Cellular FLICE-inhibitory protein regulates chemotherapy-induced apoptosis in breast cancer cells

Combination treatment regimens that include topoisomerase-II-targeted drugs, such as doxorubicin, are widely used in the treatment of breast cancer. Previously, we demonstrated that IFN-� and doxorubicin co-treatment synergistically induced apoptosis in MDA435 breast cancer cells in a STAT1-dependent manner. In this study, we found that this synergy was caspase 8-dependent. In addition, we found that IFN-γ down-regulated the expression of the caspase 8 inhibitor c-FLIP. Furthermore, IFN-� down-regulated c-FLIP in a manner that was dependent on the transcription factors STAT1 and IRF1. However, IFN-� had no effect on c-FLIP mRNA levels, indicating that c-FLIP was down-regulated at a post-transcriptional level following IFN-� treatment. Characterisation of the functional significance of c-FLIP modulation by siRNA gene silencing and stable over-expression studies, revealed it to be a key regulator of IFN-γ- and doxorubicin-induced apoptosis in MDA435 cells. Analysis of a panel of breast cancer cell lines indicated that c-FLIP was an important general determinant of doxorubicin- and IFN-�-induced apoptosis in breast cancer cells. Furthermore, c-FLIP gene silencing sensitised MDA435 cells to other chemotherapies, including etoposide, mitoxantrone and SN-38. These results suggest that c-FLIP plays a pivotal role in modulating drug-induced apoptosis in breast cancer cells.

c-FLIP: A Key Regulator of Colorectal Cancer Cell Death

c-FLIP is an inhibitor of apoptosis mediated by the death receptors Fas, DR4 and DR5 and is expressed as long (c-FLIPL) and short (c-FLIPS) splice forms. We found that siRNA-mediated silencing of c-FLIP induced spontaneous apoptosis in a panel of p53 wild-type, mutant and null colorectal cancer (CRC) cell lines and that this apoptosis was mediated by caspase 8 and FADD. Further analyses indicated the involvement of DR5 and/or Fas (but not DR4) in regulating apoptosis induced by c-FLIP siRNA. Interestingly, these effects were not dependent on activation of DR5 or Fas by their ligands TRAIL and FasL. Overexpression of c-FLIPL, but not c-FLIPS, significantly decreased spontaneous and chemotherapy-induced apoptosis in HCT116 cells. Further analyses with splice form-specific siRNAs indicated that c-FLIPL was the more important splice form in regulating apoptosis in HCT116, H630 and LoVo cells, although specific knock down of c-FLIPS induced more apoptosis in the HT29 cell line. Importantly, intra-tumoral delivery of c-FLIP-targeted siRNA duplexes induced apoptosis and inhibited the growth of HCT116 xenografts in Balb/c SCID mice. In addition, the growth of c-FLIPL overexpressing CRC xenografts was more rapid than control xenografts, an effect that was significantly enhanced in the presence of chemotherapy. These results indicate that c-FLIP inhibits spontaneous death ligand-independent, death receptor-mediated apoptosis in CRC cells and that targeting c-FLIP may have therapeutic potential for the treatment of colorectal cancer.

Calibration of assessment instruments – classification in mathematics at the Universidad Jorge Tadeo Lozano

Item Response Theory, IRT, is a valuable methodology for analyzing the quality of the instruments utilized in assessment of academic achievement. This article presents an implementation of the mentioned theory, particularly of the Rasch model, in order to calibrate items and the instrument used in the classification test for the Basic Mathematics subject at Universidad Jorge Tadeo Lozano. 509 responses chains of students, obtained in the june 2011 application, were analyzed with a set of 45 items, through eight case studies that are showing progressive steps of calibration. Criteria of validity of items and of whole instrument were defined and utilized, to select groups of responses chains and items that were finally used in the determination of parameters which then allowed the classification of assessed students by the test.

Conventional free radical polymerization in room temperature ionic liquids: a green approach to commodity polymers with practical advantages

Free-radical polymerization of methyl methacrylate and styrene using conventional organic initiators in the room temperature ionic liquid, 1-butyl-3-methylimidazolium hexafluorophosphate ([ C(4)mim][PF6]) is rapid and produces polymers with molecular weights up to 10x higher than from benzene; both polymerization and isolation of products were achieved without using VOCs, offering economic as well as environmental advantages.

Identical extraction behavior and coordination of trivalent or hexavalent f-element cations using ionic liquid and molecular solvents

The extraction of both UO22+ and trivalent lanthanide and actinide ions (Am3+, Nd3+, Eu3+) by dialkylphosphoric or dialkylphosphinic acids from aqueous solutions into the ionic liquid, 1-decyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide has been studied and compared to extractions into dodecane. Radiotracer partitioning measurements show comparable patterns of distribution ratios for both the ionic liquid/aqueous and dodecane/aqueous systems, and the limiting slopes at low acidity indicate the partitioning of neutral complexes in both solvent systems. The metal ion coordination environment, elucidated from EXAFS and UV-visible spectroscopy measurements, is equivalent in the ionic liquid and dodecane solutions with coordination of the uranyl cation by two hydrogen-bonded extractant dimers, and of the trivalent cations by three extractant dimers. This is the first definitive report of a system where both the biphasic extraction equilibria and metal coordination environment are the same in an ionic liquid and a molecular organic solvent.

Advanced glycation end products accumulate in the reproductive tract of men with diabetes

Light microscopic studies comparing sperm parameters show little association between diabetes and male fertility. However, with the introduction of new analytical techniques, evidence is now emerging of previously undetectable effects of diabetes on sperm function. Specifically, a recent study has found a significantly higher sperm nuclear DNA fragmentation in diabetic men. As advanced glycation end products (AGEs) are important instigators of oxidative stress and cell dysfunction in numerous diabetic complications, we hypothesized that these compounds could also be present in the male reproductive tract. The presence and localization of the most prominent AGE, carboxymethyl-lysine (CML), in the human testis, epididymis and sperm was determined by immunohistochemistry. Parallel ELISA and Western blot analyses were performed to ascertain the amount of CML in seminal plasma and sperm from 13 diabetic and nine non-diabetic subjects. CML immunoreactivity was found throughout the seminiferous epithelium, the nuclei of spermatogonia and spermatocytes, in the basal and principle cells cytoplasm and nuclei of the caput epididymis and on most sperm tails, mid pieces and all cytoplasmic droplets. The acrosomal cap, especially the equatorial band, was prominently stained in diabetic samples only. The amount of CML was significantly higher (p = 0.004) in sperm from non-diabetic men. Considering the known detrimental actions of AGEs in other organs, the presence, location and quantity of CML, particularly the increased expression found in diabetic men, suggest that these compounds may play a hitherto unrecognized role in male infertility.

Increased concentrations of the oxidative DNA adduct 7, 8-dihydro-8-oxo-2-deoxyguanosine in the germ-line of men with type 1 diabetes

The effects of diabetes mellitus on male reproductive health have not been clearly defined. A previous publication from this group reported significantly higher levels of nuclear DNA fragmentation and mitochondrial DNA deletions in spermatozoa from men with type 1 diabetes. This study compared semen profiles, sperm DNA fragmentation and levels of oxidative DNA modification in spermatozoa of diabetic and non-diabetic men. Semen samples from 12 non-diabetic, fertile men and 11 type 1 diabetics were obtained and subjected to conventional light microscopic semen analysis. Nuclear DNA fragmentation was assessed using an alkaline Comet assay and concentrations of 7,8-dihydro-8-oxo-2-deoxyguanosine (8-OHdG), an oxidative adduct of the purine guanosine, were assessed by high-performance liquid chromatography. Conventional semen profiles were similar in both groups, whilst spermatozoa from type 1 diabetics showed significantly higher levels of DNA fragmentation (44% versus 27%; P < 0.05) and concentrations of 8-OHdG (3.6 versus 2.0 molecules of 8-OHdG per 105 molecules of deoxyguanosine; P < 0.05). Furthermore, a positive correlation was observed between DNA fragmentation and concentrations of 8-OHdG per 105 molecules of deoxyguanosine (rs = 0.7, P < 0.05). The genomic damage evident in spermatozoa of type 1 diabetics may have important implications for their fertility and the outcome of pregnancies fathered by these individuals.

Unusual voltammetry of the reduction of O-2 in [C(4)dmim][N(Tf)(2)] reveals a strong interaction of O-2(center dot-) with the [C(4)dmim](+) cation

Voltammetric studies of the reduction of oxygen in the room temperature ionic liquid [C(4)dmim][N(Tf)(2)] have revealed a significant positive shift in the back peak potential, relative to that expected for a simple electron transfer. This shift is thought to be due to the strong association of the electrogenerated superoxide anion with the solvent cation. In this work we quantitatively simulate the microdisc electrode voltammetry using a model based upon a one-electron reduction followed by a reversible chemical step, involving the formation of the [C(4)dmim](+)center dot center dot center dot O-2(center dot-) ion-pair, and in doing so we extract a set of parameters completely describing the system. We have simulated the voltammetry in the absence of a following chemical step and have shown that it is impossible to simultaneously fit both the forward and reverse peaks. To further support the parameters extracted from fitting the experimental voltammetry, we have used these parameters to independently simulate the double step chronoamperometric response and found excellent agreement. The parameters used to describe the association of the O-2(center dot-) with the [C(4)dmim](+) were k(f) = 1.4 x 10(3) s(-1) for the first-order rate constant and K-eq = 25 for the equilibrium constant.

Electrode kinetics and mechanism of iodine reduction in the room-temperature ionic liquid [C(4)mim][NTf2]

The fast electrochemical reduction of iodine in the RTIL 1-butyl-3-methylimidazolium bis(trifluoromethyl-sulfonyl)imide, [C(4)mim][NTf2], is reported and the kinetics and mechanism of the process elucidated. Two reduction peaks were observed. The first reduction peak is assigned to the process

Extraction of electrode kinetic parameters from microdisc voltammetric data measured under transport conditions intermediate between steady-state convergent and transient linear diffusion as typically applies to room temperature ionic liquids

The extraction of electrode kinetic parameters for electrochemical couples in room-temperature ionic liquids (RTILs) is currently an area of considerable interest. Electrochemists typically measure electrode kinetics in the limits of either transient planar or steady-state convergent diffusion for which the voltammetic response is well understood. In this paper we develop a general method allowing the extraction of this kinetic data in the region where the diffusion is intermediate between the planar and convergent limits, such as is often encountered in RTILs using microelectrode voltammetry. A general working surface is derived, allowing the inference of Butler-Volmer standard electrochemical rate constants for the peak-to-peak potential separation in a cyclic voltammogram as a function of voltage scan rate. The method is applied to the case of the ferrocene/ferrocenium couple in [C(2)mim][N(Tf)(2)] and [C(4)mim][N(Tf)(2)].

Electrooxidation of the iodides [C(4)mim]I, LiI, NaI, KI, RbI, and CsI in the room temperature ionic liquid [C(4)mim][NTf2]

The electrochemical oxidation of 1-butyl-3-methylimidazolium iodide, [C(4)mim]I, has been investigated by cyclic voltammetry at a platinum microelectrode at varying concentrations in the RTIL 1-butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide, [C(4)mim][NTf2]. Two oxidation peaks were observed. The first peak is assigned to the oxidation of iodide to triiodide, in an overall two-electron process: 3I(-)- 2e(-) -> I-3(-). At higher potentials, the electrogenerated triiodide oxidizes to iodine, in an overall one-electron process: I-3(-) - e(-) -> 3/2I(2). An average diffusion coefficient, D, for I- of 1.55 x 10(-11) m(2) s(-1) was obtained. A digital simulation program was used to simulate the voltammetric response, and kinetic parameters were successfully extracted. The parameters deduced from the simulation include D for I-, I-3(-), and I-2 and K-eq,K-2, the equilibrium constant for the reaction of iodide and iodine to form triiodide. Values for these parameters are of the same order as those previously published for the oxidation of Br- in the same RTIL [Allen et al. J. Electroanal. Chem. 2005, 575, 311]. Next, the cyclic voltammetry of five different inorganic iodide salts was studied by dissolving small amounts of the solid in [C(4)mim][NTf2]. Similar oxidation peaks were observed, revealing diffusion coefficients of ca. 0.55, 1.14, 1.23, 1.44, and 1.33 x 10(-11) m(2) s(-1) and solubilities of 714, 246, 54, 83, and 36 mM for LiI, NaI, KI, RbI, and CsI, respectively. The slightly smaller diffusion coefficients for the XI salts (compared to [C(4)mim]I) may indicate that I- is ion-paired with Li+, Na+, K+, Rb+, and Cs+ in the RTIL medium.

Voltammetric characterization of the ferrocene vertical bar ferrocenium and cobaltocenium vertical bar cobaltocene redox couples in RTILs

Ferrocene, Fc, and cobaltocenium hexafluorophosphate, CcPF(6), have been recommended for use as internal reference redox couples in room-temperature ionic liquids (RTILs), as well as in more conventional aprotic solvents. In this study, the electrochemical behavior of Fc and CcPF(6) is reported in eight commonly used RTILs; [C(2)mim][NTf2], [C(4)mim][NTf2], [C(4)mim][BF4], [C(4)mim][PF6], [C(4)mim][OTf], [C(4)mim][NO3], [C(4)mpyrr][NTf2], and [P-14,P-6.6,P-6][FAP], where [C(n)mim](+) = 1-butyl-3-methylimidazolium, [NTf2](-) = bis(trifluoromethylsulfonyl)imide, [BF4](-) = tetrafluoroborate, [PF6](-) = hexafluorophosphate, [OTf](-) = trifluoromethylsulfonate, [NO3](-) = nitrate, [C(4)mpyrr](+) = N-butyl-N-methylpyrrolidinium, [P-14,P-6,P-6,P-6](+) = tris(ri-hexyl)-tetradecylphosphonium and [FAP](-) = trifluorotris(pentafluoroethyl)phosphate, over a range of concentrations and temperatures. Solubilities and diffusion coefficients, D, of both the charged and neutral species were determined using double potential-step chronoamperometry, and CcPF(6) (36.5-450.0 mM) was found to be Much more Soluble than Fc (27.5-101.8 mM). It was observed that classical Stokes-Einstein diffusional behavior applies for Fc and CcPF(6) in all eight RTILs. Diffusion coefficients of Fc and CcPF(6) were calculated at a range of temperatures, and activation energies calculated. It was also determined that D for Fc and CcPF(6) does not change significantly with concentration. This supports the use of both Fe and CcPF(6) to provide a well-characterized and model redox couple for use as a voltammetric internal potential reference in RTILs contrary to previous literature reports in the former case.