text:
Circulating endothelial microparticles (EMPs) are emerging as biomarkers of chronic obstructive pulmonary disease (COPD) in individuals exposed to cigarette smoke (CS), but their mechanism of release and function remain unknown. We assessed biochemical and functional characteristics of EMPs and circulating microparticles (cMPs) released by CS. CS exposure was sufficient to increase microparticle levels in plasma of humans and mice and in supernatants of primary human lung microvascular endothelial cells. CS-released EMPs contained predominantly exosomes that were significantly enriched in let-7d, miR-191; miR-126; and miR125a, microRNAs that reciprocally decreased intracellular in CS-exposed endothelium. CS-released EMPs and cMPs were ceramide-rich and required the ceramide-synthesis enzyme acid sphingomyelinase (aSMase) for their release, an enzyme which was found to exhibit significantly higher activity in plasma of COPD patients or of CS-exposed mice. The ex vivo or in vivo engulfment of EMPs or cMPs by peripheral blood monocytes-derived macrophages was associated with significant inhibition of efferocytosis. Our results indicate that CS, via aSMase, releases circulating EMPs with distinct microRNA cargo and that EMPs affect the clearance of apoptotic cells by specialized macrophages. These targetable effects may be important in the pathogenesis of diseases linked to endothelial injury and inflammation in smokers.
The release of heterogeneous (0.1–5 μm) microparticles (MPs) from plasma membranes into the extracellular space following cell activation or apoptosis contributes to inter-cellular communication, with implications in inflammation, thrombosis and angiogenesis1,2,3. Endothelial microparticles (EMPs) are increased in the blood of individuals exposed to cigarette smoke (CS) who develop emphysema, a form of COPD characterized by permanent enlargement of airspaces4,5,6. Since not all smokers develop emphysema, these findings implicate EMPs as potential biomarker of disease risk, phenotype, or severity. However, a precise structural and functional characterization of EMPs released in response to CS is lacking and the mechanism of EMPs release is not known.
Circulating MPs (cMPs) comprise endothelium- as well as leukocyte- or platelet-derived MPs. EMPs released in response to CS may include exosomes (30–150 nm), membrane particles (100 nm–1 μm), and/or apoptotic vesicles (1–5 μm)7. Along with size, several other markers distinguish these populations. For example, exosomes, unlike apoptotic vesicles, originate intracellular and while devoid of histones1, they express CD63 and transport RNA/ microRNAs8,9,10. Whereas externalization of phosphatidylserine (PS) has been identified on both apoptotic bodies and exosomes, it is absent on membrane particles. A precise characterization of MPs types released following exposure to soluble components of CS that reach circulation following CS inhalation has not been reported.
Since excessive apoptosis of pulmonary capillary endothelial cells in response to CS is a central mechanism of alveolar wall destruction11,12, we interrogated the role of signaling pathways typically involved in endothelial apoptosis in EMPs release, such as the activation of acid sphingomyelinase (aSMase). This interest was further spurred by a recent report demonstrating that aSMase was necessary and sufficient for MPs release from glial cells13. aSMase is responsible for the production of ceramide in cholesterol-rich membrane microdomains by catalyzing the hydrolysis of sphingomyelin. Typically activated by various stressors, including oxidative stress and CS14,15, ceramide production is involved in both apoptosis11,16 and inflammatory activation12 of lung endothelial cells. Furthermore, recent reports implicate ceramide production in plasma membrane repair such as extrusion and elimination of patches of damaged plasma membranes, in an attempt to mitigate injury17,18. This led us to interrogate the role of aSMase in the mechanism of CS-induced EMPs release.
The biological function of CS-induced EMPs is unknown. The release of ceramide-enriched EMPs may account for increased ceramides in the plasma of smokers19 or in other conditions characterized by endothelial apoptosis. In turn, miRNA-carrying exosomes could account for paracrine or epigenetic signaling associated with pulmonary and systemic vascular damage or other biological activities relevant to COPD pathogenesis20. Similar to apoptotic cells, the burden of cMPs plasma levels is determined by the balance between MPs release and MPs clearance by the monocyte-macrophage system21,22. MPs themselves may impact monocyte-macrophage function, but this effect appears to be specific to the cell type from which MPs originate. For example, MPs released from apoptotic platelets promote an anti-inflammatory macrophage phenotype22, whereas MPs released from monocytes enhance peripheral blood monocytes derived macrophage (PBMDM)’s engulfment of antibody-coated red blood cells23, a pro-inflammatory trait. Since the role of CS-induced EMPs has not been reported yet, we focused our investigation on their impact on specialized phagocytes. Others and we have shown that CS and ceramides decrease apoptotic cell clearance by macrophages (efferocytosis), via alterations of the cytoskeleton and impaired intercellular membranes fusion24. We therefore hypothesized that MPs released during CS exposure will inhibit PBMDM efferocytosis.
Using human primary human lung endothelial cells, mouse models and plasma samples from human subjects, we demonstrate that endothelial cells release ceramide-rich apoptotic and exosomal EMPs in response to CS via a mechanism that involves the activation of aSMase. Furthermore, CS-induced EMPs and mouse cMPs carry specific miRNA cargo signatures and are functionally active, impairing PBMDMs and splenic macrophages efferocytosis function.
Microparticles released by CS are enriched in exosomes and membrane particles
We first determined that our methodology of MPs isolation was consistent with previous reports of increased EMP in plasma of COPD patients4,5,6. Under an IRB-approved protocol, we isolated MPs using freshly collected plasma from healthy non-smoker controls (n = 8) and smokers with or without COPD, stratified by severity using GOLD 2007 (n = 17, details in Supplementary Table 1). Following platelet depletion and an ultraspeed centrifugation step, we quantified EMPs using flow cytometry, defining EMPs as CD31+/CD42b− events. In this small cohort, we identified significant increases in circulating EMPs in smokers and those with mild COPD, when compared to healthy non-smokers (Fig. 1A). Consistent with previous reports using the largest and best characterized patient cohort6, those with advanced COPD had fewer circulating EMPs. In addition to the increased absolute values, we also measured marked relative increases of EMPs (as % of total cMPs) in smokers and mild COPD compared to healthy individuals (Fig. 1A inset).
We next investigated in cell cultures and mouse models whether direct exposure to soluble CS components expected to be absorbed in the circulation is sufficient to release EMPs from lung endothelium, independent of complex circulatory mediators that may be related to other structural alveolar cell (e.g. epithelial) injury, inflammation, or other factors associated with a chronic condition such as COPD. We exposed primary human lung microvascular endothelial cells (HLMVEC) obtained from non-smokers to soluble components of CS at previously determined non-lethal concentrations of aqueous CS extract25. Compared to cells exposed to ambient air extract as control (AC), CS exposure (2–5%, 2 h) doubled the EMPs release from both HLMVEC and from primary human pulmonary artery endothelial cells (HPAEC) (Fig. 1B). We then used time-lapse microscopy to directly visualize the EMPs release during CS exposure (1 h) of HLMVEC. Following transfection with red fluorescence protein targeting the myristoylation/palmitoylation sequence of lymphocyte-specific protein tyrosine kinase, we could visualize in red the plasma membrane of endothelial cells (Fig. 1C–E and Supplementary videos 1–3). In contrast to control cells that occasionally extended lamellipodia following addition of AC, those exposed to CS contracted their body, releasing EMPs mostly from tips of retracting filopodia-like structures. Similarly, cMPs isolated from the plasma of volunteers visualized by transmission electron microscopy (TEM; Fig. 1F) demonstrated membrane-bordered structures in the submicron size range. While these methods allowed visualization of MPs released by CS, they may not be sufficiently sensitive to detect exosomes. To more accurately define the size of CS-induced EMPs, we used NanoSight NS300 technology, which quantified the vast majority of CS-induced EMPs from HLMVEC as having diameters of 50 nm to 200 nm with a mode of 129 nm, which overlaps with exosomes size range of 30–150 nm (Fig. 1G). We also detected a doubling in larger size microparticles released in response to CS, e.g. 262–277 nm, 372–387 nm and 790–820 nm (Fig. 1G and inset), classifying them as membrane particles1, consistent with our microscopy data. Taken together, these results suggest that CS exposure releases a mixed population of both exosomes and membrane particles.
To ensure that findings obtained in primary humans cells were not cell-culture specific we corroborated these data with a controlled in vivo CS exposure model in mice and ex-vivo mouse lung endothelial cell (MLEC) studies, that allowed for further mechanistic interrogations. The cMPs abundance in plasma of C57BL6/J mice markedly and significantly increased following CS exposure, compared to littermate mice exposed to ambient air (Supplementary Figure 1A). Since mouse cMPs encompass circulating EMPs and MPs released by other cells (e.g. inflammatory cells), we investigated if, similarly to human cells, primary mouse lung endothelial cells (MLEC) release more EMPs in response to CS exposure. There was a significant increase in EMPs released from 10%CS-exposed MLEC which reached ~3-fold after 24 h (Supplementary Figure 1B). The population of EMPs released from MLEC likely contained a mixed population as it did in human cells, based on several complementary methods. First, sizing Nile-Red labeled EMPs against fluorescent polystyrene microbeads using flow cytometry (Supplementary Figure 1B Inset) identified membrane particles. Flow cytometry indicated that CS-induced EMPs had markedly increased histone expression (Supplementary Figure 1C), phosphatidylserine (PS) externalization (detected by annexin V staining, Supplementary Figure 1D) and CD63 (Supplementary Figure 1E). Whereas during control conditions, only half of the CD63-expressing EMPs were positive for PS (Supplementary Figure 1E inset), following CS exposure, almost all CD63-expressing EMPs co-expressed PS (Supplementary Figure 1E inset). These results suggest that CS exposure of MLEC, similar to that of HLMVEC releases a mixed population of apoptotic membrane particles and exosomes.
Microparticles released by CS are enriched in miRs let-7d, −126, −125-5p, −22 and −191
To further confirm the intracellular origin of the majority of CS-induced EMPs, we investigated if they carry microRNA (miRNA) and whether CS distinctly packages certain miRNA for export in EMPs. EMPs were isolated from supernatants of CS-exposed HLMVEC (n = 5 non-smoking donors, 5%, 2 h) and miRNA was detected in EMPs following total RNA isolation and hybridization to miRNA arrays. CS exposure of HLMVEC significantly increased the abundance of several miRNAs released in EMPs (Supplementary Table 2; heat map in Fig. 2A), the most affected being let-7d, which increased by more than 2-fold in EMPs released by CS, compared to control EMPs. Using a similar approach, we identified that the most upregulated circulating miRNAs in the mouse cMPs fraction following CS (heat map in Fig. 2B) were miR-142, miR-126 and miR-706 (Supplementary Table 3). We next interrogated which exosomal miRNAs that originate from the pulmonary endothelium are significantly enriched in the systemic circulation. Since miRNAs are highly conserved and lung-specific EMPs cannot be isolated from a pool of cMPs, we performed meta-analysis of microarray data sets obtained from human cell EMPs and mouse cMPs. The metaanalysis identified the miRNAs let-7d, −126, −125-5p and −22, as the most significantly up-regulated by CS exposure in both lung cell-derived EMPs and mouse cMPs (Table 1), suggesting their potential pulmonary endothelial origin in the systemic circulation. Several miRNAs, such as miR-3960 were not regulated by CS, but were highly abundant in both EMPs and cMPs (Supplementary Table 3). Validation of CS-induced increases in miR let-7d, 126, 125-5p and 22 in EMPs detected by microarrays was accomplished with real time qPCR, to which we added measurement of miR-191, the circulating levels of which were reported increased by CS in human plasma26. Since control (housekeeping) miRNAs have not been validated yet for exosomes, we used the same starting RNA material (mass), in which we spiked-in Caenorhabditis elegans miRNA cel-mir-39, as endogenous control. For the detection of all miRNAs, a CT < 35 was set as cut-off value. All the above miRNAs were selectively enriched in EMPs, but not intracellular, where their levels were actually significantly decreased (Fig. 2C). This suggested that CS-induced miRNAs dispatching in EMPs is selective, rather than nonspecifically reflecting intracellular miRNA profiles.
Microparticles released by CS are enriched in C16- and C24-ceramides
We have previously shown that CS increases ceramides in mouse lungs27 and in cultured HLMVEC and MLEC11,16, where they act as mediators or effectors of cell stress responses, including apoptosis. Moreover, we unexpectedly detected lipophilic ceramide species in aqueous cell cultures supernatants and acellular BAL fluid28. We hypothesized that ceramides released from cells may be carried in aqueous environments by MPs. Interestingly, CS-released EMPs from MLEC were >10-fold enriched in ceramides compared to control EMPs (Fig. 2D). The relative increase in ceramide in EMPs (10.8-fold) following CS was significantly larger than their intracellular increase (1.6 fold), measured within the same timeframe (Fig. 2E), suggesting enrichment of ceramide in EMPs. The ceramide species identified in EMPs were similar to those within cells, with C16-ceramide and C24-ceramide predominaing (Supplementary Table 4). The distribution of ceramide species in cMPs isolated from human plasma was similar to that in whole (unfractionated) human plasma (Supplementary Figure 2A–C), suggesting that cMPs may be important carriers of ceramides in plasma of smokers and COPD individuals.
Acid sphingomyelinase (aSMase) is required for CS-induced microparticle release
The increase in EMP ceramides and the rapid kinetics of cMPs release following CS exposure in vivo led us to investigate the aSMase pathway of ceramide-synthesis, an enzymatic pathway that is rapidly activated by stress. Coinciding with the timing of cMPs release, we measured increased soluble aSMase activity in plasma of human subjects (Fig. 3A) and mice exposed to CS (Fig. 3B). Since endothelial cells exhibit the most abundant levels of aSMase and can contribute to soluble aSMase levels in plasma, we interrogated whether endothelial aSMase is involved in CS-induced EMP release. Treatment with an aSMase inhibitor (imipramine) markedly decreased CS-induced EMPs from MLEC (Fig. 3C). We next isolated primary MLEC from Smpd1−/− mice or wild type littermate and propagated them in culture. Following ex vivo CS exposure (10%, 24 h), aSMase-null endothelial cells produced significantly fewer EMPs compared to wild type endothelial cells (Fig. 3D). In vivo, transgenic mice with complete loss of aSMase (Smpd1−/−) released significantly fewer cMPs in plasma following CS exposure (3 h) compared to wild type mice (Smpd1+/+) (Fig. 3E). In contrast, cMP abundance was increased in plasma of mice following endothelial-specific conditional transgenic overexpression of aSMase (Fig. 3F). These findings suggest that aSMase activation is necessary for CS-induced EMP release.
Microparticles inhibit macrophages efferocytosis
Since PS-positive MPs can engage specialized receptors for apoptotic cell engulfment, EMPs may compete with other PS-expressing targets such as apoptotic cells, for efferocytotic clearance. To investigate this effect, we used PMA-stimulated human monocytes (THP-1 cell line) co-incubated ex vivo with apoptotic cells (UV-irradiated human Jurkat T-cell line) in the presence or absence of EMPs or cMPs. When co-incubated with phagocytic cells and apoptotic cells, all EMPs inhibited efferocytosis, but those shed by CS-exposed HLMVEC had, at the same concentration, a more potent effect (Fig. 4A). To determine if this effect is cell-of-origin-specific, we compared EMPs with monocyte-derived MPs, which failed to significantly inhibit efferocytosis at equal or even 4-fold higher concentrations (Fig. 4A). The EMPs inhibitory effect was related to their abundance, since aSMase inhibition reduced the number of EMPs released by CS and the EMPs collected from imipramine-treated HMLVECs had significantly less inhibitory effect of efferocytosis (Fig. 4B). Similar to EMPs, cMPs isolated from individuals with COPD significantly inhibited apoptotic cells efferocytosis by monocytes (Fig. 4C). To determine if this effect of EMPs is recapitulated in vivo, we employed a murine model described by Subramanian et al.29. In this model, Cell Tracker Green-labeled apoptotic splenocytes are injected intravenously into mice and their in situ engulfment by splenic macrophages is quantified using flow cytometry of spleen tissue homogenates (Fig. 4D). Mice injected with EMPs collected from CS-exposed MLEC supernatants had significantly decreased efferocytosis of apoptotic targets by splenic F4/80+/CD11b+ macrophages, when compared to mice injected with a similar number of EMPs derived from AC-exposed MLEC or with vehicle control (PBS) (Fig. 4D–E). Similarly, mice injected with cMPs isolated from CS-exposed wild type littermate mice had decreased in situ splenic macrophage efferocytosis, when compared with mice injected with cMPs isolated from the same volume of plasma from ambient air-exposed mice. Unlike cMPs from wild-type mice and consistent with the EMPs effects on ex vivo efferocytosis, cMPs isolated from CS-exposed aSMase (Smpd1−/−) mice had a lesser impact on in situ splenic macrophages efferocytosis (Fig. 4F).
Our results indicate that acute exposure to soluble components of CS is sufficient to stimulate the release of EMPs consisting of a mixed population of exosomes and membrane particles of much smaller size than apoptotic bodies (3–5 μm). CS-induced EMPs required aSMase activation for their release and encompassed exosomes that carried specific miRNA signatures. In addition, CS-induced EMPs were enriched in ceramides and thus may represent major carriers of these sphingolipids in plasma. The significant inhibitory effect of EMPs on efferocytosis suggests that their clearance may compete with that of apoptotic cells, implicating these membrane structures in conditions associated with excessive apoptosis.
The release of EMPs as a cellular stress response to CS exposure is not surprising, since human umbilical vein endothelial cells release EMPs after activation by inflammatory cytokines, LPS and oxidative stress30; and circulating EMPs are increased during multiple conditions characterized by systemic endothelial cell damage31. Our findings indicate that smoking exposure itself releases EMPs, yet further investigations are needed to explain why EMPs were found (both in our small and other much larger cohorts) to be present in higher numbers in the plasma of those smokers with early COPD/emphysema compared to smokers with advanced disease4,5,6. One possibility is that in advanced disease there is loss of pulmonary capillary beds with loss of vascular membrane surface area, or that signals leading to MPs shedding are lost with advanced disease.
Our experimental methods differed from earlier studies by incorporating ultraspeed centrifugation (which eliminates debris and protein aggregates) and depletion of platelet-derived cMPs from plasma for both experimental and clinical samples. Using this methodology, we found that cMPs generated during the acute CS exposure models shared several features with cMPs isolated from subjects with COPD, such as a relative increase in C16-ceramide and functional inhibition of PBMDMs efferocytosis. Given the potential importance of EMPs to clinical phenotyping (for example identifying a subset of at-risk smokers32), it will be useful for the field to standardize EMPs and cMPs isolation and quantification.
Our finding of ceramide-containing cMPs provides evidence for alternative carriers of these highly lipophilic molecules in aqueous acellular environments, such as plasma or bronchoalveolar lavage fluid. The existence of bioactive paracellular ceramides has long been suspected11,27 and attributed to (lipo)protein carriers. It remains to be demonstrated if ceramide-rich cMPs exert ceramide-specific biological effects, which could render them a target for therapy for unwanted outside-in ceramide signaling such as that involved in lung endothelial barrier dysfunction and apoptosis16,25. The release of ceramide-rich EMPs during CS-exposure by HLMVEC coincided with surface PS and histone expression in EMPs and was dependent on aSMase activation, an enzyme critical for stress-induced apoptosis in multiple cell types. This would be consistent with an apoptotic origin of CS-released EMPs, further supported by reports of EMPs shed in the circulation that parallel endothelial apoptosis4, by excessive pulmonary endothelial apoptosis in emphysema11 and by expression of apoptosis and endothelial markers on cMPs from smokers and COPD patients4,5. However, MPs may be released by alternative mechanisms related to inflammatory cell activation33,34, or to the elimination of the patches of damaged plasma membranes during repair35. Indeed, concomitantly with or preceding apoptosis, CS exposure causes endothelial cells inflammatory activation25 and induces ER stress36. We have not detected the endothelial activation marker CD62E on CS-released EMPs (data not shown), but identified, using NanoSight sizing, a marked release of exosomes. Although the majority of CS-induced EMP stained positive with Annexin V, non-apoptotic MPs such as exosomes have also been shown to externalize PS. Furthermore, only a small proportion of EMPs were the size of membrane particles, which may include the apoptotic vesicles. These results suggest that apoptosis is not the sole or predominant mechanism of CS-induced EMP release. Furthermore, we were unable to detect apoptotic vesicles following acute CS exposures in vitro or in vivo and aSMase activation was indispensable to the release of cMPs in vivo (where few apoptotic MPs were detected). These findings may indicate that although signaling shared with apoptosis was required, the completion of apoptosis may not be necessary for CS-induced MP release. Indeed, our time-lapse microscopy study showed release of EMPs early during cellular contraction, simultaneous with retraction/resolution of filopodia, without appreciable cell death.
Our results revealed a new function of aSMase, that of release of ceramide-rich EMPs in response to CS exposure. The mechanisms by which aSMase may exert this effect at the plasma membrane, where it breaks down sphingomyelin and enriches the outer membrane leaflet in cholesterol37, is via promoting membrane fluidity, destabilization and blebbing38, a mechanism which may be instrumental in EMPs release. Although our transgenic approaches implicate the lysosomal form of aSMase (which can translocate to the plasma membrane), they do not rule out soluble aSMase involvement. This notion is supported by our novel finding of significantly increased levels of circulating aSMase in COPD patients plasma. In addition to involvement in generating EMPs, circulating aSMase may breakdown circulating sphingomyelin, which would be consistent with decreased plasma levels of this sphingolipid that we recently described in a large COPD cohort19. Several explanations may exist to reconcile the findings of increased plasma aSMase activity in COPD of all severities, while we and others6 have found EMPs elevations in smokers and individuals with mild COPD, rather than severe COPD. It is possible that the measurements of aSMase activity are more sensitive than EMPs counts to detect COPD; that EMPs abundance is more closely related to certain disease phenotypes that are not linked to GOLD severity; or that a plateau is reached in aSMase-dependent EMPs release which may be proportional to the lung capillary surface area, (lost with increasing COPD severity).
It is widely recognized that exosomes represent the main transporters of circulating miRNAs and the effects of exosomes in the recipient cells are dependent on the transfer of functional miRNAs8,10,39. Identification and validation of increased hsa-let-7d and hsa-mir-125a, −126 and −191 in CS-induced EMPs are novel and may be important in predicting potential function of exosomes as paracrine effectors in COPD. For example, the release of miRNAs by microvascular pulmonary endothelial cells may modulate the phenotype of distant endothelial cells of large systemic- or pulmonary arteries via exosomes uptake. Although we did not interrogate the role of this miR signature in the inhibition of macrophage efferocytosis by EMPs, it is conceivable that miRNAs could modulate this function, given the known roles of let-7d in reducing cell motility40,41 and that of mir-126 in the cytokine secretion of activated macrophages42. Predictions based on the validated targets and recognized functions of the identified miRNAs in our studies include vascular and tissue remodeling, inflammation and injury repair. For example, let-7d induces cellular apoptosis43; mir-126 modulates the repair of damaged endothelium44; and circulating mir-191 is increased in smokers26 and individuals with pulmonary hypertension45. In addition, the relative intracellular loss of respective miRNAs may untowardly affect the function of parent endothelial cells.
We are reporting a new function of CS-released EMPs and cMPs, of hindrance of apoptotic cell clearance (efferocytosis). By impairing reparative macrophage efferocytosis in the systemic or pulmonary microcirculation, circulating EMPs may contribute to the pathogenesis of inflammatory diseases. In principle, this mechanism could affect marginated, but not yet transmigrated monocyte-derived macrophages, which have been recently identified by intravital microscopy in several microcirculatory beds in the kidney, mesentery and lung46,47,48. Through these effects, EMPs can actively impact local inflammatory responses. The mechanism by which EMPs inhibit efferocytosis may be linked to competition for PS-binding receptors, high ceramide content, or posttranscriptional modifications via miRNA cargo. The inhibition of efferocytosis by high concentrations of AC-derived EMPs together with the weaker effects of CS-derived EMPs on efferocytosis during conditions of aSMase loss of function suggest a competitive effect of EMPs with apoptotic cells for phagocyte engulfment. In addition, critical levels of ceramide in EMPs may hinder efferocytosis, which is consistent with our report that increasing ceramide content in a target lipid vesicle bilayer significantly inhibited macrophages efferocytosis, due to increased membrane stiffness and impaired intercellular membrane fusion24. We did detect sphingosine-1 phosphate (S1P) within cMPs (ranging from 0.83 to 1.65 pmoles/cMP in healthy to 1.02 to 2.38 pmoles/cMP in COPD individuals). The differences between these two groups were not statistically significant (data not shown). Therefore, the net result of CS-released cMPs on efferocytosis may be due to higher ceramide content without proportional increase in S1P. This is consistent with our recent report that plasma S1P was not increased in COPD patients enrolled in the COPDGene cohort and was rather negatively correlated with emphysema severity and risk of COPD acute exacerbation19. In contrast to our findings in cMPs, an interesting study identified hepatocyte-derived exosomes that were devoid of S1P, but contained enzymes from the S1P synthetic machinery that were then transferred to injured mouse hepatocytes to increase recipient cell S1P content and restore homeostasis following ischemia/reperfusion injury49. Although we did not measure sphingosine kinases in CS-released endothelial exosomes, their miRNA content predicts that upon uptake by recipient monocytes, these miRNAs may actually downregulate S1P synthesis enzymes. Compared to healthy individuals, human cMPs from COPD patients contained higher levels of miR124 and miR101 that are predicted to inhibit sphingosine kinase50,51. These results may reflect the specificity of exosome structure and function to the cell of origin (hepatocyte vs. endothelial cell) and to the condition in which they are generated (baseline vs. CS-exposure).
Future investigations will have to interrogate whether EMPs directly contribute to COPD pathogenesis, such as lung parenchyma remodeling or airway disease, a hypothesis which is already supported by correlations between EMPs number and lung function decline in murine models52 or COPD exacerbations in humans5. In addition, EMPs may contribute to all or some of the systemic manifestations of COPD, known as COPD co-morbidities that include cardiovascular events, bone marrow dysfunction, malignancies, impaired cognitive function and skeletal muscle wasting. A link between EMPs and these manufestations is supported by findings of increased circulating MPs in those with lung cancer cachexia53, or in smokers with impaired glucose tolerance, metabolic syndrome and cardiovascular disease54.
In conclusion, we revealed that absorbed soluble components of CS directly increase the number of cMPs in mild COPD patients and in murine and cell culture models of CS exposure, by triggering activation of aSMase, an enzyme classically associated with stress-induced apoptosis. Apoptotic and exosomal EMPs released by CS carry ceramides and specific miRNAs in circulation with potential widespread effects in the vasculature of target organs. In addition, EMPs and cMPs are cleared by phagocytes and compete with apoptotic cells for efferocytosis. These novel findings suggest that EMPs may be a useful indicator of the functional state of the endothelium in smokers and may play key roles in inflammation and vascular remodeling changes in response to CS.
Materials and Methods
Plasma from apparently healthy non-smokers and COPD subjects was obtained after informed consent, using methods that were carried out in accordance with the approved guidelines. All protocols were approved by the IRB at the Indiana University School of Medicine.
Chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO), unless otherwise stated.
HLMVEC, HPAEC, or MLEC were grown in 100 mm dishes to 98–100% confluence and exposed as indicated in low-serum (2% FBS) media. Cell supernatants were centrifuged twice (350g, 8 min, 22oC; 1500g, 25 min, 22oC), to remove floating cells and debris, respectively and underwent a final centrifugation at 100,000g, 2 h, 4oC to isolate EMPs.
Human THP-1 cell line monocytes were plated in 100 mm dishes (2 × 106 cells/dish) in media with PMA (5 ng/mL; 48 h). Following differentiation, cell supernatant was collected and subjected to similar centrifugation steps as described for EMPs.
cMP isolation from human plasma
Healthy non-smokers (n = 8) and COPD (n = 17) subjects were asked to participate in the study under IRB approved clinical protocols. Clinical details are described in the Online Supplement. Blood was drawn from consenting, fasting and resting volunteers using atraumatic butterfly needle system. The first 2–3 mL of blood were discarded and the rest collected in BD Vacutainer Cell Preparation Tubes with sodium citrate. During centrifugation (1600 g, 20 min, 23oC) the gradient gel inside the tube separated the mononuclear cells and plasma from the denser blood components. Next the plasma was further centrifuged (1550 g, 25 min, 23oC) to obtain platelet-poor plasma. Within minutes the platelet-poor plasma was transferred in BD ultracentrifugation tubes. After ultracentrifugation (100,000g, 4oC, 2h) cMPs were used for in various assays: EMPs quantification after CD31 and CD42b immunostaining, ceramides measurements, or efferocytosis assays.
Cell culture experiments
Primary mouse lung endothelial cells (MLEC) were kindly provided by Dr. P. Lee (Yale) or isolated in our laboratory from mouse lung using a magnetic cell separation (MACS) procedure from Miltenyi Biotec (Cologne, Germany)55. Primary HLMVEC and HPAEC were from Lonza (Walkersville, MD). Culture conditions are detailed in Supplementary Material.
Preparation of soluble cigarette smoke (CS) extract is detailed in Supplementary Material.
Time-lapse intravital imaging of cultured cells
HLMVEC were transduced overnight with CellLight® Plasma Membrane-RFP, BacMam2.0 (Molecular Probes). Microscopy was performed as detailed in Supplementary Material. EMP characterization by TEM is detailed in the Supplementary material.
EMPs characterization by NanoSight NS300
EMPs isolated by ultracentrifugation were subjected to a PBS wash to eliminate contamination of proteins and other small cell debris before a second ultracentrifugation step. Final EMPs suspension was analyzed using NanoSight NS300, at a rate of 10 μm/min. Three 60-s videos per sample were used to calculate EMPs mode, mean and total concentration via NTA 3.1 software.
Microparticle miRNA isolation and analysis are described in Supplementary Material.
Ceramide determination and aSMase assays were performed as previously described12 and are detailed in the Online Supplement.
Animal experiments were all approved by and conducted in compliance with the guidelines of the Institutional Animal Care and Use Committees of Indiana University School of Medicine and National Jewish Health. Experimental details are provided in Supplementary Material.
In-vivo and ex-vivo efferocytosis assays are detailed in Supplementary Material. Briefly, mice (C57Bl6, female, 3 month-old) received sequential IV administrations (tail vein) of MLEC-derived EMPs, cMPs, or PBS (60 ul) and, within 15–20 min, fluorescently labeled apoptotic splenocytes (2 × 107). MLEC-derived EMPs were isolated from cultured cell supernatants. To inject the same number of microparticles, EMPs derived from two AC-treated T150 flasks or one CS-treated T150 flask were re-suspended in 60 ul sterile PBS and within minutes administered intravenously into recipient mice. cMPs were isolated from 300 uL plasma of wild type and Smpd1−/− mice exposed to CS for 3 h.To generate apoptotic targets, splenocytes were harvested one-day prior from spleens of healthy C57Bl/6 mice, fluorescently-labeled with Cell Tracker Green (10 ul, 1 mM) and maintained under low serum conditions for 16 h, to undergo spontaneous apoptosis (>85% total cells, detected by Annexin V/7AAD staining and FACS). Recipient animals were sacrificed 3 h after administration of labeled apoptotic splenocytes, spleens were harvested, mechanically digested and the splenic macrophages/dendritic cell population was identified using membrane antigen immunostaining with F4/80 (1:200, clone CI:A3-1 conjugated with AF647) and CD11b (1:160, clone M1/70 eFlour 450) via flow cytometry (BD-LSR Fortesa cytometer with BD FACSDiva 6.0 software).
Statistical Analysis was performed with Prism (GraphPad Software, San Diego, CA), using unpaired Student t-test or ANOVA, as appropriate. When ANOVA was utilized, post-hoc inter-group comparisons were analyzed for statistical significant differences using either Tukey’s (all groups compared to each other), Dunnet’s (groups compared to a control group), or Sidak’s (comparisons of only select pairs of groups) methodology, as appropriate. Statistical significance was accepted at p < 0.05.
How to cite this article: Serban, K. A. et al. Structural and functional characterization of endothelial microparticles released by cigarette smoke. Sci. Rep. 6, 31596; doi: 10.1038/srep31596 (2016).
Thery, C., Ostrowski, M. & Segura, E. Membrane vesicles as conveyors of immune responses. Nature reviews. Immunology 9, 581–593 (2009).
Dignat-George, F. & Boulanger, C. M. The many faces of endothelial microparticles. Arteriosclerosis, thrombosis and vascular biology 31, 27–33 (2011).
Takahashi, T. & Kubo, H. The role of microparticles in chronic obstructive pulmonary disease. International journal of chronic obstructive pulmonary disease 9, 303–314 (2014).
Gordon, C. et al. Circulating endothelial microparticles as a measure of early lung destruction in cigarette smokers. American journal of respiratory and critical care medicine 184, 224–232 (2011).
Takahashi, T. et al. Increased circulating endothelial microparticles in COPD patients: a potential biomarker for COPD exacerbation susceptibility. Thorax 67, 1067–1074 (2012).
Thomashow, M. A. et al. Endothelial microparticles in mild chronic obstructive pulmonary disease and emphysema. The Multi-Ethnic Study of Atherosclerosis Chronic Obstructive Pulmonary Disease study. American journal of respiratory and critical care medicine 188, 60–68 (2013).
Gyorgy, B. et al. Membrane vesicles, current state-of-the-art: emerging role of extracellular vesicles. Cellular and molecular life sciences: CMLS 68, 2667–2688 (2011).
Valadi, H. et al. Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nature cell biology 9, 654–659 (2007).
Mause, S. F. & Weber, C. Microparticles: protagonists of a novel communication network for intercellular information exchange. Circulation research 107, 1047–1057 (2010).
Diehl, P. et al. Microparticles: major transport vehicles for distinct microRNAs in circulation. Cardiovasc Res 93, 633–644 (2012).
Petrache, I. et al. Ceramide upregulation causes pulmonary cell apoptosis and emphysema-like disease in mice. Nature medicine 11, 491–498 (2005).
Petrusca, D. N. et al. Smoking exposure induces human lung endothelial cell adaptation to apoptotic stress. American journal of respiratory cell and molecular biology 50, 513–525 (2014).
Bianco, F. et al. Acid sphingomyelinase activity triggers microparticle release from glial cells. The EMBO journal 28, 1043–1054 (2009).
Mathias, S., Pena, L. A. & Kolesnick, R. N. Signal transduction of stress via ceramide. The Biochemical journal 335 (Pt 3), 465–480 (1998).
Levy, M., Khan, E., Careaga, M. & Goldkorn, T. Neutral sphingomyelinase 2 is activated by cigarette smoke to augment ceramide-induced apoptosis in lung cell death. American journal of physiology. Lung cellular and molecular physiology 297, L125–133 (2009).
Medler, T. R. et al. Apoptotic sphingolipid signaling by ceramides in lung endothelial cells. American journal of respiratory cell and molecular biology 38, 639–646 (2008).
Fernandes, M. C. et al. Trypanosoma cruzi subverts the sphingomyelinase-mediated plasma membrane repair pathway for cell invasion. J Exp Med 208, 909–921 (2011).
Babiychuk, E. B., Monastyrskaya, K., Potez, S. & Draeger, A. Intracellular Ca(2+) operates a switch between repair and lysis of streptolysin O-perforated cells. Cell death and differentiation 16, 1126–1134 (2009).
Bowler, R. P. et al. Plasma sphingolipids associated with chronic obstructive pulmonary disease phenotypes. American journal of respiratory and critical care medicine 191, 275–284 (2015).
Tuder, R. M. & Petrache, I. Pathogenesis of chronic obstructive pulmonary disease. The Journal of clinical investigation 122, 2749–2755 (2012).
Elliott, M. R. & Ravichandran, K. S. Clearance of apoptotic cells: implications in health and disease. The Journal of cell biology 189, 1059–1070 (2010).
Vasina, E. M. et al. Microparticles from apoptotic platelets promote resident macrophage differentiation. Cell death & disease 2, e211 (2011).
Ismail, N. et al. Macrophage microvesicles induce macrophage differentiation and miR-223 transfer. Blood 121, 984–995 (2013).
Justice, M. J. et al. Effects of lipid interactions on model vesicle engulfment by alveolar macrophages. Biophysical journal 106, 598–609 (2014).
Schweitzer, K. S. et al. Mechanisms of lung endothelial barrier disruption induced by cigarette smoke: role of oxidative stress and ceramides. American journal of physiology. Lung cellular and molecular physiology 301, L836–846 (2011).
Takahashi, K. et al. Cigarette smoking substantially alters plasma microRNA profiles in healthy subjects. Toxicology and applied pharmacology 272, 154–160 (2013).
Petrache, I. et al. Superoxide dismutase protects against apoptosis and alveolar enlargement induced by ceramide. American journal of physiology. Lung cellular and molecular physiology 295, L44–53 (2008).
Petrusca, D. N. et al. Sphingolipid-mediated inhibition of apoptotic cell clearance by alveolar macrophages. The Journal of biological chemistry 285, 40322–40332 (2010).
Subramanian, M. et al. An AXL/LRP-1/RANBP9 complex mediates DC efferocytosis and antigen cross-presentation in vivo. The Journal of clinical investigation 124, 1296–1308 (2014).
Combes, V. et al. In vitro generation of endothelial microparticles and possible prothrombotic activity in patients with lupus anticoagulant. The Journal of clinical investigation 104, 93–102 (1999).
Martin, S. et al. Shed membrane particles from T lymphocytes impair endothelial function and regulate endothelial protein expression. Circulation 109, 1653–1659 (2004).
Dinkla, S., Brock, R., Joosten, I. & Bosman, G. J. Gateway to understanding microparticles: standardized isolation and identification of plasma membrane-derived vesicles. Nanomedicine 8, 1657–1668 (2013).
Martinez, M. C. & Freyssinet, J. M. Deciphering the plasma membrane hallmarks of apoptotic cells: phosphatidylserine transverse redistribution and calcium entry. BMC cell biology 2, 20 (2001).
Cordazzo, C. et al. Rapid shedding of proinflammatory microparticles by human mononuclear cells exposed to cigarette smoke is dependent on Ca2+ mobilization. Inflammation research: official journal of the European Histamine Research Society … [et al.] 63, 539–547 (2014).
Abid Hussein, M. N., Boing, A. N., Sturk, A., Hau, C. M. & Nieuwland, R. Inhibition of microparticle release triggers endothelial cell apoptosis and detachment. Thrombosis and haemostasis 98, 1096–1107 (2007).
Malhotra, D. et al. Expression of concern: Heightened endoplasmic reticulum stress in the lungs of patients with chronic obstructive pulmonary disease: the role of Nrf2-regulated proteasomal activity. American journal of respiratory and critical care medicine 180, 1196–1207 (2009).
Slotte, J. P., Hedstrom, G., Rannstrom, S. & Ekman, S. Effects of sphingomyelin degradation on cell cholesterol oxidizability and steady-state distribution between the cell surface and the cell interior. Biochimica et biophysica acta 985, 90–96 (1989).
Chang, M. P., Mallet, W. G., Mostov, K. E. & Brodsky, F. M. Adaptor self-aggregation, adaptor-receptor recognition and binding of alpha-adaptin subunits to the plasma membrane contribute to recruitment of adaptor (AP2) components of clathrin-coated pits. The EMBO journal 12, 2169–2180 (1993).
Hunter, M. P. et al. Detection of microRNA expression in human peripheral blood microvesicles. PloS one 3, e3694 (2008).
Huleihel, L. et al. Let-7d microRNA affects mesenchymal phenotypic properties of lung fibroblasts. American journal of physiology. Lung cellular and molecular physiology 306, L534–542 (2014).
Curry, E., Safranski, T. J. & Pratt, S. L. Differential expression of porcine sperm microRNAs and their association with sperm morphology and motility. Theriogenology 76, 1532–1539 (2011).
Laffont, B. et al. Platelet microparticles reprogram macrophage gene expression and function. Thrombosis and haemostasis 115 (2015).
Johnson, C. D. et al. The let-7 microRNA represses cell proliferation pathways in human cells. Cancer research 67, 7713–7722 (2007).
Jansen, F. et al. Endothelial microparticle uptake in target cells is annexin I/phosphatidylserine receptor dependent and prevents apoptosis. Arterioscler Thromb Vasc Biol 32, 1925–1935 (2012).
Wei, C. et al. Circulating miRNAs as potential marker for pulmonary hypertension. PloS one 8, e64396 (2013).
Carlin, L. M. et al. Nr4a1-dependent Ly6C(low) monocytes monitor endothelial cells and orchestrate their disposal. Cell 153, 362–375 (2013).
Carlin, L. M., Auffray, C. & Geissmann, F. Measuring intravascular migration of mouse Ly6C(low) monocytes in vivo using intravital microscopy. Current protocols in immunology /edited by John E. Coligan … [et al.] Chapter 14, Unit 14 33 11–16 (2013).
Presson, R. G. Jr. et al. Two-photon imaging within the murine thorax without respiratory and cardiac motion artifact. The American journal of pathology 179, 75–82 (2011).
Nojima, H. et al. Hepatocyte exosomes mediate liver repair and regeneration via sphingosine-1-phosphate. Journal of hepatology 64, 60–68 (2016).
Xia, J. et al. miR-124 inhibits cell proliferation in gastric cancer through down-regulation of SPHK1. The Journal of pathology 227, 470–480 (2012).
Chen, M. B. et al. MicroRNA-101 down-regulates sphingosine kinase 1 in colorectal cancer cells. Biochemical and biophysical research communications 463, 954–960 (2015).
Liu, H., Ding, L., Zhang, Y. & Ni, S. Circulating endothelial microparticles involved in lung function decline in a rat exposed in cigarette smoke maybe from apoptotic pulmonary capillary endothelial cells. Journal of thoracic disease 6, 649–655 (2014).
He, W. A. et al. Microvesicles containing miRNAs promote muscle cell death in cancer cachexia via TLR7. Proceedings of the National Academy of Sciences of the United States of America 111, 4525–4529 (2014).
Kranendonk, M. E. et al. Extracellular vesicle markers in relation to obesity and metabolic complications in patients with manifest cardiovascular disease. Cardiovasc Diabetol 13, 37 (2014).
Kamocki, K. et al. RTP801 is required for ceramide-induced cell-specific death in the murine lung. American journal of respiratory cell and molecular biology 48, 87–93 (2013).
Kumar, M. et al. Let-7 microRNA-mediated regulation of IL-13 and allergic airway inflammation. The Journal of allergy and clinical immunology 128, 1077–1085 e1071-1010 (2011).
Pandit, K. V. et al. Inhibition and role of let-7d in idiopathic pulmonary fibrosis. American journal of respiratory and critical care medicine 182, 220–229 (2010).
Kishore, S. et al. A quantitative analysis of CLIP methods for identifying binding sites of RNA-binding proteins. Nature methods 8, 559–564 (2011).
Fish, J. E. et al. miR-126 regulates angiogenic signaling and vascular integrity. Developmental cell 15, 272–284 (2008).
Wang, S. et al. The endothelial-specific microRNA miR-126 governs vascular integrity and angiogenesis. Developmental cell 15, 261–271 (2008).
Hergenreider, E. et al. Atheroprotective communication between endothelial cells and smooth muscle cells through miRNAs. Nature cell biology 14, 249–256 (2012).
Zhou, J. et al. Regulation of vascular smooth muscle cell turnover by endothelial cell-secreted microRNA-126: role of shear stress. Circulation research 113, 40–51 (2013).
Meng, S. et al. Downregulation of microRNA-126 in endothelial progenitor cells from diabetes patients, impairs their functional properties, via target gene Spred-1. J Mol Cell Cardiol 53, 64–72 (2012).
Svensson, D. et al. Inhibition of microRNA-125a promotes human endothelial cell proliferation and viability through an antiapoptotic mechanism. Journal of vascular research 51, 239–245 (2014).
Zhu, W. Y. et al. Differential expression of miR-125a-5p and let-7e predicts the progression and prognosis of non-small cell lung cancer. Cancer investigation 32, 394–401 (2014).
Banerjee, S. et al. miR-125a-5p regulates differential activation of macrophages and inflammation. The Journal of biological chemistry 288, 35428–35436 (2013).
Liu, B. et al. MiR-26a enhances metastasis potential of lung cancer cells via AKT pathway by targeting PTEN. Biochimica et biophysica acta 1822, 1692–1704 (2012).
Yu, S. H., Zhang, C. L., Dong, F. S. & Zhang, Y. M. miR-99a suppresses the metastasis of human non-small cell lung cancer cells by targeting AKT1 signaling pathway. Journal of cellular biochemistry 116, 268–276 (2015).
Zhao, B. et al. MicroRNA let-7c inhibits migration and invasion of human non-small cell lung cancer by targeting ITGB3 and MAP4K3. Cancer letters 342, 43–51 (2014).
Goedeke, L. et al. A regulatory role for microRNA 33* in controlling lipid metabolism gene expression. Molecular and cellular biology 33, 2339–2352 (2013).
Wang, Y. Y., Ren, T., Cai, Y. Y. & He, X. Y. MicroRNA let-7a inhibits the proliferation and invasion of nonsmall cell lung cancer cell line 95D by regulating K-Ras and HMGA2 gene expression. Cancer biotherapy & radiopharmaceuticals 28, 131–137 (2013).
Xu, D. et al. miR-22 represses cancer progression by inducing cellular senescence. The Journal of cell biology 193, 409–424 (2011).
Yang, J. et al. MicroRNA-22 targeting CBP protects against myocardial ischemia-reperfusion injury through anti-apoptosis in rats. Molecular biology reports 41, 555–561 (2014).
We acknowledge Dr. Rajesh Khanna (Stark Neurosciences Research Institute at Indiana University School of Medicine), Yuan Gu and Mary VanDemark for expert technical assistance; Drs Dan and Sanda Predescu (Rush University) for useful discussions and assistance with microparticle isolation, Dr. Erich Gulbins, MD (Univ of Essen, Germany) for generating and sharing Smpd1 transgenic mice and Dr. John Carpenter (University of Colorado) for assistance with NanoSight NS300 system and Nanoparticle Tracking Analysis (NTA) software. The microarray experiments were carried out using the facilities of the Center for Medical Genomics at Indiana University School of Medicine, which was funded in part by a grant to HJE from the Indiana 21st Century Research and Technology Fund and by the Indiana Genomics Initiative (INGEN; INGEN is supported in part by the Lilly Endowment). RO1HL077328 (IP), T32 5T32HL091816 and AHA Postdoctoral Grant (SR); the 2012 Alpha-1 Foundation and CHEST Foundation Junior Investigator Clinical Research grant (KAS).
The authors declare no competing financial interests.
About this article
Cite this article
Serban, K., Rezania, S., Petrusca, D. et al. Structural and functional characterization of endothelial microparticles released by cigarette smoke. Sci Rep 6, 31596 (2016). https://doi.org/10.1038/srep31596
This article is cited by
European Journal of Medical Research (2023)
Hypertension Research (2023)
Respiratory Research (2022)
Tumor endothelial cell-derived extracellular vesicles contribute to tumor microenvironment remodeling
Cell Communication and Signaling (2022)
Characterization of pulmonary vascular remodeling and MicroRNA-126-targets in COPD-pulmonary hypertension
Respiratory Research (2022)


text:
To Our Customers:
As you are aware, on March 1, 2013 a series of automatic federal spending cuts, known as sequestration, are set to begin. As a valued customer, we wanted you to be aware this is an issue we are closely monitoring, as its possible mandatory spending cuts — should they begin on March 1 — could have an impact on the physical delivery and cash settlement mechanisms of certain CME livestock and dairy products. In addition, the budget cuts could result in the disruption of USDA reports that are used in the daily calculation of the CME Feeder Cattle Index and CME Lean Hog Index and the monthly calculations used to determine settlement prices for CME Dairy futures products.
As the CME Live Cattle contract utilizes USDA grading/inspection in the delivery process, a furlough of USDA staff may require the Exchange to modify in accordance with Exchange rules the current operational process around delivery/settlement of these products. In addition, CME Group's cash-settled livestock and dairy products could also be impacted in the event the data used to compile these indexes is unavailable. Finally, CME Group's spot call dairy markets could be impacted in the event USDA grading/inspection staff is unavailable effective on March 1, 2013.
The list of CME livestock and dairy contracts that may be impacted, include:
- Live Cattle futures Feb 2013 contract
- Lean Hog futures and options, April 2013 and subsequent contracts
- Feeder Cattle Futures, March 2013 and subsequent contracts
- Milk (Class III and IV), Butter, Cheese, Non-Fat Dry Milk, and Whey March 2013 and subsequent contracts
- Spot Call (Butter, Cheese and Non-Fat Dry Milk), March 1 and subsequent trading days
We continue to closely monitor the potential for sequestration and the effect on our contracts and will keep you apprised of any potential changes to the delivery mechanism or settlement procedures for CME livestock and dairy contracts. Current procedures for the physical delivery of CME livestock and dairy futures can be found in our CME Rulebook - http://www.cmegroup.com/rulebook/CME/.
All customer inquiries should be directed to Tom Clark at 312.930.4595. Media inquiries should be directed to Chris Grams at 312.930.3435.

domain: economics


BitNami Typo Stack 5.3-0 (windows)
Windows AllPlatform :
97.24 MBFile Size :
8/31/2011Date Added :
BitNami Typo Stack Native Installer is an easy-to-install distribution of the Typo application. It includes pre-configured, ready-to-run versions of Apache, MySQL, Ruby, Rails and Subversion so users can get a Typo installation up and running in minutes after answering a few questions. Windows, Linux and Mac OS X operating systems are supported.
DOWNLOADS FROM BITROCK
[ More downloads from Bitrock
store your files on high volume Internet serv
- Web Archive Downloader
A brand new software to download from the web archive.
- BitNami WordPress Stack
- A Powerful CMS Unleashed
A step by step guide with 35 videos showing how to create a website with features like shopping cart, blog, image galleries, surveys, polls. Comes with free software.
- Lanap BotDetect for ASP
ASP CAPTCHA control to prevent automated registrations (spambot) on your website
- SEO in Practice
SEO in Practice is the ultimate must-read free SEO guide for every webmaster
Wizard to Easily Move tables from PC to mySQL Server and keep tables updated.
- Phantom EZ Form
Phantom EZ Form its a webform creator.
- Link Check
Checking your reciprocal link partners made easy
keep track of downloads and page views.


text:
Tag Archives: doctors
A huge variety of negative gastrointestinal symptoms are caused by hidden allergies to gluten, the type of protein found in wheat, rye, and barley.
Tennis elbow, or lateral epicondylitis, as doctors refer to it, is the inflammation of the carpi radialis brevis extensor or the tendon fibers of the forearm connected to joint of the elbow. Because this syndrome is so common, many people are searching for how to cure tennis elbow symptoms. This usually occurs due to the [...]
For your overall health, there are many benefits of vitamin B complex supplements. Altogether, there are eight of them that we need and they are all soluble in water. A B complex vitamin is a nutritional supplement that contains all of them in on tablet or capsule. You must be careful taking these supplements, as [...]
There are many different brands of Human Growth Hormone supplements on the market today. One of them is Sytropin HGH. Human growth hormone is sometimes used to lose weight, to postpone the effect of aging and to build muscle mass. Fighting off the effects of getting old is something that almost everybody wants today. Being [...]
There are many different types of quick weight loss diets available today, and their principles are often incompatible with each other. One of the major problems that dieters experience is failing to stick to any single diet wholeheartedly. In this article we will explore the reasons for this problem, and outline a plan for more [...]
A lot of people get frustrated with dieting and exercise and want additional weight loss help in the form of diet pills. This is often a dicey proposition because there is a fairly shadowy world of marketing surrounding these types of supplements and it can be very difficult to become reasonably informed as to what [...]

domain: medical


text:
Surfers. The word connotes sun, fun, waves, and . . . Drucker on management? In a recent Wall Street Journal article, Peter Drucker extolled the virtues of nonprofit organizations, suggesting they are at their best when held to the same standards as the rest of American business: the ability to produce results. That's been the guiding philosophy of the Surfrider Foundation, an eight-year-old, 16,000-member not-for-profit group of surfing environmentalists headquartered in Huntington Beach, Calif., that scours the coasts, fights coastal pollution, and increases public access to beaches. With a paid staff of just five, executive director Jake Grubb, 42, relies upon a legion of volunteers, and a board of directors that reads like a Who's Who of surfing legends and environmental activists. Eighty-one percent of Surfrider's annual $700,000 budget is supported by membership dues, with the remainder coming from corporate sponsorships (from the likes of Patagonia and Esprit), grants, endowments, and fund-raising events. Fund-raising has not posed much of a problem. "We are offered a lot more money than we take," says Grubb, who so far has turned away donations from, among others, an oil company and a liquor company, which seemed at odds with Surfrider's mission. Sponsors are eager to be identified with recent programs like the Blue Water Task Force, instituted last year, in which local beach users monitor coastal pollution. Surfrider's results-oriented approach has enabled it to grow by 800 members a month and has spurred the formation of 16 chapters in nine states as well as in Australia, England, and France.
-- Alessandra Bianchi n* * *

domain: news


text:
She underlined the need for both sides to exchange initiatives and experience in judicial reform, court system improvement, and legal procedure modernization, noting that Vietnam’s Supreme People’s Procuracy and the DPRK Central Procurator’s Office signed a cooperation pact.
The two countries signed an agreement on judicial assistance, Thinh said, adding that a host of important agreements have been reached across the fields of culture, science-technology, health care, trade, navigation, and civil aviation transportation.
The Vice President stressed that the DPRK was one of the first countries to set up diplomatic ties with Vietnam in 1950, just behind China and the former Soviet Union. Over the years, the bilateral friendship has been strengthened through high-level visits by the two countries’ leaders, she said.
Kang echoed Thinh’s views as saying that his visit is designed to foster the fruitful friendship between the two nations.
He also hailed Vietnam’s socio-economic-cultural achievements and voiced his hope that apart from traditional cooperation areas, the two countries will share experience in judicial affairs towards the stronger development of the judicial sector in each country.

domain: law


text:
This Sunday the Buccaneers will take on the New Orleans Saints, and considering all the things that have gone wrong, they certainly have a tough test, even if they are at home. But even though the Bucs may be underdogs at home, are missing their starting RB, and looked downright awful last week against the 49ers, there is still a positive outlook considering how they have played the Saints in the past. The Bucs have done well against the Saints in the past and head coach Raheem Morris was asked about how his team has had this success.
“We’ve done a nice job of preparing well. The Saints are an awesome team to watch. They’re an awesome team to play against. The competition is very high. They’re a division rival. It’s all those things that are going to be a part of this game that make it a great game and we can’t wait to get out there and play these guys again.” Certainly playing a division rival will help the Bucs get emotionally ready for a game, which even though so early in the season, is a must in game in my opinion.
After starting off the season with such high hopes the Bucs now stand at 3-2, and although that record is considerably good considering the way other teams have started off, it doesn’t help the Bucs because of the division they are in. The NFC South with the New Orleans Saints, always a formidable contender, the Atlanta Falcons, a team who has been down but more than likely will find it’s way and get back on track, and the Carolina Panthers, a team that with Cam Newton is on the rise, is always so competitive.
The Bucs certainly are in one of the tougher divisions in football, and the fact that they have to play the Saints at a time when mostly everything is going wrong doesn’t help at all. The Bucs, in every game this year, have gotten off to a slow start on offense and defense but last week in San Francisco it was just downright embarrassing. The Bucs couldn’t get anything going and Josh Freeman’s 2 interceptions certainly didn’t help. If the Bucs want to have any chance this weekend they will need to start off quickly on both offense and defense and avoid costly turnovers.
DON’T FORGET TO FOLLOW ME ON TWITTER @SportsBeast24

domain: sports


text:
'a pack of lies'
Brazil revives fraud case against embattled U.S. GOP congressman-elect George Santos
George Santos will be sworn in Tuesday at the new Republican congressman representing New York's 3rd Congressional District despite admitting that much of his résumé and biography is a lie, a federal investigation into his finances, local inquiries into his multiple fabrications, and as of Monday, a criminal fraud investigation in his native Brazil, The New York Times reports.
A Brazilian judge approved charges against Santos in 2011 after he admitted to using a stolen checkbook and a false name to make fraudulent purchases near Rio de Janeiro in 2008 — he wrote at least two stolen checks to buy about $1,300 worth of goods, The Wall Street Journal reports. That case was suspended because police could not locate Santos, who moved to New York in 2011, the Times reports, but the Rio de Janeiro prosecutor's office said the case has been reopened now that he has been located.
Prosecutors in Rio will now formally ask Santos, 34, to respond to the charges via a "rogatory letter" sent through Brazil's Justice Ministry to the U.S. Justice Department, the Times reports. Santos can't be compelled to respond, but once he is notified, the case can proceed, and if "Santos does not present a defense in the Brazilian case, he will be tried in absentia." If found guilty, he could get up to five years in jail and a fine.
Santos has admitted to lying about graduating from college and working at Goldman Sachs and Citigroup, acknowledged he owes thousands of dollars in unpaid rent, and has been shown to have lied about being Jewish and the child of Holocaust survivors, among other biographical details. But he recently told the New York Post he is "not a criminal here — not here or in Brazil or any jurisdiction in the world."
The federal investigation of Santos reportedly involves his sudden acquisition of wealth, the more than $700,000 he lent to his 2022 campaign, rent money he may have illegally paid to a company that shares his home address, and a series of other unusual campaign expenditures, including 37 recorded payments of $199.99, one penny below the level requiring a receipt under federal election law.
A handful of Republican officials have withdrawn support for Santos or called for him to resign. "It's clear that his whole biography is a pack of lies," Nassau County Executive Bruce Blakeman, the highest-ranking GOP elected official in the 3rd District, told the Journal. "Obviously, we've all lost confidence in him."
But incoming House Majority Leader Kevin McCarthy (R), who is scrambling for votes to be elected speaker, has repeatedly dodged questions about Santos.


Under – Odd and heavy
Goodbye, Autumn blues – We are back to heavy music. This progressive metal song is not for the faint-hearted. Another headbanging tune.
February 2022. When I teamed up with Christopher to write some prog metal, we unintentionally wrote two songs on the same day. You may know “At Your Command” from my album “Going My Way” – And the other song I wrote that day is called “Under“. Both songs are written in odd meters and have plenty of meter changes. It’s been fun to work on drumming patterns in 8/8 and 6/8 and phrasing the melodies.
Christopher did an excellent job writing lyrics to my moody composition. We are super lucky that Robert joined as guitarist and bassist. His indie rock and punk rock influenced style just gave the song the rough heaviness it needed. Lyrically, “Under” describes a person in a mental emergency. How would it feel like to be unconscious to the outside world, but awake and alert inside? Would it be terrifying seeing the world fall apart in front of you, and losing control?
We are very pleased to show you the result of an awesome collaboration. The song turned out heavier than my initial draft. I had a hard time mixing, because these guitars and screams made me bang my head! You can find the song on SoundCloud and YouTube. More heavy music will follow soon!


Herbal recipes for abortion are unproven & can be dangerous, contrary to social media posts
After the Supreme Court overturned Roe v. Wade on June 24 — dismantling federal protection of the right to an abortion — posts began spreading online with home remedies and herbal concoctions that would purportedly cause a “miscarriage.”
Many of the posts — which are circulating on major platforms, including Facebook, Instagram and TikTok — provide potentially dangerous ingredients without specifying dosage amounts, giving the impression that abortions can be induced with herbs.
One Instagram post detailed a list of homeopathic ingredients and then said, with a winking emoji, “I mean how else will we avoid accidental miscarriages? You’re smart enough to do this too.”
“The myth of the safe, herbal abortion is pervasive,” Dr. Jen Gunter, an obstetrician/gynecologist who has written books on women’s health, explained in a Substack post addressing the issue. “It is a combination of two fallacies, appealing to the belief in the magical power of ancient remedies and that of course, natural is best.”
But, she said, “there are no safe, effective methods of inducing abortion with botanicals.”
While some of the concoctions might result in an abortion, that may just be a byproduct of poisoning in the woman’s body, she explained, since “many herbal abortifacients are quite literally poisons.”
For example, one of the most commonly promoted plants is pennyroyal, a type of mint introduced to the U.S. from Europe. As with many persistent claims on the internet, there’s a grain of truth here. Pennyroyal has historically been associated with abortions, dating back to ancient Greece.
But there’s insufficient evidence that pennyroyal can actually cause an abortion, according to the National Institutes of Health. Rather, the NIH’s National Library of Medicine says that it is likely unsafe to ingest pennyroyal oil because it can cause “serious liver and kidney damage, as well as nervous system damage.”
Further, the NIH says, “There is some evidence that pennyroyal oil can cause abortions by causing the uterus to contract. But the dose needed in order to cause an abortion could kill the mother or cause life-long kidney and liver damage.”
Another plant that’s been included in the lists on social media is parsley, which may sound innocuous but can be toxic if consumed in concentrated doses and is sometimes recommended to be inserted. A woman in Argentina reportedly died from septic shock and infection after trying to end a pregnancy using parsley in 2018.
Generally, doctors and health experts recommend using tested, regulated medications for abortions. Medical abortions, which are done by taking medicine rather than having a physical procedure, accounted for about 28% of early abortions in 2016, according to a recent report from the Centers for Disease Control and Prevention. They are commonly performed by taking two drugs — mifepristone and misoprostol — which can be done at home.
“People have been self-managing their abortions for decades with the support of community organizations and medical experts,” Dr. Nisha Verma, a fellow at the American College of Obstetricians and Gynecologists, said in a statement provided by the organization to FactCheck.org.
“We have seen that people can safely self-manage their abortions with medications like misoprostol. However, some people may also turn to unsafe abortion methods when they feel they have no other option or based on information they are gathering on social media,” she said.
“It is important for people to understand that social media posts can be unreliable and can sometimes propagate misinformation. Misinformation can be harmful because it may lead people to try to end their pregnancies in an unsafe way, potentially exposing them to serious bodily harm. While people can self-manage their abortions in a safe way, spreading misinformation about unsafe methods of abortion is incredibly dangerous,” Verma said.


text:
The Eastern Corridor road constructed through Nkwanta to kpasss during the Mahama administration has been left uncompleted which is making travelling along those areas difficult especially when it comes to the raining season.
Reporters on trying to know what happened and that portion of the road has been abandoned by interacting with the incumbent Member of Parliament of the Nkwanta North Constituency, Honorable Oti Bless, he indicated that “roads are constructed by the central government and since the current NPP government has no interest in the Eastern Corridor road, there’s nothing one can do until they are voted out of power”.
He concluded by saying the NPP government led by Akuffo Addo has abandoned their roads and before we can have good access to road network, then we should vote the NDC to power coming 2020 general elections.
LISTEN TO DJ FLY LIVE ALWAYS ON FLY FM GH AND ENJOY UNADULTERATED NONSTOP MIXES. FLY FM WE ARE THE MASTERS ON UR AIRWAVES.
CLICK THE LINK TO DOWNLOAD
Honorable Oti Bless is a young,hardworking and vibrant incumbent Member of Parliament of the National Democratic Congress (NDC) in the Nkwanta North Constituency and yet standing to be voted as the Member of Parliament come 2020 general elections.
Report filed by Kingsley Sachapou, Oti regional correspondent

domain: politics


text:
On the shores of Moosehead , the little town of Greenville exploits the local fauna with several moose-themed stores. Moosin’ Around Maine (Pritham Ave., Greenville, 207/695-3929) is a mandatory stop for a moose T-shirt, mug, or magnet; it also features made-in-Maine pottery and blown glass.
More unique finds can be unearthed at Kamp Kamp (Pritham Ave., Greenville, 207/695-0789, www.moosehead  lakeindianstore.com, daily Jun.–mid-Aug.; weekends only mid-Apr.–May and Sept.–Dec.), an old time “Indian store” re-created in exquisitely kitschy detail by the owners, who visited the lake frequently as children. In addition to camp furniture and souvenirs, it sells raccoon hats, rattlesnake eggs, slingshots, and other oddities.

domain: geography


European markets are sharply down at the open on Wednesday, after some bad news from China.
Inflation eased in September to 1.6% from a year earlier. The increase, well below the 2% pace of August, was below expectations for a rise of 1.8%.
The print is reflective of a slowdown in China’s economy and opens the prospect for additional monetary policy easing from the PBOC in the months ahead.
The FTSE is off around 1% as of 8:30 a.m. UK time.
Asian markets are down after a tough session in the US. Japan’s Nikkei fell 1.89%.
Glencore continues its trend downwards after recovering from a sub-70 pence plunge at the end of last month. The mining company’s stock is down almost 5%, trading at around 112 pence.
Most of the major European share markets are heading south at the start of trading.
Here’s the scoreboard as of 8:30 a.m. UK time:
Euro Stoxx 50: -1.11%
Germany’s DAX: -1.14%
France’s CAC40: -1.13%
Spain’s IBEX: -1.28%
Business Insider Emails & Alerts
Site highlights each day to your inbox.


text:
Andy Serkis, well known for his role as Gollum and immortalizing the phrase “My Precious” in The Lord of the Rings, is scheduled to narrate a new audiobook series based on J.R.R. Tolkien’s classic works.
On September 16, a new audiobook version of the classic trilogy will be released, after Serkis’ recent readathon of The Hobbit.
Serkis raised £280,000 last year during the first coronavirus shutdown by reading the whole J.R.R. Tolkien book The Hobbit in one sitting as part of a charity readathon.
The actor finished the book in just under 11 hours, and he was watched by almost 650,000 people.
The funds raised will be distributed equally between NHS Charities Together and Best Beginnings, a children’s organization for whom Serkis is an ambassador.
Serkis said, “My only hope, now, is that I have done it justice and that the listening experience conveys the power and beauty of J.R.R. Tolkien’s masterpiece”.
Serkis also admitted that he used to “go on all fours offset when we were filming Lord of the Rings” to get into character as Gollum, calling it a “pretty method.”
Photo Credit: https://medium.com/linagora-engineering/docker-my-precious-6efbce900dcb

domain: entertainment


text:
|Posted on Sunday, March 26, 2006 - 05:12 pm: ||
G'day Folks. The Australian Bush Poets Association has defined Bush Poetry as rhyming verse with good metre and rhyme about Australia, it's people, places, things and way of life.
We invite you to doss down at Bush Poetry Ballads and Yarns and share a little of our Aussie Culture. Throw a log on the fire, pour yourself a cuppa from the billy and give old Blue a pat just to show that you're friendly. Goondoya mates.

domain: artistic


text:
The Wheeling Water Department has issued an
INTERRUPTION OF THE WATER SERVICE
Tuesday, December 4, 2018
Beginning at 9 AM to be shut off all day
There will be an interruption of the water service due to the installation of meters.
In case of inclement weather or water line breaks, the alternate day for the project will be December 5.
After service is restored, there will be a 48-hour boil order in effect.
The following addresses will be affected:
All of Washington Farms


text:
1103B - flag thanks letterpress card - set of 6
Style - 1103B
These B7 (3.25" x 5.25") cards are letterpress printed using hand-mixed ultra lava and fluoro pink inks on luxe 100% cotton (tree free!) paper. Blank inside, they with 7 coordinating ultra lava envelopes, specially sized for these unique cards.
:::Due to the nature of letterpress printing slight variations in color & registration might occur:::


text:
A roadside station 「Jyomon Roman Minami Kayabe」was opened in 2011. Hakodate city Jyomon cultural exchange center is located next to this station. A variety of ancient relics like earthenware and stoneware which have been unearthed from Minamikayabe Jyomon remains and other remains in Hakodate city area are exhibited. They include an exquisite full-length hollow earthen figurine which was designated as the first national treasure in Hokkaido. The unique in the world Japanese Jyomon-culture , lasted for over 10,000 years and a wide variety of tools like earthenware, stoneware and bonetools of this culture are exhibited. You can also learn about their vertical cave housing and storage pits and the culture’s spirit with belief in regeneration and circulation.
The center offers visitors a good opportunity to know about ancient Hokkaido culture and was awarded the 15th excellent public building award in 2016.

domain: history


text:
Welcome to the Montana Cowboy Hall of Fame and Western Heritage Center
"Preserving the Montana Cowboy, Western Heritage and Indian Culture for Future Generations."
We wish to create and maintain a tribute that all Montanans can be proud of...a place where we can reflect back on the roots of our beginnings and be glad of where we came from. Our aim is to showcase the lives of true Montana cowboys and how they entertwined with the rich cultural heritage of Native Americans and settlers of Montana.
The Montana Cowboy Hall of Fame & Western Heritage Center will be another way in which we, as Montanans, will preserve a way of life we love and cherish. Please join us in building this lasting tribute!
IMMEDIATE RELEASEGILHOUSEN’S AND MONTANA COWBOY HALL OF FAME ANNOUNCE $100,000 CHALLENGE GIFT CAMPAIGN


Rockfish Caught off Fukushima Still Exceeds Radioactivity Limits
Radiation continues to plague local fisheries near Fukushima, Japan, a decade after a massive earthquake caused a meltdown at the Fukushima Daiichi nuclear power plant. On Tuesday, Japanese health officials said that they would halt all consignments of black rockfish from the region because testing in January found excessive levels of radioactivity.
It is not the first time: in February 2021, rockfish catches near Fukushima tested out with five times the permitted radiation level allowed by the Japanese government, registering 500 Becquerels of radioactive cesium per kilo. The fish were caught about four nm off the coast. By comparison, West Coast bluefin tuna test out at less than one Becquerel per kilo.
The landscape surrounding the plant was heavily contaminated with cesium from fallout from the reactor meltdown in 2011. While the Japanese government has remediated human-occupied areas by removing the topsoil, forests and rural areas have not been treated, according to researchers - leaving an abundant reserve of dangerous radioactive isotopes in the surface soil layer. This contamination washes down into streams when it rains, entering the food web.
Plans for additional discharge
Japanese response management officials at the Fukushima disaster site have long sought to start dumping radioactive cooling water into the sea. The plant is partially flooded, and about 150 tonnes of contaminated water is pumped out every day for storage in a massive tank farm. The strategy is not sustainable, officials say: there are already more than 1,000 large tanks on site, and the plant will eventually run out of room for water storage.
Instead, site managers want to release treated water into the ocean. The wastewater has been processed to remove everything except for radioactive tritium, an isotope of hydrogen found in elevated levels in irradiated water. It is virtually impossible to remove.
The site's managers have already begun construction of the infrastructure for discharging the water. The plan calls for a series of storage pits to hold the water temporarily, along with an undersea tunnel that would carry the flow about half a nautical mile offshore. Work on drilling the tunnel has been delayed until June amidst opposition to the project.
Tepco - the plant's operator - believes that releasing the water into the sea should be safe, as the contaminants would be heavily diluted and radioactivity in the area would quickly return to background levels. Local fishermen vehemently oppose the plan, as their livelihoods are already affected by radiation from the plant. "If you insist on the safety of treated water, why don't you spray it in your garden or dump it in a river flowing into Tokyo Bay?" fisherman Toru Takahashi told Asahi Shimbun.


text:
This is very strange. Michele Bachmann has said batshitcrazy stuff before and there was never a push back by the GOP party stalwarts. But there is now, following her remarks about top Clinton aide Huma Abedin. Is there something about calling for the investigation of a particular individual who happens to be a Democrat working for the Obama administration that is worse, in the minds of Republicans, than calling for the investigation of every Democrat in Congress?
Or, is there something going on with Bachmann right now that is causing this unusual negative reaction to Bachmann’s claims. For example, is the Muslim Brotherhood in cahoots with the GOP? (Don’t laugh, it is quite possible.) Or is there talk by the Romney Campaign that Bachmann could be considered as a running mate pick (laugh all you want, but that is even more possible) and establishment Republicans want to make sure that doesn’t happen either to avoid a Palinesque disaster or because they have someone else in mind? Or has the time come, in the Republican Party (and the time always comes for this, eventually) for them to start eating their own? That, I think, is unlikely becuase one usually eats one’s own when one is on the ropes or totally in charge, and it is not clear that they are quite in either state at the moment.
Or, maybe it has to do with Money. I’m pretty sure Bachmann has a lot of money and she probably doesn’t need it to beat the guy who’s running against her right now. He’s OK and all, but has zero experience and the election season is weeks old and he’s not scored a punch yet. Maybe the Republicans want Bachmann to transfer some of her war chest elsewhere and she’s not buying it. Or maybe there’s some sort of negative Romney-Bachmann thing going on.
In any event, today, House Speaker John Boehner, who is essentially the top Republican in office at the moment, two heart beats away from the Presidency of the United States, called her remarks “dangerous.” That’s serious.
Interesting question at the end. Interesting non-answer, too. Of course, its related. Batshitcrazy does not belong on one of the most important committees in the US Congress.


Event: Mithun Chakraborty, Udit Narayan and many more celebs at the music launch of CHAL CHALEIN at Cinemax, Mumbai.
Guest List: Shilpa Shukla, Mithun Chakraborty, Udit Narayan, Ujjwal Singh, Yogesh Lakhani, Sadhana Sargam, Anup Soni
Venue: Cinemax, Mumbai.
Bollywood actor Shah Rukh Khan was snapped at the unveiling of Karan Johar's book 'An Unsuitable Boy'. Also spotted at the event were Kayoze Irani, Shweta Bachchan Nanda, Ayan Mukerji, Sidharth Malhot...
Bollywood actress Malaika Arora Khan and Amrita Arora Ladak were snapped outside Kareena Kapoor Khan's house.
Bollywood actress Kajal Aggarwal was spotted at the Mumbai airport.
Bollywood actress Karishma Kapoor was snapped at the airport.
Bollywood actress Preity Zinta was snapped at the airport.


text:
What would you ask Marvel's 'Luke Cage' star Mike Colter?
- May 15 '17
Portlandia co-creators & stars Fred Armisen & Carrie Brownstein dish about their favorite characters, ad-libbing, & assure Larry they’re not secretly a couple. Plus, Carrie on the rumored Sleater-Kinney reunion, & the impression Fred couldn’t master.
More videos from this Episode


text:
Top 1000 Classic Rock Songs of All Time
REAL TALK ABOUT REAL FUCKIN FOOD
I recently ran into a couple of reading lists (I'll share them at the end) and realized that I LOVE reading book recommendations. I can't get enough of them.
On Twitter, Pixar storyboard artist Emma Coats has compiled nuggets of narrative wisdom she's received working for the animation studio over the years.
Hear, see and learn guitar tabs online. Switch instruments, slow down playback, enjoy realistic guitar sound.


I just installed the HyperLynx v9.0 and opened a demo design and exported a 3D via into HyperLynx 3D EM. However, the "Simulate" under menu "Process" is greyed out, so I can not simualte this 3D via model. What is this problem? Does the HyperLynx 3D EM require extra license?
Linesim GHz license support 3D via extraction with Hyperlynx environment. If you wanna invoke standalone Mgrid applicaiton to run via solver you have to have Hyperlynx 3D EM license. I guess you may have only Hyperlynx 3D EM designer license.


text:
Your Favourite Photoshop Tutorials in One Place
Painting Different Kinds of Clouds for Different Timing
Under Drawing Effect, Photoshop Tutorial
Sky is always a wonderful background. Here will show you some tactics to create clouds and different timing of skies such as at night time and sunset. Therefore, you can use the ideas to create different skies to suit for your design easily.
This tutorial will show you the steps of making a nice cartoon dragon and a lady in an easy way. You can create your own cartoon artwork based on this skill. It will demonstrate you how to use brushes to carry out the painting works.
You may wonder how designers can develop these fantastic cartoons on the comic or cartoon movie. You will have a chance to read through the process of these. It really needs lots of photoshop skills to do so.


text:
Mississippi July 09, 2016
These 10 Mississippi Restaurants Have Home Style Cooking To Die For
Soul food, comfort food, southern cookin’, home style – no matter what you call it, it’s absolutely amazing. And fortunately for us residents, Mississippi is loaded with restaurants that specialize in this type of fare. Serving up perfectly fried chicken and catfish, all sorts of vegetables, and cornbread to die for, these 10 restaurants have some of the best home style cooking in the entire state.
1. Southern or Soul, Carthage
When you’re craving some authentic southern cooking, Southern or Soul in Carthage has got you covered. For only $10, you can fill up on a buffet loaded with classics, like fried green tomatoes, macaroni and cheese, mashed potatoes and gravy, and just about everything else you can think of.
2. Stub’s, Yazoo City
Stub’s Restaurant has been serving Yazoo City for over 50 years. The cafeteria-style lunches and dinners include an array of southern favorites, like country fried steak, fried chicken, pork chops, and the restaurant’s specialty dessert chocolate chess pie.
3. Whistle Stop Café, Wiggins
With over 40 items on the menu that range from “good old country meals” to burgers and po’ boys, it’s easy to see why customers claim the Whistle Stop has the best food in Stone County. Feeling overwhelmed by all the mouthwatering options? The blue plate specials, fried seafood, and burgers are said to be among the café’s top items.
4. The Tracks, Winona
This train-depot-turned-award-winning-restaurant serves up some of the best home cooking around. The Tracks’ daily specials and extensive menu have won the restaurant award after award, including “Best Restaurant” and “Best Chefs.”
5. Pap’s Place, Ackerman
At one time a five and dime store, Pap’s is now known for its extensive buffet of southern favorites. Head to Pap’s and load your plate with catfish, hushpuppies, coleslaw, turnip greens, and barbecue.
6. Checker Board, Meridian
Over the years, this “southern kitchen” has become a local favorite. According to customer reviews, the buffet is always “hot, delicious, plentiful, and definitely southern.” Another tip – get there early because the Checker Board is known to fill up fast.
7. Country Platter Restaurant, Cleveland
The Country Platter has been in the business of whipping up classic soul food for years now, with past diners including several Civil Rights Leaders. The all-you-can-eat buffet is full of authentic southern favorites, like smothered pork steak, fried chicken, and butter beans. Even better? Country Platter is open seven days a week.
8. Mama Hamil’s, Madison
Mama Hamil’s buffet, which includes perfectly seasoned and fried chicken among other southern classics, has earned the Madison restaurant numerous accolades, including “Best Buffet” and “Best Soul Food.”
9. Bovina Café, Vicksburg
This local favorite is the place to go for a true southern soul food experience. Grab a blue plate special, which includes an entrée, three vegetables, salad, dessert, and a drink, for only $10. Be sure to bring cash because credit and debit cards aren’t accepted.
10. Jean’s Restaurant, Meridian
Located in Meridian’s historic downtown, Jean’s serves up a traditional southern fare in a classic lunch counter setting. The more than reasonably priced menu includes everything from chicken salad sandwiches to chicken and dumplings.
Have you dined at any of these restaurants? If you thought these restaurants looked good, be sure to check out “
10 Mom & Pop Restaurants In Mississippi That Serve Home Cooked Meals To Die For.“


text:
Diljit Dosanjh donates Rs 1 crore to farmers Who Are Protesting
Singer Diljit Dosanjh who have been making headlines for getting into an ugly spat with Kangana Ranaut, on Saturday had addressed the protesting farmers at Singhu border. He urged the government to meet their demands. He is also said to have donated Rs. 1 crore to purchase warm clothes for the protestors.
Punjabi singer Singga took to his Instagram stories to appreciate Diljit for his stand and made the revelation that the actor has secretly donated Rs 1 crore to buy blankets and other warm clothes for farmers who are protesting at the Delhi border.
“Hats off to all of you farmers, you have created a new history. This history would be narrated to future generations. Farmers’ issues shouldn’t be diverted by anyone. We have only one request from the Centre… please fulfil the demands of our farmers. Everyone is sitting here peacefully and the entire country is with farmers. This agitation is about farmers. Things are twisted on Twitter but the truth is that farmers are protesting peacefully, nobody is talking about bloodshed here.” Diljit.


text:
WILLIAMSTOWN — A new apartment construction project is scheduled to bring more affordable housing to town by summer 2024.
The River Lofts at Cable Mills project will consist of 54 apartments, including 27 affordable units, within walking distance of downtown. Construction is expected to begin in early 2023.
The team behind the project, which is owned by Mitchell Properties, is seeking $400,000 in Community Preservation Act money from the town to augment the $26 million in construction costs. This would be the third and final phase of the project.
“We’re running out of land," said developer David Traggorth.
The 65,300-square-foot project includes the construction of a four-story building at 250 Water St. Twenty-seven of the 54 units will be limited to tenants who have incomes that are 30 to 60 percent of the area median income — or $40,000 to $50,000 per year — with monthly rents ranging from $400 to $1,000 for a one-bedroom unit.
The other 27 will be market-rate units, with monthly rates starting at about $2,000, according to Traggorth. Construction will include landscaping improvements to the 3-acre parcel to enhance the riverwalk.
The applicant notes that some local money is required in order to receive additional state money.
In the application, Mitchell Properties described the project as a “vibrant, well-designed, mixed-income community consisting of 54 rental apartments, below-grade and surface parking, indoor and outdoor residential amenities such as a community room, and outdoor grilling area with firepit.”
The applicant explained that there is “considerable unmet demand in Williamstown and Berkshire County for both the affordable and market-rate units.”
The River Lofts are an extension of the Cable Mills project, which Mitchell Properties completed in 2016, building eight condos along the riverside with plans to construct 12 more. Four more are under construction. Three of them already have been sold, Traggorth said.
Cable Mills has been more than 95 percent full — with a waiting list for the market-rate apartments — since opening.
Mitchell Properties estimated that there are 2,340 income-qualified renter households in Berkshire County for the 27 market-rate units and 9,408 income-qualified renter households qualified for the 27 restricted affordable units. It also will offer covered parking for its tenants, and outdoor decks overlooking the river.
“Cable Mills showed us the strength of the rental market in Williamstown,” Traggorth said.
Local permitting has been completed, and the architectural and engineering design, conducted by ICON Architecture, is at 75 percent of completion.
The request for $400,000 from the Community Preservation Act would be paid out over several years. If approved by the Community Preservation Committee in February, it would have to also be approved by voters at town meeting this spring.
The original mill was built in 1873, as the A Loop & Co. twine factory, but it closed 10 years later. By 1892, Boston Finishing Works had begun a bleaching dye operation, which ended in 1906. It later was taken over by the Boyd Corduroy Co. in 1912, as a weaving and finishing mill.
It held on until it closed as a result of the Great Depression in 1930. In 1936, the plant was bought and enhanced by Cornish Wire, which sold it to General Cable Co. in 1984. General Cable closed in the early 1990s.
In 2003, Robert H. Kuehn's company, Keen Development Corp., bought the property from General Cable for about $400,000. Demo work had been started when Kuehn suffered a heart attack and died in 2006.
It was sold to Mitchell Properties in 2007 for $3 million.
Already, there are about 136 people residing at Cable Mills. The new building will house about 120 new tenants, Traggorth noted.


The International Army of the Territorial Defense of Ukraine, or as you may also call it, the Ukrainian Foreign Legion, is a foreign military unit of the Regional Defense Forces of Ukraine. It was created by the Ukrainian government on 27 February 2022 at the request of President Volodymyr Zelensky to fight against the Russian invasion of the country. In establishing the force, Ukraine joins more than 90 countries that have recruited generals and raised foreign armies over the past two centuries.
Ukraine's Foreign Minister Dmitro Kuleba claimed that as of 6 March 2022, more than 20,000 volunteers from 52 countries had enlisted to fight for Ukraine, with several thousand more reportedly joining after the announcement. Kuleba withheld further demographic details, as many nations forbid their citizens from fighting for foreign governments. As of September 2022, Army volunteers were recruited from 58 countries. This military team prepared for the defense of Ukraine, which will fight Russia and provide security to Ukraine. We will tell you the rules and regulations for enlisting in it.
Training and Selection:-
The first step would be for the candidates to contact the Defense Attache of the Ukrainian Embassy in their respective country.
Listed criteria for joining include having prior military or medical experience and submitting documents as proof of military service to an official of the Ministry of Defense at the Ukrainian Embassy. People were asked not to bring their weapons to Ukraine. Lviv arrived, and most of the volunteers who applied were accepted.
Ukraine's First Deputy Interior Minister, Yevgen Yesenin, announced in March 2022 that volunteers from the International Army would be eligible for Ukrainian citizenship if desired. The required probation period is the war period.
A British volunteer stated that the contract required to pay 7,000 per month to join the range of the International Army and extended for the duration of the war. However, some volunteers were allowed to leave after signing.
It has also been said that the initial training and selection of volunteers had led to "unequal" performance and that the initial intake had already been rejected by Ukrainian authorities, with an unnamed Ukrainian general stating, "we only need experienced combat veterans." The lesson we are learning is that others don't know what they are doing, and when they find out, they want to go home".
Unique Forces Wing:-
A special forces wing, known as the Legionnaires' Special Service Group, was composed of foreign fighters and commissioned by the Intelligence Directorate (GUR) of the Ukrainian Defense Ministry. This regiment differs from the regular International Army but recruits from the force. The group has been successful in carrying out several attacks behind enemy lines. Operations use small groups, which covertly move in and out of enemy territory through jungle routes and seek their targets.
Here are the names of the different units of the army:-
Canadian Ukrainian Brigade.
Kastus Kalinowski Regiment
Russian army independence
Zhokhar Dudayev Battalion
Khamzat Gelayev Battalion
Ichkerian Special Purpose Battalion
Russian Volunteer Corps


text: I have had a life long love affair with books and stories. At the age of eleven a work mate of my Fathers moved to Israel. Back then post cards were still a big thing and he sent us a few from his new home. Inspired by the wonderful pictures of the arid land with flat roofed buildings I tried to pen my first novel, a story of a pre-teen Zambian girl who moves to Israel and is overwhelmed by culture shock. Anyone who has written a novel will tell you how important research is. Beyond the post cards I had no means of research and failed to finish my book. Over the years I started but never completed many more stories. In 2014 I got an idea for a book. Three years and six rewrites later, here we are. I hope you enjoyed The Drivers Secret.

domain: hobby