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Jhunsi, India

Morsi R.M.Y.,FTRI | EL-Tahan N.R.,Home Economics Faculty Minufiya University | El-Tobgy K.,HRI
Australian Journal of Basic and Applied Sciences | Year: 2012

Benzene, a carcinogen that can cause cancer in humans, may form at nanogram per gram levels in some beverages containing both benzoate salts and ascorbic acid. Elevated temperatures and light stimulate these reactions. This experiment was performed to determine the effect of the length of incubation time and temperature (20, 45 and 90°C) on the generation of benzene from ascorbic acid and sodium benzoate in some products of soft drinks (orange color), of 75 samples (five Brands) of carbonated soft drinks sampled on the Egyptian market. A calibration curve was generated by creating dilutions of benzene in deionized water; the range of detection was between 0.52 ng/ mL and 20.0 ng/ mL. The deuterated isotopes of the same hydrocarbons were used for quantitative determination by the internal standard technique. A survey of benzene contamination of samples, using headspace sampling combined with gas chromatography and mass spectrometry (HS-GC/MS) with a quantification limit of 0.52 ng/ mL, was conducted. GC/MS method was developed and validated for the determination of benzene in beverages. The GC/MS technique was suitable for this study as it is a very accurate, effective and reliable protocol for the monitoring of benzene in sodium benzoatecontaining Ascorbic acid. The results showed values higher than the limits recommended by the WHO for benzene in non-Alcohol carbonated soft drinks, also a presence of benzene in all beverages analyzed which gives food for thought about a possible cumulative effect, taking the intake of other contaminated foods and environmental pollution into account. Further research on the precursors of heat-induced benzene formation is very necessary, should be in Egypt (Arab countries) that seeks to set limits in line with international legislation in the various levels of benzene in soft drinks allowed. Especially, no existences of standardization limits for benzene in food products in Egypt till now. The proposed analysis method of benzene in carbonated soft drinks by HSGC/MS at 20, 45, and 90°C is an accurate and universal method for the monitoring of benzene without false-positive identification. Source


News Article
Site: http://www.nature.com/nature/current_issue/

The immune system can sense pathogens through pathogen recognition receptors2, but emerging evidence suggests that it can also sense and respond to environmental changes that cause cellular stress3. The ISR is an evolutionarily ancient mechanism that enables eukarytoic cells to sense and respond to diverse stress signals, such as amino acid starvation and endoplasmic reticulum stress4. The four known sensors of the ISR include: GCN2, protein kinase R (PKR), haem-regulated inhibitor (HRI) and PKR-like endoplasmic reticulum kinase (PERK)4. GCN2 senses amino acid depletion, PERK senses endoplasmic reticulum stress, and PKR can recognize viral double-stranded RNA4. Activation of HRI is induced by haem deficiency5, and is important for the survival of erythroid precursors. Activation of each of these four sensors results in phosphorylation of eukaryotic initiation factor 2α (eIF2α), leading to the initiation of global translational arrest4. Recent evidence suggests a crosstalk between the ISR and the immune system3. Thus, our recent systems-based analysis of immune responses to the yellow fever vaccine (YF-17D) in humans revealed a correlation between the expression of GCN2 in the blood and the magnitude of the later CD8+ T-cell response6. Furthermore, YF-17D induced GCN2 activation in dendritic cells, resulting in enhanced autophagy and antigen presentation7. Whether GCN2 can modulate immune responses during conditions of amino acid restriction remains unexplored. This is particularly relevant in the intestine, where the immune system has to endure dynamic changes in nutrient bioavailability. We thus determined whether GCN2 impacts immune homeostasis in the intestine. Phosphorylated eIF2α was detected in intestinal dendritic cells, macrophages and epithelial cells under steady-state and inflammatory conditions (Extended Data Fig. 1a). Furthermore, expression of phosphorylated PKR, PERK, eIF2α and GCN2 could be detected in tissues from healthy and inflamed human colon (Extended Data Fig. 1b). Analysis of public gene expression databases revealed that the expression of genes encoding GCN2 and other eIF2α kinases was highest in the colon, relative to other organs (Extended Data Fig. 1c). Interestingly, there was a higher expression of genes encoding GCN2, PERK and PKR in ulcerative colitis and Crohn’s disease, relative to healthy controls8, 9 (Extended Data Fig. 1d). To investigate the functions of GCN2 in vivo, we analysed the structure and morphology of gut tissue isolated from the Gcn2−/− mice. Ki-67 and chromogranin A staining in small and large intestines were unaffected in Gcn2−/− mice, suggesting that GCN2 is not required for steady-state cell differentiation and proliferation in the intestine (Extended Data Fig. 2a, b, d). Gcn2−/− mice had normal Paneth cell granules, as evident with lysozyme staining (Extended Data Fig. 2c), and did not exhibit any spontaneous gut inflammation up to 45 weeks of age. We then assessed the impact of GCN2 deficiency on acute colitis by challenging the mice with 2% dextran sodium sulfate (DSS), a chemical irritant that induces inflammation with the clinical and histological features of inflammatory bowel disease in mice10. Upon DSS administration, Gcn2−/− mice exhibited enhanced severity of colitis compared with littermates, including greater weight loss, inflammation, T 17 responses and colon shortening (Fig. 1a–c and Extended Data Fig. 3a–c). Histopathological analysis revealed severe mucosal epithelial erosion, displacement and crypt loss (Extended Data Fig. 3a). Consistent with enhanced gut inflammation, we observed a severely impaired epithelial barrier, evidenced by increased intestinal permeability (Extended Data Fig. 3d). These differences were not due to differences in the expression of antimicrobial defensins between wild-type and Gcn2−/− mice (Extended Data Fig. 3e). To assess potential roles for APCs versus epithelial cells in mediating the effects of GCN2, we generated mice lacking GCN2 specifically in epithelial cells (Gcn2fl/fl villin cre+; referred to as Gcn2Δvillin hereafter) (Fig. 1d–f and Extended Data Fig. 3a–c), or in CD11c+ APCs (Gcn2fl/fl Cd11c cre+; referred to as Gcn2ΔAPC hereafter) (Fig. 1g–i and Extended Data Fig. 3a–c). DSS induced enhanced colitis in both strains, evidenced by weight loss, colon shortening and increased T 17 responses (a surrogate readout of intestinal inflammation) relative to littermate controls (Fig. 1 and Extended Data Fig. 3b, c). Consistent with a role for GCN2 in APCs, isolated intestinal dendritic cells from Gcn2−/− mice could stimulate enhanced IL-17 production from antigen-specific CD4+ T cells, in vitro (Extended Data Fig. 3f). Collectively, these findings demonstrate that GCN2 deficiency in both epithelial cells and APCs results in enhanced inflammation and DSS-induced colitis. Since PERK activation by endoplasmic reticulum stress is also known to be an important component of the host ISR11, we generated mice lacking PERK in epithelial cells (Perkfl/fl villin cre+ (Perk also known as Eif2ak3); referred to as PerkΔvillin hereafter) or APCs (Perkfl/fl Cd11c cre+; referred to as PerkΔAPC hereafter) to study the role of PERK in intestinal inflammation (Extended Data Fig. 4). PERK-deficient strains were challenged with 2% DSS, and their symptoms and pathology were compared with littermate controls. Both PerkΔvillin and PerkΔAPC strains exhibited little or no differences relative to littermates in intestinal inflammation induced by DSS (Extended Data Fig. 4). As ISR kinases exert their function by phosphorylating Ser51 on eIF2α, we assessed the impact of eIF2α on intestinal inflammation. We generated mice conditionally lacking Ser51 eIF2α phosphorylation in epithelial cells (Eif2afl/fl villin cre+; referred to as Eif2aΔvillin hereafter) as previously described12, 13 and APCs (Eif2afl/fl Cd11c cre+; referred to as Eif2aΔAPC hereafter) (Extended Data Fig. 5). Eif2aΔvillin mice exhibited enhanced weight loss and elevated T 17 response relative to littermate controls, and Eif2aΔAPC mice exhibited enhanced T 17 responses (Extended Data Fig. 5), consistent with a recent report on a role for eIF2α in mediating protection against gut inflammation13. However, these effects were more modest than those observed in Gcn2−/− mice (Fig. 1), suggesting additional eIF2α-independent mechanisms. Recent studies indicate a role for GCN2 in promoting autophagy7, 14. Given its importance in regulating inflammation at mucosal sites15, we hypothesized that defective autophagy may mediate enhanced gut inflammation in Gcn2−/− mice. We analysed expression of the autophagy protein LC3 using a knock-in reporter strain16, and observed high LC3 expression in colonic APCs and epithelial cells, which was indicative of constitutive autophagy (Extended Data Fig. 6a). To study the role of GCN2 in mediating autophagy at mucosal sites, we generated GCN2-deficient autophagic reporter mice (Gcn2−/− × LC3–GFP), and additionally examined expression of p62, another marker for autophagy. In naive mice, expression of both LC3 and p62 were similar in the intestinal APCs and epithelial cells of Gcn2−/− mice and littermates (Fig. 1j, k). However, we observed a significant increase in the number of LC3–GFP puncta in the crypts of wild-type mice compared with the Gcn2−/− mice after oral administration of DSS (Fig. 1j). Additionally, we observed that intestinal APCs and epithelial cells from Gcn2−/− mice have lower levels of LC3B and p62 relative to cells from wild-type mice (Fig. 1k and Extended Data Fig. 6b). To determine whether the observed reduction in autophagy in Gcn2−/− mice was due to impaired induction or enhanced degradation of autophagosomes, we assessed LC3B and p62 levels in intestinal APCs and epithelial cells from wild-type or Gcn2−/− mice with or without chloroquine, an inhibitor of autophagy degradation (Fig. 1k and Extended Data Fig. 6b). Blocking the degradation of autophagosomes with chloroquine in wild-type mice resulted in greater accumulation of LC3B and p62 at 12 h after DSS (Fig. 1k and Extended Data Fig. 6b). Importantly, the accumulated form (LC3B and p62) was significantly lower in Gcn2−/− cells, indicating reduced autophagy flux relative to wild-type mice (Fig. 1k and Extended Data Fig. 6b). Similar results were observed at 24 h, although chloroquine-treated Gcn2−/− mice displayed enhanced accumulation of LC3B and p62, albeit at lower levels than wild-type mice (Fig. 1k and Extended Data Fig. 6b). The specificity of the LC3B antibody to LC3BII was confirmed using digitonin to retain specifically the membrane-bound LC3II within cells17 (Extended Data Fig. 6c, d). These findings suggested that defective autophagy in intestinal cells may mediate enhanced inflammation in Gcn2−/− mice. We therefore generated Atg5fl/fl Cd11c cre (Atg5ΔAPC) and Atg7fl/fl Cd11c cre (Atg7ΔAPC) mice that are conditionally deficient in the autophagy proteins Atg5 and Atg7 in CD11c+ APCs (Extended Data Fig. 7). Upon treatment with 2% DSS, both the Atg5ΔAPC and Atg7ΔAPC strains exhibited greater weight loss (Fig. 1l), enhanced shortening of colon length, T 17 responses and immunopathology (Extended Data Fig. 7) compared with littermate controls, indicating a role for APC-intrinsic autophagy in regulating inflammation in Gcn2−/− mice. Autophagy can limit ROS abundance during colitis15, and impaired autophagy results in abnormal mitochondrial function and oxidative stress15, which is a characteristic feature of inflammatory bowel disease18. Hence we studied the role of ROS in mediating DSS-induced inflammation using cell-permeant 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA), a fluorescent probe that reacts with numerous types of ROS19. After oxidation by ROS, the non-fluorescent H2DCFDA is converted to fluorescent 2′,7′-dichlorofluorescein (DCF)19, which was detected in intestinal APCs and epithelial cells (Fig. 2a and Extended Data Fig. 8a, b) by flow cytometry. Gcn2−/− mice exhibited significantly higher levels of ROS compared with littermate controls, indicating enhanced oxidative stress in the colon (Fig. 2a) and small intestine (Extended Data Fig. 8a, b). We also analysed the levels of mitochondrial ROS in the large and small intestine using MitoSOX, a fluorogenic dye that specifically detects mitochondrial superoxide20. Gcn2−/− mucosal cells produced excess mitochondrial ROS in comparison with the littermate controls in the colon (Fig. 2a) and small intestine (Extended Data Fig. 8a, b). To determine whether autophagy regulated mitochondrial ROS, we analysed superoxide levels in colonic cells isolated from Atg5ΔAPC and Atg7ΔAPC mice after DSS treatment. As in the Gcn2−/− strain, there was higher production of mitochondrial ROS in Atg5ΔAPC and Atg7ΔAPC mice compared with littermate controls (Fig. 2b). Furthermore, blockade of ROS via administration of the antioxidant N-acetyl-L-cysteine (NAC) in vivo led to reduced disease severity, and reduction of T 17 responses in Gcn2−/− mice (Fig. 2c–f). Thus, these data demonstrate a key role for ROS in mediating the enhanced inflammation observed in Gcn2−/−. Oxidative mitochondrial stress is known to be involved in the activation of the inflammasome pathway1, 15. We therefore hypothesized that excess ROS enhanced inflammasome activation in the Gcn2−/− cells under inflammatory conditions. Gcn2−/− dendritic cells produced excess amounts of cleaved IL-1β and cleaved caspases when subjected to amino acid starvation (Fig. 3a). Additionally, there was higher production of pro-IL-1β in the colonic macrophages and dendritic cells isolated from DSS-treated Gcn2−/− mice in the large (Fig. 3b) and small intestine (Extended Data Fig. 8b). In vivo blockade of IL-1β with a neutralizing antibody in Gcn2−/− mice ameliorated the deleterious effects of DSS (Fig. 3c, d and Extended Data Fig. 8c), and significantly reduced intestinal T 17 response (Fig. 3e). However, there were no detectable effects on histopathology, possibly due to incomplete neutralization of IL-1β (Extended Data Fig. 8c). Additionally, we observed that increased inflammation and T 17 responses in the Gcn2−/− mice were negated by the deletion of the inflammasome adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC) (Fig. 3f–i), demonstrating a clear role for inflammasome activation in mediating the enhanced inflammation in Gcn2−/− mice. Given the importance of GCN2 in sensing amino acid starvation, we hypothesized that mice fed an amino-acid-deprived diet might display enhanced activation of ISR in intestinal cells, resulting in dampened inflammation. Therefore we fed mice a reduced amino acid diet (2% protein/weight versus 16% in control mice) and observed rapid activation of phosphorylated (p)-eIF2α on intestinal APCs and epithelial cells (Fig. 4a). Intestinal cells isolated from wild-type mice that were on a low protein diet rapidly upregulated autophagy in comparison to those from Gcn2−/− mice (Fig. 4b). Consistent with this, mass spectrometric analysis of free cytosolic amino acids revealed reduced levels of specific amino acids in colonic epithelial cells and APCs isolated from mice on a 2% low protein diet, relative to the corresponding cell types from control mice, as well as in mice on DSS (data not shown). Interestingly, diets lacking in selective amino acids can also preferentially activate the GCN2 pathway21, 22, 23. We next asked whether lowering of proteins (2% protein) or selective depletion of individual essential amino acids such as leucine (Leu−) impacted intestinal inflammation (Fig. 4). Thus wild-type or Gcn2−/− mice were fed amino-acid-restricted or control diets (16% protein) and subsequently challenged with 3% DSS in their drinking water (Fig. 4c–e). Three per cent DSS was administered to induce enhanced inflammation in wild-type mice, so as to be able to reveal the putative protective effects of amino acid starvation on inflammation. Mice on protein-restricted diets and normal diets had similar weights before DSS (data not shown). After DSS, wild-type mice on protein-restricted diets weighed significantly less than those on control diet (Fig. 4c), but this was not the case in Gcn2−/− mice, indicating that GCN2 protected against gut inflammation (Fig. 4c). The colon lengths and histopathology (epithelial integrity, cellular infiltration, crypt loss) were similar (Extended Data Fig. 8e and data not shown). By contrast, mice on protein-modified diets showed a reduced incidence of ‘bloody diarrhoea’, compared with control diet mice (Fig. 4d). Remarkably, the frequencies of colonic T 17 cells were significantly lower in wild-type mice on modified diets compared with mice on control diets (Fig. 4e). In contrast, there were no significant differences in the T 1 responses or regulatory T cells, indicating that amino-acid-restricted diets selectively impair T 17 responses (Extended Data Fig. 8f). Notably, there were no differences in the T 17 frequencies among Gcn2−/− mice on various diets, indicating that this effect is GCN2 dependent (Fig. 4e). Together, these data demonstrate that amino acid starvation protects the symptoms of colitis and limits T 17 cells via a GCN2-dependent mechanism. We demonstrate that GCN2 suppresses intestinal inflammation and T 17 responses via a mechanism dependent on autophagy and sequestration of ROS, which is a trigger for inflammasome activation (Extended Data Fig. 9a). Gcn2−/− mice displayed enhanced ROS and inflammasome activation, leading to increased inflammation and T 17 responses (Figs 1, 2, 3). Thus, blockade of ROS and IL-1β led to lower inflammation and T 17 responses in Gcn2−/− mice (Figs 2 and 3). In addition, Gcn2−/− mice were deficient in autophagy, which sequesters ROS, and consistent with this there was enhanced ROS and T 17 inflammation in Atg5ΔAPC and Atg7ΔAPC mice (Fig. 1 and Extended Data Fig. 7). Future studies aimed at the functional reconstitution of a constitutively active autophagy pathway specifically in intestinal APCs and epithelial cells in Gcn2−/− mice should provide greater insight into the extent to which the observed phenotype in Gcn2−/− mice is due to impaired autophagy. Consistent with these results it is known that halofuginone, a compound that activates the amino acid starvation response, selectively inhibits mouse and human T 17 differentiation24. Remarkably, we observed that a low protein diet, which activates the amino acid starvation response pathway, reduces the symptoms of colitis and colonic T 17 responses. Although prolonged protein deficiency impairs critical immune functions25, short-term protein restriction can enhance immunity to pathogens26, 27, 28 and cancer29. Also, pharmacological activation of GCN2 protected mice against ischaemia reperfusion injury30. It is tempting to speculate on the evolutionary significance of coupling amino acid starvation with control of inflammation. Tissue injury and cell death, which occur during inflammation, inevitably result in tissue regeneration. Tissue regeneration, in turn, is accompanied by protein synthesis, which could lead to amino acid depletion in the cytosol. The consequent activation of GCN2 will suppress inflammasome activation through the mechanisms described here, in effect representing a negative feedback mechanism that limits the inflammation (Extended Data Fig. 9b). Our results show a role for GCN2 in protecting mice against intestinal inflammation. Thus, targeting the GCN2 pathway may provide new strategies for pharmacological intervention for the amelioration of inflammatory bowel disease and other inflammatory disorders.


Das T.K.,HRI | Czerny B.,Nicolaus Copernicus Astronomical Center
Monthly Notices of the Royal Astronomical Society: Letters | Year: 2012

The Blandford-Znajek (BZ) mechanism has usually been studied in the literature for accretion with considerably high angular momentum leading to the formation of either a cold Keplerian disc or a hot and geometrically thick sub-Keplerian flow as described within the framework of advection-dominated accretion flow/radiatively inefficient accretion flow. However, in nearby elliptical galaxies, as well as for our own Galactic Centre, accretion with very low angular momentum is prevalent. Such quasi-spherical strongly sub-Keplerian accretion has complex dynamical features and can accommodate stationary shocks. In this Letter, we present our calculation for the maximum efficiency obtainable through the BZ mechanism for complete general relativistic weakly rotating axisymmetric flow in the Kerr metric. Both shocked and shock-free flows have been studied in detail for rotating andcounter-rotating accretion. Such a study has never been done in the literature before.We find that the energy extraction efficiency is low, about 0.1 per centr, and inceases by afactor of 15 if the ram pressure is included. Such an efficiency is still much higher than the radiative efficiency of such optically thin flows. For the BZ mechanism, shocked flow produces a higher efficiency than the shock-freesolutions and retrograde flow providesa slightly larger value of the efficiency than that for the prograde flow. © 2011 The Authors, MNRAS 421, L24-L28 Monthly Notices of the Royal Astronomical Society. © 2011 RAS. Source


Das T.K.,HRI | Czerny B.,Nicolaus Copernicus Astronomical Center
Monthly Notices of the Royal Astronomical Society | Year: 2011

The observation of the bright Seyfert 1 galaxy REJ1034+396 is believed to demonstrate a drift in the central period of the quasi-periodic oscillation (QPO) linearly correlated with the temporary X-ray luminosity. We show, using a specific scenario of the oscillation mechanism in a black hole accretion disc, that modelling such correlated trends puts very strong constraints on the nature of this oscillation and the characteristic features of the hot flow in active galactic nuclei. In our model, QPO oscillations are due to the oscillations of the shock formed in the low angular momentum hot accretion flow, and the variation of the shock location corresponds to the observed changes in the QPO period and the X-ray flux. In this scenario, a change in the shock location caused by perturbation of the flow angular momentum is compatible with the trends observed in REJ1034+396, whereas the perturbation of the specific flow energy results in too strong a flux response to the change in the oscillation period. Using a complete general relativistic framework to study the accretion flow in the Kerr metric, we discuss the role of the black hole spin in the period drift. Future missions are expected to bring more active galaxies with time-resolved quasi-periodic oscillations, so a similar quantitative study for other QPO scenarios will be necessary. © 2011 The Authors Monthly Notices of the Royal Astronomical Society © 2011 RAS. Source


Grant
Agency: National Science Foundation | Branch: | Program: STTR | Phase: Phase I | Award Amount: 100.00K | Year: 2006

This Small Business Technology Transfer (STTR) Phase I research program proposes a novel concept for actuating artificial human-like skin. The application of the artificial skin in numerous applications such as robotic faces, prosthetics and medical simulation devices, animatronics, and high-end toys has been limited because of the lack of adequate muscle-like technologies. A solution to this problem is a novel composite actuator, a hybrid of micro piezoelectric actuators and a porous elastomer which will exhibit several characteristics of natural muscle tissues. The key to these advances is a new technique called structured porosity elastomer manufacturing (SPEM). The objectives are to determine the effect of pore geometry on the material properties in porous elastomers, use this understanding for optimization of the porous network, to fabricate the optimized pore-structure in elastomers by developing a hybrid of rapid-prototyping and injection molding processes, to identify the synthesis issues required for embedding of the piezoelectric actuators in the porous elastomer, and to fabricate the robotic face integrated with novel motion control electronics for driving micro piezoelectric actuators and ultrasonic motors. This research will lead to the fundamental understanding of piezo-actuated structured porous elastomer composite actuators as artificial muscles, their manufacturing technologies, and supporting technologies including drivers, wiring and anchoring. This understanding will be helpful in producing a wide array of bio-inspired mechanical devices that are actuated in the manner of muscles. The development of hybrid of rapid-prototyping of 3D sacrificial material and micro-arrays of piezoactuators, by means of micro-robotic gripper arrayswill provide a practical solution for mass production of active, synthetic soft tissues.

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