text:
- Research article
- Open Access
Bacillus anthracis-derived edema toxin (ET) counter-regulates movement of neutrophils and macromolecules through the endothelial paracellular pathway
© Nguyen; licensee BioMed Central Ltd. 2011
- Received: 27 August 2011
- Accepted: 9 January 2012
- Published: 9 January 2012
A common finding amongst patients with inhalational anthrax is a paucity of polymorphonuclear leukocytes (PMNs) in infected tissues in the face of abundant circulating PMNs. A major virulence determinant of anthrax is edema toxin (ET), which is formed by the combination of two proteins produced by the organism, edema factor (EF), which is an adenyl cyclase, and protective antigen (PA). Since cAMP, a product of adenyl cyclase, is known to enhance endothelial barrier integrity, we asked whether ET might decrease extravasation of PMNs into tissues through closure of the paracellular pathway through which PMNs traverse.
Pretreatment of human microvascular endothelial cell(EC)s of the lung (HMVEC-L) with ET decreased interleukin (IL)-8-driven transendothelial migration (TEM) of PMNs with a maximal reduction of nearly 60%. This effect required the presence of both EF and PA. Conversely, ET did not diminish PMN chemotaxis in an EC-free system. Pretreatment of subconfluent HMVEC-Ls decreased transendothelial 14 C-albumin flux by ~ 50% compared to medium controls. Coadministration of ET with either tumor necrosis factor-α or bacterial lipopolysaccharide, each at 100 ng/mL, attenuated the increase of transendothelial 14 C-albumin flux caused by either agent alone. The inhibitory effect of ET on TEM paralleled increases in protein kinase A (PKA) activity, but could not be blocked by inhibition of PKA with either H-89 or KT-5720. Finally, we were unable to replicate the ET effect with either forskolin or 3-isobutyl-1-methylxanthine, two agents known to increase cAMP.
We conclude that ET decreases IL-8-driven TEM of PMNs across HMVEC-L monolayers independent of cAMP/PKA activity.
- Protective Antigen
- Endothelial Cell Monolayer
- Human Microvascular Endothelial Cell
- Lethal Toxin
- Edema Factor
Anthrax refers to those clinical syndromes caused by the spore-forming, Gram-positive organism, Bacillus anthracis [1]. Classically, anthrax presents as one of three syndromes: cutaneous, gastrointestinal, and pulmonary [1]. Pulmonary anthrax is among the most feared of infectious diseases; once clinical symptoms have developed, mortality remains high even with appropriate treatment. Much of the pathogenesis of anthrax is currently attributed to two toxins, each of which is produced from two of three proteins synthesized by the bacillary form of the organism: protective antigen (PA), edema factor (EF), and lethal factor (LF) [1]. PA combines with either LF to form lethal toxin (LT), or with EF to form edema toxin (ET) [1]. LT received its name as it was thought to be the principal virulence determinant responsible for the most deleterious sequelae of anthrax infection [1]. ET was so named as it caused localized edema, in vivo, upon subcutaneous injection [1].
The mechanisms through which ET elicits host cell responses are incompletely understood. PA is the receptor binding moiety of the toxin complex. After binding to one of two surface receptors, endothelial marker-8 (TEM-8)/anthrax receptor 1 (ANTXR1) or capillary morphogenesis protein-2 (CMG-2)/anthrax receptor 2 (ANTXR2), PA is cleaved into a 63 kDa fragment by surface proteases, such as furin [2, 3]. ANTXR1 is present in the epithelial cells lining the respiratory pathway, skin, and gastrointestinal tract, as well as being selectively upregulated in endothelial cell(EC)s during angiogenesis and tumorigenesis [4]. In contrast, ANTXR2 is ubiquitously expressed in most human tissues [5]. These PA fragments oligomerize into ring-shaped heptamers, to which EF binds [2]. The entire complex then undergoes receptor-mediated endocytosis [2]. This endosome is acidified, resulting in conformational changes, which in turn, permit insertion of the multiprotein complex comprised of EF and the PA cleavage product into the endosomal membrane [2]. EF is then translocated to the cytosol, where it exerts its biological effects [2]. EF is one of four known bacterial products that are intrinsic adenyl cyclases [6]. Its catalytic rate is 100-fold higher than any mammalian equivalent [6]. The current understanding is that most of the effects of EF are due to elevated levels of mislocalized cAMP [1]. ET has been demonstrated to increase cAMP in a variety of cell types, including Jurkat cells, Hela cells, monocytes, and most relevant to the current studies, ECs and polymorphonuclear leukocyte(PMN)s [3, 7–9]. cAMP is a ubiquitous secondary messenger with multiple downstream effectors, including protein kinase A (PKA) and protein activated by cAMP (EPAC), a guanine nucleotide exchange factor (GEF) for Ras-related protein 1 (RAP1) [10]. There are two EPAC variants, EPAC1 and EPAC2, each of which has a distinct domain structure and tissue-specific expression [10]. The EPAC1-RAP1 pathway has been implicated in such cellular processes as vascular endothelial (VE)-cadherin-mediated cell-cell adhesion [11–13], integrin mediated adhesion [14], monocyte chemotaxis [15], Ca2+-induced exocytosis [16], and Fcγ-receptor mediated phagocytosis [17]. Whether ET might also exert biological effects independent of cAMP is unknown.
Highly purified, recombinant ET is lethal to mice [18] at lower doses than is LT [19]. Curiously, edema was absent in these mice at the microscopic level [18]. ET suppresses the T-lymphocyte secretion of the PMN chemoattractant, interleukin (IL)-8 [20]. ET also impairs PMN phagocytosis and superoxide production [21]. In EC-free systems, investigators have demonstrated that ET increases PMN chemotaxis [22], whereas others have shown an inhibitory effect [9]. Of relevance to the current report, ET also decreases EC chemotaxis [7].
In 2001, renewed interest in pulmonary anthrax was generated when 11 bioterrorism-related cases were described [23, 24]. A unifying feature of these cases was a normal to slightly elevated circulating leukocyte count in the face of relatively high levels of bacteremia [24]. Although circulating PMNs were abundant, lung tissues from these patients were notable for a lack of intra-alveolar inflammatory infiltrates [25]. The pleural fluid of several patients contained scant PMNs. Similarly, in African Green Monkeys exposed to anthrax spores, the pulmonary interstitium was expanded by fibrin and edema, but contained few PMNs [26]. These combined data suggest an impaired delivery of circulating PMNs to extravascular sites of infection. Since PMNs are an essential host defense against bacterial infection, a survival advantage would be conferred to any infecting organism that could disable these phagocytic cells.
From its name, most observers would intuit that ET increases edema formation, i.e., the paracellular passage of fluid and macromolecules. However, agents that increase intracellular cAMP are known to enhance EC-EC adhesion, tighten the paracellular pathway, and promote barrier integrity [11, 27–32]. He et al found that basal levels of cAMP are necessary to maintain barrier function under resting conditions [30]. Multiple investigators have demonstrated that pharmacologic agents which increase cAMP or behave as cAMP analogues in ECs enhance barrier function [11, 27, 28, 31–33]. Prior studies which have looked at ET effects on PMN chemotaxis have done so in EC-free systems, and therefore did not examine whether ET might impair PMN migration to target tissues through an effect on the endothelium. In the current study, we have defined a novel mechanism through which a bacteria-derived toxin, ET, may indirectly, through the counter-regulation of the endothelial paracellular pathway, impair extravasation of PMNs into tissues.
ET protects against IL-8-stimulated transendothelial migration (TEM) of PMNs
ET acts at the level of the EC to decrease IL-8-driven TEM of PMNs
To establish whether the ability of ET to decrease IL-8-driven TEM of PMNs was mediated indirectly through the EC response, we measured the effect of ET on movement of a permeability tracer across the endothelia. In a subconfluent HMVEC-L monolayers, where the average baseline transendothelial 14 C-albumin flux was 0.0256 (+/- 0.0147) pmol/h, ET, at increasing concentrations, dose-dependently decreased mean (+/- SEM) transendothelial 14 C-albumin flux compared to the simultaneous medium controls (Figure 2C). ET concentrations as low as 100 ng/mL:100 ng/mL diminished transendothelial 14 C-albumin flux. These data indicate that ET restricts passage of macromolecules through the same endothelial paracellular pathway through which PMNs migrate. To provide additional evidence that ET decreases IL-8-driven TEM of PMNs through the EC response, we tested whether ET might protect against agonist-induced barrier disruption. In confluent HMVEC-Ls where the mean (+/- SEM) baseline transendothelial 14 C-albumin flux was 0.01 (+/- 0.006) pmol/h, both human recombinant tumor necrosis factor (TNF)-α and bacterial lipopolysaccharide (LPS), each at 100 ng/mL, increased 14 C-albumin flux > 2-fold compared to the simultaneous medium controls (Figure 2D). When LPS and TNF-α were coadministered with ET at 1000 ng/mL:200 ng/mL, the increase in transendothelial 14 C-albumin flux in response to either LPS or TNF-α was decreased by ≥ 60% and ~ 45%, respectively, compared to albumin flux in response to each respective agonist alone (Figure 2D). These data indicate that ET provides partial protection against both endogenous host and exogenous bacteria-derived mediators of endothelial barrier disruption through its action on ECs.
The effect of ET on IL-8 driven TEM of PMNs is PKA-independent
Forskolin (FSK) and 3-isobutyl-1-methylxanthine (IBMX) fail to reproduce the ET effect on IL-8-driven TEM of PMNs
In our studies, we have found that ET decreases IL-8-driven TEM of PMNs across human lung microvascular endothelia. We asked whether the observed ET effect could be attributed to action on either the PMN and/or endothelium. We found that ET blocked TEM even when PMNs were not directly exposed to ET (Figure 1A) and required the presence of both EF and PA (Figure 1B). At the same concentrations, ET did not inhibit PMN chemotaxis in an EC-free system (Figure 2A, B). In contrast, we found that ET decreased 14 C-albumin flux across preconfluent endothelia (Figure 2C). Further, ET attenuated the increase in 14 C-albumin flux provoked by both endogenous (TNF-α) and exogenous (LPS) mediators of barrier disruption (Figure 2D). Prior inhibition of PKA with H-89 or KT-5720 did not reverse the ET effect on TEM (Figure 4C), and agents demonstrated to elevate intracellular levels of cAMP in HMVEC-Ls (Figure 5A, B, Additional File 1: Figure S1A, B) could not reconstitute the ET effect (Figure 5C, and Additional File 1: Figure S1C). These combined data indicate that ET diminishes TEM of PMNs at the level of the endothelial paracellular pathway and does so independent of via cAMP/PKA activity.
Several studies have examined the direct effect of ET on in vitro PMN function. O'Brien et al found that ET inhibited PMN phagocytosis of opsonized B. anthracis [21]. Pretreatment of PMNs with ET profoundly reduced superoxide production in response to either LPS or muramyl dipeptide. Crawford et al demonstrated that ET impaired PMN NADPH oxidase activation and downstream N-formyl-methionine-leucine-phenylalanine (fMLP)-induced superoxide production [37]. Taken together, these studies indicate that ET down-regulates PMN phagocytic and oxidative functions. Other studies have focused on the impact of ET on PMN chemotaxis and migration [9, 22]. In the current studies, ET did not alter the PMN chemotactic response to IL-8 in an EC-free system (Figure 2A). To address concerns that calcein is a Ca2+-binder and would interfere with any Ca2+-mediated ET effect, these experiments were performed in the absence of the fluoroprobe. Even in the absence of calcein, ET had no effect on IL-8 chemotaxis of PMNs (Figure 2B). Chemotaxis was not as vigorous in the latter experiment, and this may be secondary to differences in methodology; mainly the use of a modified Boyden chambers, a shorter incubation time, as well as a different means of measuring PMN migration.
Wade et al found that ET stimulated directed neutrophil migration without having any effect on unstimulated random migration [22]. They also found that although ET increased cAMP in PMNs, the absolute level of that increase was < 1% of that caused by the Bordetella pertussis toxin. In contrast, Szarowicz et al found that ET reduces chemoattractant-stimulated PMN actin assembly, chemokinesis, chemotaxis and polarization [9]. In PMNs, ET provoked a > 50-fold increase in cAMP and a 4-fold increase in PKA phosphorylation. The differences between our findings and these other reports may be attributed to dissimilar techniques. For instance, Wade et al measured chemotaxis of PMNs preincubated for 1 h with ET in an agarose-gel based system, both of which were EC-free [22], whereas Szarowicz's group utilized video microscopy to study adherence of PMNs preincubated for 2 h with ET to a fibronectin-coated surface [9]. To our knowledge, none of these previous reports studied PMN migration in the context of the endothelial paracellular pathway. Another potential explanation for these disparities may be due to differences in potency of various EF preparations and their abilities to generate cAMP. Of note, the EF preparation offered by List Biologics is the least potent (personal communication, Dr. Erik Hewlett, University of Virginia, Charlottesville).
Far less is known about the direct effect of ET on ECs. Hong et al demonstrated that ET reorganizes the cytoskeleton and inhibits chemotaxis of human microvascular ECs [7]. Tessier's group found that ET induces a gradual increase in transendothelial electrical resistance (TEER) across human umbilical vein EC monolayers cultured on collagen-coated inserts. They concluded that ET-induced edema could not be accounted for by the direct effect of ET on the endothelium [38]. Of interest, in our experimental systems for both TEM of PMNs and transendothelial 14 C-albumin flux, the ECs were similarly cultured on collagen-impregnated filters. Although Tessier et al studied TEER, their experiments did not include transendothelial flux of a permeability tracer or TEM of PMNs.
ET is an intrinsic adenyl cyclase that increases cAMP [1]. Data exists to support a cAMP-mediated mechanism underlying the ET effect on TEM of PMNs. Moy et al found that cAMP agonists attenuated the ability of thrombin to increase permeability [27]. Similarly, Fukuhara et al found that cAMP agonists decreased cell permeability and enhanced vascular EC-EC adhesion [11]. In ECs, cAMP targets multiple downstream signaling molecules that might promote endothelial barrier integrity, including PKA [39] and EPAC1 [40, 41].
One key effector of cAMP is PKA [10]. PKA has been shown to inhibit myosin-based contractility through phosphorylation of myosin-light-chain-kinase, thereby decreasing its activity [10]. PKA also inhibits RhoA activity, stabilizes microtubules, reorganizes cortical actin and strengthens tight junctions through phosphorylation of vasodilator stimulated protein (VASP) [10]. In our studies, we found that ET activates PKA in HMVEC-Ls in a dose- and time- dependent manner (Figure 3A, B). Although ET increases EC PKA activity, its inhibitory effect on TEM could not be ascribed to PKA activity. Two structurally dissimilar pharmacologic inhibitors of PKA, H-89 and KT-5720, each failed to attenuate the ET-induced decrease in IL-8-driven TEM of PMNs (Figure 4C). Further, we were unable to reproduce the ET effect on TEM with either of two structurally and functionally distinct pharmacologic agents each known to increase cAMP, FSK or IBMX (Figure 5C). Taken together, these data indicate that the mechanism through which ET counter-regulates IL-8-driven TEM of PMNs cannot be explained solely through cAMP/PKA activation.
Another downstream target for cAMP is EPAC1, which is a GEF for the ras GTPase, RAP1 [10]. Like PKA activity, the EPAC1-RAP1 pathway also enhances endothelial barrier function [11, 12, 42–44]. The EPAC1-specific analog 8CPT-2'O-Me-cAMP, which directly activates EPAC1 while bypassing PKA, has been shown to decrease permeability of endothelial cell monolayers, an effect which is ablated by prior siRNA-induced EPAC1 knockdown [12]. Birukova et al [44] and Fukuhara et al [11] both demonstrated that activation of EPAC1 attenuated thrombin-induced increases in permeability. As in the case of PKA, the mechanism(s) by which EPAC1 improves barrier function is still being elucidated. Potential EPAC1 targets include activation of VASP, as well as activation of ARAP3, which in turn is a GEF for RhoA, and vinculin, which supports EC-EC adherens junctions through association with α-catenin [10]. As PKA inhibition did not impair the ET effect on TEM (Figure 4C), one potential pathway is through EPAC1-RAP1 and its effectors.
Since ET evokes biological responses in both PMNs and ECs, it was unclear as to whether the ability of ET to regulate TEM of PMNs could be ascribed to its impact on PMNs, ECs, or both. Although prior studies had demonstrated that ET directly influenced PMN chemotaxis, in our experiments, it did not (Figure 2A). Further, ET diminished TEM of PMNs never exposed to ET (Figure 1A). Finally, not only did ET decrease the paracellular movement of PMNs (Figure 1A), but of a permeability tracer as well (Figure 2B, C). These combined data indicate that ET counter-regulates PMN diapedesis exclusively through its effects on the endothelium. Further support of this concept is offered by Wittchen et al, who reported direct activation of RAP1 in EC monolayers decreased both their permeability as well as TEM of leukocytes [43].
In conclusion, we have found that anthrax-derived ET impedes IL-8 driven movement of PMNs across an EC monolayer, as well as attenuates the increase of transendothelial 14 C albumin flux induced by TNF-α and LPS, likely as a direct effect of ET on EC-EC adhesion. This ability to counter-regulate paracellular pathway function could not be ascribed to cAMP/PKA activity. Whether this novel pathophysiology for anthrax can be extended to other pathogenic bacteria and their toxins requires further study.
H-89 and KT-5720 in-solution were purchased from Calbiochem (Gibbstown, NJ). LPS derived from E. coli 0111:B4, FSK, and IBMX were purchased from Sigma (St. Louis, MO). EF and PA were purchased from List Biologics (Campbell, CA). Human TNF-α was purchased from R&D Systems, Inc. (Minneapolis, MN). Biotinylated rabbit monoclonal anti-pCREB, murine monoclonal anti-CREB antibodies, horseradish peroxidase (HRP)-conjugated streptavidin, HRP-conjugated goat anti-rabbit IgG, and HRP-conjugated horse anti-murine IgG antibodies were purchased from Cell Signaling Technology (Danvers, MA). Unconjugated murine monoclonal anti-β-tubulin was purchased from Invitrogen (Carlsbad, CA).
EC culture
Human microvascular endothelial cells from the lung (HMVEC-Ls), purchased from Promocell (Heidelberg, Germany) were cultured in EC growth medium MV-2 (Promocell) containing 5% fetal bovine serum, human recombinant epidermal growth factor (5 ng/mL), human recombinant insulin-like growth-factor-1 (20 ng/mL), human basic fibroblast growth factor (10 ng/mL), vascular endothelial growth factor (0.5 ng/mL), hydrocortisone (0.2 μg/mL), ascorbic acid (1 μg/mL), gentamicin (30 μg/mL), and amphotericin B (15 ng/mL) [45]. Only ECs in passages 6-8 were studied.
Preparation and fluorescent labeling of PMNs
Whole peripheral blood from healthy human volunteers was collected under a protocol approved by the University of Maryland, Baltimore, Institutional Review Board, into acid citrate dextran (Sigma) solution, and PMNs were isolated by dextran erythrocyte sedimentation and density gradient centrifugation through Ficoll-Hypaque (Sigma) as previously described [46]. PMNs were resuspended in Hank's balanced salt solution (HBSS) without divalent cations (HBSS-) at 5 × 105 PMNs/ml and were incubated with 5 μM calcein-AM (Invitrogen) for 30 min at 37°C [46]. PMNs were washed three times with HBSS- after which their purity was > 95% and viability 98% by trypan blue dye exclusion. PMNs were resuspended in HBSS with divalent cations (HBSS+) immediately prior to use.
Assay for TEM of PMNs
TEM of PMNs was assayed as previously described [46]. Briefly, gelatin-impregnated polycarbonate filters (13 mm diameter, 3 μm pore size; Nucleopore, Pleasanton, CA) were mounted in polysterene chemotactic chambers (ADAPS, Dedham, MA), and sterilized overnight with UV irradiation. These chambers, which serve as the upper compartment for each assay chamber, were inserted into the wells of 24-well plates, each well serving as the lower compartment of the assay chamber and containing 1.5 mL of medium. Each upper compartment was seeded with 2.0 × 105 HMVEC-Ls/chamber in 0.5 mL and cultured to confluence (48 h, 37°C, 5% CO2). The EC monolayers cultured on filter supports were treated for 4 h with either ET at increasing concentrations or medium alone. In other experiments, the EC monolayers were treated for either 0.5 h or 4 h with either FSK (10 μM), IBMX (1 mM), or medium alone. These same chambers were then inserted into wells containing IL-8 (10 ng/mL) or medium alone. Calcein-AM-labeled PMNs (5 × 105 cells/well) were introduced into the upper compartments of assay chambers, incubated for 2 h at 37°C, after which time the contents of each lower compartment were fluorometrically assayed in a Thermo Scientific Fluoroskan Ascent fluorometer (excitation 485 nm, emission 530 nm). The fluorescence of 5 × 105 calcein-AM labeled PMNs was used to generate total fluorescence. % TEM was expressed as fluorescence signal in the lower chamber/total fluorescence signal in the upper compartment × 100%.
Chemotaxis of PMNs
Chemotaxis of PMNs was assayed as described [47]. Briefly, gelatin-impregnated polycarbonate filters were mounted in chemotactic chambers, and the chambers inserted into the wells of 24-well plates containing IL-8 (10 ng/mL) or medium alone, as described above. Calcein-AM-labeled PMNs (5 × 105 cells/well) were suspended in either medium alone versus medium containing increasing concentrations of ET before being placed into the upper compartment of assay chambers and incubated for 2 h at 37°C. The lower compartment was then sampled and fluorometrically assayed. The fluorescence of 5 × 105 calcein-AM-labeled PMNs was used to generate total fluorescence. % chemotaxis was then expressed as fluorescence signal in the lower chamber/total fluorescence signal in the upper compartment × 100%. In other experiments, unlabeled PMNs were introduced into the upper compartment of a modified Boyden chemotaxis chamber (Neuroprobe Inc., Gaithersburg, MD) while each lower compartment contained IL-8 (10 ng/mL). After 0.5 h, filters were removed, fixed, and washed. PMNs adherent to filters were stained with crystal violet, washed again, and the top surface of each filter scraped free of stained PMNs. The crystal violet was then extracted from each filter with 0.1 M citric acid in 50% ethanol for 5 min and the A560 nm of extracts measured, as described [48].
Assay of transendothelial albumin flux
Transendothelial 14 C-bovine serum albumin (BSA) flux was assayed as described [45], with minor modifications. Briefly, gelatin-impregnated polycarbonate filters (13 mm diameter, 0.4 μm pore size) were mounted on chemotactic chambers, sterilized, and inserted into the wells of 24-well plates. HMVEC-Ls were cultured in the upper compartment of each assay chamber. The baseline barrier function of each monolayer was established by introducing an equivalent concentration of the permeability tracer, 14 C-BSA (1.1 pmol, i.e., 4800-6200 dpm/0.5 ml) (Sigma; St. Louis, MO), to each upper compartment for 1 h, after which 0.5 ml from the lower compartment was mixed with 4.5 ml of Optifluor Scintillation fluid (Packard Instruments, Downers Grove, IL) and counted in a liquid scintillation counter (Beckman, Fullerton, CA). In selected experiments, ECs were seeded at 1 × 105 cells/chamber and cultured overnight to 80-90% confluence. Here, monolayers were cultured to subconfluence because baseline permeability in postconfluent monolayers was so low as to make detection of any further decreases difficult to measure in our assay system. The monolayers were then exposed for 6 h to increasing concentrations of ET, each with a fixed ratio of EF to PA of 1 ng/mL:1 ng/mL, or medium alone, after which transendothelial 14 C-BSA flux was assayed. In other experiments, ECs were seeded at 2 × 105 cells/chamber and cultured to confluence over 48 h. The baseline barrier function of each monolayer was established and only those chambers which retained ≥ 97% of the permeability tracer were studied. The monolayers were then exposed for 6 h to LPS (100 ng/mL), TNF-α (100 ng/mL), either LPS or TNF-α in the presence of increasing concentrations of ET, with a fixed ratio of EF to PA of 5 ng/mL:1 ng/mL, or medium alone. Transendothelial 14 C-BSA flux was again assayed and was expressed in pmol/h.
ELISA for PKA activity
PKA activity was measured in HMVEC-Ls using an ELISA (Stressgen, Plymouth Meeting, PA) for the screening of activators and inhibitors of PKA, according to the manufacturer's instructions [49]. Briefly, HMVEC-Ls were seeded into 10 cm dishes and cultured to 80-90% confluence. The pharmacological agent of interest was added for the indicated time, after which cells were lysed. The lysates were then added to the microtiter plate, whose wells were pre-coated with a substrate that can be phosphorylated by PKA. ATP was added and the reaction was allowed to proceed for 90 min at 30°C. The contents of each well were decanted before rabbit polyclonal antibody specific for the phosphorylated PKA substrate was added. The plate was incubated for 60 min at room temperature, washed four times, incubated for 30 min with HRP-conjugated anti-rabbit IgG, again washed, and incubated with tetramethylbenzidine (TMB) substrate. After 1 h, the stop solution was added and A450 nm measured. A standard curve was generated using purified PKA provided by the manufacturer.
pCREB, CREB, and β-tubulin immunoblotting for PKA activity
Postconfluent HMVEC-Ls were exposed to ET (1000 ng/mL:1000 ng/mL), ET + H-89 (10 μM), ET + KT-5720 (10 μM), FSK (10 μM), IBMX (1 mM), or medium alone, after which they were lysed with ice-cold modified radioimmunoprecipitation assay buffer, containing 50 mM Tris-HCl, pH 7.4, 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EGTA, 100 mg/ml type-1 DNase, 1 mM sodium orthovanadate, 1 mM NaF, 1 mg/ml pepstatin A, 10 mM pyrophosphate, and 1 mM phenylarsine oxide (all purchased from Sigma), and 1 tablet of complete protease inhibitor mixture (Roche Applied Science) per 20 ml of lysate as described [50]. The lysates were centrifuged, and the supernatants were assayed for protein concentration with a Bradford protein assay kit (Bio-Rad). The samples were resolved by 8-16% gradient SDS-PAGE and transferred onto PVDF membranes. The blots were blocked with membrane blocking solution (Zymed Laboratories Inc., San Francisco, CA) and were incubated with biotinylated rabbit anti-pCREB antibodies (Cell Signaling), followed by streptavidin HRP (Cell Signaling), after which they were developed with enhanced chemoluminescence (ECL). To control for protein loading and transfer, the blots were stripped and reprobed with either murine anti-CREB and/or murine anti-β-tubulin (Invitrogen), and each pCREB band was normalized to total CREB and/or β-tubulin signal in the same lane on the same blot.
One-way analysis of variance, followed by post hoc comparisons using Tukey-Kramer's multiple pairwise comparison test, was used to compare the mean responses among experimental and control groups for all experiments. SAS 9.2 was used for the analyses (SAS Institute Inc., Cary, NC, USA). A p value of < 0.05 was considered significant.
This work was supported in part by grant HL089179 from the NIH (SEG) and MARCE (ASC).
We would also like to thank Lei Zhang, MD, and Grish Ramachandra, PhD, for assisting in the purification of PMNs.
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This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


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Publicationsshow recent
Eastern Threat Center researchers and cooperating scientists have authored and co-authored numerous publications describing research that supports natural resource management and decision making. Select a title to read the publication, or use the search feature to find publications by author, title, or keyword.
- Alterations in leaf nitrogen metabolism indicated the structural changes of subtropical forest by canopy addition of nitrogen
- An improved water budget for the El Yunque National Forest, Puerto Rico, as determined by the Water Supply Stress Index model
- Bulk deposition of base cationic nutrients in China's forests: Annual rates and spatial characteristics
- Characterizing the dynamics of cone production for longleaf pine forests in the southeastern United States
- Cold tolerance and invasive potential of the redbay ambrosia beetle (Xyleborus glabratus) in the eastern United States
- Do United States protected areas effectively conserve forest tree rarity and evolutionary distinctiveness?
- Drought and thinning have limited impacts on evapotranspiration in a managed pine plantation on the southeastern United States coastal plain
- Forest, Trees and Water on a Changing Planet: How Contemporary Science Can Inform Policy and Practice
- Implications of upstream flow availability for watershed surface water supply across the conterminous United States
- Landscape correlates of forest plant invasions: A high-resolution analysis across the eastern United States

subset: commoncrawl


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I have had more then a few innkeepers ask me for a list of sites I read regularly, have bookmarked and find useful for information for innkeepers on, and decided to finally put together a list.
Unfortunately many of the sites and blogs out there dedicated solely to B&Bs are either offline (many for good) or they have not been updated in a year plus, with a relatively few number of posts on them. After much web browsing, I found not much more in the way of online resources written specifically for B&Bs either, that are not mass marketing articles written by non B&B people, people with no experience in the industry or link farm articles.
There does seem to be a dearth of Hotel marketing blogs out there, but as B&B people know, there is a vast divide between hotel marketing and small lodging marketing. Some of it is applicable and I listed several at the bottom of the post that are crossovers, but most are not.
B&Bs just can't afford to spend $15,000 a month for a piece of software or a social media monitoring system, unless of course it can make breakfast, answer the phone, never take a day off, change the linens for 8 rooms, as well weeding the flower beds and chatting up potential guests on twitter. Hotel software pitchers, please notice the sarcasm here!
I am neither endorsing or recommending these resources but only listing them, as I have found they have provided useful and helpful, marketing and other operational advice to B&Bs.
Blogs and Sites:
About.com B&Bs
Many (but not all) of the articles in here are written by innkeepers
About the Inn
From Scott at the Brewster House B&B
On twitter @AboutTheInn
Acorn Internet
On twitter @AcornInternet
The B&B Coach
Susan from 40 Bay StreetBed & Breakfast
On twitter @TheBBCoach
Bed and Breakfast Academy
From Karen at Hopton House B&B
On twitter @HoptonHouseBnB
Bedandbreakfast.com: Innkeepers Resource Library
(unfortunately it seems like the PDFs in this link are AWOL)
Blizzard Internet
On twitter @bnbedition
Forfeng's Blog: Resources and Marketing for Lodging
On twitter @forfeng (yes that's me 🙂
Guest House Coach
On twitter @GuestHouseCoach
The Innkeeping Blog (from PAII)
On twitter @paiiceo
Inn Marketing
Lay My Hat (contributed by @latreille24)
http://www.laymyhat.com/forum/ and http://www.laymyhat.com/newsletters/newsletterList.php
Sage Blossom Consulting
Quantum Hospitality Blog
This hasn't been updated since they moved to Florida, I hope Howard does start writing again, because there are some great articles in the Archives.
Innspiring Forums (these are public, so if you do jump in the conversation you may want to be cautious about what information you put in your personal profile, especially if you are discussing past guests.
PAII forums (you do have to become a PAII member but especially for new/beginning innkeepers, the forums alone are worth the membership price)
Linkedin Groups
While these are primarily aimed at hotels, there is enough cross-over where the posts can be applicable to B&Bs as well and some of them Do work with B&Bs.
Alain Classe (visit the categories across the top)
On twitter @AlainClasse
Are Morch
on twitter @AreMorch
Daniel Edward Craig
On twitter @dcraig
On twitter @iVisitorGuide
Review Pro
On twitter @reviewpro
If you have come across additional sites that you have found helpful or even particular or specific articles, please let me know in the comments and I will add them to the list. Thank you and Cheers!


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Mostly about dogs, but books as well. And sometimes I have other stuff on my mind. And now: a blog about my ovarian cancer.
Thursday, 25 February 2010
I have noticed since the last couple of chemo sessions, that I feel fine the day afterwards. I do things, even, like translations or trying to design our new kitchen. This lasts for 2 or 3 days and then I start to go downhill. At least I have hope that one day I may actually do some chores around the house. And that they won't feel like chores.
Yesterday the nurse asked why I was wearing my moon boots. "They're the only shoes I can get into," I said. "And now my hands and face are affected, too." My face, so far, just has a few red blotches, but I anticipate the day when I won't be able to go anywhere without a veil.
She took one look at my hands and went to find the doctor. I now have two types of cortisone creams, one for my hands and feet and one for my face.
Whatever happened to white gloves now that I need them?
Posted by Margot at 15:47 1 comment:
Saturday, 20 February 2010
Nick does stuff, too
He's been laying the floor in the workshop machine room and taking down the ceiling to make a sturdy floor for the living room above.
Posted by Margot at 14:48 3 comments:
Thursday, 18 February 2010
Good Day in Hospital - II
The things I forgot when writing last post.
Told the doctor my neighbour made me up some homoeopathic oil for my feet and hands.
"It won't hurt," he said. Then, "As long as it doesn't cost too much. I hate it when it costs a lot." I reassured him that it was a gift. He felt better.
I have the impression he believes it's harmless -- and ineffective. And so far it isn't doing anything, but we'll go through another three weeks of chemo and decide.
I'm still on shots to augment my white blood counts. Told the doctor I still have leftover EPO for my red blood counts. "Save it," he said. "Don't be selling it to any cyclists." Told him I was going to be a cyclist. "Not yet," he said.
He asked me if the doctor in charge of my program in Lyon explained things to me in French or English. "French," I replied. . . pause. . . "Does he speak English?" "He must," said my new doctor.
Live and learn. But I think the doctors prefer to speak French to make sure they don't screw anything up. If I screw up, nothing lost.
Posted by Margot at 13:26 3 comments:
Labels: chemo, feet, homeopathy
Wednesday, 17 February 2010
Good Day in Hospital
My doctor is on vacation, so I had to see another one today. He was lovely. (He is, actually.) First he said, "Everything seems to be working for you, doesn't it?" That was cheery.
Then he said, "You're 70, aren't you?" "In July," I said. "You don't look it," he said. See? Lovely. "I've got the good genes and the bad genes," I said. "That's how it goes," he said, or some French equivalent.
My big toenail on the left foot is almost off. Infectious nephritis. I'll recover when the chemo is finished. The good part is that my left food doesn't hurt so much now. Now if the right one would go.
Then he said, "You're 70, aren't you?" "In July," I said. "You don't look it," he said. See? Lovely. "I've got the good genes and the bad genes," I said. "That's how it goes," he said, or some French equivalent.
My big toenail on the left foot is almost off. Infectious nephritis. I'll recover when the chemo is finished. The good part is that my left food doesn't hurt so much now. Now if the right one would go.
Posted by Margot at 21:51 6 comments:
Thursday, 4 February 2010
More Happy Dance
Went to the vet this morning and had another blood test. Nala's white blood count has fallen from just over 35 to just under 29. The amoxillin is working! Yea!
Back on Monday. Or Saturday if need be.
Back on Monday. Or Saturday if need be.
Wednesday, 3 February 2010
Half a Happy
As I mentioned in my previous post, Nala developped pyrometra. As I also said, if you know what that is, fine; if you don't, don't ask unless you have an unspayed bitch. In that case, it would be a good idea to look it up.
At any rate, the cure is to spay. So Nala was spayed and given a course of antibiotics: Cephalexlin, to be specific. When we got to the vet on Monday for her post-surgery check up, she had eaten very little, but had eaten some, so the vet sent us home, again.
Tuesday morning, she still hadn't eaten and the incision was seeping blood. We went back to the vet. Her white blood count was up from 34+ (double the normal limit) to 35+. We changed antibiotics, now she's on Amoxicillin.
Today, she ate breakfast! At first she didn't want it, but Nick took her out for a few minutes and when she came back, she ate.
As we were leaving for the hospital, I decided to walk her for another couple of minutes. When she heard her collar (chain), she woke up, came into the kitchen. . .
and danced! Afterwards she sulked because I would only let her walk about 25 meters and made her come home.
This evening, she wouldn't eat, again. So we just gave her her pills. Then she ate -- about a quarter of her meal. I guess she thinks eating means a dessert of pills and since she'd already had them, it was save to eat.
You know how people think Chows are stupid because you can't train them and they won't do anything you say? It has occurred to me that Chows must think people are pretty stupid because they can't train us to do things their way.
She seems reasonably lively. (Is it possible to sleep lively?)
As for me, great day at the hospital. Arrived a bit early for my 1:30 appointment and, for the first time ever, did not have to go to the waiting room, do not pass Go. Was given a room immediately. The nurses arrived 15 minutes later after their lunch break. I was alone, too. In fact, most of the rooms only had one patient or were empty. They must have recovered from the holiday disruption.
One of the nurses, I discovered, has 2 cats and a German Shepherd. She showed absolutely no interest when I said I fed raw. So I went to sleep.
Posted by Margot at 19:40 3 comments:
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Every day we have minor victories, right? We get to the bus stop just as the bus is pulling up, we hit Starbucks when the line isn't out the door, the sun comes out on our day off just so we can enjoy some warmth.
For me, this week was filled with many small, fear-based victories—and three major ones.
Major Victory #1: Sharing My Writing With a "Real" Writer
For all writers, the bane of our existence is the comparisons we make between ourselves and those we consider "real" writers. Our definition of "real" usually reflects the type of writing we want to do or think we should be doing.
For me, the definition of "real" starts with major published authors who have books and novels celebrated by the literary community and ends with journalists who write well-researched and documented stories about politics and science and corruption. In my head, all "real" writers are very serious and judgmental people.
So what does that have to do with me and this challenge? Well, for whatever reason, I do not consider myself a "real" writer. At least not yet. And I am very shy about sharing my writing with those who I consider real writers because I am deeply afraid they will read my words and say, "This is crap." In fact, the thought of showing one of these very serious, judgmental, and wildly successful writers my work makes my stomach gurgle and turn.
But last weekend, I wasn't given a choice. I met my friend's husband for the first time and from the moment he opened his mouth, I was enthralled. He's a journalist in New York City and told stories of controversy and cover-ups, getting arrested in the name of a story, and it sounded so glamorous, I almost cried. No joke, if you stumbled upon our conversation, you would have thought I wanted to have this guy's babies.
For the record, I don't, but I do want his career. So when he asked where he could find my writing, my website URL got caught in my throat.
"Oh well, you can find my stuff on The Daily Muse… and on my website… "
"What about The Atlantic stuff?" my boyfriend interjected.
"Oh, that was like a year ago, so I'm sure you don't want to read that," I practically apologized.
"What are you talking about, it was really interesting," my boyfriend continued, clearly confused by my shyness.
"Oh, I… I… sure, so you can find The Atlantic stuff on my website, too. But don't feel like you have to read any of it."
I was stammering and blubbering, sounding like the biggest fool, all the while hoping he would forget all about it. The next day, it was all I could think about.
"What if he hates my writing?" I asked anyone who wasn't already sick of hearing me talk about it. "What if he thinks I'm an idiot? What if he thinks I can't write?"
"Why do you care?" was the universal response.
"Because he's a real writer and his opinion matters."
"So are you, and so does yours."
To that, all I could do was smile and say, "Thank you."
Major Victory #2: Meeting The Ex
Meeting The Ex is never a fun experience. In fact, I would rather stand naked in front of a room full of dudes trying to draw my figure than meet any boyfriend's ex-girlfriend. But if it has to happen, I want it to be just after I've had my hair done or when I'm wearing my favorite outfit so that at least I stand a little taller while she's sizing me up.
Unfortunately, luck did not smile upon me this week.
On Tuesday night, in greasy hair and an oversized sweatshirt, I met The Ex against my will. Not because we ran into her at a restaurant or at a mutual friend's wedding, but because my boyfriend was dog-sitting for her.
We were making dinner when he got a text message. "Oh yeah, Sara's dropping off the dog tonight," he said, very casually.
"When?" I asked, wondering how I could make myself scarce.
Then the doorbell rang.
"Uh, now?" The sheepish look on his face made my insides scream, and as he headed for the front door, I started walking to his bedroom. I figured if I could just hide out until the drop-off was done, I could avoid the awkwardness until I felt better prepared to face it. But then, I turned around.
This is never going to be easy. I told myself. You might as well get it over with now.
So I walked back to the kitchen just as the dog came bounding into the apartment, the voice of her owner not far behind. Not sure what to do, I picked up a chopping knife (because that's normal) and began to cut onions with force and precision.
"Hi!" I looked up to see a petite perky brunette in yoga pants and a hoodie.
"Hi," I offered, with the most authentic smile I could muster.
"Sara, this is my girlfriend, Lauren," my boyfriend said, his voice slightly shaky.
Again, I forced a smile, put down the chopping knife and shook her hand. I even pretended to listen as she rattled on and on about her dog and her upcoming trip and, "Oh, what are you guys making? That sounds good!"
It was painful and I wanted to punch her in the face, but I got through it. And the past few days, I've even walked her dog.
Major Victory #3: Climbing to the Top of a Rock Climbing Wall
Have you ever been high up in a skyscraper, put your forehead to the window and looked down on the ant-like creatures below you? You know that tingling of excitement and fear you get? That flipping in the pit of your stomach?
Well, I get that when I'm on the third story.
Heights are not my thing. I don't just fear them; I hate them. In fact, if I could throw rocks at them, I would.
So what did I do this week? I joined a rock-climbing gym.
After the hour it took for me to take my belay test, I stood below what seemed like a mini skyscraper and stared at the brightly colored holds, the pulleys, the ropes, and the people dangling like spiders from mid-air.
I swallowed a little bit of vomit.
"Are you ready? Which one do you want to do first?" My friend was enthusiastic and encouraging.
"Um, how about this one?" Too panicked to think, I pointed to the route directly in front of me.
"This looks like a fun one!" I resisted the urge to spew bile all over his shoes.
With my friend's help, I tied the rope to my harness, dipped my hands in the chalk bag around my waist and approached the wall with weak and shaking limbs. I turned to give him a final look that said, "If I die, you can have my shoe collection," but all he gave me was another enthusiastic thumbs up.
I smiled grotesquely and turned to put my hands and feet on the wall.
Hold by hold, I slowly climbed higher and after what felt like an eternity, I looked down to check my progress. Bad idea. I was only about halfway, but I felt like I was grasping the ledge of a 25th-story window. My hands started to sweat.
And then they started to slip.
I'm going to die.
If you're thinking, "But aren't you strapped in?" The answer is, yes, I was, and I was perfectly safe. But the irrational part of my brain took over, convincing every rational cell in my body that I was about to tumble to my death below.
I secured my right hand on a large handhold while I reached for the chalk bag with my left. Then my legs started to shake.
I threw my left hand around another large hold and groped for chalk with my right.
I have to get up this wall before I become slippery beyond repair.
With shaking legs and hands messy with sweat-soaked chalk, I began to run up the wall. At least that's what it felt like to me.
By the time I reached the top, I was so sweaty and scared that I couldn't speak—a problem because I had to tell my friend to lower me, lest I be stuck up there all day. I turned, gave him a thumbs up, and as I leaned back and watched as the earth rose to meet my feet, I felt the tension in my body start to loosen.
My hands still looked like I had Parkinson's, and when I touched ground, my friend had to untie my rope. But when I looked up at my accomplishment, I felt a sense of pride that I hadn't felt in a long time.
TopicsLifestyle , 30 day challenge
Lauren Hargrave is a freelance writer, former finance stiff and a fan of all things funny. She copywrites to pay the bills and has contributed to the The Atlantic, Active.com, and various business and technology blogs. She also finds time to tell you funny stories on her personal blog at WriterLauren.com.More from this Author


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The Events team go Inter-dimensional
Last week, the PrettyGreen events team went in search of immersive inspiration by experiencing the latest offering from the team at Gingerline.
Inter-dimensional exploration accompanied by multi-dimensional gastronomy was the order of the evening. On the day of our booking, we received a text message at 4:30pm with details of the secret location – it's always a short walk from one of the stations on the London Overground, or Ginger line, hence the name!
Once we'd found the secret location, been briefed on the evening's activity (and carefully considered the various drinks packages) we were pushed into the bowels of 'the machine'.
Unfortunately, until you become a member of The Chambers alumni our lips remain sealed about what actually took place (we're a bit scared they might come after us!) and so all we can say is that we were treated to five courses of mouthwatering cuisine, each taking place in very surreal surroundings.
Along the way various characters added to our adventure and helped us travel through each dimension, particular favourites from our night were Lord Ian, Cassandra the Mermaid and Captain McBastard.
If you can get a ticket (they are seriously hot property) we highly recommend a trip into the Chambers!


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Vous êtes ici
Palmarès01/06/2006 - par Mark Tungate
Do we need another advertising competition ? The adland calendar already seems to be stuffed with awards, from Cannes to the Clios, Epica, Cresta, Eurobest, the New York Festivals... Excuse me if I've forgotten four or five. And of course we recently had the respected D&AD Awards in London. (Congratulations to Yellow Pencil winners Fallon London, DDB London, Euro RSCG Flagship Thailand and JWT London, among others - you can see their winning entries at dandad.org).
Now it seems agencies are going to get yet another chance to prove their creative worth. There are rumours that veteran adman Neil French, formerly global creative adviser at WPP, is to create The World Press Awards. Apparently he's working in partnership with Barbara Levy, owner of the London International Awards. (Sorry, forgot those earlier. I went to the ceremony once - it was extremely long.) Connoisseurs of advertising gossip may remember that French quit WPP shortly after making supposedly sexist remarks at a conference, when he brutally dismissed the idea that women creatives could balance work with family time. It provoked a swirl of controversy on the Internet.
Whatever his views, there's no doubt that French knows great creative when he sees it. But is an awards scheme an ideal arena for his talent ? I accept that awards provide a barometer of creativity, motivate staff, and attract clients. But, as every good ad man knows, it's difficult to succeed in a saturated market.


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The Insecurity of Things:
Part 1 – Look into the Mirai – An overview of what happened
Part 2 – Wagging the Dog – What Mirai is really about – security and secure passwords
Part 3 – A Manufactured Problem – The 'root' of this lies with the manufacturers – Here's what they're doing, and what they need to do
This is the final piece in my three-part series about the Internet of Things and the DDoS attacks that have taken place in the last month. I've saved this post for last because I feel it's the most essential. As I'd said in my last piece, we, as users, need to create secure passwords and credentials for all aspects of our online life. I focused on what consumers can do to improve their security, but it doesn't stop with them. We need to hold manufacturers to account. Manufacturers have the biggest responsibility in this.
In the attack on Dyn, a majority of the devices used could be sourced back to one manufacturer, Hangzhou Xiongmai Technology Co Ltd. They make parts for cameras, DVRs and storage devices. You've may not have heard of them because they 'white-label' a lot of their products. They also make components used in products and some of those components were open to attack. The reason I'm distinguishing here is I want to make clear that your devices are only as secure as your weakest piece. I should make it clear that Xiongmai has issued a recall for some of their devices, but this is complicated by the fact that, as a company who white-labels, you may have one of the devices and may not know it.
In order to provide perspective, let me cover some of the problems these manufacturers have.
Security Issues
In my second piece of this series, I covered what consumers can do with passwords. I called that piece 'Wagging the Dog' because, to me, IoT is the dog and credentials are the tail. Now, I aimed that piece at users and talked about what they could do to improve their security. I want to be clear, however, that for these DDoS attacks, a lot of the blame goes on the manufacturers. The devices in question had default or easy to guess credentials that users of the devices COULDN'T change if they wanted to. You might have seen the list compiled by Brian Krebs below:
This list is compiled from the source code for Mirai, the software used to attack devices. It's pretty shocking to me to see some of the passwords and accounts listed here, honestly. For those who may not be familiar with servers and deeper computer usage terminology, let me say to you that seeing the user 'root' on so many of these is scary. Root is the main user of a system. It's superadmin with all permissions. That means that anyone with those credentials can do whatever they want to that device. But that's not all, you're note that at least one of these devices just required the username of 'root' and NO password.
The one that really got me though is Xerox. For almost all of their printers, the default user is 'admin' and the password is '1111'. I decided to see if I could find these listed in documentation on their site. I wanted to see if it would be hard for me to get this information. Unfortunately, it wasn't. Here's what I did:
- I searched from my search engine 'Logging in as system administrator on your Xerox printer'.
- I found the first unpaid result to be the link very similar to the link listed above.
- When I got to the page, this is what I found:
- I clicked on the support page link and searched for a model number.
- I clicked on a link to a pdf for the model in question.
- I searched the term 'password'.
- I found the username and password for the copier. Here's a screenshot:
Okay. That was way too easy. Now, I'm not divulging any secret here or hacking any system to get this information. Xerox is only an example of the problem. Their devices weren't named in the Mirai attacks, BUT their credentials were found in the source code. I'm taking information you could get by reading an article, performing a search and voila! What can Xerox do about this? There are several things:
- Don't use admin/1111 as the default credentials. Give each new customer a randomly generated way of authenticating.
- Password protect any system administrator documents on their website. Require a 'customer id' number along with credentials.
- Remove the display of ANY credentials from PDFs. Instead, put a 'customer support' number there, where a person has to call in to get credentials or have a remote authentication mechanism as part of the customer support.
So, I know what you're thinking. Why doesn't the user of this printer just change the password? In fact, in the screenshot from Xerox's site, they encourage users to do that. That can be easier said than done. That password is required in multiple places for support and maintenance. Also, changing the password can be an onerous task. The keyboards on copiers and printers are not the friendliest to use, so creating a more complicated password can be time consuming and having to reenter it all the time could be a nuisance. I will say, though, end user, you should think hard about this. How often do you need to access admin for your system? What constraints does it put on you to change that password? My answer is, do it. Don't think about it, just do it.
So, it appears to me that admin/1111 is used for convenience of systems support. This lies at the manufacturer's door. To me, this type of thing is essential to customer care. Build security into your device and work to educate them as to why this is essential to their business. As a non-user of a product, it should not be this easy for me to get this information. Period.
So, now that we've looked at passwords, let's move on to hardware.
Hardware Issues
Security expert Bruce Schneier first called out the issues with hardware in his excellent piece from 2014. In fact, this was the piece that inspired me to push CRT into the IoT space. He helped me see that we need to protect our members and their clients as these devices were ramping up for the home. He literally 'peels back the onion' on the hardware and software and all the challenges wrought. Briefly, I'll try to paint a picture of the challenge using Schneier's paints. In order to make an internet-enabled device, you have to pull together a number of smaller components.
As the product manufacturer, it's most likely you don't make those components because they require specialized equipment and knowledge. They are also relatively cheap, so, economically, it's better to buy than build. When you put these components together from various manufacturers, you now have a mash-up of pieces. Some of these pieces are essentially mini-computers and have software running on them. Now, each one of these components with firmware or running some low-level software are a risk because, as we know from owning computers for the last 40 years, software has bugs. Once a vulnerability is discovered in the software on these components, you now have a chink in the armor. The question then becomes, how does one get an update for the firmware for a component in a device you bought and expect to just work? It's not easy. Does the component manufacturer step up and release the patch? Does the manufacturer then deploy the patch? How does a user of the hardware know when there's a needed critical patch? Why not just release an update over the air?
Manufacturers need to have a plan on how to work with components companies to deliver better, more secure products. I do understand this is much easier said than done. But, we need to get there. Getting a product to market before anyone else does shouldn't happen unless you've considered all the issues and what you can do to fix them. One company I want to highlight as doing very well at the Internet of Things is Canary. They make a security camera for the home. We've been in contact with them several times and have talked about these issues and how they're approaching them. Out of all the companies we've met, Canary strikes me as taking these issues the most seriously.
First, they take their devices to a hacker conference called Defcon every year. They want to see if they can be hacked so they can fix any problems before they make a large impact. We've spoken to several people there who've said that they work really hard to make sure this device is secure because it's gathering sensitive data. It's a video camera in your home. They want to make sure it's as secure as can be.
Second, look at the security measures they are taking, including hardware encryption:
What that tells you is that they've looked at the potential vulnerabilities in their device and are making sure they are covered. More like this, please.
Apple's Homekit is another example of taking encryption seriously. People get upset with Apple because of their 'walled garden' approach to their systems, but there is a method to their madness. In order for your device to become a Homekit certified device, you need to have one of their encrypted chips in the device. You also need to use some 'bleeding-edge' security protocols for connecting to their system. Now, we've spoken to several manufacturers who've said it's a pretty intense process, both in time and resources. Mirai highlights the need for these measures.
The answer to making other companies follow suit really comes down to putting pressure on the manufacturers and their suppliers. Do we ask the government to intervene? Do we wait for hardware manufacturers to take action like Canary and Apple? The way we see it, there needs to be a way to either certify or validate these devices. At NAR, we are investigating how we could be a part of something like this. We've had conversations with companies like Underwriters Laboratories (UL, LLC) and Trusource Labs, public-interest groups like The Online Trust Alliance, Future of Privacy Forum, and Center for Democracy & Technology and some vendors, about how to proceed. We are actively working on setting up a certification/validation type system. We feel like we can help be a part of the solution as we have no economic interest in these devices, yet have interest in the best possible experience in owning or living in a home. As more of these devices are released, more issues will arise. We want to mitigate as many as possible, so a standardization of this process can help to clean this up.
But we have the problem now. What can we do in the meantime? To start, the US-CERT (United States Computer Emergency Readiness Team) provides a list of ways to mitigate and prevent these takeovers of IoT devices. At NAR, we worked with the Online Trust Alliance and issued a statement that 100% of IoT vulnerabilities are preventable in recent attacks. In fact, we released this a few weeks before the Mirai attacks. There are a number of simple steps manufacturers can take to improve their hardware security. What I recommend you do as a user of these technologies, before you buy any product, do a search of the product name along with the phrases 'security issues' or 'hacked'. Search devices you have now in the home as well. Update software regularly. Also, keep following us. We are here for our members. So, if you've made it through the three pieces I wrote…what thoughts do you have? Share in the comments below.
- Bruce Schneier's piece from 2014 pointing to the coming of this problem of internet security & the Internet of Things
- Scheier's latest piece on the botnet attack
- Dark Reading's piece ''Root' & the New Age of IoT-Based Internet Attacks'
- Sophos Security's analysis of the attack
Image from New Old Stock. Original source for the image here.


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So I think I mentioned in my last post (two months ago?) that some buddies and I were working on a tower defense game that we hoped to demo at an upcoming conference in September. As you may have noticed, September has almost come and gone but I haven't posted any stellar updates detailing the success of our demo at said conference.
Part of this is due to an unavoidable stall in development progress, which brings me to my first lesson learned. When I realized the ever-increasing scope of the game I would soon be writing, I should have pulled in more developers. As it is, I am currently the only programmer among a group of two 3D artists, two concept artists, and one musician. Although there is a certain amount of pride in being able to take full ownership of the (increasingly large) code base, there is also much to be said for having a fellow to bounce ideas off of and having someone to share the load when you're not available.
Another thing that's bitten us in the development of our game is not understanding core requirements in the beginning. We think we have a great idea for a game and started prototyping the basics right away. For a simple tower defense game, you have enemies moving from one end of the screen to the other, and that's what we created initially as a 2D game. Once we had this basic prototype, we realized that it was pretty darn difficult to make projectiles with faux-depth in a 2D isometric view look right. So we had to make the leap to the third dimension, which involved converting our 2D sprites into 3D rigged/animated/textured models. We started creating 3D animated models right away before adding concept artists to the team; so the models had to be recreated. But once we had our concepts, we needed to make specific decisions regarding Art Style - the overall game appearance - and this required new models yet again. As a programmer I occasionally overlook these finer details; I mean, if all the characters on screen are moving to their correct destinations... it looks good! But the second lesson I've learned is that Art Style is something that should be seriously considered early on in a game's development.
Performance is also a major consideration for our game. Although we want to have the best-looking game we can build, we also want it to be playable for as many people as possible, so limiting it to the highest-end graphics cards didn't seem right. And given the fact that I was developing the game on a laptop with only 32MB of dedicated video memory at the time, I had a big problem creating a game I wouldn't be able to play! So we took a step back and realized that our 3000-polygon models probably weren't going to cut the mustard. But even with reduced-poly models, we wanted to do some fun stuff in our game like using outlines on our toon-lit models, particle effects, etc. And some of these special effects are expensive operations that quickly drop frame rate and can cause the game to lag. So as our designer likes to say it, the final lesson for today is to "prototype everything." Although he doesn't really mean to prototype everything. What should really be prototyped is all of the core systems of a game that could be rendered/executing together at the busiest point of the game's interaction: music, sound effects, lighting, particle effects, blur effects, and so on. And provide a way to instantly, with the push of a button, drop a hundred or more models on the screen to simulate the game at its most computationally-intensive point to see how it will perform in the worst-case scenario. Doing this type of prototyping early on can help identify where you need to save polygons or cut features that could prevent your intended audience from playing your game.
The cool thing about my team is that we're all learning as we go, and sometimes part of that involves looking back and saying "You know, we could have..." So I hope this helps as you start your next project.


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I just wanted to pause in my blog writing and wish everyone a very Happy Thanksgiving. As we all pause and give thanks for everything good in our lives, let's all please remember the less fortunate in our world today. For those of you who may not celebrate Thanksgiving, please know that I am thinking of you and hope that this has been a wonderful year for you. I am very thankful that you are following my blog and hope that its content has helped you at least in some small way.
Bob Sproull


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Cold Snap
Last night was COLD. It apparently hit minus 12. I went to bed at 11 and within five minutes of turning off the electric blanket I was shivering, and that's while I was under three heavy blankets including a down comforter! I decided to take a chance and sleep with the blanket on all night.
Next thing I knew, it was 9AM (!!!), I had two cats curled up tight against me, and I was snug as a bug. Love that little 12V wonder! I'm glad I now know I can sleep with it on all night if I need to. I wouldn't do that when boondocking, but when I'm on shore power it's more cost effective (and quiet) than running the furnace off and on.
Now, I'm waiting for things to get a little warmer (and less slick) before I go out and do my fliers. I need to do all six routes today since Jody and I are road-tripping again tomorrow. It's going to be a long day, but the sun is out so if the wind cooperates the job shouldn't be too onerous. I finally have my routes laid out in the most efficient way possible so my goal is to shave fifteen minutes off each to finish all six in four and a half hours instead of six hours.Share on Facebook


text: We're proud to announce that our website has a new look! Thanks to Jason Johnson and Charles Suggs of FullSteam Labs for all their hard work. Stay tuned for blog posts, facebook updates, and watch our twitter feed! If you'd like to sign up for our newsletter, please use the form at the bottom of the page.


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At S Capital Management we focus on those areas in which we have earned particular expertise
The Primary Areas of S Capital Investment Expertise
- Private Wealth Management and Asset Allocation: S Capital Management principals are widely known in the area of private wealth management and asset allocation. We are aware that private wealth management is based on individual needs and that taxes and asset transfer issues are critical components of assisting our clients. To that end, we have developed a list of professional that specialize in the areas of taxation and business and estate planning. We work with them or CPAs and attorneys of your choice to ensure independent and coordinated advice.
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Thursday, July 15, 2010
Google a la Josef Frank
8 comments:
Those fabrics are wonderful - so joyful and colourful, and yet full of grown-up flair. I would have anything of his in my house.ReplyDelete
That's a wonderful daily theme for Google. I love the images you shared here.ReplyDelete
Please wrap that armoire up and send it to me right away. Gorgeous and so much more soulful than run of the mill mid-c (not that I don't own plenty of that).ReplyDelete
I have to admit, I saw that google, and didn't get the reference! Glad you explained it!ReplyDelete
Very hard to believe he made these prints so long ago. They look very fresh.ReplyDelete
Yes, isn't that armoire fabulous?ReplyDelete
And I only recognized Frankel thanks to an abiding addiction to design blogs.
I agree - it's astonishing that he was born 125 years ago. These designs are so 'now'. (Unlike me who posted about the doodle a day late when everyone else had already covered it! Oops!)ReplyDelete
yummo design! Those look so modern, i can't believe my eyes if you don't mention those prints are older than my grandmother!ReplyDelete


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This site will look much better in a browser that supports web standards, but it is accessible to any browser or Internet device.
commercial law, business transactions, real estate,
employment law and probate
Ms. Maxwell was born in Waukegan, Illinois on May 5, 1951. She attended the University of Illinois at Champaign-Urbana and graduated with a B.A. in 1973 and a Masters in 1976. Ms. Maxwell obtained her Ph.D. from the University of Chicago in 1987, and graduated from the University of Tennessee at Knoxville with a J.D. in 1996. She was admitted to the State Bar of Tennessee in 1996. Ms. Maxwell is a member of the Order of the Coif and was the Articles Editor for the Tennessee Law Review in for 1995 through 1996. She is also an Adjunct Professor for the University of Tennessee College of Law. Ms. Maxwell is currently a member of the Knoxville Bar Association, the Tennessee Bar Association and the American Bar Association.
Ms. Maxwell practices in the areas of employment law, probate administration, business transactions and real estate.


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On April 13, 2013, Robert Prechter and a notable group of academics, scientists and finance professionals gathered in Atlanta to share the latest findings in social mood research. Every bit as groundbreaking as the previous two Social Mood Conferences, the third annual event was a smashing success.
Experience what the packed-house audience saw and heard during the 2013 Social Mood Conference: the speakers; their charts and slides; plus the Q&A sessions between the speakers and audience. Enjoy it at your own pace, from the comfort and convenience of your home or office.


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Prince William, Kate Middleton and baby Prince George were pictured at Sydney airport boarding a flight to Wellington, New Zealand, where they are starting their three week royal tour.
Kate was seen walking through Sydney's airport holding 8-month-old George, whilst Prince William was carrying the bags, including George's kangaroo bakpack.
Following their arrival at Wellington, Prince William and Kate will head out to their first engagement of the tour to Wellington's Government House on Monday afternoon. The Duke and Duchess will be treated to a traditional Maori ceremonial welcome, also known as the Powhiri, where they will enjoy speeches, dancing and singing. The couple will also be presented with an inspection of the Guard of Honour


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Another dream about Joan
By Rick
I had another dream about Joan. I saw her Saturday at the restaurant we had bought our niece and nephew there for dinner before they went to college. Joan was even better looking Saturday than she usually is. I suppose that could be just the fact that I appreciate her more since I have been thinking about her. She is a very vivacious woman with a great smile and eyes. I just wonder why it has taken me so long t o appreciate her. Anyway here is the dream.
Joan and I agree to meet one afternoon for some reason she wants to do a video of us. She says that it is just something that would be a turn on for her. I agree and we think that the best place for us to do this would be at my house. The chance of getting caught is a turn on for both of us. We set the meting up for a Wednesday. My wife has an appointment that day and her husband won't be home until late. Some times things just click.
"Rich I don't know why but playing with someone else just makes me feel like a kid again."
"You know Joan it does the same for me! The fact that you are younger than me helps in my case. I guess it makes us remember our unattached days. What ever the reason as long as things are good for the both of us and we are both enjoying each other why not continue?"
"I'm enjoying myself so I'll see you Wednesday at your place at three."
Wednesday came and I set up the camcorder in my bedroom and was ready when she came at three. I had her park her car in the garage so my nosy neighbors wouldn't see it.
I let her in and gave her a glass of wine and we went right up to the bedroom. I turned on the camcorder and videoed her taking off her clothes and she videoed me taking mine off. The I put the camcorder on a tripod and we were making out on the bed. I was sucking on her nipples and fingering her and she was stroking my cock. She came twice before I had a went down and sucked her clit and tongue fucked her.
"Rich you can eat me whenever you like. Live it!"
"Well I love making you happy and I have always loved the flavor of pussy. You're the best tasting woman I know."
"We French women always taste great!"
"May We Madam! That's all the French I know."
"That's ok as long as you know how to get me off you can speak any language you like."
She came very hard at that point. Her hips were gyrating and her nipples as hard as rock and she pushed her nice wet pussy harder into my face.
"Shit that's nice! It beats the hell out of watching TV!"
"Remember to smile for he camera , I said, pointing to the camcorder."
"I forgot about it for a second. This is a testimony I love having Rich suck on my pussy it makes me cum and feel great!"
We both laughed and I continued to finger her pussy and suck on her clit until she said uncle and needed a rest. Then I put a condom on and she pushed me down on the bed and got on top of me and rode my cock with her facing the camcorder. We both came at the same time and when my cock was so limp I couldn't keep it in her any longer she got off of me. I took the condom off and flushed it down the toilet. I took the tape out of the camcorder and gave it to Joan.
"Thanks that was a huge turn on we have to add to this tape next time we'll do it out doors maybe in the woods. Will the battery last long enough to do that?"
"Yes it's good for a couple of hours."
"Oh shit I have to get going I have things to do when I get home."
She got dressed and gave me a good bye kiss and left. I put everything away and put the sheets in the washer before my wife got home. I also cleaned up the wine glasses and took a shower. She got home later than I thought she would.
"What's in the dryer dear?'
"Oh I did the sheets I know how much you like clean sheets."
"Your such a good husband."
"I try dear."


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- Department of Inorganic Chemistry (2003- present)
- PhD.: Freie University of Berlin, Germany (2005- 2009)
- MSc.: VNU University of Science (2001-2003)
B.Sc.: VNU University of Science (1996-2000)
1. Bio-Sketch and current research areas
Particular Information:
Full name: Nguyen Hung Huy
Date of Birth: 1978
Place of Birth: Ha Noi
Research Interests:
- Transition metal complexes of chelating thiorea ligands
- Bio-active metal complexes
- Multi-nuclear complexes and supramolecules.
2. Five most significant publications
- Hung Huy Nguyen, Canh Dinh Le, Chien Thang Pham, Thi Nguyet Trieu, Adelheid Hagenbach, Ulrich Abram. Ni(II), Pd(II) and Cu(II) Complexes with N (dialkylthiocarbamoyl)-N'-picolylbenzamidines: Structure and Activity against Human MCF-7 Breast Cancer Cells. Polyhedron 2012, 48, 181-188.
- Hung Huy Nguyen, Juan Daniel Castillo Gomez, Ulrich Abram. ReVN and TcVN Complexes with a Novel Tetradentate HybridBenzamidine/Thiosemicarbazone Ligand. Inorganic Chemistry Communications. 2012, 26, 72-76.
- Hung Huy Nguyen, Thi Nguyet Trieu, Ulrich Abram. Syntheses and Structures of Nitridorhenium(V) and Nitrido technetium(V) Complexes with N,N-[(Dialkylamino)(thiocarbonyl)]-N'-(2- hydroxyphenyl)benzamidines. ZAAC- Journal of Inorganic and General Chemistry 2011, 637, 1330-1333.
- Hung Huy Nguyen, Pham Chien Thang, Axel Rodenstein, Reinhard Kirmse, Ulrich Abram. Bipodal Acylthiourea Ligands as Building Blocks for Bi-, Tetra-, and Polynuclear Oxorhenium(V) Complexes. Inorganic Chemistry 2011, 50, 590-596.
- Hung Huy Nguyen, Khatera Hazin, Ulrich Abram. Synthesis and Characterization of Unusual Oxidorhenium(V) Cores. European Journal of Inorganic Chemistry 2011, 1, 78-82.
3. Awards
- Certificate of merit by Minister of Education and Training (2014)


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Naked Dark-Cruising
Getting increasingly poplar every month because of instant HOT action upon entry. Of course only after you've taken ALL your clothes off.Naked Dark-Cruising!! Do we need to say more?!?! Come in and find out for yourself about this HOT event!!
Open from 16:00-20:00
Entrance 7 Euro
Free Coat Check
Quand ?
Dim. 11 novembre 2018 de 16h00 à 20h00
Ou ?
1012 JH Amsterdam NL


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Get HoPiKo for Free Right Now
2016-02-24T11:15:16+01001 min read
Get HoPiKo for Free Right NowJim Squires on Feb 24, 2016 at 11:15 AM
One of the coolest little puzzle platformers of 2015 was HoPiKo – a game about freeing the helpful gremlins that... Continue reading
HoPiKo Review: Gotta Go Faster, Faster
2015-09-28T15:00:40+01003 min read
HoPiKo Review: Gotta Go Faster, Faster
Children raised during the '80s had only one mortal fear. The troubled economy? No. The depleting ozone layer? No. The... Continue reading
Hopiko is All About Lightning-Fast Platforming Action
2015-04-06T14:30:56+01001 min read
Hopiko is All About Lightning-Fast Platforming ActionNadia Oxford on Apr 6, 2015 at 2:30 PM
Fans of challenges straight from planet Crazy Reflexes might recall Alone, a high-speed endless flying game released last year. Alone's... Continue reading


text: I came upon a question today that I am curious to know the answer to. So who here likes to get a few more minutes of playing in while they do there business? I mean the acoustics in the bathroom are amazing at my house. I'm sure we've all done it a time or 2, or you're like me and do it everyday. So let's hear from the bathroom rockers!!


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May 3
Posted by Hans
Hi there!
A free version of aurora magificus is now available for those who would like to check it out.
The pixlemuchies HQ has now moved to beautiful Bali. There are worse places to do android development.
Stay tuned for our next app: Facelab!
All the best!
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My Stavast side cousins…
(You can click on the picture and it will enlarge in your browser.)
L-R back (number next to name is their parent – shown below)
Robert Johnston (2) , Ron Stavast (11), Tie Hoekstra (12), Wil Hoekstra (12), Denis de Jong (9), Myrna de Jong Maheax (9) , Matthew Stavast (5), Shirley Stavast Tityk (5), Keith Johnston (2) – Middle : Wm. James A. Stavast (Jim) (8), Deena de Jong (9), Karen Stavast (10), Helen Johnston Forster (2) , Lynda Hiemstra Gall (1), Leonard Stavast (6) – Front: Vivian Rever Nagy (3), Betty-Lou Stavast Grey (6), Evelyn Stavast Stoutenberg (10), Elsie Hiemstra Myszakowski (1)
No representatives of 2 of the siblings: Johanna Helps (4) had one son, Sydney Stavast, who is deceased. Annie Stavast Clausen (7) had 3 sons – Alf Clausen is still living, Eddie and Henry (Hank) Clausen deceased.
Missing a few living first cousins that come to mind instantly for me: Robert Stavast (10), Me (taking the picture), Ken (8), Barbara (8), Ray de Jong (9), Dick de Jong (9) – Someone said it was 2/3 of the living cousins.
With some spouses and 2 of the last 3 living spouses of the 12 siblings in my Dad's family.
Front – left – Doreen Jessie Walkden Stavast (Andrew -8 ) and Front – right – Florence Larson (Henry – 10)
Uncle Eric (Eelke) Hoekstra (Margaret Stavast – 12) came by one night and joined us at dinner. (photo)
Left 6 – Denis De Jong hands w/ Leigh De Jong, Wanda Babott Hoekstra, Tie Hoekstra, Ereck Sundmark (plaid), Vivian Rever Nagy.
Bold shirt – Jim Stavast, Blonde to his left is Valerie Reierson Hoekstra, Helen Stavast, Wil Hoekstra, Ron Stavast, Karen Stavast, Doug Ellis (plaid), Betty-Lou Gray (print w/ black slacks between 2 seated women)
Behind Florence Stavast – Helen Johnston Forster, Deena de Jong, Andre Laliberte, Myrna de Jong Maheux, Lynda Hiemstra Gall, Elsie Myszakowski (print dress) .
Back, Matthew Stavast, Vikki Stavast, Keith Johnston, Terry Gall (suspenders), Doug Stoutenburg (kneeling), Wally Tityk, Shirley Stavast Tityk, Val Johnston (widow of Ray Johnston (2)), Ev Stavast-Stoutenburg, Vera Stavast (widow of Norman Stavast (6)), Leonard Stavast and wife Marion Stavast. Dark haired behind Shirley is Robert Johnston.
The grandparents Came to Canada from the Netherlands in 1907 (with Jan Andries Stavast) :
Jantinus Stavast b. 9 July 1875 Appelscha, Friesland (Jan Andries Stavast and Janke Jurgens Krol)
Aaltje de Jong b. 5 January 1882 Sint Nicolaasga, Friesland (Johannes Haijes de Jong and Antje Heins Cnossen)
Married 28 Nov 1902 Oostellingwerf, Friesland
Their Children and reference # for place in birth:
- Janke ( "Yanke") b. 27 May 1903 – Appelscha, Friesland
- Antje ("Angie") b. 6 Oct 1904 – Appelscha, Friesland
- Geertje ("Gertie") b. 29 May 1906 – Appelscha, Friesland
- Johanna b. 23 April 1908 – Swan River, Manitoba
- John Andrew b. 19 May 1910 – Swan River, Manitoba
- Jurjen Gordon b. 16 October 1911 – Swan River, Manitoba
- Anna ("Annie") b. 2 Nov 1915 – Whitebeech, Saskatchewan
- Andrew b. 14 May 1917 – Whitebeech, Saskatchewan
- Cornelia Edna ("Kay") b. 29 July 1919 – Whitebeech, Saskatchewan
- Henry Thomas 30 May 1921 Whitebeech, Saskatechewan
- Harry William b. 31 October 1924 – Whitebeech, Saskatchewan
- Margaret Alice b. 3 May 1926 – Durban, Manitoba
(note: Great Grandfather – Jan Andries Stavast was widowed from Janke Jurgens Krol's 1st cousin – Janke DOUWES Krol)
Here are 2 genealogies listed on the web which cover the family going back. I have not confirmed all the data on these for our direct line, but will in July and August 2016. (The key questionable point is the listing near the top of a 1709 death of someone later listed has having several children with a second wife.)
Here are 2 very extensive PDF files which are the contribution of Harm-Jan Stavast. We descend from 2 brothers (of 8 siblings) born around 1800; he from Albert Jans Stavast b. 17-Jan-1802, the Jantinus Stavast's in Canada from Andries Jans Stavast b. 16-Sep-1798.
Various individuals updated info on a previous version and those are incorporated here.
If you have additions/corrections I can handle them for you with him.
(current in 2014, added Jul 2016)
Harm-Jan Stavast's compilation (2014)