Neuronal Adhesion Research Articles

ROCK Inhibitor Enhances Neurite Outgrowth In Vitro and Corneal Sensory Nerve Reinnervation In Vivo.

The intraepithelial corneal nerves are essential to corneal health. Rho kinase or ROCK inhibitors (RIs) have been reported to play a role in neuron survival after injury. Here we assess integrin and extracellular matrix expression in primary mouse neurons and determine whether treating cells with RI impacts neurite outgrowth in vitro and reinnervation after trephine and debridement injury in mice in vivo. Cocultures of human corneal limbal epithelial cells and E11.5 mouse trigeminal neurons and neurons alone were grown on glass coverslips. High-resolution imaging was performed to localize integrins and laminin on neurons and to determine whether RI impacts neurite outgrowth in vitro and in vivo after both 1.5-mm trephine and 1.5-mm debridement injuries. Several integrin α (α3, α6, αv) chains as well as β4 integrin are expressed on neuron axons and growth cones in cocultures. RI treatment of isolated neurons, cocultures, and in conditioned media increases neurite outgrowth. In vivo, RI positively impacts sensory nerve reinnervation after trephine and debridement injury. These studies are the first to demonstrate expression of β4 integrin on trigeminal sensory neurons and preferential adhesion of neurons to the laminin-enriched matrices found in footprints deposited by human corneal limbal epithelial cells. In addition, we also document for the first time the positive impact of RI on neurite outgrowth in vitro and reinnervation in vivo.

Novel 3D printed bioactive SiC orthopedic screw promotes bone growth associated activities by macrophages, neurons, and osteoblasts.

Ceramic additive manufacturing currently relies on binders or high-energy lasers, each with limitations affecting final product quality and suitability for medical applications. To address these challenges, our laboratory has devised a surface activation technique for ceramic particles that eliminates the necessity for polymer binders or high-energy lasers in ceramic additive manufacturing. We utilized this method to 3D print bioactive SiC orthopedic screws and evaluated their properties. The study's findings reveal that chemical oxidation of SiC activated its surface, enabling 3D printing of orthopedic screws in a binder jet printer. Post-processing impregnation with NaOH and/or NH4OH strengthened the scaffold by promoting silica crystallization or partial conversion of silicon oxide into silicon nitride. The silica surface of the SiC 3D printed orthopedic screws facilitated osteoblast and neuron adhesion and extensive axon synthesis. The silicate ions released from the 3D printed SiC screws favorably modulated macrophage immune responses toward an M1 phenotype as indicated by the inhibition of TNFα secretions and of reactive oxygen species (ROS) expression along with the promotion of IL6R shedding. In contrast, under the same experimental conditions, Ti ions released from Ti6Al4V discs promoted macrophage TNFα secretion and ROS expression. In vivo tests demonstrated direct bone deposition on the SiC scaffold and a strong interfacial bond between the implanted SiC and bone. Immunostaining showed innervation, mineralization, and vascularization of the newly formed bone at the interface with SiC. Taken altogether, the 3D printed SiC orthopedic screws foster a favorable environment for wound healing and bone regeneration. The novel 3D printing method, based on ceramic surface activation represents a significant advancement in ceramic additive manufacturing and is applicable to a wide variety of materials.

Understanding the function of Pax5 in development of docetaxel-resistant neuroendocrine-like prostate cancers

Resistance to the current Androgen Receptor Signaling Inhibitor (ARSI) therapies has led to higher incidences of therapy-induced neuroendocrine-like prostate cancer (t-NEPC). This highly aggressive subtype with predominant small-cell-like characteristics is resistant to taxane chemotherapies and has a dismal overall survival. t-NEPCs are mostly treated with platinum-based drugs with a combination of etoposide or taxane and have less selectivity and high systemic toxicity, which often limit their clinical potential. During t-NEPC transformation, adenocarcinomas lose their luminal features and adopt neuro-basal characteristics. Whether the adaptive neuronal characteristics of t-NEPC are responsible for such taxane resistance remains unknown. Pathway analysis from patient gene-expression databases indicates that t-NEPC upregulates various neuronal pathways associated with enhanced cellular networks. To identify transcription factor(s) (TF) that could be important for promoting the gene expression for neuronal characters in t-NEPC, we performed ATAC-Seq, acetylated-histone ChIP-seq, and RNA-seq in our NE-like cell line models and analyzed the promoters of transcriptionally active and significantly enriched neuroendocrine-like (NE-like) cancer-specific genes. Our results indicate that Pax5 could be an important transcription factor for neuronal gene expression and specific to t-NEPC. Pathway analysis revealed that Pax5 expression is involved in axonal guidance, neurotransmitter regulation, and neuronal adhesion, which are critical for strong cellular communications. Further results suggest that depletion of Pax5 disrupts neurite-mediated cellular communication in NE-like cells and reduces surface growth factor receptor activation, thereby, sensitizing them to docetaxel therapies. Moreover, t-NEPC-specific hydroxymethylation of Pax5 promoter CpG islands favors Pbx1 binding to induce Pax5 expression. Based on our study, we concluded that continuous exposure to ARSI therapies leads to epigenetic modifications and Pax5 activation in t-NEPC, which promotes the expression of genes necessary to adopt taxane-resistant NE-like cancer. Thus, targeting the Pax5 axis can be beneficial for reverting their taxane sensitivity.

The role of elasticity on adhesion and clustering of neurons on soft surfaces

The question of whether material stiffness enhances cell adhesion and clustering is still open to debate. Results from the literature are seemingly contradictory, with some reports illustrating that adhesion increases with surface stiffness and others suggesting that the performance of a system of cells is curbed by high values of elasticity. To address the role of elasticity as a regulator in neuronal cell adhesion and clustering, we investigated the topological characteristics of networks of neurons on polydimethylsiloxane (PDMS) surfaces - with values of elasticity (E) varying in the 0.55–2.65 MPa range. Results illustrate that, as elasticity increases, the number of neurons adhering on the surface decreases. Notably, the small-world coefficient – a topological measure of networks – also decreases. Numerical simulations and functional multi-calcium imaging experiments further indicated that the activity of neuronal cells on soft surfaces improves for decreasing E. Experimental findings are supported by a mathematical model, that explains adhesion and clustering of cells on soft materials as a function of few parameters - including the Young’s modulus and roughness of the material. Overall, results indicate that – in the considered elasticity interval – increasing the compliance of a material improves adhesion, improves clustering, and enhances communication of neurons.

334 Identification of novel plasma protein biomarkers for diagnosis and prediction of Alzheimer’s disease in African Americans

OBJECTIVES/GOALS: To identify novel panel of plasma protein biomarkers to improve prediction and diagnosis of Alzheimer’s disease (AD) for African Americans (AA), who are at greater risk of developing AD compared to non-Hispanic White individuals but are underrepresented in AD research. METHODS/STUDY POPULATION: Pre-existing plasma samples from 460 AA individuals with clinical diagnoses of AD, cognitively unimpaired (CU), mild cognitive impairment (MCI), or dementia with Lewy bodies (DLB) will undergo untargeted proteomics using the SomaScan assay, where modified single stranded DNA aptamers bind to protein targets which are quantified by DNA microarray. Protein expression levels will be compared between diagnostic groups to identify differentially expressed proteins. Additional clinical, genetic, and lifestyle factors will be compared with protein expression when available. Proteins of interest, identified by differential protein expression analysis results, will be included in receiver operating characteristic analyses to identify the optimal set of proteins for diagnostic classification. RESULTS/ANTICIPATED RESULTS: A pilot experiment utilizing plasma from 40 individuals identified multiple differentially expressed proteins (DEPs) between AD and non-AD groups. Eight proteins were nominated from the differential protein analysis into a receiver operating characteristic (ROC) analysis based on pvalue and previous implication in AD genome wide association studies. Proteins involved in microglial activation, neuronal adhesion, cell proliferation, and innate immunity were nominated. The ROC model achieved 100% classification accuracy of AD and CU groups using age, sex, and the eight nominated proteins. It is expected that there will be more significant associations when utilizing the full cohort of 460 AA and that DEPs between AD, CU, MCI, and DLB will be identified. DISCUSSION/SIGNIFICANCE: The nomination of a novel panel of plasma biomarkers developed from an AA cohort will directly serve the AA community by improving access to an early and accurate diagnosis of AD. Access to improved prediction and diagnosis will likely improve disease management, thus improving patient outcomes and decreasing burden on families and caregivers.

Abstract 3589: Understanding the role of Pax5 in development of taxane-resistant NE-like prostate cancer

Abstract Background: Resistance to the existing Androgen Receptor Signaling Inhibitor (ARSI) therapies led to higher incidences of aggressive therapy-induced neuroendocrine-like prostate cancer (t-NEPC) with predominant small cell-like characteristics, dismal overall survival, and an ultimate resistance to taxane chemotherapies. t-NEPCs are mostly treated with platinum-based drugs with etoposide and/or taxane but have less selectivity and high systemic toxicity, which often limits their clinical and therapeutic potential. During t-NEPC transformation, upon chromatin reprogramming associated with epigenetic alterations and altered transcriptional activity, adenocarcinoma loses its luminal features to adopt neurobasal characteristics. The following study aims to unravel the mechanism of developing t-NEPC taxane resistance upon acquiring such adaptive neuronal characteristics. Objective: The primary objective is to delineate how chromatin modifications along with epigenetic regulation facilitates the adaptive neuronal characteristics and dictates the development of t-NEPC taxane resistance. Methods: We generated various t-NEPC cell lines either by genetic modification (DKD) or therapeutic pressure (C4-2BER). We performed RNA-Seq, ATAC-Seq and acetylated Histone (H3K18 and H3K27) ChIP-Seq in our developed t-NEPC models and compared with adenocarcinoma. We next compared ATAC Seq and Acetylated histone ChIP Seq with RNA Seq in C4-2B and C4-2BER and performed functional assays. Results: Our ATAC Seq and acetylated histone footprints (Ac H3K18 and Ac H3K27 ChIP Seq) analyses revealed an enhanced chromatin accessibility during t-NEPC transformation. Overlapping RNA seq suggested newly transcribed neuronal genes with higher extent of promoter accessibility. Transcription Factor mapping among newly accessible gene promoters revealed preferential Pax5 binding, whose expression occurs selectively in t-NEPC unlike adenocarcinoma. Pathway analysis of differentially expressed neuronal genes in t-NEPC suggested an involvement of Pax5 in axonal guidance, neurotransmitter regulation, and neuronal adhesion, which are essential for strong cellular communication. Furthermore, Pax5 depletion disrupts cellular interaction in t-NEPC, thereby, sensitizing them to taxane therapies. Conclusions: Our study concludes that continuous AR ablation with ARSIs, leads to chromatin alterations with epigenetic changes, thereby allowing Pax5 transcriptional activation in t-NEPC. This altered transcriptional activity promotes gene expression necessary to govern taxane-resistant t-NEPC development. Citation Format: Sreyashi Bhattacharya, Ridwan Islam, Sanika Bodas, Juhi Mishra, Dipanwita Das, Kaustubh Datta, Samikshan Dutta. Understanding the role of Pax5 in development of taxane-resistant NE-like prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3589.

Fast Electrodeposition of MXene/PDA Composites for High‐Performance Bioelectronic Interfaces: An In Vitro Evaluation

AbstractBioelectrode is critical to many biomedical researches. However, traditional materials (typically noble metals) and manufacturing techniques limit the large‐scale production of bioelectrodes. Herein, a fast electrochemical approach is proposed to deposit versatile MXene/polydopamine (PDA) composites on a metalized substrate. PDA coating can improve the adhesion between MXene and the substrate, while MXene provides rough surfaces with unique micro/nanostructure and outstanding electrical/optical/thermal performance. The impedance of the as‐prepared bioelectrode at 1 kHz is down to 8.48 Ω cm2. The corresponding cathodic charge storage capacity (CSCc) and charge injection capacity (CIC) are up to ≈250 and 6.59 mC cm−2 respectively, much superior to that of bare Pt and other conventional material‐based electrodes. The MXene/PDA composites also demonstrate robust stability under continuous electrostimulation for 1 × 108 pulse cycles and 1000 CV cycles. Moreover, MXene/PDA composites show a high and rapid photothermal response. Photoelectrochemical activity is also observed with high photocurrent, ≈40 folds larger than that of bare Pt. The utility of this new electrode in ascorbic acid sensing is demonstrated. Excellent biocompatibility is verified via neuron adhesion test and viability assay.

Block Sequence Effects on the Self-Assembly Behaviors of Polypeptide-Based Penta-Block Copolymer Hydrogels.

Peptide-based hydrogels have great potential for applications in tissue engineering, drug delivery, and so on. We systematically synthesize, characterize, and investigate the self-assembly behaviors of a series of polypeptide-based penta-block copolymers by varying block sequences and lengths. The copolymers contain hydrophobic blocks of poly(γ-benzyl-l-glutamate) (PBG, Bx) and two kinds of hydrophilic blocks, poly(l-lysine) (PLL, Ky) and poly(ethylene glycol) (PEG, EG34), where x and y are the number of repeating units of each block, where PBG and PLL blocks have unique functions for nerve regeneration and cell adhesion. It shows that a sufficient length of the middle hydrophilic segment capped with hydrophobic end PBG blocks is required. They first self-assemble into flower-like micelles and sequentially form transparent hydrogels (as low as 2.3 wt %) with increased polymer concentration. The hydrogels contain a microscale porous structure, a desired property for tissue engineering to facilitate the access of nutrient flow for cell growth and drug delivery systems with high efficiency of drug storage. We hypothesize that the structure of Bx-Ky-EG34-Ky-Bx agglomerates is beyond micron size (transparent), while that of Ky-Bx-EG34-Bx-Ky is on the submicron scale (opaque). We establish a working strategy to synthesize a polypeptide-based block copolymer with a wide window of sol-gel transition. The study offers insight into rational polypeptide hydrogel design with specific morphology, exploring the novel materials as potential candidates for neural tissue engineering.

An optimized method of culturing neurons based on polyacrylamide gel.

Substrate stiffness is a microenvironment with a certain stiffness constructed by the extracellular matrix and adjacent cells, which plays an important role in the growth and development of cells and tissue formation. Studies have indicated that the stiffness of the brain is about 0.1-1 kPa. The physiological and pathological processes of the nervous system are mediated by the substrate stiffness that the neurons suffer. However, how substrate stiffness regulates these processes remains to be studied. Culturing neurons on substrates with different stiffness in vitro is one of the best methods to study the role of stiffness in regulating neuronal development and activity. In this study, by changing the preparation time and the activation time of polyacrylamide gel, we provide an improved method that achieves a low toxic substrate environment for better primary neuron adhesion and development. Hope that this method is convenient for those studying the role of substrate stiffness in neurons.

The role of fibrinogen in neurodegeneration during a neuroinflammatory disease

AbstractBackgroundFibrinogen is not only a marker of inflammation, but also a cause of inflammatory responses. We previously showed that when elevated during traumatic brain injury (TBI), fibrinogen increased cerebrovascular permeability mainly via caveolar transcytosis and, after extravasation and deposition in vasculo‐astrocyte interface, it activated astrocytes causing neurodegeneration that was associated with memory reduction.MethodTo define the possible mechanisms of fibrinogen‐induced activation of neurons, primary mouse brain cortical neurons from C57BL/6 mice were plated in poly‐d‐lysine‐ and laminin‐coated cell culture plates. Cells were treated for 1 hour with fibrinogen (0.5 or 1 mg/mL) in the presence or absence of a nuclear factor‐κB (NF‐kB) blocker, caffeic acid phenethyl ester (CAPE), or media alone (control). All groups contained hirudin to prevent the conversion of fibrinogen to fibrin. Levels of interleukin‐6 (IL‐6) and chemokine (C‐C motif) ligand 2 (CCL2) were assessed by qRT‐PCR. NF‐kB activity was measured in neuronal nuclear protein and cell extracts by the TransAMTM NF‐kB p65 protein assay and Western blot (WB). In parallel, using a cortical contusion injury (CCI), a mouse model of mild‐to‐moderate TBI was created in C576J mice. NF‐kB was detected with immunocytochemistry in cultured cells and brain samples collected 14 days after CCI. The specific association of fibrinogen with its neuronal receptors intercellular adhesion molecule‐1 (ICAM‐1) and cellular prion protein (PrPC) was assessed with a proximity ligation assay in cells.ResultFibrinogen specifically bound to neurons via its receptors ICAM‐1 and PrPC and increased NF‐kB levels. Fibrinogen caused upregulation of pro‐inflammatory cytokines IL‐6 and CCL2 mRNA. ELISA and WB NF‐kB p65 analysis demonstrated increased DNA binding by p65 and protein expression in fibrinogen‐treated neurons compared to that in control and CAPE groups. Fibrinogen caused translocation of NF‐kB p65 to the nucleus.ConclusionResults suggest that a specific interaction of fibrinogen with neurons results in neuroinflammation through activation of NF‐kB.

Single-cell RNA sequencing analysis of the retina under acute high intraocular pressure.

JOURNAL/nrgr/04.03/01300535-202419110-00032/figure1/v/2024-03-08T184507Z/r/image-tiff High intraocular pressure causes retinal ganglion cell injury in primary and secondary glaucoma diseases, yet the molecular landscape characteristics of retinal cells under high intraocular pressure remain unknown. Rat models of acute hypertension ocular pressure were established by injection of cross-linked hyaluronic acid hydrogel (Healaflow®). Single-cell RNA sequencing was then used to describe the cellular composition and molecular profile of the retina following high intraocular pressure. Our results identified a total of 12 cell types, namely retinal pigment epithelial cells, rod-photoreceptor cells, bipolar cells, Müller cells, microglia, cone-photoreceptor cells, retinal ganglion cells, endothelial cells, retinal progenitor cells, oligodendrocytes, pericytes, and fibroblasts. The single-cell RNA sequencing analysis of the retina under acute high intraocular pressure revealed obvious changes in the proportions of various retinal cells, with ganglion cells decreased by 23%. Hematoxylin and eosin staining and TUNEL staining confirmed the damage to retinal ganglion cells under high intraocular pressure. We extracted data from retinal ganglion cells and analyzed the retinal ganglion cell cluster with the most distinct expression. We found upregulation of the B3gat2 gene, which is associated with neuronal migration and adhesion, and downregulation of the Tsc22d gene, which participates in inhibition of inflammation. This study is the first to reveal molecular changes and intercellular interactions in the retina under high intraocular pressure. These data contribute to understanding of the molecular mechanism of retinal injury induced by high intraocular pressure and will benefit the development of novel therapies.

PEDOT-Citrate/SIKVAV modified bioaffinity microelectrode arrays for detecting theta rhythm cells in the retrosplenial cortex of rats under sensory conflict

In scenarios where visual and vestibular inputs are dissociated, humans’ spatial cognitive and task performance can deteriorate. To investigate the neural basis, microelectrode arrays (MEA) with good chronic recording performance are needed. In this study, we proposed a novel approach to modify alkaline laminin onto neural electrodes and constructed an MEA using laminin (SIKVAV) modified for single-neuron tracking detecting. The modified interface has excellent hydrophilicity and biocompatibility and is conducive to enhancing the adhesion of neurons. Moreover, the MEAs exhibits excellent performance on signal-to-noise ratio, neuron detection amount, and neuron tracking effects in chronic recording experiment. Utilizing this tool, we record the electrophysiological activities in the rats' brains under sensory conflict. We observed an apparent response of pyramidal neurons to sensory conflict. Notably, this conflict disrupted the theta rhythmic firing of theta cells and local field potentials (LFP) theta oscillations in the retrosplenial cortex (RSC), which is associated with cognition, and both required a certain amount of time for recovery. This study provides a method for chronic detection of neurons in vivo and reveals part of the mechanism of cognitive impairment of patients with sensory conflict, which may provide theoretical support for future treatment strategies.

Increased peripheral of brain-derived neurotrophic factor levels in phenylketonuric patients treated with l-carnitine

Phenylketonuria (PKU) is the most common inherited metabolic disorders caused by severe deficiency or absence of phenylalanine hydroxylase activity that converts phenylalanine (Phe) to tyrosine. PKU patients were treated with a Phe restricted diet supplemented with a special formula containing l-carnitine (L-car), well-known antioxidant compound. The lack of treatment can cause neurological and cognitive impairment, as severe mental retardation, neuronal cell loss and synaptic density reduction. Although Phe has been widely demonstrated to be involved in PKU neurotoxicity, the mechanisms responsible for the CNS injury are still not fully known. In this work, we evaluated markers of neurodegeneration, namely BDNF (brain-derived neurotrophic factor), PAI-1 total (Plasminogen activator inhibitor-1 total), Cathepsin D, PDGF AB/BB (platelet-derived growth factor), and NCAM (neuronal adhesion molecule) in plasma of PKU patients at early and late diagnosis and under treatment. We found decreased Phe levels and increased L-car concentrations in PKU patients treated with L-car compared to the other groups, indicating that the proposed treatment was effective. Furthermore, we found increased BDNF levels in the patients under treatment compared to patients at early diagnosis, and a positive correlation between BDNF and L-car and a negative correlation between BDNF and Phe. Our results may indicate that in PKU patients treated with L-car there is an attempt to adjust neuronal plasticity and recover the damage suffered, reflecting a compensatory response to brain injury.

Abstract 036: The Single-cell Gene Expression Landscape Of The Hypothalamus During The Development Of Hypertension In Three Major Models

The hypothalamus plays a key role in blood pressure regulation through coordinating and integrating signals in the central nervous system. However, it’s remains underexplored how the cell signaling network in the hypothalamus changes during the progression of hypertension, the key regulators involved, and whether shared or distinct pathophysiological mechanisms underlie the development of hypertension triggered by various genetic susceptibility or environmental factors. To address these questions, we applied the single-nucleus RNA sequencing technology to analyze hypothalamus samples from three well-established models of hypertension with distinct pathogenesis - mice treated with Ang II, the Dahl salt-sensitive rat, and the spontaneous hypertensive rat - along with their respective controls. Data were obtained from 238,445 nuclei from 24 samples under 12 experimental conditions. By characterizing the cellular composition under different disease conditions, we discovered an elevated glia-to-neuron ratio in Ang II-induced hypertension, with increases of 3.1-fold in astrocytes, 4.0-fold in microglia, and 3.4-fold in oligodendrocytes. Moreover, astrocytes underwent a transition from the resting state to a reactive mode associated with neuroinflammation and stronger neuron adhesion. Additionally, multiple neuronal signal communication pathways were overactivated in response to Ang II, including neurexin-neuroligin signaling, nitric oxide-guanylate cyclase pathway, and glutamate and its receptors. In the salt-sensitive hypertension, a 2.3-fold expansion of pro-inflammatory microglia via Cd74 activation was observed after a short-term high-sodium diet, which leveled off thereafter. With a prolonged high-salt intake, there was a 1.7-fold increase in the proportion of Plagl1+ GABA-ergic neurons. In 26 weeks old spontaneously hypertensive rats, a distinctive 1.4-fold increase in the proportion of Slit3+ Glu-ergic neurons was observed, setting them apart from WKY rats. These findings provide new insights into how the hypothalamus may be involved in the development of hypertension under different disease conditions.

A simple method for poly-D-lysine coating to enhance adhesion and maturation of primary cortical neuron cultures in vitro.

Glass coverslips are used as a substrate since Harrison's initial nerve cell culture experiments in 1910. In 1974, the first study of brain cells seeded onto polylysine (PL) coated substrate was published. Usually, neurons adhere quickly to PL coating. However, maintaining cortical neurons in culture on PL coating for a prolonged time is challenging. A collaborative study between chemical engineers and neurobiologists was conducted to find a simple method to enhance neuronal maturation on poly-D-lysine (PDL). In this work, a simple protocol to coat PDL efficiently on coverslips is presented, characterized, and compared to a conventional adsorption method. We studied the adhesion and maturation of primary cortical neurons with various morphological and functional approaches, including phase contrast microscopy, immunocytochemistry, scanning electron microscopy, patch clamp recordings, and calcium imaging. We observed that several parameters of neuronal maturation are influenced by the substrate: neurons develop more dense and extended networks and synaptic activity is enhanced, when seeded on covalently bound PDL compared to adsorbed PDL. Hence, we established reproducible and optimal conditions enhancing maturation of primary cortical neurons in vitro. Our method allows higher reliability and yield of results and could also be profitable for laboratories using PL with other cell types.

Transcriptional profiles in zebrafish atp7a mutants and responses of atp7a mutants to Cu stress

As a copper (Cu) transport ATPase, ATP7A plays an important role in maintaining Cu homeostasis in the body, but the developmental and physiological roles of atp7a in zebrafish embryogenesis are rarely studied. In this study, normal morphological phenotypes of atp7a−/− homozygous zebrafish were observed at both embryonic and adult stages, however, atp7a−/− larvae exhibited delayed touch response and obvious transcriptome changes. Compared with the WT (wild type), differentially expressed genes (DEGs) in atp7a−/− larvae showed the enrichment in gene ontology (GO) terms related to several processes including ATPase activity, oxidoreductase activity, active transmembrane transporter activity, ion binding, and the citrate cycle. Furthermore, decreases in both ATP content and Na+/K+-ATPase activity in atp7a−/− embryos and larvae were unveiled. 57 overlapping DEGs were found both in WT stressed with Cu and in WT mutated with atp7a, and GO term analysis indicated the enrichment in the genes related to neurexin family protein binding and neuronal cell-cell adhesion. Moreover, 42 overlapping DEGs in Cu stressed WT and Cu stressed atp7a−/− were identified. GO term analysis showed an enrichment in the genes related to heme binding, implying that Cu was independent of the integral function of atp7a to affect heme binding. In addition, genes involved in the negative regulation of angiogenesis were down-regulated in atp7a−/− mutants with and without Cu stress, which failed to occur in WT, implying that the integral function of atp7a is required for maintaining the normal expression of angiogenesis genes. The integrative data in this study demonstrated that atp7a is required for ion transport and angiogenesis, and for Cu-induced neurexin family protein binding defects, rather than for Cu-induced heme binding defects, during zebrafish embryogenesis. These findings provide possible clues for human diseases with ATP7A dysfunction and imbalanced Cu homeostasis.

Matrilin-2 within a three-dimensional lysine-modified chitosan porous scaffold enhances Schwann cell migration and axonal outgrowth for peripheral nerve regeneration.

Background: Matrilin-2 is a key extracellular matrix protein involved in peripheral nerve regeneration. We sought to develop a biomimetic scaffold to enhance peripheral nerve regeneration by incorporating matrilin-2 within a chitosan-derived porous scaffold. We hypothesized that the use of such a novel biomaterial delivers microenvironmental cues to facilitate Schwann cell (SC) migration and enhance axonal outgrowth during peripheral nerve regeneration. Materials and Methods: The effect of matrilin-2 on SC migration was evaluated with agarose drop migration assay on matrilin-2 coated dishes. SC adhesion was determined with SCs cultured atop tissue culture dishes coated with matrilin-2. Various formulations of chitosan vs matrilin-2 in scaffold constructs were examined with scanning electron microscopy. The effect of the matrilin-2/chitosan scaffold on SC migration in the collagen conduits was determined by capillary migration assays. Neuronal adhesion and axonal outgrowth were evaluated with three-dimensional (3D) organotypic assay of dorsal root ganglions (DRG). DRG axonal outgrowth within the scaffolds was determined by immunofluorescence staining of neurofilaments. Results: Matrilin-2 induced SC migration and enhanced its adhesion. A formulation of 2% chitosan with matrilin-2 demonstrated an optimal 3D porous architecture for SC interaction. Matrilin-2/chitosan scaffold enabled SCs to migrate against gravity within conduits. Chemical modification of chitosan with lysine (K-chitosan) further improved DRG adhesion and axonal outgrowth than the matrilin-2/chitosan scaffold without lysine modification. Conclusion: We developed a matrilin-2/K-chitosan scaffold to mimic extracellular matrix cues and provide a porous matrix to enhance peripheral nerve regeneration. Taking advantage of matrilin-2's capability to stimulate SC migration and adhesion, we formulated a porous matrilin-2/chitosan scaffold to support axongal outgrowth. Chemical modification of chitosan with lysine further improved matrilin-2 bioactivity in the 3D scaffold. The 3D porous matrilin-2/K-chitosan scaffolds have high potential for enhancing nerve repair by stimulating SC migration, neuronal adhesion, and axonal outgrowth.

4D label-free proteomic analysis of vitreous from patients with rhegmatogenous retinal detachment.

To identify metabolites, proteins, and related pathways involved in the etiology of rhegmatogenous retinal detachment (RRD) for use as biomarkers in diagnosing and treating RRD. Vitreous specimens were collected and liquid chromatography-tandem mass spectrometry analysis was performed using the four-dimensional label-free technique. Statistically significant differentially expressed proteins, gene ontology (GO) terms, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway representations, and protein interactions were analyzed. Nine specimens were subjected to proteomic analysis. In total, 161 proteins were identified as differentially expressed proteins (DEPs), including 53 upregulated proteins and 108 downregulated proteins. GO functional analysis revealed that some DEPs were enriched in neuron-related terms and membrane protein terms. Moreover, KEGG analysis indicated that the cell adhesion molecule metabolic pathway was associated with the greatest number of DEPs. Finally, the evaluation of protein-protein interaction network revealed that DEPs were clustered in neuronal adhesion, apoptosis, inflammation and immune responses, correct protein folding, and glycolysis. Proteomic profiling is useful for the exploration of molecular mechanisms that underlie RRD. This study reveals increased expression levels of proteins related to heat shock protein content, glycolysis, and inflammatory responses in RRD. Knowledge regarding biomarkers of RRD pathogenesis may help to prevent the occurrence of RRD in the future.

Genetic association study between Astrotactin-2 (ASTN2) rs10817999 gene polymorphism and attention deficit hyperactivity disorder in Korean children

ASTN2 is an integral membrane protein whose role is necessary to neuronal migration's neural-glial adhesion. It has been reported its association of ASTN2 with attention deficit hyperactivity disorder (ADHD). ADHD is a heterogeneous disorder influenced by various environmental and genetic factors. Therefore, we analyzed the genetic association of ASTN2 rs10817999 and rs12376789, with ADHD in Korean children with ADHD and children without ADHD. A total of 472 samples (150 ADHD children and 322 controls) were analyzed in this study. There were no significant results of genotype and allele distribution in rs12376789. By contrast, rs10817999 showed significant genotype and allele frequency differences between the control and the cases (p = 0.003 and 0.001, respectively). The OR of ADHD children with the rs10817999 C/T genotype compare with control was 0.335 (95 % CI 0.126–0.893, p = 0.023) and rs10817999 T/T genotype compare with control was 4.406 (95 % CI 1.662–11.680, p = 0.001), and the OR with rs10817999 T allele versus C allele was 1.658 (95 % CI 1.217–2.260, p = 0.001). Significant associations were also observed in the dominant (p = 0.005) and the recessive (p = 0.008) genetic models. In the stratified analyses of ADHD subtypes and sex, we also observed a significant association of rs10817999 and ADHD (p < 0.05). In summary, we identified that the rs10817999 was associated with ADHD in Korean children. These findings may be used as a basis of replication in other ethnic groups or populations to clarify the possible role of this genetic variant in ADHD.

Investigating NRP1 and VEGFR2 association using number and brightness.

Neuropilin-1 (NRP1) is a surface glycoprotein originally found to play a role in neuronal adhesion. It has since been discovered that NRP1 is involved in vascular development, as well as tumorigenesis. NRP1 has been previously shown to exist in a monomer-dimer equilibrium in the plasma membrane, in the presence of vascular endothelial growth factor (VEGF). With the addition of vascular endothelial growth factor receptor 2 (VEGFR2), NRP1 can also form heterodimers in the presence or absence of VEGF. The aim of my project is to further probe NRP1 homo-interactions and hetero-interactions with VEGFR2 in the presence and absence of VEGFA-165, using the number and brightness fluorescence fluctuation method (N&B). Using N&B, I hope to quantify the oligomer size of NRP1 under different conditions.