Construction and characterization of a functional variant hFGF7 with enhanced properties by circular permutation.

Human fibroblast growth factor 7 (hFGF7) is a member of the paracrine-acting FGF family and mediates various reactions such as wound healing, tissue homeostasis, and liver regeneration. These activities make it a plausible candidate for pharmaceutical applications as a drug. However, the low expression level and stability of the recombinant hFGF7 were known to be major hurdles for further applications. Here, the expression level and stability of hFGF7 were attempted to improve by changing the order of amino acids through circular permutation (CP), thereby expecting an alternative fate according to the N-end rule. CP-hFGF7 variants were constructed systematically by using putative amino acid residues in the loop region that avoided the disruption of the structural integrity especially in the functional motif. Among them, cp-hFGF7115-114 revealed a relatively higher expression level in the soluble fraction than the wild-type hFGF7 and was efficiently purified (7 mg L-1) to apparent homogeneity. The activity and stability of the purified variant cp-hFGF7115-114 were comparable or superior to that of the wild-type hFGF7, thereby strongly suggesting that CP could be an alternative tool for the functional expression of hFGF7 in Escherichia coli.

One-step metal affinity purification of recombinant hFGF19 without using tags.

Human fibroblast growth factor 19 (hFGF19) belongs to the endocrine FGF19 superfamily and is considered a potential agent to treat severe or relapsing nonalcoholic fatty liver disease. Numerous studies have confirmed the beneficial effects of this hormone on the related symptoms of the disease and attempts at producing recombinant proteins in various hosts are steadily proliferating. Recently, we reported that authentic hFGF19 can be solubly expressed through combining synonymous codon substitutions and co-expression with disulfide-bond isomerase (DsbC) in Escherichia coli. However, during purification, hFGF19 without the His-tag occasionally co-eluted with His-tagged DsbC when using metal affinity chromatography, thereby requiring auxiliary purification steps to achieve apparent homogeneity. This phenomenon provides evidence that hFGF19 specifically interacts with immobilized Ni2+, which can thus be used as an alternative tool for the purification of hFGF19. Consequently, we could simply and reproducibly purify hFGF19 from cell lysates by using Ni2+-immobilized metal affinity chromatography and stepwise gradient elution with imidazole.

OSBPL2 mutations impair autophagy and lead to hearing loss, potentially remedied by rapamycin.

Intracellular accumulation of mutant proteins causes proteinopathies, which lack targeted therapies. Autosomal dominant hearing loss (DFNA67) is caused by frameshift mutations in OSBPL2. Here, we show that DFNA67 is a toxic proteinopathy. Mutant OSBPL2 accumulated intracellularly and bound to macroautophagy/autophagy proteins. Consequently, its accumulation led to defective endolysosomal homeostasis and impaired autophagy. Transgenic mice expressing mutant OSBPL2 exhibited hearing loss, but osbpl2 knockout mice or transgenic mice expressing wild-type OSBPL2 did not. Rapamycin decreased the accumulation of mutant OSBPL2 and partially rescued hearing loss in mice. Rapamycin also partially improved hearing loss and tinnitus in individuals with DFNA67. Our findings indicate that dysfunctional autophagy is caused by mutant proteins in DFNA67; hence, we recommend rapamycin for DFNA67 treatment.Abbreviations: ABR: auditory brainstem response; ACTB: actin beta; CTSD: cathepsin D; dB: decibel; DFNA67: deafness non-syndromic autosomal dominant 67; DPOAE: distortion product otoacoustic emission; fs: frameshift; GFP: green fluorescent protein; HsQ53R-TG: human p.Q53Rfs*100-transgenic: HEK 293: human embryonic kidney 293; HFD: high-fat diet; KO: knockout; LAMP1: lysosomal associated membrane protein 1; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; NSHL: non-syndromic hearing loss; OHC: outer hair cells; OSBPL2: oxysterol binding protein-like 2; SEM: scanning electron microscopy; SGN: spiral ganglion neuron; SQSTM1/p62: sequestosome 1; TEM: transmission electron microscopy; TG: transgenic; WES: whole-exome sequencing; YUHL: Yonsei University Hearing Loss; WT: wild-type.

COCH-related autosomal dominant nonsyndromic hearing loss: a phenotype-genotype study.

This phenotype-genotype study aimed to investigate the extent of audioprofile variability related to cochlin major domains and to identify potential ethnic-specific differences associated with COCH-related hearing loss. Eight Korean families (26 cases) were diagnosed with COCH-related hearing loss by exome sequencing. Audiometric test results were combined with those from nine published East Asian families (20 cases) and compared with those from 38 European-descent families (277 cases). Audioprofiles were created by grouping audiometric test results into age ranges by age at testing and then averaging hearing loss thresholds by frequency within age ranges. The functional impact of the identified variants was assessed in vitro by examining the intracellular trafficking, secretion, and cleavage of cochlin. In both East Asian and European-descent families segregating COCH-related hearing loss, deafness-associated variants in non-LCCL domains of cochlin were associated with hearing loss that was more severe earlier in life than hearing loss caused by variants in the LCCL domain. Consistent with this phenotypic difference, functional studies demonstrated distinct pathogenic mechanisms for COCH variants in a domain-dependent manner; specifically, a cytotoxic effect was observed for the p.Phe230Leu variant, which is located in the vWFA1 domain. No ethnic-specific differences in hearing loss progression were observed, except for those attributable to an overrepresentation of presymptomatic cases in the European-descent cohort.

Mental Health and Quality of Life for Healthcare Workers in a University Hospital Under COVID-19.

OBJECTIVE: The aim of this study was to assess the psychosocial characteristics of the employees working at a university hospital and investigated the factors affecting their quality of life (QOL) under COVID-19. METHODS: This study enrolled 1,191 healthcare workers from a university hospital, including doctors, nurses, administrative officer and technicians. Besides demographic information, depression, anxiety, somatization, insomnia, resilience, and QOL were assessed. RESULTS: The nurses presented significantly higher scores for anxiety, depression and showed significantly higher insomnia scores and significantly lower resilience scores. The occupations showed significant differences in the QOL and sub-groups, including the overall quality of life and general health (F=4.774, p<0.001), psychological domain (F=6.230, p<0.001), and environment domain (F=5.254, p<0.001). There was a positive correlation between the QOL and resilience (r=0.608, p<0.01). However, depression (r=-0.502, p<0.01), anxiety (r=-0.425, p<0.01), somatization (r=-0.364, p<0.01), and insomnia (r=-0.385, p<0.01) showed negative correlations with the QOL. Resilience was the most important factor influencing the QOL. CONCLUSION: The results of this study showed that low resilience adversely affected the QOL and the mental health of the healthcare workers, which consequently had a direct effect on the quality of medical care given to patients.

A designed fusion tag for soluble expression and selective separation of extracellular domains of fibroblast growth factor receptors.

Fibroblast growth factor receptors (FGFRs) generate various transduction signals by interaction with fibroblast growth factors (FGFs) and are involved in various biological functions such as cell proliferation, migration, and differentiation. Malfunction of these proteins may lead to the development of various diseases, including cancer. Accordingly, FGFRs are considered an alternative therapeutic target for protein and/or gene therapy. However, the screening of antagonists or agonists of FGFRs is challenging due to their complex structural features associated with protein expression. Herein, we conducted the development of a protease-free cleavable tag (PFCT) for enhancing the solubility of difficult-to express protein by combining maltose-binding protein (MBP) and the C-terminal region of Npu intein. To validate the availability of the resulting tag for the functional production of extracellular domains of FGFRs (Ec_FGFRs), we performed fusion of PFCT with the N-terminus of Ec_FGFRs and analyzed the expression patterns. Almost all PFCT-Ec_FGFR fusion proteins were mainly detected in the soluble fraction except for Ec_FGFR4. Upon addition of the N-terminal region of Npu intein, approximately 85% of the PFCT-Ec_FGFRs was separated into PFCT and Ec_FGFR via intein-mediated cleavage. Additionally, the structural integrity of Ec_FGFR was confirmed by affinity purification using heparin column. Taken together, our study demonstrated that the PFCT could be used for soluble expression and selective separation of Ec_FGFRs.

The Korean Version of Fear of COVID-19 Scale: Psychometric Validation in the Korean Population.

OBJECTIVE: Coronavirus disease 2019 (COVID-19) has psychological effects such as anxiety and depression as well as direct infection in people. The Fear of COVID-19 scale is a scale that can measure anxiety related to COVID-19 in a short time. The purpose of this study was to verify the reliability and validity the Korean version of Fear of COVID-19 scale (KF-COVID-19S). METHODS: The data of total 186 normal adults and 17 patients were finally used for the statistical analysis. For internal consistency, Cronbach's α was calculated. For concurrent and discriminant validity, the correlations with the Hospital Anxiety and Depression scale (HADS), Patient Health Questionnaire-15 (PHQ-15), World Health Organization Quality of Life Assessment Instrument Brief Form (WHOQOLBREF) were analyzed. For construct validity, exploratory and confirmatory factor analysis were conducted. RESULTS: Cronbach alpha was 0.88. The two-factor model (factor 1: Physical fear, factor 2: Emotional fear) showed significantly positive correlations and appeared to be "good" fitness (CFI=0.906, IFI=0.907, NFI=0.902). CONCLUSION: The KF-COVID-19S can be a useful scale that can measure the physical and emotional fears associated with COVID-19 in a short time. Because the psychiatric patients are a more vulnerable group to the fear, it is thought that the KF-COVID-19S will help to determine the patient's level of anxiety and make a therapeutic plan for the underlying mental disorder.

Heterogeneity of MYO15A variants significantly determine the feasibility of acoustic stimulation with hearing aid and cochlear implant.

Autosomal recessive nonsyndromic hearing loss 3 (DFNB3) mainly leads to congenital and severe-to-profound hearing impairment, which is caused by variants in MYO15A. However, audiological heterogeneity in patients with DFNB3 hinders precision medicine in hearing rehabilitation. Here, we aimed to elucidate the heterogeneity of the auditory phenotypes of MYO15A variants according to the affected domain and the feasibilities for acoustic stimulation. We conducted whole-exome sequencing for 10 unrelated individuals from seven multiplex families with DFNB3; 11 MYO15A variants, including the novel frameshift c.900delT (p.Pro301Argfs*143) and nonsense c.4879G > T (p.Glu1627*) variants, were identified. In seven probands, residual hearing at low frequencies was significantly higher in the groups with one or two N-terminal frameshift variants in trans conformation compared to that in the group without these variants. This is consistent with the 56 individuals from the previously published reports that carried a varying number of N-terminal truncating variants in MYO15A. In addition, patients with missense variants in the second FERM domain had better hearing at low frequencies than patients without these variants. Subsequently, acoustic stimulation provided by devices such as hearing aids or cochlear implants was feasible in patients with one or two N-terminal truncating variants or a second FERM missense variant. In conclusion, N-terminal or second FERM variants in MYO15A allow the practical use of acoustic stimulation through hearing aids or electroacoustic stimulation for aural rehabilitation.

Soluble Expression of hFGF19 without Fusion Protein through Synonymous Codon Substitutions and DsbC Co-Expression in E. coli.

Human fibroblast growth factor 19 (hFGF19) is a difficult-to-express protein that is frequently fused with another protein for soluble expression. However, residual amino acids after cleavage with protease represent one of the major problems in therapeutic protein development. Here, we introduced synonymous codon substitutions in the N-terminal region encoding sequence of hFGF19 and co-expressed disulfide bond isomerase (ΔssDsbC) to functionally express hFGF19 without any fusion protein. Synonymous codon substitution significantly increased hFGF19 expression. Subsequent co-expression of ΔssDsbC with a selected variant of hFGF19 (scvhFGF19) further increased the proportion of soluble hFGF19 expression in Escherichia coli XL1-Blue. Both total and soluble scvhFGF19 expression increased remarkably in the alternative host, E. coli Origami 2 with mutated thioredoxin reductase and glutathione reductase. scvhFGF19 purification by anion exchange and heparin affinity chromatography resulted in a yield of 6.5 mg/L under normal induction conditions in flask culture. As such, a high cell density culture is expected to achieve an even higher yield. The biological activities of purified scvhFGF19 were assessed based on its ability to activate ERK1/2 signaling pathway in HepG2 hepatocarcinoma cells. In conclusion, the strategy described here may represent an efficient alternative process for the production of hFGF19 and/or related proteins.

DNAJC14 Ameliorates Inner Ear Degeneration in the DFNB4 Mouse Model.

The His723Arg (H723R) mutation in SLC26A4, encoding pendrin, is the most prevalent mutation in East Asia, resulting in DFNB4, an autosomal recessive type of genetic hearing loss. Although the main pathological mechanism of H723R was identified as a protein-folding defect in pendrin, there is still no curative treatment for associated hearing loss. Here, we show that H723R-pendrin expression and activity are rescued by activation of the chaperonin DNAJC14. In vitro, DNAJC14 was activated via Japanese encephalitis virus (JEV) inoculation, and toxin-attenuated JEV rescued the surface expression and anion exchange activity of H723R-pendrin. Human H723R-pendrin transgenic mice (hH723R Tg) were established in a mouse slc26a4 knockout background, in which only hH723R-pendrin was expressed in the inner ear (Pax2-Cre dependent) to mimic human DFNB4 pathology. Crossing hH723R Tg with DNAJC14-overexpressing mice resulted in reduced cochlear hydrops and more preserved outer hair cells in the cochlea compared to those in hH723R Tg mice. Furthermore, the stria vascularis and spiral ligament were thicker and KCNJ10 expression was increased with DNAJC14 overexpression; however, hearing function and enlarged endolymphatic hydrops were not recovered. These results indicate that DNAJC14 overexpression ameliorates the cochlear degeneration caused by misfolded pendrin and might be a potential therapeutic target for DFNB4.

Genetic Inheritance of Late-Onset, Down-Sloping Hearing Loss and Its Implications for Auditory Rehabilitation.

OBJECTIVES: Late-onset, down-sloping sensorineural hearing loss has many genetic and nongenetic etiologies, but the proportion of this commonly encountered type of hearing loss attributable to genetic causes is not well known. In this study, the authors performed genetic analysis using next-generation sequencing techniques in patients showing late-onset, down-sloping sensorineural hearing loss with preserved low-frequency hearing, and investigated the clinical implications of the variants identified. DESIGN: From a cohort of patients with hearing loss at a tertiary referral hospital, 18 unrelated probands with down-sloping sensorineural hearing loss of late onset were included in this study. Down-sloping hearing loss was defined as a mean low-frequency threshold at 250 Hz and 500 Hz less than or equal to 40 dB HL and a mean high-frequency threshold at 1, 2, and 4 kHz greater than 40 dB HL. The authors performed whole-exome sequencing and segregation analysis to identify the genetic causes and evaluated the outcomes of auditory rehabilitation in the patients. RESULTS: There were nine simplex and nine multiplex families included, in which the causative variants were found in six of 18 probands, demonstrating a detection rate of 33.3%. Various types of variants, including five novel and three known variants, were detected in the MYH14, MYH9, USH2A, COL11A2, and TMPRSS3 genes. The outcome of cochlear and middle ear implants in patients identified with pathogenic variants was satisfactory. There was no statistically significant difference between pathogenic variant-positive and pathogenic variant-negative groups in terms of onset age, family history of hearing loss, pure-tone threshold, or speech discrimination scores. CONCLUSIONS: The proportion of patients with late-onset, down-sloping hearing loss identified with potentially causative variants was unexpectedly high. Identification of the causative variants will offer insights on hearing loss progression and prognosis regarding various modes of auditory rehabilitation, as well as possible concomitant syndromic features.

Soluble overexpression of a flagellin derivative from Salmonella enterica using synonymous codon substitutions of 5'-coding region in Escherichia coli.

OBJECTIVE: To obtain a recombinant flagellin derivative CBLB502, expressed in functionally soluble form, the technology of library construction and screening of synonymous codon variants was employed, and its expression, solubility, and activity were assessed. RESULTS: We screened several synonymous codon variants scvCBLB502s with the enhanced solubility from the constructed library, harboring the random substitutions of the first ten amino acid residues of the parental CBLB502 with synonymous codons. Among them, scvCBLB502-5 was purified (> 8.4 mg/l) by single step procedure using an affinity chromatography without any ancillary treatment with protease inhibitor cocktail solution and/or boiling at 90 °C. Subsequent study showed that the recombinant protein scvCBLB502-5 distinctly induced the TLR5 (Toll-Like Receptor 5)-mediated NF-κB activation and also IL-8 production in HEK293-hTLR5 cells. CONCLUSION: Results showed that scvCBLB502-5, engineered through the synonymous codon substitutions, was easily expressed in functionally soluble form and maintained the proper folding to be recognized by TLR5, as an inducer for pathogen-associated molecular pattern (PAMP).

Rare KCNQ4 variants found in public databases underlie impaired channel activity that may contribute to hearing impairment.

KCNQ4 is frequently mutated in autosomal dominant non-syndromic hearing loss (NSHL), a typically late-onset, initially high-frequency loss that progresses over time (DFNA2). Most KCNQ4 mutations linked to hearing loss are clustered around the pore region of the protein and lead to loss of KCNQ4-mediated potassium currents. To understand the contribution of KCNQ4 variants to NSHL, we surveyed public databases and found 17 loss-of-function and six missense KCNQ4 variants affecting amino acids around the pore region. The missense variants have not been reported as pathogenic and are present at a low frequency (minor allele frequency < 0.0005) in the population. We examined the functional impact of these variants, which, interestingly, induced a reduction in potassium channel activity without altering expression or trafficking of the channel protein, being functionally similar to DFNA2-associated KCNQ4 mutations. Therefore, these variants may be risk factors for late-onset hearing loss, and individuals harboring any one of these variants may develop hearing loss during adulthood. Reduced channel activity could be rescued by KCNQ activators, suggesting the possibility of medical intervention. These findings indicate that KCNQ4 variants may contribute more to late-onset NSHL than expected, and therefore, genetic screening for this gene is important for the prevention and treatment of NSHL.

A novel early truncation mutation in OTOG causes prelingual mild hearing loss without vestibular dysfunction.

OTOG was identified as a nonsyndrmoic hearing loss gene in 2012 in two families with nonprogressive mild-to-moderate hearing loss. However, no further literature have this gene for nonsyndromic hearing loss. Furthermore, it is still unclear whether vestibular impairment is involved or not in patients with mutations in OTOG. This study presents a validated second report for homozygous causative mutations in OTOG of mild hearing loss. Whole exome sequencing (WES) was performed in a five-year-old male proband with mild hearing loss. The analysis of WES revealed a homozygous truncating mutation (c.330C > G; p.Tyr110*) in OTOG. The identified novel mutation, p.Tyr110*, leads to a null allele based on the fact that early truncated protein contains no functional domain of otogelin. While defects in otogelin previously reported to result in hearing loss and vestibular dysfunction, p.Tyr110* only caused nonsydromic and nonprogressive hearing loss without any vestibular impairment, indicating that vestibular phenotype would be variable. Given that mild hearing loss is not easy to be detected early, mutations of OTOG may be more prevalent than reported. Therefore, genetic evaluation for OTOG should be considered in children with mild hearing loss with/without vestibular dysfunction.

A recurrent mutation in KCNQ4 in Korean families with nonsyndromic hearing loss and rescue of the channel activity by KCNQ activators.

Mutations in potassium voltage-gated channel subfamily Q member 4 (KCNQ4) are etiologically linked to nonsyndromic hearing loss (NSHL), deafness nonsyndromic autosomal dominant 2 (DFNA2). To identify causative mutations of hearing loss in 98 Korean families, we performed whole exome sequencing. In four independent families with NSHL, we identified a cosegregating heterozygous missense mutation, c.140T>C (p.Leu47Pro), in KCNQ4. Individuals with the c.140T>C KCNQ4 mutation shared a haplotype flanking the mutated nucleotide, suggesting that this mutation may have arisen from a common ancestor in Korea. The mutant KCNQ4 protein could reach the plasma membrane and interact with wild-type (WT) KCNQ4, excluding a trafficking defect; however, it exhibited significantly decreased voltage-gated potassium channel activity and fast deactivation kinetics compared with WT KCNQ4. In addition, when co-expressed with WT KCNQ4, mutant KCNQ4 protein exerted a dominant-negative effect. Interestingly, the channel activity of the p.Leu47Pro KCNQ4 protein was rescued by the KCNQ activators MaxiPost and zinc pyrithione. The c.140T>C (p.Leu47Pro) mutation in KCNQ4 causes progressive NSHL; however, the defective channel activity of the mutant protein can be rescued using channel activators. Hence, in individuals with the c.140T>C mutation, NSHL is potentially treatable, or its progression may be delayed by KCNQ activators.

Whole-exome sequencing identifies two novel mutations in KCNQ4 in individuals with nonsyndromic hearing loss.

Mutations in potassium voltage-gated channel subfamily Q member 4 (KCNQ4) are etiologically linked to a type of nonsyndromic hearing loss, deafness nonsyndromic autosomal dominant 2 (DFNA2). We performed whole-exome sequencing for 98 families with hearing loss and found mutations in KCNQ4 in five families. In this study, we characterized two novel mutations in KCNQ4: a missense mutation (c.796G>T; p.Asp266Tyr) and an in-frame deletion mutation (c.259_267del; p.Val87_Asn89del). p.Asp266Tyr located in the channel pore region resulted in early onset and moderate hearing loss, whereas p.Val87_Asn89del located in the N-terminal cytoplasmic region resulted in late onset and high frequency-specific hearing loss. When heterologously expressed in HEK 293 T cells, both mutant proteins did not show defects in protein trafficking to the plasma membrane or in interactions with wild-type (WT) KCNQ4 channels. Patch-clamp analysis demonstrated that both p.Asp266Tyr and p.Val87_Asn89del mutant channels lost conductance and were completely unresponsive to KCNQ activators, such as retigabine, zinc pyrithione, and ML213. Channels assembled from WT-p.Asp266Tyr concatemers, like those from WT-WT concatemers, exhibited conductance and responsiveness to KCNQ activators. However, channels assembled from WT-p.Val87_Asn89del concatemers showed impaired conductance, suggesting that p.Val87_Asn89del caused complete loss-of-function with a strong dominant-negative effect on functional WT channels. Therefore, the main pathological mechanism may be related to loss of K+ channel activity, not defects in trafficking.

ST-Elevation Myocardial Infarction in Coronary Ectasia: A Case Report.

Coronary artery ectasia (CAE) is localized or diffuse dilatation of the coronary artery lumen exceeding the diameter of adjacent healthy reference segments by 1.5 times. It is a rare phenomenon and incidence ranges from 1 to 5% in patients undergoing angiography. We report a case of a 58-year-old man with atherosclerotic CAE who experienced ST-elevation myocardial infarction (STEMI) despite prophylactic antiplatelet therapy. He was successfully treated with IV eptifibatide and aspiration thrombectomy. We reviewed the literature of CAE presentation, etiology and treatment and discussed the most appropriate antithrombotic therapy to prevent STEMIs in patients with CAE. While the current literature appears to favour prophylactic antiplatelet and anticoagulant in these patients, more studies are needed to determine the optimal form and duration of antithrombotic therapy. Currently, there is no gold standard treatment for CAE and further prospective and randomized-controlled studies are needed to guide recommendations.

Inhibition of Wntless/GPR177 suppresses gastric tumorigenesis.

Wntless/GPR177 functions as WNT ligand carrier protein and activator of WNT/ß-catenin signaling, however, its molecular role in gastric cancer (GC) has remained elusive. We investigated the role of GPR177 in gastric tumorigenesis and provided the therapeutic potential of a clinical development of anti-GPR177 monoclonal antibodies. GPR177 mRNA expression was assessed in GC transcriptome data sets (GSE15459, n = 184; GSE66229, n = 300); protein expression was assessed in independent patient tumor tissues (Yonsei TMA, n = 909). GPR177 expression were associated with unfavorable prognosis [log-rank test, GSE15459 (P = 0.00736), GSE66229 (P = 0.0142), and Yonsei TMA (P = 0.0334)] and identified as an independent risk predictor of clinical outcomes: GSE15459 [hazard ratio (HR) 1.731 (95% confidence interval; CI; 1.103- 2.715), P = 0.017], GSE66229 [HR 1.54 (95% CI, 1.10-2.151), P = 0.011], and Yonsei TMA [HR 1.254 (95% CI, 1.049- 1.500), P = 0.013]. Either antibody treatment or GPR177 knockdown suppressed proliferation of GC cells and sensitized cells to apoptosis. And also inhibition of GPR177 suppresses in vitro and in vivo tumorogenesis in GC cells and inhibits WNT/ß-catenin signaling. Finally, targeting and inhibition of GPR177 with antibody suppressed tumorigenesis in PDX model. Together, these results suggest GPR177 as a novel candidate for prognostic marker as well as a promising target for treatment of GC patients. [BMB Reports 2018; 51(5): 255-260].

Survival of Cancer Stem-Like Cells Under Metabolic Stress via CaMK2α-mediated Upregulation of Sarco/Endoplasmic Reticulum Calcium ATPase Expression.

Purpose: Cancer cells grow in an unfavorable metabolic milieu in the tumor microenvironment and are constantly exposed to metabolic stress such as chronic nutrient depletion. Cancer stem-like cells (CSC) are intrinsically resistant to metabolic stress, thereby surviving nutrient insufficiency and driving more malignant tumor progression. In this study, we aimed to demonstrate the potential mechanisms by which CSCs avoid Ca2+-dependent apoptosis during glucose deprivation.Experimental Design: We investigated cell viability and apoptosis under glucose deprivation, performed genome-wide transcriptional profiling of paired CSCs and parental cells, studied the effect of calcium/calmodulin-dependent protein kinase 2 alpha (CaMK2α) gene knockdown, and investigated the role of nuclear factor kappa B (NFκB) in CSCs during time-dependent Ca2+-mediated and glucose deprivation-induced apoptosis. We also observed the effect of combined treatment with 2-deoxy-d-glucose, a metabolic inhibitor that mimics glucose deprivation conditions in mouse xenograft models, and thapsigargin, a specific inhibitor of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA).Results: We demonstrated the coordinated upregulation of SERCA in CSCs. SERCA, in turn, is transcriptionally regulated by CaMK2α via NFκB activation. Combined treatment with 2-deoxy-d-glucose and thapsigargin, a specific inhibitor of SERCA, significantly reduced tumor growth compared with that in untreated control animals or those treated with the metabolic inhibitor alone.Conclusions: The current study provides compelling evidence that CaMK2α acts as a key antiapoptosis regulator in metabolic stress-resistant CSCs by activating NFκB. The latter induces expression of SERCA, allowing survival in glucose-deprived conditions. Importantly, our combination therapeutic strategy provides a novel approach for the clinical application of CSC treatment. Clin Cancer Res; 24(7); 1677-90. ©2017 AACR.

Whole-exome sequencing identified a missense mutation in WFS1 causing low-frequency hearing loss: a case report.

BACKGROUND: Low-frequency nonsyndromic hearing loss (LF-NSHL) is a rare, inherited disorder. Here, we report a family with LF-NSHL in whom a missense mutation was found in the Wolfram syndrome 1 (WFS1) gene. CASE PRESENTATION: Family members underwent audiological and imaging evaluations, including pure tone audiometry and temporal bone computed tomography. Blood samples were collected from two affected and two unaffected subjects. To determine the genetic background of hearing loss in this family, genetic analysis was performed using whole-exome sequencing. Among 553 missense variants, c.2419A → C (p.Ser807Arg) in WFS1 remained after filtering and inspection of whole-exome sequencing data. This missense mutation segregated with affected status and demonstrated an alteration to an evolutionarily conserved amino acid residue. Audiological evaluation of the affected subjects revealed nonprogressive LF-NSHL, with early onset at 10 years of age, but not to a profound level. CONCLUSION: This is the second report to describe a pathological mutation in WFS1 among Korean patients and the second to describe the mutation in a different ethnic background. Given that the mutation was found in independent families, p.S807R possibly appears to be a "hot spot" in WFS1, which is associated with LF-NSHL.