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Pathogenic loss-of-function variants in BGN, an X-linked gene encoding biglycan, are associated with Meester-Loeys syndrome (MRLS), a thoracic aortic aneurysm/dissection syndrome. Since the initial publication of five probands in 2017, we have considerably expanded our MRLS cohort to a total of 18 probands (16 males and 2 females). Segregation analyses identified 36 additional BGN variant-harboring family members (9 males and 27 females). The identified BGN variants were shown to lead to loss-of-function by cDNA and Western Blot analyses of skin fibroblasts or were strongly predicted to lead to loss-of-function based on the nature of the variant. No (likely) pathogenic missense variants without additional (predicted) splice effects were identified. Interestingly, a male proband with a deletion spanning the coding sequence of BGN and the 5' untranslated region of the downstream gene (ATP2B3) presented with a more severe skeletal phenotype. This may possibly be explained by expressional activation of the downstream ATPase ATP2B3 (normally repressed in skin fibroblasts) driven by the remnant BGN promotor. This study highlights that aneurysms and dissections in MRLS extend beyond the thoracic aorta, affecting the entire arterial tree, and cardiovascular symptoms may coincide with non-specific connective tissue features. Furthermore, the clinical presentation is more severe and penetrant in males compared to females. Extensive analysis at RNA, cDNA, and/or protein level is recommended to prove a loss-of-function effect before determining the pathogenicity of identified BGN missense and non-canonical splice variants. In conclusion, distinct mechanisms may underlie the wide phenotypic spectrum of MRLS patients carrying loss-of-function variants in BGN., (© 2024. The Author(s).)

The imprinted genes Grb10 and Nesp influence impulsive behavior on a delay discounting task in an opposite manner. A recently developed theory suggests that this pattern of behavior may be representative of predicted effects of imprinted genes on tolerance to risk. Here we examine whether mice lacking paternal expression of Grb10 show abnormal behavior across a number of measures indicative of risk-taking. Although Grb10 +/p mice show no difference from wild type (WT) littermates in their willingness to explore a novel environment, their behavior on an explicit test of risk-taking, namely the Predator Odor Risk-Taking task, is indicative of an increased willingness to take risks. Follow-up tests suggest that this risk-taking is not simply because of a general decrease in fear, or a general increase in motivation for a food reward, but reflects a change in the trade-off between cost and reward. These data, coupled with previous work on the impulsive behavior of Grb10 +/p mice in the delayed reinforcement task, and taken together with our work on mice lacking maternal Nesp, suggest that maternally and paternally expressed imprinted genes oppositely influence risk-taking behavior as predicted., (© 2020 The Authors. Genes, Brain and Behavior published by International Behavioural and Neural Genetics Society and John Wiley & Sons Ltd.)

Imprinted genes are expressed from one parental allele only as a consequence of epigenetic events that take place in the mammalian germ line and are thought to have evolved through intragenomic conflict between parental alleles. We demonstrate, for the first time, oppositional effects of imprinted genes on brain and behavior. Specifically, we show that mice lacking paternal Grb10 make fewer impulsive choices, with no dissociable effects on a separate measure of impulsive action. Taken together with previous work showing that mice lacking maternal Nesp55 make more impulsive choices, this suggests that impulsive choice behavior is a substrate for the action of genomic imprinting. Moreover, the contrasting effect of these two genes suggests that impulsive choices are subject to intragenomic conflict and that maternal and paternal interests pull this behavior in opposite directions. Finally, these data may also indicate that an imbalance in expression of imprinted genes contributes to pathological conditions such as gambling and drug addiction, where impulsive behavior becomes maladaptive., (Copyright © 2018 Dent et al.)

Pregnancy is a state of high metabolic demand. Fasting diverts metabolism to fatty acid oxidation, and the fasted response occurs much more rapidly in pregnant women than in non-pregnant women. The product of the imprinted DLK1 gene (delta-like homolog 1) is an endocrine signaling molecule that reaches a high concentration in the maternal circulation during late pregnancy. By using mouse models with deleted Dlk1, we show that the fetus is the source of maternal circulating DLK1. In the absence of fetally derived DLK1, the maternal fasting response is impaired. Furthermore, we found that maternal circulating DLK1 levels predict embryonic mass in mice and can differentiate healthy small-for-gestational-age (SGA) infants from pathologically small infants in a human cohort. Therefore, measurement of DLK1 concentration in maternal blood may be a valuable method for diagnosing human disorders associated with impaired DLK1 expression and to predict poor intrauterine growth and complications of pregnancy.

Genomic imprinting is the process whereby germline epigenetic events lead to parent-of-origin specific monallelic expression of a number of key mammalian genes. The imprinted gene Nesp is expressed from the maternal allele only and encodes for Nesp55 protein. In the brain, Nesp55 is found predominately in discrete areas of the hypothalamus and midbrain. Previously, we have shown that loss of Nesp55 gives rise to alterations in novelty-related behaviour. Here, we extend these findings and demonstrate, using the Nesp m/+ mouse model, that loss of Nesp55 leads to impulsive choices as measured by a delayed-reinforcement task, whereby Nesp m/+ mice were less willing to wait for a delayed, larger reward, preferring instead to choose an immediate, smaller reward. These effects were highly specific as performance in another component of impulsive behaviour, the ability to stop a response once started as assayed in the stop-signal reaction time task, was equivalent to controls. We also showed changes in the serotonin system, a key neurotransmitter pathway mediating impulsive behaviour. First, we demonstrated that Nesp55 is co-localized with serotonin and then went on to show that in midbrain regions there were reductions in mRNA expression of the serotonin-specific genes Tph2 and Slc6a4, but not the dopamine-specific gene Th in Nesp m/+ mice; suggesting an altered serotonergic system could contribute, in part, to the changes in impulsive behaviour. These data provide a novel mode of action for genomic imprinting in the brain and may have implications for pathological conditions characterized by maladaptive response control., (© 2016 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.)

The recent discovery of somatically acquired CALR mutations in a substantial proportion of patients with myeloproliferative neoplasms has provided a new marker of clonal disease, advancing both diagnosis and prognosis in these previously difficult to characterise disorders. The mutations, which can be challenging to detect on a routine basis, are heterogeneous insertions/deletions (indels) in exon 9 with mutant allele burden that vary substantially between patients. We evaluated four genetic screening methods for their ability to detect a series of different CALR mutations; Sanger sequencing, fragment analysis PCR, high resolution melt (HRM) and targeted next generation sequencing (NGS). The limit of detection (LoD) of each assay was tested using serial dilution series made with DNA from CALR positive sample DNA and a cell line, MARIMO, found to carry a heterozygous 61 nucleotide CALR deletion. All methods were capable of detecting each mutation; HRM and fragment analysis PCR were better at detecting low mutation levels compared to Sanger sequencing but targeted NGS had the lowest LoD at a 1% mutation burden., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Impulsive behavior is a key constituent of many psychiatric illnesses, with maladaptive response control being a feature of disorders such as ADHD, schizophrenia, mania, and addiction. In order to understand the neurological underpinnings of impulsivity, a number of behavioral tasks have been developed for use with animal models. Data from studies with rats and other animals have led to the idea of the existence of dissociable components of impulsivity, which in turn informs studies of human disorders and potentially the development of specific therapies. Increasingly, mouse models are being used to investigate the known genetic contribution to psychiatric disorders in which abnormal response control leads to altered impulsive behaviors. In order to maximize the potential of these mouse models, it is important that researchers take into account the non-unitary nature of response control and impulsivity. In this article, we briefly review the tasks available to behavioral neuroscientists and how these can be used in order to tease apart the contribution of a specific genetic lesion into the discrete aspects of impulsive behavior., (Copyright © 2014 John Wiley & Sons, Inc.)

Assessing risk is an essential part of human behaviour and may be disrupted in a number of psychiatric conditions. Currently, in many animal experimental designs the basis of the potential 'risk' is loss or attenuation of reward, which fail to capture 'real-life' risky situations where there is a trade-off between a separate cost and reward. The development of rodent tasks where two separate and conflicting factors are traded against each other has begun to address this discrepancy. Here, we discuss the merits of these risk-taking tasks and describe the development of a novel test for mice - the 'predator-odour risk-taking' task. This paradigm encapsulates a naturalistic approach to measuring risk-taking behaviour where mice have to balance the benefit of gaining a food reward with the cost of exposure to a predator odour using a range of different odours (rat, cat and fox). We show that the 'predator-odour risk-taking' task was sensitive to the trade-off between cost and benefit by demonstrating reduced motivation to collect food reward in the presence of these different predator odours in two strains of mice and, also, if the value of the food reward was reduced. The 'predator-odour risk-taking' task therefore provides a strong platform for the investigation of the genetic substrates of risk-taking behaviour using mouse models, and adds a further dimension to other recently developed rodent tests., (© 2013 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Imprinted genes are defined by their parent-of-origin-specific monoallelic expression. Although the epigenetic mechanisms regulating imprinted gene expression have been widely studied, their functional importance is still unclear. Imprinted genes are associated with a number of physiologies, including placental function and foetal growth, energy homeostasis, and brain and behaviour. This review focuses on genomic imprinting in the brain and on two imprinted genes in particular, Nesp and paternal Grb10, which, when manipulated in animals, have been shown to influence adult behaviour. These two genes are of particular interest as they are expressed in discrete and overlapping neural regions, recognised as key "imprinting hot spots" in the brain. Furthermore, these two genes do not appear to influence placental function and/or maternal provisioning of offspring. Consequently, by understanding their behavioural function we may begin to shed light on the evolutionary significance of imprinted genes in the adult brain, independent of the recognised role in maternal care. In addition, we discuss the potential future directions of research investigating the function of these two genes and the behavioural role of imprinted genes more generally.

Imprinted, maternally silenced insulin-like growth factor-2 is expressed in both the foetus and placenta and has been shown to have roles in foetal and placental development in animal models. Here we compared mice engineered to be null for the placenta-specific P0 transcript (insulin-like growth factor-2-P0 KO) to mice with disruptions of all four insulin-like growth factor-2 transcripts, and therefore null for insulin-like growth factor-2 in both placenta and foetus (insulin-like growth factor-2-total KO). Both models lead to intrauterine growth restriction but dissociate between a situation where there is an imbalance between foetal demand and placental supply of nutrients (the insulin-like growth factor-2-P0 KO) and one where demand and supply is more balanced (the insulin-like growth factor-2-total KO). Increased reactivity to anxiety-provoking stimuli is manifested later in life only in those animals where there is a mismatch between placental supply and foetal demand for nutrients during gestation. Our findings further distinguish placental dysfunction from intrauterine growth restriction and reveal a role for the placenta in long-term programming of emotional behaviour.

Background: Analysis of cell free fetal (cff) DNA in maternal plasma is used routinely for non invasive prenatal diagnosis (NIPD) of fetal sex determination, fetal rhesus D status and some single gene disorders. True positive results rely on detection of the fetal target being analysed. No amplification of the target may be interpreted either as a true negative result or a false negative result due to the absence or very low levels of cffDNA. The hypermethylated RASSF1A promoter has been reported as a universal fetal marker to confirm the presence of cffDNA. Using methylation-sensitive restriction enzymes hypomethylated maternal sequences are digested leaving hypermethylated fetal sequences detectable. Complete digestion of maternal sequences is required to eliminate false positive results., Methods: cfDNA was extracted from maternal plasma (n = 90) and digested with methylation-sensitive and insensitive restriction enzymes. Analysis of RASSF1A, SRY and DYS14 was performed by real-time PCR., Results: Hypermethylated RASSF1A was amplified for 79 samples (88%) indicating the presence of cffDNA. SRY real time PCR results and fetal sex at delivery were 100% accurate. Eleven samples (12%) had no detectable hypermethylated RASSF1A and 10 of these (91%) had gestational ages less than 7 weeks 2 days. Six of these samples were male at delivery, five had inconclusive results for SRY analysis and one sample had no amplifiable SRY., Conclusion: Use of this assay for the detection of hypermethylated RASSF1A as a universal fetal marker has the potential to improve the diagnostic reliability of NIPD for fetal sex determination and single gene disorders.

Dyggve-Melchior-Clausen syndrome (DMC), a severe autosomal recessive skeletal disorder with mental retardation, is caused by mutation of the gene encoding Dymeclin (DYM). Employing patient fibroblasts with mutations characterized at the genomic and, for the first time, transcript level, we identified profound disruption of Golgi organization as a pathogenic feature, resolved by transfection of heterologous wild-type Dymeclin. Collagen targeting appeared defective in DMC cells leading to near complete absence of cell surface collagen fibers. DMC cells have an elevated apoptotic index (P< 0.01) likely due to a stress response contingent upon Golgi-related trafficking defects. We performed spatiotemporal mapping of Dymeclin expression in zebrafish embryos and identified high levels of transcript in brain and cartilage during early development. Finally, in a chondrocyte cDNA library, we identified two novel secretion pathway proteins as Dymeclin interacting partners: GOLM1 and PPIB. Together these data identify the role of Dymeclin in secretory pathways essential to endochondral bone formation during early development., (© 2011 Wiley-Liss, Inc.)

Proteomic analysis has revealed potential early biomarkers of acute kidney injury (AKI) in children undergoing cardiopulmonary bypass (CPB), the most prominent one with a mass-to-charge ratio of 6.4 kDa. The objective of this study was to identify this protein and test its utility as a biomarker of AKI. Trypsin-digested protein bands were analyzed by tandem mass spectrometry (MS/MS) to identify the protein in urine samples. Surface-enhanced laser desorption/ionization time-of-flight analysis and a functional activity assay were performed to quantify urinary levels in a pilot study of 106 pediatric patients undergoing CPB. The protein was identified as aprotinin. Urinary aprotinin levels 2 h after initiation of CPB were predictive of AKI (for functional assay: 92% sensitivity, 96% specificity, area under the curve of 0.98). By multivariate analysis, the urinary aprotinin level 2 h after CPB was an independent predictor of AKI (beta = 0.001, P < 0.0001). The 2 h urinary aprotinin level correlated with serum creatinine, duration of AKI, and length of hospital stay. We concluded that urinary aprotinin levels 2 h after initiation of CPB predict the development of AKI and adverse clinical outcomes.

Acute kidney injury (AKI) is a major complication in children who undergo cardiopulmonary bypass surgery. We performed metabonomic analyses of urine samples obtained from 40 children that underwent cardiac surgery for correction of congenital cardiac defects. Serial urine samples were obtained from each patient prior to surgery and at 4 h and 12 h after surgery. AKI, defined as a 50% or greater rise in baseline level of serum creatinine, was noted in 21 children at 48-72 h after cardiac surgery. The principal component analysis of liquid chromatography/mass spectrometry (LC/MS) negative ionization data of the urine samples obtained 4 h and 12 h after surgery from patients who develop AKI clustered away from patients who did not develop AKI. The LC/MS peak with mass-to-charge ratio (m/z) 261.01 and retention time (tR) 4.92 min was further analyzed by tandem mass spectrometry (MS/MS) and identified as homovanillic acid sulfate (HVA-SO4), a dopamine metabolite. By MS single-reaction monitoring, the sensitivity was 0.90 and specificity was 0.95 for a cut-off value of 24 ng/microl for HVA-SO4 at 12 h after surgery. We concluded that urinary HVA-SO4 represents a novel, sensitive, and predictive early biomarker of AKI after pediatric cardiac surgery.

Background and Objectives: The authors have previously shown that urine neutrophil gelatinase-associated lipocalin (NGAL), measured by a research ELISA, is an early predictive biomarker of acute kidney injury (AKI) after cardiopulmonary bypass (CPB). In this study, whether an NGAL immunoassay developed for a standardized clinical platform (ARCHITECT analyzer, Abbott Diagnostics Division, Abbott Laboratories, Abbott Park, IL) can predict AKI after CPB was tested., Design, Setting, Participants, & Measurements: In a pilot study with 136 urine samples (NGAL range, 0.3 to 815 ng/ml) and 6 calibration standards (NGAL range, 0 to 1000 ng/ml), NGAL measurements by research ELISA and by the ARCHITECT assay were highly correlated (r = 0.99). In a subsequent study, 196 children undergoing CPB were prospectively enrolled and serial urine NGAL measurements obtained by ARCHITECT assay. The primary outcome was AKI, defined as a > or = 50% increase in serum creatinine., Results: AKI developed in 99 patients (51%), but the diagnosis using serum creatinine was delayed by 2 to 3 d after CPB. In contrast, mean urine NGAL levels increased 15-fold within 2 h and by 25-fold at 4 and 6 h after CPB. For the 2-h urine NGAL measurement, the area under the curve was 0.95, sensitivity was 0.82, and the specificity was 0.90 for prediction of AKI using a cutoff value of 100 ng/ml. The 2-h urine NGAL levels correlated with severity and duration of AKI, length of stay, dialysis requirement, and death., Conclusions: Accurate measurements of urine NGAL are obtained using the ARCHITECT platform. Urine NGAL is an early predictive biomarker of AKI severity after CPB.

A change in the serum creatinine is not sensitive for an early diagnosis of acute kidney injury. We evaluated urinary levels of matrix metalloproteinase-9 (MMP-9), N-acetyl-beta-D-glucosaminidase (NAG), and kidney injury molecule-1 (KIM-1) as biomarkers for the detection of acute kidney injury. Urine samples were collected from 44 patients with various acute and chronic kidney diseases, and from 30 normal subjects in a cross-sectional study. A case-control study of children undergoing cardio-pulmonary bypass surgery included urine specimens from each of 20 patients without and with acute kidney injury. Injury was defined as a greater than 50% increase in the serum creatinine within the first 48 h after surgery. The biomarkers were normalized to the urinary creatinine concentration at 12, 24, and 36 h after surgery with the areas under the receiver-operating characteristic curve compared for performance. In the cross-sectional study, the area under the curve for MMP-9 was least sensitive followed by KIM-1 and NAG. Combining all three biomarkers achieved a perfect score diagnosing acute kidney injury. In the case-control study, KIM-1 was better than NAG at all time points, but combining both was no better than KIM-1 alone. Urinary MMP-9 was not a sensitive marker in the case-control study. Our results suggest that urinary biomarkers allow diagnosis of acute kidney injury earlier than a rise in serum creatinine.

Although the diagnostic criteria for acute rheumatic fever (ARF) are well known, a high index of suspicion is necessary in order to assure timely diagnosis and appropriate treatment. We present a case of an 8-year-old child who presented with unilateral pulmonary edema secondary to acute mitral insufficiency due to ARF. ARF should be considered in the differential diagnosis of unilateral pulmonary edema in children.

The arterial switch operation has become the preferred procedure for surgical management of transposition, defined on the basis of concordant atrioventricular and discordant ventriculo-arterial connections. We conducted a retrospective evaluation of our experience in 61 infants with this segmental combination, seen from January, 1997, to July, 2003, in order to determine the factors that are associated with a prolonged postoperative course. Factors independently associated with a prolonged postoperative stay in the cardiac intensive care unit included prematurity, difficulty in feeding, capillary leak, need for preoperative inotropic support, and postoperative infectious complications. Future research is warranted designed to minimize the impact of capillary leak and postoperative infectious complications. In addition, based on these results, our practice has evolved to initiate enteral feedings in the preoperative period if feasible, with such enteral feedings resumed as soon as possible following surgery.

Small-molecule-regulated gene expression offers the promise of titrating the dose and duration of action of DNA-based therapies. To this end, we show that engineered zinc-finger protein transcription factors (ZFP TFs) can be coupled with a drug-inducible regulatory domain to permit small-molecule control of endogenous gene transcription. We constructed a drug-responsive ZFP TF via the fusion of a ZFP DNA-binding domain (DBD) targeting the human VEGF-A gene and an effector domain containing a truncated progesterone receptor ligand-binding domain linked to the NFkappaB p65 activation domain. Introduction of this engineered ZFP TF into human or murine cells allowed expression of the chromosomal VEGF-A gene to be induced upon addition of mifepristone, a synthetic steroid analog. Mifepristone-dependent VEGF-A induction was rapid, dose-dependent and reversible. Moreover, stable lines expressing the drug-responsive ZFP TF could be maintained in a state of continuous induction for at least 30 days without loss of viability. Potent VEGF-A induction was demonstrated using different engineered ZFP DBDs, thus this approach may represent a general solution to small-molecule regulation of targeted endogenous genes.

Introduction: Acute kidney injury (AKI) is a frequent complication of cardiopulmonary bypass (CPB). The lack of early biomarkers has impaired our ability to intervene in a timely manner. We previously showed in a small cohort of patients that plasma neutrophil gelatinase-associated lipocalin (NGAL), measured using a research enzyme-linked immunosorbent assay, is an early predictive biomarker of AKI after CPB. In this study we tested whether a point-of-care NGAL device can predict AKI after CPB in a larger cohort., Method: First, in a cross-sectional pilot study including 40 plasma samples (NGAL range 60 to 730 ng/ml) and 12 calibration standards (NGAL range 0 to 1,925 ng/ml), NGAL measurements by enzyme-linked immunosorbent assay and by Triage NGAL Device (Biosite Inc., San Diego, CA, USA) were highly correlated (r = 0.94). Second, in a subsequent prospective uncontrolled cohort study, 120 children undergoing CPB were enrolled. Plasma was collected at baseline and at frequent intervals for 24 hours after CPB, and analyzed for NGAL using the Triage(R) NGAL device. The primary outcome was AKI, which was defined as a 50% or greater increase in serum creatinine., Results: AKI developed in 45 patients (37%), but the diagnosis using serum creatinine was delayed by 2 to 3 days after CPB. In contrast, mean plasma NGAL levels increased threefold within 2 hours of CPB and remained significantly elevated for the duration of the study. By multivariate analysis, plasma NGAL at 2 hours after CPB was the most powerful independent predictor of AKI (beta = 0.004, P < 0.0001). For the 2-hour plasma NGAL measurement, the area under the curve was 0.96, sensitivity was 0.84, and specificity was 0.94 for prediction of AKI using a cut-off value of 150 ng/ml. The 2 hour postoperative plasma NGAL levels strongly correlated with change in creatinine (r = 0.46, P < 0.001), duration of AKI (r = 0.57, P < 0.001), and length of hospital stay (r = 0.44, P < 0.001). The 12-hour plasma NGAL strongly correlated with mortality (r = 0.48, P = 0.004) and all measures of morbidity mentioned above., Conclusion: Accurate measurements of plasma NGAL are obtained using the point-of-care Triage(R) NGAL device. Plasma NGAL is an early predictive biomarker of AKI, morbidity, and mortality after pediatric CPB.

We describe an open-chest epicardial approach to transcatheter pulmonary artery stenting in a critically ill infant following heart transplantation. Technical considerations, indications, and feasibility are discussed. This case provides another example of the value of a "hybrid" approach (combining surgery and interventional cardiology) to complex congenital heart disease.

Background: Transplant coronary arteriopathy (TCAD) limits graft survival after heart transplantation in adult and pediatric heart transplant recipients. Intravascular ultrasound (IVUS) provides a highly sensitive technique to detect TCAD. However, its use to determine factors associated with TCAD in pediatric recipients has been limited and its utility in surveillance for symptomatic TCAD in this population is uncertain., Methods: One hundred fifty-eight IVUS studies from 66 patients (27 <1 year and 39 >1 year at time of transplant) were performed 12 to 144 months after transplantation within the routine surveillance for TCAD. Maximal intimal thickness (MIT) and intimal index (II) were measured, and the Stanford classification was utilized to grade overall severity of disease. Mixed repeated-measures linear regression models were used to investigate the main and interaction effects of age at transplant, age at time of study, time since transplant and rejection events., Results: Age at catheterization (p = 0.0002), transplantation at age >12 months (p = 0.014), increasing time after transplantation (p = 0.021) and the combination of late rejection and hemodynamic compromising rejection (p = 0.05) were significantly associated with increasing MIT. Age at catheterization (p = 0.0149), transplantation at age >12 months (p = 0.016), time from transplantation (p = 0.0076) and rejection with hemodynamic compromise (p = 0.01) were significantly associated with increased II. Nine patients developed evidence of severe (Stanford Class 4) TCAD by IVUS, but only 2 (22%) developed symptomatic TCAD, with a median follow-up of 44 months. Four of the 7 patients who developed symptomatic TCAD had no or minimal TCAD (Stanford Class 0 or 1) on a surveillance examination within 18 months of the onset of symptoms., Conclusions: Increasing time after transplantation, recipient age and age at transplantation as well as rejection history, especially rejection with hemodynamic compromise, are associated with the development of TCAD as detected by IVUS in pediatric heart transplant recipients. Severe TCAD detected by IVUS does not often rapidly progress to symptomatic TCAD. Symptomatic TCAD may develop rapidly even in patients with little or no TCAD detected by IVUS.

Nuclear migration and positioning within cells are critical for many developmental processes and are governed by the cytoskeletal network. Although mechanisms of nuclear-cytoskeletal attachment are unclear, growing evidence links a novel family of nuclear envelope (NE) proteins that share a conserved C-terminal SUN (Sad1/UNC-84 homology) domain. Analysis of Caenorhabditis elegans mutants has implicated UNC-84 in actin-mediated nuclear positioning by regulating NE anchoring of a giant actin-binding protein, ANC-1. Here, we report the identification of SUN1 as a lamin A-binding protein in a yeast two-hybrid screen. We demonstrate that SUN1 is an integral membrane protein located at the inner nuclear membrane. While the N-terminal domain of SUN1 is responsible for detergent-resistant association with the nuclear lamina and lamin A binding, lamin A/C expression is not required for SUN1 NE localization. Furthermore, SUN1 does not interact with type B lamins, suggesting that NE localization is ensured by binding to an additional nuclear component(s), most likely chromatin. Importantly, we find that the luminal C-terminal domain of SUN1 interacts with the mammalian ANC-1 homologs nesprins 1 and 2 via their conserved KASH domain. Our data provide evidence of a physical nuclear-cytoskeletal connection that is likely to be a key mechanism in nuclear-cytoplasmic communication and regulation of nuclear position.

Objectives: Neurologic deficits are common after the Norwood procedure for hypoplastic left heart syndrome. Because of the association of deep hypothermic circulatory arrest with adverse neurologic outcome, regional low-flow cerebral perfusion has been used to limit the period of intraoperative brain ischemia. To evaluate the impact of this technique on brain ischemia, we performed serial brain magnetic resonance imaging in a cohort of infants before and after the Norwood operation using regional cerebral perfusion., Methods: Twenty-two term neonates with hypoplastic left heart syndrome were studied with brain magnetic resonance imaging before and at a median of 9.5 days after the Norwood operation. Results were compared with preoperative, intraoperative, and postoperative risk factors to identify predictors of neurologic injury., Results: Preoperative magnetic resonance imaging (n = 22) demonstrated ischemic lesions in 23% of patients. Postoperative magnetic resonance imaging (n = 15) demonstrated new or worsened ischemic lesions in 73% of patients, with periventricular leukomalacia and focal ischemic lesions occurring most commonly. Prolonged low postoperative cerebral oximetry (<45% for >180 minutes) was associated with the development of new or worsened ischemia on postoperative magnetic resonance imaging (P = .029)., Conclusions: Ischemic lesions occur commonly in neonates with hypoplastic left heart syndrome before surgery. Despite the adoption of regional cerebral perfusion, postoperative cerebral ischemic lesions are frequent, occurring in the majority of infants after the Norwood operation. Long-term follow-up is necessary to assess the functional impact of these lesions.

Objectives: Neurologic deficits are common after the Norwood procedure for hypoplastic left heart syndrome. Because of the association of deep hypothermic circulatory arrest with adverse neurologic outcome, regional low-flow cerebral perfusion has been used to limit the period of intraoperative brain ischemia. To evaluate the effect of this technique on brain ischemia, we performed serial brain magnetic resonance imaging in a cohort of infants before and after the Norwood operation using regional cerebral perfusion., Methods: Twenty-two term neonates with hypoplastic left heart syndrome were studied with brain magnetic resonance imaging before and at a median of 9.5 days after the Norwood operation. Results were compared with preoperative, intraoperative, and postoperative risk factors to identify predictors of neurologic injury., Results: Preoperative magnetic resonance imaging (n = 22) demonstrated ischemic lesions in 23% of patients. Postoperative magnetic resonance imaging (n = 15) demonstrated new or worsened ischemic lesions in 73% of patients, with periventricular leukomalacia and focal ischemic lesions occurring most commonly. Prolonged low postoperative cerebral oximetry (<45% for >180 minutes) was associated with the development of new or worsened ischemia on postoperative magnetic resonance imaging (P = .029)., Conclusions: Ischemic lesions occur commonly in neonates with hypoplastic left heart syndrome before surgical intervention. Despite the adoption of regional cerebral perfusion, postoperative cerebral ischemic lesions are frequent, occurring in the majority of infants after the Norwood operation. Long-term follow-up is necessary to assess the functional effect of these lesions.

Aims: The lack of early biomarkers for acute renal failure (ARF) has crippled our ability to launch potentially effective therapeutic measures. We tested the hypothesis that urinary proteomics could identify novel early biomarker patterns for ischemic renal injury., Methods: Sixty patients undergoing cardiopulmonary bypass (CPB) were enrolled. Urine samples obtained at 2 and 6 h post CPB were analyzed by Surface-Enhanced Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (SELDI-TOF-MS). The primary outcome variable was ARF, defined as a 50% or greater increase in serum creatinine., Results: Fifteen patients (25%) developed ARF 2-3 days after CPB. SELDI-TOF-MS analysis of urine from the ARF group at baseline versus at 2 and 6 h post-CPB consistently showed a marked and statistically significant enhancement of protein biomarkers with m/z of 6.4, 28.5, 43 and 66 kDa. The same biomarkers were enhanced when comparing control versus ARF groups at 2 and 6 h post-CPB. The sensitivity and specificity of the 28.5-, 43- and 66-kDa biomarkers for the prediction of ARF at 2 h following CPB was 100%. The receiver operating characteristic curves revealed an area under the curve of 0.98., Conclusion: SELDI-TOF-MS is a novel, non-invasive, sensitive, highly predictive, reproducible, rapid method for the prediction of acute renal injury following CPB., (Copyright 2005 S. Karger AG, Basel.)

The type I interferons, interferon-alpha (IFN-alpha) and interferon-beta (IFN-beta), are situated on the short arm of chromosome 9, specifically 9p21-22. This locus lies very close to an area that is deleted or rearranged in nearly half of all melanomas tested. The identification of 9p rearrangements in both melanoma precursor lesions (dysplastic naevi) and primary lesions has implicated the 9p locus in the early stages of melanoma development. Recent evidence has demonstrated that metastatic melanoma cell lines have a specific loss of IFN-alpha gene expression, a defect that appears to occur at the level of transcription. In this study, we examined the expression of IFN-alpha in cell lines isolated from the various stages of melanoma progression, with a view to determine the prevalence of the IFN-alpha transcription defects exhibited by malignant melanoma, and to assess whether the loss of IFN-alpha expression was particular to a certain stage of melanoma progression. We showed that all the melanoma cell lines tested (n=20) demonstrated an inability to express IFN-alpha, a defect that was reflected in the apparent inactivity of the IFN-alpha promoter. These defects were found to occur in cells isolated from early melanomas, lending support to the hypothesis that IFN-alpha has a role in the aetiology of malignant melanoma.

Although biogeochemistry is an integrative discipline, terrestrial and aquatic subdisciplines have developed somewhat independently of each other. Physical and biological differences between aquatic and terrestrial ecosystems explain this history. In both aquatic and terrestrial biogeochemistry, key questions and concepts arise from a focus on nutrient limitation, ecosystem nutrient retention, and controls of nutrient transformations. Current understanding is captured in conceptual models for different ecosystem types, which share some features and diverge in other ways. Distinctiveness of subdisciplines has been appropriate in some respects and has fostered important advances in theory. On the other hand, lack of integration between aquatic and terrestrial biogeochemistry limits our ability to deal with biogeochemical phenomena across large landscapes in which connections between terrestrial and aquatic elements are important. Separation of the two approaches also has not served attempts to scale up or to estimate fluxes from large areas based on plot measurements. Understanding connectivity between the two system types and scaling up biogeochemical information will rely on coupled hydrologic and ecological models, and may be critical for addressing environmental problems associated with locally, regionally, and globally altered biogeochemical cycles.

The patient with single-ventricle physiology presents a significant challenge to the intensive care team at all stages of management. An integrated approach that applies a working knowledge of cardiac anatomy, cardiopulmonary physiology, and the basic principles of intensive care is essential to guide management for each individual patient. This management requires cooperative and constructive involvement of surgeons, cardiologists, and intensivists, as well as a nursing and respiratory care team experienced in the management of single-ventricle patients. The outcome of each stage of palliation for single-ventricle lesions should continue to improve as new ideas are developed and as older ideas are subjected to rigorous scientific analyses.

Nonlinear models of ecosystem dynamics that incorporate positive feedbacks and multiple, internally reinforced states have considerable explanatory power. However, linear models may be adequate, particularly if ecosystem behaviour is primarily controlled by external processes. In lake ecosystems, internal (mainly biotic) processes are thought to have major impacts on system behaviour, whereas in rivers, external (mainly physical) factors have traditionally been emphasized. We consider the hypothesis that models that exhibit multiple states are useful for understanding the behaviour of lake ecosystems, but not as useful for understanding stream ecosystems. Some of the best-known examples of multiple states come from lake ecosystems. We review some of these examples, and we also describe examples of multiple states in rivers. We conclude that the hypothesis is an oversimplification; the importance of physical forcing in rivers does not eliminate the possibility of internal feedbacks that create multiple states, although in rivers these feedbacks are likely to include physical as well as biotic processes. Nonlinear behaviour in aquatic ecosystems may be more common than current theory indicates.

Abnormalities of diastolic function (DF) precede systolic dysfunction in diabetic cardiomyopathy. Transmitral Doppler flow analysis is the primary method for noninvasively assessing DF. We used model-based Doppler E-wave analysis to evaluate diastolic function differences between normal and diabetic rat hearts. Control rats and those with diabetes underwent echocardiography with analysis by traditional Doppler indexes and by the parameterized diastolic filling (PDF) formalism, generating 3 parameters, x0, c, and k, that uniquely characterize each E-wave. Significant intergroup differences in the E/A ratios (P <.01), isovolumic relaxation times (P <.01), and the modeling parameter c (P <.05) were found. There were no significant differences in shortening fraction, deceleration time, myocardial collagen content, or the parameters x0 and k between diabetic and control rats. These results indicate that differences in diastolic function may be noninvasively quantified and that diabetic hearts may exhibit defects in uncoupling of the contractile apparatus without concomitant increases in chamber stiffness.

Type I interferons constitute an important part of the innate immune response against viral infection. Unlike the expression of interferon (IFN) B gene, the expression of IFNA genes is restricted to the lymphoid cells. Both IFN regulatory factor 3 and 7 (IRF-3 and IRF-7) were suggested to play positive roles in these genes expression. However, their role in the differential expression of individual subtypes of human IFNA genes is unknown. Using various IFNA reporter constructs in transient transfection assay we found that overexpression of IRF-3 in virus infected 2FTGH cells selectively activated IFNA1 VRE, whereas IRF-7 was able to activate IFNA1, A2, and A4. The binding of recombinant IRF-7 and IRF-3 to these VREs correlated with their transcriptional activation. Nuclear proteins from infected and uninfected IRF-7 expressing 2FTGH cells formed multiple DNA-protein complexes with IFNA1 VRE, in which two unique DNA-protein complexes containing IRF-7 were detected. In 2FTGH cells, virus stimulated expression of IFNB gene but none of the IFNA genes. Reconstitution of IRF-7 synthesis in these cells resulted, upon virus infection, in the activation of seven endogenous IFNA genes in which IFNA1 predominated. These studies suggest that IRF-7 is a critical determinant for the induction of IFNA genes in infected cells.

Background: Transplant coronary artery disease (TxCAD) contributes to a large percentage of late morbidity and mortality among adult heart transplant recipients. Intracoronary ultrasound (ICUS) is a sensitive tool in the diagnosis of TxCAD in adult patients and has allowed analysis of factors contributing to disease development. Experience with ICUS in pediatrics, however, has been limited. By using ICUS we sought to determine the overall prevalence of TxCAD in pediatrics and to characterize factors associated with its development in this population., Methods: Eighty-six studies were performed in 51 pediatric patients a median of 3.4 years after heart transplantation. Evaluation included angiography and ICUS in 83 and angiography alone in 3 studies. Donor and recipient characteristics were obtained. The ICUS images were analyzed for intimal thickening and compared with coronary angiograms. The presence of any intimal thickening on ICUS was considered TxCAD. An intimal index and point of maximal intimal thickening (MIT) were measured. Vessel disease was graded 0 to 4 based on these results. Four patients had evidence of vasculopathy by angiography, whereas 32 patients (63%) had evidence of intimal proliferation by ICUS. Grade 2 or greater disease was present in 19 (37%) patients. A positive correlation was found when comparing time from transplant with intimal index and MIT (p < 0.001). No other factors were found to predict the development of disease. The overall prevalence of disease was 74% in patients studied at least 5 years after transplant. Intracoronary ultrasound can be performed safely in pediatric patients. Transplant coronary artery disease is common in infants and children after heart transplantation, although its prevalence appears to be less than in adult recipients at similar time intervals. We found no factor other than time from transplant was associated with development of disease.

Myocardial edema has been associated with impaired ventricular compliance and diastolic filling. To determine the sensitivity of high-frequency (40 MHz) ultrasound to myocardial edema, we employed a model in which myocardial edema was induced by immersion of tissue in isotonic saline. The effect of freezing tissue on edema formation was also evaluated. Rat hearts were arrested at end-diastole and insonified fresh within 15 min of excision (n = 5) or following being frozen for 24 h and thawed (n = 4). Measurements of attenuation, backscatter, tissue thickness and speed of sound were performed at baseline and hourly for 4 h, and compared with direct measurements of myocardial edema. Fresh tissue demonstrated a greater propensity for the development of edema than frozen tissue. Integrated backscatter increased in both tissues, whereas the magnitude and slope of attenuation decreased as edema evolved. We conclude that high-frequency ultrasound sensitively detects myocardial edema, and we propose that the extension of these methods to clinical frequencies may prove useful for monitoring and treatment of cardiac edematous disease states.

The progressive increase in stiffening of the myocardium associated with the aging process and abetted by comorbid conditions such as diabetes may be linked to an excessive number of collagen cross links within the myocardial extra-cellular matrix. To determine whether ultrasound can delineate changes in the physical properties of heart tissue undergoing cross linking, the authors employed a model in which increased cross linking was induced by treating rat myocardial tissue with specific chemical fixatives. Rat hearts (n=5 each group) were arrested at end-diastole, insonified (30 to 50 MHz) fresh within a few minutes of excision in a phosphate buffered solution, placed in a fixative (10% formalin or 2.5% glutaraldehyde) and insonified at 30-minute intervals thereafter for 24 hours. Ultrasonic attenuation increased in tissues cross linked with formalin (maximal change: 27.2+/-3.4 dB/cm) and glutaraldehyde (maximal change: 40.2+/-5.6 dB/cm) over a 24-hour period. The frequency dependence of the attenuation coefficient increased as a function of the extent of collagen cross links in formalin (maximal change: 0.8+/-0.3 dB/cm-MHz) and glutaraldehyde (maximal change: 0.9+/-0.6 dB/cm-MHz). This study represents the first time that the precise time course of myocardial protein cross linking in situ has been characterized by using real time monitoring, and the physiologic effect has been delineated on microscopic material properties.

This review recounts recent advances in the understanding and treatment of the processes that cause pulmonary hypertension in infancy and childhood. New discoveries have begun to unveil connections between the basic physiological mechanisms responsible for the regulation of pulmonary vascular tone and the abnormal responses of the pulmonary vasculature in a variety of disease conditions. These discoveries raise hope for new therapeutic interventions that may improve the high mortality and morbidity of both children and adults with pulmonary vascular disease. In the meantime, treatment efforts continue to be focused on the relief of pulmonary vasoconstriction with inhaled nitric oxide and intravenous prostacyclin in the short term and oral calcium channel blockers as the mainstay of long-term therapy. Lung transplantation often remains as the only viable option for continued survival when the pulmonary vascular disease is progressive.

Background: Perioperative myocardial injury is a major determinant of postoperative cardiac dysfunction for congenital heart disease, but its assessment during this period is difficult. The objective of this study was to determine the suitability of using postoperative serum concentrations of cardiac troponin I (cTnI) for this purpose., Methods: Cardiac troponin I levels were measured serially in the serum of patients undergoing uncomplicated repairs of atrial septal defect (n = 23), ventricular septal defect (n = 16) or tetralogy of Fallot (n = 16). The concentrations were correlated with intraoperative parameters (cardiopulmonary bypass time, aortic cross-clamp time, and cardiac bypass temperature), and postoperative parameters (magnitude of inotropic support, duration of intubation, and postoperative intensive care and hospital stay)., Results: Postoperative absolute cTnI levels were lesion specific, with a pattern of increase and decrease similar for each lesion. For the total cohort, significant correlations between postoperative cTnI levels at all times (r = 0.43 to 0.83, p < 0.05) until 72 hours were noted for all parameters, except for cardiac bypass temperature. When evaluated as individual procedure groups, no significant relationships were noted in the atrial septal defect group, whereas postoperative cTnI levels were more strongly correlated with all intraoperative and postoperative parameters in the ventricular septal defect group than in the tetralogy of Fallot group., Conclusions: This study suggests that cTnI values immediately after operation reflect the extent of myocardial damage from both incisional injury and intraoperative factors. Cardiac tropinin I levels in the first hours after operation for congenital heart disease are a potentially useful prognostic indicator for difficulty of recovery.

Type-I interferons are encoded by a multigene family, the major members of which are at least 13 IFN A subtypes and a single IFN B gene. IFNs A and B are induced in response to similar stimuli, such as virus infection and double-stranded RNA, but in different cell types: the induction of IFN A is almost exclusively restricted to cells of lymphoid origin, while IFN B has been found to be induced in a variety of cell types including fibroblasts. The virus-responsive enhancer element in the promoter region of IFN A family members is largely responsible for the differential expression of individual subtypes in responsive cells. In this paper we describe experiments which address the issue of the differential expression of IFN A and IFN B in different cell types. We show that IFN-beta is induced in a variety of cells of different origin, while not all of these are able to secrete IFN-alpha. By transfection of reporter gene constructs comprising the virus-responsive enhancer from the IFN A1 and IFN B genes, we show that this differential response is mediated at the level of transcription via these control elements. More detailed analysis of the function of these regions identifies specific sequences within the IFN A1 virus response element that has an inhibitory effect on expression in cells that are normally inducible, and is also implicated in the overall suppression of IFN A induction in non-inducible cells.

This paper addresses the role of transcriptional regulation in the determination of the levels of expression of different interferon-alpha subtypes secreted from Namalwa cells following infection with Sendai virus. Using RT-PCR to determine the relative abundance of mRNA species coding for the various subtypes, we found a general correlation with corresponding protein levels, indicative of a role for transcriptional control in the determination of levels of individual subtypes. We have used reporter gene constructs to compare the inducibility of the virus-response elements from the IFNA1, A2, A4, and A14 subtype genes cloned upstream of a secreted alkaline phosphatase gene. The inducibility of these reporter gene constructs broadly correlated with the relative mRNA abundances in both transiently and stably transfected Namalwa cells. During work with stable cell lines, we found that G418, the drug used for the selection of transfected cells, inhibited the induction of interferon by both Sendai virus and double-stranded RNA. This inhibition was reversible when G418 was removed from the medium 24 h before the addition of virus.

Transactivation of the herpes simplex virus (HSV) immediate-early (IE) genes is dependent upon the formation of a complex between the viral protein Vmw65 and the cellular transactivation factor Oct-1. Differentiation of the proliferating ND7 neuronal cell to a nondividing phenotype results in a large fall in the amount of Oct-1 to a level characteristic of nondividing neuronal cells but does not dramatically affect the level of IE gene expression following infection or the ability of Vmw65 to transactivate the IE promoter in transfection experiments. This suggests that the low levels of Oct-1 in nonproliferating neuronal cells do not play a key role in the failure of IE gene expression following initial infection of these cells, which is an essential step in the establishment of latent infections with HSV.

The upstream regulatory region (URR) of the tumorigenic human papillomaviruses HPV 16 and 18 contains an octamer binding site which is located adjacent to a binding site for the ubiquitous transcription factor NFI. The octamer site binds both the constitutively expressed transcription factor Oct-1 and a novel cervical octamer binding protein. In contrast the URR of the non-tumorigenic viruses HPV6 and HPV11 lacks the octamer binding site although the adjacent NFI site is conserved. Inactivation of the octamer binding site results in a higher level of gene expression in cells which contain only Oct-1 and a lower level in cells containing the cervical octamer binding protein indicating that that whilst Oct-1 binding reduces promoter activity, the cervical protein increases it. In agreement with this, over-expression of Oct-1 reduces the level of gene activity directed by this region of the HPV 16/18 URR and inhibits its activation by NFI whilst having no effect on the corresponding region of the HPV 6/11 URR. The significance of these effects is discussed in terms of the cervical-specific activity of the HPV16/18 URR and its role in HPV-mediated transformation.

POU proteins are a class of homeobox-containing transcription factors that regulate tissue-specific gene expression and influence cell differentiation and function. We have investigated the possible role of such factors in mediating the actions of nerve growth factor (NGF) on sensory neurons. NGF has been found to have differential effects on the levels of three POU protein transcription factors that are expressed in adult rat sensory neurons. A sensory neuron octamer-binding protein with the properties of the transcription factor Oct-2 is up-regulated 3-4-fold by NGF, as measured by mobility shift assays using nuclear extracts from adult rat dorsal root ganglion neurons grown in the presence or absence of NGF. Quantitation of Oct-2 mRNA by polymerase chain reaction amplification of RNA from such cells shows a parallel increase in Oct-2 mRNA levels. In contrast, the levels of mRNA encoding the ubiquitous POU protein Oct-1 or the neuron-specific POU protein Brn-3, also present in sensory neurons, are unaffected by NGF. These observations suggest a role for Oct-2 in mediating transcriptional effects induced by NGF. In particular, as Oct-2 is known to inhibit herpes simplex virus immediate-early gene expression in neuronal cells, these findings provide a mechanism for the known action of NGF in the maintenance of latent herpes virus infections in sensory neurons.

Immortalized cell lines derived from sensory neurons are relatively non-permissive for lytic infection with herpes simplex virus (HSV) and fail to transcribe the viral immediate-early genes following infection. Treatment of these cells with agents which raise the intra-cellular level of cyclic AMP results in increased activity of the IE1 gene which contains a cyclic AMP response element within its promoter and produces a consequent increase in permissivity for HSV infection. The significance of these effects for the regulation of HSV infection of neuronal cells are discussed in the light of the finding that cyclic AMP treatment can reactivate latent HSV infections.

Treatment of Daudi cells with alpha-interferon (alpha-IFN) results in a considerable decrease in the levels of the octamer-binding DNA replication/transcription factors Oct-1 and Oct-2 and specifically inhibits gene expression by octamer-containing promoters. The inhibitory effect on octamer-binding proteins also occurs after culturing cells with phorbol 12-myristate 13-acetate but it does not occur following alpha-IFN treatment of an alpha-IFN-resistant variant of the Daudi cell line or of HeLa cells. We discuss the potential role of the decreased levels of octamer-binding proteins in the inhibition of cell proliferation.

Transcription of herpes simplex virus (HSV) immediate-early (IE) genes does not occur in sensory neurons latently infected with the virus or following infection of neuronal cell lines. In neuronal cell lines this inability results from the weak activity of the viral IE promoters, which is caused by a neuron-specific repressor factor that binds specifically to the TAATGARAT motif in these promoters and to related octamer elements. Cells expressing this repressor contain an additional octamer-binding protein that is absent from permissive cells. We identify this factor as the lymphocyte- and neuron-specific octamer-binding protein Oct-2 and show that Oct-2 mRNA is also present in dorsal root ganglion neurons, the natural site of HSV latency in vivo. Moreover, artificially elevated expression of Oct-2 can repress the IE promoter. The potential role of Oct-2 in the initiation and maintenance of in vivo latent infection with HSV is discussed.