Evolutionarily conserved function of the even-skipped ortholog in insects revealed by gene knock-out analyses in Gryllus bimaculatus.

Comparing the developmental mechanisms of segmentation among insects with different modes of embryogenesis provides insights on how the function of segmentation genes evolved. Functional analysis of eve by genetic mutants shows that the Drosophila pair-rule gene, even-skipped (eve), contributes to initial segmental patterning. However, eve orthologs tends to have diverse functions in other insects. To compare the evolutionary functional divergence of this gene, we evaluated eve function in a phylogenetically basal insect, the cricket Gryllus bimaculatus. To investigate the phenotypic effects of eve gene knock-out, we generated CRISPR/Cas9 system-mediated mutant strains of the cricket. CRISPR/Cas9 mutagenesis of multiple independent sites in the eve coding region revealed that eve null mutant embryos were defective in forming the gnathal, thoracic, and abdominal segments, consequently shortening the anterior-posterior axis. In contrast, the structures of the anterior and posterior ends (e.g., antenna, labrum, and cercus) formed normally. Hox gene expression in the gnathal, thoracic, and abdominal segments was detected in the mutant embryos. Overall, this study showed that Gryllus eve plays an important role in embryonic elongation and the formation of segmental boundaries in the gnathal to abdominal region of crickets. In the light of studies on other species, the eve function shown in Gryllus might be ancestral in insects.

Auditory hair cell replacement and hearing improvement by Atoh1 gene therapy in deaf mammals.

In the mammalian auditory system, sensory cell loss resulting from aging, ototoxic drugs, infections, overstimulation and other causes is irreversible and leads to permanent sensorineural hearing loss. To restore hearing, it is necessary to generate new functional hair cells. One potential way to regenerate hair cells is to induce a phenotypic transdifferentiation of nonsensory cells that remain in the deaf cochlea. Here we report that Atoh1, a gene also known as Math1 encoding a basic helix-loop-helix transcription factor and key regulator of hair cell development, induces regeneration of hair cells and substantially improves hearing thresholds in the mature deaf inner ear after delivery to nonsensory cells through adenovectors. This is the first demonstration of cellular and functional repair in the organ of Corti of a mature deaf mammal. The data suggest a new therapeutic approach based on expressing crucial developmental genes for cellular and functional restoration in the damaged auditory epithelium and other sensory systems.

Gene transfer into guinea pig cochlea using adeno-associated virus vectors.

BACKGROUND: Several genes are candidates for treating inner ear diseases. For clinical applications, minimally invasive approaches to the inner ear are desirable along with minimal side-effects. METHODS: Adeno-associated virus (AAV) was used as a vector into the guinea pig inner ear. Six AAV-cytomegalovirus hybrids (AAV-2/1, -2/2, -2/5, -2/7, -2/8 and -2/9) were infused into perilymph of the cochlea basal turn, an approach that could be used in cochlear implant surgery. At 7 days after injection, distribution of gene expression, hearing and morphology were evaluated. Adenoviral vector was also used to compare distributions of gene expression. Moreover, distribution of cell surface receptors of AAV in the cochlea was examined using immunohistochemistry. RESULTS: Using the perilymphatic approach, adenovirus could be transferred to mesothelial cells lining the perilymph, but not sensory cells. Conversely, all AAV serotypes displayed tissue tropism to inner hair cells, with AAV-2/2 showing particularly efficient transfer to sensory cells. This tissue tropism of AAV could not be explained by the distribution of AAV receptors. Hearing and morphology were largely unaffected. CONCLUSIONS: Our results indicate that AAV vector can be safely applied to the inner ear and AAV-2/2 offers a good tool for transferring transgenes into sensory cells of the inner ear efficiently without toxicity.

Characterization and evaluation of graphene oxide scaffold for periodontal wound healing of class II furcation defects in dog.

INTRODUCTION: The 3-dimensional scaffold plays a key role in volume and quality of repair tissue in periodontal tissue engineering therapy. We fabricated a novel 3D collagen scaffold containing carbon-based 2-dimensional layered material, named graphene oxide (GO). The aim of this study was to characterize and assess GO scaffold for periodontal tissue healing of class II furcation defects in dog. MATERIALS AND METHODS: GO scaffolds were prepared by coating the surface of a 3D collagen sponge scaffold with GO dispersion. Scaffolds were characterized using cytotoxicity and tissue reactivity tests. In addition, GO scaffold was implanted into dog class II furcation defects and periodontal healing was investigated at 4 weeks postsurgery. RESULTS: GO scaffold exhibited low cytotoxicity and enhanced cellular ingrowth behavior and rat bone forming ability. In addition, GO scaffold stimulated healing of dog class II furcation defects. Periodontal attachment formation, including alveolar bone, periodontal ligament-like tissue, and cementum-like tissue, was significantly increased by GO scaffold implantation, compared with untreated scaffold. CONCLUSION: The results suggest that GO scaffold is biocompatible and possesses excellent bone and periodontal tissue formation ability. Therefore, GO scaffold would be beneficial for periodontal tissue engineering therapy.

Administration of amitriptyline attenuates noise-induced hearing loss via glial cell line-derived neurotrophic factor (GDNF) induction.

Antidepressant treatments have been described to induce neurotrophic factors (NTFs) and reverse the cell loss observed in rodent stress models. Amitriptyline (AT), a tricyclic antidepressant agent, has been reported in recent studies to induce glial cell line-derived neurotrophic factor (GDNF) synthesis and release in rat C6 glioblastoma cells. GDNF has shown protection against acoustic trauma in previous studies. Therefore, we investigated whether AT could induce GDNF synthesis in the cochlea and attenuate cochlea damage against acoustic trauma. We used Hartley guinea pigs and injected AT (30 mg/kg) or saline into the peritoneum. Subjects were exposed to 117 dB SPL octave band noise centered at 4 kHz for 24 h. Noise-induced hearing loss (NIHL) was assessed with auditory brain stem response (ABR) at 4, 8 and 16 kHz measured prior to the injection, 3 days and 7 days after noise exposure. For histological assessment, we observed the sensory epithelium using a surface preparation technique and assessed the quantitative hair cell (HC) damage. We evaluated GDNF synthesis with or without intense noise exposure at 3, 12 and 24 h after the administration of AT in the cochlea using Western blot analysis. GDNF expression was shown 3 h and 12 h after the injection without noise, whereas with noise the GDNF expression lasted for 24 h. The AT-administrated group showed significantly reduced ABR threshold shift and less HC damage than the saline-administrated group. These findings suggest that the administration of AT-induced GDNF levels in the cochlea and attenuated cochlea damage from NIHL.

Manipulating cell cycle regulation in the mature cochlea.

Sensorineural hearing loss, which is often caused by degeneration of hair cells in the auditory epithelium, is permanent because lost hair cells are not replaced. Several conceptual approaches can be used to place new hair cells in the auditory epithelium. One possibility is to enhance proliferation of non-sensory cells that remain in the deaf ear and induce transdifferentiation of some of these cells into the hair cell phenotype. Several genes, including p27(Kip1), have been shown to regulate proliferation and differentiation in the developing auditory epithelium. The role of p27(Kip1) in the mature ear is not well characterized. We now show that p27(Kip1) is present in the nuclei of non-sensory cells of the mature auditory epithelium. We determined that forced expression of Skp2 using a recombinant adenovirus vector, resulted in presence of BrdU-positive cells in the auditory epithelium. When SKP2 over-expression was combined with forced expression of Atoh1, ectopic hair cells were found in the auditory epithelium in greater numbers than were seen with Atoh1 alone. Skp2 over-expression alone did not result in ectopic hair cells. These findings suggest that the p27(Kip1) protein remains in the mature auditory epithelium and therefore p27(Kip1) can serve as a target for gene manipulation. The data also suggest that induced proliferation, by itself, does not generate new hair cells in the cochlea.

PCTK3/CDK18 regulates cell migration and adhesion by negatively modulating FAK activity.

PCTAIRE kinase 3 (PCTK3) is a member of the cyclin dependent kinase family, but its physiological function remains unknown. We previously reported that PCTK3-knockdown HEK293T cells showed actin accumulation at the leading edge, suggesting that PCTK3 is involved in the regulation of actin reorganization. In this study, we investigated the physiological function and downstream signal transduction molecules of PCTK3. PCTK3 knockdown in HEK293T cells increased cell motility and RhoA/Rho-associated kinase activity as compared with control cells. We also found that phosphorylation at residue Tyr-397 in focal adhesion kinase (FAK) was increased in PCTK3-knockdown cells. FAK phosphorylation at Tyr-397 was increased in response to fibronectin stimulation, whereas its phosphorylation was suppressed by PCTK3. In addition, excessive expression of PCTK3 led to the formation of filopodia during the early stages of cell adhesion in HeLa cells. These results indicate that PCTK3 controls actin cytoskeleton dynamics by negatively regulating the FAK/Rho signaling pathway.

Dose effects of beta-tricalcium phosphate nanoparticles on biocompatibility and bone conductive ability of three-dimensional collagen scaffolds.

Three-dimensional collagen scaffolds coated with beta-tricalcium phosphate (ß-TCP) nanoparticles reportedly exhibit good bioactivity and biodegradability. Dose effects of ß-TCP nanoparticles on biocompatibility and bone forming ability were then examined. Collagen scaffold was applied with 1, 5, 10, and 25 wt% ß-TCP nanoparticle dispersion and designated TCP1, TCP5, TCP10, and TCP25, respectively. Compressive strength, calcium ion release and enzyme resistance of scaffolds with ß-TCP nanoparticles applied increased with ß-TCP dose. TCP5 showed excellent cell-ingrowth behavior in rat subcutaneous tissue. When TCP10 was applied, osteoblastic cell proliferation and rat cranial bone augmentation were greater than for any other scaffold. The bone area of TCP10 was 7.7-fold greater than that of non-treated scaffold. In contrast, TCP25 consistently exhibited adverse biological effects. These results suggest that the application dose of ß-TCP nanoparticles affects the scaffold bioproperties; consequently, the bone conductive ability of TCP10 was remarkable.

Transfer of accelerated presbycusis by transplantation of bone marrow cells from senescence-accelerated mice.

Until now, there has been no effective therapy for chronic sensorineural hearing impairment. This study investigated the role of bone marrow cells (BMCs) in cochlear dysfunction. BALB/c mice (2 months of age), a non-presbycusis-prone mouse strain, were lethally irradiated and then transplanted with BMCs from SAMP1 mice (2 months of age), a presbycusis-prone mouse strain. Acceleration of age-related hearing loss, early degeneration of spiral ganglion cells (SGCs) and impairment of immune function were observed in the recipient mice as well as in the SAMP1 mice. However, no spiral ganglion cells of donor (SAMP1) origin were detected in the recipient mice. These results indicated that accelerated presbycusis, cochlear pathology, and immune dysfunction of SAMP1 mice can be transferred to BALB/c recipient mice using allogeneic bone marrow transplantation (BMT). However, although the BMCs themselves cannot differentiate into the spiral ganglion cells (SGCs), they indirectly cause the degeneration of the SGCs. Further studies into the relationship between the inner ear cells and BMCs are required.

p27(Kip1) deficiency causes organ of Corti pathology and hearing loss.

p27(Kip1) (p27) has been shown to inhibit several cyclin-dependent kinase molecules and to play a central role in regulating entry into the cell cycle. Once hair cells in the cochlea are formed, p27 is expressed in non-sensory cells of the organ of Corti and prevents their re-entry into the cell cycle. In one line of p27 deficient mice (p27(-/-)), cell division in the organ of Corti continues past its normal embryonic time, leading to continual production of cells in the organ of Corti. Here we report on the structure and function of the inner ear in another line of p27 deficient mice originating from the Memorial Sloan-Kettering Cancer Center. The deficiency in p27 expression of these mice is incomplete, as they retain expression of amino acids 52-197. We determined that mice homozygote for this mutation had severe hearing loss and their organ of Corti exhibited an increase in the number of inner and outer hair cells. There also was a marked increase in the number of supporting cells, with severe pathologies in pillar cells. These data show similarities between this p27(Kip1) mutation and another, previously reported null allele of this gene, and suggest that reducing the inhibition on the cell cycle in the organ of Corti leads to pathology and dysfunction. Manipulations to regulate the time and place of p27 inhibition will be necessary for inducing functionally useful hair cell regeneration.

Math1 gene transfer generates new cochlear hair cells in mature guinea pigs in vivo.

Hair cell loss in the mammalian cochlea is irreversible and results in permanent hearing loss. Math1, the basic helix-loop-helix transcription factor homolog of the Drosophila atonal gene, is a positive regulator of hair cell differentiation during cochlear development. Developing hair cells express Math1, and nonsensory cells do not. We set out to determine the outcome of overexpression of Math1 in nonsensory cells of the cochlea on the phenotype of these cells. We demonstrate that in vivo inoculation of adenovirus with the Math1 gene insert into the endolymph of the mature guinea pig cochlea results in Math1 overexpression in nonsensory cochlear cells, as evident from the presence of Math1 protein in supporting cells of the organ of Corti and in adjacent nonsensory epithelial cells. Math1 overexpression leads to the appearance of immature hair cells in the organ of Corti and new hair cells adjacent to the organ of Corti in the interdental cell, inner sulcus, and Hensen cell regions. Axons are extended from the bundle of auditory nerve toward some of the new hair cells, suggesting that the new cells attract auditory neurons. We conclude that nonsensory cells in the mature cochlea retain the competence to generate new hair cells after overexpression of Math1 in vivo and that Math1 is necessary and sufficient to direct hair cell differentiation in these mature nonsensory cells.

Sebaceous carcinoma of the parotid gland: a case report.

BACKGROUND: Primary sebaceous carcinoma of the parotid gland is extremely rare, and because of its rarity, clinicopathological characteristics and histogenesis are not fully understood. METHODS: Here, we report a patient who presented with a left infra-auricular painless mass. We present the histological features and discuss possible optimal treatments based on previous literature. RESULTS: The mass was suspected to be a myoepithelial tumor or possibly a pleomorphic adenoma. Initially, the mass was resected with preservation of the facial nerve, but this caused facial palsy. Because the histological examination showed a sebaceous carcinoma and a part of the mass could be remaining on the facial nerve, additional surgery was performed, and the facial nerve was reconstructed with cervical nerve. Follow-up after 7 months showed no sign of recurrence of metastasis. CONCLUSION: We encountered a rare sebaceous carcinoma of the parotid gland. Additional surgery was performed because preoperative diagnosis was difficult.

Glial cell line-derived neurotrophic factor and chronic electrical stimulation prevent VIII cranial nerve degeneration following denervation.

As with other cranial nerves and many CNS neurons, primary auditory neurons degenerate as a consequence of loss of input from their target cells, the inner hair cells (IHCs). Electrical stimulation (ES) of spiral ganglion cells (SGCs) has been shown to enhance their survival. Glial cell line-derived neurotrophic factor (GDNF) has also been shown to increase survival of SGCs following IHC loss. In this study, the combined effects of the GDNF transgene delivered by adenoviral vectors (Ad-GDNF) and ES were tested on SGCs after first eliminating the IHCs. Animal groups received Ad-GDNF or ES or both. Ad-GDNF was inoculated into the cochlea of guinea pigs after deafening, to overexpress human GDNF. ES-treated animals were implanted with a cochlear implant electrode and chronically stimulated. A third group of animals received both Ad-GDNF and ES (GDNF/ES). Electrically evoked auditory brainstem responses were recorded from ES-treated animals at the start and end of the stimulation period. Animals were sacrificed 43 days after deafening and their ears prepared for evaluation of IHC survival and SGC counts. Treated ears exhibited significantly greater SGC survival than nontreated ears. The GDNF/ES combination provided significantly better preservation of SGC density than either treatment alone. Insofar as ES parameters were optimized for maximal protection (saturated effect), the further augmentation of the protection by GDNF suggests that the mechanisms of GDNF- and ES-mediated SGC protection are, at least in part, independent. We suggest that GDNF/ES combined treatment in cochlear implant recipients will improve auditory perception. These findings may have implications for the prevention and treatment of other neurodegenerative processes. .

Strategies for replacing lost cochlear hair cells.

The auditory sensory epithelium is a mosaic composed of sensory (hair) cells and several types of non-sensory (supporting) cells. All these cells are highly differentiated in their structure and function. Mosaic epithelia (and other complex tissues) are generally formed by differentiation of distinct and specialized cell types from common progenitors. Most types of epithelial tissues maintain a population of undifferentiated (basal) cells which facilitate turnover (renewal) and repair, but this is not the case for the organ of Corti in the cochlea. Therefore, when cochlear hair cells are lost they cannot be replaced. Consequently, sensorineural hearing loss is permanent. In designing therapy for sensorineural deafness, the most important task is to find a way to generate new cochlear hair cells to replace lost cells.

Gene transfer into supporting cells of the organ of Corti.

To utilize the rapidly accumulating genetic information for developing new therapeutic technologies for inner ear disease, it is necessary to design technologies for expressing transgenes in the inner ear, especially in the organ of Corti. We examined the outcome of an adenovirus gene transfer into the organ of Corti via the scala media in guinea pigs. The transgene insert is the bacterial lacZ gene driven by a cytomegalovirus promoter. We demonstrate that the inoculation is detrimental to the hair cells that surround the site of inoculation, but the supporting cells in the organ of Corti survive and retain the ability to express the reporter transgene beta-gal. The ability to deliver transgenes that are expressed in the supporting cells is an important step in the development of clinically applicable treatments that involve hair cell regeneration.

Gene-based therapy for inner ear disease.

Environmental inner ear insults often lead to hair cell injury and loss. Therapeutic measures for the prevention of hair cell loss are currently limited. Several reports have demonstrated the applicability of growth factors for hair cell protection. The goal of the experiments presented here was to assess the protective capability of the human GDNF transgene against noise trauma in the guinea pig cochlea. The left ears of guinea pigs were inoculated with a recombinant adenovirus with a human GDNF insert (Ad.GDNF). Four days later, animals were exposed to noise trauma. One week later, animals were sacrificed and hair cells counted in the left (inoculated) and right (non-inoculated) ears. Auditory brainstem thresholds were measured before the inoculation and just prior to sacrifice. Control groups included inoculation with a reporter gene vector (Ad.lacZ) and Ad.GDNF in normal ears with no noise exposure. The results show that intracochlear inoculation with adenovirus into normal ears does not compromise hair cell counts and ABR thresholds. Both Ad.GDNF and Ad.lacZ vectors can protect the cochlear hair cells and hearing from the noise insult. The difference between the protection afforded by Ad.GDNF and that of the Ad.lacZ vector is not statistically significant. The mechanism of Ad.lacZ protection needs to be elucidated. The data demonstrate the general feasibility of gene therapy for over-expression of neurotrophic factors against noise trauma, and emphasize the complexity of the technique and the problems of variability between subjects.

Downregulation of N-methyl-D-aspartate receptor ζ1 subunit (GluN1) gene in inferior colliculus with aging.

Presbycusis is the impairment of auditory function associated with aging, which stems from peripheral cochlear lesions and degeneration of the central auditory process. The effect of age-induced peripheral hearing loss on the central auditory process is not fully understood. C57Bl/6 (C57) mice present accelerated peripheral hearing loss, which is well developed by middle-age and mimics the human presbycusis pattern. The aim of this study was to elucidate the molecular effects of peripheral hearing loss in the inferior colliculus (IC) with age between young and middle-aged C57 mice using cDNA microarray. Glutamate receptor ionotropic NMDA ζ1 (GluN1) exhibited the greatest decrease in the middle-aged group as determined using cDNA microarray and by further assessment using real-time PCR (qPCR). Histological assessment with in situ hybridization of GluN1 showed significantly decreased expression in all IC subdivisions of the middle-aged group. GluN1 is a receptor for excitatory neurotransmission, and significant downregulation of this gene may be subsequent to the decline of afferent input from the cochlea in aging C57 mice. Consequently, using the combination of microarray, qPCR, and in situ hybridization, we showed that the decline of GluN1 in the IC of aging animals might have a key role in the pathogenesis of presbycusis.

Spontaneous hair cell regeneration in the mouse utricle following gentamicin ototoxicity.

Whereas most epithelial tissues turn-over and regenerate after a traumatic lesion, this restorative ability is diminished in the sensory epithelia of the inner ear; it is absent in the cochlea and exists only in a limited capacity in the vestibular epithelium. The extent of regeneration in vestibular hair cells has been characterized for several mammalian species including guinea pig, rat, and chinchilla, but not yet in mouse. As the fundamental model species for investigating hereditary disease, the mouse can be studied using a wide variety of genetic and molecular tools. To design a mouse model for vestibular hair cell regeneration research, an aminoglycoside-induced method of complete hair cell elimination was developed in our lab and applied to the murine utricle. Loss of utricular hair cells was observed using scanning electron microscopy, and corroborated by a loss of fluorescent signal in utricles from transgenic mice with GFP-positive hair cells. Regenerative capability was characterized at several time points up to six months following insult. Using scanning electron microscopy, we observed that as early as two weeks after insult, a few immature hair cells, demonstrating the characteristic immature morphology indicative of regeneration, could be seen in the utricle. As time progressed, larger numbers of immature hair cells could be seen along with some mature cells resembling surface morphology of type II hair cells. By six months post-lesion, numerous regenerated hair cells were present in the utricle, however, neither their number nor their appearance was normal. A BrdU assay suggested that at least some of the regeneration of mouse vestibular hair cells involved mitosis. Our results demonstrate that the vestibular sensory epithelium in mice can spontaneously regenerate, elucidate the time course of this process, and identify involvement of mitosis in some cases. These data establish a road map of the murine vestibular regenerative process, which can be used for elucidating the molecular events that govern this process.