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Edwardsiella tarda is an important pathogenic bacterium that can replicate in macrophages. However, how the intramacrophage infection process affects the virulence of this bacterium is essentially unknown. Here, we show that E. tarda replicates and induces a caspase-1-dependent cell pyroptosis in a murine macrophage model. Via pyroptosis, intracellular E. tarda escapes to the extracellular milieu, forming a unique bacterial population. Being different from the bacteria cultured alone, this unique population possesses a reprogrammed transcriptional profile, particularly with upregulated type III secretion system (T3SS)/T6SS cluster genes. Subsequent studies revealed that the macrophage-released population gains enhanced infectivity for host epithelial cells and increases resistance to multiple host defenses and hence displays significantly promoted virulence in vivo. Further studies indicated that T3SS is essentially required for the macrophage infection process, while T6SS contributes to infection-induced bacterial virulence. Altogether, this work demonstrates that E. tarda can utilize macrophages as a niche for virulence priming and for spreading infection, suggesting a positive role for intramacrophage infection in bacterial pathogenesis. [ABSTRACT FROM AUTHOR]

Short contact time catalytic partial oxidation (CPO) of methane has received increasing attention in industrial applications. Understanding of the catalytic system under high pressure and in the presence of sulfur represents a key challenge of this technology. This paper discusses CPO performance using a Rh−Ce catalyst supported on washcoat alumina foam under industrial relevant operating conditions (pressure up to 160 psig and different sulfur levels up to 16 ppm in methane). A long-term experimental run cycling between sulfur-free and sulfur injection conditions was conducted to examine the influence of sulfur on the catalyst. Sulfur effects were also evaluated under different sulfur levels and with different feed compositions. A dual zone reaction model was used to explain the effects of pressure, sulfur level, and the O/C ratio on CPO catalytic performance. Engineering challenges and requirements of the CPO process for industrial applications were discussed on the basis of recent advances in the understanding of CPO chemistry. [ABSTRACT FROM AUTHOR]

In this study, a network of reactions involved in the ethanol steam reforming (ESR) over supported Co catalysts has been investigated using temperature-programmed reaction (TPRxn) and isotopic labeling techniques. The products and intermediates in the gas phase have been monitored by mass spectrometry (MS) as a function of temperature. Ethanol conversion and product distribution obtained in steady-state reaction experiments have also been presented. The results point to a complex network of reactions that directly impact the selectivity and H2yield that can be obtained in ethanol steam reforming. The reaction network is governed by the temperature range as well as the type of catalyst used, with catalysts exhibiting higher oxygen mobility favoring complete oxidation of ethanol to CO2and leading to increased hydrogen yield as well as improved stability. [ABSTRACT FROM AUTHOR]

Cyclic nucleotide phosphodiesterase (POE) isoforms can influence disease pathogenesis and be novel therapeutic targets. Because lower cAMP levels may contribute to the decreased apoptosis that occurs in chronic lymphocytic leukemia (CLL), we assessed the expression levels of POE isoforms in peripheral blood mononuclear cells (PBMC) of healthy adults and patients with CLL. We found a unique POE mRNA signature in CLI: higher levels than in normal PBMC of PDE7B (increased ≈23-fold) and lower levels of PDE3B, 4D, 5A, and 9A mRNA (each decreased ≈30-fold). Increased POE7B mRNA in CLL correlates with a 10-fold-higher expression of PDE7B protein and results in an increased contribution of PDE7 to total PDE activity. Consistent with the higherlevel of PDE7B expression, inhibitors of PDE7 (BRL-50481, IR-202) and a dual PDE4/PDE7 inhibitor (IR-284) selectively increase apoptosis in CLI cells compared with normal PBMC or B cells. Apoptosis of CLI cells promoted by inhibitors of PDE7 and PDE4I7 is attenuated by PKA inhibition, occurs via a mitochondrial-dependent process, and is associated with increased cAMP accumulation and down-regulation of the antiapoptotic protein survivin and of PDE7B. The increase in PDE7B expression and PDE7 inhibitor-promoted apoptosis implicates PDE7B as a drug target in CLI. Our findings identify a unique PDE signature in CLL and illustrate the utility of broad analyses of POE isoform expression in human disease. [ABSTRACT FROM AUTHOR]

The second messenger cAMP acts via protein kinase A (PKA) to induce apoptosis by mechanisms that are poorly understood. Here, we assessed a role for mitochondria and analyzed gene expression in cAMP/PKA-promoted apoptosis by comparing wild-type (WI) S49 lymphoma cells and the S49 variant, D- (cAMP-deathless), which lacks cAMP-promoted apoptosis but has wild-type levels of PKA activity and cAMP-promoted G1 growth arrest. Treatment of WI, but not D-, S49 cells with 8-CPT-cAMP (8-(4-chlorophenylthio)-adenosine-3′ :5′-cyclic monophosphate) for 24 h induced loss of mitochondrial membrane potential, mitochondrial release of cytochrome c and SMAC, and increase in caspase-3 activity. Gene expression analysis (using Affymetrix 430 2.0 arrays) revealed that WT and D- cells incubated with 8-CPT-cAMP have similar, but non-identical, extents of cAMP-regulated gene expression at 2 h (-800 transcripts) and 6 h (~1000 transcripts) (∣Fold∣ > 2, p < 0.06); by contrast, at 24 h, ~2500 and ~1100 transcripts were changed in WT and D- cells, respectively. Using an approach that combined regression analysis, clustering, and functional annotation to identify transcripts that showed differential expression between WT and D- cells, we found differences in cAMP-mediated regulation of mRNAs involved in transcriptional repression, apoptosis, the cell cycle, RNA splicing, Golgi, and lysosomes. The two cell lines differed in cAMP-response element-binding protein (CREB) phosphorylation and expression of the transcriptional inhibitor ICER (inducible cAMP early repressor) and in cAMP-regulated expression of genes in the inhibitor of apoptosis (lAP) and Bcl families. The findings indicate that cAMP/PKA-promoted apoptosis of lymphoid cells occurs via mitochondrial-mediated events and imply that such apoptosis involves gene networks in multiple biochemical pathways. [ABSTRACT FROM AUTHOR]

Organosilicon compounds can form novel electrolytes having a number of unusual properties, including high electrochemical stability, high thermal stability, and low viscosity. Vertically aligned carbon nanofibers can act as exceptionally good nanostructured electrodes, with the vertical orientation and presence of edge-plane graphite along the sidewalls providing desirable electrochemical properties. We have explored the electrical properties of the interfaces of VACNF electrodes with a model organosilicon electrolyte solution. The compound 1-(3-2-2-(2-methoxy-ethoxy)-ethoxy-ethoxy-propyl)-1,1,3,3,3-pentamethyl-disiloxane (2SM3) with added lithium bis-oxalato-borate (LiBOB) was used. Forests of vertically aligned carbon nanofibers (VACNFs) ∼80 nm in diameter grown by plasma-enhanced chemical vapor deposition (PECVD) were used as electrodes. Our results show that the resulting interfaces yield interfacial capacitances equal to the increase in geometric area, indicating full electrochemical accessibility of the nanofiber sidewalls. Measurements as a function of potential perfect stability at applied voltages up to 3 V. Some initial Faradaic reactions arise near 3.5 V, but these decrease within a few cycles to produce interfaces that exhibit excellent stability up to 5 V while yielding >1000 F/cm2from nanofibers only 2 m long. The results show that the organosilicon electrolyte combined with VACNFs provides excellent electrochemical properties. The results have implications for understanding the ability to integrate organosilicon electrolytes with high-surface-area carbon materials for applications such as electrochemical double-layer capacitors and lithium-ion batteries. [ABSTRACT FROM AUTHOR]

The effects of synthesis parameters on the activity of Co–ZrO2 catalysts in bio-ethanol steam reforming (BESR) were investigated. The supported catalysts were prepared by incipient wetness impregnation (IWI) and characterized through N2 physisorption, H2 chemisorption, X-ray diffraction, X-ray photoelectron spectroscopy, temperature programmed calcination, temperature programmed reduction, temperature programmed oxidation, thermogravimetric analysis, ethanol pulse chemisorption, and temperature programmed reaction techniques. The synthesis parameters examined include calcination temperature, reduction temperature, and reduction time. The catalyst evolution at different stages of the synthesis process was investigated. The ethanol adsorption and temperature programmed reaction experiments indicated a strong correlation between the catalytic activity and metallic cobalt surface area for the 10 wt% Co–ZrO2 catalyst. [ABSTRACT FROM AUTHOR]

Although a substantial number of hormones and drugs increase cellular CAMP levels, the global impact of CAMP and its major effector mechanism, protein kinase A (PKA). on gene expression is not known. Here we show that treatment of murine wild-type 549 lymphoma cells for 24 h with 8-(4-chlorophenylthio)-cAMP (8-CPT- CAMP), a PKA-selective cAMP analog, alters the expression of ≈4,500 of ≈ 13,600 unique genes. By contrast, gene expression was unaltered in Kin- 549 cells (that lack PKA) incubated with 8-CPT- CAMP. Changes in mRNA and protein expression of several cell- cycle regulators accompanied cAMP-induced G1-phase cell-cycle arrest of wild-type S49 cells. Within 2 h, 8-CPT-cAMP altered expression of 152 genes that contain evolutionarily conserved cAMP-response elements within 5 kb of transcriptional start sites, including the circadian clock gene Pen. Thus, CAMP through its activation of PKA produces extensive transcriptional regulation in eukaryotic cells. These transcriptional networks include a primary group of cAMP-response element-containing genes and secondary networks that include the circadian clock. [ABSTRACT FROM AUTHOR]

Examines the role of Bcl-2 in protecting lymphoma cells from apoptosis. Insignificance of Bcl-2 in protecting growth arrest by cAMP and dexamethasone; Decrease in cell number without affecting viability; Manifestation of cell growth pattern different from wild-type S49 cells.

Examines the impact of Bcl-2, an antiapoptic protein on S49 cell growth and death promoted by cyclic adenylic acid and dexamethasone. Assessment on the morphological appearance and DNA laddering; Reflection of apoptic cell death; Incubation of wild type cells.

Edwardsiella piscicida is an intracellular pathogen within a broad spectrum of hosts. Essential to E. piscicida's virulence is its ability to invade and replicate inside host cells, yet the survival mechanisms and the nature of the replicative compartment remain unknown. Here, we characterized its intracellular lifestyle in nonphagocytic cells and showed that the intracellular replication of E. piscicida in nonphagocytic cells is dependent on its type III secretion system (T3SS) but not its type VI secretion system. Following internalization, E. piscicida is contained in vacuoles that transiently mature into early endosomes but subsequently bypasses the classical endosome pathway and fusion with lysosomes, which depend on its T3SS. Following rapid escape from the degradative pathway, E. piscicida was found to create a specialized replication-permissive niche characterized by endoplasmic reticulum (ER) markers. Furthermore, we found that a T3SS effector, EseJ, is responsible for the intracellular replication of E. piscicida by preventing endosome/lysosome fusion. In vivo experiments also confirmed that EseJ is necessary for bacterial colonization by E. piscicida in the epithelial layer, followed by systemic dissemination in both zebrafish and mice. Thus, this work elucidates the tactics used by E. piscicida to survive and proliferate within host nonphagocytic cells. [ABSTRACT FROM AUTHOR]

Edwardsiella piscicida is an important pathogen that infects a wide range of hosts from fish to human. Recent studies demonstrated that E. piscicida can invade and survive within multiple nonphagocytic cells, but the internalization mechanism remains poorly understood. Here, we used HeLa cells as a nonphagocytic cell model to investigate the endocytic strategy used by the pathogenic E. piscicida isolate EIB202. Using a combination of optical and electron microscopy, we observed obvious membrane ruffles and F-actin rearrangements in HeLa cells after EIB202 infection. We also revealed that EIB202 internalization significantly depended on the activity of Na+/H+ exchangers and multiple intracellular signaling events related to macropinocytosis, suggesting that E. piscicida utilizes the host macropinocytosis pathway to enter HeLa cells. Further, using inhibitory drugs and shRNAs to block specific endocytic pathways, we found that a caveolin-dependent but not clathrin-dependent pathway is involved in E. piscicida entry and that its entry requires dynamin and membrane cholesterol. Together, these data suggest that E. piscicida enters nonphagocytic cells via macropinocytosis and caveolin-dependent endocytosis involving cholesterol and dynamin, improving the understanding of how E. piscicida interacts with nonphagocytic cells. [ABSTRACT FROM AUTHOR]

In the crystal structure of the title compound, C7H18NO2+·I−, cations and anions are linked into ion pairs by means of a long O—H...I hydrogen-bonding interaction [O...I = 3.5299 (11) Å and O—H...I = 169.8 (19)°]. [ABSTRACT FROM AUTHOR]

Mitochondrial morphology is regulated by fusion and fission machinery. Impaired mitochondria dynamics cause various diseases, including Alzheimer's disease. Appoptosin (encoded by SLC25A38) is a mitochondrial carrier protein that is located in the mitochondrial inner membrane. Appoptosin overexpression causes overproduction of reactive oxygen species (ROS) and caspase-dependent apoptosis, whereas appoptosin downregulation abolishes β-amyloid-induced mitochondrial fragmentation and neuronal death during Alzheimer's disease. Herein, we found that overexpression of appoptosin resulted in mitochondrial fragmentation in a manner independent of its carrier function, ROS production or caspase activation. Although appoptosin did not affect levels of mitochondrial outer-membrane fusion (MFN1 and MFN2), inner-membrane fusion (OPA1) and fission [DRP1 (also known as DNM1L) and FIS1] proteins, appoptosin interacted with MFN1 and MFN2, as well as with the mitochondrial ubiquitin ligase MITOL (also known as MARCH5) but not OPA1, FIS1 or DRP1. Appoptosin overexpression impaired the interaction between MFN1 and MFN2, and mitochondrial fusion. By contrast, co-expression ofMFN1,MITOL and a dominant-negative form of DRP1, DRP1K38A, partially rescued appoptosin-induced mitochondrial fragmentation and apoptosis, whereas co-expression of FIS1 aggravated appoptosin-induced apoptosis. Together, our results demonstrate that appoptosin can interact with mitochondrial outer-membrane fusion proteins and regulates mitochondrial morphology. [ABSTRACT FROM AUTHOR]

Background: Delivery of antigens by live bacterial carriers can elicit effective humoral and cellular responses and may be an attractive strategy for live bacterial vaccine production through introduction of a vector that expresses an exogenous protective antigen. To overcome the instability and metabolic burden associated with plasmid introduction, alternative strategies, such as the use of in vivo-inducible promoters, have been proposed. However, screening an ideal in vivo-activated promoter with high efficiency and low leak expression in a particular strain poses great challenges to many researchers. Results: In this work, we constructed an in vivo antigen-expressing vector suitable for Edwardsiella tarda, an enteric Gram-negative invasive intracellular pathogen of both animals and humans. By combining quorum sensing genes from Vibrio fischeri with iron uptake regulons, a synthetic binary regulation system (ironQS) for E. tarda was designed. In vitro expression assay demonstrated that the ironQS system is only initiated in the absence of Fe2+ in the medium when the cell density reaches its threshold. The ironQS system was further confirmed in vivo to present an in vivo-triggered and cell density-dependent expression pattern in larvae and adult zebrafish. A recombinant E. tarda vector vaccine candidate WED(ironQS-G) was established by introducing gapA34, which encodes the protective antigen glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the fish pathogen Aeromonas hydrophila LSA34 into ironQS system, and the immune protection afforded by this vaccine was assessed in turbot (Scophtalmus maximus). Most of the vaccinated fish survived under the challenge with A. hydrophila LSA34 (RPS = 67.0%) or E. tarda EIB202 (RPS = 72.3%). Conclusions: Quorum sensing system has been extensively used in various gene structures in synthetic biology as a well-functioning and population-dependent gene circuit. In this work, the in vivo expression system, ironQS, maintained the high expression efficiency of the quorum sensing circuit and achieved excellent expression regulation of the Fur box. The ironQS system has great potential in applications requiring in vivo protein expression, such as vector vaccines. Considering its high compatibility, ironQS system could function as a universal expression platform for a variety of bacterial hosts. [ABSTRACT FROM AUTHOR]

To define the proteins whose expression is regulated by cAMP and protein kinase A (PKA), we used a quantitative proteomics approach in studies of wild-type (WT) and kin- (PKA-null) S49 murine T lymphoma cells. We also compared the impact of endogenous increases in the level of cAMP [by forskolin (Fsk) and the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX)] or by a cAMP analogue (8-CPT-cAMP). We identified 1056 proteins in WT and kin- S49 cells and found that 8-CPT-cAMP and Fsk with IBMX produced differences in protein expression. WT S49 cells had a correlation coefficient of 0.41 between DNA microarray data and the proteomics analysis in cells incubated with 8-CPT-cAMP for 24 h and a correlation coefficient of 0.42 between the DNA microarray data obtained at 6 h and the changes in protein expression after incubation with 8-CPT-cAMP for 24 h. Glutathione reductase (Gsr) had a higher level of basal expression in kin- S49 cells than in WT cells. Consistent with this finding, kin- cells are less sensitive to cell killing and generation of malondialdehyde than are WT cells incubated with H2O2. Cyclic AMP acting via PKA thus has a broad impact on protein expression in mammalian cells, including in the regulation of Gsr and oxidative stress. [ABSTRACT FROM AUTHOR]

The olfactory system receives extensive serotonergic inputs from the dorsal raphe, a nucleus involved in control of behavior, regulation of mood, and modulation of sensory processing. Although many studies have investigated how serotonin modulates the olfactory bulb, few have focused on the anterior piriform cortex (aPC), a region important for olfactory learning and encoding of odor identity and intensity. Specifically, the mechanism and functional significance of serotonergic modulation of the aPC remain largely unknown. Here we used pharmacologic, optogenetic, and fiber photometry techniques to examine the serotonergic modulation of neural activity in the aPC in vitro and in vivo. We found that serotonin (5-HT) reduces the excitability of pyramidal neurons directly via 5-HT2C receptors, phospholipase C, and calcium-activated potassium (BK) channels. Furthermore, endogenous serotonin attenuates odor-evoked calcium responses in aPC pyramidal neurons. These findings identify the mechanism underlying serotonergic modulation of the aPC and shed light on its potential role. [ABSTRACT FROM AUTHOR]