A Protocol for Transverse Cardiac Slicing and Optical Mapping in Murine Heart.

Thin living tissue slices have recently emerged as a new tissue model for cardiac electrophysiological research. Slices can be produced from human cardiac tissue, in addition to small and large mammalian hearts, representing a powerful in vitro model system for preclinical and translational heart research. In the present protocol, we describe a detailed mouse heart transverse slicing and optical imaging methodology. The use of this technology for high-throughput optical imaging allows study of electrophysiology of murine hearts in an organotypic pseudo two-dimensional model. The slices are cut at right angles to the long axis of the heart, permitting robust interrogation of transmembrane potential (Vm) and calcium transients (CaT) throughout the entire heart with exceptional regional precision. This approach enables the use of a series of slices prepared from the ventricles to measure Vm and CaT with high temporal and spatial resolution, allowing (i) comparison of successive slices which form a stack representing the original geometry of the heart; (ii) profiling of transmural and regional gradients in Vm and CaT in the ventricle; (iii) characterization of transmural and regional profiles of action potential and CaT alternans under stress (e.g., high frequency pacing or ß-adrenergic stimulation) or pathological conditions (e.g., hypertrophy). Thus, the protocol described here provides a powerful platform for innovative research on electrical and calcium handling heterogeneity within the heart. It can be also combined with optogenetic technology to carry out optical stimulation; aiding studies of cellular Vm and CaT in a cell type specific manner.

Effect of non-invasive prenatal testing as a contingent approach on the indications for invasive prenatal diagnosis and prenatal detection rate of Down's syndrome.

INTRODUCTION: In Hong Kong, universal combined first-trimester screening for Down's syndrome was started as a 'free service' in July 2010. Non-invasive prenatal testing was available as a self-financed item in August 2011. This study aimed to determine whether the introduction of non-invasive prenatal testing as a contingent approach influenced the indications for invasive prenatal diagnosis and the consequent prenatal detection of Down's syndrome. METHODS: This historical cohort study was conducted at the Prenatal Diagnosis Clinic of Queen Elizabeth Hospital in Hong Kong. We compared the indications for invasive prenatal diagnosis and prenatal detection of Down's syndrome in singleton pregnancies 1 year before and 2 years following the availability of non-invasive prenatal testing as a contingent test after a positive aneuploidy test. All pregnant women who attended our hospital for counselling about universal Down's syndrome screening between August 2010 and July 2013 were recruited. RESULTS: A total of 16 098 women were counselled. After the introduction of non-invasive prenatal testing, the invasive prenatal diagnosis rate for a positive aneuploidy screening reduced from 77.7% in 2010-11 to 68.8% in 2012-13. The new combined conventional plus non-invasive prenatal testing strategy was associated with a lower false-positive rate (6.9% in 2010-11 vs 5.2% in 2011-12 and 4.9% in 2012-13). There was no significant increase in invasive prenatal diagnosis for structural anomalies over the years. There was no significant trend in the overall prenatal detection rate of Down's syndrome (100% 1 year before vs 89.1% 2 years after introduction of non-invasive prenatal testing). Four (2.6%) of 156 women who underwent non-invasive prenatal testing for a screen-positive result had a high-risk result for trisomy 21, which was subsequently confirmed by invasive prenatal diagnosis. There were no false-negative cases. CONCLUSION: The introduction of non-invasive prenatal testing as a contingent approach reduced the invasive prenatal diagnosis rate for a positive aneuploidy screening without affecting the invasive prenatal diagnosis rate for structural anomalies or the overall detection rate of fetal Down's syndrome.

An Unusual Hydrops Fetalis Associated with Compound Heterozygosity for Krüppel-like Factor 1 mutations.

Hydrops fetalis is commonly due to Hb Bart's (γ4) disease in South East Asia. Here, we report an unusual case of hydrops fetalis due to congenital dyserythropoietic anemia (CDA) associated with compound heterozygosity for Krüppel-like factor 1 (KLF1) gene mutations. Fetal cardiomegaly was first detected on routine mid-trimester scan in a pregnant woman with normal mean corpuscular volume (MCV) and Rhesus positive status. The fetus subsequently developed hydrops fetalis, and cordocentesis showed severe fetal anemia with a hemoglobin (Hb) level of 3.4 g/dL. Common causes of fetal anemia including Hb Bart's disease, parvovirus infection, and red cell antibodies were excluded. In view of the marked increase in erythroblasts at various stages of erythropoiesis, the diagnosis of CDA was suspected. We screened the couple for previously reported KLF1 gene mutations, showing that the mother was heterozygous for the c.525_526insCGGCGCC, p.Gly176Argfs*179 mutation, and her husband heterozygous for c.1012C>A, p.Pro338Thr mutation. The fetus was a compound heterozygote for these two KLF1 mutations. After counseling, repeated intrauterine transfusions were given at 27, 29, and 34 weeks' gestation; the hydrops fetalis was resolved. The baby was delivered at 34 weeks' gestation and required monthly blood transfusions but was otherwise thriving. Bone marrow aspiration at 10 months of age showed the features of ineffective erythropoiesis, compatible with CDA. In conclusion, hydrops fetalis can rarely be due to CDA associated with a compound heterozygous mutation for KLF1 gene mutations, and be managed by repeated intrauterine transfusions. Our present report adds to the wide clinical spectrum of KLF1 mutations.

Network-analysis-guided synthesis of weisaconitine D and liljestrandinine.

General strategies for the chemical synthesis of organic compounds, especially of architecturally complex natural products, are not easily identified. Here we present a method to establish a strategy for such syntheses, which uses network analysis. This approach has led to the identification of a versatile synthetic intermediate that facilitated syntheses of the diterpenoid alkaloids weisaconitine D and liljestrandinine, and the core of gomandonine. We also developed a web-based graphing program that allows network analysis to be easily performed on molecules with complex frameworks. The diterpenoid alkaloids comprise some of the most architecturally complex and functional-group-dense secondary metabolites isolated. Consequently, they present a substantial challenge for chemical synthesis. The synthesis approach described here is a notable departure from other single-target-focused strategies adopted for the syntheses of related structures. Specifically, it affords not only the targeted natural products, but also intermediates and derivatives in the three families of diterpenoid alkaloids (C-18, C-19 and C-20), and so provides a unified synthetic strategy for these natural products. This work validates the utility of network analysis as a starting point for identifying strategies for the syntheses of architecturally complex secondary metabolites.

Tandem rhodium catalysis: exploiting sulfoxides for asymmetric transition-metal catalysis.

Sulfoxides are uncommon substrates for transition-metal catalysis due to their propensity to inhibit catalyst turnover. In a collaborative effort with Ken Houk, we developed the first dynamic kinetic resolution (DKR) of allylic sulfoxides using asymmetric rhodium-catalyzed hydrogenation. A detailed mechanistic analysis of this transformation using both experimental and theoretical methods revealed rhodium to be a tandem catalyst that promoted both hydrogenation of the alkene and racemization of the allylic sulfoxide. Using a combination of deuterium labelling and DFT studies, a novel mode of allylic sulfoxide racemization via a Rh(III)-π-allyl intermediate was identified.

Uptake of Noninvasive Prenatal Testing in Chinese Women following Positive Down Syndrome Screening.

OBJECTIVES: To investigate how the introduction of noninvasive prenatal testing (NIPT) influenced women's testing choices following a positive Down syndrome screening. METHODS: A retrospective study was conducted to compare differences in the uptake rates of invasive prenatal diagnosis (IPD) or no testing in one public hospital 1 year before (pre-NIPT) and 1 and 2 years after the introduction of NIPT in private in August 2011 using descriptive analysis and a χ² test. Conventional screening was funded publicly, but NIPT was not. Multivariable binary logistic regression was used to determine factors affecting choices. RESULTS: In pre-NIPT and in years 1 and 2 after the introduction of NIPT, 306, 362 and 401 women who screened positive were seen, respectively. In year 1 and year 2, 12.6 and 26.7% of them underwent NIPT while IPD was decreased by 16.3 and 25.6%, respectively (p < 0.001). Both chorionic villus sampling and amniocentesis decreased in year 1, but only the former in year 2. However, the rate of declining further testing was similar before and after NIPT (p = 0.213). In multivariable analysis, first trimester screening, nulliparity and working women were significant predictors of accepting NIPT, while only nulliparity was a predictor of declining IPD (OR = 0.61). CONCLUSIONS: Introduction of NIPT resulted in a significant decrease in IPD for 2 consecutive years..

Periostin levels correlate with disease severity and chronicity in patients with atopic dermatitis.

BACKGROUND: Recent findings indicate that periostin, an extracellular matrix protein induced by T helper 2 cytokines, plays a critical role in the pathogenesis of atopic dermatitis (AD). OBJECTIVES: To determine whether serum periostin level is associated with clinical phenotype in adult patients with AD. METHODS: An enzyme-linked immunosorbent assay was performed to determine serum periostin levels in 257 adult patients with AD, 66 patients with psoriasis vulgaris (PV) as a disease control and 25 healthy controls. Serum periostin levels were analysed together with clinical characteristics and laboratory parameters, including thymus and activation-regulated chemokine (TARC), lactate dehydrogenase (LDH), blood eosinophil count and total IgE. Immunohistochemical analysis evaluated the expression of periostin in association with various clinical phenotypes of AD. The effect of treatment on serum periostin level was also assessed. RESULTS: Serum periostin was significantly higher in patients with AD than in patients with PV and healthy controls. Periostin level was found to be positively correlated with disease severity, TARC level, LDH level and eosinophil count, but not with IgE level. Higher serum periostin level was observed in patients with extrinsic AD compared with patients with intrinsic AD; the positive correlation of disease severity disappeared in patients with intrinsic AD. Robust expression of periostin was detected in the dermis of patients with AD with erythroderma, lichenification and, to a lesser extent, scaly erythema. Serial measurement of serum periostin revealed decreased levels of periostin after treatment for AD. CONCLUSIONS: Periostin may play a critical role in disease severity and chronicity in the pathogenesis of AD.

Two unusual cases of haemoglobin Bart's hydrops fetalis due to uniparental disomy or non-paternity.

The authors present 2 unusual cases of haemoglobin (Hb) Bart's hydrops fetalis and highlight the problem of a screening system for α-thalassaemia which focuses on maternal and paternal mean corpuscular volume (MCV) alone. Normal paternal MCV may not preclude fetal Hb Bart's disease because of the rare occurrence of maternal uniparental disomy or non-paternity. During a mid-trimester anomaly scan, with fetal cardiomegaly or hydrops in a woman with low MCV but normal paternal MCV, obstetricians should remain alert for fetal Hb Bart's disease. This is very important and relevant for national screening systems in South-East Asia, where a routine mid-trimester scan may not be available. A routine mid-trimester anomaly scan should therefore be implemented and in high prevalence areas, sonographers should be sensitive to the cardio-thoracic ratio even if screening shows that pregnancy is unlikely to be at risk.

Identification of Hic-5 as a novel regulatory factor for integrin αIIbß3 activation and platelet aggregation in mice.

BACKGROUND: Integrin αIIbß3 plays key roles in platelet aggregation and subsequent thrombus formation. Hydrogen peroxide-inducible clone-5 (Hic-5), a member of the paxillin family, serves as a focal adhesion adaptor protein associated with αIIbß3 at its cytoplasmic strand. OBJECTIVES: Hic-5 function in αIIbß3 activation and subsequent platelet aggregation remains unknown. To address this question, platelets from Hic-5(-/-) mice were analyzed. METHODS AND RESULTS: Hic-5(-/-) mice displayed a significant hemostatic defect and resistance to thromboembolism, which were explained in part by weaker thrombin-induced aggregation in Hic-5(-/-) platelets. Mechanistically, Hic-5(-/-) platelets showed limited activation of αIIbß3 upon thrombin treatment. Morphological alteration in Hic-5(-/-) platelets after thrombin stimulation on fibrinogen plates was also limited. As a direct consequence, the quantity of actin co-immunoprecipitating with the activated αIIbß3 was smaller in Hic-5(-/-) platelets than in wild-type platelets. CONCLUSION: We identified Hic-5 as a novel and specific regulatory factor for thrombin-induced αIIbß3 activation and subsequent platelet aggregation in mice.

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.

[Long-term outcome after extracorporeal shock wave lithotripsy for gallstones].

We retrospectively reviewed 289 cases followed for a long time after extracorporeal shock wave lithotripy (ESWL) for gallstones. Follow-up periods ranged from 0.5 years to 9.2 years, with a median of 4.1 years. The complete resolution rate was 45.6%. A solitary gallstone, non-calcified gallstones on CT, and Tsuchiya's sonographic type Ia and Ib gallstones were found to be statistically significant variable for resolution of the disease. With a maximum follow-up period of 9 years, the cumulative recurrence rate was 30.7%. Among the 156 patients whose gallstones did not resolve, 70 were symptomatic and the other 86 were asymptomatic. Thirty-eight of the symptomatic patients (54.3%) became symptom-free, while 23 asymptomatic patients (29.1%) became symptomatic. The cumulative rate of occurrence of colic attacks and/or acute cholecystitis was significantly higher in the cases with lithotripsy fragments 4 mm or more in size than in the cases wih fragments 3 mm or less in size (51.8% vs. 16.7%; p < 0.05). ESWL was followed by surgery in 23.1% because symptoms developed or became aggravated after ESWL in 58.3% of them. Gallbladder cancer was discovered in one case. Patients undergoing ESWL should be followed by focusing on postoperative recurrence, symptoms, and gallbladder cancer.

[Usefulness of DIC-CT in choledocholithiasis].

In order to assess the efficacy of helical CT in drip-infusion cholangiography (DIC-CT) for diagnosis of choledocholithiasis, 82 patients with biliary diseases, including 25 patients with a definite diagnosis of choledocholithiasis obtained by direct cholangiography, were investigated by DIC-CT and EUS. Comparative investigation showed that, of the 25 cases, 94.7% could be imaged by DIC-CT and 87.5% by EUS, with respective sensitivities of 94.7% and 87.5%. The specificities in both cases were 100% and accuracies were 97.8% with DIC-CT and 96% with EUS respectively. Therefore, in diagnosis the choledocholithiasis, DIC-CT displays similar diagnostic efficiency as EUS or ERC, and can be recognized as the non-invasive and useful procedure for pre-operative diagnosis of cholecystolithiasis.

Bladder origin neuroblastoma detected by mass screening.

Pelvic neuroblastoma is relatively rare and most are a presacral mass. We present a case of asymptomatic neuroblastoma arising from the dome of the bladder wall, detected by a mass screening program. Only 1 case of neuroblastoma originating from the bladder has been previously reported. Both the reported case and our present case arose from the dome of the bladder wall. The differential diagnosis for bladder tumor in children is discussed; although uncommon, neuroblastoma should be considered a probable choice, especially for tumors arising from the dome of the bladder wall.

[Immunomodulation by TYB-2285 of Dermatophagoides farinae (Df) antigen-induced IFN-gamma and IL-4 production in lymphocytes from children with bronchial asthma].

Effect of TYB-2285 and its metabolites on immune responses by peripheral blood mononuclear cells (PBMC) from children with bronchial asthma was investigated. TYB-2285 and its metabolites have immunosuppressive activity for the proliferation by Df-stimulated patients' lymphocytes. Concanavalin A (Con A)-activated peripheral blood mononuclear cells (PBMC) from the patients were not affected by the same treatment. The results indicate that TYB-2285 and its metabolites are capable of suppressing antigen-induced 3H-thymidine (3H-TdR) uptake but not the response induced by Con A. Interferon-gamma (IFN-gamma) production by Df-stimulated PBMC from patients with active asthma, which was lower than that of normal lymphocytes, were reversed beyond the levels of that in normal subjects. Thus reduced production of IFN-gamma by Df-stimulated patients' lymphocytes was increased by TYB-2285 and its metabolites in a dose-dependent manner. This phenomenon was not observed in lymphocytes from normal subjects. Furthermore, TYB-2285 inhibited IL-4 production induced by Df antigen in asthmatic patients' lymphocytes. Taken, together, TYB-2285 could work as a weak immunosuppressant to modify lymphocytes' responses to allergen in patients with bronchial asthma. These data underscore the potential benefit for the patients with bronchial asthma.

[Correlation of interleukin-2 (IL-2) responsiveness by egg white-stimulated lymphocytes with hen egg oral provocation test in atopic children].

One hundred and twenty five cases of atopic children such as atopic dermatitis and bronchial asthma were orally provocated with rare hen egg every 20 minutes one by one upto the whole amount. In one week observation 75 cases showed any symptoms of allergy including eruption and exacerbation of atopic eczema in an immediate, late, and/or delayed responses. Frequency of positive egg white-induce IL-2 responsiveness test in patients with positive oral provocation was 90.7% (68 out of 75 cases; sensitivity). That of negative test in patients with negative provocation was 84.0% (42 out of 50 cases; specificity). In contrast, specificity of IgE RAST for egg white were 88.0% comparable to the value of antigen-specific IL-2 responsiveness (AIR) test, but the specificity was lower value (37.3%) for screening the etiological antigens as compared to that of AIR test. High frequency of positive egg white-induced IL-2 responsiveness test was observed over an immediate, late and delayed responses, while low frequency of positive IgE RAST for hen egg was observed largely in patients showing delayed but not immediate response. The results indicate that IgE RAST in this study reflects IgE-mediated immediate type hypersensitivity, whereas AIR test reflects, in addition to immediate responses, late and delayed type hypersensitivity. The combined results suggest that AIR test in hen egg allergy is a useful method in vitro for both screening and determining etiological allergens, and might be able to substitute for provocation test in vivo for which many times, labours, expenses, and patients' risks are required, and to cover IgE RAST which fails to determine etiological allergens in 62.7% of patients with positive oral provocation.

Pattern of cytokine production by T cells from adolescents with asthma in remission, after stimulation with Dermatophagoides farinae antigen.

Children with asthma usually become asymptomatic by the time they reach age 20 y. To clarify the immunologic mechanisms responsible for this phenomenon, we studied patients in remission and others who still had frequent asthma attacks. Patients were grouped by clinical status, and three variables were measured: serum levels of IgE, production of IL4 and interferon (IFN)-gamma, and the activation of T cells induced by Dermatophagoides farinae (Df) antigen. Df-induced activation of T cells (as measured by antigen-induced IL2 responsiveness) or IL2 synthesis itself was induced in patients with active asthma but not in normal subjects. These responses were much weaker in patients in remission. When stimulated by Df antigen in vitro, lymphocytes from patients with active asthma produced much more IL4 than did the cells from normal subjects, and cells from patients in remission produced only a small amount. In contrast, under similar conditions lymphocytes from patients with active asthma produced less IFN-gamma than did the cells from normal subjects. Production of IFN-gamma stimulated by Df antigen was high in patients in remission but not in normal subjects. Thus, upregulated IFN-gamma production after exposure to Df antigen might reduce IL4 secretion, which would suppress IgE production and would improve clinical status. Df antigen may suppress Df-induced allergic responses in patients with asthma in remission.

Rapid activation of insulin-like growth factor binding protein-5 gene transcription during myoblast differentiation.

Insulin-like growth factor binding proteins (IGFBPs) comprise a family of secreted proteins that bind insulin-like growth factors-I and -II (IGF-I and -II) with high affinity and potentially modulate their biological effects. We have demonstrated previously that IGFBP-5, the most conserved of the six known IGFBPs, is expressed in muscle cells in the developing embryo and during the terminal differentiation of several myogenic cell lines. In this study we show that an IGF-I analog that binds minimally to IGFBPs potently enhances the differentiation of the stringently controlled inducible C2 myoblast (C2l) cell line and identify IGFBP-5 as the sole IGFBP secreted during C2l differentiation. We find that induction of IGFBP-5 mRNA and protein is coincident with the onset of myogenin gene expression and occurs secondary to the rapid activation of IGFBP-5 gene transcription. By transient gene transfer experiments we demonstrate that a 1004 base pair segment of the IGFBP-5 promoter is very active in directing expression of the reporter gene luciferase in C2l myoblasts. A promoter fragment containing only 156 nucleotides of 5'-flanking DNA retained more than 70% of maximal activity and mediated at least part of the differentiation-dependent rise in IGFBP-5 gene transcription. Within this active segment are several potential binding sites for muscle-enriched transcription factors. Our results show that induction of IGFBP-5 expression is an early event in the myogenic differentiation of the C2l cell line and suggest that one function of this IGFBP is to modulate IGF-induced differentiation. C2l cells are thus an excellent in vitro model for elucidating the developmental factors that control IGFBP-5 gene transcription and action in skeletal muscle.

Structure and function of the mouse insulin-like growth factor binding protein 5 gene promoter.

The actions of insulin-like growth factors I and II (IGF-I and -II) are modulated by interactions with one or more of a family of secreted IGF binding proteins (IGFBPs). IGFBP-5, the most conserved of the six known IGFBPs, is a 252-amino-acid protein that has been shown both to potentiate and inhibit IGF action. In previous studies, we have cloned and characterized the mouse IGFBP-5 gene and demonstrated that it is expressed in a hierarchical pattern in different adult mouse tissues and during rodent embryonic development. In this report, we describe the initial analysis of the IGFBP-5 gene promoter. By transient gene transfer studies, we show the orientation-specific activity of DNA fragments containing from 31 to 4,100 bp from the 5'-flanking region of the mouse IGFBP-5 gene in directing expression of the heterologous reporter gene luciferase in Hep G2 cells. DNA fragments with only 156 bp of 5'-flanking sequence mediated over 60% of maximal promoter activity, and a segment containing the TATA box and the first 120 bp of exon 1 still conferred some promoter function. Within the highly active 156-bp region, we identified a 37-bp segment from -70 to -34 that exhibited specific binding in DNase I footprinting and gel-mobility shift experiments with Hep G2 nuclear protein extracts. The footprinted region, which is almost completely conserved in the rat and human IGFBP-5 genes, was responsible for at least 70% of the activity of the intact promoter, as evidenced by the deleterious consequences of small internal deletions within this sequence on promoter function.(ABSTRACT TRUNCATED AT 250 WORDS)