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Alveolar leukocyte recruitment is a hallmark of acute lung inflammation and involves transmigration of leukocytes through endothelial and epithelial layers. The disintegrin and metalloproteinase (ADAM) 8 is expressed on human isolated leukocytic cells and can be further upregulated on cultured endothelial and epithelial cells by proinflammatory cytokines. By shRNA-mediated knockdown we show that leukocytic ADAM8 is required on monocytic THP-1 cells for chemokine-induced chemotaxis as well as transendothelial and transepithelial migration. Furthermore, ADAM8 promotes α L -integrin upregulation and THP-1 cell adhesion to endothelial cells. On endothelial cells ADAM8 enhances transendothelial migration and increases cytokine-induced permeability. On epithelial cells the protease facilitates migration in a wound closure assay but does not affect transepithelial leukocyte migration. Blood leukocytes and bone marrow-derived macrophages (BMDM) from ADAM8-deficient mice show suppressed chemotactic response. Intranasal application of LPS to mice is accompanied with ADAM8 upregulation in the lung. In this model of acute lung inflammation ADAM8-deficient mice are protected against leukocyte infiltration. Finally, transfer experiments of BMDM in mice indicate that ADAM8 exerts a promigratory function predominantly on leukocytes. Our study provides in vitro and in vivo evidence that ADAM8 on leukocytes holds a proinflammatory function in acute lung inflammation by promoting alveolar leukocyte recruitment., (Copyright © 2017 the American Physiological Society.)

A broad spectrum of diseases is characterized by myelin abnormalities and/or oligodendrocyte pathology. In most, if not all, of these diseases, early activation of microglia occurs. Our knowledge regarding the factors triggering early microglia activation is, however, incomplete. In this study, we used the cuprizone model to investigate the temporal and causal relationship of oligodendrocyte apoptosis and early microglia activation. Genome-wide gene expression studies revealed the induction of distinct chemokines, among them Cxcl10, Ccl2, and Ccl3 in cuprizone-mediated oligodendrocyte apoptosis. Early microglia activation was unchanged in CCL2- and CCL3-deficient knockouts, but was significantly reduced in CXCL10-deficient mice, resulting in an amelioration of cuprizone toxicity at later time points. Subsequent in vitro experiments revealed that recombinant CXCL10 induced migration and a proinflammatory phenotype in cultured microglia, without affecting their phagocytic activity or proliferation. In situ hybridization analyses suggest that Cxcl10 mRNA is mainly expressed by astrocytes, but also oligodendrocytes, in short-term cuprizone-exposed mice. Our results show that CXCL10 actively participates in the initiation of microglial activation. These findings have implications for the role of CXCL10 as an important mediator during the initiation of neuroinflammatory processes associated with oligodendrocyte pathology., (Copyright © 2015 by The American Association of Immunologists, Inc.)

A major goal of nanotechnology is to develop the capability to arrange matter at will by placing individual atoms at desired locations in a predetermined configuration to build a nanostructure with specific properties or function. The scanning tunneling microscope has demonstrated the ability to arrange the basic building blocks of matter, single atoms, in two-dimensional configurations. An array of various nanostructures has been assembled, which display the quantum mechanics of quantum confined geometries. The level of human interaction needed to physically locate the atom and bring it to the desired location limits this atom assembly technology. Here we report the use of autonomous atom assembly via path planning technology; this allows atomically perfect nanostructures to be assembled without the need for human intervention, resulting in precise constructions in shorter times. We demonstrate autonomous assembly by assembling various quantum confinement geometries using atoms and molecules and describe the benefits of this approach.

Calcium (Ca(2+)) is a secondary messenger in cells and Ca(2+) flux initiated from endoplasmic reticulum (ER) stores via inositol 1,4,5-triphosphate (IP3) binding to the IP3 receptor (IP3R) is particularly important for the activation and function of immune cells. Previous studies demonstrated that genetic deletion of selenoprotein K (Selk) led to decreased Ca(2+) flux in a variety of immune cells and impaired immunity, but the mechanism was unclear. Here we show that Selk deficiency does not affect receptor-induced IP3 production, but Selk deficiency through genetic deletion or low selenium in culture media leads to low expression of the IP3R due to a defect in IP3R palmitoylation. Bioinformatic analysis of the DHHC (letters represent the amino acids aspartic acid, histidine, histidine, and cysteine in the catalytic domain) family of enzymes that catalyze protein palmitoylation revealed that one member, DHHC6, contains a predicted Src-homology 3 (SH3) domain and DHHC6 is localized to the ER membrane. Because Selk is also an ER membrane protein and contains an SH3 binding domain, immunofluorescence and coimmunoprecipitation experiments were conducted and revealed DHHC6/Selk interactions in the ER membrane that depended on SH3/SH3 binding domain interactions. DHHC6 knockdown using shRNA in stably transfected cell lines led to decreased expression of the IP3R and impaired IP3R-dependent Ca(2+) flux. Mass spectrophotometric and bioinformatic analyses of the IP3R protein identified two palmitoylated cysteine residues and another potentially palmitoylated cysteine, and mutation of these three cysteines to alanines resulted in decreased IP3R palmitoylation and function. These findings reveal IP3R palmitoylation as a critical regulator of Ca(2+) flux in immune cells and define a previously unidentified DHHC/Selk complex responsible for this process.

Cancer-associated fibroblasts (CAFs) drive tumour progression, but the emergence of this cell state is poorly understood. A broad spectrum of metalloproteinases, controlled by the Timp gene family, influence the tumour microenvironment in human cancers. Here, we generate quadruple TIMP knockout (TIMPless) fibroblasts to unleash metalloproteinase activity within the tumour-stromal compartment and show that complete Timp loss is sufficient for the acquisition of hallmark CAF functions. Exosomes produced by TIMPless fibroblasts induce cancer cell motility and cancer stem cell markers. The proteome of these exosomes is enriched in extracellular matrix proteins and the metalloproteinase ADAM10. Exosomal ADAM10 increases aldehyde dehydrogenase expression in breast cancer cells through Notch receptor activation and enhances motility through the GTPase RhoA. Moreover, ADAM10 knockdown in TIMPless fibroblasts abrogates their CAF function. Importantly, human CAFs secrete ADAM10-rich exosomes that promote cell motility and activate RhoA and Notch signalling in cancer cells. Thus, Timps suppress cancer stroma where activated-fibroblast-secreted exosomes impact tumour progression.

Inflammation is a key process in various diseases, characterized by leukocyte recruitment to the inflammatory site. This study investigates the role of a disintegrin and a metalloproteinase (ADAM) 10 and ADAM17 for leukocyte migration in vitro and in a murine model of acute pulmonary inflammation. Inhibition experiments or RNA knockdown indicated that monocytic THP-1 cells and primary human neutrophils require ADAM10 but not ADAM17 for efficient chemokine-induced cell migration. Signaling and adhesion events that are linked to cell migration such as p38 and ρ GTPase-family activation, F-actin polymerization, adhesion to fibronectin, and up-regulation of α5 integrin were also dependent on ADAM10 but not ADAM17. This was confirmed with leukocytes isolated from mice lacking either ADAM10 or ADAM17 in all hematopoietic cells (vav 1 guanine nucleotide exchange factor [Vav]-Adam10(-/-) or Vav-Adam17(-/-) mice). In lipopolysaccharide-induced acute pulmonary inflammation, alveolar recruitment of neutrophils and monocytes was transiently increased in Vav-Adam17(-/-) but steadily reduced in Vav-Adam10(-/-) mice. This deficit in alveolar leukocyte recruitment was also observed in LysM-Adam10(-/-) mice lacking ADAM10 in myeloid cells and correlated with protection against edema formation. Thus, with regard to leukocyte migration, leukocyte-expressed ADAM10 but not ADAM17 displays proinflammatory activities and may therefore serve as a target to limit inflammatory cell recruitment., (© 2014 by The American Society of Hematology.)

Acute lung injury (ALI) is associated with increased vascular permeability, leukocyte recruitment, and pro-inflammatory mediator release. We investigated the role of the metalloproteinase ADAM17 in endotoxin-induced ALI with focus on endothelial ADAM17. In vitro, endotoxin-mediated induction of endothelial permeability and IL-8-induced transmigration of neutrophils through human microvascular endothelial cells required ADAM17 as shown by inhibition with GW280264X or shRNA-mediated knockdown. In vivo, ALI was induced by intranasal endotoxin-challenge combined with GW280264X treatment or endothelial adam17-knockout. Endotoxin-triggered upregulation of ADAM17 mRNA in the lung was abrogated in knockout mice and associated with reduced ectodomain shedding of the junctional adhesion molecule JAM-A and the transmembrane chemokine CX3CL1. Induced vascular permeability, oedema formation, release of TNF-α and IL-6 and pulmonary leukocyte recruitment were all markedly reduced by GW280264X or endothelial adam17-knockout. Intranasal application of TNF-α could not restore leukocyte recruitment and oedema formation in endothelial adam17-knockout animals. Thus, activation of endothelial ADAM17 promotes acute pulmonary inflammation in response to endotoxin by multiple endothelial shedding events most likely independently of endothelial TNF-α release leading to enhanced vascular permeability and leukocyte recruitment., (Copyright © 2012 EMBO Molecular Medicine.)

We describe the design, development and performance of a scanning probe microscopy (SPM) facility operating at a base temperature of 10 mK in magnetic fields up to 15 T. The microscope is cooled by a custom designed, fully ultra-high vacuum (UHV) compatible dilution refrigerator (DR) and is capable of in situ tip and sample exchange. Subpicometer stability at the tip-sample junction is achieved through three independent vibration isolation stages and careful design of the dilution refrigerator. The system can be connected to, or disconnected from, a network of interconnected auxiliary UHV chambers, which include growth chambers for metal and semiconductor samples, a field-ion microscope for tip characterization, and a fully independent additional quick access low temperature scanning tunneling microscope (STM) and atomic force microscope (AFM) system. To characterize the system, we present the cooling performance of the DR, vibrational, tunneling current, and tip-sample displacement noise measurements. In addition, we show the spectral resolution capabilities with tunneling spectroscopy results obtained on an epitaxial graphene sample resolving the quantum Landau levels in a magnetic field, including the sublevels corresponding to the lifting of the electron spin and valley degeneracies.

The surface-expressed transmembrane CX3C chemokine ligand 1 (CX3CL1/fractalkine) induces firm adhesion of leukocytes expressing its receptor CX3CR1. After shedding by the disintegrins and metalloproteinases (ADAM) 10 and 17, CX3CL1 also acts as soluble leukocyte chemoattractant. Here, we demonstrate that transmembrane CX3CL1 expressed on both endothelial and epithelial cells induces leukocyte transmigration. To investigate the underlying mechanism, we generated CX3CR1 variants lacking the intracellular aspartate-arginine-tyrosine (DRY) motif or the intracellular C-terminus which led to a defect in intracellular calcium response and impaired ligand uptake, respectively. While both variants effectively mediated firm cell adhesion, they failed to induce transmigration and rather mediated retention of leukocytes on the CX3CL1-expressing cell layer. Targeting of ADAM10 led to increased adhesion but reduced transmigration in response to transmembrane CX3CL1, while transmigration towards soluble CX3CL1 was not affected. Thus, transmembrane CX3CL1 mediates leukocyte transmigration via the DRY motif and C-terminus of CX3CR1 and the activity of ADAM10.

Syndecans are cell surface proteoglycans that bind and modulate various proinflammatory mediators and can be proteolytically shed from the cell surface. Within the lung, syndecan-1 and -4 are expressed as transmembrane proteins on epithelial cells and released in the bronchoalveolar fluid during inflammation. We here characterize the mechanism leading to the generation of soluble syndecan-1 and -4 in cultured epithelial cells and murine lung tissue. We show that the bladder carcinoma epithelial cell line ECV304, the lung epithelial cell line A459 and primary alveolar epithelial cells express and constitutively release syndecan-1 and -4. This release involves the activity of the disintegrin-like metalloproteinase ADAM17 as demonstrated by use of specific inhibitors and lentivirally transduced shRNA. Stimulation of epithelial cells with PMA, thrombin, or proinflammatory cytokines (TNFalpha/IFNgamma) led to the down-regulation of surface-expressed syndecan-1 and -4, which was associated with a significant increase of soluble syndecans and cell-associated cleavage fragments. The enhanced syndecan release was not related to gene induction of syndecans or ADAM17, but rather due to increased ADAM17 activity. Soluble syndecan-1 and -4 were also released into the bronchoalveolar fluid of mice. Treatment with TNFalpha/IFNgamma increased ADAM17 activity and syndecan release in murine lungs. Both constitutive and induced syndecan shedding was prevented by the ADAM17 inhibitor. ADAM17 may therefore be an important regulator of syndecan functions on inflamed lung epithelium.

We report an experimental study of adsorbed films of C(2)F(6) on graphite by using infrared reflection absorption spectroscopy supplemented by ellipsometry. The vibrational C-F stretch modes nu(5) (parallel to the molecular axis) and nu(7) (perpendicular) in the film are strongly blueshifted by dynamic dipole coupling, and these shifts are sensitive to lattice spacing and molecular tilt. The relative strength of the absorption peaks mainly depends on the tilt angle relative to the surface normal. We use the strength data to estimate the tilt angle across the known monolayer phases, information that is difficult to obtain by other techniques. Although only the surface-normal component of the induced dipole moment appreciably couples to the external infrared field, surface-parallel components contribute to the intralayer coupling and hence to the frequency shifts for tilted molecules. Comparison to model calculations for a range of herringbone tilt configurations allows us to draw conclusions regarding the pattern of tilt azimuths. On this basis, we offer a revised interpretation of the origin of the Ising-type ordering transition found by Arndt et al. [Phys. Rev. Lett. 80, 1686 (1998)] in heat capacity measurements. Our phase boundaries for monolayer phases above 80 K are in good agreement with earlier results of the Saarbrucken group. We identify three distinct bilayer phases near saturation in isothermal pressure scans from ellipsometric steps and spectroscopic signatures. In temperature scans, we find evidence for several monolayer phases more dense than the well-established 2 x 2 commensurate phase and for a stable trilayer phase below about 60 K.

A new dinitrogen complex, deep blue-green {[NPN]*Zr(THF)}(2)(mu-eta(2):eta(2)-N(2)) ([NPN]* = {[N-(2,4,6-Me(3)C(6)H(2))(2-N-5-MeC(6)H(3))](2)PPh}), was prepared in high yield by the reduction of [NPN]*ZrCl(2) with 2.2 equiv of KC(8) in THF under N(2). The solid-state molecular structure shows that N(2) is strongly activated (N-N bond length: 1.503(6) A) and bound side-on to two Zr atoms. Coordinated THF can be readily replaced by adding pyridine (py) or PMe(2)R (R = Me, Ph) to the complex to obtain {[NPN]*Zr(py)}(2)(mu-eta(2):eta(2)-N(2)) or {[NPN]*Zr(PMe(2)R)}(mu-eta(2):eta(2)-N(2)){Zr[NPN]*} in high yield. X-ray diffraction experiments show that the N(2) moiety is strongly activated and remains side-on bound to Zr for the py and PMe(2)Ph adducts; interestingly, only one PMe(2)Ph coordinates to the Zr(2)N(2) unit. {[NPN]*Zr(PMe(2)R)}(mu-eta(2):eta(2)-N(2)){Zr[NPN]*} reacts slowly with H(2) to provide {[NPN]*Zr(PMe(2)R)}(mu-H)(mu-eta(2):eta(2)-N(2)H){Zr[NPN]*}, as determined by isotopic labeling, and multinuclear NMR spectroscopy. The THF adduct does not react with H(2) even after an extended period, whereas the pyridine adduct does undergo a reaction with H(2), but to a mixture of products.

A method is described for in situ hybridization of riboprobes to free-floating brain sections. Brain sections are hybridized and processed free-floating in buffer, i.e., without attachment to a support such as a slide. To withstand the extra wear compared with sections processed on-slide, the brain tissue must be well fixed (4% paraformaldehyde) and sections cut at thickness of typically 40 microm. Sections were exposed to a prehybridization treatment before a riboprobe is added to form the hybridization solution. Riboprobes were prepared from cDNA via an in vitro transcription reaction and are labeled with digoxigenin. The sections are subsequently processed to remove nonspecific binding and the digoxigenin label detected via an antibody conjugated to alkaline phosphatase. This method may be readily combined with neuronal tracing and is ideal for further processing by immunohistochemistry to detect specific proteins.

In situ prepared chromium catalysts containing bulky diphosphinoamine (PNP) ligands, upon activation with MAO, are extremely efficient catalysts for the trimerisation of ethylene to 1-hexene.

Linear alpha-olefins, such as 1-hexene and 1-octene, are important comonomers in the production of linear low-density polyethylene (LLDPE). The conventional method of producing 1-hexene and 1-octene is by oligomerization of ethylene, which yields a wide spectrum of linear alpha-olefins (LAOs). While there exists several processes for producing 1-hexene via ethylene trimerization, a similar route for the selective production of 1-octene has so far been elusive. We now, for the first time, report an unprecedented ethylene tetramerization reaction that produces 1-octene in selectivities exceeding 70%, using an aluminoxane-activated chromium/((R2)2P)2NR1 catalyst system.

Equilibrium conductivity fluctuations of mesoscopic domains are found in film and bulk single-crystal manganite colossal magnetoresistive material. Temperature and field dependences of the Boltzmann factors for a collection of two-state fluctuators give measures of the magnetic moment and entropy differences between the states, and of the fluctuator volumes. The large resistance step size implies dramatic current inhomogeneities. Occasional anomalous temperature dependences indicate that the film inhomogeneous phase is stabilized by a repulsive interaction between conducting regions.

Humidification, unlike nebulization, does not produce water droplets in the process of producing desiccated gases, and therefore is considered low risk in terms of potential infection sources. However, direct inoculation of bacteria into these units has been shown to produce contaminated effluents. Therefore, the present study was designed to determine if the airborne route of contamination was a potential source of bacterial inoculum for these units and to determine the maximum number of days units remained free of bacterial contamination. Disposable humidifiers were placed in both high and low volume patient areas of the recovery room. Units were used on multiple patients with only the cannula replaced between patients. Our results showed that these units do have the potential for becoming inoculated through the airborne route, but did not produce contaminated effluents greater than background air. Units remained sterile during the entire time in use in both high and low volume areas. In conclusion, our data suggests that each institution should evaluate the usage of disposable humidifiers based on patient parameters and environment rather than manufacturer's guidelines.

In a previous paper we reported that serum, urine and fecal zinc levels fell markedly in women taking a combination oral contraceptive agent (+OCA) and in women with normal menstrual cycles (-OCA) when they consumed a low-zinc diet (less than 0.2 mg/day) for 35 days. We evaluated other biochemical and clinical data in order to determine if depletion of accessible body zinc and/or physiologic adjustment to conserve body zinc stores had occurred. Neither low zinc intake nor oral contraceptive use appeared to influence nitrogen balance or body weight. Use of contraceptive drugs appeared to influence the response of blood parameters to zinc depletion. Serum transferrin and cholesterol declined significantly in the -OCA group, whereas alkaline phosphatase and gamma-globulin changed significantly in both groups. Clinical problems developed in all the subjects with serum zinc levels below 50 microgram/dl during the study; three of the six with serum zinc levels above 50 microgram/dl also complained of clinical symptoms. The results suggest that zinc deficiency through depletion of accessible body zinc stores developed during the 35-day study.

Zinc excretion under conditions of negligible zinc intake (0.17 mg/day) was measured in women taking a combination oral contraceptive agent (+OCA) and in women with normal menstrual cycles (-OCA). A semipurified, constant formula diet, providing negligible amounts of zinc, copper and iron, but adequate levels of other essential nutrients, was fed. During the 35--day study, serum and urinary zinc declined more markedly in the +OCA than -OCA groups. Serum zinc dropped 47% in the +OCA and 21% in the -OCA; urinary zinc declined 83% and 62% in the two groups. After pre-study, zinc was cleared from the gut, fecal zinc decreased about 40% in both groups. Whole body integumental zinc losses were similar for both groups, about 0.7 mg/day. For -OCA, zinc losses via this route were higher during the luteal phase than during menstruation. Menstrual fluid zinc contents were negligible and similar for both groups, about 5 microgram/day. The data suggest that accessible zinc stores are not extensive and that depletion of these stores, as a result of the low-zinc diet, caused the fall in serum zinc. The use of OCA influenced the response to the low-zinc diet, but endogenous zinc losses calculated for day 1 were nearly the same for both groups, about 1.6 mg/day.