The cellular responses to steroids are mediated by 2 general mechanisms: genomic and rapid/nongenomic effects. Identification of the mechanisms underlying aldosterone (ALDO)'s rapid vs their genomic actions is difficult to study, and these mechanisms are not clearly understood. Recent data suggest that striatin is a mediator of nongenomic effects of estrogen. We explored the hypothesis that striatin is an intermediary of the rapid/nongenomic effects of ALDO and that striatin serves as a novel link between the actions of the mineralocorticoid and estrogen receptors. In human and mouse endothelial cells, ALDO promoted an increase in phosphorylated extracellular signal-regulated protein kinases 1/2 (pERK) that peaked at 15 minutes. In addition, we found that striatin is a critical intermediary in this process, because reducing striatin levels with small interfering RNA (siRNA) technology prevented the rise in pERK levels. In contrast, reducing striatin did not significantly affect 2 well-characterized genomic responses to ALDO. Down-regulation of striatin with siRNA produced similar effects on estrogen's actions, reducing nongenomic, but not some genomic, actions. ALDO, but not estrogen, increased striatin levels. When endothelial cells were pretreated with ALDO, the rapid/nongenomic response to estrogen on phosphorylated endothelial nitric oxide synthase (peNOS) was enhanced and accelerated significantly. Importantly, pretreatment with estrogen did not enhance ALDO's nongenomic response on pERK. In conclusion, our results indicate that striatin is a novel mediator for both ALDO's and estrogen's rapid and nongenomic mechanisms of action on pERK and phosphorylated eNOS, respectively, thereby suggesting a unique level of interactions between the mineralocorticoid receptor and the estrogen receptor in the cardiovascular system.

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Physical Unclonable Functions (PUFs) are used for securing electronic designs across the implementation spectrum ranging from lightweight FPGA to server-class ASIC designs. However, current PUF implementations are vulnerable to model-building attacks; they often incur significant design overheads and are challenging to configure based on application-specific requirements. These factors limit their application, primarily in the case of the system on chip (SoC) designs used in diverse applications. In this work, we propose MeL-PUF -Memory-in-Logic PUF, a low-overhead, distributed, and synthesizable PUF that takes advantage of existing logic gates in a design and transforms them to create cross-coupled inverters (i.e. memory cells) controlled by a PUF control signal. The power-up states of these memory cells are used as the source of entropy in the proposed PUF architecture. These on-demand memory cells can be distributed across the combinational logic of various intellectual property (IP) blocks in a system on chip (SoC) design. They can also be synthesized with a standard logic synthesis tool to meet the area/power/performance constraints of a design. By aggregating the power-up states from multiple such memory cells, we can create a PUF signature or digital fingerprint of varying size. We evaluate the MeL-PUF signature quality with both circuit-level simulations as well as with measurements in FPGA devices. We show that MeL-PUF provides high-quality signatures in terms of uniqueness, randomness, and robustness, without incurring large overheads. We also suggest additional optimizations that can be leveraged to improve the performance of MeL-PUF .

Activation of the mineralocorticoid receptor (MR), a critical component of the Renin-Angiotensin-Aldosterone (ALDO)-System (RAAS), has been shown to play an important role in inflammatory and vascular endothelial responses in addition to its well-described effects on sodium and water homeostasis. Activationof endothelial cells leads to, among other factors, increased endothelin-1 (ET-1) and protein disulfide isomerase (PDI) release. PDI and ET-1 contribute to vascular inflammation and are increased in patients with SCD and sickle transgenic mouse models. The MR is a member of the steroid family of nuclear receptors and transcription factors that upon activation binds to hormone response elements of edn1, the gene for ET-1, leading to increased ET-1 expression.In vivo, blockade of MR has been shown to reduce circulating ET-1 levels and ET-1 mRNA expression. However, the role of MR in SCD is unclear. We hypothesized that MR blockade in sickle transgenic mice would reduce PDI activity and improve hematological parameters and inflammation. We first studied EA.hy926 (EA) cells, a human endothelial cell line that expresses MR. We incubated EA cells with ALDO (10-8 M), an MR agonist, for 24 hr and observed a rise in PDI mRNA levels by qRT-PCR (P<0.01, n=5), an event that was blocked by pre-incubation of EA cells with 1 μM canrenoic acid (CA), an MR antagonist (P<0.05, n=5). We then measured PDI activity in the supernatant of ALDO-stimulated EA cells using a Di-E-GSSH fluorescent marker and observed a rise in PDI activity following ALDO (10-8 M) when compared to vehicle treatment (P<0.05; n=5). To test the in vivo effects of MR activation, we studied Berkeley Sickle Transgenic (BERK) mice that were randomized to receive either normal rodent chow or chow containing eplerenone (156 mg/kg per day), an MR antagonist (MRA), for 14 days. We observed significantly lower plasma PDI activity in mice treated with MRA than those on regular chow (63.7 ± 8.7 control diet to 47.9 ± 2.4 eplerenone, Relative Fluorescence Units (RFU); P<0.005, n=6 and 9, respectively). Treatment with MRA was associated with reduced plasma ET-1 and myeloperoxidase (MPO) levels in BERK mice. We also studied RBC Gardos channel activity in these mice and observed a significant reduction in clotrimazole-sensitive K+ efflux following MR blockade (2.49±0.5 control and 1.37±0.3 mmol/1013 cells x hr; P<0.04 n= 5 and 7 respectively). Consistent with these results, MR blockade was associated with increases in both erythrocyte MCV (41.3±2.5 vs 47.4±1.1 fL, P<0.03, n=7) and reticulocyte MCV (53.6.3±2.8 vs 60.1±0.6 fL, P<0.02, n=7). We also studied gene expression by qRT-PCR in heart tissue from these mice and observed that MR blockade reduced mRNA expression of: ET-1 (0.654 ± 0.233, P<0.05, n=5 and n=7); PDI (0.546 ± 0.063, P<0.01, n=5 and n=7); and Tumor Necrosis Factor Receptor Superfamily Member 1A mRNA (0.464 ± 0.061, P<0.01, n=5 and n=7). Thus, our results suggest a novel role for RAAS and, in particular, MR activation in SCD. Disclosures No relevant conflicts of interest to declare.

Activation of the minerolocorticoid receptor (MR) by aldosterone (ALDO) has been shown to play an important role in inflammatory vascular responses in addition to its well described effects on sodium homeostasis. Steroid responses are mediated by well-known genomic and less known rapid/nongenomic responses. However, characterization of the mechanisms underlying ALDO’s rapid/nongenomic actions have been difficult to study and are not clearly understood. We recently reported that in vivo and in vitro activation of MR leads to increases of striatin levels in endothelial cells, aortas and heart tissue (Pojoga, Amer J Hypertens, 2012) and that MR forms a complex with caveolin-1 and striatin within caveolae in endothelial cells. We hypothesized that striatin is a critical intermediary of the rapid effects of ALDO and that striatin serves as a novel link for MR regulation in endothelial cells activation. Endothelial cell activation promotes, among other factors, increased levels of reactive oxygen species (ROS) and protein disulfide isomerase (PDI), a redox modifying enzyme that catalyze disulfide interchange reactions. We studied EA.hy926 cells (EA), a human endothelial cell line that expresses MR, striatin and maintains its caveolae while in culture. We incubated EA cells with ALDO (10–9–10–7M) for 60 min and observed a dose-dependent rise in ROS production (P<0.001, n=4) using the oxidative fluorescent indicator dye 5-6-chloromethyl-2ʼ,7ʼ-dichlorodihydrofluorescein diacetate (CM-H2DCFDA) that peaked at around 10-8M ALDO, an event that was blocked by pre-incubation of EA cells for 30 mins with 1μM canrenoic acid (CA), an MR antagonist (P<0.03, n=3). Time course analyses showed ALDO stimulated ROS responses that increased for up to 3 hours following the addition of ALDO. As there are no known inhibitors for striatin we then used siRNA technology to down regulate striatin in these cells. EA cells were transfected with striatin siRNA and subsequently stimulated with ALDO and ROS production measured. The transfection process itself did not modify baseline levels of ROS significantly, as assessed in cells transfected with scrambled siRNA and non-transfected cells, which had nearly identical ROS levels, basally and in response to ALDO. In addition, in the presence of lower levels of striatin protein the ALDO-stimulated ROS response was abrogated, supporting the concept that striatin is necessary for the rapid effects of ALDO. We also measured phosphorylated ERK-1/2 (pERK) levels that peaked within 10 minutes in EA as estimated by western blot analyses. Consistent with these observations pre-incubation of EA cells with 10-6M PD0325901, a selective MEK-1/2 inhibitor was associated with greater than 90% reduction of the ALDO-stimulated ROS responses (7244.3±497 vs 4386.6±586 RFU, P<0.02, n=3). Qualitatively similar responses were observed using another MEK inhibitor, U0126 [10-5M] (P<0.01, n=3). We then tested the effects of ALDO on PDI secretion. Incubation of EA cells with ALDO (10-7M) led to PDI increases when compared to vehicle treated cells (P<0.01, n=3). We also tested the effects of low levels of striatin using siRNA on PDI activity in EA cells. We found that PDI secretion was reduced by 62% in striatin knockdown conditions. We then tested the effects of Methyl-β-cyclodextrin to disrupt caveolae in these cells and observed a blunted 10nM ALDO–stimulated PDI response (530±117 to 215±99 RFU/mg protein, n=3, P<0.01). We then isolated early cultures of mouse aortic endothelial cells (MAEC) from endothelial-specific caveolin-1 knockout mice and measured PDI activity following 24 hrs of incubation in 0.4% fetal bovine serum. Our results show that MAEC from caveolin-1 knockout mice had lower PDI secretion when compared to cells from WT mice (99.4±16 vs 129.9±35, n=5, P<0.03). These results suggest that striatin is a novel mediator for ALDO’s rapid effects on PDI and ROS, thereby suggesting a unique level of interaction between the MR and striatin in endothelial cell activation. Supported by NIH R01HL090632 (AR), R01HL104032 (LHP) and R01HL096518 (JRR). Disclosures: No relevant conflicts of interest to declare.

Increased endothelin‐1 (ET‐1) levels in patients with sickle cell disease (SCD) and transgenic mouse models of SCD contribute to disordered hematological, vascular, and inflammatory responses. Mineralocorticoid receptor (MR) activation by aldosterone, a critical component of the Renin‐Angiotensin‐Aldosterone‐System, modulates inflammation and vascular reactivity, partly through increased ET‐1 expression. However, the role of MR in SCD remains unclear. We hypothesized that MR blockade in transgenic SCD mice would reduce ET‐1 levels, improve hematological parameters, and reduce inflammation. Berkeley SCD (BERK) mice, a model of severe SCD, were randomized to either sickle standard chow or chow containing the MR antagonist (MRA), eplerenone (156 mg/Kg), for 14 days. We found that MRA treatment reduced ET‐1 plasma levels (p = .04), improved red cell density gradient profile (D50; p < .002), and increased mean corpuscular volume in both erythrocytes (p < .02) and reticulocytes (p < .024). MRA treatment also reduced the activity of the erythroid intermediate‐conductance Ca2+‐activated K+ channel – KCa3.1 (Gardos channel, KCNN4), reduced cardiac levels of mRNAs encoding ET‐1, Tumor Necrosis Factor Receptor‐1, and protein disulfide isomerase (PDI) (p < .01), and decreased plasma PDI and myeloperoxidase activity. Aldosterone (10−8 M for 24 h in vitro) also increased PDI mRNA levels (p < .01) and activity (p < .003) in EA.hy926 human endothelial cells, in a manner blocked by pre‐incubation with the MRA canrenoic acid (1 μM; p < .001). Our results suggest a novel role for MR activation in SCD that may exacerbate SCD pathophysiology and clinical complications.

Background Disordered endothelial cell activation plays an important role in the pathophysiology of atherosclerosis, cancer, sepsis, viral infections, and inflammatory responses. There is interest in developing novel therapeutics to regulate endothelial cell function in atherothrombotic, metabolic, vascular, and hematological diseases. Extracts from leaves of the Syzygium jambos (L.) Alston (S. jambos) trees have been proposed to treat cardiovascular diseases and diabetes through unclear mechanisms. We investigated the effects of the S. jambos extract on biomarkers of endothelial dysfunction and immune responses in the human endothelial cell line, EA.hy926. Methods Leaves of S. jambos were collected, concocted and lyophilized. To study the effects of S. jambos on endothelial cell activation, we used the human endothelial cell line. IL-6 levels were measured using qPCR and ELISA. PDI activity was measured using Insulin Turbidity and Di-E-GSSG assays. CM-H2DCFDA was used to study ROS levels. Migration assay was used to study S. jambos effect on ex vivo human polymorphonuclear and human mononuclear cells. Results Our results show that incubation of EA.hy926 cells with ET-1 led to a 6.5 ± 1.6 fold increase in IL-6 expression by qPCR, an event that was blocked by S. jambos. Also, we observed that ET-1 increased extracellular protein disulfide isomerase (PDI) activity that was likewise dose-dependently blocked by S. jambos (IC50 = 14 μg/mL). Consistent with these observations, ET-1 stimulated ex vivo human polymorphonuclear and mononuclear cell migration that also was dose-dependently blocked by S. jambos. In addition, ET-1 stimulation led to significant increases in ROS production that were sensitive to S. jambos. Conclusion Our results suggest that the S. jambos extract represents a novel cardiovascular protective pharmacological approach to regulate endothelial cell activation, IL-6 expression, and immune-cell responses.

<p>The Field Programmable Gate Array (FPGA) market has seen significant growth due to the low cost and reduced time to market when compared to ASIC circuits. However, FPGAs' reconfigurable nature introduces security vulnerabilities that can be exploited by adversaries to obtain sensitive information. Physical Unclonable Functions (PUFs) have shown to be valuable security primitives. By leveraging manufacturing variations in a device one can generate unique signatures that can be used for authentication and generation of secret keys. However, PUF implementation can be costly, taking up FPGA resources and requiring long design times. In this paper, we propose a method which takes advantage of the FPGA Look Up Table (LUT) architecture to embed the PUF into functional logic circuits, reducing the cost and design time. We provide detailed implementation guidelines and evaluate the PUF's signature quality and stability under different environmental variations using a set of testbench circuits. Our results demonstrate the effectiveness of our method in securing FPGA designs while reducing implementation costs and design time. </p>