Search

The development of reliable methods to diagnose acute kidney injury is essential to allow the adoption of early therapeutic interventions and evaluate their effectiveness. Based on the fact that kidney injury molecule-1 (KIM-1) expression levels in kidneys are markedly upregulated early after a damage event, here we developed a noninvasive KIM-1-based molecular imaging technique to detect kidney injury. First, we took advantage of a phage-display platform to select small peptides demonstrating a specific high binding affinity to KIM-1. The promising candidate was conjugated with fluorescent probes, and its imaging potential was validated

Targeting specific cell types in the mammalian inner ear is important for treating genetic hearing loss due to the different cell type-specific functions. Adeno-associated virus (AAV) is an efficient in vivo gene transfer vector, and it has demonstrated promise for treating genetic hearing loss. Although more than 100 AAV serotypes have been identified, few studies have investigated whether AAV can be distributed to specific inner ear cell types. Here we screened three EGFP-AAV reporter constructs (serotypes DJ, DJ8, and PHP.B) in the neonatal mammalian inner ear by injection via the round window membrane to determine the cellular specificity of the AAV vectors. Sensory hair cells, supporting cells, cells in Reissner’s membrane, interdental cells, and root cells were successfully transduced. Hair cells in the cochlear sensory epithelial region were the most frequently transduced cell type by all tested AAV serotypes. The recombinant DJ serotype most effectively transduced a range of cell types at a high rate. Our findings provide a basis for improving treatment of hereditary hearing loss using targeted AAV-mediated gene therapy. Keywords: gene therapy, genetic hearing loss, inner ear, adeno-associated virus, serotype

Hereditary hearing loss (HHL) is a common genetically heterogeneous disorder, which follows Mendelian inheritance in humans. Because of this heterogeneity, the identification of the causative gene of HHL by linkage analysis or Sanger sequencing have shown economic and temporal limitations. With recent advances in next-generation sequencing (NGS) techniques, rapid identification of a causative gene via massively parallel sequencing is now possible. We recruited a Korean family with three generations exhibiting autosomal dominant inheritance of hearing loss (HL), and the clinical information about this family revealed that there are no other symptoms accompanied with HL. To identify a causative mutation of HL in this family, we performed whole-exome sequencing of 4 family members, 3 affected and an unaffected. As the result, A novel splicing mutation, c.763+1G>T, in the solute carrier family 17, member 8 (SLC17A8) gene was identified in the patients, and the genotypes of the mutation were co-segregated with the phenotype of HL. Additionally, this mutation was not detected in 100 Koreans with normal hearing. Via NGS, we detected a novel splicing mutation that might influence the hearing ability within the patients with autosomal dominant non-syndromic HL. Our data suggests that this technique is a powerful tool to discover causative genetic factors of HL and facilitate diagnoses of the primary cause of HHL.

The human body consists of 37 trillion single cells represented by over 50 organs that are stitched together to make us who we are, yet we still have very little understanding about the basic units of our body: what cell types and states make up our organs both compositionally and spatially. Previous efforts to profile a wide range of human cell types have been attempted by the FANTOM and GTEx consortia. Now, with the advancement in genomic technologies, profiling the human body at single-cell resolution is possible and will generate an unprecedented wealth of data that will accelerate basic and clinical research with tangible applications to future medicine. To date, several major organs have been profiled, but the challenges lie in ways to integrate single-cell genomics data in a meaningful way. In recent years, several consortia have begun to introduce harmonization and equity in data collection and analysis. Herein, we introduce existing and nascent single-cell genomics consortia, and present benefits to necessitate single-cell genomic consortia in a regional environment to achieve the universal human cell reference dataset., Genomics: Collaborative efforts to map the human body—single cell at a time Profiling the human body at the single cell resolution will reveal a map to enable researchers to compare cell types that are found in healthy and disease tissues at a much finer resolution. Large international projects such as FANTOM (Functional ANnoTation Of the Mammalian genome) and GTEx (Genotype-Tissue Expression) have profiled gene expression in hundreds of human cell types. However, the data were obtained from ‘bulk’ samples. The possibility of sequencing large numbers of single cells at a reasonable cost offers the opportunity to create a much more detailed human cell reference map. Jay Shin, at the RIKEN Institute, Yokohama, Japan, and colleagues review rapidly growing single cell genomics consortia and highlight the advantages of initiating national collaborations to harmonize procedures and to obtain a more accurate representation of regional ethnic diversity.

ObjectiveThis study aimed to develop a new oral paclitaxel formulation (DHP23002) and to evaluate its absorption and antitumor effects in a pancreatic tumor mouse model.MethodsTo investigate the oral absorption of DHP23002, a newly developed lipid-based orally active paclitaxel formulation, a pharmacokinetic study of DHP23002, was conducted in mice (62.5 and 125 mg/kg). Moreover, to evaluate the antitumor effect of DHP23002 in pancreatic cancer treatment, the drug was administered to female athymic nude mice at 0 (vehicle), 25, 62.5, and 125 mg/kg on alternate days; the efficacy of the agent was compared with the efficacy of intravenous Taxol® injections at 10 mg/kg once per week. After 3 weeks of administration, tumor growth in mice belonging to each group was further monitored for 4 weeks after discontinuing medication. Moreover, to examine paclitaxel (DHP23002) accumulation in the tumor tissue, the amount of paclitaxel in tumor/blood was quantified using liquid chromatography with quadruple-TOF mass spectrometry.ResultsIn the mouse pharmacokinetic study, oral Taxol® showed a negligible absorption, whereas DHP23002 showed a high absorption rate dependent on dosage, with a bioavailability of approximately 40% at a dose of 62.5 mg/kg. In efficacy-related studies, DHP23002 administration at a dose of 25, 62.5, or 125 mg/kg on alternate days for 3 weeks showed a superior tumor inhibitory effect of 80%, 92%, and 97% in a xenograft mouse model, respectively, after 7 weeks. Paclitaxel accumulation in tumors persisted for >24 h in mice, when orally administered once at doses of 25, 62.5, and 125 mg/kg DHP23002.ConclusionOral chemotherapy with DHP23002 showed excellent absorption in animals owing to a strong antitumor activity in a pancreatic cancer mouse model. This demonstrates that paclitaxel is largely distributed and persists for a prolonged period at the tumor site owing to oral DHP23002 administration.

Aims: Methionine sulfoxide reductase B3 (MsrB3), which stereospecifically repairs methionine-R-sulfoxide, is an important Msr protein that is associated with auditory function in mammals. MsrB3 deficiency leads to profound congenital hearing loss due to the degeneration of stereociliary bundles and the apoptotic death of cochlear hair cells. In this study, we investigated a fundamental treatment strategy in an MsrB3 deficiency mouse model and confirmed the biological significance of MsrB3 in the inner ear using MsrB3 knockout (MsrB3−/−) mice. Results: We delivered a recombinant adeno-associated virus encoding the MsrB3 gene directly into the otocyst at embryonic day 12.5 using a transuterine approach. We observed hearing recovery in the treated ears of MsrB3−/− mice at postnatal day 28, and we confirmed MsrB3 mRNA and protein expression in cochlear extracts. Additionally, we demonstrated that the morphology of the stereociliary bundles in the rescued ears of MsrB3−/− mice was similar to those in MsrB3+/+ mice. Innovation: To our knowledge, this is the first study to demonstrate functional and morphological rescue of the hair cells of the inner ear in the MsrB3 deficiency mouse model of congenital genetic sensorineural hearing loss using an in utero, virus-mediated gene therapy approach. Conclusion: Our results provide insight into the role of MsrB3 in hearing function and bring us one step closer to hearing restoration as a fundamental therapy. Antioxid. Redox Signal. 24, 590–602.

Pannexins (Panx) are a family of proteins that share sequences with the invertebrate gap junction proteins, innexins, and have a similar structure to that of the vertebrate gap junction proteins, connexins. To date, the Panx family consists of 3 members, but their genetic sequences have only been completely determined in a few vertebrate species. Moreover, expression of the Panx family has been reported in several rodent tissues: Panx1 is ubiquitously expressed in mammals, whereas Panx2 and Panx3 expressions are more restricted. Although members of the Panx family have been detected in mammals, their genetic sequences in avian species have not yet been fully elucidated. Here, we obtained the full-length mRNA sequences of chicken PANX genes and evaluated the homology of the amino acids from these sequences with those of other species. Furthermore, PANX gene expression in several chicken tissues was investigated based on mRNA levels. PANX1 was detected in the brain, cochlea, chondrocytes, eye, lung, skin, and intestine, and PANX2 was expressed in the brain, eye, and intestine. PANX3 was observed in the cochlea, chondrocytes, and bone. In addition, expression of PANX3 was higher than PANX1 in the cochlea. Immunofluorescent staining revealed PANX1 in hair cells, as well as the supporting cells, ganglion neurons, and the tegmentum vasculosum in chickens, whereas PANX3 was only detected in the bone surrounding the cochlea. Overall, the results of this study provide the first identification and characterization of the sequence and expression of the PANX family in an avian species, and fundamental data for confirmation of Panx function.

Viral respiratory infections are common in horses, notably equine herpesvirus infection and equine influenza, which primarily initiate secondary bacterial respiratory infections such as strangles caused by Streptococcus equi equi. A decline in the production of stallions has been associated with these respiratory diseases leading to adverse financial implications. This study investigated the antibody responses against respiratory diseases in horses from Jeju Island a year after vaccination. A low level of equine herpesvirus type 1 (EHV-1) (11.36%) antibodies was detected from stallions, however a high level of EHV-4 (95.84%) antibodies was detected from horses without vaccination against this infection suggesting that EHV-4 is ubiquitous in this horse population. In case of equine influenza, ranch stallions showed low positive rate (12.06%) whereas stallions from Subtropical Livestock Research Institute displayed higher positive rate (81.32%). Antibody responses against equine influenza and strangles revealed positive rates of 26.32% and 55.12%, respectively. These findings may draw attention towards the importance of developing an improved disease prevention and/or immunization program that will effectively control respiratory diseases in horses.

The methionine sulfoxide reductase (Msr) family of proteins is a class of repair enzymes that reduce methionine-S (MsrA) or methionine-R (MsrB) sulfoxide to methionine. Recent studies have reported that mutations in the MSRB3 gene cause autosomal recessive hearing loss in humans, and in mice MsrB3 deficiency leads to profound hearing loss due to hair cell apoptosis and stereocilia degeneration. However, apart from MsrB3, studies on Msr proteins in the inner ear have not yet been reported. In this study, we identified and characterized Msr expression in the cochlea and vestibule. First, we confirmed RNA expression levels of Msr family members in the cochlea and vestibule using reverse transcription PCR and detected Msr family members in both tissues. We also conducted immunohistochemical staining to localize Msr family members within the cochlea and vestibule. In the cochlea, MsrA was detected in supporting cells, spiral ligament, spiral limbus, Reissner's membrane and the spiral ganglion. MsrB1 was specifically expressed in hair cells and the spiral ganglion. MsrB2 was noted in the spiral ganglion, tectorial membrane and stria vascularis. In the vestibule, MsrA and MsrB1 were detected in hair cells and the vestibular ganglion, while MsrB2 was restricted to the vestibular ganglion. In this study, we identified distinct distributions of Msr family members in the organ of Corti and hypothesized that MsrA, MsrB1 and MsrB2 protect proteins in the organ of Corti from oxidative stress.

Methionine sulfoxide reductase B3 (MsrB3) is a protein repair enzyme that specifically reduces methionine-R-sulfoxide to methionine. A recent genetic study showed that the MSRB3 gene is associated with autosomal recessive hearing loss in human deafness DFNB74. However, the precise role of MSRB3 in the auditory system and the pathogenesis of hearing loss have not yet been determined. This work is the first to generate MsrB3 knockout mice to elucidate the possible pathological mechanisms of hearing loss observed in DFNB74 patients. We found that homozygous MsrB3(-/-) mice were profoundly deaf and had largely unaffected vestibular function, whereas heterozygous MsrB3(+/-) mice exhibited normal hearing similar to that of wild-type mice. The MsrB3 protein is expressed in the sensory epithelia of the cochlear and vestibular tissues, beginning at E15.5 and E13.5, respectively. Interestingly, MsrB3 is densely localized at the base of stereocilia on the apical surface of auditory hair cells. MsrB3 deficiency led to progressive degeneration of stereociliary bundles starting at P8, followed by a loss of hair cells, resulting in profound deafness in MsrB3(-/-) mice. The hair cell loss appeared to be mediated by apoptotic cell death, which was measured using TUNEL and caspase 3 immunocytochemistry. Taken together, our data suggest that MsrB3 plays an essential role in maintaining the integrity of hair cells, possibly explaining the pathogenesis of DFNB74 deafness in humans caused by MSRB3 deficiency.

Auditory neuropathy spectrum disorder (ANSD) is caused by dys-synchronous auditory neural response as a result of impairment of the functions of the auditory nerve or inner hair cells, or synapses between inner hair cells and the auditory nerve. To identify a causative gene causing ANSD in the Korean population, we conducted gene screening of the OTOF, DIAPH3, and PJVK genes in 19 unrelated Korean patients with ANSD. A novel nonsense mutation (p.Y1064X) and a known pathogenic mutation (p.R1939Q) of the OTOF gene were identified in a patient as compound heterozygote. Pedigree analysis for these mutations showed co-segregation of mutation genotype and the disease in the family, and it supported that the p.Y1064X might be a novel genetic cause of autosomal recessive ANSD. A novel missense variant p.K1017R (c.3050A>G) in the DIAPH3 gene was also identified in the heterozygous state. In contrast, no mutation was detected in the PJVK gene. These results indicate that no major causative gene has been reported to date in the Korean population and that pathogenic mutations in undiscovered candidate genes may have an effect on ANSD.

Cell culture systems reproducing virus replication can serve as unique models for the discovery of novel bioactive molecules. Here, using a hepatitis C virus (HCV) cell culture system, we identified neoechinulin B (NeoB), a fungus-derived compound, as an inhibitor of the liver X receptor (LXR). NeoB was initially identified by chemical screening as a compound that impeded the production of infectious HCV. Genome-wide transcriptome analysis and reporter assays revealed that NeoB specifically inhibits LXR-mediated transcription. NeoB was also shown to interact directly with LXRs. Analysis of structural analogs suggested that the molecular interaction of NeoB with LXR correlated with the capacity to inactivate LXR-mediated transcription and to modulate lipid metabolism in hepatocytes. Our data strongly suggested that NeoB is a novel LXR antagonist. Analysis using NeoB as a bioprobe revealed that LXRs support HCV replication: LXR inactivation resulted in dispersion of double-membrane vesicles, putative viral replication sites. Indeed, cells treated with NeoB showed decreased replicative permissiveness for poliovirus, which also replicates in double-membrane vesicles, but not for dengue virus, which replicates via a distinct membrane compartment. Together, our data suggest that LXR-mediated transcription regulates the formation of virus-associated membrane compartments. Significantly, inhibition of LXRs by NeoB enhanced the activity of all known classes of anti-HCV agents, and NeoB showed especially strong synergy when combined with interferon or an HCV NS5A inhibitor. Thus, our chemical genetics analysis demonstrates the utility of the HCV cell culture system for identifying novel bioactive molecules and characterizing the virus-host interaction machinery. IMPORTANCE Hepatitis C virus (HCV) is highly dependent on host factors for efficient replication. In the present study, we used an HCV cell culture system to screen an uncharacterized chemical library. Our results identified neoechinulin B (NeoB) as a novel inhibitor of the liver X receptor (LXR). NeoB inhibited the induction of LXR-regulated genes and altered lipid metabolism. Intriguingly, our results indicated that LXRs are critical to the process of HCV replication: LXR inactivation by NeoB disrupted double-membrane vesicles, putative sites of viral replication. Moreover, NeoB augmented the antiviral activity of all known classes of currently approved anti-HCV agents without increasing cytotoxicity. Thus, our strategy directly links the identification of novel bioactive compounds to basic virology and the development of new antiviral agents.

Taste sensation plays a crucial role in selecting and ingesting foods with different qualities which convey information about their nutrient content and/or safety. Sweetness is one of the five modalities in humans and serves as an energy resource for metabolism. There are reports on allelic polymorphisms which influence perception of sweetness in mice and humans. Since the influence of genetic factors on taste disorder has not been studied, we investigated the association of genetic polymorphisms in TAS1R3 and guanine nucleotide binding protein, alpha transducing 3 (GNAT3) genes and taste disorder. A total of 150 individuals composed of 50 patients with taste disorder and 100 healthy controls were recruited for the study and PCR-mediated directing sequencing method was used to genotype for two different single nucleotide polymorphisms (SNPs) - rs307355 (T>C) and rs35744813 (T>C) in the TAS1R3 gene, and rs7792845 (T>C) and rs1524600 (C>T) in the the GNAT3 gene. The allele and genotype frequencies of rs307355 and rs35744813 in the TAS1R3 gene showed a significant association between patients with taste disorder (p

Sweetness plays an important role in providing calories and promoting appetite for food. Since it has been known that genetic factor(s) is involved in individual differences in taste sensitivity in humans, this study aimed to examine genetic variations of the TAS1R2 gene, one of the components for tasting sweet compounds, by using DNA sequencing analysis from 98 unrelated Korean subjects. As a result, 12 different single nucleotide polymorphisms (SNPs) were identified in the hTAS1R2 gene and most of them were nonsynonymous. Also, two novel SNPs were found for the first time in this study. It was noted that the frequencies of these SNPs were common in the Korean population. 20 different haplotypes with coding SNPs (cSNPs) were also found in this study. Three out of these haplotypes were common, showing frequencies of > 10%. The repertoire and frequencies of cSNPs and haplotypes in the hTAS1R2 gene will provide information that will help identify a functional ligand receptor common in the Korean population.

Mutations in the DFNA5 gene are known to cause autosomal dominant non-syndromic hearing loss (ADNSHL). To date, five DFNA5 mutations have been reported, all of which were different in the genomic level. In this study, we ascertained a Korean family with autosomal dominant, progressive and sensorineural hearing loss and performed linkage analysis that revealed linkage to the DFNA5 locus on chromosome 7. Sequence analysis of DFNA5 identified a 3-bp deletion in intron 7 (c.991-15_991-13del) as the cause of hearing loss in this family. As the same mutation had been reported in a large Chinese family segregating DFNA5 hearing loss, we compared their DFNA5 mutation-linked haplotype with that of the Korean family. We found a conserved haplotype, suggesting that the 3-bp deletion is derived from a single origin in these families. Our observation raises the possibility that this mutation may be a common cause of autosomal dominant progressive hearing loss in East Asians.

Hereditary nonsyndromic hearing loss (NSHL) is a highly heterogeneous disorder in humans. Mutations of the transmembrane channel-like (TMC1) gene have been identified as the genetic cause for both autosomal recessive (DFNB7/11) and autosomal dominant (DFNA36) nonsyndromic hearing loss. To evaluate the spectrum and frequency of mutation(s) caused by TMC1 gene in the Korean population, we have performed sequencing analysis of the PCR products amplified from genomic DNA of each proband in 193 unrelated families showing 30 autosomal dominant and 163 autosomal recessive inheritance patterns. As a result, we identified eight different novel sequence variations for the first time in this study, respectively. However, none of these showed co-segregation of phenotype in the families. Therefore, our study suggests that the TMC1 gene is not the cause of nonsyndromic hearing loss in the Korean population.

Mutations in five unconventional myosin genes have been associated with genetic hearing loss (HL). These genes encode the motor proteins myosin IA, IIIA, VI, VIIA and XVA. To date, most mutations in myosin genes have been found in the Caucasian population. In addition, only a few functional studies have been performed on the previously reported myosin mutations. We performed screening and functional studies for mutations in the MYO1A and MYO6 genes in Korean cases of autosomal dominant non-syndromic HL. We identified four novel heterozygous mutations in MYO6 . Three mutations (p.R825X, p.R991X and Q918fsX941) produce a premature truncation of the myosin VI protein. Another mutation, p.R205Q, was associated with diminished actin-activated ATPase activity and actin gliding velocity of myosin VI in an in vitro analysis. This finding is consistent with the results of protein modelling studies and corroborates the pathogenicity of this mutation in the MYO6 gene. One missense variant, p.R544W, was found in the MYO1A gene, and in silico analysis suggested that this variant has deleterious effects on protein function. This finding is consistent with the results of protein modelling studies and corroborates the pathogenic effect of this mutation in the MYO6 gene.

Pendred syndrome (PS) is an autosomal recessive disorder characterized by congenital bilateral sensorineural hearing loss, goiter, and incomplete iodide organification. Patients with PS also have structural anomalies of the inner ear such as enlarged vestibular aqueducts (EVA) and Mondini's malformation. The goiter, which is a major clinical manifestation of PS, usually develops around adolescence. PS is caused by biallelic mutations of the SLC26A4 gene, while nonsyndromic bilateral EVA is associated with zero or one SLC26A4 mutant allele. We report here a Korean family including a young female with PS who had goiter and progressive, fluctuating sensorineural hearing loss that could be partially recovered by oral steroid treatment. Genetic investigation revealed compound heterozygous mutations for p.R677AfsX11, a novel frameshift mutation, and p.H723R in the SLC26A4 gene. Our findings provide detailed information regarding the distribution of mutant alleles for PS and may serve as a foundation for studies to comprehend the genetic portion of syndromic hearing loss.

Background Mutations in the neurofibromatosis type 2 (NF2) tumor-suppressor gene have been identified in not only NF2-related tumors but also sporadic vestibular schwannomas (VS). This study investigated the genetic and epigenetic alterations in tumors and blood from 30 Korean patients with sporadic VS and correlated these alterations with tumor behavior. Methodology/Principal Findings NF2 gene mutations were detected using PCR and direct DNA sequencing and three highly polymorphic microsatellite DNA markers were used to assess the loss of heterozygosity (LOH) from chromosome 22. Aberrant hypermethylation of the CpG island of the NF2 gene was also analyzed. The tumor size, the clinical growth index, and the proliferative activity assessed using the Ki-67 labeling index were evaluated. We found 18 mutations in 16 cases of 30 schwannomas (53%). The mutations included eight frameshift mutations, seven nonsense mutations, one in-frame deletion, one splicing donor site, and one missense mutation. Nine patients (30%) showed allelic loss. No patient had aberrant hypermethylation of the NF2 gene and correlation between NF2 genetic alterations and tumor behavior was not observed in this study. Conclusions/Significance The molecular genetic changes in sporadic VS identified here included mutations and allelic loss, but no aberrant hypermethylation of the NF2 gene was detected. In addition, no clear genotype/phenotype correlation was identified. Therefore, it is likely that other factors contribute to tumor formation and growth.

Background Otologic manifestations are one of the most consistent findings of CHARGE syndrome found in more than 90%. Since genetic analysis of the CHD7 gene has rarely been performed in previous reports dealing with ear abnormalities, the genotypic spectrum of CHD7 mutations was analyzed in deaf patients with CHARGE syndrome, and the clinical considerations concerning auditory rehabilitation were investigated. Methods Nine Korean patients with CHARGE syndrome showing profound hearing loss and semicircular canal aplasia were included. All 38 exons of CHD7 were analyzed by direct sequencing. For splice site variations, in silico and exon-trapping analyses were performed to verify the pathogenicity of nucleotide variations. Clinical features and the outcome of auditory rehabilitation were also analyzed. Results Eight of 9 patients revealed alterations of the CHD7 gene including 3 frameshift, 2 nonsense, 2 splice site, and 1 missense mutations. Five of 9 patients were clinically diagnosed as atypical CHARGE syndrome but demonstrated various mutations of the CHD7 gene. One familial case showed intra-familial variability. Radiologic findings suggesting cochleovestibular nerve deficiency were identified in most of the patients. Of the 8 patients who underwent cochlear implantation, 5 patients demonstrated favorable outcome. Larger diameter of the cochleovestibular nerve on imaging and absence of severe mental retardation were factors related to better outcome after cochlear implantation rather than the type of CHD7 mutations. Auditory brainstem implantation was performed in two patients who did not benefit from cochlear implantation. Conclusions Genetic analysis of the CHD7 gene should be performed in cases with semicircular canal aplasia even when other typical features of CHARGE syndrome are absent. For auditory rehabilitation in CHARGE syndrome, cochlear implantation should be strongly recommended in selected cases with favorable prognostic factors. Auditory brainstem implantation may be a viable option in patients with CHARGE syndrome who have failed to benefit from cochlear implantation.

Received December 26, 2012Revised February 25, 2013Accepted March 8, 2013Addressor f correspondenceIn Seok Moon, MD, PhDDepartment of Otorhinolaryngology, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-752, KoreaTel +82-2-2228-3627Fax +82-2-393-0580E-mail ismoonmd@yuhs.ac