Early-stage antibody kinetics after the third dose of BNT162b2 mRNA COVID-19 vaccination measured by a point-of-care fingertip whole blood testing.

Amid the Coronavirus Disease 2019 pandemic, we aimed to demonstrate the accuracy of the fingertip whole blood sampling test (FWT) in measuring the antibody titer and uncovering its dynamics shortly after booster vaccination. Mokobio SARS-CoV-2 IgM & IgG Quantum Dot immunoassay (Mokobio Biotechnology R&D Center Inc., MD, USA) was used as a point-of-care FWT in 226 health care workers (HCWs) who had received two doses of the BNT162b2 mRNA vaccine (Pfizer-BioNTech) at least 8 months prior. Each participant tested their antibody titers before and after the third-dose booster up to 14-days. The effect of the booster was observed as early as the fourth day after vaccination, which exceeded the detection limit (> 30,000 U/mL) by 2.3% on the fifth day, 12.2% on the sixth day, and 22.5% after the seventh day. Significant positive correlations were observed between the pre- and post-vaccination (the seventh and eighth days) antibody titers (correlation coefficient, 0.405; p < 0.001). FWT is useful for examining antibody titers as a point-of-care test. Rapid response of antibody titer started as early as the fourth day post-vaccination, while the presence of weak responders to BNT162b2 vaccine was indicated.

Numerical evaluation reveals the effect of branching morphology on vessel transport properties during angiogenesis.

Blood flow governs transport of oxygen and nutrients into tissues. Hypoxic tissues secrete VEGFs to promote angiogenesis during development and in tissue homeostasis. In contrast, tumors enhance pathologic angiogenesis during growth and metastasis, suggesting suppression of tumor angiogenesis could limit tumor growth. In line with these observations, various factors have been identified to control vessel formation in the last decades. However, their impacts on the vascular transport properties of oxygen remain elusive. Here, we take a computational approach to examine the effects of vascular branching on blood flow in the growing vasculature. First of all, we reconstruct a 3D vascular model from the 2D confocal images of the growing vasculature at postnatal day 5 (P5) mouse retina, then simulate blood flow in the vasculatures, which are obtained from the gene targeting mouse models causing hypo- or hyper-branching vascular formation. Interestingly, hyper-branching morphology attenuates effective blood flow at the angiogenic front, likely promoting tissue hypoxia. In contrast, vascular hypo-branching enhances blood supply at the angiogenic front of the growing vasculature. Oxygen supply by newly formed blood vessels improves local hypoxia and decreases VEGF expression at the angiogenic front during angiogenesis. Consistent with the simulation results indicating improved blood flow in the hypo-branching vasculature, VEGF expression around the angiogenic front is reduced in those mouse retinas. Conversely, VEGF expression is enhanced in the angiogenic front of hyper-branching vasculature. Our results indicate the importance of detailed flow analysis in evaluating the vascular transport properties of branching morphology of the blood vessels.

Digenic inheritance of mutations in EPHA2 and SLC26A4 in Pendred syndrome.

Enlarged vestibular aqueduct (EVA) is one of the most commonly identified inner ear malformations in hearing loss patients including Pendred syndrome. While biallelic mutations of the SLC26A4 gene, encoding pendrin, causes non-syndromic hearing loss with EVA or Pendred syndrome, a considerable number of patients appear to carry mono-allelic mutation. This suggests faulty pendrin regulatory machinery results in hearing loss. Here we identify EPHA2 as another causative gene of Pendred syndrome with SLC26A4. EphA2 forms a protein complex with pendrin controlling pendrin localization, which is disrupted in some pathogenic forms of pendrin. Moreover, point mutations leading to amino acid substitution in the EPHA2 gene are identified from patients bearing mono-allelic mutation of SLC26A4. Ephrin-B2 binds to EphA2 triggering internalization with pendrin inducing EphA2 autophosphorylation weakly. The identified EphA2 mutants attenuate ephrin-B2- but not ephrin-A1-induced EphA2 internalization with pendrin. Our results uncover an unexpected role of the Eph/ephrin system in epithelial function.

Regulation of programmed cell death ligand 1 expression by atypical protein kinase C lambda/iota in cutaneous angiosarcoma.

The expression of immune checkpoint proteins such as programmed cell death protein 1 (PD-1) and its ligand (PD-L1) has been shown to correlate with patient prognosis in many malignant cancers. The expression of PD-L1 is controlled by c-Myc; however, further upstream regulation of PD-L1 expression is largely unknown. We have previously shown that atypical protein kinase C lambda/iota (aPKCλ) phosphorylates the Forkhead box protein O1 (FoxO1) transcription factor at Ser218 to suppress its DNA-binding ability, thereby regulating c-Myc expression and controlling physiologic and pathologic endothelial proliferation. The presence of phosphorylation of FoxO1 at Ser218 (pSer218 FoxO1) in cutaneous angiosarcoma (CAS) strongly correlates with poor patient prognosis. Here, we reported that patients with PD-L1+ cells in CAS lesions showed significantly worse prognosis compared to those that were PD-L1- . Expression of PD-L1 correlated with that of aPKCλ or the presence of pSer218FoxO1. Moreover, suppression of aPKCλ expression or inhibition of its activity in HUVECs or AS-M, an established human angiosarcoma cell line, resulted in decreased PD-L1 expression. Our results suggest that combined treatment with immune checkpoint inhibitors and aPKCλ inhibitors could be a novel treatment strategy for CAS patients.

aPKC controls endothelial growth by modulating c-Myc via FoxO1 DNA-binding ability.

Strict regulation of proliferation is vital for development, whereas unregulated cell proliferation is a fundamental characteristic of cancer. The polarity protein atypical protein kinase C lambda/iota (aPKCλ) is associated with cell proliferation through unknown mechanisms. In endothelial cells, suppression of aPKCλ impairs proliferation despite hyperactivated mitogenic signaling. Here we show that aPKCλ phosphorylates the DNA binding domain of forkhead box O1 (FoxO1) transcription factor, a gatekeeper of endothelial growth. Although mitogenic signaling excludes FoxO1 from the nucleus, consequently increasing c-Myc abundance and proliferation, aPKCλ controls c-Myc expression via FoxO1/miR-34c signaling without affecting its localization. We find this pathway is strongly activated in the malignant vascular sarcoma, angiosarcoma, and aPKC inhibition reduces c-Myc expression and proliferation of angiosarcoma cells. Moreover, FoxO1 phosphorylation at Ser218 and aPKC expression correlates with poor patient prognosis. Our findings may provide a potential therapeutic strategy for treatment of malignant cancers, like angiosarcoma.

PAR-3 controls endothelial planar polarity and vascular inflammation under laminar flow.

Impaired cell polarity is a hallmark of diseased tissue. In the cardiovascular system, laminar blood flow induces endothelial planar cell polarity, represented by elongated cell shape and asymmetric distribution of intracellular organelles along the axis of blood flow. Disrupted endothelial planar polarity is considered to be pro-inflammatory, suggesting that the establishment of endothelial polarity elicits an anti-inflammatory response. However, a causative relationship between polarity and inflammatory responses has not been firmly established. Here, we find that a cell polarity protein, PAR-3, is an essential gatekeeper of GSK3ß activity in response to laminar blood flow. We show that flow-induced spatial distribution of PAR-3/aPKCλ and aPKCλ/GSK3ß complexes controls local GSK3ß activity and thereby regulates endothelial planar polarity. The spatial information for GSK3ß activation is essential for flow-dependent polarity to the flow axis, but is not necessary for flow-induced anti-inflammatory response. Our results shed light on a novel relationship between endothelial polarity and vascular homeostasis highlighting avenues for novel therapeutic strategies.

Detachment of Chain-Forming Neuroblasts by Fyn-Mediated Control of cell-cell Adhesion in the Postnatal Brain.

In the rodent olfactory system, neuroblasts produced in the ventricular-subventricular zone of the postnatal brain migrate tangentially in chain-like cell aggregates toward the olfactory bulb (OB) through the rostral migratory stream (RMS). After reaching the OB, the chains are dissociated and the neuroblasts migrate individually and radially toward their final destination. The cellular and molecular mechanisms controlling cell-cell adhesion during this detachment remain unclear. Here we report that Fyn, a nonreceptor tyrosine kinase, regulates the detachment of neuroblasts from chains in the male and female mouse OB. By performing chemical screening and in vivo loss-of-function and gain-of-function experiments, we found that Fyn promotes somal disengagement from the chains and is involved in neuronal migration from the RMS into the granule cell layer of the OB. Fyn knockdown or Dab1 (disabled-1) deficiency caused p120-catenin to accumulate and adherens junction-like structures to be sustained at the contact sites between neuroblasts. Moreover, a Fyn and N-cadherin double-knockdown experiment indicated that Fyn regulates the N-cadherin-mediated cell adhesion between neuroblasts. These results suggest that the Fyn-mediated control of cell-cell adhesion is critical for the detachment of chain-forming neuroblasts in the postnatal OB.SIGNIFICANCE STATEMENT In the postnatal brain, newly born neurons (neuroblasts) migrate in chain-like cell aggregates toward their destination, where they are dissociated into individual cells and mature. The cellular and molecular mechanisms controlling the detachment of neuroblasts from chains are not understood. Here we show that Fyn, a nonreceptor tyrosine kinase, promotes the somal detachment of neuroblasts from chains, and that this regulation is critical for the efficient migration of neuroblasts to their destination. We further show that Fyn and Dab1 (disabled-1) decrease the cell-cell adhesion between chain-forming neuroblasts, which involves adherens junction-like structures. Our results suggest that Fyn-mediated regulation of the cell-cell adhesion of neuroblasts is critical for their detachment from chains in the postnatal brain.

PlexinD1 signaling controls morphological changes and migration termination in newborn neurons.

Newborn neurons maintain a very simple, bipolar shape, while they migrate from their birthplace toward their destinations in the brain, where they differentiate into mature neurons with complex dendritic morphologies. Here, we report a mechanism by which the termination of neuronal migration is maintained in the postnatal olfactory bulb (OB). During neuronal deceleration in the OB, newborn neurons transiently extend a protrusion from the proximal part of their leading process in the resting phase, which we refer to as a filopodium-like lateral protrusion (FLP). The FLP formation is induced by PlexinD1 downregulation and local Rac1 activation, which coincide with microtubule reorganization and the pausing of somal translocation. The somal translocation of resting neurons is suppressed by microtubule polymerization within the FLP The timing of neuronal migration termination, controlled by Sema3E-PlexinD1-Rac1 signaling, influences the final positioning, dendritic patterns, and functions of the neurons in the OB These results suggest that PlexinD1 signaling controls FLP formation and the termination of neuronal migration through a precise control of microtubule dynamics.

Disrupted-in-schizophrenia 1 regulates transport of ITPR1 mRNA for synaptic plasticity.

Disrupted-in-schizophrenia 1 (DISC1) is a susceptibility gene for major psychiatric disorders, including schizophrenia. DISC1 has been implicated in neurodevelopment in relation to scaffolding signal complexes. Here we used proteomic analysis to screen for DISC1 interactors and identified several RNA-binding proteins, such as hematopoietic zinc finger (HZF), that act as components of RNA-transporting granules. HZF participates in the mRNA localization of inositol-1,4,5-trisphosphate receptor type 1 (ITPR1), which plays a key role in synaptic plasticity. DISC1 colocalizes with HZF and ITPR1 mRNA in hippocampal dendrites and directly associates with neuronal mRNAs, including ITPR1 mRNA. The binding potential of DISC1 for ITPR1 mRNA is facilitated by HZF. Studies of Disc1-knockout mice have revealed that DISC1 regulates the dendritic transport of Itpr1 mRNA by directly interacting with its mRNA. The DISC1-mediated mRNA regulation is involved in synaptic plasticity. We show that DISC1 binds ITPR1 mRNA with HZF, thereby regulating its dendritic transport for synaptic plasticity.

Speed control for neuronal migration in the postnatal brain by Gmip-mediated local inactivation of RhoA.

Throughout life, new neurons generated in the ventricular-subventricular zone take the long journey to the olfactory bulb. The intracellular mechanisms that precisely control the neurons' migration speed, enabling their well-organized movement, remain unclear. Rho signalling is known to affect the morphology and movement of various cell types, including neurons. Here we identify Gem-interacting protein (Gmip), a RhoA-specific GTPase-activating protein, as a key factor in saltatory neuronal migration. RhoA is activated at the proximal leading process of migrating neurons, where Gmip is also localized and negatively regulates RhoA. Gmip controls the saltatory movement of neurons that regulate their migration speed and 'stop' positions in the olfactory bulb, thereby altering the neural circuitry. This study demonstrates that Gmip serves as a brake for the RhoA-mediated movement of neuronal somata, and highlights the significance of speed control in the well-organized neuronal migration and the maintenance of neuronal circuits in the postnatal brain.

Rac1-mediated indentation of resting neurons promotes the chain migration of new neurons in the rostral migratory stream of post-natal mouse brain.

New neurons generated in the ventricular-subventricular zone in the post-natal brain travel toward the olfactory bulb by using a collective cell migration process called 'chain migration.' These new neurons show a saltatory movement of their soma, suggesting that each neuron cycles through periods of 'rest' during migration. Here, we investigated the role of the resting neurons in chain migration using post-natal mouse brain, and found that they undergo a dynamic morphological change, in which a deep indentation forms in the cell body. Inhibition of Rac1 activity resulted in less indentation of the new neurons in vivo. Live cell imaging using a Förster resonance energy transfer biosensor revealed that Rac1 was activated at the sites of contact between actively migrating and resting new neurons. On the cell surface of resting neurons, Rac1 activation coincided with the formation of the indentation. Furthermore, Rac1 knockdown prevented the indentation from forming and impaired migration along the resting neurons. These results suggest that Rac1 regulates a morphological change in the resting neurons, which allows them to serve as a migratory scaffold, and thereby non-cell-autonomously promotes chain migration.

Proteomic analysis of Girdin-interacting proteins in migrating new neurons in the postnatal mouse brain.

Neural stem cells continuously generate new neurons in the ventricular-subventricular zone (V-SVZ) of the postnatal and adult mammalian brain. New neurons born in the rodent V-SVZ migrate toward the olfactory bulb (OB), where they differentiate into interneurons. To reveal novel intracellular molecular mechanisms that control postnatal neuronal migration, we performed a global proteomic search for proteins interacting with Girdin, an essential protein for postnatal neuronal migration. Using GST pull-down and LC-MS/MS shotgun analysis, we identified cytoskeletal proteins, cytoskeleton-binding proteins, and signal-transduction proteins as possible participants in neuronal migration. Our results suggest that Girdin and Girdin-interacting proteins control neuronal migration by regulating actin and/or microtubule dynamics.

Growth factors released from gelatin hydrogel microspheres increase new neurons in the adult mouse brain.

Recent studies have shown that new neurons are continuously generated by endogenous neural stem cells in the subventricular zone (SVZ) of the adult mammalian brain. Some of these new neurons migrate to injured brain tissues and differentiate into mature neurons, suggesting that such new neurons may be able to replace neurons lost to degenerative disease or injury and improve or repair neurological deficits. Here, we tested whether delivering growth factors via gelatin hydrogel microspheres would support neurogenesis in the SVZ. Insulin-like growth factor-1 (IGF-1)-containing microspheres increased the number of new neurons in the SVZ. Hepatocyte growth factor (HGF)-containing microspheres increased the number of new neurons migrating from the SVZ towards the injured striatum in a stroke model in mouse. These results suggest that the strategy of using gelatin hydrogel microspheres to achieve the sustained release of growth factors holds promise for the clinical regeneration of damaged brain tissues from endogenous neural stem cells in the adult SVZ.

Girdin is an intrinsic regulator of neuroblast chain migration in the rostral migratory stream of the postnatal brain.

In postnatally developing and adult brains, interneurons of the olfactory bulb (OB) are continuously generated at the subventricular zone of the forebrain. The newborn neuroblasts migrate tangentially to the OB through a well defined pathway, the rostral migratory stream (RMS), where the neuroblasts undergo collective migration termed "chain migration." The cell-intrinsic regulatory mechanism of neuroblast chain migration, however, has not been uncovered. Here we show that mice lacking the actin-binding Akt substrate Girdin (a protein that interacts with Disrupted-In-Schizophrenia 1 to regulate neurogenesis in the dentate gyrus) have profound defects in neuroblast chain migration along the RMS. Analysis of two gene knock-in mice harboring Girdin mutants identified unique amino acid residues in Girdin's C-terminal domain that are responsible for the regulation of neuroblast chain migration but revealed no apparent requirement of Girdin phosphorylation by Akt. Electron microscopic analyses demonstrated the involvement of Girdin in neuroblast cell-cell interactions. These findings suggest that Girdin is an important intrinsic factor that specifically governs neuroblast chain migration along the RMS.

Immunohistochemical study of vesicle monoamine transporter 2 in the hippocampal region of genetic animal model of schizophrenia.

Recent research in the etiology of schizophrenia revealed that there may be some neurodevelopmental failures such as neuronal network incompetence in the brain of this disease, and neurotransmitters cannot function accurately or adequately. But, it is unknown precisely what kinds of deficit in neurotransmission may be existed histopathologically. We investigated the expression of vesicle monoamine transporter 2 (VMAT2), which has a significant role in neurotransmission, in the hippocampal formation of the animal model of schizophrenia, 14-3-3 epsilon hetero knockout (KO) mouse, using an immunohistochemical staining technique to clarify the neuronal abnormalities in the model animal. As a result, the expression of VMAT2 was increased significantly in the hippocampal formation of 14-3-3 epsilon hetero KO mice compared to that of the wild-type littermates. In conclusion, these findings might be related the pathophysiology of this disease includes a monoaminergic transmission abnormality, based on the investigation in a genetically-modified mouse as schizophrenic model.

Dysfunction of dopamine release in the prefrontal cortex of dysbindin deficient sandy mice: an in vivo microdialysis study.

Dystrobrevin binding protein-1 gene (DTNBP1), which encodes dysbindin protein, has been identified as a schizophrenia susceptibility gene. Dysbindin has been shown to contribute to the regulation of exocytosis and formation of synaptic vesicles. Although hypofrontality in schizophrenia underlies its pathophysiology, the molecular function of dysbindin in synaptic neurotransmission remains unclear. In the present study, we investigated depolarization-evoked dopamine (DA) and serotonin (5-HT) release in the prefrontal cortex (PFC) of sandy (sdy) mice, which have a deletion mutation in the gene encoding DTNBP1. In vivo microdialysis analysis revealed that extracellular DA levels in the PFC of wild-type mice were increased by 60mM KCl stimulation, and the KCl-evoked DA release was significantly decreased in sdy mice compared with wild-type mice. Extracellular 5-HT levels in the PFC of wild-type mice were also increased by 60mM KCl stimulation. The KCl-evoked 5-HT release did not differ between wild-type and sdy mice. There was no difference in basal levels of DA and 5-HT before the stimulation between two groups. Behavioral sensitization after repeated methamphetamine (METH) treatment was significantly reduced in sdy mice compared with wild-type mice whereas no difference was observed in METH-induced hyperlocomotion between two groups. These results suggest that dysbindin may have a role in the regulation of depolarization-evoked DA release in the PFC and in the development of behavioral sensitization induced by repeated METH treatment.

Proteomic analysis reveals novel binding partners of dysbindin, a schizophrenia-related protein.

Schizophrenia is a complex mental disorder with fairly high level of heritability. Dystrobrevin binding protein 1, a gene encoding dysbindin protein, is a susceptibility gene for schizophrenia that was identified by family-based association analysis. Recent studies revealed that dysbindin is involved in the exocytosis and/or formation of synaptic vesicles. However, the molecular function of dysbindin in synaptic transmission is largely unknown. To investigate the signaling pathway in which dysbindin is involved, we isolated dysbindin-interacting molecules from rat brain lysate by combining ammonium sulfate precipitation and dysbindin-affinity column chromatography, and identified dysbindin-interacting proteins by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and liquid chromatography-tandem mass spectrometry. Proteins involved in protein localization process, including Munc18-1, were identified as dysbindin-interacting proteins. Munc18-1 was co-immunoprecipitated with dysbindin from rat brain lysate, and directly interacted with dysbindin in vitro. In primary cultured rat hippocampal neurons, a part of dysbindin was co-localized with Munc18-1 at pre-synaptic terminals. Our result suggests a role for dysbindin in synaptic vesicle exocytosis via interaction with Munc18-1.

Association analysis between schizophrenia and the AP-3 complex genes.

A susceptibility gene for schizophrenia, dysbindin, is a component of BLOC-1, which interacts with the adaptor protein (AP)-3 complex. As a direct interaction between dysbindin and AP-3 complex was reported, we examined a possible association between 16 SNPs in the AP3 complex genes and schizophrenia using 432 cases and 656 controls. Nominal association between rs6688 in the AP3M1 gene and schizophrenia (chi(2)=6.33, P=0.012, odds ratio=0.80) was no longer positive after correction for multiple testing (corrected P=0.192). The present results suggest that AP3 complex genes might not play a major role in the pathogenesis of schizophrenia in this population.

Direct interaction of Dysbindin with the AP-3 complex via its mu subunit.

Genetic factors are important in the etiology of schizophrenia. Recent studies have revealed the association between genetic variation of Dysbindin (DTNBP1) and schizophrenia. Dysbindin is one of the essential components of the biogenesis of lysosome-related organelles complex 1 (BLOC-1). BLOC-1 physically interacts with the adaptor protein (AP)-3 complex, which is essential for vesicle or protein sorting. However, it remains largely unknown how BLOC-1 interacts with the AP-3 complex. To investigate the binding mode of BLOC-1 and the AP-3 complex, we examined the relation between Dysbindin and the AP-3 complex and found that Dysbindin formed a complex with the AP-3 complex through the direct binding to its mu subunit. Dysbindin partially co-localized with the AP-3 complex in CA1 and CA3 of mouse hippocampus, and at presynaptic terminals and axonal growth cones of cultured hippocampal neurons. Suppression of Dysbindin results in the reduction of presynaptic protein expression and glutamate release. Thus, Dysbindin appears to participate in the exocytosis or sorting of the synaptic vesicle via direct interaction with the AP-3 complex.