Animesh Tandon

Regulation of SMN Protein Stability

ABSTRACT Spinal muscular atrophy (SMA) is caused by mutations of the survival of motor neuron (SMN1) gene and deficiency of full-length SMN protein (FL-SMN). All SMA patients retain one or more copies of the SMN2 gene, but the principal protein product of SMN2 lacks exon 7 (SMNΔ7) and is unable to compensate for a deficiency of FL-SMN. SMN is known to oligomerize and form a multimeric protein complex; however, the mechanisms regulating stability and degradation of FL-SMN and SMNΔ7 proteins have been largely unexplored. Using pulse-chase analysis, we characterized SMN protein turnover and confirmed that SMN was ubiquitinated and degraded by the ubiquitin proteasome system (UPS). The SMNΔ7 protein had a twofold shorter half-life than FL-SMN in cells despite similar intrinsic rates of turnover by the UPS in a cell-free assay. Mutations that inhibited SMN oligomerization and complex formation reduced the FL-SMN half-life. Furthermore, recruitment of SMN into large macromolecular complexes as well as increased association with several Gemin proteins was regulated in part by protein kinase A. Together, our data indicate that SMN protein stability is modulated by complex formation. Promotion of the SMN complex formation may be an important novel therapeutic strategy for SMA.

Risk factors for aortic valve disease in bicuspid aortic valve: A family‐based study

Bicuspid aortic valve (BAV) is the most common cardiovascular malformation and is a risk factor for aortic valve disease (AVD). AVD typically manifests later in life, and the majority of cases have BAV. The purpose of this study was to identify risk factors for AVD in individuals with BAV. Families enriched for BAV were identified in a pediatric population, and echocardiography was performed on all family members. AVD was identified as stenosis and/or insufficiency, and BAV morphology was defined as right–left (RL), right–non (RN) or indeterminate. Heritability (h2) of AVD and BAV morphology was estimated using variance components analysis (SOLAR). To assess AVD risk over time, we used Generalized Estimating Equations methodology (SAS) adjusting for age and gender. A total of 1,128 individuals from 226 families were evaluated. BAV was identified in 281 individuals (25%), and AVD was identified in 167 (59%) individuals with BAV. Previously, we identified a high heritability for BAV (h2 = 0.89 ± 0.06, P < 0.00001), but the heritability of AVD in the present study (0.07 ± 0.17, P = 0.33) was low. AVD was significantly associated with BAV morphology (P = 0.0027) and age (P = 0.0068). Children with RN BAV and adults with RL BAV were more likely to develop AVD. BAV is determined largely by genetic effects, but the phenotypic variability of AVD is primarily determined by nongenetic factors. BAV morphology may have predictive value for the time course of AVD.

Temporal summation of heat pain in humans: Evidence supporting thalamocortical modulation

&NA; Noxious cutaneous contact heat stimuli (48 °C) are perceived as increasingly painful when the stimulus duration is extended from 5 to 10 s, reflecting the temporal summation of central neuronal activity mediating heat pain. However, the sensation of increasing heat pain disappears, reaching a plateau as stimulus duration increases from 10 to 20 s. We used functional magnetic resonance imaging (fMRI) in 10 healthy subjects to determine if active central mechanisms could contribute to this psychophysical plateau. During heat pain durations ranging from 5 to 20 s, activation intensities in the bilateral orbitofrontal cortices and the activation volume in the left primary (S1) somatosensory cortex correlated only with perceived stimulus intensity and not with stimulus duration. Activation volumes increased with both stimulus duration and perceived intensity in the left lateral thalamus, posterior insula, inferior parietal cortex, and hippocampus. In contrast, during the psychophysical plateau, both the intensity and volume of thalamic and cortical activations in the right medial thalamus, right posterior insula, and left secondary (S2) somatosensory cortex continued to increase with stimulus duration but not with perceived stimulus intensity. Activation volumes in the left medial and right lateral thalamus, and the bilateral mid‐anterior cingulate, left orbitofrontal, and right S2 cortices also increased only with stimulus duration. The increased activity of specific thalamic and cortical structures as stimulus duration, but not perceived intensity, increases is consistent with the recruitment of a thalamocortical mechanism that participates in the modulation of pain‐related cortical responses and the temporal summation of heat pain.

Rhodobacter capsulatus nifA1 Promoter: High-GC −10 Regions in High-GC Bacteria and the Basis for Their Transcription

It was previously shown that the Rhodobacter capsulatus NtrC enhancer-binding protein activates the R. capsulatus housekeeping RNA polymerase but not the Escherichia coli RNA polymerase at the nifA1 promoter. We have tested the hypothesis that this activity is due to the high G+C content of the -10 sequence. A comparative analysis of R. capsulatus and other alpha-proteobacterial promoters with known transcription start sites suggests that the G+C content of the -10 region is higher than that for E. coli. Both in vivo and in vitro results obtained with nifA1 promoters with -10 and/or -35 variations are reported here. A major conclusion of this study is that alpha-proteobacteria have evolved a promiscuous sigma factor and core RNA polymerase that can transcribe promoters with high-GC -10 regions in addition to the classic E. coli Pribnow box. To facilitate studies of R. capsulatus transcription, we cloned and overexpressed all of the RNA polymerase subunits in E. coli, and these were reconstituted in vitro to form an active, recombinant R. capsulatus RNA polymerase with properties mimicking those of the natural polymerase. Thus, no additional factors from R. capsulatus are necessary for the recognition of high-GC promoters or for activation by R. capsulatus NtrC. The addition of R. capsulatus sigma(70) to the E. coli core RNA polymerase or the use of -10 promoter mutants did not facilitate R. capsulatus NtrC activation of the nifA1 promoter by the E. coli RNA polymerase. Thus, an additional barrier to activation by R. capsulatus NtrC exists, probably a lack of the proper R. capsulatus NtrC-E. coli RNA polymerase (protein-protein) interaction(s).

Cardiogenic shock as the initial presentation of neonatal systemic hypertension

Neonatal systemic hypertension is an underappreciated etiology of cardiac failure. We present a series of three neonates who presented in cardiogenic shock secondary to severe hypertension, recognized after initial resuscitation efforts. Although the underlying etiology of the hypertension varied, all three patients had improved hemodynamics after their blood pressure was controlled. These cases suggest that neonates presenting in cardiogenic shock with hypertension, and without structural heart disease, may benefit from a thorough renal evaluation and institution of anti-hypertensive therapy.

The effect of right ventricular compression on cardiac function in pediatric pectus excavatum

Background Pectus excavatum can cause right ventricular (RV) dysfunction due to extrinsic compression. Assessment of ventricular function by echocardiography is often suboptimal due to technical limitations. We utilized cardiac magnetic resonance imaging (CMR) to assess pectus severity and RV systolic function in relation to the site of RV compression in a large pediatric pectus cohort. Methods All CMR studies performed for clinical evaluation of pediatric pectus excavatum at our institution were retrospectively reviewed. CMR analysis included evaluation of Haller index, left ventricular (LV) and RV ejection fractions (EF), and indexed LV and RV end-diastolic and end-systolic volumes. The site of maximal compression of the right ventricle (no compression, atrioventricular (AV) groove, or free wall) was assessed. The relationships of age, BSA, Haller index, and ln(Haller) to LVEF, RVEF, and indexed RV end-diastolic volume were assessed using linear regression. The relationships of patient gender and RV compression site to LVEF, RVEF, and indexed RV end-diastolic volume were evaluated using ANOVA with post-hoc Tukey comparisons. Results We analyzed CMR studies from 197 patients with pectus excavatum (163 males, 34 females, age 2.67-24.9 yrs). 28 patients (14.2%) had RVEF<50%. 75 patients had no RV compression, 104 had compression of the RV free wall, and 18 had compression of the AV groove. The Haller index, ln(Haller), and age did not show linear relationships to LVEF, RVEF, or indexed RV end-diastolic volume. The LVEF was significantly lower in patients

Co-occurring Duchenne muscular dystrophy and hypertrophic cardiomyopathy in an adult with atypical cardiac phenotype

We present the case of a 29-year-old man with mutation-positive Duchenne muscular dystrophy and mutation-positive hypertrophic cardiomyopathy. His cardiac phenotype has characteristics of both disorders; he manifests sub-epicardial left ventricular free wall late gadolinium enhancement that is consistent with Duchenne cardiomyopathy, as well as asymmetric ventricular septal hypertrophy, hyperdynamic left ventricular systolic function, and septal mid-myocardial late gadolinium enhancement, which are characteristic of hypertrophic cardiomyopathy.

A LONGITUDINAL EXAMINATION OF CARDIAC DYSFUNCTION IN PATIENTS WITH DUCHENNE AND BECKER MUSCULAR DYSTROPHY VIA CMRI

Duchenne and Becker muscular dystrophy (D/BMD) typically exhibit cardiac dysfunction, but the temporal evolution is not well understood. Evaluation of potential therapies for cardiac dysfunction in D/BMD patients requires accurate, quantitative, longitudinal data; therefore, our study examined the