Characterization of Residual Stress in SOI Wafers by Using MEMS Cantilever Beams.

Silicon-on-insulator (SOI) wafers are crucial raw materials in the manufacturing process of microelectromechanical systems (MEMS). Residual stresses generated inside the wafers during the fabrication process can seriously affect the performance, reliability, and yield of MEMS devices. In this paper, a low-cost method based on mechanical modeling is proposed to characterize the residual stresses in SOI wafers in order to calculate the residual stress values based on the deformation of the beams. Based on this method, the residual strain of the MEMS beam, and thus the residual stress in the SOI wafer, were experimentally determined. The results were also compared with the residual stress results calculated from the deflection of the rotating beam to demonstrate the validity of the results obtained by this method. This method provides valuable theoretical reference and data support for the design and optimization of devices based on SOI-MEMS technology. It provides a lower-cost solution for the residual stress measurement technique, making it available for a wide range of applications.

BATF2 promotes HSC myeloid differentiation by amplifying IFN response mediators during chronic infection.

Basic leucine zipper ATF-like transcription factor 2 (BATF2), an interferon-activated immune response regulator, is a key factor responsible for myeloid differentiation and depletion of HSC during chronic infection. To delineate the mechanism of BATF2 function in HSCs, we assessed Batf2 KO mice during chronic infection and found that they produced less pro-inflammatory cytokines, less immune cell recruitment to the spleen, and impaired myeloid differentiation with better preservation of HSC capacity compared to WT. Co-IP analysis revealed that BATF2 forms a complex with JUN to amplify pro-inflammatory signaling pathways including CCL5 during infection. Blockade of CCL5 receptors phenocopied Batf2 KO differentiation defects, whereas treatment with recombinant CCL5 was sufficient to rescue IFNγ-induced myeloid differentiation and recruit more immune cells to the spleen in Batf2 KO mice. By revealing the mechanism of BATF2-induced myeloid differentiation of HSCs, these studies elucidate potential therapeutic strategies to boost immunity while preserving HSC function during chronic infection.

Lunar Dust-Mitigation Behavior of Aluminum Surfaces with Multiscale Roughness Prepared by a Composite Etching Method.

Reducing lunar dust adhesion to various material surfaces is important for protecting equipment from damage during lunar exploration missions. In this study, we investigate the lunar dust-mitigation ability and dust adhesion force of aluminum (Al) substrates prepared using different etching methods. Among them, composite etching methods (combining chemical and electrochemical steps) can result in multiscale structures with micro- and nanoroughness, reducing the contact area between the substrate and thus decreasing lunar dust adhesion. After composite etching, the dust adhesion force of the Al substrate was significantly reduced by 80% from 45.53 to 8.89 nN. The dust adhesion force of Al substrates dominates their dust-mitigation performance in floating dust environments. The lunar dust coverage (2.19%) of the Al substrate modified by composite etching (placed with a tilt angle of 90°) was 4-fold lower than that of the pristine Al substrate (9.11%), indicating excellent lunar-dust repellence. In addition, other factors such as tilt angle of the substrate and dust loading significantly affect dust-mitigation performance of the modified Al substrates. The Al substrate with an excellent dust-mitigation ability highlights good potential for lunar exploration missions.

Research Progress of MEMS Inertial Switches.

As a typical type of MEMS acceleration sensor, the inertial switch can alter its on-off state while the environmental accelerations satisfy threshold value. An exhaustive summary of the design concept, performance aspects, and fabrication methods of the micro electromechanical system (MEMS) inertial switch is provided. Different MEMS inertial switch studies were reviewed that emphasized acceleration directional and threshold sensitivity, contact characteristics, and their superiorities and disadvantages. Furthermore, the specific fabrication methods offer an applicability reference for the preparation process for the designed inertial switch, including non-silicon surface micromachining technology, standard silicon micromachining technology, and the special fabrication method for the liquid inertial switch. At the end, the main conclusions of the current challenges and prospects about MEMS inertial switches are drawn to assist with the development of research in the field of future engineering applications.

Conformational Changes of RORγ During Response Element Recognition and Coregulator Engagement.

The retinoic acid receptor-related orphan receptor γ (RORγ) is a ligand-dependent transcription factor of the nuclear receptor super family that underpins metabolic activity, immune function, and cancer progression. Despite being a valuable drug target in health and disease, our understanding of the ligand-dependent activities of RORγ is far from complete. Like most nuclear receptors, RORγ must recruit coregulatory protein to enact the RORγ target gene program. To date, a majority of structural studies have been focused exclusively on the RORγ ligand-binding domain and the ligand-dependent recruitment of small peptide segments of coregulators. Herein, we examine the ligand-dependent assembly of full length RORγ:coregulator complexes on cognate DNA response elements using structural proteomics and small angle x-ray scattering. The results from our studies suggest that RORγ becomes elongated upon DNA recognition, preventing long range interdomain crosstalk. We also determined that the DNA binding domain adopts a sequence-specific conformation, and that coregulatory protein may be able to 'sense' the ligand- and DNA-bound status of RORγ. We propose a model where ligand-dependent coregulator recruitment may be influenced by the sequence of the DNA to which RORγ is bound. Overall, the efforts described herein will illuminate important aspects of full length RORγ and monomeric orphan nuclear receptor target gene regulation through DNA-dependent conformational changes.

Acetylation of histone H3K27 signals the transcriptional elongation for estrogen receptor alpha.

As approximately 70% of human breast tumors are estrogen receptor α (ERα)-positive, estrogen and ERα play essential roles in breast cancer development. By interrupting the ERα signaling pathway, endocrine therapy has been proven to be an effective therapeutic strategy. In this study, we identified a mechanism by which Transcription Start Site (TSS)-associated histone H3K27 acetylation signals the Super Elongation Complex (SEC) to regulate transcriptional elongation of the ESR1 (ERα) gene. SEC interacts with H3K27ac on ESR1 TSS through its scaffold protein AFF4. Depletion of AFF4 by siRNA or CRISPR/Cas9 dramatically reduces expression of ESR1 and its target genes, consequently inhibiting breast cancer cell growth. More importantly, a AFF4 mutant which lacks H3K27ac interaction failed to rescue ESR1 gene expression, suggesting H3K27 acetylation at TSS region is a key mark bridging the transition from transcriptional initiation to elongation, and perturbing SEC function can be an alternative strategy for targeting ERα signaling pathway at chromatin level.

Refolding Proteins from Inclusion Bodies using Differential Scanning Fluorimetry Guided (DGR) Protein Refolding and MeltTraceur Web.

Differential Scanning Fluorimetry Guided Refolding (DGR) is a simple methodology that can be used to rapidly screen for and identify conditions capable of accurately refolding protein preparations, such as those obtained from Escherichia coli inclusion bodies. It allows for the production in E. coli of functional proteins that would otherwise require far more expensive production methods. This unit describes how to set up a DGR refolding assay, perform DGR refolding trials in microplate format, use MeltTraceur Web software to interactively analyze the resulting data, scale-up protein production via refolding, and lastly, validate that the protein is properly folded. © 2018 by John Wiley & Sons, Inc.

Pyrazolo[1,5-a]pyrimidine TRPC6 antagonists for the treatment of gastric cancer.

Transient receptor potential canonical 6 (TRPC6) proteins form receptor-operated Ca2+-permeable channels, which have been thought to bring benefit to the treatment of diseases, including cancer. However, selective antagonists for TRPC channels are rare and none of them has been tested against gastric cancer. Compound 14a and analogs were synthesized by chemical elaboration of previously reported TRPC3/6/7 agonist 4o. 14a had very weak agonist activity at TRPC6 expressed in HEK293 cells but exhibited strong inhibition on both 4o-mediated and receptor-operated activation of TRPC6 with an IC50 of about 1 µM. When applied to the culture media, 14a suppressed proliferation of AGS and MKN45 cells with IC50 values of 17.1 ±â€¯0.3 and 18.5 ±â€¯1.0 µM, respectively, and inhibited tube formation and migration of cultured human endothelial cells. This anti-tumor effect on gastric cancer was further verified in xenograft models using nude mice. This study has found a new tool compound which shows excellent therapeutic potential against human gastric cancer most likely through targeting TRPC6 channels.

Structure-activity relationships of 2-substituted phenyl-N-phenyl-2-oxoacetohydrazonoyl cyanides as novel antagonists of exchange proteins directly activated by cAMP (EPACs).

Exchange proteins directly activated by cAMP (EPACs) are critical cAMP-dependent signaling pathway mediators that play important roles in cancer, diabetes, heart failure, inflammations, infections, neurological disorders and other human diseases. EPAC specific modulators are urgently needed to explore EPAC's physiological function, mechanism of action and therapeutic applications. On the basis of a previously identified EPAC specific inhibitor hit ESI-09, herein we have designed and synthesized a novel series of 2-substituted phenyl-N-phenyl-2-oxoacetohydrazonoyl cyanides as potent EPAC inhibitors. Compound 31 (ZL0524) has been discovered as the most potent EPAC inhibitor with IC50 values of 3.6 µM and 1.2  µM against EPAC1 and EPAC2, respectively. Molecular docking of 31 onto an active EPAC2 structure predicts that 31 occupies the hydrophobic pocket in cAMP binding domain (CBD) and also opens up new space leading to the solvent region. These findings provide inspirations for discovering next generation of EPAC inhibitors.

A cell-based, quantitative and isoform-specific assay for exchange proteins directly activated by cAMP.

Extensive functional studies of the exchange protein directly activated by cAMP (EPAC) family of signaling molecules have demonstrated that EPAC proteins play a fundamental role in several physiological and pathophysiological responses, therefore are attractive drug targets. In this report, the development of a cell-based, medium to high throughput screening assay that is capable of monitoring EPAC-mediated activation of cellular Rap1 in an isoform-specific manner is described. This assay adapts a conventional ELISA format with immobilized RalGDS-RBD as a bait to selectively capture GTP-bound active Rap1. As a result, it fills an urgent need for a cell-based EPAC assay that can be conveniently performed using microtiter plates for the discovery and/or validation of isoform-specific EPAC agonists and antagonists.

Pyrazolopyrimidines as Potent Stimulators for Transient Receptor Potential Canonical 3/6/7 Channels.

Transient receptor potential canonical 3/6/7 (TRPC3/6/7) are highly homologous receptor-operated nonselective cation channels. Despite their physiological significance, very few selective and potent agonists are available for functional examination of these channels. Using a cell-based high throughput screening approach, a lead compound with the pyrazolopyrimidine skeleton was identified as a TRPC6 agonist. Synthetic schemes for the lead and its analogues were established, and structural-activity relationship studies were carried out. A series of potent and direct agonists of TRPC3/6/7 channels were identified, and among them, 4m-4p have a potency order of TRPC3 > C7 > C6, with 4n being the most potent with an EC50 of <20 nM on TRPC3. Importantly, these compounds exhibited no stimulatory activity on related TRP channels. The potent and selective compounds described here should be suitable for evaluation of the roles of TRPC channels in the physiology and pathogenesis of diseases, including glomerulosclerosis and cancer.

Identification of novel 2-(benzo[d]isoxazol-3-yl)-2-oxo-N-phenylacetohydrazonoyl cyanide analoguesas potent EPAC antagonists.

Two series of novel EPAC antagonists are designed, synthesized and evaluated in an effort to develop diversified analogues based on the scaffold of the previously identified high-throughput (HTS) hit 1 (ESI-09). Further SAR studies reveal that the isoxazole ring A of 1 can tolerate chemical modifications with either introduction of flexible electron-donating substitutions or structurally restrictedly fusing with a phenyl ring, leading to identification of several more potent and diversified EPAC antagonists (e.g., 10 (NY0617), 14 (NY0460), 26 (NY0725), 32 (NY0561), and 33 (NY0562)) with low micromolar inhibitory activities. Molecular docking studies on compounds 10 and 33 indicate that these two series of compounds bind at a similar site with substantially different interactions with the EPAC proteins. The findings may serve as good starting points for the development of more potent EPAC antagonists as valuable pharmacological probes or potential drug candidates.

Structure-Activity Relationship Studies with Tetrahydroquinoline Analogs as EPAC Inhibitors.

EPAC proteins are therapeutic targets for the potential treatment of cardiac hypertrophy and cancer metastasis. Several laboratories use a tetrahydroquinoline analog, CE3F4, to dissect the role of EPAC1 in various disease states. Here, we report SAR studies with tetrahydroquinoline analogs that explore various functional groups. The most potent EPAC inhibitor 12a exists as a mixture of inseparable E (major) and Z (minor) rotamers. The rotation about the N-formyl group indeed impacts the activity against EPAC.

Functionalized N,N-Diphenylamines as Potent and Selective EPAC2 Inhibitors.

N,N-Diphenylamines were discovered as potent and selective EPAC2 inhibitors. A study was conducted to determine the structure-activity relationships in a series of inhibitors of which several compounds displayed submicromolar potencies. Selectivity over the related EPAC1 protein was also demonstrated. Computational modeling reveals an allosteric site that is distinct from the cAMP binding domain shared by both EPAC isoforms, providing a theory with regards to subtype selectivity.

A Terrestrial Microbial Fuel Cell for Powering a Single-Hop Wireless Sensor Network.

Microbial fuel cells (MFCs) are envisioned as one of the most promising alternative renewable energy sources because they can generate electric current continuously while treating waste. Terrestrial Microbial Fuel Cells (TMFCs) can be inoculated and work on the use of soil, which further extends the application areas of MFCs. Energy supply, as a primary influential factor determining the lifetime of Wireless Sensor Network (WSN) nodes, remains an open challenge in sensor networks. In theory, sensor nodes powered by MFCs have an eternal life. However, low power density and high internal resistance of MFCs are two pronounced problems in their operation. A single-hop WSN powered by a TMFC experimental setup was designed and experimented with. Power generation performance of the proposed TMFC, the relationships between the performance of the power generation and the environment temperature, the water content of the soil by weight were measured by experiments. Results show that the TMFC can achieve good power generation performance under special environmental conditions. Furthermore, the experiments with sensor data acquisition and wireless transmission of the TMFC powering WSN were carried out. We demonstrate that the obtained experimental results validate the feasibility of TMFCs powering WSNs.

Critical role for Epac1 in inflammatory pain controlled by GRK2-mediated phosphorylation of Epac1.

cAMP signaling plays a key role in regulating pain sensitivity. Here, we uncover a previously unidentified molecular mechanism in which direct phosphorylation of the exchange protein directly activated by cAMP 1 (EPAC1) by G protein kinase 2 (GRK2) suppresses Epac1-to-Rap1 signaling, thereby inhibiting persistent inflammatory pain. Epac1(-/-) mice are protected against inflammatory hyperalgesia in the complete Freund's adjuvant (CFA) model. Moreover, the Epac-specific inhibitor ESI-09 inhibits established CFA-induced mechanical hyperalgesia without affecting normal mechanical sensitivity. At the mechanistic level, CFA increased activity of the Epac target Rap1 in dorsal root ganglia of WT, but not of Epac1(-/-), mice. Using sensory neuron-specific overexpression of GRK2 or its kinase-dead mutant in vivo, we demonstrate that GRK2 inhibits CFA-induced hyperalgesia in a kinase activity-dependent manner. In vitro, GRK2 inhibits Epac1-to-Rap1 signaling by phosphorylation of Epac1 at Ser-108 in the Disheveled/Egl-10/pleckstrin domain. This phosphorylation event inhibits agonist-induced translocation of Epac1 to the plasma membrane, thereby reducing Rap1 activation. Finally, we show that GRK2 inhibits Epac1-mediated sensitization of the mechanosensor Piezo2 and that Piezo2 contributes to inflammatory mechanical hyperalgesia. Collectively, these findings identify a key role of Epac1 in chronic inflammatory pain and a molecular mechanism for controlling Epac1 activity and chronic pain through phosphorylation of Epac1 at Ser-108. Importantly, using the Epac inhibitor ESI-09, we validate Epac1 as a potential therapeutic target for chronic pain.

Structure-Activity Relationship Studies of Substituted 2-(Isoxazol-3-yl)-2-oxo-N'-phenyl-acetohydrazonoyl Cyanide Analogues: Identification of Potent Exchange Proteins Directly Activated by cAMP (EPAC) Antagonists.

Exchange proteins directly activated by cAMP (EPAC) as guanine nucleotide exchange factors mediate the effects of the pivotal second messenger cAMP, thereby regulating a wide variety of intracellular physiological and pathophysiological processes. A series of novel 2-(isoxazol-3-yl)-2-oxo-N'-phenyl-acetohydrazonoyl cyanide EPAC antagonists was synthesized and evaluated in an effort to optimize properties of the previously identified high-throughput (HTS) hit 1 (ESI-09). Structure-activity relationship (SAR) analysis led to the discovery of several more active EPAC antagonists (e.g., 22 (HJC0726), 35 (NY0123), and 47 (NY0173)) with low micromolar inhibitory activity. These inhibitors may serve as valuable pharmacological probes to facilitate our efforts in elucidating the biological functions of EPAC and developing potential novel therapeutics against human diseases. Our SAR results have also revealed that further modification at the 3-, 4-, and 5-positions of the phenyl ring as well as the 5-position of the isoxazole moiety may allow for the development of more potent EPAC antagonists.

Rp-cAMPS Prodrugs Reveal the cAMP Dependence of First-Phase Glucose-Stimulated Insulin Secretion.

cAMP-elevating agents such as the incretin hormone glucagon-like peptide-1 potentiate glucose-stimulated insulin secretion (GSIS) from pancreatic ß-cells. However, a debate has existed since the 1970s concerning whether or not cAMP signaling is essential for glucose alone to stimulate insulin secretion. Here, we report that the first-phase kinetic component of GSIS is cAMP-dependent, as revealed through the use of a novel highly membrane permeable para-acetoxybenzyl (pAB) ester prodrug that is a bioactivatable derivative of the cAMP antagonist adenosine-3',5'-cyclic monophosphorothioate, Rp-isomer (Rp-cAMPS). In dynamic perifusion assays of human or rat islets, a step-wise increase of glucose concentration leads to biphasic insulin secretion, and under these conditions, 8-bromoadenosine-3',5'-cyclic monophosphorothioate, Rp-isomer, 4-acetoxybenzyl ester (Rp-8-Br-cAMPS-pAB) inhibits first-phase GSIS by up to 80%. Surprisingly, second-phase GSIS is inhibited to a much smaller extent (≤20%). Using luciferase, fluorescence resonance energy transfer, and bioluminescence resonance energy transfer assays performed in living cells, we validate that Rp-8-Br-cAMPS-pAB does in fact block cAMP-dependent protein kinase activation. Novel effects of Rp-8-Br-cAMPS-pAB to block the activation of cAMP-regulated guanine nucleotide exchange factors (Epac1, Epac2) are also validated using genetically encoded Epac biosensors, and are independently confirmed in an in vitro Rap1 activation assay using Rp-cAMPS and Rp-8-Br-cAMPS. Thus, in addition to revealing the cAMP dependence of first-phase GSIS from human and rat islets, these findings establish a pAB-based chemistry for the synthesis of highly membrane permeable prodrug derivatives of Rp-cAMPS that act with micromolar or even nanomolar potency to inhibit cAMP signaling in living cells.

Biochemical and pharmacological characterizations of ESI-09 based EPAC inhibitors: defining the ESI-09 "therapeutic window".

The cAMP signaling cascade is one of the most frequently targeted pathways for the development of pharmaceutics. A plethora of recent genetic and pharmacological studies suggest that exchange proteins directly activated by cAMP (EPACs) are implicated in multiple pathologies. Selective EPAC inhibitors have been recently developed. One specific inhibitor, ESI-09, has been shown to block EPAC activity and functions, as well as to recapitulate genetic phenotypes of EPAC knockout mice when applied in vivo. However, a recent study raised concern that ESI-09 might act as a non-specific protein denaturant. Herein, we present a detailed biochemical and pharmacological characterization, as well as a structure-activity relationship (SAR) analysis of ESI-09. Our studies show that ESI-09 dose-dependently inhibits activity of both EPAC1 and EPAC2 with apparent IC50 values well below the concentrations shown to induce "protein denaturation". Moreover, the ESI-09's action towards EPAC proteins is highly sensitive to minor modifications of the 3-chlorophenyl moiety. Taken together, these results demonstrate that ESI-09 indeed acts as an EPAC specific antagonist and does not significantly destabilize/denature proteins at pharmacological effective concentrations. This conclusion is further supported by NMR data showing that ESI-09 induces residue-dependent chemical shift changes at low concentrations, while preserving well dispersed peaks.

Identification and optimization of 2-aminobenzimidazole derivatives as novel inhibitors of TRPC4 and TRPC5 channels.

BACKGROUND AND PURPOSE: Transient receptor potential canonical (TRPC) channels play important roles in a broad array of physiological functions and are involved in various diseases. However, due to a lack of potent subtype-specific inhibitors the exact roles of TRPC channels in physiological and pathophysiological conditions have not been elucidated. EXPERIMENTAL APPROACH: Using fluorescence membrane potential and Ca(2+) assays and electrophysiological recordings, we characterized new 2-aminobenzimidazole-based small molecule inhibitors of TRPC4 and TRPC5 channels identified from cell-based fluorescence high-throughput screening. KEY RESULTS: The original compound, M084, was a potent inhibitor of both TRPC4 and TRPC5, but was also a weak inhibitor of TRPC3. Structural modifications of the lead compound resulted in the identification of analogues with improved potency and selectivity for TRPC4 and TRPC5 channels. The aminobenzimidazole derivatives rapidly inhibited the TRPC4- and TRPC5-mediated currents when applied from the extracellular side and this inhibition was independent of the mode of activation of these channels. The compounds effectively blocked the plateau potential mediated by TRPC4-containing channels in mouse lateral septal neurons, but did not affect the activity of heterologously expressed TRPA1, TRPM8, TRPV1 or TRPV3 channels or that of the native voltage-gated Na(+) , K(+) and Ca(2) (+) channels in dissociated neurons. CONCLUSIONS AND IMPLICATIONS: The TRPC4/C5-selective inhibitors developed here represent novel and useful pharmaceutical tools for investigation of physiological and pathophysiological functions of TRPC4/C5 channels.