[ECG-gated chest CT angiography: value for atypical chest pain evaluation]

PURPOSE: The aim of this study was to evaluate ECG-gated whole chest CTA as a routine triage tool for patients with acute chest pain. MATERIAL AND METHODS: Whole chest CTA with retrospective ECG-gating was performed in 30 patients with acute atypical chest pain. The ten main segments of the coronary arteries, the pulmonary arteries, the aorta, and the myocardium (function, morphology) were independently analyzed by a resident and two senior radiologists. The inter-observer agreement between resident and senior radiologists was calculated. A final diagnosis was determined by consensus. RESULTS: Thirty patients were included. The coronary artery segments, myocardium and pulmonary arteries were considered analyzable in 84%, 90% and 97% of cases respectively. A final diagnosis for the cause of pain was retained in 19 patients: significant coronary artery stenosis (5), pulmonary embolus (5), aortic dissection (1), hypokinetic cardiomyopathy (2), lung parenchymal abnormalities (5), and hiatus hernia (1). Inter-observer agreement ranged from 0.76 to 1 between senior radiologists and from 0.76 to 1 between resident and senior radiologists. The average time of image interpretation ranged from 14 to 15 minutes. CONCLUSION: ECG-gated whole chest CT angiography appears as a promising tool for the evaluation of acute chest pain. Combined evaluation of appearance and function of the myocardium can reveal additional interesting information.

β1- and β3- voltage-gated sodium channel subunits modulate cell surface expression and glycosylation of Nav1.7 in HEK293 cells

Voltage-gated sodium channels (Navs) are glycoproteins composed of a pore-forming α-subunit and associated β-subunits that regulate Nav α-subunit plasma membrane density and biophysical properties. Glycosylation of the Nav α-subunit also directly affects Navs gating. β-subunits and glycosylation thus comodulate Nav α-subunit gating. We hypothesized that β-subunits could directly influence α-subunit glycosylation. Whole-cell patch clamp of HEK293 cells revealed that both β1- and β3-subunits coexpression shifted V ½ of steady-state activation and inactivation and increased Nav1.7-mediated I Na density. Biotinylation of cell surface proteins, combined with the use of deglycosydases, confirmed that Nav1.7 α-subunits exist in multiple glycosylated states. The α-subunit intracellular fraction was found in a core-glycosylated state, migrating at ~250 kDa. At the plasma membrane, in addition to the core-glycosylated form, a fully glycosylated form of Nav1.7 (~280 kDa) was observed. This higher band shifted to an intermediate band (~260 kDa) when β1-subunits were coexpressed, suggesting that the β1-subunit promotes an alternative glycosylated form of Nav1.7. Furthermore, the β1-subunit increased the expression of this alternative glycosylated form and the β3-subunit increased the expression of the core-glycosylated form of Nav1.7. This study describes a novel role for β1- and β3-subunits in the modulation of Nav1.7 α-subunit glycosylation and cell surface expression.

Dysregulation of voltage-gated sodium channels by ubiquitin ligase NEDD4-2 in neuropathic pain

Peripheral neuropathic pain is a disabling condition resulting from nerve injury. It is characterized by the dysregulation of voltage-gated sodium channels (Navs) expressed in dorsal root ganglion (DRG) sensory neurons. The mechanisms underlying the altered expression of Na(v)s remain unknown. This study investigated the role of the E3 ubiquitin ligase NEDD4-2, which is known to ubiquitylate Navs, in the pathogenesis of neuropathic pain in mice. The spared nerve injury (SNI) model of traumatic nerve injury-induced neuropathic pain was used, and an Na(v)1.7-specific inhibitor, ProTxII, allowed the isolation of Na(v)1.7-mediated currents. SNI decreased NEDD4-2 expression in DRG cells and increased the amplitude of Na(v)1.7 and Na(v)1.8 currents. The redistribution of Na(v)1.7 channels toward peripheral axons was also observed. Similar changes were observed in the nociceptive DRG neurons of Nedd4L knockout mice (SNS-Nedd4L(-/-)). SNS-Nedd4L(-/-) mice exhibited thermal hypersensitivity and an enhanced second pain phase after formalin injection. Restoration of NEDD4-2 expression in DRG neurons using recombinant adenoassociated virus (rAAV2/6) not only reduced Na(v)1.7 and Na(v)1.8 current amplitudes, but also alleviated SNI-induced mechanical allodynia. These findings demonstrate that NEDD4-2 is a potent posttranslational regulator of Na(v)s and that downregulation of NEDD4-2 leads to the hyperexcitability of DRG neurons and contributes to the genesis of pathological pain.

Ubiquitylation of voltage-gated sodium channels.

Ion channel proteins are regulated by different types of posttranslational modifications. The focus of this review is the regulation of voltage-gated sodium channels (Navs) upon their ubiquitylation. The amiloride-sensitive epithelial sodium channel (ENaC) was the first ion channel shown to be regulated upon ubiquitylation. This modification results from the binding of ubiquitin ligase from the Nedd4 family to a protein-protein interaction domain, known as the PY motif, in the ENaC subunits. Many of the Navs have similar PY motifs, which have been demonstrated to be targets of Nedd4-dependent ubiquitylation, tagging them for internalization from the cell surface. The role of Nedd4-dependent regulation of the Nav membrane density in physiology and disease remains poorly understood. Two recent studies have provided evidence that Nedd4-2 is downregulated in dorsal root ganglion (DRG) neurons in both rat and mouse models of nerve injury-induced neuropathic pain. Using two different mouse models, one with a specific knockout of Nedd4-2 in sensory neurons and another where Nedd4-2 was overexpressed with the use of viral vectors, it was demonstrated that the neuropathy-linked neuronal hyperexcitability was the result of Nav1.7 and Nav1.8 overexpression due to Nedd4-2 downregulation. These studies provided the first in vivo evidence of the role of Nedd4-2-dependent regulation of Nav channels in a disease state. This ubiquitylation pathway may be involved in the development of symptoms and diseases linked to Nav-dependent hyperexcitability, such as pain, cardiac arrhythmias, epilepsy, migraine, and myotonias.

p.L1612P, a Novel Voltage-gated Sodium Channel Nav1.7 Mutation Inducing a Cold Sensitive Paroxysmal Extreme Pain Disorder

BACKGROUND Mutations in the SCN9A gene cause chronic pain and pain insensitivity syndromes. We aimed to study clinical, genetic, and electrophysiological features of paroxysmal extreme pain disorder (PEPD) caused by a novel SCN9A mutation. METHODS Description of a 4-generation family suffering from PEPD with clinical, genetic and electrophysiological studies including patch clamp experiments assessing response to drug and temperature. RESULTS The family was clinically comparable to those reported previously with the exception of a favorable effect of cold exposure and a lack of drug efficacy including with carbamazepine, a proposed treatment for PEPD. A novel p.L1612P mutation in the Nav1.7 voltage-gated sodium channel was found in the four affected family members tested. Electrophysiologically the mutation substantially depolarized the steady-state inactivation curve (V1/2 from -61.8 ± 4.5 mV to -30.9 ± 2.2 mV, n = 4 and 7, P < 0.001), significantly increased ramp current (from 1.8% to 3.4%, n = 10 and 12) and shortened recovery from inactivation (from 7.2 ± 5.6 ms to 2.2 ± 1.5 ms, n = 11 and 10). However, there was no persistent current. Cold exposure reduced peak current and prolonged recovery from inactivation in wild-type and mutated channels. Amitriptyline only slightly corrected the steady-state inactivation shift of the mutated channel, which is consistent with the lack of clinical benefit. CONCLUSIONS The novel p.L1612P Nav1.7 mutation expands the PEPD spectrum with a unique combination of clinical symptoms and electrophysiological properties. Symptoms are partially responsive to temperature but not to drug therapy. In vitro trials of sodium channel blockers or temperature dependence might help predict treatment efficacy in PEPD.

ATP-gated ionotropic P2X7 receptor controls follicular T helper cell numbers in Peyer's patches to promote host-microbiota mutualism.

Microbial colonization of the gut induces the development of gut-associated lymphoid tissue (GALT). The molecular mechanisms that regulate GALT function and result in gut-commensal homeostasis are poorly defined. T follicular helper (Tfh) cells in Peyer's patches (PPs) promote high-affinity IgA responses. Here we found that the ATP-gated ionotropic P2X7 receptor controls Tfh cell numbers in PPs. Lack of P2X7 in Tfh cells enhanced germinal center reactions and high-affinity IgA secretion and binding to commensals. The ensuing depletion of mucosal bacteria resulted in reduced systemic translocation of microbial components, lowering B1 cell stimulation and serum IgM concentrations. Mice lacking P2X7 had increased susceptibility to polymicrobial sepsis, which was rescued by Tfh cell depletion or administration of purified IgM. Thus, regulation of Tfh cells by P2X7 activity is important for mucosal colonization, which in turn results in IgM serum concentrations necessary to protect the host from bacteremia.

Cellular hyper-excitability caused by mutations that alter the activation process of voltage-gated sodium channels.

Voltage-gated sodium channels (Nav) are widely expressed as macro-molecular complexes in both excitable and non-excitable tissues. In excitable tissues, the upstroke of the action potential is the result of the passage of a large and rapid influx of sodium ions through these channels. NaV dysfunction has been associated with an increasingly wide range of neurological, muscular and cardiac disorders. The purpose of this review is to summarize the recently identified sodium channel mutations that are linked to hyper-excitability phenotypes and associated with the alteration of the activation process of voltage gated sodium channels. Indeed, several clinical manifestations that demonstrate an alteration of tissue excitability were recently shown to be strongly associated with the presence of mutations that affect the activation process of the Nav. These emerging genotype-phenotype correlations have expanded the clinical spectrum of sodium channelopathies to include disorders which feature a hyper-excitability phenotype that may or may not be associated with a cardiomyopathy. The p.I141V mutation in SCN4A and SCN5A, as well as its homologous p.I136V mutation in SCN9A, are interesting examples of mutations that have been linked to inherited hyperexcitability myotonia, exercise-induced polymorphic ventricular arrhythmias and erythromelalgia, respectively. Regardless of which sodium channel isoform is investigated, the substitution of the isoleucine to valine in the locus 141 induces similar modifications in the biophysical properties of the Nav by shifting the voltage-dependence of steady state activation toward more negative potentials.

Post-translational modifications of voltage-gated sodium channels in chronic pain syndromes.

In the peripheral sensory nervous system the neuronal expression of voltage-gated sodium channels (Navs) is very important for the transmission of nociceptive information since they give rise to the upstroke of the action potential (AP). Navs are composed of nine different isoforms with distinct biophysical properties. Studying the mutations associated with the increase or absence of pain sensitivity in humans, as well as other expression studies, have highlighted Nav1.7, Nav1.8, and Nav1.9 as being the most important contributors to the control of nociceptive neuronal electrogenesis. Modulating their expression and/or function can impact the shape of the AP and consequently modify nociceptive transmission, a process that is observed in persistent pain conditions. Post-translational modification (PTM) of Navs is a well-known process that modifies their expression and function. In chronic pain syndromes, the release of inflammatory molecules into the direct environment of dorsal root ganglia (DRG) sensory neurons leads to an abnormal activation of enzymes that induce Navs PTM. The addition of small molecules, i.e., peptides, phosphoryl groups, ubiquitin moieties and/or carbohydrates, can modify the function of Navs in two different ways: via direct physical interference with Nav gating, or via the control of Nav trafficking. Both mechanisms have a profound impact on neuronal excitability. In this review we will discuss the role of Protein Kinase A, B, and C, Mitogen Activated Protein Kinases and Ca++/Calmodulin-dependent Kinase II in peripheral chronic pain syndromes. We will also discuss more recent findings that the ubiquitination of Nav1.7 by Nedd4-2 and the effect of methylglyoxal on Nav1.8 are also implicated in the development of experimental neuropathic pain. We will address the potential roles of other PTMs in chronic pain and highlight the need for further investigation of PTMs of Navs in order to develop new pharmacological tools to alleviate pain.

Prospective gated 64 slice computed tomography in assessment of coronary artery disease: A single center experience and relationship of body mass index to outcomes

Coronary artery disease (CAD) is the most common cause of morbidity and mortality in the United States. While Coronary Angiography (CA) is the gold standard test to investigate coronary artery disease, Prospective gated-64 Slice Computed Tomography (Prosp-64CT) is a new non-invasive technology that uses the 64Slice computed tomography (64CT) with electrocardiographic gating to investigate coronary artery disease. The aim of the current study was to investigate the role of Body Mass Index (BMI) as a factor affecting occurrence of CA after a Prosp-64CT, as well as the quality of the Prosp-64CT. Demographic and clinical characteristics of the study population were described. A secondary analysis of data on patients who underwent a Prosp-64CT for evaluation of coronary artery disease was performed. Seventy seven patients who underwent Prosp-64CT for evaluation for coronary artery disease were included. Fifteen patients were excluded because they had missing data regarding BMI, quality of the Prosp-64CT or CA. Thus, a total of 62 patients were included in the final analysis. The mean age was 56.2 years. The mean BMI was 31.3 kg/m 2. Eight (13%) patients underwent a CA within one month of Prosp-64CT. Eight (13%) patients had a poor quality Prosp-64CT. There was significant association of higher BMI as a factor for occurrence of CA post Prosp-64CT (P<0.05). There was a trend, but no statistical significance was observed for the association of being obese and occurrence of CA (P=0.06). BMI, as well as obesity, were not found to be significantly associated with poor quality of Prosp-64CT (P=0.19 and P=0.76, respectively). In conclusion, BMI was significantly associated with occurrence of CA within one month of Prosp-64CT. Thus, in patients with a higher BMI, diagnostic investigation with both tests could be avoided; rather, only a CA could be performed. However, the relationship of BMI to quality of Prosp-64CT needs to be further investigated since the sample size of the current study was small.^

Verification of the clinical implementation of the respiratory gated beam delivery technique with synchrotron-based proton irradiation

The clinical advantage for protons over conventional high-energy x-rays stems from their unique depth-dose distribution, which delivers essentially no dose beyond the end of range. In order to achieve it, accurate localization of the tumor volume relative to the proton beam is necessary. For cases where the tumor moves with respiration, the resultant dose distribution is sensitive to such motion. One way to reduce uncertainty caused by respiratory motion is to use gated beam delivery. The main goal of this dissertation is to evaluate the respiratory gating technique in both passive scattering and scanning delivery mode. Our hypothesis for the study was that optimization of the parameters of synchrotron operation and respiratory gating can lead to greater efficiency and accuracy of respiratory gating for all modes of synchrotron-based proton treatment delivery. The hypothesis is tested in two specific aims. The specific aim #1 is to assess the efficiency of respiratory-gated proton beam delivery and optimize the synchrotron operations for the gated proton therapy. A simulation study was performed and introduced an efficient synchrotron operation pattern, called variable Tcyc. In addition, the simulation study estimated the efficiency in the respiratory gated scanning beam delivery mode as well. The specific aim #2 is to assess the accuracy of beam delivery in respiratory-gated proton therapy. The simulation study was extended to the passive scattering mode to estimate the quality of pulsed beam delivery to the residual motion for several synchrotron operation patterns with the gating technique. The results showed that variable Tcyc operation can offer good reproducible beam delivery to the residual motion at a certain phase of the motion. For respiratory gated scanning beam delivery, the impact of motion on the dose distributions by scanned beams was investigated by measurement. The results showed the threshold for motion for a variety of scan patterns and the proper number of paintings for normal and respiratory gated beam deliveries. The results of specific aims 1 and 2 provided supporting data for implementation of the respiratory gating beam delivery technique into both passive and scanning modes and the validation of the hypothesis.