Microtia-atresia is a rare craniofacial malformation primarily affecting the first and second pharyngeal arches, leading to the deformity of the auricle and atresia of the external ear canal. Its etiology is heterogenous and largely unknown, including both genetic and environmental factors. The HOXA4 gene has been identified as potentially pathogenetic for microtia-atresia in three twin families. A hoxa4a mosaic knockdown zebrafish model was constructed using CRISPR/Cas9. hoxa4a was expressed in the mandible during early development in zebrafish, while the F0 mosaic knockdowns exhibited craniofacial malformations with abnormal chondrocyte morphologies. Specifically, hoxa4a knockdown reduced cranial neural crest cell proliferation while increasing apoptosis, markedly downregulating chondrogenic markers sox9a and col2a1a. Consequently, pharyngeal arch chondrocytes exhibited disorganized arrangement and morphological abnormalities, resulting in mandibular hypoplasia. Our findings provide important insights into the role of hoxa4a in zebrafish mandibular development and the pathology of microtia-atresia caused by HOXA4 gene mutations in humans.

Microtia-atresia is a rare type of congenital craniofacial malformation causing severe damage to the appearance and hearing ability of affected individuals. The genetic factors associated with microtia-atresia have not yet been determined. The AMER1 gene has been identified as potentially pathogenic for microtia-atresia in two twin families. An amer1 mosaic knockdown zebrafish model was constructed using CRISPR/Cas9. The phenotype and the development process of cranial neural crest cells of the knockdown zebrafish were examined. Components of the Wnt/β-catenin pathway were examined by qPCR, Western blotting, and immunofluorescence assay. IWR-1-endo, a reversible inhibitor of the Wnt/β-catenin pathway, was applied to rescue the abnormal phenotype. The present study showed that the development of mandibular cartilage in zebrafish was severely compromised by amer1 knockdown using CRISPR/Cas9. Specifically, amer1 knockdown was found to affect the proliferation and apoptosis of cranial neural crest cells, as well as their differentiation to chondrocytes. Mechanistically, amer1 exerted an antagonistic effect on the Wnt/β-catenin pathway. The application of IWR-1-endo could partially rescue the abnormal phenotype. We demonstrated that amer1 was essential for the craniofacial development of zebrafish by interacting with the Wnt/β-catenin pathway. These findings provide important insight into the role of amer1 in zebrafish mandibular development and the pathology of microtia-atresia caused by AMER1 gene mutations in humans.

HYPOTHESIS:The current study employed a skull-simulator verification method to assess whether the output of softband bone conduction hearing devices (BCHDs) at the manufacturer's default settings deviated widely from the target determined by the fitting formula. BACKGROUND:Real ear analysis is utilized for the verification of the fitting of air conduction hearing devices (ACHDs) in a variety of institutions. This procedure, however, has not been used in the fitting of BCHDs, largely due to the difficulty of testing the output of these devices to temporal bones. Despite the availability of skull simulators, they have not been utilized clinically to measure BCHD output. MATERIALS AND METHODS:This prospective, single-center study enrolled 42 subjects, aged 3 months to 10 years, with microtia-atresia-associated mild-to-severe bilateral conductive hearing loss. Hearing sensitivity was evaluated behaviorally by pure tone audiometry (PTA) in 22 subjects 4 years or older (the PTA group), and by auditory brainstem response (ABR) in 20 subjects younger than 4 years (the ABR group). Following 6 months of subjects wearing the prescribed softband BCHDs, their dial level (DL) thresholds were reassessed while using their own BCHDs, configured with zero gain across all frequencies, functioning solely as a bone vibrator. These DL thresholds were inputted into the fitting formula, desired sensation level-bone conduction devices (DSL-BCD) for children, to obtain the target values of BCHD output. The simulator output of the BCHD programmed at the manufacturer's default setting was measured in response to speech presented at 55, 65, and 80 dB SPL, followed by gain adjustment based on the differences between the simulator output and the target. Aided speech intelligibility index (SII) was measured before and after the gain adjustment. RESULTS:The softband BCHDs at the manufacturer's settings generally had lower output than the prescribed target values. This difference was larger at low frequencies and low levels. Across the 12 points tested (four frequencies from 500 to 4000 Hz multiplied by three levels), 22 (52.3%) and 42 (100%) BCHDs had deviations of +7 and +5 dB, respectively, at one point or more. The gain adjustments reduced the deviation and improved the SII values at the two lower levels of speech presented. CONCLUSION:The simulator output of softband bone conduction hearing devices (BCHDs) with the manufacturer's settings may exhibit significant deviations from the formula. Objective output verification should be considered a beneficial step in BCHD fitting and is recommended when applicable.

BACKGROUND:In vertebrates, the development of the inner ear is a delicate process, whereas its relating molecular pathways are still poorly understood. LMO4, an LIM domain-only transcriptional regulator, is drawing an increasing amount of interest for its multiple roles regarding human embryonic development and the modulation of ototoxic side effects of cisplatin including cochlear apoptosis and hearing loss. The aim of the present study is to further explore the role of lmo4a in zebrafish inner ear development and thus explore its functional role.METHODS:The Spatial Transcript Omics DataBase was referred to in order to evaluate the expression of lmo4a during the first 24 h of zebrafish development. In situ hybridization was applied to validate and extend the expression profile of lmo4a to 3 days post-fertilization. The morpholino (MO) knockdown and CRISPR/Cas9 knockout (KO) of lmo4a was applied. Morphological analyses of otic vesical, hair cells, statoacoustic ganglion and semicircular canals were conducted. The swimming pattern of lmo4a KO and MO zebrafish was tracked. In situ hybridization was further applied to verify the expression of genes of the related pathways. Rescue of the phenotype was attempted by blockage of the bmp pathway via heat shock and injection of Dorsomorphin.RESULTS:lmo4a is constitutively expressed in the otic placode and otic vesicle during the early stages of zebrafish development. Knockdown and knockout of lmo4a both induced smaller otocysts, less hair cells, immature statoacoustic ganglion and malformed semicircular canals. Abnormal swimming patterns could be observed in both lmo4a MO and KO zebrafish. eya1 in preplacodal ectoderm patterning was downregulated. bmp2 and bmp4 expressions were found to be upregulated and extended in lmo4a morphants, and blockage of the Bmp pathway partially rescued the vestibular defects.CONCLUSIONS:We concluded that lmo4a holds a regulative effect on the Bmp pathway and is required for the normal development of zebrafish inner ear. Our study pointed out the conservatism of LMO4 in inner ear development between mammals and zebrafish as well as shed more light on the molecular mechanisms behind it. Further research is needed to distinguish the relationships between lmo4 and the Bmp pathway, which may lead to diagnostic and therapeutic approaches towards human inner ear malformation.

Hemifacial microsomia (HFM), a rare disorder of first- and second-pharyngeal arch development, has been linked to a point mutation in VWA1 (von Willebrand factor A domain containing 1), encoding the protein WARP in a five-generation pedigree. However, how the VWA1 mutation relates to the pathogenesis of HFM is largely unknown. Here, we sought to elucidate the effects of the VWA1 mutation at the molecular level by generating a vwa1-knockout zebrafish line using CRISPR/Cas9. Mutants and crispants showed cartilage dysmorphologies, including hypoplastic Meckel's cartilage and palatoquadrate cartilage, malformed ceratohyal with widened angle, and deformed or absent ceratobranchial cartilages. Chondrocytes exhibited a smaller size and aspect ratio and were aligned irregularly. In situ hybridization and RT-qPCR showed a decrease in barx1 and col2a1a expression, indicating abnormal cranial neural crest cell (CNCC) condensation and differentiation. CNCC proliferation and survival were also impaired in the mutants. Expression of FGF pathway components, including fgf8a, fgfr1, fgfr2, fgfr3, fgfr4, and runx2a, was decreased, implying a role for VWA1 in regulating FGF signaling. Our results demonstrate that VWA1 is essential for zebrafish chondrogenesis through effects on condensation, differentiation, proliferation, and apoptosis of CNCCs, and likely impacts chondrogenesis through regulation of the FGF pathway.

Aging-associated microbial dysbiosis exacerbates various disorders and dysfunctions, and is a major contributor to morbidity and mortality in the elderly, but the underlying cause of this aging-related syndrome is confusing. SIRT6 knockout (SIRT6 KO) mice undergo premature aging and succumb to death by 4 weeks, and are therefore useful as a premature aging research model. Here, fecal microbiota transplantation from SIRT6 KO mice into wild-type (WT) mice phenocopies the gut dysbiosis and premature aging observed in SIRT6 KO mice. Conversely, an expanded lifespan was observed in SIRT6 KO mice when transplanted with microbiota from WT mice. Antibiotic cocktail treatment attenuated inflammation and cell senescence in KO mice, directly suggesting that gut dysbiosis contributes to the premature aging of SIRT6 KO mice. Increased Enterobacteriaceae translocation, driven by the overgrowth of Escherichia coli, is the likely mechanism for the premature aging effects of microbiome dysregulation, which could be reversed by a high-fat diet. Our results provide a mechanism for the causal link between gut dysbiosis and aging, and support a beneficial effect of a high-fat diet for correcting gut dysbiosis and alleviating premature aging. This study provides a rationale for the integration of microbiome-based high-fat diets into therapeutic interventions against aging-associated diseases.

Background:Various amplification options are available for patients with congenital bilateral conductive hearing loss. Unilateral bone conduction hearing device (BCHD) is widely used for these patients, whereas benefits of bilateral BCHDs in certain subgroups of patients require further exploration. Objectives:To evaluate functional and directional hearing in patients with unilateral Bonebridge (MEDEL) and contralateral ADHEAR (MEDEL) devices. Materials and methods:This study included 32 patients (20 males, 12 females), of mean age 11.8 years (range 7-27 years). Hearing thresholds, speech perception and sound localization were tested three months after activation of the Bonebridge under three conditions: unaided, unilateral BHCD (Bonebridge) and bilateral BHCDs (Bonebridge plus contralateral ADHEAR). Patient acceptance of these devices in daily life was evaluated by questionnaire. Results:Compared with unaided, the mean hearing thresholds (0.5, 1, 2, and 4 kHz) and speech perception with unilateral BCHD and bilateral BCHDs were improved significantly (p < .05 each). Markers of directional hearing ability, including percentages of accurate responses, bias angles and RMS errors, were significantly better with bilateral BCHDs than unilateral BHCD (p < .05 each). Questionnaire revealed high patient satisfaction with both unilateral and bilateral devices. Conclusions:Functional hearing and sound localization abilities were better with bilateral BCHDs than unilateral BCHD.

Objective We used data from twins and their families to probe the genetic factors contributing to microtia-atresia, in particular, early post-twinning variations that potentially account for the discordant phenotypes of monozygotic twin pairs. Methods Six families of monozygotic twins discordant for congenital microtia-atresia were recruited for study. The six patients shared a consistent clinical phenotype of unilateral microtia-atresia. Whole-exome sequencing (WES) was performed for all six twin pairs and their parents. Family segregation and multiple bioinformatics methods were applied to identify suspicious mutations in all families. Recurring mutations commonly detected in at least two families were highlighted. All variants were validated via Sanger sequencing. Gene Ontology (GO) analysis was performed to identify candidate gene sets and related pathways. Copy number variation (CNV), linkage analysis, association analysis and machine learning methods were additionally applied to isolate candidate mutations, and comparative genomics and structural modeling tools used to evaluate their potential roles in onset of microtia-atresia. Results Our analyses revealed 61 genes with suspected mutations associated with microtia-atresia. Five (HOXA4, MUC6, CHST15, TBX10, and AMER1) contained 7 de novo mutations that appeared in at least two families, which have been previously reported as pathogenic for other diseases. Among these, HOXA4 (c.920A>C, p.H307P) was determined as the most likely pathogenic variant for microtia-atresia. GO analysis revealed four gene sets involving 11 pathways potentially related to underlying pathogenesis of the disease. CNVs in three genes (UGT2B17, OVOS, and KATNAL2) were detected in at least two families. Linkage analysis disclosed 13 extra markers for the disease, of which two (FGFR1 and EYA1) were validated via machine learning analysis as plausible candidate genes for the disease. Conclusion Based on comprehensive genetic and bioinformatic analyses of WES data from six families of discordant monozygotic twins with microtia-atresia, we identified multiple candidate genes that may function in post-twinning onset of the disease. The collective findings provide novel insights into the pathogenesis of congenital microtia-atresia.

Mammalian sirtuin 6 (SIRT6) is involved in the regulation of many essential processes, especially metabolic homeostasis. SIRT6 knockout mice undergo premature aging and die at age ~4 weeks. Severe glycometabolic disorders have been found in SIRT6 knockout mice, and whether a dietary intervention can rescue SIRT6 knockout mice remains unknown. In our study, we found that at the same calorie intake, a high-fat diet dramatically increased the lifespan of SIRT6 knockout mice to 26 weeks (males) and 37 weeks (females), reversed multi-organ atrophy, and reduced body weight, hypoglycemia, and premature aging. Furthermore, the high-fat diet partially but significantly normalized the global gene expression profile in SIRT6 knockout mice. Regarding the mechanism, excessive glucose uptake and glycolysis induced by the SIRT6 deficiency were attenuated in skeletal muscle through inhibition of insulin and IGF1 signaling by the high-fat diet. Similarly, fatty acids but not ketone bodies inhibited glucose uptake, glycolysis, and senescence in SIRT6 knockout fibroblasts, whereas PI3K inhibition antagonized the effects of a high-fatty-acid medium in vitro. Overall, the high-fat diet dramatically reverses numerous consequences of SIRT6 deficiency through modulation of insulin and IGF1 signaling, providing a new basis for elucidation of SIRT6 and fatty-acid functions and supporting novel therapeutic approaches against metabolic disorders and aging-related diseases.

Background Hemifacial microsomia (HFM) is a type of rare congenital syndrome caused by developmental disorders of the first and second pharyngeal arches that occurs in one out of 5,600 live births. There are significant gaps in our knowledge of the pathogenic genes underlying this syndrome. Methods Whole exome sequencing (WES) was performed on five patients, one asymptomatic carrier, and two marry-in members of a five-generation pedigree. Structure of WARP (product of VWA1) was predicted using the Phyre2 web portal. In situ hybridization and vwa1-knockdown/knockout studies in zebrafish using morpholino and CRISPR/Cas9 techniques were performed. Cartilage staining and immunofluorescence were carried out. Results Through WES and a set of filtration, we identified a c.G905A:p.R302Q point mutation in a novel candidate pathogenic gene, VWA1. The Phyre2 web portal predicted alterations in secondary and tertiary structures of WARP, indicating changes in its function as well. Predictions of protein-to-protein interactions in five pathways related to craniofacial development revealed possible interactions with four proteins in the FGF pathway. Knockdown/knockout studies of the zebrafish revealed deformities of pharyngeal cartilage. A decrease of the proliferation of cranial neural crest cells (CNCCs) and alteration of the structure of pharyngeal chondrocytes were observed in the morphants as well. Conclusion Our data suggest that a mutation in VWA1 is functionally linked to HFM through suppression of CNCC proliferation and disruption of the organization of pharyngeal chondrocytes.

OBJECTIVE:The ability of early intervention with softband bone conducted hearing device (BCHD) to ensure normal development of speech, language and psychosocial situations remains undetermined. We aimed to evaluate auditory and speech development, as well as psychosocial situations of children with bilateral microtia fitted with a softband BCHD for 3-5 years. METHODS:The study included 53 patients with bilateral microtia and 53 sex- and age-matched children with normal hearing. Auditory development was evaluated using the Meaningful Auditory Integration Scale (MAIS) and Categories of Auditory Performance (CAP). Speech Intelligibility Rating (SIR) and Meaningful Use of Speech Scale (MUSS) were used to assess speech development. The psychometric properties of these patients were evaluated using Achenbach's Child Behavior Checklist (CBCL), and subjective benefits were measured using the Glasgow Children's Benefit Inventory (GCBI) questionnaire. RESULTS:The average unaided and aided hearing thresholds measured using VRA were 73.8 ± 5.1 dB HL and 30.5 ± 6.0 dB HL, respectively. The total MAIS scores of the patients were 89.6 ± 9.6% and 93.0 ± 8.8% of normal hearing children at the last follow-up. The CAP scores of the two groups were 6.5 ± 1.3 and 6.9 ± 0.3, respectively. The mean MUSS score of the patients and the control group were 31.9 ± 7.0 and 34.3 ± 6.0, respectively. The mean SIR score of the two groups were 4.6 ± 0.7 and 4.8 ± 0.4. CBCL found that only two patients could be considered problematic psychosocially. The average benefit score on the GCBI was 32.9 ± 29.3. CONCLUSIONS:Softband BCHD significantly improved auditory development in patients with bilateral microtia, with speech development reaching the level of normal hearing peers. No significant behavioral problems were found in the patients, with subjective evaluations showing that softband BCHD improved patient quality of life.

Sirtuin 6 (SIRT6), as a NAD + -dependent deacetylase, plays an indispensable role in the regulation of health and physiology. Loss of SIRT6 causes spontaneous colitis in mice and makes intestinal epithelial cells prone to stress. However, whether SIRT6 overexpression increases resistance to colitis remains unknown. Here, in vivo results demonstrated that SIRT6 overexpression attenuates DSS-induced colitis in terms of clinical manifestations, histopathological damage, loss of tight junction function and imbalanced intestinal microenvironment. Additionally, we also found that the activation of NF-κB and c-Jun induced by DSS is diminished by SIRT6 overexpression. Furthermore, SIRT6 may regulate TAK1 to inhibit NF-κB and c-Jun signaling. Thus, our findings highlight the protective effect of SIRT6 on colon, further supporting the perspective that SIRT6 may be a therapeutic target for intestine injury under stress.

Photoresist grating was fabricated by holography, and it was used in the mask of ion etching. The groovy depth of the etched glass grating was 1.6 ��m. The glass waveguide was formed by K +/Na + ion exchanging. The laser beam of 633nm was coupled in the waveguide by a prism at one end, then, it passed through the grating and came out of the waveguide at other end. In the experiment, the Bragg diffraction with several orders was observed. The first order Bragg diffraction had the highest efficiency of 90 percent.