Assessing Nordihydroguaiaretic Acid Therapeutic Effect for Glioblastoma Multiforme.

In this study, we demonstrate that Raman microscopy combined with computational analysis is a useful approach to discriminating accurately between brain tumor bio-specimens and to identifying structural changes in glioblastoma (GBM) bio-signatures after nordihydroguaiaretic acid (NDGA) administration. NDGA phenolic lignan was selected as a potential therapeutic agent because of its reported beneficial effects in alleviating and inhibiting the formation of multi-organ malignant tumors. The current analysis of NDGA's impact on GBM human cells demonstrates a reduction in the quantity of altered protein content and of reactive oxygen species (ROS)-damaged phenylalanine; results that correlate with the ROS scavenger and anti-oxidant properties of NDGA. A novel outcome presented here is the use of phenylalanine as a biomarker for differentiating between samples and assessing drug efficacy. Treatment with a low NDGA dose shows a decline in abnormal lipid-protein metabolism, which is inferred by the formation of lipid droplets and a decrease in altered protein content. A very high dose results in cell structural and membrane damage that favors transformed protein overexpression. The information gained through this work is of substantial value for understanding NDGA's beneficial as well as detrimental bio-effects as a potential therapeutic drug for brain cancer.

Pharmacodynamic biomarkers in metronomic chemotherapy: multiplex cytokine measurements in gastrointestinal cancer patients.

Metronomic chemotherapy has shown promising antitumor activity in a number of malignancies. We previously reported a phase II clinical trial of metronomic UFT (a 5-fluorouracil prodrug; 100 mg/twice per day p.o.) and cyclophosphamide (CTX; 500 mg/m2 i.v. bolus on day 1 and then 50 mg/day p.o.) plus celecoxib (200 mg/twice a day p.o.) in 38 patients with advanced refractory gastrointestinal tumors. The mechanisms of action of metronomic chemotherapy include inhibition of angiogenesis, direct cytotoxic effects on cancer cells, and, at least for drugs such as CTX, activation of the immune system. To further evaluate the latter, we carried out an immune system multiplex 14-cytokine profiling of plasma samples that were available (for day 0, day 28, and day 56) from 31 of the 38 patients in the above-noted clinical trial. Our results show that pre-treatment plasma-level cutoffs of interferon gamma (> 12.84 pg/ml), sCD40L (< 2168 pg/ml), interferon alpha 2 (> 55.11 pg/ml), and IL-17a (< 15.1 pg/ml) were predictive markers for those patients with better progression-free survival (p < .05 for each cytokine). After 28 days of metronomic therapy, the plasma levels of sCD40L, IL-17a, and IL-6 (< 130 pg/ml) could serve as predictors of improved progression-free survival, as could levels interferon gamma and sCD40L after 56 days of therapy. We observed minimal changes in cytokine profiles, from baseline, as a consequence of the metronomic therapy, with the exception of an elevation of IL-6 and IL-8 levels 28 days (and 56 days) after treatment started (p < 0.05). Our results indicate that a selective cytokine elevation involves IL-6 and IL-8, following metronomic chemotherapy administration. In addition, interferon gamma and sCD40L may be potential biomarkers for gastrointestinal cancer patients that are likely to benefit from metronomic chemotherapy. Our study contributes to our understanding of the mechanisms of action of metronomic chemotherapy, and the cytokine profiling we describe may guide future selection of gastrointestinal cancer patients for UFT/CTX/celecoxib combination metronomic chemotherapy.

Assessment of Renal Osteodystrophy via Computational Analysis of Label-free Raman Detection of Multiple Biomarkers.

Accurate clinical evaluation of renal osteodystrophy (ROD) is currently accomplished using invasive in vivo transiliac bone biopsy, followed by in vitro histomorphometry. In this study, we demonstrate that an alternative method for ROD assessment is through a fast, label-free Raman recording of multiple biomarkers combined with computational analysis for predicting the minimally required number of spectra for sample classification at defined accuracies. Four clinically relevant biomarkers: the mineral-to-matrix ratio, the carbonate-to-matrix ratio, phenylalanine, and calcium contents were experimentally determined and simultaneously considered as input to a linear discriminant analysis (LDA). Additionally, sample evaluation was performed with a linear support vector machine (LSVM) algorithm, with a 300 variable input. The computed probabilities based on a single spectrum were only marginally different (~80% from LDA and ~87% from LSVM), both providing an unacceptable classification power for a correct sample assignment. However, the Type I and Type II assignment errors confirm that a relatively small number of independent spectra (7 spectra for Type I and 5 spectra for Type II) is necessary for a p < 0.05 error probability. This low number of spectra supports the practicality of future in vivo Raman translation for a fast and accurate ROD detection in clinical settings.

Simultaneous Detection of Dopamine and Serotonin-A Comparative Experimental and Theoretical Study of Neurotransmitter Interactions.

With the goal of accurately detecting and quantifying the amounts of dopamine (DA) and serotonin (5-HT) in mixtures of these neurotransmitters without using any labelling, we present a detailed, comparative computational and Raman experimental study. Although discrimination between these two analytes is achievable in such mixtures for concentrations in the millimolar range, their accurate quantification remains unattainable. As shown for the first time in this work, the formation of a new composite resulting from their interactions with each other is the main reason for this lack of quantification. While this new hydrogen-bonded complex further complicates potential analyte discrimination and quantification at concentrations characteristic of physiological levels (i.e., nanomolar concentrations), it can also open new avenues for its use in drug delivery and pharmaceutical research. This remark is based not only on chemical interactions analyzed here from both theoretical and experimental approaches, but also on biological relationship, with consideration of both functional and neural proximity perspectives. Thus, this research constitutes an important contribution toward better understanding of neural processes, as well as toward possible future development of label-free biosensors.

Optimization of biomarkers-based classification scores as progression-free survival predictors: an intuitive graphical representation.

Aim: To construct classification scores based on a combination of cancer patient plasma biomarker levels, for predicting progression-free survival. Methods: The approach is based on the optimization of the biomarker cut-off values, which maximize the statistical differences between the groups with values lower or larger than the cut-offs, respectively. An intuitive visualization of the quality of the classification score is also proposed. Results: Even if there are only weak correlations between individual biomarker levels and progression-free survival, scores based on suitably chosen combination of three biomarkers have classification power comparable with the Response Evaluation Criteria in Solid Tumors criteria classification of response to treatments in solid tumors. Conclusion: Our approach has the potential to improve the selection of the patients who will benefit from a given anticancer treatment.

Raman Spectroscopic and Microscopic Analysis for Monitoring Renal Osteodystrophy Signatures.

Defining the pathogenesis of renal osteodystrophy (ROD) and its treatment efficacy are difficult, since many factors potentially affect bone quality. In this study, confocal Raman microscopy and parallel statistical analysis were used to identify differences in bone composition between healthy and ROD bone tissues through direct visualization of three main compositional parametric ratios, namely, calcium content, mineral-to-matrix, and carbonate-to-matrix. Besides the substantially lower values found in ROD specimens for these representative ratios, an obvious accumulation of phenylalanine is Raman spectroscopically observed for the first time in ROD samples and reported here. Thus, elevated phenylalanine could also be considered as an indicator of the disease. Since the image results are based on tens of thousands of spectra per sample, not only are the average ratios statistically significantly different for normal and ROD bone, but the method is clearly powerful in distinguishing between the two types of samples. Furthermore, the statistical outcomes demonstrate that only a relatively small number of spectra need to be recorded in order to classify the samples. This work thus opens the possibility of future development of in vivo Raman sensors for assessment of bone structure, remodeling, and mineralization, where different biomarkers are simultaneously detected with unprecedented accuracy.

Detection of Biased Rating of Medical Students by Standardized Patients: Opportunity for Improvement.

OBJECTIVES: To assess the inter-rater reliability of standardized patients (SPs) as they assess the clinical skills of medical students and to detect possible rating bias in SPs. METHODS: The ratings received by 6 students examined in 4 clinical stations by 13 SPs were examined. Each SP contributed at least 3 and at most 10 pairwise ratings, with an average of approximately 5 ratings per SP. The standard Cohen' kappa statistic was calculated and the distribution of scores among SPs was compared via both ANOVA the Kruskal-Wallis H test (one-way ANOVA by ranks). Furthermore, the number of discrepancies between pairwise raters (showing either "positive" or "negative" bias in the rating) were analyzed using ANOVA and a χ2 goodness-of-fit test. RESULTS: The conventional method, which compared the statistics of kappa scores of the raters (including the prevalence-adjusted-bias adjusted kappa scores) did not reject the null hypothesis, that the raters (SPs) are similar. However, the analysis of the distribution of the discrepancies among the raters revealed that the differences between raters cannot be attributed to chance, particularly when a distinction was made between their overall "positive" and "negative" bias. A strong (p<0.001) "negative" bias was detected, and the SPs responsible for this bias have been identified. CONCLUSIONS: The statistical method suggested here, which takes into account explicitly the "positive" and the "negative" bias of the raters, is more sensitive than the conventional method (Cohens' kappa). Since the outliers (the biased SPs) affect the fairness of the grading of the medical students, it is important to detect any statistically-significant bias in the rating and to adjust correspondingly the SP's assessment.

Impact of CTLA-4 blockade in conjunction with metronomic chemotherapy on preclinical breast cancer growth.

BACKGROUND: Although there are reports that metronomic cyclophosphamide (CTX) can be immune stimulating, the impact of its combination with anti-CTLA-4 immunotherapy for the treatment of cancer remains to be evaluated. METHODS: Murine EMT-6/P breast cancer, or its cisplatin or CTX-resistant variants, or CT-26 colon, were implanted into Balb/c mice. Established tumours were monitored for relative growth following treatment with anti-CTLA-4 antibody alone or in combination with; (a) metronomic CTX (ldCTX; 20 mg kg-1 day-1), b) bolus (150 mg kg-1) plus ldCTX, or (c) sequential treatment with gemcitabine (160 mg kg-1 every 3 days). RESULTS: EMT-6/P tumours responded to anti-CTLA-4 therapy, but this response was less effective when combined with bolus plus ldCTX. Anti-CTLA-4 could be effectively combined with either ldCTX (without a bolus), or with regimens of either sequential or concomitant gemcitabine, including in orthotopic EMT-6 tumours, and independently of the schedule of drug administration. Tumour responses were confirmed with CT-26 tumours but were less pronounced in drug-resistant EMT-6/CTX or EMT-6/DDP tumour models than in the parent tumour. A number of tumour bearing mice developed spontaneous metastases under continuous therapy. The majority of cured mice rejected tumour re-challenges. CONCLUSIONS: Metronomic CTX can be combined with anti-CTLA-4 therapy, but this therapy is impaired by concomitant bolus CTX. Sequential therapy of anti-CTLA-4 followed by gemcitabine is effective in chemotherapy-naive tumours, although tumour relapses can occur, in some cases accompanied by the development of spontaneous metastases.

On the surface tension and Zeta potential of electrolyte solutions.

The distribution of ions in the vicinity of the air/water interface is still a matter of strong debate, with numerous calculations and experiments providing contradictory results, even regarding the preference of simple ions (such as H+ and OH-) for interfacial or bulk water. When short range interactions between ions and the interface are assumed independent of bulk concentrations, if they are compatible with the surface tension data, they underpredict the experimental Zeta potentials by orders of magnitude. If they are compatible with Zeta potential data, they are in strong disagreement with surface tension experiments. It is suggested that these observations might be a result of the relatively low number of interfacial water molecules available to hydrate the ions and the competition between various ions for adsorption sites. Therefore, whereas at low bulk concentrations, the Structure-Breaking ions prefer the interface, at sufficiently large bulk concentrations the surface adsorptions of these ions become saturated, and their interfacial concentrations may become lower than in the bulk. Consequently, the total interactions of ions with the interface can be strongly attractive at low bulk concentrations, and less attractive (or even repulsive), at high concentrations. To model this effect, the interactions between ions and interface are taken into account via modified Langmuir adsorption expressions for OH- and Cl-, while the H+ ions are considered to be attached to any interfacial water molecule, even if the latter participate in the hydration of anions. The simple model of adsorption employed here is in agreement with both experiments on Zeta potential and on surface tension, and might reveal the conditions under which a given ion exhibits propensity for either the air/water interface, or for bulk water.

Label-Free Raman Imaging to Monitor Breast Tumor Signatures.

Although not yet ready for clinical application, methods based on Raman spectroscopy have shown significant potential in identifying, characterizing, and discriminating between noncancerous and cancerous specimens. Real-time and accurate medical diagnosis achievable through this vibrational optical method largely benefits from improvements in current technological and software capabilities. Not only is the acquisition of spectral information now possible in milliseconds and analysis of hundreds of thousands of data points achieved in minutes, but Raman spectroscopy also allows simultaneous detection and monitoring of several biological components. Besides demonstrating a significant Raman signature distinction between nontumorigenic (MCF-10A) and tumorigenic (MCF-7) breast epithelial cells, our study demonstrates that Raman can be used as a label-free method to evaluate epidermal growth factor activity in tumor cells. Comparative Raman profiles and images of specimens in the presence or absence of epidermal growth factor show important differences in regions attributed to lipid, protein, and nucleic acid vibrations. The occurrence, which is dependent on the presence of epidermal growth factor, of new Raman features associated with the appearance of phosphothreonine and phosphoserine residues reflects a signal transduction from the membrane to the nucleus, with concomitant modification of DNA/RNA structural characteristics. Parallel Western blotting analysis reveals an epidermal growth factor induction of phosphorylated Akt protein, corroborating the Raman results. The analysis presented in this work is an important step toward Raman-based evaluation of biological activity of epidermal growth factor receptors on the surfaces of breast cancer cells. With the ultimate future goal of clinically implementing Raman-guided techniques for the diagnosis of breast tumors (e.g., with regard to specific receptor activity), the current results just lay the foundation for further label-free optical tools to diagnose the disease.

A Drude model analysis of conductivity and free carriers in boron-doped diamond films and investigations of their internal stress and strain.

Boron-doped diamond (BDD) has seen a substantial increase in interest for use as electrode coating material for electrochemistry and studies of deep brain stimulation mechanism. In this study, we present an alternative method for determining important characteristics, including conductivity, carrier concentration, and time constant, of such material by the signature of Drude-like metallic behavior in the far-infrared (IR) spectral range. Unlike the direct determination of conductivity from the four-point probe method, using far-IR transmittance provides additional information, such as whether the incorporation of boron results in a large concentration of carriers or in inducing defects in the diamond lattice. The slightly doped to medium-doped BDD samples that were produced using chemical vapor deposition and analyzed in this work show conductivities ranging between 5.5 and 11 (Ω cm)-1. Different growth conditions demonstrate that increasing boron concentration results in an increase in the carrier concentration, with values between 7.2 × 1016 and 2.5 × 1017 carriers/cm3. Addition of boron, besides leading to a decrease in the resistivity, also resulted in a decrease in the time constant, limiting BDD conductivity. Investigations, by confocal Raman mapping, of the induced stress in the material due to interaction with the substrate or to the amount of doping are also presented and discussed. The induced tensile stress, which was distributed closer to the film-substrate interface decreased slightly with doping.

Size-dependent transitions in grafted polymer brushes.

The partition function of a grafted polymer brush was calculated as a sum over all possible configurations, each of them being an ensemble of n1, n2, ...n(i), ... loops having 2, 4, ..., 2i, ... segments. A system of equations for the most likely configuration has been obtained, and it was concluded that no solution exists for an infinite chain, with nonvanishing interactions between segments and surface. This implies that an infinite chain is either collapsed on the surface (for attractive interactions) or is forming a stretched brush (for repulsive interactions). However, for finite chains, a solution could be found. When the attractive segment-surface interaction becomes sufficiently strong, the brush collapses on the surface (the "loops" to "trains" transition). When the interaction is repulsive and sufficiently strong, the brush becomes stretched, with most segments belonging to open loops which do not return to the surface (the "loops" to "tails" transition). The critical values of the segment-surface interaction, for which the above transitions occur, depend on the length of the polymer chain as well as on other physical properties of the brush, such as the segment-segment and segment-solvent interactions and the grafting density.

Loops, tails and trains: A simple model for structural transformations of grafted adsorbing neutral polymer brushes.

The structure of grafted adsorbing polymers on surfaces is described as a statistical ensemble of loops generated by an one-dimensional random walk perpendicular to the surface. The configuration of each chain is considered as a succession of closed loops ended by an open loop (a tail). The probability of formation of each individual loop is the product between the probability of first return to the surface and a Boltzmann factor containing the free energy of the Flory-Huggins kind, which is approximated by the minimum free energy of all possible configurations of that loop. At high grafting densities, the attractive interactions between monomers and surface control the fraction of polymer belonging to either closed loops or tails, hence the formation of a stretched grafted brush. At low grafting densities, the increase of that interaction above a critical value generates an abrupt collapse of the brush on the surface. Whereas for long polymers (with more than about 100 Kuhn segments), the structure of the brush can be determined, in general, only via Monte-Carlo sampling, it is argued that the two structural transitions indicated above can be well predicted by simple approximations.

Robust megavoltage x-ray spectra estimation from transmission measurements.

Megavoltage X-ray sources are commonly used for therapy planning, and knowledge of their spectral distribution is important for accurate dose calculations. There are many methods that could provide reasonable estimations of Megavoltage X-ray spectra, when very accurate attenuation data or at least very good set of initial guesses of the spectra are available. We present here a novel method, which can be used for accurate Megavoltage spectral reconstruction without any prior knowledge of spectral distribution; the method performs well even when the available transmission data are affected by noise. The method is based on a search for a smooth function that minimizes the differences between measured and calculated attenuation data. The algorithm is compared with well-known existing algorithms, using computer simulated data, both error-free and containing added random Gaussian noise. The reconstructed spectra are subsequently used to calculate the transmission through 50 cm of bone, muscle or fat tissue. It is shown that the relative errors in dose calculations, using the spectra reconstructed via this method, are significantly smaller than those obtained via well-established reconstruction algorithms--Truncated Singular Value Decomposition (TSVD) and Expectation Maximization (EM). These results suggest that the novel algorithm might be practical for routine Megavoltage therapy X-ray source calibration.

On possible microscopic origins of the swelling of neutral lipid bilayers induced by simple salts.

It was recently suggested that the swelling of neutral multilipid bilayers upon addition of a salt can be simply explained only by the electrolyte screening of the van der Waals attractions, while assuming that the hydration force and the repulsion due to thermal undulations of membranes are unaffected by the salt. While we agree that the screening of the van der Waals interactions plays a role, we suggest that the increase in the hydration force upon addition of a salt has also to be taken into account. In a statistical model, which accounts for the membrane undulations, parameters could be found to explain the multibilayer swelling even when the van der Waals attraction is considered unaffected by the electrolyte screening. These results point out that the decrease by a factor of three of the Hamaker constant upon addition of a salt, suggested recently to be responsible for the swelling of neutral multilipid bilayers, is perhaps too large, and a smaller decrease in Hamaker constant, coupled with the above mentioned effects might explain the swelling.

Polarization model for poorly-organized interfacial water: hydration forces between silica surfaces.

The main goal of this paper is to review the theoretical models which can be used to describe the interactions between silica surfaces and to show that a model proposed earlier by the authors (the polarization model), which accounts concomitantly for double layer and hydration forces, can be adapted to explain recent experiments in this direction. When the water molecules near the interface were considered to have an ice-like structure, a strong coupling between the double layer and hydration forces (described by the correlation length between neighboring dipoles, lambda(m)) generates long range interactions, larger than the experimentally determined interactions between silica surfaces. Arguments are brought that a gel layer is likely to be formed on the surface of silica, which, by generating disorder in the interfacial water layers, can decrease strongly the value of lambda(m). Since the prediction of lambda(m) involves a choice for the microscopic structure of water, which is often unknown, the polarization model is also presented here as a phenomenological theory, in which lambda(m) is used as a fitting parameter. Two extreme cases are considered. In one of them, the water molecules near the interface are considered to have an ice-like structure, whereas in the other they are considered randomly distributed. In the first case, the dipole correlation length lambda(m)=14.9 Angstrom. In the second limiting case, lambda(m) can be of the order of 1 Angstrom. It is shown that, for lambda(m)=4 Angstrom, a more than qualitative agreement with the experiment could be obtained, for reasonable values of the parameters involved (e.g. surface dipole strength and density, dipole location, surface charge).

Ions at the air/water interface.

In a recent review of this topic [B.C. Garett, Science 303 (2004) 1146] the emphasis was on some recent experiments, in which it was found that some anions accumulate at the air/water interface and not in the bulk, as usually happens to the cations, and on some simulations which explained those positive surface adsorption excesses. Because a large number of these experiments could be explained on the basis of some simple physical models proposed by the authors for the interaction between the ions and the air/water interface [M. Manciu, E. Ruckenstein, Adv. Colloid Interface Sci. 105 (2003) 63; Adv. Colloid Interface Sci. 112 (2004) 109; Langmuir 21 (2005) 11312], those models are reviewed in the present note, the goal being to draw attention to them.

Polarization of water near dipolar surfaces: a simple model for anomalous dielectric behavior.

A model for the electrostatic interactions in water in the vicinity of a surface is suggested, which accounts, within the Poisson-Boltzmann mean field approach, for the screening of the charges and for the coupling interactions between neighboring dipoles. When the water molecules near a solid surface are assumed to be organized in icelike layers, the polarization is not a continuous function but exists only at the discrete positions of the water molecules. The particular positions of the water molecules in the icelike structure govern the manner in which the average water dipoles align with each other. On the basis of this model, one could explain the nonmonotonic behavior of the polarization and the electrical potential as well as the anomalous dielectric response of water (the nonproportionality of the polarization and the macroscopic electric field), which were obtained recently via molecular dynamics simulations.

On the interactions of ions with the air/water interface.

In the vicinity of a charged interface, the Poisson-Boltzmann approach considers that the ions obey Boltzmann distributions in a mean electrical field that satisfies the Poisson equation. However, the boundary between two dielectrics generates additional interactions between ions and the interface. The traditional models of ion hydration interactions, that assume that water is a homogeneous dielectric, predict that these interactions are repulsive for all kinds of ions, since all ions should prefer the medium with a larger dielectric constant, where they are better hydrated. In reality, the interactions between the ions and the neighboring water molecules can generate additional short-range ion-hydration interactions, which are either repulsive (for structure-making ions) or attractive (for structure-breaking ions). In the present paper, various models for the ion-hydration forces are examined and compared with the results of molecular dynamics simulations. At large ionic strengths, the latter results could be reproduced qualitatively only when short-ranged attractions between the structure-breaking ions and the interface were taken into account.

On the restabilization of protein-covered latex colloids at high ionic strengths.

Recent experiments on restabilization of protein-covered latex colloids at high ionic strengths reported by Lopez-Leon et al.(1) revealed strong specific anion effects. The same authors also emphasized that a recent polarization model, which involves both hydration and double layer forces, can account only for some of their experimental results but are in disagreement with other experimental results. The aim of the present paper is to show that most experimental results of ref 1 can be described, more than qualitatively, when the association equilibria for all the ions (with both the acidic and basic sites of the protein) are taken into account. As the traditional Poisson-Boltzmann approach, the polarization model neglects additional interactions between ions, and ions and surfaces, not included in the "mean field" electrical potential; therefore, a complete quantitative agreement should not be expected. While many of the discrepancies between calculations and experiment occur at low ionic strengths (10(-)(4)-10(-)(2) M), in the range of validity of the traditional DLVO theory, the latter can neither explain them. It is suggested that the structural modifications of the protein configuration induced by the electrolyte are responsible for some of the disagreements between experiment and calculations.