A Unidirectional Cell Switching Gate by Engineering Grating Length and Bending Angle

On a microgrooved substrate, cells migrate along the pattern, and at random positions, reverse their directions. Here, we demonstrate that these reversals can be controlled by introducing discontinuities to the pattern. On “V-shaped grating patterns”, mouse osteogenic progenitor MC3T3-E1 cells reversed predominately at the bends and the ends. The patterns were engineered in a way that the combined effects of angle- and length-dependence could be examined in addition to their individual effects. Results show that when the bend was placed closer to one end, migration behaviour of cells depends on their direction of approach. At an obtuse bend (135°), more cells reversed when approaching from the long segment than from the short segment. But at an acute bend (45°), this relationship was reversed. Based on this anisotropic behaviour, the designed patterns effectively allowed cells to move in one direction but blocked migrations in the opposing direction. This study demonstrates that by the strategic placement of bends and ends on grating patterns, we can engineer effective unidirectional switching gates that can control the movement of adherent cells. The knowledge developed in this study could be utilised in future cell sorting or filtering platforms without the need for chemotaxis or microfluidic control.     

Structure based virtual screening of anticanerous polyphenolic phytocompounds against G-protein coupled receptor and identification of potent antagonist ligand(s) through molecular docking

Design of potential drug-like candidates for cancer is of interest in recent years. We used 60 compounds which are known to have the potential to down regulate Nuclear Factor kappaB (NFκB) for this study. The compounds were assessed for Lipinski's RO5 and ADMET properties. Allixin, anethole, capsaicin, linearol and syringic acid satisfied both Lipinski's RO5 and ADMET properties. These compounds showed strong molecular interaction with receptor GPCR55 indicating they have ability to block GPCR55. Thus, their role in anticellular proliferation and induction of apoptosis is implied.     

Development of a novel spatiotemporal depletion system for cellular cholesterol

Cholesterol is an essential component of mammalian cell membranes whose subcellular concentration and function are tightly regulated by de novo biosynthesis, transport, and storage. Although recent reports have suggested diverse functions of cellular cholesterol in different subcellular membranes, systematic investigation of its site-specific roles has been hampered by the lack of a methodology for spatiotemporal manipulation of cellular cholesterol levels. Here, we report the development of a new cholesterol depletion system that allows for spatiotemporal manipulation of intracellular cholesterol levels. This system utilizes a genetically encoded cholesterol oxidase whose intrinsic membrane binding activity is engineered in such a way that its membrane targeting can be controlled in a spatiotemporally specific manner via chemically induced dimerization. In combination with in situ quantitative imaging of cholesterol and signaling activity measurements, this system allows for unambiguous determination of site-specific functions of cholesterol in different membranes, including the plasma membrane and the lysosomal membrane.     

Self-assembly processes in the prebiotic environment

An important question guiding research on the origin of life concerns the environmental conditions where molecular systems with the properties of life first appeared on the early Earth. An appropriate site would require liquid water, a source of organic compounds, a source of energy to drive polymerization reactions and a process by which the compounds were sufficiently concentrated to undergo physical and chemical interactions. One such site is a geothermal setting, in which organic compounds interact with mineral surfaces to promote self-assembly and polymerization reactions. Here, we report an initial study of two geothermal sites where mixtures of representative organic solutes (amino acids, nucleobases, a fatty acid and glycerol) and phosphate were mixed with high-temperature water in clay-lined pools. Most of the added organics and phosphate were removed from solution with half-times measured in minutes to a few hours. Analysis of the clay, primarily smectite and kaolin, showed that the organics were adsorbed to the mineral surfaces at the acidic pH of the pools, but could subsequently be released in basic solutions. These results help to constrain the range of possible environments for the origin of life. A site conducive to self-assembly of organic solutes would be an aqueous environment relatively low in ionic solutes, at an intermediate temperature range and neutral pH ranges, in which cyclic concentration of the solutes can occur by transient dry intervals.     

Detection of domoic acid in northern anchovies and California sea lions associated with an unusual mortality event

The occurrence of an unusual mortality event involving California sea lions (Zalophus californianus) along the central California coast in May 1998 was recently reported. The potent neurotoxin domoic acid (DA), produced naturally by the diatom Pseudo-nitzschia australis and transmitted to the sea lions via planktivorous northern anchovies (Engraulis mordax), was identified as the probable causative agent. Details of DA analyses for anchovy tissues and sea lion feces are described. Domoic acid levels were estimated in anchovy samples by HPLC-UV, and in sea lion feces using the same method as well as a microplate receptor binding assay, with absolute confirmation by tandem mass spectrometry. The highest DA concentrations in anchovies occurred in the viscera (223 +/- 5 microg DA g(-1)), exceeding values in the body tissues by seven-fold and suggesting minimal bioaccumulation of DA in anchovy tissue. HPLC values for DA in sea lion fecal material (ranging from 152 to 136.5 microg DA g(-1)) required correction for interference from an unidentified compound. Inter-laboratory comparisons of HPLC data showed close quantitative agreement. Fecal DA activity determined using the receptor binding assay corresponded with HPLC values to within a factor of two. Finally, our detection of P. australis frustules, via scanning electron microscopy, in both anchovy viscera and fecal material from sea lions exhibiting seizures provides corroborating evidence that this toxic algal species was involved in this unusual sea lion mortality event.     

Effects of tributyltin and benzo[a]pyrene on the immune-associated activities of hemocytes and recovery responses in the gastropod abalone, Haliotis diversicolor

Our previous study reports that short-term exposure to sublethal concentrations of benzo[a]pyrene (BaP) induces immunomodulation in the gastropod abalone, Haliotis diversicolor. In the present study, it was further observed that long-term chronic exposure to sublethal concentrations of BaP modulated the immunocompetence of abalones in terms of the change in activity of the antioxidant and immune associated parameters tested. In addition, the effect of tributyltin (TBT), another important genotoxicant in the aquatic environment, was investigated. Exposure of abalones to sublethal concentrations of TBT and BaP for 21 days resulted in significant decrease of total hemocyte count, phagocytosis, membrane stability and lysozyme activity. Conversely induction of extra and intra cellular superoxide generation, nitric oxide, nitric oxide synthase and myeloperoxidase activity was present when the abalones were exposed to TBT and BaP. Most of the immune associated parameters tested showed clear time dependent response to both toxicants. Within 14 days after the 21 day exposure to BaP, recovery was observed as evidenced by most of the parameters returning to their normal level. However, no recovery was observed within 14 days after the 21 day exposure to TBT as evidenced by continued elevation of intra cellular superoxide and nitrite production and decrease in THC, membrane stability and lysozyme activity. This suggested a prolonged TBT-induced impact on the immune reaction and possibly more damage than that caused by BaP. Overall the results suggest that chronic exposure to sublethal concentrations of TBT or BaP causes modulations in the immunocompetence of abalones with most of the immune associated parameters tested being stimulated, and this might be harmful to the host.     

Differences in the reducing power along the rat GI tract: Lower antioxidant capacity of the colon

The ability of the gastrointestinal (GI) tract, as well as other tissues, to cope with reactive oxygen species (ROS) efflux in pathological events is determined partly by epithelial antioxidant levels. These levels are comprized of tissue antioxidant enzymes and low molecular weight antioxidants (LMWA). While glutathione levels and the activity of enzymatic antioxidants along the GI tract have been studied, the contribution of the overall LMWA to the total antioxidant capacity has not yet been determined. In this study the overall antioxidant activity in the mucosa/submucosa and muscularis/serosa of various sections along the small intestine and colon of the rat was evaluated by determining the reducing power, which reflects the total antioxidant activity derived from LMWA, using cyclic voltammetry. The activity of the antioxidant enzymes superoxide dismutase (SOD) and catalase was also measured. The reducing power (total antioxidant activity) was higher in the mucosa/submucosa of the small intestine as compared to the mucosa/submucosa of the colon. Similarly, catalase and SOD activity in the mucosa/submucosa of the small intestine was significantly higher than in the mucosa/submucosa of the colon. Differences were also observed in the reducing power and SOD activity in the muscularis/serosa of the rat small intestine as compared to the colon. The low antioxidant capacity in the colon may facilitate reactive oxygen species (ROS)-mediated injury and lead to inflammatory diseases such as ulcerative colitis, specifically in the colon.     

Optical reversal of halothane-induced immobility in C. elegans

Volatile anesthetics (VAs) cause profound neurological effects, including reversible loss of consciousness and immobility. Despite their widespread use, the mechanism of action of VAs remains one of the unsolved puzzles of neuroscience [ [1] and [2] ]. Genetic studies in Caenorhabditis elegans [ [3] and [4] ], Drosophila [ [3] and [5] ], and mice [ [6] , [7] , [8] and [9] ] indicate that ion channels controlling the neuronal resting membrane potential (RMP) also control anesthetic sensitivity. Leak channels selective for K⁺ [ [10] , [11] , [12] and [13] ] or permeable to Na⁺ [14] are critical for establishing RMP. We hypothesized that halothane, a VA, caused immobility by altering the neuronal RMP. In C. elegans, halothane-induced immobility is acutely and completely reversed by channelrhodopsin-2 based depolarization of the RMP when expressed specifically in cholinergic neurons. Furthermore, hyperpolarizing cholinergic neurons via halorhodopsin activation increases sensitivity to halothane. The sensitivity of C. elegans to halothane can be altered by 25-fold by either manipulation of membrane conductance with optogenetic methods or generation of mutations in leak channels that set the RMP. Immobility induced by another VA, isoflurane, is not affected by these treatments, thereby excluding the possibility of nonspecific hyperactivity. The sum of our data indicates that leak channels and the RMP are important determinants of halothane-induced general anesthesia.