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In July 2023, cases of highly pathogenic avian influenza (HPAI) were reported at 2 shelters for stray cats in Seoul, South Korea. The cause of infection was suspected to be improperly sterilized raw food made from domestic duck meat, which was manufactured in South Korea. All viruses isolated from cats at the shelters and from the raw food belonged to HPAI A(H5N1) clade 2.3.4.4b. The gene constellation of all viruses was most similar to that of viruses isolated in Korea in November 2022. Of note, the viruses isolated from infected cats harbored mutations E627K or D701N in polymerase basic 2, which are indicative of adaptation to mammals. Postmortem examination revealed systemic pathologic lesions and the presence of widespread virus in different tissues. Thus, consumption of raw duck meat contaminated with HPAI virus likely caused systemic symptoms and death in cats, indicating the introduction of mammal-adapted mutations of the virus.

Atopic dermatitis (AD), marked by intense itching and eczema-like lesions, is a globally increasing chronic skin inflammation. Kahweol, a diterpene that naturally occurs in coffee beans, boasts anti-inflammatory, antioxidative, and anti-cancer properties. This research explores the anti-inflammatory action of kahweol on HaCaT human keratinocytes stimulated by tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ), focusing on key signal transduction pathways. Our results demonstrate that kahweol markedly reduces the production of IL-1β, IL-6, C-X-C motif chemokine ligand 8, and macrophage-derived chemokine in TNF-α/IFN-γ-activated HaCaT cells. Furthermore, it curtails the phosphorylation of key proteins in the mitogen-activated protein kinase (MAPK) pathways, including c-Jun N-terminal kinase, extracellular signal-regulated kinase, and p38. Additionally, kahweol impedes the phosphorylation and nuclear translocation of the NF-κB p65 subunit and constrains its DNA-binding capability. It also hampers the phosphorylation, nuclear translocation, and DNA-binding activities of signal transducer and activator of transcription 1 (STAT1) and STAT3. Collectively, these findings suggest that kahweol hinders the generation of cytokines and chemokines in inflamed keratinocytes by inhibiting the MAPK, NF-κB, and STAT cascades. These insights position kahweol as a promising agent for dermatological interventions, especially in managing inflammatory skin conditions such as AD.

Currently, pest control work using speed sprayers results in increasing numbers of safety accidents such as worker pesticide poisoning and rollover of vehicles during work. To address this, there is growing interest in autonomous driving technology for speed sprayers. To commercialize and rapidly expand the use of self-driving speed sprayers, an economically efficient self-driving speed sprayer using a minimum number of sensors is essential. This study developed an orchard passage map using location data acquired from positioning sensors to generate autonomous driving paths, without installing additional sensors. The method for creating the orchard passage map presented in this study was to create paths using location data obtained by manually driving the speed sprayer and merging them. In addition, to apply the orchard passage map when operating autonomously, a method is introduced for generating an autonomous driving path for the work start point movement path, work path, and return point movement path.

In people living with HIV, Kaposi Sarcoma (KS), a vascular neoplasm caused by KS herpesvirus (KSHV/HHV-8), remains one of the most common malignancies worldwide. Individuals living with HIV, receiving otherwise effective antiretroviral therapy, may present with extensive disease requiring chemotherapy. Hence, new therapeutic approaches are needed. The Wilms' tumor 1 (WT1) protein is overexpressed and associated with poor prognosis in several hematologic and solid malignancies and has shown promise as an immunotherapeutic target. We found that WT1 was overexpressed in >90% of a total 333 KS biopsies, as determined by immunohistochemistry and image analysis. Our largest cohort from ACTG, consisting of 294 cases was further analyzed demonstrating higher WT1 expression was associated with more advanced histopathologic subtypes. There was a positive correlation between the proportion of infected cells within KS tissues, assessed by expression of the KSHV-encoded latency-associated nuclear antigen (LANA), and WT1 positivity. Areas with high WT1 expression showed sparse T-cell infiltrates, consistent with an immune evasive tumor microenvironment. We show that major oncogenic isoforms of WT1 are overexpressed in primary KS tissue and observed WT1 upregulation upon de novo infection of endothelial cells with KSHV. KSHV latent viral FLICE-inhibitory protein (vFLIP) upregulated total and major isoforms of WT1, but upregulation was not seen after expression of mutant vFLIP that is unable to bind IKKƴ and induce NFκB. siRNA targeting of WT1 in latent KSHV infection resulted in decreased total cell number and pAKT, BCL2 and LANA protein expression. Finally, we show that ESK-1, a T cell receptor-like monoclonal antibody that recognizes WT1 peptides presented on MHC HLA-A0201, demonstrates increased binding to endothelial cells after KSHV infection or induction of vFLIP expression. We propose that oncogenic isoforms of WT1 are upregulated by KSHV to promote tumorigenesis and immunotherapy directed against WT1 may be an approach for KS treatment., Competing Interests: TD is a consultant at Eureka Therapeutics, Inc. DAS is a consultant to, or has equity in, or is on the Board of: Sellas, Eureka, Pfizer, Repertoire, Sapience, Iovance, Actinium, and Atengen; MSK has filed for patent protections on his behalf for related work and some of these patents are licensed. There are no other relationships or activities that could appear to have influenced the submitted work. The other authors have no competing interests to report., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

Purpose: Primary ciliary dyskinesia (PCD) is a genetically heterogeneous disorder that leads to secondary ciliary dysfunction. PCD is a rare disease, and data on it are limited in Korea. This study systematically evaluated the clinical symptoms, diagnostic characteristics, and treatment modalities of pediatric PCD in Korea., Methods: This Korean nationwide, multicenter study, conducted between January 2000 and August 2022, reviewed the medical records of pediatric patients diagnosed with PCD. Prospective studies have been added to determine whether additional genetic testing is warranted in some patients., Results: Overall, 41 patients were diagnosed with PCD in 15 medical institutions. The mean age at diagnosis was 11.8 ± 5.4 years (range: 0.5 months-18.9 years). Most patients (40/41) were born full term, 15 (36.6%) had neonatal respiratory symptoms, and 12 (29.3%) had a history of admission to the neonatal intensive care unit. The most common complaint (58.5%) was chronic nasal symptoms. Thirty-three patients were diagnosed with transmission electron microscopy (TEM) and 12 patients by genetic studies. TEM mostly identified outer dynein arm defects (alone or combined with inner dynein arm defects, n = 17). The genes with the highest mutation rates were DNAH5 (3 cases) and DNAAF1 (3 cases). Rare genotypes ( RPGR , HYDIN , NME5 ) were found as well. Chest computed tomography revealed bronchiectasis in 33 out of 41 patients. Among them, 15 patients had a PrImary CiliAry DyskinesiA Rule score of over 5 points., Conclusions: To our knowledge, this is the first multicenter study to report the clinical characteristics, diagnostic methods, and genotypes of PCD in Korea. These results can be used as basic data for further PCD research., Competing Interests: There are no financial or other issues that might lead to conflict of interest., (Copyright © 2023 The Korean Academy of Asthma, Allergy and Clinical Immunology • The Korean Academy of Pediatric Allergy and Respiratory Disease.)

Background: Silicone implant augmentation rhinoplasty along with various tip plasties are commonly performed in Asian patients, but a revision rhinoplasty is frequently required because of various problems. Secondary rhinoplasties are often performed using silicone, dermofat, costal cartilage block, or diced rib cartilage, but often result in unsatisfactory outcomes. This study assessed the surgical outcomes and complications of cross-linked acellular dermal matrix (ADM) as an alternative biological substitute for silicone implant in secondary rhinoplasty., Methods: The authors prospectively studied 56 patients with a minimum follow-up of 1 year among 104 patients who underwent secondary rhinoplasty in their clinic between January of 2015 and December of 2018. Silicone implant, capsule, and scar tissue removal; dorsal augmentation with ADM; and tip plasty using autogenous cartilage were performed for all of them. The results were assessed using the modified Rhinoplasty Outcome Evaluation, consisting of a 10-item questionnaire completed preoperatively, 6 months postoperatively, and over 1 year postoperatively., Result: One infection and three cases of excessive resorption were noted, with no other major complications. The mean modified Rhinoplasty Outcome Evaluation score was 31.7 on preoperative evaluation, 77.3 at 6 months postoperatively, and 81.4 at 1 year postoperatively (mean difference, 45.6 and 49.7, respectively; P < 0.001)., Conclusions: Various problems that occur after primary rhinoplasty using silicone implants can be resolved successfully with secondary rhinoplasty by dorsal augmentation using the cross-linked human ADM along with various nasal tip work using autogenous cartilage. Surgical outcome showed favorable resolution of contracture deformities, a low infection rate, firm fixation of the implant, good skin texture/thickness of the skin/soft-tissue envelope, and gain of desired height and dorsal line., Clinical Question/level of Evidence: Therapeutic, IV., (Copyright © 2023 The Authors. Published by Wolters Kluwer Health, Inc. on behalf of the American Society of Plastic Surgeons.)

Background: Human coronaviruses (HCoV) cause mild upper respiratory infections; however, in 2019, a novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged, causing an acute respiratory disease pandemic. Coronaviruses exhibit marked epidemiological and clinical differences., Purpose: This study compared the clinical, laboratory, and radiographic findings of children infected with SARS-CoV-2 versus HCoV., Methods: SARS-CoV-2 data were obtained from the Korea Disease Control and Prevention Agency (KDCA) registry and 4 dedicated coronavirus disease 2019 (COVID-19) hospitals. Medical records of children admitted with a single HCoV infection from January 2015 to March 2020 were collected from 10 secondary/tertiary hospitals. Clinical data included age, sex, underlying disease, symptoms, test results, imaging findings, treatment, and length of hospital stay., Results: We compared the clinical characteristics of children infected with HCoV (n=475) to those of children infected with SARS-CoV-2 (272 from KDCA, 218 from COVID-19 hospitals). HCoV patients were younger than KDCA patients (older than 9 years:3.6% vs. 75.7%; P<0.001) and patients at COVID-19 hospitals (2.0±2.9 vs 11.3±5.3; P<0.001). Patients with SARS-CoV-2 infection had a lower rate of fever (26.6% vs. 66.7%; P<0.001) and fewer respiratory symptoms than those with HCoV infection. Clinical severity, as determined by oxygen therapy and medication usage, was worse in children with HCoV infection. Children and adolescents with SARS-CoV-2 had less severe symptoms., Conclusion: Children and adolescents with COVID-19 had a milder clinical course and less severe disease than those with HCoV in terms of symptoms at admission, examination findings, and laboratory and radiology results.

To address problems such as pesticide poisoning and accidents during pest control work and to enable efficient work in this area, the development of a competitively prices speed sprayer with autonomous driving is required. Accordingly, in order to contribute to developing the commercialization of a low-cost autonomous driving speed sprayer, we developed a positioning algorithm and an autonomous driving-based spraying algorithm by using two low-cost global navigation satellite system (GNSS) modules and a low-cost motion sensor. In order to provide stable navigation solutions from the autonomous driving hardware despite disturbances from the electromagnetic field generated by the spraying device, the proposed positioning algorithm, a moving baseline (MB) real-time kinematic (RTK)/motion sensor-integrated positioning algorithm, was developed using a loosely coupled extended Kalman filter. To compare the yaw estimation performance provided by the MB RTK positioning technique, yaw was calculated by post-processing with two types of positioning algorithms: the MB RTK/motion sensor-integrated positioning algorithm and the GNSS RTK/motion sensor-integrated positioning algorithm. In the static test, the precision of the yaw provided by the MB RTK/motion sensor-integrated positioning algorithm was 0.14°, but with the GNSS RTK/motion sensor-integrated positioning algorithm, the precision of the yaw was 4.53°. The static test results confirmed that the proposed positioning algorithm using the yaw provided by the MB RTK positioning technique based on two GNSS modules for measurement, precisely estimated the yaw even when the spray engine was operating. To perform autonomous driving and spraying, an autonomous driving-based spraying algorithm was developed using the MB RTK/motion sensor-integrated positioning algorithm. As a result of two performance tests based on the proposed algorithm in an orchard, autonomous driving and spraying were stably performed according to the set autonomous driving route and spraying method, and the root mean square (RMS) of the path-following error was 0.06 m.

Introduction: Macrolide-resistant Mycoplasma pneumoniae (MRMP) has become prevalent in children. This study investigated the clinical and laboratory variables of MRMP and macrolide-sensitive M. pneumoniae (MSMP) and identified factors associated with prolonged hospital admission in children., Methods: A prospective multicenter study was conducted in 1063 children <18 years old in July 2018-June 2020. The 454 had a positive M. pneumoniae polymerase chain reaction assay., Results: Most subjects had MRMP (78.4%), and all mutated strains had the A2063G transition. We defined MRMP* (n = 285) as MRMP pneumonia requiring admission and MSMP* (n = 72) as MSMP pneumonia requiring admission. Patients with MRMP pneumonia were older, more likely to have segmental/lobar pneumonia, and had more febrile days than those with MSMP pneumonia. C-reactive protein (CRP), lactate dehydrogenase (LDH), and percentage neutrophils were more strongly associated with MRMP* than MSMP* groups. Percentage neutrophils, CRP, and alanine aminotransferase significantly changed between admission and follow-up measurements in patients with MRMP* (P < 0.05). The duration of admission positively correlated with the number of febrile days after initiation of antibiotic medication and laboratory variables (white blood cell count, CRP, and aspartate aminotransferase [AST]) (P < 0.05). Random forest analysis indicated that the number of febrile days after initiation of antibiotic medication, AST, and percentage neutrophils at admission was over five., Conclusions: This study indicated that children with M. pneumoniae pneumonia with a higher number of febrile days after initiation of antibiotic medication, AST, and percentage neutrophils at admission were more likely to have prolonged admission duration., (© 2022 The Authors. The Clinical Respiratory Journal published by John Wiley & Sons Ltd.)

Objective: Mycoplasma pneumoniae (M. pneumoniae) pneumonia is the second-most common cause of community-acquired pneumonia (CAP). This study aimed at investigating into the prevalence of macrolide-resistant M. pneumoniae (MRMP) with respiratory virus co-infection and the antibiotic prescriptions in children with CAP in four provinces in Korea, and to assess the variations in the findings across regions and throughout the year., Patients and Methods: This prospective study was conducted in 29 hospitals in Korea between July 2018 and June 2020. Among the enrolled 1,063 children with CAP, all 451 patients with M. pneumoniae underwent PCR assays of M. pneumoniae and respiratory viruses, and the presence of point mutations of residues 2063 and 2064 was evaluated., Results: Gwangju-Honam (88.6%) showed the highest prevalence of MRMP pneumonia, while Daejeon-Chungcheong (71.3%) showed the lowest, although the differences in prevalence were not significant (p=0.074). Co-infection of M. pneumoniae pneumonia and respiratory virus was observed in 206 patients (45.4%), and rhinovirus co-infection (101 children; 22.2%) was the most frequent. The prevalence of MRMP pneumonia with respiratory virus co-infection and the antibiotic prescriptions differed significantly among the four provinces (p < 0.05). The monthly rate of MRMP pneumonia cases among all cases of M. pneumoniae pneumonia and tetracycline or quinolone prescriptions did not differ significantly among the four regions (trend p > 0.05) during the study period., Conclusions: The prevalence of M. pneumoniae pneumonia with virus co-infection and antibiotic prescriptions could differ according to region, although the MRMP pneumonia rate showed no difference within Korea.

Background: Respiratory infections among children, particularly community-acquired pneumonia (CAP), is a major disease with a high frequency among outpatient and inpatient visits. The causes of CAP vary depending on individual susceptibility, the epidemiological characteristics of the community, and the season. We performed this study to establish a nationwide surveillance network system and identify the causative agents for CAP and antibiotic resistance in Korean children with CAP., Methods: The monitoring network was composed of 28 secondary and tertiary medical institutions. Upper and lower respiratory samples were assayed using a culture or polymerase chain reaction (PCR) from August 2018 to May 2020., Results: A total of 1023 cases were registered in patients with CAP, and PCR of atypical pneumonia pathogens revealed 422 cases of M. pneumoniae (41.3%). Respiratory viruses showed a positivity rate of 65.7% by multiplex PCR test, and human rhinovirus was the most common virus, with 312 cases (30.5%). Two hundred sixty four cases (25.8%) were isolated by culture, including 131 cases of S. aureus (12.8%), 92 cases of S. pneumoniae (9%), and 20 cases of H. influenzae (2%). The cultured, isolated bacteria may be colonized pathogen. The proportion of co-detection was 49.2%. The rate of antibiotic resistance showed similar results as previous reports., Conclusions: This study will identify the pathogens that cause respiratory infections and analyze the current status of antibiotic resistance to provide scientific evidence for management policies of domestic respiratory infections. Additionally, in preparation for new epidemics, including COVID-19, monitoring respiratory infections in children and adolescents has become more important, and research on this topic should be continuously conducted in the future., (© 2022. The Author(s).)

Biliary fibrosis is an important pathological indicator of hepatobiliary damage. Cholangiocyte is the key cell type involved in this process. To reveal the pathogenesis of biliary fibrosis, it is essential to understand the normal development as well as the aberrant generation and proliferation of cholangiocytes. Numerous reports suggest that the Wnt signaling pathway is implicated in the physiological and pathological processes of cholangiocyte development and ductular reaction. In this review, we summarize the effects of Wnt pathway in cholangiocyte development from embryonic stem cells, as well as the underlying mechanisms of cholangiocyte responses to adult ductal damage. Wnt signaling pathway is regulated in a step-wise manner during each of the liver differentiation stages from embryonic stem cells to functional mature cholangiocytes. With the modulation of Wnt pathway, cholangiocytes can also be generated from adult liver progenitor cells and mature hepatocytes to repair liver damage. Non-canonical Wnt signaling is triggered in the active ductal cells during biliary fibrosis. Targeted control of the Wnt signaling may hold the great potential to reduce and/or reverse the biliary fibrogenic process.

Mycoplasma pneumoniae is a major causative pathogen of community-acquired pneumonia in children, and the treatment of choice is macrolides. There is an increasing trend in reports of refractory clinical responses despite macrolide treatment due to the emergence of macrolide-resistant M. pneumoniae . Early discrimination of macrolide-refractory M. pneumoniae pneumonia (MrMP) from macrolide-sensitive M. pneumoniae pneumonia (MSMP) is vital; however, testing for macrolide susceptibility at the time of admission is not feasible. This study aimed to identify the characteristics of MrMP in Korean children, in comparison with those of MSMP. In this multicenter study, board-certified pediatric pulmonologists at 22 tertiary hospitals reviewed the medical records from 2010 to 2015 of 5294 children who were hospitalized with M. pneumoniae pneumonia and administered macrolides as the initial treatment. One-way analysis of variance and the Kruskal-Wallis test were used to compare differences between groups. Of 5294 patients (mean age, 5.6 years) included in this analysis, 240 (4.5%), 925 (17.5%), and 4129 (78.0%) had MrMP, macrolide-less effective M. pneumoniae pneumonia, and MSMP, respectively. Compared with the MSMP group, the MrMP group had a longer fever duration, overall (13.0 days) and after macrolide use (8.0 days). A higher proportion of MrMP patients had respiratory distress, pleural effusion, and lobar pneumonia. The mean aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, and C-reactive protein levels were the highest in the MrMP group, along with higher incidences of extrapulmonary manifestations and atelectasis (during and post infection). Pre-existing conditions were present in 17.4% ( n = 725/4159) of patients, with asthma being the most common ( n = 334/4811, 6.9%). This study verified that MrMP patients show more severe initial radiographic findings and clinical courses than MSMP patients. MrMP should be promptly managed by agents other than macrolides.

Regenerative medicine promises a bright future where damaged body parts can be restored, rejuvenated, and replaced. The application of regenerative medicine is interdisciplinary and covers nearly all fields of medical sciences and molecular engineering. This review provides a road map on how regenerative medicine is applied on the levels of cell, tissue, and organ and summarizes the advantages and limitation of human pluripotent stem cells in disease modeling and regenerative application., (© 2021. Springer Nature Switzerland AG.)

To address the problems of inefficient agricultural production and labor shortages, there has been active research to develop autonomously driven agricultural machines, using advanced sensors and ICT technology. Autonomously driven speed sprayers can also reduce accidents such as the pesticide poisoning of farmers, and vehicle overturn that frequently occur during spraying work in orchards. To develop a commercial, autonomously driven speed sprayer, we developed a prototype of an autonomously driven agricultural vehicle, and conducted performance evaluations in an orchard environment. A prototype of the agricultural vehicle was created using a rubber-tracked vehicle equipped with two AC motors. A prototype of the autonomous driving hardware consisted of a GNSS module, a motion sensor, an embedded board, and an LTE module, and it was made for less than $1000. Additional software, including a sensor fusion algorithm for positioning and a path-tracking algorithm for autonomous driving, were implemented. Then, the performance of the autonomous driving agricultural vehicle was evaluated based on two trajectories in an apple farm. The results of the field test determined the RMS, and the maximums of the path-following errors were 0.10 m, 0.34 m, respectively.

Background: Viral load and shedding duration are highly associated with the transmission of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. However, limited studies have reported on viral load or shedding in children and adolescents infected with sudden acute respiratory syndrome coronavirus 2 (SARS-CoV-2)., Purpose: This study aimed to investigate the natural course of viral load in asymptomatic or mild pediatric cases., Methods: Thirty-one children (<18 years) with confirmed SARS-CoV-2 infection were hospitalized and enrolled in this study. Viral loads were evaluated in nasopharyngeal swab samples using real-time reverse transcription polymerase chain reaction (E, RdRp, N genes). cycle threshold (Ct) values were measured when patients met the clinical criteria to be released from quarantine., Results: The mean age of the patients was 9.8 years, 18 (58%) had mild disease, and 13 (42%) were asymptomatic. Most children were infected by adult family members, most commonly by their mothers. The most common symptoms were fever and sputum (26%), followed by cough and runny nose. Nine patients (29%) had a high or intermediate viral load (Ct value≤30) when they had no clinical symptoms. Viral load showed no difference between symptomatic and asymptomatic patients. Viral rebounds were found in 15 cases (48%), which contributed to prolonged viral detection. The mean duration of viral detection was 25.6 days. Viral loads were significantly lower in patients with viral rebounds than in those with no rebound (E, P=0.003; RdRp, P=0.01; N, P=0.02)., Conclusion: Our study showed that many pediatric patients with coronavirus disease 2019 (COVID-19) experienced viral rebound and showed viral detection for more than 3 weeks. Further studies are needed to investigate the relationship between viral rebound and infectiousness in COVID-19.

Background: Bronchial hyperresponsiveness (BHR), an important physiological feature of asthma, is a prognostic marker of childhood asthma., Purpose: We aimed to investigate the factors associated with BHR in adolescents with childhood asthma., Methods: Two hundred and fifteen adolescents (≥13 years of age; 149 males, 66 females) who were diagnosed with asthma during childhood were enrolled, underwent methacholine challenge tests, and were divided into the BHR group (<25 mg/mL of provocation concentration causing a 20% fall in forced expiratory volume in 1 second [FEV1] [PC20], n=113) or non-BHR group (≥25 mg/mL of PC20, n=102). We examined longitudinal changes in BHR and the risk factors for its persistence in the 108 adolescents for whom baseline data, including methacholine PC20 at age 6 years, were available. Multivariate logistic regression analyses were performed to assess the factors associated with BHR in adolescents., Results: Mold sensitization (adjusted odds ratio [aOR], 5.569; P=0.005) and increased blood eosinophil count (aOR, 1.002; P=0.026) were independently associated with BHR in boys but not girls. The odds of BHR decreased by 32% with each 1-year increase in age in boys (aOR, 0.683; P=0.010) but not girls. A reduced FEV1/forced vital capacity ratio (<90%) was independently related with BHR in female patients only (aOR, 7.500; P=0.007). BHR decreased with age throughout childhood. A low methacholine PC20 at age 6 years was independently associated with persistent BHR throughout childhood in male and female patients, whereas early mold sensitization was a risk factor for persistent BHR in male patients only (aOR, 7.718; P=0.028)., Conclusion: Our study revealed sex-specific differences in the factors associated with BHR in adolescents with childhood asthma. Our findings suggest the risk factors that might affect asthma transition from childhood to adolescence and adulthood.

The last decade has seen many exciting technological breakthroughs that greatly expanded the toolboxes for biological and biomedical research, yet few have had more impact than induced pluripotent stem cells and modern-day genome editing. These technologies are providing unprecedented opportunities to improve physiological relevance of experimental models, further our understanding of developmental processes, and develop novel therapies. One of the research areas that benefit greatly from these technological advances is the three-dimensional human organoid culture systems that resemble human tissues morphologically and physiologically. Here we summarize the development of human pluripotent stem cells and their differentiation through organoid formation. We further discuss how genetic modifications, genome editing in particular, were applied to answer basic biological and biomedical questions using organoid cultures of both somatic and pluripotent stem cell origins. Finally, we discuss the potential challenges of applying human pluripotent stem cell and organoid technologies for safety and efficiency evaluation of emerging genome editing tools.

Background: Residential greenness has been reported to be positively associated with health benefits for children, including improved cognitive function. We investigated the association between the intelligence quotient (IQ) of 6-year-old children in Seoul, South Korea and surrounding greenness currently and during the mothers' pregnancy. We also analyzed whether these effects differed by the type of greenness, such as natural or built greenness., Methods: This study considered 189 mother-child dyads from the Environment and Development of Children Cohort study, who lived in Seoul during the prenatal period and when the child was 6 years old. We defined surrounding greenness using Landsat image data from Korean Arirang satellite images with buffers within 100 m - 2000 m of the radius of each participant's residential address. We separately analyzed two types of greenness, namely natural and built greenness. The children's IQ (total, verbal, and performance IQ) was measured using the Korean Educational Developmental Institute's Wechsler Intelligence Scale for Children., Results: Prenatal exposure to built greenness in 500 m and 1000 m buffers was associated with children's total IQ in a full model [difference in IQ (95% CI): 3.46(0.68, 6.24) and 3.42 (0.53, 6.31) per interquartile increase in proportion of greenness]. However, postnatal exposure to built greenness in all buffers was associated in children's total IQ. We found a stronger association between children's total IQ and built greenness rather than natural greenness., Conclusions: We found that 6-year-old children tended to score higher on total IQ if they lived in greener neighborhoods. The results provide further evidence of the health benefits of greenness and provide support for urban planning and public health to build healthy urban cities for children and pregnant women., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Fractional exhaled nitric oxide (FeNO) is a non-invasive test for evaluating the degree of airway inflammation and for the diagnosis, evaluation, and treatment of asthma. We attempted to measure FeNO levels in Korean children with asthma and determine its cutoff value for diagnosing asthma. We enrolled 176 children and adolescents between the ages of 5 and 18 years, who visited for the evaluation of chronic cough, shortness of breath, and wheezing. Among them, 138 patients who underwent skin prick tests or inhalation Immuno CAP (UniCAP; Pharmacia, Uppsala, Sweden) tests for allergy testing together with a pulmonary function test were included. FeNO was measured using a NIOX MINO (Aerocrine AB, Solna, Sweden) instrument according to the American Thoracic Society/European Respiratory Society (ATS/ERS) guidelines. There were 29 patients with asthma, 43 with rhinitis, and 38 with asthma and allergic rhinitis. In the asthma group, FeNO levels significantly correlated with total immunoglobulin E (r = 0.572, p < 0.001), but did not show significant correlation with pulmonary function test parameters (forced vital capacity-FVC, forced expiratory volume in one second-FEV1, FEV1/FVC) or PC20 (provocative concentration of methacholine causing a 20% fall in FEV1). The FeNO cutoff values obtained in the asthma and asthma rhinitis groups were 16.5 ppb and 18.5 ppb, respectively. Hence, we provide a FeNO cutoff value according to the presence or absence of rhinitis in pediatric patients with asthma.

Background: South Korea's aggressive responses to the coronavirus disease 2019 (COVID-19) have greatly slowed the epidemic without regional lockdowns., Methods: The Korean Centers for Disease Control and Prevention's daily briefings were thoroughly reviewed. Information about hospital countermeasures and government coordination was collected via telephone interviews with 4 infection control team leaders, 1 emergency department nurse, and 1 infectious disease physician in Korea., Results: After the 2015 Middle East Respiratory Syndrome outbreak, the government and hospitals prepared for the inevitable outbreak of emerging infectious diseases by reforming the epidemic preparedness system. As a result, COVID-19 diagnostic test kits were quickly developed, enabling extensive early detection of potential cases. Other key steps were tracking cases, finding exposed individuals, coordinating case assignments with health care facilities, and selective clinic screenings for visitors' entering hospitals with mandatory mask wearing. Consequently, after overcoming the initial peak of the outbreak, which was related to a religious group, Korea has been able to maintain daily new cases at around 100 and to less than 50 daily cases in the second week of April., Conclusions: To counter the COVID-19 pandemic, which may persist, long-term, sustained response strategies must be prepared along with coordination between government and health systems., (Copyright © 2020 Association for Professionals in Infection Control and Epidemiology, Inc. Published by Elsevier Inc. All rights reserved.)

Combining photothermal and photodynamic modalities has shown encouraging therapeutic efficacy against various malignant cancers. Developing a delivery method for targeting and penetrating tumors is still a major focus for advancing this therapeutic approach. Herein, we report a novel strategy involving the utilization of stem cells as a live carrier to codeliver photothermal and photodynamic agents for cancer therapy. To this end, a novel gold nanorod (AuNR)-PEG-PEI (APP)/chlorin e6 (Ce6)-loaded adipose-derived stem cell (ADSC) system is proposed in which AuNRs and Ce6 act as the photothermal and photodynamic agents, respectively. To integrate with stem cells, the APP/Ce6 nanocomplexes exhibit advantages of low drug leakage, low cytotoxicity, efficient cellular uptake, and redox-responsive release. After loading of APP/Ce6 nanocomplexes, the ADSCs still maintained good tumor tropism and were capable of penetrating into the tumor spheroids. The photothermal effect induced by exposure to near-infrared light irradiation at 808 nm promoted the release of Ce6 from the stem cells into the surroundings and hence increased its availability to treat cancer cells. APP/Ce6-loaded ADSCs exerted effective dose-dependent in vitro anticancer activities via anticipated photothermal and photodynamic effects. In a murine CT26 colon cancer model, APP/Ce6 delivered by ADSCs resulted in superior tumor suppression compared to other delivery strategies. It was also noted that in vivo applications of APP/Ce6-loaded ADSCs did not induce noticeable detrimental effects on normal tissues/organs.

In vitro studies of drug toxicity and drug-drug interactions are crucial for drug development efforts. Currently, the utilization of primary human hepatocytes (PHHs) is the de facto standard for this purpose, due to their functional xenobiotic response and drug metabolizing CYP450 enzyme metabolism. However, PHHs are scarce, expensive, require laborious maintenance, and exhibit lot-to-lot heterogeneity. Alternative human in vitro platforms include hepatic cell lines, which are easy to access and maintain, and induced pluripotent stem cell (iPSC) derived hepatocytes. In this study, we provide a direct comparison of drug induced CYP3A4 and PXR expression levels of PHHs, hepatic cell lines Huh7 and HepG2, and iPSC derived hepatocyte like cells. Confluently cultured Huh7s exhibited an improved CYP3A4 expression and were inducible by up to 4.9-fold, and hepatocytes differentiated from human iPSCs displayed a 3.3-fold CYP3A4 induction. In addition, an increase in PXR expression levels was observed in both hepatic cell lines and iPSC derived hepatocytes upon rifampicin treatment, whereas a reproducible increase in PXR expression was not achieved in PHHs. Our results indicate that both hepatoma originated cell lines and iPSCs may provide alternative sources to primary hepatocytes, providing reliable and reproducible results for CYP3A4/PXR metabolism, upon in vitro maturation. This study may serve as a guide for the selection of suitable and feasible in vitro platforms for drug-drug interaction and toxicology studies., Competing Interests: The authors have declared that no competing interests exist.

Background: Community-acquired pneumonia (CAP) is one of the leading worldwide causes of childhood morbidity and mortality. Its disease burden varies by age and etiology and is time dependent. We aimed to investigate the annual and seasonal patterns in etiologies of pediatric CAP requiring hospitalization., Methods: We conducted a retrospective study in 30,994 children (aged 0-18 years) with CAP between 2010 and 2015 at 23 nationwide hospitals in South Korea. Mycoplasma pneumoniae (MP) pneumonia was clinically classified as macrolide-sensitive MP, macrolide-less effective MP (MLEP), and macrolide-refractory MP (MRMP) based on fever duration after initiation of macrolide treatment, regardless of the results of in vitro macrolide sensitivity tests., Results: MP and respiratory syncytial virus (RSV) were the two most commonly identified pathogens of CAP. With the two epidemics of MP pneumonia (2011 and 2015), the rates of clinical MLEP and MRMP pneumonia showed increasing trends of 36.4% of the total MP pneumonia. In children < 2 years of age, RSV (34.0%) was the most common cause of CAP, followed by MP (9.4%); however, MP was the most common cause of CAP in children aged 2-18 years of age (45.3%). Systemic corticosteroid was most commonly administered for MP pneumonia. The rate of hospitalization in intensive care units was the highest for RSV pneumonia, and ventilator care was most commonly needed in cases of adenovirus pneumonia., Conclusions: The present study provides fundamental data to establish public health policies to decrease the disease burden due to CAP and improve pediatric health.

DNA base editors have enabled genome editing without generating DNA double strand breaks. The applications of this technology have been reported in a variety of animal and plant systems, however, their editing specificity in human stem cells has not been studied by unbiased genome-wide analysis. Here we investigate the fidelity of cytidine deaminase-mediated base editing in human induced pluripotent stem cells (iPSCs) by whole genome sequencing after sustained or transient base editor expression. While base-edited iPSC clones without significant off-target modifications are identified, this study also reveals the potential of APOBEC-based base editors in inducing unintended point mutations outside of likely in silico-predicted CRISPR-Cas9 off-targets. The majority of the off-target mutations are C:G->T:A transitions or C:G->G:C transversions enriched for the APOBEC mutagenesis signature. These results demonstrate that cytosine base editor-mediated editing may result in unintended genetic modifications with distinct patterns from that of the conventional CRISPR-Cas nucleases.

The purpose of this study was to determine the anti-inflammatory and in vitro bone formation effects of Garcinia mangostana L. (mangosteen) and propolis extracts. Immortalized human gingival fibroblasts (hTERT-hNOF) cells were treated with Porphyromonas gingivalis KCOM 2804 lipopolysaccharide followed by treatment with mangosteen and propolis extract alone or in combination. Expression levels of inflammatory cytokines were evaluated by enzyme-linked immunosorbent assay. Effect of mangosteen and/or propolis extracts on mineralization of MG-63 cells was evaluated by alkaline phosphatase activity and alizarin red S staining. Group mangosteen extract complex 1:34 (1 µg/ml mangosteen extract and 34 µg/ml propolis extract) significantly reduced expression levels of IL-6, IL-8, and PGE 2 . It had higher than other groups in vitro bone formation effect on MG63 cells. These results suggest that mangosteen and propolis extract complex could be used in the prevention and treatment of periodontal disease., Competing Interests: Conflict of interestDae Sung Lee is a co-register on patent (No. 1018793980000, Korea) entitled “Pharmaceutical composition for enhancing osteogenesis comprising garcinia mangostana extract and propolis extract” and on patent (Nos. US 10,022,412 B2 and JP 6387467 B2) entitled “Composition for preventing or alleviating periodontal diseases, containing, as active ingredients, mangosteen extract or α- or γ-mangosteen. The authors declare that they have no conflict of interest., (© The Korean Society of Food Science and Technology 2019.)

Impact Statement: In this review, we address the potential of human-induced pluripotent stem cell-based hepatobiliary differentiation technology as a means to study human liver development and cell fate determination, and to model liver diseases in an effort to develop a new human-relevant preclinical platform for drug development.

Background: Bronchiectasis is a chronic pulmonary disease characterized by progressive and irreversible bronchial dilatation. The aim of the present study was to investigate the etiologies and clinical features of bronchiectasis in Korean children., Methods: We performed a retrospective review of the medical records for children diagnosed with bronchiectasis between 2000 and 2017 at 28 secondary or tertiary hospitals in South Korea., Results: A total of 387 cases were enrolled. The mean age at diagnosis was 9.2 ± 5.1 years and 53.5% of the patients were boys. The most common underlying cause of bronchiectasis was preexisting respiratory infection (55.3%), post-infectious bronchiolitis obliterans (14.3%), pulmonary tuberculosis (12.3%), and heart diseases (5.6%). Common initial presenting symptoms included chronic cough (68.0%), recurrent pneumonia (36.4%), fever (31.1%), and dyspnea (19.7%). The most predominantly involved lesions were left lower lobe (53.9%), right lower lobe (47.1%) and right middle lobe (40.2%). No significant difference was observed in the distribution of these involved lesions by etiology. The forced expiratory volume in 1 s (FEV 1 ) levels were lowest in cases with interstitial lung disease-associated bronchiectasis, followed by those with recurrent aspiration and primary immunodeficiency., Conclusions: Bronchiectasis should be strongly considered in children with chronic cough and recurrent pneumonia. Long-term follow-up studies on pediatric bronchiectasis are needed to further clarify the prognosis and reduce the disease burden in these patients., (Copyright © 2019. Published by Elsevier Ltd.)

Directed differentiation of human induced pluripotent stem cells (iPSCs) toward hepatobiliary lineages has been increasingly used as models of human liver development/diseases. As protein kinases are important components of signaling pathways regulating cell fate changes, we sought to define the key molecular mediators regulating human liver development using inhibitors targeting tyrosine kinases during hepatic differentiation of human iPSCs. A library of tyrosine kinase inhibitors was used for initial screening during the multistage differentiation of human iPSCs to hepatic lineage. Among the 80 kinase inhibitors tested, only Src inhibitors suppressed endoderm formation while none had significant effect on later stages of hepatic differentiation. Transient inhibition of c-Src during endodermal induction of human iPSCs reduced endodermal commitment and expression of endodermal markers, including SOX17 and FOXA2, in a dose-dependent manner. Interestingly, the transiently treated cells later developed into profibrogenic cholangiocyte-like cells expressing both cholangiocyte markers, such as CK7 and CK19, and fibrosis markers, including Collagen1 and smooth muscle actin. Further analysis of these cells revealed colocalized expression of collagen and yes-associated protein (YAP; a marker associated with bile duct proliferation/fibrosis) and an increased production of interleukin-6 and tumor necrosis factor-α. Moreover, treatment with verteporfin, a YAP inhibitor, significantly reduced expression of fibrosis markers. In summary, these results suggest that c-Src has a critical role in cell fate determination during endodermal commitment of human iPSCs, and its alteration in early liver development in human may lead to increased production of abnormal YAP expressing profibrogenic proinflammatory cholangiocytes, similar to those seen in livers of patients with biliary fibrosis. Stem Cells 2019;37:306-317., (© AlphaMed Press 2018.)

Biliary atresia (BA) is the most common cause of pediatric end-stage liver disease and the etiology is poorly understood. There is no effective therapy for BA partly due to lack of human BA models. Towards developing in vitro human models of BA, disease-specific induced pluripotent stem cells (iPSCs) from 6 BA patients were generated using non-integrating episomal plasmids. In addition, to determine the functional significance of BA-susceptibility genes identified by genome-wide association studies (GWAS) in biliary development, a genome-editing approach was used to create iPSCs with defined mutations in these GWAS BA loci. Using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system, isogenic iPSCs deficient in BA-associated genes (GPC1 and ADD3) were created from healthy iPSCs. Both the BA patient-iPSCs and the knock out (KO) iPSCs were studied for their in vitro biliary differentiation potential. These BA-specific iPSCs demonstrated significantly decreased formation of ductal structures, decreased expression of biliary markers including CK7, EpCAM, SOX9, CK19, AE2, and CFTR and increased fibrosis markers such as alpha smooth muscle actin, Loxl2, and Collagen1 compared to controls. Both the patient- and the KO-iPSCs also showed increased yes-associated protein (YAP, a marker of bile duct proliferation/fibrosis). Collagen and YAP were reduced by treatment with the anti-fibrogenic drug pentoxifylline. In summary, these BA-specific human iPSCs showed deficiency in biliary differentiation along with increased fibrosis, the 2 key disease features of BA. These iPSCs can provide new human BA models for understanding the molecular basis of abnormal biliary development and opportunities to identify drugs that have therapeutic effects on BA.

The cellular redox state of organisms continues to fluctuate during the metabolism. All organisms have various sensors that help detect and adapt to changes in the redox state. Nicotinamide adenine dinucleotides (NAD + /NADH), which are involved in various cellular metabolic activities as cofactors, have been revealed as the key molecules sensing the intra-cellular redox state. The Rex family members are well conserved transcriptional repressors that regulate the expression of respiratory genes by sensing the redox state according to the intra-cellular NAD + /NADH balance. Herein, we reported crystal structures of apo and NAD + - and NADH-bound forms of Rex from Thermotoga maritima to analyse the structural basis of transcriptional regulation depending on either NAD + or NADH binding. The different orientation of the reduced nicotinamide group to helix α9 caused the rearrangement of N-terminal DNA binding domain, thus resulting in closed form of Rex to dissociate from cognate DNA. The structural data of Rex from T. maritima also support the previous redox-sensing mechanism models of Rex homologues.

Glutamine synthetase (GS) is an enzyme that regulates nitrogen metabolism and synthesizes glutamine via glutamate, ATP, and ammonia. GS is a homo-oligomeric protein of eight, ten, or twelve subunits, and each subunit-subunit interface has its own active site. GS can be divided into GS I, GS II, and GS III. GS I and GS III form dodecamer in bacteria and archaea, whereas GS II form decamer in eukaryotes. GS I can be further subdivided into GS I-α and GS I-β according to its sequence and regulatory mechanism. GS is an essential protein for the survival of Helicobacter pylori which its infection could promote gastroduodenal diseases. Here, we determined the crystal structures of the GS from H. pylori (Hpy GS) in its apo- and substrate-bound forms at 2.8 Å and 2.9 Å resolution, respectively. Hpy GS formed a dodecamer composed of two hexameric rings stacked face-to-face. Hpy GS, which belongs to GS I, cannot be clearly classified as either GS I-α or GS I-β based on its sequence and regulatory mechanism. In this study, we propose that Hpy GS could be classified as a new GS-I subfamily and provide structural information on the apo- and substrate-bound forms of the protein.

Biliary atresia (BA) is a common cause of pediatric end-stage liver disease. While its etiology is not yet clear, evidence has suggested that BA results from interactions between genetic susceptibility and environmental factors. Disease relevant human cellular models of BA will facilitate identification of both genetic and environmental factors that are important for disease prevention and treatment. Here we report the generation of a human induced pluripotent stem cell line from a BA patient using episomal vectors. Patient-specific BA iPSC lines provide valuable tools for disease mechanism study and drug development., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

Children with post-infectious bronchiolitis obliterans (PIBO) are frequently hospitalized with acute exacerbation, but clinical differentiation of PIBO exacerbation from acute bronchiolitis is often challenging, which may result in treatment delay and chronic lung function impairment. We aimed to examine whether serum YKL-40 and growth factors could be markers for PIBO exacerbation. Thirty-seven children admitted with acute exacerbation of PIBO were enrolled and studied retrospectively. Diagnosis of PIBO was based on clinical history of acute respiratory infection followed by persistent airway obstruction and characteristic findings in high-resolution computed tomography. Serum levels of YKL-40, vascular endothelial growth factor (VEGF), transforming growth factor (TGF)-β1, and platelet-derived growth factor (PDGF)-BB were measured on admission. The biomarkers were also examined in children admitted with acute bronchiolitis serving as positive controls (N = 30) and in age-matched controls (N = 20). Only YKL-40 levels were found to be significantly higher in PIBO patients with exacerbation compared with that in bronchiolitis patients and showed a positive correlation with the severity of disease before diagnosis of PIBO., Conclusion: Our results suggest that measuring serum YKL-40 levels might help distinguish exacerbation of PIBO from acute bronchiolitis in young children. What is Known: • The children with post-infectious BO (PIBO) usually have recurrent wheezing and need frequent hospitalization due to acute exacerbation during the first disease years. • Clinical differentiation of PIBO exacerbation from acute bronchiolitis in young children is often challenging, which may result in treatment delay and chronic lung function impairment. What is New: • Measuring serum YKL-40 levels might help distinguish exacerbation of PIBO from acute bronchiolitis in young children.

Background Aims: Few studies have examined the migration pattern of natural killer (NK) cells, especially after radiation treatment for cancer. We investigated whether irradiation can modulate the expression of chemokines in cancer cells and the migration of NK cells to irradiated tumor cells., Methods: The expression of chemokine receptors (CXCR3, CXCR4 and CXCR6) on interleukin-2 (IL-2)/IL-15-activated NK cells was assessed using flow cytometry. Related chemokine ligands (CXCL11, CXCL12 and CXCL16) in human breast cancer cell lines (MCF7, SKBR3 and MDA-MB231) irradiated at various doses were assessed using reverse transcription-polymerase chain reaction (RT-PCR), fluorescence-activated cell sorting (FACS) and enzyme-linked immunosorbent assay (ELISA). The cell-free culture supernatant was collected 96 h after irradiation of breast cancer cell lines for migration and blocking assays., Results: The activated NK cells expressed CXCR6. Expression of the CXCR6 ligand CXCL16 increased in a time- and dose-dependent manner in all analyzed cancer cell lines. CXCL16 expression was statistically significantly enhanced in all breast cancer cell lines on day 3 after 20 Gy irradiation. Activated NK cells migration correlated with CXCL16 concentration (R 2  = 0.91; P <0.0001). Significantly enhanced migration of NK cells to irradiated cancer cells was observed for a dose of 20 Gy in MCF7 (P = 0.043) and SKBR3 (P = 0.043) cells, but not in MDA-MB231 (P = 0.225) cells. A blocking assay using a CXCR6 antibody showed a significant decrease in the migration of activated NK cells in all cancer cell lines., Conclusions: Our data indicate that irradiation induces CXCL16 chemokine expression in cancer cells and enhances the migration of activated NK cells expressing CXCR6 to irradiated breast cancer cells. These results suggest that radiation would improve the anti-tumor effect of NK cells through enhanced migration of NK cells to tumor site for the treatment of patients with breast cancer., (Copyright © 2016 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

The discovery that mature cells can be reprogrammed to become pluripotent and the development of engineered endonucleases for enhancing genome editing are two of the most exciting and impactful technology advances in modern medicine and science. Human pluripotent stem cells have the potential to establish new model systems for studying human developmental biology and disease mechanisms. Gene correction in patient-specific iPSCs can also provide a novel source for autologous cell therapy. Although historically challenging, precise genome editing in human iPSCs is becoming more feasible with the development of new genome-editing tools, including ZFNs, TALENs, and CRISPR. iPSCs derived from patients of a variety of diseases have been edited to correct disease-associated mutations and to generate isogenic cell lines. After directed differentiation, many of the corrected iPSCs showed restored functionality and demonstrated their potential in cell replacement therapy. Genome-wide analyses of gene-corrected iPSCs have collectively demonstrated a high fidelity of the engineered endonucleases. Remaining challenges in clinical translation of these technologies include maintaining genome integrity of the iPSC clones and the differentiated cells. Given the rapid advances in genome-editing technologies, gene correction is no longer the bottleneck in developing iPSC-based gene and cell therapies; generating functional and transplantable cell types from iPSCs remains the biggest challenge needing to be addressed by the research field.

Due to the limitations of research using human embryos and the lack of a biological model of human liver development, the roles of the various markers associated with liver stem or progenitor cell potential in humans are largely speculative, and based on studies utilizing animal models and certain patient tissues. Human pluripotent stem cell-based in vitro multistage hepatic differentiation systems may serve as good surrogate models for mimicking normal human liver development, pathogenesis and injury/regeneration studies. Here, we describe the implications of various liver stem or progenitor cell markers and their bipotency (i.e. hepatocytic- and biliary-epithelial cell differentiation), based on the pluripotent stem cell-derived model of human liver development. Future studies using the human cellular model(s) of liver and biliary development will provide more human relevant biological and/or pathological roles of distinct markers expressed in heterogeneous liver stem/progenitor cell populations., (© 2016 by the Society for Experimental Biology and Medicine.)

While in vitro liver tissue engineering has been increasingly studied during the last several years, presently engineered liver tissues lack the bile duct system. The lack of bile drainage not only hinders essential digestive functions of the liver, but also leads to accumulation of bile that is toxic to hepatocytes and known to cause liver cirrhosis. Clearly, generation of bile duct tissue is essential for engineering functional and healthy liver. Differentiation of human induced pluripotent stem cells (iPSCs) to bile duct tissue requires long and/or complex culture conditions, and has been inefficient so far. Towards generating a fully functional liver containing biliary system, we have developed defined and controlled conditions for efficient 2D and 3D bile duct epithelial tissue generation. A marker for multipotent liver progenitor in both adult human liver and ductal plate in human fetal liver, EpCAM, is highly expressed in hepatic spheroids generated from human iPSCs. The EpCAM high hepatic spheroids can, not only efficiently generate a monolayer of biliary epithelial cells (cholangiocytes), in a 2D differentiation condition, but also form functional ductal structures in a 3D condition. Importantly, this EpCAM high spheroid based biliary tissue generation is significantly faster than other existing methods and does not require cell sorting. In addition, we show that a knock-in CK7 reporter human iPSC line generated by CRISPR/Cas9 genome editing technology greatly facilitates the analysis of biliary differentiation. This new ductal differentiation method will provide a more efficient method of obtaining bile duct cells and tissues, which may facilitate engineering of complete and functional liver tissue in the future.

Alcohol consumption has long been a global problem affecting human health, and has been found to influence both fetal and adult liver functions. However, how alcohol affects human liver development and liver progenitor cells remains largely unknown. Here, we used human induced pluripotent stem cells (iPSCs) as a model to examine the effects of alcohol, on multi-stage hepatic cells including hepatic progenitors, early and mature hepatocyte-like cells derived from human iPSCs. While alcohol has little effect on endoderm development from iPSCs, it reduces formation of hepatic progenitor cells during early hepatic specification. The proliferative activities of early and mature hepatocyte-like cells are significantly decreased after alcohol exposure. Importantly, at a mature stage of hepatocyte-like cells, alcohol treatment increases two liver progenitor subsets, causes oxidative mitochondrial injury and results in liver disease phenotypes (i.e., steatosis and hepatocellular carcinoma associated markers) in a dose dependent manner. Some of the phenotypes were significantly improved by antioxidant treatment. This report suggests that fetal alcohol exposure may impair generation of hepatic progenitors at early stage of hepatic specification and decrease proliferation of fetal hepatocytes; meanwhile alcohol injury in post-natal or mature stage human liver may contribute to disease phenotypes. This human iPSC model of alcohol-induced liver injury can be highly valuable for investigating alcoholic injury in the fetus as well as understanding the pathogenesis and ultimately developing effective treatment for alcoholic liver disease in adults., Competing Interests: The authors have declared that no competing interest exists.

Alcohol is one of the most common etiologies of liver disease, and alcoholic liver disease overall is the second most common indication for liver transplantation in the United States. It encompasses a spectrum of disease, including fatty liver disease, alcoholic hepatitis (AH), and alcoholic cirrhosis. AH can range from mild to severe disease, with severe disease being defined as: Discriminant Function (DF) ≥ 32, or Model for End-stage Liver Disease (MELD) ≥ 21, or presence of hepatic encephalopathy. Management of the mild disease consists mainly of abstinence and supportive care. Severe AH is associated with significant mortality. Currently, there is no ideal medical treatment for this condition. Besides alcohol cessation, corticosteroids have been used with conflicting results and are associated with an inherent risk of infection. Overall steroids have shown short term benefit when compared to placebo, but they have no obvious long term benefits. Pentoxifylline does not improve survival in patients with severe AH and is no longer recommended based on the results of the STOPAH (Steroid Or Pentoxifylline for Alcoholic Hepatitis) trial. Anti-tumor necrosis factor (TNF) agents are associated with increased risk of life threatening infections and death. Currently, early stage trials are underway, mainly targeting novel pathways based on disease pathogenesis, including modulation of innate immune system, inhibition of gut-liver axis and cell death pathways, and activation of transcription factor farnesyl X receptor (FXR). Future treatment may lie in human induced pluripotent stem cell (iPSC) technology, which is currently under investigation for the study of pathogenesis, drug discovery, and stem cell transplantation. Liver transplantation has been reported with good results in highly selected patients but is controversial due to limited organ supply.

The purpose of this study was to investigate the effects of brain-computer interface (BCI)-controlled functional electrical stimulation (FES) training on shoulder subluxation of patients with stroke. Twenty subjects were randomly divided into two groups: the BCI-FES group (n = 10) and the FES group (n = 10). Patients in the BCI-FES group were administered conventional therapy with the BCI-FES on the shoulder subluxation area of the paretic upper extremity, five times per week during 6 weeks, while the FES group received conventional therapy with FES only. All patients were assessed for shoulder subluxation (vertical distance, VD; horizontal distance, HD), pain (visual analogue scale, VAS) and the Manual Function Test (MFT) at the time of recruitment to the study and after 6 weeks of the intervention. The BCI-FES group demonstrated significant improvements in VD, HD, VAS and MFT after the intervention period, while the FES group demonstrated significant improvements in HD, VAS and MFT. There were also significant differences in the VD and two items (shoulder flexion and abduction) of the MFT between the two groups. The results of this study suggest that BCI-FES training may be effective in improving shoulder subluxation of patients with stroke by facilitating motor recovery. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

Alcohol consumption has long been associated with a majority of liver diseases and has been found to influence both fetal and adult liver functions. In spite of being one of the major causes of morbidity and mortality in the world, currently, there are no effective strategies that can prevent or treat alcoholic liver disease (ALD), due to a lack of human-relevant research models. Recent success in generation of functionally active mature hepatocyte-like cells from human-induced pluripotent cells (iPSCs) enables us to better understand the effects of alcohol on liver functions. Here, we describe the method and effect of alcohol exposure on multistage hepatic cell types derived from human iPSCs, in an attempt to recapitulate the early stages of liver tissue injury associated with ALD. We exposed different stages of iPSC-induced hepatic cells to ethanol at a pathophysiological concentration. In addition to stage-specific molecular markers, we measured several key cellular parameters of hepatocyte injury, including apoptosis, proliferation, and lipid accumulation.

The advent of induced pluripotent stem cell (iPSC) technology has enabled the modeling of an array of specific human disease phenotypes, aiding in the increasingly important and indispensable understanding of disease progression and pathogenesis. Pluripotent stem cell-derived hepatocytes present a new avenue for drug screening and personalized drug testing toward precision medicine. CYP450 microsomal enzymes play a critical role in drug metabolism. Hence, CYP activity measurement of iPSC-derived hepatocytes is a vital prerequisite, to ensure metabolic functionality before proceeding to drug testing. Herein, we describe the protocol for measurement of different CYP450 enzyme activities in human iPSC-derived hepatocytes.

Genome editing is the process in which DNA sequences at precise genomic locations are modified. In the past three decades, genome editing by homologous recombination has been successfully performed in mouse for generating genetic models. The low efficiency of this process in human cells, however, had prevented its clinical application until the recent advancements in designer endonuclease technologies. The significantly improved genome editing efficiencies aided by ZFN, TALEN, and CRISPR systems provide unprecedented opportunities not only for biomedical research, but also for developing novel therapies. Applications based on these genome editing tools to disrupt deleterious genes, correct genetic mutations, deliver functional transgenes more effectively or even modify the epigenetic landscape are being actively investigated for gene and cell therapy purposes. Encouraging results have been obtained in limited clinical trials in the past two years. While most of the applications are still in proof-of-principle or preclinical development stages, it is anticipated that the coming years will see increasing clinical success in novel therapies based on the modern genome editing technologies. It should be noted that critical issues still remain before the technologies can be translated into more reliable therapies. These key issues include off-target evaluation, establishing appropriate preclinical models and improving the currently low efficiency of homology-based precise gene replacement. In this review we discuss the preclinical and clinical studies aiming at translating the genome editing technologies as well as the issues that are important for more successful translation.

Efficient and precise genome editing is crucial for realizing the full research and therapeutic potential of human induced pluripotent stem cells (iPSCs). Engineered nucleases including CRISPR/Cas9 and transcription activator like effector nucleases (TALENs) provide powerful tools for enhancing gene-targeting efficiency. In this study, we investigated the relative efficiencies of CRISPR/Cas9 and TALENs in human iPSC lines for inducing both homologous donor-based precise genome editing and nonhomologous end joining (NHEJ)-mediated gene disruption. Significantly higher frequencies of NHEJ-mediated insertions/deletions were detected at several endogenous loci using CRISPR/Cas9 than using TALENs, especially at nonexpressed targets in iPSCs. In contrast, comparable efficiencies of inducing homologous donor-based genome editing were observed at disease-associated loci in iPSCs. In addition, we investigated the specificity of guide RNAs used in the CRISPR/Cas9 system in targeting disease-associated point mutations in patient-specific iPSCs. Using myeloproliferative neoplasm patient-derived iPSCs that carry an acquired JAK2-V617F point mutation and α1-antitrypsin (AAT) deficiency patient-derived iPSCs that carry an inherited Z-AAT point mutation, we demonstrate that Cas9 can specifically target either the mutant or the wild-type allele with little disruption at the other allele differing by a single nucleotide. Overall, our results demonstrate the advantages of the CRISPR/Cas9 system in allele-specific genome targeting and in NHEJ-mediated gene disruption.

[Purpose] The purpose of this study was to determine the effects of brain-computer interface (BCI)-based functional electrical stimulation (FES) on balance and gait function in patients with stroke. [Subjects] Subjects were randomly allocated to a BCI-FES group (n=5) and a FES group (n=5). [Methods] The BCI-FES group received ankle dorsiflexion training with FES according to a BCI-based program for 30 minutes per day for 5 days. The FES group received ankle dorsiflexion training with FES for the same duration. [Results] Following the intervention, the BCI-FES group showed significant differences in Timed Up and Go test value, cadence, and step length on the affected side. The FES group showed no significant differences after the intervention. However, there were no significant differences between the 2 groups after the intervention. [Conclusion] The results of this study suggest that BCI-based FES training is a more effective exercise for balance and gait function than FES training alone in patients with stroke.

Background: Lambda interferons (IFNLs) have potent antiviral activity against HCV, and polymorphisms within the IFNL gene cluster near the IFNL3 gene strongly predict spontaneous- and treatment-related HCV infection outcomes. The mechanism(s) linking IFNL polymorphisms and HCV control is currently elusive., Methods: IFNL induction was studied in primary human hepatocytes (PHH) from 18 human donors, peripheral blood mononuclear cells (PBMCs) from 18 human donors, multiple cell lines and induced pluripotent stem cell-derived hepatocyte-like cells (iPSC-hepatocytes) from 7 human donors. After stimulation with intracellular RNA and infectious HCV, quantitative PCR (qPCR) primers and probes were designed to distinguish and quantify closely related IFNL messenger (m)RNAs from IFNL1, IFNL2 and IFNL3., Results: PHH demonstrated the most potent induction of IFNLs, although had lower pre-stimulation levels compared to PBMCs, monocytes and cell lines. PHH stimulation with cytoplasmic poly I:C induced >1,000-fold expression of IFNL1, IFNL2 and IFNL3. PHH from donors who were homozygous for the favourable IFNL3 allele (IFNL3-CC) had higher IFNL3 induction compared to PHH from IFNL3-TT donors (P=0.03). Baseline IFNL mRNA expression and induction was also tested in iPSC-hepatocytes: iPSC-hepatocytes had significantly higher baseline expression of IFNLs compared to PHH (P<0.0001), and IFNL3 induction was marginally different in iPSC-hepatocytes by IFNL genotype (P=0.07)., Conclusions: Hepatocytes express IFNLs when stimulated by a synthetic viral RNA that signals the cell through the cytoplasm. IFNL induction may be greater in persons with the favourable IFNL3 allele. These data provide insight into the strong linkage between IFNL3 genetics and control of HCV infection.

Background Aims: Interleukin-21 (IL-21) can enhance the effector function of natural killer (NK) cells but also limits their proliferation when continuously combined with IL-2/IL-15. Paradoxically, membrane-bound (mb)-IL-21 has been shown to improve human NK cell proliferation when cultured with IL-2/mb-IL-15. To clarify the role of IL-21, we investigated the effect of the timing of IL-21 addition to NK cell culture., Methods: IL-2/IL-15-activated NK cells were additionally treated with IL-21 according to the following schedules; (i) control (without IL-21); (ii) first week (day 0 to day 7); (iii) intermittent (the first 3 days of each week for 7 weeks); (iv) after 1 week (day 8 to day 14); and (v) continuous (day 0 to day 49). The expression of NK receptors, granzyme B, perforin, CD107a, interferon-γ, telomere length and NK cell death were measured by flow cytometry., Results: Compared with the control (2004.2-fold; n = 10 healthy donors) and intermittent groups (2063.9-fold), a strong proliferative response of the NK cells on day 42 was identified in the "first week" group (3743.8-fold) (P < 0.05). NK cells treated with IL-21 in the "first week" group showed cytotoxicity similar to that in control cells. On day 28, there was a significant increase in cytotoxicity of "first week" NK cells that received IL-21 treatment for an additional 2 days compared with the "first week" NK cells (P < 0.05)., Conclusions: These data suggest that controlling temporal exposure of IL-21 during NK cell proliferation can be a critical consideration to improve the yields and cytotoxicity of NK cells., (Copyright © 2014 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.)