Serial cloning of pigs by somatic cell nuclear transfer: restoration of phenotypic normality during serial cloning.

Somatic cell nuclear transfer (scNT) is a useful way to create cloned animals. However, scNT clones exhibit high levels of phenotypic instability. This instability may be due to epigenetic reprogramming and/or genomic damage in the donor cells. To test this, we produced transgenic pig fibroblasts harboring the truncated human thrombopoietin (hTPO) gene and used them as donor cells in scNT to produce first-generation (G1) cloned piglets. In this study, 2,818 scNT embryos were transferred to 11 recipients and five G1 piglets were obtained. Among them, a clone had a dimorphic facial appearance with severe hypertelorism and a broad prominent nasal bridge. The other clones looked normal. Second-generation (G2) scNT piglets were then produced using ear cells from a G1 piglet that had an abnormal nose phenotype. We reasoned that, if the phenotypic abnormality of the G1 clone was not present in the G2 and third-generation (G3) clones, or was absent in the G2 clones but reappeared in the G3 clones, the phenotypic instability of the G1 clone could be attributed to faulty epigenetic reprogramming rather than to inherent/accidental genomic damage to the donor cells. Blastocyst rates, cell numbers in blastocyst, pregnancy rates, term placenta weight and ponderal index, and birth weight between G1 and G2 clones did not differ, but were significantly (P < 0.05) lower than control age- and sex-matched piglets. Next, we analyzed global methylation changes during development of the preimplantation embryos reconstructed by donor cells used for the production of G1 and G2 clones and could not find any significant differences in the methylation patterns between G1 and G2 clones. Indeed, we failed to detect the phenotypic abnormality in the G2 and G3 clones. Thus, the phenotypic abnormality of the G1 clone is likely to be due to epigenetic dysregulation. Additional observations then suggested that expression of the hTPO gene in the transgenic clones did not appear to be the cause of the phenotypic abnormality in the G1 clones and that the abnormality was acquired by only a few of the G1 clone's cells during its gestational development.     

Association analyses of DNA methyltransferase-1 (<Emphasis Type="Italic">DNMT1</Emphasis>) polymorphisms with systemic lupus erythematosus

The etiology of systemic lupus erythematosus (SLE) is very complex, and genetic factors appear to play a significant role in susceptibility to SLE, in determining the disease expression, and in the autoantibody profiles of individuals with SLE. DNA methyltransferase-1 (DNMT1) is a major enzyme that determines genomic methylation patterns and both maintains methyltransferase and exhibits de novo DNA methylation activity in vivo. In order to clarify the association of DNMT1 polymorphisms with SLE, we scrutinized the genetic polymorphisms in exons and their boundaries of DNMT1, including the –1,500 bp promoter region, by direct sequencing in 24 Korean individuals. Twenty-nine sequence variants were identified: two in 5UTR, six in exons, and 21 in introns. Eight of these polymorphisms were selected for a larger-scale genotyping (n=680) by considering their allele frequencies, haplotype-tagging status, and linkage disequilibrium coefficiencies (LDs) among polymorphisms. The associations between DNMT1 polymorphisms and the clinical profiles of SLE were analyzed. No significant associations with the risk of SLE were detected. However, further analyses of association with autoantibody production among SLE patients revealed that one nonsynonymous SNP, +14463G>C (V120L) in exon 4, was weakly associated with an increased risk of anti-La antibody production (P=0.04), although the significance could not be retained after correction of multiple tests. The DNMT1 variations and haplotypes clarified in this study would provide valuable information for future genetic studies of other autoimmune diseases.     

Modulation of large conductance calcium-activated K+ channel by membrane-delimited protein kinase and phosphatase activities

Large conductance Ca²⁺-activated K⁺ channel was identified and studied in excised inside-out membrane patches of freshly dispersed smooth muscle cells from rabbit gastric antrum. The current-voltage relationship of the single channel was linear from -80 to +80 mV of pipette voltage in which single channel conductance was 249±17.8 pS (n=19) in symmetrical concentration of K⁺ (145mM) across the patch. Activity of the channel (NPo) depended not only on cytoplasmic calcium concentration but also on membrane potential. MgATP increased NPo in a dose-dependent manner and Mg²⁺ was prerequisite for the effect. Okadaic acid (l00nM), inhibitor of protein phosphatases, increased NPo further in the presence of MgATP. Therefore, it would be concluded that activity of the calcium-activated K⁺ channel in gastric smooth muscle cells was controlled by phosphorylation state of the channel protein and the state is further modulated by membrane-delimited protein kinase and protein phosphatase activities.     

The lifetime of automatic semantic priming effects may exceed two seconds

The N400 component of event-related potentials (ERPs) was obtained in a modified version of the Neely [J.H. Neely, Semantic priming and retrieval from lexical memory: Roles of inhibitionless spreading activation and limited-capacity attention, Journal of Experimental Psychology: General, Vol. 106 (1977), pp. 226-254.] paradigm which permits unconfounding of semantic priming effects due to automatic and attentional processes. It was found that a short stimulus onset asynchrony (SOA) of 250 ms between the prime and the target was associated with automatic but not expectancy effects on the amplitude of N400. At a long SOA of 2000 ms between prime and target, semantic priming effects on N400 were obtained associated with both automatic and expectancy processes. Moreover, there was no significance difference in the magnitude of the automatic effects at the two SOAs, suggesting that automatic processing had not decayed within the 2000 ms interval between the prime and target. The results support the two-processing interpretation of semantic priming advanced by Posner and Synder [M.I. Posner, C.R.R. Snyder, Attention and cognitive control, in: R.L. Solso (Ed.), Information Processing and Cognition: The Loyola Symposium, Erlbaum, Hillsdale, NJ (1975).] and concur with the results of Neely [J.H. Neely, Semantic priming and retrieval from lexical memory: Roles of inhibitionless spreading activation and limited-capacity attention, Journal of Experimental Psychology: General, Vol. 106 (1977), pp. 226-254], with the exception of indicating a longer persistence of automatic processes.     

Adipocyte signals in energy balance and digestive diseases]

For the regulation of energy balance in various internal organs including gut, pancreas and liver, visceral adipose tissue and brain perform important sensing and signaling roles via neural and endocrine pathway. Among these, adipose tissue has been known as a simple energy-storing organ, which stores excess energy in triglyceride. However, it became apparent that adipocytes have various receptors related to energy homeostasis, and secrete adipocytokines by endocrine, paracrine and autocrine mechanisms. In this review, basic roles of adipocytes in energy homeostasis and the correlation between adipocyte signals and digestive diseases are discussed.     

Effectiveness and safety of single transseptal ablation for atrial fibrillation in real‐word practice

BackgroundWe have previously reported that unilateral groin‐single transseptal (ST) ablation in patients with paroxysmal atrial fibrillation (AF) was safe and significantly reduced patient discomfort compared with bilateral groin‐double transseptal (DT) ablation.HypothesisIn the present study, we hypothesized that ST ablation would be as effective and safe as DT ablation in real‐world practice like previous study. Among the 1765 consecutive patients in the Yonsei AF ablation cohort from October 2015 to January 2020, 1144 patients who underwent radiofrequency ablation were included for the analysis. Among them, 450 underwent ST ablation and 694 underwent DT ablation.ResultsThe total procedure time, ablation time, and fluoroscopy time were longer in the ST group than in the DT group (p < .05 for all). The hospital stay after catheter ablation was 1.3 ± 1.1 days which was longer in DT group than ST group (p = .001). No significant difference was observed in the complication rate (p = .263) and AF‐free survival rate (log‐rank p = .19) between the groups. However, after excluding patients who used antiarrhythmic drugs when AF recurred, the AF‐free survival rates were lower in the DT group than in the ST group before and after propensity score matching (log‐rank p = .026 and .047, respectively).ConclusionAlthough the ST approach increases the procedure time compared with the DT approach owing to the need for more frequent catheter exchanges, the ST approach is a feasible and safe strategy for AF ablation in terms of rhythm outcomes and risk of complications.     

Cytokine balance in kidney tissue from lupus nephritis patients

OBJECTIVE: To identify the balance of Th1/Th2 cytokine expression in the kidney and evaluate the difference in cytokine balance between patients with lupus nephritis WHO classes IV and V. METHODS: The expression of the CD40 molecule on cultured human mesangial cells was assessed by flow cytometry after stimulation with interferon gamma (IFN-gamma) or other cytokines. Frozen sections of kidney tissue from 10 patients with lupus nephritis and two non-SLE patients (with minimal-change disease) were stained with monoclonal antibodies for interleukin (IL)-4, IL-10, IL-12, IFN-gamma, CD4, CD8, CD40, CD68 and CD40L. RESULTS: CD40 expression of cultured mesangial cells was up-regulated by IFN-gamma, but was not down-regulated in the presence of the Th2 cytokines IL-4 and IL-10. In the glomeruli, CD40 expression and the ratios of IFN-gamma-/IL-10-, IL-12-/IL-4- and (IFN-gamma+IL-12)/(IL-4+IL-10)-positive cells were significantly higher in class IV than in class V lupus nephritis (P < 0.05). Also CD40, IFN-gamma and the activity index derived from the renal biopsy were closely correlated. CONCLUSION: IFN-gamma may contribute to the pathogenesis of proliferative glomerulonephritis by the up-regulation of CD40 and the activation of the cellular immune response in human lupus.     

Quality Indicators for Evaluating the Health Care of Patients with Rheumatoid Arthritis: a Korean Expert Consensus

BackgroundThere is increasing interest in the quality of health care and considerable efforts are being made to improve it. Rheumatoid arthritis (RA) is a disease that can result in favorable outcomes when appropriate diagnosis and treatment are provided. However, several studies have shown that RA is often managed inappropriately. Therefore, the Korean College of Rheumatology aimed to develop quality indicators (QIs) to evaluate and improve the health care of patients with RA.MethodsPreliminary QIs were derived based on the existing guidelines and QIs for RA. The final QIs were determined through two separate consensus meetings of experts. The consensus was achieved through a panel of experts who voted using the modified Delphi method.ResultsFourteen final QIs were selected among 70 preliminary QIs. These included early referral to and regular follow-up with a rheumatologist, radiographs of the hands and feet, early initiation and maintenance of disease-modifying anti-rheumatic drug (DMARD) therapy, periodic assessment of disease activity, screening for drug safety and comorbidities, including viral hepatitis and tuberculosis before biologic DMARD therapy, periodic laboratory testing, supplementation with folic acid, assessment of the risk for cervical spine instability before general anesthesia, patient education, and specialized nurse.ConclusionThese QIs can be used to assess and improve the quality of health care for patients with RA.