Divergence of Intracellular Trafficking of Sphingosine Kinase 1 and Sphingosine-1-Phosphate Receptor 3 in MCF-7 Breast Cancer Cells and MCF-7-Derived Stem Cell-Enriched Mammospheres

Breast cancer MCF-7 cell-line-derived mammospheres were shown to be enriched in cells with a CD44+/CD24– surface profile, consistent with breast cancer stem cells (BCSC). These BCSC were previously reported to express key sphingolipid signaling effectors, including pro-oncogenic sphingosine kinase 1 (SphK1) and sphingosine-1-phosphate receptor 3 (S1P3). In this study, we explored intracellular trafficking and localization of SphK1 and S1P3 in parental MCF-7 cells, and MCF-7 derived BCSC-enriched mammospheres treated with growth- or apoptosis-stimulating agents. Intracellular trafficking and localization were assessed using confocal microscopy and cell fractionation, while CD44+/CD24- marker status was confirmed by flow cytometry. Mammospheres expressed significantly higher levels of S1P3 compared to parental MCF-7 cells (p < 0.01). Growth-promoting agents (S1P and estrogen) induced SphK1 and S1P3 translocation from cytoplasm to nuclei, which may facilitate the involvement of SphK1 and S1P3 in gene regulation. In contrast, pro-apoptotic cytokine tumor necrosis factor α (TNFα)-treated MCF-7 cells demonstrated increased apoptosis and no nuclear localization of SphK1 and S1P3, suggesting that TNFα can inhibit nuclear translocation of SphK1 and S1P3. TNFα inhibited mammosphere formation and induced S1P3 internalization and degradation. No nuclear translocation of S1P3 was detected in TNFα-stimulated mammospheres. Notably, SphK1 and S1P3 expression and localization were highly heterogenous in mammospheres, suggesting the potential for a large variety of responses. The findings provide further insights into the understanding of sphingolipid signaling and intracellular trafficking in BCs. Our data indicates that the inhibition of SphK1 and S1P3 nuclear translocation represents a novel method to prevent BCSCs proliferation.     

Characterisation of surface oxides on carbon and their influence on dynamic adsorption

The aim of this work is to gain a better fundamental understanding of the nature of surface oxide sites present on carbon surfaces, and their role in the adsorption process. A number of model carbon substrates with different degrees of surface oxidation and similar textural properties were prepared using a wide range of solution and gaseous phase oxidation techniques. Some of the carbons were characterised using established techniques including flow microcalorimetry, thermogravimetric analysis (TGA) and X-ray photoelectron spectroscopy (XPS). The results showed that both carboxylic acid (-CO₂H) and ether/hydroxyl (C-O) surface oxygen complexes were introduced to all of these carbons as a result of the oxidation processes. The number, strength and thermal stability of the surface groups formed were dependent on the nature of the base material and the oxidation conditions employed. The dynamic adsorption performance of the carbons against hexane, under humid conditions, was found to be mainly determined by the quantity of acidic surface functional groups. However, the location, strength of interaction and availability of the surface oxygen complexes to the adsorbate molecules, are also thought to affect the breakthrough characteristics of the carbons used in this work.     

A semiquantitative analysis of the effects of cisplatin on the rat stria vascularis

Cisplatin (CDDP) is a very effective chemotherapeutic agent but is highly ototoxic. Most studies have focused on the effects of CDDP on the outer hair cells. The purpose of this study was to examine changes in the stria vascularis in cisplatin treated male Wistar rats and to provide semiquantitative analysis of the results. We removed a section of the stria vascularis from the basal turn of five control and five CDDP (16 mg/kg) treated rats. Using transmission electron microscopy (TEM) we analyzed: (1) changes to the strial tissue as a whole; and (2) intracellular changes in the marginal cells. We also subjected the samples to semiquantitative analysis using the MCID, focusing on three aspects of strial profile abnormalities; the number of abnormal marginal cells in CDDP treated tissue, intracellular strial edema and densitometry. Controls appeared normal, but many pathologic changes were apparent in the experimental group. Results from the semiquantitative analysis indicate cisplatin has a deleterious effect on the stria vascularis including strial edema; bulging, rupture and/or compression of the marginal cells and depletion of the cytoplasmic organelles.     

INTELLIGOLD - AN EXPERT SYSTEM FOR GOLD PLANT PROCESS DESIGN

Gold mining projects are a rare opportunity in the minerals industry. They require relatively small capital and give high profitability and fast return on investment compared with other mineral projects. To expand or maintain gold production, continuous development of new deposits and fast implementation of new mining sites are needed. Process design is one of the major issues. As simple and easily extractable ores are almost all exhausted, there is a need for a consistent approach to deal with increasing complexity and decreasing or stagnant gold prices. Process design must consider ore genesis, mineralogical characteristics, ore behavior in available metallurgical processes, linkage with the mining method, environmental impact, and economic issues. The type of work and environment involved makes this application ideal for using AI tools such as expert systems, fuzzy logic, and neural networks. This paper presents Intelligold, an expert system for project development teams to use at the preliminary evaluation and conceptual project stages. Information and knowledge from geology mineralogy, processing, and economics are organized, and recommendations on process options and estimated costs and revenue are given. The "knowledge-building" method is described, together with implementation and verification. Success in building this system suggests application to other ores such as copper and complex base metals.     

Adenoviral-mediated gene transfer in lymphocytes

Although adenovirus can infect a wide range of cell types, lymphocytes are not generally susceptible to adenovirus infection, in part because of the absence of the expression of the cellular receptor for the adenoviral fiber protein. The cellular receptor for adenovirus and coxsackievirus (CAR) recently was cloned and shown to mediate adenoviral entry by interaction with the viral fiber protein. We show that the ectopic expression of CAR in various lymphocyte cell lines, which are almost completely resistant to adenovirus infection, is sufficient to facilitate the efficient transduction of these cells by recombinant adenoviruses. Furthermore, this property of CAR does not require its cytoplasmic domain, consistent with the idea that CAR primarily serves as a high affinity binding site for the adenoviral fiber protein, and that viral entry is mediated by interaction of the viral penton base proteins with cellular integrins. As a demonstration of their functional utility, we used CAR-expressing lymphocytes transduced with an adenovirus expressing Fas ligand to efficiently kill Fas receptor-expressing tumor cells. The ability to efficiently manipulate gene expression in lymphocyte cells by using adenovirus vectors should facilitate the functional characterization of pathways affecting lymphocyte physiology.     

WAR,PEACE, AND FUZZY LOGIC

Our world is controlled by the Darwinian principle of survival of the fittest. In the causal equations of events, peace is a “trivial solution” that becomes lost, like a needle in a haystack, among war-bound “general solutions.” To find peace, we propose two routes. The first and perhaps preferred option is to leave the Darwinian universe of conflict and attempt to lay out our problems in a different setting in which the ruling principle is concord rather than conflict. The second path is to salvage peace from its triviality by compromising its purity and moving into a universe that redefines concord and conflict in a fuzzy way. We will show that the first approach (that we call transcendental) and the second track (that we call pragmatic) can overlap to define a realistic “Pathway to Peace.” The transcendental approach provides boundary conditions without which the generation of pragmatic rules becomes too complex and confusing.

We have used the universal laws of thermodynamics as a framework to understand the nature of conflict and to bridge between the hard and soft sciences. To this framework, we have added some fuzzy rules to pave the passage where transcendental and pragmatic realms of peace-making overlap. Though unity and equal relationships are underlying moral themes in conflict resolution, we show that peace cannot and should not be achieved by compromising diversity.     

Influence of impact velocity on fragmentation and the energy efficiency of comminution

Comminution processes such as crushing and grinding are essential stages in mining and mineral processing operations to reduce the size of ore and rock, and to liberate the valuable mineral for beneficiation. Comminution is energy-intensive and responsible for most of the energy used during mineral recovery. Energy efficiency is very low since almost all the energy is dissipated as heat instead of generating new surface area. This paper reports on studies conducted on strain rates achieved by various velocities of impacts that enhance energy efficiency and mineral liberation. The research focuses on understanding comminution fracture mechanics and on quantifying the distribution of energy with respect to generating new surface area. In interpreting breakage energy phenomena, accurate measurements of surface roughness and surface area are essential. A novel approach to measure surface roughness and surface area based on a fractal analysis procedure has been developed. Changes in surface roughness of broken specimens under variable loading rates were studied using a laser probe to generate 3D topographical maps of the fracture surfaces. The results indicate that surface roughness and hence, specific surface area, increase with increasing loading rate by several orders of magnitude as particle size decreases to 1 μm. Below this limit, surface roughness begins to diminish from particle–particle attrition. An apparatus to measure the quantitative parameters of impact at different velocities on aggregated rock samples is proposed. Experiments are being carried out at projectile velocities up to 500 m s⁻¹ utilizing a compressed-air device. The results suggest possible efficiency improvements in breakage under the velocity of impact.     

D-methionine provides excellent protection from cisplatin ototoxicity in the rat

Cisplatin (CDDP) is a widely used chemotherapeutic agent. Unfortunately, CDDP is highly ototoxic. We tested D-methionine (D-Met), a sulfur containing compound, as an otoprotectant in male Wistar rats. Complete data sets were obtained for five groups of five animals each, including a treated control group (16 mg/kg CDDP), an untreated control group (administered an equivalent volume of saline) and three groups that received either 75, 150, or 300 mg/kg D-Met 30 min prior to the 16 mg/kg CDDP dosing. Auditory brainstem response (ABR) thresholds were obtained in response to clicks, and 1 kHz, 4 kHz, 8 kHz, and 14 kHz toneburst stimuli, before and 3 days after drug administration. Scanning electron microscopy (SEM) was used to examine the outer hair cells of the apical, middle and basal turns of the cochlea. Animal weight was measured on the first and final day. D-Met provided excellent otoprotection even at the lowest level with complete otoprotection obtained for the 300 mg/kg dosing as measured by both ABR and SEM. D-Met also markedly reduced weight loss and mortality. All animals receiving D-Met (15/15) survived to the end of the study period as opposed to only 5/10 of the treated controls.     

The application of fluorescence decay measurements in studies of biological systems

Synchronously pumped, cavity dumped dye lasers provide moderately intense stable pulses of circa 10 ps duration, at a high repetition rate. Such a laser system is an ideal excitation source for the measurement of energy-resolved fluorescence decay profiles. When used in conjunction with photon counting electronics and well tested deconvolution procedures the system is capable of measuring complex (multi-component) fluorescence decays in the time range of < 100 ps- >100 ns. Different fluorophores, when incorporated into membranes or proteins (i.e., extrinsic probing), exhibit different types of interactions with each system. Many simple aromatics are relatively insensitive to the medium, thus the fluorescence decay contains little information of interest. However, in these cases the decay of the anisotropy, which may be extracted from the fluorescence decays, will yield information about the environment. Many heteroaromatic systems are more polar in the first excited singlet state than in the ground state. Consequently there is a strong interaction between the excited state and the surrounding field, this results in a relaxation of the environment about the excited state, resulting in a shift in the fluorescence spectrum to lower energy. When the relaxation is slow, the spectral shift can be time resolved, which can give further information about the structure of the probed system, usually a biological membrane. These types of measurements are discussed, as are some of the problems involved in extrinsic probing, such as multiple siting of the probe.