Molecular differences between neonatal and adult stria vascularis from organotypic explants and transcriptomics.

Summary: The stria vascularis (SV), part of the blood-labyrinth barrier, is an essential component of the inner ear that regulates the ionic environment required for hearing. SV degeneration disrupts cochlear homeostasis, leading to irreversible hearing loss, yet a comprehensive understanding of the SV, and consequently therapeutic availability for SV degeneration, is lacking. We developed a whole-tissue explant model from neonatal and adult mice to create a robust platform for SV research. We validated our model by demonstrating that the proliferative behaviour of the SV in vitro mimics SV in vivo, providing a representative model and advancing high-throughput SV research. We also provided evidence for pharmacological intervention in our system by investigating the role of Wnt/ß-catenin signaling in SV proliferation. Finally, we performed single-cell RNA sequencing from in vivo neonatal and adult mouse SV and revealed key genes and pathways that may play a role in SV proliferation and maintenance. Together, our results contribute new insights into investigating biological solutions for SV-associated hearing loss. Significance: Hearing loss impairs our ability to communicate with people and interact with our environment. This can lead to social isolation, depression, cognitive deficits, and dementia. Inner ear degeneration is a primary cause of hearing loss, and our study provides an in depth look at one of the major sites of inner ear degeneration: the stria vascularis. The stria vascularis and associated blood-labyrinth barrier maintain the functional integrity of the auditory system, yet it is relatively understudied. By developing a new in vitro model for the young and adult stria vascularis and using single cell RNA sequencing, our study provides a novel approach to studying this tissue, contributing new insights and widespread implications for auditory neuroscience and regenerative medicine. Highlights: - We established an organotypic explant system of the neonatal and adult stria vascularis with an intact blood-labyrinth barrier. - Proliferation of the stria vascularis decreases with age in vitro , modelling its proliferative behaviour in vivo . - Pharmacological studies using our in vitro SV model open possibilities for testing injury paradigms and therapeutic interventions. - Inhibition of Wnt signalling decreases proliferation in neonatal stria vascularis.- We identified key genes and transcription factors unique to developing and mature SV cell types using single cell RNA sequencing.

Chlamydomonas dynein-preassembly-deficient mutants exhibit characteristic ciliary responses to viscous media.

Ciliary-dynein preassembly in the cytoplasm is critical for the assembly and movement of motile cilia, organelles that function under viscous conditions. Defects in preassembly often lead to a reduction in specific types of ciliary dyneins. Here, we investigated how environmental viscosity affects the motility of preassembly-deficient cilia in the alga Chlamydomonas. We found that, depending on the type of ciliary dynein deficiency, each Chlamydomonas mutant displays a characteristic phenotype in cell propulsion. Our results highlight not only the unique function(s) of each dynein species, but also the importance of functional coordination between dyneins for ciliary motility under viscous conditions.

EBF1 Limits the Numbers of Cochlear Hair and Supporting Cells and Forms the Scala Tympani and Spiral Limbus during Inner Ear Development.

Early B-cell factor 1 (EBF1) is a basic helix-loop-helix transcription factor essential for the differentiation of various tissues. Our single-cell RNA sequencing data suggest that Ebf1 is expressed in the sensory epithelium of the mouse inner ear. Here, we found that the murine Ebf1 gene and its protein are expressed in the prosensory domain of the inner ear, medial region of the cochlear duct floor, otic mesenchyme, and cochleovestibular ganglion. Ebf1 deletion in mice results in incomplete formation of the spiral limbus and scala tympani, increased number of cells in the organ of Corti and Kölliker's organ, and aberrant course of the spiral ganglion axons. Ebf1 deletion in the mouse cochlear epithelia caused the proliferation of SOX2-positive cochlear cells at E13.5, indicating that EBF1 suppresses the proliferation of the prosensory domain and cells of Kölliker's organ to facilitate the development of appropriate numbers of hair and supporting cells. Furthermore, mice with deletion of cochlear epithelium-specific Ebf1 showed poor postnatal hearing function. Our results suggest that Ebf1 is essential for normal auditory function in mammals.

Functional and structural significance of the inner-arm-dynein subspecies d in ciliary motility.

Motile cilia play various important physiological roles in eukaryotic organisms including cell motility and fertility. Inside motile cilia, large motor-protein complexes called "ciliary dyneins" coordinate their activities and drive ciliary motility. The ciliary dyneins include the outer-arm dyneins, the double-headed inner-arm dynein (IDA f/I1), and several single-headed inner-arm dyneins (IDAs a, b, c, d, e, and g). Among these single-headed IDAs, one of the ciliary dyneins, IDA d, is of particular interest because of its unique properties and subunit composition. In addition, defects in this subspecies have recently been associated with several types of ciliopathies in humans, such as primary ciliary dyskinesia and multiple morphologic abnormalities of the flagellum. In this mini-review, we discuss the composition, structure, and motor properties of IDA d, which have been studied in the model organism Chlamydomonas reinhardtii, and further discuss the relationship between IDA d and human ciliopathies. In addition, we provide future perspectives and discuss remaining questions regarding this intriguing dynein subspecies.

Effects of neurofeedback on standing postural control task with combined imagined and executed movements.

Introduction: Motor imagery (MI) is a method of imagining movement without actual movement, and its use in combination with motor execution (ME) enhances the effects of motor learning. Neurofeedback (NFB) is another method that promotes the effects of MI. This study aimed to investigate the effects of NFB on combined MI and ME (MIME) training in a standing postural control task. Methods: Sixteen participants were randomly divided into MIME and MIME + NFB groups and performed 10 trials of a postural control task on an unstable board, with nine trials of MI in between. Electroencephalogram was assessed during MI, and the MIME + NFB group received neurofeedback on the degree of MI via auditory stimulation. A postural control task using an unstable board was performed before and after the MIME task, during which postural instability was evaluated. Results: Postural instability was reduced after the MIME task in both groups. In addition, the root mean square, which indicates the sway of the unstable board, was significantly reduced in the MIME + NFB group compared to that in the MIME group. Conclusion: Our results indicate that MIME training is effective for motor learning of standing postural control. Furthermore, when MI and ME are combined, the feedback on the degree of MI enhances the learning effect.

Effects of θ High Definition-Transcranial Alternating Current Stimulation in the Anterior Cingulate Cortex on the Dominance of Attention Focus in Standing Postural Control.

Attention focus affects performance in postural control while standing, and it is divided into internal focus (IF) and external focus (EF). Each individual has a predominant attention focus, and research has revealed that the dominance of attention focus may be an acquired trait. However, the impact of non-invasive brain stimulation on attention-focus dominance remains unexplored in the current literature. Here, we examined the effect of high-definition transcranial alternating current stimulation (HD-tACS) on θ waves in the anterior cingulate cortex (ACC) on standing postural control tasks in an EF condition for IF- and EF-dominant groups. The effect of θ HD-tACS on the ACC differed between IF- and EF-dominant groups, and θ HD-tACS in the IF-dominant group decreased the performance of standing postural control under the EF condition. The forced activation of the ACC with θ HD-tACS may have conversely reduced the activity of brain regions normally activated by the IF-dominant group. Additionally, the activation of ACC prioritized visual information processing and suppressed the superficial sensory processing that is normally potentially prioritized by the IF-dominant group. These results highlight the importance of changing the type of rehabilitation and sports training tasks to account for the individual's dominance of attention focus.

Effects of neurofeedback training combined with transcranial direct current stimulation on motor imagery: A randomized controlled trial.

Introduction: Neurofeedback (NFB) training and transcranial direct current stimulation (tDCS) have been shown to individually improve motor imagery (MI) abilities. However, the effect of combining both of them with MI has not been verified. Therefore, the aim of this study was to examine the effect of applying tDCS directly before MI with NFB. Methods: Participants were divided into an NFB group (n = 10) that performed MI with NFB and an NFB + tDCS group (n = 10) that received tDCS for 10 min before MI with NFB. Both groups performed 60 MI trials with NFB. The MI task was performed 20 times without NFB before and after training, and µ-event-related desynchronization (ERD) and vividness MI were evaluated. Results: µ-ERD increased significantly in the NFB + tDCS group compared to the NFB group. MI vividness significantly increased before and after training. Discussion: Transcranial direct current stimulation and NFB modulate different processes with respect to MI ability improvement; hence, their combination might further improve MI performance. The results of this study indicate that the combination of NFB and tDCS for MI is more effective in improving MI abilities than applying them individually.

Stepwise fate conversion of supporting cells to sensory hair cells in the chick auditory epithelium.

In contrast to mammals, the avian cochlea, specifically the basilar papilla, can regenerate sensory hair cells, which involves fate conversion of supporting cells to hair cells. To determine the mechanisms for converting supporting cells to hair cells, we used single-cell RNA sequencing during hair cell regeneration in explant cultures of chick basilar papillae. We identified dynamic changes in the gene expression of supporting cells, and the pseudotime trajectory analysis demonstrated the stepwise fate conversion from supporting cells to hair cells. Initially, supporting cell identity was erased and transition to the precursor state occurred. A subsequent gain in hair cell identity progressed together with downregulation of precursor-state genes. Transforming growth factor ß receptor 1-mediated signaling was involved in induction of the initial step, and its inhibition resulted in suppression of hair cell regeneration. Our data provide new insights for understanding fate conversion from supporting cells to hair cells in avian basilar papillae.

Repetitive Peripheral Magnetic Stimulation Combined with Motor Imagery Changes Resting-State EEG Activity: A Randomized Controlled Trial.

Repetitive peripheral magnetic stimulation is a novel non-invasive technique for applying repetitive magnetic stimulation to the peripheral nerves and muscles. Contrarily, a person imagines that he/she is exercising during motor imagery. Resting-state electroencephalography can evaluate the ability of motor imagery; however, the effects of motor imagery and repetitive peripheral magnetic stimulation on resting-state electroencephalography are unknown. We examined the effects of motor imagery and repetitive peripheral magnetic stimulation on the vividness of motor imagery and resting-state electroencephalography. The participants were divided into a motor imagery group and motor imagery and repetitive peripheral magnetic stimulation group. They performed 60 motor imagery tasks involving wrist dorsiflexion movement. In the motor imagery and repetitive peripheral magnetic stimulation group, we applied repetitive peripheral magnetic stimulation to the extensor carpi radialis longus muscle during motor imagery. We measured the vividness of motor imagery and resting-state electroencephalography before and after the task. Both groups displayed a significant increase in the vividness of motor imagery. The motor imagery and repetitive peripheral magnetic stimulation group exhibited increased ß activity in the anterior cingulate cortex by source localization for electroencephalography. Hence, combined motor imagery and repetitive peripheral magnetic stimulation changes the resting-state electroencephalography activity and may promote motor imagery.

Reliability and validity of the Japanese movement imagery questionnaire-revised second version.

OBJECTIVE: Developing a Japanese version of the Movement Imagery Questionnaire-Revised Second Version (MIQ-RS) is essential for widespread evaluation and treatment based on motor imagery in physically disabled persons and patients in rehabilitation. This study aimed to investigate the reliability and validity of the Movement Imagery Questionnaire-Revised Second Version (MIQ-RS), which assesses motor imagery ability, by translating it into Japanese. RESULTS: This study enrolled twenty healthy participants (10 men and 10 women, mean age 21.17 ± 1.10 years). Reliability was examined for internal consistency using Cronbach's alpha coefficient. Spearman's rank correlation coefficient was used to examine the criterion-related validity of the MIQ-RS and the Kinesthetic and Visual Imagery Questionnaire (KVIQ-20). Results showed that Cronbach's alpha coefficients for the MIQ-RS were 0.81 and 0.82 for visual and kinesthetic imagery, respectively. Significant positive correlations were found between each visual and kinesthetic imagery score, and each total on the MIQ-RS and KVIQ-20 scores (r = 0.73, p < 0.01; r = 0.84, p < 0.01; r = 0.80, p < 0.01, respectively). This study suggests that the Japanese version of the MIQ-RS is a reliable and valid method of assessing motor imagery ability.

Composition and function of ciliary inner-dynein-arm subunits studied in Chlamydomonas reinhardtii.

Motile cilia (also interchangeably called "flagella") are conserved organelles extending from the surface of many animal cells and play essential functions in eukaryotes, including cell motility and environmental sensing. Large motor complexes, the ciliary dyneins, are present on ciliary outer-doublet microtubules and drive movement of cilia. Ciliary dyneins are classified into two general types: the outer dynein arms (ODAs) and the inner dynein arms (IDAs). While ODAs are important for generation of force and regulation of ciliary beat frequency, IDAs are essential for control of the size and shape of the bend, features collectively referred to as waveform. Also, recent studies have revealed unexpected links between IDA components and human diseases. In spite of their importance, studies on IDAs have been difficult since they are very complex and composed for several types of IDA motors, each unique in composition and location in the axoneme. Thanks in part to genetic, biochemical, and structural analysis of Chlamydomonas reinhardtii, we are beginning to understand the organization and function of the ciliary IDAs. In this review, we summarize the composition of Chlamydomonas IDAs particularly focusing on each subunit, and discuss the assembly, conservation, and functional role(s) of these IDA subunits. Furthermore, we raise several additional questions/challenges regarding IDAs, and discuss future perspectives of IDA studies.

A dynein-associated photoreceptor protein prevents ciliary acclimation to blue light.

Light-responsive regulation of ciliary motility is known to be conducted through modulation of dyneins, but the mechanism is not fully understood. Here, we report a novel subunit of the two-headed f/I1 inner arm dynein, named DYBLUP, in animal spermatozoa and a unicellular green alga. This subunit contains a BLUF (sensors of blue light using FAD) domain that appears to directly modulate dynein activity in response to light. DYBLUP (dynein-associated BLUF protein) mediates the connection between the f/I1 motor domain and the tether complex that links the motor to the doublet microtubule. Chlamydomonas lacking the DYBLUP ortholog shows both positive and negative phototaxis but becomes acclimated and attracted to high-intensity blue light. These results suggest a mechanism to avoid toxic strong light via direct photoregulation of dyneins.

Light-Triggered, Non-Centrosymmetric Self-Assembly of Aqueous Arylazopyrazoles at the Air-Water Interface and Switching of Second-Harmonic Generation.

Trans-p-methoxy arylazopyrazole spontaneously forms non-centrosymmetric polar crystals, which reversibly undergo liquefaction upon photoisomerization to the cis-isomer. This liquid cis-isomer has a large electric dipole moment and is highly soluble in water (solubility up to ≈58 mM), which is remarkably higher than that of the trans-isomer (690 µM). Vis-light illumination of the aqueous cis-isomer generates macroscopically oriented, non-centrosymmetric crystals at the air-water interface. Polar crystals are also formed in sandwich glass cells (spacing, 20 µm) upon photo-induced crystallization of the liquid cis-isomer. The trans-crystals thus formed showed second harmonic generation (SHG) whose intensity is switched on/off in response to the photo-induced phase transition.

In silico analysis of inner ear development using public whole embryonic body single-cell RNA-sequencing data.

The inner ear comprises four epithelial domains: the cochlea, vestibule, semicircular canals, and endolymphatic duct/sac. These structures are segregated at embryonic day 13.5 (E13.5). However, these four anatomical structures remain undefined at E10.5. Here, we aimed to identify lineage-specific genes in the early developing inner ear using published data obtained from single-cell RNA-sequencing (scRNA-seq) of embryonic mice. We downloaded 5000 single-cell transcriptome data, named 'auditory epithelial trajectory', from the Mouse Organogenesis Cell Atlas. The dataset was supposed to include otic epithelial cells at E9.5-13.5. We projected the 5000 â€‹cells onto a two-dimensional space encoding the transcriptional state and visualised the pattern of otic epithelial cell differentiation. We identified 15 clusters, which were annotated as one of the four components of the inner ear epithelium using known genes that characterise the four different tissues. Additionally, we classified 15 clusters into sub-regions of the four inner ear components. By comparing transcriptomes between these 15 clusters, we identified several candidates of lineage-specific genes. Characterising these new candidate genes will help future studies about inner ear development.

Initiation of Supporting Cell Activation for Hair Cell Regeneration in the Avian Auditory Epithelium: An Explant Culture Model.

Sensorineural hearing loss is a common disability often caused by the loss of sensory hair cells in the cochlea. Hair cell (HCs) regeneration has long been the main target for the development of novel therapeutics for sensorineural hearing loss. In the mammalian cochlea, hair cell regeneration is limited, but the auditory epithelia of non-mammalian organisms retain the capacity for hair cell regeneration. In the avian basilar papilla (BP), supporting cells (SCs), which give rise to regenerated hair cells, are usually quiescent. Hair cell loss induces both direct transdifferentiation and mitotic division of supporting cells. Here, we established an explant culture model for hair cell regeneration in chick basilar papillae and validated it for investigating the initial phase of hair cell regeneration. The histological assessment demonstrated hair cell regeneration via direct transdifferentiation of supporting cells. Labeling with 5-ethynyl-2'-deoxyuridine (EdU) revealed the occurrence of mitotic division in the supporting cells at specific locations in the basilar papillae, while no EdU labeling was observed in newly generated hair cells. RNA sequencing indicated alterations in known signaling pathways associated with hair cell regeneration, consistent with previous findings. Also, unbiased analyses of RNA sequencing data revealed novel genes and signaling pathways that may be related to the induction of supporting cell activation in the chick basilar papillae. These results indicate the advantages of our explant culture model of the chick basilar papillae for exploring the molecular mechanisms of hair cell regeneration.

Mutations in PIH proteins MOT48, TWI1 and PF13 define common and unique steps for preassembly of each, different ciliary dynein.

Ciliary dyneins are preassembled in the cytoplasm before being transported into cilia, and a family of proteins containing the PIH1 domain, PIH proteins, are involved in the assembly process. However, the functional differences and relationships between members of this family of proteins remain largely unknown. Using Chlamydomonas reinhardtii as a model, we isolated and characterized two novel Chlamydomonas PIH preassembly mutants, mot48-2 and twi1-1. A new allele of mot48 (ida10), mot48-2, shows large defects in ciliary dynein assembly in the axoneme and altered motility. A second mutant, twi1-1, shows comparatively smaller defects in motility and dynein assembly. A double mutant mot48-2; twi1-1 displays greater reduction in motility and in dynein assembly compared to each single mutant. Similarly, a double mutant twi1-1; pf13 also shows a significantly greater defect in motility and dynein assembly than either parent mutant. Thus, MOT48 (IDA10), TWI1 and PF13 may define different steps, and have partially overlapping functions, in a pathway required for ciliary dynein preassembly. Together, our data suggest the three PIH proteins function in preassembly steps that are both common and unique for different ciliary dyneins.

Deep learning prediction of falls among nursing home residents with Alzheimer's disease.

AIM: This study aimed to use a convolutional neural network (CNN) to investigate the associations between the time of falling and multiple complicating factors, including age, dementia severity, lower extremity strength and physical function, among nursing home residents with Alzheimer's disease. METHODS: A total of 42 people with Alzheimer's disease were enrolled. We evaluated falling events from nursing home admission (baseline) to 300 days later. We assessed the knee extension strength and Functional Independence Measure locomotion item and carried out the Mini-Mental State Examination at baseline. To predict falling, participants were categorized into three classes: those who fell within the first 150 (or 300) days from baseline or those who did not experience a fall within the study period. For each class, 1000 bootstrap datasets were generated using 42 actual sample datasets, and were used to propose a CNN algorithm and cross-validate the algorithm. RESULTS: Eight (19.0%), 11 (26.2%) and 31 participants (73.8%) fell within 150 or 300 days after the baseline assessment or did not fall until 300 days or later, respectively. The highest accuracy rate of the CNN classification was 0.647 in the factor combination extracted from the Mini-Mental State Examination score, knee extension strength and Functional Independence Measure locomotion item score. CONCLUSIONS: A CNN based on multiple complicating factors could predict the time of falling in nursing home residents with Alzheimer's disease. Geriatr Gerontol Int 2020; ••: ••-••.

Enhanced Electric Polarization and Polar Switching of Dipolar Aromatic Liquids Confined in Supramolecular Gel Networks.

Dipolar aromatic liquids confined in the interstitial domains of chiral organogels show significantly enhanced electric polarization, as compared with those of pure liquids alone or organogels formed with nonpolar liquids. Intriguingly, nitrobenzene gels showed a supramolecular polar switching phenomenon; i.e., hysteresis in the polarization (P)-electric field (E) curves was observed for the gel above the melting point of the solvent. This indicates that the nitrobenzene molecules confined in the chiral nanofibrous gel networks exert macroscopic polarization whose direction is inversed depending on the direction of the external electric field. The anomalously enhanced electric polarization and polar switching phenomenon of supramolecular gels in varied solvents are scrutinized by the positive-up-negative-down (PUND) measurements, and the interactions between the gel nanofibers and the polar solvent molecules play crucial roles for the emergence of the polar switching phenomenon. This work presents for the first time that dipolar liquid molecules filling the interstitial space of supramolecular gels exhibit a significant confinement effect. It provides a new perspective to design electric-field-responsive soft materials based on the functional liquid domains confined in their porous networks.

Assessing a polluted river environment by oxidative stress biomarker responses in caddisfly larvae.

The effluents of wastewater treatment plants (WWTPs) contain various anthropogenic pollutants that produce negative effects in river ecosystems. Although the oxidative stress responses in aquatic organisms are useful tools for assessing such effects, the responses of aquatic insects to WWTP effluents are poorly understood. This work investigated the responses of antioxidants (superoxide dismutase, catalase, and oxy-radical absorbance capacity), oxidative damage (lipid peroxidation), and energy reserves in caddisfly (Stenopsyche marmorata) larvae caused by the WWTP effluent in two parts of the Chikumagawa River during different seasons. It was found that effluent strongly influenced the antioxidants and oxidative damage and depleted the energy reserves. Hence, both the oxidative stress biomarkers and energy reserves in aquatic insects can be used for assessing the impacts of wastewater effluents. Lipid peroxidation proceeded more intensely at some reference sites than at the effluent-impacted sites, indicating that the use of a single biomarker could lead to a misunderstanding of the effect of pollutant mixtures in field studies. To mitigate this issue, a new reference-impacted approach based on the integrated biomarker response (IBRRI) method has been developed to assess anthropogenic impacts while considering spatiotemporal fluctuations due to the natural variations in a river system. This approach produced larger IBRRI values at higher concentrations of anthropogenic pollutants, which correlated with higher ammonium and nitrate concentrations. Therefore, IBRRI is a potentially useful tool for assessing the impact of WWTP effluents under variable spatiotemporal conditions.

Bulk ferromagnetic tips for spin-polarized scanning tunneling microscopy.

We characterized the performance of electrochemically etched bulk Fe and Ni tips as a probe of spin-polarized scanning tunneling microscopy (SP-STM). Through the observation of the striped contrast on the conical spin-spiral structure formed in Mn double layers on a W(110) substrate, the capability of both the tips to detect the magnetic signal was clarified. We also confirmed that the magnetized direction of the Fe and Ni tips can be flipped between the two out-of-plane directions by external magnetic fields. Our results demonstrate that the ex-situ prepared tips are reliable in SP-STM for the samples that are not susceptible to a stray magnetic field.