Detailed Structural Analysis of Lipids Directly on Tissue Specimens Using a MALDI-SpiralTOF-Reflectron TOF Mass Spectrometer

Direct tissue analysis using a novel tandem time-of-flight (TOF-TOF) mass spectrometer is described. This system consists of a matrix-assisted laser desorption/ionization ion source, a spiral ion trajectory TOF mass spectrometer “SpiralTOF (STOF)”, a collision cell, and an offset parabolic reflectron (RTOF). The features of this system are high precursor ion selectivity due to a 17-m flight path length in STOF and elimination of post-source decay (PSD) ions. The acceleration energy is 20 keV, so that high-energy collision-induced dissociation (HE-CID) is possible. Elimination of PSD ions allows observation of the product ions inherent to the HE-CID process. By using this tandem TOF instrument, the product ion spectrum of lipids provided detailed structural information of fatty acid residues

Expression profile of the N-myc Downstream Regulated Gene 2 (NDRG2) in human cancers with focus on breast cancer

<p>Abstract</p> <p>Background</p> <p>Several studies have shown that <it>NDRG2 </it>mRNA is down-regulated or undetectable in various human cancers and cancer cell-lines. Although the function of <it>NDRG2 </it>is currently unknown, high <it>NDRG2 </it>expression correlates with improved prognosis in high-grade gliomas, gastric cancer and hepatocellular carcinomas. Furthermore, <it>in vitro </it>studies have revealed that over-expression of NDRG2 in cell-lines causes a significant reduction in their growth. The aim of this study was to examine levels of <it>NDRG2 </it>mRNA in several human cancers, with focus on breast cancer, by examining affected and normal tissue.</p> <p>Methods</p> <p>By labelling a human Cancer Profiling Array with a radioactive probe against <it>NDRG2</it>, we evaluated the level of <it>NDRG2 </it>mRNA in 154 paired normal and tumor samples encompassing 19 different human cancers. Furthermore, we used quantitative real-time RT-PCR to quantify the levels of <it>NDRG2 </it>and <it>MYC </it>mRNA in thyroid gland cancer and breast cancer, using a distinct set of normal and tumor samples.</p> <p>Results</p> <p>From the Cancer Profiling Array, we saw that the level of <it>NDRG2 </it>mRNA was reduced by at least 2-fold in almost a third of the tumor samples, compared to the normal counterpart, and we observed a marked decreased level in colon, cervix, thyroid gland and testis. However, a Benjamini-Hochberg correction showed that none of the tissues showed a significant reduction in <it>NDRG2 </it>mRNA expression in tumor tissue compared to normal tissue. Using quantitative RT-PCR, we observed a significant reduction in the level of <it>NDRG2 </it>mRNA in a distinct set of tumor samples from both thyroid gland cancer (p = 0.02) and breast cancer (p = 0.004), compared with normal tissue. <it>MYC </it>mRNA was not significantly altered in breast cancer or in thyroid gland cancer, compared with normal tissue. In thyroid gland, no correlation was found between <it>MYC </it>and <it>NDRG2 </it>mRNA levels, but in breast tissue we found a weakly significant correlation with a positive r-value in both normal and tumor tissues, suggesting that <it>MYC </it>and <it>NDRG2 </it>mRNA are regulated together.</p> <p>Conclusion</p> <p>Expression of <it>NDRG2 </it>mRNA is reduced in many different human cancers. Using quantitative RT-PCR, we have verified a reduction in thyroid cancer and shown, for the first time, that <it>NDRG2 </it>mRNA is statistically significantly down-regulated in breast cancer. Furthermore, our observations indicate that other tissues such as cervix and testis can have lower levels of <it>NDRG2 </it>mRNA in tumor tissue compared to normal tissue.</p

Neuroinflammation, Mast Cells, and Glia: Dangerous Liaisons

The perspective of neuroinflammation as an epiphenomenon following neuron damage is being replaced by the awareness of glia and their importance in neural functions and disorders. Systemic inflammation generates signals that communicate with the brain and leads to changes in metabolism and behavior, with microglia assuming a pro-inflammatory phenotype. Identification of potential peripheral-to-central cellular links is thus a critical step in designing effective therapeutics. Mast cells may fulfill such a role. These resident immune cells are found close to and within peripheral nerves and in brain parenchyma/meninges, where they exercise a key role in orchestrating the inflammatory process from initiation through chronic activation. Mast cells and glia engage in crosstalk that contributes to accelerate disease progression; such interactions become exaggerated with aging and increased cell sensitivity to stress. Emerging evidence for oligodendrocytes, independent of myelin and support of axonal integrity, points to their having strong immune functions, innate immune receptor expression, and production/response to chemokines and cytokines that modulate immune responses in the central nervous system while engaging in crosstalk with microglia and astrocytes. In this review, we summarize the findings related to our understanding of the biology and cellular signaling mechanisms of neuroinflammation, with emphasis on mast cell-glia interactions

Application of Graphene within Optoelectronic Devices and Transistors

Scientists are always yearning for new and exciting ways to unlock graphene's true potential. However, recent reports suggest this two-dimensional material may harbor some unique properties, making it a viable candidate for use in optoelectronic and semiconducting devices. Whereas on one hand, graphene is highly transparent due to its atomic thickness, the material does exhibit a strong interaction with photons. This has clear advantages over existing materials used in photonic devices such as Indium-based compounds. Moreover, the material can be used to 'trap' light and alter the incident wavelength, forming the basis of the plasmonic devices. We also highlight upon graphene's nonlinear optical response to an applied electric field, and the phenomenon of saturable absorption. Within the context of logical devices, graphene has no discernible band-gap. Therefore, generating one will be of utmost importance. Amongst many others, some existing methods to open this band-gap include chemical doping, deformation of the honeycomb structure, or the use of carbon nanotubes (CNTs). We shall also discuss various designs of transistors, including those which incorporate CNTs, and others which exploit the idea of quantum tunneling. A key advantage of the CNT transistor is that ballistic transport occurs throughout the CNT channel, with short channel effects being minimized. We shall also discuss recent developments of the graphene tunneling transistor, with emphasis being placed upon its operational mechanism. Finally, we provide perspective for incorporating graphene within high frequency devices, which do not require a pre-defined band-gap.Comment: Due to be published in "Current Topics in Applied Spectroscopy and the Science of Nanomaterials" - Springer (Fall 2014). (17 pages, 19 figures

K+ channel openers restore verapamil-inhibited lung fluid resolution and transepithelial ion transport

<p>Abstract</p> <p>Background</p> <p>Lung epithelial Na⁺channels (ENaC) are regulated by cell Ca²⁺signal, which may contribute to calcium antagonist-induced noncardiogenic lung edema. Although K⁺channel modulators regulate ENaC activity in normal lungs, the therapeutical relevance and the underlying mechanisms have not been completely explored. We hypothesized that K⁺channel openers may restore calcium channel blocker-inhibited alveolar fluid clearance (AFC) by up-regulating both apical and basolateral ion transport.</p> <p>Methods</p> <p>Verapamil-induced depression of heterologously expressed human αβγ ENaC in <it>Xenopus </it>oocytes, apical and basolateral ion transport in monolayers of human lung epithelial cells (H441), and <it>in vivo </it>alveolar fluid clearance were measured, respectively, using the two-electrode voltage clamp, Ussing chamber, and BSA protein assays. Ca²⁺signal in H441 cells was analyzed using Fluo 4AM.</p> <p>Results</p> <p>The rate of <it>in vivo </it>AFC was reduced significantly (40.6 ± 6.3% of control, <it>P </it>< 0.05, n = 12) in mice intratracheally administrated verapamil. K_Ca3.1(1-EBIO) and K_ATP(minoxidil) channel openers significantly recovered AFC. In addition to short-circuit current (Isc) in intact H441 monolayers, both apical and basolateral Isc levels were reduced by verapamil in permeabilized monolayers. Moreover, verapamil significantly altered Ca²⁺signal evoked by ionomycin in H441 cells. Depletion of cytosolic Ca²⁺in αβγ ENaC-expressing oocytes completely abolished verapamil-induced inhibition. Intriguingly, K_V(pyrithione-Na), K _Ca3.1(1-EBIO), and K_ATP(minoxidil) channel openers almost completely restored the verapamil-induced decrease in Isc levels by diversely up-regulating apical and basolateral Na⁺and K⁺transport pathways.</p> <p>Conclusions</p> <p>Our observations demonstrate that K⁺channel openers are capable of rescuing reduced vectorial Na⁺transport across lung epithelial cells with impaired Ca²⁺signal.</p

Acute and Persistent Mycobacterium tuberculosis Infections Depend on the Thiol Peroxidase TPX

The macrophage is the natural niche of Mycobacterium tuberculosis infection. In order to combat oxidative and nitrosative stresses and persist in macrophages successfully, M. tuberculosis is endowed with a very efficient antioxidant complex. Amongst these antioxidant enzymes, TpX is the only one in M. tuberculosis with sequence homology to thiol peroxidase. Previous reports have demonstrated that the M. tuberculosis TpX protein functions as a peroxidase in vitro. It is the dominant antioxidant which protects M. tuberculosis against oxidative and nitrosative stresses. The level of the protein increases in oxidative stress. To determine the roles of tpx gene in M. tuberculosis survival and virulence in vivo, we constructed an M. tuberculosis strain lacking the gene. The characteristics of the mutant were examined in an in vitro stationary phase model, in response to stresses; in murine bone marrow derived macrophages and in an acute and an immune resistant model of murine tuberculosis. The tpx mutant became sensitive to H2O2 and NO compared to the wild type strain. Enzymatic analysis using bacterial extracts from the WT and the tpx mutant demonstrated that the mutant contains reduced peroxidase activity. As a result of this, the mutant failed to grow and survive in macrophages. The growth deficiency in macrophages became more pronounced after interferon-γ activation. In contrast, its growth was significantly restored in the macrophages of inducible nitric oxide synthase (iNOS or NOS2) knockout mice. Moreover, the tpx mutant was impaired in its ability to initiate an acute infection and to maintain a persistent infection. Its virulence was attenuated. Our results demonstrated that tpx is required for M. tuberculosis to deal with oxidative and nitrosative stresses, to survive in macrophages and to establish acute and persistent infections in animal tuberculosis models