Generation and Characterization of Patient-Specific iPSC Model for Cardiovascular Disease.

Advances in differentiation of cardiomyocytes from human induced pluripotent stem cell (hiPSC) were emerged as a tool for modeling of cardiovascular disease that recapitulates the phenotype for the purpose of drug screening, biomarker discovery, and testing of single-nucleotide polymorphism (SNP) as a modifier for disease stratification. Here, we describe the (1) retroviral reprogramming strategies in the generation of human iPSC, (2) methodology in characterization of iPSC in order to identify the stem cell clones with the best quality, and (3) protocol of cardiac differentiation by modulation of Wnt signaling and ß-catenin pathway.

Motion corrected LV quantification based on 3D modelling for improved functional assessment in cardiac MRI.

Cine MRI is a clinical reference standard for the quantitative assessment of cardiac function, but reproducibility is confounded by motion artefacts. We explore the feasibility of a motion corrected 3D left ventricle (LV) quantification method, incorporating multislice image registration into the 3D model reconstruction, to improve reproducibility of 3D LV functional quantification. Multi-breath-hold short-axis and radial long-axis images were acquired from 10 patients and 10 healthy subjects. The proposed framework reduced misalignment between slices to subpixel accuracy (2.88 to 1.21 mm), and improved interstudy reproducibility for 5 important clinical functional measures, i.e. end-diastolic volume, end-systolic volume, ejection fraction, myocardial mass and 3D-sphericity index, as reflected in a reduction in the sample size required to detect statistically significant cardiac changes: a reduction of 21-66%. Our investigation on the optimum registration parameters, including both cardiac time frames and number of long-axis (LA) slices, suggested that a single time frame is adequate for motion correction whereas integrating more LA slices can improve registration and model reconstruction accuracy for improved functional quantification especially on datasets with severe motion artefacts.

Granzyme M targets topoisomerase II alpha to trigger cell cycle arrest and caspase-dependent apoptosis.

Cytotoxic lymphocyte protease granzyme M (GrM) is a potent inducer of tumor cell death. The apoptotic phenotype and mechanism by which it induces cell death, however, remain poorly understood and controversial. Here, we show that GrM-induced cell death was largely caspase-dependent with various hallmarks of classical apoptosis, coinciding with caspase-independent G2/M cell cycle arrest. Using positional proteomics in human tumor cells, we identified the nuclear enzyme topoisomerase II alpha (topoIIα) as a physiological substrate of GrM. Cleavage of topoIIα by GrM at Leu(1280) separated topoIIα functional domains from the nuclear localization signals, leading to nuclear exit of topoIIα catalytic activity, thereby rendering it nonfunctional. Similar to the apoptotic phenotype of GrM, topoIIα depletion in tumor cells led to cell cycle arrest in G2/M, mitochondrial perturbations, caspase activation, and apoptosis. We conclude that cytotoxic lymphocyte protease GrM targets topoIIα to trigger cell cycle arrest and caspase-dependent apoptosis.

Developing and testing the effectiveness of a novel health qigong for frail elders in Hong Kong: a preliminary study.

Eight-Section Brocades and Yijin Jing consist of some routine movements that are too difficult for frail elders. A novel health qigong protocol was developed and its effectiveness for frail elders was examined using a randomized clinical trial (RCT). An expert panel performed functional anatomy analysis and safety field test prior to the RCT. The experimental group (n = 61, 83 ± 6 yr) was given a 12-week qigong exercise program, while the comparison group (n = 55, 84 ± 6 yr) participated in a newspaper reading program with the same duration and frequency. Pre-, mid-, post-, and follow-up assessments were conducted. At 12 weeks, the qigong group had significant improvements in thinking operations (F = 4.05, P = .02) and significant reduction of resting heart rate (F = 3.14, P = .045) as compared to the newspaper reading group. A trend of improvements in grip strength and a decreasing trend of depression levels were observed among the qigong group. Significant perceived improvements in physical health (F = 13.01, P = .001), activities of daily living (F = 5.32, P = .03), and overall health status (F = 15.26, P = .0001) were found. There are improvements in some aspects of psychosocial, cognitive, physical, and physiological domains. Clinical applications and possibilities for further research are discussed.

Granzyme M targets host cell hnRNP K that is essential for human cytomegalovirus replication.

Human cytomegalovirus (HCMV) is the most frequent viral cause of congenital defects and HCMV infection in immunocompromised patients may trigger devastating disease. Cytotoxic lymphocytes control HCMV by releasing granzymes towards virus-infected cells. In mice, granzyme M (GrM) has a physiological role in controlling murine CMV infection. However, the underlying mechanism remains poorly understood. In this study, we showed that human GrM was expressed by HCMV-specific CD8(+) T cells both in latently infected healthy individuals and in transplant patients during primary HCMV infection. We identified host cell heterogeneous nuclear ribonucleoprotein K (hnRNP K) as a physiological GrM substrate. GrM most efficiently cleaved hnRNP K in the presence of RNA at multiple sites, thereby likely destroying hnRNP K function. Host cell hnRNP K was essential for HCMV replication not only by promoting viability of HCMV-infected cells but predominantly by regulating viral immediate-early 2 (IE2) protein levels. Furthermore, hnRNP K interacted with IE2 mRNA. Finally, GrM decreased IE2 protein expression in HCMV-infected cells. Our data suggest that targeting of hnRNP K by GrM contributes to the mechanism by which cytotoxic lymphocytes inhibit HCMV replication. This is the first evidence that cytotoxic lymphocytes target host cell proteins to control HCMV infections.

Gate dependent photo-responses of carbon nanotube field effect phototransistors.

Gate dependent photoconductivity of carbon nanotube (CNT) field effect phototransistors (FEPs) was systematically investigated in this study. The photo-response comparisons of CNT FEPs with symmetric and asymmetric metal structures connecting to the same CNT revealed that the gate effect contributed to a sensitivity improvement with a lower dark current, a higher photocurrent, and an enhanced photovoltage. A functionalized asymmetric FEP, fabricated by partially doping the CNT utilizing a polyethylene imine (PEI) polymer, verified that FEPs delivered a better performance by using asymmetric structures. A multi-gate FEP, with three pairs of side-gates that can electrostatically dope different sections of a CNT independently, was fabricated to examine the gate structure dependent photo-responses. Experimental measurements showed an unconventional photocurrent improvement that was weakly dependent on the gate location, which was attributed to the unique charge distribution of one-dimensional semiconductors.

Knowledge of cardiopulmonary resuscitation among the public in Hong Kong: telephone questionnaire survey.

OBJECTIVES: To evaluate the knowledge of basic life-support and training experience in cardiopulmonary resuscitation among the public in Hong Kong and to identify areas for improvement in public education. DESIGN: Telephone interview using a structured multiple-choice questionnaire. SETTING: Random cross-section of the Hong Kong public, from mid-March to May 2002. PARTICIPANTS: Men and women aged 16 years and older selected using random telephone dialling. MAIN OUTCOME MEASURE: Overall score in the cardiopulmonary resuscitation knowledge questionnaire. RESULTS: Of the 357 participants, approximately 12% had received cardiopulmonary resuscitation training. Cardiopulmonary resuscitation knowledge in Hong Kong was poor, even among the previously trained and especially with regard to circulatory maintenance. The most common reason for not taking cardiopulmonary resuscitation training was lack of time. CONCLUSION: The degree of citizen preparedness in initiating cardiopulmonary resuscitation is very poor in Hong Kong. Intensified educational efforts and exploration of new approaches to improve this first stage in the chain of survival are warranted.

Changes in heart rate and R-wave amplitude with posture.

BACKGROUND: Effect of gravity on heart rate and blood pressure are well documented but the effect of posture on R-wave amplitude has not been studied. AIM: To investigate the effect of posture on the heart rate (HR) and R-wave amplitude (RWA). METHODS: The electrocardiograph (ECG) was recorded in 20 young subjects on two occasions. A 5 minute recording of resting ECG was taken with the subject adopting the following postural cycle: lying, sitting, standing, sitting and lying positions. RESULTS: The standing HR was significantly higher than that in sitting and lying positions, but the RWA was significantly lower in standing compared to the sitting and lying postures. The HR significantly increased at the initial phase of postural changes, irrespective of the position. For example, the HR increased from lying to sitting (66.6+/-2.3 to 85.0+/-10.9 beat x min(-1)) but also increased when changed from sitting to lying (70.6+/-10.6 to 85.2+/-8.7 beat x min(-1)). CONCLUSION: Body posture has an effect on HR and RWA. The changes are probably related to orthostatic haemodynamic stress as well as changes in cardiac electrical axis. Whether the cause of the initial increase in the HR during postural change was attributed to skeletal muscle reflex or venous return requires further investigation.

Inhibitor of apoptosis proteins and ovarian dysfunction in galactosemic rats.

Galactosemia is a genetic disease with deficiency of galactose-1-uridyltransferase, resulting in the accumulation of galactose or galactose-1-phosphate in the blood and tissues. Rats were fed with normal rat chow and with a high-galactose diet for 4 weeks to give control and galactosemic groups, and their ovarian function was studied. The two groups of rats were injected with pregnant mare's serum gonadotrophin (PMSG) and were killed at different time points after human chorionic gonadotrophin (hCG) injection. The number of oocytes ovulated in the controls was significantly higher than in the galactosemic group. Morphometric studies of the ovaries also showed a higher number of corpora lutea in the controls. Western blot analysis of granulosa cells showed that the overall expressions of Fas and FasL were lower in the control group and their expressions of inhibitor of apoptosis proteins (IAPs) were higher than in the galactosemic group, especially at 8 h post hCG injection. TDT-mediated dUTP-biotin nick end-labeling (TUNEL) and immunohistochemical staining of ovarian sections with Ki-67 and IAPs showed more apoptotic granulosa cells in the galactosemic group and the expressions of IAPs in granulosa cells also confirmed the result of the Western blot. These findings support our hypothesis that ovarian dysfunction in galactosemic rats is due to increased apoptosis in granulosa cells of maturing follicles.

Plasma nitric oxide level in heart failure secondary to left ventricular diastolic dysfunction.

Nitric oxide (NO) is a free radical that is elevated in the plasma of patients with systolic heart failure. However, its relation to diastolic function is unknown. This study investigated the relation between the level of stable end-products of plasma NO (NOx level) and diastolic function in patients with heart failure. We performed echocardiographic Doppler studies in 76 patients (mean age of 66 +/- 10 years, 75% men) with congestive heart failure. Left ventricular (LV) diastolic dysfunction was classified as either a restrictive (RFP) or nonrestrictive filling pattern (non-RFP). Same day venous total nitrite plus nitrate levels were measured by chemiluminscence. Both patients with isolated diastolic heart failure (ejection fraction >50%) (77 +/- 9 micromol/L, n = 33) and systolic failure (ejection fraction < or = 50%) (115 +/- 17 micromol/L, n = 43) had significantly higher plasma NOx levels than controls (37 +/- 2 micromol/L, both p <0.001). RFP coexists mostly in patients with systolic heart failure (15 of 18), and these patients had a higher NOx level than patients with systolic failure and a non-RFP (n = 28) (163 +/- 35 vs 88 +/- 16 micromol/L, p <0.05). Patients who were not on oral nitrate drugs had insignificant lower plasma NOx levels than those on regular nitrate therapy, although it was still higher than controls. Plasma NOx level did not correlate with LV ejection fraction. Stepwise multiple regression analysis confirmed that the presence of RFP was the only independent predictor of NOx, and hence NO production. Plasma NOx level is elevated in patients with isolated diastolic heart failure. In addition, in patients with LV systolic failure, the severity of LV diastolic dysfunction determines the amount of NO production.

Methylation of p16INK4A in primary gynecologic malignancy.

The p16INK4A gene mapped on band p21 of chromosome 9 can be inactivated via multiple mechanisms including homozygous deletion, point mutation and promoter hypermethylation in various human tumors. A polymerase chain reaction (PCR) based analysis was performed to examine methylation of the p16INK4A gene promoter in 196 primary gynecologic malignancies including 98 cervical, 49 endometrial and 49 ovarian carcinomas. Methylation of p16INK4A was detected in 31% of cervical, 20% of endometrial, and 4% of ovarian carcinomas, respectively. The incidence of p16INK4A methylation in patients with cervical and endometrial carcinomas at advanced stages (stages III-IV) was statistically higher than those at early stages (stages I-II). There were also significant differences in the incidence of p16INK4A methylation in both cancers between the patients who had died of their disease or were alive with evidence of disease, and those without evidence of disease. The results indicate that methylation of the p16INK4A gene is present in a proportion of primary gynecologic malignancies and this alteration may be associated with poor outcome in cervical and endometrial carcinomas.

A comparative study of a digital radiography system.

The present study aimed to examine the efficiency of a new digital radiography system that was installed in the Royal Adelaide Hospital in September 1997, as compared to the existing conventional radiography system. A total of 55 examinations were observed over a 3-week period in January, and these consisted of 18 digital and 10 conventional chest examinations, and 27 conventional orthopaedic examinations. These were combined with 18 digital orthopaedic examinations recorded from a prior study. Total examination time was broken into several components, of which reporting time was of the most interest. The mean reporting times for digital and conventional chest examinations were 17 and 25 min, respectively, a significant (P < 0.1) 8-min difference. The orthopaedic examinations revealed mean reporting times of 8 and 26 min for digital and conventional systems, respectively; a significant (P < 0.001) 18-min difference. These results demonstrate that the digital system is a faster, more efficient system for the reporting of X-rays.

p16INK4 and p15INK4B alterations in primary gynecologic malignancy.

Chromosome 9 abnormalities have been found in primary tumors and cell lines from human gynecologic malignancy. Alterations of p16INK4 and p15INK4B genes mapped on the band p21 of chromosome 9 have been detected in various human tumors, but the role of these genes as tumor suppressors in vivo appear to be dependent on tumor type. Polymerase chain reaction (PCR)-based analysis was performed to search for lesions of these genes in 202 primary gynecologic malignancies. Homozygous deletions of p16INK4 were detected in 7 of 128 (5%) cervical, 1 of 41 (2%) endometrial, 2 of 27 (7%) ovarian, and 3 of 6 (50%) vulvar carcinomas, while homozygous deletions of p15INK4B were detected in 19 of 128 (15%) cervical, 1 of 41 (2%) endometrial, 9 of 27 (33%) ovarian, and 3 of 6 (50%) vulvar carcinomas, respectively. No mutations were found in exon 2 of p16INK4 from 161 cases of gynecologic malignancy without deletion of p16INK4. All 3 cases of vulvar carcinoma showing homozygous deletions of p16INK4 and p15INK4B were at advanced clinical stage (stage III-IV), while all 7 cases of cervical carcinoma and 2 cases of ovarian carcinoma showing homozygous deletion of p16INK4 were at early stage (stage I-II). The results indicate that homozygous deletions of p16INK4 and/or p15INK4B genes may play a role in a subset of primary gynecologic malignancy.

Sister-chromatid exchanges in lymphocytes from methimazole-induced hypothyroid mice.

The inhibitory effect of an antithyroid drug on mouse T lymphocytes was investigated. Inbred C57BL/6 mice were provided with an antithyroid drug, methimazole, for 2, 4 and 6 weeks and the in vitro responses of the lymphocytes were studied. The proliferative responses of T lymphocytes from the spleen of methimazole (MMI)-treated mice significantly (p < 0.05) decreased following concanavalin A stimulation, and the inhibitory effect became prominent with the increased duration of MMI treatment. A concomitant increase in the frequency of induced sister-chromatid exchanges was also observed in these T lymphocytes. When the splenocytes were stimulated with concanavalin A for 24 h, their ability to produce interleukin-2 (IL-2) was significantly decreased (p < 0.05). The results indicated that methimazole interfered with the normal proliferation of T lymphocytes by suppressing the production of IL-2, a cytokine also known as T cell growth factor, as well as inducing a higher incidence of sister-chromatid exchange during cell division.