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BACKGROUND: The discovery of effective treatments for major depressive disorder (MDD) may help target different brain pathways. Invasive vagus nerve stimulation (VNS) is an effective neuromodulation technique for the treatment of MDD; however, the effectiveness of the noninvasive technique, transauricular VNS (taVNS), remains unknown. Moreover, a mechanistic understanding of the neural effects behind its biological and therapeutic effects is lacking. This review aimed to evaluate the clinical evidence and the neural and anti-inflammatory effects of taVNS in MDD. METHODS: Two searches were conducted using a systematic search strategy reviewed the clinical efficacy and neural connectivity of taVNS in MDD in humans and evaluated the changes in inflammatory markers after taVNS in humans or animal models of depression. A risk of bias assessment was performed in all human studies. RESULTS: Only 5 studies evaluated the effects of taVNS in patients with depression. Although the studies demonstrated the efficacy of taVNS in treating depression, they used heterogeneous methodologies and limited data, thus preventing the conduct of pooled quantitative analyses. Pooled analysis could not be performed for studies that investigated the modulation of connectivity between brain areas; of the 6 publications, 5 were based on the same experiment. The animal studies that analyzed the presence of inflammatory markers showed a reduction in the level of pro-inflammatory cytokines or receptor expression. CONCLUSIONS: Data on the clinical efficacy of taVNS in the treatment of MDD are limited. Although these studies showed positive results, no conclusions can be drawn regarding this topic considering the heterogeneity of these studies, as in the case of functional connectivity studies. Based on animal studies, the application of taVNS causes a decrease in the level of inflammatory factors in different parts of the brain, which also regulate the immune system. Therefore, further studies are needed to understand the effects of taVNS in patients with MDD.
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Transtorno Depressivo Maior , Estimulação do Nervo Vago , Animais , Humanos , Transtorno Depressivo Maior/terapia , Estimulação do Nervo Vago/métodos , Encéfalo , Resultado do Tratamento , Nervo Vago/fisiologiaRESUMO
Bilateral transcutaneous auricular vagus nerve stimulation (taVNS) - a non-invasive neuromodulation technique - has been investigated as a safe and feasible technique to treat many neuropsychiatric conditions. such as epilepsy, depression, anxiety, and chronic pain. Our aim is to investigate the effect of taVNS on neurophysiological processes during emotional and Go/No-Go tasks, and changes in frontal alpha asymmetry. We performed a randomized, double-blind, sham-controlled trial with 44 healthy individuals who were allocated into two groups (the active taVNS group and the sham taVNS group). Subjects received one session of taVNS (active or sham) for 60 min. QEEG was recorded before and after the interventions, and the subjects were assessed while exposed to emotional conditions with sad and happy facial expressions, followed by a Go/No-Go trial. The results demonstrated a significant increase in N2 amplitude in the No-Go condition for the active taVNS post-intervention compared to the sham taVNS after adjusting by handedness, mood, and fatigue levels (p = 0.046), significantly reduced ERD during sad conditions after treatment (p = 0.037), and increased frontal alpha asymmetry towards the right frontal hemisphere during the emotional task condition (p = 0.046). Finally, we observed an interesting neural signature in this study that suggests a bottom-up modulation from brainstem/subcortical to cortical areas as characterized by improved lateralization of alpha oscillations towards the frontal right hemisphere, and changes in ERP during emotional and Go/No-Go tasks that suggests a better subcortical response to the tasks. Such bottom-up effects may mediate some of the clinical effects of taVNS.
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Eletroencefalografia , Emoções , Estimulação do Nervo Vago , Humanos , Masculino , Feminino , Adulto , Emoções/fisiologia , Estimulação do Nervo Vago/métodos , Método Duplo-Cego , Adulto Jovem , Eletroencefalografia/métodos , Função Executiva/fisiologia , Ritmo alfa/fisiologia , Potenciais Evocados/fisiologia , Estimulação Elétrica Nervosa Transcutânea/métodos , Inibição PsicológicaRESUMO
BACKGROUND: Tinnitus is a bothersome condition associated with various symptoms. However, the mechanisms of tinnitus are still uncertain, and a standardized assessment of the diagnostic criteria for tinnitus is required. We aimed to reach a consensus on diagnosing tinnitus with professional experts by conducting a Delphi study with systematic review of the literature. METHODS: Twenty-six experts in managing tinnitus in Korea were recruited, and a two-round modified Delphi study was performed online. The experts evaluated the level of agreement of potential criteria for tinnitus using a scale of 1-9. After the survey, a consensus meeting was held to establish agreement on the results obtained from the Delphi process. Consensus was defined when over 70% of the participants scored 7-9 (agreement) and fewer than 15% scored 1-3 (disagreement). To analyze the responses of the Delphi survey, the content validity ratio and Kendall's coefficient of concordance were evaluated. RESULTS: Consensus was reached for 22 of the 38 statements. For the definition of tinnitus, 10 out of 17 statements reached consensus, with three statements achieving complete agreement including; 1) Tinnitus is a conscious perception of an auditory sensation in the absence of a corresponding external stimulus, 2) Tinnitus can affect one's quality of life, and 3) Tinnitus can be associated with hearing disorders including sensorineural hearing loss, vestibular schwannoma, Meniere's disease, otosclerosis, and others. For the classification of tinnitus, 11 out of 18 statements reached consensus. The participants highly agreed with statements such as; 1) Vascular origin is expected in pulse-synchronous tinnitus, and 2) Tinnitus can be divided into acute or chronic tinnitus. Among three statements on the diagnostic tests for tinnitus only Statement 3, "There are no reliable biomarkers for sensory or emotional factors of tinnitus." reached consensus. All participants agreed to perform pure-tone audiometry and tinnitus questionnaires, including the Tinnitus Handicap Inventory and Tinnitus Questionnaire. CONCLUSION: We used a modified Delphi method to establish a consensus-based definition, a classification, and diagnostic tests for tinnitus. The expert panel reached agreement for several statements, with a high level of consensus. This may provide practical information for clinicians in managing tinnitus.
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Consenso , Técnica Delphi , Zumbido , Zumbido/diagnóstico , Humanos , República da Coreia , Inquéritos e Questionários , Qualidade de Vida , Feminino , MasculinoRESUMO
OBJECTIVE: Vagus nerve stimulation (VNS) has recently been reported to exert additional benefits for functional recovery in patients with brain injury. However, the mechanisms underlying these effects have not yet been elucidated. This study examined the effects of transcutaneous auricular VNS (taVNS) on cortical excitability in healthy adults. MATERIALS AND METHODS: We recorded subthreshold and suprathreshold single- and paired-pulse motor-evoked potentials (MEPs) in the right-hand muscles of 16 healthy adults by stimulating the left primary motor cortex. Interstimulus intervals were set at 2 milliseconds and 3 milliseconds for intracortical inhibition (ICI), and 10 milliseconds and 15 milliseconds for intracortical facilitation (ICF). taVNS was applied to the cymba conchae of both ears for 30 minutes. The intensity of taVNS was set to a maximum tolerable level of 1.95 mA. MEPs were measured before stimulation, 20 minutes after the beginning of the stimulation, and 10 minutes after the cessation of stimulation. RESULTS: The participants' age was 33.25 ± 7.08 years, and nine of 16 were male. No statistically significant changes were observed in the mean values of the single-pulse MEPs before, during, or after stimulation. Although the ICF showed an increasing trend after stimulation, the changes in ICI and ICF were not significant, primarily because of the substantial interindividual variability. CONCLUSIONS: The effect of taVNS on cortical excitability varied in healthy adults. An increase in ICF was observed after taVNS, although the difference was not statistically significant. Our findings contribute to the understanding of the mechanisms by which taVNS is effective in patients with brain disorders.
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OBJECTIVE: We aimed to conduct a systematic review and meta-analysis assessing the antiinflammatory effects of various VNS methods while exploring multiple antiinflammatory pathways. MATERIALS AND METHODS: We included clinical trials that used electrical stimulation of the vagus nerve and assessed inflammatory markers up to October 2022. We excluded studies lacking control groups, those with combined interventions, or abstracts without full text. We adhered to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines and the Cochrane Handbook for Systematic Reviews. For each inflammatory marker, a random-effects meta-analysis using the inverse variance method was performed. Methods used include transcutaneous auricular VNS (taVNS), transcutaneous cervical VNS (tcVNS), invasive cervical VNS (iVNS), and electroacupuncture VNS (eaVNS). Main reported outcomes included tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-1ß, C-reactive protein (CRP), and IL-10. Risk of bias was evaluated using the Cochrane Collaboration Tool (RoB 2.0). RESULTS: This review included 15 studies, involving 597 patients. No statistically significant general VNS effect was observed on TNF-α, IL-6, and IL-1ß. However, CRP, IL-10, and interferon (IFN)-γ were significantly modulated by VNS across all methods. Subgroup analysis revealed specific stimulation techniques producing significant results, such as taVNS effects in IL-1ß and IL-10, and iVNS in IL-6, whereas tcVNS and eaVNS did not convey significant pooled results individually. Cumulative exposure to VNS, higher risk of bias, study design, and pulse width were identified as effect size predictors in our meta-regression models. CONCLUSIONS: Pooling all VNS techniques indicated the ability of VNS to modulate inflammatory markers such as CRP, IL-10, and IFN-γ. Individually, methods such as taVNS were effective in modulating IL-1ß and IL-10, whereas iVNS modulated IL-6. However, different VNS techniques should be separately analyzed in larger, homogeneous, and powerful studies to achieve a clearer and more consistent understanding of the effect of each VNS method on the inflammatory system.
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Trophoblasts play an important role in the regulation of the development and function of the placenta. Our recent study demonstrated the skin regeneration capacity of trophoblast-derived extracellular vesicles (EV). Here, we aimed to determine the potential of trophoblast-derived conditioned medium (TB-CM) in enhancing the osteogenic differentiation of bone marrow mesenchymal stem cells (MSCs). We found that TB-CM promoted the osteogenic differentiation of MSCs in a dose-dependent manner. Furthermore, it inhibited adipogenesis of MSCs. We also found that the primary trophoblast-derived conditioned medium (PTB-CM) significantly enhanced the proliferation and osteogenic differentiation of human MSCs. Our study demonstrated the regulatory mechanisms underlying the TB-CM-induced osteogenesis in MSCs. An upregulation of genes associated with cytokines/chemokines was observed. The treatment of MSCs with TB-CM stimulated osteogenesis by activating several biological processes, such as mitogen-activated protein kinase (MAPK) and bone morphogenetic protein 2 (BMP2) signaling. This study demonstrated the proliferative and osteogenic efficacies of the trophoblast-derived secretomes, suggesting their potential for use in clinical interventions for bone regeneration and treatment.
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Células-Tronco Mesenquimais , Osteogênese , Fosfatase Alcalina/metabolismo , Diferenciação Celular/genética , Células Cultivadas , Meios de Cultivo Condicionados/metabolismo , Meios de Cultivo Condicionados/farmacologia , Humanos , Células-Tronco Mesenquimais/metabolismo , Osteogênese/genética , Trofoblastos/metabolismoRESUMO
Native extracellular matrix (ECM) exhibits dynamic change in the ligand position. Herein, the ECM-emulating control and real-time monitoring of stem cell differentiation are demonstrated by ligand nanoassembly. The density of gold nanoassembly presenting cell-adhesive Arg-Gly-Asp (RGD) ligand on Fe3 O4 (magnetite) nanoparticle in nanostructures flexibly grafted to material is changed while keeping macroscale ligand density invariant. The ligand nanoassembly on the Fe3 O4 can be magnetically attracted to mediate rising and falling ligand movements via linker stretching and compression, respectively. High ligand nanoassembly density stimulates integrin ligation to activate the mechanosensing-assisted stem cell differentiation, which is monitored via in situ real-time electrochemical sensing. Magnetic control of rising and falling ligand movements hinders and promotes the adhesion-mediated mechanotransduction and differentiation of stem cells, respectively. These rising and falling ligand states yield the difference in the farthest distance (≈34.6 nm) of the RGD from material surface, thereby dynamically mimicking static long and short flexible linkers, which hinder and promote cell adhesion, respectively. Design of cytocompatible ligand nanoassemblies can be made with combinations of dimensions, shapes, and biomimetic ligands for remotely regulating stem cells for offering novel methodologies to advance regenerative therapies.
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Fenômenos Magnéticos , Mecanotransdução Celular , Adesão Celular , Diferenciação Celular , LigantesRESUMO
A Gram-stain-negative, strictly aerobic, non-flagellated, rod-shaped bacterium, designated GSB7T, was isolated from seawater collected at the Yellow Sea coast of South Korea. Catalase and oxidase activities were positive. Growth occurred at pH 6.0-9.0 (optimum pH 7.0), 10-40 °C (optimum 30 °C) and with 0-8% NaCl (optimum 1-2%). Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain GSB7T belonged to the genus Marivivens, showing the sequence similarities of 96.3, 96.1, and 96.0% with Marivivens niveibacter HSLHS2T, Limimaricola hongkongensis DSM17492T, and Marivivens donghaensis AM-4T, respectively. The respiratory quinone was ubiquinone-10 and the major fatty acids were summed feature 8 (C18:1 ω7c and/or C18:1 ω6c), C18:1 ω7c 11-methyl, C16:0 and C10:0 3-OH. The polar lipids comprised phosphatidylglycerol, diphosphatidylglycerol, one unidentified aminolipid, and five unidentified lipids. The DNA G + C content calculated from the whole-genome sequence was 60.6 mol%. On the basis of phenotypic, chemotaxonomic and genotypic characteristics presented in this study, strain GSB7T is suggested to represent a novel species of the genus Marivivens, for which the name Marivivens aquimaris sp. nov. is proposed. The type strain is GSB7T (= KCTC 82026T = JCM 34042T).
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Rhodobacteraceae , Água do Mar , Ácidos Graxos/análise , Fosfolipídeos/análise , Filogenia , RNA Ribossômico 16S/genética , República da Coreia , Rhodobacteraceae/classificação , Rhodobacteraceae/genética , Rhodobacteraceae/metabolismo , Água do Mar/microbiologia , Especificidade da EspécieRESUMO
Macrophages can associate with extracellular matrix (ECM) demonstrating nanosequenced cell-adhesive RGD ligand. In this study, we devised barcoded materials composed of RGD-coated gold and RGD-absent iron nanopatches to show various frequencies and position of RGD-coated nanopatches with similar areas of iron and RGD-gold nanopatches that maintain macroscale and nanoscale RGD density invariant. Iron patches were used for substrate coupling. Both large (low frequency) and externally positioned RGD-coated nanopatches stimulated robust attachment in macrophages, compared with small (high frequency) and internally positioned RGD-coated nanopatches, respectively, which mediate their regenerative/anti-inflammatory M2 polarization. The nanobarcodes exhibited stability in vivo. We shed light into designing ligand-engineered nanostructures in an external position to facilitate host cell attachment, thereby eliciting regenerative host responses.
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Macrófagos , Oligopeptídeos , Anti-Inflamatórios , Adesão Celular , Ouro/farmacologia , Ligantes , Oligopeptídeos/farmacologiaRESUMO
Developing materials with remote controllability of macroscale ligand presentation can mimic extracellular matrix (ECM) remodeling to regulate cellular adhesion in vivo. Herein, we designed charged mobile nanoligands with superparamagnetic nanomaterials amine-functionalized and conjugated with polyethylene glycol linker and negatively charged RGD ligand. We coupled negatively a charged nanoligand to a positively charged substrate by optimizing electrostatic interactions to allow reversible planar movement. We demonstrate the imaging of both macroscale and in situ nanoscale nanoligand movement by magnetically attracting charged nanoligand to manipulate macroscale ligand density. We show that in situ magnetic control of attracting charged nanoligand facilitates stem cell adhesion, both in vitro and in vivo, with reversible control. Furthermore, we unravel that in situ magnetic attraction of charged nanoligand stimulates mechanosensing-mediated differentiation of stem cells. This remote controllability of ECM-mimicking reversible ligand variations is promising for regulating diverse reparative cellular processes in vivo.
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Adesão Celular , Fenômenos Magnéticos , Oligopeptídeos , Células-Tronco , Diferenciação Celular , Matriz ExtracelularRESUMO
To date, placental trophoblasts have been of interest in the fields of obstetrics and gynecology, mainly due to their involvement in the formation of a connection between the mother and fetus that aids in placental development and fetal survival. However, the regenerative capacities of trophoblasts for application in regenerative medicine and tissue engineering are poorly understood. Here, we aim to determine the skin regeneration and anti-aging capacities of trophoblast-derived conditioned medium (TB-CM) and exosomes (TB-Exos) using human normal dermal fibroblasts (HNDFs). TB-CM and TB-Exos treatments significantly elevated the migration and proliferation potencies of HNDF cells in a dose- and time-dependent manner. When RNA sequencing (RNA-seq) was used to investigate the mechanism underlying TB-CM-induced cell migration on scratch-wounded HNDFs, the increased expression of genes associated with C-X-C motif ligand (CXCL) chemokines, toll-like receptors, and nuclear factor-kappa B (NF-κB) signaling was observed. Furthermore, treatment of intrinsically/extrinsically senescent HNDFs with TB-CM resulted in an enhanced rejuvenation of HNDFs via both protection and restoration processes. Gene expression of extracellular matrix components in the skin dermis significantly increased in TB-CM- and TB-Exos-treated HNDFs. These components are involved in the TB-CM and Exo-mediated regeneration and anti-aging of HNDFs. Thus, this study demonstrated the regenerative and anti-aging efficacies of trophoblast-derived secretomes, suggesting their potential for use in interventions for skin protection and treatment.
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Fibroblastos/citologia , Pele/citologia , Trofoblastos/citologia , Western Blotting , Linhagem Celular , Proliferação de Células/efeitos da radiação , Meios de Cultivo Condicionados/farmacologia , Exossomos/efeitos dos fármacos , Exossomos/metabolismo , Vesículas Extracelulares/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/efeitos da radiação , Humanos , RNA-Seq , Reação em Cadeia da Polimerase em Tempo Real , Trofoblastos/efeitos dos fármacos , Trofoblastos/efeitos da radiação , Raios UltravioletaRESUMO
BACKGROUND: Recent interest has been focused on the production of platform chemicals from renewable biomass due to increasing concerns on global warming and depletion of fossil fuel reserves. Microbial production of platform chemicals in biorefineries has been suggested to be a promising solution for these problems. Gamma-aminobutyrate (GABA), a versatile bulk chemical used in food and pharmaceutical industry, is also used as a key monomer for nylon 4. GABA can be biologically produced by decarboxylation of glutamate. RESULTS: In this study, we examined high glutamate-producing Corynebacterium glutamicum strains as hosts for enhanced production of GABA from glucose and xylose as carbon sources. An Escherichia coli gadB mutant with a broad pH range of activity and E. coli xylAB genes were expressed under the control of a synthetic H36 promoter. When empty fruit bunch (EFB) solution was used as carbon source (45 g/L glucose and 5 g/L xylose), 12.54 ± 0.07 g/L GABA was produced by recombinant C. glutamicum H36GD1852 expressing E. coli gadB mutant gene and xylAB genes. Batch fermentation of the same strain resulted in the production of 35.47 g/L of GABA when EFB solution was added to support 90 g/L glucose and 10 g/L xylose. CONCLUSIONS: This is the first report of GABA production by recombinant C. glutamicum strains from co-utilization of glucose and xylose from EFB solution. Recombinant C. glutamicum strains developed in this study should be useful for an efficient and sustainable production of GABA from lignocellulosic biomasses.
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Corynebacterium glutamicum/metabolismo , Frutas/química , Ácido gama-Aminobutírico/metabolismo , Fermentação , Ácido gama-Aminobutírico/biossínteseRESUMO
BACKGROUND: Dizziness is one of the most common complaints in medicine and a frequent symptom among older people. Dizziness has a considerable impact on life quality and is associated with high economic costs. The purpose of this study was to evaluate the prevalence of dizziness in the general population and to describe its clinical characteristics and associated factors. METHODS: The Korea National Health and Nutrition Examination Survey (KNHANES) is a cross-sectional survey of the civilian, non-institutionalized population of South Korea. We evaluated data for 12,653 participants (5,450 men and 7,203 women), aged 40 years and above, whom participated in the KNHANES between 2010 and 2012. RESULTS: In the age group over 40 years old, the 1-year prevalence of dizziness was 20.10%. Dizziness was more prevalent among women (25.18%) than among men (14.57%; P < 0.001) and the prevalence rate increased with age (P < 0.001). In multivariable analysis, female sex, older age, serum triglyceride level, experience of depression, limited functional status owing to visual acuity impairment, limited physical performance, smoking, alcohol consumption, and perception of stress were independently associated with dizziness. CONCLUSIONS: In our study, the prevalence of dizziness in the general population was 20.10%. There was a stronger relationship between dizziness and physical performance, chronic diseases, and health behaviors compared to that with otologic diseases. Interventions for dizziness should be approached in a multifactorial manner and an understanding of various factors is necessary for the prevention and management of this condition.
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Tontura/epidemiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos Transversais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Prevalência , República da Coreia/epidemiologia , Fatores de RiscoRESUMO
BACKGROUND: Inpatient suicide is one of the major issues related with hospital patient safety. Although there are many studies addressing suicide in the psychiatric unit, little is known about suicide in a medical setting. This study included the development and validation of a screening tool for the assessment of suicide risk, specialized for medical inpatients. METHODS: The preliminary questionnaire was based on review of previous suicide ideation scales, mood scales, and clinical experiences of psychiatrists and clinical psychologists in Korea. Initially, the questionnaire consisted of 12 questions. One hundred adult medical inpatients were asked to perform the questionnaire. Explorative factor analysis was used to examine construct validity. Concurrent validity was evaluated by comparison with the Korean versions of the Beck Scale for Suicide Ideation (BSI), the Beck Hopelessness Scale (BHS), and the Hospital Anxiety and Depression Scale (HADS). RESULTS: Five questions were removed from the preliminary questionnaire by explorative factor analysis and seven questions remained to comprise the Suicide Risk Scale for Medical inpatients (SRSM). Explorative factor analysis showed that the SRSM is composed of a single factor. SRSM was highly reliable in terms of internal consistency (Cronbach's alpha 0.91). Concurrent validity with the BSI, BHS, and HADS was statistically significant. The proposed cut-off score of the SRSM was five. CONCLUSION: In conclusion, the SRSM is a valid and reliable measure for screening suicide risks in medical inpatients.
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Desenvolvimento de Programas , Suicídio/psicologia , Adulto , Idoso , Doença Crônica , Análise Fatorial , Feminino , Humanos , Pacientes Internados , Masculino , Pessoa de Meia-Idade , Atenção Primária à Saúde , Escalas de Graduação Psiquiátrica , Encaminhamento e Consulta , República da Coreia , Risco , Ideação Suicida , Inquéritos e Questionários , Prevenção do SuicídioRESUMO
This is the first study reporting on the incidence and clinical aspects of sudden sensorineural hearing loss (SSNHL) in South Korea. Using Health Insurance Review and Assessment Service data from 2011 to 2015, the monthly incidence of unilateral SSNHL and incidence according to patients' sex, age, and month of diagnosis were investigated. The monthly incidence of unilateral SSNHL increased over the 5-year study period, with a mean annual incidence of 17.76 cases/ 100,000 of the population. The incidence increased with age, with most patients presenting in their 60s. There was a slight female preponderance, with a male-to-female ratio of 1: 1.35. Most new patients were diagnosed in October, and the fewest in January. In conclusion, this large-scale study indicates that unilateral SSNHL has a higher incidence among the elderly, women, and in autumn (i.e., along with colder weather).
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Aspergillus terreus cadA, encoding cis-aconitate decarboxylase, is an essential gene for itaconic acid (IA) biosynthesis, but it is primarily expressed as insoluble aggregates in most industrial hosts. This has been a hurdle for the development of recombinant strategies for IA production. Here, we created a library of synonymous codon variants (scv) of the cadA gene containing synonymous codons in the first 10 codons (except ATG) and screened it in Escherichia coli. Among positive clones, E. coli scvCadA_No8 showed more than 95% of expressed CadA in the soluble fraction, and in production runs, produced threefold more IA than wild-type E. coli in Luria-Bertani broth supplemented with 0.5% glucose. In M9 minimal media containing 0.85 g/L citrate and 1% glycerol, E. coli scvCadA_No8 produced 985.6 ± 33.4 mg/L IA during a 72-h culture after induction with isopropyl ß-D-1-thiogalactopyranoside. In a 2-L fed-batch fermentation consisting of two stages (growth and nitrogen limitation conditions), we obtained 7.2 g/L IA by using E. coli by introducing only the scv_cadA gene and optimizing culture conditions for IA production. These results could be combined with metabolic engineering and generate an E. coli strain as an industrial IA producer. Biotechnol. Bioeng. 2016;113: 1504-1510. © 2015 Wiley Periodicals, Inc.
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Proteínas de Bactérias/metabolismo , Carboxiliases/metabolismo , Escherichia coli/metabolismo , Glicerol/metabolismo , Engenharia Metabólica/métodos , Mutação Silenciosa/genética , Succinatos/metabolismo , Proteínas de Bactérias/genética , Carboxiliases/genética , Escherichia coli/genética , Succinatos/análiseRESUMO
Cell-free protein synthesis utilizes translational machinery isolated from the cells for in vitro expression of template genes. Because it produces proteins without gene cloning and cell cultivation steps, cell-free protein synthesis can be used as a versatile platform for high-throughput expression of enzyme libraries. Furthermore, the open nature of cell-free protein synthesis allows direct integration of enzyme synthesis with subsequent screening steps. However, the presence of high concentration of chemical buffers in the conventional reaction mixture makes it difficult to streamline cell-free protein synthesis with pH-based assay of the synthesized enzymes. In this study, we have implemented an enzyme-assisted bacterial acid resistance mechanism into an Escherichia coli (E.coli) extract-based cell-free protein synthesis system in place of chemical buffers. When deployed in the reaction mixture for cell-free synthesis of enzymes, through proton-consuming conversion of glutamate into γ-aminobutyric acid (GABA), an engineered glutamate decarboxylase (GADß) was able to maintain the pH of reaction mixture during enzyme synthesis. Because the reaction mixture becomes free of buffering capacity upon the depletion of glutamate, synthesized enzyme could be directly assayed without purification steps. The designed method was successfully applied to the screening of mutant library of sialyltransferase genes to identify mutants with improved enzymatic activity.
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Sistema Livre de Células , Biossíntese de Proteínas , Sialiltransferases/biossíntese , Sialiltransferases/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Glutamato Descarboxilase/metabolismo , Ácido Glutâmico/metabolismo , Concentração de Íons de Hidrogênio , Programas de Rastreamento/métodos , Sialiltransferases/genética , Ácido gama-Aminobutírico/metabolismoRESUMO
Recent studies using heterologous protein expression systems suggest that synonymous codons affect not only the expression but also the properties of the expressed protein. However, practical application of this information is challenging, and to date, efforts to employ bioinformatics tools to design synonymous codon mixes have been only marginally successful. Here, we sought to enhance the functional expression of heterologous protein in Escherichia coli through completely random substitution of the first ten codons with synonymous codons, using a previously isolated exocellulase CelEdx-SF301 as the model protein. Synonymous codon variants were generated by PCR using forward primers with mixed nucleotides at the third position in each codon and a conventional reverse primer. The resulting PCR products were inserted upstream of the fluorescent protein mCherry without linkers. After transformation and cultivation, colonies exhibiting red fluorescence were selected, and the activity of SF301-mCherry fusion proteins was tested. Synonymous codon variant fusion proteins exhibited 35- to 530-fold increases in functional expression compared with wild-type controls. Unlike results from other reports, we found that the stability of mRNA secondary structure in the 5' untranslated region and codon rarity were not correlated with functional expression level. Our work demonstrates that a completely random mixed of synonymous codons effectively enhances functional expression levels without the need for amino acid substitutions.
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Celulases/biossíntese , Celulases/genética , Expressão Gênica , Mutação Puntual , Escherichia coli/genética , Mutagênese , Proteínas Mutantes/biossíntese , Proteínas Mutantes/genética , Reação em Cadeia da Polimerase , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genéticaRESUMO
OBJECTIVE: The objective of this study was to assess the relationship between superior semicircular canal dehiscence (SSCD) and hearing impairment. METHODS: We retrospectively compared the prevalence of SSCD in the ears classified as conductive hearing loss (CHL), mixed hearing loss (MHL), and normal hearing status using submillimetric temporal bone computed tomography (TBCT) on the basis of coronal and additional reformatted planes dedicated to SSCD. RESULTS: From the patients with CHL (n = 127) and MHL (n = 45), the overall prevalence of SSCD in the ears classified as CHL, MHL, and normal hearing status were 6.6%, 7.2%, and 3.0%, respectively. Furthermore, the odds ratio for SSCD in the absence of any cause of hearing loss (eg, dysfunction of the tympanic membrane or middle ear, TBCT abnormalities, otosclerosis, trauma, surgery) was 5.35 in MHL (4/27; P = 0.037, 95% confidence interval, 1.1-25.81) and 3.31 in CHL (5/61; P = 0.115, 95% confidence interval, 0.75-14.63), compared with normal hearing status. CONCLUSIONS: Bony covering of the SSC should be specifically evaluated in patients with hearing impairment using submillimetric TBCT.
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Perda Auditiva Condutiva/diagnóstico por imagem , Perda Auditiva Condutiva-Neurossensorial Mista/diagnóstico por imagem , Canais Semicirculares/diagnóstico por imagem , Osso Temporal/diagnóstico por imagem , Tomografia Computadorizada por Raios X/métodos , Adolescente , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Prevalência , Estudos Retrospectivos , Canais Semicirculares/patologia , Osso Temporal/patologiaRESUMO
NecroX-5, one of the derivatives of NecroX series compounds, is a mitochondrial reactive oxygen species and reactive nitrogen species scavenger that inhibits cell death against various kinds of oxidative stresses. The objective of the present study was to evaluate the effects of NecroX-5 on neomycin-induced ototoxicity in transgenic zebrafish (Brn3C: EGFP). Five days post-fertilization, zebrafish larvae were exposed to 125 µM neomycin and one of the following NecroX-5 concentrations for 1 h: 10, 25, 50, and 75 µM. Hair cells within the neuromasts of the supraorbital (SO1 and SO2), otic (O1), and occipital (OC1) lateral lines were analyzed using fluorescence microscopy (n = 10). The terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay and 2-[4-(dimethylamino) styryl]-N-ethylpyridiniumiodide (DASPEI) assay were performed for evaluation of apoptosis and mitochondrial damage. Ultrastructural changes were evaluated using scanning electron microscopy. NecroX-5 decreased neomycin-induced hair cell loss in the neuromasts (NecroX-5 50 µM: 13.4 ± 2.0 cells, 125 µM neomycin only: 8.1 ± 1.2 cells; n = 10, P < 0.05) and decreased the TUNEL reaction. The ultrastructural analysis showed that the structures of mitochondria and hair cells within the neuromasts were preserved in zebrafish exposed to 125 µM neomycin and 50 µM NecroX-5. NecroX-5 decreased apoptosis and mitochondrial damage. In conclusion, NecroX-5 attenuated neomycin-induced hair cell loss in zebrafish.