Visualization of the subcellular localization of SARS-CoV-2 proteins in lung patient material of COVID-19 patients is important for the understanding of this new virus. We detected viral proteins in the context of the ultrastructure of infected cells and tissues and discovered that some viral proteins accumulate in novel, lipid-filled compartments.

AIMS:We aim to evaluate if the myelin pathology observed in epilepsy-associated focal cortical dysplasia type 2B (FCD2B) and-histologically indistinguishable-cortical tubers of tuberous sclerosis complex (TSC) is primarily related to the underlying malformation or constitutes a secondary phenomenon due to the toxic microenvironment created by epileptic seizures. We also aim to investigate the possible beneficial effect of the mTOR pathway regulator everolimus on white matter pathology.METHODS:Primary mixed glial cell cultures derived from epilepsy surgery specimens of one TSC and seven FCD2B patients were grown on polycaprolactone fibre matrices and analysed using immunofluorescence and electron microscopy. Unaffected white matter from three age-matched epilepsy patients with mild malformations of cortical development (mMCD) and one with FCD3D served as controls. Additionally, TSC2 knock-out was performed using an oligodendroglial cell line. Myelination capacities of nanofibre grown cells in an inflammatory environment after mTOR-inhibitor treatment with everolimus were further investigated.RESULTS:Reduced oligodendroglial turnover, directly related to a lower myelin content was found in the patients' primary cells. In our culture model of myelination dynamics, primary cells grown under 'inflammatory condition' showed decreased myelination, that was repaired by treatment with everolimus.CONCLUSIONS:Results obtained in patient-derived primary oligodendroglial and TSC2 knock-out cells suggest that maturation of oligodendroglia and production of a proper myelin sheath seem to be impaired as a result of mTOR pathway disturbance. Hence, oligodendroglial pathology may reflect a more direct effect of the abnormal genetic programme rather than to be an inactive bystander of chronic epilepsy.

INTRODUCTION 217 I. TEXT MESSAGE PRICING 218 A. History of Text Message Pricing 218 B. Text Messaging Price Comparison 220 II. TEXT MESSAGING MARKET BACKGROUND 223 III. COMPETITIVE, MONOPOLY, AND OLIGOPOLY MARKETS ... 225 IV. ANTITRUST LAW AND THE OLIGOPOLY PROBLEM 227 A. The Scope of Agreement Under the Sherman Act 227 B. Economics-Based Approaches to Antitrust Enforcement 230 V. ANTITRUST ANALYSIS OF TEXT MESSAGING 231 A. Text Messaging Market Definition 231 B. An Economic Analysis of the Text Messaging Market 233 1. The Text Messaging Market’s Susceptibility to Collusion 234 2. Direct Economic Evidence of Collusion in the Text Messaging Market 238 VI. FCC REGULATION OF TEXT MESSAGE PRICING 241 CONCLUSION 242

We have recently shown (1) that bestrophin‐3, a member of the Best family, is essential for the cGMP‐dependent Ca2+‐activated Cl− current (I(Ca, cGMP)Cl) (2) which has been shown in different types of smooth muscles (3). The I(Ca, cGMP)Cl has similar characteristics as the currents produced by heterologous expression of bestrophins and distributs similarly to Best‐3 expression. Furthermore, downregulation of Best‐3 with siRNA was associated with a significant reduction of the I(Ca, cGMP)Cl.To study the physiological significance of Best‐3 immunohistochemical (IHC) and siRNA approaches were used. Wistar rats were used in accordance with national guidelines for animal research.We characterized the Best‐3 specific antibody by expression of eGFP‐fusion‐protein with the epitope containing peptide. IHC showed a broad expression of Best‐3 (e.g. kidney, heart, brain and mesentery) primarily in the vasculature. Within the vascular wall Best‐3 was shown to be strongly expressed in both smooth muscle and endothelial cells. This was supported by PCR cloning. Specific siRNAs significantly suppressed Best‐3 expression and the I(Ca, cGMP)Cl.Our study demonstrates the functional significance of Best‐3 protein for the cGMP‐dependent Ca2+‐activated Cl− channel in vascular tissue.1. Matchkov et al., Circ Res 20082. Matchkov et al., J Gen Physiol 20043. Matchkov et al., Pflugers Arch 2005

Although the biophysical fingerprints (ion selectivity, voltage- dependence, kinetics, etc) of Ca2+-activated Cl- currents are well established, their molecular identity is still controversial. Several molecular candidates have been suggested; however, none of them has been fully accepted. We have recently characterized a cGMP- dependent Ca2+-activated Cl- current with unique characteristics in smooth muscle cells. This novel current has been shown to coexist with a "classic" (cGMP- independent) Ca2+-activated Cl- current and to have characteristics distinct from those previously known for Ca2+-activated Cl- currents. Here, we suggest that a bestrophin, a product of the Best gene family, is responsible for the cGMP- dependent Ca2+-activated Cl- current based on similarities between the membrane currents produced by heterologous expressions of bestrophins and the cGMP- dependent Ca2+-activated Cl- current. This is supported by similarities in the distribution pattern of the cGMP- dependent Ca2+-activated Cl- current and bestrophin-3 (the product of Best-3 gene) expression in different smooth muscle. Furthermore, downregulation of Best-3 gene expression with small interfering RNA both in cultured cells and in vascular smooth muscle cells in vivo was associated with a significant reduction of the cGMP- dependent Ca2+-activated Cl- current, whereas the magnitude of the classic Ca2+-activated Cl- current was not affected. The majority of previous suggestions that bestrophins are a new Cl- channel family were based on heterologous expression in cell culture studies. Our present results demonstrate that at least 1 family member, bestrophin-3, is essential for a well-defined endogenous Ca2+-activated Cl- current in smooth muscles in the intact vascular wall.

OBJECTIVE:We investigated whether weight loss episodes were associated with the metabolic syndrome and diabetes in elderly men.DESIGN AND SUBJECTS:Men residing in Oslo and born in 1923-32 (n = 16,209) were screened for cardiovascular diseases and risk factors in 1972-73. Those who resided in the same area in the year 2000 were invited to a repeat physical and laboratory examination, attended by 6 410 men (mean age 72.5 years). Weight, height and blood pressure were measured both times, and a non-fasting measurements of total serum cholesterol, triglycerides and glucose were obtained. Weight loss, leisure time physical activity, smoking habits, and educational attendance were self-reported.RESULTS:The proportion that reported one or more episodes of weight loss at age 25-50 years was 15.6% (n = 3,564 respondents) while 26.8% reported weight loss after age 50 (n = 3,473 respondents). Age-specific weight loss scores based on the number of the episodes of weight loss or on the total amount of weight loss were strongly associated with the presence of obesity, the metabolic syndrome and diabetes in the year 2000. The risk of the metabolic syndrome and diabetes in 2000 increased with increasing number of weight loss episodes also adjusted for BMI in 1972-73 and other potential confounders. The odds ratio for the metabolic syndrome for one standard deviation change in the number of weight loss episodes after age 50 was 1.43 (95% confidence limits 1.30-1.57). The corresponding odds ratio for diabetes was 1.25 (95% confidence limits 1.14-1.37). Similar results were found using a score for the total amount of weight loss.CONCLUSION:Among elderly men the number of episodes or amount of weight loss after age 50 was associated with the metabolic syndrome and diabetes, but this study cannot establish the causality of the association.

We have recently characterized in smooth muscle cells a unique cGMP-dependent Ca2+-activated Cl− current (ICl(cGMP-Ca)) that co-exists with a “classical” Ca2+-activated Cl− current. We hypothesized that bestrophin-4 (a product of the VMD2-like 3 gene) could be responsible for the ICl(cGMP-Ca) based on similarities between membrane current produced by bestrophin heterologous expression and this endogenous current. This was supported by a similar distribution pattern of the ICl(cGMP-Ca) and the bestrophin expression. To test this hypothesis siRNA-mediated downregulation of gene expression was used. Cultured aortic smooth muscle cells (A7r5) were transfected with siRNA directed against bestrophin-4 and cultured for 3 days. The efficiency of transfection was demonstrated by specific perinuclear fluorescence of Cy3-labelled siRNA. The downregulation of targeted protein expression was controlled by qPCR and Western blot. The downregulation of bestrophin-4 expression (by 88% in mRNA) with siRNA was a associated with significant reduction (by 83%) of the ICl(cGMP-Ca) while the “classical” Ca2+-activated Cl− current was not affected. Our studies provide evidence that bestrophin-4 is responsible for the ICl(cGMP-Ca) in smooth muscle cells. This study presents a novel efficient technique for specific downregulation of gene expression in blood vessels, much needed in studies of vascular function.

BACKGROUND:Data are scarce on the long term relationship between leisure time physical activity, smoking and development of metabolic syndrome and diabetes. We wanted to investigate the relationship between leisure time physical activity and smoking measured in middle age and the occurrence of the metabolic syndrome and diabetes in men that participated in two cardiovascular screenings of the Oslo Study 28 years apart.METHODS:Men residing in Oslo and born in 1923-32 (n = 16 209) were screened for cardiovascular diseases and risk factors in 1972/3. Of the original cohort, those who also lived in same area in 2000 were invited to a repeat screening examination, attended by 6 410 men. The metabolic syndrome was defined according to a modification of the National Cholesterol Education Program criteria. Leisure time physical activity, smoking, educational attendance and the presence of diabetes were self-reported.RESULTS:Leisure time physical activity decreased between the first and second screening and tracked only moderately between the two time points (Spearman's rho = 0.25). Leisure time physical activity adjusted for age and educational attendance was a significant predictor of both the metabolic syndrome and diabetes in 2000 (odds ratio for moderately vigorous versus sedentary/light activity was 0.65 [95% CI, 0.54-0.80] for the metabolic syndrome and 0.68 [0.52-0.91] for diabetes) (test for trend P < 0.05). However, when adjusted for more factors measured in 1972/3 including glucose, triglycerides, body mass index, treated hypertension and systolic blood pressure these associations were markedly attenuated. Smoking was associated with the metabolic syndrome but not with diabetes in 2000.CONCLUSION:Physical activity during leisure recorded in middle age prior to the current waves of obesity and diabetes had an independent predictive association with the presence of the metabolic syndrome but not significantly so with diabetes 28 years later in life, when the subjects were elderly.