Self-reported chronic conditions and COVID-19 public health measures among Canadian adults: an analysis of the Canadian longitudinal study on aging.

OBJECTIVES: During the COVID-19 pandemic, public health measures were used to reduce the spread of COVID-19; it is unknown whether people with chronic conditions differentially adhered to public health measures. The objectives of this study were to evaluate the association between chronic conditions and adherence and to explore effect modification by sex, age, and income. STUDY DESIGN: An analysis of data from the Canadian Longitudinal Study on Aging COVID-19 Questionnaires (from April to September 2020) was conducted among middle-aged and older adults aged 50-96 years (n = 28,086). METHODS: Self-reported chronic conditions included lung disease, diabetes, heart disease, cancer, obesity, anxiety, and depression. Multinomial logistic regression was used to estimate odds ratios (ORs) and 95% confidence intervals (CIs) for the association between chronic conditions and low, medium, and high levels of adherence. Effect modification was evaluated using statistical interaction and stratification. RESULTS: Most people (n = 17,435; 62%) had at least one chronic condition, and 2866 (10%) had three to seven chronic conditions. Among those with high adherence to public health measures, 69% had one or more chronic condition (n = 2266). Having three to seven chronic conditions, compared with none, was associated with higher adherence to public health measures (OR: 2.14; 95% CI: 1.12-1.42). Higher adherence was also noted across chronic conditions, for example, those with diabetes had higher adherence (OR: 1.72; 95% CI: 1.53-1.93). There was limited evidence of effect modification by sex, age, or income. CONCLUSIONS: Canadians with chronic conditions were more likely to adhere to public health measures; however, future research is needed to understand whether adherence helped to prevent adverse COVID-19 outcomes and if adherence had unintended consequences.

Predictive Value of Cardiac CTA, Cardiac MRI, and Transthoracic Echocardiography for Cardioembolic Stroke Recurrence.

BACKGROUND. Transthoracic echocardiography (TTE) is the standard of care for initial evaluation of patients with suspected cardioembolic stroke. Although TTE is useful for assessing certain sources of cardiac emboli, its diagnostic capability is limited in the detection of other sources, including left atrial thrombus and aortic plaques. OBJECTIVE. The purpose of this article was to investigate sensitivity, specificity, and predictive value of cardiac CTA (CCTA), cardiac MRI (CMRI), and TTE for recurrence in patients with suspected cardioembolic stroke. METHODS. We retrospectively included 151 patients with suspected cardioembolic stroke who underwent TTE and either CMRI (n = 75) or CCTA (n = 76) between January 2013 and May 2017. We evaluated for the presence of left atrial thrombus, left ventricular thrombus, vulnerable aortic plaque, cardiac tumors, and valvular vegetation as causes of cardioembolic stroke. The end point was stroke recurrence. Sensitivity, specificity, PPV, and NPV for recurrent stroke were calculated; the diagnostic accuracy of CMRI, CCTA, and TTE was compared between and within groups using AUC. RESULTS. Twelve and 14 recurrent strokes occurred in the CCTA and CMRI groups, respectively. Sensitivity, specificity, PPV, and NPV were 33.3%, 93.7%, 50.0%, and 88.2% for CCTA; 14.3%, 80.3%, 14.3%, and 80.3% for CMRI; 14.3%, 83.6%, 16.7%, and 80.9% for TTE in the CMRI group; and 8.3%, 93.7%, 20.0%, and 84.5% for TTE in the CCTA group. Accuracy was not different (p > .05) between CCTA (AUC = 0.63; 95% CI, 0.49-0.77), CMRI (0.53; 95% CI, 0.42-0.63), TTE in the CMRI group (0.51; 95% CI, 0.40-0.61), and TTE in the CCTA group (0.51; 95% CI, 0.42-0.59). In the CCTA group, atrial and ventricular thrombus were detected by CCTA in three patients and TTE in one patient; in the CMRI group, thrombus was detected by CMRI in one patient and TTE in two patients. CONCLUSION. CCTA, CMRI, and TTE showed comparably high specificity and NPV for cardioembolic stroke recurrence. CCTA and CMRI may be valid alternatives to TTE. CCTA may be preferred given potentially better detection of atrial and ventricular thrombus. CLINICAL IMPACT. CCTA and CMRI have similar clinical performance as TTE for predicting cardioembolic stroke recurrence. This observation may be especially important when TTE provides equivocal findings.

A fully automated software platform for structural mitral valve analysis.

OBJECTIVE: To evaluate a novel fully automated mitral valve analysis software platform for cardiac computer tomography angiography (CCTA)-based structural heart therapy procedure planning. METHODS: The study included 52 patients (25 women; mean age, 66.9 ± 12.4 years) who had undergone CCTA prior to transcatheter mitral valve replacement (TMVR) or surgical mitral valve intervention (replacement or repair). Therapeutically relevant mitral valve annulus parameters (projected area, circumference, trigone-to-trigone (T-T) distance, anterior-posterior (AP) diameter, and anterolateral-posteromedial (AL-PM) diameter) were measured. Results of the fully automated mitral valve analysis software platform with and without manual adjustments were compared with the reference standard of a user-driven measurement program (3mensio, Pie Medical Imaging). Measurements were compared between the fully automated software, both with and without manual adjustment, and the user-driven program using intraclass correlation coefficients (ICC). A secondary analysis included the time to obtain all measurements. RESULTS: Fully automated measurements showed a good to excellent agreement (circumference, ICC = 0.70; projected area, ICC = 0.81; T-T distance, ICC = 0.64; AP, ICC = 0.62; and AL-PM diameter, ICC = 0.78) compared with the user-driven analysis. There was an excellent agreement between fully automated measurement with manual adjustments and user-driven analysis regarding circumference (ICC = 0.91), projected area (ICC = 0.93), T-T distance (ICC = 0.80), AP (ICC = 0.78), and AL-PM diameter (ICC = 0.79). The time required for mitral valve analysis was significantly lower using the fully automated software with manual adjustments compared with the standard assessment (134.4 ± 36.4 s vs. 304.3 ± 77.7 s) (p < 0.01). CONCLUSION: The fully automated mitral valve analysis software, when combined with manual adjustments, demonstrated a strong correlation compared with the user-driven software while reducing the total time required for measurement. KEY POINTS: • The novel software platform allows for a fully automated analysis of mitral valve structures. • An excellent agreement was found between the fully automated measurement with manual adjustments and the user-driven analysis. • The software showed quicker measurement time compared with the standard analysis of the mitral valve.

Comparison of Artificial Intelligence-Based Fully Automatic Chest CT Emphysema Quantification to Pulmonary Function Testing.

OBJECTIVE. The purpose of this study was to evaluate an artificial intelligence (AI)-based prototype algorithm for fully automated quantification of emphysema on chest CT compared with pulmonary function testing (spirometry). MATERIALS AND METHODS. A total of 141 patients (72 women, mean age ± SD of 66.46 ± 9.7 years [range, 23-86 years]; 69 men, mean age of 66.72 ± 11.4 years [range, 27-91 years]) who underwent both chest CT acquisition and spirometry within 6 months were retrospectively included. The spirometry-based Tiffeneau index (TI; calculated as the ratio of forced expiratory volume in the first second to forced vital capacity) was used to measure emphysema severity; a value less than 0.7 was considered to indicate airway obstruction. Segmentation of the lung based on two different reconstruction methods was carried out by using a deep convolution image-to-image network. This multilayer convolutional neural network was combined with multilevel feature chaining and depth monitoring. To discriminate the output of the network from ground truth, an adversarial network was used during training. Emphysema was quantified using spatial filtering and attenuation-based thresholds. Emphysema quantification and TI were compared using the Spearman correlation coefficient. RESULTS. The mean TI for all patients was 0.57 ± 0.13. The mean percentages of emphysema using reconstruction methods 1 and 2 were 9.96% ± 11.87% and 8.04% ± 10.32%, respectively. AI-based emphysema quantification showed very strong correlation with TI (reconstruction method 1, ρ = -0.86; reconstruction method 2, ρ = -0.85; both p < 0.0001), indicating that AI-based emphysema quantification meaningfully reflects clinical pulmonary physiology. CONCLUSION. AI-based, fully automated emphysema quantification shows good correlation with TI, potentially contributing to an image-based diagnosis and quantification of emphysema severity.

Nonbinary quantification technique accounting for myocardial infarct heterogeneity: Feasibility of applying percent infarct mapping in patients.

BACKGROUND: Binary threshold-based quantification techniques ignore myocardial infarct (MI) heterogeneity, yielding substantial misquantification of MI. PURPOSE: To assess the technical feasibility of MI quantification using percent infarct mapping (PIM), a prototype nonbinary algorithm, in patients with suspected MI. STUDY TYPE: Prospective cohort POPULATION: Patients (n = 171) with suspected MI referred for cardiac MRI. FIELD STRENGTH/SEQUENCE: Inversion recovery balanced steady-state free-precession for late gadolinium enhancement (LGE) and modified Look-Locker inversion recovery (MOLLI) T1 -mapping on a 1.5T system. ASSESSMENT: Infarct volume (IV) and infarct fraction (IF) were quantified by two observers based on manual delineation, binary approaches (2-5 standard deviations [SD] and full-width at half-maximum [FWHM] thresholds) in LGE images, and by applying the PIM algorithm in T1 and LGE images (PIMT1 ; PIMLGE ). STATISTICAL TEST: IV and IF were analyzed using repeated measures analysis of variance (ANOVA). Agreement between the approaches was determined with Bland-Altman analysis. Interobserver agreement was assessed by intraclass correlation coefficient (ICC) analysis. RESULTS: MI was observed in 89 (54.9%) patients, and 185 (38%) short-axis slices. IF with 2, 3, 4, 5SDs and FWHM techniques were 15.7 ± 6.6, 13.4 ± 5.6, 11.6 ± 5.0, 10.8 ± 5.2, and 10.0 ± 5.2%, respectively. The 5SD and FWHM techniques had the best agreement with manual IF (9.9 ± 4.8%) determination (bias 1.0 and 0.2%; P = 0.1426 and P = 0.8094, respectively). The 2SD and 3SD algorithms significantly overestimated manual IF (9.9 ± 4.8%; both P < 0.0001). PIMLGE measured significantly lower IF (7.8 ± 3.7%) compared to manual values (P < 0.0001). PIMLGE , however, showed the best agreement with the PIMT1 reference (7.6 ± 3.6%, P = 0.3156). Interobserver agreement was rated good to excellent for IV (ICCs between 0.727-0.820) and fair to good for IF (0.589-0.736). DATA CONCLUSION: The application of the PIMLGE technique for MI quantification in patients is feasible. PIMLGE , with its ability to account for voxelwise MI content, provides significantly smaller IF than any thresholding technique and shows excellent agreement with the T1 -based reference. LEVEL OF EVIDENCE: 2 Technical Efficacy: Stage 1 J. Magn. Reson. Imaging 2018.

Non-contrast-enhanced magnetic resonance angiography: a reliable clinical tool for evaluating transplant renal artery stenosis.

PURPOSE: To evaluate image quality of non-contrast-enhanced magnetic resonance angiography (MRA) and compare transplant renal artery stenosis (TRAS) seen by non-contrast-enhanced MRA with digital subtraction angiography (DSA) as the gold standard. MATERIALS AND METHODS: 330 patients receiving 369 non-contrast-enhanced MRA examinations from July 2014 to June 2017 were included. Thirty patients received at least two MRA examinations. Image quality was independently assessed by two radiologists. Inter-observer agreement was analyzed. Transplant renal artery anatomy and complications were evaluated and compared with DSA. If possible, accuracy was calculated on a per-artery basis. RESULTS: Good or excellent image quality was found in 95.4 % (352/369) of examinations with good inter-observer agreement (K=0.760). Twenty-two patients with DSA had 28 non-contrast-enhanced MRA examinations within a 2-month period. Of these, 19 patients had TRAS, two patients had pseudoaneurysms, and one patient had a normal transplant renal artery but an occluded external iliac artery. Non-contrast-enhanced MRA correctly detected 19 TRAS and nine normal arteries, giving 96.6 % accuracy on a per-artery basis. CONCLUSIONS: Non-contrast-enhanced MRA demonstrates a good depiction of the transplanted renal artery and shows good correlation with DSA in cases where there was TRAS. KEY POINTS: • Good or excellent image quality was found in 95.4 % of examinations. • Non-contrast-enhanced MRA can clearly map transplant renal artery anatomy. • Non-contrast-enhanced MRA is a reliable tool to detect TRAS.

Quantitative Assessment of Population Variability in Hepatic Drug Metabolism Using a Perfused Three-Dimensional Human Liver Microphysiological System.

In this work, we first describe the population variability in hepatic drug metabolism using cryopreserved hepatocytes from five different donors cultured in a perfused three-dimensional human liver microphysiological system, and then show how the resulting data can be integrated with a modeling and simulation framework to accomplish in vitro-in vivo translation. For each donor, metabolic depletion profiles of six compounds (phenacetin, diclofenac, lidocaine, ibuprofen, propranolol, and prednisolone) were measured, along with metabolite formation, mRNA levels of 90 metabolism-related genes, and markers of functional viability [lactate dehydrogenase (LDH) release, albumin, and urea production]. Drug depletion data were analyzed with mixed-effects modeling. Substantial interdonor variability was observed with respect to gene expression levels, drug metabolism, and other measured hepatocyte functions. Specifically, interdonor variability in intrinsic metabolic clearance ranged from 24.1% for phenacetin to 66.8% for propranolol (expressed as coefficient of variation). Albumin, urea, LDH, and cytochrome P450 mRNA levels were identified as significant predictors of in vitro metabolic clearance. Predicted clearance values from the liver microphysiological system were correlated with the observed in vivo values. A population physiologically based pharmacokinetic model was developed for lidocaine to illustrate the translation of the in vitro output to the observed pharmacokinetic variability in vivo. Stochastic simulations with this model successfully predicted the observed clinical concentration-time profiles and the associated population variability. This is the first study of population variability in drug metabolism in the context of a microphysiological system and has important implications for the use of these systems during the drug development process.

Spontaneous dormancy of metastatic breast cancer cells in an all human liver microphysiologic system.

BACKGROUND: Metastatic outgrowth in breast cancer can occur years after a seeming cure. Existing model systems of dormancy are limited as they do not recapitulate human metastatic dormancy without exogenous manipulations and are unable to query early events of micrometastases. METHODS: Here, we describe a human ex vivo hepatic microphysiologic system. The system is established with fresh human hepatocytes and non-parenchymal cells (NPCs) creating a microenvironment into which breast cancer cells (MCF7 and MDA-MB-231) are added. RESULTS: The hepatic tissue maintains function through 15 days as verified by liver-specific protein production and drug metabolism assays. The NPCs form an integral part of the hepatic niche, demonstrated within the system through their participation in differential signalling cascades and cancer cell outcomes. Breast cancer cells intercalate into the hepatic niche without interfering with hepatocyte function. Examination of cancer cells demonstrated that a significant subset enter a quiescent state of dormancy as shown by lack of cell cycling (EdU(-) or Ki67(-)). The presence of NPCs altered the cancer cell fraction entering quiescence, and lead to differential cytokine profiles in the microenvironment effluent. CONCLUSIONS: These findings establish the liver microphysiologic system as a relevant model for the study of breast cancer metastases and entry into dormancy.

A method to generate perfusable physiologic-like vascular channels within a liver-on-chip model.

The human vasculature is essential in organs and tissues for the transport of nutrients, metabolic waste products, and the maintenance of homeostasis. The integration of vessels in in vitro organs-on-chip may, therefore, improve the similarity to the native organ microenvironment, ensuring proper physiological functions and reducing the gap between experimental research and clinical outcomes. This gap is particularly evident in drug testing and the use of vascularized models may provide more realistic insights into human responses to drugs in the pre-clinical phases of the drug development pipeline. In this context, different vascularized liver models have been developed to recapitulate the architecture of the hepatic sinusoid, exploiting either porous membranes or bioprinting techniques. In this work, we developed a method to generate perfusable vascular channels with a circular cross section within organs-on-chip without any interposing material between the parenchyma and the surrounding environment. Through this technique, vascularized liver sinusoid-on-chip systems with and without the inclusion of the space of Disse were designed and developed. The recapitulation of the Disse layer, therefore, a gap between hepatocytes and endothelial cells physiologically present in the native liver milieu, seems to enhance hepatic functionality (e.g., albumin production) compared to when hepatocytes are in close contact with endothelial cells. These findings pave the way to numerous further uses of microfluidic technologies coupled with vascularized tissue models (e.g., immune system perfusion) as well as the integration within multiorgan-on-chip settings.

A prospective, randomized, controlled trial of autologous haematopoietic stem cell transplantation for aggressive multiple sclerosis: a position paper.

BACKGROUND: Haematopoietic stem cell transplantation (HSCT) has been tried in the last 15 years as a therapeutic option in patients with poor-prognosis autoimmune disease who do not respond to conventional treatments. Worldwide, more than 600 patients with multiple sclerosis (MS) have been treated with HSCT, most of them having been recruited in small, single-centre, phase 1-2 uncontrolled trials. Clinical and magnetic resonance imaging outcomes from case series reports or Registry-based analyses suggest that a major response is achieved in most patients; quality and duration of response are better in patients transplanted during the relapsing-remitting phase than in those in the secondary progressive stage. OBJECTIVES: An interdisciplinary group of neurologists and haematologists has been formed, following two international meetings supported by the European and American Blood and Marrow Transplantation Societies, for the purpose of discussing a controlled clinical trial, to be designed within the new scenarios of evolving MS treatments. CONCLUSIONS: Objectives of the trial, patient selection, transplant technology and outcome assessment were extensively discussed. The outcome of this process is summarized in the present paper, with the goal of establishing the background and advancing the development of a prospective, randomized, controlled multicentre trial to assess the clinical efficacy of HSCT for the treatment of highly active MS.

Low-kV coronary artery calcium scoring with tin filtration using a kV-independent reconstruction algorithm.

PURPOSE: To investigate the accuracy of Agatston scoring and potential for radiation dose reduction of a coronary artery calcium scoring (CACS) CT protocol at 100 kV with tin filtration (Sn100kV) and kV-independent iterative reconstruction, compared to standard 120 kV acquisitions. MATERIALS AND METHODS: With IRB approval and in HIPAA compliance, 114 patients (61.8 ± 9.6 years; 66 men) underwent CACS using a standard 120 kV protocol and an additional Sn100kV CACS scan. The two datasets were reconstructed using a medium sharp convolution algorithm and in addition the Sn100kV scans were reconstructed iteratively based on a kV-independent algorithm. Agatston scores and radiation dose values were compared between the Sn100kV and the standard 120 kV protocol. RESULTS: Median Agatston scores derived from the Sn100kV protocol with the kV-independent algorithm and the standard 120 kV were 21.4 (IQR, 0-173.8) and 24.7 (IQR, 0-171.1) respectively, with no significant differences (p=0.18). Agatston scores derived from the two different protocols had an excellent correlation (r = 0.99). The dose-length-product was 11.5 ± 4.1 mGy × cm using Sn100kV and 50.4 ± 24.9 mGy × cm using the standard 120 kV protocol (p < 0.01), resulting in a significantly lower (77%) effective dose at Sn100kV (0.16 ± 0.06 mSv vs. 0.71 ± 0.35 mSv, p < 0.01). Additionally, 99% of the patients were classified into the same risk category (0, 1-10, 11-100, 101-400, or >400) using the Sn100kV protocol. CONCLUSION: CACS at Sn100kV using the kV-independent iterative algorithm is feasible and provides high accuracy when compared to standard 120 kV scanning. Furthermore, radiation dose can be significantly reduced for this screening application in a priori healthy individuals.

Myocardial extracellular volume fraction to differentiate healthy from cardiomyopathic myocardium using dual-source dual-energy CT.

OBJECTIVE: To evaluate the feasibility of dual-energy CT (DECT)-based iodine quantification to estimate myocardial extracellular volume (ECV) fraction in patients with and without cardiomyopathy (CM), as well as to assess its ability to distinguish healthy myocardial tissue from cardiomyopathic, with the goal of defining a threshold ECV value for disease detection. METHODS: Ten subjects free of heart disease and 60 patients with CM (mean age 66.4 ±â€¯9.4; 59 males and 11 females; 40 ischemic and 20 non-ischemic CM) underwent late iodine enhanced DECT imaging. Myocardial iodine maps were obtained using 3-material decomposition. ECV of the left ventricle was estimated from hematocrit levels and the iodine maps using the AHA 16-segment model. Receiver operating characteristic curve analysis was performed, with corresponding area under the curve, along with Youden's index assessment, to establish a threshold for CM detection. RESULTS: The median ECV for healthy myocardium, non-ischemic CM, and ischemic CM were 25.4% (22.9-27.3), 38.3% (33.7-43.0), and 36.9% (32.4-41.1), respectively. Healthy myocardium showed significantly lower ECV values compared to ischemic and non-ischemic CM (p < 0.001). From Youden's index analysis, an ECV>29.5% would indicate the presence of CM in the myocardium (sensitivity = 90.3; specificity = 90.3); the AUC for this criterion was 0.950 (p < 0.001). CONCLUSION: The findings of this study resulted in a statistically significant distinction between healthy myocardium and CM ECVs. This led to the establishment of a promising threshold ECV value that could facilitate the differentiation between healthy and diseased myocardium, and highlights the potential of this DECT methodology to detect cardiomyopathic tissue.

Value of Machine Learning-based Coronary CT Fractional Flow Reserve Applied to Triple-Rule-Out CT Angiography in Acute Chest Pain.

PURPOSE: To evaluate the additional value of noninvasive artificial intelligence (AI)-based CT-derived fractional flow reserve (CT FFR), derived from triple-rule-out coronary CT angiography for acute chest pain (ACP) in the emergency department (ED) setting. MATERIALS AND METHODS: AI-based CT FFR from triple-rule-out CT angiography data sets was retrospectively obtained in 159 of 271 eligible patients (102 men; mean age, 57.0 years ± 9.7 [standard deviation]) presenting to the ED with ACP. The agreement between CT FFR (≤ 0.80) and stenosis at triple-rule-out CT angiography (≥ 50%), as well as downstream cardiac diagnostic testing, was investigated. Furthermore, the predictive value of CT FFR for coronary revascularization and major adverse cardiac events (MACE) was assessed over a 1-year follow-up period. RESULTS: CT FFR and triple-rule-out CT angiography demonstrated agreement in severity of coronary artery disease (CAD) in 52% (82 of 159) of all cases. CT FFR of 0.80 and less served as a better predictor for coronary revascularization and MACE than stenosis of 50% and greater at triple-rule-out CT angiography (odds ratio, 3.4; 95% confidence interval: 1.4, 8.2 vs odds ratio, 2.2; 95% confidence interval: 0.9, 5.3) (P < .01). In the subgroup of patients with additional noninvasive cardiac testing (94 of 159), there was higher agreement as to the presence or absence of significant disease with CT FFR (55%) than with coronary triple-rule-out CT angiography (47%) (P = .23). CONCLUSION: CT FFR derived from triple-rule-out CT angiography was a better predictor for coronary revascularization and MACE and showed better agreement with additional diagnostic testing than triple-rule-out CT angiography. Therefore, CT FFR may improve the specificity in identifying patients with ACP with significant CAD in the ED setting and reduce unnecessary downstream testing.© RSNA, 2020See also the commentary by Ihdayhid and Ben Zekry in this issue.

Evidence for actin filament-microtubule interaction mediated by microtubule-associated proteins.

We have used low shear viscometry and electron microscopy to study the interaction between pure actin filaments and microtubules. Mixtures of microtubules having microtubule-associated proteins (MAPs) with actin filament have very high viscosities compared with the viscosities of the separate components. MAPs themselves also cause a large increase in the viscosity of actin filaments. In contrast, mixtures of actin filaments with tubulin polymers lacking MAPs have low viscosities, close to the sum of the viscosities of the separate components. Our interpretation of these observations is that there is an interaction between actin filaments and microtubules which requires MAPs. This interaction is inhibited by ATP and some related compounds. Electron micrographs of thin sections through mixtures of actin and microtubules show numerous close associations between the two polymers which may be responsible for their high viscosity.

Ca2+-dependent phosphorylation of tyrosine hydroxylase in PC12 cells.

Ca2+-dependent protein phosphorylation has been detected in numerous tissues and may mediate some of the effects of hormones and other extracellular stimuli on cell function. In this paper we demonstrate that a Ca2+/calmodulin-dependent protein kinase similar to the enzyme previously purified and characterized from rat brain is present in PC12, a rat pheochromocytoma cell line. We show that Ca2+ influx elicited by various forms of cell stimulation leads to increased 32P incorporation into tyrosine hydroxylase (TH), a major phosphoprotein in these cells. Several other unidentified proteins are either phosphorylated or dephosphorylated as a result of Ca2+ influx. Acetylcholine stimulates TH phosphorylation by activation of nicotinic receptors. K+-induced depolarization stimulates TH phosphorylation in a Ca2+-dependent manner, presumably by opening voltage-dependent Ca2+ channels. Ca2+ influx that results from the direct effects of the ionophore A23187 also leads to TH phosphorylation. Phosphorylation of TH is accompanied by an activation of the enzyme. These Ca2+-dependent effects are independent of cyclic AMP and thus implicate a Ca2+-dependent protein kinase as a mediator of both hormonal and electrical stimulation of PC12 cells.

Myosin light chain kinase and myosin light chain phosphatase from Dictyostelium: effects of reversible phosphorylation on myosin structure and function.

We have partially purified myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) from Dictyostelium discoideum. MLCK was purified 4,700-fold with a yield of approximately 1 mg from 350 g of cells. The enzyme is very acidic as suggested by its tight binding to DEAE. Dictyostelium MLCK has an apparent native molecular mass on HPLC G3000SW of approximately 30,000 D. Mg2+ is required for enzyme activity. Ca2+ inhibits activity and this inhibition is not relieved by calmodulin. cAMP or cGMP have no effect on enzyme activity. Dictyostelium MLCK is very specific for the 18,000-D light chain of Dictyostelium myosin and does not phosphorylate the light chain of several other myosins tested. Myosin purified from log-phase amebas of Dictyostelium has approximately 0.3 mol Pi/mol 18,000-D light chain as assayed by glycerol-urea gel electrophoresis. Dictyostelium MLCK can phosphorylate this myosin to a stoichiometry approaching 1 mol Pi/mol 18,000-D light chain. MLCP, which was partially purified, selectively removes phosphate from the 18,000-D light chain but not from the heavy chain of Dictyostelium myosin. Phosphatase-treated Dictyostelium myosin has less than or equal to 0.01 mol Pi/mol 18,000-D light chain. Phosphatase-treated myosin could be rephosphorylated to greater than or equal to 0.96 mol Pi/mol 18,000-D light chain by incubation with MLCK and ATP. We found myosin thick filament assembly to be independent of the extent of 18,000-D light-chain phosphorylation when measured as a function of ionic strength. However, actin-activated Mg2+-ATPase activity of Dictyostelium myosin was found to be directly related to the extent of phosphorylation of the 18,000-D light chain. MLCK-treated myosin moved in an in vitro motility assay (Sheetz, M. P., and J. A. Spudich, 1983, Nature (Lond.), 305:31-35) at approximately 1.4 micron/s whereas phosphatase-treated myosin moved only slowly or not at all. The effects of phosphatase treatment on the movement were fully reversed by subsequent treatment with MLCK.

Epidermal growth factor (EGF)-like repeats of human tenascin-C as ligands for EGF receptor.

Signaling through growth factor receptors controls such diverse cell functions as proliferation, migration, and differentiation. A critical question has been how the activation of these receptors is regulated. Most, if not all, of the known ligands for these receptors are soluble factors. However, as matrix components are highly tissue-specific and change during development and pathology, it has been suggested that select growth factor receptors might be stimulated by binding to matrix components. Herein, we describe a new class of ligand for the epidermal growth factor (EGF) receptor (EGFR) found within the EGF-like repeats of tenascin-C, an antiadhesive matrix component present during organogenesis, development, and wound repair. Select EGF-like repeats of tenascin-C elicited mitogenesis and EGFR autophosphorylation in an EGFR-dependent manner. Micromolar concentrations of EGF-like repeats induced EGFR autophosphorylation and activated extracellular signal-regulated, mitogen-activated protein kinase to levels comparable to those induced by subsaturating levels of known EGFR ligands. EGFR-dependent adhesion was noted when the ligands were tethered to inert beads, simulating the physiologically relevant presentation of tenascin-C as hexabrachion, and suggesting an increase in avidity similar to that seen for integrin ligands upon surface binding. Specific binding to EGFR was further established by immunofluorescence detection of EGF-like repeats bound to cells and cross-linking of EGFR with the repeats. Both of these interactions were abolished upon competition by EGF and enhanced by dimerization of the EGF-like repeat. Such low affinity behavior would be expected for a matrix-"tethered" ligand; i.e., a ligand which acts from the matrix, presented continuously to cell surface EGF receptors, because it can neither diffuse away nor be internalized and degraded. These data identify a new class of "insoluble" growth factor ligands and a novel mode of activation for growth factor receptors.

Endoplasmic reticulum in rat renal interstitial cells: molecular rearrangement after water deprivation.

Cylindrical bodies in renal interstitial cells of dehydrated rats are confluent with membranes of endoplasmic reticulum. The cylinder walls, composed of helically arranged pentagonal tubules, may represent a molecular rearrangement of the membrane structure. The cylinders may represent a morphologic expression of altered ergastoplasmic function possibly related to the production of concentrated urine.

Regulatory phosphorylation of AMPA-type glutamate receptors by CaM-KII during long-term potentiation.

Long-term potentiation (LTP), a cellular model of learning and memory, requires calcium-dependent protein kinases. Induction of LTP increased the phosphorus-32 labeling of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors (AMPA-Rs), which mediate rapid excitatory synaptic transmission. This AMPA-R phosphorylation appeared to be catalyzed by Ca2+- and calmodulin-dependent protein kinase II (CaM-KII): (i) it correlated with the activation and autophosphorylation of CaM-KII, (ii) it was blocked by the CaM-KII inhibitor KN-62, and (iii) its phosphorus-32 peptide map was the same as that of GluR1 coexpressed with activated CaM-KII in HEK-293 cells. This covalent modulation of AMPA-Rs in LTP provides a postsynaptic molecular mechanism for synaptic plasticity.

Feasibility of extracellular volume quantification using dual-energy CT.

ObjectiveTo assess the feasibility of dual energy CT (DECT) to derive myocardial extracellular volume (ECV) and detect myocardial ECV differences without a non-contrast acquisition, compared to single energy CT (SECT). MethodsSubjects (n = 35) with focal fibrosis (n = 17), diffuse fibrosis (n = 10), and controls (n = 9) underwent non-contrast and delayed acquisitions to calculate SECT-ECV. DECT-ECV was calculated using the delayed acquisition and the derived virtual non-contrast images. In the control and diffuse fibrotic groups, the entire myocardium of the left ventricle was used to calculate ECV. Two ROIs were placed in the focal fibrotic group, one in normal and one in fibrotic myocardium. ResultsMedian ECV was 33.4% (IQR, 30.1-37.4) using SECT and 34.9% (IQR, 31.2-39.2) using DECT (p = 0.401). For both techniques, focal and diffuse fibrosis had significantly higher ECV values (all p < 0.021) than normal myocardium. There was no systematic bias between DECT and SECT (p = 0.348). SECT had a higher radiation dose (1.1 mSv difference) than DECT (p < 0.001). ConclusionECV can be measured using a DECT approach with only a delayed acquisition. The DECT approach provides similar results at a lower radiation dose compared to SECT.