Comorbid functional dyspepsia reflects IL-33-mediated airway neuronal dysfunction in asthma.

BACKGROUND: Neuronal dysfunction is implicated in the pathophysiology of asthma and functional dyspepsia (FD). However, the relationship between these diseases remains unclear. OBJECTIVE: This study aimed to clarify the clinical implications of comorbid FD in asthma and to explore the unified pathway between asthma and FD by focusing on airway neuronal dysfunction. METHODS: Clinical indices and biomarkers, including capsaicin cough sensitivity (C-CS), were compared between patients with asthma with and without FD. C-CS was determined based on the capsaicin concentration that induced at least two (C2) or five coughs (C5). Additionally, the associations of airway inflammation with airway innervation and gastrointestinal motility were evaluated in mouse models of type 2 airway inflammation. RESULTS: Patients with asthma with FD had worse asthma control and cough severity and lower C2 and C5 thresholds than those without FD. The severity of FD symptoms was negatively correlated with C2 and C5 thresholds. FD and poor asthma control were predictors of heightened C-CS (defined as C5 of ≤ 2.44 µM) in asthma. A mouse model of papain-induced airway inflammation developed airway hyperinnervation and gastrointestinal dysmotility, and both pathologies were ameliorated by an anti-interleukin (IL)-33 antibody. Moreover, papain-induced gastrointestinal dysmotility was mitigated by silencing the airway sensory neurons using QX-314, a sodium channel blocker. Furthermore, sputum IL-33 levels were significantly elevated in patients with asthma with FD or heightened C-CS compared with those in their counterparts. CONCLUSION: FD is significantly associated with airway neuronal dysfunction in asthma. IL-33-mediated airway neuronal dysfunction may contribute to the interaction between asthma and FD.

High-density lipoproteins mediate small RNA intercellular communication between dendritic cells and macrophages.

HDL are dynamic transporters of diverse molecular cargo and play critical roles in lipid metabolism and inflammation. We have previously reported that HDL transport both host and nonhost small RNAs (sRNA) based on quantitative PCR and sRNA sequencing approaches; however, these methods require RNA isolation steps which have potential biases and may not isolate certain forms of RNA molecules from samples. HDL have also been reported to accept functional sRNAs from donor macrophages and deliver them to recipient endothelial cells; however, using PCR to trace HDL-sRNA intercellular communication has major limitations. The present study aims to overcome these technical barriers and further understand the pathways involved in HDL-mediated bidirectional flux of sRNAs between immune cells. To overcome these technical limitations, SYTO RNASelect, a lipid-penetrating RNA dye, was used to quantify a) overall HDL-sRNA content, b) bidirectional flux of sRNAs between HDL and immune cells, c) HDL-mediated intercellular communication between immune cells, and d) HDL-mediated RNA export changes in disease. Live cell imaging and loss-of-function assays indicate that the endo-lysosomal system plays a critical role in macrophage storage and export of HDL-sRNAs. These results identify HDL as a substantive mediator of intercellular communication between immune cells and demonstrate the importance of endocytosis for recipient cells of HDL-sRNAs. Utilizing a lipid-penetrating RNA-specific fluorescence dye, we were able to both quantify the absolute concentration of sRNAs transported by HDL and characterize HDL-mediated intercellular RNA transport between immune cells.

A gain-of-function mutation in the acid-sensing ion channel 2a induces marked cerebellar maldevelopment in rats.

Specific amino acid substitutions in degenerin mechano-gated channels (DEGs) of C. elegans convert these channels into constitutively active mutants that induce the degeneration of neurons where DEGs are expressed. Acid-sensing ion channel-2a (ASIC2a), a proton-gated cation channel predominantly expressed in central neurons, is a mammalian ortholog of DEGs, and it can remain unclosed to be cytotoxic once the same mutations as the DEG mutants are introduced into its gene. Here we show that heterozygous transgenic (Tg) rats expressing ASIC2a-G430F (ASIC2aG430F), the most active form of the gain-of-function mutants, under the control of the intrinsic ASIC2a promoter exhibited marked cerebellar maldevelopment with mild whole-brain atrophy. The Tg rats were small and developed an early-onset ataxic gait, as evidenced by rotarod and footprint tests. The overall gross-anatomy of the Tg brain was normal just after birth, but a reduction in brain volume, especially cerebellar volume, gradually emerged with age. Histological examination of the adult Tg brain revealed that the cell-densities of cerebellar Purkinje and granule cells were markedly reduced, while the cytoarchitecture of other brain regions was not significantly altered. RT-PCR and immunoblot analyses demonstrated that ASIC2aG430F transcripts and proteins were already present in various regions of the neonatal Tg brain before the deforming cerebellum became apparent. These results suggest that, according to the spatiotemporal pattern of the wild-type (WT) ASIC2a gene expression, the ASIC2aG430F channel induced lethal degeneration in Tg brain neurons expressing both ASIC2aG430F and ASIC2a channels.

AITC inhibits fibroblast-myofibroblast transition via TRPA1-independent MAPK and NRF2/HO-1 pathways and reverses corticosteroids insensitivity in human lung fibroblasts.

BACKGROUND: Little is known on the role of transient receptor potential ankyrin 1 (TRPA1) in fibroblast-myofibroblast transition (FMT) that can lead to airway remodeling which is a major problem for severe asthma and fibrosis. Thus, this study investigated the effect of TRPA1 modulators on transforming growth factor beta 1(TGF-ß1) -treated lung fibroblasts. METHODS: MRC-5 cells were preincubated with TGF-ß1 for 24 h. TRPA1 agonist or antagonist were added and further incubated for 24 h. The changes in TRPA1 and alpha-smooth muscle actin (α-SMA) expressions by stimuli were evaluated using qRT-PCR, western blot and immunohistochemical analyses. Statistical significance was determined by using one- or two-way ANOVA, followed by Bonferroni's post hoc analysis for comparison of multiple groups and paired 2-tailed Student's t-test between 2 groups. RESULTS: MRC-5 cells treated by TGF-ß1 significantly upregulated α-SMA mRNA expressions (P < 0.01), but downregulated TRPA1 gene expression (P < 0.001). Post-treatment of TRPA1 activator, allyl isothiocyanate (AITC), after TGF-ß1 significantly downregulated the α-SMA gene induction (P < 0.01 at 24 h), protein expression (P < 0.05) and immunoreactivity with stress fibers (P < 0.05). On the other hand, TRPA1 antagonist HC-030031 did not prevent this effect, and instead tended to facilitate the suppressive effect of AITC when co-stimulated. AITC significantly increased phosphorylated- extracellular signal-regulated kinase (ERK) 1/2 and heme oxygenase (HO)-1 protein expressions (P < 0.05) in TGF-ß1-treated cells. Combined inhibition with ERK1/2 mitogen-activated protein kinase (MAPK) and nuclear factor erythroid 2-related factor (NRF2) almost completely reversed AITC-induced α-SMA suppression (P < 0.05). Dexamethasone was not able to inhibit the upregulated α-SMA induction by TGF-ß1. However, AITC improved dexamethasone-insensitive myodifferentiation in the presence of the corticosteroid (P < 0.01). CONCLUSION: We found that AITC exerts protective effect on TGF-ß1-induced α-SMA induction by activating ERK1/2 MAPK and NRF2/HO-1 pathways in lung fibroblasts. It also overcomes corticosteroids insensitivity in TGF-ß1-induced α-SMA induction. TRPA1 antagonist modulates the suppressive effect, but not prevent it. AITC and TRPA1 antagonist may be therapeutic agents in treating chronic respiratory diseases.

Dynamic Changes in Ultrastructure of the Primary Cilium in Migrating Neuroblasts in the Postnatal Brain.

New neurons, referred to as neuroblasts, are continuously generated in the ventricular-subventricular zone of the brain throughout an animal's life. These neuroblasts are characterized by their unique potential for proliferation, formation of chain-like cell aggregates, and long-distance and high-speed migration through the rostral migratory stream (RMS) toward the olfactory bulb (OB), where they decelerate and differentiate into mature interneurons. The dynamic changes of ultrastructural features in postnatal-born neuroblasts during migration are not yet fully understood. Here we report the presence of a primary cilium, and its ultrastructural morphology and spatiotemporal dynamics, in migrating neuroblasts in the postnatal RMS and OB. The primary cilium was observed in migrating neuroblasts in the postnatal RMS and OB in male and female mice and zebrafish, and a male rhesus monkey. Inhibition of intraflagellar transport molecules in migrating neuroblasts impaired their ciliogenesis and rostral migration toward the OB. Serial section transmission electron microscopy revealed that each migrating neuroblast possesses either a pair of centrioles or a basal body with an immature or mature primary cilium. Using immunohistochemistry, live imaging, and serial block-face scanning electron microscopy, we demonstrate that the localization and orientation of the primary cilium are altered depending on the mitotic state, saltatory migration, and deceleration of neuroblasts. Together, our results highlight a close mutual relationship between spatiotemporal regulation of the primary cilium and efficient chain migration of neuroblasts in the postnatal brain.SIGNIFICANCE STATEMENT Immature neurons (neuroblasts) generated in the postnatal brain have a mitotic potential and migrate in chain-like cell aggregates toward the olfactory bulb. Here we report that migrating neuroblasts possess a tiny cellular protrusion called a primary cilium. Immunohistochemical studies with zebrafish, mouse, and monkey brains suggest that the presence of the primary cilium in migrating neuroblasts is evolutionarily conserved. Ciliogenesis in migrating neuroblasts in the rostral migratory stream is suppressed during mitosis and promoted after cell cycle exit. Moreover, live imaging and 3D electron microscopy revealed that ciliary localization and orientation change during saltatory movement of neuroblasts. Our results reveal highly organized dynamics in maturation and positioning of the primary cilium during neuroblast migration that underlie saltatory movement of postnatal-born neuroblasts.

Isozyme-Specific Role of SAD-A in Neuronal Migration During Development of Cerebral Cortex.

SAD kinases regulate presynaptic vesicle clustering and neuronal polarization. A previous report demonstrated that Sada-/- and Sadb-/- double-mutant mice showed perinatal lethality with a severe defect in axon/dendrite differentiation, but their single mutants did not. These results indicated that they were functionally redundant. Surprisingly, we show that on a C57BL/6N background, SAD-A is essential for cortical development whereas SAD-B is dispensable. Sada-/- mice died within a few days after birth. Their cortical lamination pattern was disorganized and radial migration of cortical neurons was perturbed. Birth date analyses with BrdU and in utero electroporation using pCAG-EGFP vector showed a delayed migration of cortical neurons to the pial surface in Sada-/- mice. Time-lapse imaging of these mice confirmed slow migration velocity in the cortical plate. While the neurites of hippocampal neurons in Sada-/- mice could ultimately differentiate in culture to form axons and dendrites, the average length of their axons was shorter than that of the wild type. Thus, analysis on a different genetic background than that used initially revealed a nonredundant role for SAD-A in neuronal migration and differentiation.

Efficient method for isolation of exosomes from raw bovine milk.

OBJECTIVE: This study aimed to establish a rapid and simple method for isolating exosomes from raw bovine milk and to compare the quality of the isolated exosomes with those isolated by a standard method involving ultracentrifugation (UC). METHODS: To remove caseins, which are major milk proteins consisting more than 80% of milk protein (35% in human breast milk) and hamper isolation and purification of exosomes, hydrochloride (HCl) was added to milk for isoelectric precipitation (IP). The effects of acidification on morphological features, particle size distribution, surface charge, and exosome surface proteins were analyzed by electron microscopy, tunable resistive pulse sensing (TRPS), and Western blot (WB) analysis, respectively. RESULTS: Electron microscopy showed that some of the exosomes isolated using IP had rough surfaces; most exosomes were successfully isolated without breakage, and their morphological features were similar to those of exosomes isolated by UC. TRPS showed that their surface charge and peaks (mode) for particle size distribution did not significantly differ between both methods. WB analysis using antibodies against the exosome surface marker proteins - milk fat globule-epidermal growth factor 8 (MFG-E8) and CD63 - revealed that the structures of exosome surface proteins were not affected by adding HCl. CONCLUSIONS: IP can be used to remove caseins to reduce operation time. This method will be useful for efficient isolation and purification of bovine milk exosomes and contribute to progression of research on health management of dairy cattle and drug delivery systems in human medicine, which require large amounts of milk exosomes.

High salt loading induces urinary storage dysfunction via upregulation of epithelial sodium channel alpha in the bladder epithelium in Dahl salt-sensitive rats.

We aimed to investigate whether high salt intake affects bladder function via epithelial sodium channel (ENaC) by using Dahl salt-resistant (DR) and salt-sensitive (DS) rats. Bladder weight of DR + high-salt diet (HS, 8% NaCl) and DS + HS groups were significantly higher than those of DR + normal-salt diet (NS, 0.3% NaCl) and DS + NS groups after one week treatment. We thereafter used only DR + HS and DS + HS group. Systolic and diastolic blood pressures were significantly higher in DS + HS group than in DR + HS group after the treatment period. Cystometrogram showed the intercontraction intervals (ICI) were significantly shorter in DS + HS group than in DR + HS group during infusion of saline. Subsequent infusion of amiloride significantly prolonged ICI in DS + HS group, while no intra-group difference in ICI was observed in DR + HS group. No intra- or inter-group differences in maximum intravesical pressure were observed. Protein expression levels of ENaCα in the bladder were significantly higher in DS + HS group than in DR + HS group. ENaCα protein was localized at bladder epithelium in both groups. In conclusion, high salt intake is considered to cause urinary storage dysfunction via upregulation of ENaC in the bladder epithelium with salt-sensitive hypertension, suggesting that ENaC might be a candidate for therapeutic target for urinary storage dysfunction.

Expression profiling of microRNA in cryptorchid testes: miR-135a contributes to the maintenance of spermatogonial stem cells by regulating FoxO1.

PURPOSE: We elucidated the mechanism of spermatogonial stem cell disturbance of cryptorchidism and investigated the expression of miRNAs and their target genes in undescended testes. MATERIALS AND METHODS: Using microarray analysis we compared total miRNA expression in unilateral undescended testes with that in contralateral descended and normal testes in a rat model of cryptorchidism. The model was derived by administering flutamide to pregnant Sprague Dawley® rats. We identified mRNA targets of miRNAs by bioinformatic analysis, followed by in situ hybridization and immunohistochemistry to localize candidate miRNAs and mRNAs, respectively. We also investigated whether miRNAs could inhibit target protein expression in vitro. RESULTS: Microarray analysis and subsequent quantitative reverse transcriptase-polymerase chain reaction showed that only miR-135a was expressed at a lower level in undescended testes. We identified its target as FoxO1, which is essential for stem cell maintenance. miR-135a and FoxO1 localized to spermatogonial stem cells. FoxO1 localized to the spermatogonial stem cell nucleus less frequently in undescended testes, indicating that the activity of FoxO1, which acts as a transcription factor, is altered in undescended testes. Finally, miR-135a transfection into spermatogonia in vitro resulted in down-regulation of FoxO1 expression. CONCLUSIONS: In cryptorchid testes there is a decreased number of spermatogonial stem cells in which FoxO1 is activated, indicating that failure of spermatogonial stem cell maintenance results in spermatogenesis alteration. We also noted interaction between miR-135a and FoxO1, and propose that miR-135a contributes to spermatogonial stem cell maintenance through modulation of FoxO1 activity.

Vascular endothelial growth factor-A is involved in intramuscular carrageenan-induced cutaneous mechanical hyperalgesia through the vascular endothelial growth factor-A receptor 1 and transient receptor potential vanilloid 1 pathways.

OBJECTIVES: Vascular endothelial growth factor-A (VEGF-A) plays a leading role in angiogenesis and pain hypersensitivity in cancer and chronic pain. It is not only induced by ischemic conditions but is also highly correlated with proalgesic cytokines, both of which are prominent in inflammatory muscle pain. However, the molecular basis of the involvement of VEGF-A in muscle pain remains unknown. METHODS: In the present study, we performed behavioral and pharmacological analyses to determine the possible involvement of VEGF-A in the development of inflammatory muscle pain and the associated signal transduction pathway. RESULTS: Unilateral intramuscular injection of carrageenan, a classical model of inflammatory muscle pain, increased VEGF-A gene expression in the tissues surrounding the injection site. Intramuscular administration of recombinant VEGF-A165 on the same side induced cutaneous mechanical hyperalgesia during the acute and subacute phases. The application of a specific VEGFR1 antibody on the same side significantly reduced the mechanical hyperalgesia induced by carrageenan or VEGF-A165 injection, whereas both a VEGFR2-neutralizing antibody and a VEGFR2 antagonist showed limited effects. Local preinjection of capsazepine, a transient receptor potential vanilloid 1 (TRPV1) antagonist, also inhibited VEGF-A165-induced hyperalgesia. Finally, intramuscular VEGF-A165-induced mechanical hyperalgesia was not found in TRPV1 knockout mice during the subacute phase. CONCLUSIONS: These findings suggest that inflammatory stimuli increase interstitial VEGF-A165, which in turn induces cutaneous mechanical pain via the VEGFR1-mediated TRPV1 nociceptive pathway during inflammatory muscle pain. VEGFR1 could be a novel therapeutic target for inflammation-induced muscle pain.

Indispensable role of factor for adipocyte differentiation 104 (fad104) in lung maturation.

Factor for adipocyte differentiation 104 (fad104) is a regulator of adipogenesis and osteogenesis. Our previous study showed that fad104-deficient mice died immediately after birth, suggesting fad104 to be essential for neonatal survival. However, the cause of this rapid death is unclear. Here, we demonstrate the role of fad104 in neonatal survival. Phenotypic and morphological analyses showed that fad104-deficient mice died due to cyanosis-associated lung dysplasia including atelectasis. Furthermore, immunohistochemistry revealed that FAD104 was strongly expressed in ATII cells in the developing lung. Most importantly, the ATII cells in lungs were immature, and impaired the expression of surfactant-associated proteins. Collectively, these results indicate that fad104 has an indispensable role in lung maturation, especially the maturation and differentiation of ATII cells.

The iodide transporter Slc26a7 impacts thyroid function more strongly than Slc26a4 in mice.

SLC26A4 is a known iodide transporter, and is localized at the apical membrane of thyrocytes. Previously, we reported that SLC26A7 is also involved in iodide transport and that Slc26a7 is a novel causative gene for congenital hypothyroidism. However, its detailed role in vivo remains to be elucidated. We generated mice that were deficient in Slc26a7 and Slc26a4 to delineate differences and associations in their roles in iodide transport. Slc26a7-/- mice showed goitrous congenital hypothyroidism and mild growth failure on a normal diet. Slc26a7-/- mice with a low iodine environment showed marked growth failure. In contrast, Slc26a4-/- mice showed no growth failure and hypothyroidism in the same low iodine environment. Double-deficient mice showed more severe growth failure than Slc26a7-/- mice. RNA-seq analysis revealed that the number of differentially expressed genes (DEGs) in Slc26a7-/- mice was significantly higher than that in Slc26a4-/- mice. These indicate that SLC26A7 is more strongly involved in iodide transport and the maintenance of thyroid function than SLC26A4.

Human Lung Fibroblasts Exhibit Induced Inflammation Memory via Increased IL6 Gene Expression and Release.

Fibroblasts of different origins are known to possess stromal memory after inflammatory episodes. However, there are no studies exploring human lung fibroblast memory which may predict a subsequent inflammatory response in chronic respiratory diseases and COVID-19. MRC-5 and HF19 human lung fibroblast cell lines were treated using different primary and secondary stimulus combinations: TNFα-WD-TNFα, Poly (I:C)-WD-TNFα, TNFα-WD-Poly (I:C), or LPS-WD-TNFα with a 24-h rest period (withdrawal period; WD) between the two 24-h stimulations. TLR3 and NF-κB inhibitors were used to determine pathways involved. The effect of SARS-Cov-2 spike protein to inflammatory response of lung fibroblasts was also investigated. mRNA expressions of genes and IL6 release were measured using qRT-PCR and ELISA, respectively. Statistical significance was determined by using one- or two-way ANOVA, followed by Bonferroni's post hoc analysis for comparison of multiple groups. Preexposure with Poly (I:C) significantly increased TNFα-induced IL6 gene expression and IL6 release in both cell lines, while it affected neither gene expressions of IL1B, IL2, IL8, and MMP8 nor fibrosis-related genes: ACTA2, COL1A1, POSTN, and TGFB1. Inhibition of TLR3 or NF-κB during primary stimulation significantly downregulated IL6 release. Simultaneous treatment of MRC-5 cells with SARS-CoV-2 spike protein further increased TNFα-induced IL6 release; however, preexposure to Poly (I:C) did not affect it. Human lung fibroblasts are capable of retaining inflammatory memory and showed an augmented response upon secondary exposure. These results may contribute to the possibility of training human lung fibroblasts to respond suitably on inflammatory episodes after viral infection.

The TRPV4 channel is a novel regulator of intracellular Ca2+ in human esophageal epithelial cells.

The esophageal epithelium has sensory properties that enable it to sustain normal barrier function. Transient receptor potential vanilloid 4 (TRPV4) is a Ca(2+)-permeable channel that is activated by extracellular hypotonicity, polyunsaturated fatty acids, phorbol esters, and elevated temperature. We found that TRPV4 is expressed in both human esophageal tissue and in HET-1A cells, a human esophageal epithelial cell line. Specific activation of TRPV4 by the phorbol ester 4α-phorbol 12,13-didecanoate (4α-PDD) increased intracellular Ca(2+) in a subset of HET-1A cells. Elevated temperature strongly potentiated this effect at low concentrations of 4α-PDD, and all of the responses were inhibited by the TRPV antagonist ruthenium red. TRPV4 activation differentially affected cell proliferation and cell viability; HET-1A cell proliferation was increased by 1 µM 4α-PDD, whereas higher concentrations (10 µM and 30 µM) significantly decreased cell viability. Transient TRPV4 activation triggered ATP release in a concentration-dependent manner via gap-junction hemichannels, including pannexin 1 and connexin 43. Furthermore, TRPV4 activation for 24 h did not increase the production of interleukin 8 (IL-8) but reduced IL-1ß-induced IL-8 production. Small-interference RNA targeted to TRPV4 significantly attenuated all of the 4α-PDD-induced responses in HET-1A cells. Collectively, these findings suggest that TRPV4 is a novel regulator of Ca(2+)-dependent signaling pathways linked to cell proliferation, cell survival, ATP release, and IL-8 production in human esophageal epithelial cells.

Additive Effects of L-Ornithine on Preferences to Basic Taste Solutions in Mice.

In addition to the taste receptors corresponding to the six basic taste qualities-sweet, salty, sour, bitter, umami, and fatty-another type of taste receptor, calcium-sensing receptor (CaSR), is found in taste-bud cells. CaSR is called the 'kokumi' receptor because its agonists increase sweet, salty and umami tastes to induce 'koku', a Japanese word meaning the enhancement of flavor characters such as thickness, mouthfulness, and continuity. Koku is an important factor for enhancing food palatability. However, it is not well known whether other kokumi-receptors and substances exist. Here, we show that ornithine (L-ornithine but not D-ornithine) at low concentrations that do not elicit a taste of its own, enhances preferences to sweet, salty, umami, and fat taste solutions in mice. Increased preference to monosodium glutamate (MSG) was the most dominant effect. Antagonists of G-protein-coupled receptor family C group 6 subtype A (GPRC6A) abolished the additive effect of ornithine on MSG solutions. The additive effects of ornithine on taste stimuli are thought to occur in the oral cavity, and are not considered post-oral events because ornithine's effects were confirmed in a brief-exposure test. Moreover, the additive effects of ornithine and the action of the antagonist were verified in electrophysiological taste nerve responses. Immunohistochemical analysis implied that GPRC6A was expressed in subsets of type II and type III taste cells of mouse circumvallate papillae. These results are in good agreement with those reported for taste modulation involving CaSR and its agonists. The present study suggests that ornithine is a kokumi substance and GPRC6A is a newly identified kokumi receptor.

Functional expression of transient receptor potential vanilloid 3 (TRPV3) in corneal epithelial cells: involvement in thermosensation and wound healing.

Transient receptor potential vanilloid 3 (TRPV3), a member of the calcium-permeable thermosensitive TRP (thermoTRP) subfamily of receptors, is an important cutaneous sensor that detects thermal and chemical stimuli. TRPV3 is activated by innocuous warm temperature stimuli (>33 degrees C) and a variety of physiologically active substances. While the corneal epithelium is known to respond to such stimuli, it is unknown whether TRPV3 is involved in this phenomenon. We show here that TRPV3 mRNA and protein are abundantly expressed in the epithelial cells of human and mouse cornea. Carvacrol, an agonist of TRPV3, elevated cytosolic Ca2+ concentration in both primary mouse corneal epithelial cells and cultured human corneal epithelial cells (HCE-T cells). The response to carvacrol was inhibited by ruthenium red, a TRPV channel antagonist. Moreover, repetitive agonist stimulation sensitized the response with gradually increasing amplitude, suggesting that the TRPV3 in the cornea has similar physiological and pharmacological characteristics to that in skin keratinocytes. Finally, a wound healing assay revealed that appropriate calcium ion influx via activated TRPV3 in corneal epithelial cells accelerated their proliferation. Thus, functional TRPV3 is present in corneal epithelial cells and may play a role not only in thermosensation, but also in the regulation of cell proliferation.

In vitro Time-lapse Imaging of Primary Cilium in Migrating Neuroblasts.

Neuronal migration is a critical step for the development of neuronal circuits in the brain. Immature new neurons (neuroblasts) generated in the postnatal ventricular-subventricular zone (V-SVZ) show a remarkable potential to migrate for a long distance at a high speed in the postnatal mammalian brain, and are thus a powerful model to analyze the molecular and cellular mechanisms of neuronal migration. Here we describe a methodology for in vitro time-lapse imaging of the primary cilium and its related structures in migrating V-SVZ-derived neuroblasts using confocal or superresolution laser-scanning microscopy. The V-SVZ tissues are dissected from postnatal day 0-1 (P0-1) mouse brains and dissociated into single cells by trypsinization and gentle pipetting. These cells are then transduced with a plasmid(s) encoding a gene(s) of interest, aggregated by centrifugation, and cultured for 2 days in Matrigel. Time-lapse images of migratory behaviors of cultured neuroblasts and their ciliary structures, including the ciliary membrane and basal body, are acquired by confocal or superresolution laser-scanning microscopy. This method provides information about the spatiotemporal dynamics of neuroblasts' morphology and ciliary structures, and is widely applicable to various types of migrating neuronal and nonneuronal cells in various species.

Prebiotic effect of fructo-oligosaccharides on the inner ear of DBA/2 J mice with early-onset progressive hearing loss.

Nutrition and dietary habits contribute to the onset and progression of sensorineural hearing loss (SNHL). Fructo-oligosaccharides (FOS) are non-digestible oligosaccharides and are known as prebiotics, which enhance short-chain fatty acid (SCFA) production and antioxidant activity. Although a substantial number of studies have shown that FOS play a role in the prevention of lifestyle-related diseases as prebiotics, little is known about the effects on the inner ear. The purpose of this study is to investigate the effect of FOS on gene expression and spiral ganglion neuron (SGN) protection in the inner ear of DBA/2 J mice, which is a model for early-onset progressive hearing loss. DBA/2 J mice were fed either control diet or FOS diet contained 10% (w/w) of FOS for 8 weeks. Analysis of mice fed the FOS diet revealed a change in intestinal flora including an inversion of the ratio of Bacteroidetes and Firmicutes, which was followed by a significant increase in SCFAs in the cecum and a decrease in an oxidative stress marker in the serum. In the inner ear, gene expression of neurotrophin, brain-derived neurotrophic factor (BDNF), its receptor, tyrosine kinase receptor b (Trkb), and the SCFA receptor, free fatty acid receptor 3 (FFAR3), were increased by FOS. In addition, the survival rate of SGNs in the inner ear was maintained in FOS-fed mice. Altogether, these results suggest that a compositional variation of the intestinal flora due to a prebiotic effect may be involved in the progression of SNHL.

Two mouse models carrying truncating mutations in Magel2 show distinct phenotypes.

Schaaf-Yang syndrome (SYS) is a neurodevelopmental disorder caused by truncating variants in the paternal allele of MAGEL2, located in the Prader-Willi critical region, 15q11-q13. Although the phenotypes of SYS overlap those of Prader-Willi syndrome (PWS), including neonatal hypotonia, feeding problems, and developmental delay/intellectual disability, SYS patients show autism spectrum disorder and joint contractures, which are atypical phenotypes for PWS. Therefore, we hypothesized that the truncated Magel2 protein could potentially produce gain-of-function toxic effects. To test the hypothesis, we generated two engineered mouse models; one, an overexpression model that expressed the N-terminal region of Magel2 that was FLAG tagged with a strong ubiquitous promoter, and another, a genome-edited model that carried a truncating variant in Magel2 generated using the CRISPR/Cas9 system. In the overexpression model, all transgenic mice died in the fetal or neonatal period indicating embryonic or neonatal lethality of the transgene. Therefore, overexpression of the truncated Magel2 could show toxic effects. In the genome-edited model, we generated a mouse model carrying a frameshift variant (c.1690_1924del; p(Glu564Serfs*130)) in Magel2. Model mice carrying the frameshift variant in the paternal or maternal allele of Magel2 were termed Magel2P:fs and Magel2M:fs, respectively. The imprinted expression and spatial distribution of truncating Magel2 transcripts in the brain were maintained. Although neonatal Magel2P:fs mice were lighter than wildtype littermates, Magel2P:fs males and females weighed the same as their wildtype littermates by eight and four weeks of age, respectively. Collectively, the overexpression mouse model may recapitulate fetal or neonatal death, which are the severest phenotypes for SYS. In contrast, the genome-edited mouse model maintains genomic imprinting and distribution of truncated Magel2 transcripts in the brain, but only partially recapitulates SYS phenotypes. Therefore, our results imply that simple gain-of-function toxic effects may not explain the patho-mechanism of SYS, but rather suggest a range of effects due to Magel2 variants as in human SYS patients.

Polo-like kinase 4 and Stromal antigen 3 are not associated with recurrent pregnancy loss caused by embryonic aneuploidy.

No genetic association with recurrent pregnancy loss (RPL) caused by embryonic aneuploidy has been found. Recent studies have indicated that the common genetic variant rs2305957, surrounding the PLK4 gene, contributes to mitotic-origin aneuploidy risk during human early embryo development. The decrease in meiosis-specific cohesin causes predivision of sister chromatids in the centromere and chromosome segregation errors. STAG3 is a component of cohesin and is a meiosis-specific gene. Our case-control study included 184 patients with RPL whose previous products of conception (POC) exhibited aneuploidy and 190 fertile control women without a history of miscarriage. We performed a genetic association study to examine the genotype distribution at PLK4 (rs2305957) and STAG3 in patients with RPL caused by aneuploidy compared with controls. Regarding STAG3, SNPs with a minor allele frequency (MAF) threshold > 0.05 that were predicted to be binding sites of transcription factors and that showed significant associations in expression quantitative trait locus (e-QTL) analysis were selected. No significant differences in the MAF or distribution in any model of PLK4 (rs2305957) and 5 selected tag SNPs in STAG3 were found between the patients and controls. A further genome-wide association study is needed since a combination of genetic risk alleles might be useful in predicting future age-dependent RPL caused by aneuploidy.