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Key Points Patients with AML who received IC were compared with those who received ven to investigate predictors of outcomes.Presence of RUNX1 mutations is associated with better outcomes for ven/aza compared with IC., Visual Abstract, Venetoclax (ven) plus azacitidine (aza) is the standard of care for patients with newly diagnosed acute myeloid leukemia (AML) who are not candidates for intensive chemotherapy (IC). Some patients who are IC candidates instead receive ven/aza. We retrospectively analyzed patients with newly diagnosed AML who received ven/aza (n = 143) or IC (n = 149) to compare outcomes, seek variables that could predict response to 1 therapy or the other, and ascertain whether treatment recommendations could be refined. The response rates were 76.9% for ven/aza and 70.5% for IC. The median overall survival (OS) was 884 days for IC compared with 483 days for ven/aza (P = .0020). A propensity-matched cohort was used to compare outcomes in the setting of equivalent baseline variables, and when matched for age, biological risk, and transplantation, the median OS was 705 days for IC compared with not reached for ven/aza (P = .0667). Variables that favored response to ven/aza over IC included older age, secondary AML, and RUNX1 mutations. AML M5 favored response to IC over ven/aza. In the propensity-matched cohort analyzing OS, older age, adverse risk, and RUNX1 mutations favored ven/aza over IC, whereas intermediate risk favored IC over ven/aza. In conclusion, patients receiving IC have improved OS compared with those receiving ven/aza. However, in a propensity-matched cohort of patients with equivalent baseline factors, there was a trend toward favorable OS for ven/aza. Specific variables, such as RUNX1 mutations, reported here for the first time, can be identified that favor ven/aza or IC, helping to guide treatment decisions for patients who may be eligible candidates for either therapy.

Allogeneic hematopoietic stem cell transplantation (SCT) after a patient with acute myeloid leukemia (AML) achieves a remission from intensive chemotherapy (IC) is given with curative intent. Recently, venetoclax-based regimens have become the standard of care for patients with newly diagnosed AML who are unfit for IC. If these patients achieve remission, they may also be considered for potentially curative consolidation with SCT. There are limited data comparing outcomes after SCT with these different induction strategies. The purpose of the current study was to evaluate depth of remission before SCT and outcomes after SCT in adults with nonrelapsed/refractory AML receiving pre-SCT therapy with either venetoclax/azacitidine (ven/aza) or IC. This was a retrospective, single-institution analysis of 169 patients receiving SCT in first remission after IC or ven/aza. Patient demographics and AML risk features were collected, as well as pre-SCT measurable residual disease (MRD) assessed by flow cytometry and molecular methods. Relapse, transplantation-related mortality, incidence of acute and chronic graft-versus-host-disease (GVHD), and death from any cause were also recorded. Descriptive and survival statistics were applied to these data to compare IC and ven/aza groups. Cox proportional hazard models were used for univariate and multivariate analyses. We demonstrate that despite differences in baseline factors between these groups, outcomes were similar. Relapse-free and overall survival, as well as cumulative incidences of transplantation-related mortality, relapse, and acute and chronic GVHD were comparable between groups. Exploring survival in younger (65 years) versus older (≥65 years) patients by treatment group did not alter these results. Finally, although pre-SCT MRD by flow cytometry was significantly predictive of post-SCT relapse and survival in the IC + SCT patients, it was not significantly predictive of relapse and survival in the ven/aza + SCT patients. Although these findings require prospective validation in a larger cohort of patients, they suggest that ven/aza followed by SCT is a reasonable management strategy for transplantation candidates at any age.

Traffic accidents in an urban road network are inevitable as a result claims and disputes arise among different road users. It is imperative to estimate the likelihood of traffic accidents resulting from different factors that contribute to loss of life, property and health of road users. There is a pressing need to reduce traffic accidents by identifying the location of accident hotspots using suitable analysis methods and examining them which is essential for the safety of road users. In this research traffic accident hotspots are identified using two spatial clustering analysis methods namely Getis-Ord Gi* and Nearest Neighborhood Hierarchy (NNH). These methods are compared and evaluated using the Prediction Accuracy Index (PAI) for their degree of accuracy. In this study, a cumulative traffic accident data of Hyderabad city of Telangana state over four years is researched upon and considered. Getis-Ord Gi* analysis measures the concentration ratio based on Z score identified as high (positive Z-values) and low values (negative Z-values). NNH analysis is another spatial clustering method which displays hotspot regions in the form of Convex hulls and Ellipses. The choice of the above two clustering methods represents the significance of the precision required. The findings of the study reveal that NNH method performed better compared to Getis-Ord Gi* method in its ability to detect hotspots. The above research methodology can be performed to any size of road network area globally having relevant accident data for the identification of hotspots for reducing the traffic accidents.

The use of polymer matrix composites has been increased exponentially for the past. Marine, aeronautical, automobile, and space structures are the areas were fiber-reinforced composites are very widely used for their specific properties like high tensile strength, high strength to weight, inert to the environment, etc. These conventional fiber-reinforced polymers (FRP) fall shortage of both compressive strength and impact strength. To overcome these challenges, an attempt is being made to develop the properties of FRP composites (glass/epoxy) by adding nanofiller. Three different types of nanoparticles are used: (i) multi-wall carbon nanotube (MWCNT), (ii) Nanosilica (NS), (iii) Nano-Iron oxide (NI) with four different weight percentages 0.1, 0.2, 0.5, and 1.0%. The experiments reveal a significant enhancement in the compression and impact behavior for Glass/Epoxy FRP when modified with nanofillers.

Background: Venetoclax-based therapy regimens are now FDA-approved for treatment of acute myeloid leukemia (AML) in older patients or those unfit to tolerate intensive chemotherapy (IC). Remission rates are high at 60-70% but relapses do frequently occur. Outcomes for newly-diagnosed patients who receive venetoclax-based therapies and proceed to a potentially curative allogeneic stem cell transplant (SCT) have largely been unreported. In the current study we compare outcomes of patients who received SCT following either IC or venetoclax + azacitidine (ven/aza) at the University of Colorado Hospital. Methods: Patients 18 years or older who received SCT in first remission of AML between 2010-2020 were included in the analysis. Patients were stratified into the IC arm if they initially received a backbone of cytarabine and an anthracycline; some patients in this cohort received IC in combination with other targeted agents. Patients who received ven/aza as first-line therapy followed by SCT were grouped in a separate cohort. Demographic and clinical information - including flow cytometry-based (MCF) MRD - was extracted from the electronic medical record. Comparisons of demographic and clinical variables between IC and ven/aza groups were made with t-test, Chi-squared, or Fisher's exact test depending on the nature of the variable. Relapse-free (RFS) and overall (OS) survival were calculated from the day of SCT to the respective endpoint or last documented follow-up using log-rank statistics. Finally, a Cox proportional hazards model was used to assess the interplay between variables pre- and post-SCT. P-values Results: We identified 179 patients who received SCT for AML in first remission. Of these patients, 151 received IC and 28 received ven/aza prior to SCT. Patients in the ven/aza group had higher median age than those in the IC group, as well as a higher proportion with adverse ELN genetic risk scoring. Patients in the ven/aza group received less intensive conditioning regimens. Sex, rates of MCF MRD negativity pre-BMT, incidence of severe acute or chronic GVHD, and causes of death were not significantly different between the two groups. There was no difference between the two groups in post-transplant RFS or OS (Figure 1). In a multivariate Cox model of pre-transplant variables predicting OS, the only factor that achieved significance was pre-SCT MCF MRD; the induction regimen was not a multivariate factor. Negative MCF MRD going into SCT was associated with decreased likelihood of relapse, GVHD, and death, respectively. Conclusions: In our cohort of AML patients receiving SCT, we found that ven/aza as a pre-transplant therapy yielded equivalent post-transplant outcomes compared to IC, in a population of older age and with higher ELN genetic risk. MCF MRD pre-SCT was confirmed as a key prognostic factor for post-SCT outcome. These findings support ongoing use of ven/aza as a first line therapy for elderly patients with AML as well as its exploration as a candidate therapy for younger patients. Figure 1 Figure 1. Disclosures Pollyea: Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other; Aprea: Honoraria, Membership on an entity's Board of Directors or advisory committees; Kiadis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Syndax: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria; Agios: Other, Research Funding; Karyopharm: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Teva: Research Funding; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Curis, Servier: Other; Pfizer: Research Funding; Syros: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Astellas: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Other: advisory board; Foghorn: Honoraria, Membership on an entity's Board of Directors or advisory committees; Genentech: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy, Honoraria, Other: advisory board; Jazz: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: advisory board.

Chronic myeloid leukemia (CML) is a heterogeneous disease, initiated by reciprocal translocation of chromosome 9 and 22, resulting in the generation of a BCR-ABL fusion protein and constitutive activation of the ABL kinase. ABL tyrosine kinase inhibitors (TKIs) have been very successful in suppressing CML disease. However, TKIs may not eliminate leukemia stem cells (LSCs), as evidenced by the frequent re-emergence of the disease upon TKI discontinuation. Moreover, blast phase CML (bpCML) remains a formidable challenge in disease management. Recent clinical evidence suggests that the BCL2 inhibitor venetoclax (Ven) in combination with ABL-targeting tyrosine kinase inhibitors (TKI) can eradicate bpCML LSCs. However, the exact mechanism by which this combination may targets LSCs is not known. In this report, we confirm the efficacy and LSC-targeting capacity of Ven/TKI combination in preclinical models of bpCML and we further identify that inhibition of free fatty acid (FFA) mobilization pathways may provide enhanced efficacy against LSCs. To establish the efficacy of Ven/TKI combination, we treated bpCML samples with Ven+Dasatinib (Das) combination for 24h, this resulted in a significant decrease in the viability of bulk and primitive populations (CD34+, CD38+). Patient-derived xenografts of bpCML samples in NSGS-mice, were treated with Ven/Das as well as single agents. The result showed a significant decrease in leukemia burden in the combination treated group, compared to either drug alone, albeit, some resistant cells survived in the combo treated group. Furthermore, using a syngeneic mouse model of bpCML, co-expressing Bcr-Abl and Nup98-HOXA9 translocations, the mouse leukemic cells treated with Ven/Dasatinib combination demonstrated a significant loss of viability of the bulk as well as phenotypically defined LSCs (Lin-/Sca1+). Treatment of leukemic mice with Ven/Das had a significant survival benefit and remained disease free at 80 days post treatment. We also showed significant survival benefits of Ven/ponatinib in NSGS-mice harboring syngeneic bpCML cells with the T315I gatekeeper mutation. Treatment of normal mice with Ven/Das combo did not affect the colony forming ability of LSK cells from the bone marrow, indicating a leukemia-specific response. Based on these results, we conclude that Ven/TKI combination effects were due to direct targeting of the LSC population. To investigate the potential mechanism of Ven/TKI activity in LSC targeting, we performed gene expression studies using RNA-seq based methods after short term treatment. Our findings indicated that the LSC population from Ven/TKI-treated mice showed enrichment of a gene signature associated with lysosome biology. Pre-treatment of mouse leukemia cells with bafilomycin, an inhibitor of lysosome function, resulted in increased sensitivity to Ven/TKI combo. Intriguingly, we also found significant induction of lysosomal acid lipase (LIPA), an enzyme involved in the generation of free fatty acids for energy needs. Metabolomic analysis of LSCs isolated after short term treatment with Ven/TKI, showed that a number of fatty acids were up-regulated in the Ven/Das treated group compared to control. Knocking down Lipa using CRISPR technology resulted in increased sensitivity to Ven/TKI combination, whereas overexpression of Lipa resulted in decreased sensitivity to the Ven/TKI combination, implicating Lipa upregulation and a resultant increase in free fatty acids as a protective response to Ven/TKI treatment. Furthermore, knocking down CPT1A, an important free fatty acid mitochondrial transporter, resulted in increased sensitivity to Ven/TKI combination both in mouse and primary human leukemic cells, leading to the hypothesis that activation of fatty acid processing through enhanced Lipa activity may represent a compensatory response to venetoclax based therapies in bpCML. In summary, we demonstrate the preclinical efficacy of Ven/TKI combination therapies for targeting of bpCML LSCs. Furthermore, our data suggest that blocking upregulation of free fatty acids through mechanisms such as inhibition of LIPA activity, might synergize with Ven/TKI combinations to eradicate LSCs, allowing for more durable response. Our findings provide a therapeutic rationale for blocking pathways involved in free fatty acids generation, as a potential strategy for increasing remission duration. Disclosures Pollyea: Amgen: Consultancy; Janssen: Consultancy; Genentech: Consultancy; AbbVie: Consultancy, Research Funding; Syndax: Consultancy; Daiichi Sankyo: Consultancy; Takeda: Consultancy; Pfizer: Consultancy; Celgene/BMS: Consultancy; Agios: Consultancy; Karyopharm: Consultancy; Novartis: Consultancy; Glycomimetics: Other. Smith: Syros: Research Funding; Kura: Research Funding; Argenx: Research Funding.

Multistoried buildings with open ground floor are inherently vulnerable to collapse due to earthquake load, their construction is still widespread in the developing nations due to social and functional need for provide car parking space at ground level. Engineering community warned against such buildings from time to time. Dynamic analysis shall be performed to obtain the design seismic force, and its distribution to different levels along the height of the building. It should be performed for both regular and irregular building. These are provisions in IS 1893 (part 1) 2002, with respect to height of building and according to irregularity of the building. In regular building greater than 40m height in zone IV and V is required and greater than 90m height in zone II and III. In irregular building greater than 12m height in zone IV and V is required and greater than 40m height in zone II and III. In this case study R.C.C. building is modeled and analyzed in four cases. I) Model with bare framed. II) Model with infill walls. III) Model with shear walls at bottom storey. IV) Model with steel bracing system at bottom storey

The goal of this project was to identify and target metabolic vulnerabilities of leukemia stem cells (LSCs) to improve therapeutic outcomes for patients with AML. We have previously shown that primary human LSCs reside in a unique metabolic condition characterized by a relatively low oxidative state (termed "ROS-low") and increased levels of glutathione (Lagadinou et al. Cell Stem Cell, 2013). Cells in this condition are highly dependent on oxidative phosphorylation for survival, in striking contrast to many tumor cells which often rely on glycolysis; indicating that LSCs are governed by distinct metabolic properties. To further elucidate key metabolic properties of LSCs, we measured differences in the global metabolome of ROS-Low LSCs in comparison to ROS-high AML blasts. Our preliminary data demonstrated that ROS-low LSCs have higher levels of amino acids and require amino acid catabolism for survival. We hypothesized that certain individual amino acids may be more important for LSC survival. If true, then targeting specific amino acids may be an avenue towards improved AML therapy. To determine if any individual amino acid is essential for LSC survival, we analyzed AML cells from five patients that were systematically cultured in media lacking one of the twenty amino acids. Cysteine depletion was consistently the most cytotoxic, showing decreased cell viability and colony forming potential of LSCs. We next determined the effect of an engineered human enzyme that selectively degrades cysteine and cystine (AEB3103, Aeglea BioTherapeutics, Inc.) on LSC viability and colony forming potential. We found that AEB3103 treatment decreased viability of LSCs in all AML specimens tested and significantly decreased colony formation (p To determine how AEB3103 decreased LSC viability we investigated the metabolic changes that occur upon AEB3103 treatment by mass spectroscopy. We found that AEB3103 treatment decreased the abundance of metabolites involved in glutathione synthesis (cysteine, cystine, glutathione, taurine, and Cys-Gly) in ROS-low LSCs. Tracing of cysteine13C315N in LSCs demonstrated that all detectable heavy cysteine was metabolized to cystine and glutathione. Furthermore, pretreatment with cell permeable glutathione rescued cell viability and colony-forming potential upon AEB3103 treatment. These data suggest that modulation of glutathione is central to the mechanism by which AEB3103 kills LSCs. Glutathione is a well-characterized antioxidant, therefore, we measured ROS levels and the expression of genes known to be expressed upon ROS induction in LSCs. Surprisingly, we did not observe changes in ROS induction or the expression of ROS induced genes upon AEB3103 treatment. Next, we interrogated cellular functions of glutathione that are independent from the role of glutathione as an antioxidant. Glutathione has previously been shown to mediate electron transport chain complex II function via posttranslational glutathione modification, glutathionylation. Therefore, we hypothesized that decreased glutathione levels upon AEB3103 treatment could result in decreased complex II activity and therefore decreased levels of OXPHOS and ATP production. To test this hypothesis, we measured complex II activity, complex II glutathionylation, oxidative phosphorylation, and ATP levels upon AEB3103 treatment. We observed AEB3103 treatment significantly decreased glutathionylation, complex II activity, oxidative phosphorylation, and ATP levels in AML cells (p Disclosures Pollyea: AbbVie: Consultancy, Research Funding; Argenx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Celyad: Consultancy, Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Consultancy; Curis: Membership on an entity's Board of Directors or advisory committees.

Epidemiological studies indicate that obese populations have an increased risk for leukemia. However, the mechanism underlying this phenomenon remains unclear. In the current study, utilizing both murine AML models and human AML samples, we observe that AML pathogenesis leads to aberrancies in adipose tissue, pancreatic function, and gut/microbiome, all of which contribute to an insulin resistant phenotype. We demonstrate that through induction of insulin resistance, leukemic disease alters systemic metabolism and thereby redirects systemic glucose to be preferentially available to malignant cells. We found that insulin effect on leukemic mice was significantly impaired as shown by insulin tolerance tests (ITT) (Fig.1) as well as by reduced glucose utilization in both adipose and muscle tissues. Interestingly, glucose utilization in leukemia cells was not affected by insulin. Several mechanisms were found to underlie this insulin resistant phenotype. First, leukemia induced a high-level production of IGFBP1 from adipose tissue and led to a 100-fold increase in circulating IGFBP1, which impaired insulin sensitivity (Fig.1). Blocking IGFBP1 partially restored insulin sensitivity and reduced leukemic burden, whereas pretreating with IGFBP1 facilitated leukemic progression. Second, a 95% reduction in the gut-derived circulating serotonin led to a significant decrease in insulin secretion from pancreas in leukemic mice (Fig.2). Serotonin supplementation partially restored serum insulin levels and decreased leukemic burden by 50% (Fig.2). Finally, the profile of gut microbiota in leukemic mice was distinct from normal mice. Leukemia-associated microbiota functionally contributed to the insulin resistant phenotype and therefore promoted disease progression. Mechanistically, decreased productions of microbiota-derived short chain fatty acids (SCFAs) i.e. butyrate and propionate, were found in leukemic fecal materials and butyrate supplementation reduced leukemic burden by 50% (Fig.2). Of note, there was interplay between these mechanisms. For example, agents increasing insulin levels resulted in a decreased IGFBP1 production in leukemic mice. Further, leukemia-associated microbiota also contributed to the elevated IGFBP1 level and the reduced insulin level in leukemic mice. To test the therapeutic relevance of our findings, we combined serotonin supplementation with butyrate, and found this combination (hereafter termed 'Ser-Bu') reduced leukemic burden by 80% (Fig.3) and provided survival benefits. More importantly, multiple assays including PET-CT scanning (Fig.3), 3H-2-DG labeling, and 13C-NMR labeling were employed to demonstrate that Ser-Bu treatment increased glucose uptake/utilization in adipose and muscle tissues by 200% and 50% respectively and reduced glucose uptake/utilization in leukemia cells by 70% (Fig.3). Therefore, leukemia progression can be significantly attenuated solely by modulation of systemic glucose metabolism. To examine the human relevance of our findings, we analyzed serum samples from normal controls, MDS and AML patients and found that a 6-fold and a 14-fold increase of IGFBP1 in MDS and AML serum respectively compared to normal controls (Fig.4). Additionally, other insulin resistance indicators such as serum free fatty acids, inflammatory cytokines and serum Leptin and Resistin levels were all elevated in AML serum. An insulin resistant phenotype and elevated serum IGFBP1 were also observed in a primary human leukemia specimen xenograft model. Further, a 65% reduction in serotonin was found in AML serum compared to normal controls (Fig.4). Impressively, analyses of paired diagnostic, remission and relapsed BM specimens showed that IGFBP1 was decreased in remission samples and rebounded in the relapsed state, whereas serotonin level showed the opposite pattern (Fig.5). Together, these data support that an insulin resistant phenotype is also evident in AML patients. Collectively, our studies suggest that leukemic tumors gain a competitive advantage by co-opting multiple mechanisms to induce a diabetes-like physiological condition and thereby subvert systemic glucose metabolism to facilitate disease progression. Our studies demonstrate that restoration of normal glucose regulation may be a feasible strategy to suppress systemic growth of malignant cell types. Disclosures Pollyea: Gilead: Consultancy; Curis: Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Celyad: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Consultancy, Research Funding; Argenx: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Activation of RhoA/Rho-associated kinase (ROCK) pathway and the associated changes in actin cytoskeleton induced by thrombin are crucial for activation of NF-kappaB and expression of its target gene ICAM-1 in endothelial cells. However, the events acting downstream of RhoA/ROCK to mediate these responses remain unclear. Here, we show a central role of cofilin-1, an actin-binding protein that promotes actin depolymerization, in linking RhoA/ROCK pathway to dynamic alterations in actin cytoskeleton that are necessary for activation of NF-kappaB and thereby expression of ICAM-1 in these cells. Stimulation of human umbilical vein endothelial cells with thrombin resulted in Ser(3) phosphorylation/inactivation of cofilin and formation of actin stress fibers in a ROCK-dependent manner. RNA interference knockdown of cofilin-1 stabilized the actin filaments and inhibited thrombin- and RhoA-induced NF-kappaB activity. Similarly, constitutively inactive mutant of cofilin-1 (Cof1-S3D), known to stabilize the actin cytoskeleton, inhibited NF-kappaB activity by thrombin. Overexpression of wild type cofilin-1 or constitutively active cofilin-1 mutant (Cof1-S3A), known to destabilize the actin cytoskeleton, also impaired thrombin-induced NF-kappaB activity. Additionally, depletion of cofilin-1 was associated with a marked reduction in ICAM-1 expression induced by thrombin. The effect of cofilin-1 depletion on NF-kappaB activity and ICAM-1 expression occurred downstream of IkappaBalpha degradation and was a result of impaired RelA/p65 nuclear translocation and consequently, RelA/p65 binding to DNA. Together, these data show that cofilin-1 occupies a central position in RhoA-actin pathway mediating nuclear translocation of RelA/p65 and expression of ICAM-1 in endothelial cells.

We have shown that the mammalian target of rapamycin (mTOR) down-regulates thrombin-induced ICAM-1 expression in endothelial cells by suppressing the activation of NF-kappaB. However, the mechanisms by which mTOR is activated to modulate these responses remain to be addressed. Here, we show that thrombin engages protein kinase C (PKC)-delta and phosphattidylinositol 3-kinase (PI3K)/Akt pathways to activate mTOR and thereby dampens NF-kappaB activation and intercellular adhesion molecule 1 (ICAM-1) expression. Stimulation of human vascular endothelial cells with thrombin induced the phosphorylation of mTOR and its downstream target p70 S6 kinase in a PKC-delta- and PI3K/Akt-dependent manner. Consistent with this, thrombin-induced phosphorylation of p70 S6 kinase was defective in embryonic fibroblasts from mice with targeted disruption of PKC-delta (Pkc-delta(-)(/)(-)), p85alpha and p85beta subunits of the PI3K (p85alpha(-)(/)(-)beta(-)(/)(-)), or Akt1 and Akt2 (Akt1(-)(/)(-)2(-)(/)(-)). Furthermore, we observed that expression of the constitutively active form of PKC-delta or Akt was sufficient to induce NF-kappaB activation and ICAM-1 expression, and that co-expression of mTOR suppressed these responses. In reciprocal experiments, inhibition/depletion of mTOR augmented NF-kappaB activation and ICAM-1 expression induced by PKC-delta or Akt. In control experiments, increasing or impairing mTOR signaling by the above approaches produced similar effects on NF-kappaB activation and ICAM-1 expression induced by thrombin. Thus, these data reveal an important role of PKC-delta and PI3K/Akt pathways in activating mTOR as an endogenous modulator to ensure a tight regulation of NF-kappaB signaling of ICAM-1 expression in endothelial cells.

Protein kinase C-delta (PKC-delta) plays a pivotal role in mediating thrombin-induced NF-kappaB activation and ICAM-1 expression in endothelial cells. However, the downstream mechanisms mediating its function are unclear. In this study, we show that PKC-delta-mediated activation of protein-tyrosine kinase Syk plays an important role in thrombin signaling of NF-kappaB activation and intercellular adhesion molecule-1 (ICAM-1) expression in endothelial cells. Stimulation of human vascular endothelial cells with thrombin resulted in a time-dependent phosphorylation of Syk on tyrosine 525 and 526, an indication of Syk activation. Inhibition of PKC-delta by pharmacological and genetic approaches prevented Syk activation by thrombin. These results place Syk downstream of PKC-delta in transmitting thrombin-activated signaling in endothelial cells. Consistent with this, thrombin-induced NF-kappaB activity and ICAM-1 expression were prevented by the expression of a kinase-defective mutant or RNA interference knockdown of Syk. Similarly, inhibiting Syk also impaired NF-kappaB activity and ICAM-1 expression induced by a constitutively active mutant of PKC-delta. Analysis of the NF-kappaB pathway showed that Syk contributes to thrombin-induced NF-kappaB activation by controlling its transactivation potential and that this response is associated with tyrosine phosphorylation of RelA/p65. Thus, these data unveil a novel pathway in which Syk signals downstream of PKC-delta to mediate thrombin induced ICAM-1 expression in endothelial cells by increasing transcriptional capacity of NF-kappaB via a mechanism that relies on tyrosine phosphorylation of RelA/p65.

Activation of the transcription factor NF-kappaB involves its release from the inhibitory protein IkappaBalpha in the cytoplasm and subsequently, its translocation to the nucleus. Whereas the events responsible for its release have been elucidated, mechanisms regulating the nuclear transport of NF-kappaB remain elusive. We now provide evidence for actin cytoskeleton-dependent and -independent mechanisms of RelA/p65 nuclear transport using the proinflammatory mediators, thrombin and tumor necrosis factor alpha, respectively. We demonstrate that thrombin alters the actin cytoskeleton in endothelial cells and interfering with these alterations, whether by stabilizing or destabilizing the actin filaments, prevents thrombin-induced NF-kappaB activation and consequently, expression of its target gene, ICAM-1. The blockade of NF-kappaB activation occurs downstream of IkappaBalpha degradation and is associated with impaired RelA/p65 nuclear translocation. Importantly, thrombin induces association of RelA/p65 with actin and this interaction is sensitive to stabilization/destabilization of the actin filaments. In parallel studies, stabilizing or destabilizing the actin filaments fails to inhibit RelA/p65 nuclear accumulation and ICAM-1 expression by tumor necrosis factor alpha, consistent with its inability to induce actin filament formation comparable with thrombin. Thus, these studies reveal the existence of actin cytoskeleton-dependent and -independent pathways that may be engaged in a stimulus-specific manner to facilitate RelA/p65 nuclear import and thereby ICAM-1 expression in endothelial cells.

We addressed the regulatory function of mammalian target of rapamycin (mTOR) in the mechanism of thrombin-induced ICAM-1 gene expression in endothelial cells. Pretreatment of HUVECs with rapamycin, an inhibitor of mTOR, augmented thrombin-induced ICAM-1 expression. Inhibition of mTOR by this approach promoted whereas over-expression of mTOR inhibited thrombin-induced transcriptional activity of NF-κB, an essential regulator of ICAM-1 transcription. Analysis of the NF-κB signaling pathway revealed that inhibition of mTOR potentiated IκB kinase activation resulting in a rapid and persistent phosphorylation of IκBα on Ser32 and Ser36, a requirement for IκBα degradation. Consistent with these data, we observed a more efficient and stable nuclear localization of RelA/p65 and, subsequently, the DNA binding activity of NF-κB by thrombin following mTOR inhibition. These data define a novel role of mTOR in down-regulating thrombin-induced ICAM-1 expression in endothelial cells by controlling a delayed and transient activation of NF-κB.

The main objective of this study was to deduce and assess crack propagation parameters of twelve asphalt mixtures including: eight conventional dense graded, two polymer-modified gap graded, and two asphalt-rubber gap graded mixtures using the EN 12697-44:2010 based monotonic semi-circular bending (SCB) test. The asphalt mixes were prepared using different binder types, binder contents, and air voids levels totaling 72 samples with two replicates per mix type. Dense graded mixes had higher fracture toughness than rubber- and polymer-modified mixes at various temperatures. Total fracture energy and residual energy were higher for modified mixes than dense mixes. The share of residual energy in rubber-modified mixtures was 80 % of total fracture energy, indicating that even though a crack initiates in these mixes, it will take much more time to completely fail those materials. Predictive models for crack propagation parameters were developed and were based upon material properties. All three models had very good statistical goodness of fit measures (R2adj ≥ 0.80, and Se/Sy ≤ 0.38). Overall, extensive experimentation and analytical methodologies provided a first of its kind comparative understanding of fracture crack propagation mechanism of conventional versus modified asphalt mixtures based on rationally established SCB test technique using a linear elastic fracture mechanics approach.

The procoagulant thrombin promotes polymorphonuclear leukocyte (PMN) adhesion to endothelial cells by a mechanism involving expression of intercellular adhesion molecule-1 (ICAM-1) via an NF-κB-dependent pathway. We now provide evidence that activation of c-Src is crucial in signaling thrombin-induced ICAM-1 expression via tyrosine phosphorylation of RelA/p65. Stimulation of human umbilical vein endothelial cells with thrombin resulted in a time-dependent activation of c-Src, with maximal activation occurring at 30 min after thrombin challenge. Inhibition of c-Src by pharmacological and genetic approaches impaired thrombin-induced NF-κB-dependent reporter activity and ICAM-1 expression. Analysis of the NF-κB pathway revealed that the effect of c-Src inhibition occurred independently of IκBα degradation and NF-κB DNA binding function and was not associated with exchange of NF-κB dimers. Phosphorylation of RelA/p65 at Ser536, an event mediating the transcriptional activity of DNA-bound RelA/p65, was also insensitive to c-Src inhibition. Interestingly, thrombin induced association of c-Src with RelA/p65, and inhibition of c-Src prevented this response, indicating that this interaction is contingent on activation of c-Src. We also observed that thrombin induced tyrosine phosphorylation of RelA/p65, and this phosphorylation was lost upon inhibition of c-Src, consistent with the requirement of activated c-Src for interaction with RelA/p65. These data implicate an important role of c-Src in phosphorylating RelA/p65 to promote the transcriptional activity of NF-κB and thereby ICAM-1 expression in endothelial cells.

We investigated the mechanisms by which elevated intracellular cAMP concentration inhibits the thrombin-induced ICAM-1 expression in endothelial cells. Exposure of human umbilical vein endothelial cells to forskolin or dibutyryl cAMP, which increase intracellular cAMP by separate mechanisms, inhibited the thrombin-induced ICAM-1 expression. This effect of cAMP was secondary to inhibition of NF-kappaB activity, the key regulator of thrombin-induced ICAM-1 expression in endothelial cells. The action of cAMP occurred downstream of IkappaBalpha degradation and was independent of NF-kappaB binding to the ICAM-1 promoter. We observed that cAMP interfered with thrombin-induced phosphorylation of NF-kappaB p65 (RelA) subunit, a crucial event promoting the activation of the DNA-bound NF-kappaB. Because p38 MAPK can induce transcriptional activity of RelA/p65 without altering the DNA binding function of NF-kappaB, we addressed the possibility that cAMP antagonizes thrombin-induced NF-kappaB activity and ICAM-1 expression by preventing the activation of p38 MAPK. We observed that treating cells with forskolin blocked the activation of p38 MAPK, and inhibition of p38 MAPK interfered with phosphorylation of RelA/p65 induced by thrombin. Our data demonstrate that increased intracellular cAMP concentration in endothelial cells prevents thrombin-induced ICAM-1 expression by inhibiting p38 MAPK activation, which in turn prevents phosphorylation of RelA/p65 and transcriptional activity of the bound NF-kappaB.