An All-Dielectric Metamaterial Terahertz Biosensor for Cytokine Detection.

In this paper, we report an all-dielectric metamaterial terahertz biosensor, which exhibits a high Q factor of 35 at an 0.82 resonance peak. A structure with an electromagnetically induced transparency effect was designed and fabricated to perform a Mie resonance for the terahertz response. The biosensor exhibits a limit of detection of 100 pg/mL for cytokine interleukin 2 (IL-2) and a linear response for the logarithm of the concentration of IL-2 in the range of 100 pg/mL to 1 µg/mL. This study implicates an important potential for the detection of cytokines in serum and has potential application in the clinical detection of cytokine release syndrome.

Effect of Sintering Temperature on Adhesion Property and Electrochemical Activity of Pt/YSZ Electrode.

The (Pt/YSZ)/YSZ sensor unit is the basic component of the NOx sensor, which can detect the emission of nitrogen oxides in exhaust fumes and optimize the fuel combustion process. In this work, the effect of sintering temperature on adhesion property and electrochemical activity of Pt/YSZ electrode was investigated. Pt/YSZ electrodes were prepared at different sintering temperatures. The microstructure of the Pt/YSZ electrodes, as well as the interface between Pt/YSZ electrode and YSZ electrolyte, were observed by SEM. Chronoamperometry, linear scan voltammetry, and AC impedance were tested by the electrochemical workstation. The results show that increasing the sintering temperature (≤1500 °C) helped to improve adhesion property and electrochemical activity of the Pt/YSZ electrode, which benefited from the formation of the porous structure of the Pt/YSZ electrode. For the (Pt/YSZ) electrode/YSZ electrolyte system, O2- in YSZ is converted into chemisorbed O2 on Pt/YSZ, which is desorbed into the gas phase in the form of molecular oxygen; this process could be the rate-controlling step of the anodic reaction. Increasing the sintering temperature (≤1500 °C) could reduce the reaction activation energy of the Pt/YSZ electrode. The activation energy reaches the minimum value (1.02 eV) when the sintering temperature is 1500 °C.

Hydrophobic Wafer-Scale High-Reproducibility SERS Sensor Based on Silicon Nanorods Arrays Decorated with Au Nanoparticles for Pesticide Residue Detection.

High sensitivity and reproducibility are highly desirable to a SERS sensor in diverse detection applications. Moreover, it is a great challenge to determine how to promote the target molecules to be more concentrated on the hotspots of the SERS substrate by engineering a surface with switching interfacial wettability. Along these lines, wafer-scale uniformly hydrophobic silicon nanorods arrays (SiNRs) decorated with Au nanoparticles were designed as the SERS substrate. Typically, the SERS substrate was fabricated by enforcing the polystyrene (PS) sphere self-assembly, as well as the plasma etching and the magnetron sputtering techniques. Consequently, the SERS substrate was treated by soaking within a n-dodecyl mercaptan (NDM) solution at different times in order to obtain adjustable wettabilities. By leveraging the electromagnetic enhancement resulted from the Au nanostructures and enrichment effect induced by the hydrophobicity, the SERS substrate is endowed with efficient SERS capabilities. During the detection of malachite green (MG), an ultralow relative standard deviation (RSD) 4.04-6.14% is achieved and the characteristic signal of 1172 cm-1 can be detected as low as 1 ng/mL. The proposed SiNRs' structure presents outstanding SERS activity with sensitivity and reproducibility rendering thus an ideal candidate for potential application in analytical detection fields.

Numerical Analysis and Experimental Studies on the Residual Stress of W/2024Al Composites.

W/2024Al composites can be used for radiation shielding with desirable mechanical properties such as high strength, excellent corrosion resistance, and low density. The quench-induced residual stresses in W/2024Al composites were studied by experimental measurements and numerical analysis using ABAQUS software. Due to the accurate calculation of heat transfer coefficients and the established constitutive equation for description of the variation of yield stress at elevated temperature with different strain rates, the prediction of residual stresses in as-quenched composite blocks achieved by finite element method (FEM) is reliable. Moreover, X-ray diffraction and crack-compliance method were carried out to measure the stresses that developed at the surface and interior of the composites to validate the simulation results. Quenching residual stresses of composite blocks were investigated by taking the influence of quenching medium temperature into consideration. In addition, a comparative study on residual stress magnitudes of as-quenched 2024Al and W/2024Al composites was conducted, and the results show that stress magnitudes of W/2024Al composites are lower than that of 2024Al due to lower thermal gradients during the quenching process.

Interfacial Microstructure in W/2024Al Composite and Inhibition of W-Al Direct Reaction by CeO2 Doping: Formation and Crystallization of Al-Ce-Cu-W Amorphous Layers.

In this work, interfacial microstructure in W/2024Al composite and inhibition of the W-Al direct reaction by CeO2 doping were investigated. The composites were prepared through powder sintering, and after preparation the composites were treated by annealing at 823 K. For the prepared W/2024Al composite, a multi-phase thin layer composed of WAl12 and WAl5 compounds were formed at the interface due to the W-Al direct reaction. While doping CeO2 in the composite, Al-Ce-Cu-W amorphous substituting of W-Al compounds were formed at the interfacial reaction layer. In an annealed state, the composite with CeO2 doping shows a significant inhibitory effect on W-Al compounds, which was attributed to the crystallized layer that evolved from Al-Ce-Cu-W amorphous as an interfacial obstacle. The crystallization product for Al-Ce-Cu-W amorphous layer was identified as bcc-structure Al-Ce-Cu-W phase without any binary/ternary Ce-containing phases. Therefore, by doping CeO2 in W/2024Al composite, W-Al direct reaction was markedly inhibited during both preparation and annealing.

Linking stochastic fluctuations in chromatin structure and gene expression.

The number of mRNA and protein molecules expressed from a single gene molecule fluctuates over time. These fluctuations have been attributed, in part, to the random transitioning of promoters between transcriptionally active and inactive states, causing transcription to occur in bursts. However, the molecular basis of transcriptional bursting remains poorly understood. By electron microscopy of single PHO5 gene molecules from yeast, we show that the "activated" promoter assumes alternative nucleosome configurations at steady state, including the maximally repressive, fully nucleosomal, and the maximally non-repressive, nucleosome-free, configuration. We demonstrate that the observed probabilities of promoter nucleosome configurations are obtained from a simple, intrinsically stochastic process of nucleosome assembly, disassembly, and position-specific sliding; and we show that gene expression and promoter nucleosome configuration can be mechanistically coupled, relating promoter nucleosome dynamics and gene expression fluctuations. Together, our findings suggest a structural basis for transcriptional bursting, and offer new insights into the mechanism of transcriptional regulation and the kinetics of promoter nucleosome transitions.

In vivo role for the chromatin-remodeling enzyme SWI/SNF in the removal of promoter nucleosomes by disassembly rather than sliding.

Analysis of in vivo chromatin remodeling at the PHO5 promoter of yeast led to the conclusion that remodeling removes nucleosomes from the promoter by disassembly rather than sliding away from the promoter. The catalytic activities required for nucleosome disassembly remain unknown. Transcriptional activation of the yeast PHO8 gene was found to depend on the chromatin-remodeling complex SWI/SNF, whereas activation of PHO5 was not. Here, we show that PHO8 gene circles formed in vivo lose nucleosomes upon PHO8 induction, indicative of nucleosome removal by disassembly. Our quantitative analysis of expression noise and chromatin-remodeling data indicates that the dynamics of continual nucleosome removal and reformation at the activated promoters of PHO5 and PHO8 are closely similar. In contrast to PHO5, however, activator-stimulated transcription of PHO8 appears to be limited mostly to the acceleration of promoter nucleosome disassembly with little or no acceleration of promoter transitions following nucleosome disassembly, accounting for the markedly lower expression level of PHO8.

Occlusion of regulatory sequences by promoter nucleosomes in vivo.

Nucleosomes are believed to inhibit DNA binding by transcription factors. Theoretical attempts to understand the significance of nucleosomes in gene expression and regulation are based upon this assumption. However, nucleosomal inhibition of transcription factor binding to DNA is not complete. Rather, access to nucleosomal DNA depends on a number of factors, including the stereochemistry of transcription factor-DNA interaction, the in vivo kinetics of thermal fluctuations in nucleosome structure, and the intracellular concentration of the transcription factor. In vitro binding studies must therefore be complemented with in vivo measurements. The inducible PHO5 promoter of yeast has played a prominent role in this discussion. It bears two binding sites for the transcriptional activator Pho4, which at the repressed promoter are positioned within a nucleosome and in the linker region between two nucleosomes, respectively. Earlier studies suggested that the nucleosomal binding site is inaccessible to Pho4 binding in the absence of chromatin remodeling. However, this notion has been challenged by several recent reports. We therefore have reanalyzed transcription factor binding to the PHO5 promoter in vivo, using 'chromatin endogenous cleavage' (ChEC). Our results unambiguously demonstrate that nucleosomes effectively interfere with the binding of Pho4 and other critical transcription factors to regulatory sequences of the PHO5 promoter. Our data furthermore suggest that Pho4 recruits the TATA box binding protein to the PHO5 promoter.

Quantitative analysis of the transcription control mechanism.

Gene transcription requires a sequence of promoter state transitions, including chromatin remodeling, assembly of the transcription machinery, and clearance of the promoter by RNA polymerase. The rate-limiting steps in this sequence are regulated by transcriptional activators that bind at specific promoter elements. As the transition kinetics of individual promoters cannot be observed, the identity of the activator-controlled steps has remained a matter of speculation. In this study, we investigated promoter chromatin structure, and the intrinsic noise of expression over a wide range of expression values for the PHO5 gene of yeast. Interpretation of our results with regard to a stochastic model of promoter chromatin remodeling and gene expression suggests that the regulatory architecture of the gene expression process is measurably reflected in its intrinsic noise profile. Our chromatin structure and noise analyses indicate that the activator of PHO5 transcription stimulates the rates of promoter nucleosome disassembly, and assembly of the transcription machinery after nucleosome removal, but no other rates of the expression process.

Targeted mutation of the mouse Grp94 gene disrupts development and perturbs endoplasmic reticulum stress signaling.

Glucose-regulated protein 94 (GRP94) is one of the most abundant endoplasmic reticulum (ER) resident proteins and is the ER counterpart of the cytoplasmic heat shock protein 90 (HSP90). GRP94, a component of the GRP78 chaperone system in protein processing, has pro-survival properties with implicated function in cancer progression and autoimmune disease. Previous studies on the loss of GRP94 function showed that it is required for embryonic development, regulation of toll-like receptors and innate immunity of macrophages. Here we report the creation of mouse models targeting exon 2 of the Grp94 allele that allows both traditional and conditional knockout (KO) of Grp94. In this study, we utilized the viable Grp94+/+ and +/- mice, as well as primary mouse embryonic fibroblasts generated from them as experimental tools to study its role in ER chaperone balance and ER stress signaling. Our studies reveal that while Grp94 heterozygosity reduces GRP94 level it does not alter ER chaperone levels or the ER stress response. To study the effect of complete loss of GRP94 function, since homozygous GRP94 KO leads to embryonic lethality, we generated Grp94-/- embryonic stem cells. In contrast to Grp94 heterozygosity, complete knockout of GRP94 leads to compensatory upregulation of the ER chaperones GRP78, calnexin and calreticulin but not protein disulphide isomerase. Unexpectedly, loss of GRP94 leads to significant decrease in the level of ER-stress induced spliced form of XBP-1 protein, a downstream target of the IRE1 signaling pathway. Furthermore, from analysis of microarray database and immunohistochemical staining, we present predictions where GRP94 may play an important role in specific adult organ homeostasis and function.

Critical role of the stress chaperone GRP78/BiP in tumor proliferation, survival, and tumor angiogenesis in transgene-induced mammary tumor development.

The unfolded protein response (UPR) is an evolutionarily conserved mechanism that activates both proapoptotic and survival pathways to allow eukaryotic cells to adapt to endoplasmic reticulum (ER) stress. Although the UPR has been implicated in tumorigenesis, its precise role in endogenous cancer remains unclear. A major UPR protective response is the induction of the ER chaperone GRP78/BiP, which is expressed at high levels in a variety of tumors and confers drug resistance in both proliferating and dormant cancer cells. To determine the physiologic role of GRP78 in in situ-generated tumor and the consequence of its suppression on normal organs, we used a genetic model of breast cancer in the Grp78 heterozygous mice where GRP78 expression level was reduced by about half, mimicking anti-GRP78 agents that achieve partial suppression of GRP78 expression. Here, we report that Grp78 heterozygosity has no effect on organ development or antibody production but prolongs the latency period and significantly impedes tumor growth. Our results reveal three major mechanisms mediated by GRP78 for cancer progression: enhancement of tumor cell proliferation, protection against apoptosis, and promotion of tumor angiogenesis. Importantly, although partial reduction of GRP78 in the Grp78 heterozygous mice substantially reduces the tumor microvessel density, it has no effect on vasculature of normal organs. Our findings establish that a key UPR target GRP78 is preferably required for pathophysiologic conditions, such as tumor proliferation, survival, and angiogenesis, underscoring its potential value as a novel therapeutic target for dual antitumor and antiangiogenesis activity.

Endoplasmic reticulum stress.

Stress is the imbalance of homeostasis, which can be sensed even at the subcellular level. The stress-sensing capability of various organelles including the endoplasmic reticulum (ER) has been described. It has become evident that acute or prolonged ER stress plays an important role in many human diseases; especially those involving organs/tissues specialized in protein secretion. This article summarizes the emerging role of ER stress in diverse human pathophysiological conditions such as carcinogenesis and tumor progression, cerebral ischemia, plasma cell maturation and apoptosis, obesity, insulin resistance, and type 2 diabetes. Certain components of the ER stress response machinery are identified as biomarkers of the diseases or as possible targets for therapeutic intervention.

GRP78/BiP is required for cell proliferation and protecting the inner cell mass from apoptosis during early mouse embryonic development.

GRP78, also known as BiP, is a central regulator of endoplasmic reticulum (ER) homeostasis due to its multiple functional roles in protein folding, ER calcium binding, and controlling of the activation of transmembrane ER stress sensors. ER stress induction of GRP78/BiP represents a major prosurvival arm of the unfolded protein response (UPR). However, the physiological role of GRP78 in development is not known. Using a transgenic approach, we discovered that the Grp78 promoter is activated in both the trophectoderm and inner cell mass (ICM) of embryos at embryonic day 3.5 via a mechanism requiring the ER stress elements. To reveal the function of the GRP78 in vivo, we created a tri-loxP Grp78 mutant allele, which was further crossed with EIIA-cre to create a knockout allele. The Grp78+/- mice, which express 50% of the wild-type level of the GRP78 protein, are viable. Interestingly, the heterozygous Grp78 cells up-regulate the ER proteins GRP94 and protein disulfide isomerase at both the transcript and protein levels, while other UPR targets such as CHOP and XBP-1 are not affected. Further studies revealed that mouse embryonic fibroblasts from Grp78+/- mice are capable of responding to ER stress. However, Grp78-/- embryos that are completely devoid of GRP78 lead to peri-implantation lethality. These embryos do not hatch from the zona pellucida in vitro, fail to grow in culture, and exhibit proliferation defects and a massive increase in apoptosis in the ICM, which is the precursor of embryonic stem cells. These findings provide the first evidence that GRP78 is essential for embryonic cell growth and pluripotent cell survival.

In vivo regulation of Grp78/BiP transcription in the embryonic heart: role of the endoplasmic reticulum stress response element and GATA-4.

The transcriptional activation of GRP78, which controls multiple signaling pathways of the unfolded protein response, has been used extensively as an indicator for the onset of endoplasmic reticulum stress in tissue culture systems. Here we investigate the mechanism of Grp78 induction during mouse embryonic development. Our results reveal that in transgenic mouse models, reporter gene activity driven by the Grp78 promoter is strongly activated during early embryonic heart development but subsides in later stages. This activation is strictly dependent on a 100-base pair region of the Grp78 promoter containing the endoplasmic reticulum stress response elements (ERSEs). Previous studies establish that endoplasmic reticulum stress induces in vivo binding of YY1 and the nuclear form of ATF6 to the ERSE. Since the expression of YY1 as well as ATF6 is ubiquitous in the mouse embryo, activation of the Grp78 promoter in the early embryonic heart may involve a specific mechanism. Here we report that GATA-4, a transcription factor essential for heart development, binds to the Grp78 promoter in vivo and activates the ERSE, which does not contain a consensus GATA binding site. GATA-4 cooperatively activates the Grp78 promoter with YY1, and the DNA binding domain of YY1 is necessary and sufficient for this cooperation. In addition, GATA-4 activation of the Grp78 promoter is enhanced by the nuclear form of ATF6, and this synergy is further potentiated by YY1. These results suggest that during early heart organogenesis, Grp78 can be activated through cooperation between the cell type-specific transcription factors and ERSE-binding factors.

Endoplasmic reticulum stress triggers an acute proteasome-dependent degradation of ATF6.

ATF6, a 670 amino acid endoplasmic reticulum (ER) transmembrane glycoprotein with the electrophoretic mobility of a 90 kDa protein, is a key transcriptional activator of the unfolded protein response (UPR) that allows mammalian cells to maintain cellular homeostasis when the cells are subjected to a variety of environmental and physiological stress. Previous studies have established that ATF6 is a short-lived protein, the activation of which involves relocation from the ER to the Golgi where it is cleaved by the S1P/S2P protease system to generate a nuclear form that acts as a transcriptional activator for ER-stress inducible target genes such as Grp78/BiP. We report here that in addition to this process, ER-stress mediated by thapsigargin triggers an acute proteasomal degradation of the pre-existing pool of p90ATF6 independent of S1P/S2P cleavage. We showed that ATF6 is a direct target of proteasome-ubiquitin pathway, and this process can be suppressed by proteasome inhibitors, ALLN and MG115. We further observed that in non-stressed cells, p90ATF6 can be stabilized by MG115 but not ALLN and that treatment of cells with MG115 results in Grp78 induction in the absence of ER stress. These studies suggest that ER-stress induced acute, transit degradation of p90ATF6 could represent a novel cellular defense mechanism to prevent premature cell death resulting from p90ATF6 activation. Further, inhibition of proteasome activity can result in chaperone protein gene induction through stabilization of p90ATF6 as well as accumulation of malfolded proteins.

Endoplasmic reticulum chaperone protein GRP78 protects cells from apoptosis induced by topoisomerase inhibitors: role of ATP binding site in suppression of caspase-7 activation.

A large number of correlative studies have established that the activation of the unfolded protein response (UPR) alters the cell's sensitivity to chemotherapeutic agents. Although the induction of the glucose-regulated proteins (GRPs) is commonly used as an indicator for the UPR, the direct role of the GRPs in conferring resistance to DNA damaging agents has not been proven. We report here that without the use of endoplasmic reticulum (ER) stress inducers, specific overexpression of GRP78 results in reduced apoptosis and higher colony survival when challenged with topoisomerase II inhibitors, etoposide and doxorubicin, and topoisomerase I inhibitor, camptothecin. While investigating the mechanism for the GRP78 protective effect against etoposide-induced cell death, we discovered that in contrast to the UPR, GRP78 overexpression does not result in G1 arrest or depletion of topoisomerase II. Caspase-7, an executor caspase that is associated with the ER, is activated by etoposide. We show here that specific expression of GRP78 blocks caspase-7 activation by etoposide both in vivo and in vitro, and this effect can be reversed by addition of dATP in a cell-free system. Recently, it was reported that ectopically expressed GRP78 and caspases-7 and -12 form a complex, thus coupling ER stress to the cell death program. However, the mechanism of how GRP78, a presumably ER lumen protein, can regulate cytosolic effectors of apoptosis is not known. Here we provide evidence that a subpopulation of GRP78 can exist as an ER transmembrane protein, as well as co-localize with caspase-7, as confirmed by fluorescence microscopy. Co-immunoprecipitation studies further reveal endogenous GRP78 constitutively associates with procaspase-7 but not with procaspase-3. Lastly, a GRP78 mutant deleted of its ATP binding domain fails to bind procaspase-7 and loses its protective effect against etoposide-induced apoptosis.

Cancer-inducible transgene expression by the Grp94 promoter: spontaneous activation in tumors of various origins and cancer-associated macrophages.

A major challenge in treating cancer is the difficulty of bringing therapy to poorly perfused areas of solid tumors, which are often most resistant to chemotherapy and radiation. GRP94 is a chaperone protein localized in the endoplasmic reticulum with antiapoptotic properties. We report here that in vitro the proximal murine grp94 promoter is regulated differently from the hypoxia response element fused to the SV40 minimal promoter, with glucose starvation as an inducer of grp94 but a potent repressor of the hypoxia response element/SV40 fusion promoter. Through the use of transgenic mouse models, we showed that LacZ transgene expression driven by the grp94 promoter was strongly activated not only in spontaneous but also in a variety of chemically induced tumors. We additionally discovered that macrophages in the vicinity of malignant tumor showed a high level of transgene expression, consistent with intense beta-galactosidase staining at boundaries between viable tumor cells and necrotic areas. Isolated macrophages also showed grp94 mRNA and transgene activation under glucose starvation in vitro. In contrast, transgene activity was not detected in the normal tissue counterparts of any of the malignant tumors examined or macrophages associated with normal organs. These studies provide direct evidence that the tumor microenvironment is a potent physiological inducer of the grp94 promoter. The unique properties of the grp94 promoter suggest that it may offer a novel tool for directing transcription of therapeutic agents to tumors particularly in resistant regions bordering necrotic areas, delivered through standard vectors or macrophages.