TDP-43 represses cryptic exon inclusion in the FTD-ALS gene UNC13A.

A hallmark pathological feature of the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is the depletion of RNA-binding protein TDP-43 from the nucleus of neurons in the brain and spinal cord1. A major function of TDP-43 is as a repressor of cryptic exon inclusion during RNA splicing2-4. Single nucleotide polymorphisms in UNC13A are among the strongest hits associated with FTD and ALS in human genome-wide association studies5,6, but how those variants increase risk for disease is unknown. Here we show that TDP-43 represses a cryptic exon-splicing event in UNC13A. Loss of TDP-43 from the nucleus in human brain, neuronal cell lines and motor neurons derived from induced pluripotent stem cells resulted in the inclusion of a cryptic exon in UNC13A mRNA and reduced UNC13A protein expression. The top variants associated with FTD or ALS risk in humans are located in the intron harbouring the cryptic exon, and we show that they increase UNC13A cryptic exon splicing in the face of TDP-43 dysfunction. Together, our data provide a direct functional link between one of the strongest genetic risk factors for FTD and ALS (UNC13A genetic variants), and loss of TDP-43 function.

Gabapentin receptor alpha2delta-1 is a neuronal thrombospondin receptor responsible for excitatory CNS synaptogenesis.

Synapses are asymmetric cellular adhesions that are critical for nervous system development and function, but the mechanisms that induce their formation are not well understood. We have previously identified thrombospondin as an astrocyte-secreted protein that promotes central nervous system (CNS) synaptogenesis. Here, we identify the neuronal thrombospondin receptor involved in CNS synapse formation as alpha2delta-1, the receptor for the anti-epileptic and analgesic drug gabapentin. We show that the VWF-A domain of alpha2delta-1 interacts with the epidermal growth factor-like repeats common to all thrombospondins. alpha2delta-1 overexpression increases synaptogenesis in vitro and in vivo and is required postsynaptically for thrombospondin- and astrocyte-induced synapse formation in vitro. Gabapentin antagonizes thrombospondin binding to alpha2delta-1 and powerfully inhibits excitatory synapse formation in vitro and in vivo. These findings identify alpha2delta-1 as a receptor involved in excitatory synapse formation and suggest that gabapentin may function therapeutically by blocking new synapse formation.

Discordant clinical features of identical hypertrophic cardiomyopathy twins.

Hypertrophic cardiomyopathy (HCM) is a disease of heart muscle, which affects ∼1 in 500 individuals and is characterized by increased left ventricular wall thickness. While HCM is caused by pathogenic variants in any one of eight sarcomere protein genes, clinical expression varies considerably, even among patients with the same pathogenic variant. To determine whether background genetic variation or environmental factors drive these differences, we studied disease progression in 11 pairs of monozygotic HCM twins. The twin pairs were followed for 5 to 14 y, and left ventricular wall thickness, left atrial diameter, and left ventricular ejection fraction were collected from echocardiograms at various time points. All nine twin pairs with sarcomere protein gene variants and two with unknown disease etiologies had discordant morphologic features of the heart, demonstrating the influence of nonhereditable factors on clinical expression of HCM. Whole genome sequencing analysis of the six monozygotic twins with discordant HCM phenotypes did not reveal notable somatic genetic variants that might explain their clinical differences. Discordant cardiac morphology of identical twins highlights a significant role for epigenetics and environment in HCM disease progression.

Genetic Inhibition of APOL1 Pore-Forming Function Prevents APOL1-Mediated Kidney Disease.

SIGNIFICANCE STATEMENT: African Americans are at increased risk of CKD in part due to high-risk (HR) variants in the apolipoprotein L1 ( APOL1 ) gene, termed G1/G2. A different APOL1 variant, p.N264K , reduced the risk of CKD and ESKD among carriers of APOL1 HR variants to levels comparable with individuals with APOL1 low-risk variants in an analysis of 121,492 participants of African ancestry from the Million Veteran Program (MVP). Functional genetic studies in cell models showed that APOL1 p.N264K blocked APOL1 pore-forming function and ion channel conduction and reduced toxicity of APOL1 HR mutations. Pharmacologic inhibitors that mimic this mutation blocking APOL1 -mediated pore formation may be able to prevent and/or treat APOL1 -associated kidney disease. BACKGROUND: African Americans are at increased risk for nondiabetic CKD in part due to HR variants in the APOL1 gene. METHODS: We tested whether a different APOL1 variant, p.N264K , modified the association between APOL1 HR genotypes (two copies of G1/G2) and CKD in a cross-sectional analysis of 121,492 participants of African ancestry from the MVP. We replicated our findings in the Vanderbilt University Biobank ( n =14,386) and National Institutes of Health All of Us ( n =14,704). Primary outcome was CKD and secondary outcome was ESKD among nondiabetic patients. Primary analysis compared APOL1 HR genotypes with and without p.N264K . Secondary analyses included APOL1 low-risk genotypes and tested for interaction. In MVP, we performed sequential logistic regression models adjusting for demographics, comorbidities, medications, and ten principal components of ancestry. Functional genomic studies expressed APOL1 HR variants with and without APOL1 p.N264K in cell models. RESULTS: In the MVP cohort, 15,604 (12.8%) had two APOL1 HR variants, of which 582 (0.5%) also had APOL1 p.N264K . In MVP, 18,831 (15%) had CKD, 4177 (3%) had ESKD, and 34% had diabetes. MVP APOL1 HR, without p.N264K , was associated with increased odds of CKD (odds ratio [OR], 1.72; 95% confidence interval [CI], 1.60 to 1.85) and ESKD (OR, 3.94; 95% CI, 3.52 to 4.41). In MVP, APOL1 p.N264K mitigated the renal risk of APOL1 HR, in CKD (OR, 0.43; 95% CI, 0.28 to 0.65) and ESKD (OR, 0.19; CI 0.07 to 0.51). In the replication cohorts meta-analysis, APOL1 p.N264K mitigated the renal risk of APOL1 HR in CKD (OR, 0.40; 95% CI, 0.18 to 0.92) and ESKD (OR, 0.19; 95% CI, 0.05 to 0.79). In the mechanistic studies, APOL1 p.N264K blocked APOL1 pore-forming function and ion channel conduction and reduced toxicity of APOL1 HR variants. CONCLUSIONS: APOL1 p.N264K is associated with reduced risk of CKD and ESKD among carriers of APOL1 HR to levels comparable with individuals with APOL1 low-risk genotypes.

Deciphering the super relaxed state of human ß-cardiac myosin and the mode of action of mavacamten from myosin molecules to muscle fibers.

Mutations in ß-cardiac myosin, the predominant motor protein for human heart contraction, can alter power output and cause cardiomyopathy. However, measurements of the intrinsic force, velocity, and ATPase activity of myosin have not provided a consistent mechanism to link mutations to muscle pathology. An alternative model posits that mutations in myosin affect the stability of a sequestered, super relaxed state (SRX) of the protein with very slow ATP hydrolysis and thereby change the number of myosin heads accessible to actin. Here we show that purified human ß-cardiac myosin exists partly in an SRX and may in part correspond to a folded-back conformation of myosin heads observed in muscle fibers around the thick filament backbone. Mutations that cause hypertrophic cardiomyopathy destabilize this state, while the small molecule mavacamten promotes it. These findings provide a biochemical and structural link between the genetics and physiology of cardiomyopathy with implications for therapeutic strategies.

Proteins and small molecules for cellular regenerative medicine.

Regenerative medicine seeks to understand tissue development and homeostasis and build on that knowledge to enhance regeneration of injured tissues. By replenishing lost functional tissues and cells, regenerative medicine could change the treatment paradigm for a broad range of degenerative and ischemic diseases. Multipotent cells hold promise as potential building blocks for regenerating lost tissues, but successful tissue regeneration will depend on comprehensive control of multipotent cells-differentiation into a target cell type, delivery to a desired tissue, and integration into a durable functional structure. At each step of this process, proteins and small molecules provide essential signals and, in some cases, may themselves act as effective therapies. Identifying these signals is thus a fundamental goal of regenerative medicine. In this review we discuss current progress using proteins and small molecules to regulate tissue regeneration, both in combination with cellular therapies and as monotherapy.

Characterization of WWOX expression and function in canine mast cell tumors and malignant mast cell lines.

BACKGROUND: The WW domain-containing oxidoreductase (WWOX) tumor suppressor gene is frequently lost in a variety of solid and hematopoietic malignancies in humans. Dysregulation of WWOX has been implicated as playing a key role in tumor cell survival, DNA damage repair, and genomic stability. The purpose of this study was to characterize WWOX expression in spontaneous canine mast cell tumors (MCTs) and malignant cell lines and investigate the potential contribution of WWOX loss on malignant mast cell behavior. METHODS/RESULTS: WWOX expression is decreased in primary canine MCTs and malignant mast cell lines compared to normal canine bone marrow-cultured mast cells. In transformed canine mastocytoma cell lines, overexpression of WWOX or WWOX knockdown had no effect on mast cell viability. Inhibition of WWOX enhanced clonogenic survival following treatment with ionizing radiation in the C2 mast cell line. Lastly, immunohistochemistry for WWOX was performed using a canine MCT tissue microarray, demonstrating that WWOX staining intensity and percent of cells staining for WWOX is decreased in high-grade MCTs compared to low-grade MCTs. CONCLUSIONS: These data suggest that WWOX expression is attenuated or lost in primary canine MCTs and malignant mast cell lines. Given the observed increase in clonogenic survival in WWOX-deficient C2 mast cells treated with ionizing radiation, further investigation of WWOX and its role in mediating the DNA damage response in malignant mast cells is warranted.

Percutaneous microwave ablation of solitary presumptive pulmonary metastases in two dogs with appendicular osteosarcoma.

OBJECTIVE: To describe percutaneous microwave ablation (MWA) of presumptive pulmonary metastases and the outcome of two dogs. ANIMALS: Two dogs with pulmonary lesions after treatment of spontaneously occurring appendicular osteosarcoma. STUDY DESIGN: Preliminary prospective clinical study. METHODS: Two large-breed dogs were referred from tertiary veterinary hospitals 146 and 217 days after limb amputation to pursue MWA as an alternative therapy to metastasectomy. Both dogs had been receiving chemotherapy protocols at their respective referral centers. RESULTS: A novel percutaneous approach for MWA with ultrasonographic or computed tomographic (CT) guidance was successfully performed. The only complications consisted of pneumothoraxes, requiring treatment in one dog. In the weeks after their procedures, both dogs were reported to do well at home. Dog 1 died and dog 2 was euthanized 82 and 19 days, respectively, after their MWA of confirmed (dog 1) or presumed (dog 2) metastatic disease. CONCLUSION: Percutaneous MWA of pulmonary nodules was technically feasible in two dogs without major complications. CLINICAL SIGNIFICANCE: Percutaneous MWA may provide a minimally invasive option for treatment of osteosarcoma pulmonary metastases. Additional studies are required to evaluate the benefits of MWA on survival and confirm histologic cell death within pulmonary neoplastic lesions.

Genotype and Lifetime Burden of Disease in Hypertrophic Cardiomyopathy: Insights from the Sarcomeric Human Cardiomyopathy Registry (SHaRe).

Background: A better understanding of the factors that contribute to heterogeneous outcomes and lifetime disease burden in hypertrophic cardiomyopathy (HCM) is critically needed to improve patient management and outcomes. The Sarcomeric Human Cardiomyopathy Registry (SHaRe) was established to provide the scale of data required to address these issues, aggregating longitudinal datasets curated by eight international HCM specialty centers. Methods: Data on 4591 HCM patients (2763 genotyped), followed for a mean of 5.4±6.9 years (24,791 patient-years; median [interquartile range] 2.9 [0.3-7.9] years) were analyzed regarding cardiac arrest, cardiac transplantation, appropriate implantable cardioverter-defibrillator (ICD) therapy, all-cause death, atrial fibrillation, stroke, New York Heart Association Functional Class III/IV symptoms (all comprising the overall composite endpoint), and left ventricular ejection fraction (LVEF)<35%. Outcomes were analyzed individually and as composite endpoints. Results: Median age of diagnosis was 45.8 [30.9-58.1] years and 37% of patients were female. Age of diagnosis and sarcomere mutation status were predictive of outcomes. Patients <40 years old at diagnosis had a 77% [95% confidence interval: 72%, 80%] cumulative incidence of the overall composite outcome by age 60, compared to 32% [29%, 36%] by age 70 for patients diagnosed >60 years. Young HCM patients (20-29 years) had 4-fold higher mortality than the general United States population at a similar age. Patients with pathogenic/likely pathogenic sarcomere mutations had two-fold greater risk for adverse outcomes compared to patients without mutations; sarcomere variants of uncertain significance were associated with intermediate risk. Heart failure and atrial fibrillation were the most prevalent adverse events, although typically not emerging for several years after diagnosis. Ventricular arrhythmias occurred in 32% [23%, 40%] of patients <40 years at diagnosis, but in 1% [1%, 2%] >60 years. Conclusions: The cumulative burden of HCM is substantial and dominated by heart failure and atrial fibrillation occurring many years following diagnosis. Young age of diagnosis and the presence of a sarcomere mutation are powerful predictors of adverse outcomes. These findings highlight the need for close surveillance throughout life, and the need to develop disease-modifying therapies.

Multidimensional structure-function relationships in human ß-cardiac myosin from population-scale genetic variation.

Myosin motors are the fundamental force-generating elements of muscle contraction. Variation in the human ß-cardiac myosin heavy chain gene (MYH7) can lead to hypertrophic cardiomyopathy (HCM), a heritable disease characterized by cardiac hypertrophy, heart failure, and sudden cardiac death. How specific myosin variants alter motor function or clinical expression of disease remains incompletely understood. Here, we combine structural models of myosin from multiple stages of its chemomechanical cycle, exome sequencing data from two population cohorts of 60,706 and 42,930 individuals, and genetic and phenotypic data from 2,913 patients with HCM to identify regions of disease enrichment within ß-cardiac myosin. We first developed computational models of the human ß-cardiac myosin protein before and after the myosin power stroke. Then, using a spatial scan statistic modified to analyze genetic variation in protein 3D space, we found significant enrichment of disease-associated variants in the converter, a kinetic domain that transduces force from the catalytic domain to the lever arm to accomplish the power stroke. Focusing our analysis on surface-exposed residues, we identified a larger region significantly enriched for disease-associated variants that contains both the converter domain and residues on a single flat surface on the myosin head described as the myosin mesa. Notably, patients with HCM with variants in the enriched regions have earlier disease onset than patients who have HCM with variants elsewhere. Our study provides a model for integrating protein structure, large-scale genetic sequencing, and detailed phenotypic data to reveal insight into time-shifted protein structures and genetic disease.

Canine urinary bladder transitional cell carcinoma tumor volume is dependent on imaging modality and measurement technique.

Transitional cell carcinoma is the most common cancer of the canine urinary tract. The inconsistent appearance of transitional cell carcinoma in patients introduces error if applying mathematic models for extrapolating total tumor volume from linear measurements. Reliable techniques to assess tumor size are important for monitoring treatment response. A method comparison study was performed comparing four techniques for calculating tumor volume were compared: (1 and 2) contoured tracing of tumor margins using serial computed tomography (CT) images using pre-(1) and postintravenous (2) contrast medium studies, (3) longest three linear dimensions using CT, and (4) longest three linear dimensions on abdominal ultrasound. Volumes of the transitional cell carcinoma tumor calculated by CT tracing techniques were significantly smaller than volumes calculated with an ellipsoid mathematic model using the linear measurements (P < 0.01). Intravenous contrast medium did not significantly change the volumes calculated from tracing tumor margins on CT for observer B; however, volumes differed for observer A. The volumes extrapolated from linear measurements using CT and ultrasound did not differ significantly. The interobserver reliability was highest for the precontrast CT contoured technique and was lowest using the ultrasound linear technique. Tumor volumes differed significantly between techniques of contoured tracing of the tumor margins on serial CT images compared to calculation of tumor volume from linear dimensions. The calculated volume of a transitional cell carcinoma depends upon the technique used. Characterizing the response of urinary bladder transitional cell carcinoma tumor size to therapy differs based on the method and modality used.

A pilot study of toceranib/vinblastine therapy for canine transitional cell carcinoma.

BACKGROUND: Effective therapies for transitional cell carcinoma (TCC) are limited, with objective response rates to most chemotherapeutic regimens below 20%. The purpose of this study was to investigate the biologic activity of combined toceranib phosphate and vinblastine chemotherapy for treatment of TCC. A secondary objective was to compare the utility of Computed Tomography (CT) and abdominal ultrasound (AUS) in tumor response assessments. RESULTS: Dogs with TCC received vinblastine at 1.6 mg/m2 every 2 weeks and toceranib at 2.5-2.75 mg/kg on Monday/Wednesday/Friday. Tumor monitoring was achieved through CT and AUS. Five patients completed the 16-week study. Based on AUS assessments, 3 dogs experienced biologic response to therapy including partial responses (PR, n = 2) and stable disease (SD, n = 1). Based on CT, 5 dogs experienced a biologic response (n = 2 PR, n = 3 SD). Both imaging modalities (ultrasound and CT) were found to provide repeatable measurements between operators, however agreement between operator measurements was greater when CT images were used to assess tumor size. CONCLUSIONS: The combination of toceranib and vinblastine did not result in improved response rates. While agreement in tumor volume assessments between both AUS and CT were excellent between operators, this did not extend to assessment of tumor response. The higher rate of concordance between operators when assessing response to treatment with CT suggests that CT should be considered for future clinical trials involving canine bladder TCC to improve the accuracy and repeatability of tumor measurement. The data suggest that response to therapy as assessed by AUS or CT do not predict duration of clinical response.

COMPARISON OF COMPUTED TOMOGRAPHY AND ABDOMINAL RADIOGRAPHY FOR DETECTION OF CANINE MECHANICAL INTESTINAL OBSTRUCTION.

Vomiting, often caused by mechanical intestinal obstruction, is common in dogs. Equivocal radiographic signs often necessitate repeat radiographs or additional imaging procedures. For our prospective, case-controlled, accuracy study, we hypothesized the following: (1) using computed tomography (CT), radiologists will be more sensitive and specific for detecting mechanical intestinal obstruction and recommending surgery compared to using radiographs; and (2) using measurements, radiologists will be more sensitive and specific using radiographs or CT for detecting mechanical intestinal obstruction and recommending surgery. Twenty dogs had abdominal radiographs and abdominal CT. Seventeen dogs had abdominal surgery and three dogs were not obstructed based on clinical follow-up. Confidence levels (five-point scale) of three experienced radiologists for mechanical intestinal obstruction and recommending surgery were recorded before and after making selected measurements. Eight dogs had surgically confirmed mechanical intestinal obstruction, and 12 dogs did not have obstruction. For detecting mechanical intestinal obstruction, CT was more sensitive (95.8% vs. 79.2%) and specific (80.6% vs. 69.4%) compared to radiographs, but the difference was not statistically significant. For recommending surgery, radiography was more sensitive (91.7% vs. 83.3%) and specific (83.3% vs. 72.2%) than using CT, but differences were not statistically significant. We reported objective CT measurements for predicting small mechanical intestinal obstruction. By incorporating these objective data, the diagnosis of mechanical intestinal obstruction changed in five of 120 instances (radiographs and CT). In no instance (0/120), did the objective data change the recommendation for surgery. Using CT or abdominal radiographs for the detection of canine mechanical intestinal obstruction is sensitive and specific when evaluated by experienced veterinary radiologists.

Small-molecule inhibition of glycogen synthase 1 for the treatment of Pompe disease and other glycogen storage disorders.

Glycogen synthase 1 (GYS1), the rate-limiting enzyme in muscle glycogen synthesis, plays a central role in energy homeostasis and has been proposed as a therapeutic target in multiple glycogen storage diseases. Despite decades of investigation, there are no known potent, selective small-molecule inhibitors of this enzyme. Here, we report the preclinical characterization of MZ-101, a small molecule that potently inhibits GYS1 in vitro and in vivo without inhibiting GYS2, a related isoform essential for synthesizing liver glycogen. Chronic treatment with MZ-101 depleted muscle glycogen and was well tolerated in mice. Pompe disease, a glycogen storage disease caused by mutations in acid α glucosidase (GAA), results in pathological accumulation of glycogen and consequent autophagolysosomal abnormalities, metabolic dysregulation, and muscle atrophy. Enzyme replacement therapy (ERT) with recombinant GAA is the only approved treatment for Pompe disease, but it requires frequent infusions, and efficacy is limited by suboptimal skeletal muscle distribution. In a mouse model of Pompe disease, chronic oral administration of MZ-101 alone reduced glycogen buildup in skeletal muscle with comparable efficacy to ERT. In addition, treatment with MZ-101 in combination with ERT had an additive effect and could normalize muscle glycogen concentrations. Biochemical, metabolomic, and transcriptomic analyses of muscle tissue demonstrated that lowering of glycogen concentrations with MZ-101, alone or in combination with ERT, corrected the cellular pathology in this mouse model. These data suggest that substrate reduction therapy with GYS1 inhibition may be a promising therapeutic approach for Pompe disease and other glycogen storage diseases.

Severe polycystic liver disease in a cat.

Case summary: Ductal plate malformations (DPMs) are poorly documented in the veterinary literature, particularly those of the polycystic liver disease (PCLD) phenotype. A 13-year-old female spayed cat presented with progressive icterus, abdominal distension, weight loss and elevated liver enzymes. Initial empirical treatment consisting of amoxicillin/clavulanate, ursodiol and later prednisolone was attempted; however, clinical signs progressed. On abdominal ultrasound, numerous large hepatic cystic masses were noted, characterized by an anechoic center with a heterogeneous, hyperechoic wall. A post-mortem examination confirmed numerous hepatic cysts, the larger of which resulted in hemorrhage and subsequent hemoabdomen. Histologically, these cysts were determined to be of biliary origin, and a diagnosis of PCLD was assigned. Relevance and novel information: Herein, we present a detailed report of clinical, gross and histologic findings in a cat clinically affected by PCLD. This case demonstrates that cysts present in this congenital disease can ultimately lead to hepatobiliary malfunction and clinical decline via marked expansion of cysts, compression of the liver and hemoabdomen from cyst rupture. DPMs, specifically PCLD, should be considered in cats presenting with multifocal large hepatic cysts.

Management of acute right ventricular failure in the intensive care unit.

Acute failure of the right ventricle is a common challenge in the intensive care unit that is associated with significant morbidity and mortality. Although often a complication of left ventricular failure, right ventricular failure is a distinct clinical entity, both in terms of its hemodynamic abnormalities and response to treatment. Effective management of right ventricular failure must consider the unique properties of the right ventricle and the pulmonary circulation, and their response to common pharmacologic and mechanical interventions. In this review, we present a contemporary approach to patients with acute failure of the right ventricle including strategies for mechanical ventilation, hemodynamic management, and mechanical circulatory support.

Utility of cardiac MRI to diagnose myocardial ischemia and fibrosis in dogs with cardiomegaly secondary to myxomatous mitral valve disease.

OBJECTIVE: To assess whether cardiac MRI or various biomarkers can be used to detect myocardial ischemia and fibrosis in dogs with cardiomegaly secondary to myxomatous mitral valve disease (MMVD). ANIMALS: 6 dogs with cardiomegaly secondary to naturally occurring stage B2 MMVD being treated only with pimobendan with or without enalapril and 6 control dogs with no cardiac disease. All dogs were ≥ 5 years old with no systemic illness. PROCEDURES: Serum cardiac troponin I and concentrations were measured, and dogs were anesthetized for cardiac MRI with ECG-triggered acquisition of native T1- and T2-weighted images. Gadolinium contrast was administered to evaluate myocardial perfusion and late gadolinium enhancement (LGE). Mean T1 and T2 values and regions of LGE were measured with dedicated software. Extracellular volume (ECV) was estimated on the basis of Hct and T1 values of myocardium and surrounding blood. Subjective analysis for myocardial perfusion deficits was performed. RESULTS: Dogs with MMVD had significantly (P = .013) higher cardiac troponin I concentrations than control dogs, but galectin-3 concentrations did not differ (P = .08) between groups. Myocardial fibrosis was detected in 4 dogs with MMVD and 3 control dogs; no dogs had obvious myocardial perfusion deficits. Native T1 and T2 values, postcontrast T1 values, and ECV values were not significantly different between groups (all P > .3). CLINICAL RELEVANCE: Results suggest that some dogs with cardiomegaly secondary to MMVD may not have clinically relevant myocardial fibrosis.

The tumor suppressor eIF3e mediates calcium-dependent internalization of the L-type calcium channel CaV1.2.

Voltage-gated calcium channels (VGCCs) convert electrical activity into calcium (Ca2+) signals that regulate cellular excitability, differentiation, and connectivity. The magnitude and kinetics of Ca2+ signals depend on the number of VGCCs at the plasma membrane, but little is known about the regulation of VGCC surface expression. We report that electrical activity causes internalization of the L-type Ca2+ channel (LTC) CaV1.2 and that this is mediated by binding to the tumor suppressor eIF3e/Int6 (eukaryotic initiation factor 3 subunit e). Using total internal reflection microscopy, we identify a population of CaV1.2 containing endosomes whose rapid trafficking is strongly regulated by Ca2+. We define a domain in the II-III loop of CaV1.2 that binds eIF3e and is essential for the activity dependence of both channel internalization and endosomal trafficking. These findings provide a mechanism for activity-dependent internalization and trafficking of CaV1.2 and provide a tantalizing link between Ca2+ homeostasis and a mammalian oncogene.

Intra- and interobserver variability of board-certified veterinary radiologists and veterinary general practitioners for pulmonary nodule detection in standard and inverted display mode images of digital thoracic radiographs of dogs.

OBJECTIVE: To determine intra- and interobserver variability of 2 veterinary radiologists and 2 veterinary general practitioners for detection of pulmonary nodules in standard and inverted (reversed grayscale) displays of digital thoracic radiographs of dogs. DESIGN: Evaluation study. SAMPLE: 114 sets of 3-view (right lateral, left lateral, and ventrodorsal or dorsoventral views) digital thoracic radiographs from 114 dogs. PROCEDURES: 2 experienced board-certified veterinary radiologists and 2 experienced veterinary general practitioners individually evaluated 114 randomized sets of radiographs. Pulmonary nodules were present in radiographs of 60 of 114 dogs. Each reviewer examined all images in standard or inverted display mode and scored nodule detection on a confidence scale of 1 to 5. After ≥ 2 months, the same individuals evaluated the same images in the remaining display mode. Intraobserver agreement for each display mode was determined via a κ statistic; results between the 2 groups of reviewers were compared via receiver operator curve analysis. RESULTS: There was no significant intraobserver variability in pulmonary nodule detection between the 2 display modes. Detection accuracy for board-certified radiologists was significantly greater than that of veterinary general practitioners for both display modes. Near-perfect intraobserver agreement was detected between the 2 display modes for board-certified radiologists, whereas moderate to slight intraobserver agreement was detected for the veterinary general practitioners. CONCLUSIONS AND CLINICAL RELEVANCE: Detection of pulmonary nodules in digital thoracic radiographs was comparable, whether a standard or inverted mode was used for evaluations. However, the board-certified radiologists had greater detection accuracy than did veterinary general practitioners.