Development of reverse phase protein microarrays for clinical applications and patient-tailored therapy

While genomics provide important information about the somatic genetic changes, and RNA transcript profiling can reveal important expression changes that correlate with outcome and response to therapy, it is the proteins that do the work in the cell. At a functional level, derangements within the proteome, driven by post-translational and epigenetic modifications, such as phosphorylation, is the cause of a vast majority of human diseases. Cancer, for instance, is a manifestation of deranged cellular protein molecular networks and cell signaling pathways that are based on genetic changes at the DNA level. Importantly, the protein pathways contain the drug targets in signaling networks that govern overall cellular survival, proliferation, invasion and cell death. Consequently, the promise of proteomics resides in the ability to extend analysis beyond correlation to causality. A critical gap in the information knowledge base of molecular profiling is an understanding of the ongoing activity of protein signaling in human tissue: what is activated and “in use” within the human body at any given point in time. To address this gap, we have invented a new technology, called reverse phase protein microarrays, that can generate a functional read-out of cell signaling networks or pathways for an individual patient obtained directly from a biopsy specimen. This “wiring diagram” can serve as the basis for both, selection of a therapy and patient stratification.

Use of reverse phase protein microarrays and reference standard development for molecular network analysis of metastatic ovarian carcinoma

Cancer can be defined as a deregulation or hyperactivity in the ongoing network of intracellular and extracellular signaling events. Reverse phase protein microarray technology may offer a new opportunity to measure and profile these signaling pathways, providing data on post-translational phosphorylation events not obtainable by gene microarray analysis. Treatment of ovarian epithelial carcinoma almost always takes place in a metastatic setting since unfortunately the disease is often not detected until later stages. Thus, in addition to elucidation of the molecular network within a tumor specimen, critical questions are to what extent do signaling changes occur upon metastasis and are there common pathway elements that arise in the metastatic microenvironment. For individualized combinatorial therapy, ideal therapeutic selection based on proteomic mapping of phosphorylation end points may require evaluation of the patient's metastatic tissue. Extending these findings to the bedside will require the development of optimized protocols and reference standards. We have developed a reference standard based on a mixture of phosphorylated peptides to begin to address this challenge.

Pt(II) bipyridyl complexes bearing substituted fluorenyl motif on the bipyridyl and acetylide ligands : synthesis, photophysics, and reverse saturable absorption

A series of Pt(II) diimine complexes bearing benzothiazolylfluorenyl (BTZ-F8), diphenylaminofluorenyl (NPh2- F8), or naphthalimidylfluorenyl (NI-F8) motifs on the bipyridyl or acetylide ligands (Pt-4−Pt-8), (i.e., {4,4′-bis[7-R1-F8-(≡)n-]bpy}Pt(7- R2-F8- ≡ -)2, where F8 = 9,9′-di(2-ethylhexyl)fluorene, bpy = 2,2′- bipyridine, Pt-4: R1 = R2 = BTZ, n = 0; Pt-5: R1 = BTZ, R2 = NI, n = 0; Pt-6: R1 = R2 = BTZ, n = 1; Pt-7: R1 = BTZ, R2 = NPh2, n = 1; Pt- 8: R1 = NPh2, R2 = BTZ, n = 1) were synthesized. Their ground-state and excited-state properties and reverse saturable absorption performances were systematically investigated. The influence of these motifs on the photophysics of the complexes was investigated by spectroscopic methods and simulated by time-dependent density functional theory (TDDFT). The intense absorption bands below 410 nm for these complexes is assigned to predominantly 1π,π* transitions localized on either the bipyridine or the acetylide ligands; while the broad low-energy absorption bands between 420 and 575 nm are attributed to essentially 1MLCT (metal-to-ligand charge transfer)/ 1LLCT (ligand-to-ligand charge transfer) transitions, likely mixed with some 1ILCT (intraligand charge transfer) transition for Pt-4−Pt-7, and predominantly 1ILCT transition admixing with minor 1MLCT/1LLCT characters for Pt-8. The different substituents on the acetylide and bipyridyl ligands, and the degrees of π-conjugation in the bipyridyl ligand influence both the 1π,π* and charge transfer transitions pronouncedly. All complexes are emissive at room temperature. Upon excitation at their respective absorption band maxima, Pt-4, Pt-6, and Pt-8 exhibit acetylide ligand localized 1π,π* fluorescence and 3MLCT/3LLCT phosphorescence in CH2Cl2, while Pt-5 manifests 1ILCT fluorescence and 3ILCT phosphorescence. However, only 1LLCT fluorescence was observed for Pt-7 at room temperature. The nanosecond transient absorption study was carried out for Pt-4−Pt-8 in CH3CN. Except for Pt-7 that contains NPh2 at the acetylide ligands, Pt-4−Pt-6 and Pt-8 all exhibit weak to moderate excited-state absorption in the visible spectral region. Reverse saturable absorption (RSA) of these complexes was demonstrated at 532 nm using 4.1 ns laser pulses in a 2 mm cuvette. The strength of RSA follows this trend: Pt-4 > Pt-5 > Pt-7 > Pt-6 > Pt-8. Incorporation of electron-donating substituent NPh2 on the bipyridyl ligand significantly decreases the RSA, while shorter π-conjugation in the bipyridyl ligand increases the RSA. Therefore, the substituent at either the acetylide ligands or the bipyridyl ligand could affect the singlet and triplet excited-state characteristics significantly, which strongly influences the RSA efficiency.

Research on injury compensation and health outcomes : ignoring the problem of reverse causality led to a biased conclusion

Objective This study highlights the serious consequences of ignoring reverse causality bias in studies on compensation-related factors and health outcomes and demonstrates a technique for resolving this problem of observational data. Study Design and Setting Data from an English longitudinal study on factors, including claims for compensation, associated with recovery from neck pain (whiplash) after rear-end collisions are used to demonstrate the potential for reverse causality bias. Although it is commonly believed that claiming compensation leads to worse recovery, it is also possible that poor recovery may lead to compensation claims—a point that is seldom considered and never addressed empirically. This pedagogical study compares the association between compensation claiming and recovery when reverse causality bias is ignored and when it is addressed, controlling for the same observable factors. Results When reverse causality is ignored, claimants appear to have a worse recovery than nonclaimants; however, when reverse causality bias is addressed, claiming compensation appears to have a beneficial effect on recovery, ceteris paribus. Conclusion To avert biased policy and judicial decisions that might inadvertently disadvantage people with compensable injuries, there is an urgent need for researchers to address reverse causality bias in studies on compensation-related factors and health.

Identifying and forecasting the reverse salient in video game consoles: A performance gap ratio comparative analysis

This study uses the reverse salient methodology to contrast subsystems in video game consoles in order to discover, characterize, and forecast the most significant technology gap. We build on the current methodologies (Performance Gap and Time Gap) for measuring the magnitude of Reverse Salience, by showing the effectiveness of Performance Gap Ratio (PGR). The three subject subsystems in this analysis are the CPU Score, GPU core frequency, and video memory bandwidth. CPU Score is a metric developed for this project, which is the product of the core frequency, number of parallel cores, and instruction size. We measure the Performance Gap of each subsystem against concurrently available PC hardware on the market. Using PGR, we normalize the evolution of these technologies for comparative analysis. The results indicate that while CPU performance has historically been the Reverse Salient, video memory bandwidth has taken over as the quickest growing technology gap in the current generation. Finally, we create a technology forecasting model that shows how much the video RAM bandwidth gap will grow through 2019 should the current trend continue. This analysis can assist console developers in assigning resources to the next generation of platforms, which will ultimately result in longer hardware life cycles.

Determining reverse salient types and evolutionary dynamics of technology systems with performance disparities

Technological system evolution is marked by the uneven evolution of constituent sub-systems. Subsequently, system evolution is hampered by the resulting state of unevenness, or reverse salience, which results from the presence of the sub-system that delivers the lowest level of performance with respect to other sub-systems, namely, the reverse salient. In this paper, we develop absolute and proportional performance gap measures of reverse salience and, in turn, derive a typology of reverse salients that distinguishes alternative dynamics of change in the evolving system. We subsequently demonstrate the applicability of the measures and the typology through an illustrative empirical study of the PC (personal computer) technological system that functions as a gaming platform. Our empirical analysis demonstrates that patterns of temporal dynamics can be distinguished with the measurement of reverse salience, and that distinct paths of technological system evolution can be identified as different types of reverse salients emerge over time.

Bibliometric study of the reverse salient concept

In the development of technological systems the focus of system analysis is often on the sub-system that delivers insufficient performance – i.e. the reverse salient – which subsequently limits the performance of the system in its entirety. The reverse salient is therefore a useful concept in the study of technological systems and while the literature holds numerous accounts of its use, it is not known how often, in which streams of literature, and in what type of application the concept has been utilized by scholars since its introduction by Thomas Hughes in 1983. In this paper we employ bibliometric citation analysis between 1983 and 2008, inclusively, to study the impact of the reverse salient concept in the literature at large as well as study the dissemination of the concept into different fields of research. The study results show continuously growing number of concept citations in the literature over time as well as increasing concept diffusion into different research areas. The analysis of article contents additionally suggests the opportunity for scholars to engage in deeper conceptual application. Finally, the continuing increase in the number of citations highlights the importance of the reverse salient concept to scholars and practitioners.

Dynamics of reverse salience as technological performance gap: An empirical study of the personal computer technology system

The evolution of technological systems is hindered by systemic components, referred to as reverse salients, which fail to deliver the necessary level of technological performance thereby inhibiting the performance delivery of the system as a whole. This paper develops a performance gap measure of reverse salience and applies this measurement in the study of the PC (personal computer) technological system, focusing on the evolutions of firstly the CPU (central processing unit) and PC game sub-systems, and secondly the GPU (graphics processing unit) and PC game sub-systems. The measurement of the temporal behavior of reverse salience indicates that the PC game sub-system is the reverse salient, continuously trailing behind the technological performance of the CPU and GPU sub-systems from 1996 through 2006. The technological performance of the PC game sub-system as a reverse salient trails that of the CPU sub-system by up to 2300 MHz with a gradually decreasing performance disparity in recent years. In contrast, the dynamics of the PC game sub-system as a reverse salient trails the GPU sub-system with an ever increasing performance gap throughout the timeframe of analysis. In addition, we further discuss the research and managerial implications of our findings.

Solution chemistry impacts on the seawater neutralisation process: Benefits of nanofiltered seawater and reverse osmosis brine

It is well known that the neutralisation of Bayer liquor with seawater causes the precipitation of stable alkaline products and a reduction in pH and dissolved metal concentrations in the effluent. However, there is limited information available on solution chemistry effects on the stability and reaction kinetics of these precipitates. This investigation shows the influence of reactive species (magnesium and calcium) in seawater on precipitate stabilities and volumetric efficiencies during the neutralisation of bauxite refinery residues. Correlations between synthetic seawater solutions and real samples of seawater (filtered seawater, nanofiltered seawater and reverse osmosis brine) have been made. These investigations have been used to confirm that alternative seawater sources can be used to increase the productivity potential of the neutralisation process with minimal implications on the composition and stability of precipitates formed. The volume efficiency of the neutralisation process using synthetic analogues has been shown to be almost directly proportional with the concentration of magnesium. This was further confirmed in the nanofiltered seawater and reverse osmosis brine that showed increases in the efficiency of neutralisation by factors of 3 and 2 compared to seawater, which corresponds with relatively the same increase in the concentration of magnesium in these alternative seawater sources. An assessment of the chemical stability of the precipitates, volumetric efficiency, and discharge water quality have been determined using numerous techniques that include pH, conductivity, inductively coupled plasma optical emission spectroscopy, infrared spectroscopy, thermogravimetric analysis coupled to mass spectrometry and X-ray diffraction. Correlations between synthetic solution compositions and alternative seawater sources have been used to determine if alternative seawater sources are potential substitutes for seawater based on improvements in productivity, implementation costs, savings to operations and environmental benefits.

5-year Chlamydia vaccination programme could reverse disease-related koala population decline: Predictions from a mathematical model using field data

BACKGROUND Many koala populations around Australia are in serious decline, with a substantial component of this decline in some Southeast Queensland populations attributed to the impact of Chlamydia. A Chlamydia vaccine for koalas is in development and has shown promise in early trials. This study contributes to implementation preparedness by simulating vaccination strategies designed to reverse population decline and by identifying which age and sex category it would be most effective to target. METHODS We used field data to inform the development and parameterisation of an individual-based stochastic simulation model of a koala population endemic with Chlamydia. The model took into account transmission, morbidity and mortality caused by Chlamydia infections. We calibrated the model to characteristics of typical Southeast Queensland koala populations. As there is uncertainty about the effectiveness of the vaccine in real-world settings, a variety of potential vaccine efficacies, half-lives and dosing schedules were simulated. RESULTS Assuming other threats remain constant, it is expected that current population declines could be reversed in around 5-6 years if female koalas aged 1-2 years are targeted, average vaccine protective efficacy is 75%, and vaccine coverage is around 10% per year. At lower vaccine efficacies the immunological effects of boosting become important: at 45% vaccine efficacy population decline is predicted to reverse in 6 years under optimistic boosting assumptions but in 9 years under pessimistic boosting assumptions. Terminating a successful vaccination programme at 5 years would lead to a rise in Chlamydia prevalence towards pre-vaccination levels. CONCLUSION For a range of vaccine efficacy levels it is projected that population decline due to endemic Chlamydia can be reversed under realistic dosing schedules, potentially in just 5 years. However, a vaccination programme might need to continue indefinitely in order to maintain Chlamydia prevalence at a sufficiently low level for population growth to continue.

In vivo evaluation of folate decorated cross-linked micelles for the delivery of platinum anticancer drugs

The biodistribution of micelles with and without folic acid targeting ligands were studied using a block copolymer consisting of acrylic acid (AA) and polyethylene glycol methyl ether acrylate (PEGMEA) blocks. The polymers were prepared using RAFT polymerization in the presence of a folic acid functionalized RAFT agent. Oxoplatin was conjugated onto the acrylic acid block to form amphiphilic polymers which, when diluted in water, formed stable micelles. In order to probe the in vivo stability, a selection of micelles were cross-linked using 1,8-diamino octane. The sizes of the micelles used in this study range between 75 and 200 nm, with both spherical and worm-like conformation. The effects of cross-linking, folate conjugation and different conformation on the biodistribution were studied in female nude mice (BALB/c) following intravenous injection into the tail vein. Using optical imaging to monitor the fluorophore-labeled polymer, the in vivo biodistribution of the micelles was monitored over a 48 h time-course after which the organs were removed and evaluated ex vivo. These experiments showed that both cross-linking and conjugation with folic acid led to increased fluorescence intensities in the organs, especially in the liver and kidneys, while micelles that are not conjugated with folate and not cross-linked are cleared rapidly from the body. Higher accumulation in the spleen, liver, and kidneys was also observed for micelles with worm-like shapes compared to the spherical micelles. While the various factors of cross-linking, micelle shape, and conjugation with folic acid all contribute separately to prolong the circulation time of the micelle, optimization of these parameters for drug delivery devices could potentially overcome adverse effects such as liver and kidney toxicity.

Transition from disc to rod-like shape of 16-3-16 dimeric micelles in aqueous solutions

Small-angle neutron scattering (SANS) measurements from bis-cationic C16H33N+(CH3)(2)-(CH2)(3)-N+ (CH3)(2)C16H33 2Br(-) dimeric surfactant, referred to as 16-3-16, at different concentrations and temperatures, are reported. It is seen that micelles are disc-like for concentrations C = 2.5 and 10 mM at temperature T = 30 degrees C. At low concentration C = 0.5 mM micelles are rod-like. Similarly, there is a disc to rod-like transition of micelles on increasing the temperature. For C = 2.5 mM, micelles are rod-like at T = 45 and 70 degrees C.

A Jacobian-Based Algorithm for Planning Attitude Maneuvers Using Forward and Reverse Rotations

Algorithms for planning quasistatic attitude maneuvers based on the Jacobian of the forward kinematic mapping of fully-reversed (FR) sequences of rotations are proposed in this paper. An FR sequence of rotations is a series of finite rotations that consists of initial rotations about the axes of a body-fixed coordinate frame and subsequent rotations that undo these initial rotations. Unlike the Jacobian of conventional systems such as a robot manipulator, the Jacobian of the system manipulated through FR rotations is a null matrix at the identity, which leads to a total breakdown of the traditional Jacobian formulation. Therefore, the Jacobian algorithm is reformulated and implemented so as to synthesize an FR sequence for a desired rotational displacement. The Jacobian-based algorithm presented in this paper identifies particular six-rotation FR sequences that synthesize desired orientations. We developed the single-step and the multiple-step Jacobian methods to accomplish a given task using six-rotation FR sequences. The single-step Jacobian method identifies a specific FR sequence for a given desired orientation and the multiple-step Jacobian algorithm synthesizes physically feasible FR rotations on an optimal path. A comparison with existing algorithms verifies the fast convergence ability of the Jacobian-based algorithm. Unlike closed-form solutions to the inverse kinematics problem, the Jacobian-based algorithm determines the most efficient FR sequence that yields a desired rotational displacement through a simple and inexpensive numerical calculation. The procedure presented here is useful for those motion planning problems wherein the Jacobian is singular or null.

Inhibition of avian myeloblastosis virus reverse transcriptase and virus inactivation by metal complexes of isonicotinic acid hydrazide

The cupric and ferric complexes of isonicotinic acid hydrazide (INH) inhibit the DNA synthesis catalysed by avian myeloblastosis virus (AMV) reverse transcriptase. The inhibition was to the extent of 95% by 50 μM of cupric-INH complex and 55% by 100 μM of ferric-INH complex. These complexes have been found to bind preferentially to the enzyme than to the template-primer. Kinetic analysis showed that the cupric-INH complex is a non-competitive inhibitor with respect to dTTP. The time course of inhibition has revealed that the complexes are inhibitory even after the initiation of polynucleotide synthesis. In vivo toxicity studies in 1-day-old chicks have shown that the complexes are not toxic up to a concentration of 500 μg per chick. Infection of the 1-day-old chicks with AMV pretreated with 150 μg of either of the complexes prevented symptoms of leukemia due to virus inactivation.

A H-1-NMR STUDY OF BILIRUBIN IX-ALPHA SOLUBILIZATION BY CHOLATE MICELLES - APPLICATION OF NUCLEAR OVERHAUSER EFFECTS

The solubilization of bilirubin IX-Alpha in aqueous solution by sodium cholate micelles has been examined by 270 MHz 1H-NMR spectroscopy. Incorporation of bilirubin into the micelles is accompanied by specific shifts of bilirubin vinyl and bridgehead protons and the C18 and C19 methyl groups of the steroid. The observed chemical shifts show a monotonic concentration dependence suggesting that changes in aggregation size are continuous. Nuclear Overhauser effects (NOE) have been shown to be a useful probe or micellization. A 4:1 cholate/bilirubin mixture has been investigated by difference NOE spectroscopy. The observation of intermolecular nuclear Overhauser effects between peripheral protons of bilirubin and cholate are diagnostic of spatially proximate groups. Inter-cholate nuclear Overhauser effects increase in magnitude upon bilirubin incorporation suggesting closer packing of steroid molecules on solubilization of the pigment. Intramolecular nuclear Overhauser effects observed for solubilized bilirubin are consistent with a compact intramolecularly hydrogen-bonded conformation resembling that determined for bilirubin in the solid state.