate tissue is approximately 10-20 mV lower than that of RodBCs of that observed in wild type retina. Results presented here suggest that although RodBCs in RPC1 show early, altered morphology compared to RC1, the lower membrane potential is primarily a consequence of reduced rod photoreceptor input to RodBCs in the degenerated retina. Frequency response and step input analyses suggest that individual cell responses of RodBCs in either healthy or early-degenerated retina, prior to substantial photoreceptor cell loss, do not differ significantly.

Natural immunoglobulin M-based delivery of a complement alternative pathway inhibitor in mouse models of retinal degeneration.

PURPOSE: Age-related macular degeneration is a slowly progressing disease. Studies have tied disease risk to an overactive complement system. We have previously demonstrated that pathology in two mouse models, the choroidal neovascularization (CNV) model and the smoke-induced ocular pathology (SIOP) model, can be reduced by specifically inhibiting the alternative complement pathway (AP). Here we report on the development of a novel injury-site targeted inhibitor of the alternative pathway, and its characterization in models of retinal degeneration. METHODS: Expression of the danger associated molecular pattern, a modified annexin IV, in injured ARPE-19 cells was confirmed by immunohistochemistry and complementation assays using B4 IgM mAb. Subsequently, a construct was prepared consisting of B4 single chain antibody (scFv) linked to a fragment of the alternative pathway inhibitor, fH (B4-scFv-fH). ARPE-19 cells stably expressing B4-scFv-fH were microencapsulated and administered intravitreally or subcutaneously into C57BL/6 J mice, followed by CNV induction or smoke exposure. Progression of CNV was analyzed using optical coherence tomography, and SIOP using structure-function analyses. B4-scFv-fH targeting and AP specificity was assessed by Western blot and binding experiments. RESULTS: B4-scFv-fH was secreted from encapsulated RPE and inhibited complement in RPE monolayers. B4-scFv-fH capsules reduced CNV and SIOP, and western blotting for breakdown products of C3α, IgM and IgG confirmed a reduction in complement activation and antibody binding in RPE/choroid. CONCLUSIONS: Data supports a role for natural antibodies and neoepitope expression in ocular disease, and describes a novel strategy to target AP-specific complement inhibition to diseased tissue in the eye. PRECIS: AMD risk is tied to an overactive complement system, and ocular injury is reduced by alternative pathway (AP) inhibition in experimental models. We developed a novel inhibitor of the AP that targets an injury-specific danger associated molecular pattern, and characterized it in disease models.

A pathoconnectome of early neurodegeneration: Network changes in retinal degeneration.

Connectomics has demonstrated that synaptic networks and their topologies are precise and directly correlate with physiology and behavior. The next extension of connectomics is pathoconnectomics: to map neural network synaptology and circuit topologies corrupted by neurological disease in order to identify robust targets for therapeutics. In this report, we characterize a pathoconnectome of early retinal degeneration. This pathoconnectome was generated using serial section transmission electron microscopy to achieve an ultrastructural connectome with 2.18nm/px resolution for accurate identification of all chemical and gap junctional synapses. We observe aberrant connectivity in the rod-network pathway and novel synaptic connections deriving from neurite sprouting. These observations reveal principles of neuron responses to the loss of network components and can be extended to other neurodegenerative diseases.

Unilateral Epidural Targeting of Resiniferatoxin Induces Bilateral Neurolysis of Spinal Nociceptive Afferents.

OBJECTIVE: This study modeled image-guided epidural drug delivery to test whether intraprocedural distribution of pre-injected contrast reliably predicts the neuroanatomical reach of resiniferatoxin-mediated nociceptive neurolysis. METHODS: Swine (N = 12) received unilateral L4-S2 computed tomography fluoroscopy injections by a blinded neuroradiologist; 0.25 mL of contrast was pre-injected to confirm dorsal periganglionic targeting, followed by a 0.5-mL injection of 5 µg of resiniferatoxin/Tween80 or vehicle control. Epidural contrast distribution was graded according to maximum medial excursion. Spinal cord substance P immunostaining quantified the magnitude and anatomical range of resiniferatoxin activity. RESULTS: Periganglionic injection was well tolerated by all animals without development of neurological deficits or other complications. Swine were a suitable model of human clinical spinal intervention. The transforaminal approach was used at all L4 and 50% of L5 segments; the remaining segments were approached by the interlaminar route. All injections were successful with unilateral contrast distribution for all resiniferatoxin injections (N = 28). Immunohistochemistry showed bilateral ablation of substance P+ fibers entering the spinal cord of all resiniferatoxin-treated segments. The intensity of substance P immunostaining in treated segments fell below the lower 99% confidence interval of controls, defining the knockout phenotype. Substance P knockout occurred over a narrow range and was uncorrelated to the anatomical distribution of pre-injected contrast. CONCLUSIONS: Periganglionic resiniferatoxin/Tween80 induced bilateral ablation of spinal cord substance P despite exclusively unilateral targeting. These data suggest that the location of pre-injected contrast is an imperfect surrogate for the neuroanatomical range of drugs delivered to the dorsal epidural compartment that may fail to predict contralateral drug effects.

Pathoconnectome Analysis of Müller Cells in Early Retinal Remodeling.

Glia play important roles in neural function, including but not limited to amino acid recycling, ion homeostasis, glucose metabolism, and waste removal. During retinal degeneration and subsequent retinal remodeling, Müller cells (MCs) are the first cells to show metabolic and morphological alterations in response to stress. Metabolic alterations in MCs chaotically progress in retina undergoing photoreceptor degeneration; however, what relationship these alterations have with neuronal stress, synapse maintenance, or glia-glia interactions is currently unknown. The work described here reconstructs a MC from a pathoconnectome of early retinal remodeling retinal pathoconnectome 1 (RPC1) and explores relationships between MC structural and metabolic phenotypes in the context of neighboring neurons and glia. Here we find variations in intensity of osmication inter- and intracellularly, variation in small molecule metabolic content of MCs, as well as morphological alterations of glial endfeet. RPC1 provides a framework to analyze these relationships in early retinal remodeling through ultrastructural reconstructions of both neurons and glia. These reconstructions, informed by quantitative metabolite labeling via computational molecular phenotyping (CMP), allow us to evaluate neural-glial interactions in early retinal degeneration with unprecedented resolution and sensitivity.

Future ozone-related acute excess mortality under climate and population change scenarios in China: A modeling study.

BACKGROUND: Climate change is likely to further worsen ozone pollution in already heavily polluted areas, leading to increased ozone-related health burdens. However, little evidence exists in China, the world's largest greenhouse gas emitter and most populated country. As China is embracing an aging population with changing population size and falling age-standardized mortality rates, the potential impact of population change on ozone-related health burdens is unclear. Moreover, little is known about the seasonal variation of ozone-related health burdens under climate change. We aimed to assess near-term (mid-21st century) future annual and seasonal excess mortality from short-term exposure to ambient ozone in 104 Chinese cities under 2 climate and emission change scenarios and 6 population change scenarios. METHODS AND FINDINGS: We collected historical ambient ozone observations, population change projections, and baseline mortality rates in 104 cities across China during April 27, 2013, to October 31, 2015 (2013-2015), which included approximately 13% of the total population of mainland China. Using historical ozone monitoring data, we performed bias correction and spatially downscaled future ozone projections at a coarse spatial resolution (2.0° × 2.5°) for the period April 27, 2053, to October 31, 2055 (2053-2055), from a global chemistry-climate model to a fine spatial resolution (0.25° × 0.25°) under 2 Intergovernmental Panel on Climate Change Representative Concentration Pathways (RCPs): RCP4.5, a moderate global warming and emission scenario where global warming is between 1.5°C and 2.0°C, and RCP8.5, a high global warming and emission scenario where global warming exceeds 2.0°C. We then estimated the future annual and seasonal ozone-related acute excess mortality attributable to both climate and population changes using cause-specific, age-group-specific, and season-specific concentration-response functions (CRFs). We used Monte Carlo simulations to obtain empirical confidence intervals (eCIs), quantifying the uncertainty in CRFs and the variability across ensemble members (i.e., 3 predictions of future climate and air quality from slightly different starting conditions) of the global model. Estimates of future changes in annual ozone-related mortality are sensitive to the choice of global warming and emission scenario, decreasing under RCP4.5 (-24.0%) due to declining ozone precursor emissions but increasing under RCP8.5 (10.7%) due to warming climate in 2053-2055 relative to 2013-2015. Higher ambient ozone occurs under the high global warming and emission scenario (RCP8.5), leading to an excess 1,476 (95% eCI: 898 to 2,977) non-accidental deaths per year in 2053-2055 relative to 2013-2015. Future ozone-related acute excess mortality from cardiovascular diseases was 5-8 times greater than that from respiratory diseases. Ozone concentrations increase by 15.1 parts per billion (10-9) in colder months (November to April), contributing to a net yearly increase of 22.3% (95% eCI: 7.7% to 35.4%) in ozone-related mortality under RCP8.5. An aging population, with the proportion of the population aged 65 years and above increased from 8% in 2010 to 24%-33% in 2050, will substantially amplify future ozone-related mortality, leading to a net increase of 23,838 to 78,560 deaths (110% to 363%). Our analysis was mainly limited by using a single global chemistry-climate model and the statistical downscaling approach to project ozone changes under climate change. CONCLUSIONS: Our analysis shows increased future ozone-related acute excess mortality under the high global warming and emission scenario RCP8.5 for an aging population in China. Comparison with the lower global warming and emission scenario RCP4.5 suggests that climate change mitigation measures are needed to prevent a rising health burden from exposure to ambient ozone pollution in China.

Assessing the Accuracy of Multi-Temporal Built-Up Land Layers across Rural-Urban Trajectories in the United States.

Global data on settlements, built-up land and population distributions are becoming increasingly available and represent important inputs to a better understanding of key demographic processes such as urbanization and interactions between human and natural systems over time. One persistent drawback that prevents user communities from effectively and objectively using these data products more broadly, is the absence of thorough and transparent validation studies. This study develops a validation framework for accuracy assessment of multi-temporal built-up land layers using integrated public parcel and building records as validation data. The framework is based on measures derived from confusion matrices and incorporates a sensitivity analysis for potential spatial offsets between validation and test data as well as tests for the effects of varying criteria of the abstract term built-up land on accuracy measures. Furthermore, the framework allows for accuracy assessments by strata of built-up density, which provides important insights on the relationship between classification accuracy and development intensity to better instruct and educate user communities on quality aspects that might be relevant to different purposes. We use data from the newly-released Global Human Settlement Layer (GHSL), for four epochs since 1975 and at fine spatial resolution (38m), in the United States for a demonstration of the framework. The results show very encouraging accuracy measures that vary across study areas, generally improve over time but show very distinct patterns across the rural-urban trajectories. Areas of higher development intensity are very accurately classified and highly reliable. Rural areas show low degrees of accuracy, which could be affected by misalignment between the reference data and the data under test in areas where built-up land is scattered and rare. However, a regression analysis, which examines how well GHSL can estimate built-up land using spatially aggregated analytical units, indicates that classification error is mainly of thematic nature. Thus, caution should be taken in using the data product in rural regions. The results can be useful in further improving classification procedures to create measures of the built environment. The validation framework can be extended to data-poor regions of the world using map data and Volunteered Geographic Information.

Target engagement and histopathology of neuraxial resiniferatoxin in dog.

OBJECTIVE: To evaluate target engagement of intracisternally (IC) delivered TRPV1 agonist, resiniferatoxin (RTX), as measured by primary afferent and dorsal horn substance P immunoreactivity (sP-IR), histopathology and thermal escape latencies in dogs. STUDY DESIGN: Prospective experimental trial. ANIMALS: Fourteen adult male Beagle dogs, weighing 10.3-13.2 kg; 11 dogs surviving to scheduled euthanasia. METHODS: Anesthetized dogs were randomly assigned to be administered IC RTX (3.6 µg, 0.1 mL kg-1) in a hyperbaric (hRTX, n = 6), normobaric (nRTX, n = 4) vehicle or a hyperbaric vehicle (hVehicle, n = 4). Over 16 days, animals were examined for thoracic and pelvic limb paw thermal withdrawal latencies and neurologic function. Spinal cords, trigeminal ganglia and dorsal root ganglia (DRGs) were assessed for morphologic changes and sP-IR. RESULTS: IC RTX in anesthetized dogs resulted in a < 1 hour increase in blood pressure. Acute reactions leading to euthanasia within 8 hours occurred in three dogs (two hRTX, one nRTX). All other animals recovered with normal neurologic, bowel and bladder function. Final groups were: vehicle n = 4, hRTX n = 4 and nRTX n = 3. Animals in nRTX and hRTX showed increases in escape latencies in thoracic paws and, to a lesser extent, in pelvic paws, correlating to a loss of sP-IR in cervical cord with smaller reductions in thoracic and lumbar cord. In animals surviving to euthanasia, thickening of the arachnoid membrane (predominantly in the cervical region) was the most consistent change. This change, present in controls, was interpreted to be vehicle related. There was no evidence of structural changes in brain and spinal cord. CONCLUSIONS AND CLINICAL RELEVANCE: IC RTX produced localized loss of spinal and DRG sP with a corresponding thermal analgesia, absent motor impairment or spinal pathology. Loss of three animals emphasizes the need to refine the use of this promising therapeutic modality in managing companion animal pain.