Biology subjects

Liu, M.

Publications and source records attributed to Liu, M..

At least 415 records · Page 23Linked to original sources

bioRxiv2020

Potentiating CD8+ T cell antitumor activity by targeting the PCSK9/LDLR axis

Metabolic regulation has proven to play a critical role in T cell antitumor immunity. Cholesterol metabolism is a key component of this response but remains largely unexplored. Herein, we found that the LDL receptor (LDLR), which has been previously identified as a transporter for cholesterol and fatty acids, plays a pivotal role in regulating CD8+ T cell antitumor activity, with the genetic ablation of LDLR significantly attenuating CD8+ T cell activation and clonal expansion. Additionally, we found that LDLR interacts with the T-cell receptor (TCR) signalosome and regulates TCR signaling, facilitating CD8+ T cell activation and effector function. Furthermore, we found that the tumor microenvironment downregulates CD8+ T cell LDLR levels and TCR signaling via tumor cell-derived PCSK9, which binds and prevents the recycling of LDLR and TCR into the plasma membrane. Our findings indicate that genetic deletion or pharmacological inhibition of PCSK9 in tumor cells can enhance the antitumor activity of CD8+ T cells by alleviating the tumor microenvironments suppressive effect on CD8+ T cells and consequently inhibit tumor progression. While previously established as a hyperlipidemia target, this study highlights PCSK9 as a potential target for cancer immunotherapy as well.

immunology↗

bioRxiv2020

Controlling Cellular Arrangements via Stretched Bioprinting

Bioprinting is a common method to replicate geometrical architecture of native tissues. However, it usually fails to modulate cellular arrangements, which is critical for the tissues functionality. To our knowledge, no method has successfully addressed this challenge. Here, we report a method of controlling cellular orientation during the bioprinting process by integrating a stretch process into a modified bioprinting frame. We demonstrate that the cellular orientation is a result of cells sensing and responding to the tensile stress, instead of shear stress or topographical patterns. Moreover, our method shows a potent capability to induce myoblast differentiation, fusion and maturation without the presence of differentiation medium. As a potential clinical application, we demonstrate that aligned myofibers directly printed onto injured muscle in vivo, can not only repair the structure of damaged tissue, but also recover the muscle functionalities effectively. This study shows that the new method can produce tissues with precise control of cellular arrangements and more clinically viable functionalities. Significance StatementDue to no method could reproduce the exact cellular arrangements of native tissues in engineered tissues, tissue engineering facing difficult in fabricating 3D tissues that possess desirable biological and mechanical functionalities for biomedical applications. For the first time, we report a method of controlling cellular orientation during 3D bio-printing process. This method can be used to produce engineered tissues with controlled cellular arrangement with several different cell types. Moreover, this method shows a potent capability of fabricating fully mature and aligned myofibers in vitro in the absence of differentiation medium. As potential clinical applications, with this method, engineered tissues could be directly printed in vivo with high efficacy of tissue repair and function recovery.

cell biology↗

bioRxiv2020

Plot diversity differentially affects the chemical composition of leaves, roots and root exudates in four subtropical tree species

O_LIPlants produce thousands of compounds, collectively called the metabolome, which mediate interactions with other organisms. The metabolome of an individual plant may change according to the number and nature of these interactions. We tested the hypothesis that tree diversity level affects the metabolome of four subtropical tree species in a biodiversity ecosystem-functioning experiment, BEF-China. We postulated that the chemical diversity of leaves, roots and root exudates increases with tree diversity. We expected the strength of this diversity effect to differ among leaf, root and root exudates samples. Considering their role in plant competition, we expected to find the strongest effects in root exudates. C_LIO_LIIn an ecometabolomics approach, roots, root exudates and leaves of four tree species (Cinnamomum camphora, Cyclobalanopsis glauca, Daphniphyllum oldhamii, Schima superba) were sampled from selected plots in BEF-China. Samples were extracted and analysed using Liquid Chromatography-Time of Flight-Mass Spectrometry. The exudate metabolomes were normalized over their non-purgeable organic carbon level. Multivariate analyses were applied to identify the effect of both neighbouring (local) trees and plot diversity on tree metabolomes. The species and sample specific metabolites were assigned to major compound classes using the ClassyFire tool, whereas m/z features related to diversity effects were annotated manually. C_LIO_LIIndividual tree species showed distinct leaf, root and root exudate metabolomes. The main compound class in leaves were the flavonoids, whereas carboxylic acids, prenol lipids and specific alkaloids were most prominent in root exudates and roots. Overall plot diversity had a stronger effect on metabolome profiles than the diversity of local, directly neighbouring trees. Leaf metabolomes responded more often to tree diversity level than exudates, whereas root metabolomes varied the least. We found not overall correlation between metabolite richness or diversity and tree diversity. C_LIO_LISynthesis: Classification of metabolites supported initial ecological interpretation of differences among species and organs. Particularly the metabolomes of leaves and root exudates respond to differences in tree diversity. These responses were neither linear nor uniform and individual metabolites showed different dynamics. More controlled interaction experiments are needed to dissect the causes and consequences of the observed shifts in plant metabolomes. C_LI

ecology↗

bioRxiv2020

Hippocampal Asymmetry of Regional Development and Structural Covariance in Preterm Neonates

Premature birth is associated with high prevalence of neurodevelopmental impairments in surviving infants. The hippocampus is known to be critical for learning and memory, the putative role of hippocampus dysfunction remains poorly understood in preterm neonates. Particularly, hemispherical asymmetry of the hippocampus has been well-noted, either structurally or functionally. How the preterm birth impairs the hippocampal development, and to what extent the hippocampus was impaired by preterm birth asymmetrical has not been well studied. In this study, we compared regional and local hippocampal development in term born neonates (n=361) and prematurely born infants at term-born equivalent age on MRI studies (n = 53) using T2 MRI images collected from the Developing Human Connectome Project (dHCP); We compared 1) volumetric growth; 2) shape development in the hippocampal hemispheres using Laplace-Beltrami eigen-projection and boundary deformation between the two groups; and 3) structural covariance between hippocampal vertices and the cortical thickness in cerebral cortex regions. We demonstrated that premature infants have smaller volume for the right hippocampi, while no difference was observed for the left hippocampi. Lower thickness was observed in the hippocampal head in both hemispheres for preterm neonates compared to full-term peers, while an accelerated hippocampal thickness growth rate was found in left hippocampus only. Structural covariance analysis demonstrated that in premature infants, the structural covariance between hippocampi and limbic lobe were severely impaired compared to healthy term neonates only in left hemisphere. These data suggest that the development of the hippocampus during the third trimester may be altered following early extrauterine exposure, with high degree of asymmetry. These findings suggested that the hippocampus shows high degree of vulnerability, particularly asymmetrical vulnerability or plasticity, in preterm neonates at the term-born equivalent age compared to full-term healthy controls.

neuroscience↗

bioRxiv2020

Human Identical Sequences of SARS-CoV-2 Promote Clinical Progression of COVID-19 by Upregulating Hyaluronan via NamiRNA-Enhancer Network

The COVID-19 pandemic is a widespread and deadly public health crisis. The pathogen SARS-CoV-2 replicates in the lower respiratory tract and causes fatal pneumonia. Although tremendous efforts have been put into investigating the pathogeny of SARS-CoV-2, the underlying mechanism of how SARS-CoV-2 interacts with its host is largely unexplored. Here, by comparing the genomic sequences of SARS-CoV-2 and human, we identified five fully conserved elements in SARS-CoV-2 genome, which were termed as "human identical sequences (HIS)". HIS are also recognized in both SARS-CoV and MERS-CoV genome. Meanwhile, HIS-SARS-CoV-2 are highly conserved in the primate. Mechanically, HIS-SARS-CoV-2, behaving as virus-derived miRNAs, directly target to the human genomic loci and further interact with host enhancers to activate the expression of adjacent and distant genes, including cytokines gene and angiotensin converting enzyme II (ACE2), a well-known cell entry receptor of SARS-CoV-2, and hyaluronan synthase 2 (HAS2), which further increases hyaluronan formation. Noteworthily, hyaluronan level in plasma of COVID-19 patients is tightly correlated with severity and high risk for acute respiratory distress syndrome (ARDS) and may act as a predictor for the progression of COVID-19. HIS antagomirs, which downregulate hyaluronan level effectively, and 4-Methylumbelliferone (MU), an inhibitor of hyaluronan synthesis, are potential drugs to relieve the ARDS related ground-glass pattern in lung for COVID-19 treatment. Our results revealed that unprecedented HIS elements of SARS-CoV-2 contribute to the cytokine storm and ARDS in COVID-19 patients. Thus, blocking HIS-involved activating processes or hyaluronan synthesis directly by 4-MU may be effective strategies to alleviate COVID-19 progression.

molecular biology↗

bioRxiv2020

Genetic determinants of COVID-19 drug efficacy revealed by genome-wide CRISPR screens

Immunomodulatory agents dexamethasone and colchicine, antiviral drugs remdesivir, favipiravir and ribavirin, as well as antimalarial drugs chloroquine phosphate and hydroxychloroquine are currently used in the combat against COVID-191-16. However, whether some of these drugs have clinical efficacy for COVID-19 is under debate. Moreover, these drugs are applied in COVID-19 patients with little knowledge of genetic biomarkers, which will hurt patient outcome. To answer these questions, we designed a screen approach that could employ genome-wide sgRNA libraries to systematically uncover genes crucial for these drugs action. Here we present our findings, including genes crucial for the import, export, metabolic activation and inactivation of remdesivir, as well as genes that regulate colchicine and dexamethasones immunosuppressive effects. Our findings provide preliminary information for developing urgently needed genetic biomarkers for these drugs. Such biomarkers will help better interpret COVID-19 clinical trial data and point to how to stratify COVID-19 patients for proper treatment with these drugs.

pharmacology and toxicology↗

bioRxiv2020

Novel Asgard archaea phylum Hermodarchaeota degrade alkanes and aromatics via alkyl/benzyl-succinate synthase and benzoyl-CoA pathway

Asgard superphylum is composed of a group of uncultivated archaea that are deemed the closest relatives of eukaryotes. These archaea are widely distributed in anaerobic environments and suggested to be important players in carbon cycling of sediments. Alkanes and aromatics are refractory organic compounds and abundant in sediments. However, little is known about degradation of these compounds by Asgard archaea to date. Here, we describe a previously unrecognized archaeal phylum, Hermodarchaeota, affiliated with the Asgard superphylum. The genomes of these archaea were recovered in metagenomes from mangrove sediments, and were found to encode alkyl/benzyl-succinate synthases and their activating enzymes that are similar to those found in alkanes-degrading sulfate-reducing bacteria. Hermodarchaeota also encode enzymes for alkyl-coenzyme A and benzoyl-coenzyme A oxidation, and the Wood-Ljungdahl pathway, as well as nitrate reductases. Furthermore, transcripts for these enzymes have been frequently detected in metatranscriptomes from mangrove sediments. This indicates that members of this phylum are able to anaerobically oxidize alkanes and aromatic compounds, coupling the reduction of nitrate. Genes encoding 16S rRNA and alkyl/benzyl-succinate synthases analogous to those in Hermodarchaeota were identified in a range of marine and freshwater sediments. These findings suggest that Asgard archaea capable of degrading alkanes and aromatics via formation of alkyl/benzyl-substituted succinates are ubiquitous in sediments.

genomics↗

bioRxiv2020

Multivariate genomic analysis of 1.5 million people identifies genes related to addiction, antisocial behavior, and health

Behaviors and disorders related to self-regulation, such as substance use, antisocial conduct, and ADHD, are collectively referred to as externalizing and have a shared genetic liability. We applied a multivariate approach that leverages genetic correlations among externalizing traits for genome-wide association analyses. By pooling data from ~1.5 million people, our approach is statistically more powerful than single-trait analyses and identifies more than 500 genetic loci. The identified loci were enriched for genes expressed in the brain and related to nervous system development. A polygenic score constructed from our results captures variation in a broad range of behavioral and medical outcomes that were not part of our genome-wide analyses, including traits that until now lacked well-performing polygenic scores, such as opioid use disorder, suicide, HIV infections, criminal convictions, and unemployment. Our findings are consistent with the idea that persistent difficulties in self-regulation can be conceptualized as a neurodevelopmental condition.

genetics↗

bioRxiv2020

CTCF-binding element regulates ESC differentiation via orchestrating long-range chromatin interaction between enhancers and HoxA

Proper expressions of Homeobox A cluster genes (HoxA) are essential for embryonic stem cells (ESC) differentiation and individual development. However, the mechanisms underlying controlling precise spatiotemporal expressions of HoxA during early ESC differentiation remain poorly understood. Herein, we identified a functional CTCF-binding element (CBE+47) closest to the 3-end of HoxA within the same topologically associated domain (TAD) in ESC. CRISPR-Cas9 mediated the CBE+47 deletion significantly promotes retinoic acid (RA)-induced HoxA expression and early ESC abnormal differentiation. In delineating its underlying mechanisms, we find that CBE+47 can precisely organize chromatin interactions between its adjacent enhancers and HoxA by using chromosome conformation capture assay (Capture-C). Furthermore, we also find that its adjacent enhancers as enhancer-enhancer interaction complex (EEIC) are required for RA-induced HoxA activation. Collectively, these results provide a new insight for RA-induced HoxA expression, also highlight the unique and precise regulatory roles of CBE in ESC differentiation.

developmental biology↗

bioRxiv2020

Connexin 43 confers chemoresistance through activating PI3K

Circumventing chemoresistance is crucial for effectively treating glioblastoma due to limited therapeutic options. The gap junction protein connexin 43 (Cx43) renders glioblastoma resistant to the frontline chemotherapy temozolomide; however, targeting Cx43 is difficult because mechanisms underlying Cx43-mediated chemoresistance remain elusive. Here we show that Cx43, but not other connexins, is highly expressed in glioblastoma and strongly correlates with poor patient prognosis and chemoresistance, making Cx43 the prime therapeutic target among all connexins. The intracellular carboxyl terminus of Cx43 binds to phosphatidylinositol 3-kinase (PI3K) catalytic subunit {beta} (PIK3CB, also called PI3K{beta} or p110{beta}), thereby activating PI3K signaling independent of Cx43-channels and subsequently inducing temozolomide resistance. A combination of CT1, a Cx43-targeting peptide inhibitor, and PIK3CB-selective inhibitors restores temozolomide sensitivity in vitro and in vivo. This study not only reveals novel mechanistic insights into chemoresistance in glioblastoma, but also demonstrates that targeting Cx43 and PIK3CB/p110{beta} is an effective approach for overcoming chemoresistance.

cancer biology↗

bioRxiv2020

Single-dose intranasal administration of AdCOVID elicits systemic and mucosal immunity against SARS-CoV-2 in mice

The coronavirus disease 2019 (COVID-19) pandemic has highlighted the urgent need for effective preventive vaccination to reduce burden and spread of severe acute respiratory syndrome (SARS) coronavirus 2 (SARS-CoV-2) in humans. Intranasal vaccination is an attractive strategy to prevent COVID-19 as the nasal mucosa represents the first-line barrier to SARS-CoV-2 entry before viral spread to the lung. Although SARS-CoV-2 vaccine development is rapidly progressing, the current intramuscular vaccines are designed to elicit systemic immunity without conferring mucosal immunity. Here, we show that AdCOVID, an intranasal adenovirus type 5 (Ad5)-vectored vaccine encoding the receptor binding domain (RBD) of the SARS-CoV-2 spike protein, elicits a strong and focused immune response against RBD through the induction of mucosal IgA, serum neutralizing antibodies and CD4+ and CD8+ T cells with a Th1-like cytokine expression profile. Therefore, AdCOVID, which promotes concomitant systemic and local mucosal immunity, represents a promising COVID-19 vaccine candidate.

immunology↗

bioRxiv2020

Discriminative SKP2 interactions with CDK-cyclin complexes support a cyclin A-specific role in p27KIP1 degradation

The SCFSKP2 ubiquitin ligase relieves G1 checkpoint control of CDK-cyclin complexes by promoting p27KIP1 degradation. We describe reconstitution of stable complexes containing SKP1-SKP2 and CDK1-cyclin B or CDK2-cyclin A/E, mediated by the CDK regulatory subunit CKS1. We further show that a direct interaction between a SKP2 N-terminal motif and cyclin A can stabilize SKP1-SKP2-CDK2-cyclin A complexes in the absence of CKS1. We identify the SKP2 binding site on cyclin A and demonstrate the site is not present in cyclin B or cyclin E. This site is distinct from but overlapping with features that mediate binding of p27KIP1 and other G1 cyclin regulators to cyclin A. We propose that the capacity of SKP2 to engage with CDK2-cyclin A by more than one structural mechanism provides a way to fine tune the degradation of p27KIP1 and distinguishes cyclin A from other G1 cyclins to ensure orderly cell cycle progression.

biochemistry↗

bioRxiv2020

Structural basis for repurposing a 100-years-old drug suramin for treating COVID-19

The COVID-19 pandemic by non-stop infections of SARS-CoV-2 has continued to ravage many countries worldwide. Here we report the discovery of suramin, a 100-year-old drug, as a potent inhibitor of the SARS-CoV-2 RNA dependent RNA polymerase (RdRp) through blocking the binding of RNA to the enzyme. In biochemical assays, suramin and its derivatives are at least 20-fold more potent than remdesivir, the currently approved nucleotide drug for COVID-19. The 2.6 [A] cryo-EM structure of the viral RdRp bound to suramin reveals two binding sites of suramin, with one site directly blocking the binding of the RNA template strand and the other site clash with the RNA primer strand near the RdRp catalytic active site, therefore inhibiting the viral RNA replication. Furthermore, suramin potently inhibits SARS-CoV-2 duplication in Vero E6 cells. These results provide a structural mechanism for the first non-nucleotide inhibitor of the SARS-CoV-2 RdRp and a rationale for repurposing suramin for treating COVID-19. One Sentence SummaryDiscovery and mechanism of suramin as potent SARS-CoV-2 RNA polymerase inhibitor and its repurposing for treating COVID-19.

molecular biology↗

bioRxiv2020

A Probabilistic and Anisotropic Failure Metric for Ascending Thoracic Aortic Aneurysm Risk Stratification

Experimental studies have shown that aortic wall tensile strengths in circumferential and longitudinal directions are different (i.e., anisotropic), and vary significantly among patients with aortic aneurysm. To assess aneurysm rupture and dissection risk, material failure metric of the aortic wall needs to be accurately defined and determined. Previously such risk assessment methods have largely relied on deterministic or isotropic failure metric. In this study, we develop a novel probabilistic and anisotropic failure metric for risk stratification of ascending thoracic aortic aneurysm (ATAA). To this end, uniaxial tensile tests were performed using aortic tissue samples of 84 ATAA patients, from which a joint probability distribution of the anisotropic wall strengths was obtained. Next, the anisotropic failure probability (FP) based on the Tsai-Hill (TH) failure criterion was derived. The novel FP metric, which incorporates uncertainty in the anisotropic failure properties, can be evaluated after the aortic wall stresses are computed from patient-specific biomechanical analysis. For method validation, "ground-truth" risks of additional 41 ATAA patients were numerically-reconstructed using corresponding CT images and tissue testing data. Performance of different risk stratification methods (e.g., with and without patient-specific hyperelastic properties) was compared using p-value and receiver operating characteristic (ROC) curve. The results show that: (1) the probabilistic FP metric outperforms the deterministic TH metric; and (2) patient-specific hyperelastic properties can help to improve the performance of probabilistic FP metric in ATAA risk stratification.

bioengineering↗

bioRxiv2020

CircRNA may not be "circular"

Circular RNA (circRNA) is a novel regulatory non-coding RNA and participates in diverse physiological and pathological processes. However, the structures and molecular mechanisms of circRNAs remain unclear. In this study, we took advantage of circRNA array and bioinformatics analysis and found lots of internal complementary base-pairing sequences (ICBPS) existed in plenty of circRNAs, especially in extremely long circRNAs (el-circRNAs, > 5,000 nt). The result indicated that circRNA may not be a simple circular structure. In addition, we put forward the hypothesis of "open-close effect " in the transition for specific circRNA from normal state to morbid state. Taken together, our results not only expand the knowledge of circRNAs, but also highlight the potential molecular mechanism of circRNAs.

molecular biology↗

bioRxiv2020

Telmisartan potentiates insulin secretion via ion channels, independent of the AT1 receptor and PPARγ

Angiotensin II type 1 receptor blockers (ARBs), as antihypertensive drugs, have drawn attention for their benefits to individuals with diabetes and prediabetes. However, the effects of ARBs on insulin secretion remain unclear. Here, we investigated the insulinotropic effects of ARBs (telmisartan, valsartan, and irbesartan) and the underlying electrophysiological mechanism in rat islets. We found that only telmisartan among the three ARBs exhibited an insulin secretagogue role. Distinct from other ARBs, telmisartan exerted effects on ion channels including voltage-gated potassium (Kv) channels and voltage-gated Ca2+ channels to promote extracellular Ca2+ influx, thereby potentiating insulin secretion in a glucose-dependent manner. We observed that the peroxisome proliferator-activated receptor {gamma} pathway was not involved in these telmisartan-induced effects. Furthermore, we identified that telmisartan at least directly inhibited Kv2.1 channel through construction of a Chinese hamster ovary cell line with Kv2.1 channel overexpression. Acute exposure of type 2 diabetes model (db/db) mice to a telmisartan dose equivalent to therapeutic doses in humans resulted in lower blood glucose and increased plasma insulin concentration in the oral glucose tolerance test. We further observed the telmisartan-induced insulinotropic and electrophysiological effects on pathological pancreatic islets isolated from db/db mice. Collectively, our results establish an important function of telmisartan distinct from other ARBs in the treatment of diabetes.

cell biology↗

bioRxiv2020

TIN2 facilitates TRF1-mediated trans- and cis-interactions on physiologically relevant long telomeric DNA

The shelterin complex consisting of TRF1, TRF2, RAP1, TIN2, TPP1, and POT1, functions to prevent false recognition of telomeres as double-strand DNA breaks, and to regulate telomerase and DNA repair protein access. TIN2 is a core component linking double-stranded telomeric DNA binding proteins (TRF1 and TRF2) and proteins at the 3 overhang (TPP1-POT1). Since knockdown of TIN2 also removes TRF1 and TRF2 from telomeres, determining TIN2s unique mechanistic function has been elusive. Here, we investigated DNA molecular structures promoted by TRF1-TIN2 using complementary single-molecule imaging platforms, including atomic force microscopy (AFM), total internal reflection fluorescence microscopy (TIRFM), and the DNA tightrope assay. We demonstrate that TIN2S and TIN2L isoforms facilitate TRF1-mediated DNA compaction (cis-interactions) and DNA-DNA bridging (trans-interactions) in a telomeric sequence- and length-dependent manner. On the short telomeric DNA substrate (6 TTAGGG repeats), the majority of TRF1 mediated telomeric DNA-DNA bridging events are transient with a lifetime of ~1.95 s. On longer DNA substrates (270 TTAGGG), TIN2 forms multi-protein complexes with TRF1 and stabilizes TRF1-mediated DNA-DNA bridging events that last for at least minutes. Preincubation of TRF1 with its regulator protein Tankyrase 1 significantly reduces TRF1-TIN2 mediated DNA-DNA bridging, whereas TIN2 protects the disassembly of TRF1-TIN2 mediated DNA-DNA bridging upon Tankyrase 1 addition. Our study provides evidence that TIN2 functions to promote TRF1 mediated trans-interactions of telomeric DNA, leading to new mechanistic insight into sister telomere cohesion.

biophysics↗

bioRxiv2020

Alleviation of neuropathic pain by over-expressing a soluble colony-stimulating factor 1 receptor

In this study, we aim to alleviate neuropathic pain by suppressing microgliosis and macrophage accumulation, which is achieved by over-expressing a non-functional soluble colony-stimulating factor 1 receptor using adeno-associated virus 9 vectors (AAV9/sCSF1R). AAV9/sCSF1R and AAV9/GFP were intrathecally administered into mouse lumbar spine. Two weeks later, these mice underwent partial sciatic nerve ligation to induce neuropathic pain. GFP and sCSF1R were highly expressed in dorsal root ganglia (DRG) and spinal cords in AAV9-injected mice. Nerve ligation alone or pre-treated with AAV9/GFP led to significant microgliosis in the lumbar spinal cords and macrophage accumulation in DRG and sciatic nerves. In nerve-ligated mice pre- treated with AAV9/sCSF1R the microglia densities in the dorsal and ventral horns and macrophage densities in DRG and sciatic nerves were significantly lower compared to nerve-ligated mice pre-treated with AAV9/GFP. Behavioural tests showed that nerve-ligated mice pre- treated with AAV9/sCSF1R had a significantly higher paw withdrawal threshold, indicating the alleviation of neuropathic pain. The results implicate that viral vector-mediated expression of sCSF1R may represent a novel strategy in long-term alleviation of neuropathic pain.

neuroscience↗