Sufficient water intake maintains the gut microbiota and immune homeostasis and promotes pathogen elimination.

Water is the most abundant substance in the human body and plays a pivotal role in various bodily functions. While underhydration is associated with the incidence of certain diseases, the specific role of water in gut function remains largely unexplored. Here, we show that water restriction disrupts gut homeostasis, which is accompanied by a bloom of gut microbes and decreased numbers of immune cells, especially Th17 cells, within the colon. These microbial and immunological changes in the gut are associated with an impaired ability to eliminate the enteric pathogen Citrobacter rodentium. Moreover, aquaporin 3, a water channel protein, is required for the maintenance of Th17 cell function and differentiation. Taken together, adequate water intake is critical for maintaining bacterial and immunological homeostasis in the gut, thereby enhancing host defenses against enteric pathogens.

Pediatric acute disseminated encephalomyelitis following asymptomatic coronavirus disease 2019.

After the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) epidemic emerged, the virus spread rapidly worldwide, and outbreaks continued to occur intermittently. Here, we present the case of a 5-year-old boy with acute disseminated encephalomyelitis (ADEM) and initial symptoms of dysphoria and pain in the right lower extremity. Around the time of this episode, the patient exhibited no fever or respiratory symptoms. Brain magnetic resonance imaging (MRI) revealed multiple T2-weighted image/fluid-attenuated inversion recovery high-signal areas bilaterally subcortical to the deep white matter, corpus callosum, and bilateral basal ganglia. MRI of the cervical and thoracic regions indicated a long lesion with continuous T2WI high signal intensity in the central gray matter. Serum aquaporin-4 antibody and serum myelin oligodendrocyte glycoprotein antibody tests were negative and positive, respectively. A polymerase chain reaction test using nasopharyngeal swab fluid upon admission was positive for SARS-CoV-2. Patients with severe coronavirus disease 2019 (COVID-19) in the acute phase may show central nervous system symptoms. There have been no previous reports of ADEM in the subacute phase of COVID-19, lacking symptoms in the acute phase, as in the present case. Notably, ADEM can develop in the subacute phase of asymptomatic COVID-19 infection.

Lengthened circadian rhythms in mice with self-controlled ambient light intensity.

Laboratory animals are typically maintained under 12-h light and 12-h dark (12:12 LD) conditions with a daytime light intensity of ~ 200 lx. In this study, we designed an apparatus that allowed mice to self-select the room light intensity by nose poking. We measured the behavioral rhythms of the mice under this self-controlled light regimen. The mice quickly learned the relationship between their nose pokes and the resulting changes in the light intensity. Under these conditions, the mice exhibited free-running circadian behavior with a period of 24.5 ± 0.4 h. This circadian period was ~ 1 h longer than that of the same strain of mice when they were kept in constant darkness (DD) after 12:12 LD entrainment, and the lengthened period lasted for at least 30 days. The rhythm of the light intensity controlled by the mice also exhibited a similar period, but the phase of the illuminance rhythm preceded the phase of the locomotor activity rhythm. Mice that did not have access to the light controller were also entrained to the illuminance cycle produced by the mice that did have access to the light controller, but with a slightly delayed phase. The rhythm was likely controlled by the canonical circadian clock because mice with tau mutations in the circadian clock gene CSNK1E exhibited short periods of circadian rhythm under the same conditions. These results indicate that the free-running period of mice in the wild may differ from what they exhibit if they are attuned by forced light cycles in laboratories because mice in their natural habitats can self-control their exposure to ambient light, similar to our experimental conditions.

Safety and efficacy of long-term nicotinamide mononucleotide supplementation on metabolism, sleep, and nicotinamide adenine dinucleotide biosynthesis in healthy, middle-aged Japanese men.

Obesity and aging are major risk factors for several life-threatening diseases. Accumulating evidence from both rodents and humans suggests that the levels of nicotinamide adenine dinucleotide (NAD+), a regulator of many biological processes, declines in multiple organs and tissues with aging and obesity. Administration of an NAD+ intermediate, nicotinamide mononucleotide (NMN), replenishes intracellular NAD+ levels and mitigates aging- and obesity-associated derangements in animal models. In this human clinical study, we aimed to investigate the safety and effects of 8-week oral administration of NMN on biochemical, metabolic, ophthalmologic, and sleep quality parameters as well as on chronological alterations in NAD+ content in peripheral tissues. An 8-week, single-center, single-arm, open-label clinical trial was conducted. Eleven healthy, middle-aged Japanese men received two 125-mg NMN capsules once daily before breakfast. The 8-week NMN supplementation regimen was well-tolerated; NAD+ levels in peripheral blood mononuclear cells increased over the course of NMN administration. In participants with insulin oversecretion after oral glucose loading, NMN modestly attenuated postprandial hyperinsulinemia, a risk factor for coronary artery disease (n = 3). In conclusion, NMN overall safely and effectively boosted NAD+ biosynthesis in healthy, middle-aged Japanese men, showing its potential for alleviating postprandial hyperinsulinemia.

Localized amyloidosis of the ureter: a case report.

BACKGROUND: Amyloidosis is a collection of disorders characterized by the extracellular deposition of amyloid, a specialized fibrous protein, in diverse tissues, leading to functional impairments. CASE PRESENTATION: A 70-year old Asian-Japanese female was referred to our department for further examination of her left hydronephrosis come from lower ureteral obstruction. Contrast enhanced CT and retrograde pyelo-nephrography revealed left ureteral tumor. Though ureteroscropic biopsy did not show malignant pathological findings, ureteroscopic image suspected malignant disease, thus nephroureterectomy was performed. Pathological findings revealed localized ureteral amyloidosis. Whole body examination including gastro endoscopy and cardio ultrasonography could not reveal amyloidosis except ureter. She was free from recurrence 9 months postoperatively. CONCLUSION: We herein report a rare case of localized ureteral amyloidosis.

GABBR2 as a Downstream Effector of the Androgen Receptor Induces Cisplatin Resistance in Bladder Cancer.

The precise molecular mechanisms responsible for resistance to cisplatin-based chemotherapy in patients with bladder cancer remain elusive, while we have indicated that androgen receptor (AR) activity in urothelial cancer is associated with its sensitivity. Our DNA microarray analysis in control vs. AR-knockdown bladder cancer sublines suggested that the expression of a GABA B receptor GABBR2 and AR was correlated. The present study aimed to determine the functional role of GABBR2 in modulating cisplatin sensitivity in bladder cancer. AR knockdown and dihydrotestosterone treatment considerably reduced and induced, respectively, GABBR2 expression, and the effect of dihydrotestosterone was at least partially restored by an antiandrogen hydroxyflutamide. A chromatin immunoprecipitation assay further revealed the binding of AR to the promoter region of GABBR2 in bladder cancer cells. Meanwhile, GABBR2 expression was significantly elevated in a cisplatin-resistant bladder cancer subline, compared with control cells. In AR-positive bladder cancer cells, knockdown of GABBR2 or treatment with a selective GABA B receptor antagonist, CGP46381, considerably enhanced the cytotoxic activity of cisplatin. However, no additional effect of CGP46381 on cisplatin-induced growth suppression was seen in GABBR2-knockdown cells. Moreover, in the absence of cisplatin, CGP46381 treatment and GABBR2 knockdown showed no significant changes in cell proliferation or migration. These findings suggest that GABBR2 represents a key downstream effector of AR signaling in inducing resistance to cisplatin treatment. Accordingly, inhibition of GABBR2 has the potential of being a means of chemosensitization, especially in patients with AR/GABBR2-positive bladder cancer.

Silent spinal epidural abscess due to facet joint arthritis.

Spinal epidural abscess (SEA) is an abscess that forms between the dura mater and vertebrae. SEA is characterized by back pain and neuropathy associated with fever, of which early diagnosis and treatment are necessary to avoid irreversible neurological sequelae. However, its diagnosis is often difficult because specific symptoms are rarely present in the early stages of the disease. A 25-month-old boy, healthy by nature and free of risk factors, was referred and admitted for fever symptoms only, without back pain or neurological symptoms. We focused on the residual activation of the coagulation-fibrinolytic system, which was contrary to the response to therapy, and were able to establish a diagnosis of SEA. After the initiation of antibiotics, the patient responded well to treatment and made a mild recovery without the need for surgical intervention. To date, there are no reported cases of SEA with only febrile symptoms without localized spinal cord tenderness. SEA is easily overlooked and should be considered in the differential diagnosis of pediatric fever of unknown origin. Although imaging studies have drawbacks, such as radiation exposure and sedation, they should be immediately performed if SEA is suspected.

Stimulated Raman scattering microscopy reveals a unique and steady nature of brain water dynamics.

The biological activities of substances in the brain are shaped by their spatiotemporal dynamics in brain tissues, all of which are regulated by water dynamics. In contrast to solute dynamics, water dynamics have been poorly characterized, owing to the lack of appropriate analytical tools. To overcome this limitation, we apply stimulated Raman scattering multimodal multiphoton microscopy to live brain tissues. The microscopy system allows for the visualization of deuterated water, fluorescence-labeled solutes, and cellular structures at high spatiotemporal resolution, revealing that water moves faster than fluorescent molecules in brain tissues. Detailed analyses demonstrate that water, unlike solutes, diffuses homogeneously in brain tissues without differences between the intra- and the extracellular routes. Furthermore, we find that the water dynamics are steady during development and ischemia, when diffusions of solutes are severely affected. Thus, our approach reveals routes and uniquely robust properties of water diffusion in brain tissues.

Aquaporin 3 inhibition suppresses the mitochondrial respiration rate and viability of multiple myeloma cells.

Aquaporin 3 (AQP3) is a member of the aquaporin water channel family expressed by numerous cell types, including some cancer cells. Accumulating evidence suggests that AQP3 inhibition may impede cancer progression, but drugs targeting AQP3 are still in the early pre-clinical stage of development. Here, we examined the effect of AQP3 inhibition on multiple myeloma (MM), an incurable plasma cell malignancy. Four MM cell lines were cultured in the presence of an anti-AQP3 monoclonal antibody (mAb), the AQP3 inhibitor DFP00173, or corresponding controls, and the effects on cell viability, proliferation, apoptosis, and mitochondrial respiration capacity were compared. Both anti-AQP3 mAb and DFP00173 reduced cell growth, mitochondrial respiration rate, and electron transport chain complex I activity. Both agents also potentiated the antiproliferative efficacy of the anticancer drug venetoclax. Administration of the anti-AQP3 mAb to immunodeficient mice inoculated with RPMI8226 or KMS-11 MM cells significantly suppressed tumor growth. These data provide evidence that AQP3 blockade can suppress MM cell growth in vitro and tumor growth in mice. Thus, AQP3 inhibition may be an effective therapeutic strategy for MM.

Vitamin D Receptor Antagonist MeTC7 Inhibits PD-L1.

Small-molecule inhibitors of PD-L1 are postulated to control immune evasion in tumors similar to antibodies that target the PD-L1/PD-1 immune checkpoint axis. However, the identity of targetable PD-L1 inducers is required to develop small-molecule PD-L1 inhibitors. In this study, using chromatin immunoprecipitation (ChIP) assay and siRNA, we demonstrate that vitamin D/VDR regulates PD-L1 expression in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) cells. We have examined whether a VDR antagonist, MeTC7, can inhibit PD-L1. To ensure that MeTC7 inhibits VDR/PD-L1 without off-target effects, we examined competitive inhibition of VDR by MeTC7, utilizing ligand-dependent dimerization of VDR-RXR, RXR-RXR, and VDR-coactivators in a mammalian 2-hybrid (M2H) assay. MeTC7 inhibits VDR selectively, suppresses PD-L1 expression sparing PD-L2, and inhibits the cell viability, clonogenicity, and xenograft growth of AML cells. MeTC7 blocks AML/mesenchymal stem cells (MSCs) adhesion and increases the efferocytotic efficiency of THP-1 AML cells. Additionally, utilizing a syngeneic colorectal cancer model in which VDR/PD-L1 co-upregulation occurs in vivo under radiation therapy (RT), MeTC7 inhibits PD-L1 and enhances intra-tumoral CD8+T cells expressing lymphoid activation antigen-CD69. Taken together, MeTC7 is a promising small-molecule inhibitor of PD-L1 with clinical potential.

Protocol to image deuterated propofol in living rat neurons using multimodal stimulated Raman scattering microscopy.

Propofol is a widely used anesthetic important in clinics, but like many other bioactive molecules, it is too small to be tagged and visualized by fluorescent dyes. Here, we present a protocol to visualize deuterated propofol in living rat neurons using stimulated Raman scattering (SRS) microscopy with carbon-deuterium bonds serving as a Raman tag. We describe the preparation and culture of rat neurons, followed by optimization of the SRS system. We then detail neuron loading and real-time imaging of anesthesia dynamics. For complete details on the use and execution of this protocol, please refer to Oda et al.1.

Recent advances in the understanding of urothelial tumorigenesis.

INTRODUCTION: Patients with non-muscle-invasive bladder tumor suffer from disease recurrence following transurethral surgery even with intravesical pharmacotherapy, while muscle-invasive disease is often deadly. It is therefore critical to elucidate the underlying molecular mechanisms responsible for not only bladder tumor progression but also its tumorigenesis. Indeed, various molecules and/or signaling pathways have been suggested to contribute to the pathogenesis of bladder cancer. AREAS COVERED: We summarize the progress during the last few years on the initiation or development, but not progression, of urothelial cancer. The clinical implications of these available data, including prognostic significance and possible application for the prevention of the recurrence of non-muscle-invasive bladder tumors, are also discussed. EXPERT OPINION: Bladder cancer is a heterogeneous group of neoplasms. The establishment of personalized therapeutic options based on the molecular profile in each case should thus be considered. On that account, further accumulation of data on urothelial tumorigenesis is warranted to identify promising targets for the prevention of postoperative tumor recurrence or tumor development in otherwise high-risk patients.

Mammals sustain amino acid homochirality against chiral conversion by symbiotic microbes.

Mammals exhibit systemic homochirality of amino acids in L-configurations. While ribosomal protein synthesis requires rigorous chiral selection for L-amino acids, both endogenous and microbial enzymes convert diverse L-amino acids to D-configurations in mammals. However, it is not clear how mammals manage such diverse D-enantiomers. Here, we show that mammals sustain systemic stereo dominance of L-amino acids through both enzymatic degradation and excretion of D-amino acids. Multidimensional high performance liquidchromatography analyses revealed that in blood, humans and mice maintain D-amino acids at less than several percent of the corresponding L-enantiomers, while D-amino acids comprise ten to fifty percent of the L-enantiomers in urine and feces. Germ-free experiments showed that vast majority of D-amino acids, except for D-serine, detected in mice are of microbial origin. Experiments involving mice that lack enzymatic activity to catabolize D-amino acids showed that catabolism is central to the elimination of diverse microbial D-amino acids, whereas excretion into urine is of minor importance under physiological conditions. Such active regulation of amino acid homochirality depends on maternal catabolism during the prenatal period, which switches developmentally to juvenile catabolism along with the growth of symbiotic microbes after birth. Thus, microbial symbiosis largely disturbs homochirality of amino acids in mice, whereas active host catabolism of microbial D-amino acids maintains systemic predominance of L-amino acids. Our findings provide fundamental insight into how the chiral balance of amino acids is governed in mammals and further expand the understanding of interdomain molecular homeostasis in host-microbial symbiosis.

Sodium benzoate attenuates 2,8-dihydroxyadenine nephropathy by inhibiting monocyte/macrophage TNF-α expression.

Sodium benzoate (SB), a known D-amino acid oxidase (DAO) enzyme inhibitor, has an anti-inflammatory effect, although its role in renal damage has not been explored. 2,8-dihydroxyadenine crystal induced chronic kidney disease, in which TNF-α is involved in the pathogenesis, was established by oral adenine administration in C57BL/6JJcl mice (AdCKD) with or without SB to investigate its renal protective effects. SB significantly attenuated AdCKD by decreasing serum creatinine and urea nitrogen levels, and kidney interstitial fibrosis and tubular atrophy scores. The survival of AdCKD mice improved 2.6-fold by SB administration. SB significantly decreased the number of infiltrating macrophages observed in the positive F4/80 immunohistochemistry area and reduced the expression of macrophage markers and inflammatory genes, including TNF-α, in the kidneys of AdCKD. Human THP-1 cells stimulated with either lipopolysaccharide or TNF-α showed increased expression of inflammatory genes, although this was significantly reduced by SB, confirming the anti-inflammatory effects of SB. SB exhibited renal protective effects in AdCKD in DAO enzyme deficient mice, suggesting that anti-inflammatory effect of SB was independent of DAO enzyme activity. Moreover, binding to motif DNA sequence, protein level, and mRNA level of NF-κB RelB were significantly inhibited by SB in AdCKD kidneys and lipopolysaccharide treated THP-1 cells, respectively. We report that anti-inflammatory property of SB is independent of DAO enzymatic activity and is associated with down regulated NF-κB RelB as well as its downstream inflammatory genes such as TNF-α in AdCKD.

A tyrosine-based YXXΦ motif regulates the degradation of aquaporin-4 via both lysosomal and proteasomal pathways and is functionally inhibited by a 10-amino-acid sequence within its C-terminus.

Aquaporin-4 (AQP4) is a dominant water channel in the brain and is expressed on astrocytic end-feet, mediating water homeostasis in the brain. AQP4 is a target of an inflammatory autoimmune disease, neuromyelitis optica spectrum disorders (NMOSD), that causes demyelination. An autoantibody recognizing the extracellular domains of AQP4, called NMO-IgG, is critically implicated in the pathogenesis of the disease. Complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) in astrocytes are the primary causes of the disease, preceding demyelination and neuronal damage. Additionally, some cytotoxic effects of binding of NMO-IgG to AQP4, independent of CDC/ADCC, have been proposed. Antibody-induced endocytosis of AQP4 is thought to be involved in CDC/ADCC-independent cytotoxicity induced by the binding of NMO-IgG to AQP4. To clarify the mechanism responsible for antibody-induced endocytosis of AQP4, we investigated the subcellular localization and trafficking of AQP4, focusing on its C-terminal domain, by making a variety of deletion and substitution mutants of mouse AQP4. We found that a tyrosine-based YXXΦ motif in the C-terminal domain of AQP4 plays a critical role in the steady-state subcellular localization/turnover and antibody-induced endocytosis/lysosomal degradation of AQP4. Our results indicate that the YXXΦ motif has to escape the inhibitory effect of the C-terminal 10-amino-acid sequence and be located at an appropriate distance from the plasma membrane to act as a signal for lysosomal degradation of AQP4. In addition to lysosomal degradation, we demonstrated that the YXXΦ motif also functions as a signal to degrade AQP4 using proteasomes under specific conditions.

Prolonged Light Exposure Induces Circadian Impairment in Aquaporin-4-Knockout Mice.

Astrocytes are densely present in the suprachiasmatic nucleus (SCN), which is the master circadian oscillator in mammals, and are presumed to play a key role in circadian oscillation. However, specific astrocytic molecules that regulate the circadian clock are not yet well understood. In our study, we found that the water channel aquaporin-4 (AQP4) was abundantly expressed in SCN astrocytes, and we further examined its circadian role using AQP4-knockout mice. There was no prominent difference in circadian behavioral rhythms between Aqp4-/- and Aqp4+/+ mice subjected to light-dark cycles and constant dark conditions. However, exposure to constant light induced a greater decrease in the Aqp4-/- mice rhythmicity. Although the damped rhythm in long-term constant light recovered after transfer to constant dark conditions in both genotypes, the period until the reappearance of original rhythmicity was severely prolonged in Aqp4-/- mice. In conclusion, AQP4 absence exacerbates the prolonged light-induced impairment of circadian oscillations and delays their recovery to normal rhythmicity.

Endogenous d-serine exists in the mammalian brain independent of synthesis by serine racemase.

Activation of N-methyl-d-aspartate receptors (NMDARs) requires binding of a co-agonist in addition to l-glutamate. d-serine binds to the co-agonist site on GluN1 subunits of NMDARs and modulates glutamatergic neurotransmission. While loss of GluN1 subunits in mice results in neonatal death due to respiratory failure, animals that lack a d-serine synthetic enzyme, serine racemase (SR), show grossly normal growth. However, SR-independent origins of d-serine in the brain remain unclarified. In the present study, we investigated the origin of brain d-serine in mice. Loss of SR significantly reduced d-serine in the cerebral cortex, but a portion of d-serine remained in both neonates and adults. Although d-serine was also produced by intestinal bacteria, germ-free experiments did not influence d-serine levels in the cerebral cortex. In addition, treatment of SR-knockout mice with antibiotics showed a significant reduction of intestinal d-serine, but no reduction in the brain. On the other hand, restriction of dietary intake reduced systemic circulation of d-serine and resulted in a slight decrease of d-serine in the cerebral cortex, but did not account for brain d-serine found in the SR-knockout mice. Therefore, our findings show that endogenous d-serine of non-SR origin exists in the brain. Such previously unrecognized, SR-independent, endogenous d-serine may contribute baseline activity of NMDARs, especially in developing brain, which has minimal SR expression.

AQP9 transports lactate in tumor-associated macrophages to stimulate an M2-like polarization that promotes colon cancer progression.

Macrophages play a major role in the immune defense against pathogenic factors; however, they can lead to tumor exacerbation and metastasis, as the tumor microenvironment (TME) polarizes tumor-associated macrophages (TAMs) into the M2 subtype. Lactate, a metabolite produced by carcinoma cells at high concentrations in the TME, induces an M2-polarization in macrophages, which ultimately leads to the secretion of factors, such as vascular endothelial growth factor (VEGF), and promotes tumor progression. However, the effect of TAM lactate import on tumor progression has not been fully elucidated. Aquaporin 9 (AQP9) is a transporter of water and glycerol expressed in macrophages. Here, we used a tumor allograft mouse model to show that AQP9 knockout (AQP9-/-) mice were more resistant against tumor cell growth and exhibited a suppressive M2-like polarization in tumor tissue than wild-type mice. Moreover, we discovered that the primary bone marrow-derived macrophages from AQP9-/- mice were less sensitive to lactate stimulation and exhibited reduced M2-like polarization as well as decreased VEGF production. To further investigate the role of AQP9 in macrophage polarization, we overexpressed AQP9 in Chinese hamster ovary cells and found that AQP9 functioned in lactate import. In contrast, primary AQP9-/- macrophages and AQP9 knockdown RAW264.7 cells exhibited a reduced lactate transport rate, suggesting the involvement of AQP9 in lactate transport in macrophages. Together, our results reveal the mechanism by which the TME modifies the polarization and function of tumor-infiltrating macrophages via AQP9 transport function.

Probing the Spatiotemporal Dynamics of Oxytocin in the Brain Tissue Using a Simple Peptide Alkyne-Tagging Approach.

The dynamics of oxytocin and its site of action in the brain are poorly understood due to the lack of appropriate tools, despite the interest in the central action of oxytocin signaling. Here, we develop and apply an oxytocin analogue probe by conjugating it with an alkyne via a widely applicable simple coupling reaction. Alkyne-tagged oxytocin behaves similarly to endogenous oxytocin while allowing specific and highly sensitive detection of extracellularly applied oxytocin. Using this probe, we find the existence of high-affinity specific binding sites of oxytocin in the hippocampus. Furthermore, characterization of oxytocin dynamics reveals the cellular basis of its volume transmission in the brain tissue. Finally, we show the wide applicability of this technique for other centrally acting peptides. Thus, the alkyne tagging strategy provides a unique opportunity to characterize the spatiotemporal dynamics of oxytocin and other small-sized peptides in the brain tissue.

Aquaporin-4 in Neuromyelitis Optica Spectrum Disorders: A Target of Autoimmunity in the Central Nervous System.

Since the discovery of a specific autoantibody in patients with neuromyelitis optica spectrum disorder (NMOSD) in 2004, the water channel aquaporin-4 (AQP4) has attracted attention as a target of autoimmune diseases of the central nervous system. In NMOSD, the autoantibody (NMO-IgG) binds to the extracellular loops of AQP4 as expressed in perivascular astrocytic end-feet and disrupts astrocytes in a complement-dependent manner. NMO-IgG is an excellent marker for distinguishing the disease from other inflammatory demyelinating diseases, such as multiple sclerosis. The unique higher-order structure of AQP4-called orthogonal arrays of particles (OAPs)-as well as its subcellular localization may play a crucial role in the pathogenesis of the disease. Recent studies have also demonstrated complement-independent cytotoxic effects of NMO-IgG. Antibody-induced endocytosis of AQP4 has been suggested to be involved in this mechanism. This review focuses on the binding properties of antibodies that recognize the extracellular region of AQP4 and the characteristics of AQP4 that are implicated in the pathogenesis of NMOSD.