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Picornaviruses induce dramatic rearrangements of endomembranes in the cells that they infect to produce dedicated platforms for viral replication. These structures, termed replication organelles (ROs), have been well characterized for the Enterovirus genus of the Picornaviridae. However, it is unknown whether the diverse RO morphologies associated with enterovirus infection are conserved among other picornaviruses. Here, we use serial electron tomography at different stages of infection to assess the three-dimensional architecture of ROs induced by encephalomyocarditis virus (EMCV), a member of the Cardiovirus genus of the family of picornaviruses that is distantly related. Ultrastructural analyses revealed connections between early single-membrane EMCV ROs and the endoplasmic reticulum (ER), establishing the ER as a likely donor organelle for their formation. These early single-membrane ROs appear to transform into double-membrane vesicles (DMVs) as infection progresses. Both single- and double-membrane structures were found to support viral RNA synthesis, and progeny viruses accumulated in close proximity, suggesting a spatial association between RNA synthesis and virus assembly. Further, we explored the role of phosphatidylinositol 4-phosphate (PI4P), a critical host factor for both enterovirus and cardiovirus replication that has been recently found to expedite enterovirus RO formation rather than being strictly required. By exploiting an EMCV escape mutant, we found that low-PI4P conditions could also be overcome for the formation of cardiovirus ROs. Collectively, our data show that despite differences in the membrane source, there are striking similarities in the biogenesis, morphology, and transformation of cardiovirus and enterovirus ROs, which may well extend to other picornaviruses.
IMPORTANCE Like all positive-sense RNA viruses, picornaviruses induce the rearrangement of host cell membranes to form unique structures, or replication organelles (ROs), that support viral RNA synthesis. Here, we investigate the architecture and biogenesis of cardiovirus ROs and compare them with those induced by enteroviruses, members of the well-characterized picornavirus genus Enterovirus. The origins and dynamic morphologies of cardiovirus ROs are revealed using electron tomography, which points to the endoplasmic reticulum as the donor organelle usurped to produce single-membrane tubules and vesicles that transform into double-membrane vesicles. We show that PI4P, a critical lipid for cardiovirus and enterovirus replication, is not strictly required for the formation of cardiovirus ROs, as functional ROs with typical morphologies are formed under phosphatidylinositol 4-kinase type III alpha (PI4KA) inhibition in cells infected with an escape mutant. Our data show that the transformation from single-membrane structures to double-membrane vesicles is a conserved feature of cardiovirus and enterovirus infections that likely extends to other picornavirus genera.
The cardioviruses comprise an important genus of the Picornaviridae family of positive-sense RNA (+RNA) viruses. The type species Cardiovirus A, or encephalomyocarditis virus (EMCV), ostensibly infects mice but is capable of infecting a range of mammals, including humans. Severe disease in wild or farm animals occurs primarily in swine (1, 2), but concerns for human safety have followed from studies demonstrating the potential for pig-to-human EMCV transmission during organ xenotransplantation (3, 4), which will become more imminent as the technique matures. The Cardiovirus B species is represented by Theiler's murine encephalomyocarditis virus (TMEV), the study of which has led to critical contributions to our understanding of demyelinating diseases (reviewed in reference 5). Unlike TMEV, the closely related Saffold virus is capable of infecting humans. Saffold virus was discovered only recently in a patient with a fever of unknown origin (6) but appears to be widespread and commonly found in coinfections with other viruses. Among these coinfecting viruses are the enteroviruses (7, 8), another genus of picornaviruses that includes poliovirus, the coxsackieviruses, rhinoviruses, and enteroviruses A71 and D68. Although the consequences of enterovirus and Saffold virus infections in coinfected patients are entwined, there are suggestions that Saffold virus could cause or contribute to diseases as diverse as gastroenteritis, encephalitis, myocarditis, and nonpolio acute flaccid paralysis (reviewed in reference 9), which are typically associated with enterovirus infections.
Understanding the conserved requirements for picornavirus replication is an important strategy for developing broadly acting antiviral therapies. Currently, vaccines are available to prevent infections with hepatitis A virus, poliovirus, and enterovirus 71 (EV71) (available only in China), but there are no approved antiviral therapies to treat picornavirus infections. Substantial efforts have been invested in developing directly acting antivirals that target viral proteins, but the spectrum of antiviral activity of these agents is limited (10–12). Another strategy, for which interest has been growing in recent years, is the development of inhibitors targeting host factors that are conserved among different genera (13). One host factor required for the replication of both enteroviruses and cardioviruses is the lipid phosphatidylinositol 4-phosphate (PI4P). Cellular production of PI4P is orchestrated by phosphatidylinositol 4-kinases (PI4Ks), which reside in various subcellular compartments and generate local pools of PI4P (reviewed in references 14 and 15). Enteroviruses rely upon the III beta class of PI4K (PI4KB) (16, 17), which predominantly localizes to the Golgi apparatus, while cardioviruses require the largely endoplasmic reticulum (ER)-based III alpha class of PI4K (PI4KA) (18). Interestingly, as with cardioviruses and enteroviruses, PI4P is also required by the more distantly related hepatitis C virus (HCV), which depends upon PI4KA for its replication (19).
A role for PI4P in expediting the formation of enterovirus replication organelles (ROs) has recently been described (20), which could in part explain its importance during viral replication. ROs are virus-induced membrane rearrangements formed during infections of all eukaryote-infecting +RNA viruses. These structures serve as a hub for viral replication in the cell, and their unique properties are believed to confer intrinsic benefits on the viruses that generate them. The expansion of membranes that support the viral RNA synthesis machinery may increase replication efficiency, while the unique morphology of some ROs could serve to compartmentalize this process or its products and limit host innate immune sensing. The three-dimensional (3D) morphology of enterovirus ROs is well characterized. Early in infection, enteroviruses induce the formation of single-membrane tubular ROs that gradually transform into double-membrane vesicles (DMVs) and multilamellar vesicles over the course of infection (21, 22). Our understanding of cardiovirus ROs, however, is more limited. Different studies have identified single- or double-membrane structures using two-dimensional (2D) electron microscopy (EM) of chemically fixed samples (23–25), and it is unclear whether these disparate observations reflect differences in sample preparation or truly distinct structures, perhaps found at different stages of infection. It remains to be established how membrane modifications arise and develop across cardiovirus infection, what their 3D morphology is, which structures are capable of supporting RNA synthesis, and, furthermore, whether PI4KA plays a role in cardiovirus RO formation.
Here, we use serial electron tomography (ET) to unravel the 3D morphology of EMCV ROs across an extensive time course that encompasses their formation and development. Samples were prepared using high-pressure freezing and freeze substitution (HPF-FS) as an alternative to chemical fixation, which better preserves structures sensitive to artifacts. We provide evidence that early EMCV ROs consist of single-membrane tubules (SMTs) and vesicles (SMVs) that emerge from the ER. These single-membrane structures, which arise during the exponential phase of viral RNA synthesis, appear to serve as the primary platform for genome replication. However, double-membrane vesicles (DMVs), which largely emerge later in infection, were also capable of supporting RNA synthesis. Remarkably, putative transition states between single-membrane tubules and DMVs were found that mirror the transformation intermediates observed for enterovirus ROs (21). This chronological progression of RO morphology contextualizes the previous observations of single- or double-membrane EMCV ROs and represents a striking parallel between cardiovirus and enterovirus DMV formation that may represent a conserved mechanism among picornaviruses. Given this cohesion in RO morphology between enteroviruses and EMCV, we then investigated whether PI4KA plays a role in the formation of EMCV ROs, as PI4KB does for enterovirus ROs (20). To this end, we studied the effects of PI4KA inhibition on an EMCV mutant that harbors a single substitution in its 3A protein (3A-A32V), which confers resistance to PI4KA inhibitors (26). Interestingly, PI4KA inhibition neither affected RO morphology nor significantly delayed RO formation. This indicates that, similarly to enteroviruses, a lack of high PI4P levels does not pose an unsurmountable barrier for cardiovirus RO formation.
EMCV-induced membrane rearrangements with different morphologies arise over the course of infection.We first set out to determine the relationship between EMCV replication and the emergence of ROs. Samples for quantitative PCR (qPCR), viral titer determination, immunofluorescence analysis, and electron microscopy (EM) (chemical fixation) were generated within a single experiment. HeLa cells infected with EMCV (strain Mengovirus) at a multiplicity of infection (MOI) of 50 were processed every hour up to 9 h postinfection (hpi) for qPCR of viral RNA and for the determination of viral titer. Samples for immunofluorescence and electron microscopy (EM) analyses were fixed between 3 and 9 hpi. While data from these chemically fixed EM samples were assessed to quantify the emergence of ROs, representative images are presented from a subsequent experiment using an improved sample preparation method (high-pressure freezing and freeze substitution) that more faithfully preserves RO morphology. The exponential phase of RNA synthesis spanned 3 to 6 hpi, while the increase in viral titer lagged by ~2 h (Fig. 1A). These RNA replication dynamics aligned well with increasing double-stranded RNA (dsRNA) immunofluorescence signal, which provides a secondary indication of viral replication. The first fluorescent signal was detected at 4 hpi, and levels increased steadily each hour before reaching a plateau at 7 hpi. Between 4 and 5 hpi, dsRNA signal was found at small foci distributed throughout the cytoplasm, which became larger and more clustered as infection progressed (Fig. 1B).
The first virus-induced membrane modifications were detected in EM cell sections of cells fixed at 5 hpi, revealing a complex but sparse assortment of virus-induced single-membrane structures (Fig. 2A, arrowheads) and, very occasionally, double-membrane structures (Fig. 2A, asterisk). From 6 hpi, membrane modifications became increasingly clustered into large regions that dominated the cytosol, and the abundance of double-membrane structures increased (Fig. 2B, asterisks), although single-membrane structures persisted (Fig. 2B, arrowheads). Late in infection, double-membrane vesicles became more dominant (Fig. 2C, white asterisks), and multilamellar structures were also found (Fig. 2C, black asterisks and inset). Between 5 and 8 hpi, the RO density in the cytoplasm greatly increased (from 0.11 to 0.63 ROs per µm2), and the proportion of DMVs (as a percentage of total ROs) grew from less than 1% to an average of 35%, although in some cells DMVs rather than single-membrane structures were the predominant RO morphology (Fig. 2D). A number of cells with evident signs of lysis (e.g., disrupted plasma membrane and extracted cytosolic content) could also be found later in infection, which may explain the lack of immunofluorescence labeling in some cells by 7 hpi.
Electron tomography reveals the 3D architecture and transformation of ROs.One drawback of 2D EM cell section analysis is the similarity in cross section between vesicles and tubules running perpendicular to the section plane. Additionally, features like membrane connections or small openings may be obscured by the superposition of structures in a 2D projection image. To more unambiguously characterize the morphology of EMCV ROs, cells at early and late stages of infection were prepared for electron tomography (ET). Tomograms from serial cell sections of the same region of interest were combined to form large 3D volumes.
Single-membrane structures identified in the 2D analysis at early times postinfection were found to comprise a mixed population of single-membrane vesicles (SMVs) and single-membrane tubules (SMTs) (Fig. 3A, red and blue, respectively; see also Movie S1 in the supplemental material). Unlike the tubules formed during enterovirus infections, EMCV SMTs did not form tightly packed clusters. Both SMVs and SMTs were smooth-membrane structures and were frequently found connected to rough ER (green) by neck-like membrane connections (Fig. 3B and C, arrowheads), like the ER-derived ROs of nidoviruses (27–29) and HCV (30). In contrast, DMVs, which were predominantly found at late stages of infection, appeared in all instances as separate compartments, with no membrane connections to other structures (Fig. 4A, yellow; Movie S2). Some DMVs seemed to be partially enwrapped by additional cisternae late in infection (Fig. 4B, purple), to form multilamellar structures. Virus particles were also detectable in the EM data as dense hexagonal profiles of ~25 nm (Fig. 4C, arrowhead), which were frequently found in the cytosol within 30 nm of ROs (Fig. S1). These particles (Fig. 4D, black arrowhead) were distinguishable from ribosomes (Fig. 4D, white arrowheads) by their symmetry and well-defined edges. A closer spatial association was found between virus particles and RO membranes (SMVs, SMTs, or DMVs) than between randomly generated coordinates and RO membranes (Fig. S1, P < 0.001). This could reflect localized clustering of virions following their formation or a spatial connection between RNA synthesis and virion assembly.
Copyright © 2018 Melia et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
Copyright © 2018 Melia et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
Copyright © 2018 Melia et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
Ultrastructural investigations of enterovirus ROs have revealed putative transition structures that may represent the transformation of SMTs into DMVs. This process appears to involve the pairing of membrane tubules to form flattened cisternae, which curve to enwrap a small volume of cytosol, ultimately forming an open DMV in a vase-like configuration. The opening then seals to form a complete, closed DMV (21, 22). Similar structures were found in tomograms of EMCV-infected cells, including paired and highly curved tubules (Fig. 5A, i and ii, white arrowheads), which could represent an early stage of DMV formation from SMTs, and DMVs with small openings (Fig. 5A, iii, arrowhead). Tubular extensions of the outer membranes of DMVs were also found (Fig. 5B, indicated by white arrowheads in consecutive sections) that could represent the partial transformation of SMTs to DMVs. Supporting this idea, similar electron-dense material was observed both in the lumen of single-membrane structures (Fig. 5A, black arrowheads) and within the intermembrane space created by these tubular extensions (Fig. 5B, black arrowhead). To further examine the possibility of a single- to double-membrane structure transformation, the surface areas of SMVs, SMTs, and DMVs were estimated using the measured average sizes for each structure (Fig. 5C, top). These figures provide a guide as to whether SMTs or SMVs are likely candidate precursor structures for DMVs (as described in reference 21) as, unless other lipid sources contribute to DMV membranes, any precursor structure should have at least the same surface area as the DMV that forms from it. The estimated surface areas generated allow for a scenario where DMVs are formed by SMTs (Fig. 5C, bottom) and are compatible with the possibility that some (smaller) DMVs could also be derived from SMVs. In this scenario, the multilamellar vesicles seen at late time points may arise from SMTs that enwrap or partially enwrap existing DMVs, rather than enwrapping the cytosol to form a new DMV. DMVs were occasionally found to contain virions in samples analyzed at 7 hpi (pooled data from four tomograms; example shown in Fig. 5D) but at a low frequency (7% of 68 DMVs), and particles within DMVs represented a small fraction of total virus particles detected (2% of 339 particles). An enwrapping mechanism of SMT-to-DMV transformation could explain the presence of virus particles within a subset of DMVs, as virions may be enclosed by chance during the process.
RNA synthesis occurs at virus-induced single- and double-membrane structures.To investigate whether all or some of these virus-induced membrane modifications support RNA synthesis, metabolic labeling and EM autoradiography (31) were performed. Cells were infected with EMCV and fixed at 5 or 7 hpi. During the 2 h prior to fixation, cells were incubated with 10 µg/ml dactinomycin to limit cellular transcription. During the 45 min prior to fixation, cells were additionally treated with tritiated uridine to label newly synthesized RNA. After chemical fixation and preparation for EM, sections were processed for autoradiography.
While only larger clusters of electron-dense grains are good indicators of underlying viral RNA synthesis given the limited resolution of EM autoradiography (32), substantial autoradiography signal could be found clustered around areas containing exclusively single-membrane tubules and vesicles (Fig. 6A). This demonstrates their ability to support viral RNA synthesis. Newly synthesized RNA was also evident at RO foci comprised predominantly of DMVs at later stages of infection (Fig. 6B), indicating that the transformation from single-membrane structure to DMV does not impede continued genome replication.
PI4KA inhibition of a PI4K-resistant EMCV mutant does not alter RO development or morphology.While there may be divergence in the origins of enterovirus and cardiovirus ROs, the development of SMTs that transform into DMVs appears to be a common feature of both. We next investigated whether common requirements for RO biogenesis might also exist between enteroviruses and cardioviruses. Specific roles for the enterovirus host factor PI4KB, a PI4P-generating kinase, have recently been identified. In addition to facilitating efficient polyprotein processing (20, 33–35), PI4KB accelerates the formation of enterovirus ROs (20). To investigate possible roles during EMCV RO formation of PI4KA, an essential PI4P-generating kinase for cardiovirus replication, we utilized the 3A-A32V mutant (26). This mutant, generated through serial passaging of wild-type (wt) EMCV in cells with a stable knockdown of PI4KA, is capable of establishing replication even in the presence of the PI4KA inhibitor A1. At a concentration of 10 nM, this compound potently inhibits wt EMCV replication in HeLa cells, with no effect on cell viability (26).
In the absence of A1 treatment, infections by the EMCV 3A-A32V mutant in HeLa cells produced ROs whose morphology was indistinguishable from those of wt EMCV (Fig. S2). SMTs and SMVs predominated at earlier time points, while DMVs were found only occasionally (Fig. S2A, asterisk). Later in infection, DMVs proliferated and ROs became more clustered (Fig. S2B, DMVs indicated by asterisks). Together, these results indicate that the 3A-A32V substitution does not affect RO development or the RO general architecture. PI4KA inhibition under A1 treatment led to a more clustered, perinuclear dsRNA signal compared to uninhibited infections with EMCV 3A-A32V or wt EMCV (Fig. 7A) (26). Despite this different staining pattern under PI4KA inhibition, the morphology of ROs and time postinfection at which ROs were first detected were not affected. EMCV 3A-A32V infection under PI4KA inhibition produced single- and double-membrane cardiovirus ROs (Fig. 7B, left, white arrowheads and asterisks, respectively), typically in proximity to the ER (black arrowheads). Importantly, while the clustered dsRNA signal found under inhibition partially localized in the perinuclear Golgi region (Fig. S3), connections between single-membrane ROs and the ER were still found (Fig. 7B, right, white arrowheads), and ROs produced under PI4KA inhibition also retained their ability to support RNA synthesis (Fig. 7C). Taken together, our data demonstrate that, while PI4KA/PI4P availability may govern the subcellular location of EMCV ROs, it does not significantly affect their emergence, morphology, or ability to serve as platforms for viral RNA synthesis.
Copyright © 2018 Melia et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
Copyright © 2018 Melia et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
Here, we reveal the 3D morphology of cardiovirus ROs and present a comprehensive overview of their formation and development throughout infection. At early stages of EMCV infection, virus-induced SMTs and SMVs were found, which appear to transform into DMVs over the course of infection. This progression bears a striking resemblance to that of enterovirus ROs, suggesting a universal mechanism for DMV formation in picornaviruses. Despite differences in the cellular origins of enterovirus and cardiovirus ROs, our data establish that escape mutants from both genera are able to bypass any role of PI4P in RO formation.
Single-membrane ROs which were capable of supporting viral RNA synthesis predominated at early stages of EMCV infection. Interestingly, while enteroviruses invariably produce SMTs early in infection (21, 22), also in HeLa cells (22), EMCV produced both SMTs and SMVs. These structures were of different sizes and did not form packed clusters like the SMTs of enteroviruses. Many of the SMTs and SMVs visualized at early stages of infection were also found connected to the ER, providing compelling evidence that cardiovirus ROs are ER derived. This finding is supported by the reported reliance of cardioviruses on ER-associated proteins like PI4KA and the observed colocalization of calreticulin and RO-associated viral proteins (18). Unlike cardioviruses, enteroviruses depend on Golgi apparatus-based proteins like PI4KB and GBF1 (16, 17, 36) and likely utilize Golgi membranes to generate their ROs. Interestingly, membrane connections between donor organelle and enterovirus ROs were not detected in the tomography studies (21, 22), suggesting that the process of initial enterovirus RO budding and release from the donor organelle is rapid compared to that of cardioviruses.
Despite these divergences in origin and morphology, our findings suggest that the transformation of single-membrane ROs into DMVs is conserved among enteroviruses and cardioviruses. For both genera, DMV formation appears to occur via membrane pairing of single-membrane ROs to form curved cisternae that ultimately enwrap small volumes of cytosol (21, 22). Given that transport from the DMV interior to the cell cytosol across the double membrane of a closed DMV is problematic, these structures may represent a means of sequestering viral products at late stages of infection. Interestingly, a small population of DMVs were also found with openings to the cytosol. While these open DMVs could reflect an intermediate step in their transformation from single-membrane structures to closed DMVs, it is also possible that a small population of DMVs retain openings to the cytosol for the duration of infection. Given that viral replication is likely to occur on the cytosolic face of early single-membrane ROs (37, 38), the interior of open DMVs could represent a shielded environment for replication at later stages of infection, with access to the cytosol for virion packing and/or export. A similar enwrapping mechanism could also underlie the formation of the sparse double-membrane structures among abundant single-membrane structures identified in 2D EM investigations of foot-and-mouth disease virus infection (genus Aphthovirus) (39) and may represent the means by which DMVs form during infections with Picornaviridae more broadly. Evidence for a similar mechanism for DMV biogenesis has been presented for the arterivirus equine arteritis virus and Middle East respiratory syndrome (MERS) coronavirus (order Nidovirales), which in this case would occur not through single-membrane intermediate ROs but by direct pairing and curving of the ER membranes (40, 41). While this enwrapping mechanism could represent a general route for DMV RO formation in +RNA viruses, it is intriguing that DMV formation is possible both via enwrapping of Golgi apparatus- or ER-derived single-membrane ROs in the case of picornaviruses and via direct enwrapping of cytosol by ER membranes in the case of nidoviruses. The formation of DMVs from these diverse progenitor membranes could suggest that their development depends primarily on viral machinery or recruited host factors. The cellular functions of these host factors may also provide clues about their role during infection. For instance, LC3 has been found in conjunction with enterovirus ROs (42, 43). In uninfected cells, lipidated LC3 is hypothesized to induce the membrane curvature underlying omegasome formation during autophagy (44), and its recruitment to enterovirus ROs could suggest a similar role in the formation of DMVs, whose development appears to resemble that of autophagosomes.
While direct actors have yet to be established, it is likely that cardiovirus RO formation is dependent upon specific, membrane-associated host factors. PI4P is an essential lipid for the replication of picornaviruses (16, 18, 45) and is generated at the ER or the Golgi apparatus by PI4KA and PI4KB, respectively. Interestingly, PI4Ks are also important for HCV replication, where they appear to play a role in RO formation (19, 46). Expression of a mutated HCV NS5A, whose ability to recruit PI4KA is impaired, has been shown to produce ROs with irregular morphologies (19). A role for PI4Ks during RO biogenesis has also been demonstrated for enteroviruses, as RO formation was delayed under PI4KB inhibition during infections of the PI4KB inhibitor-resistant virus coxsackievirus B3 (CVB3) 3A-H57Y. Under these conditions, ROs were not detected at the peak of viral RNA synthesis, and this process occurred instead on apparently intact Golgi membranes (20). In our study, PI4KA inhibition shifted the subcellular distribution of EMCV dsRNA but did not affect the time postinfection at which ROs were first detected or alter their observed morphology, connectivity with the ER, or competence to support viral RNA synthesis. These different observations in the two genera could arise from differences in the efficiency of the escape mechanism conferred by the specific resistance mutations or alternatively suggest that, unlike for enteroviruses, PI4P does not play a significant role in cardiovirus RO formation. In either case, both mutants were able to (ultimately) produce ROs with normal morphology under PI4K inhibition, showing that any role of high PI4P levels in RO formation can be circumvented by members of both picornavirus genera. PI4P is also required for efficient polyprotein processing in enteroviruses, although this requirement is circumvented in PI4KB inhibitor escape mutants (20, 33, 34). Whether EMCV requires PI4P for polyprotein processing remains to be established.
ROs are believed to confer inherent advantages on the viruses that generate them, but it is unclear whether the different morphologies generated provide specific benefits. Although both single- and double-membrane ROs were found to support cardiovirus RNA synthesis, single-membrane structures predominated during the peak hours of genome replication. While this suggests that DMVs are largely superfluous for RNA synthesis, they may provide other benefits. DMVs could selectively sequester viral products to prevent their detection by cellular innate immune sensors. In the case of enteroviruses, it has been suggested that DMVs may also be utilized for the nonlytic release of viral progeny (47). However, virions were only rarely detected within EMCV DMVs, which likely reflects random incorporation of virus particles and other cytosolic material when DMVs arise by enwrapping. Intriguingly, we found a large proportion of EMCV virions adjacent to ROs, suggesting close spatial coordination between RNA synthesis and capsid assembly. In enteroviruses, viral RNA synthesis and capsid assembly have been shown to be directly coupled, likely through interactions between the RNA replication machinery and the viral structural proteins (48, 49).
Collectively, our data reveal striking similarities between the ROs produced by enteroviruses and cardioviruses and raise the possibility of a conserved mechanism of picornavirus RO biogenesis to form single-membrane structures that can transform into DMVs. In this regard, picornaviruses appear to be unique, as other DMV-forming viruses, like coronaviruses or HCV, generate DMVs directly from the cellular donor membrane. Given that all +RNA viruses to date have been found to produce spherules or DMVs, viruses tend to be classified as producing one or the other RO type. However, while picornaviruses do produce DMVs, RNA synthesis transpires predominantly on the early single-membrane structures. Thus, in addition to the negative-curvature spherules of alphaviruses and flaviviruses and the DMVs formed by coronaviruses and hepaciviruses, positive-curvature single-membrane structures appear to represent a third major form of RO.
Cell lines and reagents.HeLa R19 cells were maintained in Dulbecco's modified Eagle's medium (Gibco) supplemented with 10% fetal calf serum, penicillin, and streptomycin and grown at 37°C in 5% CO2. The PI4KA inhibitor A1 (50) was kindly provided by T. Balla (National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD) and used at a concentration of 10 nM.
Virus infections.Cells were inoculated for 1 h with EMCV wt (strain Mengovirus) or EMCV 3A-A32V (described in reference 26) at an MOI of 50, after which the inoculum was removed and fresh medium (with drug A1 where indicated) was added. At specified time points after infection, cells were prepared for qPCR, viral titer determination, immunofluorescence, or electron microscopy.
Quantitative PCR.A NucleoSpin RNA kit (Macherey-Nagel) was used to isolate RNA from lysed cells. Random hexamer primers were used with a TaqMan reverse transcription reagent kit (Roche) to synthesize cDNA. Quantitative PCR was carried out using the LightCycler 480 SYBR green I master kit (Roche) for 45 cycles (5 s at 95°C, 10 s at 60°C, and 20 s at 72°C) on a LightCycler 480 (Roche). Resulting threshold cycle (CT) values were expressed as fold increase with the value at t = 2 h set as 1.
Viral titer determination.Total virus titers (of intra- and extracellular particles) were determined by freeze-thaw lysis of infected cells and endpoint titration.
Immunofluorescence microscopy.Cells were fixed with 3% paraformaldehyde and permeabilized with phosphate-buffered saline (PBS) containing 0.1% Triton X-100. Cells were then labeled with a mouse monoclonal antibody against dsRNA (J2 antibody; English and Scientific Consulting) and secondary Alexa Fluor 488-conjugated goat anti-mouse IgG, alongside a nuclear stain (Hoechst 33342). Imaging was performed using a wide-field DM5500 (Leica) fluorescence microscope.
Electron microscopy. (i) Metabolic labeling and autoradiography.Infected cells were preincubated with 10 µg/ml dactinomycin for 1 h to inhibit cellular transcription and then labeled for 45 min with tritiated uridine ([5-3H]uridine; 1 mCi/ml) (PerkinElmer) also containing dactinomycin. Cells were then washed several times to remove unincorporated label and fixed for 1 h in 1.5% glutaraldehyde in 0.1 M cacodylate buffer. Mock-infected control samples were similarly prepared. Postfixation consisted of a 1-h incubation with 1% osmium tetroxide in 0.1 M cacodylate buffer followed by a 1-h incubation with 1% uranyl acetate solution. Samples were then washed and dehydrated in ethanol and infiltrated and embedded in epoxy resin LX-112 before polymerization at 60°C. Sections of 50 nm were collected on Formvar-coated EM grids, poststained with lead citrate and uranyl acetate, and then carbon coated and prepared for autoradiography. For this, a thin layer of silver halide photographic emulsion (Ilford L4) was applied in the dark to the cell sections. Samples were incubated in the dark and developed after several weeks for autoradiography as described in reference 20. During incubation, the radiolabeled uridine incorporated during viral RNA synthesis decays, and the beta radiation emitted reduces the silver halide to atomic silver. These events manifest as small, electron-dense grains dispersed around the original radioactive source that can be visualized during EM imaging.
(ii) High-pressure freezing and freeze substitution.Cells were high-pressure frozen using an EM PACT2 (Leica). Samples were maintained at −90°C in an AFS2 (Leica) freeze substitution device for 44 h in a solution of 20% H2O, 2% osmium tetroxide, and 1% anhydrous glutaraldehyde. A high water content was used as this was found to improve the contrast of RO membranes. The temperature was then raised to 0°C over a period of 22 h through a series of controlled warming phases (identical to those described in reference 21). Samples were washed with acetone, infiltrated with epoxy resin LX-112 (Ladd Research), and polymerized at 60°C. Sections of 70 nm were collected on EM grids and poststained with uranyl acetate and lead citrate. For tomography, thicker sections of 200 nm were collected. Before poststaining, both sides of the EM grid were incubated with 10-nm colloidal gold beads to serve as fiducial markers during tomogram reconstruction.
(iii) Electron microscopy imaging.Thin-section (50- or 70-nm) 2D images were collected (binning mode 2) on a Tecnai12 BioTwin or Twin electron microscope at 120 kV using an Eagle 4k slow-scan charge-coupled device (CCD) camera (Thermo Fisher Scientific [formerly FEI]) or OneView 4k high-frame-rate camera (Gatan), respectively. For the semiautomated collection of larger areas, meshes of overlapping images were acquired across the entire region of interest and later reconstituted into a single composite image (as described in reference 51).
(iv) Electron tomography.Dual-axis tilt series of 200-nm sections each covering 130 to 140° around the region of interest were collected using an F20 electron microscope (Thermo Fisher Scientific [formerly FEI]) at 200 kV with zero-loss energy filtering using a 2k camera (Gatan) and 1.27-nm pixel size. Automated tilt series acquisition was performed using Xplore3D software (Thermo Fisher Scientific). Tilt series alignment and tomogram reconstruction by weighted back-projection were carried out in IMOD (52) (version 4.7.15). In the process, the gold fiducial markers were digitally erased to be eliminated from the final 3D reconstruction. To determine the sizes of ROs spanning multiple sections, tilt series of the same region across 2 to 5 consecutive sections were collected and combined in IMOD to build tomogram volumes with a z-thickness of ~400 to 1,000 nm. The sizes of ROs were estimated by measuring the SMTs, SMVs, and DMVs within reconstructed volumes in IMOD. The maximum widths of SMTs, SMVs, and DMVs were considered the RO diameters. For all calculations, SMTs were approximated to a cylinder, and the inner DMV membrane surface area was considered an approximation of the outer.
To generate models of features of interest, image segmentations were made from tomograms using Amira 6.0.1 (Thermo Fisher Scientific). Before segmentation, tomograms were postprocessed to enhance edges by nonlinear anisotropic diffusion filtering (53) as implemented in IMOD using 5 iterations and K = 1,000 and then binned by 2 in x and y. Membranes were selected in manually masked areas by thresholding and then refined by removing islands and smoothing. Surfaces that were poorly represented (e.g., top and bottom surfaces of vesicles and tubules) due to the missing-wedge effect were manually repaired.
This work was supported by research grants from the Netherlands Organization for Scientific Research (NWO-VENI-863.12.005 to H.M.V.D.S., NWO-VICI-91812628 to F.J.M.V.K., ERASysApp project "SysVirDrug" ALW project number 832.14.003 to F.J.M.V.K. and E.J.S., and NWO-MEERVOUD 863.10.003 to M.B.) and from the European Union (7th Framework, EUVIRNA Marie Curie Initial Training Network, grant agreement number 264286 to F.J.M.V.K. and E.J.S.). The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
- Received 20 March 2018
- Accepted 22 March 2018
- Published 17 April 2018
- Copyright © 2018 Melia et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.


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Dasher5227Full Member
Content Count81
Last visited
About Dasher5227
WTB Power Play.. offering my dignity etc. for a working PP
Props to the caniacs still in their seats at the end of that game
Surgery Scheduled for November 10th!
Dasher5227 replied to BigMiike's topic in Hurricanes TalkBest of luck!
Dasher5227 joined the community
who thinks that they did good
Dasher5227 replied to 10dilpickle's topic in Hurricanes TalkYes, They did well.
I come in Peace!
Dasher5227 replied to Gonch55-ch1's topic in Hurricanes TalkWe have Pitt fans who wear Steelers jerseys to canes games that do not involve pittsburg... at all.
Player Sightings
Dasher5227 replied to its-staal-good-num-12's topic in Hurricanes TalkI was just chilling in the airport parking lot, as usual, the other night, and all of the canes drove by me at once!
key to further playoff sucess
Dasher5227 replied to meckcountycane's topic in Hurricanes TalkI think Joni wants the cup as badly as any player on the team... It's important to remember that the man has ice in his veins. If he's playing well and dominating it appears he is cool and in complete control of the situation. If he screws up, the exact same facial expression makes him look like he doesn't care. Ever seen Joni pushing and shoving in the crease? The opponent always has a look of anger or frustration on his face, and Joni is as calm as ever... I agree, he has made his fair share of mistakes... but that man's natural composure makes him the kind of person you want to have on the ice during the playoffs.
playoff fan/seating/standing/cheering etiquette
Dasher5227 replied to wilmingtoncanesfan's topic in Hurricanes TalkOh yeah, I completely understand if traffic or work gets into the way and people arrive late to the arena. It drives me crazy though when people come in late to the 2nd, 3rd, and yes... sadly, even the overtime period.
playoff fan/seating/standing/cheering etiquette
Dasher5227 replied to wilmingtoncanesfan's topic in Hurricanes TalkWith you, I agree. It's also frustrating when people come back to the game 7 or 8 minutes into the period. I don't think I saw a single person come in that late who was wearing a Canes jersey in the last game, but there were plenty with suites and designer purses (thus my earlier stereotyping). It's annoying that these people didn't show up until the playoffs. I just don't see why you would miss 1/3 of a period of hockey that you paid for... to go buy a 10 dollar beer. If the beer is more important than the hockey game, stay home .
playoff fan/seating/standing/cheering etiquette
Dasher5227 replied to wilmingtoncanesfan's topic in Hurricanes TalkI've got a few words for you... -- Read the initial post and notice that the complaining began before the puck was dropped. -- The poster is a STH and this is the first time they've had to deal with the complaint (also the 1st home playoff game). -- It seems the poster did not recognize the person complaining, and given that the poster is a STH, this suggests the "whiner" was not an STH. -- In addition to reading the posts that come before yours, I recommend trying to stand for once. The ensuing fallout will, no doubt, ensure that Brind'amour is no longer the only Rod in the arena, and hopefully it will prevent your digestive system from backing up into your throat and relieving itself in the form of an accusatory, self-righteous, not to mention downright jerky forum post. [Edit] P.S. You sound like an owl ---- Who?
playoff fan/seating/standing/cheering etiquette
Dasher5227 replied to wilmingtoncanesfan's topic in Hurricanes TalkNow that the Canes have won a cup and are back in the playoffs, there are going to be "new" fans that come to the arena. They most likely haven't attended a regular season game, but that won't keep them from yelling at anyone else for doing what's completely acceptable. Don't let them get in the way of your fun. If they get to whinny, turn around and teach them a few words that their wannabe blue-blooded, cheap seat superiority complex having, fat, lazy *edit* had forgotten in their world of Paul Harvey, Wayne Brady, and quiet hockey games (provided there are no kids around). You know the words I mean I sat in the lower bowl for Game 3, and when the few people around me with designer purses and/or (lol) nice suites began to whine or give me a mean look, I let them have it. I kept trying to cheer at a pitch that was just a few cents flatter than the pitch of the guy next to me. When done correctly, it gives your combined cheers that annoying oscillation in volume (beat frequency), that allows you to really drive those fair-weather fans crazy. An Understanding of Physics: The Anti-Fairweather Fan
Airport Ritual - Playoffs Canes Style
Dasher5227 replied to caniac-97's topic in Hurricanes TalkWow, thats bull... My friends and I decided to go and greet the Canes and had to fly down to RDU to make it on time. When we got there we saw the cops blocking the entrance and the line of canes fans there. We got to sit and chill, watching the players leave, and then to drive by the crowd and get cheered for wearing our canes jerseys.
We're starting to get noticed (and respected)
Dasher5227 replied to Law-Dawg's topic in Hurricanes TalkI figured it was because the team with home ice won all three series.
FIRST ROUND- vs. Devils - your thoughts
Dasher5227 replied to coastal_caniac's topic in Hurricanes TalkIgnoring the streak before the last 5, the Devil's last 5 games were against: Lightning, Sabres, Leafs, Senators, Canes Given that only one of these teams made the playoffs, I believe it's safe to say that the Devils are a higher quality team than their average opponent in the last 5... but hey, I like numbers so let's screw around with them anyway. Assuming that the Devils were perfectly matched with each opponent (50-50 chance, win-lose), the probability of them winning at least 4 of their 5 final games stands at just beneath 20%. Hardly out of this world, especially when you consider the quality of their schedule. But hey, let's dig deeper... Devils Last 5 Games: GF: 14 GA: 14 One of their victories was won in a shootout. (no good in POs, but Devils might have pulled out win in OT so no use speculating) Another came on the wings of two odd bounces. (perfectly good in the playoffs, but I just like bringing it up) The one loss was at the hands of the leafs... I'm not saying the Devils haven't pulled out of the slump, and I'm perfectly aware that a win is a win and GF and GA can be totally worthless unless we're referring to just one game. However, I feel that the conditions of the wins and their statistics cast a fair amount doubt on the "late season run" as evidence that the Devils have pulled out of their nosedive (that's for the playoffs to decide). Personally, I think this series is going to rock... (no pun intended)
FIRST ROUND- vs. Devils - your thoughts
Dasher5227 replied to coastal_caniac's topic in Hurricanes TalkI realized that I haven't said anything constructive in some time, and it's always easier to critique than to create. So here goes nothing: I think this series is going to be awesome. When the (knowledgeable) fans from each team are confident in their team's ability to pull out a win, it generally means we are in for a treat. I would rather have the Devils than any of the other draws we could possibly get; however, not for the same reasons that others seem to have wanted them. Here's My 2e2 hundredths of a cent. (Everyone else was making up their own way of saying 2 cents) : 1)Both of these teams' current systems will work well in the playoffs. 2)Not only do each of these teams possess several dynamic star players, but they also add a depth at the foward position that will allow goals to be scored, irregardless of which lines are currently on the ice. 3)Given MartyB's half-god herritage (child of Zeus and a brick wall), Ward's excellent play of late, and both teams' scoring prowess, I suspect there will be no shortage of highlight reel saves. 4)Nether the Devils, nor the Canes have a history of cheapshots, so whoever pulls out the W can advance without losing too many players (*Knocking on Wood*). 5)The Jersey fanbase is as knowledgeable as any, yet they do not have the annoying aura of self-entitlement that often emanates from the fans of other more traditional market teams. (C'mon... the Rangers were the laughingstock of the original six, but some of their fans will still try to act like they invented hockey). 6)No Crosby/Ovechkin Rule (see Jordan Rule)

subset: commoncrawl


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This step-by-step guide to preventing panic attacks provides simple strategies for stopping a panic attack on the spot and overcoming them in the long-term. Sharing her personal and professional experience of panic and anxiety disorders, clinical psychologist Sandra Scheinbaum presents tried-and-tested methods, including new cutting-edge approaches based on functional medicine.
You will learn how to:
- Understand what panic attacks are and recognise symptoms
- Trust your body's own calming methods to control panic without the need for medication
- Find your abdominal breath and relaxed breathing patterns
- Let go of clenching and muscle tension through relaxation exercises and postural awareness
- Use positive mental imagery and avoid irrational thinking
- Find deep belly laughter, even in the midst of a panic episode
- Eat well with calming foods and advice on supplements.
The ten easy steps in this book will help readers to control their fight or flight response and overcome panic attacks naturally and permanently.
- Prof. Cary L. Cooper, Distinguished Professor of Organizational Psychology and Health, Lancaster University
'A superb, streamlined, simplistic approach to the overwhelming fear and frenzy of the internal tsunami of panic! Open the book and immediately dive into a solution to reverse the tide of feeling out of control. An excellent navigation guide in those choppy waters of chaos!'
- Deanna Minich, PhD, FACN, CNS, author and Functional Medicine nutritionist, founder of Food & Spirit™ (www.foodandspirit.com)


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Our goal is to provide you with a quick access to the content of the user manual for Kodak 1759836. Using the online preview, you can quickly view the contents and go to the page where you will find the solution to your problem with Kodak 1759836.
If looking through the Kodak 1759836 user manual directly on this website is not convenient for you, there are two possible solutions:
Many people prefer to read the documents not on the screen, but in the printed version. The option to print the manual has also been provided, and you can use it by clicking the link above - Print the manual. You do not have to print the entire manual Kodak 1759836 but the selected pages only. paper.
Below you will find previews of the content of the user manuals presented on the following pages to Kodak 1759836. If you want to quickly view the content of pages found on the following pages of the manual, you can use them.
Camera / M522
Extended user guide
For help with your camera:
Eastman Kodak Company Rochester, New York 14650 © Eastman Kodak Company, 2011 All screen images are simulated. Kodak, EasyShare, Perfect Touch, and Pulse are trademarks of Eastman Kodak Company. 4H7438_en
Product features Quick start Charge until the 1 Load the battery. 2 light stops blinking. 3 4 Set language, date/time. Take pictures, videos. 5 Install software, then share. Keep learning! Take and share your best pictures, videos! www.kodak.com/go/support i
Product features Front view Shutter Mode Self-timer, Power Video, Battery Speaker Charging light Flash Microphone Lens Tripod socket Battery compartment SD/SDHC Card slot ii www.kodak.com/go/support
Back view LCD Zoom USB port Flash button Strap post OK Delete Menu Info Review Share www.kodak.com/go/support iii
Table of contents 1 1 Setting up your camera .........................................................................1 Attaching the strap .......................................................................................1 Loading the KLIC-7006 battery......................................................................1 Charging the battery .....................................................................................2 Turning on the camera .......................................
Table of contents A smarter way to find and enjoy your pictures..............................................26 Understanding the review icons...................................................................27 Displaying pictures/videos on a television.....................................................28 Using the menu button in review.................................................................29 Cropping pictures..........................................................................
1 Setting up your camera Attaching the strap Loading the KLIC-7006 battery All batteries lose power over time–even if they're not being used. For longer life, fully charge the battery prior to use. Purchase extra batteries www.kodak.com/go/accessories Charge the battery See page 2 Watch the battery level See page 9 1 www.kodak.com/go/support
Setting up your camera Charging the battery If your package included Then see An in-camera battery charger Using the in-camera battery charger An external battery charger Using the external battery charger Using the in-camera battery charger You can charge the Li-Ion battery whenever necessary. Battery Charging light: • Blinking: charging • Steady On: finished charging 1 Turn off the camera. Connect the KODAK Camera USB Cable, Micro B / 5-Pin and 2 Your adapter charger. (Use only the cable in
Setting up your camera Using the external battery charger You can charge the Li-Ion battery whenever necessary. If you have this charger... Your plug may differ. Battery Charging light: • Orange: charging • Green: finished charging If you have this charger... Your plug may differ. Battery Charging light: • Flashing green: charging • Steady On green: finished charging Purchase extra batteries www.kodak.com/go/accessories Watch the battery level See page 9 3 www.kodak.com/go/support
Setting up your camera Charge via your computer's USB port You can charge the Li-Ion battery whenever necessary. IMPORTANT: The camera charges when the computer is on and awake. Turn off the computer's hibernation, sleep, and standby modes. 1 Turn off the camera. Connect the KODAK Camera USB Cable, 2 Micro B / 5-Pin and charger. Use only the cable included with this camera. Battery Charging light: • Blinking: charging • Steady: finished charging Purchase extra batteries www.kodak.com/go/acces
Setting up your camera Turning on the camera Press the Power button. Setting the language and date/time Language: to change. OK to accept. Date/Time: At the prompt, press OK. to change current field. for previous/next field. OK to accept. NOTE: To change language in the future, use the Setup Menu (see page 36). 5 www.kodak.com/go/support
Setting up your camera Storing pictures on an accessory SD or SDHC Card Your camera has internal memory. We strongly recommend purchasing an SD or SDHC Card to conveniently store more pictures/videos. CAUTION: A card can only be inserted one way; forcing it may cause damage. Inserting or removing a card while the camera is on may damage the pictures, the card, or the camera. Turn off the camera. 1 SD/SDHC Card (Accessory) 2 Insert the card until it clicks. 3 Turn on the camera. To eject the c
2 Taking pictures/videos Taking pictures 1 The camera turns on in Smart Capture mode. (If it's in a different mode, press the Mode button, then until the LCD displays Smart Capture mode , then press OK.) 2 Compose the picture. Press the Shutter button halfway to focus and set exposure. If the framing marks are not focused on the desired subject, release the Shutter button and recompose the scene. 3 Press the Shutter button completely down. Framing marks 7 www.kodak.com/go/support
Taking pictures/videos Taking a video 1 Press the Mode button. 2 Press until the LCD displays Video , then press OK. 3 To take a video, press the Shutter button completely down, then release it. To stop recording, press and release the Shutter button again. To review and work with videos, see page 23. Reviewing the picture/video just taken After you take a picture/video, the LCD briefly displays a quickview—the picture just taken or the first frame of a video. While a picture/video is disp
Taking pictures/videos Understanding the picture-taking icons Focus mode Self timer Exposure compensation Flash mode Pictures/time remaining Capture mode Zoom indicator Battery level Watch the battery level Don't miss out on great pictures and videos—keep your battery fully charged. Full Three quarters Half Critical AC Power Charge the battery see page 2 Purchase extra batteries www.kodak.com/go/accessories 9 www.kodak.com/go/support
Taking pictures/videos Using the optical zoom 1 Use the LCD to frame your subject. 2 To zoom in, press Telephoto (T). To zoom out, press Wide Angle (W). 3 Take the picture or video. NOTE: For focus distances, see Specifications starting on page 46. Using digital zoom Use digital zoom to get up to 5X magnification beyond optical zoom. (Digital zoom is not available in Smart Capture mode.) Press Telephoto (T) until the slider on the LCD is at the end of the optical zoom range. Release Telephoto
Taking pictures/videos Using the flash Press the Flash button repeatedly until the LCD displays the desired flash mode. Flash button Flash modes Flash fires Auto When the lighting conditions require it. Fill (Flash On) Every time you take a picture, regardless of the lighting conditions. Use when the subject is in shadow or is "backlit" (when the light is behind the subject). Off Never Red-eye A preflash fires, shrinking the eye pupil, before the picture is Reduction taken. 11 www.kodak.com/g
Taking pictures/videos Using different modes Press the Mode button, then until the LCD displays the mode that best suits your subjects and surroundings, then press OK. Mode button Use this mode For Smart General picture taking—it offers excellent image quality and ease Capture of use. ■ Faces get focus and exposure priority. ■ The scene is analyzed for other content and for lighting. You can choose the best picture size and aspect ratio for your needs—just press the Menu button and choose
Taking pictures/videos Using Scene (SCN) modes Choose a Scene mode—for great pictures in practically any situation. 1 Press the Mode button, then until the LCD displays SCN, then press OK. 2 Press to choose a mode and read its description. 3 Press OK to enter that Scene mode. The camera is now in the scene mode that you chose. SCN mode For Portrait Full-frame portraits of people. Sport Action pictures. Landscape Distant scenes. Close-up Close range. Use available light instead of flash, if p
|User manual
|Kodak 1278829
|Digital Camera
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|Digital Camera
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|Digital Camera


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Coverage of Nonpharmacologic Treatments for Low Back Pain Among US Public and Private Insurers
SOURCE: JAMA Netw Open. 2018 (Oct 5); 1 (6): e183044
James Heyward, MPH; Christopher M. Jones, PharmD, MPH; Wilson M. Compton, MD, MPE; et al
Center for Drug Safety and Effectiveness,
Johns Hopkins Bloomberg School of Public Health,
Baltimore, Maryland.
IMPORTANCE: Despite epidemic rates of addiction and death from prescription opioids in the United States, suggesting the importance of providing alternatives to opioids in the treatment of pain, little is known regarding how payers' coverage policies may facilitate or impede access to such treatments.
OBJECTIVE: To examine coverage policies for 5 nonpharmacologic approaches commonly used to treat acute or chronic low back pain among commercial and Medicare Advantage insurance plans, plus an additional 6 treatments among Medicaid plans.
DESIGN, SETTING, AND PARTICIPANTS: Cross-sectional study of 15 commercial, 15 Medicaid, and 15 Medicare Advantage health plans for the 2017 calendar year in 16 states representing more than half of the US population. Interviews were conducted with 43 senior medical and pharmacy health plan executives from representative plans.
MAIN OUTCOMES AND MEASURES: Medical necessity and coverage status for the treatments examined, as well as the use of utilization management tools and cost-sharing magnitude and structure.
RESULTS: Commercial and Medicare insurers consistently regarded physical and occupational therapy as medically necessary, but policies varied for other therapies examined.
Payers most commonly covered
physical therapy (98% [44 of 45 plans]),
occupational therapy (96% [43 of 45 plans]), and
chiropractic care (89% [40 of 45 plans]),
while transcutaneous electrical nerve stimulation (67% [10 of 15 plans]) and steroid injections (60% [9 of 15 plans]) were the most commonly covered among the therapies examined for Medicaid plans only.
Despite evidence in the literature to support use of acupuncture and psychological interventions, these therapies were either not covered by plans examined (67% of all plans [30 of 45] did not cover acupuncture) or lacked information about coverage (80% of Medicaid plans [12 of 15] lacked information about coverage of psychological interventions). Utilization management tools, such as prior authorization, were common, but criteria varied greatly with respect to which conditions and what quantity and duration of services were covered. Interviewees represented 6 Medicaid managed care organizations, 2 Medicare Advantage or Part D plans, 9 commercial plans, and 3 trade organizations (eg, Blue Cross Blue Shield Association). Interviews with plan executives indicated a low level of integration between the coverage decision-making processes for pharmacologic and nonpharmacologic therapies for chronic pain.
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CONCLUSIONS AND RELEVANCE: Wide variation in coverage of nonpharmacologic treatments for low back pain may be driven by the absence of best practices, the administrative complexities of developing and revising coverage policies, and payers' economic incentives. Such variation suggests an important opportunity to improve the accessibility of services, reduce opioid use, and ultimately improve the quality of care for individuals with chronic, noncancer pain while alleviating the burden of opioid addiction and overdose.
From the FULL TEXT Article:
Opioid overdose deaths in the United States have risen to epidemic proportions, driven by an approximate 4–fold increase in prescription opioid sales that occurred between 1999 and 2010. [1, 2] While deaths from heroin and highly potent synthetic opioids such as illicit fentanyl have increased dramatically since 2010, prescription opioids remain a major contributor to overdose deaths in the United States. In 2016, 42,249 people died from overdoses due to prescription or illicit opioids in the United States, with 17,087 deaths attributed to prescription opioids [3, 4] and more than 2 million individuals estimated to have a prescription opioid use disorder. [5] Projections suggest that an even greater number of Americans will be found to have died from an opioid overdose in 2017. [6]
Although many factors have contributed to increased use of opioids during the past 2 decades, 1 important contributor has been their overuse for the treatment of chronic, noncancer pain in the absence of data demonstrating long-term benefit. [7–9] This is important because opioids are but 1 of many pharmacologic and nonpharmacologic treatments for pain from chronic conditions such as lower back pain, headache, and fibromyalgia. In 2016, the Centers for Disease Control and Prevention released its Guideline for Prescribing Opioids for Chronic Pain, [10] recommending the use of nonopioid and nonpharmacologic therapies as first-line treatment for chronic pain. Consistent with other recent clinical practice guidelines, [11–13] the Centers for Disease Control and Prevention also advises that if opioids are prescribed, they should be combined with nonpharmacologic and nonopioid therapies. An increasing volume of evidence and consensus demonstrates the role of many of these approaches in clinical practice, underscoring the opportunities that exist to simultaneously improve the quality of care for those with pain while reducing exposure to and overreliance on prescription opioids. [14–17]
Recognizing the importance of nonopioid therapies, the US Department of Health and Human Services' National Pain Strategy urges changes to insurers' coverage policies to enable greater access and adherence to nonopioid therapies for pain. [9] Although coverage policies are an important determinant of health services utilization, little is known regarding payers' coverage of nonpharmacologic treatments for chronic, noncancer pain. This information gap constrains the policy development and implementation process.
To address this gap, we examined 2017 policies for these treatment modalities for low back pain — 1 of the most common pain-related conditions — among a diverse sample of 45 commercial, Medicaid, and Medicare Advantage plans. We focused on 5 common nonpharmacologic therapies for pain across all plans (physical therapy, occupational therapy, chiropractic care, acupuncture, and therapeutic massage), and, owing to the greater availability of information in public Medicaid coverage documents, examined an additional 6 therapies among Medicaid plans (transcutaneous electrical nerve stimulation [TENS], psychological interventions, steroid injections, facet injections, laminectomy, and diskectomy). We also conducted key informant interviews with 43 senior executives from the examined plans to better understand how plans have determined coverage and utilization management criteria for the treatments of interest. The aim of the study was to describe the current landscape of coverage for these therapies, and we did not conduct any formal hypothesis testing or infer causal pathways regarding coverage policies among the sampled plans.
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Decades ago, there were legitimate barriers that made it difficult for companies to achieve operational efficiency. Whether it was a challenge with communicating with employees or increasing speed in manufacturing, there were valid impediments. Today, it's an entirely different story because technology has completely reshaped the workplace. There are few challenges that cannot be overcome by implementing appropriate systems for your operations. Any Conversational Interface Platform Software is a great example of how innovation has boosted business capabilities in ways that were unimaginable a few decades ago.
Communication in the workplace is critical. In fact, it can make the difference in whether or not your business succeeds or fails. It's the reason why there has always been a concentrated focus on providing different methods for communicating with internal and external stakeholders. A couple of decades ago, email communication was transformed by Smartphones that enabled you to send messages from wherever you were in the world. It' actually hard to imagine a time when you couldn't send someone at work an email message. It's the number one way in which people communicate today.
In addition to email, companies have become more relaxed about the way in which text messaging is used. While it was once frowned upon as an informal way to communicate, it's now understood that sending a text message is not only convenient, it also helps by being able to quickly reference important information. The same applies to video calls that are increasingly popular for use in the business world. You can have a video chat with a colleague without any hassles, unlike in years past where videoconferencing was a masterful undertaking that often required assistance from an audiovisual crew.
The transformative nature of technology in business has always removed some of the barriers to entry. Small business owners are able to compete in the marketplace with vigor because they can access the same or similar technology used by large enterprises. You don't need a million dollar budget to market or run your business. You simply need to identify the technologies that work well and meet your needs. This includes marketing your business and building brand equity. Technology has transformed the way that process works. While it's helpful to have a robust advertising budget, you can enter the market and build a brand from the ground up much faster than has ever been possible in the past.
Another key way in which technology has reshaped business is by offering many different ways to provide security. Both physical and data security is important for every business because both can be costly if they are not appropriately managed. The loss of physical assets can be greatly diminished by using electronic surveillance systems that enable a company to monitor facilities from all angles. While retailers still deal with shrinkage, they are able to fight back much more effectively. When it comes to data security, the technology available is tremendous and it's continually improving, to the delight of business owners around the world.


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Read this tip to make your life smarter, better, faster and wiser. LifeTips is the place to go when you need to know about Freelance Design Jobs and other Freelance Design topics.
If you work for yourself, chances are you already know that freelance design jobs can be challenging to find. Having some tools to help raise your visibility can be very helpful. Business cards are an important thing to have, and if you're a designer, you'll want to make sure that they look sharp. People will be evaluating your skills as they look at the card, so take the time to make them shine.
The same thing applies to your web presence. Whether you have a full website or just a blog, make sure that it's visually pleasing and represents your style and abilities. If you opt for an online portfolio, display only your very best work.
One tool that designers often overlook is their biography. If you aren't a writer, you may want to hire one to make sure that it reads well. Be sure to include a bit about your design philosophy and background. Some people limit the biography to schooling and job history, but that doesn't tell a potential client much about who you are as a designer.
|Guru Spotlight
|Alexis Niki


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Jake Gardiner – Out, Travis Dermott – Out, Frederick Andersen – resting. Thing's are a little lean on the back end for the Toronto Maple Leafs as they visit Tavares' old home in Long Island.
New Third Pair
Ahead of the second game in the back-to-back the Leafs are shuffling their defensive lines a little more and mixing in players as much as they can it would seem. Martin Marincin has been called up to pair with Justin Holl as the bottom two for the Leafs defense.
It's an interesting move for Mike Babcock, to not pair each new player off with a veteran, but rather pair them together. I understand, however, the desire to keep your greenest defenders on a pair that you can minute-manage for the entirety of the game.
The Gardiner injury saw Igor Ozhiganov return to the lineup after a months absence and he played well. Ozhiganov finished with an empty stat line and 15:08 if ice time. While underwhelming, it's a positive; Igor showed he can seamlessly return to the team and put in a solid effort.
Justin Holl
Ozhiganov did well, but there is no need to rush him into what looks to be a high intensity back-to-back. Justin Holl, the Leafs OTHER extra defenseman will get the right-side duties tonight on the bottom pairing.
Holl impressed last season after getting called up and scoring in back-to-back games from the blue line. He impressed in those two games with the two goals and, more importantly, his +5 rating in that stretch.
There is a possible problem with injecting Holl into the lineup tonight. That issue is rust. Holl has played in just 2 NHL games this season and due to having waivers attached to him, has had no AHL games to keep him in game shape.
I would have liked to have seen Holl in the first game against Edmonton in Gardiner's absence but that's only due to the hindsight gained after the Dermott injury. Holl will have a tired team in front of him but no doubt they'll be motivated for JT.
Martin Marincin
I'm seriously impressed that Martin Marincin is still a Maple Leaf right now. He played a big part in the Leafs' playoff defensive woes last season and offers little to nothing from the back. This begs the question: why is Marincin getting the call up?
Let's be honest here, Marincin is in because Calle Rosen is injured.
The Leafs did have Marincin on the roster earlier this season. Once Ennis returned from injury, however, there was no roster space available for him and he was waived. Despite my crossed fingers, Marincin (and his gaudy $700,000 contract) wasn't claimed by anyone and he's been in the AHL since.
In his 10 games with the Leafs this season, Marincin has chipped in 2 assists with a -1 rating. In his 8 AHL games, he has 1 goal and 3 assists for a 0+/-.
Scheduled Sparks gets the Start
As is tradition this season, Garret Sparks gets the start for the Leafs second game in the back-to-back. It's good for Sparks to get into a game that will have a playoff-like intensity, providing him meaningful NHL hockey experience while still in the regular season.
There is little to no pressure on Sparks, whose job is basically to take whatever the opposition throws at him. He's tasked with playing and knowing there is little to no chance of Frederick Andersen coming off the bench to relieve him.
Sparks hasn't looked the most steady in net this season, but his stats show he's doing a fine job as a backup. Throughout his 13 games played this season, Sparks is 7-3 with a .908% save percentage and GAA of 2.86.
He's improved on his numbers in 15/16 when he first came up which is terrific. The next step in Sparks' development is to learn how to stay more composed in net, which he can absorb from Andersen's style of play.
It'll be a tough game, regardless of Toronto's bottom pairing and backup goaltender getting the start. Hopefully, the added pressure on the Leafs to come together for Tavares can inspire a solid performance.


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Very recently, more and more condominium and homeowner associations find themselves as potential defendants in Federal Fair Housing Act (the "FHA") discrimination litigation due to the association's website. It is alleged that the failure to make the website easily accessible to those with visual impairments or who are blind is discriminatory. In short, the FHA prohibits making, printing or publishing, with respect to the sale or rental of a dwelling, anything that indicates any preference, limitation, or discrimination based on a handicap, or the intention to make such preference, limitation, or discrimination. Thus, the FHA covers all written and oral notices or statements by a person engaged in the sale or rental of a dwelling. Therefore, as the argument presented is explained, if the association's website is providing information regarding the sale or rental of units or lots, and proper precautions are not taken to ensure that the website can be "listened to" rather than "read" by an individual who is visually impaired or blind, then that association could be a prime target for the threat of a federal discrimination lawsuit.
Victim's Awareness, Inc. is a national not-for-profit corporation whose membership consists of persons with disabilities and others who are committed to equal access, equal opportunity and equal rights for protected classes. Employees of this company, along with its constituent members, troll the internet searching for websites offering housing for sale or lease that do not provide a mechanism for those who are visually impaired or blind to have the content of the website automatically read to them. In order to have this functionality, what is technically referred to as a "widget" must be installed by the website host.
Typically, organizations such as Victim's Awareness, Inc. will send a demand letter including a letter of explanation, demand for evidence preservation, and a draft copy of the to-be-filed federal lawsuit and complaint demanding that the association immediately retrofit its website to ensure equal access by the visually impaired and blind. Failure to do so guarantees a lawsuit will be filed in Washington, D.C. against the association. Typically, this type of lawsuit is extremely expensive to defend. If liability results, the damages can easily be in the tens, if not hundreds, of thousands of dollars.
Sadly, even immediate compliance may not be sufficient to avoid monetary penalties. Because the demand brings about the desired change, the would-be plaintiff, in this case, Victims Awareness, Inc., argues that they are entitled to their attorneys' fees and costs for their preparation of the demand letter, preservation of evidence demand, and draft complaint. Therefore, even if an association complies with the demand by making its website accessible to those who are visually impaired, Victims Awareness, Inc.'s asserts that its attorneys' fees and costs will need to be satisfied. If an association refuses, then, even though the website is now FHA compliant, Victim's Awareness, Inc. suggests that they can still file the lawsuit to collect its attorney's fees and costs.
Because discrimination lawsuits is one of the few areas where board members can have individual liability it is likely that most associations will fold their hand and agree to the would-be plaintiff's demands. It will be interesting to see the results should an association decide to fight such demands on the basis that the FHA also provides that reasonable modifications must be granted by an association in response to a handicapped person's request so long as the modification is paid for by the person making the request. It remains to be seen whether such an argument pierce the demands made by groups such as Victim's Awareness, Inc.?
A community association risks being in harm's way when it operates a website that promotes sales and leasing activities and is open to the public at large. In this instance the ol' adage remains true- "an ounce of prevention is worth a pound of cure". Thus, to find additional information on the "widget" to bring your website into compliance and to learn more about this issue you can visit www.userway.com. In addition, consider discussing this important matter with your association's attorney.


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Hence, our Android application development process creates projects that are successful from the get-go. Our low-code Android mobile app development process aids the development of fully-stacked mobile apps that are easy to use and configured to work on all Android-based smart devices, regardless of type of device, screen size or pixel density.
Every Android app development requirement goes through stages of thorough research (UI/UX, business logic, and competitor analysis), required technology solutions (API integrations) and a quality assurance process that is more stringent than any other. Additionally, our 24*7 customer support and full-lifecycle maintenance means that we are co-partners in your Android application process through the app's entire lifecycle.
With a perfect mix of technology access, experience, and programming language expertise, HokuApps creates the most well-received Android apps, which have seen more than a million app downloads (cumulative).


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I didn't get as much done today as I would have liked as I've been in full on stressed out mode for most of the day. And what do you know, there it goes again! My Internet hasn't been working hardly at all the last few days. Off and on, off and on. I've written about it on here before. It just went off again, and I'm hoping I can save this post and get it done whenever it decides to come back on.
I felt like giving up on this creative habits project today because of all the stress. And besides, if I can't post my content online and share it with the world, then why bother? Who's going to keep me accountable?
One of my goals on Patreon is to upgrade my Internet to something more reliable. I already have it all figured out, I just need to work even harder to find a way to bring in some income. I was hoping Patreon was the answer, but either I'm just not good at selling myself, my artwork isn't nearly good enough, or maybe I'm just not reaching the right kind of people.
Then I got thinking that maybe I should just cancel my Internet service and find a way to do things differently. I mean, I'm fully committed to using the Internet to get noticed. Maybe sell some of my work online. Get some financial support through Patreon. Maybe I can find another way to create my artwork and get noticed locally. Hell, my work would certainly stand out a lot more! I mean, on the Internet you're basically up against every single artist on the planet.
I don't know, I'm absolutely sure that anything I feel right now is surely not really how I feel on a normal day. I'm just angry. Frustrated. I'll feel differently once things get figured out and settled down. I really should take the time to really think about whether I should make a big change though. Giving up my home Internet is a massive change though!
I'm so close to finishing my year…
EDIT: 10:00PM, as of this moment my Internet is finally back on. I tried to post this hours ago 😛
Help support my creative habit by liking, sharing, or commenting on my posts! Visit me on the Facebooks! Or, if you like, click right here! to help support me and my creative habit financially. Any and all assistance is greatly appreciated! 🙂


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|How To Lead Others
How To Lead Others
You also may be called upon to be a leader. The fact is, leading others does not have an age limit. So, regardless of how young or old you are, you can learn how to lead and value being a positive leader. Sooner or later everyone is asked to lead in one way or another. You may be asked to lead a team project, or you may be chosen as a lead supervisor, so be prepared.
One of the keys to learning how to lead is to maintain your positive character. Knowing how to be a positive leader can help you get yourself and others out of a bad situation. You may suddenly find yourself in a situation where everyone else is doing the wrong thing.
Sometimes making a difference means taking the initiative and having the courage to stand up for what is right. You never know when, through your positive leadership, you may encourage someone else to make a right choice.
Not born a leader? Join the crowd. Most people aren't either; but, don't let that discourage you from learning how to lead. There are many things we are born not knowing how to do, such as reading, writing, or even riding a bike.
Learning these skills may have been difficult at first, but you not only learned how to do them, you learned how to do them well.Leading others requires personality, character and skills, but to be the very best, you must develop a positive character and a harmless, helpful personality.
You already know that you must maintain and develop a positive character on a daily basis. As for skills, well, you can learn the skills relevant to leadership in the same way you learned all of your other skills. You practice, practice, practice.
A few important steps to keep in mind includes: setting your mind in advance that you can be a positive leader; valuing how learning to lead can help you maintain a positive character and finally, don't quit trying.
If you have experienced being in a leadership position, then you are well aware of how much responsibility it takes to be a leader. Remember, there are both positive and negative leaders. What category does your leadership style fall into?
Do you motivate others to apply their positive character traits to achieve their goals? Do you treat those who look to you for guidance with respect and compassion? Think carefully about these questions, because being a positive leader includes all of these and more.


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Property Showcase
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Being informed is the key to a smooth and successful home purchase. We want to arm you with the knowledge to make the best decisions and save you valuable time, money, and eliminate the stress. A brief phone conversation with us may be life-changing. Whether you are looking to purchase a home for you and your family to live in, or if you're looking for an investment property, connect with us today.
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What Others Say About Us!
From: Pat H
To: TMC Property Solutions
Date: 20 April 2015
Dear Mr. Cook,
Earlier this year I purchased a home from your company on the west side of Fort Worth. I am writing to let you know how much I appreciate all of the help and support that you gave me which allowed me to purchase this home. The home is a dream come true for me and has greatly improved the quality of my life. I am convinced that your diligence and willingness to help out in any way that you could made it possible for me to be living in this lovely home. I also appreciate your quick response to any questions that I have had or any small issues that have come up even after living in the home for several months. Not once in this whole process have I ever received anything but quick, kind and courteous help from TMC Property Solutions.
Thank you for all of your help and support,
Pat H
From: Jim and Lee R
To: TMC Property Solutions
Date: 19 Sept 2015
My wife and I purchased our new home recently. Closing on our new home was a catastrophe, it took 12 weeks!!! Although this was completely due to the finance company, we did not want to chance another drawn out affair and the thought of making house payments on a home we did not live in until it sold was troubling. I had seen Tim's truck from TMC Property Solutions around town so I called him out of the blue. He quickly responded to me with several offers and we came to an agreement on the spot. There was always an open line of communication and we were kept updated at all times. After the first meeting we closed in short order. I feel that Tim along with the work of his very pleasant team saved us several thousands of dollars that we would have spent on mortgage payments, electricity, water, taxes, etc. TMC Property Solutions comes highly recommended from us.
Jim and Lee R


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Wayne S. Walker is a homeschooling father and a book reviewer. His book reviewing began several years ago when his older son, then probably ten or twelve, picked up a book at the library and asked if he could read it. It was written by a modern author but said to be based on a famous series of children's books from the earlier part of the twentieth century that they liked, and it had a recommendation on the back from a well known children's author. So Wayne and his wife said yes. Well, after their son read it, Wayne picked it up to read it and found that it was NOT for children (or should not be for children) because it had a very vulgar scene in it with quite explicit language. Therefore, he determined that he would preview every book that their children read (unless they had specific recommendations fromtrusted sources).
Wayne began posting his reviews of books on a homeschooling e-mail list and people started to express appreciation for them. He was editing (and still continues to edit) a free e-mail homeschooling newsletter called Biblical Homeschooling ( firstname.lastname@example.com or http://groups.yahoo.com/group/biblicalhomeschooling ) and began including his reviews, along with those of others, in the newsletter each month. This led to his being asked to provide reviews for several homeschool-related publications and websites, such as The Old Schoolhouse and Home School Enrichment magazines, and Homeschoolbuzz. Then, for the last couple of years, he has been a regular book reviewer for Stories for Children Magazine, an on-line web-zine for young people ages 3-12.
Recently, SFC has closed its book review/library donation program for the time being. However, because of Wayne's interest in and love of good children's literature, and because he is a homeschooling father who understands the fact that homeschooling families love to read, he therefore wants to bring to the homeschooling community information about new children's books and other good children's literature, so he has started a book review webblog which will be entitled Home School Book Review at http://www.homeschoolblogger.com/homeschoolbookreview/ . He plans to continue the book review/library donation program of SFC.
The way the program works is that when Wayne finishes reading and reviewing a book that is donated, he will post the review on his weblog ( http://www.homeschoolblogger.com/homeschoolbookreview/ ) and also send an e-mail with a copy to let the author/publisher know that it has been posted, along with permission to go ahead and use any part or all of the review for marketing efforts. He will also post it on Amazon.com and Barnes & Noble if the book is available from those sources. Finally, he will donate the book to the children's section of their local library, the Bryan-Bennett Public Library of Salem, IL.
Therefore, if any authors, publishers, literary agents, etc., want Wayne to review any children's books and post the reviews on the new blog, he will be happy to do so. You can contact him at firstname.lastname@example.com for more information about the program and how to participate. You may also feel free to add this e-mail address to your list of those to receive notices about new books to review.
Any books for children, ages 3-12, and for teens, whether fiction or non-fiction are welcome (including picture books, adventure, fantasy, science fiction, historical fiction, mystery, and biography); also books on topics related to homeschooling or parenting and to apologetics will be accepted. Please be advised that all books will be reviewed from a Biblical Judaeo-Christian worldview. Again, the contact e-mail address is email@example.com.
New Home School Book Review Site
Our friend Wayne Walker has been a frequent contributor to the children's book reviews on this site. Now Wayne has decided to strike out on his own with a new website devoted to homeschool book reviews. We wish Wayne the best of success in this new venture. Here is his recent press release:


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Blood Pressure (BP) Monitor
What Is A BP Monitor?
- A blood pressure monitor measures and records your blood pressure over a 24 hour period.
How Is A BP Monitor Performed?
- At your appointment a technician will apply the monitor to you for you to take home for the 24 hour period.
- The monitor will take your BP every 30minutes during the day and every hour at night.
- While you are wearing the monitor you are required to do everything as per normal in your day to day life except for showering.
- You cannot shower while you have the monitor on, nor should you remove the monitor.
- When you note the monitor pumping up, if safe to do so, relax your arm down by your side while the monitor takes your BP.
What to Expect?
- As the monitor records your BP every 30 minutes during the day and every hour at night, you may not have the most restful 24 hours.
- You are required to return the BP monitor the following day.
- We need time to analyse the recordings, as such, you will not be given the results immediately.
- If referred by your GP, your results will be sent to your GP the following week.
- If your cardiologist ordered the test, your cardiologist will discuss the results with you at your next appointment.


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Broccoli and Bean Sprouts Stir Fry
- 7 Months ago
- 441
- 4.0
Bean sprouts are rich in nutrients like vitamin C, protein, and fiber which keep you full and improve the function of insulin in the body. Mix these nutritious sprouts with colorful vegetables to make a low calorie snack that is both tasty and nutritious.
Preparation Time: 10 minutes
Cooking Time: 5 minutes
Makes: 4 servings
Bean Sprouts- 1 cup
Broccoli (Roughly Chopped)- 1 cup
Thinly-Sliced Capsicum- ½ cup
Sliced Baby Corn- ¼ cup
Finely-Chopped Garlic- 2 tsp
Dried Thyme- 2 tsp
Oil- 1 ½ tsp
Chili Flakes- To taste
Salt- To taste
• Blanch broccoli and baby corn in boiling water and keep aside.
• Heat 1 ½ tsp oil in a wok or a non-stick kadai, on high flame.
• Add garlic and sauté for a minute.
• Now add the bean sprouts, broccoli, capsicum and baby corn and stir fry on high flame for a few seconds.
• Add the thyme, chili flakes, and salt, and cook for 2 minutes.
• Serve hot.
You can also make use chicken/ mushrooms/paneer or tofu in the stir fry.Remember to cook the vegetables on a very high flame to prevent mushrooms from letting out any water.