Enhanced genome editing efficiency of CRISPR PLUS: Cas9 chimeric fusion proteins.

Efforts to improve CRISPR-Cas9 genome editing systems for lower off-target effects are mostly at the cost of its robust on-target efficiency. To enhance both accuracy and efficiency, we created chimeric SpyCas9 proteins fused with the 5'-to-3' exonuclease Recombination J (RecJ) or with GFP and demonstrated that transfection of the pre-assembled ribonucleoprotein of the two chimeric proteins into human or plant cells resulted in greater targeted mutagenesis efficiency up to 600% without noticeable increase in off-target effects. Improved activity of the two fusion proteins should enable editing of the previously hard-to-edit genes and thus readily obtaining the cells with designer traits.

Human Leukocyte Antigen Class I Pseudo-Homozygous Mesenchymal Stem Cells Derived from Human Induced Pluripotent Stem Cells.

Mesenchymal stem cells (MSC) are an important type of cell that are highly recognized for their safety and efficacy as a cell therapy agent. In order to obtain MSC, primary tissues (adipose tissue, bone marrow, and umbilical cord blood) must be used; however, these tissues, especially umbilical cord blood, are difficult to obtain due to various reasons, such as the low birth rate trend. In addition, to maximize the safety and efficacy of MSC as allogenic cell therapeutic agents, it is desirable to minimize the possibility of an immune rejection reaction after in vivo transplantation. This study tried to establish a novel method for producing induced pluripotent stem cells (iPSC)-derived MSC in which the human leukocyte antigen (HLA)-class I gene is knocked out. To do so, dermal fibroblast originated iPSC generation using Yamanaka 4-factor, HLA class I gene edited iPSC generation using CRISPR/Cas9, and differentiation from iPSC to MSC using MSC culture medium was utilized. Through this, HLA-A, B, and C pseudo-homozygous iPSC-derived MSC (KO iMSC) were produced by monoallelically knocking out the polymorphic HLA-A, B, and C genes, which are the major causes of immune rejection during allogenic cell transplantation. Produced KO iMSC possesses multipotency and it was safe in vivo to be able to be differentiated to cartilage. In addition, it was not attacked by natural killer cells unlike HLA class I null cells. In conclusion, KO iMSC that do not induce immune rejection during allogenic cell transplantation can be produced. In the future, KO iMSC can be successfully utilized as allogenic cell therapeutic agents for many recipients through HLA screening.

Combinatorial Strategies for Long-term Control of HIV Infection.

AIDS is a disease caused by a chronic infection of HIV. Recently, long-term control of HIV infection has been demonstrated through the bone marrow transplantation of hematopoietic stem cells (HSC), in which the C-C chemokine receptor type 5 (CCR5) gene is mutated innately. However, it is very difficult to obtain CCR5 mutant HSC that match human leukocyte antigen between donor and recipient. To solve this problem, this review will summarize and discuss various reports related to the generation of patient-specific CCR5 geneedited HSC. The fusion of current gene editing (zinc-finger nuclease, transcription activator-like effector nuclease, and clustered regulatory interspaced short palindromic repeats) and cellular reprogramming technology (somatic cell nuclear transfer, induced pluripotent stem cells technology, and direct phenotypic conversion) enables the generation of patient-specific CCR5 edited HSC. These cells can be useful as valuable therapeutic agents for long-term control of HIV-infected patients in the future.

Dual growth factor-immobilized bioactive injection material for enhanced treatment of glottal insufficiency.

With increasing demand for treatment of glottal insufficiency, several injection materials have been examined. However, biological resorption, degradation of injected materials, and the subsequent need to perform multiple injections still remain major clinical problems. In this study, we fabricated two different growth factor (GF) [single basic fibroblast growth factor (bFGF), single hepatocyte growth factor (HGF), or dual bFGF/HGF]-immobilized polycaprolactone (PCL)/Pluronic F127 microspheres. These materials were investigated for their potential use as bioactive injection laryngoplasty agents. HGF was found to be continuously released over 20 days and the bFGF was found to be continuously released over 25 days, as demonstrated by ELISA assay. Human vocal fold fibroblasts (hVFFs) showed significantly higher proliferative ability on dual GF-immobilized microspheres. GF-immobilized microspheres (bFGF, HGF, and dual GF) were injected into paralyzed vocal folds of New Zealand white rabbits. Through endoscopic observation and H&E staining, we identified that the microspheres remained localized at the injection site, resulting in constant volume augmentation of the paralyzed vocal fold without significant loss of the initial volume after 4 weeks. The expression of genes related to the extracellular matrix (ECM) in the vocal fold was upregulated by dual GF-immobilized microspheres. Furthermore, dual GF-immobilized microspheres inhibited muscle degeneration and upregulation of myogenic-related genes. In conclusion, dual GF-immobilized microspheres passively augmented the volume of the paralyzed vocal fold while actively inducing ECM synthesis at the injected vocal fold and preserving muscle tissue. Dual GF-immobilized microspheres could be a new and promising injection material for paralyzed vocal folds. STATEMENT OF SIGNIFICANCE: Limitation of prolonged augmentation of vocal fold and degeneration of vocal fold tissue still remain as major clinical problems in the treatment of vocal fold paralysis. Herein, we fabricated the polycaprolactone (PCL)/Pluronic F127 microspheres to augment volume of paralyzed vocal folds. On top of that, we additionally immobilized the growth factors (bFGF, HGF, or dual bFGF/HGF) on the surface of these microspheres. We highlight the efficacy of the dual GF-immobilized microspheres which augmented the volume of the paralyzed vocal fold passively, induced ECM synthesis actively at the injected vocal fold and preserved laryngeal muscle tissue. Our results suggest that the dual GF-immobilized microsphere could be a new promising injection material for injection laryngoplasty to treat paralyzed vocal fold.

A Case of Segmental Vitiligo Along Blaschko's Lines

No abstract available.

Non-cultured Epidermal Cell Suspension Transplantation Using Suction Blisters to Treat Refractory Vitiligo: A Retrospective Study of 20 Cases / 대한피부과학회지

BACKGROUND: As nonsurgical interventions for vitiligo are not always successful, various surgical modalities have been used in patients with refractory vitiligo. Of these, non-cultured epidermal suspension transplantation (NCES) was recently introduced to treat large recipient sites using cells from small donor tissue. OBJECTIVE: We assessed the effectiveness and safety of NCES as a surgical treatment for patients with refractory vitiligo. METHODS: We retrospectively reviewed 20 cases in 17 patients (11 females; median age 25 years) who underwent NCES from July 2015 through March 2018. Suction blisters (20 mm in diameter) were collected from the patient's inner thigh at a donor-to-recipient area ratio of 1:5. After the addition of 5 mL recombinant trypsin solution to the suction blisters, followed by incubation at 37℃ for 60 min, epidermal cells were manually scraped off the blister surface, and epidermal cell suspension was obtained by centrifugation at 1,500 RPM for 5 min. The suspension was applied to the vitiligo regions after epidermal ablation of those regions. Phototherapy resumed 1 month later. Treatment success was defined as ≥75% repigmentation of the surgical site, and all adverse events were noted. RESULTS: Overall, 85.0% of cases (17/20) exhibited treatment success. Adverse events included hyperpigmentation (20%) and surgical site infection (5%), but the treatment was tolerable in all cases. CONCLUSION: NCES is a reliable surgical option for patients with vitiligo refractory to nonsurgical treatment. Large areas of vitiligo can be treated by NCES, and use of this technique should be encouraged in Korea.

Prevention of polydimethylsiloxane microsphere migration using a mussel-inspired polydopamine coating for potential application in injection therapy.

The use of injectable bulking agents is a feasible alternative procedure for conventional surgical therapy. In this study, poly(dimethylsiloxane) (PDMS) microspheres coated with polydopamine (PDA) were developed as a potential injection agent to prevent migration in vocal fold. Uniform PDMS microspheres are fabricated using a simple fluidic device and then coated with PDA. Cell attachment test reveals that the PDA-coated PDMS (PDA-PDMS) substrate favors cell adhesion and attachment. The injected PDA-PDMS microspheres persist without migration on reconstructed axial CT images, whereas, pristine PDMS locally migrates over a period of 12 weeks. The gross appearance of the implants retrieved at 4, 8, 12 and 34 weeks indicates that the PDA-PDMS group maintained their original position without significant migration until 34 weeks after injection. By contrast, there is diffuse local migration of the pristine PDMS group from 4 weeks after injection. The PDA-coated PDMS microspheres can potentially be used as easily injectable, non-absorbable filler without migration.

Treatment of Laser Therapy-Induced Punctate Leukoderma Using a 308-nm Excimer Laser

Punctate leukoderma presents as numerous, distinct, round or oval depigmented spots. Recently, laser therapy-induced punctate leukoderma associated with various Q-switched laser and carbon dioxide laser have been reported. A 25-year-old man presented with numerous, discrete, round, confetti-like, depigmented macules on his left neck. He had undergone 3 sessions of 532-nm Q-switched Neodymium: Yttrium-Aluminum-Garnet laser treatment for café-au-lait macules three years ago. After the last laser treatment session, the punctate leukoderma had been developed. We started treatment with the 308-nm excimer laser twice a week. After 7 months of treatment duration, complete repigmentation was achieved without serious adverse effects. We recommend the 308-nm excimer laser as an effective treatment modality for laser therapy-induced punctate leukoderma.

Association Between Obesity, Abdominal Obesity, and Adiposity and the Prevalence of Atopic Dermatitis in Young Korean Adults: the Korea National Health and Nutrition Examination Survey 2008-2010

PURPOSE: Whether obesity is a risk factor for atopic dermatitis (AD) remains unclear. The aim of the present study was to investigate the association between obesity and AD in Korean young adults. METHODS: We included nationally representative data of 5,202 Korean adults aged 19-40 years, obtained from the cross-sectional Korea National Health and Nutrition Examination Survey 2008-2010. RESULTS: Single (unmarried) status was more frequently observed in AD patients (male, [P=0.0002] and female, [P or =25 kg/m2, WC > or =80 cm, and highest quartile (Q4) of total BF percentage had the highest prevalence of AD. The odds ratio (OR) for participants with both BMI > or =25 kg/m2 and WC > or =80 cm was 3.29 (95% confidence interval [CI] 1.71-3.55); therefore, having both general and abdominal obesity was considered a prominent risk factor for AD in young women. After adjustment for confounding factors, including age, smoking, alcohol drinking, exercise, vitamin D, income level, and single status, high BMI (> or =30 kg/m2) (OR=4.08, 95% CI: 1.53-10.93), high WC (> or =80 cm) (OR=2.05, 95% CI: 1.07-3.94), and high BF percentage (Q4) (OR=2.10, 95% CI: 1.24-3.57) were shown to be significantly associated with AD in young adult women. CONCLUSIONS: In this large-scale nation-wide study of Korean adults, obesity was positively related to the presence of AD in women. Our findings suggest that weight management may help prevent AD.

Hydrogel-laden paper scaffold system for origami-based tissue engineering.

In this study, we present a method for assembling biofunctionalized paper into a multiform structured scaffold system for reliable tissue regeneration using an origami-based approach. The surface of a paper was conformally modified with a poly(styrene-co-maleic anhydride) layer via initiated chemical vapor deposition followed by the immobilization of poly-l-lysine (PLL) and deposition of Ca(2+). This procedure ensures the formation of alginate hydrogel on the paper due to Ca(2+) diffusion. Furthermore, strong adhesion of the alginate hydrogel on the paper onto the paper substrate was achieved due to an electrostatic interaction between the alginate and PLL. The developed scaffold system was versatile and allowed area-selective cell seeding. Also, the hydrogel-laden paper could be folded freely into 3D tissue-like structures using a simple origami-based method. The cylindrically constructed paper scaffold system with chondrocytes was applied into a three-ring defect trachea in rabbits. The transplanted engineered tissues replaced the native trachea without stenosis after 4 wks. As for the custom-built scaffold system, the hydrogel-laden paper system will provide a robust and facile method for the formation of tissues mimicking native tissue constructs.

Comparing Nutrition Knowledge, Eating Habits, and Neonatal Health Status of Primipara for Pregnant Women of Advanced Maternal Age Compared to Those of Younger Ages.

PURPOSE: The aim of this study was to examine differences in nutrition knowledge, eating habits during pregnancy, and neonatal health status between primipara for pregnant women of advanced maternal age in comparison to those under the age of 35. METHODS: This study used a comparative survey design. Data were collected through self-report questionnaires and patients medical records. A total of 127 participants, mothers after delivery were recruited from metropolitan city B. RESULTS: Primipara in advanced maternal age (n=32) reported significantly higher scores of eating habits (Z=-2.96, p=.003) than younger ages (n=95). There were no significant differences in scores of pregnancy nutrition knowledge (Z=-0.44, p=.660), duration of gestation (Z=-0.28, p=.778), neonatal birth height (Z=-0.10, p=.924), neonatal birth weight (Z=-0.28, p=.777), Apgar score 1 minute (Z=-0.53, p=.599) and 5 minutes (Z=-0.23, p=.816) between two groups. CONCLUSION: It concludes that age is not the obstacle to the best nutritional status of women and their newborns.

Postpartum Depressive Score and Related Factors Pre- and Post-delivery.

PURPOSE: This study aimed to identify the scores of postpartum depression(PPD) on the first day, 1st week, and 6th week after the delivery and to explore their related factors before and after delivery in postpartum women. METHODS: With a survey design, 293 postpartum women were recruited from a postpartum unit, Ilsin Christian hospital in Pusan via convenience sampling and were followed at 1st week and 6th week in the outpatient clinic. RESULTS: Results showed that the scores of PPD(EPDS score) were low at postpartum 1st day, 1st week and 6th week but prevalence of PPD(EPDS > or =13) was 3.1% at 1st day, 8.2% at 1st week and 7.5% at 6th week, respectively. The pre-delivery factors were experience of depression, and the post-delivery factors were baby's sex(1st day), no caregiver for baby(1st week), and no help and concern for taking care of baby from husband and family(1st day and 6th week). The greater satisfaction with becoming a mother and her life, and greater maternal attachment were related to lower level of PPD at the three time points. CONCLUSION: Regular screening for postpartum depression and supportive and informative education is needed for postpartum women visiting the outpatient clinic for follow-up.

Should we consider the ethnic difference in selecting size of intraaortic balloon by commercial guideline?

To evaluate the appropriateness of commercial guideline in selecting the size of balloon for intraaortic counterpulsation in Asian patients, we analyzed the relationship between aortic dimensions measured from computed tomography and body indexes. There were 125 patients who underwent the computed tomography. They were divided into three height groups [<152 cm (I), 152-162 cm (II), and 163-183 cm (III)]. Aortic length and diameter were obtained from the origin of left subclavian artery to the orifice of renal artery. We compared those measurements with the balloon size according to the commercial guideline. Aortic lengths were measured as 250 +/- 15 (233-289), 262 +/-16 (227-298), and 285 +/- 19 (239-334) mm in each group, respectively. As the guideline, if we insert the balloon of 25 ml/191 mm, 34 ml/236 mm, and 40 ml/280 mm, there will be 29 of 66 patients (44%) in group III, who have shorter aortic length, with increased risk of obstructing renal artery. In addition, the younger the patient the higher is the risk (p = 0.038). We should pay attention to proper positioning of intraaortic balloon in younger Asian patients with 163-183 cm height, in applying the commercial guideline to prevent renal arterial obstruction.

Clinical observation of 12 farmers who believe themselves to have suffered from chronic pesticide intoxication.

BACKGROUND/AIM: We assessed twelve cases of suspected chronic pesticide intoxication, with medically unexplained physical symptoms. METHODS: Complete blood cell count (CBC), blood chemistry, routine urinalysis, chest X-ray, ECG, gastrofiberscopy, abdominal ultrasonography, neuroselective sensory nerve conduction threshold, and psychological assessment were performed on 12 farmers who believe themselves to have suffered from chronic pesticide intoxication. RESULTS: No specific abnormalities were observed on CBC, routine urinalysis, chest X-ray, ECG, gastroscopy, abdominal ultrasonography, or peripheral nerve conduction velocity test. They persistently manifested helplessness, depression, and anxiety. The results of both psychological assessment and general physical examination revealed the following clinical features: depression (8 cases), multiple chemical hypersensitivity syndrome (2 cases), alcoholism (1 case), and religious preoccupation (1 case). CONCLUSION: In those living in the western rural area of South Korea, depression is a prominent ongoing presentation in pesticide-exposed farmers, in addition to unexplainable physical symptoms.

Effects of the early administration of heparin in patients with ST-elevation myocardial infarction treated by primary angioplasty.

BACKGROUND: The effect of adjunctive heparin for primary angioplasty in patients with ST-elevation myocardial infarction (STEMI) is not well established, so the authors investigated the effect of early heparin administration in the emergency room (ER) on initial patency of the infarct-related artery (IRA) and on the clinical outcome in STEMI patients. METHODS AND RESULTS: One hundred and twenty consecutive patients who presented with STEMI less than 12 h from pain onset and who were eligible for primary percutaneous coronary intervention were allocated to an early heparin group (heparin administered in ER) or a late heparin group (heparin administered after angiography). In the early heparin group, unfractionated heparin (60 U/kg bolus IV, then 14 U . kg(-1) . h(-1) IV infusion) or enoxaparin (1 mg/kg bolus SC) were administered 144+/-95 min before angioplasty. No significant differences in baseline characteristics were observed between the early heparin group (n=56) and the late heparin group (n=64). However, initial Thrombolysis In Myocardial Infarction (TIMI) flow grade in the IRA was significantly different between the 2 groups (frequency of TIMI 0/1/2/3; 48/4/7/41% vs 70/8/11/11%, early vs late respectively, p=0.002). TIMI 2 or 3 flow was significantly more frequent in the early heparin group than in the late heparin group (48% vs 22%, p=0.002). However, no significant differences were noted between the 2 groups in terms of in-hospital major adverse cardiac events (7% vs 11%, p=0.472) and TIMI major bleeding (2% vs 3%, p=0.639). CONCLUSIONS: In STEMI patients, early heparin therapy administered in the ER improves coronary patency, despite not reaching clinical benefit.

Pesticide-initiated idiopathic environmental intolerance in South Korean farmers.

This study was designed to study patients with intolerance to pesticide smells. Ten subjects chosen were complaining of vague symptoms such as headache, dizziness, fatigue, nausea, vomiting, abdominal pain, myalgia, flu-like symptoms, etc., whenever exposed to the pesticide smells even at low intensity. To determine whether the etiology of this kind of pesticide hypersensitivity was of organic or psychiatric nature, all the subjects underwent tests as follows: complete blood cell count, urinalysis, and blood chemistry as routine tests; esophogastroduodenoscopy and abdomen ultrasonography for the gastrointestinal symptoms; chest x-ray, pulmonary function tests, and electrocardiography for the respiratory and/or cardiac symptoms; nerve conduction velocity and brain magnetic resonance imaging (MRI) for peripheral and central nerve system symptoms; and K-WAIS, Rey-Kim memory test, Rorschach, Mini Mental State Examination (MMSE), and Minnesota Multiphasic Personality Inventory (MMPI) for psychoanalysis. Of the 10 cases in which the chief complaint was headache, symptoms of two cases were caused by maxillary sinusitis. Another two showed typical multiple chemical sensitivity (MCS) or idiopathic environmental intolerance (IEI). Six out of the 10 cases, whose symptoms closely resembled the others, did not conclusively meet the criteria of classic MCS or IEI. The subjects of this case shared vague fears, both fear of pesticides and hypochondriasis. Some subjects faced financial insecurity and social uncertainty; others felt uneasy about the future of their farming life. Thus, to help verify the causes of MCS or IEI, which is strongly suggestive of pesticide smells, diagnosis needs a dual approach: on the anima and soma. Psychoanalysis can delve into the mental status of the patients to see whether the patients are aware of their symptoms. Clinical tests can see through the physical structure and functions of the organs on which patients' complaints are centered.

Nitric oxide and tumor necrosis factor-alpha production by Ixeris dentata in mouse peritoneal macrophages.

Using mouse peritoneal macrophages, we have examined the mechanism by which Ixeris dentata (IXD) regulates nitric oxide (NO) production. When IXD was used in combination with recombinant interferon-gamma (rIFN-gamma), there was a marked cooperative induction of NO production. However, IXD had no effect on NO production by itself. The increased production of NO from rIFN-gamma plus IXD-stimulated cells was almost completely inhibited by pre-treatment with pyrrolidine dithiocarbamate (PDTC), an inhibitor of nuclear factor kappa B (NF-kappaB). Furthermore, treatment with IXD alone or rIFN-gamma plus IXD in peritoneal macrophages caused a significant increase in tumor necrosis factor-alpha (TNF-alpha) production. PDTC decreased TNF-alpha production induced by IXD significantly. These findings demonstrate that IXD increases the production of NO and TNF-alpha by rIFN-gamma-primed macrophages and suggest that NF-kappaB plays a critical role in mediating these effects of IXD.