The clustered regularly interspaced short palindromic repeats (CRISPR) system, a rapidly advancing genome editing technology, allows DNA alterations into the genome of organisms. Gene editing using the CRISPR system enables more precise and diverse editing, such as single nucleotide conversion, precise knock-in of target sequences or genes, chromosomal rearrangement, or gene disruption by simple cutting. Moreover, CRISPR systems comprising transcriptional activators/repressors can be used for epigenetic regulation without DNA damage. Stem cell DNA engineering based on gene editing tools has enormous potential to provide clues regarding the pathogenesis of diseases and to study the mechanisms and treatments of incurable diseases. Here, we review the latest trends in stem cell research using various CRISPR/Cas technologies and discuss their future prospects in treating various diseases.

Background: The prevalence of depression is much higher in people with chronic disease than in the general population. Depression exacerbates existing physical conditions, resulting in a higher-than-expected death rate from the physical condition itself. In our aging society, the prevalence of multimorbid patients is expected to increase; the resulting mental problems, especially depression, should be considered. Using a large-scale cohort from the Korean Longitudinal Study of Aging (KLoSA), we analyzed the combined effects of depression and chronic disease on all-cause mortality. Methods: We analyzed 10-year (2006–2016) longitudinal data of 9,819 individuals who took part in the KLoSA, a nationwide survey of people aged 45–79 years. We examined the association between multimorbidity and depression using chi-square test and logistic regression. We used the Cox proportional hazard model to determine the combined effects of multimorbidity and depression on the all-cause mortality risk. Results: During the 10-year follow up, 1,574 people (16.0%) died. The hazard ratio associated with mild depression increased from 1.35 (95% confidence interval [CI], 1.05–1.73) for no chronic disease to 1.25 (95% CI, 0.98–1.60) for 1 chronic disease, and to 2.00 (95% CI, 1.58–2.52) for multimorbidity. The hazard ratio associated with severe depression increased from 1.73 (95% CI, 1.33–2.24) for no chronic disease, to 2.03 (95% CI, 1.60–2.57) for 1 chronic disease, and to 2.94 (95% CI, 2.37–3.65) for multimorbidity. Conclusion Patients with coexisting multimorbidity and depression are at an increased risk of all-cause mortality than those with chronic disease or depression alone.

Background: The prevalence of depression is much higher in people with chronic disease than in the general population. Depression exacerbates existing physical conditions, resulting in a higher-than-expected death rate from the physical condition itself. In our aging society, the prevalence of multimorbid patients is expected to increase; the resulting mental problems, especially depression, should be considered. Using a large-scale cohort from the Korean Longitudinal Study of Aging (KLoSA), we analyzed the combined effects of depression and chronic disease on all-cause mortality. Methods: We analyzed 10-year (2006–2016) longitudinal data of 9,819 individuals who took part in the KLoSA, a nationwide survey of people aged 45–79 years. We examined the association between multimorbidity and depression using chi-square test and logistic regression. We used the Cox proportional hazard model to determine the combined effects of multimorbidity and depression on the all-cause mortality risk. Results: During the 10-year follow up, 1,574 people (16.0%) died. The hazard ratio associated with mild depression increased from 1.35 (95% confidence interval [CI], 1.05–1.73) for no chronic disease to 1.25 (95% CI, 0.98–1.60) for 1 chronic disease, and to 2.00 (95% CI, 1.58–2.52) for multimorbidity. The hazard ratio associated with severe depression increased from 1.73 (95% CI, 1.33–2.24) for no chronic disease, to 2.03 (95% CI, 1.60–2.57) for 1 chronic disease, and to 2.94 (95% CI, 2.37–3.65) for multimorbidity. Conclusion Patients with coexisting multimorbidity and depression are at an increased risk of all-cause mortality than those with chronic disease or depression alone.

Collagen-induced arthritis (CIA) is mediated by self-reactive CD4+ T cells that produce inflammatory cytokines. TGF-beta2-treated tolerogenic antigen-presenting cells (Tol-APCs) are known to induce tolerance in various autoimmune diseases. In this study, we investigated whether collagen-specific Tol-APCs could induce suppression of CIA. We observed that Tol-APCs could suppress the development and severity of CIA and delay the onset of CIA. Treatment of Tol-APCs reduced the number of IFN-gamma- and IL-17-producing CD4+ T cells and increased IL-4- and IL-5-producing CD4+ T cells upon collagen antigen stimulation in vitro. The suppression of CIA conferred by Tol-APCs correlated with their ability to selectively induce IL-10 production. We also observed that treatment of Tol-APCs inhibited not only cellular immune responses but also humoral immune responses in the process of CIA. Our results suggest that in vitro-generated Tol-APCs have potential therapeutic value for the treatment of rheumatoid arthritis as well as other autoimmune diseases.
Animals ; Antigen-Presenting Cells/*drug effects/*immunology ; Arthritis, Experimental/*immunology ; Chickens ; Collagen Type II/immunology ; Immune Tolerance/drug effects ; Mice ; Mice, Inbred BALB C ; Ovalbumin/immunology ; Th1 Cells/drug effects/immunology ; Th2 Cells/*drug effects/*immunology ; Transforming Growth Factor beta2/*pharmacology

Background/Aims: Intrahepatic cholangiocarcinoma (iCCA) is a subgroup of cholangiocarcinoma and is the second- most-common primary hepatic tumor. Several predictive and prognostic factors have been analyzed; however, in this study we focused on the influence of age. Our aim was to use real-world results to determine the influence of age in iCCA patients. Methods: A retrospective analysis of patients treated between 2005 and 2016 at Konkuk University Medical Center. In total, 133 patients with iCCA were identified. The mass-forming, periductal-infiltrating, and intraductal-growth types were included; patients with extrahepatic or hilar-type cholangiocarcinoma were excluded. We defined two groups: a younger group, age < 65 years, and an older group, age ≥ 65 years. Statistical analyses using univariate and multivariate Cox regression analyses, including the Kaplan-Meier method, were conducted. Results: In total, 114 patients were enrolled. The two groups differed with regard to treatment options such as surgery with adjuvant chemotherapy or palliative chemotherapy (p = 0.012, p < 0.001). The younger group had significantly longer survival than the older group (p = 0.017). In the younger group, patients who received therapy had longer survival than those who did not (hazard ratio, 3.942; 95% confidence interval, 2.053 to 7.569; p < 0.001). Multivariate analysis indicated that younger age, lower bilirubin, low CA 19-9, and no lymph-node involvement were independent factors for improved survival. Conclusions Younger patients and those who underwent surgery with adjuvant chemotherapy had longer survival. The younger the patient, the more treatments received, including palliative chemotherapy.

Purpose: This study aimed to evaluate the feasibility of measuring liver fat using the volumetric measurement method (Fatvol) by comparing it with the conventional 27-regions of interest drawing method (Fatroi). Materials and Methods: This retrospective study included 67 patients who underwent liver magnetic resonance imaging with fat quantification in August or September 2020.Two experienced abdominal radiologists measured the proton density fat fraction (PDFF) of the liver using the mDIXON-Quant sequence for each of two methods. The PDFF was measured twice with each method at intervals of at least 4 weeks to avoid recall bias. Measurement times were recorded. The intra-class correlation coefficient (ICC) was calculated for intra-exam repeatability, inter-reviewer reproducibility, and inter-exam agreement. Results: Measurement times for Fatvol were significantly shorter than for Fatroi. Measurement times for Fatroi and Fatvol, respectively, for reviewer A were 209.4 ± 55.1 s and 137.2 ± 51.5 s in session 1, and 180.9 ± 37.3 s and 127.0 ± 46.1 s in session 2. For reviewer B, the times were 190.7 ± 30.1 s and 74.8 ± 27.4 s in session 1, and 174.6 ± 21.8 s and 64.1 ± 17.5 s in session 2. In all cases, p < 0.001. The mean PDFF values were 7.2% ± 6.4% and 7.2% ± 6.5% (sessions 1 and 2, respectively) for Fatroi and 7.4% ± 6.0% and 7.3% ± 6.1% for Fatvol for reviewer A. For reviewer B, they were 7.1% ± 6.6% and 7.1% ± 6.6% for Fatroi and 7.4% ± 5.8% and 7.4% ± 5.8% for Fatvol. The ICCs between measurement methods (0.998 and 0.995 for reviewers A and B, respectively), for Fatvol within each reviewer (0.999 and 1.000 in sessions 1 and 2, respectively), and between reviewers (0.999) were excellent. Conclusion The measurement time could be significantly reduced using Fatvol compared to Fatroi while maintaining the consistency of the liver fat measurement values.

As a part of our ongoing search for a safe and efficient anti-tumor vaccine, we attempted to determine whether the molecular nature of certain tumor antigens would influence immune responses against tumor cells. As compared with freeze-thawed or formaldehyde-fixed tumor antigens, heat-denatured tumor antigens elicited profound anti-tumor immune responses and greatly inhibited the growth of live tumor cells. The heat-denatured tumor antigens induced a substantial increase in the anti-tumor CTL response in the absence of any adjuvant material. This response appears to be initiated by strong activation of the antigen-presenting cells, which may recognize heat-denatured protein antigens. Upon recognition of the heat-denatured tumor antigens, macrophages and dendritic cells were found to acutely upregulate the expression of co-stimulatory molecules such as B7.2, as well as the secretion of inflammatory cytokines such as IL-12 and TNF-alpha. The results of this study indicate that heat-denatured tumor extracts might elicit protective anti-tumor adaptive immune responses and also raise the possibility that a safe and efficient adjuvant-free tumor vaccine might be developed in conjunction with a dendritic cell-based tumor vaccine.
Adjuvants, Immunologic ; Animals ; Antibodies, Neoplasm/immunology ; Antibody Specificity/immunology ; Antigens, Neoplasm/immunology ; Cancer Vaccines/*immunology ; Cell Line, Tumor ; Cell Proliferation ; Cytokines/biosynthesis ; Cytotoxicity, Immunologic/immunology ; Dendritic Cells/immunology ; *Hot Temperature ; Immunity, Cellular/immunology ; Immunization ; Immunologic Memory/immunology ; Macrophages, Peritoneal/immunology ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Neoplasms/*immunology/*pathology ; Survival Analysis ; T-Lymphocytes, Cytotoxic/immunology

In this study, we report the development of a new dual reporter vector system for the analysis of promoter activity. This system employs green fluorescence emitting protein, EGFP, as a reporter, and uses red fluorescence emitting protein, DsRed, as a transfection control in a single vector. The expression of those two proteins can be readily detected via flow cytometry in a single analysis, with no need for any further manipulation after transfection. As this system allows for the simultaneous detection of both the control and reporter proteins in the same cells, only transfected cells which express the control protein, DsRed, can be subjected to promoter activity analysis, via the gating out of all un-transfected cells. This results in a dramatic increase in the promoter activity detection sensitivity. This novel reporter vector system should prove to be a simple and efficient method for the analysis of promoter activity.
Enzyme Multiplied Immunoassay Technique ; Flow Cytometry ; Fluorescence ; Luminescent Proteins ; Proteins ; Transfection

Objective: Overcrowding in the emergency department (ED) has been a long-standing global problem, but has yet to be resolved. This study was undertaken to investigate whether expansion of the ED can affect overcrowding. Methods: This was a retrospective study comparing two 10-month periods: before (September 2015 to June 2016) and after (September 2017 to June 2018) the ED expansion in an urban tertiary hospital. The ED expansion included expansion of the ambulatory area and establishment of a 25-bedded emergency ward dedicated to patients admitted through the ED. Results: Comparing the two study periods, we noted an increase in the number of patients visiting the ED, from 77,078 to 87,027. Moreover, the proportion of patients who returned home untreated significantly decreased from 11.5% to 0.9% (P<0.001). The number of adult patients increased from 40,814 to 60,720; in particular, the number of ambulatory patients increased from 18,648 to 42,944. Conversely, waiting time for X-ray and computed tomography increased (10.0 to 17.0 minutes, and 35.0 to 48.0 minutes, respectively). Other areas with increased time duration include median ED length of stay of total patients (193.0 minutes to 205.8 minutes), and time from consultation to admission decision (122.3 to 161.4 minutes). However, the boarding time decreased from 239.2 to 190.9 minutes. Conclusion The ED expansion allowed more patients to be treated, and the boarding time of admitted patients was reduced through operation of the emergency ward. However, due to increase in the number of visiting patients, the time required for medical treatment increased concurrently.

Phthalates widely used in the manufacture of plastics have deeply penetrated into our everyday lives. Recently, a concern over the toxicity of phthalates on thyroid, has been raised but in most of cases, the doses employed were unrealistically high. To investigate the effects of phthalates on thyroid, we investigated the effects of the repeated oral exposure to low to high doses (0.3, 3, 30 and 150 mg/kg) di-2-ethylhexylphthalate (DEHP) from weaning to maturity for 90 days in juvenile rats on the thyroid. The histological examination revealed that DEHP significantly induced hyperplasia in the thyroid from the doses of 30 mg/kg, which was confirmed with Ki67 staining. In line with this finding, increased mRNA expression of thyrotropin releasing hormone (Trh) was observed in the thyroid of female at 0.3 mg/kg and 150 mg/kg as determined by RNAseq analysis. Moreover, significantly increased expression of parathyroid hormone (Pth) in the female at 0.3 mg/kg, and thyroglobulin (Tg) and thyroid hormone responsive (Thrsp) in the male at 0.3 mg/kg were noted in the blood, of which changes were substantially attenuated at 150 m/kg, alluding the meaningful effects of low dose DEHP on the thyroid hormone regulation. Urinary excretion of mono-2-ethylhexyl-phthalate (MEHP), a major metabolite of DEHP was determined to be 4.10 and 12.26 ppb in male, 6.65 and 324 ppb in female at 0.3 and 30 mg/kg DEHP, respectively, which fell within reported human urine levels. Collectively, these results suggest a potential adverse effects of low dose phthalates on the thyroid.
Animals ; Diethylhexyl Phthalate* ; Female ; Humans ; Hyperplasia ; Male ; Parathyroid Hormone ; Plastics ; Rats* ; RNA, Messenger ; Thyroglobulin ; Thyroid Gland* ; Thyrotropin-Releasing Hormone ; Weaning