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This article presents a method for extracting the optical constants of homogeneous isotropic materials using the infrared spectra of that material. The method is based on using the harmonic oscillator model of molecular polarizability to obtain optical constants, then calculating the spectrum, comparing the calculated spectrum to an experimental spectrum of the material, and adjusting the model parameters until a close fit between the spectra is obtained. Corrections that need to be made to the experimental spectra in order to remove instrumental distortions are also briefly described. The remainder of the article centers on describing how the optical constants can be used to simulate spectra of that material in different experimental arrangements and the benefits that spectral simulations afford to experimentalists.

The ability of anti-heat shock protein 90 (Hsp90) drugs to attenuate NF-κB-mediated transcription is the major basis for their anti-inflammatory properties. While the molecular mechanisms underlying this effect are not clear, they appear to be distinct in human endothelial cells. We now show for the first time that type 2 sirtuin (Sirt-2) histone deacetylase binds human NF-κB target gene promoter and prevents the recruitment of NF-κB proteins and subsequent assembly of RNA polymerase II complex in human lung microvascular endothelial cells. Hsp90 inhibitors stabilize the Sirt-2/promoter interaction and impose a "transcriptional block," which is reversed by either inhibition or downregulation of Sirt-2 protein expression. Furthermore, this process is independent of NF-κB (p65) Lysine 310 deacetylation, suggesting that it is distinct from known Sirt-2-dependent mechanisms. We demonstrate that Sirt-2 is recruited to NF-κB target gene promoter via interaction with core histones. Upon inflammatory challenge, chromatin remodeling and core histone H3 displacement from the promoter region removes Sirt-2 and allows NF-κB/coactivator recruitment essential for RNA Pol II-dependent mRNA induction. This novel mechanism may have important implications in pulmonary inflammation., (Copyright © 2016 the American Physiological Society.)

The self-assembly of medium chain length alkanethiol monolayers on polycrystalline Sn electrodes has been investigated by cyclic voltammetry and coulometry. These studies have been performed in order to ascertain the conditions under which their oxidative deposition can be achieved directly on the oxide-free Sn surface, and the extent to which these electrochemically prepared self-assembled monolayers (SAMs) act as barriers to surface oxide growth. This work has shown that the potentials for their oxidative deposition are more cathodic (by 100-200 mV) than those for Sn surface oxidation and that the passivating abilities of these SAMs improve with increasing film thickness (or chain length). Oxidative desorption potentials for these films have been observed to shift more positively, and in a highly linear fashion, with increasing film thickness (~75 mV/CH2). Although reductive desorption potentials for the SAMs are in close proximity to those for reduction of the surface oxide (SnOx), little or no SnOx formation occurs unless the potential is made sufficiently anodic that the monolayers start to be removed oxidatively. Our coulometric data indicate that the charge involved with alkanethiol reductive desorption or oxidative deposition is consistent with the formation of a close-packed monolayer, given uncertainties attributable to surface roughness and heterogeneity phenomena. These experiments also reveal that the quantity of charge passed during oxidative desorption is significantly larger than what would be predicted for simple alkylsulfinate or alkylsulfonate formation, suggesting that oxidative removal involves a more complex oxidation mechanism. Analogous chronocoulometric experiments for short-chain alkanethiols on polycrystalline Au electrodes have evidenced similar oxidative charge densities. This implies that the mechanism for oxidative desorption on both surfaces may be very similar, despite the significant differences in the inherent dissolution characteristics of the two materials at the anodic potentials employed.

High-amplitude electric pulses of nanosecond duration, also known as nanosecond pulsed electric field (nsPEF), are a novel modality with promising applications for cell stimulation and tissue ablation. However, key mechanisms responsible for the cytotoxicity of nsPEF have not been established. We show that the principal cause of cell death induced by 60- or 300-ns pulses in U937 cells is the loss of the plasma membrane integrity ("nanoelectroporation"), leading to water uptake, cell swelling, and eventual membrane rupture. Most of this early necrotic death occurs within 1-2 hr after nsPEF exposure. The uptake of water is driven by the presence of pore-impermeable solutes inside the cell, and can be counterbalanced by the presence of a pore-impermeable solute such as sucrose in the medium. Sucrose blocks swelling and prevents the early necrotic death; however the long-term cell survival (24 and 48 hr) does not significantly change. Cells protected with sucrose demonstrate higher incidence of the delayed death (6-24 hr post nsPEF). These cells are more often positive for the uptake of an early apoptotic marker dye YO-PRO-1 while remaining impermeable to propidium iodide. Instead of swelling, these cells often develop apoptotic fragmentation of the cytoplasm. Caspase 3/7 activity increases already in 1 hr after nsPEF and poly-ADP ribose polymerase (PARP) cleavage is detected in 2 hr. Staurosporin-treated positive control cells develop these apoptotic signs only in 3 and 4 hr, respectively. We conclude that nsPEF exposure triggers both necrotic and apoptotic pathways. The early necrotic death prevails under standard cell culture conditions, but cells rescued from the necrosis nonetheless die later on by apoptosis. The balance between the two modes of cell death can be controlled by enabling or blocking cell swelling.

Cell permeabilization by electric pulses (EP), or electroporation, is widely used for intracellular delivery of drugs and plasmids, as well as for tumour and tissue ablation. We found that cells pre-treated with 100-μs EP develop delayed hypersensitivity to subsequent EP applications. Sensitizing B16 and CHO cells by splitting a single train of eight 100-μs EP into two trains of four EP each (with 5-min. interval) decreased the LD(50) 1.5-2 times. Sensitization profoundly enhanced the electroporation-assisted uptake of bleomycin, a cell-impermeable cytotoxic agent accepted for killing tumours by electrochemotherapy. EP exposures that were not lethal per se caused cell death in the presence of bleomycin and proportionally to its concentration. Sensitizing cells by a split-dose EP exposure increased bleomycin-mediated lethality to the same extent as a 10-fold increase in bleomycin concentration when using a single EP dose. Likewise, sensitization by a split-dose EP exposure (without changing the overall dose, pulse number, or amplitude) enhanced the electroporative uptake of propidium up to fivefold. Enhancement of the electroporative uptake appears a key mechanism of electrosensitization and may benefit electrochemotherapy and numerous applications that employ EP for cell permeabilization., (© 2012 The Authors. Published by Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.)

Finding optimal experimental conditions for generating stable negative ion electrospray ionization ion trap mass spectra (ESI-IT-MS) of alkanethiol-derived species is critical for quantitatively characterizing multicomponent alkanethiol-based self-assembled monolayers by this technique. Since alkanethiolates slowly oxidize in solution, purposeful oxidation of alkanethiols to their fully oxidized form (alkanesulfonates) is advantageous: sulfonates are chemically stable and have little affinity for covalent binding to metal surfaces. We have used ESI-IT-MS to characterize the products of H(2)O(2) oxidation of simple n-alkanethiols in solution and have observed monomeric alkanesulfonate species as well as alkanesulfonic acid/alkanesulfonate adducts, yielding gas-phase dimers and trimers. MS intensities of both monomers and adducts exhibit a dependence on the ion transfer capillary temperature that is alkyl-chain-length-dependent and that appears to be correlated with C-S bond cleavage. The trend in optimal capillary temperatures indicates that entropic effects lead to lower thermal decomposition temperatures for short-chain species relative to the longer-chain homologues. MS calibration data from alkanesulfonate mixtures are characterized by large linear dynamic ranges (10(-6)-10(-3) M) and detection limits influenced by their thermal decomposition. The high degree of precision in the calibration data should facilitate distinguishing among mixed SAMs having similar compositions.

Background: Electroporation is a method of disrupting the integrity of cell membrane by electric pulses (EPs). Electrical modeling is widely employed to explain and study electroporation, but even most advanced models show limited predictive power. No studies have accounted for the biological consequences of electroporation as a factor that alters the cell's susceptibility to forthcoming EPs., Methodology/principal Findings: We focused first on the role of EP rate for membrane permeabilization and lethal effects in mammalian cells. The rate was varied from 0.001 to 2,000 Hz while keeping other parameters constant (2 to 3,750 pulses of 60-ns to 9-µs duration, 1.8 to 13.3 kV/cm). The efficiency of all EP treatments was minimal at high rates and started to increase gradually when the rate decreased below a certain value. Although this value ranged widely (0.1-500 Hz), it always corresponded to the overall treatment duration near 10 s. We further found that longer exposures were more efficient irrespective of the EP rate, and that splitting a high-rate EP train in two fractions with 1-5 min delay enhanced the effects severalfold., Conclusions/significance: For varied experimental conditions, EPs triggered a delayed and gradual sensitization to EPs. When a portion of a multi-pulse exposure was delivered to already sensitized cells, the overall effect markedly increased. Because of the sensitization, the lethality in EP-treated cells could be increased from 0 to 90% simply by increasing the exposure duration, or the exposure dose could be reduced twofold without reducing the effect. Many applications of electroporation can benefit from accounting for sensitization, by organizing the exposure either to maximize sensitization (e.g., for sterilization) or, for other applications, to completely or partially avoid it. In particular, harmful side effects of electroporation-based therapies (electrochemotherapy, gene therapies, tumor ablation) include convulsions, pain, heart fibrillation, and thermal damage. Sensitization can potentially be employed to reduce these side effects while preserving or increasing therapeutic efficiency.

Background: Nanosecond electric pulses (EP) disrupt cell membrane and organelles and cause cell death in a manner different from the conventional irreversible electroporation. We explored the cytotoxic effect of 10-ns EP (quantitation, mechanisms, efficiency, and specificity) in comparison with 300-ns, 1.8- and 9-μs EP., Methods: Effects in Jurkat and U937 cells were characterized by survival assays, DNA electrophoresis and flow cytometry., Results: 10-ns EP caused apoptotic or necrotic death within 2-20 h. Survival (S, %) followed the absorbed dose (D, J/g) as: S=alphaD((-K)), where coefficients K and alpha determined the slope and the "shoulder" of the survival curve. K was similar in all groups, whereas alpha was cell type- and pulse duration-dependent. Long pulses caused immediate propidium uptake and phosphatidylserine (PS) externalization, whereas 10-ns pulses caused PS externalization only., Conclusions: 1.8- and 9-μs EP cause cell death efficiently and indiscriminately (LD₅₀ 1-3 J/g in both cell lines); 10-ns EP are less efficient, but very selective (LD₅₀ 50-80 J/g for Jurkat and 400-500 J/g for U937); 300-ns EP show intermediate effects. Shorter EP open propidium-impermeable, small membrane pores ("nanopores"), triggering different cell death mechanisms., General Significance: Nanosecond EP can selectively target certain cells in medical applications like tumor ablation., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Background: In this study, proteomic changes were examined in response to paclitaxel chemotherapy or 5-fluorouracil, doxorubicin, and cyclophosphamide (FAC) chemotherapy in plasma from patients with Stage I-III breast carcinoma. The authors also compared the plasma profiles of patients with cancer with the plasma profiles of healthy women to identify breast carcinoma-associated protein markers., Methods: Sixty-nine patients and 15 healthy volunteers participated in the study. Plasma was sampled on Day 0 before chemotherapy and on Day 3 posttreatment in the 69 patients or 3 days apart in the 15 healthy women. Twenty-nine patients received preoperative chemotherapy, and 40 received postoperative chemotherapy. Surface-enhanced laser desorption/ionization mass spectrometry was used to generate protein mass profiles., Results: Few changes were observed in plasma during treatment. Only 1 protein peak was identified (mass/charge ratio [m/z], 2790) that was induced by paclitaxel and, to a lesser extent, by FAC chemotherapy. This proteomic response was detectable in 80% of patients who were treated preoperatively but also was present with lesser intensity in approximately 40% of patients treated postoperatively. There was no clear correlation between induction of m/z 2790 during a single course of treatment and final tumor response to preoperative chemotherapy. Five other peaks also were identified that discriminated between plasma from patients with breast carcinoma and plasma from normal women. These same peaks also were detectable in a subset of patients who already had undergone surgery to remove their tumors., Conclusions: A single chemotherapy-inducible SELDI-MS peak and five other peaks that distinguished plasma obtained from patients with breast carcinoma from plasma obtained from normal, healthy women were identified. The (as yet unsequenced) proteins represented by these peaks are candidate markers of micrometastatic disease after surgery., (Copyright 2004 American Cancer Society.)

The estrogenic activities of 17beta-estradiol, biphenyl, chlorinated biphenyls, and Aroclor mixtures 1221, 1242, and 1248 were measured with a modified recombinant yeast estrogen assay (i.e., a Saccharomyces cerevisiae-based lac-Z (beta-galactosidase) reporter assay). Modifications of the assay included the use of glass vials instead of plastic microtiter plates and the addition of the medium and yeast before the test substrate. 14C-labeled compounds were used to follow improvements in the assay procedures. 14C-17beta-estradiol recovery from plastic microtiter plates and glass vials using the standard or the modified procedure was approximately 89%. However, 14C-4-CB (4-chlorobiphenyl) recovery was considerably less, ranging from 3% in plastic microtiter plates using the standard procedure to 26% in vials using the modified procedure. These results suggest that the toxicity of strongly hydrophobic chemicals may be underestimated. Using the modified yeast estrogen assay, full agonist activity was observed for 4-CB, 2,4,6-CB, and 2,5-CB while each of the Aroclor mixtures were only partial agonists. The equivalent EC50 values in ppm were in environmentally relevant concentrations for biphenyl (19 ppm), 4-CB (4.5 ppm), 2,5-CB (21 ppm), 2,4,6-CB (0.8 ppm), Aroclor 1221 (2.9 ppm), Aroclor 1242 (0.65 ppm), and Aroclor 1248 (2.3 ppm). Estrogen receptor binding for the individual PCB congeners was 25- to 650-fold less than the reported estrogen binding for the corresponding hydroxylated PCB metabolite. Gas chromatographic/mass spectrometric analysis of yeast extracts indicated that S. cerevisiae hydroxylated the individual PCB congeners in the ppb range. With the exception of biphenyl, the concentration of hydroxylated metabolites obtained from incubation of S. cerevisiae with PCB congeners was consistent with the concentration necessary to elicit a positive estrogen receptor-binding response. This work provides evidence that S. cerevisiae are capable of metabolic transformation of PCBs and that estrogen receptor binding of PCBs is mediated through the hydroxylated metabolite rather than through the direct interaction of the PCB congeners with the estrogen receptor., (Copyright 2002 Elsevier Science (USA).)

Toxicity data for the 50% growth inhibitory concentration against Tetrahymena pyriformis (log (IGC50-1)) for 42 alkyl- and halogen-substituted nitro- and dinitrobenzenes were obtained experimentally. Log (IGC50-1) along with the hydrophobicity, the logarithm of the 1-octanol/water partition coefficient (log Kow), and the molecular orbital properties, the lowest unoccupied molecular orbital energy (Elumo) and maximum acceptor superdelocalizability (Amax), were used to develop quantitative structure-activity relationships (QSARs). All the nitroaromatic compounds tested had toxicity in excess of baseline, nonpolar narcosis. The nitrobenzenes were thought to elicit their toxic response through multiple (and mixed) mechanisms. No high-quality relationship was observed between toxicity and hydrophobicity, or Elumo, individually. However, a strong relationship ¿log (IGC50-1) = 16.4(Amax) - 4.64; n = 42, r2 = 0.847, s = 0.279, F = 229¿ was obtained. In an effort to improve predictability, two-parameter QSAR, or response surface, analyses were performed. These analyses resulted in the following QSARs: ¿log (IGC50-1) = 0.206(log Kow) - 16.0(Amax) - 5.04; n = 42, r2 = 0.897, s = 0.229, F = 180¿ and ¿log (IGC50-1) = 0.467(log Kow) - 1.60(Elumo) - 2.55; n = 42, r2 = 0.881, s = 0.246, F = 154¿.

This study describes effects of selected nonpolar narcotics of varying hydrophobicity (quantified by the 1-octanol-water partition coefficient, log Kow) and molecular structure on the population growth kinetics of the freshwater ciliate Tetrahymena pyriformis. The response of Tetrahymena exposed to different nonpolar narcotics varied from a change in generation time to a change in lag phase with similar generation time compared to control. Two narcotics with high (>3.00), intermediate (>0.00 and <3.00), and low log Kow (<0. 00) values were tested. Growth of Tetrahymena inhibited up to 85% by the high log Kow toxicants (2-decanone and butylbenzene) grew with similar rates as the control, but exhibited increased lag time, suggesting that the protozoan became acclimated to toxicant stress. Results from growth of Tetrahymena in the low log Kow toxicants (ethanol and acetone) indicate an increased generation time with increasing concentration. Cells inhibited by the intermediate log Kow chemicals, 1-pentanol and anisole, exhibited a response that was a combination of the previously mentioned two contrary responses. Cells inhibited <35% with 1-pentanol and <50% with anisole grew with similar generation times as control flasks, whereas in cells inhibited >35% or >50%, respectively, the doubling times were longer than control growth.

The growth kinetics of preexposed and naive Tetrahymena pyriformis grown in the presence of one hydrophilic and one hydrophobic nonpolar narcotic (acetone and 2-decanone, respectively) have been evaluated. The response of naive Tetrahymena exposed to nonpolar narcotics varied from a change in generation time upon exposure to hydrophilic chemical to a change in lag phase with similar generation time compared to control upon exposure to hydrophobic compounds. Tetrahymena grown in the presence of low concentrations of 2-decanone and then transferred to higher concentrations acclimated to the presence of the toxicant. Acclimation was demonstrated by reduced lag phases compared to naive cells. Results of population growth studies of Tetrahymena grown in the presence of low concentrations of acetone and then transferred to higher concentrations of acetone exhibit the same pattern, an increased generation time with increasing concentration with no lag time, as naive populations. Additionally, the observed generation times in acetone were cumulative relative to the transfer concentration as well as the acclimation concentration. The most feasible explanation for this phenomenon is the interaction of the toxicants with the plasma membrane.

In preparation for a clinical trial using GM-CSF on days 4-10 of sequential high-dose cytarabine (ara-C) and asparaginase (ASNase) on days 1-3 and 8-10, potential interactions between the protein synthesis inhibitor ASNase and GM-CSF were evaluated. Granulocyte-macrophage colony-stimulating factor (GM-CSF) can stimulate acute myeloid leukemia (AML) cells to proliferate in vitro and in vivo. Log phase HL-60 cells were exposed to ara-C (10 microM x 3 h) and/or ASNase (10 U/ml during the last 2 h of ara-C). Ara-C and/or ASNase was removed and cells were incubated with or without GM-CSF (10 ng/ml). After 24, 48 and 72 h of GM-CSF there was no significant difference in the S phase fraction of cells exposed to ASNase prior to GM-CSF. Soft agar cloning efficiency was determined after retreatment with ara-C +/- ASNase 24 h into the GM-CSF incubation. GM-CSF enhanced cytotoxicity for all combinations, although this effect was of borderline significance (P = 0.0621); addition of ASNase to the treatment regimen significantly (P = 0.0229) enhanced cytotoxicity without any evidence of a negative interaction with GM-CSF. In addition, ara-C metabolism was assessed during simultaneous exposure to ara-C (10 microM x 3 h) +/- ASNase (10 U/ml the last 2 h) +/- GM-CSF (10 ng/ml beginning 24 h prior to ara-C). Ara-C incorporated into DNA (P = 0.0302) and ara-CTP formation (P = 0.0084 and P = 0.0003 at 2 and 3 h timepoints, respectively) were both increased significantly by GM-CSF, with modest non-significant increases with ASNase exposures. Neither ASNase nor GM-CSF inhibited the effects of the other in this in vitro model. Therefore, when appropriately scheduled, both GM-CSF and ASNase may potentiate ara-C cytotoxicity.

Bone marrow leukemia cells from eight adults with untreated acute myeloid leukemia (AML) were evaluated before and after three daily leukaphereses to determine if mechanical cytoreduction can modulate the cell cycle distribution. The percentage of cells in S-phase and the proliferative fraction (PF = %S + %G2M) were determined by flow cytometry after dual labeling with bromodeoxyuridine and propidium iodide. Prior to pheresis the median %S and PF were 5.4 and 15.4%, respectively. The median change in %S was +2.5% (range -5.5 to +18.8) with increases greater than or equal to 3.7% in 4/8 patients. The median change in PF was +6.1% (range -13.8 to +25.3) with an increase of greater than or equal to 3.6% in 6/8 patients. The median absolute changes of 2.5 and 6.1% represent increases of 47% for %S and 40% for PF compared to the day 1 (pre-pheresis) median values. As the number of nucleoside transporters in the cell membrane [nitrobenzylmercaptopurine riboside (NBMPR) binding sites] has been related to the percentage of cells in S-phase and to cytosine arabinoside (ara-C) cellular pharmacology, these were also measured before and after leukapheresis. Changes in the number of NBMPR binding sites varied widely with a median increase of 365 sites per cell (range -26,061 to +10,396). The change in NBMPR sites was significantly and positively correlated with changes in %S (r = 0.829, p = 0.042). These data suggest that mechanical cytoreduction by leukapheresis can increase the fraction of leukemia cells in S-phase and the PF in some patients with AML. The increase in %S is accompanied by an increase in NBMPR binding sites per cell. These changes in leukemia cell characteristics would be expected to result in an increase in efficacy of ara-C or other S-phase specific agents.

The expression of nucleoside carrier [nitrobenzylmercaptopurine riboside (NBMPR) binding] sites has been related to proliferative fraction in cell lines and in patient myeloid and lymphoid blasts. This correlation was examined in patients with untreated acute myeloid leukemia (AML). Bone marrow blasts were incubated with 8 microM bromodeoxyuridine (BrdUrd) and dual-labeled with propidium iodide and anti-BrdUrd monoclonal antibody. Flow cytometry was used to determine the percentage of cells with detectable BrdUrd incorporation into DNA (%S) and the proliferative fraction (PF = %S+%G2M) in 63 patients; NBMPR binding sites were quantitated in samples from 29 patients. The median %S was 6.1% (range 0.6-25.9%) and the median PF was 13.0% (range 2.4-36.1%), with a median of 7243 NBMPR binding sites per cell (range 1716-27247). In contrast to a previous report which included bone marrow and peripheral blood blasts, %S in marrow blasts did not correlate with NBMPR binding sites per cell (r = 0.005, p = 0.979). Similarly, PF did not correlate with NBMPR sites per cell (r = 0.190, p = 0.325). This lack of correlation between leukemia cell proliferation and NBMPR binding sites per cell suggests that DNA synthesis in AML blasts depends primarily on de novo nucleoside synthesis rather than the usage of salvage pathways.

Therapy of acute myelogenous leukemia (AML) with sequential high-dose ara-C and asparaginase (HiDAC----ASNase) on a day 1 and 8 schedule was designed to exploit potential recruitment of residual leukemia cells following initial cytoreduction from day 1 treatment. DNA flow cytometry was used to evaluate the proliferative index (%S + G2M) of bone marrow leukemia cells from pretreatment and day 8 marrow samples. The proliferative index on day 1, day 8, and incremental change (day 8 minus day 1) were analyzed for their correlation with bone marrow aplasia on day 15 and with the attainment of subsequent complete remission. Pretreatment (day 1) and the change in proliferative index did not correlate (p greater than 0.10) with day 15 marrow aplasia or with clinical outcome. However, the magnitude of the day 8 proliferative index did relate to the attainment of bone marrow aplasia on day 15 (p = 0.05) and the attainment of complete remission (p = 0.002). Recruitment of residual leukemia cells into the proliferative phases of the cell cycle may contribute to the unique efficacy of the day 1 and 8 schedule of HIDAC----ASNase. Additionally, the cytokinetics of residual leukemia after initial chemotherapy may be predictive of outcome and could be useful as a marker for the design of optimal therapeutic regimens.

Leukemia blasts isolated from bone marrow aspirates of 44 adults with acute leukemia were incubated for 1 h with 0.008-32 microM bromodeoxyuridine (Brd-Urd). After dual labeling with monoclonal anti-BrdUrd antibodies and propidium iodide, the cells were analyzed by flow cytometry. Percent labeled cells and intensity of labeling were similar over concentrations of BrdUrd ranging from 0.8-32 microM--a 40-fold range. Therefore, despite potential interpatient variability in nucleoside pharmacokinetics, commonly used doses of BrdUrd which are intended to achieve steady-state plasma concentrations in the 8.0 microM range can be expected to provide a reliable estimate of the S-phase fraction.