Interaction between the TBC1D24 TLDc domain and the KIBRA C2 domain is disrupted by two epilepsy-associated TBC1D24 missense variants.

Mutations of human TBC1D24 are associated with either deafness, epilepsy or DOORS syndrome (deafness, onychodystrophy, osteodystrophy, cognitive disability, seizures). The causal relationships between TBC1D24 variants and the different clinical phenotypes are not understood. Our hypothesis is that phenotypic heterogeneity of missense mutations of TBC1D24 results, in part, from perturbed binding of different protein partners. To discover novel protein partners of TBC1D24, we conducted a yeast two-hybrid (Y2H) screen using mouse full-length (FL) TBC1D24 as bait. KIBRA, a scaffold protein encoded by Wwc1, was identified as a partner of TBC1D24. KIBRA functions in the Hippo signaling pathway and is important for human cognition and memory. The TBC1D24 TLDc domain binds to KIBRA FL and to its C2 domain, confirmed by Y2H assays. No interaction was detected with Y2H assays between the KIBRA C2 domain and TLDc domains of NCOA7, MEAK7 and OXR1. Moreover, the C2 domains of other WWC family proteins do not interact with the TLDc domain of TBC1D24, demonstrating specificity. The mRNAs encoding TBC1D24 and KIBRA proteins in mouse are coexpressed at least in a subset of hippocampal cells indicating availability to interact in vivo. As two epilepsy-associated recessive variants (Gly511Arg and Ala515Val) in the TLDc domain of human TBC1D24 disrupt the interaction with human KIBRA C2 domain, this study reveals a pathogenic mechanism of TBC1D24-associated epilepsy, linking the TBC1D24 and KIBRA pathways. The interaction of TBC1D24-KIBRA is physiologically meaningful and necessary to reduce the risk of epilepsy.

Temporal Bone Histopathology of Atypical Cogan Syndrome.

Cogan's syndrome is a rare disorder first clinically defined in 1945, characterized by nonsyphilitic interstitial keratitis and progressive audiovestibular symptoms. Later, patients with audiovestibular dysfunction and various types of inflammatory eye disease were classified as having atypical Cogan's syndrome. The etiology and pathogenesis of Cogan's syndrome remain largely unknown. Here, we report a case of atypical Cogan's syndrome with a histological assessment of the temporal bone during the acute disease period. Temporal bone histology was compared to age- and gender-matched normal control, and our findings revealed endolymphatic hydrops and degenerative changes in various parts of the inner ear. Laryngoscope, 2024.

Live-cell single-molecule fluorescence microscopy for protruding organelles reveals regulatory mechanisms of MYO7A-driven cargo transport in stereocilia of inner ear hair cells.

Stereocilia are unidirectional F-actin-based cylindrical protrusions on the apical surface of inner ear hair cells and function as biological mechanosensors of sound and acceleration. Development of functional stereocilia requires motor activities of unconventional myosins to transport proteins necessary for elongating the F-actin cores and to assemble the mechanoelectrical transduction (MET) channel complex. However, how each myosin localizes in stereocilia using the energy from ATP hydrolysis is only partially understood. In this study, we develop a methodology for live-cell single-molecule fluorescence microscopy of organelles protruding from the apical surface using a dual-view light-sheet microscope, diSPIM. We demonstrate that MYO7A, a component of the MET machinery, traffics as a dimer in stereocilia. Movements of MYO7A are restricted when scaffolded by the plasma membrane and F-actin as mediated by MYO7A's interacting partners. Here, we discuss the technical details of our methodology and its future applications including analyses of cargo transportation in various organelles.

Live-cell single-molecule fluorescence microscopy for protruding organelles reveals regulatory mechanisms of MYO7A-driven cargo transport in stereocilia of inner ear hair cells.

Stereocilia are unidirectional F-actin-based cylindrical protrusions on the apical surface of inner ear hair cells and function as biological mechanosensors of sound and acceleration. Development of functional stereocilia requires motor activities of unconventional myosins to transport proteins necessary for elongating the F-actin cores and to assemble the mechanoelectrical transduction (MET) channel complex. However, how each myosin localizes in stereocilia using the energy from ATP hydrolysis is only partially understood. In this study, we develop a methodology for live-cell single-molecule fluorescence microscopy of organelles protruding from the apical surface using a dual-view light-sheet microscope, diSPIM. We demonstrate that MYO7A, a component of the MET machinery, traffics as a dimer in stereocilia. Movements of MYO7A are restricted when scaffolded by the plasma membrane and F-actin as mediated by MYO7A's interacting partners. Here, we discuss the technical details of our methodology and its future applications including analyses of cargo transportation in various organelles.

Immune responses in the mammalian inner ear and their implications for AAV-mediated inner ear gene therapy.

Adeno-associated virus (AAV)-mediated inner ear gene therapy is a promising treatment option for hearing loss and dizziness. Several studies have shown that AAV-mediated inner ear gene therapy can be applied to various mouse models of hereditary hearing loss to improve their auditory function. Despite the increase in AAV-based animal and clinical studies aiming to rescue auditory and vestibular functions, little is currently known about the host immune responses to AAV in the mammalian inner ear. It has been reported that the host immune response plays an important role in the safety and efficacy of viral-mediated gene therapy. Therefore, in order for AAV-mediated gene therapy to be successfully and safely translated into patients with hearing loss and dizziness, a better understanding of the host immune responses to AAV in the inner ear is critical. In this review, we summarize the current knowledge on host immune responses to AAV-mediated gene therapy in the mammalian inner ear and other organ systems. We also outline the areas of research that are critical for ensuring the safety and efficacy of AAV-mediated inner ear gene therapy in future clinical and translational studies.

Dual-AAV vector-mediated expression of MYO7A improves vestibular function in a mouse model of Usher syndrome 1B.

Usher syndrome is the most common cause of deafness-blindness in the world. Usher syndrome type 1B (USH1B) is associated with mutations in MYO7A. Patients with USH1B experience deafness, blindness, and vestibular dysfunction. In this study, we applied adeno-associated virus (AAV)-mediated gene therapy to the shaker-1 (Myo7a4626SB/4626SB) mouse, a model of USH1B. The shaker-1 mouse has a nonsense mutation in Myo7a, is profoundly deaf throughout life, and has significant vestibular dysfunction. Because of the ∼6.7-kb size of the MYO7A cDNA, a dual-AAV approach was used for gene delivery, which involves splitting human MYO7A cDNA into 5' and 3' halves and cloning them into two separate AAV8(Y733F) vectors. When MYO7A cDNA was delivered to shaker-1 inner ears using the dual-AAV approach, cochlear hair cell survival was improved. However, stereocilium organization and auditory function were not improved. In contrast, in the vestibular system, dual-AAV-mediated MYO7A delivery significantly rescued hair cell stereocilium morphology and improved vestibular function, as reflected in a reduction of circling behavior and improved vestibular sensory-evoked potential (VsEP) thresholds. Our data indicate that dual-AAV-mediated MYO7A expression improves vestibular function in shaker-1 mice and supports further development of this approach for the treatment of disabling dizziness from vestibular dysfunction in USH1B patients.

AAV8BP2 and AAV8 transduce the mammalian cochlear lateral wall and endolymphatic sac with high efficiency.

Inner ear gene therapy using adeno-associated viruses (AAVs) has been successfully applied to several mouse models of hereditary hearing loss to improve their auditory function. While most inner ear gene therapy studies have focused on the mechanosensory hair cells and supporting cells in the organ of Corti, the cochlear lateral wall and the endolymphatic sac have not garnered much attention. The cochlear lateral wall and the endolymphatic sac play critical roles in inner ear ionic and fluid homeostasis. Mutations in genes expressed in the cochlear lateral wall and the endolymphatic sac are present in a large percentage of patients with hereditary hearing loss. In this study, we examine the transduction patterns and efficiencies of conventional (AAV2 and AAV8) and synthetic (AAV2.7m8, AAV8BP2, and Anc80L65) AAVs in the mouse inner ear. We found that AAV8BP2 and AAV8 are capable of transducing the marginal cells and intermediate cells in the stria vascularis. These two AAVs can also transduce the epithelial cells of the endolymphatic sac. Our data suggest that AAV8BP2 and AAV8 are highly useful viral vectors for gene therapy studies targeting the cochlear lateral wall and the endolymphatic sac.

Refining surgical techniques for efficient posterior semicircular canal gene delivery in the adult mammalian inner ear with minimal hearing loss.

Hearing loss is a common disability affecting the world's population today. While several studies have shown that inner ear gene therapy can be successfully applied to mouse models of hereditary hearing loss to improve hearing, most of these studies rely on inner ear gene delivery in the neonatal age, when mouse inner ear has not fully developed. However, the human inner ear is fully developed at birth. Therefore, in order for inner ear gene therapy to be successfully applied in patients with hearing loss, one must demonstrate that gene delivery can be safely and reliably performed in the mature mammalian inner ear. In this study, we examine the steps involved in posterior semicircular canal gene delivery in the adult mouse inner ear. We find that the duration of perilymphatic leakage and injection rate have a significant effect on the post-surgical hearing outcome. Our results show that although AAV2.7m8 has a lower hair cell transduction rate in adult mice compared to neonatal mice at equivalent viral load, AAV2.7m8 is capable of transducing the adult mouse inner and outer hair cells with high efficiency in a dose-dependent manner.

Analyses of cytochrome b mutations in Plasmodium falciparum isolates in Thai-Myanmar border.

BACKGROUND: The combination of atovaquone and proguanil (Malarone) has been established as a drug of choice to prevent and treat multi-drug resistant Plasmodium (P.) falciparum malaria in travelers. However, several cases of resistance against Malarone have been reported in some parts of Africa, and many of the cases are believed to be associated with mutations at the codon 268 of cytochrome b gene in mitochondria of P. falciparum. The aim of the study was to estimate the effectiveness of Malarone in treatment and prophylaxis for the travelers to Thai-Myanmar border where multi-drug resistant malaria is highly endemic. METHODS: Seventy P. falciparum samples obtained from patients from Thai-Myanmar border were sequenced to detect mutations around the codon 268. The same samples were also sequenced to detect P. falciparum chloroquine resistance transporter mutation (PfCRT K76T). RESULTS: All the 70 samples showed no mutations at the codon 268 of cytochrome b gene. Whereas, 50 samples, whose pfcrt genes were sequenced successfully, had an identical genotype for K76T mutation. CONCLUSION: In Asian countries, even in the multi-drug resistant areas in the great Mekong region, no case of Malarone resistance has been reported clinically or genetically thus far. In this study, all the P. falciparum parasites tested successfully were shown to be chloroquine resistant but atovaquone susceptible genetically. The more the usefulness of Malarone increases for both treatment and prophylaxis, the wider the drug-resistance against Malarone may spread in the region. Although the total number of samples examined is not large, it is concluded from these findings that Malarone should be recommended for prophylaxis of malaria for travelers to the Mekong region.

Nystagmus-sensation dissociation in spinocerebellar degeneration.

OBJECTIVE: To study nystagmus-sensation dissociation (NSD), i.e. caloric nystagmus without the sensation of vertigo, in patients with spinocerebellar degeneration (SCD). MATERIAL AND METHODS: The neuro-otological and neuro-radiological records of 179 patients clinically diagnosed as having SCD (91 males, 88 females; age range 20-89 years) and 48 patients diagnosed as having peripheral vestibular disorders (15 males, 33 females; age range 21-80 years) were reviewed. Subjects underwent caloric tests using cold water (20 degrees C) in a totally dark room. Immediately after each test session, subjects were asked if they had experienced spinning or moving sensations during the test. Maximum slow phase eye velocities (SPEVs) were measured using electronystagmography. RESULTS: Among patients with peripheral vestibular disorders, all patients with SPEVs > 15 degrees/s experienced spinning or moving sensations during testing. Among the 179 patients with SCD, 21 (39 sides) showed maximum SPEVs > 15 degrees/s without spinning or moving sensations. In particular, 8 patients (10 sides) had maximum SPEVs > 25 degrees/s without spinning or moving sensations. Common lesions in the cerebral cortex could not be detected using either MRI or single-photon emission CT. CONCLUSION: Patients with SCD may also have NSD, which may be attributable to subcortical lesions in the ascending pathways to the vestibular cortex.

Antiplasmodial triterpenoids from Ekebergia capensis.

From the stem bark of Ekebergia capensis, 10 new triterpenoid compounds, ekeberins A (1), B (2), C1 (3), C2 (4), C3 (5), D1 (6), D2 (7), D3 (8), D4 (9), and D5 (10), were isolated together with 17 known compounds. The structures of these new compounds were elucidated on the basis of the results of spectroscopic analysis, and the absolute configuration of compounds 6-10 were determined by partial synthesis from known compounds and using the Mosher ester method. Several of these compounds were screened in vitro against both chloroquine (CQ)-sensitive and -resistant Plasmodium falciparum isolates and were found to exhibit moderate antiplasmodial activity, with compounds 20 (7-deacetoxy-7-oxogedunin) and 27 (2-hydroxymethyl-2,3,22,23-tetrahydroxy-2,6,10,15,19,23-hexamethyl-6,10,14,18-tetracosatetraene) showing IC50 values of 6 and 7 microM, respectively. Compound 27 at a dose of 500 mg/kg showed moderate parasitemia suppression of 52.9% against P. berghei NK 65 in a mouse model.

A case of Cronkhite-Canada syndrome with vestibular disturbances.

A 66-year-old Japanese man with Cronkhite-Canada syndrome (CCS) presented with complaints of long-lasting dysequilibrium. On neuro-otological examination, he showed gaze-evoked nystagmus at the rightward and leftward gaze, and saccadic pursuit. On the caloric test, he showed no response in either side, and on the head-impulse test he showed bilateral loss of vestibule-ocular reflexes around the yaw axis, while he had bilateral normal responses on the vestibular-evoked myogenic potential testing. Neuro-otological findings suggested that he had lesions in the peripheral vestibular system as well as the central nervous system. Neurological disorders such as sensory neuropathy have been reported in patients with CCS. This patient's balance problems could be due to CCS itself.