TY - JOUR. T1 - Evaluation of polymeric gene delivery nanoparticles by nanoparticle tracking analysis and high-throughput flow cytometry.. AU - Shmueli, Ron B.. AU - Bhise, Nupura S.. AU - Green, Jordan J.. PY - 2013. Y1 - 2013. N2 - Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases(1) and as a technology for regenerative medicine(2). Unlike viruses, which have significant safety issues, polymeric nanoparticles can be designed to be non-toxic, non-immunogenic, non-mutagenic, easier to synthesize, chemically versatile, capable of carrying larger nucleic acid cargo and biodegradable and/or environmentally responsive. Cationic polymers self-assemble with negatively charged DNA via electrostatic interaction to form complexes on the order of 100 nm that are commonly termed polymeric nanoparticles. Examples of biomaterials used to form nanoscale polycationic gene delivery nanoparticles include polylysine, ...

The technology provides numerous systems and methods to detect stress in stem cells by measuring potency factors (and markers) and prioritized differentiation factors (and markers). Potency factors can be thought of as brake pedals for differentiation. That is, their expression and activity is associated with toti- or pluri-potency. Differentiation factors can be thought of as accelerator pedals for differentiation. That is, their expression and activity is associated with normal differentiation that occurs when potency-maintaining extracellular growth factors are removed. Under stress conditions, however, potency factors can decrease and differentiation factors can increase even in the presence of potency-maintaining extracellular growth factors. This type of differentiation is referred to as compensatory or prioritized differentiation. That is, under compensatory or prioritized differentiation, stem cells can be induced by stress from a toti- or pluripotent state into a more ...

To screen for chemical toxicity in human or mouse stem cell lines (undifferentiated or differentiated) by quantitative high throughput screening (qHTS) at NCGC, or by using lower throughput assays at NIEHS. Initially, the project focuses on fostering collaborations with stem cell technology providers and assessing control compounds and subsets of NTP chemicals using various assay approaches. Stem cell technology platforms and model systems shown to be useful for in vitro toxicology screening will be employed with larger sets of chemicals for hazard identification and chemical prioritization for toxicity testing. Data have been generated on a library of 80 predominantly developmental neurotoxicants evaluated for effects on neurite outgrowth in a human stem cell-derived neural cell population, cytotoxicity in different neural populations derived from human stem cells, and effects on the beating of human stem cell derived cardiomyocytes. Dose-response analysis has been carried out on the data from ...

Drugs currently available for leishmaniasis treatment often show parasite resistance, highly toxic side effects and costs that are prohibitive commonly for patients from the tropical endemic countries. There is an urgent need for new drugs as a treatment solution for this neglected disease. Here the authors describe the development and implementation of an automated high-throughput viability screening assay for the discovery of new drugs against Leishmania. Assay validation was done with Leishmania promastigote forms, and included the screening of 4000 compounds with known pharmacological properties. In an attempt to find new compounds with leishmanicidal properties, 26,500 structurally diverse chemical compounds were screened. A cut-off of 70% growth inhibition in the primary screening led to the identification of 567 active compounds. Cellular toxicity and selectivity were responsible for the exclusion of 78% of the pre-selected compounds. The activity of the remaining 124 compounds was ...

Patients with hereditary diffuse gastric cancer (HDGC), a cancer predisposition syndrome associated with germline mutations of the CDH1 (E-cadherin) gene, have few effective treatment options. Despite marked differences in natural history, histopathology, and genetic profile to patients afflicted by sporadic gastric cancer, patients with HDGC receive, in large, identical systemic regimens. The lack of a robust preclinical in vitro system suitable for effective drug screening has been one of the obstacles to date which has hampered therapeutic advances in this rare disease. In order to identify therapeutic leads selective for the HDGC subtype of gastric cancer, we compared gene expression profiles and drug phenotype derived from an oncology library of 1912 compounds between gastric cancer cells established from a patient with metastatic HDGC harboring a c.1380delA CDH1 germline variant and sporadic gastric cancer cells. Unsupervised hierarchical cluster analysis shows select gene expression alterations

Synthetic lethality is an attractive strategy for the design of novel therapies for cancer. Using this approach we have previously demonstrated that inhibition of the DNA repair protein, PARP1, is synthetically lethal with deficiency of either of the breast cancer susceptibility proteins, BRCA1 and …

The WHO gold standard method for malaria diagnosis is microscopy. It is considered inexpensive and field adapted, even though it is time-consuming and requires specifically trained personnel. Molecular detection methods achieve much higher detection sensitivities [1, 26], and they are better adapted to automation of the process and objective reading of results by machines. This potential makes them a valuable option for large-scale epidemiologic studies. Unfortunately few efforts have been spent on developing the high-throughput approaches needed for such studies.. The 18S rRNA gene is the most frequently cited marker for malaria detection. It is composed of highly conserved regions which can be targeted for a qualitative detection of Plasmodium spp., and of variable zones allowing species identification [4-7]. However, the 18S rRNA genes in Plasmodium spp. also have unusual properties, such as the existence of three stage-specific A-, S- and O-types, as well as copy number and strain-specific ...

We present a novel automated methodology that compensates for the effect of cell morphology on flow cytometry data, and thereby enables a quantitative analysis of high‐throughput flow cytometry data. The algorithm normalizes the effect of the physical characteristics of cell size and cell granularity on the fluorescence intensity, thereby enabling the analysis of fluorescence intensities (protein abundance) in the presence of different morphological characteristics of cells in a population. In contrast to traditional gating, which discards the large majority of cells, the regression model retains all cells and thereby provides more accurate statistics, higher consistency across replicates and the ability to handle biological samples that contain far fewer cells (at least 10‐fold), allowing for faster and cheaper data acquisition. This is relevant when one is looking for rare cells (e.g., stem cells), or when performing high‐throughput screens where only a few hundred cells per experimental ...

To understand the function of cellular protein networks, spatial and temporal context is essential. Fluorescence correlation spectroscopy (FCS) is a single-molecule method to study the abundance, mobility and interactions of fluorescence-labeled biomolecules in living cells. However, manual acquisition and analysis procedures have restricted live-cell FCS to short-term experiments of a few proteins. Here, we present high-throughput (HT)-FCS, which automates screening and time-lapse acquisition of FCS data at specific subcellular locations and subsequent data analysis. We demonstrate its utility by studying the dynamics of 53 nuclear proteins. We made 60,000 measurements in 10,000 living human cells, to obtain biophysical parameters that allowed us to classify proteins according to their chromatin binding and complex formation. We also analyzed the cell-cycle-dependent dynamics of the mitotic kinase complex Aurora B/INCENP and showed how a rise in Aurora concentration triggers two-step complex ...

In collaboration with Dr. Lisa DeLouise, the Kobie Lab has developed patented nanowell array technology called microbubbles, which allow for the long term culture and functional interrogation of B cells at the single cell level. This high-throughput platform enables the rapid screening of millions of individual B cells to obtain precise resolution of cellular diversity and isolation of rare B cells with desired functional profiles. This platform is particularly advantageous for high-content single cell analysis and therapeutic monoclonal antibody development.. « back to all projects. ...

In contrast to upstream optimization, which focuses mainly on preservation of cells and proteins as they grow, downstream optimization focuses primarily on achieving maximum yield from each lot, and ensuring that the resulting product remains as concentrated and high-quality as possible. These improvements may be achieved by implementing additional filtration and purification steps, introducing new high-throughput methods, and/or re-engineering existing processes to streamline the pipeline from cell harvest to final product.. Chromatographic separation methods are often used to isolate mAbs (or other desired proteins) from the fermentation broth after harvesting. Chromatographic separation is highly selective, resulting in a significantly higher flow and purity rates than conventional filtration techniques. In fact, recent advances have helped further integrate chromatographic separation into the production process, allowing for fine-tuned adjustment of elution conditions, thus effectively ...

The virologic synapse (VS), which is formed between a virus-infected and uninfected cell, plays a central role in the transmission of certain viruses, such as HIV and HTLV-1. During VS formation, HTLV-1-infected T-cells polarize cellular and viral proteins toward the uninfected T-cell. This polarization resembles anterior-posterior cell polarity induced by immunological synapse (IS) formation, which is more extensively characterized than VS formation and occurs when a T-cell interacts with an antigen-presenting cell. One measure of cell polarity induced by both IS or VS formation is the repositioning of the microtubule organizing center (MTOC) relative to the contact point with the interacting cell. Here we describe an automated, high throughput system to score repositioning of the MTOC and thereby cell polarity establishment. The method rapidly and accurately calculates the angle between the MTOC and the IS for thousands of cells. We also show that the system can be adapted to score anterior-posterior

Assessment of autoantibody responses in cancer patients, 978-3-639-85018-5, Cancer insistently remains among the leading causes of death worldwide, and identification of novel biomarkers that could aid in early diagnosis of this disease is of great importance. Circulating antibodies found in cancer patients blood have been described to have promising biomarker qualities. This work describes approaches that were successfully applied to identify novel antigens eliciting antibody formation in several types of cancer. One of the approaches, T7 phage-display SEREX approach that was elaborated within this study, resulted in the identification of 1064 antigens, which were further used for the development of an antigen microarray. This high-throughput platform enabled systematic comparison of antibody responses in cancer patients and healthy controls, and the results revealed that signatures of autoantibody responses holds great promise to be used for cancer diagnostics. Apart from the experimental part, the

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The combined effect of multiple mutations on protein function is hard to predict; thus, the ability to functionally assess a vast number of protein sequence variants would be practically useful for protein engineering. Here we present a high-throughput platform that enables scalable assembly and parallel characterization of barcoded protein variants with combinatorial modifications. We demonstrate this platform, which we name CombiSEAL, by systematically characterizing a library of 948 combination mutants of the widely used Streptococcus pyogenes Cas9 (SpCas9) nuclease to optimize its genome-editing activity in human cells. The ease with which the editing activities of the pool of SpCas9 variants can be assessed at multiple on- and off-target sites accelerates the identification of optimized variants and facilitates the study of mutational epistasis. We successfully identify Opti-SpCas9, which possesses enhanced editing specificity without sacrificing potency and broad targeting range. This ...

Innate Immunity has long been regarded as the non-specific arm of immune response, acting immediately and in a generic way to defend the host from infections. In the post genomic era, our knowledge of the innate immune system is enriched by findings on the specificity of innate immune reactions as well as to novel functions that do not strictly correlate with immunological defense and surveillance, immune modulation or inflammation. The advent of high-throughput platforms for genome and proteome-wide profiling, together with the enormous quantity of raw genetic information that has accumulated in the databases, have stirred new expectations in biomedical research, and led scientists to revisit established biological systems from a global and integrative perspective. Innate Immunity research now faces the challenge of integrating isolated biochemical pathways into complex gene and protein regulatory circuits. This volume collects topics on natural killer cells, mast cells, phagocytes, toll-like ...

Centrosome amplification (CA) is a hallmark of virtually all types of cancers including solid tumors and hematological malignancies. Cancer cells with extra centrosomes use centrosome clustering (CC) to allow for successful division. Because normal cells do not rely on this mechanism, CC is regarded as a promising target to selectively eradicate cells harboring supernumerary centrosomes. To identify novel inhibitors of CC, we developed a cell-based high-throughput screen that reports differential drug cytotoxicity for isogenic cell populations with different centrosome contents. We identified CP-673451 and crenolanib, two chemically related compounds originally developed for inhibition of PDGFR-β, as robust inhibitors of CC with selective cytotoxicity for cells with extra centrosomes. We demonstrate that these compounds induce mitotic spindle multipolarity by activation of the actin severing protein cofilin, leading to destabilization of the cortical actin network, and provide evidence that ...

Forward transfection is a widely used method that works well for adherent cell types; however, if one is using suspension cells or a high-throughput format, reverse transfection, where cells are added to the plated transfection reagent complex, is a more appropriate approach. This method can be used to increase the throughput and reproducibility of a CRISPR-Cas9 screen by optimizing gene editing efficiency as demonstrated in our recently published application note: Optimization of reverse transfection of Dharmacon Edit-R synthetic crRNA and tracrRNA components with DharmaFECT transfection reagent in a Cas9-expressing cell line.. An un-cleavable ubiquitin moiety fused to EGFP allows constitutive degradation of the EGFP protein, while disruption of the proteasome components by functional protein knockout leads to accumulation of EGFP and detectable fluorescence. This functional knockout data shows that determining optimal transfection conditions prior to arrayed screening leads to high gene ...

Here we describe a rapid high-throughput method for performing RNA interference (RNAi) in moss, in which phenotyping is performed within 1 week after transformation. The moss Physcomitrella patens is a great plant model system for reverse genetic studies due to its amenability to homologous recombination as well as RNAi. Our lab has developed a rapid RNAi assay to screen for growth phenotypes in moss protonemal tissue. Here we describe how we have recently further facilitated this assay by modifying the PEG-mediated transformation protocol allowing for transformations to be carried out in a semiautomated fashion in a 96-well plate format ...

Cellular senescence plays an important role in organismal aging and age-related diseases. However, it is challenging to isolate low numbers of senescent cells from small volumes of biofluids for downstream analysis. Furthermore, there is no technology that could selectively remove senescent cells in a high-throughput manner. In this work, we developed a novel microfluidic chip platform, termed senescence chip, for ultrahigh-throughput isolation and removal of senescent cells. The core component of our senescence chip is a slanted and tunable 3D micropillar array with a variety of shutters in the vertical direction for rapid cell sieving, taking advantage of the characteristic cell size increase during cellular senescence. The 3D configuration achieves high throughput, high recovery rate, and device robustness with minimum clogging. We demonstrated proof-of-principle applications in isolation and enumeration of senescent mesenchymal stem cells (MSCs) from undiluted human whole blood, and ...

Video articles in JoVE about retinoblastoma protein include In Vivo Detection and Analysis of Rb Protein SUMOylation in Human Cells, Workflow for High-content, Individual Cell Quantification of Fluorescent Markers from Universal Microscope Data, Supported by Open Source Software, Analysis of Cell Cycle Position in Mammalian Cells, Induction of Protein Deletion Through In Utero Electroporation to Define Deficits in Neuronal Migration in Transgenic Models, Whole Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea, In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration, An In Vitro Model for Studying Cellular Transformation by Kaposi Sarcoma Herpesvirus, A Protein Preparation Method for the High-throughput Identification of Proteins Interacting with a Nuclear Cofactor Using LC-MS/MS Analysis, Genome-wide Analysis using ChIP to Identify Isoform-specific Gene Targets, Assessing Replication and Beta Cell Function in Adenovirally-transduced

Sigma-Aldrich offers abstracts and full-text articles by [Lily Mahapatra, Chengjian Mao, Neal Andruska, Chen Zhang, David J Shapiro].

The mucin MUC16 and the glycosylphosphatidylinositol anchored glycoprotein mesothelin likely facilitate the peritoneal metastasis of ovarian tumors. The biochemical basis and the kinetics of the binding between these two glycoproteins are not clearly understood. Here we have addressed this deficit and provide further evidence supporting the role of the MUC16-mesothelin interaction in facilitating cell-cell binding under conditions that mimic the peritoneal environment. In this study we utilize recombinant-Fc tagged human mesothelin to measure the binding kinetics of this glycoprotein to MUC16 expressed on the ovarian tumor cell line OVCAR-3. OVCAR-3 derived sublines that did not express MUC16 showed no affinity for mesothelin. In a flow cytometry-based assay mesothelin binds with very high affinity to the MUC16 on the OVCAR-3 cells with an apparent Kd of 5-10 nM. Maximum interaction occurs within 5 mins of incubation of the recombinant mesothelin with the OVCAR-3 cells and significant binding is

Our main objectives are to identify key actors of RNA degradation pathways in plants, and to determine their impact on genome expression, development or stress response. Our studies currently focus on new co-factors of the RNA exosome, on enzymes that adenylate or uridylate RNAs and on new factors associated to P-bodies and decapping activators. Finally, we address the roles of RNA degradation pathways during viral infections.. Our main experimental strategies include forward and reverse genetics in the model plant Arabidopsis thaliana, protein biochemistry approaches coupled to mass spectrometry analyses, and new high-throughput techniques based on Illumina and Nanopore sequencing to identify 3 modifications of transcripts.. Key funding of our current research includes the NetRNA LabEx (2011-2028) and the ANR grants 3modRN (2015-2021) and URIVir (2021-2025).. ...

As a valuable aid in the construction of AOPs, toxicogenomics can be used to identify toxicity pathways or perturbed biology in response to different classes of substances. This knowledge can then be used to derive dose-response, identifying appropriate biological models pertinent to assessing the adverse outcome, and establishment of high throughput assays for screening or prioritizing.. ...

Using a novel screening platform to rapidly evaluate the cellular effects of 1,000 chemical compounds, a team led by UC San Francisco scientists has identified eight drugs that may stimulate nervous system repair in multiple sclerosis (MS).. All eight compounds have previously been approved by the U.S. Food and Drug Administration (FDA) for the treatment of other conditions. One of the most promising agents is an antihistamine, though the scientists caution that MS patients should not use the drug until clinical trials have established whether it can safely and effectively treat MS, and if it does, what the proper dosages and treatment regimens would be. Because of the drugs emergence as a clear front-runner in the new study, a Phase 2 clinical trial to evaluate its effectiveness in MS is already underway at UCSF.. A major unmet need in the development of therapeutics for repair in MS has been the ability to screen compounds in a high-throughput manner, said Jonah Chan, PhD, the Debbie and ...

In this project, we develop single-cell manipulation surfaces on which cells are rapidly adhered or released on/from desired positions at a single-cell level. On the surfaces coated with photo-responsive materials, multiple cells can be freely patterned and arrayed in a light-guided manner, and accordingly, the properties and interactions of individual cells are accurately investigated in a high-throughput manner. Furthermore, only the desired cells are selectively obtained by light irradiation. This technology is expected to be applied to isolation of cancer cells from blood samples for assessment of cancer therapy and purification of functional cells for cell therapy and tissue engineering.. ...

Previous efforts toward preparing multicellular aggregates (spheroids) have been made in traditional rocker-plate [1], porous foam block [2], and microarray chip cultures [3] in order to maintain liver-specific functions in vitro. These approaches all employ static culture methods and thus physiological flow conditions could not be simulated. Furthermore, the ability to analyze cell viability and function in a high-throughput manner is hindered due to the opaque substrates used in all three systems. To effectively coalesce otherwise monolayer liver cells seeded into microfluidic channels,we present a high-density hepatic spheroid trapping array chip for rapid three-dimensional spheroid self-assembly. Microfabricated 3-D bioreactors address many of the issues associated with traditional liver cell culture such as providing more in vivo-like spatial cell-cell interactions [4]. A platform that closely mimics the in vivo architecture of the human liver has yet to be achieved. We aim to bring hepatic ...

Mass-spectrometry-based proteomics enables the high-throughput identification and quantification of proteins, including sequence variants and post-translational modifications (PTMs) in biological samples. However, most workflows require that such variations be included in the search space used to analyze the data, and doing so remains challenging with most analysis tools. In order to facilitate the search for known sequence variants and PTMs, the Proteomics Standards Initiative (PSI) has designed and implemented the PSI extended FASTA format (PEFF). PEFF is based on the very popular FASTA format but adds a uniform mechanism for encoding substantially more metadata about the sequence collection as well as individual entries, including support for encoding known sequence variants, PTMs, and proteoforms. The format is very nearly backward compatible, and as such, existing FASTA parsers will require little or no changes to be able to read PEFF files as FASTA files, although without supporting any of the

RNA sequencing (RNASeq) provides the ability to comprehensively assay the transcriptome in a high-throughput manner. Current there are a variety of library preparation methodologies for measuring and sequencing the transcriptome depending on (a) the sample source and (b) outcomes of interest. Beyond protocol selection, the requisite computational tools and resources are significant considerations in processing, analyzing and reporting the experimental results. While there are many resources readily available to effectively perform RNA-seq experiments, optimal protocols and analysis tools for the cancer domain remain to be developed.. We have developed and characterized a set of protocols and analysis procedures that comprise an RNA-seq pipeline that can effectively be used in a cancer research setting. The analysis pipeline consists of a sequence of functions and tools to process and clean the raw data, generate quality control and summary metrics, and perform secondary analyses that include ...

Methods are provided for detecting biomolecular interactions. The use of labels is not required and the methods can be performed in a high-throughput manner. The invention also relates to optical devices.

TY - JOUR. T1 - YAP and TAZ regulate cell volume. AU - Perez-Gonzalez, Nicolas A.. AU - Rochman, Nash D.. AU - Yao, Kai. AU - Tao, Jiaxiang. AU - Le, Minh Tam Tran. AU - Flanary, Shannon. AU - Sablich, Lucia. AU - Toler, Ben. AU - Crentsil, Eliana. AU - Takaesu, Felipe. AU - Lambrus, Bram. AU - Huang, Jessie. AU - Fu, Vivian. AU - Chengappa, Pragati. AU - Jones, Tia M.. AU - Holland, Andrew J.. AU - An, Steven. AU - Wirtz, Denis. AU - Petrie, Ryan J.. AU - Guan, Kun Liang. AU - Sun, Sean X.. N1 - Funding Information: This work has been funded in part by National Institutes of Health grants R01GM114674 and U54CA210172. The authors declare no competing financial interests. Publisher Copyright: © 2019 Perez-Gonzalez et al.. PY - 2019/10/7. Y1 - 2019/10/7. N2 - How mammalian cells regulate their physical size is currently poorly understood, in part due to the difficulty in accurately quantifying cell volume in a high-throughput manner. Here, using the fluorescence exclusion method, we demonstrate ...

TY - JOUR. T1 - Introducing discrete frequency infrared technology for high-throughput biofluid screening. AU - Hughes, Caryn. AU - Clemens, Graeme. AU - Bird, Benjamin. AU - Dawson, Timothy. AU - Ashton, Katherine M.. AU - Jenkinson, Michael D.. AU - Brodbelt, Andrew. AU - Weida, Miles. AU - Fotheringham, Edeline. AU - Barre, Matthew. AU - Rowlette, Jeremy. AU - Baker, Matthew J.. PY - 2016/2/4. Y1 - 2016/2/4. N2 - Accurate early diagnosis is critical to patient survival, management and quality of life. Biofluids are key to early diagnosis due to their ease of collection and intimate involvement in human function. Large-scale mid-IR imaging of dried fluid deposits offers a high-throughput molecular analysis paradigm for the biomedical laboratory. The exciting advent of tuneable quantum cascade lasers allows for the collection of discrete frequency infrared data enabling clinically relevant timescales. By scanning targeted frequencies spectral quality, reproducibility and diagnostic potential ...

Colorectal tumor (CRC) is the third most common cancer and the fourth leading cause of cancer death in the world. as FGFR EGFR CD44 EpCAM CA IX PPARγ and COX-2) overexpressed by cancerous cells have also been shown to be effective. This review aims to put forth an overview of drug delivery technologies that have been and may be developed for the treatment of CRC. [19] found that treatment with a combination of Irinotecan (Camptosar Pharmacia) a potent inhibitor of topoisomerase I 5 and Leucovorin resulted in significantly longer progression-free survival (median 7 4.3 months; = 0.004) greater confirmed response (39% 21% < 0.001) and longer overall survival (median 14.8 12.6 months; = 0.04) than 5-FU/Leucovorin alone. LY335979 An extensive body of data shows that Fluoropyrimidines Irinotecan and Oxaliplatin have emerged as cornerstones of chemotherapy for CRC. However these traditional pharmaceutical therapeutic regimens are usually accompanied by severe mucositis myelosuppression and cumulative ...

Data Availability StatementThe datasets used and/or analyzed through the present research are available through the corresponding writer on reasonable demand. no factor between organizations B and C (P 0.05). Tumor mass in organizations B, C and D was considerably less than that in group A (P 0.05), which in group D was significantly less than that in organizations B and C (P 0.05), whereas there MK-1775 cost is no factor between organizations B and C (P 0.05). Weighed against organizations C and B, mice in group D got considerably lower IL-6 level (P 0.05), but significantly higher IL-12 level (P 0.05). There is no factor in IL-6 and IL-12 amounts between organizations B and C (P 0.05). To conclude, erlotinib coupled with cisplatin KRT20 can inhibit the tumor development of mice with LLC, and inhibition of IL-6 known level and upregulation of IL-12 level could be among its therapeutic systems. (10), there is absolutely no factor in effectiveness between erlotinib and chemotherapy MK-1775 cost ...

Protein interactions with low molecular weight ligand have great implication in biology for both allosteric regulation and enzymatic activity. Another important...

An awful lot of drug discovery comes down (sooner or later) to screening compound collections. This has been true for a long time now, and it doesnt look like its going away, either. So with that in mind, whats in your collection? Did you buy a bunch of stuff from the vendors to fill it out? If so, your chemical

High-throughput plasma separation based on atomic mass holds promise for offering unique solutions to a variety of high-impact societal applications. Through the mass differential effects they exhibit, crossed-field configurations can in principle be exploited in various ways to separate ions based on atomic mass. Here, we review some of the E x B mass filter concepts proposed to date and underline how the practicality of these concepts is conditioned upon the ability to sustain a suitable perpendicular electric field in a plasma for parameters compatible with high-throughput operation. We show that while the limited present predictive capabilities do not make it possible to confirm this possibility, past experimental results suggest that end-electrode biasing may be effective, at least for certain electric field values. We conclude that a better understanding of cross-field conductivity is needed to confirm these results and confirm the potential of crossed-field configurations for high-throughput

Protein-protein interactions (PPIs) play a critical role in all cellular processes, ranging from cellular division to apoptosis. Elucidating and analyzing PPIs is thus essential to understanding the underlying mechanisms in biology. Indeed, this has been a major focus of research in recent years, providing a wealth of experimental data about protein associations [1-9]. Current PPI networks have been constructed using a number of techniques, such as yeast-two-hybrid (Y2H), co-immunopurification or coaffinity purification, followed by mass spectroscopy and curation of published low-throughput experiments [10-16]. Despite this tremendous push, the current coverage of PPIs is still rather poor (for example, , 10% of interactions in humans) [17]. Additionally, despite considerable improvements in high-throughput (HTP) techniques, they are still prone to spurious errors and systematic biases, yielding a significant number of false-positives and false-negatives [18-21]. This limitation impedes our ...

TY - JOUR. T1 - Probabilistic modeling of personalized drug combinations from integrated chemical screen and molecular data in sarcoma.. AU - Berlow, Noah. AU - Rikhi, R. AU - Geltzeiler, M. AU - Abraham, J. AU - Svalina, M N. AU - Davis, L E. AU - Wise, E. AU - Mancini, M. AU - Noujaim, J. AU - Mansoor, A. AU - Quist, M J. AU - Matlock, Kevin. AU - Goros, M W. AU - Hernandez, B S. AU - Doung, Y C. AU - Thway, K. AU - Tsukahara, T. AU - Nishio, J. AU - Huang, E T. AU - Airhart, S. AU - Bult, C J. AU - Gandour-Edwards, R. AU - Maki, R G. AU - Jones, R L. AU - Michalek, J E. AU - Milovancev, M. AU - Ghosh, Souparno. AU - Pal, Ranadip. AU - Keller, C. PY - 2019/6/17. Y1 - 2019/6/17. M3 - Article. SP - 593. JO - Default journal. JF - Default journal. ER - ...

Stem cell platform recreates the human intestinal epithelium. Altis Biosystems, Inc. is a preclinical contract research organization whose patent-pending stem cell platform recreates the human intestinal epithelium in a high-throughput format for drug screening and microbiome research. Altis provides a variety of services for customers, including toxicology, permeability, ELISA, transport, custom cell isolation, and more. In addition to providing services utilizing its platform, Altis is able to ship its platform to customers as well.. ...

High-throughput screening of cellular effects of RNA interference (RNAi) libraries is now being increasingly applied to explore the role of genes in specific cell biological processes and disease states. However, the technology is still limited to specialty laboratories, due to the requirements for robotic infrastructure, access to expensive reagent libraries, expertise in high-throughput screening assay development, standardization, data analysis and applications. In the future, alternative screening platforms will be required to expand functional large-scale experiments to include more RNAi constructs, allow combinatorial loss-of-function analyses (e.g. genegene or gene-drug interaction), gain-of-function screens, multi-parametric phenotypic readouts or comparative analysis of many different cell types. Such comprehensive perturbation of gene networks in cells will require a major increase in the flexibility of the screening platforms, throughput and reduction of costs. As an alternative for ...

Workstation, apparatuses and methods for the high-throughput synthesis, screening and/or characterization of combinatorial libraries. The invention relates to an array, which permits various high-throughput methods for synthesis, screening and/or characterization in the same array, without requiring sample transfer from the array. In a preferred embodiment, the synthesis, screening, and/or characterization steps are carried out in a highly parallel fashion, where more than one compound is synthesized, screened, and/or characterized at the same time. The invention may be practiced at the microscale. The array may comprise thermal channels, for regulating the temperature of the wells in the array. The wells of the array may comprise a membrane, which is used in various screening and characterization methods. The invention also relates to a covered array, comprising the array and an array cover, as well as an apparatus comprising the array, which comprises the array, an array cover and a stage. The array,

Fernández did not need to go on a hiking trip to find the answers. Instead, together with a number of other PhD fellows, she conducted a two-week clinical study at the ACTA (Academic Centre for Dentistry Amsterdam) research labs - one of the scientific partners in the TiFN Oral Health project. Sixty-one volunteers agreed to abstain from all oral hygiene activities during the trial period, and had their saliva tested regularly. Fernández developed and optimised an automated high-throughput scratch assay connected to a mathematical model. The aim was to measure metabolite levels in saliva over time, and link them to re-epithelialization - an essential component of wound healing - in the gums, she explains. Setting up the testing procedure was not easy, she stresses: I was the only one in our project team working with high-throughput microscopy, so I had to learn all about it and start up the procedure by myself.. Fernández succeeded in identifying the metabolite signatures of 10 ...

PubMed Identifiers (PMIDs) point to literature references that support an interaction. A PMID may be used to support more than one interaction. The lpr score (lowest PMID re-use) is the lowest number of distinct interactions (RIGIDs: see column 35) that any one PMID (supporting the interaction in this row) is used to support. A value of one indicates that at least one of the PMIDs supporting this interaction has never been used to support any other interaction. This likely indicates that only one interaction was described by that reference and that the present interaction is not derived from high throughput methods. The hpr score (highest PMID re-use) is the highest number of interactions (RIGIDs: see column 35) that any one PMID (supporting the interaction in this row) is used to support. A high value (e.g. greater than 50) indicates that one PMID describes at least 50 other interactions and it is more likely that high-throughput methods were used. The np score (number PMIDs) is the total ...

BioAssay record AID 1256 submitted by The Scripps Research Institute Molecular Screening Center: Counterscreen assay for antagonists of neuropeptide Y receptor Y2 (NPY-Y2): Cell-based high throughput assay to measure NPY-Y1 antagonism..

Despite significant public health impact, there is no specific antiprotozoal therapy for prevention and treatment of Acanthamoeba castellanii infection. There is a need for new and efficient anti-Acanthamoeba drugs that are less toxic and can reduce treatment duration and frequency of administration. In this context a new, rapid and sensitive assay is required for high-throughput activity testing and screening of new therapeutic compounds. A colorimetric assay based on sulforhodamine B (SRB) staining has been developed for anti-Acanthamoeba drug susceptibility testing and adapted to a 96-well microtiter plate format. Under these conditions chlorhexidine was tested to validate the assay using two clinical strains of A. castellanii (Neff strain, T4 genotype [IC50 4.68±0.6 _M] and T3 genotype [IC50 5.69±0.9 _M]). These results were in good agreement with those obtained by the conventional Alamar Blue assay, OCR cytotoxicity assay and manual cell counting method. Our new assay offers an ...

Development of high throughput assays is a crucial step in developing more efficient techniques that aid in many important areas of research today such as drug development or identification of protein structure function relationships. Integration of high throughput assays into more research efforts could drastically decrease the time and cost it takes for a new drug to hit the market. Protein Kinase A (PKA) is an extensively studied protein as it is highly upregulated in cancer and is a hot spot for drug targeting. In this work, azide-tagged PKA is covalently attached to magnetic beads using azide-alkyne cycloaddition, a well-known click chemistry reaction that selectively and covalently links two compounds. Modified PKA is attached to magnetic beads and the activity of the covalently bound PKA is determined. Significant levels of PKA activity can open the door to development of more efficient drug screening processes. It is anticipated that the azide-PKA conjugated beads will have significantly more

Hepatitis C virus (HCV) poses a major health threat to the world. The recent development of direct-acting antivirals (DAAs) against HCV has markedly improved the response rate of HCV and reduced the side effects in comparison to the interferon-based therapy. Despite this therapeutic advance, there is still a need to develop new inhibitors that target different stages of the HCV life cycle because of various limitations of the current regimens. In this study, we performed a quantitative high throughput screening of the Molecular Libraries Small Molecule Repository (MLSMR) of ∼350,000 chemicals for novel HCV inhibitors using our previously developed cell-based HCV infection assay. Following confirmation and structural clustering analysis, we narrowed down to 158 compounds from the initial ∼3000 molecules that showed inhibitory activity for further structural and functional analyses. We were able to assign the majority of these compounds to specific stage(s) in the HCV life cycle. Three of them ...

Menthol is a cooling compound derived from mint leaves and is extensively used as a flavoring chemical. Menthol activates transient receptor potential melastatin 8 (TRPM8), an ion channel also activated by cold, voltage and phosphatidylinositol-4,5-bisphosphate (PIP2). Here we investigated the mechanism by which menthol activates mouse TRPM8. Using a new high-throughput approach, we screened a random mutant library consisting of approximately 14,000 individual TRPM8 mutants for clones that are affected in their response to menthol while retaining channel function. We identified determinants of menthol sensitivity in two regions: putative transmembrane segment 2 (S2) and the C-terminal TRP domain. Analysis of these mutants indicated that activation by menthol involves a gating mechanism distinct and separable from gating by cold, voltage or PIP2. Notably, TRP domain mutations mainly attenuated menthol efficacy, suggesting that this domain influences events downstream of initial binding. In ...

L. David Sibley at Washington University in St. Louis in the U.S. is developing a long-term in vitro intestinal epithelial culture system for the intracellular parasite Cryptosporidium, which causes severe diarrheal disease in both humans and animals, and is refractory to many anti-parasitic drugs. Currently, Cryptosporidium can only be grown in infected calves or in short-term in vitro cultures, which cannot be used for the high-throughput chemical screens needed to identify new drugs. In Phase I, they optimized the in vitro culture of isolated intestinal stem cells from human and mouse biopsies, and identified factors to control their differentiation into primary epithelial monolayers, which can better support the growth of intestinal pathogens. This led to around a five-fold increase in the rate of asexual replication of Cryptosporidium, which was enough to successfully test a chemical growth inhibitor. In Phase II, they will further improve culture conditions to support longer-term in vitro ...

L. David Sibley at Washington University in St. Louis in the U.S. is developing a long-term in vitro intestinal epithelial culture system for the intracellular parasite Cryptosporidium, which causes severe diarrheal disease in both humans and animals, and is refractory to many anti-parasitic drugs. Currently, Cryptosporidium can only be grown in infected calves or in short-term in vitro cultures, which cannot be used for the high-throughput chemical screens needed to identify new drugs. In Phase I, they optimized the in vitro culture of isolated intestinal stem cells from human and mouse biopsies, and identified factors to control their differentiation into primary epithelial monolayers, which can better support the growth of intestinal pathogens. This led to around a five-fold increase in the rate of asexual replication of Cryptosporidium, which was enough to successfully test a chemical growth inhibitor. In Phase II, they will further improve culture conditions to support longer-term in vitro ...

Because of its permeability to small molecules, zebrafish can be used for testing and screening of drugs affecting different biological processes.50-52 Single and multiple compounds tests can be easily and successfully achieved on both zebrafish embryos and adults.53,54 The reason for this resides in the fact that (1) a high quantity of zebrafish embryos (≈5000) can be obtained synchronously, and (2) the small size of the embryos allows them to fit in a 384-well plate and thus allows high-throughput analyses. In this way, libraries of thousands of compounds can be screened for their effects in a reasonable time frame (eg, weeks). Progresses for drug screening in zebrafish embryos are also subjected to automatic readout for phenotypic effects. An automated high-throughput platform for in vivo chemical screenings on zebrafish embryos has been developed aiming for the highest possible throughput and minimization of human error. It includes an automated method for embryo collection and ...

Orlando Fla. (PRWEB) January 14 2013 Genedata a leading provider of advanced software solutions for drug discovery and life science research today announced that more than half of the worlds top 25 pharmaceutical companies have adopted Genedata Screener as their plate-based screening analysis platform. Also used by biotechnology companies contract researc,Genedata,Screener,Adopted,for,all,Plate-based,Screening,by,Major,Pharmas,biological,advanced biology technology,biology laboratory technology,biology device technology,latest biology technology

Recently developed technologies have enabled multi-well measurement of O2 consumption, facilitating the rate of mitochondrial research, particularly regarding the mechanism of action of drugs and proteins that modulate metabolism. Among these technologies, the Seahorse XF24 Analyzer was designed for use with intact cells attached in a monolayer to a multi-well tissue culture plate. In order to have a high throughput assay system in which both energy demand and substrate availability can be tightly controlled, we have developed a protocol to expand the application of the XF24 Analyzer to include isolated mitochondria. Acquisition of optimal rates requires assay conditions that are unexpectedly distinct from those of conventional polarography. The optimized conditions, derived from experiments with isolated mouse liver mitochondria, allow multi-well assessment of rates of respiration and proton production by mitochondria attached to the bottom of the XF assay plate, and require extremely small quantities

Hosted by the International Society for Computational Biology (ISCB) and the Centre for Genomic Regulation (CRG), the Next Generation Sequencing Conference 2014 (NGS 2014) is a dedicated meeting on cutting-edge approaches to the processing and analysis of Next Generation Sequencing data. The goal of this conference is to bring together bioinformatics researchers and biologists facing new high-throughput sequencing challenges. The conference will feature presentations showing how current platforms can be used to address key biological questions and what is the current state of the art for data analysis. Sizeable space will also be dedicated to emerging and future trends in high-throughput sequencing and their associated computational challenges.. see more at http://www.iscb.org/ngs2014. ...

Small-molecule microarrays composed of tens of thousands of distinct synthetic molecules, natural products, and their combinations/modifications provide a high-throughput platform for studying protein-ligand interactions. Immobilization of small molecule compounds on solid supports remains a challenge as widely varied small molecules generally lack unique chemical groups that readily react with singly or even multiply functionalized solid support. We explored two strategies for immobilizing small molecule compounds on epoxy-functionalized glass surface using primary-aminecontaining macromolecular scaffolds: bovine serum albumin (BSA) and amine-modified poly-vinyl alcohol (PVA). Small molecules with N-hydroxysuccinimide (NHS) groups were conjugated to BSA or amine-modified PVA. Small-molecule-BSA conjugates and small-molecule-PVA conjugates were subsequently immobilized on epoxy-functionalized glass slides through amine-epoxy reactions. Using an oblique-incidence reflectivity difference (OI-RD) ...

Deregulation of c-Myc at the transcriptional level involves mutation, translocation, and amplification of the myc gene, which makes targeting c-Myc at the gene expression level not an easy task. Regulation of c-Myc activity at the protein level is an attractive approach, but it is often indirect, as the protein lacks a clear ligand-binding domain for direct interaction (9). Thus, an effective targeting of c-Myc protein requires good understanding of its signaling pathways and regulation networks. However, there are currently more than 700 binary interactions of c-Myc according to the EBI IntAct, a comprehensive molecular interaction database, whereas new interactions keep adding to the list (10). Therefore, to identify an effective indirect target out of its complex network by examining the effect of each interaction is clearly labor intensive and often misses the network effect. Systematic profiling of drug-inhibitory effect on c-Myc not only can help identify inhibitors of a specific ...

Laromustine is an experimental sulfonylhydrazine prodrug used in late-stage clinical studies against acute myeloid leukemia (AML) and glioblastoma multiforme (GBM). Despite initial promise for both indications, clinical trials for GBM have not been as successful as those for AML. To investigate methods for improving the effectiveness of laromustine in GBM and to learn more about the mechanism of action of laromustine, a chemical genetic screen will be conducted to identify agents that sensitize GBM cells to the anti-proliferative effects of laromustine. The library, which will include approximately 450 FDA-approved drugs, will be screened using a newly optimized high throughput assay based on the Click-iT EdU Microplate Assay kit (Molecular Probes). Optimization of the assay has required determining the proper cell seed density, drug concentration and incubation time, and fluorescent substrate concentration, among other variables. It was determined that low cell seed densities allow for maximal

The selection, acquisition, and use of high‐quality small molecule libraries for screening is an essential aspect of drug discovery and chemical biology programs

Utility of WGAs for genomic analysis. We used a strategy for mapping transcription units that included both a traditional cDNA cloning and DNA sequencing approach and a novel high-throughput approach that utilizes tiling array technology (12, 13, 25). Sequencing ESTs and fl-cDNAs verified 40% of the annotated genes, dramatically improved genome annotation, and allowed the construction of 30% of the ORFeome. As with other large-scale cDNA-based gene collection projects (8, 15, 18), the traditional approach of sequencing fl-cDNAs reaches a point of diminishing returns: two-thirds of the total annotated Arabidopsis genes still have no corresponding fl-cDNA. The WGA technology allowed us to identify transcripts for the ANE genes and enabled construction of the remaining 30% of the Arabidopsis ORFeome. We were able to detect ∼60% of the total annotated genes (AE and ANE) using only a limited number of RNA samples (four). A more comprehensive survey of tissues and cell types should enable a more ...

Author SummaryPathogenic bacteria that can be transferred from animals to humans represent a highly potent human health hazard. Understanding the ecology of these pathogens in the animal host is of fundamental importance. A major analytical challenge, however, is the fact that individual animal hosts can be colonised by multiple strains of a given pathogen. We have addressed this challenge by developing a novel high-throughput approach for analyses of mixed strain infections. We chose Campylobacter jejuni colonisation of the chicken gastrointestinal (GI) tract as a model. C. jejuni is a major cause of food-borne disease in humans, and chickens are considered a main reservoir from which this bacterium may enter the food chain. We analysed the co-colonisation of seven C. jejuni strains in two groups of chickens with very different background GI microfloras. We found that mainly two of the C. jejuni strains colonised the chickens, with a shift in the dominant coloniser during the infection period. The C.

The 1000 Genomes Project aims to provide a deep characterization of human genome sequence variation as a foundation for investigating the relationship between genotype and phenotype. Here we present results of the pilot phase of the project, designed to develop and compare different strategies for genome-wide sequencing with high-throughput platforms. We undertook three projects: low-coverage whole-genome sequencing of 179 individuals from four populations; high-coverage sequencing of two mother-father-child trios; and exon-targeted sequencing of 697 individuals from seven populations. We describe the location, allele frequency and local haplotype structure of approximately 15 million single nucleotide polymorphisms, 1 million short insertions and deletions, and 20,000 structural variants, most of which were previously undescribed. We show that, because we have catalogued the vast majority of common variation, over 95% of the currently accessible variants found in any individual are present in this data

The 1000 Genomes Project aims to provide a deep characterization of human genome sequence variation as a foundation for investigating the relationship between genotype and phenotype. Here we present results of the pilot phase of the project, designed to develop and compare different strategies for genome-wide sequencing with high-throughput platforms. We undertook three projects: low-coverage whole-genome sequencing of 179 individuals from four populations; high-coverage sequencing of two mother-father-child trios; and exon-targeted sequencing of 697 individuals from seven populations. We describe the location, allele frequency and local haplotype structure of approximately 15 million single nucleotide polymorphisms, 1 million short insertions and deletions, and 20,000 structural variants, most of which were previously undescribed. We show that, because we have catalogued the vast majority of common variation, over 95% of the currently accessible variants found in any individual are present in this data

Protein microarrays provide a high-throughput platform that enables the profiling of serum antibodies to a large number of protein antigens. The value of AAb biomarkers in diagnosis, prognosis and treatment is well recognized in autoimmune diseases including T1D. We performed a systematic screening …

DNS has identified protein targets for drug discovery in two complementary ways. The first approach was to develop a high-throughput cell-based assay to screen for compounds that modulated a CRE-luciferse reporter gene in human neuroblastoma cell lines. From a 180,000 compound library, hundreds of confirmed hits were found and 10 with novel structures were selected for further study. Biochemical analyses revealed that 8 out of 10 of these compounds were inhibitors of PDE4, and one was an inhibitor of MAO-B. The second approach was to use DNA microarrays to identify genes that responded transcriptionally during long-term memory formation. GlyT1 and most of the novel targets derive from this approach.. ...

Project Title: Development of high throughput assay for screening of novel DNA replication inhibitors for therapeutic purposes Supervisors: Professor Christian Speck, Professor David Rueda Funding: Tuition fees plus £21,000 pa stipend for 3.5 years Date posted: 09 July 2021 Closing date: 04 August 2021 The student will develop a fluorescence-based assay to identify novel DNA replication inhibitors for anti-cancer therapy. Inhibitors will be consequently characterised for their impact on the multi-step DNA replication process and on cancer cell growth. This interdisciplinary project will train the student in biochemistry, biophysics and drug screening. Project details , LMS 3.5-year Studentships , Apply ...

The effects of a novel high intensity exercise intervention on established markers of cardiovascular disease and health in Scottish adolescent youth

About 71% of UK land is committed to agriculture and farmers are the primary managers of land. There is little data on how farming practice affects the biological capacity and function of soils. Currently the majority of existing tests for soil management focus on physical and chemical parameters and disregard biological function. However soil biology is not only intrinsically linked to the physical and chemical properties of soil, but also vital for healthy soils to function as living systems.. Through this KESS 2 funded project we aim to tailor and validate a commercial scale, high throughput assay to help Welsh farmers assess the quality and health of their soils. We will evaluate relationships between the abundance of microbial functional groups (using GCMS- Fatty Acid Methyl Ester (FAME) based tests) and the soil health status by measuring field performance across a wide range of soil types, crops and farming systems. Ultimately seeking the provision of a robust test to identify microbial ...

BACKGROUND: Human cystatin C is a cysteine protease inhibitor produced by all nucleated cells in the body and the protein is present in all body fluids. The concentration in cerebrospinal fluid (CSF) is considerably higher than in plasma. Cystatin C levels seem to influence the development of Alzheimer disease (AD) and low levels in the brain are associated with an increased risk for AD. The aim of this study was to develop a high throughput assay for the quantification of cystatin C in CSF. METHODS: Antigen excess, imprecision, interference, linearity, recovery, sample stability and reference values were evaluated on Architect ci8200 (Abbott Laboratories, Abbott Park, IL, USA). RESULTS: The assay had an antigen-excess limit at 23 mg/L and was linear over the range of 0.84 to 8.33 mg/L. Results , 8.33 mg/L were automatically rerun in a higher dilution. Within-run coefficient of variation (CV) was 1.71, 1.10 and 0.79%, between day CV was 1.71, 0.39 and 1.45%, between-run CV was 0.58, 0.66 and ...

Human saliva has been used as an auxiliary biological fluid for disease diagnosis (1-3), because the biomolecular composition of saliva changes in a disease state. Total RNA extracted from saliva is useful in determining gene expression profiles in cancer, as well as in genetic studies (4, 5). Salivary RNA is prone to degradation by RNases and other nucleases present in saliva, and therefore care should be taken to alleviate or minimize sample degradation before downstream transcriptomic analysis. Although the number of methods for isolating RNA from saliva has markedly increased over the years, most of the protocols remain relatively costly, owing to the predominant use of commercial kits (6) and, more importantly, the relatively low RNA yields. Furthermore, a large body of literature on transcriptomic studies of saliva has considered only the cell-free salivary supernatant as a biological source for isolating RNA, as opposed to the salivary cellular pellet (7-10). New high-throughput ...

A screening tool from the U.S. Department of Energys National Renewable Energy Laboratory (NREL) eases and greatly quickens one of the thorniest tasks in the biofuels industry: determining cell wall chemistry to find plants with ideal genes.. NRELs new High-Throughput Analytical Pyrolysis tool (HTAP) can thoroughly analyze hundreds of biomass samples a day and give an early look at the genotypes that are most worth pursuing. Analysis of a sample that previously took two weeks can now be done in two minutes. That is potentially game changing for tree nurseries and the biomass industry.. When it comes to making fuels out of trees, crops, grasses, or algae, its all about the cell walls of the plants. Will they make it hard or easy for enzymes to turn the biomass into sugars? Differences in cell walls are enormous, and choosing the right ones can make the difference between a profit and a loss for tree growers, or between a fruitful or fruitless feedstock line for biomass companies.. Finding that ...

The PiggyBac (PB) transposon has emerged as a novel mutagenesis tool for understanding gene function and for phenotypic screening in eukaryotes

Extracellular vesicles (EVs) are nanosized lipid bilayer-bound vesicles that are naturally secreted from most cell types as a communication mechanism to deliver proteins, lipids, and genetic material. Despite the therapeutic potential of EVs, there is limited information on EV uptake kinetics and specificity. Here, we optimized an imaging flow cytometry (IFC)-based platform to quantitatively assess dose, time, and recipient cell specificity effects on human embryonic kidney cell (HEK293T) EV internalization in a high-throughput manner. We found that HEK293T EV uptake is an active process that is dose and time dependent. Further, the selectivity of EV uptake was quantified in vitro, and we found that HEK293T EVs were internalized at higher quantities by cells of the same origin. Lastly, neural stem cells internalized significantly more HEK293T EVs relative to mature neurons, suggesting that stem cells or progenitors, which are more metabolically active than terminally differentiated cells, may have

Hypertrophic scar (HS) formation is associated with the fibrosis of fibrocytes caused by excessive extracellular matrix (ECM) synthesis and deposition, the initial event of HS formation. Our high throughput screen of miRNA expression profiles identified hsa-miR31-5p, whose transcription level was most differentially in normal skin fibroblasts (NS) and HS among other miRNAs. The level of hsa-miR31-5p in HS was significantly higher than in NS. In-vitro functional experiments showed hsa-miR31-5p knockdown remarkably suppressed the proliferation of hypertrophic scar fibroblasts (HSFBs) under hypoxia, promoted cell invasion, and inhibited the expression of Collagen I and III and Fibronectin (FN), suggesting that hsa-miR31-5p knockdown effectively reduces HS formation caused by excessive ECM synthesis and deposition in HSFBs under hypoxia ...

|br /> Shiga toxin-producing |em>E. coli|/em> (STEC) causes approximately 176,000 human illnesses in the US each year. These organisms reside and propagate in the gastrointestinal tract of cattle and serve as a major source of food and water contamination when they are shed in the feces. The top 7 STEC serogroups causing human infections are O157, O26, O103, O111, O121, O45, and O145. Four major virulence factors are commonly associated with more severe infections: Shiga toxin 1 and 2 (|em>stx|/em>1, |em>stx|/em>2), intimin (|em>eae|/em>), and enterohemolysin (|em>ehx|/em>A). This webinar will discuss the development of an 11-gene multiplex endpoint PCR assay to detect and differentiate the top 7 STEC serogroups and the 4 major virulence factors in a high-throughput manner.|br /> |br /> Most multiplex real-time PCR assays are limited to 2–3 targets due to design difficulties. End-point PCR can detect more targets than real-time PCR, but is laborious due to the need to visualize results

TABLE-US-00002 TABLE 2 Sugars used in the sugar-drug conjugates of the present invention. D-lyxoside D-lactoside 6-deoxy-D-glucoside L-rhamnoside 3-deoxy-D-glucoside D-maltoside D-xyloside N-acetyl-D-glucosaminoside D-riboside D-cellobioside 2-deoxy-D-galactoside 6-deoxy-6-chloro-D-galactoside L-lyxoside 6-deoxy-6-bromo-D-galactoside L-glucoside 6-deoxy-6-azido-D-galactoside D-fucoside 4-deoxy-4-azido-D-glucoside L-mannoside D-glucorono-6,3-lactonide D-alloside 2-deoxy-2-amino-D-glucoside L-galactoside 3-O-methyl-D-glucoside maltotrioside 2,3,4-tri-O-acetyl-L-rhamnoside L-fucoside mycaroside D-glucuronoside 2,3,4,6-tetra-O-benzyl-D- glucopyranoside 2-deoxy-D-glucoside 2,3,4-tri-O-benzyl -L-fucopyranoside L-xyloside 2,3,5-tri-O-benzyl -D- arabinofuranoside 3-amino-3-deoxy-L-xyloside 2,3,5-tri-O-benzyl-D-ribofuranoside 3-azido-3-deoxy-L-xyloside 6-deoxy-6-fluoro-D-glucoside 3-thio-3-deoxy-L-xyloside 4-O-(quadrature-D-galacto pyranosyl-D- mannopyranoside) D-Galacturonoside ...

The interpretation of genome sequences requires reliable and standardized methods to assess protein function at high throughput. Here we describe a fast and reliable pipeline to study protein function in mammalian cells based on protein tagging in bacterial artificial chromosomes (BACs). The large size of the BAC transgenes ensures the presence of most, if not all, regulatory elements and results in expression that closely matches that of the endogenous gene. We show that BAC transgenes can be rapidly and reliably generated using 96-well-format recombineering. After stable transfection of these transgenes into human tissue culture cells or mouse embryonic stem cells, the localization, protein-protein and/or protein-DNA interactions of the tagged protein are studied using generic, tag-based assays. The same high-throughput approach will be generally applicable to other model systems.

High-throughput screening remains one of the most powerful, unbiased approaches for small molecule drug discovery.. Today, the toolbox for high-throughput screening features traditional label-dependent approaches and novel label-free technologies. Researchers determine which methodology offers the most promising path forward for their target, taking into account assay sensitivity, data quality, costs, and speed. Moreover, many companies opt to outsource drug discovery efforts to contract research organizations, which offer a particular expertise and an established discovery infrastructure to lead hit identification and hit-to-lead programs.. In this white paper, we discuss important aspects to consider when choosing a methodology for drug discovery. ...

Although most cases of chronic lymphocytic leukemia (CLL, one of the most common forms of adult leukemia) are sporadic, perhaps 10 to 20% are familial (see Debatin). Noting that aberrant DNA methylation--and thereby abnormal gene silencing--was emerging as a factor in CLL, Raval et al. performed quantitative high-throughput analysis to investigate DNA methylation in the CpG island of DAPK1 (death-associated protein kinase 1). DNA methylation of DAPK1 gene, which encodes a serine/threonine kinase implicated in promoting apoptosis in response to Fas, interferon-γ, and TNF-α, was increased in peripheral blood mononuclear cells and CD19+ B cells from people with CLL compared with that in cells from healthy volunteers. Reverse transcription polymerase chain reaction analysis indicated that DAPK1 expression was decreased in CD19+ CLL cells compared with that in B lymphocytes, and methylation reduced activity of a gene reporter containing a region of the DAPK1 promoter. Genome-wide linkage analysis ...

The objective of this Cancer Prevention and Research Institute of Texas (CPRIT) project is to identify and target lung cancer acquired vulnerabilities (synthetic lethalities) that have arisen during lung cancer pathogenesis and that can be used as new, cancer specific therapeutic targets with associated molecular enrollment biomarkers for personalized treatment.. When oncogenic changes (mutations and epigenetic changes) arise during lung cancer development they represent absolute differences from normal tissues that have the potential to become lung cancer specific therapeutic targets. While notable examples of this are acquired mutations in oncogenes such as the EGFR receptor and its targeting by EGFR tyrosine kinase inhibitors, these actually represent only a minority of cases and targeting these by themselves are not curative. By contrast, our genome-wide knockout (RNAi) screens, as well as our large compound library screens (220,000 synthetic chemicals and natural product extracts) have ...

BioAssay record AID 1273 submitted by Columbia University Molecular Screening Center: Primary screen for compounds that inhibit Insulin promoter activity in TRM-6 cells.

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Currently, drug screening relies on cell-based experiments or on animal models to confirm biological effects. The mammalian system is considered too time-consuming, expensive and complex to perform high-throughput drug screening. There is a gap between in vitro cell-based models and the in vivo mammalian models. The zebrafish is an ideal model that could link preclinical toxicity screening with the drug development pipeline. Taking advantage of a highly conservative genomic, rapid development, large number of offspring, low cost and easy manipulation, zebrafish has been considered an excellent animal model for disease-based drug screening. In this study, zebrafish embryos were incubated with small molecular compounds that potentially affected bone mineralization in microplates. Two compounds of alendronate and dorsomorphin were used as positive and negative controls, respectively. The level of osteogenic mineralization was measured and quantified by using ImageJ software with fluorescent calcein

Laboratory automation impacts nearly all aspects of high throughput RNAi screens. It is particularly relevant when considering library format and storage, and also when planning high throughput transfection protocols. In situations where libraries are stored as screening copies that are used for just-in-time dispensing, automation can be utilized as a tool to accommodate diverse assay and cell types and can enable forward or reverse transfections into a variety of different microplates. Automation has the ability to increase the feasibility and decrease consumable costs for assays that require a large number of replicates. It is also an important tool when considering more complex assays, such as those that utilize non-standard plate types or electroporation, as automation increases reliability and can improve assay performance. This chapter highlights important considerations for library formatting and ways in which laboratory automation can be implemented to facilitate RNAi high throughput ...

Little is known on the subject of variations between induced pluripotent stem cells produced from tissues originating from the same germ coating. growth-arrested. BJ1 cells communicate GFP and FGF2 protein were perepared in the iSTEM platform. hES culture medium was KO/DMEM (Invitrogen) supplemented with 20% knockout serum alternative (KSR) (Invitrogen) 0.1 mM nonessential amino acids (Invitrogen) 2 mM glutamax (Invitrogen) 50 μM β-mercaptoethanol (Invitrogen) 100 UI/ml penicillin/streptomycin (Invitrogen). hES cell medium for MEF feeder was supplemented by 10 ng/ml fibroblast growth element FGF2 (Invitrogen). The iPS cells were passaged every 7 days. Retroviral Transduction Cryovial of Platinum-A (PlatA) cells (Cell Biolabs) were utilized for transient computer virus packaging. 3×106 PlatA cells were plated per 60 mm gelatine-coated dish (80% confluent) in PlatA medium of DMEM+Glutamax II (Invitrogen) comprising 10% foetal calf serum 1 mM SMER28 sodium pyruvate (Invitrogen) and 50 mM ...

History/Goals: Even though the Cl- efflux assays are relatively straightforward, their capability to assess the efficiency of phenotypic modification in cystic fibrosis (CF) tissues or cells might end up being small. wild-type (wt), the 36Cl efflux assay was no reliable much longer. 115256-11-6 manufacture Polarized CFBE41o-cells, homozygous for the Y508 mutation also, had been utilized in the Ussing step research. Ussing evaluation discovered cAMP-dependent Cl- currents in blends with 1% wild-type cells suggesting that Ussing evaluation is normally even more delicate than 36Cd efflux evaluation for recognition of useful CFTR. A conclusion: Evaluation of CFTR function by Ussing evaluation is definitely more sensitive than 36Cl efflux analysis. Ussing analysis shows that cell mixes comprising 10% 16HBecome14o- cells showed 40C50% of normal cAMP-dependent Cl- transport that drops off exponentially between 10-1% wild-type cells. cell systems. Materials and Methods Cell tradition and cell lines The ...