ABSTRACT
Deafness is the prevailing sensory impairment among humans, impacting every aspect of one's existence. Half of congenital deafness cases are attributed to genetic factors. Studies have shown that Luzp2 is expressed in hair cells (HCs) and supporting cells of the inner ear, but its specific role in hearing remains unclear. To determine the importance of Luzp2 in auditory function, we generated mice deficient in Luzp2. Our results revealed that Luzp2 has predominant expression within the HCs and pillar cells. However, the loss of Luzp2 did not result in any changes in auditory threshold. HCs or synapse number and HC stereocilia morphology in Luzp2 knockout mice did not show any notable distinctions. This was the first study of the role of Luzp2 in hearing in mice, and our results provide important guidance for the screening of deafness genes.
ABSTRACT
There are no clear boundaries between internal tissues in sheep Computerized Tomography images, and it is difficult for traditional methods to meet the requirements of image segmentation in application. Deep learning has shown excellent performance in image analysis. In this context, we investigated the Loin CT image segmentation of sheep based on deep learning models. The Fully Convolutional Neural Network (FCN) and 5 different UNet models were applied in image segmentation on the data set of 1471 CT images including the Loin part from 25 Australian White rams and Dolper rams using the method of 5-fold cross validation. After 10 independent runs, different evaluation metrics were applied to assess the performances of the models. All models showed excellent results in terms evaluation metrics. There were slight differences among the results from the six models, and Attention-UNet outperformed others methods with 0.998±0.009 in accuracy, 4.391±0.338 in AVER_HD, 0.90±0.012 in MIOU and 0.95±0.007 in DICE, respectively, while the optimal value of LOSS was 0.029±0.018 from Channel-UNet, and the running time of ResNet34-UNet is the shortest.
Subject(s)
Deep Learning , Animals , Male , Sheep , Australia , Benchmarking , Image Processing, Computer-Assisted , Sheep, Domestic , Tomography, X-Ray Computed , MeatABSTRACT
Cingulin (CGN) is a cytoskeleton-associated protein localized at the apical junctions of epithelial cells. CGN interacts with major cytoskeletal filaments and regulates RhoA activity. However, physiological roles of CGN in development and human diseases are currently unknown. Here, we report a multi-generation family presenting with autosomal dominant non-syndromic hearing loss (ADNSHL) that co-segregates with a CGN heterozygous truncating variant, c.3330delG (p.Leu1110Leufs*17). CGN is normally expressed at the apical cell junctions of the organ of Corti, with enriched localization at hair cell cuticular plates and circumferential belts. In mice, the putative disease-causing mutation results in reduced expression and abnormal subcellular localization of the CGN protein, abolishes its actin polymerization activity, and impairs the normal morphology of hair cell cuticular plates and hair bundles. Hair cell-specific Cgn knockout leads to high-frequency hearing loss. Importantly, Cgn mutation knockin mice display noise-sensitive, progressive hearing loss and outer hair cell degeneration. In summary, we identify CGN c.3330delG as a pathogenic variant for ADNSHL and reveal essential roles of CGN in the maintenance of cochlear hair cell structures and auditory function.
Subject(s)
Deafness , Hearing Loss , Animals , Humans , Mice , Cytoskeletal Proteins , Deafness/genetics , Hair Cells, Auditory/metabolism , Hearing/physiology , Hearing Loss/genetics , Hearing Loss/metabolismABSTRACT
Orthosteric binding sites of olfactory receptors have been well understood for ligand-receptor interactions. However, a lack of explanation for subtle differences in ligand profile of olfactory receptors even with similar orthosteric binding sites promotes more exploration into the entry tunnels of the receptors. An important question regarding entry tunnels is the number of entry tunnels, which was previously believed to be one. Here, we used TAAR9 that recognizes important biogenic amines such as cadaverine, spermine, and spermidine as a model for entry tunnel study. We identified two entry tunnels in TAAR9 and described the residues that form the tunnels. In addition, we found two vestibular binding pockets, each located in one tunnel. We further confirmed the function of two tunnels through site-directed mutagenesis. Our study challenged the existing views regarding the number of entry tunnels in the subfamily of olfactory receptors and demonstrated the possible mechanism how the entry tunnels function in odorant recognition.
Subject(s)
Olfactory Receptor Neurons , Receptors, G-Protein-Coupled/chemistry , Receptors, Odorant/chemistry , Animals , Binding Sites , Biogenic Polyamines/chemistry , Mice , Mutagenesis, Site-Directed , Receptors, G-Protein-Coupled/genetics , Receptors, Odorant/metabolismABSTRACT
Background: Sensorineural hearing loss can be cured by cochlear implantation (CI), but complications can occur. Based on when the complications develop, they are categorized as intraoperative complications, early postoperative complications, or delayed postoperative complications (>3 months after the surgery).Aims/objectives: We aimed to investigate the occurrence of delayed complications after CI surgery, and identify appropriate management methods.Material and methods: We analyzed 624 sensorineural hearing loss patients who had been consecutively treated with CI using the conventional surgical technique in our institution and had been followed-up until September 2017.Results: A total of 43 (6.86%) patients out of the 624 CIs (627 ears) reported complications, and 9 (1.44%) were major complications and 34 (5.42%) were minor complications. Wound infection and device failure were the most common major complications, and hematoma was the most common minor complication.Conclusions and significance: CI surgery is a relatively mature technology; the incidence of complications is low, and with early diagnosis and treatment most complications have a good prognosis. Head trauma was the main reason for children's complications, and patients and guardians should be given good education preoperatively about how to manage the CI postoperatively.
Subject(s)
Cochlear Implantation/adverse effects , Cochlear Implants , Postoperative Complications/epidemiology , Prosthesis Failure , Adolescent , Adult , Aged , Child , Child, Preschool , Cochlear Implants/adverse effects , Female , Follow-Up Studies , Hearing Loss, Sensorineural/surgery , Humans , Infant , Male , Middle Aged , Seroma/etiology , Surgical Wound Infection/epidemiology , Time Factors , Young AdultABSTRACT
Atg7 is an indispensable factor that plays a role in canonical nonselective autophagy. Here we show that genetic ablation of Atg7 in outer hair cells (OHCs) in mice caused stereocilium damage, somatic electromotility disturbances, and presynaptic ribbon degeneration over time, which led to the gradual wholesale loss of OHCs and subsequent early-onset profound hearing loss. Impaired autophagy disrupted OHC mitochondrial function and triggered the accumulation of dysfunctional mitochondria that would otherwise be eliminated in a timely manner. Atg7-independent autophagy/mitophagy processes could not compensate for Atg7 deficiency and failed to rescue the terminally differentiated, non-proliferating OHCs. Our results show that OHCs orchestrate intricate nonselective and selective autophagic/mitophagy pathways working in concert to maintain cellular homeostasis. Overall, our results demonstrate that Atg7-dependent autophagy plays a pivotal cytoprotective role in preserving OHCs and maintaining hearing function.
Subject(s)
Autophagy-Related Protein 7/metabolism , Deafness/metabolism , Hair Cells, Auditory, Outer/metabolism , Animals , Autophagy , Autophagy-Related Protein 7/genetics , Cell Differentiation/physiology , Deafness/genetics , Deafness/pathology , Hair Cells, Auditory, Outer/pathology , Mice , Microscopy, Electron, ScanningABSTRACT
Genetic hearing loss is a common health problem with no effective therapy currently available. DFNA15, caused by mutations of the transcription factor POU4F3, is one of the most common forms of autosomal dominant non-syndromic deafness. In this study, we established a novel mouse model of the human DFNA15 deafness, with a Pou4f3 gene mutation (Pou4f3Δ) identical to that found in a familial case of DFNA15. The Pou4f3(Δ/+) mice suffered progressive deafness in a similar manner to the DFNA15 patients. Hair cells in the Pou4f3(Δ/+) cochlea displayed significant stereociliary and mitochondrial pathologies, with apparent loss of outer hair cells. Progression of hearing and outer hair cell loss of the Pou4f3(Δ/+) mice was significantly modified by other genetic and environmental factors. Using Pou4f3(-/+) heterozygous knockout mice, we also showed that DFNA15 is likely caused by haploinsufficiency of the Pou4f3 gene. Importantly, inhibition of retinoic acid signaling by the aldehyde dehydrogenase (Aldh) and retinoic acid receptor inhibitors promoted Pou4f3 expression in the cochlear tissue and suppressed the progression of hearing loss in the mutant mice. These data demonstrate Pou4f3 haploinsufficiency as the main underlying cause of human DFNA15 deafness and highlight the therapeutic potential of Aldh inhibitors for treatment of progressive hearing loss.
Subject(s)
Aldehyde Dehydrogenase/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Hair Cells, Auditory/pathology , Hearing Loss/drug therapy , Hearing Loss/etiology , Homeodomain Proteins/genetics , Transcription Factor Brn-3C/genetics , Animals , Benzaldehydes/pharmacology , Disease Models, Animal , Haploinsufficiency/genetics , Hearing Loss/genetics , Hearing Loss/pathology , Homeodomain Proteins/metabolism , Humans , Mice, Inbred C57BL , Mice, Inbred Strains , Mice, Knockout , Noise/adverse effects , Quinolines/pharmacology , Transcription Factor Brn-3C/metabolism , Tretinoin/pharmacology , para-Aminobenzoates/pharmacologyABSTRACT
Epithelial-mesenchymal transition (EMT) allows neoplastic cells to gain the invasive phenotype and become migratory, which is required for cancer progression and metastasis. In the present study, the expression of EMT-associated biomarkers and their association with clinicopathological parameters in laryngeal squamous cell carcinoma (LSCC) was investigated. E-cadherin, N-cadherin, ß-catenin and zinc finger E-box binding homeobox 2 (ZEB2) protein expression was evaluated with immunohistochemistry in a cohort of 76 patients with operable LSCC. The association between these transition markers, clinicopathological parameters and their prognostic impact in LSCC was analyzed. Immunohistochemical analysis revealed that EMT-associated proteins were differentially expressed between LSCC and adjacent non-neoplastic laryngeal tissue. Negative E-cadherin expression and positive N-cadherin, ß-catenin and ZEB2 expression were associated with a later tumor (T) stage, decreasing tumor differentiation and a reduced overall survival (OS) time (OS: E-cadherin, P=0.016; N-cadherin, P=0.003; ß-catenin, P=0.002; ZEB2, P=0.0003). E-cadherin/ß-catenin co-expression was significantly associated with the majority of clinicopathological parameters assessed, including lymph node metastases, T stage and tumor cell differentiation (P=0.004, P=0.005, and P<0.001, respectively). Multivariate analysis indicated that T stage and the positive expression of ß-catenin and ZEB2 were independent risk factors for OS in LSCC (P=0.014, P=0.025 and P=0.003, respectively). It was concluded that EMT mediates tumor progression, and reduces OS time in patients with LSCC. E-cadherin/ß-catenin co-expression may be associated with clinicopathological parameters. T stage, and the positive co-expression of ß-catenin and ZEB2 may be independent predictors of prognosis in LSCC.
ABSTRACT
Enlarged vestibular aqueduct (EVA)associated hearing loss is frequently detected in individuals carrying the SLC26A4 mutation in the Chinese population. The present study aimed to identify the causative SLC26A4 coding mutations in a patient group with nonsyndromic hearing loss (NSHL) and EVA. Genomic DNA was extracted from blood samples obtained from 52 NSHL patients with EVA and from 60 normal controls. The mutation analysis for 20 coding exons of SLC26A4 was performed by direct sequencing. The results of the mutational analysis showed that there were two probands from two separate families suffering from bilateral sensorineural hearing loss with EVA, carrying the same novel compound heterozygous mutation of SLC26A4 (c.1644_1645insA and c.2168A>G). Other members of the two families had heterozygous monoallelic mutations with normal hearing. However, neither of these mutations were detected in the 60 normal controls. These results are the first, to the best of our knowledge, to link the compound heterozygote mutation, c.1644_1645insA and c.2168A>G, in the SLC26A4 gene to NSHL patients with EVA. The two mutations identified in the present study were located in the antisigma factor antagonist domain, the core region for plasma membrane targeting of anion transporters, which suggested that the reduced or complete loss of SLC26A4 function was the direct cause of hearing loss in the two patients. These results provide a foundation for further elucidating the genetic factors responsible for EVAassociated NSHL.
Subject(s)
Deafness/genetics , Genetic Predisposition to Disease , Membrane Transport Proteins/genetics , Mutation/genetics , Vestibular Aqueduct/pathology , Audiometry, Pure-Tone , Base Sequence , Case-Control Studies , Child , DNA Mutational Analysis , Deafness/diagnostic imaging , Exons/genetics , Family , Female , Heterozygote , Humans , Male , Pedigree , Sulfate Transporters , Vestibular Aqueduct/diagnostic imagingABSTRACT
Polyethylenimine (PEI) has been demonstrated to be an effective nonviral synthetic polymer for efficient gene delivery amongst various cell types in vitro and in vivo. In the present study, 25 kDa linear PEI (LPEI) was used to transfer plasmid DNA (pDNA), encoding the enhanced green fluorescent protein reporter gene, into the cultured cochlear epithelium of neonatal mice. The 25 kDa LPEI/pDNA nanoparticles were generated in phosphatebuffered saline prior to transfection. Sensory epithelial cells were transfected using an appropriate weight ratio of LPEI and pDNA. However, the use of higher LPEI/pDNA ratios, which result in the generation of a greater number of nanoparticles, induced significant damage to the sensory epithelial cells, as demonstrated by immunofluorescent and transmission electron microscopy analyses. These results indicated that precautionary measures are required with regard to the use of PEI nanoparticles in nanomedicine, and emphasized the requirement for thorough physicochemical characterization and toxicity testing for each polymer vector prior to the construction of nanotechnology systems for use in clinical applications. The development of effective management techniques for potential nanoototoxicity risks is of considerable significance to the clinical applications of nanoparticles.
Subject(s)
Epithelium/metabolism , Nanoparticles/chemistry , Nanoparticles/toxicity , Plasmids/chemistry , Plasmids/genetics , Polyethyleneimine/chemistry , Animals , Animals, Newborn , Cochlea/metabolism , Epithelium/ultrastructure , Female , Gene Expression , Gene Transfer Techniques , Hair Cells, Auditory/metabolism , Hair Cells, Auditory/pathology , Hair Cells, Auditory/ultrastructure , Male , Mice , Nanoparticles/ultrastructure , TransfectionABSTRACT
An eightbase pair (bp) deletion in the Pou4f3 gene in hair cells is associated with DFNA15, a hereditary form of hearing loss. To explore the pathological mechanisms underlying the development of DFNA15, the effect of the mutation in Pou4f3 on the activity of the myosin VI (Myo6) promoter, was investigated. The upstream regulatory sequence of Myo6 (2625 bp), consisting of an 1899 bp upstream sequence and a 727 bp intron 1 sequence, was amplified using polymerase chain reaction and subcloned into the pGL3Basic vector expressing firefly luciferase. For verification of inserted fragments, plasmids were subjected to restriction analysis and then sequenced. HEK293T human embryonic kidney cells were transiently transfected with renilla luciferasethymidine kinase vectors expressing Renilla luciferase and the Myo6 promoterdriven firefly luciferase expressing vectors along with pIRES2enhanced green fluorescent protein (EGFP)Pou4f3 (expressing wildtype Pou4f3) or pIRES2EGFPPou4f3 (expressing the truncation mutant of Pou4f3). The relative luciferase activities were measured to determine the activity of the Myo6 promoter. The Myo6 promoter activity was not affected by coexpression of wildtype Pou4f3, as indicated by the comparable relative luciferase activities in the presence of the pIRES2EGFPPou4f3 and the empty control vectors. However, coexpression of mutated Pou4f3 significantly inhibited the activity of the Myo6 promoter to almost half of that of the control (P<0.001). The data suggests that mutated Pou4f3 has a negative role in the promoter activity of Myo6, and by extension, the expression of myosin VI, and this may be an underlying mechanism of DFNA15 hearing loss.
Subject(s)
Gene Expression Regulation , Hair Cells, Auditory/metabolism , Mutation/genetics , Myosin Heavy Chains/genetics , Transcription Factor Brn-3C/genetics , Transcription Factor Brn-3C/metabolism , Animals , Base Sequence , Genes, Reporter , Genetic Vectors/metabolism , Humans , Luciferases/metabolism , Mice , Mice, Inbred C57BL , Molecular Sequence Data , Myosin Heavy Chains/metabolism , Promoter Regions, Genetic/genetics , Recombinant Proteins/metabolismABSTRACT
Myosin light chain kinase (MLCK) phosphorylates myosin regulatory light chains to facilitate its interaction with actin filaments and produce contractile activity. The outer hair cells (OHCs) in the ear contain large amounts of actin and a variety myosins. The stereociliary and somatic motility of OHCs are closely related to hearing. It appears likely that MLCK may play an important role in acoustic trans-duction. In this study, we analyzed, both in vivo and in vitro, the OHCs of mice bearing a specific deletion of the MLCK gene and the OHCs of control mice. The phenotype was assessed by auditory function [acoustic brainstem responses (ABRs) and distortion product otoacoustic emissions (DPOAEs)], inner ear morphology and histology. MLCK-deficient mice aged 6-7 months showed impaired hearing, a 5- to 10-dB sound pressure level (SPL) increase in the ABR thresholds, when responding to clicks and tones of different frequencies (8 and 16 kHz) (P<0.05). The DPOAE amplitudes of 3-month-old MLCK-deficient mice decreased significantly (>10 dB SPL) at low frequencies (4, 5 and 6 kHz). The OHCs in the MLCK-deficient mice increased with abnormal stereocilia. The staining of F-actin and the phosphorylation of the regulatory light chain in MLCK-deficient OHCs was weak. Our results indicate that MLCK may regulate the structure and the motility of stereocilia through F-actin polymerization.
Subject(s)
Actins/metabolism , Cytoskeleton/metabolism , Hair Cells, Auditory, Outer/enzymology , Hair Cells, Auditory, Outer/physiology , Hearing/physiology , Myosin-Light-Chain Kinase/metabolism , Animals , Auditory Threshold/physiology , Evoked Potentials, Auditory, Brain Stem/physiology , Fluorescent Antibody Technique , Hair Cells, Auditory, Outer/cytology , Hair Cells, Auditory, Outer/ultrastructure , Mice , Mice, Knockout , Myosin Light Chains/metabolism , Myosin-Light-Chain Kinase/deficiency , Otoacoustic Emissions, Spontaneous/physiology , PhosphorylationABSTRACT
OBJECTIVE: To investigate the function of myosin light chain kinase (MLCK) in hearing in mouse by generating inner hair cell-specific Mlck knockout mice and analyze the effect on their hearing. METHODS: Cross Mlck floxed mice with IHC-Cre mice, the genotype and knockout efficiency were confirmed by PCR. We used auditory brain stem response (ABR) to evaluate mice hearing function at different frequencies. RESULTS: Mlck knockout mice were selected by mice tail DNA genotyping and confirmed the deletion of the target gene by isolated inner hair cell DNA genotyping. Mlck-deficient mice showed impaired hearing with a rise in ABR threshold response to click and three different pure tones (8 kHz, 16 kHz, 32 kHz), and the rise was over 20 dB at high-frequency(32 kHz). Further analyses of waveforms showed that wave-I amplitudes on 60 dB SPL, 50 dB SPL and 40 dBSPL in response to tone (16 kHz) were less than control group(P < 0.05) on average, but the ratio of wave I/II and I/III were not difference (P > 0.05). CONCLUSIONS: Mlck is successfully deleted in inner hair cell-specific Mlck knockout mice. Mlck knockout mice display a significantly higher threshold in response to click and tones, especially in high-frequencies.
Subject(s)
Evoked Potentials, Auditory, Brain Stem , Hair Cells, Auditory, Inner/metabolism , Hearing Loss/genetics , Myosin-Light-Chain Kinase/genetics , Animals , Audiometry, Pure-Tone , Auditory Threshold , Mice , Mice, Inbred C57BL , Mice, KnockoutABSTRACT
OBJECTIVE: To study the risk factors and interaction of nasal septal perforation (NSP) in rats. METHOD: Animals (n=120) that underwent unilateral nasal obstruction using Merocel nasal packing or gelfoam with/without standard staphylococcus aureus inoculation were observed for the formation of NSP at 2, 3, 5, and 7 days after operation by endoscope system. Following sacrifice at 7 days, the obtained nasal secretions were prepared for bacterial culture. Experimental interventions were compared with normal controls (n=10). RESULT: Perforation of nasal septum was observed in 80% of the animals accepted nasal obstruction using Merocel nasal packing with standard staphylococcus aureus inoculation in 3 days (P < 0.01), while in 70% of those using abacterial Merocel nasal packing in 5 days (P < 0.05) and no significant difference than that of before (P > 0.05). There was a weak region in anteroinferior nasal septum in rats, which the almost NSPs located in. The position of NSP does not overlap Merocel. CONCLUSION: The interaction of risk factors contributes to NSP. The occurrence of NSP mainly depends on the construction of nasal septum, while dysaemia is also necessary. Obstruction of nasal drainage and infection promote the development of NSP.
Subject(s)
Nasal Obstruction/pathology , Nasal Septal Perforation/etiology , Nasal Septum/anatomy & histology , Animals , Causality , Disease Models, Animal , Male , Nasal Obstruction/microbiology , Rats , Rats, Sprague-Dawley , Risk Factors , Staphylococcal Infections/pathologyABSTRACT
The structural homeostasis of the cochlear hair cell membrane is critical for all aspects of sensory transduction, but the regulation of its maintenance is not well understood. In this report, we analyzed the cochlear hair cells of mice with specific deletion of myosin light chain kinase (MLCK) in inner hair cells. MLCK-deficient mice showed impaired hearing, with a 5- to 14-dB rise in the auditory brainstem response (ABR) thresholds to clicks and tones of different frequencies and a significant decrease in the amplitude of the ABR waves. The mutant inner hair cells produced several ball-like structures around the hair bundles in vivo, indicating impaired membrane stability. Inner hair cells isolated from the knockout mice consistently displayed less resistance to hypoosmotic solution and less membrane F-actin. Myosin light-chain phosphorylation was also reduced in the mutated inner hair cells. Our results suggest that MLCK is necessary for maintaining the membrane stability of inner hair cells.