Pediatric Cardiology Fellowship Standards for Training in Exercise Medicine and Curriculum Outline.

Over the past 2 decades, fundamentals of exercise medicine, including clinical exercise testing, assessment and promotion of physical activity, exercise prescription, and supervised exercise training/rehabilitation programming have demonstrated considerable clinical value in the management of children and adolescents with congenital and acquired heart disease. Although the principles of exercise medicine have become an integral component in pediatric cardiology, there are no standardized training recommendations for exercise physiology during pediatric cardiology fellowship at this time. Thus, the Pediatric Cardiology Exercise Medicine Curriculum Committee (PCEMCC) was formed to establish core and advanced exercise physiology training recommendations for pediatric cardiology trainees. The PCEMCC includes a diverse group of pediatric cardiologists, exercise physiologists, and fellowship program directors. The expert consensus training recommendations are by no means a mandate and are summarized herein, including suggestions for achieving the minimum knowledge and training needed for general pediatric cardiology practice.

Emergence of New Delhi metallo-beta-lactamase 1 and other carbapenemase-producing Acinetobacter calcoaceticus-baumannii complex among patients in hospitals in Ha Noi, Viet Nam.

Acinetobacter baumannii is an important cause of multidrug-resistant hospital acquired infections in the world. Here, we investigate the presence of NDM-1 and other carbapenemases among carbapenem-resistant A. baumannii isolated between August 2010 and December 2014 from three large hospitals in Hanoi, Vietnam. We identified 23/582 isolates (4 %) (11 from hospital A, five from hospital B, and seven from hospital C) that were NDM-1 positive, and among them 18 carried additional carbapenemase genes, including seven isolates carrying NDM-1, IMP-1, and OXA-58 with high MICs for carbapenems. Genotyping indicated that NDM-1 carrying A. baumannii have expanded clonally in these hospitals. Five new STs (ST1135, ST1136, ST1137, ST1138, and ST1139) were identified. One isolate carried NDM-1 on a plasmid belonging to the N-repA replicon type; no NDM-1-positive plasmids were identified in the other isolates. We have shown the extent of the carbapenem resistance and the local clonal spread of A. baumannii carrying NDM-1 in these hospitals; coexistence of NDM-1 and IMP-1 is reported for the first time from Vietnam here, and this will further seriously limit future therapeutic options.

Detection of tubule boundaries based on circular shortest path and polar-transformation of arbitrary shapes.

In studies of germ cell transplantation, counting cells and measuring tubule diameters from different populations using labelled antibodies are important measurement processes. However, it is slow and sanity grinding to do these tasks manually. This paper proposes a way to accelerate these processes using a new image analysis framework based on several novel algorithms: centre points detection of tubules, tubule shape classification, skeleton-based polar-transformation, boundary weighting of polar-transformed image, and circular shortest path smoothing. The framework has been tested on a dataset consisting of 27 images which contain a total of 989 tubules. Experiments show that the detection results of our algorithm are very close to the results obtained manually and the novel approach can achieve a better performance than two existing methods.

Segmentation of clustered nuclei based on concave curve expansion.

Segmentation of nuclei from images of tissue sections is important for many biological and biomedical studies. Many existing image segmentation algorithms may lead to oversegmentation or undersegmentation for clustered nuclei images. In this paper, we proposed a new image segmentation algorithm based on concave curve expansion to correctly and accurately extract markers from the original images. Marker-controlled watershed is then used to segment the clustered nuclei. The algorithm was tested on both synthetic and real images and better results are achieved compared with some other state-of-the-art methods.

Automatic nuclear bud detection using ellipse fitting, moving sticks or top-hat transformation.

Micronucleus assays are extensively used by biologists to assess genotoxicity and to monitor human exposure to genotoxic materials. As recent studies suggested that nuclear buds can be a new source of micronuclei formed in interphase, the quantification of nuclear buds, which are micronucleus like objects that are attached to the nuclei in interphase, in normal and control group is needed. Three automatic nuclear bud detection algorithms fit for different situations are proposed in this paper. One is based on ellipse fitting, one is based on a stick model and the other is based on the top-hat transform. Comparison of the three methods is also given in this paper. Experimental results showed that the proposed algorithms are all effective and efficient for nuclear bud detection.

Heat-stable spores of carotenoid-producing Bacillus marisflavi and non-pigmented Bacillus subtilis cooperatively promote growth, quality, and gut microbiota of white-leg shrimp.

We evaluated the benefits of heat-stable carotenoid-producing Bacillus marisflavi SH8 spores individually and in combination with non-pigmented Bacillus subtilis SH23 spores on growth, colour change, nutritional content, innate immunity, and gut microbiota of white-leg shrimp. White-leg shrimp (Litopenaeus vannamei; n = 30 per tank; 2 tanks per group) were provided feed without (control group) or with SH8, SH23, or mixed spores (total, 1 × 106 cfu/g pellet) for 28 d. The SH8 and SH8-23 combination groups had significantly higher specific growth rates (9.6 and 11.0%), improved red-colour score (4 scores), astaxanthin concentration (1.8- and 2.3-fold), lipid contents (30 and 50%), and superoxidase dismutase activity (8.5 and 12.3%) than that of the control group. Analysis of shrimp's gut microbiome using 16S rRNA metagenome sequencing revealed increased abundance of four useful species and reduced abundance of four harmful species in the combination group than in the control group. Heat-stable Bacillus spore combination improved growth parameters, nutrient content, red-colour score, live counts, and abundance of useful bacteria in the gut of L. vannamei. This is the first study to show the benefits of combining highly heat-stable pigmented and non-pigmented Bacillus spores and their possible mechanisms in a shrimp model.

Ostracods as pollution indicators in Lap An Lagoon, central Vietnam.

Southeast Asia is particularly susceptible to the negative impacts of increasing coastal pollution as coastal populations and cities grow at unprecedented rates. Although water chemistry can be monitored, there are greater advantages in using bioindicators as reflectors of the combined effect of multiple pollution types on coastal ecosystem health and for early detection of the negative impacts of pollutants on biotic systems. This study explores the utility and application of ostracods as pollution bioindicators and examines the response of ostracod assemblages to variable pollution in Lap An Lagoon, central Vietnam. From 14 sites within the lagoon, 79 species of 46 genera were identified and sediment grain size, total organic carbon, organic matter and heavy metal concentration were measured. Cluster analysis, detrended correspondence analysis and canonical correspondence analysis identified four distinct ostracod biofacies that were highly correlated to the physical environmental variables (salinity, depth, sediment type, heavy metal concentrations, total organic carbon and organic matter) and are shown to be the main factors controlling ostracod biofacies. Low ostracod diversities were found in silty sediments with heavy metal concentrations likely toxic. Sinocytheridea impressa was indicative of a marginally polluted environment within the lagoon. This study provides evidence for the potential for Southeast Asian ostracods to be used in water quality assessments and the data collected can be used as a baseline for future pollution monitoring.

Live birth rate after human chorionic gonadotropin priming in vitro maturation in women with polycystic ovary syndrome.

BACKGROUND: In vitro maturation (IVM) has some advantages over conventional in vitro fertilization (IVF), particularly in polycystic ovary syndrome (PCOS) where the risk of ovarian hyperstimulation is high. We studied the live birth rate in a large series of PCOS women undergoing human chorionic gonadotropin (hCG)-priming IVM. METHODS: This retrospective study included women with PCOS aged 18-42 years undergoing IVM with hCG priming. We reported live birth rate after the first embryo transfer and cumulative live birth rate from embryos obtained in the IVM cycle. We also performed logistic regression to assess which factors predicted number of oocytes and live birth. RESULTS: We included 921 women (age 28.9±3.5 years, body mass index 21.8±3.1 kg/m2, infertility duration 3.7±2.6 years, 81% primary infertility, 88% first IVF attempt, 94% ovulation induction failure). Live birth rate after the first embryo transfer was 31.7%, with a cumulative live birth rate from the cycle of 33.7%. High anti-Müllerian hormone levels predicted a high number of oocytes and a high oocyte maturation rate while the opposite was the case when luteinizing hormone levels were high. CONCLUSIONS: In women with PCOS, hCG priming IVM was feasible and resulted in acceptable live birth rates.

High-fat diet induces systemic B-cell repertoire changes associated with insulin resistance.

The development of obesity-associated insulin resistance is associated with B-lymphocyte accumulation in visceral adipose tissue (VAT) and is prevented by B-cell ablation. To characterize potentially pathogenic B-cell repertoires in this disorder, we performed high-throughput immunoglobulin (Ig) sequencing from multiple tissues of mice fed high-fat diet (HFD) and regular diet (RD). HFD significantly changed the biochemical properties of Ig heavy-chain complementarity-determining region-3 (CDRH3) sequences, selecting for IgA antibodies with shorter and more hydrophobic CDRH3 in multiple tissues. A set of convergent antibodies of highly similar sequences found in the VAT of HFD mice but not RD mice showed significant somatic mutation, suggesting a response shared between mice to a common antigen or antigens. These findings indicate that a simple high-fat dietary intervention has a major impact on mouse B-cell repertoires, particularly in adipose tissues.

Enteric dopaminergic neurons: definition, developmental lineage, and effects of extrinsic denervation.

The existence of enteric dopaminergic neurons has been suspected; however, the innervation of the gut by sympathetic nerves, in which dopamine (DA) is the norepinephrine precursor, complicates analyses of enteric DA. We now report that transcripts encoding tyrosine hydroxylase (TH) and the DA transporter (DAT) are present in the murine bowel (small intestine > stomach or colon; proximal colon > distal colon). Because sympathetic neurons are extrinsic, transcripts encoding TH and DAT in the bowel are probably derived from intrinsic neurons. TH protein was demonstrated immunocytochemically in neuronal perikarya (submucosal >> myenteric plexus; small intestine > stomach or colon). TH, DA, and DAT immunoreactivities were coincident in subsets of neurons (submucosal > myenteric) in guinea pig and mouse intestines in situ and in cultured guinea pig enteric ganglia. Surgical ablation of sympathetic nerves by extrinsic denervation of loops of the bowel did not affect DAT immunoreactivity but actually increased numbers of TH-immunoreactive neurons, expression of mRNA encoding TH and DAT, and enteric DOPAC (the specific dopamine metabolite). The fetal gut contains transiently catecholaminergic (TC) cells. TC cells are the proliferating crest-derived precursors of mature neurons that are not catecholaminergic and, thus, disappear after embryonic day (E) 14 (mouse) or E15 (rat). TC cells appear early in ontogeny, and their development/survival is dependent on mash-1 gene expression. In contrast, the intrinsic TH-expressing neurons of the murine bowel appear late (perinatally) and are mash-1 independent. We conclude that the enteric nervous system contains intrinsic dopaminergic neurons that arise from a mash-1-independent lineage of noncatecholaminergic precursors.

Neurotrophin-3 is required for the survival-differentiation of subsets of developing enteric neurons.

Neurotrophin-3 (NT-3) promotes enteric neuronal development in vitro; nevertheless, an enteric nervous system (ENS) is present in mice lacking NT-3 or TrkC. We thus analyzed the physiological significance of NT-3 in ENS development. Subsets of neurons developing in vitro in response to NT-3 became NT-3 dependent; NT-3 withdrawal led to apoptosis, selectively in TrkC-expressing neurons. Antibodies to NT-3, which blocked the developmental response of enteric crest-derived cells to exogenous NT-3, did not inhibit neuronal development in cultures of isolated crest-derived cells but did so in mixed cultures of crest- and non-neural crest-derived cells; therefore, the endogenous NT-3 that supports enteric neuronal development is probably obtained from noncrest-derived mesenchymal cells. In mature animals, retrograde transport of (125)I-NT-3, injected into the mucosa, labeled neurons in ganglia of the submucosal but not myenteric plexus; injections of (125)I-NT-3 into myenteric ganglia, the tertiary plexus, and muscle, labeled neurons in underlying submucosal and distant myenteric ganglia. The labeling pattern suggests that NT-3-dependent submucosal neurons may be intrinsic primary afferent and/or secretomotor, whereas NT-3-dependent myenteric neurons innervate other myenteric ganglia and/or the longitudinal muscle. Myenteric neurons were increased in number and size in transgenic mice that overexpress NT-3 directed to myenteric ganglia by the promoter for dopamine beta-hydroxylase. The numbers of neurons were regionally reduced in both plexuses in mice lacking NT-3 or TrkC. A neuropoietic cytokine (CNTF) interacted with NT-3 in vitro, and if applied sequentially, compensated for NT-3 withdrawal. These observations indicate that NT-3 is required for the normal development of the ENS.

Time of origin of neurons in the murine enteric nervous system: sequence in relation to phenotype.

The hypothesis was tested that developing enteric neurons withdraw from the cell cycle in a sequence related to their phenotype. The birthdays of immunocytochemically identified myenteric and submucosal neurons were determined in the murine duodenum and jejunum. [3H]thymidine ([3H]TdR) was injected into timed pregnant mice or pups at 4-8 hour intervals over a 24 hour period. Pups were killed on postnatal day 30 (P30). [3H]TdR incorporation was detected by radioautography in enteric neurons, which were phenotypically identified by the simultaneous detection of the immunoreactivities of 5-hydroxytryptamine (5-HT), choline acetyl transferase (ChAT), neuropeptide Y (NPY), enkephalin (ENK), calcitonin gene-related peptide (CGRP), and vasoactive intestinal peptide (VIP). The dates of the earliest withdrawal from the cell cycle of neurons containing these markers were determined, as well as the length of time during which the identified neurons continued to be born, and the date on which their rate of birth was maximal. The birthdates of myenteric neurons that contained 5-HT (E8-E14, peak at E10) or ChAT (E8-E15, peak at E12) tended to be earlier than those that contained ENK (E10-E18, peak at E14), NPY (E10-E18, peak at E15), VIP (E10-P5, peak at E15), or CGRP (E10-P3, peak at E17). For any given immunocytochemically defined neuronal phenotype, submucosal neurons tended to be born later than their myenteric counterparts and submucosal neurons that contained neuropeptides were born later than those that contained only ChAT immunoreactivity. The day (E8) on which the first 5-HT- and ChAT-immunoreactive neurons became postmitotic is earlier than the day (E9) on which the colonization of the bowel by crest-derived cells has been detected. The population of neural precursors that colonizes the gut, therefore, is heterogeneous; many cells are proliferating, but a specific subset, which will ultimately give rise to serotoninergic or cholinergic neurons, is already postmitotic. Neurons continued to be born throughout fetal life and even after birth. Consequently, terminally differentiated neurons coexist in the developing enteric nervous system with dividing neural precursor cells. This observation is consistent with the idea that early developing neurons could affect the development of enteric neural precursors; moreover, they also demonstrate that it is possible to add neurons to the enteric plexuses even after the neural circuits on which the bowel depends have become functional.

Origin and morphology of nerve fibers in the aganglionic colon of the lethal spotted (ls/ls) mutant mouse.

The lethal spotted mutant mouse (ls/ls) develops congenital megacolon because of the absence of ganglia in the terminal colon. This aganglionosis results from a failure of neural crest cells to colonize this area during fetal life. We have postulated that the microenvironment of the aganglionic segment of bowel is abnormal. Our hypothesis suggests that this abnormal enteric microenvironment fosters the sprouting of neuritic processes. We further propose that neural and glial precursors cease to migrate once they have extended their definitive processes. As a result, the area distal to the site where neurite extension is favored does not become colonized by neural or glial precursors. A prediction of this hypothesis is that the aganglionic tissue should be innervated by axons from neurons located both in the more proximal ganglionated bowel and in ganglia located outside the gut. Neurons and their processes in control and ls/ls terminal gut were located by the histochemical demonstration of acetylcholinesterase (AChE) activity and their structure was classified as intrinsic (enteric) or extrinsic in type by electron microscopy. In ls/ls mice the submucosal plexus was much more severely affected than the myenteric plexus. No submucosal ganglia were found within 30 mm of the anus. In contrast, myenteric ganglia extended to within 4 mm of the anus on the mesenteric side of the gut and to within 15 mm on the antimesenteric side. Rostral to the areas that were absolutely aganglionic, both plexuses were hypoganglionic, especially the submucosal plexus, which was hypoganglionic throughout the entire colon. Both the aganglionic and caudal hypoganglionic zones of the ls/ls bowel were penetrated by large nerve trunks that had the ultrastructural characteristics of extra-enteric peripheral nerve. Unusual ganglia, outside the enteric musculature in the adventitia of the colon, were connected to these trunks. The location of the cell bodies of origin of the nerve fibers in the terminal colon of control mice and in the aganglionic segment of the bowel in ls/ls mice was determined by following the retrograde transport of tracers injected as close as possible to the anus. An extrinsic innervation originating from the inferior mesenteric ganglion and dorsal root ganglia (L6-S1) was found in both types of animal. In control but not ls/ls mice retrograde labeling was also observed in the sacral parasympathetic nucleus of the spinal cord. In addition, neuritic processes were traced to neurons in myenteric ganglia. In control mice, these labeled neurons were present in ganglia within the injection site as well as in bowel rostral and caudal to it.(ABSTRACT TRUNCATED AT 400 WORDS)

Immunoelectron microscopic localization of platelet factor 4 and fibrinogen in the granules of human platelets.

To determine the storage site of platelet fibrinogen and of platelet factor 4 (PF4) in human platelets by immunoelectron microscopic techniques, washed human platelets were briefly exposed to Karnovsky's fixative and embedded in water-soluble Durcupan. Thin sections of platelets were exposed to Fab fragments of rabbit anti-human fibrinogen or of goat anti-human PF4, followed by a peroxidase conjugate of Fab fragments of antibodies to rabbit immunoglobulin (Ig) G or to goat IgG. The technique enabled preservation of the antigenic determinants of the platelet proteins, accessibility of Fab fragments to the platelet proteins, and maintenance of the ultrastructural integrity of the platelets. Using this approach, it was directly demonstrated that platelet fibrinogen and PF4 are stored in the alpha-granules of human platelets.

Influence of SIN-1 on platelet Ca2+ handling in patients with suspected coronary artery disease: ex vivo and in vitro studies.

The 3-morpholinosydnonimine (SIN-1) generates both nitric oxide (NO) and superoxide anion (O2-). It elicits dose-dependent vasodilation in vivo, in spite of the opposite effects of its breakdown products on vascular tone and platelet aggregation. This study was designed to investigate the influence of intravenous SIN-1 injection on platelet Ca2+ handling in patients undergoing coronary angiography. SIN-1 administration reduced cytosolic [Ca2+] in unstimulated platelets by decreasing Ca2+ influx. It attenuated Ca2+ mobilization from internal stores evoked by thrombin or thapsigargin. In vitro studies were used as an approach to investigate how simultaneous productions of NO and O2- from SIN-1 modify thrombin- or thapsigargin-induced platelet Ca2+ mobilization. Superoxide dismutase, the O2- scavenger, enhanced the capacity of SIN-1 to inhibit Ca2+ mobilization but catalase had no effect. This suggests that the effects of SIN-1 on platelet Ca2+ handling resemble those of NO, but are modulated by simultaneous O2- release, independently of H2O2 formation.

Right atrial ultrastructure in congenital heart disease. I. Comparison of ventricular septal defect and endocardial cushion defect.

Ultrastructural studies were performed on portions of the operatively resected right atrium from six patients with a ventricular septal defect and six patients with an endocardial cushion defect. The six patients with a ventricular septal defect had normal right atrial mean pressure and no evidence of right atrial volume overload. Ultrastructurally, the atrial muscle cells in these patients appeared normal and measured 6 to 12 mu in diameter. The six patients with an endocardial cushion defect had elevated right atrial mean pressure and evidence of right atrial volume overload. Ultrastructurally, the atrial muscle cells in these patients were generally larger than 12 mu in diameter. The cells were irregular and had multiple and occasionally widened intercalated discs. In addition, there were degenerative changes in two patients with markedly increased atrial pressure. These changes included extensive loss of contractile elements, aggregation of small irregular mitochondria and proliferation of tubules of the sarcoplasmic reticulum. The structural changes suggest that hypertrophy of the right atrium may be secondary to volume overload of the atrium, whereas degenerative changes may be secondary to increased right atrial pressure.

Right atrial ultrastructure in congenital heart disease. II. Atrial septal defect: effects of volume overload.

Portions of operatively resected right atrium from 15 patients with atrial septal defect were studied ultrastructurally to determine whether the cell hypertrophy in the right atrium of patients with increased right atrial blood flow and increased right atrial pressure is caused by the increased blood flow. In 12 patients with normal right atrial mean pressure but increased right atrial blood flow the atrium was dilated but no atrial arrhythmias were noted clinically. Ultrastructurally, the atrial myocardial cells in these patients were normal, measuring 6 to 10 mu in diameter, and there was no evidence of cell hypertrophy or degeneration. The remaining three patients had elevated right atrial mean pressure and increased right atrial blood flow. Ultrastructurally, the atrial myocardial cells in all three patients were hypertrophied, and two patients had evidence of focal cell degeneration; the atrium was markedly dilated, but atrial arrhythmias were not noted. The lack of cell hypertrophy in the right atrium of the 12 patients with increased blood flow but normal mean pressure suggests that in congenital heart disease volume overload alone does not lead to cell hypertrophy of the right atrial myocardium.

Effects of left atrial enlargement on atrial transmembrane potentials and structure in dogs with mitral valve fibrosis.

The effects of left atrial enlargement on atrial cell electrophysiology and structure were studied in dogs with mitral valve fibrosis. Thirteen dogs (Groups I) had left atrial enlargement and intermittent atrial arrhythmias; 10 dogs (Group II) had left atrial enlargement and chronic atrial fibrillation. The resting and action potentials of cells in isolated preparations from the enlarged left atrium were found not to differ from those in the nonenlarged right atrium or in the atrium of control dogs. The resting and action potentials of cells in Group II atria did not differ significantly from those in Group I atria. Some cells (15 percent of the total studied) in the atria of dogs in Groups I and II were inexcitable, but either superfusion with acetylcholine or norepinephrine restored excitability. The structural studies showed that the left atrium of the dogs in Groups I and II had a reduced number of muscle cell layers spanning the wall with an unusually large amount of connective tissue between greatly hypertrophied cells. Very few degenerating cells were seen. Dramatic abnormalities of cell electrophysiology may not be involved in the genesis of arrhythmias in the enlarged canine atrium, and the altered morphologic features of the atrium in these dogs may be important in the genesis of persistent atrial arrhythmias.

Histological and ultrastructural findings in a case of the Sézary syndrome.

This paper deals with the histological and ultrastructural findings in a case of the Sézary syndrome. The striking nuclear and cytoplasmic features of the Sézary cell are illustrated, and the similarities of this cell to the mycosis fungoides cell are once more stressed.

Detection of avian leukosis virus in albumen of chicken eggs using reverse transcription polymerase chain reaction.

A reverse transcriptase polymerase chain (RT-PCR) assay was developed to detect avian leukosis retrovirus (ALV) in egg albumen. Eggs of Single Comb White Leghorns were from a commercial breeder (stock F) and from a pathogen-free flock (stock N). RT-PCR was undertaken on isolated RNA from 20 unfertilized egg samples using seven sets of primers that correspond to the ALV gp85 envelope glycoprotein which determines the ALV subgroup classification. An ELISA assay for ALV gs antigen of egg albumen was positive for all stock F birds tested and negative for all stock N birds. Virus isolation was undertaken by inoculating egg albumen, feather pulp, or blood from five stock F chickens onto cultures of chicken embryo fibroblasts (C/E). IFA analysis of the inoculated C/E cultures indicated that all stock F birds tested contained infectious ALV. For the virus-positive stock F chickens, RT-PCR analyses using primers designed to detect all ALV subgroups detected ALV in 15/15 (100%) egg albumen samples, while primers designed to detect subgroup A ALV were positive for 12/15 (80%) egg albumen samples. RT-PCR products were not detected from five egg albumen samples from five stock N chickens by any primer sets. Direct sequencing using primers specific for subgroup A ALV verified the viral subgroup in the RT-PCR amplification products. The combined use of RT-PCR and direct sequencing of the RT-PCR product provides a new approach for identifying ALV-infected poultry.