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PubMedDate not supplied

C-peptide enhances insulin-mediated cell growth and protection against high glucose-induced apoptosis in SH-SY5Y cells.

BACKGROUND: We have previously reported that C-peptide exerts preventive and therapeutic effects on diabetic neuropathy in type 1 diabetic BB/Wor-rats and that it prevents duration-dependent hippocampal apoptosis in the same animal model. In the present study, we examined human neuroblastoma SH-SY5Y cells to examine whether C-peptide stimulates cell proliferation/neurite outgrowth and whether it has antiapoptotic effects. METHODS: For neurite outgrowth, serum-starved cultures were treated with C-peptide and/or insulin or IGF-1. Neurites were visualized with NF-L antibody and measured morphometrically. Cell numbers were determined using an electronic cell counter. Scrambled C-peptide was used as a negative control. For assessment of apoptosis, SH-SY5Y cells were incubated with 100 mM glucose for 24 h, and the effects of C-peptide and/or insulin or IGF-1 were examined. Apoptosis was demonstrated by transferase-mediated dUTP nick-end labeling (TUNEL)/4,6-diamidino-2-phenylindole (DAPI) stainings, flow cytometry and changes in the expression of Bcl2. Activation of insulin signaling intermediaries was determined by Western blots. Translocation of NF-kappaB was demonstrated immunocytochemically. RESULTS: C-peptide but not scrambled C-peptide stimulated cell proliferation and neurite outgrowth. In the presence of 4 nM insulin, 3 nM C-peptide significantly increased autophosphorylation of the insulin receptor (IR) but not that of the insulin-like growth factor 1 receptor (IGF-1R). It stimulated phosphoinositide 3-kinase (PI-3 kinase) and p38 mitogen-activated protein (MAP) kinase activation, enhanced the expression and translocation of nuclear factor-kappaB (NF-kappaB), promoted the expression of Bcl2 and reduced c-jun N-terminal kinase (JNK) phosphorylation in excess of that of insulin alone. CONCLUSIONS: C-peptide in the presence of insulin exerts synergistic effects on cell proliferation, neurite outgrowth and has in the presence of insulin an antiapoptotic effect on high glucose-induced apoptosis but less so on hyperosmolar-induced apoptosis. These effects are likely to be mediated via interactions with the insulin signaling pathway.

Apoptosis↗

PubMed1990

The importance of the 3'-enhancer region in immunoglobulin kappa gene expression.

The first enhancers to be identified in the immunoglobulin gene loci are located in the J-C intron. However, deletion of the immunoglobulin kappa intron-enhancer has little effect on the transcription of kappa transgenes. Here we ask whether the second kappa enhancer which we recently identified at the 3'-end of the locus plays a role in kappa gene expression. We show that its omission leads to 20-40 fold lower expression of kappa transgenes and to poor allelic exclusion. Transfection experiments show that activity of the 3'-enhancer, like that of the kappa-intron enhancer, can be induced in a pre-B cell line by incubation with bacterial lipopolysaccharide. Whereas induction of the kappa-intron enhancer is due to induction of NF-kappa B activity, deletion mapping of the 3'-enhancer localises its activity to a 50 nucleotide region that lacks an NF-kappa B site; indeed the 3'-enhancer allows kappa expression in a cell line which lacks NF-kappa B. Thus, both the 3'- and intron-enhancers can be induced at the same stage of differentiation but by distinct pathways. Furthermore, unlike the intron-enhancer, the 3'-enhancer plays a critical role in the transcription of rearranged immunoglobulin kappa genes.

Animals↗

PubMed2006

Mapping iso-orientation columns by contrast agent-enhanced functional magnetic resonance imaging: reproducibility, specificity, and evaluation by optical imaging of intrinsic signal.

Activation resembling ocular dominance or orientation columns has been mapped with high-resolution functional magnetic resonance imaging (fMRI). However, the neuronal interpretation of these functional maps is unclear because of the poor sensitivity of fMRI, unknown point spread function (PSF), and lack of comparison with independent techniques. Here we show that cerebral blood volume (CBV)-weighted fMRI with a blood plasma contrast agent (monocrystalline iron oxide nanoparticles), in combination with continuous temporally encoded stimulation, can map columnar neuronal activity in the cat primary visual cortex with high sensitivity, selectivity, and reproducibility. We examined hemodynamic response PSF by comparing these CBV-based signals with oxygen metabolism-based negative blood oxygenation level-dependent signals. A significant positive correlation exists between CBV- and metabolism-based iso-orientation maps, suggesting that the hemodynamic PSF is narrower than intercolumn distances. We also compared CBV-based fMRI with optical intrinsic signal (OIS) imaging, a technique that identifies sites of increased neuronal activity, to investigate neuronal correlation. Iso-orientation maps obtained by fMRI and OIS were well matched, indicating that areas of the highest orientation-selective CBV signals correspond to sites of increased neural activity. Using CBV-based fMRI, we successfully mapped orientation-selective functional architecture in the medial bank of the visual cortex, an area inaccessible to OIS imaging. Thus, we conclude that contrast agent-based fMRI, in combination with continuous temporally encoded stimulation, is a highly sensitive technique capable of mapping neural activity at the resolution of functional columns without depth limitation.

Animals↗

PubMed1993

Doppler color flow mapping of epicardial coronary arteries: initial observations.

OBJECTIVES: We addressed the hypothesis that blood flow could be imaged by Doppler color flow mapping of the coronary arteries and characteristic patterns described in normal and diseased vessels. BACKGROUND: Echocardiographic imaging of the epicardial coronary arteries has been suggested as a useful adjunct to their intraoperative evaluation. Addition of Doppler color flow mapping could potentially enhance this evaluation by displaying the flow disturbance produced by anatomic lesions whose physiologic significance may otherwise be uncertain. In experimental models, such displays could also potentially provide insights into the pathophysiology of coronary blood flow and stenosis. METHODS: Epicardial coronary arteries were examined with a high resolution 7-MHz linear phased-array transducer both in vivo and in vitro. 1) The coronary arteries were studied in the beating hearts of 10 open chest dogs in which experimental stenoses were also created; the maximal extent of the arterial tree in which flow could be seen in the most ideal setting was also examined in four additional excised perfused canine hearts. 2) Six excised human coronary arteries were perfused in a pulsatile manner to determine whether abnormal flow patterns could be prospectively identified and subsequently correlated with pathologic evidence of stenosis. RESULTS: All normal coronary artery segments studied showed homogeneous flow without evidence of flow disturbance. In the excised heart, flow could be visualized to the distal extent of the epicardial vessels; in the open chest model, visualization of the proximal 5 to 6 cm was comparable, although surrounding structures limited access to the terminal portions of the vessels. The stenotic lesions created in the canine hearts (n = 9) showed recognizable alterations in the flow pattern: localized aliasing, proximal blood flow acceleration, distal flow disturbance and recirculatory flow. In the excised human arteries, these features identified 12 lesions, all of which corresponded to areas of > or = 50% lumen narrowing by pathologic examination. CONCLUSION: Blood flow in the epicardial coronary arteries can be imaged by Doppler color flow mapping and characteristic flow patterns described in normal and diseased vessels.

Animals↗

PubMed1995

Stimulation of receptor protein-tyrosine phosphatase alpha activity and phosphorylation by phorbol ester.

Receptor Protein-Tyrosine Phosphatase alpha (RPTP alpha) is a transmembrane protein with two cytoplasmic catalytic protein-tyrosine phosphatase (PTP) domains and a relatively short (123 amino acids) extracellular domain. Here we report that treatment of transfected cells that express RPTP alpha with the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate, a direct activator of protein kinase C, induced a rapid, transient increase in RPTP alpha activity due to a 2- to 3-fold increase in substrate affinity. A transient increase in RPTP alpha serine phosphorylation was concomitant with the enhanced activity. Tryptic phosphopeptide mapping of RPTP alpha demonstrated that phosphorylation of three tryptic peptides was enhanced in response to phorbol ester. In vitro dephosphorylation of RPTP alpha from phorbol ester-treated cells reduced RPTP alpha activity to prestimulation levels, indicating that enhanced serine phosphorylation directly accounted for the increase in activity. Our results demonstrate that serine phosphorylation may play a key role in the regulation of the activity of transmembrane PTPs.

Cell Line↗

PubMed2001

Visual activation in functional magnetic resonance imaging at very high field (4 Tesla).

OBJECTIVES: Functional magnetic resonance imaging (fMRI) at very high field strengths provides functional brain mapping with the enhanced signal to noise ratio and the larger blood oxygenation level-dependent (BOLD) effect. We report activated areas in the standard space detected by fMRI at 4 Tesla (T) during simple visual stimulation. MATERIALS AND METHODS: Twelve healthy young subjects were scanned using a 4 T scanner during binocular flashing visual stimulation. Functional images were realigned to the first scan and then spatially normalized. Individual and group data analyses were performed to identify areas of visual activation. RESULTS: Activation of the bilateral primary visual cortex (V1/V2) was observed along the entire calcarine fissure in all subjects. The activated area extended to the extrastriate cortex in all subjects. Activation of the bilateral lateral geniculate nucleus (LGN) was detected in all subjects. The group data showed activation of the bilateral primary visual cortex and the bilateral lateral geniculate nucleus. CONCLUSIONS: Robust activation of the vision-related areas was successfully obtained in all subjects using a 4 T magnetic resonance scanner. These results suggest that fMRI at very high field strengths may be effective in showing visual system physiology, and that it can be a promising method to assess visual function of human subjects.

Adult↗

PubMed2002

Statistical parametric mapping of (99m)Tc-HMPAO-SPECT images for the diagnosis of Alzheimer's disease: normalizing to cerebellar tracer uptake.

BACKGROUND: For a quantitative comparison of images obtained during (99m)Tc-hexamethylpropylene amine oxime (HMPAO) single-photon emission computed tomography (SPECT), brain activity values are usually normalized to a reference region. In studies of Alzheimer-type dementia (ATD), the cerebellum is often used as a reference region, assuming that it is spared any major pathological involvement. Statistical parametric mapping (SPM) may enhance the evaluation of SPECT scans in ATD patients. However, current SPM software only allows scaling to average whole brain activity (i.e., global normalization). The aim of this study was to develop an easily applied, objective, and reproducible method for determining average cerebellar tracer uptake so that images can be scaled specifically to cerebellar activity prior to the performance of SPM analysis. We also investigated whether cerebellar normalization increases the sensitivity and specificity of SPM analysis of ATD patients compared with global normalization. METHODS: Image files were taken from a parallel study investigating the use of SPECT as a diagnostic tool for early onset of ATD. Two methods for determining cerebellar activity were developed: one manually, using templates, the other automated, using specified coordinates entered into a Matlab routine. Group comparison of ATD patients versus controls (= healthy volunteers and depressed patients) was performed on a voxel-by-voxel basis using SPM 96 on Windows 95. Receiver operator characteristics (ROC) were computed for 20 student raters examining patient and control scans with and without single-subject SPMs. RESULTS: The reduction of cerebral blood flow in the group of ATD patients appeared 1.7 times greater in spatial extent when the tracer uptake was normalized to cerebellum rather than to average whole brain activity. Computing the reverse contrast (reductions in the control group compared with ATD patients) produced clusters of significance in globally normalized images which were not manifest after normalizing to cerebellum. This is consistent with the notion that the cerebellum is spared in ATD. Analysis of the area under the ROC curve showed that cerebellar-normalized SPM produced significantly improved accuracy over perfusion scans alone. CONCLUSION: An easily applied, objective, reproducible method was developed for normalizing images to cerebellum prior to the performance of SPM analysis. Cerebellar normalization produced more extensive abnormalities in SPM analyses of ATD patients than global normalization. Furthermore, cerebellar normalization produced marginally more accurate diagnostic results in single-scan SPM analysis of ATD patients than did global normalization.

Aged↗

PubMed2005

CD40 ligand influences platelet release of reactive oxygen intermediates.

OBJECTIVE: Soluble CD40 ligand (sCD40L) has been recently implicated in the pathogenesis of atherosclerosis. Elevated levels of sCD40L in acute coronary syndrome patients suggests enhanced platelet function; however, the exact mechanism by which this occurs is unknown. In this study, we examined the effect of sCD40L on platelet function and reactive oxygen and nitrogen species (RONS) generation. METHODS AND RESULTS: Platelet stimulation in the presence of recombinant sCD40L (rsCD40L) led to enhanced generation of RONS as measured by DCFHDA oxidation and confocal microscopy. Incubation with rsCD40L led to enhanced platelet P-selectin expression, aggregation, and platelet-leukocyte conjugation. Platelets isolated from CD40L-deficient mice had decreased agonist-induced NO release as compared with wild-type mice. Incubation of platelets with rsCD40L enhanced stimulation-induced p38 MAP kinase and Akt phosphorylation. CONCLUSIONS: Soluble CD40L enhances platelet activation, aggregation, and platelet-leukocyte conjugation, as well as increases stimulation-induced platelet release of RONS through activation of Akt and p38 MAP kinase signaling pathways. These data suggest that sCD40L regulates platelet-dependent inflammatory and thrombotic responses that contribute to the pathogenesis of atherothrombosis.

Animals↗

PubMed1990

Insulin and the stimulation of glycogen synthesis. The road from glycogen structure to glycogen synthase to cyclic AMP-dependent protein kinase to insulin mediators.

The enhanced phosphorylations via cAMP, Ca2+ mobilization, and diacyl glycerol formation via the activation of the respective kinases is now classical. The decreased phosphorylation via inhibition of adenylate cyclase via the alpha adrenergic receptor is also becoming understood. What the insulin studies on the control of glycogen synthesis have taught us is that the rate limiting enzyme glycogen synthase is regulated by multiple covalent phosphorylation in an elegant but complex manner. The overall pattern of dephosphorylation is influenced by effecting both phosphatase and kinase activities in a set of interrelated mechanisms. In the presence of glucose, in muscle, fat, and liver under physiological conditions G-6-P acts as a signal to stimulate the phosphatase. An additional stimulation could occur via a novel insulin phosphatase stimulatory mediator. The phosphatase is also stimulated by at least three covalent mechanisms involving altered phosphorylation state. In one there is a decreased phosphorylation of the phosphatase inhibitor 1 potentially related to decreased cAMP-dependent protein kinase activity. In the second, there is decreased phosphorylation of the deinhibitor also potentially related to decreased cAMP-dependent protein kinase phosphorylation. In the third, an increased activity of casein kinase 2 could activate the ATP-Mg dependent phosphatase by an increased phosphorylation of phosphatase inhibitor 2 (modulatory subunit). In the liver, allosteric control of the phosphatase by G-6-P and nucleotides is of great importance. Insulin also stimulates the phosphatase in long-term experiments via increased protein synthesis. It is clear that future work will be required to determine which species of the various classes of phosphatases are regulated in short-term and long-term regulation by insulin. In terms of kinases, the effects of insulin to inactivate and desensitize the cAMP-dependent protein kinase are established. The molecular mechanisms of this effect remain to be worked out. The enhanced activity of MAP and S-6 kinase would appear to be part of a cascade of reactions perhaps originating in the autophosphorylation and activation of the insulin receptor tyrosine kinase. The mechanism of the short-term activation of casein kinase 2 remains to be elucidated. A cAMP-dependent protein kinase inhibitory mediator, which also inhibits adenylate cyclase is an important element in the regulation of kinase and adenylate cyclase activity by insulin. Its physiological significance must be established in the future, in terms of its control of glycogen synthase activation by insulin. Clearly this kinase inhibitor as well as the phosphatase stimulator are potential regulators of glycogen synthase activity by insulin.

Amino Acid Sequence↗

PubMed2003

Sodium dodecyl sulfate versus acid-labile surfactant gel electrophoresis: comparative proteomic studies on rat retina and mouse brain.

A long-chain derivative of 1,3-dioxolane sodium propyloxy sulfate, with similar denaturing and electrophoretic properties as SDS, and facilitated protein identification following polyacrylamide gel electrophoresis (PAGE) for Coomassie-stained protein bands, has been tested. Comparative acid-labile surfactant/sodium dodecyl sulfate two-dimensional (ALS/SDS 2-D)-PAGE experiments of lower abundant proteins from the proteomes of regenerating rat retina and mouse brain show that peptide recovery for mass spectrometry (MS) mapping is significantly enhanced using ALS leading to more successful database searches. ALS may influence some procedures in proteomic analysis such as the determination of protein content and methods need to be adjusted to that effect. The promising results of the use of ALS in bioanalytics call for detailed physicochemical investigations of surfactant properties.

Acids↗

PubMed2001

Elucidating protein secondary structures using alpha-carbon recurrence quantifications.

Secondary structures of proteins were studied by recurrence quantification analysis (RQA). High-resolution, 3-dimensional coordinates of alpha-carbon atoms comprising a set of 68 proteins were downloaded from the Protein Data Bank. By fine-tuning four recurrence parameters (radius, line, residue, separation), it was possible to establish excellent agreement between percent contribution of alpha-helix and beta-sheet structures determined independently by RQA and that of the DSSP algorithm (Define Secondary Structure of Proteins). These results indicate that there is an equivalency between these two techniques, which are based upon totally different pattern recognition strategies. RQA enhances qualitative contact maps by quantifying the arrangements of recurrent points of alpha carbons close in 3-dimensional space. For example, the radius was systematically increased, moving the analysis beyond local alpha-carbon neighborhoods in order to capture super-secondary and tertiary structures. However, differences between proteins could only be detected within distances up to about 6-11 A, but not higher. This result underscores the complexity of alpha-carbon spacing when super-secondary structures appear at larger distances. Finally, RQA-defined secondary structures were found to be robust against random displacement of alpha carbons upwards of 1 A. This finding has potential import for the dynamic functions of proteins in motion.

Bacterial Proteins↗

PubMed1996

The mechanism of recruitment of the lactate dehydrogenase-B/epsilon-crystallin gene by the duck lens.

In duck, the housekeeping enzyme lactate dehydrogenase B (LDH-B) and the lens structural protein epsilon-crystallin are encoded by the same single copy gene. Transcription of the gene is initiated from two closely spaced start sites, at -28 and +1. The usage of the downstream site is greatly enhanced in lens. Deletion mapping of the promoter shows that the region -70/+18 specifies the enhanced promoter activity in the lens. A critical role is played by the consensus Sp1 binding site at -50; mutation of this site abolishes lens-preferred expression. Deletion of the +1 transcription initiation site also leads to a decrease in lens-preferred expression, which can be restored by moving the -28 transcription initiation site downstream. By band shift experiments, supershift mobility assays and methylation interference assays, Sp1 was shown to bind to the Sp1 consensus binding site at -50 using either heart or lens nuclear extracts. Co-expression of Sp1 or Sp1-like factors inhibited the activity of an LDH-B/epsilon-crystallin promoter construct by approximately 60% in lens and by 40% in heart cells. Co-expression of Pax-6, a transcription factor shown to be involved in the lens-enhanced expression of a number of other crystallin genes, did not influence the promoter activity of the -130/+650 LDH-B/epsilon-crystallin promoter construct. In contrast to other crystallin promoters, the LDH-B/epsilon-crystallin promoter does not appear to contain a lens-specific element, rather our data lead to a model in which a factor transmitting the effect of Sp1, bound at -50, to the transcription initiation complex is responsible for the lens-preferred expression of the LDH-B/epsilon-crystallin promoter.

Animals↗

PubMed1999

F1-ATP synthase beta-subunit and cytochrome c transcriptional regulation in right ventricular hemodynamic overload and hypertrophically stimulated cardiocytes.

Cardiac hypertrophic growth secondary to hemodynamic pressure overload causes changes in energy requirements that may involve the transcriptional upregulation of oxidative phosphorylation genes. Therefore, two representative nuclear-encoded genes, the mitochondrial F1-ATP synthase beta-subunit (beta-subunit) and cytochrome c (cyt c), were examined in a feline chronic pulmonary artery banded right ventricular pressure-overload model. In the hypertrophying right ventricle, beta-subunit and cyt c mRNA levels increased after two and seven days, during the peak growth response. To examine cardiac transcriptional regulation, neonatal rat cardiac myocytes (cardiocytes) were transiently transfected with beta-subunit promoter constructs ranging from -1519 nucleotides (nt) upstream of transcription initiation as well as cyt c promoter constructs ranging from -726 nt. A full-length p1519beta-subunit/Luc construct was alpha-adrenergically inducible by 275% (+/-30%) with this activation being mapped to an enhancer region between -1519 to -1480 nt. Smaller constructs containing more proximal promoter elements were not inducible. Additionally, the full-length and enhancer deleted beta-subunit constructs were also inducible in electrically stimulated cardiocytes, suggesting a different mechanism of activation. Cyt c constructs containing known constitutive elements from -191 to -167 nt and -139 to -84 nt were responsible for the majority of the reporter activity of the full-length promoter but were not inducible in the presence of phenylephrine. Hence, we show that promoter regions containing elements common in other metabolism-related gene families are active in neonatal rat cardiocytes. Once more, we have identified a beta-subunit genomic region responsive to alpha-adrenergic and electrical stimulation.

3T3 Cells↗

PubMed2002

Classical and Bayesian inference in neuroimaging: applications.

In Friston et al. ((2002) Neuroimage 16: 465-483) we introduced empirical Bayes as a potentially useful way to estimate and make inferences about effects in hierarchical models. In this paper we present a series of models that exemplify the diversity of problems that can be addressed within this framework. In hierarchical linear observation models, both classical and empirical Bayesian approaches can be framed in terms of covariance component estimation (e.g., variance partitioning). To illustrate the use of the expectation-maximization (EM) algorithm in covariance component estimation we focus first on two important problems in fMRI: nonsphericity induced by (i) serial or temporal correlations among errors and (ii) variance components caused by the hierarchical nature of multisubject studies. In hierarchical observation models, variance components at higher levels can be used as constraints on the parameter estimates of lower levels. This enables the use of parametric empirical Bayesian (PEB) estimators, as distinct from classical maximum likelihood (ML) estimates. We develop this distinction to address: (i) The difference between response estimates based on ML and the conditional means from a Bayesian approach and the implications for estimates of intersubject variability. (ii) The relationship between fixed- and random-effect analyses. (iii) The specificity and sensitivity of Bayesian inference and, finally, (iv) the relative importance of the number of scans and subjects. The forgoing is concerned with within- and between-subject variability in multisubject hierarchical fMRI studies. In the second half of this paper we turn to Bayesian inference at the first (within-voxel) level, using PET data to show how priors can be derived from the (between-voxel) distribution of activations over the brain. This application uses exactly the same ideas and formalism but, in this instance, the second level is provided by observations over voxels as opposed to subjects. The ensuing posterior probability maps (PPMs) have enhanced anatomical precision and greater face validity, in relation to underlying anatomy. Furthermore, in comparison to conventional SPMs they are not confounded by the multiple comparison problem that, in a classical context, dictates high thresholds and low sensitivity. We conclude with some general comments on Bayesian approaches to image analysis and on some unresolved issues.

Algorithms↗

PubMed1988

Photofootprint of nucleosome core DNA in intact chromatin having different structural states.

Recently, we reported that the distribution of ultraviolet light (u.v.) induced pyrimidine dimers in nucleosome core DNA has a striking 10.3(+/- 0.1) base periodicity and the regions of enhanced quantum yield map to positions where DNA strands are farthest from the core histone surface. Improvement of the mapping procedure has allowed us to analyze this distribution in more detail, and compare the distribution pattern for nucleosome cores from intact chromatin having different higher-order structures (from the 10 nm filament to the 30 nm fiber). At all levels of chromatin compaction, we observed the following. (1) The average periodicity in pyrimidine dimer yield is 10.3 bases. (2) The peak-to-peak spacing in this distribution is significantly different from 10.3 bases in the region covering three helix turns immediately 5' of the dyad axis. (3) There is a suppression of photoproduct formation in the region of the dyad axis, especially at position 84 from the 5' end. (4) The approximately 10 base ensembles have alternating peak intensities throughout core DNA. Furthermore, peak deconvolution analysis of the pyrimidine dimer pattern yielded a striking similarity in photoproduct yield for the different levels of chromatin compaction. Irradiation of isolated core DNA yields a much more random distribution of photoproducts, although a weak modulation pattern is observed (indicating that there is a non-random alignment of adjacent pyrimidines in our core DNA preparations). This pattern includes a depression in photoproduct yield near position 95, suggesting that the sequence in this region plays a role in nucleosome positioning. These results show that the u.v. photofootprint is a sensitive, diagnostic probe of core histone-DNA interactions in intact chromatin, and these interactions are not significantly altered by changes in the structural state of the chromatin fiber.

Animals↗

PubMed1996

Radiofrequency catheter ablation of right ventricular outflow tachycardia in children and adolescents.

OBJECTIVES: The current study reviews the safety and efficacy of radiofrequency catheter ablation for the treatment of right ventricular outflow tachycardia in children and adolescents and describes a modified method for mapping the tachycardia focus. BACKGROUND: Although radiofrequency catheter ablation has proved highly effective for the treatment of supraventricular tachycardia during childhood and adolescence, its application in children with idiopathic right ventricular outflow tachycardia has been limited. METHODS: Six children (mean [+/- SD] age 10.6 +/- 2.4 years, range 6 to 16) with right ventricular outflow tachycardia underwent seven radiofrequency catheter ablation procedures. The mean tachycardia cycle length was 323 +/- 24 ms (range 300 to 360). Two multipolar catheters were positioned in the right ventricular outflow tract to map the tachycardia focus. RESULTS: Radiofrequency catheter ablation was successful in five (83%) of the six children (95% confidence interval 36% to 99%). At successful ablation sites, local endocardial activation time preceded the surface QRS onset by 46 +/- 5 ms (range 37 to 57), and there was concordance of the 12-lead pace map and the electrocardiogram (ECG) in 11 (one patient) to 12 ECG leads (four patients). One patient developed complete right bundle branch block during radiofrequency catheter ablation. There were no additional complications and no clinical recurrences over a mean follow-up period of 12.7 +/- 3.8 months (range 9 to 22). CONCLUSIONS: These results suggest that radiofrequency catheter ablation is a safe and effective treatment for right ventricular outflow tachycardia during childhood and adolescence. In addition, tachycardia mapping may be enhanced by use of a multipolar right ventricular outflow catheter technique.

Adolescent↗

PubMed2026

Long-read sequencing resolves complex CYP21A2 variants and identifies 2+0 carriers in 21-hydroxylase deficiency.

The complex CYP21A2 variants arising from high homology with its pseudogene CYP21A1P challenge the diagnosis of 21-hydroxylase deficiency (21-OHD). This study systematically evaluated long-read sequencing (LRS) for identifying complex structural variants of the CYP21A2 gene in 21-OHD in comparison with conventional molecular diagnostic methods, including multiplex ligation-dependent probe amplification (MLPA), CNVplex, and SNaPshot. Twenty patients with suspected 21-OHD and defined CYP21A2 structural variants identified via initial MLPA screening were enrolled. Variants were further analyzed using CNVplex and SNaPshot, then all samples underwent LRS for comprehensive variant detection, breakpoint mapping, and haplotype resolution. LRS overcame key limitations of conventional methods. It reliably identified a novel large-fragment deletion and defined its boundaries. Notably, LRS identified "2+0" carriers, where deletions masked by duplications cause false-negatives with standard techniques. Moreover, LRS accurately distinguished CYP21A1P/CYP21A2_CH-4 and CH-9 chimera subtypes which were indistinguishable by the combined conventional assays. Furthermore, LRS enabled the precise identification and characterization of TNXA/TNXB chimeric deletions. These are frequently misclassified as CYP21A1P/CYP21A2 chimeras by conventional methods but are critical for diagnosing associated conditions such as CAH-X syndrome. LRS provides a superior, integrated solution for the molecular diagnosis of 21-OHD, offering precise structural variant characterization, accurate carrier detection, and reliable breakpoint mapping. Its application enhances diagnostic accuracy, supports advanced genetic counseling, and paves the way for genotype-informed clinical management.

Journal Article↗

PubMed1998

Liver-specific MR imaging contrast agents.

Liver-specific MR imaging contrast agents consist of iron oxide particles or specially designed paramagnetic complexes targeting either the reticulo-endothelial system of the liver or the hepatocytes. These agents enhance the relaxation of water molecules in normal liver tissue and are excluded from abnormal tissue, such as metastases. Relaxation enhancement provides a map of normal liver function, increasing conspicuity of focal abnormalities. Understanding the indications and use of these agents is a central challenge for radiologists practicing liver MR imaging.

Contrast Media↗