Alkylbenzoic and Alkyloxybenzoic Acid Blending for Expanding the Liquid Crystalline State and Improving Its Rheology.

Thermotropic mesogens typically exist as liquid crystals (LCs) in a narrow region of high temperatures, making lowering their melting point with the temperature expansion of the mesophase state an urgent task. Para-substituted benzoic acids can form LCs through noncovalent dimerization into homodimers via hydrogen bonds, whose strength and, consequently, the temperature region of the mesophase state can be potentially altered by creating asymmetric heterodimers from different acids. This work investigates equimolar blends of p-n-alkylbenzoic (kBA, where k is the number of carbon atoms in the alkyl radical) and p-n-alkyloxybenzoic (kOBA) acids by calorimetry and viscometry to establish their phase transitions and regions of mesophase existence. Non-symmetric dimerization of acids leads to the extension of the nematic state region towards low temperatures and the appearance of new monotropic and enantiotropic phase transitions in several cases. Moreover, the crystal-nematic and nematic-isotropic phase changes have a two-step character for some acid blends, suggesting the formation of symmetric and asymmetric associates from heterodimers. The mixing of 6BA and 8OBA most strongly extends the region of the nematic state towards low temperatures (from 95-114 °C and 108-147 °C for initial homodimers, respectively, to 57-133 °C for the resulting heterodimer), whereas the combination of 4OBA and 5OBA gives the most extended high-temperature nematic phase (up to 156 °C) and that of 6BA and 9OBA (or 12OBA) provides the existence of a smectic phase at the lowest temperatures (down to 51 °C).

Asphaltenes from Heavy Crude Oil as Ultraviolet Stabilizers against Polypropylene Aging.

The destruction of polymers under the influence of ultraviolet (UV) radiation is the cause of their aging and deterioration of strength properties. Asphaltenes are low-value waste products after the refining and deasphalting of heavy crude oil, which absorb UV radiation well. Asphaltenes require rational utilization, which suggests their use as UV stabilizing agents for polymers. In this work, asphaltenes were used to prevent UV aging of polypropylene (PP) by adding them in a mass fraction from 5% to 30% within an asphaltene/PP composite material. Rheometry, calorimetry, X-ray diffraction analysis, and tensile strength of PP films containing asphaltenes were performed before and after their intense UV irradiation for accelerated aging. Asphaltenes slightly reduce the viscosity, crystallinity, and mechanical strength of the initial PP due to their plasticizing effect. However, this deterioration in properties is more than compensated when studying UV-aged samples. Intense UV aging causes multiple catastrophic drops in the viscosity and strength of pure PP with the preservation of crystallinity due to the break of polymer chains and a decrease in molecular weight by approximately eight times. Asphaltenes suppress the destruction of PP, which is expressed in a significantly smaller decline in its viscosity and strength due to UV aging. The most optimal content of asphaltenes is 20%, which suppresses UV destruction by six times and best preserves the strength properties of PP.

Self-Lubricating and Shape-Stable Phase-Change Materials Based on Epoxy Resin and Vegetable Oils.

Palm or coconut oil is capable of dissolving in a mixture of bisphenol A-based epoxy resin and a high-temperature hardener (4,4'-diaminodiphenyl sulfone) when heated and then forms a dispersed phase as a result of cross-linking and molecular weight growth of the epoxy medium. Achieving the temporary miscibility between the curing epoxy matrix and the vegetable oil allows a uniform distribution of vegetable oil droplets in the epoxy medium. This novel approach to creating a dispersed phase-change material made a cured epoxy polymer containing up to 20% oil. The miscibility of epoxy resin and oil was studied by laser interferometry, and phase state diagrams of binary mixtures were calculated according to theory and experiments. A weak effect of oil on the viscosity and kinetics of the epoxy resin curing was demonstrated by rotational rheometry. According to differential scanning calorimetry and dynamic mechanical analysis, the oil plasticizes the epoxy matrix slightly, expanding its glass transition region towards low temperatures and reducing its elastic modulus. In the cured epoxy matrix, oil droplets have a diameter of 3-14 µm and are incapable of complete crystallization due to their multi-component chemical composition and non-disappeared limited miscibility. The obtained phase-change materials have relatively low specific energy capacity but can be used alternatively as self-lubricating low-noise materials due to dispersed oil, high stiffness, and reduced friction coefficient. Palm oil crystallizes more readily, better matching the creation of phase-change materials, whereas coconut oil crystallization is more suppressed, making it better for reducing the friction coefficient of the oil-containing material.

Epoxy Phase-Change Materials Based on Paraffin Wax Stabilized by Asphaltenes.

The usual problem of meltable phase-change agents is the instability in their form upon heating, which can be solved by placing them into a continuous polymer matrix. Epoxy resin is a suitable medium for dispersing molten agents, but it is necessary to make the obtained droplets stable during the curing of the formed phase-change material. This work shows that molten paraffin wax forms a Pickering emulsion in an epoxy medium and in the presence of asphaltenes extracted from heavy crude oil. Theoretical calculations revealed the complex equilibrium in the epoxy/wax/asphaltene triple system due to their low mutual solubility. Rheological studies showed the viscoplastic behavior of the obtained dispersions at 25 °C, which disappears upon the heating and melting of the paraffin phase. Wax and asphaltenes increased the viscosity of the epoxy medium during its curing but did not inhibit cross-linking or reduce the glass transition temperature of the cured polymer. As a result of curing, it is possible to obtain phase-change materials containing up to 45% paraffin wax that forms a dispersed phase with a size of 0.2-6.5 µm. The small size of dispersed wax can decrease its degree of crystallinity to 13-29% of its original value, reducing the efficiency of the phase-change material.

Nanocellulose-stabilized bitumen emulsions as a base for preparation of nanocomposite asphalt binders.

Pickering bitumen emulsions stabilized by 1 % aqueous dispersion of microfibrillated cellulose (MFC) were used to obtain micro- and nanocomposite asphalt binders. Initial bitumen emulsions are characterized by a yield stress for a bitumen content of up to 40 %, while higher bitumen amounts result in phase inversion with the formation of highly viscous inverse emulsions. Drying of emulsions leads to the production of nanocomposite bitumen binders containing from 0.6 % to 8.3 % of MFC. In this case, the MFC content of 1.5 % or more formed a microfibrillar network in the bitumen, which gives it gel-like properties and the yield stress, increasing its cohesive strength and resistance to rutting. The effect of addition of 5 % sodium dodecyl sulfate (SDS) on the properties of the bitumen emulsions and the resulting binders is considered. SDS increases the emulsifying ability of MFC, making it possible to obtain 70 % direct bitumen emulsions and reducing their effective viscosity together with the yield stress. However, the presence of SDS in the dried binders increases the aggregation of MFC, reducing the stiffness of the resulting microcomposite binders, their yield stress, and rutting resistance.

The Effect of Non-Solvent Nature on the Rheological Properties of Cellulose Solution in Diluted Ionic Liquid and Performance of Nanofiltration Membranes.

The weak point of ionic liquids is their high viscosity, limiting the maximum polymer concentration in the forming solutions. A low-viscous co-solvent can reduce viscosity, but cellulose has none. This study demonstrates that dimethyl sulfoxide (DMSO), being non-solvent for cellulose, can act as a nominal co-solvent to improve its processing into a nanofiltration membrane by phase inversion. A study of the rheology of cellulose solutions in diluted ionic liquids ([EMIM]Ac, [EMIM]Cl, and [BMIM]Ac) containing up to 75% DMSO showed the possibility of decreasing the viscosity by up to 50 times while keeping the same cellulose concentration. Surprisingly, typical cellulose non-solvents (water, methanol, ethanol, and isopropanol) behave similarly, reducing the viscosity at low doses but causing structuring of the cellulose solution and its phase separation at high concentrations. According to laser interferometry, the nature of these non-solvents affects the mass transfer direction relative to the forming membrane and the substance interdiffusion rate, which increases by four-fold when passing from isopropanol to methanol or water. Examination of the nanofiltration characteristics of the obtained membranes showed that the dilution of ionic liquid enhances the rejection without changing the permeability, while the transition to alcohols increases the permeability while maintaining the rejection.

Infrared Spectral Classification of Natural Bitumens for Their Rheological and Thermophysical Characterization.

Natural bitumens consist of many molecules whose chemical composition depends on the oilfield and determines the physicochemical properties of the bitumens as materials. Infrared (IR) spectroscopy is the fastest and least expensive method to assess the chemical structure of organic molecules, which makes it attractive in terms of rapid prediction of the properties of natural bitumens based on their composition evaluated in this way. In this work, IR spectra were measured for ten samples of natural bitumens significantly different in properties and origin. Based on the ratios of certain IR absorption bands, bitumens are proposed to be divided into paraffinic, aromatic, and resinous. In addition, the internal relationship between IR spectral characteristics of bitumens, such as polarity, paraffinicity, branchiness, and aromaticity, is shown. A study of phase transitions in bitumens by differential scanning calorimetry was carried out, and the use of a heat flow differential to find hidden points of bitumens' glass transitions is proposed. Furthermore, the dependences of the total melting enthalpy of crystallizable paraffinic compounds on the aromaticity and branchiness of bitumens are demonstrated. A detailed study of bitumens' rheology in a wide temperature range was carried out, and characteristic features of rheological behavior for different bitumen classes are revealed. Based on the viscous properties of bitumens, their glass transition points were found and compared with the calorimetric glass transition temperatures and nominal solid-liquid transition points obtained from temperature dependences of bitumens' storage and loss moduli. The dependences of viscosity, flow activation energy, and glass transition temperature of bitumens on their IR spectral characteristics are shown, which can be used to predict the rheological properties of bitumens.

Asphaltene-Stabilized Polyisobutylene Pressure-Sensitive Adhesives for Ultraviolet Protection and Surface Bonding.

The usual way to protect indoor areas from solar UV radiation is to use UV-absorbing materials, which are applied as a thin film on the surface of the windowpane. Asphaltenes are useless wastes from crude oil refining that absorb UV radiation well, which gave the idea of their use in protective coatings. Pressure-sensitive adhesives based on polyisobutylene containing from 5 to 30 wt% of asphaltenes were obtained. Deterioration of the adhesive properties with the introduction of 5-20 wt% of asphaltenes was shown by adhesion tests, which can be associated with the plasticization of the polymer matrix. At the same time, the use of 30 wt% of asphaltenes leads to the polymer matrix reinforcement with the restoration of adhesive properties to the original level or even slightly higher. The rheological study of adhesives at 25 °C and 120 °C showed the structural network formation by asphaltenes at a content of 30 wt%, explaining the increase in adhesion performance. According to microscopy, asphaltenes are flat brown glass shards in a polymer matrix. They absorb electromagnetic radiation, predominantly in the UV range, while maintaining relative translucency in the visible range. This makes it possible to obtain thin films from the asphaltene-filled adhesive for bonding glass sheets to produce UV-blocked and tinted windowpanes.

Hot-Melt and Pressure-Sensitive Adhesives Based on Styrene-Isoprene-Styrene Triblock Copolymer, Asphaltene/Resin Blend and Naphthenic Oil.

Asphaltene/resin blend (ARB) extracted from heavy crude oil was used to modify poly(styrene-block-isoprene-block-styrene) (SIS) to make it an adhesive. There were prepared double and triple mixtures containing 10-60% SIS, 10-40% ARB, and 10-50% naphthenic oil used as an additional plasticizer. The viscoelasticity of the mixtures at 25 °C and 120 °C was studied, their flow curves were obtained, and the temperature dependences of the loss tangent and the components of the complex modulus were measured. In addition, the mixtures were used as hot-melt adhesives (HMAs) and pressure-sensitive adhesives (PSAs) in the shear, peel, and pull-off tests of the adhesive bonds that they formed with steel. Both naphthenic oil and ARB act as plasticizers for SIS and make it sticky. However, only the combined use of ARB and the oil allows for achieving the best set of adhesive properties of the SIS-based mixture. High-quality HMA requires low oil content (optimal SIS/ARB/oil ratio is 50/40/10, pull-off adhesion strength (τt) of 1990 kPa), whereas a lot of the oil is needed to give SIS characteristics of a PSA (SIS/ARB/oil is 20/40/40, τt of 100 kPa). At the same time, the resulting PSA can be used as a hot-melt pressure-sensitive adhesive (HMPSA) that has many times lower viscosity than HMA (13.9 Pa·s versus 2640 Pa·s at 120 °C and 1 s-1) but provides a less strong adhesive bond (τt of 960 kPa).

The Use of Branching Agents in the Synthesis of PBAT.

Biodegradable polyesters represent an advanced alternative to polyolefin plastics in various applications. Polybutylene adipate terephthalate (PBAT) can compete with polyolefins in terms of their mechanical characteristics and melt processing conditions. The properties of PBAT depend on the molecular weight, dispersity, and architecture of the copolymer. Long-chain branching (LCB) of the PBAT backbone is an efficient method for the improvement of the copolymer characteristics. In the present work, we studied branching agents (BAs) 1-7 of different structures in the two-stage polycondensation of 1,4-butanediol, dimethyl terephthalate, and adipic acid and investigated the composition and melt rheology of the copolymers. According to the results of the research, 1,1,1-tris(hydroxymethyl)ethane 2 and 3-hydroxy-2-(hydroxymethyl)-2-methylpropanoic acid 5 outperformed glycerol 1 as BAs in terms of shear thinning behavior and viscoelasticity.

Rheological and tribological properties of low-temperature greases based on cellulose acetate butyrate gel.

A new approach to produce biodegradable low-temperature greases, based on cellulose acetate butyrate (CAB) that dissolves in the medium of acetyl tributyl citrate (ATBC) at high temperatures and produces a gel during cooling because of phase separation, is proposed. Rheological properties of CAB solutions and gels in a wide temperature range from -80 °C to 160 °C were investigated with characterization of their viscoelasticity and viscoplasticity that arise because of the sol-gel transition of CAB/ATBC systems at 55 °C. CAB gelation reduces the wear coefficient tenfold when using ATBC as a lubricant but leads to a noticeable increase in the friction coefficient. To improve tribological properties of gel greases, additives of various solid particles were used: hexagonal boron nitride, graphite, and polytetrafluoroethylene (PTFE). The introduction of 10% to 30% additives in a gel grease containing 10% CAB has shown the preference of PTFE at a concentration of 10% for improving grease tribological characteristics.

A Recursive Model of the Spread of COVID-19: Modelling Study.

BACKGROUND: The major medical and social challenge of the 21st century is COVID-19, caused by the novel coronavirus SARS-CoV-2. Critical issues include the rate at which the coronavirus spreads and the effect of quarantine measures and population vaccination on this rate. Knowledge of the laws of the spread of COVID-19 will enable assessment of the effectiveness and reasonableness of the quarantine measures used, as well as determination of the necessary level of vaccination needed to overcome this crisis. OBJECTIVE: This study aims to establish the laws of the spread of COVID-19 and to use them to develop a mathematical model to predict changes in the number of active cases over time, possible human losses, and the rate of recovery of patients, to make informed decisions about the number of necessary beds in hospitals, the introduction and type of quarantine measures, and the required threshold of vaccination of the population. METHODS: This study analyzed the onset of COVID-19 spread in countries such as China, Italy, Spain, the United States, the United Kingdom, Japan, France, and Germany based on publicly available statistical data. The change in the number of COVID-19 cases, deaths, and recovered persons over time was examined, considering the possible introduction of quarantine measures and isolation of infected people in these countries. Based on the data, the virus transmissibility and the average duration of the disease at different stages were evaluated, and a model based on the principle of recursion was developed. Its key features are the separation of active (nonisolated) infected persons into a distinct category and the prediction of their number based on the average duration of the disease in the inactive phase and the concentration of these persons in the population in the preceding days. RESULTS: Specific values for SARS-CoV-2 transmissibility and COVID-19 duration were estimated for different countries. In China, the viral transmissibility was 3.12 before quarantine measures were implemented and 0.36 after these measures were lifted. For the other countries, the viral transmissibility was 2.28-2.76 initially, and it then decreased to 0.87-1.29 as a result of quarantine measures. Therefore, it can be expected that the spread of SARS-CoV-2 will be suppressed if 56%-64% of the total population becomes vaccinated or survives COVID-19. CONCLUSIONS: The quarantine measures adopted in most countries are too weak compared to those previously used in China. Therefore, it is not expected that the spread of COVID-19 will stop and the disease will cease to exist naturally or owing to quarantine measures. Active vaccination of the population is needed to prevent the spread of COVID-19. Furthermore, the required specific percentage of vaccinated individuals depends on the magnitude of viral transmissibility, which can be evaluated using the proposed model and statistical data for the country of interest.

Formation of Microfiltration Membranes from PMP/PIB Blends: Effect of PIB Molecular Weight on Membrane Properties.

A series of microfiltration membranes were fabricated by the extraction of polyisobutylene (PIB) from its immiscible blends with polymethylpentene (PMP). Three PIB with different molecular weight of 7.5 × 104 (Oppanol B15), 34 × 104 (Oppanol B50) and 110 × 104 (Oppanol B100) g/mol, respectively, were used to evaluate the effect of molecular weight on the porous structure and transport properties of resulting PMP-based membranes. To mimic the conditions of 3D printing, the flat-sheet membranes were fabricated by means of melting of mixtures of various PMP and PIB concentrations through the hot rolls at 240 ∘ C followed by a quick cooling. The rheology study of individual components and blends at 240 ∘ C revealed that PIB B50 possessed the most close flow curve to the pure PMP, and their blends demonstrated the lowest viscosity comparing to the compositions made of PIB with other molecular weights (B15 or B100). SEM images of the cross-section PMP membranes after PIB extraction (PMP/PIB = 55/45) showed that the use of PIB B50 allowed obtaining the sponge-like porous structure, whereas the slit-shaped pores were found in the case of PIB B15 and PIB B100. Additionally, PMP/B50 blends demonstrated the optimum combinations of mechanical properties (str = 9.1 MPa, E = 0.20 GPa), adhesion to steel (adh = 0.8 kPa) and retention performance (R240 nm = 99%, R38 nm = 39%). The resulting membranes were non- or low-permeable for water if the concentration of PIB B50 in the initial blends was 40 wt.% or lower. The optimal filtration performance was observed in the case of PMP/B50 blends with a ratio of 55/45 (Pwater = 1.9 kg/m2hbar, R240 nm = 99%, R38 nm = 39%) and 50/50 (Pwater = 1100 kg/m2hbar, R240 nm = 91%, R38 nm = 36%).

The role of acoustic nonlinearity in tissue heating behind a rib cage using a high-intensity focused ultrasound phased array.

The goal of this study was to investigate theoretically the effects of nonlinear propagation in a high-intensity focused ultrasound (HIFU) field produced by a therapeutic phased array and the resultant heating of tissue behind a rib cage. Three configurations of focusing were simulated: in water, in water with ribs in the beam path and in water with ribs backed by a layer of soft tissue. The Westervelt equation was used to model the nonlinear HIFU field, and a 1 MHz phased array consisting of 254 circular elements was used as a boundary condition to the model. The temperature rise in tissue was modelled using the bioheat equation, and thermally necrosed volumes were calculated using the thermal dose formulation. The shapes of lesions predicted by the modelling were compared with those previously obtained in in vitro experiments at low-power sonications. Intensity levels at the face of the array elements that corresponded to the formation of high-amplitude shock fronts in the focal region were determined as 10 W cm(-2) in the free field in water and 40 W cm(-2) in the presence of ribs. It was shown that exposures with shocks provided a substantial increase in tissue heating, and its better spatial localization in the main focal region only. The relative effects of overheating ribs and splitting of the focus due to the periodic structure of the ribs were therefore reduced. These results suggest that utilizing nonlinear propagation and shock formation effects can be beneficial for inducing confined HIFU lesions when irradiating through obstructions such as ribs. Design of compact therapeutic arrays to provide maximum power outputs with lower intensity levels at the elements is necessary to achieve shock wave regimes for clinically relevant sonication depths in tissue.

Novel inhibitors of heat shock protein Hsp70-mediated luciferase refolding that bind to DnaJ.

Inhibitors of both heat shock proteins Hsp90 and Hsp70 have been identified in assays measuring luciferase refolding containing rabbit reticulocyte lysate or purified chaperone components. Here, we report the discovery of a series of phenoxy-N-arylacetamides that disrupt Hsp70-mediated luciferase refolding by binding to DnaJ, the bacterial homolog of human Hsp40. Inhibitor characterization experiments demonstrated negative cooperativity with respect to DnaJ and luciferase concentration, but varying the concentration of ATP had no effect on potency. Thermal shift analysis suggested a direct interaction with DnaJ, but not with Hsp70. These compounds may be useful tools for studying DnaJ/Hsp40 in various cellular processes.

Nanoliter high-throughput RT-qPCR: a statistical analysis and assessment.

Biomarkers discovered from gene expression profiles using hybridization microarrays have made great inroads in the diagnosis and development of safer and efficacious drugs. The candidate gene set is biologically validated by quantitative measurement with reverse transcriptase quantitative PCR (RT-qPCR) and is an effective strategy when implemented with microplates if the number of candidate genes and samples is small. With the trend toward informative candidate gene panels increasing from tens to hundreds of genes and sample cohorts exceeding several hundred, an alternative fluidic approach is needed that preserves the intrinsic analytical precision, large dynamic range, and high sensitivity of RT-qPCR, yet is scalable to high throughputs. We have evaluated the performance of a nanoliter fluidic system that enables up to 3072 nanoliter RT-qPCR assays simultaneously in a high-density array format. We measured the transcription from two different adult human tissues to assess measurement reproducibility across replicates, measurement accuracy, precision, specificity, and sensitivity; determined the false positive rate (FPR) and false negative rate (FNR) of the expressed transcript copies; and determined differences in kinase gene expression reflecting tissue and dosage differences. Using our methodology, we confirm the potential of this technology in advancing pharmaceutical research and development.

Considerations for target validation and industrial approaches.

Target validation in health and disease integrates the modulation of a certain molecular target with an expected biological/biochemical/physiological or pathophysiological response or effect. The current state-of-the-art in target validation requires the interface of multiple complementary approaches and technologies to define the mechanistic connectivity between a molecular target and underlying micro- and macrobiotic processes. Target validation also represents the basis for "drug target validation" with focus on therapeutic applications. The concepts of "target validation" and "drug-based therapeutic intervention" continue to coevolve as new classes of therapeutic agents and delivery systems emerge and enable us to target or modulate previously inaccessible molecular entities.

Levels of mRNA coding for alpha-, beta-, and gamma-synuclein in the brains of newborn, juvenile, and adult rats.

Synucleins are proteins known for their malfunction in a group of illnesses called synucleopathies, which includes Alzheimer's and Parkinson's disease. To learn more about the role of synucleins in the CNS, we have studied levels of message coding for alpha-, beta-, and gamma-synuclein using quantitative RT-PCR. Levels of synuclein mRNAs were studied in the cerebral cortex (left and right, anterior and posterior), hippocampus, striatum, and cerebellum, obtained from 5-d-old (newborn), 1-mo (juvenile)-, and 6-, and 9-mo (adult)-old rats. The mRNA levels for all synucleins varied significantly among structures. The rank order of mRNA levels in different structures was cortex = hippocampus > striatum > cerebellum for alpha-synuclein; cortex > hippocampus = cerebellum > striatum for beta-synuclein; and hippocampus = striatum > cortex = cerebellum for gamma-synuclein. There was significant effect of age for mRNA levels for all synucleins. The dynamics of these changes were different depending on type of synuclein and brain structure. Levels of mRNA for alpha-synuclein were significantly reduced with age in all structures except hippocampus. For beta- and gamma-synuclein, levels increased significantly only in the cerebral cortex and only from 5 d to 1 mo of age. In contrast, gamma-synuclein levels in the cerebellum were very high at 5 d and significantly reduced at 1 mo of age. The revealed pattern and dynamics of changes in the levels of mRNA coding for synucleins would support the conclusion for an important role of these molecules during development and the aging process.

Nanoliter high throughput quantitative PCR.

Understanding biological complexity arising from patterns of gene expression requires accurate and precise measurement of RNA levels across large numbers of genes simultaneously. Real time PCR (RT-PCR) in a microtiter plate is the preferred method for quantitative transcriptional analysis but scaling RT-PCR to higher throughputs in this fluidic format is intrinsically limited by cost and logistic considerations. Hybridization microarrays measure the transcription of many thousands of genes simultaneously yet are limited by low sensitivity, dynamic range, accuracy and sample throughput. The hybrid approach described here combines the superior accuracy, precision and dynamic range of RT-PCR with the parallelism of a microarray in an array of 3072 real time, 33 nl polymerase chain reactions (RT-PCRs) the size of a microscope slide. RT-PCR is demonstrated with an accuracy and precision equivalent to the same assay in a 384-well microplate but in a 64-fold smaller reaction volume, a 24-fold higher analytical throughput and a workflow compatible with standard microplate protocols.

Integrated expressional analysis: application to the drug discovery process.

Microarray technology enables high-throughput testing of gene expression to investigate various neuroscience related questions. This in turn creates a demand for scalable methods to confirm microarray results and the opportunity to use this information to discover and test novel pathways and therapeutic applications. Discovery of new central nervous system (CNS) treatments requires a comprehensive understanding of multiple aspects including the biology of a target, the pathophysiology of a disease/disorder, and the selection of successful lead compounds as well as efficient biomarker and drug disposition strategies such as absorption (how a drug is absorbed), distribution (how a drug spreads through an organism), metabolism (chemical conversion of a drug, if any, and into which substances), and elimination (how is a drug eliminated) (ADME). Understanding of the toxicity is also of paramount importance. These approaches, in turn, require novel high-content integrative assay technologies that provide thorough information about changes in cell biology. To increase efficiency of profiling, characterization, and validation, we established a new screening strategy that combines high-content image-based testing on Array Scan (Cellomics) with a confocal system and the multiplexed TaqMan RT-PCR method for quantitative mRNA expression analysis. This approach could serve as an interface between high-throughput microarray testing and specific application of markers discovered in the course of a microarray experiment. Markers could pinpoint activation or inhibition of a molecular pathway related, for instance, to neuronal viability. We demonstrate the successful testing of the same cell population in an image-based translocational assay followed by poly(A) mRNA capture and multiplexed single tube RT-PCR. In addition, Ciphergen ProteinChip analysis can be performed on the supernatant, thus allowing significant complementarity in the data output and interpretation by also including the capture and initial analysis of proteins in the integrative approach presented. We have determined various conditions including the number of cells, RT and PCR optimization, which are necessary for successful detection and consequent assay integration. We also show the successful convergence of various different approaches and multiplexing of different targets within a single real-time PCR tube. This novel integrative technological approach has utility for CNS drug discovery, target and biomarker identification, selection and characterization as well as for the study of toxicity- and adverse event-associated molecular mechanisms.