Inhibiting TGF-[Formula: see text] 1-Mediated Cellular Processes as an Effective Strategy for the Treatment of Pulmonary Fibrosis with Chinese Herbal Medicines.

Pulmonary fibrosis (PF) is a chronic and irreversible interstitial lung disease that even threatens the lives of some patients infected with COVID-19. PF is a multicellular pathological process, including the initial injuries of epithelial cells, recruitment of inflammatory cells, epithelial-mesenchymal transition, activation and differentiation of fibroblasts, etc. TGF-[Formula: see text]1 acts as a key effect factor that participates in these cellular processes of PF. Recently, much attention was paid to inhibiting TGF-[Formula: see text]1 mediated cell processes in the treatment of PF with Chinese herbal medicines (CHM), an important part of traditional Chinese medicine. Here, this review first summarized the effects of TGF-[Formula: see text]1 in different cellular processes of PF. Then, this review summarized the recent research on CHM (compounds, multi-components, single medicines and prescriptions) to directly and/or indirectly inhibit TGF-[Formula: see text]1 signaling (TLRs, PPARs, micrRNA, etc.) in PF. Most of the research focused on CHM natural compounds, including but not limited to alkaloids, flavonoids, phenols and terpenes. After review, the research perspectives of CHM on TGF-[Formula: see text]1 inhibition in PF were further discussed. This review hopes that revealing the inhibiting effects of CHM on TGF-[Formula: see text]1-mediated cellular processes of PF can promote CHM to be better understood and utilized, thus transforming the therapeutic activities of CHM into practice.

Punicalagin in Cancer Prevention-Via Signaling Pathways Targeting.

The extract of pomegranate (Punica granatum) has been applied in medicine since ancient times due to its broad-spectrum health-beneficial properties. It is a rich source of hydrolyzable tannins and anthocyanins, exhibiting strong antioxidative, anti-inflammatory, and antineoplastic properties. Anticancer activities of pomegranate with reference to modulated signaling pathways in various cancer diseases have been recently reviewed. However, less is known about punicalagin (Pug), a prevailing compound in pomegranate, seemingly responsible for its most beneficial properties. In this review, the newest data derived from recent scientific reports addressing Pug impact on neoplastic cells are summarized and discussed. Its attenuating effect on signaling circuits promoting cancer growth and invasion is depicted. The Pug-induced redirection of signal-transduction pathways from survival and proliferation into cell-cycle arrest, apoptosis, senescence, and autophagy (thus compromising neoplastic progression) is delineated. Considerations presented in this review are based mainly on data obtained from in vitro cell line models and concern the influence of Pug on human cervical, ovarian, breast, lung, thyroid, colorectal, central nervous system, bone, as well as other cancer types.

Uncovering the anti-NSCLC effects and mechanisms of gypenosides by metabolomics and network pharmacology analysis.

ETHNOPHARMACOLOGICAL RELEVANCE: Lung cancer is the chief reason of cancer death worldwide, and non-small cell lung cancer (NSCLC) make up the majority of lung cancers. Gypenosides are the main active constituents from Gynostemma pentaphyllum. Previous studies showed that they were used to remedy many cancers. The effect of gypenosides on NSCLC has never been studied from the perspective of network pharmacology and metabolomics. The mechanism is still not clear and remains to be explored. AIM OF THE STUDY: To explore the anti-NSCLC activity and mechanism of gypenosides in A549 cells. MATERIAL/METHODS: Gypenosides of G. pentaphyllum were detected by HPLC-MS. The cytotoxicity was detected by MTT assay. The migration, cell cycle and apoptosis of gypenosides were studied by wound healing assay, JC-1 assay and flow cytometry. The mechanism of gypenosides on NSCLC was studied by metabolomics and network pharmacology. Some key proteins and pathways were further confirmed by Western blot. RESULTS: Eleven gypenosides were detected by HPLC-MS. Gypenosides could suppress the proliferation of A549 cells, inhibit the migration of A549 cells, induce apoptosis and arrest cell cycle in G0/G1 phase. Metabolomics and network pharmacology approach revealed that gypenosides might affect 17 metabolite related proteins by acting on 9 candidate targets (STAT3, VEGFA, EGFR, MMP9, IL2, TYMS, FGF2, HPSE, LGALS3), thus resulting in the changes of two metabolites (uridine 5'-monophosphate, D-4'-Phosphopantothenate) and two metabolic pathways (pyrimidine metabolism; pantothenate and CoA biosynthesis). Western blotting indicated that gypenosides might inhibit A549 cells through MMP9, STAT3 and TYMS to indirectly affect the pathways of pyrimidine metabolism, pantothenate and CoA biosynthesis. CONCLUSIONS: This study revealed that metabolomics combined with network pharmacology was conducive to understand the anti-NSCLC mechanism of gypenosides.

The toxicity of Aspidosperma subincanum to MCF7 cells is related to modulation of oxidative status and proinflammatory pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Cancer is an inflammatory disease because carcinogenesis and tumor progression depend on intrinsic and extrinsic inflammatory pathways. Although species of the genus Aspidosperma are widely used to treat tumors, and there is ethnopharmacological evidence for traditional use of the species A. subincanum as an anti-inflammatory agent, its antineoplastic potential is unknown. AIM OF THE STUDY: To evaluate toxic effects of the indole alkaloid-rich fraction (IAF) of A. subincanum on the MCF7 cell line and identify some of the anti-inflammatory mechanisms involved. MATERIALS AND METHODS: Chromatographic analyses were performed by ultra-high-performance liquid chromatography with electrospray ionization mass spectrometry, and cytotoxic and antiproliferative effects of IAF were verified by MTT and clonogenic assays. Cell cycle alterations were analyzed by measuring DNA content, while propidium iodide and acridine orange staining was performed to determine the type of induced cell death. The expression of apoptosis markers and proteins involved in cell proliferation and survival pathways was analyzed by immunoblotting, RT-qPCR, and ELISAs. Interference with redox status was investigated using a DCFH-DA probe and by measuring catalase activity. RESULTS: Chromatographic analyses showed that IAF is a complex mixture containing indole alkaloids. IAF selectively exerted toxic and antiproliferative effects, elevating the Bax/Bcl-xL ratio and inducing apoptosis in MCF7 cells. IAF decreased intracellular reactive oxygen species levels and increased catalase activity, while reducing the IL-8 level and suppressing COX-2 expression. CONCLUSIONS: IAF induces apoptosis in MCF7 cells by suppressing COX-2 expression while reducing IL-8 levels and intracellular content of reactive oxygen species.

Known Triterpenes and their Derivatives as Scaffolds for the Development of New Therapeutic Agents for Cancer.

BACKGROUND: Plants produce several bioactive secondary metabolites whose are used as therapeutic agents to treat several diseases, among whom cancer. Triterpenes are secondary metabolites that exert inhibitory activity against multiple intracellular and extracellular targets in euchariotic cells. These targets are proteins involved in apoptosis, cell development and differentiation, angiogenesis, metastasis and inflammatory processes. The inhibition of their functions leads to decreased cellular growth, differentiation and migration, resulting in antitumor activity, as shown by several authors. Furthermore, during recent years synthetic triterpenoid derivatives have also been developed to implement potency and efficacy of starting compounds, allowing the obtainment of new agents having promising anticancer activity. OBJECTIVE: In this review we report the latest results regarding anticancer activity of some of the most studied triterpenes in the field, as well as of their semi-synthetic derivatives, with the aim to summarize the role of triterpenes as molecular leads for the development of new classes of antitumor agents. METHODS: We focused on the most recent literature regarding triterpenes in cancer treatment, highlighting the potential of developing new drugs starting from these natural compounds. CONCLUSION: Several "old" triterpenes as ursolic, betulinic and oleanolic acids were recently reconsidered as model compounds for the development of innovative anticancer agents. Their activity against proteins involved in tumor development enhances the opportunity to exploit these compounds as new multi-target therapeutic agents. Furthermore, the possibility to synthetize new compounds from their natural-occurring structures could be an alternative to overcome cellular resistance to drugs and could improve their therapeutic efficacy.

Antitumor and antimetastatic effects of walnut oil in esophageal adenocarcinoma cells.

BACKGROUND: Walnuts contain many components including specific fatty acids, which could be active against cancer. Even though the anticarcinogenic effect of some of the individual fatty acids in walnut oil has been described, the effect of walnut oil itself on esophageal cancer cells hasn't yet been investigated. OBJECTIVE: We aimed to investigate whether walnut oil affects tumor growth and metastatic potential in esophageal cancer cells. METHODS: The human esophageal adenocarcinoma cell line, OE19, was treated with different doses of walnut oil and cell viability, apoptosis/necrosis and cell cycle analyses were performed using WST-1 assay and flow cytometry respectively. Adhesion, colony formation and wound healing assays were performed to assess the antimetastatic effects of walnut oil. NFkB expression was evaluated with western blot analysis. RESULTS: Walnut oil decreased the cell viability of esophageal cancer cells in a dose-dependent manner. 20 mg/mL walnut oil reduced cell viability by ∼50% when compared with control. The analysis revealed that necrosis and accumulation of cells in G0/G1 phase was induced in the cells treated with high doses of walnut oil. It also down-regulated the protein levels of NFkB. Walnut oil suppressed the adhesion, migration and colony formation of the cells. CONCLUSIONS: High-dose short-term administration of walnut oil reduces the cell viability and metastatic ability of esophageal cancer cells, while exhibiting anticarcinogenic effect by inducing necrosis and cell cycle arrest at the G0/G1 phase, probably through suppression of the NFkB pathway. These data indicate that walnut oil, and by extension walnut consumption, may have beneficial effects in esophageal cancer in humans. This should be tested by clinical trials in the future.

Representing high throughput expression profiles via perturbation barcodes reveals compound targets.

High throughput mRNA expression profiling can be used to characterize the response of cell culture models to perturbations such as pharmacologic modulators and genetic perturbations. As profiling campaigns expand in scope, it is important to homogenize, summarize, and analyze the resulting data in a manner that captures significant biological signals in spite of various noise sources such as batch effects and stochastic variation. We used the L1000 platform for large-scale profiling of 978 representative genes across thousands of compound treatments. Here, a method is described that uses deep learning techniques to convert the expression changes of the landmark genes into a perturbation barcode that reveals important features of the underlying data, performing better than the raw data in revealing important biological insights. The barcode captures compound structure and target information, and predicts a compound's high throughput screening promiscuity, to a higher degree than the original data measurements, indicating that the approach uncovers underlying factors of the expression data that are otherwise entangled or masked by noise. Furthermore, we demonstrate that visualizations derived from the perturbation barcode can be used to more sensitively assign functions to unknown compounds through a guilt-by-association approach, which we use to predict and experimentally validate the activity of compounds on the MAPK pathway. The demonstrated application of deep metric learning to large-scale chemical genetics projects highlights the utility of this and related approaches to the extraction of insights and testable hypotheses from big, sometimes noisy data.

Active machine learning-driven experimentation to determine compound effects on protein patterns.

High throughput screening determines the effects of many conditions on a given biological target. Currently, to estimate the effects of those conditions on other targets requires either strong modeling assumptions (e.g. similarities among targets) or separate screens. Ideally, data-driven experimentation could be used to learn accurate models for many conditions and targets without doing all possible experiments. We have previously described an active machine learning algorithm that can iteratively choose small sets of experiments to learn models of multiple effects. We now show that, with no prior knowledge and with liquid handling robotics and automated microscopy under its control, this learner accurately learned the effects of 48 chemical compounds on the subcellular localization of 48 proteins while performing only 29% of all possible experiments. The results represent the first practical demonstration of the utility of active learning-driven biological experimentation in which the set of possible phenotypes is unknown in advance.

Cellular Biomechanics in Drug Screening and Evaluation: Mechanopharmacology.

The study of mechanobiology is now widespread. The impact of cell and tissue mechanics on cellular responses is well appreciated. However, knowledge of the impact of cell and tissue mechanics on pharmacological responsiveness, and its application to drug screening and mechanistic investigations, have been very limited in scope. We emphasize the need for a heightened awareness of the important bidirectional influence of drugs and biomechanics in all living systems. We propose that the term 'mechanopharmacology' be applied to approaches that employ in vitro systems, biomechanically appropriate to the relevant (patho)physiology, to identify new drugs and drug targets. This article describes the models and techniques that are being developed to transform drug screening and evaluation, ranging from a 2D environment to the dynamic 3D environment of the target expressed in the disease of interest.

Curcumin ameliorates high glucose-induced neural tube defects by suppressing cellular stress and apoptosis.

OBJECTIVE: Curcumin is a naturally occurring polyphenol present in the roots of the Curcuma longa plant (turmeric), which possesses antioxidant, antitumorigenic, and antiinflammatory properties. Here, we test whether curcumin treatment reduces high glucose-induced neural tube defects (NTDs), and if this occurs via blocking cellular stress and caspase activation. STUDY DESIGN: Embryonic day 8.5 mouse embryos were collected for use in whole-embryo culture under normal (100 mg/dL) or high (300 mg/dL) glucose conditions, with or without curcumin treatment. After 24 hours in culture, protein levels of oxidative stress makers, nitrosative stress makers, endoplasmic reticulum (ER) stress makers, cleaved caspase 3 and 8, and the level of lipid peroxides were determined in the embryos. After 36 hours in culture, embryos were examined for evidence of NTD formation. RESULTS: Although 10 µmol/L of curcumin did not significantly reduce the rate of NTDs caused by high glucose, 20 µmol/L of curcumin significantly ameliorated high glucose-induced NTD formation. Curcumin suppressed oxidative stress in embryos cultured under high glucose conditions. Treatment reduced the levels of the lipid peroxidation marker, 4-hydroxynonenal, nitrotyrosine-modified protein, and lipid peroxides. Curcumin also blocked ER stress by inhibiting phosphorylated protein kinase RNA-like ER kinase, phosphorylated inositol-requiring protein-1α (p-IRE1α), phosphorylated eukaryotic initiation factor 2α (p-eIF2α), C/EBP-homologous protein, binding immunoglobulin protein, and x-box binding protein 1 messenger RNA splicing. Additionally, curcumin abolished caspase 3 and caspase 8 cleavage in embryos cultured under high glucose conditions. CONCLUSION: Curcumin reduces high glucose-induced NTD formation by blocking cellular stress and caspase activation, suggesting that curcumin supplements could reduce the negative effects of diabetes on the embryo. Further investigation will be needed to determine if the experimental findings can translate into clinical settings.

Zinc homeostasis in the metabolic syndrome and diabetes.

Zinc (Zn) is an essential mineral that is required for various cellular functions. Zn dyshomeostasis always is related to certain disorders such as metabolic syndrome, diabetes and diabetic complications. The associations of Zn with metabolic syndrome, diabetes and diabetic complications, thus, stem from the multiple roles of Zn: (1) a constructive component of many important enzymes or proteins, (2) a requirement for insulin storage and secretion, (3) a direct or indirect antioxidant action, and (4) an insulin-like action. However, whether there is a clear cause-and-effect relationship of Zn with metabolic syndrome, diabetes, or diabetic complications remains unclear. In fact, it is known that Zn deficiency is a common phenomenon in diabetic patients. Chronic low intake of Zn was associated with the increased risk of diabetes and diabetes also impairs Zn metabolism. Theoretically Zn supplementation should prevent the metabolic syndrome, diabetes, and diabetic complications; however, limited available data are not always supportive of the above notion. Therefore, this review has tried to summarize these pieces of available information, possible mechanisms by which Zn prevents the metabolic syndrome, diabetes, and diabetic complications. In the final part, what are the current issues for Zn supplementation were also discussed.

Salviae Miltiorrhizae Radix and Puerariae Lobatae Radix herbal formula mediates anti-atherosclerosis by modulating key atherogenic events both in vascular smooth muscle cells and endothelial cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Salviae Miltiorrhizae Radix (Danshen) and Puerariae Lobatae Radix (Gegen) are principal herbs have long been used in combination for treating cardiovascular disease. AIMS OF STUDY: Danshen and Gegen in the ratio of 7:3 (DGW) have significantly reduced the carotid intimal-media thickening (IMT) in patients in our previous clinical study. In the present study, we have demonstrated the mechanisms on IMT reduction by investigating its key processes on both vascular smooth muscle cell (vSMC) and endothelial cells. MATERIALS AND METHODS: The anti-proliferative effects of DGW on platelet-derived growth factor (PDGF) induced vSMC proliferation were studied by cell proliferation, cell cycle distribution, p-ERK and cyclin D expression level. The anti-migratory effect of DGW was investigated by using transwell apparatus. For human umbilical endothelial cells (HUVEC), the inhibitory effects of DGW on TNF-alpha induced cell adhesion, cell adhesion molecules expression, MCP-1 and IL-6 production were investigated. RESULTS: DGW significantly inhibited A7r5 proliferation and exhibited G1/S cell cycle arrest by suppressing both p-ERK and cyclin D expression. Moreover, DGW showed anti-migratory effect against PDGF-induced A7r5 migration. In addition, DGW inhibited the cell adhesion as well as the expression of ICAM-1 and VCAM-1, the production of MCP-1 but not IL-6 in TNF-α stimulated HUVECs. CONCLUSIONS: Our study provided strong scientific evidence on IMT reduction in patients by modulating the key atherogenic events in both vSMC and endothelial cells.

Cyclopeptide RA-V inhibits angiogenesis by down-regulating ERK1/2 phosphorylation in HUVEC and HMEC-1 endothelial cells.

BACKGROUND AND PURPOSE: Anti-angiogenic agents have recently become one of the major adjuvants for cancer therapy. A cyclopeptide, RA-V, has been shown to have anti-tumour activities. Its in vitro anti-angiogenic activities were evaluated in the present study, and the underlying mechanisms were also assessed. EXPERIMENTAL APPROACH: Two endothelial cell lines, human umbilical vein endothelial cells (HUVEC) and human microvascular endothelial cells (HMEC-1), were used. The effects of RA-V on the proliferation, cell cycle phase distribution, migration, tube formation and adhesion were assessed. Western blots and real-time PCR were employed to examine the protein and mRNA expression of relevant molecules. KEY RESULTS: RA-V inhibited HUVEC and HMEC-1 proliferation dose-dependently with IC(50) values of 1.42 and 4.0 nM respectively. RA-V inhibited migration and tube formation of endothelial cells as well as adhesion to extracellular matrix proteins. RA-V treatment down-regulated the protein and mRNA expression of matrix metalloproteinase-2. Regarding intracellular signal transduction, RA-V interfered with the activation of ERK1/2 in both cell lines. Furthermore, RA-V significantly decreased the phosphorylation of JNK in HUVEC whereas, in HMEC-1, p38 MAPK was decreased. CONCLUSIONS AND IMPLICATIONS: RA-V exhibited anti-angiogenic activities in HUVEC and HMEC-1 cell lines with changes in function of these endothelial cells. The underlying mechanisms of action involved the ERK1/2 signalling pathway. However, RA-V may regulate different signalling pathways in different endothelial cells. These findings suggest that RA-V has the potential to be further developed as an anti-angiogenic agent.

Cell-based screening using high-throughput flow cytometry.

This review describes the use of high-throughput flow cytometry for performing multiplexed cell-based and bead-based screens. With the many advances in cell-based analysis and screening, flow cytometry has historically been underutilized as a screening tool largely due to the limitations in handling large numbers of samples. However, there has been a resurgence in the use of flow cytometry due to a combination of innovations around instrumentation and a growing need for cell-based and bead-based applications. The HTFC™ Screening System (IntelliCyt Corporation, Albuquerque, NM) is a novel flow cytometry-based screening platform that incorporates a fast sample-loading technology, HyperCyt®, with a two-laser, six-parameter flow cytometer and powerful data analysis capabilities. The system is capable of running multiplexed screening assays at speeds of up to 40 wells per minute, enabling the processing of a 96- and 384-well plates in as little as 3 and 12 min, respectively. Embedded in the system is HyperView®, a data analysis software package that allows rapid identification of hits from multiplexed high-throughput flow cytometry screening campaigns. In addition, the software is incorporated into a server-based data management platform that enables seamless data accessibility and collaboration across multiple sites. High-throughput flow cytometry using the HyperCyt technology has been applied to numerous assay areas and screening campaigns, including efflux transporters, whole cell and receptor binding assays, functional G-protein-coupled receptor screening, in vitro toxicology, and antibody screening.

Microfluidic cell culture systems for drug research.

In pharmaceutical research, an adequate cell-based assay scheme to efficiently screen and to validate potential drug candidates in the initial stage of drug discovery is crucial. In order to better predict the clinical response to drug compounds, a cell culture model that is faithful to in vivo behavior is required. With the recent advances in microfluidic technology, the utilization of a microfluidic-based cell culture has several advantages, making it a promising alternative to the conventional cell culture methods. This review starts with a comprehensive discussion on the general process for drug discovery and development, the role of cell culture in drug research, and the characteristics of the cell culture formats commonly used in current microfluidic-based, cell-culture practices. Due to the significant differences in several physical phenomena between microscale and macroscale devices, microfluidic technology provides unique functionality, which is not previously possible by using traditional techniques. In a subsequent section, the niches for using microfluidic-based cell culture systems for drug research are discussed. Moreover, some critical issues such as cell immobilization, medium pumping or gradient generation in microfluidic-based, cell-culture systems are also reviewed. Finally, some practical applications of microfluidic-based, cell-culture systems in drug research particularly those pertaining to drug toxicity testing and those with a high-throughput capability are highlighted.

In vitro effects of Sutherlandia frutescens water extracts on cell numbers, morphology, cell cycle progression and cell death in a tumorigenic and a non-tumorigenic epithelial breast cell line.

Sutherlandia frutescens is a South African herb traditionally used for internal cancers, diabetes, a variety of inflammatory conditions and recently to improve the overall health in cancer and HIV/AIDS patients. The in vitro effects of S. frutescens extracts were evaluated on cell numbers, morphology, cell cycle progression and cell death. Dose-dependent studies (2-10 mg/ml) revealed a decrease in malignant cell numbers when compared to their controls. S. frutescens extracts (10 mg/ml) decreased cell growth in a statistically significantly manner to 26% and 49% (P<0.001) in human breast adenocarcinoma (MCF-7) and human non-tumorigenic epithelial mammary gland cells (MCF-12A) respectively after 72 h of exposure. Cell density was significantly compromised and hypercondensed chromatin, cytoplasmic shrinking, membrane blebbing and apoptotic bodies were more pronounced in the MCF-7 cell line. Both S. frutescens-treated cell lines exhibited and increased tendency for acridine orange staining, suggesting increased lysosomal and/or autophagy activity. Flow cytometry showed an increase in the sub G(1) apoptotic fraction and an S phase arrest in both the 5 mg/ml and 10 mg/ml S. frutescens-treated cells. S. frutescens induced an increase in apoptosis in both cell lines as detected by Annexin V and propidium iodide flow cytometric measurement. At 10 mg/ml, late stages of apoptosis were more prominent in MCF-7 S. frutescens-treated cells when compared to the MCF-12A cells. Transmission electron microscopy revealed hallmarks of increased vacuolarization and hypercondensed chromatin, suggesting autophagic and apoptotic processes. The preliminary study demonstrates that S. frutescens water extracts exert a differential action mechanism in non-tumorigenic MCF-12A cells when compared to tumorigenic MCF-7 cells, warranting future studies on this multi-purpose medicinal plant in southern Africa.

Miniaturized microfluidic formats for cell-based high-throughput screening.

Cell-based high-throughput screening (HTS) has become an important method used in pharmaceutical drug discovery, and is presently carried out using robots and micro-well plates. A microfluidic-based device for cell-based HTS using a traditional cell-culture protocol would be a key enabler in miniaturization and in increasing throughput without consequent detrimental effects on the physiological significance of the screen. In this paper, we illustrate the advances in miniaturization of cell-based HTS, especially using microfabrication and microfluidics. We also detail a novel microfluidic HTS device targeted for cell-based assays using traditional non-compartmentalized agar gel as a cell-culture medium and electric control over drug dose. The basic design of this device consists of a gel layer supported by a nanoporous membrane that is bonded to microchannels underneath it. The pores of the membrane are blocked everywhere except in selected regions that serve as fluidic interfaces between the microchannel below and the gel above. Upon application of an electric field, nanopores start to act as electrokinetic pumps. By selectively switching an array of such micropumps, a number of spots containing drug molecules are created simultaneously in the gel layer. By diffusion, drugs reach the top surface of the gel where cells are to be grown. Based on this principle, a number of different devices can be fabricated using microfabrication technology. The fabricated devices include a single drug spot-forming device, a multiple drug spot-forming device, and a microarray drug spot-forming device. By controlling the pumping potential and duration, spots sizes ranging from 200 mu;m to 6 mm in diameter and having inter-spot distances of 0.4 to 10 mm have been created. The absence of diffusional transport through the nanoporous interfaces without an electric field is demonstrated. A number of representative molecules, including surrogate drug molecules (trypan blue and methylene blue) and biomolecules (DNA and protein) were selected for demonstration purposes. A dosing range of 50 to 3000 mu;g and a spot density of 156 spots/cm2 were achieved. The drug spot density was found to be limited by molecular diffusion in gel, so a numerical study was carried to determine ways to increase density. Based on this simulation, a diffusion barrier was proposed, which uses a specially dimensioned (having shallow grooves) gel sheet to reduce diffusion.

poolHiTS: a shifted transversal design based pooling strategy for high-throughput drug screening.

BACKGROUND: A key goal of drug discovery is to increase the throughput of small molecule screens without sacrificing screening accuracy. High-throughput screening (HTS) in drug discovery involves testing a large number of compounds in a biological assay to identify active compounds. Normally, molecules from a large compound library are tested individually to identify the activity of each molecule. Usually a small number of compounds are found to be active, however the presence of false positive and negative testing errors suggests that this one-drug one-assay screening strategy can be significantly improved. Pooling designs are testing schemes that test mixtures of compounds in each assay, thereby generating a screen of the whole compound library in fewer tests. By repeatedly testing compounds in different combinations, pooling designs also allow for error-correction. These pooled designs, for specific experiment parameters, can be simply and efficiently created using the Shifted Transversal Design (STD) pooling algorithm. However, drug screening contains a number of key constraints that require specific modifications if this pooling approach is to be useful for practical screen designs. RESULTS: In this paper, we introduce a pooling strategy called poolHiTS (Pooled High-Throughput Screening) which is based on the STD algorithm. In poolHiTS, we implement a limit on the number of compounds that can be mixed in a single assay. In addition, we show that the STD-based pooling strategy is limited in the error-correction that it can achieve. Due to the mixing constraint, we show that it is more efficient to split a large library into smaller blocks of compounds, which are then tested using an optimized strategy repeated for each block. We package the optimal block selection algorithm into poolHiTS. The MATLAB codes for the poolHiTS algorithm and the corresponding decoding strategy are also provided. CONCLUSION: We have produced a practical version of STD algorithm for pooled drug screens. This pooling strategy provides both assay compression and error-correction capabilities that can both accelerate and reduce the overall cost of HTS in drug discovery.

Cellular effects of common antioxidants.

Tabular information of proposed cellular effects regarding the use of various antioxidants is presented. The information was gathered from scientific sources and experimental research from Medline. It is suggested that practitioners use their best available judgment and sound reasoning when applying these supplements in clinical situations.