A critical evaluation of "leakage" at the cochlear blood-stria-barrier and its functional significance.

The blood-labyrinth-barrier (BLB) is a semipermeable boundary between the vasculature and three separate fluid spaces of the inner ear, the perilymph, the endolymph and the intrastrial space. An important component of the BLB is the blood-stria-barrier, which shepherds the passage of ions and metabolites from strial capillaries into the intrastrial space. Some investigators have reported increased "leakage" from these capillaries following certain experimental interventions, or in the presence of inflammation or genetic variants. This leakage is generally thought to be harmful to cochlear function, principally by lowering the endocochlear potential (EP). Here, we examine evidence for this dogma. We find that strial capillaries are not exclusive, and that the asserted detrimental influence of strial capillary leakage is often confounded by hair cell damage or intrinsic dysfunction of the stria. The vast majority of previous reports speculate about the influence of strial vascular barrier function on the EP without directly measuring the EP. We argue that strial capillary leakage is common across conditions and species, and does not significantly impact the EP or hearing thresholds, either on evidentiary or theoretical grounds. Instead, strial capillary endothelial cells and pericytes are dynamic and allow permeability of varying degrees in response to specific conditions. We present observations from mice and demonstrate that the mechanisms of strial capillary transport are heterogeneous and inconsistent among inbred strains.

Pneumococcal Meningitis Induces Hearing Loss and Cochlear Ossification Modulated by Chemokine Receptors CX3CR1 and CCR2.

PURPOSE: Pneumococcal meningitis is a major cause of hearing loss and permanent neurological impairment despite widely available antimicrobial therapies to control infection. Methods to improve hearing outcomes for those who survive bacterial meningitis remains elusive. We used a mouse model of pneumococcal meningitis to evaluate the impact of mononuclear phagocytes on hearing outcomes and cochlear ossification by altering the expression of CX3CR1 and CCR2 in these infected mice. METHODS: We induced pneumococcal meningitis in approximately 500 C57Bl6 adult mice using live Streptococcus pneumoniae (serotype 3, 1 × 105 colony forming units (cfu) in 10 µl) injected directly into the cisterna magna of anesthetized mice and treated these mice with ceftriaxone daily until recovered. We evaluated hearing thresholds over time, characterized the cochlear inflammatory response, and quantified the amount of new bone formation during meningitis recovery. We used microcomputed tomography (microCT) scans to quantify cochlear volume loss caused by neo-ossification. We also performed perilymph sampling in live mice to assess the integrity of the blood-perilymph barrier during various time intervals after meningitis. We then evaluated the effect of CX3CR1 or CCR2 deletion in meningitis symptoms, hearing loss, macrophage/monocyte recruitment, neo-ossification, and blood labyrinth barrier function. RESULTS: Sixty percent of mice with pneumococcal meningitis developed hearing loss. Cochlear fibrosis could be detected within 4 days of infection, and neo-ossification by 14 days. Loss of spiral ganglion neurons was common, and inner ear anatomy was distorted by scarring caused by new soft tissue and bone deposited within the scalae. The blood-perilymph barrier was disrupted at 3 days post infection (DPI) and was restored by seven DPI. Both CCR2 and CX3CR1 monocytes and macrophages were present in the cochlea in large numbers after infection. Neither chemokine receptor was necessary for the induction of hearing loss, cochlear fibrosis, ossification, or disruption of the blood-perilymph barrier. CCR2 knockout (KO) mice suffered the most severe hearing loss. CX3CR1 KO mice demonstrated an intermediate phenotype with greater susceptibility to hearing loss compared to control mice. Elimination of CX3CR1 mononuclear phagocytes during the first 2 weeks after meningitis in CX3CR1-DTR transgenic mice did not protect mice from any of the systemic or hearing sequelae of pneumococcal meningitis. CONCLUSIONS: Pneumococcal meningitis can have devastating effects on cochlear structure and function, although not all mice experienced hearing loss or cochlear damage. Meningitis can result in rapid progression of hearing loss with fibrosis starting at four DPI and ossification within 2 weeks of infection detectable by light microscopy. The inflammatory response to bacterial meningitis is robust and can affect all three scalae. Our results suggest that CCR2 may assist in controlling infection and maintaining cochlear patency, as CCR2 knockout mice experienced more severe disease, more rapid hearing loss, and more advanced cochlear ossification after pneumococcal meningitis. CX3CR1 also may play an important role in the maintenance of cochlear patency.

CHLOROPLAST UNUSUAL POSITIONING 1 is a plant-specific actin polymerization factor regulating chloroplast movement.

Plants have unique responses to fluctuating light conditions. One such response involves chloroplast photorelocation movement, which optimizes photosynthesis under weak light by the accumulation of chloroplasts along the periclinal side of the cell, which prevents photodamage under strong light by avoiding chloroplast positioning toward the anticlinal side of the cell. This light-responsive chloroplast movement relies on the reorganization of chloroplast actin (cp-actin) filaments. Previous studies have suggested that CHLOROPLAST UNUSUAL POSITIONING 1 (CHUP1) is essential for chloroplast photorelocation movement as a regulator of cp-actin filaments. In this study, we conducted comprehensive analyses to understand CHUP1 function. Functional, fluorescently tagged CHUP1 colocalized with and was coordinately reorganized with cp-actin filaments on the chloroplast outer envelope during chloroplast movement in Arabidopsis thaliana. CHUP1 distribution was reversibly regulated in a blue light- and phototropin-dependent manner. X-ray crystallography revealed that the CHUP1-C-terminal domain shares structural homology with the formin homology 2 (FH2) domain, despite lacking sequence similarity. Furthermore, the CHUP1-C-terminal domain promoted actin polymerization in the presence of profilin in vitro. Taken together, our findings indicate that CHUP1 is a plant-specific actin polymerization factor that has convergently evolved to assemble cp-actin filaments and enables chloroplast photorelocation movement.

Contribution of macrophages to neural survival and intracochlear tissue remodeling responses following cochlear implantation.

BACKGROUND: Cochlear implants (CIs) restore hearing to deafened patients. The foreign body response (FBR) following cochlear implantation (post-CI) comprises an infiltration of macrophages, other immune and non-immune cells, and fibrosis into the scala tympani, a space that is normally devoid of cells. This FBR is associated with negative effects on CI outcomes including increased electrode impedances and loss of residual acoustic hearing. This study investigates the extent to which macrophage depletion by an orally administered CSF-1R specific kinase (c-FMS) inhibitor, PLX-5622, modulates the tissue response to CI and neural health. MAIN TEXT: 10- to 12-week-old CX3CR1 + /GFP Thy1 + /YFP mice on C57BL/6J/B6 background was fed chow containing 1200 mg/kg PLX5622 or control chow for the duration of the study. 7 days after starting the diet, 3-channel cochlear implants were implanted in the ear via the round window. Serial impedance and neural response telemetry (NRT) measurements were acquired throughout the study. Electric stimulation began 7 days post-CI until 28 days post-CI for 5 h/day, 5 days/week, with programming guided by NRT and behavioral responses. Cochleae harvested at 10, 28 or 56 days post-CI were cryosectioned and labeled with an antibody against α-smooth muscle actin (α-SMA) to identify myofibroblasts and quantify the fibrotic response. Using IMARIS image analysis software, the outlines of scala tympani, Rosenthal canal, modiolus, and lateral wall for each turn were traced manually to measure region volume. The density of nuclei, CX3CR1 + macrophages, Thy1 + spiral ganglion neuron (SGN) numbers, and the ratio of the α-SMA + volume/scala tympani volume were calculated. Cochlear implantation in control diet subjects caused infiltration of cells, including macrophages, into the cochlea. Fibrosis was evident in the scala tympani adjacent to the electrode array. Mice fed PLX5622 chow showed reduced macrophage infiltration throughout the implanted cochleae across all time points. However, scala tympani fibrosis was not reduced relative to control diet subjects. Further, mice treated with PLX5622 showed increased electrode impedances compared to controls. Finally, treatment with PLX5622 decreased SGN survival in implanted and contralateral cochleae. CONCLUSION: The data suggest that macrophages play an important role in modulating the intracochlear tissue response following CI and neural survival.

Actin-binding domain of Rng2 sparsely bound on F-actin strongly inhibits actin movement on myosin II.

We report a case in which sub-stoichiometric binding of an actin-binding protein has profound structural and functional consequences, providing an insight into the fundamental properties of actin regulation. Rng2 is an IQGAP contained in contractile rings in the fission yeast Schizosaccharomyces pombe Here, we used high-speed atomic force microscopy and electron microscopy and found that sub-stoichiometric binding of the calponin-homology actin-binding domain of Rng2 (Rng2CHD) induces global structural changes in skeletal muscle actin filaments, including shortening of the filament helical pitch. Sub-stoichiometric binding of Rng2CHD also reduced the affinity between actin filaments and muscle myosin II carrying ADP and strongly inhibited the motility of actin filaments on myosin II in vitro. On skeletal muscle myosin II-coated surfaces, Rng2CHD stopped the actin movements at a binding ratio of 11%. Rng2CHD also inhibited actin movements on myosin II of the amoeba Dictyostelium, but in this case, by detaching actin filaments from myosin II-coated surfaces. Thus, sparsely bound Rng2CHD induces apparently cooperative structural changes in actin filaments and inhibits force generation by actomyosin II.

Texture-Modified Diets Need to be Improved in Vietnamese Hospitals.

Purpose : The aim of this study is to compare the nutritional status of older adult inpatients consuming Texture-Modified Diets (TMD) to older adult inpatients consuming a regular diet. Methods : The study was designed as cross-sectional and was conducted in three large hospitals in northern Vietnam. The data for 344 older adult inpatients were collected in 2021 by dietitians. Results : The result showed 104 subjects were prescribed TMD. The textures of hospital diets have still not been adequately developed. In particular, the hospitals had not yet developed pureed meals. All older adult inpatients (74.7 ±â€…6.8 years old, 52.9% female) had 28.8% malnutrition by the GLIM criteria. The TMD group had a malnutrition prevalence two times higher than the regular diet group. Total energy intake from hospital meal and outside snacks in the regular diet group was higher than in the TMD group, about 150 kcal. Although energy provided from the hospital diet in both groups was similar, hospital meal wastage in the TMD group was higher than in the regular diet group. Conclusion : The older adult inpatients on TMD may have a nutritional status worse than those consuming a regular diet. Hospital diets need to be improved in texture and quality. J. Med. Invest. 69 : 230-236, August, 2022.

Oscillatory movement of a dynein-microtubule complex crosslinked with DNA origami.

Bending of cilia and flagella occurs when axonemal dynein molecules on one side of the axoneme produce force and move toward the microtubule (MT) minus end. These dyneins are then pulled back when the axoneme bends in the other direction, meaning oscillatory back and forth movement of dynein during repetitive bending of cilia/flagella. There are various factors that may regulate the dynein activity, e.g. the nexin-dynein regulatory complex, radial spokes, and central apparatus. In order to understand the basic mechanism of dynein's oscillatory movement, we constructed a simple model system composed of MTs, outer-arm dyneins, and crosslinks between the MTs made of DNA origami. Electron microscopy (EM) showed pairs of parallel MTs crossbridged by patches of regularly arranged dynein molecules bound in two different orientations, depending on which of the MTs their tails bind to. The oppositely oriented dyneins are expected to produce opposing forces when the pair of MTs have the same polarity. Optical trapping experiments showed that the dynein-MT-DNA-origami complex actually oscillates back and forth after photolysis of caged ATP. Intriguingly, the complex, when held at one end, showed repetitive bending motions. The results show that a simple system composed of ensembles of oppositely oriented dyneins, MTs, and inter-MT crosslinkers, without any additional regulatory structures, has an intrinsic ability to cause oscillation and repetitive bending motions.

Chronic cochlear implantation with and without electric stimulation in a mouse model induces robust cochlear influx of CX3CR1+/GFP macrophages.

BACKGROUND: Cochlear implantation is an effective auditory rehabilitation strategy for those with profound hearing loss, including those with residual low frequency hearing through use of hybrid cochlear implantation techniques. Post-mortem studies demonstrate the nearly ubiquitous presence of intracochlear fibrosis and neo-ossification following cochlear implantation. Current evidence suggests post-implantation intracochlear fibrosis is associated with delayed loss of residual acoustic hearing in hybrid cochlear implant (CI) recipients and may also negatively influence outcomes in traditional CI recipients. This study examined the contributions of surgical trauma, foreign body response and electric stimulation to intracochlear fibrosis and the innate immune response to cochlear implantation and the hierarchy of these contributions. METHODS: Normal hearing CX3CR1+/GFP mice underwent either round window opening (sham), acute CI insertion or chronic CI insertion with no, low- or high-level electric stimulation. Electric stimulation levels were based on neural response telemetry (NRT), beginning post-operative day 7 for 5 h per day. Subjects (n=3 per timepoint) were sacrificed at 4 h, 1,4,7,8,11,14 and 21 days. An unoperated group (n=3) served as controls. Cochleae were harvested at each time-point and prepared for immunohistochemistry with confocal imaging. The images were analyzed to obtain CX3CR1+ macrophage cell number and density in the lateral wall (LW), scala tympani (ST) and Rosenthal's canal (RC). RESULTS: A ST peri-implant cellular infiltrate and fibrosis occurred exclusively in the chronically implanted groups starting on day 7 with a concurrent infiltration of CX3CR1+ macrophages not seen in the other groups. CX3CR1+ macrophage infiltration was seen in the LW and RC in all experimental groups within the first week, being most prominent in the 3 chronically implanted groups during the second and third week. CONCLUSIONS: The cochlear immune response was most prominent in the presence of chronic cochlear implantation, regardless of electric stimulation level. Further, the development of intracochlear ST fibrosis was dependent on the presence of the indwelling CI foreign body. An innate immune response was evoked by surgical trauma alone (sham and acute CI groups) to a lesser degree. These data suggest that cochlear inflammation and intrascalar fibrosis after cochlear implantation are largely dependent on the presence of a chronic indwelling foreign body and are not critically dependent on electrical stimulation. Also, these data support a role for surgical trauma in inciting the initial innate immune response.

Malnutrition is associated with dysphagia in Vietnamese older adult inpatients.

BACKGROUND AND OBJECTIVES: The number of older adults is increasing rapidly in Vietnam. They suffer from various health problems, including malnutrition and dysphagia. By using a simple screening questionnaire such as the Mini Nutritional Assessment - Short Form (MNA-SF) and 10-item Eating Assessment Tool (EAT-10), nutritional and dysphagic status were screened in this study. The study aimed to determine the prevalence of and relationship between malnutrition and dysphagia in Vietnamese older adult inpatients. METHODS AND STUDY DESIGN: The study was designed as a cross-sectional study and conducted in three large hospitals in northern Vietnam. The data about nutritional status and dysphagia status of 1007 older inpatients were collected by dietitians. RESULTS: There were 71.6% of subjects at malnourished (MNA-SF score <8) and risk of malnutrition (MNA-SF score: 8-11). The prevalence of dysphagia by EAT-10 was 24.6%. The risk of dysphagia was independently associated with higher risk of malnutrition, with an odds ratio of 3.21 (95% CI: 1.93-5.31, p<0.001). In addition, malnutrition was also an independent predictor for risk of dysphagia, with an odds ratio of 3.09 (95% CI: 1.84- 5.17, p<0.001). CONCLUSIONS: Malnutrition and dysphagia prevalence were high; and malnutrition and dysphagia have a strong relationship among older adult inpatients in Vietnam. Therefore, nutritional and dysphagia screening at hospital admission are very important and recommended.

Decreasing Salt in Hospital Meals Reduced Energy Intake in Elderly Japanese Inpatients.

Japanese salt consumption is high, about 10 g salt/d. Low salt intake reduces the risk of hypertension and cardiovascular disease. However, saltiness is an important taste in daily meals, greatly influencing eating habits. When hospital admission is short-term, reducing salt supply may have an adverse effect on food intake. The aim of this study is to find the effect of sudden change in dietary salt content on energy intake in elderly Japanese inpatients. The study is an observational study of 83 patients and was conducted in a hospital in Tokyo, Japan. The research period was three weeks, and included 8 g salt/d meals for the 1st week, 7 g salt/d meals for the 2nd week, and 7 g salt/d meals with total 1 g/d salt packs that patients were allowed to use freely for the 3rd week. The energy supply satisfied the individuals' energy requirements and was the same throughout the three weeks. Nutrition surveys and blood pressure measurements during the three weeks were conducted by dietitians and nurses, respectively. The results showed that energy intake of patients was reduced by about 90 kcal/d in the 2nd week compared with the 1st week and increased about 130 kcal/d in the 3rd week compared with the 2nd week. Blood pressure did not change during the research period. When high salt intake has become a habit, reducing salt supply suddenly in a short time period may lead to decreased energy intake in elderly inpatients but does not affect blood pressure.

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.

Implementation of a screening tool for primary ciliary dyskinesia (PCD) in a pediatric otolaryngology clinic.

BACKGROUND: Primary ciliary dyskinesia (PCD) is a rare genetic disease arising from motile ciliary dysfunction and associated with recurrent and chronic upper and lower respiratory tract infections. Pediatric otolaryngologists may see these patients prior to the development of lung disease. Features of PCD may overlap with other suppurative respiratory diseases, creating diagnostic challenges. A simple screening tool would be beneficial to identify potential patients who have chronic upper respiratory tract disease requiring further specialist evaluation. OBJECTIVE: To test a simple screening tool consisting of four questions to detect PCD in children with chronic otitis media and chronic rhinosinusitis seen in a tertiary otolaryngology clinic. METHODS: A prospective, single site, observational study in a tertiary care pediatric otolaryngology clinic. Children aged 3-17 years diagnosed with chronic otitis media or rhinosinusitis with onset at less than 2 years of age were recruited. All study subjects had at least one of four key clinical features for PCD as determined by answers to screening questions, while control subjects had none. All participants completed a medical history questionnaire and nasal nitric oxide measurements. Those with reduced nasal nitric oxide levels were referred to our PCD center for further evaluation. RESULTS: A total of 153 patients were screened and 62 subjects were enrolled. Of those, 35 were enrolled as study subjects and 27 as matched controls. Study subjects had mean age of 7.5 years (3.2-16.5) with pre-screening diagnosis of chronic otitis media (n = 29) or chronic rhinosinusitis (n = 6). Control subjects (n = 27) had mean age 7.2 years (3.0-16.3) with pre-screening diagnosis of chronic otitis media (n = 25), and chronic rhinosinusitis (n = 2). There were no differences in subject demographics or mean nasal nitric oxide values between the two groups (179.8 vs 210.8 nl/min). Ten individuals had low nasal nitric oxide values, 7 of which were normal on repeat testing. Three subjects failed to return for follow up evaluations. Four referrals were made for further evaluation on the basis of clinical symptoms and nasal nitric oxide results. While no new cases of PCD were detected, a subject and his sibling with recurrent sinopulmonary infections were referred for immunologic evaluation. CONCLUSION: The use of standardized screening questions can be used in an otolaryngology clinic to identify patients who require further evaluation for PCD or primary immunodeficiency.

Growth arrest specific protein 7 inhibits tau fibrillogenesis.

Here we show that Gas7 inhibits phosphorylated tau fibrillogenesis by binding to phosphorylated tau at its non-WW domain, presumably F-BAR domain. We revealed that Gas7 binds to the third repeat domain of tau, the core element of tau oligomerization and the C-terminal domain of tau and alters the conformation not to form fibrils. These results suggest that Gas7 may serve to protect against Alzheimer's disease and other tauopathies by preventing tau fibrillogenesis.

Virus-induced cochlear inflammation in newborn mice alters auditory function.

Although human cytomegalovirus (HCMV) is a known cause of sensorineural hearing loss in infants with congenital HCMV (cCMV) infections, mechanisms that contribute to sensorineural hearing loss (SNHL) in infants with cCMV infection are not well defined. Using a murine model of CMV infection during auditory development, we have shown that peripheral infection of newborn mice with murine CMV (MCMV) results in focal infection of the cochlea and virus-induced cochlear inflammation. Approximately 50%-60% of infected mice exhibited increased auditory brainstem response (ABR) thresholds across a range of sound frequencies. Histological analyses of the cochlea in MCMV-infected mice with elevated ABR thresholds revealed preservation of hair cell (HC) number and morphology in the organ of Corti. In contrast, the number of spiral ganglion neurons (SGN), synapses, and neurites connecting the cochlear HC and SGN nerve terminals were decreased. Decreasing cochlear inflammation by corticosteroid treatment of MCMV-infected mice resulted in preservation of SGN and improved auditory function. These findings show that virus-induced cochlear inflammation during early auditory development, rather than direct virus-mediated damage, could contribute to histopathology in the cochlea and altered auditory function without significant loss of HCs in the sensory epithelium.

Vesicular Glutamatergic Transmission in Noise-Induced Loss and Repair of Cochlear Ribbon Synapses.

Noise-induced excitotoxicity is thought to depend on glutamate. However, the excitotoxic mechanisms are unknown, and the necessity of glutamate for synapse loss or regeneration is unclear. Despite absence of glutamatergic transmission from cochlear inner hair cells in mice lacking the vesicular glutamate transporter-3 (Vglut3KO ), at 9-11 weeks, approximately half the number of synapses found in Vglut3WT were maintained as postsynaptic AMPA receptors juxtaposed with presynaptic ribbons and voltage-gated calcium channels (CaV1.3). Synapses were larger in Vglut3KO than Vglut3WT In Vglut3WT and Vglut3+/- mice, 8-16 kHz octave-band noise exposure at 100 dB sound pressure level caused a threshold shift (∼40 dB) and a loss of synapses (>50%) at 24 h after exposure. Hearing threshold and synapse number partially recovered by 2 weeks after exposure as ribbons became larger, whereas recovery was significantly better in Vglut3WT Noise exposure at 94 dB sound pressure level caused auditory threshold shifts that fully recovered in 2 weeks, whereas suprathreshold hearing recovered faster in Vglut3WT than Vglut3+/- These results, from mice of both sexes, suggest that spontaneous repair of synapses after noise depends on the level of Vglut3 protein or the level of glutamate release during the recovery period. Noise-induced loss of presynaptic ribbons or postsynaptic AMPA receptors was not observed in Vglut3KO , demonstrating its dependence on vesicular glutamate release. In Vglut3WT and Vglut3+/-, noise exposure caused unpairing of presynaptic ribbons and presynaptic CaV1.3, but not in Vglut3KO where CaV1.3 remained clustered with ribbons at presynaptic active zones. These results suggest that, without glutamate release, noise-induced presynaptic Ca2+ influx was insufficient to disassemble the active zone. However, synapse volume increased by 2 weeks after exposure in Vglut3KO , suggesting glutamate-independent mechanisms.SIGNIFICANCE STATEMENT Hearing depends on glutamatergic transmission mediated by Vglut3, but the role of glutamate in synapse loss and repair is unclear. Here, using mice of both sexes, we show that one copy of the Vglut3 gene is sufficient for noise-induced threshold shift and loss of ribbon synapses, but both copies are required for normal recovery of hearing function and ribbon synapse number. Impairment of the recovery process in mice having only one functional copy suggests that glutamate release may promote synapse regeneration. At least one copy of the Vglut3 gene is necessary for noise-induced synapse loss. Although the excitotoxic mechanism remains unknown, these findings are consistent with the presumption that glutamate is the key mediator of noise-induced synaptopathy.

K336I mutant actin alters the structure of neighbouring protomers in filaments and reduces affinity for actin-binding proteins.

Mutation of the Lys-336 residue of actin to Ile (K336I) or Asp (K336E) causes congenital myopathy. To understand the effect of this mutation on the function of actin filaments and gain insight into the mechanism of disease onset, we prepared and biochemically characterised K336I mutant actin from Dictyostelium discoideum. Subtilisin cleavage assays revealed that the structure of the DNase-I binding loop (D-loop) of monomeric K336I actin, which would face the adjacent actin-protomer in filaments, differed from that of wild type (WT) actin. Although K336I actin underwent normal salt-dependent reversible polymerisation and formed apparently normal filaments, interactions of K336I filaments with alpha-actinin, myosin II, and cofilin were disrupted. Furthermore, co-filaments of K336I and WT actins also exhibited abnormal interactions with cofilin, implying that K336I actin altered the structure of the neighbouring WT actin protomers such that interaction between cofilin and the WT actin protomers was prevented. We speculate that disruption of the interactions between co-filaments and actin-binding proteins is the primary reason why the K336I mutation induces muscle disease in a dominant fashion.

The role of monocytes and macrophages in the dynamic permeability of the blood-perilymph barrier.

The blood-perilymph barrier serves a critical role by separating the components of blood from inner ear fluids, limiting traffic of cells, proteins and other solutes into the labyrinth, and allowing gas (O2-CO2) exchange. Inflammation produces changes in the blood-perilymph barrier resulting in increased vascular permeability. It is commonly thought that compromise of the blood-inner ear barrier would lead to hearing impairment through loss of the endocochlear potential (EP). In fact, the effect of increasing cochlear vascular permeability on hearing function and EP is poorly understood. We used a novel method to measure the integrity of the blood-perilymph barrier and demonstrated the effects of barrier compromise on ABR threshold and EP. We also investigated the contribution of CX3CR1 cochlear macrophages and CCR2 inflammatory monocytes to barrier function after systemic exposure to lipopolysaccharide (LPS). We found that systemic LPS induced a profound change in vascular permeability, which correlated with minimal change in ABR threshold and EP. Macrophage depletion using CX3CR1-DTR mice did not alter the baseline permeability of cochlear vessels and resulted in preservation of barrier function in LPS-treated animals. We conclude that cochlear macrophages are not required to maintain the barrier in normal mice and activated macrophages are a critical factor in breakdown of the barrier after LPS. CCR2 null mice demonstrated that LPS induction of barrier leakiness occurs in the absence of CCR2 expression. Thus, enhanced aminoglycoside ototoxicity after LPS can be linked to the expression of CCR2 in inflammatory monocytes, and not to preservation of the blood-perilymph barrier in CCR2 knockout mice.

Microtubule stabilizer reveals requirement of Ca2+-dependent conformational changes of microtubules for rapid coiling of haptonema in haptophyte algae.

A haptonema is an elongated microtubule-based motile organelle uniquely present in haptophytes. The most notable and rapid movement of a haptonema is 'coiling', which occurs within a few milliseconds following mechanical stimulation in an unknown motor-independent mechanism. Here, we analyzed the coiling process in detail by high-speed filming and showed that haptonema coiling was initiated by left-handed twisting of the haptonema, followed by writhing to form a helix from the distal tip. On recovery from a mechanical stimulus, the helix slowly uncoiled from the proximal region. Electron microscopy showed that the seven microtubules in a haptonema were arranged mostly in parallel but that one of the microtubules often wound around the others in the extended state. A microtubule stabilizer, paclitaxel, inhibited coiling and induced right-handed twisting of the haptonema in the absence of Ca2+, suggesting changes in the mechanical properties of microtubules. Addition of Ca2+ resulted in the conversion of haptonematal twist into the planar bends near the proximal region. These results indicate that switching microtubule conformation, possibly with the aid of Ca2+-binding microtubule-associated proteins is responsible for rapid haptonematal coiling.

Communication pathways to and from the inner ear and their contributions to drug delivery.

The environment of the inner ear is highly regulated in a manner that some solutes are permitted to enter while others are excluded or transported out. Drug therapies targeting the sensory and supporting cells of the auditory and vestibular systems require the agent to gain entry to the fluid spaces of the inner ear, perilymph or endolymph, which surround the sensory organs. Access to the inner ear fluids from the vasculature is limited by the blood-labyrinth barriers, which include the blood-perilymph and blood-strial barriers. Intratympanic applications provide an alternative approach in which drugs are applied locally. Drug from the applied solution enters perilymph through the round window membrane, through the stapes, and under some circumstances, through thin bone in the otic capsule. The amount of drug applied to the middle ear is always substantially more than the amount entering perilymph. As a result, significant amounts of the applied drug can pass to the digestive system, to the vasculature, and to the brain. Drugs in perilymph pass to the vasculature and to cerebrospinal fluid via the cochlear aqueduct. Conversely, drugs applied to cerebrospinal fluid, including those given intrathecally, can enter perilymph through the cochlear aqueduct. Other possible routes in or out of the ear include passage by neuronal pathways, passage via endolymph and the endolymphatic sac, and possibly via lymphatic pathways. A better understanding of the pathways for drug movements in and out of the ear will enable better intervention strategies.

Benign Rolandic epilepsy presenting like paradoxical vocal fold motion.

Paradoxical vocal fold motion (PVFM) is characterized by vocal fold adduction during respiration. Benign Rolandic epilepsy (BRE) is the most common childhood epilepsy and can cause oropharyngolaryngeal or facial manifestations. A 9-year-old male presented with intermittent apnea lasting 30-60 seconds and presumed PVFM. The patient's physical and fiberoptic exam were normal. He was admitted and found to have episodes of oxygen desaturation, neck twitching, and tongue burning. An EEG revealed focal epilepsy. After starting anti-epileptic medications, he had resolution of symptoms. Our patient was eventually diagnosed with BRE, a focal onset epilepsy that can mimic primary otolaryngologic disease.