The three-dimensionality of the hiPSC-CM spheroid contributes to the variability of the field potential

Background: Field potential (FP) signals from human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) spheroid which are used for drug safety tests in the preclinical stage are different from action potential (AP) signals and require working knowledge of the multi-electrode array (MEA) system. In this study, we developed in silico three-dimensional (3-D) models of hiPSC-CM spheroids for the simulation of field potential measurement. We compared our model simulation results against in vitro experimental data under the effect of drugs E-4031 and nifedipine.Methods:In silico 3-D models of hiPSC-CM spheroids were constructed in spherical and discoidal shapes. Tetrahedral meshes were generated inside the models, and the propagation of the action potential in the model was obtained by numerically solving the monodomain reaction-diffusion equation. An electrical model of electrode was constructed and FPs were calculated using the extracellular potentials from the AP propagations. The effects of drugs were simulated by matching the simulation results with in vitro experimental data.Results: The simulated FPs from the 3-D models of hiPSC-CM spheroids exhibited highly variable shapes depending on the stimulation and measurement locations. The values of the IC50 of E-4031 and nifedipine calculated by matching the simulated FP durations with in vitro experimental data were in line with the experimentally measured ones reported in the literature.Conclusion: The 3-D in silico models of hiPSC-CM spheroids generated highly variable FPs similar to those observed in in vitro experiments. The in silico model has the potential to complement the interpretation of the FP signals obtained from in vitro experiments

Complete genome sequence of Middle East respiratory syndrome coronavirus KOR/KNIH/002_05_2015, isolated in South Korea

The full genome sequence of a Middle East respiratory syndrome coronavirus (MERS-CoV) was identified from cultured and isolated in Vero cells. The viral genome sequence has high similarity to 53 human MERS-CoVs, ranging from 99.5% to 99.8% at the nucleotide level. © 2015 Kim et al.

Efficacy of Lactic Acid Bacteria (LAB) supplement in management of constipation among nursing home residents

<p>Abstract</p> <p>Background</p> <p>Constipation is a significant problem in the elderly, specifically nursing home and/or extended-care facility residents are reported to suffer from constipation. Lactic acid bacteria (LAB) are beneficial probiotic organisms that contribute to improved nutrition, microbial balance, and immuno-enhancement of the intestinal tract, as well as diarrhea and constipation effect. The objective of this study was to investigate the efficacy of this LAB supplement in the management of nursing home residents.</p> <p>Methods</p> <p>Nineteen subjects (8M, 11F; mean age 77.1 ± 10.1) suffering with chronic constipation were assigned to receive LAB (3.0 × 10¹¹CFU/g) twice (to be taken 30 minutes after breakfast and dinner) a day for 2 weeks in November 2008. Subjects draw up a questionnaire on defecation habits (frequency of defecation, amount and state of stool), and we collected fecal samples from the subjects both before entering and after ending the trial, to investigate LAB levels and inhibition of harmful enzyme activities. Results were tested with SAS and Student's t-test.</p> <p>Results</p> <p>Analysis of questionnaire showed that there was an increase in the frequency of defecation and amount of stool excreted in defecation habit after LAB treatment, but there were no significant changes. And it also affects the intestinal environment, through significantly increase (<it>p </it>< 0.05) fecal LAB levels. In addition, tryptophanase and urease among harmful enzyme activities of intestinal microflora were significantly decreased (<it>p </it>< 0.05) after LAB treatment.</p> <p>Conclusion</p> <p>LAB, when added to the standard treatment regimen for nursing home residents with chronic constipation, increased defecation habit such as frequency of defecation, amount and state of stool. So, it may be used as functional probiotics to improve human health by helping to prevent constipation.</p

Dietary fat increases solid tumor growth and metastasis of 4T1 murine mammary carcinoma cells and mortality in obesity-resistant BALB/c mice

Introduction High-fat diets (HFDs) are known to cause obesity and are associated with breast cancer progression and metastasis. Because obesity is associated with breast cancer progression, it is important to determine whether dietary fat per se stimulates breast cancer progression in the absence of obesity. This study investigated whether an HFD increases breast cancer growth and metastasis, as well as mortality, in obesity-resistant BALB/c mice. Methods The 4-week-old, female BALB/c mice were fed HFD (60% kcal fat) or control diet (CD, 10% kcal fat) for 16 weeks. Subsequently, 4T1 mammary carcinoma cells were injected into the inguinal mammary fat pads of mice fed continuously on their respective diets. Cell-cycle progression, angiogenesis, and immune cells in tumor tissues, proteases and adhesion molecules in the lungs, and serum cytokine levels were analyzed with immunohistochemistry, Western blotting, and enzyme-linked immunosorbent assay (ELISA). In vitro studies were also conducted to evaluate the effects of cytokines on 4T1 cell viability, migration, and adhesion. Results Spleen and gonadal fat-pad weights, tumor weight, the number and volume of tumor nodules in the lung and liver, and tumor-associated mortality were increased in the HFD group, with only slight increases in energy intake and body weight. HF feeding increased macrophage infiltration into adipose tissues, the number of lipid vacuoles and the expression of cyclin-dependent kinase (CDK)2, cyclin D1, cyclin A, Ki67, CD31, CD45, and CD68 in the tumor tissues, and elevated serum levels of complement fragment 5a (C5a), interleukin (IL)-16, macrophage colony-stimulating factor (M-CSF), soluble intercellular adhesion molecule (sICAM)-1, tissue inhibitors of metalloproteinase (TIMP)-1, leptin, and triggering receptor expressed on myeloid cells (TREM)-1. Protein levels of the urokinase-type plasminogen activator, ICAM-1, and vascular cell adhesion molecule-1 were increased, but plasminogen activator inhibitor-1 levels were decreased in the lungs of the HFD group. In vitro assays using 4T1 cells showed that sICAM-1 increased viability; TREM-1, TIMP-1, M-CSF, and sICAM-1 increased migration; and C5a, sICAM-1, IL-16, M-CSF, TIMP-1, and TREM-1 increased adhesion. Conclusions Dietary fat increases mammary tumor growth and metastasis, thereby increasing mortality in obesity-resistant mice

A Multiinstitutional Consensus Study on the Pathologic Diagnosis of Endometrial Hyperplasia and Carcinoma

BACKGROUND: The purpose of this study was to examine the reproducibility of both the diagnosis of endometrial hyperplasia (EH) or adenocarcinoma, and the histologic grading (HG) of endometrioid adenocarcinoma (EC). METHODS: Ninety-three cases of EH or adenocarcinomas were reviewed independently by 21 pathologists of the Gynecologic Pathology Study Group. A consensus diagnosis was defined as agreement among more than two thirds of the 21 pathologists. RESULTS: There was no agreement on the diagnosis in 13 cases (14.0%). According to the consensus review, six of the 11 EH cases (54.5%) were diagnosed as EH, 48 of the 57 EC cases (84.2%) were EC, and 5 of the 6 serous carcinomas (SC) (83.3%) were SC. There was no consensus for the 6 atypical EH (AEH) cases. On the HG of EC, there was no agreement in 2 cases (3.5%). According to the consensus review, 30 of the 33 G1 cases (90.9%) were G1, 11 of the 18 G2 cases (61.1%) were G2, and 4 of the 4 G3 cases (100.0%) were G3. CONCLUSIONS: The consensus study showed high agreement for both EC and SC, but there was no consensus for AEH. The reproducibility for the HG of G2 was poor. We suggest that simplification of the classification of EH and a two-tiered grading system for EC will be necessary.This study was partly supported by research fund of Chungnam National University in 2007

ApoE Receptor 2 Regulates Synapse and Dendritic Spine Formation

Apolipoprotein E receptor 2 (ApoEr2) is a postsynaptic protein involved in long-term potentiation (LTP), learning, and memory through unknown mechanisms. We examined the biological effects of ApoEr2 on synapse and dendritic spine formation-processes critical for learning and memory.In a heterologous co-culture synapse assay, overexpression of ApoEr2 in COS7 cells significantly increased colocalization with synaptophysin in primary hippocampal neurons, suggesting that ApoEr2 promotes interaction with presynaptic structures. In primary neuronal cultures, overexpression of ApoEr2 increased dendritic spine density. Consistent with our in vitro findings, ApoEr2 knockout mice had decreased dendritic spine density in cortical layers II/III at 1 month of age. We also tested whether the interaction between ApoEr2 and its cytoplasmic adaptor proteins, specifically X11α and PSD-95, affected synapse and dendritic spine formation. X11α decreased cell surface levels of ApoEr2 along with synapse and dendritic spine density. In contrast, PSD-95 increased cell surface levels of ApoEr2 as well as synapse and dendritic spine density.These results suggest that ApoEr2 plays important roles in structure and function of CNS synapses and dendritic spines, and that these roles are modulated by cytoplasmic adaptor proteins X11α and PSD-95

심장독성 약물의 전기생리학적 분석을 위한 줄기세포 이용 통합 연구

학위논문 (박사)-- 서울대학교 대학원 : 의과대학 의과학과, 2016. 2. 김성준.Even though several in vitro and in vivo QT screening systems (e.g. hERG assay, telemetry in conscious animals) are currently used as standardized assays for cardiotoxicity, these testing models are deficient. The major reason for their poor predictive powers is that they cannot replicate human cardiac electrophysiology. With the development of stem cell technologies, in vitro assays using human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) is suggested as an effective candidate of drug screening platform. The aims of this study are to explore the necessity of integrative interpretation with multiple types of cardiac ion channels for the cardiotoxicity test, and to evaluate the usefulness of adopting hPSC-CMs for the electrophysiological study. Chapter 1: Comparison of electrophysiological effects of calcium channel blockers on cardiac repolarization1 It is anticipated that Ca2+ channel blockers (CCBs) would shorten action potential duration (APD), which could lead to tachycardia. Nevertheless, CCBs are widely prescribed to hypertension without serious problems of cardiac arrhythmia. Here I investigated the electrophysiological effects of dihydropyridine class of CCBs, nicardipine (NIC), isradipine (ISR), and amlodipine (AML). All the three CCBs inhibited the L-type Ca2+ currents (ICa) whereas the shortening of APD was observed only with ISR. In addition, interestingly, NIC and AML also inhibited voltage-gated K+ channels currents (IKr and IKs) at micromolar ranges while ISR did not. I interpret that the concomitant K+ channel inhibition by NIC and AML might have compensated the AP shortening effects induced by the ICa inhibition. Chapter 2: Integrative analysis of cardiac ion channel modulation by SARI class antidepressants in human stem cell-derived cardiomyocytes2 The potential usefulness of human stem cell-derived cardiomyocytes in drug toxicity testing is drawing attention to the pharmaceutical industry recently. Here I evaluated the usefulness of commercialized human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). The cardiac three types of action potentials, nodal (N)-, atrial (A)-, and ventricular (V)-type, and ion channels related cardiac AP (IKr, IKs, IK1, If, INa, ICa) were recorded in the cells. Additionally, hiPSC-CMs effectively recapitulate the electrophysiological behaviors of the major ion channel blockers, (E-4031 for hERG channel, tetrodotoxin for Na+ channel, nifedipine for calcium channel), confirming the plausibility of a platform for preclinical drug safety assessment. Then I analyzed the cardiotoxic effects of trazodone and nefazodone, serotonin antagonist and reuptake inhibitor (SARI) class antidepressants, using hiPSC-CMs or HEK293 cells overexpressing cardiac ion channel. Both drugs induced APD prolongation and early afterdepolarizations (EADs) and reduced the upstroke velocity in a dose-dependent manner. Consistent with the changes in the AP parameters, nefazodone and trazodone inhibited IKr, IKs, INa, and ICa, among them especially IKr and INa, but nefazodone had a higher inhibitory potency than trazodone. Chapter 3: Differentiation period-dependent changes in the electrophysiological properties of human stem cell-derived cardiomyocytes The assessment of functionality of human embryonic stem cell derived cardiomyocytes (hESC-CMs) at early developmental stages is essential for determining the appropriate differentiation stage for cardiotoxicity screening. In this study, to determine more suitable stage of differentiation required for the reliable pharmacological and toxicological testing, I characterized 2 week (2W) and 4 week (4W) differentiated hESC-CMs and compared their electrophysiological phenotypes and functional maturation using patch-clamp technique. The densities of functional ion channels currents, INa, ICa, IKr, IKs, and IK1, tended to increase in the 4W hESC-CMs while not significantly. In the AP recordings, the 2W hESC-CMs displayed only A-type (87.5%) and N-type (12.5%) without V-type of APs. However, the 4W hESC-CMs revealed 3 types of AP with the majority of cells revealed V-type APs (69%). The pharmacological responses for anti-arrhythmic drugs revealed that quinidine and amiodarone (Na+ and K+ channel blockers, respectively) prolonged APD at 90% (APD90) in the 4W hESC-CMs while not in the 2W hESC-CMs. Nifedipine significantly shortened APD90 only in the 4W hESC-CMs. Taken together, this study demonstrated the drug-induced cardiotoxicity has to be estimated with overall effects on multiple ion channels because of their compensatory effects between depolarizing and repolarizing currents. The hiPSC-CMs could be a valuable testbed for evaluating the proarrhythmic liability of trazodone and nefazodoneelectrophysiological properties of hiPSC-CMs and their responses faithfully reflected the changes of individual ion channel current. The hiPSC-CMs can be an effective model for detection of early drug-induced cardiotoxicity beyond the current standard assay of hERG K+ channels. However, to use stem cell-derived cardiomyocytes in drug screening, at least 4 weeks of differentiation period is required for the reliable pharmacological and toxicological testing.General Introduction 1 CHAPTER 1 Comparison of electrophysiological effects of calcium channel blockers on cardiac repolarization 6 Introduction 7 Material and Methods 11 Results 17 Discussion 29 CHAPTER 2 Integrative analysis of cardiac ion channel modulation by SARI class antidepressants in human stem cell derived cardiomyocytes 32 Introduction 33 Material and Methods 36 Results 40 Discussion 70 CHAPTER 3 Differentiation period-dependent changes in the electrophysiological properties of human embryonic stem cell-derived cardiomyocytes 75 Introduction 76 Material and Methods 78 Results 81 Discussion 94 General Discussion 96 References 99 Abstract in Korean 110Docto