129 research outputs found
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Platform for High-Throughput Testing of the Effect of Soluble Compounds on 3D Cell Cultures
In vitro 3D culture could provide an important model of tissues in vivo, but assessing the effects of chemical compounds on cells in specific regions of 3D culture requires physical isolation of cells and thus currently relies mostly on delicate and low-throughput methods. This paper describes a technique (“cells-in-gels-in-paper”, CiGiP) that permits rapid assembly of arrays of 3D cell cultures and convenient isolation of cells from specific regions of these cultures. The 3D cultures were generated by stacking sheets of 200-μm-thick paper, each sheet supporting 96 individual “spots” (thin circular slabs) of hydrogels containing cells, separated by hydrophobic material (wax, PDMS) impermeable to aqueous solutions, and hydrophilic and most hydrophobic solutes. A custom-made 96-well holder isolated the cell-containing zones from each other. Each well contained media to which a different compound could be added. After culture and disassembly of the holder, peeling the layers apart “sectioned” the individual 3D cultures into 200-μm-thick sections which were easy to analyze using 2D imaging (e.g., with a commercial gel scanner). This 96-well holder brings new utilities to high-throughput, cell-based screening, by combining the simplicity of CiGiP with the convenience of a microtiter plate. This work demonstrated the potential of this type of assays by examining the cytotoxic effects of phenylarsine oxide (PAO) and cyclophosphamide (CPA) on human breast cancer cells positioned at different separations from culture media in 3D cultures.Chemistry and Chemical Biolog
Simultaneous Embeddings with Few Bends and Crossings
A simultaneous embedding with fixed edges (SEFE) of two planar graphs and
is a pair of plane drawings of and that coincide when restricted to
the common vertices and edges of and . We show that whenever and
admit a SEFE, they also admit a SEFE in which every edge is a polygonal curve
with few bends and every pair of edges has few crossings. Specifically: (1) if
and are trees then one bend per edge and four crossings per edge pair
suffice (and one bend per edge is sometimes necessary), (2) if is a planar
graph and is a tree then six bends per edge and eight crossings per edge
pair suffice, and (3) if and are planar graphs then six bends per edge
and sixteen crossings per edge pair suffice. Our results improve on a paper by
Grilli et al. (GD'14), which proves that nine bends per edge suffice, and on a
paper by Chan et al. (GD'14), which proves that twenty-four crossings per edge
pair suffice.Comment: Full version of the paper "Simultaneous Embeddings with Few Bends and
Crossings" accepted at GD '1
Cross-sectional association of volume, blood pressures, and aortic stiffness with left ventricular mass in incident hemodialysis patients: the Predictors of Arrhythmic and Cardiovascular Risk in End-Stage Renal Disease (PACE) study
Acidic microenvironment plays a key role in human melanoma progression through a sustained exosome mediated transfer of clinically relevant metastatic molecules
Background: Microenvironment cues involved in melanoma progression are largely unknown. Melanoma is highly influenced in its aggressive phenotype by the changes it determinates in its microenvironment, such as pH decrease, in turn influencing cancer cell invasiveness, progression and tissue remodelling through an abundant secretion of exosomes, dictating cancer strategy to the whole host. A role of exosomes in driving melanoma progression under microenvironmental acidity was never described. Methods: We studied four differently staged human melanoma lines, reflecting melanoma progression, under microenvironmental acidic pHs pressure ranging between pH 6.0-6.7. To estimate exosome secretion as a function of tumor stage and environmental pH, we applied a technique to generate native fluorescent exosomes characterized by vesicles integrity, size, density, markers expression, and quantifiable by direct FACS analysis. Functional roles of exosomes were tested in migration and invasion tests. Then we performed a comparative proteomic analysis of acid versus control exosomes to elucidate a specific signature involved in melanoma progression. Results: We found that metastatic melanoma secretes a higher exosome amount than primary melanoma, and that acidic pH increases exosome secretion when melanoma is in an intermediate stage, i.e. metastatic non-invasive. We were thus able to show that acidic pH influences the intercellular cross-talk mediated by exosomes. In fact when exposed to exosomes produced in an acidic medium, pH naĂŻve melanoma cells acquire migratory and invasive capacities likely due to transfer of metastatic exosomal proteins, favoring cell motility and angiogenesis. A Prognoscan-based meta-analysis study of proteins enriched in acidic exosomes, identified 11 genes (HRAS, GANAB, CFL2, HSP90B1, HSP90AB1, GSN, HSPA1L, NRAS, HSPA5, TIMP3, HYOU1), significantly correlating with poor prognosis, whose high expression was in part confirmed in bioptic samples of lymph node metastases. Conclusions: A crucial step of melanoma progression does occur at melanoma intermediate -stage, when extracellular acidic pH induces an abundant release and intra-tumoral uptake of exosomes. Such exosomes are endowed with pro-invasive molecules of clinical relevance, which may provide a signature of melanoma advancement
Genome-Wide Gene Expression Analysis in Response to Organophosphorus Pesticide Chlorpyrifos and Diazinon in C. elegans
Organophosphorus pesticides (OPs) were originally designed to affect the nervous system by inhibiting the enzyme acetylcholinesterase, an important regulator of the neurotransmitter acetylcholine. Over the past years evidence is mounting that these compounds affect many other processes. Little is known, however, about gene expression responses against OPs in the nematode Caenorhabditis elegans. This is surprising because C. elegans is extensively used as a model species in toxicity studies. To address this question we performed a microarray study in C. elegans which was exposed for 72 hrs to two widely used Ops, chlorpyrifos and diazinon, and a low dose mixture of these two compounds. Our analysis revealed transcriptional responses related to detoxification, stress, innate immunity, and transport and metabolism of lipids in all treatments. We found that for both compounds as well as in the mixture, these processes were regulated by different gene transcripts. Our results illustrate intense, and unexpected crosstalk between gene pathways in response to chlorpyrifos and diazinon in C. elegans
Regional-scale phenology modeling based on meteorological records and remote sensing observations
Author Posting. © American Geophysical Union, 2012. This article is posted here by permission of American Geophysical Union for personal use, not for redistribution. The definitive version was published in Journal of Geophysical Research 117 (2012): G03029, doi:10.1029/2012JG001977.Changes of vegetation phenology in response to climate change in the temperate forests have been well documented recently and have important implications on the regional and global carbon and water cycles. Predicting the impact of changing phenology on terrestrial ecosystems requires an accurate phenology model. Although species-level phenology models have been tested using a small number of vegetation species, they are rarely examined at the regional level. In this study, we used remotely sensed phenology and meteorological data to parameterize the species-level phenology models. We used a remotely sensed vegetation index (Two-band Enhanced Vegetation Index, EVI2) derived from the Moderate Resolution Spectroradiometer (MODIS) 8-day reflectance product from 2000 to 2010 of New England, United States to calculate remotely sensed vegetation phenology (start/end of season, or SOS/EOS). The SOS/EOS and the daily mean air temperature data from weather stations were used to parameterize three budburst models and one senescence model. We compared the relative strengths of the models to predict vegetation phenology and selected the best model to reconstruct the “landscape phenology” in New England from year 1960 to 2010. Of the three budburst models tested, the spring warming model showed the best performance with an averaged Root Mean Square Deviation (RMSD) of 4.59 days. The Akaike Information Criterion supported the spring warming model in all the weather stations. For senescence modeling, the Delpierre model was better than a null model (the averaged phenology of each weather station, averaged model efficiency = 0.33) and has a RMSD of 8.05 days. A retrospective analysis using the spring warming model suggests a statistically significant advance of SOS in New England from 1960 to 2010 averaged as 0.143 days per year (p = 0.015). EOS calculated using the Delpierre model and growing season length showed no statistically significant advance or delay between 1960 and 2010 in this region. These results suggest the applicability of species-level phenology models at the regional level (and potentially terrestrial biosphere models) and the feasibility of using these models in reconstructing and predicting vegetation phenology.This research was supported
by the Brown University–Marine Biological Laboratory graduate program in Biological and Environmental Sciences, Brown–ECI phenology
working group, and Brown Office of International Affairs Seed Grant on
phenology.2013-03-1
MassCode Liquid Arrays as a Tool for Multiplexed High-Throughput Genetic Profiling
Multiplexed detection assays that analyze a modest number of nucleic acid targets over large sample sets are emerging as the preferred testing approach in such applications as routine pathogen typing, outbreak monitoring, and diagnostics. However, very few DNA testing platforms have proven to offer a solution for mid-plexed analysis that is high-throughput, sensitive, and with a low cost per test. In this work, an enhanced genotyping method based on MassCode technology was devised and integrated as part of a high-throughput mid-plexing analytical system that facilitates robust qualitative differential detection of DNA targets. Samples are first analyzed using MassCode PCR (MC-PCR) performed with an array of primer sets encoded with unique mass tags. Lambda exonuclease and an array of MassCode probes are then contacted with MC-PCR products for further interrogation and target sequences are specifically identified. Primer and probe hybridizations occur in homogeneous solution, a clear advantage over micro- or nanoparticle suspension arrays. The two cognate tags coupled to resultant MassCode hybrids are detected in an automated process using a benchtop single quadrupole mass spectrometer. The prospective value of using MassCode probe arrays for multiplexed bioanalysis was demonstrated after developing a 14plex proof of concept assay designed to subtype a select panel of Salmonella enterica serogroups and serovars. This MassCode system is very flexible and test panels can be customized to include more, less, or different markers
Multizone Paper Platform for 3D Cell Cultures
In vitro 3D culture is an important model for tissues in
vivo. Cells in different locations of 3D tissues are
physiologically different, because they are exposed to different concentrations
of oxygen, nutrients, and signaling molecules, and to other environmental
factors (temperature, mechanical stress, etc). The majority of high-throughput
assays based on 3D cultures, however, can only detect the
average behavior of cells in the whole 3D construct.
Isolation of cells from specific regions of 3D cultures is possible, but relies
on low-throughput techniques such as tissue sectioning and micromanipulation.
Based on a procedure reported previously (“cells-in-gels-in-paper”
or CiGiP), this paper describes a simple method for culture of arrays of thin
planar sections of tissues, either alone or stacked to create more complex 3D
tissue structures. This procedure starts with sheets of paper patterned with
hydrophobic regions that form 96 hydrophilic zones. Serial spotting of cells
suspended in extracellular matrix (ECM) gel onto the patterned paper creates an
array of 200 micron-thick slabs of ECM gel (supported mechanically by cellulose
fibers) containing cells. Stacking the sheets with zones aligned on top of one
another assembles 96 3D multilayer constructs. De-stacking the layers of the 3D
culture, by peeling apart the sheets of paper, “sections” all 96
cultures at once. It is, thus, simple to isolate 200-micron-thick
cell-containing slabs from each 3D culture in the 96-zone array. Because the 3D
cultures are assembled from multiple layers, the number of cells plated
initially in each layer determines the spatial distribution of cells in the
stacked 3D cultures. This capability made it possible to compare the growth of
3D tumor models of different spatial composition, and to examine the migration
of cells in these structures
Pre-Micro RNA Signatures Delineate Stages of Endothelial Cell Transformation in Kaposi Sarcoma
MicroRNAs (miRNA) have emerged as key regulators of cell lineage differentiation and cancer. We used precursor miRNA profiling by a novel real-time QPCR method (i) to define progressive stages of endothelial cell transformation cumulating in Kaposi sarcoma (KS) and (ii) to identify specific miRNAs that serve as biomarkers for tumor progression. We were able to compare primary patient biopsies to well-established culture and mouse tumor models. Loss of mir-221 and gain of mir-15 expression demarked the transition from merely immortalized to fully tumorigenic endothelial cells. Mir-140 and Kaposi sarcoma–associated herpesvirus viral miRNAs increased linearly with the degree of transformation. Mir-24 emerged as a biomarker specific for KS
The responding relationship between plants and environment is the essential principle for agricultural sustainable development on the globe
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