Flow Cytometry Sorting to Separate Viable Giant Viruses from Amoeba Co-culture Supernatants.

PubMed

Khalil, Jacques Y B; Langlois, Thierry; Andreani, Julien; Sorraing, Jean-Marc; Raoult, Didier; Camoin, Laurence; La Scola, Bernard

2016-01-01

Flow cytometry has contributed to virology but has faced many drawbacks concerning detection limits, due to the small size of viral particles. Nonetheless, giant viruses changed many concepts in the world of viruses, as a result of their size and hence opened up the possibility of using flow cytometry to study them. Recently, we developed a high throughput isolation of viruses using flow cytometry and protozoa co-culture. Consequently, isolating a viral mixture in the same sample became more common. Nevertheless, when one virus multiplies faster than others in the mixture, it is impossible to obtain a pure culture of the minority population. Here, we describe a robust sorting system, which can separate viable giant virus mixtures from supernatants. We tested three flow cytometry sorters by sorting artificial mixtures. Purity control was assessed by electron microscopy and molecular biology. As proof of concept, we applied the sorting system to a co-culture supernatant taken from a sample containing a viral mixture that we couldn't separate using end point dilution. In addition to isolating the quick-growing Mimivirus , we sorted and re-cultured a new, slow-growing virus, which we named "Cedratvirus." The sorting assay presented in this paper is a powerful and versatile tool for separating viral populations from amoeba co-cultures and adding value to the new field of flow virometry.

FISHIS: Fluorescence In Situ Hybridization in Suspension and Chromosome Flow Sorting Made Easy

PubMed Central

Giorgi, Debora; Farina, Anna; Grosso, Valentina; Gennaro, Andrea; Ceoloni, Carla; Lucretti, Sergio

2013-01-01

The large size and complex polyploid nature of many genomes has often hampered genomics development, as is the case for several plants of high agronomic value. Isolating single chromosomes or chromosome arms via flow sorting offers a clue to resolve such complexity by focusing sequencing to a discrete and self-consistent part of the whole genome. The occurrence of sufficient differences in the size and or base-pair composition of the individual chromosomes, which is uncommon in plants, is critical for the success of flow sorting. We overcome this limitation by developing a robust method for labeling isolated chromosomes, named Fluorescent In situ Hybridization In suspension (FISHIS). FISHIS employs fluorescently labeled synthetic repetitive DNA probes, which are hybridized, in a wash-less procedure, to chromosomes in suspension following DNA alkaline denaturation. All typical A, B and D genomes of wheat, as well as individual chromosomes from pasta (T. durum L.) and bread (T. aestivum L.) wheat, were flow-sorted, after FISHIS, at high purity. For the first time in eukaryotes, each individual chromosome of a diploid organism, Dasypyrum villosum (L.) Candargy, was flow-sorted regardless of its size or base-pair related content. FISHIS-based chromosome sorting is a powerful and innovative flow cytogenetic tool which can develop new genomic resources from each plant species, where microsatellite DNA probes are available and high quality chromosome suspensions could be produced. The joining of FISHIS labeling and flow sorting with the Next Generation Sequencing methodology will enforce genomics for more species, and by this mightier chromosome approach it will be possible to increase our knowledge about structure, evolution and function of plant genome to be used for crop improvement. It is also anticipated that this technique could contribute to analyze and sort animal chromosomes with peculiar cytogenetic abnormalities, such as copy number variations or cytogenetic aberrations. PMID:23469124

FISHIS: fluorescence in situ hybridization in suspension and chromosome flow sorting made easy.

PubMed

Giorgi, Debora; Farina, Anna; Grosso, Valentina; Gennaro, Andrea; Ceoloni, Carla; Lucretti, Sergio

2013-01-01

The large size and complex polyploid nature of many genomes has often hampered genomics development, as is the case for several plants of high agronomic value. Isolating single chromosomes or chromosome arms via flow sorting offers a clue to resolve such complexity by focusing sequencing to a discrete and self-consistent part of the whole genome. The occurrence of sufficient differences in the size and or base-pair composition of the individual chromosomes, which is uncommon in plants, is critical for the success of flow sorting. We overcome this limitation by developing a robust method for labeling isolated chromosomes, named Fluorescent In situ Hybridization In suspension (FISHIS). FISHIS employs fluorescently labeled synthetic repetitive DNA probes, which are hybridized, in a wash-less procedure, to chromosomes in suspension following DNA alkaline denaturation. All typical A, B and D genomes of wheat, as well as individual chromosomes from pasta (T. durum L.) and bread (T. aestivum L.) wheat, were flow-sorted, after FISHIS, at high purity. For the first time in eukaryotes, each individual chromosome of a diploid organism, Dasypyrum villosum (L.) Candargy, was flow-sorted regardless of its size or base-pair related content. FISHIS-based chromosome sorting is a powerful and innovative flow cytogenetic tool which can develop new genomic resources from each plant species, where microsatellite DNA probes are available and high quality chromosome suspensions could be produced. The joining of FISHIS labeling and flow sorting with the Next Generation Sequencing methodology will enforce genomics for more species, and by this mightier chromosome approach it will be possible to increase our knowledge about structure, evolution and function of plant genome to be used for crop improvement. It is also anticipated that this technique could contribute to analyze and sort animal chromosomes with peculiar cytogenetic abnormalities, such as copy number variations or cytogenetic aberrations.

Application of Multi-Objective Human Learning Optimization Method to Solve AC/DC Multi-Objective Optimal Power Flow Problem

NASA Astrophysics Data System (ADS)

Cao, Jia; Yan, Zheng; He, Guangyu

2016-06-01

This paper introduces an efficient algorithm, multi-objective human learning optimization method (MOHLO), to solve AC/DC multi-objective optimal power flow problem (MOPF). Firstly, the model of AC/DC MOPF including wind farms is constructed, where includes three objective functions, operating cost, power loss, and pollutant emission. Combining the non-dominated sorting technique and the crowding distance index, the MOHLO method can be derived, which involves individual learning operator, social learning operator, random exploration learning operator and adaptive strategies. Both the proposed MOHLO method and non-dominated sorting genetic algorithm II (NSGAII) are tested on an improved IEEE 30-bus AC/DC hybrid system. Simulation results show that MOHLO method has excellent search efficiency and the powerful ability of searching optimal. Above all, MOHLO method can obtain more complete pareto front than that by NSGAII method. However, how to choose the optimal solution from pareto front depends mainly on the decision makers who stand from the economic point of view or from the energy saving and emission reduction point of view.

Sensitivity of Breast Cancer Stem Cells to TRA-8 Anti-DR5 Monoclonal Antibody

DTIC Science & Technology

2012-02-01

cytotoxicity and reduction in BrCSC marker expression. A. 2LMP cells were sorted using flow cytometry for CD44+/CD24-/ALDHhigh. Cells were pre...cells were sorted using flow cytometry for ALDH? cells and allowed to form primary tumorspheres for 3 days. After tumorspheres were mechanically...n =5 ) Day Fig. 5 Effect of ex vivo treatment of BrCSC enriched cells on tumorgenicity in NOD/SCID mice. 2LMP cells were sorted using flow cytometry

Size-sensitive sorting of microparticles through control of flow geometry

NASA Astrophysics Data System (ADS)

Wang, Cheng; Jalikop, Shreyas V.; Hilgenfeldt, Sascha

2011-07-01

We demonstrate a general concept of flow manipulation in microfluidic environments, based on controlling the shape and position of flow domains in order to force switching and sorting of microparticles without moving parts or changes in design geometry. Using microbubble acoustic streaming, we show that regulation of the relative strength of streaming and a superimposed Poiseuille flow allows for size-selective trapping and releasing of particles, with particle size sensitivity much greater than what is imposed by the length scales of microfabrication. A simple criterion allows for quantitative tuning of microfluidic devices for switching and sorting of particles of desired size.

A revised velocity-reversal and sediment-sorting model for a high-gradient, pool-riffle stream

USGS Publications Warehouse

Thompson, D.M.; Wohl, E.E.; Jarrett, R.D.

1996-01-01

Sediment-sorting processes related to varying channel-bed morphology were investigated from April to November 1993 along a 1-km pool-riffle and step-pool reach of North Saint Vrain Creek, a small mountain stream in the Rocky Mountains of northern Colorado. Measured cross-sectional areas of flow were used to suggest higher velocities in pools than in riffles at high flow. Three hundred and sixteen tracer particles, ranging in size from 16 mm to 256 mm, were placed in two separate pool-riffle-pool sequences and used to assess sediment-sorting patterns and sediment-transport competence variations. Tracer-particle depositional evidence indicated higher sediment-transport competence in pools than in riffles at high flow. Pool-riffle sediment sorting may be created by velocity reversals, and more localized sorting results from gravitational forces along the upstream sloping portion of the channel bed located at the downstream end of pools.

Phylogenomics of species from four genera of New World monkeys by flow sorting and reciprocal chromosome painting

PubMed Central

Dumas, Francesca; Stanyon, Roscoe; Sineo, Luca; Stone, Gary; Bigoni, Francesca

2007-01-01

Background The taxonomic and phylogenetic relationships of New World monkeys (Platyrrhini) are difficult to distinguish on the basis of morphology and because diagnostic fossils are rare. Recently, molecular data have led to a radical revision of the traditional taxonomy and phylogeny of these primates. Here we examine new hypotheses of platyrrhine evolutionary relationships by reciprocal chromosome painting after chromosome flow sorting of species belonging to four genera of platyrrhines included in the Cebidae family: Callithrix argentata (silvered-marmoset), Cebuella pygmaea (pygmy marmoset), Callimico goeldii (Goeldi's marmoset) and Saimiri sciureus (squirrel monkey). This is the first report of reciprocal painting in marmosets. Results The paints made from chromosome flow sorting of the four platyrrhine monkeys provided from 42 to 45 hybridization signals on human metaphases. The reciprocal painting of monkey probes on human chromosomes revealed that 21 breakpoints are common to all four studied species. There are only three additional breakpoints. A breakpoint on human chromosome 13 was found in Callithrix argentata, Cebuella pygmaea and Callimico goeldii, but not in Saimiri sciureus. There are two additional breakpoints on human chromosome 5: one is specific to squirrel monkeys, and the other to Goeldi's marmoset. Conclusion The reciprocal painting results support the molecular genomic assemblage of Cebidae. We demonstrated that the five chromosome associations previously hypothesized to phylogenetically link tamarins and marmosets are homologous and represent derived chromosome rearrangements. Four of these derived homologous associations tightly nest Callimico goeldii with marmosets. One derived association 2/15 may place squirrel monkeys within the Cebidae assemblage. An apparently common breakpoint on chromosome 5q33 found in both Saimiri and Aotus nancymae could be evidence of a phylogenetic link between these species. Comparison with previous reports shows that many syntenic associations found in platyrrhines have the same breakpoints and are homologous, derived rearrangements showing that the New World monkeys are a closely related group of species. Our data support the hypothesis that the ancestral karyotype of the Platyrrhini has a diploid number of 2n = 54 and is almost identical to that found today in capuchin monkeys; congruent with a basal position of the Cebidae among platyrrhine families. PMID:17767727

Host lung immunity is severely compromised during tropical pulmonary eosinophilia: role of lung eosinophils and macrophages.

PubMed

Sharma, Pankaj; Sharma, Aditi; Vishwakarma, Achchhe Lal; Agnihotri, Promod Kumar; Sharma, Sharad; Srivastava, Mrigank

2016-04-01

Eosinophils play a central role in the pathogenesis of tropical pulmonary eosinophilia, a rare, but fatal, manifestation of filariasis. However, no exhaustive study has been done to identify the genes and proteins of eosinophils involved in the pathogenesis of tropical pulmonary eosinophilia. In the present study, we established a mouse model of tropical pulmonary eosinophilia that mimicked filarial manifestations of human tropical pulmonary eosinophilia pathogenesis and used flow cytometry-assisted cell sorting and real-time RT-PCR to study the gene expression profile of flow-sorted, lung eosinophils and lung macrophages during tropical pulmonary eosinophilia pathogenesis. Our results show that tropical pulmonary eosinophilia mice exhibited increased levels of IL-4, IL-5, CCL5, and CCL11 in the bronchoalveolar lavage fluid and lung parenchyma along with elevated titers of IgE and IgG subtypes in the serum. Alveolar macrophages from tropical pulmonary eosinophilia mice displayed decreased phagocytosis, attenuated nitric oxide production, and reduced T-cell proliferation capacity, and FACS-sorted lung eosinophils from tropical pulmonary eosinophilia mice upregulated transcript levels of ficolin A and anti-apoptotic gene Bcl2,but proapoptotic genes Bim and Bax were downregulated. Similarly, flow-sorted lung macrophages upregulated transcript levels of TLR-2, TLR-6, arginase-1, Ym-1, and FIZZ-1 but downregulated nitric oxide synthase-2 levels, signifying their alternative activation. Taken together, we show that the pathogenesis of tropical pulmonary eosinophilia is marked by functional impairment of alveolar macrophages, alternative activation of lung macrophages, and upregulation of anti-apoptotic genes by eosinophils. These events combine together to cause severe lung inflammation and compromised lung immunity. Therapeutic interventions that can boost host immune response in the lungs might thus provide relief to patients with tropical pulmonary eosinophilia. © Society for Leukocyte Biology.

Development of a novel cell sorting method that samples population diversity in flow cytometry.

PubMed

Osborne, Geoffrey W; Andersen, Stacey B; Battye, Francis L

2015-11-01

Flow cytometry based electrostatic cell sorting is an important tool in the separation of cell populations. Existing instruments can sort single cells into multi-well collection plates, and keep track of cell of origin and sorted well location. However currently single sorted cell results reflect the population distribution and fail to capture the population diversity. Software was designed that implements a novel sorting approach, "Slice and Dice Sorting," that links a graphical representation of a multi-well plate to logic that ensures that single cells are sampled and sorted from all areas defined by the sort region/s. Therefore the diversity of the total population is captured, and the more frequently occurring or rarer cell types are all sampled. The sorting approach was tested computationally, and using functional cell based assays. Computationally we demonstrate that conventional single cell sorting can sample as little as 50% of the population diversity dependant on the population distribution, and that Slice and Dice sorting samples much more of the variety present within a cell population. We then show by sorting single cells into wells using the Slice and Dice sorting method that there are cells sorted using this method that would be either rarely sorted, or not sorted at all using conventional single cell sorting approaches. The present study demonstrates a novel single cell sorting method that samples much more of the population diversity than current methods. It has implications in clonal selection, stem cell sorting, single cell sequencing and any areas where population heterogeneity is of importance. © 2015 International Society for Advancement of Cytometry.

Fractionation of Exosomes and DNA using Size-Based Separation at the Nanoscale

NASA Astrophysics Data System (ADS)

Wunsch, Benjamin; Smith, Joshua; Wang, Chao; Gifford, Stacey; Brink, Markus; Bruce, Robert; Solovitzky, Gustavo; Austin, Robert; Astier, Yann

Exosomes, a key target of ``liquid biopsies'', are nano-vesicles found in nearly all biological fluids. Exosomes are secreted by eukaryotic and prokaryotic cells alike, and contain information about their originating cells, including surface proteins, cytoplasmic proteins, and nucleic acids. One challenge in studying exosome morphology is the difficulty of sorting exosomes by size and surface markers. Common separation techniques for exosomes include ultracentrifugation and ultrafiltration, for preparation of large volume samples, but these techniques often show contamination and significant heterogeneity between preparations. To date, deterministic lateral displacement (DLD) pillar arrays in silicon have proven an efficient technology to sort, separate, and enrich micron-scale particles including human parasites, eukaryotic cells, blood cells, and circulating tumor cells in blood; however, the DLD technology has never been translated to the true nanoscale, where it could function on bio-colloids such as exosomes. We have fabricated nanoscale DLD (nanoDLD) arrays capable of rapidly sorting colloids down to 20 nm in continuous flow, and demonstrated size sorting of individual exosome vesicles and dsDNA polymers, opening the potential for on-chip biomolecule separation and diagnosti

Free-Flow Zone Electrophoresis of Peptides and Proteins in PDMS Microchip for Narrow pI Range Sample Prefractionation Coupled with Mass Spectrometry

PubMed Central

Song, Yong-Ak; Chan, Michael; Celio, Chris; Tannenbaum, Steven R.; Wishnok, John S.; Han, Jongyoon

2010-01-01

In this paper, we are evaluating the strategy of sorting peptides / proteins based on the charge to mass without resorting to ampholytes and / or isoelectric focusing, using a single- and two-step free-flow zone electrophoresis. We developed a simple fabrication method to create a salt bridge for free-flow zone electrophoresis in PDMS chips by surface printing a hydrophobic layer on a glass substrate. Since the surface-printed hydrophobic layer prevents plasma bonding between the PDMS chip and the substrate, an electrical junction gap can be created for free-flow zone electrophoresis. With this device, we demonstrated a separation of positive and negative peptides and proteins at a given pH in standard buffer systems, and validated the sorting result with LC/MS. Furthermore, we coupled two sorting steps via off-chip titration, and isolated peptides within specific pI ranges from sample mixtures, where the pI range was simply set by the pH values of the buffer solutions. This free-flow zone electrophoresis sorting device, with its simplicity of fabrication, and a sorting resolution of 0.5 pH unit, can potentially be a high-throughput sample fractionation tool for targeted proteomics using LC/MS. PMID:20163146

Low-frequency chimeric yeast artificial chromosome libraries from flow-sorted human chromosomes 16 and 21.

PubMed Central

McCormick, M K; Campbell, E; Deaven, L; Moyzis, R

1993-01-01

Construction of chromosome-specific yeast artificial chromosome (YAC) libraries from sorted chromosomes was undertaken (i) to eliminate drawbacks associated with first-generation total genomic YAC libraries, such as the high frequency of chimeric YACs, and (ii) to provide an alternative method for generating chromosome-specific YAC libraries in addition to isolating such collections from a total genomic library. Chromosome-specific YAC libraries highly enriched for human chromosomes 16 and 21 were constructed. By maximizing the percentage of fragments with two ligatable ends and performing yeast transformations with less than saturating amounts of DNA in the presence of carrier DNA, YAC libraries with a low percentage of chimeric clones were obtained. The smaller number of YAC clones in these chromosome-specific libraries reduces the effort involved in PCR-based screening and allows hybridization methods to be a manageable screening approach. Images PMID:8430075

Multiple sort flow cytometer

DOEpatents

Van den Engh, Ger; Esposito, Richard J.

1996-01-01

A flow cytometer utilizes multiple lasers for excitation and respective fluorescence of identified dyes bonded to specific cells or events to identify and verify multiple events to be sorted from a sheath flow and droplet stream. Once identified, verified and timed in the sheath flow, each event is independently tagged upon separation from the flow by an electrical charge of +60, +120, or +180 volts and passed through oppositely charged deflection plates with ground planes to yield a focused six way deflection of at least six events in a narrow plane.

Rare cancer cell analyzer for whole blood applications: microcytometer cell counting and sorting subcircuits.

PubMed

Lancaster, C; Kokoris, M; Nabavi, M; Clemmens, J; Maloney, P; Capadanno, J; Gerdes, J; Battrell, C F

2005-09-01

We demonstrate sorting of rare cancer cells from blood using a thin ribbon monolayer of cells within a credit-card sized, microfluidic laboratory-on-a-card ("lab card") structure. This enables higher cell throughput per minute thereby speeding up cell interrogation. In this approach, multiple cells are viewed and sorted, not individually, but as a whole cell row or section of the ribbon at a time. Gated selection of only the cell rows containing a tagged rare cell provides enrichment of the rare cell relative to background blood cells. We also designed the cell injector for laminar flow antibody labeling within 20s. The approach combines rapid laminar flow cell labeling with monolayer cell sorting thereby enabling rare cell target detection at sensitivity levels 1000 to 10,000 times that of existing flow cytometers. Using this method, total cell labeling and data acquisition time on card may be reduced to a few minutes compared to 30-60 min for standard flow methods.

AI techniques for optimizing multi-objective reservoir operation upon human and riverine ecosystem demands

NASA Astrophysics Data System (ADS)

Tsai, Wen-Ping; Chang, Fi-John; Chang, Li-Chiu; Herricks, Edwin E.

2015-11-01

Flow regime is the key driver of the riverine ecology. This study proposes a novel hybrid methodology based on artificial intelligence (AI) techniques for quantifying riverine ecosystems requirements and delivering suitable flow regimes that sustain river and floodplain ecology through optimizing reservoir operation. This approach addresses issues to better fit riverine ecosystem requirements with existing human demands. We first explored and characterized the relationship between flow regimes and fish communities through a hybrid artificial neural network (ANN). Then the non-dominated sorting genetic algorithm II (NSGA-II) was established for river flow management over the Shihmen Reservoir in northern Taiwan. The ecosystem requirement took the form of maximizing fish diversity, which could be estimated by the hybrid ANN. The human requirement was to provide a higher satisfaction degree of water supply. The results demonstrated that the proposed methodology could offer a number of diversified alternative strategies for reservoir operation and improve reservoir operational strategies producing downstream flows that could meet both human and ecosystem needs. Applications that make this methodology attractive to water resources managers benefit from the wide spread of Pareto-front (optimal) solutions allowing decision makers to easily determine the best compromise through the trade-off between reservoir operational strategies for human and ecosystem needs.

Viability and membrane integrity of spermatozoa after dilution and flow cytometric sorting in the presence or absence of seminal plasma.

PubMed

Maxwell, W M; Welch, G R; Johnson, L A

1996-01-01

Boar, bull and ram spermatozoa were examined after staining with the DNA-permeant Hoechst 33342 fluorochrome and flow cytometric sorting in the presence or absence of seminal plasma. Spermatozoa were assessed for viability with flow cytometry using the live cell nucleic acid stain SYBR-14 and propidium iodide (PI), and for membrane integrity using fluorescein isothiocyanate-conjugated Pisum sativum (FITC-PSA) and PI; motility and acrosome integrity were estimated by microscopy. Flow cytometric sorting was compared with pipette dilution of boar and bull spermatozoa into: (1) medium [boar: Test buffer containing 2% yolk (TY) or Beltsville thawing solution (BTS); bull: TY or HEPES buffer containing 0.1% bovine serum albumin (HEPES-BSA)] with or without 10% (v/v) seminal plasma; or (2) an empty tube containing no medium. Sorted spermatozoa were either not centrifuged or centrifuged before assessment during a 4-h holding period. The viability, motility and membrane integrity of boar, bull and ram spermatozoa centrifuged after sorting were also examined when seminal plasma was present or absent from the staining extender and/or the TY collection medium. The results indicate that the viability and membrane integrity of spermatozoa in vitro would be improved if: (1) seminal plasma (10%) was routinely included in the BTS and HEPES-BSA staining extenders for boar spermatozoa and ram spermatozoa, respectively, when used in preparation for flow cytometric sorting; and (2) 10% and 50% seminal plasma were included in the TY collection medium for boar or bull spermatozoa and ram spermatozoa respectively.

Identification of residual leukemic cells by flow cytometry in childhood B-cell precursor acute lymphoblastic leukemia: verification of leukemic state by flow-sorting and molecular/cytogenetic methods.

PubMed

Øbro, Nina F; Ryder, Lars P; Madsen, Hans O; Andersen, Mette K; Lausen, Birgitte; Hasle, Henrik; Schmiegelow, Kjeld; Marquart, Hanne V

2012-01-01

Reduction in minimal residual disease, measured by real-time quantitative PCR or flow cytometry, predicts prognosis in childhood B-cell precursor acute lymphoblastic leukemia. We explored whether cells reported as minimal residual disease by flow cytometry represent the malignant clone harboring clone-specific genomic markers (53 follow-up bone marrow samples from 28 children with B-cell precursor acute lymphoblastic leukemia). Cell populations (presumed leukemic and non-leukemic) were flow-sorted during standard flow cytometry-based minimal residual disease monitoring and explored by PCR and/or fluorescence in situ hybridization. We found good concordance between flow cytometry and genomic analyses in the individual flow-sorted leukemic (93% true positive) and normal (93% true negative) cell populations. Four cases with discrepant results had plausible explanations (e.g. partly informative immunophenotype and antigen modulation) that highlight important methodological pitfalls. These findings demonstrate that with sufficient experience, flow cytometry is reliable for minimal residual disease monitoring in B-cell precursor acute lymphoblastic leukemia, although rare cases require supplementary PCR-based monitoring.

On-chip cell sorting via patterned magnetic traps

NASA Astrophysics Data System (ADS)

Byvank, Tom; Prikockis, Michael; Chen, Aaron; Miller, Brandon; Chalmers, Jeffrey; Sooryakumar, Ratnasingham

2015-03-01

Due to their importance in research for the diagnosis and treatment of cancer, numerous schemes have been developed to sort rare cell populations, e.g., circulating tumor cells (CTCs), from a larger ensemble of cells. Here, we improve upon a previously developed microfluidic device (Lab Chip 13, 1172, (2013)) to increase throughput and sorting purity of magnetically labeled cells. The separation mechanism involves controlling magnetic forces by manipulating the magnetic domain structures of embedded permalloy microdisks with weak external fields. These forces move labeled cells from the input flow stream into an adjacent buffer flow stream. Such magnetically activated transfer separates the magnetic entities from their non-magnetic counterparts as the two flow streams split apart and move toward their respective outputs. Purity of the magnetic output is modulated by the withdrawal rate of the non-magnetic output relative to the inputs. A proof of concept shows that CTCs from metastatic breast cancer patients can be sorted, recovered from the device, and confirmed as CTCs using separate immunofluorescence staining and analysis. With further optimizations, the channel could become a useful device for high purity final sorting of enriched patient cell samples.

Multiple sort flow cytometer

DOEpatents

Engh, G. van den; Esposito, R.J.

1996-01-09

A flow cytometer utilizes multiple lasers for excitation and respective fluorescence of identified dyes bonded to specific cells or events to identify and verify multiple events to be sorted from a sheath flow and droplet stream. Once identified, verified and timed in the sheath flow, each event is independently tagged upon separation from the flow by an electrical charge of +60, +120, or +180 volts and passed through oppositely charged deflection plates with ground planes to yield a focused six way deflection of at least six events in a narrow plane. 8 figs.

Mesenchymal Stem Cells for Vascular Target Discovery in Breast Cancer-Associated Angiogenesis

DTIC Science & Technology

2004-09-01

Matrigel plug and sorted by flow cytometry . Sorting of these retrieved cells based on co-expression of the GFP marker and cell- surface endothelial...express the green fluorescent protein (GFP) and clonal MSC populations can be isolated and phenotypically and genotypically analyzed by flow cytometry ...monoclonal populations of these GFP+ murine MSCs and conducted flow cytometry analysis to determine their phenotype. Specifically, we determined if

Early Detection of NSCLC Using Stromal Markers in Peripheral Blood

DTIC Science & Technology

2016-09-01

circulating myeloid cells, flow cytometry, RNA -sequencing, expression profiling. 3. ACCOMPLISHMENTS:  What were the major goals of the project...Subtask 2: Flow cytometry sorting of circulating myeloid cells. Subtask 3: RNA -Sequencing Subtask 4: RNA -seq data analysis Subtask 5: Feasible RT-PCR...accomplished the patient recruitment, flow cytometry sorting of circulating myeloid cells, RNA -sequencing of the samples. During the RNA - seq data analysis, we

Flow cytometric sorting of fecal bacteria after in situ hybridization with polynucleotide probes.

PubMed

Bruder, Lena M; Dörkes, Marcel; Fuchs, Bernhard M; Ludwig, Wolfgang; Liebl, Wolfgang

2016-10-01

The gut microbiome represents a key contributor to human physiology, metabolism, immune function, and nutrition. Elucidating the composition and genetics of the gut microbiota under various conditions is essential to understand how microbes function individually and as a community. Metagenomic analyses are increasingly used to study intestinal microbiota. However, for certain scientific questions it is sufficient to examine taxon-specific submetagenomes, covering selected bacterial genera in a targeted manner. Here we established a new variant of fluorescence in situ hybridization (FISH) combined with fluorescence-activated cell sorting (FACS), providing access to the genomes of specific taxa belonging to the complex community of the intestinal microbiota. In contrast to standard oligonucleotide probes, the RNA polynucleotide probe used here, which targets domain III of the 23S rRNA gene, extends the resolution power in environmental samples by increasing signal intensity. Furthermore, cells hybridized with the polynucleotide probe are not subjected to harsh pretreatments, and their genetic information remains intact. The protocol described here was tested on genus-specifically labeled cells in various samples, including complex fecal samples from different laboratory mouse types that harbor diverse intestinal microbiota. Specifically, as an example for the protocol described here, RNA polynucleotide probes could be used to label Enterococcus cells for subsequent sorting by flow cytometry. To detect and quantify enterococci in fecal samples prior to enrichment, taxon-specific PCR and qPCR detection systems have been developed. The accessibility of the genomes from taxon-specifically sorted cells for subsequent molecular analyses was demonstrated by amplification of functional genes. Copyright © 2016 Elsevier GmbH. All rights reserved.

Critical assessment of the efficiency of CD34 and CD133 antibodies for enrichment of rabbit hematopoietic stem cells.

PubMed

Vašíček, Jaromír; Shehata, Medhat; Schnabl, Susanne; Hilgarth, Martin; Hubmann, Rainer; Jäger, Ulrich; Bauer, Miroslav; Chrenek, Peter

2018-06-08

Rabbits have many hereditary diseases common to humans and are therefore a valuable model for regenerative disease and hematopoietic stem cell (HSC) therapies. Currently, there is no substantial data on the isolation and/or enrichment of rabbit HSCs. This study was initiated to evaluate the efficiency of the commercially available anti-CD34 and anti-CD133 antibodies for the detection and potential enrichment of rabbit HSCs from peripheral blood. PBMCs from rabbit and human blood were labelled with different clones of anti-human CD34 monoclonal antibodies (AC136, 581 and 8G12) and rabbit polyclonal CD34 antibody (pCD34) and anti-human CD133 monoclonal antibodies (AC133 and 293C3). Flow cytometry showed a higher percentage of rabbit CD34 + cells labelled by AC136 in comparison to the clone 581 and pCD34 (P<0.01). A higher percentage of rabbit CD133 + cells were also detected by 293C3 compared to the AC133 clone (P<0.01). Therefore, AC136 clone was used for the indirect immunomagnetic enrichment of rabbit CD34 + cells using magnetic-activated cell sorting (MACS). The enrichment of the rabbit CD34 + cells after sorting was low in comparison to human samples (2.4% vs. 39.6%). PCR analyses confirmed the efficient enrichment of human CD34 + cells and the low expression of CD34 mRNA in rabbit positive fraction. In conclusion, the tested antibodies might be suitable for detection, but not for sorting the rabbit CD34 + HSCs and new specific anti-rabbit CD34 antibodies are needed for efficient enrichment of rabbit HSCs. This article is protected by copyright. All rights reserved. © 2018 American Institute of Chemical Engineers.

Viable cell sorting of dinoflagellates by multi-parametric flow cytometry.

USDA-ARS?s Scientific Manuscript database

Electronic cell sorting for isolation and culture of dinoflagellates and other marine eukaryotic phytoplankton was compared to the traditional method of manually picking of cells using a micropipette. Trauma to electronically sorted cells was not a limiting factor as fragile dinoflagellates, such a...

Physiology of spermatozoa at high dilution rates: the influence of seminal plasma.

PubMed

Maxwell, W M; Johnson, L A

1999-12-01

Extensive dilution of spermatozoa, as occurs during flow-cytometric sperm sorting, can reduce their motility and viability. These effects may be minimized by the use of appropriate dilution and collection media, containing balanced salts, energy sources, egg yolk and some protein. Dilution and flow-cytometric sorting of spermatozoa, which involves the removal of seminal plasma, also destabilizes sperm membranes leading to functional capacitation. This membrane destabilization renders the spermatozoa immediately capable of fertilization in vitro, or in vivo after deposition close to the site of fertilization, but shortens their lifespan, resulting in premature death if the cells are deposited in the female tract distant from the site of fertilization or are held in vitro at standard storage temperatures. This functional capacitation can be reversed in boar spermatozoa by inclusion of seminal plasma in the medium used to collect the cells from the cell sorter and, consequently, reduces their in vitro fertility. It has yet to be determined whether seminal plasma would have similar effects on flow cytometrically sorted spermatozoa of other species, and what its effects might be on the in vivo fertility of flow sorted boar.

Microfluidic Droplet Sorting with a High Frequency Ultrasound Beam

PubMed Central

Lee, Changyang; Lee, Jungwoo; Kim, Hyung Ham; Teh, Shia-Yen; Lee, Abraham; Chung, In-Young; Park, Jae Yeong; Shung, K. Kirk

2012-01-01

This paper presents experimental results demonstrating the feasibility of high frequency ultrasonic sensing and sorting for screening single oleic acid (lipid or oil) droplets under continuous flow in a microfluidic channel. In these experiments, hydrodynamically focused lipid droplets of two different diameters (50 μm and 100 μm) are centered along the middle of the channel that is filled with deionized (DI) water. A 30 MHz lithium niobate (LiNbO3) transducer, placed outside the channel, first transmits short sensing pulses to non-invasively determine acoustic scattering properties of individual droplets that are passing through the beam’s focus. Integrated backscatter (IB) coefficients, utilized as a sorting criterion, are measured by analyzing received echo signals from each droplet. When the IB values corresponding to 100 μm droplets are obtained, a custom-built LabVIEW panel commands the transducer to emit sinusoidal burst signals to commence the sorting operation. The number of droplets tested for the sorting is 139 for 50 μm droplets and 95 for 100 μm droplets. The sensing efficiencies are estimated to be 98.6 % and 99.0 %, respectively. The sorting is carried out by applying acoustic radiation forces to 100 μm droplets to direct them towards the upper sheath flow, thus separating them from the centered droplet flow. The sorting efficiencies are 99.3 % for 50 μm droplets and 85.3 % for 100 μm droplets. The results suggest that this proposed technique has the potential to be further developed into a cost-effective and efficient cell/microparticle sorting instrument. PMID:22643737

Mesenchymal Stem Cells for Vascular Target Discovery in Breast Cancer-Associated Angiogenesis

DTIC Science & Technology

2005-09-01

demonstrating this marker as demonstrated by flow cytometry . These GFP+ MSCs were subsequently analyzed for expression of commonly reported markers of...phenotypically and genotypically analyzed by flow cytometry and gene chip analysis, respectively. We have also shown that MSCs can then be stimulated to...positive MSCs retrieved by collagenase digestion of the Matrigel plug and sorted by flow cytometry . Sorting of these retrieved cells based on co-expression

Semiconductor Microcavity Flow Spectroscopy of Intracellular Protein in Human Cells

NASA Astrophysics Data System (ADS)

Gourley, Paul; Cox, Jim; Hendricks, Judy; McDonald, Anthony; Copeland, Guild; Sasaki, Darryl; Skirboll, Steve; Curry, Mark

2001-03-01

The speed of light through a biofluid or biological cell is inversely related to the biomolecular concentration of proteins and other complex molecules that modify the refractive index at wavelengths accessible to semiconductor lasers. By placing a fluid or cell into a semiconductor microcavity laser, these decreases in light speed can be sensitively recorded in picoseconds as frequency red-shifts in the laser output spectrum. This biocavity laser equipped with microfluidics for transporting cells at high speed through the laser microcavity has shown potential for rapid analysis of biomolecular mass of normal and malignant human cells in their physiologic condition without time-consuming fixing, staining, or tagging. We have used biocavity laser spectroscopy to measure the optical properties of solutions of standard biomolecules (sugars, proteins, DNA, and ions) and human cells. The technique determines the frequency shift, relative to that of water, of spontaneous or stimulated emission from cavity filled with a biomolecular solution. The shift was also measured in human glioblastoma cells that had been sorted by conventional fluorescence-activated cell sorting according to protein content. The results show a direct correlation between protein measured by fluorescence and the frequency shift observed in the microcavity laser.

Passive chip-based droplet sorting

DOEpatents

Beer, Neil Reginald; Lee, Abraham P; Hatch, Andrew C; Fisher, Jeffrey S

2015-03-03

An apparatus for passive sorting of microdroplets including a main flow channel, a flow stream of microdroplets in the main flow channel wherein the microdroplets have substantially the same diameter and wherein the flow stream of microdroplets includes first microdroplets having a first degree of stiffness and second microdroplets having a second degree of stiffness wherein the second degree of stiffness is different than the first degree of stiffness. A second flow channel is connected to the main flow channel for the second microdroplets having a second degree of stiffness. A separator separates the second microdroplets having a second degree of stiffness from the first microdroplets and directs the second microdroplets having a second degree of stiffness into the second flow channel.

Numerical and experimental evaluation of microfluidic sorting devices.

PubMed

Taylor, Jay K; Ren, Carolyn L; Stubley, G D

2008-01-01

The development of lab-on-a-chip devices calls for the isolation or separation of specific bioparticles or cells. The design of a miniaturized cell-sorting device for handheld operation must follow the strict parameters associated with lab-on-a-chip technology. The limitations include applied voltage, high efficiency of cell-separation, reliability, size, flow control, and cost, among others. Currently used designs have achieved successful levels of cell isolation; however, further improvements in the microfluidic chip design are important to incorporate into larger systems. This study evaluates specific design modifications that contribute to the reduction of required applied potential aiming for developing portable devices, improved operation reliability by minimizing induced pressure disturbance when electrokinetic pumping is employed, and improved flow control by incorporating directing streams achieving dynamic sorting and counting. The chip designs fabricated in glass and polymeric materials include asymmetric channel widths for sample focusing, nonuniform channel depth for minimizing induced pressure disturbance, directing streams to assist particle flow control, and online filters for reducing channel blockage. Fluorescence-based visualization experimental results of electrokinetic focusing, flow field phenomena, and dynamic sorting demonstrate the advantages of the chip design. Numerical simulations in COMSOL are validated by the experimental data and used to investigate the effects of channel geometry and fluid properties on the flow field.

Cellular and Molecular Effect of MEHP Involving LXRα in Human Fetal Testis and Ovary

PubMed Central

Muczynski, Vincent; Lecureuil, Charlotte; Messiaen, Sébastien; Guerquin, Marie-Justine; N’Tumba-Byn, Thierry; Moison, Delphine; Hodroj, Wassim; Benjelloun, Hinde; Baijer, Jan; Livera, Gabriel; Frydman, René; Benachi, Alexandra; Habert, René; Rouiller-Fabre, Virginie

2012-01-01

Background Phthalates have been shown to have reprotoxic effects in rodents and human during fetal life. Previous studies indicate that some members of the nuclear receptor (NR) superfamilly potentially mediate phthalate effects. This study aimed to assess if expression of these nuclear receptors are modulated in the response to MEHP exposure on the human fetal gonads in vitro. Methodology/Principal Findings Testes and ovaries from 7 to 12 gestational weeks human fetuses were exposed to 10−4M MEHP for 72 h in vitro. Transcriptional level of NRs and of downstream genes was then investigated using TLDA (TaqMan Low Density Array) and qPCR approaches. To determine whether somatic or germ cells of the testis are involved in the response to MEHP exposure, we developed a highly efficient cytometric germ cell sorting approach. In vitro exposure of fetal testes and ovaries to MEHP up-regulated the expression of LXRα, SREBP members and of downstream genes involved in the lipid and cholesterol synthesis in the whole gonad. In sorted testicular cells, this effect is only observable in somatic cells but not in the gonocytes. Moreover, the germ cell loss induced by MEHP exposure, that we previously described, is restricted to the male gonad as oogonia density is not affected in vitro. Conclusions/Significance We evidenced for the first time that phthalate increases the levels of mRNA for LXRα, and SREBP members potentially deregulating lipids/cholesterol synthesis in human fetal gonads. Interestingly, this novel effect is observable in both male and female whereas the germ cell apoptosis is restricted to the male gonad. Furthermore, we presented here a novel and potentially very useful flow cytometric cell sorting method to analyse molecular changes in germ cells versus somatic cells. PMID:23118965

Cellular and molecular effect of MEHP Involving LXRα in human fetal testis and ovary.

PubMed

Muczynski, Vincent; Lecureuil, Charlotte; Messiaen, Sébastien; Guerquin, Marie-Justine; N'tumba-Byn, Thierry; Moison, Delphine; Hodroj, Wassim; Benjelloun, Hinde; Baijer, Jan; Livera, Gabriel; Frydman, René; Benachi, Alexandra; Habert, René; Rouiller-Fabre, Virginie

2012-01-01

Phthalates have been shown to have reprotoxic effects in rodents and human during fetal life. Previous studies indicate that some members of the nuclear receptor (NR) superfamilly potentially mediate phthalate effects. This study aimed to assess if expression of these nuclear receptors are modulated in the response to MEHP exposure on the human fetal gonads in vitro. Testes and ovaries from 7 to 12 gestational weeks human fetuses were exposed to 10(-4)M MEHP for 72 h in vitro. Transcriptional level of NRs and of downstream genes was then investigated using TLDA (TaqMan Low Density Array) and qPCR approaches. To determine whether somatic or germ cells of the testis are involved in the response to MEHP exposure, we developed a highly efficient cytometric germ cell sorting approach. In vitro exposure of fetal testes and ovaries to MEHP up-regulated the expression of LXRα, SREBP members and of downstream genes involved in the lipid and cholesterol synthesis in the whole gonad. In sorted testicular cells, this effect is only observable in somatic cells but not in the gonocytes. Moreover, the germ cell loss induced by MEHP exposure, that we previously described, is restricted to the male gonad as oogonia density is not affected in vitro. We evidenced for the first time that phthalate increases the levels of mRNA for LXRα, and SREBP members potentially deregulating lipids/cholesterol synthesis in human fetal gonads. Interestingly, this novel effect is observable in both male and female whereas the germ cell apoptosis is restricted to the male gonad. Furthermore, we presented here a novel and potentially very useful flow cytometric cell sorting method to analyse molecular changes in germ cells versus somatic cells.

Interval linear programming model for long-term planning of vehicle recycling in the Republic of Serbia under uncertainty.

PubMed

Simic, Vladimir; Dimitrijevic, Branka

2015-02-01

An interval linear programming approach is used to formulate and comprehensively test a model for optimal long-term planning of vehicle recycling in the Republic of Serbia. The proposed model is applied to a numerical case study: a 4-year planning horizon (2013-2016) is considered, three legislative cases and three scrap metal price trends are analysed, availability of final destinations for sorted waste flows is explored. Potential and applicability of the developed model are fully illustrated. Detailed insights on profitability and eco-efficiency of the projected contemporary equipped vehicle recycling factory are presented. The influences of the ordinance on the management of end-of-life vehicles in the Republic of Serbia on the vehicle hulks procuring, sorting generated material fractions, sorted waste allocation and sorted metals allocation decisions are thoroughly examined. The validity of the waste management strategy for the period 2010-2019 is tested. The formulated model can create optimal plans for procuring vehicle hulks, sorting generated material fractions, allocating sorted waste flows and allocating sorted metals. Obtained results are valuable for supporting the construction and/or modernisation process of a vehicle recycling system in the Republic of Serbia. © The Author(s) 2015.

Pulsed laser activated cell sorter (PLACS) for high-throughput fluorescent mammalian cell sorting

NASA Astrophysics Data System (ADS)

Chen, Yue; Wu, Ting-Hsiang; Chung, Aram; Kung, Yu-Chung; Teitell, Michael A.; Di Carlo, Dino; Chiou, Pei-Yu

2014-09-01

We present a Pulsed Laser Activated Cell Sorter (PLACS) realized by exciting laser induced cavitation bubbles in a PDMS microfluidic channel to create high speed liquid jets to deflect detected fluorescent samples for high speed sorting. Pulse laser triggered cavitation bubbles can expand in few microseconds and provide a pressure higher than tens of MPa for fluid perturbation near the focused spot. This ultrafast switching mechanism has a complete on-off cycle less than 20 μsec. Two approaches have been utilized to achieve 3D sample focusing in PLACS. One is relying on multilayer PDMS channels to provide 3D hydrodynamic sheath flows. It offers accurate timing control of fast (2 m sec-1) passing particles so that synchronization with laser bubble excitation is possible, an critically important factor for high purity and high throughput sorting. PLACS with 3D hydrodynamic focusing is capable of sorting at 11,000 cells/sec with >95% purity, and 45,000 cells/sec with 45% purity using a single channel in a single step. We have also demonstrated 3D focusing using inertial flows in PLACS. This sheathless focusing approach requires 10 times lower initial cell concentration than that in sheath-based focusing and avoids severe sample dilution from high volume sheath flows. Inertia PLACS is capable of sorting at 10,000 particles sec-1 with >90% sort purity.

Liver repopulation by c-Met-positive stem/progenitor cells isolated from the developing rat liver.

PubMed

Suzuki, Atsushi; Zheng, Yun-wen; Fukao, Katashi; Nakauchi, Hiromitsu; Taniguchi, Hideki

2004-01-01

Self-renewing stem cells responsible for tissue or organ development and regeneration have been recently described. To isolate such cells using flow cytometry, it should be required to find molecules expressing on their cell surfaces. We have previously reported that, on cells fulfilling the criteria for hepatic stem cells, the hepatocyte growth factor receptor c-Met is expressed specifically in the developing mouse liver. In this study, to determine whether c-Met is an essential marker for hepatic stem cells in other animal strains, we examined the potential for in vivo liver-repopulation in sorted fetal rat-derived c-Met+ cells using the retrorsine model. Using flow cytometry and monoclonal antibodies for c-Met and leukocyte common antigen CD45, fetal rat liver cells were fractionated according to the expression of these molecules. Then, cells in each cell subpopulation were sorted and transplanted into the retrorsine-treated adult rats with two-third hepatectomy. At 9 months post transplant, frequency of liver-repopulation was examined by qualitative and quantitative analyses. When we transplanted c-Met+ CD45- sorted cells, many donor-derived cells formed colonies that included mature hepatocytes expressing albumin and containing abundant glycogen in their cytoplasm. In contrast, c-Met- cells and CD45+ cells could not repopulate damaged recipient livers. High enrichment of liver-repopulating cells was conducted by sorting of c-Met+ cells from the developing rat liver. This result suggests that c-Met/HGF interaction plays a crucial role for stem cell growth, differentiation, and self-renewal in rat liver organogenesis. Since the c-Met is also expressed in the fetal mouse-derived hepatic stem cells, this molecule could be expected to be an essential marker for such cell population in the various animal strains, including human.

Canonical Wnt Signaling as a Specific Marker of Normal and Tumorigenic Mammary Stem Cells

DTIC Science & Technology

2013-02-01

get enough sorted mammary cells for the transplantation experiments. We are currently working with our Flow Cytometry Core to sort Lin-/CD24+/CD49...activity our flow cytometry data suggests t here is a 2-fold increase in the number of FOG+ MEC’s in BATgal animals compared to contro ls which...this populat ion of cells is enriched for stem cell activity. Flow cytometry will determine the percentage of FOG+ cells within pre-neoplastic BATgai

Dean Flow Dynamics in Low-Aspect Ratio Spiral Microchannels

PubMed Central

Nivedita, Nivedita; Ligrani, Phillip; Papautsky, Ian

2017-01-01

A wide range of microfluidic cell-sorting devices has emerged in recent years, based on both passive and active methods of separation. Curvilinear channel geometries are often used in these systems due to presence of secondary flows, which can provide high throughput and sorting efficiency. Most of these devices are designed on the assumption of two counter rotating Dean vortices present in the curved rectangular channels and existing in the state of steady rotation and amplitude. In this work, we investigate these secondary flows in low aspect ratio spiral rectangular microchannels and define their development with respect to the channel aspect ratio and Dean number. This work is the first to experimentally and numerically investigate Dean flows in microchannels for Re > 100, and show presence of secondary Dean vortices beyond a critical Dean number. We further demonstrate the impact of these multiple vortices on particle and cell focusing. Ultimately, this work offers new insights into secondary flow instabilities for low-aspect ratio, spiral microchannels, with improved flow models for design of more precise and efficient microfluidic devices for applications such as cell sorting and micromixing. PMID:28281579

Flow Sorting of Marine Bacterioplankton after Fluorescence In Situ Hybridization

PubMed Central

Sekar, Raju; Fuchs, Bernhard M.; Amann, Rudolf; Pernthaler, Jakob

2004-01-01

We describe an approach to sort cells from coastal North Sea bacterioplankton by flow cytometry after in situ hybridization with rRNA-targeted horseradish peroxidase-labeled oligonucleotide probes and catalyzed fluorescent reporter deposition (CARD-FISH). In a sample from spring 2003 >90% of the cells were detected by CARD-FISH with a bacterial probe (EUB338). Approximately 30% of the microbial assemblage was affiliated with the Cytophaga-Flavobacterium lineage of the Bacteroidetes (CFB group) (probe CF319a), and almost 10% was targeted by a probe for the β-proteobacteria (probe BET42a). A protocol was optimized to detach cells hybridized with EUB338, BET42a, and CF319a from membrane filters (recovery rate, 70%) and to sort the cells by flow cytometry. The purity of sorted cells was >95%. 16S rRNA gene clone libraries were constructed from hybridized and sorted cells (S-EUB, S-BET, and S-CF libraries) and from unhybridized and unsorted cells (UNHYB library). Sequences related to the CFB group were significantly more frequent in the S-CF library (66%) than in the UNHYB library (13%). No enrichment of β-proteobacterial sequence types was found in the S-BET library, but novel sequences related to Nitrosospira were found exclusively in this library. These bacteria, together with members of marine clade OM43, represented >90% of the β-proteobacteria in the water sample, as determined by CARD-FISH with specific probes. This illustrates that a combination of CARD-FISH and flow sorting might be a powerful approach to study the diversity and potentially the activity and the genomes of different bacterial populations in aquatic habitats. PMID:15466568

Optimization of flow cytometric detection and cell sorting of transgenic Plasmodium parasites using interchangeable optical filters

PubMed Central

2012-01-01

Background Malaria remains a major cause of morbidity and mortality worldwide. Flow cytometry-based assays that take advantage of fluorescent protein (FP)-expressing malaria parasites have proven to be valuable tools for quantification and sorting of specific subpopulations of parasite-infected red blood cells. However, identification of rare subpopulations of parasites using green fluorescent protein (GFP) labelling is complicated by autofluorescence (AF) of red blood cells and low signal from transgenic parasites. It has been suggested that cell sorting yield could be improved by using filters that precisely match the emission spectrum of GFP. Methods Detection of transgenic Plasmodium falciparum parasites expressing either tdTomato or GFP was performed using a flow cytometer with interchangeable optical filters. Parasitaemia was evaluated using different optical filters and, after optimization of optics, the GFP-expressing parasites were sorted and analysed by microscopy after cytospin preparation and by imaging cytometry. Results A new approach to evaluate filter performance in flow cytometry using two-dimensional dot blot was developed. By selecting optical filters with narrow bandpass (BP) and maximum position of filter emission close to GFP maximum emission in the FL1 channel (510/20, 512/20 and 517/20; dichroics 502LP and 466LP), AF was markedly decreased and signal-background improve dramatically. Sorting of GFP-expressing parasite populations in infected red blood cells at 90 or 95% purity with these filters resulted in 50-150% increased yield when compared to the standard filter set-up. The purity of the sorted population was confirmed using imaging cytometry and microscopy of cytospin preparations of sorted red blood cells infected with transgenic malaria parasites. Discussion Filter optimization is particularly important for applications where the FP signal and percentage of positive events are relatively low, such as analysis of parasite-infected samples with in the intention of gene-expression profiling and analysis. The approach outlined here results in substantially improved yield of GFP-expressing parasites, and requires decreased sorting time in comparison to standard methods. It is anticipated that this protocol will be useful for a wide range of applications involving rare events. PMID:22950515

Birth of kids after artificial insemination with sex-sorted, frozen-thawed goat spermatozoa.

PubMed

Bathgate, R; Mace, N; Heasman, K; Evans, G; Maxwell, W M C; de Graaf, S P

2013-12-01

Successful sex-sorting of goat spermatozoa and subsequent birth of pre-sexed kids have yet to be reported. As such, a series of experiments were conducted to develop protocols for sperm-sorting (using a modified flow cytometer, MoFlo SX(®) ) and cryopreservation of goat spermatozoa. Saanen goat spermatozoa (n = 2 males) were (i) collected into Salamon's or Tris catch media post-sorting and (ii) frozen in Tris-citrate-glucose media supplemented with 5, 10 or 20% egg yolk in (iii) 0.25 ml pellets on dry ice or 0.25 ml straws in a controlled-rate freezer. Post-sort and post-thaw sperm quality were assessed by motility (CASA), viability and acrosome integrity (PI/FITC-PNA). Sex-sorted goat spermatozoa frozen in pellets displayed significantly higher post-thaw motility and viability than spermatozoa frozen in straws. Catch media and differing egg yolk concentration had no effect on the sperm parameters tested. The in vitro and in vivo fertility of sex-sorted goat spermatozoa produced with this optimum protocol were then tested by means of a heterologous ova binding assay and intrauterine artificial insemination of Saanen goat does, respectively. Sex-sorted goat spermatozoa bound to sheep ova zona pellucidae in similar numbers (p > 0.05) to non-sorted goat spermatozoa, non-sorted ram spermatozoa and sex-sorted ram spermatozoa. Following intrauterine artificial insemination with sex-sorted spermatozoa, 38% (5/13) of does kidded with 83% (3/5) of kids being of the expected sex. Does inseminated with non-sorted spermatozoa achieved a 50% (3/6) kidding rate and a sex ratio of 3 : 1 (F : M). This study demonstrates for the first time that goat spermatozoa can be sex-sorted by flow cytometry, successfully frozen and used to produce pre-sexed kids. © 2013 Blackwell Verlag GmbH.

How to measure RNA expression in rare senescent cells expressing any specific protein such as p16Ink4a

PubMed Central

Jeyapalan, Jessie C.; Sedivy, John M.

2013-01-01

Here we describe a carefully optimized method for the preparation of high quality RNA by flow sorting of formaldehyde fixed senescent cells immunostained for any intracellular antigen. Replicative cellular senescence is a phenomenon of irreversible growth arrest triggered by the accumulation of a discrete number of cell divisions. The underlying cause of senescence due to replicative exhaustion is telomere shortening. We document here a spontaneous and apparently stochastic process that continuously generates senescent cells in cultures fully immortalized with telomerase. In the course of studying this phenomenon we developed a preparative fluorescence activated flow sorting method based on immunofluorescent staining of intracellular antigens that can also deliver RNA suitable for quantitative analysis of global gene expression. The protocols were developed using normal human diploid fibroblasts (HDF) and up to 5×107 cells could be conveniently processed in a single experiment. The methodology is based on formaldehyde crosslinking of cells, followed by permeabilization, antibody staining, flow sorting, reversal of the crosslinks, and recovery of the RNA. We explored key parameters such as crosslink reversal that affect the fragmentation of RNA. The recovered RNA is of high quality for downstream molecular applications based on short range sequence analysis, such qPCR, hybridization microarrays, and next generation sequencing. The RNA was analyzed by Affymetrix Gene Chip expression profiling and compared to RNA prepared by the direct lysis of cells. The correlation between the data sets was very high, indicating that the procedure does not introduce systematic changes in the mRNA transcriptome. The methods presented in this communication should be of interest to many investigators working in diverse model systems. PMID:23454889

How to measure RNA expression in rare senescent cells expressing any specific protein such as p16Ink4a.

PubMed

Jeyapalan, Jessie C; Sedivy, John M

2013-02-01

Here we describe a carefully optimized method for the preparation of high quality RNA by flow sorting of formaldehyde fixed senescent cells immunostained for any intracellular antigen. Replicative cellular senescence is a phenomenon of irreversible growth arrest triggered by the accumulation of a discrete number of cell divisions. The underlying cause of senescence due to replicative exhaustion is telomere shortening. We document here a spontaneous and apparently stochastic process that continuously generates senescent cells in cultures fully immortalized with telomerase. In the course of studying this phenomenon we developed a preparative fluorescence activated flow sorting method based on immunofluorescent staining of intracellular antigens that can also deliver RNA suitable for quantitative analysis of global gene expression. The protocols were developed using normal human diploid fibroblasts (HDF) and up to 5x107 cells could be conveniently processed in a single experiment. The methodology is based on formaldehyde crosslinking of cells, followed by permeabilization, antibody staining, flow sorting, reversal of the crosslinks, and recovery of the RNA. We explored key parameters such as crosslink reversal that affect the fragmentation of RNA. The recovered RNA is of high quality for downstream molecular applications based on short range sequence analysis, such qPCR, hybridization microarrays, and next generation sequencing. The RNA was analyzed by Affymetrix Gene Chip expression profiling and compared to RNA prepared by the direct lysis of cells. The correlation between the data sets was very high, indicating that the procedure does not introduce systematic changes in the mRNA transcriptome. The methods presented in this communication should be of interest to many investigators working in diverse model systems.

High-throughput autofluorescence flow cytometry of breast cancer metabolism (Conference Presentation)

NASA Astrophysics Data System (ADS)

Shah, Amy T.; Cannon, Taylor M.; Higginbotham, Jim N.; Skala, Melissa C.

2016-02-01

Tumor heterogeneity poses challenges for devising optimal treatment regimens for cancer patients. In particular, subpopulations of cells can escape treatment and cause relapse. There is a need for methods to characterize tumor heterogeneity of treatment response. Cell metabolism is altered in cancer (Warburg effect), and cells use the autofluorescent cofactor NADH in numerous metabolic reactions. Previous studies have shown that microscopy measurements of NADH autofluorescence are sensitive to treatment response in breast cancer, and these techniques typically assess hundreds of cells per group. An alternative approach is flow cytometry, which measures fluorescence on a single-cell level and is attractive for characterizing tumor heterogeneity because it achieves high-throughput analysis and cell sorting in millions of cells per group. Current applications for flow cytometry rely on staining with fluorophores. This study characterizes flow cytometry measurements of NADH autofluorescence in breast cancer cells. Preliminary results indicate flow cytometry of NADH is sensitive to cyanide perturbation, which inhibits oxidative phosphorylation, in nonmalignant MCF10A cells. Additionally, flow cytometry is sensitive to higher NADH intensity for HER2-positive SKBr3 cells compared with triple-negative MDA-MB-231 cells. These results agree with previous microscopy studies. Finally, a mixture of SKBr3 and MDA-MB-231 cells were sorted into each cell type using NADH intensity. Sorted cells were cultured, and microscopy validation showed the expected morphology for each cell type. Ultimately, flow cytometry could be applied to characterize tumor heterogeneity based on treatment response and sort cell subpopulations based on metabolic profile. These achievements could enable individualized treatment strategies and improved patient outcomes.

Identification of microbes from the surfaces of food-processing lines based on the flow cytometric evaluation of cellular metabolic activity combined with cell sorting.

PubMed

Juzwa, W; Duber, A; Myszka, K; Białas, W; Czaczyk, K

2016-09-01

In this study the design of a flow cytometry-based procedure to facilitate the detection of adherent bacteria from food-processing surfaces was evaluated. The measurement of the cellular redox potential (CRP) of microbial cells was combined with cell sorting for the identification of microorganisms. The procedure enhanced live/dead cell discrimination owing to the measurement of the cell physiology. The microbial contamination of the surface of a stainless steel conveyor used to process button mushrooms was evaluated in three independent experiments. The flow cytometry procedure provided a step towards monitoring of contamination and enabled the assessment of microbial food safety hazards by the discrimination of active, mid-active and non-active bacterial sub-populations based on determination of their cellular vitality and subsequently single cell sorting to isolate microbial strains from discriminated sub-populations. There was a significant correlation (r = 0.97; p < 0.05) between the bacterial cell count estimated by the pour plate method and flow cytometry, despite there being differences in the absolute number of cells detected. The combined approach of flow cytometric CRP measurement and cell sorting allowed an in situ analysis of microbial cell vitality and the identification of species from defined sub-populations, although the identified microbes were limited to culturable cells.

Continuous-feed optical sorting of aerosol particles

PubMed Central

Curry, J. J.; Levine, Zachary H.

2016-01-01

We consider the problem of sorting, by size, spherical particles of order 100 nm radius. The scheme we analyze consists of a heterogeneous stream of spherical particles flowing at an oblique angle across an optical Gaussian mode standing wave. Sorting is achieved by the combined spatial and size dependencies of the optical force. Particles of all sizes enter the flow at a point, but exit at different locations depending on size. Exiting particles may be detected optically or separated for further processing. The scheme has the advantages of accommodating a high throughput, producing a continuous stream of continuously dispersed particles, and exhibiting excellent size resolution. We performed detailed Monte Carlo simulations of particle trajectories through the optical field under the influence of convective air flow. We also developed a method for deriving effective velocities and diffusion constants from the Fokker-Planck equation that can generate equivalent results much more quickly. With an optical wavelength of 1064 nm, polystyrene particles with radii in the neighborhood of 275 nm, for which the optical force vanishes, may be sorted with a resolution below 1 nm. PMID:27410570

Confirmation of Pig-a mutation in flow cytometry-identified CD48-deficient T-lymphocytes from F344 rats.

PubMed

Revollo, Javier; Pearce, Mason G; Petibone, Dayton M; Mittelstaedt, Roberta A; Dobrovolsky, Vasily N

2015-05-01

The Pig-a assay is used for monitoring somatic cell mutation in laboratory animals and humans. The assay detects haematopoietic cells deficient in glycosylphosphatidylinositol (GPI)-anchored protein surface markers using flow cytometry. However, given that synthesis of the protein markers (and the expression of their genes) is independent of the expression of the X-linked Pig-a gene and the function of its enzyme product, the deficiency of markers at the surface of the cells may be caused by a number of events (e.g. by mutation or epigenetic silencing in the marker gene itself or in any of about two dozen autosomal genes involved in the synthesis of GPI). Here we provide direct evidence that the deficiency of the GPI-anchored surface marker CD48 in rat T-cells is accompanied by mutation in the endogenous X-linked Pig-a gene. We treated male F344 rats with N-ethyl-N-nitrosourea (ENU), and established colonies from flow cytometry-identified and sorted CD48-deficient spleen T-lymphocytes. Molecular analysis confirmed that the expanded sorted cells have mutations in the Pig-a gene. The spectrum of Pig-a mutation in our model was consistent with the spectrum of ENU-induced mutation determined in other in vivo models, mostly base-pair substitutions at A:T with the mutated T on the non-transcribed strand of Pig-a genomic DNA. We also used next generation sequencing to derive a similar mutational spectrum from a pool of 64 clones developed from flow-sorted CD48-deficient lymphocytes. Our findings confirm that Pig-a assays detect what they are designed to detect-gene mutation in the Pig-a gene. © The Author 2015. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Flow cytometric characterization of the response of Fanconi's anemia cells to mitomycin C treatment.

PubMed

Kaiser, T N; Lojewski, A; Dougherty, C; Juergens, L; Sahar, E; Latt, S A

1982-03-01

DNA flow histogram analysis, using 33342 Hoechst as a stain, has been used to detect the effect of the potentially bifunctional alkylating agent, mitomycin C (MMC) on dermal fibroblasts from patients with Fanconi's anemia (FA), a hereditary human disease characterized by pancytopenia, hypersensitivity to DNA-crosslinking agents, congenital abnormalities and a predisposition for neoplasia. At 24 or 48 hr after a 2-hr exposure to 0.05 or 0.10 micrograms/ml MMC, (3)HdT incorporation was reduced to a greater extent in FA cells than in normal cells. Cells sorted from the last half of S phase showed a slightly greater inhibition of (3)HdT incorporation than did those sorted from the first half of S. Fanconi's anemia cells exhibited a marked accumulation in the G(2) + M peak of flow histograms following exposure to MMC. Twenty-four hr after treatment with .0.5 micrograms/ml MMC, the G(2) + M fraction of FA cells (eight lines) increased to more than 0.5 from a control value of approximately 0.02. Both normals (six lines) and heterozygotes (eight lines) showed, on the average, much less of a G(2) + M increment than did FA cells, even after exposure to 0.1 micrograms/ml MMC. Examination of cells sorted from the G(2) + M peak revealed that MMC-treated FA cells were blocked prior to mitosis. To determine whether the response of FA cells was specific for bifunctional alkylating agent, cells were also treated with ethylmethanesulfonate, a monofunctional agent. Twenty-four hours after exposure to 0.25 or 0.5 mg/ml ethylmethanesulfonate, FA and normal cells showed similar, small increases in the G(2) + M peak. The results suggest the utility of flow cytometry in the diagnostic evaluation of fibroblasts from patients suspected of having Fanconi's anemia.

Characterization of cancer stem-like cells derived from a side population of a human gallbladder carcinoma cell line, SGC-996

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Xin-xing; Wang, Jian, E-mail: dr_wangjian@yahoo.com.cn; Wang, Hao-lu

2012-03-23

Highlights: Black-Right-Pointing-Pointer We sorted SP cells from a human gallbladder carcinoma cell lines, SGC-996. Black-Right-Pointing-Pointer SP cells displayed higher proliferation and stronger clonal-generating capability. Black-Right-Pointing-Pointer SP cells showed more migratory and invasive abilities. Black-Right-Pointing-Pointer SP cells were more resistant and tumorigenic than non-SP counterparts. Black-Right-Pointing-Pointer ABCG2 might be a candidate as a marker for SP cells. -- Abstract: The cancer stem cell (CSC) hypothesis proposes that CSCs, which can renew themselves proliferate infinitely, and escape chemotherapy, become the root of recurrence and metastasis. Previous studies have verified that side population (SP) cells, characterized by their ability to efflux lipophilic substratemore » Hoechst 33342, to share many characteristics of CSCs in multiplying solid tumors. The purpose of this study was to sort SP cells from a human gallbladder carcinoma cell line, SGC-996 and to preliminarily identify the biological characteristics of SP cells from the cell line. Using flow cytometry we effectively sorted SP cells from the cell line SGC-996. SP cells not only displayed higher proliferative, stronger clonal-generating, more migratory and more invasive capacities, but showed stronger resistance. Furthermore, our experiments demonstrated that SP cells were more tumorigenic than non-SP counterparts in vivo. Real-time PCR analysis and immunocytochemistry showed that the expression of ATP-binding cassette subfamily G member 2 (ABCG2) was significantly higher in SP cells. Hence, these results collectively suggest that SP cells are progenitor/stem-like cells and ABCG2 might be a candidate marker for SP cells in human gallbladder cancer.« less

Concurrent Isolation of 3 Distinct Cardiac Stem Cell Populations From a Single Human Heart Biopsy.

PubMed

Monsanto, Megan M; White, Kevin S; Kim, Taeyong; Wang, Bingyan J; Fisher, Kristina; Ilves, Kelli; Khalafalla, Farid G; Casillas, Alexandria; Broughton, Kathleen; Mohsin, Sadia; Dembitsky, Walter P; Sussman, Mark A

2017-07-07

The relative actions and synergism between distinct myocardial-derived stem cell populations remain obscure. Ongoing debates on optimal cell population(s) for treatment of heart failure prompted implementation of a protocol for isolation of multiple stem cell populations from a single myocardial tissue sample to develop new insights for achieving myocardial regeneration. Establish a robust cardiac stem cell isolation and culture protocol to consistently generate 3 distinct stem cell populations from a single human heart biopsy. Isolation of 3 endogenous cardiac stem cell populations was performed from human heart samples routinely discarded during implantation of a left ventricular assist device. Tissue explants were mechanically minced into 1 mm 3 pieces to minimize time exposure to collagenase digestion and preserve cell viability. Centrifugation removes large cardiomyocytes and tissue debris producing a single cell suspension that is sorted using magnetic-activated cell sorting technology. Initial sorting is based on tyrosine-protein kinase Kit (c-Kit) expression that enriches for 2 c-Kit + cell populations yielding a mixture of cardiac progenitor cells and endothelial progenitor cells. Flowthrough c-Kit - mesenchymal stem cells are positively selected by surface expression of markers CD90 and CD105. After 1 week of culture, the c-Kit + population is further enriched by selection for a CD133 + endothelial progenitor cell population. Persistence of respective cell surface markers in vitro is confirmed both by flow cytometry and immunocytochemistry. Three distinct cardiac cell populations with individualized phenotypic properties consistent with cardiac progenitor cells, endothelial progenitor cells, and mesenchymal stem cells can be successfully concurrently isolated and expanded from a single tissue sample derived from human heart failure patients. © 2017 American Heart Association, Inc.

Quantifying the influence of flow asymmetries on glottal sound sources in speech

NASA Astrophysics Data System (ADS)

Erath, Byron; Plesniak, Michael

2008-11-01

Human speech is made possible by the air flow interaction with the vocal folds. During phonation, asymmetries in the glottal flow field may arise from flow phenomena (e.g. the Coanda effect) as well as from pathological vocal fold motion (e.g. unilateral paralysis). In this study, the effects of flow asymmetries on glottal sound sources were investigated. Dynamically-programmable 7.5 times life-size vocal fold models with 2 degrees-of-freedom (linear and rotational) were constructed to provide a first-order approximation of vocal fold motion. Important parameters (Reynolds, Strouhal, and Euler numbers) were scaled to physiological values. Normal and abnormal vocal fold motions were synthesized, and the velocity field and instantaneous transglottal pressure drop were measured. Variability in the glottal jet trajectory necessitated sorting of the data according to the resulting flow configuration. The dipole sound source is related to the transglottal pressure drop via acoustic analogies. Variations in the transglottal pressure drop (and subsequently the dipole sound source) arising from flow asymmetries are discussed.

Engineering quadrupole magnetic flow sorting for the isolation of pancreatic islets

NASA Astrophysics Data System (ADS)

Kennedy, David J.; Todd, Paul; Logan, Sam; Becker, Matthew; Papas, Klearchos K.; Moore, Lee R.

2007-04-01

Quadrupole magnetic flow sorting (QMS) is being adapted from the separation of suspensions of single cells (<15 μm) to the isolation of pancreatic islets (150-350 μm) for transplant. To achieve this goal, the critical QMS components have been modeled and engineered to optimize the separation process. A flow channel has been designed, manufactured, and tested. The quadrupole magnet assembly has been designed and verified by finite element analysis. Pumps have been selected and verified by test. Test data generated from the pumps and flow channel demonstrate that the fabricated channel and peristaltic pumps fulfill the requirements of successful QMS separation.

Interannual variability of sorted bedforms in the coastal German Bight (SE North Sea)

NASA Astrophysics Data System (ADS)

Mielck, F.; Holler, P.; Bürk, D.; Hass, H. C.

2015-12-01

Sorted bedforms are ubiquitous on the inner continental shelves worldwide. They are described as spatially-grain-size-sorted features consisting of small rippled medium-to-coarse sand and can remain stable for decades. However, the knowledge about their genesis and development is still fragmentary. For this study, a representative investigation area (water depth<15 m) located on the shelf west of the island of Sylt (SE North Sea, Germany) was periodically surveyed with hydroacoustic means (i.e. sidescan sonar, multibeam echo sounder, and sub-bottom profiler) during 2010-2014. Since this area is influenced by tidal and wind-driven currents, the aim was to detect and examine interannual variabilities in the characteristics of the prevailing sorted bedforms. Our measurements reveal sinuous stripes of rippled medium sand which are embedded in shallow symmetrical depressions. These domains are surrounded by relatively smooth fine-sand areas. These sorted bedforms were identified as flow-transverse features that are maintained by ebb and flood currents of almost equal strengths that flow in opposite directions. This bidirectional flow field generates sharp boundaries between the medium- and fine-sand domains in both current directions. Further to the north, where flood currents are dominant, asymmetric sorted bedforms were detected which show sharp boundaries only in flood-current direction. Comparisons between the measurements of the different years show no significant variations in morphology and distribution of the sorted bedforms. However, variations of the boundaries between the medium and the fine-sand domains were observed. Additionally, new minor sorted bedforms and rippled excavation marks as well as new fine-sand areas developed and disappeared occasionally. It can be supposed that such sediment winnowing and focusing processes take place during periodically recurring storm surges, which change the shapes of the features. Moreover, variations in alignments and sizes of the small ripple formations were detected. They seem to indicate the directions and intensities of previous storm events.

The effects of hoechst 33342 staining and the male sample donor on the sorting efficiency of canine spermatozoa.

PubMed

Rodenas, C; Lucas, X; Tarantini, T; Del Olmo, D; Roca, J; Vazquez, J M; Martinez, E A; Parrilla, I

2014-02-01

The aim of this study was to evaluate the influence of Hoechst 33342 (H-42) concentration and of the male donor on the efficiency of sex-sorting procedure in canine spermatozoa. Semen samples from six dogs (three ejaculates/dog) were diluted to 100 × 10(6) sperm/ml, split into four aliquots, stained with increasing H-42 concentrations (5, 7.5, 10 and 12.5 μl, respectively) and sorted by flow cytometry. The rates of non-viable (FDA+), oriented (OS) and selected spermatozoa (SS), as well as the average sorting rates (SR, sorted spermatozoa/s), were used to determine the sorting efficiency. The effects of the sorting procedure on the quality of sorted spermatozoa were evaluated in terms of total motility (TM), percentage of viable spermatozoa (spermatozoa with membrane and acrosomal integrity) and percentage of spermatozoa with reacted/damaged acrosomes. X- and Y-chromosome-bearing sperm populations were identified in all of the samples stained with 7.5, 10 and 12.5 μl of H-42, while these two populations were only identified in 77.5% of samples stained with 5 μl. The values of OS, SS and SR were influenced by the male donor (p < 0.01) but not by the H-42 concentration used. The quality of sorted sperm samples immediately after sorting was similar to that of fresh samples, while centrifugation resulted in significant reduction (p < 0.05) in TM and in the percentage of viable spermatozoa and a significant increase (p < 0.01) in the percentage of spermatozoa with damage/reacted acrosomes. In conclusion, the sex-sorting of canine spermatozoa by flow cytometry can be performed successfully using H-42 concentrations between 7.5 and 12.5 μl. The efficiency of the sorting procedure varies based on the dog from which the sperm sample derives. © 2013 Blackwell Verlag GmbH.

Raman-activated cell sorting based on dielectrophoretic single-cell trap and release.

PubMed

Zhang, Peiran; Ren, Lihui; Zhang, Xu; Shan, Yufei; Wang, Yun; Ji, Yuetong; Yin, Huabing; Huang, Wei E; Xu, Jian; Ma, Bo

2015-02-17

Raman-activated cell sorting (RACS) is a promising single-cell technology that holds several significant advantages, as RACS is label-free, information-rich, and potentially in situ. To date, the ability of the technique to identify single cells in a high-speed flow has been limited by inherent weakness of the spontaneous Raman signal. Here we present an alternative pause-and-sort RACS microfluidic system that combines positive dielectrophoresis (pDEP) for single-cell trap and release with a solenoid-valve-suction-based switch for cell separation. This has allowed the integration of trapping, Raman identification, and automatic separation of individual cells in a high-speed flow. By exerting a periodical pDEP field, single cells were trapped, ordered, and positioned individually to the detection point for Raman measurement. As a proof-of-concept demonstration, a mixture of two cell strains containing carotenoid-producing yeast (9%) and non-carotenoid-producing Saccharomyces cerevisiae (91%) was sorted, which enriched the former to 73% on average and showed a fast Raman-activated cell sorting at the subsecond level.

Phenotypic and functional characterization of earthworm coelomocyte subsets: Linking light scatter-based cell typing and imaging of the sorted populations.

PubMed

Engelmann, Péter; Hayashi, Yuya; Bodó, Kornélia; Ernszt, Dávid; Somogyi, Ildikó; Steib, Anita; Orbán, József; Pollák, Edit; Nyitrai, Miklós; Németh, Péter; Molnár, László

2016-12-01

Flow cytometry is a common approach to study invertebrate immune cells including earthworm coelomocytes. However, the link between light-scatter- and microscopy-based phenotyping remains obscured. Here we show, by means of light scatter-based cell sorting, both subpopulations (amoebocytes and eleocytes) can be physically isolated with good sort efficiency and purity confirmed by downstream morphological and cytochemical applications. Immunocytochemical analysis using anti-EFCC monoclonal antibodies combined with phalloidin staining has revealed antigenically distinct, sorted subsets. Screening of lectin binding capacity indicated wheat germ agglutinin (WGA) as the strongest reactor to amoebocytes. This is further evidenced by WGA inhibition assays that suggest high abundance of N-acetyl-d-glucosamine in amoebocytes. Post-sort phagocytosis assays confirmed the functional differences between amoebocytes and eleocytes, with the former being in favor of bacterial engulfment. This study has proved successful in linking flow cytometry and microscopy analysis and provides further experimental evidence of phenotypic and functional heterogeneity in earthworm coelomocyte subsets. Copyright © 2016 Elsevier Ltd. All rights reserved.

Isolation and Applications of Prostate Side Population Cells Based on Dye Cycle Violet Efflux

PubMed Central

Gangavarapu, Kalyan J.; Huss, Wendy J.

2011-01-01

This unit describes methods for the digestion of human prostate clinical specimens, dye cycle violet (DCV) staining procedure for the identification, isolation, and quantitation of radiolabeled dihydrotestosterone (DHT) retention of side population cells. The principle of the side population assay is based on differential efflux of DCV, a cell membrane permeable fluorescent dye, by cells with high ATP binding cassette (ABC) transporter activity. Cells with high ABC transporter activity efflux DCV and fall in the lower left quadrant of a flow cytograph are designated as “side population” cells. This unit emphasizes tissue digestion, DCV staining, flow settings for sorting side population cells and quantitation of radiolabeled DHT retention. PMID:21400686

Isolation of purified oocyst walls and sporocysts from Toxoplasma gondii.

PubMed

Everson, William V; Ware, Michael W; Dubey, J P; Lindquist, H D Alan

2002-01-01

Toxoplasma gondii oocysts are environmentally resistant and can infect virtually all warm-blooded hosts, including humans and livestock. Little is known about the biochemical basis for this resistance of oocysts, and mechanism for excystation of T. gondii sporozoites. The objective of the present study was to evaluate different methods (mechanical fragmentation, gradients, flow cytometry) to separate and purify T. gondii oocyst walls and sporocysts. Oocyst walls were successfully separated and purified using iodixanol gradients. Sporocysts were successfully separated and purified using iodixanol and Percoll gradients. Purification was also achieved by flow cytometry. Flow cytometry with fluorescence-activated cell sorting (FACS) yielded analytical quantities of oocyst walls and intact sporocysts. Flow cytometry with FACS also proved useful for quantitation of purity obtained following iodixanol gradient fractionation. Methods reported in this paper will be useful for analytical purposes, such as proteomic analysis of components unique to this life cycle stage, development of detection methods, or excystation studies.

An integrated micromechanical large particle in flow sorter (MILPIS)

NASA Astrophysics Data System (ADS)

Fuad, Nurul M.; Skommer, Joanna; Friedrich, Timo; Kaslin, Jan; Wlodkowic, Donald

2015-06-01

At present, the major hurdle to widespread deployment of zebrafish embryo and larvae in large-scale drug development projects is lack of enabling high-throughput analytical platforms. In order to spearhead drug discovery with the use of zebrafish as a model, platforms need to integrate automated pre-test sorting of organisms (to ensure quality control and standardization) and their in-test positioning (suitable for high-content imaging) with modules for flexible drug delivery. The major obstacle hampering sorting of millimetre sized particles such as zebrafish embryos on chip-based devices is their substantial diameter (above one millimetre), mass (above one milligram), which both lead to rapid gravitational-induced sedimentation and high inertial forces. Manual procedures associated with sorting hundreds of embryos are very monotonous and as such prone to significant analytical errors due to operator's fatigue. In this work, we present an innovative design of a micromechanical large particle in-flow sorter (MILPIS) capable of analysing, sorting and dispensing living zebrafish embryos for drug discovery applications. The system consisted of a microfluidic network, revolving micromechanical receptacle actuated by robotic servomotor and opto-electronic sensing module. The prototypes were fabricated in poly(methyl methacrylate) (PMMA) transparent thermoplastic using infrared laser micromachining. Elements of MILPIS were also fabricated in an optically transparent VisiJet resin using 3D stereolithography (SLA) processes (ProJet 7000HD, 3D Systems). The device operation was based on a rapidly revolving miniaturized mechanical receptacle. The latter function was to hold and position individual fish embryos for (i) interrogation, (ii) sorting decision-making and (iii) physical sorting..The system was designed to separate between fertilized (LIVE) and non-fertilized (DEAD) eggs, based on optical transparency using infrared (IR) emitters and receivers embedded in the system. Digital oscilloscope were used to distinguish the diffraction signals from IR sensors when both LIVE and DEAD embryos were flow through in the chip. Image process analysis were also used as detection module to track DEAD embryos as it flowed in the channel.

Detection and isolation of rare cells by 2-step enrichment high-speed flow cytometry/cell sorting and single cell LEAP laser ablation

NASA Astrophysics Data System (ADS)

Zordan, M. D.; Leary, James F.

2011-02-01

The clonal isolation of rare cells, especially cancer and stem cells, in a population is important to the development of improved medical treatment. We have demonstrated that the Laser-Enabled Analysis and Processing (LEAP, Cyntellect Inc., San Diego, CA) instrument can be used to efficiently produce single cell clones by photoablative dilution. Additionally, we have also shown that cells present at low frequencies can be cloned by photoablative dilution after they are pre-enriched by flow cytometry based cell sorting. Circulating tumor cells were modeled by spiking isolated peripheral blood cells with cells from the lung carcinoma cell line A549. Flow cytometry based cell sorting was used to perform an enrichment sort of A549 cells directly into a 384 well plate. Photoablative dilution was performed with the LEAPTM instrument to remove any contaminating cells, and clonally isolate 1 side population cell per well. We were able to isolate and grow single clones of side population cells using this method at greater than 90% efficiency. We have developed a 2 step method that is able to perform the clonal isolation of rare cells based on a medically relevant functional phenotype.

Live births from artificial insemination of microfluidic-sorted bovine spermatozoa characterized by trajectories correlated with fertility

PubMed Central

Nagata, Maria Portia B.; Endo, Kenji; Ogata, Kazuko; Yamanaka, Kenichi; Egashira, Junki; Katafuchi, Naoto; Yamanouchi, Tadayuki; Matsuda, Hideo; Goto, Yuki; Sakatani, Miki; Hojo, Takuo; Nishizono, Hirofumi; Yotsushima, Kenji; Takenouchi, Naoki; Hashiyada, Yutaka; Yamashita, Kenichi

2018-01-01

Selection of functional spermatozoa plays a crucial role in assisted reproduction. Passage of spermatozoa through the female reproductive tract requires progressive motility to locate the oocyte. This preferential ability to reach the fertilization site confers fertility advantage to spermatozoa. Current routine sperm selection techniques are inadequate and fail to provide conclusive evidence on the sperm characteristics that may affect fertilization. We therefore developed a selection strategy for functional and progressively motile bovine spermatozoa with high DNA integrity based on the ability to cross laminar flow streamlines in a diffuser-type microfluidic sperm sorter (DMSS). The fluid dynamics, with respect to microchannel geometry and design, are relevant in the propulsion of spermatozoa and, consequently, ultrahigh-throughput sorting. Sorted spermatozoa were assessed for kinematic parameters, acrosome reaction, mitochondrial membrane potential, and DNA integrity. Kinematic and trajectory patterns were used to identify fertility-related subpopulations: the rapid, straighter, progressive, nonsinuous pattern (PN) and the transitional, sinuous pattern (TS). In contrast to the conventional notion that the fertilizing spermatozoon is always vigorously motile and more linear, our results demonstrate that sinuous patterns are associated with fertility and correspond to truly functional spermatozoa as supported by more live births produced from predominant TS than PN subpopulation in the inseminate. Our findings ascertain the true practical application significance of microfluidic sorting of functional sperm characterized by sinuous trajectories that can serve as a behavioral sperm phenotype marker for fertility potential. More broadly, we foresee the clinical application of this sorting technology to assisted reproduction in humans. PMID:29555773

Flow cytometric sex sorting affects CD4 membrane distribution and binding of exogenous DNA on bovine sperm cells.

PubMed

Domingues, William Borges; da Silveira, Tony Leandro Rezende; Komninou, Eliza Rossi; Monte, Leonardo Garcia; Remião, Mariana Härter; Dellagostin, Odir Antônio; Corcini, Carine Dahl; Varela Junior, Antônio Sergio; Seixas, Fabiana Kömmling; Collares, Tiago; Campos, Vinicius Farias

2017-08-01

Bovine sex-sorted sperm have been commercialized and successfully used for the production of transgenic embryos of the desired sex through the sperm-mediated gene transfer (SMGT) technique. However, sex-sorted sperm show a reduced ability to internalize exogenous DNA. The interaction between sperm cells and the exogenous DNA has been reported in other species to be a CD4-like molecule-dependent process. The flow cytometry-based sex-sorting process subjects the spermatozoa to different stresses causing changes in the cell membrane. The aim of this study was to elucidate the relationship between the redistribution of CD4-like molecules and binding of exogenous DNA to sex-sorted bovine sperm. In the first set of experiments, the membrane phospholipid disorder and the redistribution of the CD4 were evaluated. The second set of experiments was conducted to investigate the effect of CD4 redistribution on the mechanism of binding of exogenous DNA to sperm cells and the efficiency of lipofection in sex-sorted bovine sperm. Sex-sorting procedure increased the membrane phospholipid disorder and induced the redistribution of CD4-like molecules. Both X-sorted and Y-sorted sperm had decreased DNA bound to membrane in comparison with the unsorted sperm; however, the binding of the exogenous DNA was significantly increased with the addition of liposomes. Moreover, we demonstrated that the number of sperm-bound exogenous DNA was decreased when these cells were preincubated with anti-bovine CD4 monoclonal antibody, supporting our hypothesis that CD4-like molecules indeed play a crucial role in the process of exogenous DNA/bovine sperm cells interaction.

Forces on particles in microstreaming flows

NASA Astrophysics Data System (ADS)

Hilgenfeldt, Sascha; Rallabandi, Bhargav; Thameem, Raqeeb

2015-11-01

In various microfluidic applications, vortical steady streaming from ultrasonically driven microbubbles is used in concert with a pressure-driven channel flow to manipulate objects. While a quantitative theory of this boundary-induced streaming is available, little work has been devoted to a fundamental understanding of the forces exerted on microparticles in boundary streaming flows, even though the differential action of such forces is central to applications like size-sensitive sorting. Contrary to other microfluidic sorting devices, the forces in bubble microstreaming act over millisecond times and micron length scales, without the need for accumulated deflections over long distances. Accordingly, we develop a theory of hydrodynamic forces on the fast time scale of bubble oscillation using the lubrication approximation, showing for the first time how particle displacements are rectified near moving boundaries over multiple oscillations in parallel with the generation of the steady streaming flow. The dependence of particle migration on particle size and the flow parameters is compared with experimental data. The theory is applicable to boundary streaming phenomena in general and demonstrates how particles can be sorted very quickly and without compromising device throughput. We acknowledge support by the National Science Foundation under grant number CBET-1236141.

MetaSort untangles metagenome assembly by reducing microbial community complexity

PubMed Central

Ji, Peifeng; Zhang, Yanming; Wang, Jinfeng; Zhao, Fangqing

2017-01-01

Most current approaches to analyse metagenomic data rely on reference genomes. Novel microbial communities extend far beyond the coverage of reference databases and de novo metagenome assembly from complex microbial communities remains a great challenge. Here we present a novel experimental and bioinformatic framework, metaSort, for effective construction of bacterial genomes from metagenomic samples. MetaSort provides a sorted mini-metagenome approach based on flow cytometry and single-cell sequencing methodologies, and employs new computational algorithms to efficiently recover high-quality genomes from the sorted mini-metagenome by the complementary of the original metagenome. Through extensive evaluations, we demonstrated that metaSort has an excellent and unbiased performance on genome recovery and assembly. Furthermore, we applied metaSort to an unexplored microflora colonized on the surface of marine kelp and successfully recovered 75 high-quality genomes at one time. This approach will greatly improve access to microbial genomes from complex or novel communities. PMID:28112173

Comparative genome map of human and cattle

DOE Office of Scientific and Technical Information (OSTI.GOV)

Solinas-Toldo, S.; Fries, R.; Lengauer, C.

Chromosomal homologies between individual human chromosomes and the bovine karyotype have been established by using a new approach termed Zoo-FISH. Labeled DNA libraries from flow-sorted human chromosomes were used as probes for fluorescence in situ hybridization on cattle chromosomes. All human DNA libraries, except the Y chromosome library, hybridized to one or more cattle chromosomes, identifying and delineating 50 segments of homology, most of them corresponding to the regions of homology as identified by the previous mapping of individual conserved loci. However, Zoo-FISH refines the comparative maps constructed by molecular gene mapping of individual loci by providing information on themore » boundaries of conserved regions in the absence of obvious cytogenetic homologies of human and bovine chromosomes. It allows study of karyotypic evolution and opens new avenues for genomic analysis by facilitating the extrapolation of results from the human genome initiative. 50 refs., 3 figs., 1 tab.« less

DOD Needs to Improve Management and Oversight of Operations at the Theater Retrograde- Camp Arifjan, Kuwait

DTIC Science & Technology

2010-09-30

only at the TRC (includes Retro Sort), Warehouse, and Bulk Yard. 2 Theater Retrograde The Theater Retrograde consists of the TRC (includes Retro Sort...through Kuwait. Figure 1. Proper Flow of Materiel from Iraq through Kuwait Note 1: We reviewed operations at the TRC, Retro Sort, Warehouse...Materiel Processing Instructions CIIC Definition 1 Highest Sensitivity - Non-nuclear missiles and rockets , launcher tube and explosive rounds 2 Highest

Static optical sorting in a laser interference field

NASA Astrophysics Data System (ADS)

Jákl, Petr; Čižmár, Tomáš; Šerý, Mojmír; Zemánek, Pavel

2008-04-01

We present a unique technique for optical sorting of heterogeneous suspensions of microparticles, which does not require the flow of the immersion medium. The method employs the size-dependent response of suspended dielectric particles to the optical field of three intersecting beams that form a fringelike interference pattern. We experimentally demonstrate sorting of a polydisperse suspension of polystyrene beads of diameters 1, 2, and 5.2μm and living yeast cells.

Trapping, focusing, and sorting of microparticles through bubble streaming

NASA Astrophysics Data System (ADS)

Wang, Cheng; Jalikop, Shreyas; Hilgenfeldt, Sascha

2010-11-01

Ultrasound-driven oscillating microbubbles can set up vigorous steady streaming flows around the bubbles. In contrast to previous work, we make use of the interaction between the bubble streaming and the streaming induced around mobile particles close to the bubble. Our experiment superimposes a unidirectional Poiseuille flow containing a well-mixed suspension of neutrally buoyant particles with the bubble streaming. The particle-size dependence of the particle-bubble interaction selects which particles are transported and which particles are trapped near the bubbles. The sizes selected for can be far smaller than any scale imposed by the device geometry, and the selection mechanism is purely passive. Changing the amplitude and frequency of ultrasound driving, we can further control focusing and sorting of the trapped particles, leading to the emergence of sharply defined monodisperse particle streams within a much wider channel. Optimizing parameters for focusing and sorting are presented. The technique is applicable in important fields like cell sorting and drug delivery.

Droplet electric separator microfluidic device for cell sorting

NASA Astrophysics Data System (ADS)

Guo, Feng; Ji, Xing-Hu; Liu, Kan; He, Rong-Xiang; Zhao, Li-Bo; Guo, Zhi-Xiao; Liu, Wei; Guo, Shi-Shang; Zhao, Xing-Zhong

2010-05-01

A simple and effective droplet electric separator microfluidic device was developed for cell sorting. The aqueous droplet without precharging operation was influenced to move a distance in the channel along the electric field direction by applying dc voltage on the electrodes beside the channel, which made the target droplet flowing to the collector. Single droplet can be isolated in a sorting rate of ˜100 Hz with microelectrodes under a required pulse. Single or multiple mammalian cell (HePG2) encapsulated in the surfactant free alginate droplet could be sorted out respectively. This method may be used for single cell operation or analysis.

Dielectrophoretic focusing integrated pulsed laser activated cell sorting

NASA Astrophysics Data System (ADS)

Zhu, Xiongfeng; Kung, Yu-Chun; Wu, Ting-Hsiang; Teitell, Michael A.; Chiou, Pei-Yu

2017-08-01

We present a pulsed laser activated cell sorter (PLACS) integrated with novel sheathless size-independent dielectrophoretic (DEP) focusing. Microfluidic fluorescence activated cell sorting (μFACS) systems aim to provide a fully enclosed environment for sterile cell sorting and integration with upstream and downstream microfluidic modules. Among them, PLACS has shown a great potential in achieving comparable performance to commercial aerosol-based FACS (>90% purity at 25,000 cells sec-1). However conventional sheath flow focusing method suffers a severe sample dilution issue. Here we demonstrate a novel dielectrophoresis-integrated pulsed laser activated cell sorter (DEP-PLACS). It consists of a microfluidic channel with 3D electrodes laid out to provide a tunnel-shaped electric field profile along a 4cmlong channel for sheathlessly focusing microparticles/cells into a single stream in high-speed microfluidic flows. All focused particles pass through the fluorescence detection zone along the same streamline regardless of their sizes and types. Upon detection of target fluorescent particles, a nanosecond laser pulse is triggered and focused in a neighboring channel to generate a rapidly expanding cavitation bubble for precise sorting. DEP-PLACS has achieved a sorting purity of 91% for polystyrene beads at a throughput of 1,500 particle/sec.

Continuous-flow separation of nanoparticles by electrostatic sieving at a micro-nanofluidic interface.

PubMed

Regtmeier, Jan; Käsewieter, Jörg; Everwand, Martina; Anselmetti, Dario

2011-05-01

Continuous-flow separation of nanoparticles (NPs) (15 and 39 nm) is demonstrated based on electrostatic sieving at a micro-nanofluidic interface. The interface is realized in a poly(dimethylsiloxane) device with a nanoslit of 525 nm laterally spanning the microfluidic channel (aspect ratio of 540:1). Within this nanoslit, the Debye layers overlap and generate an electrostatic sieve. This was exploited to selectively deflect and sort NPs with a sorting purity of up to 97%. Because of the continuous-flow operation, the sample is continuously fed into the device, immediately separated, and the parameters can be adapted in real time. For bioanalytical purposes, we also demonstrate the deflection of proteins (longest axis 6.8 nm). The continuous operation mode and the general applicability of this separation concept make this method a valuable addition to the current Lab-on-a-Chip devices for continuous sorting of NPs and macromolecules. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Divergent sorting of a balanced ancestral polymorphism underlies the establishment of gene-flow barriers in Capsella

PubMed Central

Sicard, Adrien; Kappel, Christian; Josephs, Emily B.; Lee, Young Wha; Marona, Cindy; Stinchcombe, John R.; Wright, Stephen I.; Lenhard, Michael

2015-01-01

In the Bateson–Dobzhansky–Muller model of genetic incompatibilities post-zygotic gene-flow barriers arise by fixation of novel alleles at interacting loci in separated populations. Many such incompatibilities are polymorphic in plants, implying an important role for genetic drift or balancing selection in their origin and evolution. Here we show that NPR1 and RPP5 loci cause a genetic incompatibility between the incipient species Capsella grandiflora and C. rubella, and the more distantly related C. rubella and C. orientalis. The incompatible RPP5 allele results from a mutation in C. rubella, while the incompatible NPR1 allele is frequent in the ancestral C. grandiflora. Compatible and incompatible NPR1 haplotypes are maintained by balancing selection in C. grandiflora, and were divergently sorted into the derived C. rubella and C. orientalis. Thus, by maintaining differentiated alleles at high frequencies, balancing selection on ancestral polymorphisms can facilitate establishing gene-flow barriers between derived populations through lineage sorting of the alternative alleles. PMID:26268845

People detection method using graphics processing units for a mobile robot with an omnidirectional camera

NASA Astrophysics Data System (ADS)

Kang, Sungil; Roh, Annah; Nam, Bodam; Hong, Hyunki

2011-12-01

This paper presents a novel vision system for people detection using an omnidirectional camera mounted on a mobile robot. In order to determine regions of interest (ROI), we compute a dense optical flow map using graphics processing units, which enable us to examine compliance with the ego-motion of the robot in a dynamic environment. Shape-based classification algorithms are employed to sort ROIs into human beings and nonhumans. The experimental results show that the proposed system detects people more precisely than previous methods.

Coupling Bacterial Activity Measurements with Cell Sorting by Flow Cytometry.

PubMed

Servais; Courties; Lebaron; Troussellier

1999-08-01

> Abstract A new procedure to investigate the relationship between bacterial cell size and activity at the cellular level has been developed; it is based on the coupling of radioactive labeling of bacterial cells and cell sorting by flow cytometry after SYTO 13 staining. Before sorting, bacterial cells were incubated in the presence of tritiated leucine using a procedure similar to that used for measuring bacterial production by leucine incorporation and then stained with SYTO 13. Subpopulations of bacterial cells were sorted according to their average right-angle light scatter (RALS) and fluorescence. Average RALS was shown to be significantly related to the average biovolume. Experiments were performed on samples collected at different times in a Mediterranean seawater mesocosm enriched with nitrogen and phosphorus. At four sampling times, bacteria were sorted in two subpopulations (cells smaller and larger than 0.25 µm(3)). The results indicate that, at each sampling time, the growth rate of larger cells was higher than that of smaller cells. In order to confirm this tendency, cell sorting was performed on six subpopulations differing in average biovolume during the mesocosm follow-up. A clear increase of the bacterial growth rates was observed with increasing cell size for the conditions met in this enriched mesocosm.http://link.springer-ny.com/link/service/journals/00248/bibs/38n2p180.html

Modeling the effect of dune sorting on the river long profile

NASA Astrophysics Data System (ADS)

Blom, A.

2012-12-01

River dunes, which occur in low slope sand bed and sand-gravel bed rivers, generally show a downward coarsening pattern due to grain flows down their avalanche lee faces. These grain flows cause coarse particles to preferentially deposit at lower elevations of the lee face, while fines show a preference for its upper elevations. Before considering the effect of this dune sorting mechanism on the river long profile, let us first have a look at some general trends along the river profile. Tributaries increasing the river's water discharge in streamwise direction also cause a streamwise increase in flow depth. As under subcritical conditions mean dune height generally increases with increasing flow depth, the dune height shows a streamwise increase, as well. This means that also the standard deviation of bedform height increases in streamwise direction, as in earlier work it was found that the standard deviation of bedform height linearly increases with an increasing mean value of bedform height. As a result of this streamwise increase in standard deviation of dune height, the above-mentioned dune sorting then results in a loss of coarse particles to the lower elevations of the bed that are less and even rarely exposed to the flow. This loss of coarse particles to lower elevations thus increases the rate of fining in streamwise direction. As finer material is more easily transported downstream than coarser material, a smaller bed slope is required to transport the same amount of sediment downstream. This means that dune sorting adds to river profile concavity, compared to the combined effect of abrasion, selective transport and tributaries. A Hirano-type mass conservation model is presented that deals with dune sorting. The model includes two active layers: a bedform layer representing the sediment in the bedforms and a coarse layer representing the coarse and less mobile sediment underneath migrating bedforms. The exposure of the coarse layer is governed by the rate of sediment supply from upstream. By definition the sum of the exposure of both layers equals unity. The model accounts for vertical sediment fluxes due to grain flows down the bedform lee face and the formation of a less mobile coarse layer. The model with its vertical sediment fluxes is validated against earlier flume experiments. It deals well with the transition between a plane bed and a bedform-dominated bed. Applying the model to field scale confirms that dune sorting increases river profile concavity.

Spontaneous cell sorting of fibroblasts and keratinocytes creates an organotypic human skin equivalent.

PubMed

Wang, C K; Nelson, C F; Brinkman, A M; Miller, A C; Hoeffler, W K

2000-04-01

We show that an inherent ability of two distinct cell types, keratinocytes and fibroblasts, can be relied upon to accurately reconstitute full-thickness human skin including the dermal-epidermal junction by a cell-sorting mechanism. A cell slurry containing both cell types added to silicone chambers implanted on the backs of severe combined immunodeficient mice sorts out to reconstitute a clearly defined dermis and stratified epidermis within 2 wk, forming a cell-sorted skin equivalent. Immunostaining of the cell-sorted skin equivalent with human cell markers showed patterns similar to those of normal full-thickness skin. We compared the cell-sorted skin equivalent model with a composite skin model also made on severe combined immunodeficient mice. The composite grafts were constructed from partially differentiated keratinocyte sheets placed on top of a dermal equivalent constructed of devitalized dermis. Electron microscopy revealed that both models formed ample numbers of normal appearing hemidesmosomes. The cell-sorted skin equivalent model, however, had greater numbers of keratin intermediate filaments within the basal keratinocytes that connected to hemidesmosomes, and on the dermal side both collagen filaments and anchoring fibril connections to the lamina densa were more numerous compared with the composite model. Our results may provide some insight into why, in clinical applications for treating burns and other wounds, composite grafts may exhibit surface instability and blistering for up to a year following grafting, and suggest the possible usefulness of the cell-sorted skin equivalent in future grafting applications.

Particle separation

NASA Technical Reports Server (NTRS)

Arnott, W. Patrick (Inventor); Chakrabarty, Rajan K. (Inventor); Moosmuller, Hans (Inventor)

2011-01-01

Embodiments of a method for selecting particles, such as based on their morphology, is disclosed. In a particular example, the particles are charged and acquire different amounts of charge, or have different charge distributions, based on their morphology. The particles are then sorted based on their flow properties. In a specific example, the particles are sorted using a differential mobility analyzer, which sorts particles, at least in part, based on their electrical mobility. Given a population of particles with similar electrical mobilities, the disclosed process can be used to sort particles based on the net charge carried by the particle, and thus, given the relationship between charge and morphology, separate the particles based on their morphology.

Particle separation

DOEpatents

Moosmuller, Hans [Reno, NV; Chakrabarty, Rajan K [Reno, NV; Arnott, W Patrick [Reno, NV

2011-04-26

Embodiments of a method for selecting particles, such as based on their morphology, is disclosed. In a particular example, the particles are charged and acquire different amounts of charge, or have different charge distributions, based on their morphology. The particles are then sorted based on their flow properties. In a specific example, the particles are sorted using a differential mobility analyzer, which sorts particles, at least in part, based on their electrical mobility. Given a population of particles with similar electrical mobilities, the disclosed process can be used to sort particles based on the net charge carried by the particle, and thus, given the relationship between charge and morphology, separate the particles based on their morphology.

The 15 September 1991 pyroclastic flows at Unzen Volcano (Japan): a flow model for associated ash-cloud surges

NASA Astrophysics Data System (ADS)

Fujii, Toshitsugu; Nakada, Setsuya

1999-04-01

Large-scale collapse of a dacite dome in the late afternoon of 15 September 1991 generated a series of pyroclastic-flow events at Unzen Volcano. Pyroclastic flows with a volume of 1×10 6 m 3 (as DRE) descended the northeastern slope of the volcano, changing their courses to the southeast due to topographic control. After they exited a narrow gorge, an ash-cloud surge rushed straight ahead, detaching the main body of the flow that turned and followed the topographic lows to the east. The surge swept the Kita-Kamikoba area, which had been devastated by the previous pyroclastic-flow events, and transported a car as far as 120 m. Following detachment, the surge lost its force after it moved several hundred meters, but maintained a high temperature. The deposits consist of a bottom layer of better-sorted ash (unit 1), a thick layer of block and ash (unit 2), and a thin top layer of fall-out ash (unit 3). Unit 2 overlies unit 1 with an erosional contact. The upper part of unit 2 grades into better-sorted ash. At distal block-and-ash flow deposits, the bottom part of unit 2 also consists of better-sorted ash, and the contact with the unit 1 deposits becomes ambiguous. Video footage of cascading pyroclastic flows during the 1991-1995 eruption, traveling over surfaces without any topographic barriers, revealed that lobes of ash cloud protruded intermittently from the moving head and sides, and that these lobes surged ahead on the ground surface. This fact, together with the inspection by helicopter shortly after the events, suggests that the protruded lobes consisted of better-sorted ash, and resulted in the deposits of unit 1. The highest ash-cloud plume at the Oshigadani valley exit, and the thickest deposition of fall-out ash over Kita-Kamikoba and Ohnokoba, indicate that abundant ash was also produced when the flow passed through a narrow gorge. In the model presented here, the ash clouds from the pyroclastic flows were composed of a basal turbulent current of high concentration (main body), an overriding and intermediate fluidization zone, and an overlying dilute cloud. Release of pressurized gas in lava block pores, due to collisions among blocks and the resulting upward current, caused a zone of fluidization just above the main body. The mixture of gas and ash sorted in the fluidization zone moved ahead and to the side of the main body as a gravitational current, where the ash was deposited as surge deposits. The main body, which had high internal friction and shear near its base, then overran the surge deposits, partially eroding them. When the upward current of gas (fluidization) waned, better-sorted ash suspended in the fluidization zone was deposited on block-and-ash deposits. In the distal places of block-and-ash deposits, unit 2 probably was deposited in non-turbulent fashion without any erosion of the underlying layer (unit 1).

Differential Interaction of Platelet-Derived Extracellular Vesicles with Leukocyte Subsets in Human Whole Blood.

PubMed

Weiss, René; Gröger, Marion; Rauscher, Sabine; Fendl, Birgit; Eichhorn, Tanja; Fischer, Michael B; Spittler, Andreas; Weber, Viktoria

2018-04-26

Secretion and exchange of biomolecules via extracellular vesicles (EVs) are crucial mechanisms in intercellular communication, and the roles of EVs in infection, inflammation, or thrombosis have been increasingly recognized. EVs have emerged as central players in immune regulation and can enhance or suppress the immune response, depending on the state of donor and recipient cells. We investigated the interaction of blood cell-derived EVs with leukocyte subpopulations (monocytes and their subsets, granulocytes, B cells, T cells, and NK cells) directly in whole blood using a combination of flow cytometry, imaging flow cytometry, cell sorting, and high resolution confocal microscopy. Platelet-derived EVs constituted the majority of circulating EVs and were preferentially associated with granulocytes and monocytes, while they scarcely interacted with lymphocytes. Further flow cytometric differentiation of monocyte subsets provided clear indications for a preferential association of platelet-derived EVs with intermediate (CD14 ++ CD16 + ) monocytes in whole blood.

A Quality Sorting of Fruit Using a New Automatic Image Processing Method

NASA Astrophysics Data System (ADS)

Amenomori, Michihiro; Yokomizu, Nobuyuki

This paper presents an innovative approach for quality sorting of objects such as apples sorting in an agricultural factory, using an image processing algorithm. The objective of our approach are; firstly to sort the objects by their colors precisely; secondly to detect any irregularity of the colors surrounding the apples efficiently. An experiment has been conducted and the results have been obtained and compared with that has been preformed by human sorting process and by color sensor sorting devices. The results demonstrate that our approach is capable to sort the objects rapidly and the percentage of classification valid rate was 100 %.

Blood flow velocity measurements in chicken embryo vascular network via PIV approach

NASA Astrophysics Data System (ADS)

Kurochkin, Maxim A.; Stiukhina, Elena S.; Fedosov, Ivan V.; Tuchin, Valery V.

2018-04-01

A method for measuring of blood velocity in the native vasculature of a chick embryo by the method of micro anemometry from particle images (μPIV) is improved. A method for interrogation regions sorting by the mask of the vasculature is proposed. A method for sorting of the velocity field of capillary blood flow is implemented. The in vitro method was evaluated for accuracy in a glass phantom of a blood vessel with a diameter of 50 μm and in vivo on the bloodstream of a chicken embryo, by comparing the transverse profile of the blood velocity obtained by the PIV method with the theoretical Poiseuille laminar flow profile.

Using Functional Flow Diagrams to Enhance Technical Systems Understanding.

ERIC Educational Resources Information Center

Satchwell, Richard E.

1997-01-01

A treatment group of 20 aviation students used training manuals that presented functional flow diagrams before schematic diagrams. Comparison of data from 10 controls on a card-sort task showed that functional flow diagrams enhanced understanding of technical systems. (SK)

Acoustic bubble sorting for ultrasound contrast agent enrichment.

PubMed

Segers, Tim; Versluis, Michel

2014-05-21

An ultrasound contrast agent (UCA) suspension contains encapsulated microbubbles with a wide size distribution, with radii ranging from 1 to 10 μm. Medical transducers typically operate at a single frequency, therefore only a small selection of bubbles will resonate to the driving ultrasound pulse. Thus, the sensitivity can be improved by narrowing down the size distribution. Here, we present a simple lab-on-a-chip method to sort the population of microbubbles on-chip using a traveling ultrasound wave. First, we explore the physical parameter space of acoustic bubble sorting using well-defined bubble sizes formed in a flow-focusing device, then we demonstrate successful acoustic sorting of a commercial UCA. This novel sorting strategy may lead to an overall improvement of the sensitivity of contrast ultrasound by more than 10 dB.

IB-LBM simulation on blood cell sorting with a micro-fence structure.

PubMed

Wei, Qiang; Xu, Yuan-Qing; Tian, Fang-bao; Gao, Tian-xin; Tang, Xiao-ying; Zu, Wen-Hong

2014-01-01

A size-based blood cell sorting model with a micro-fence structure is proposed in the frame of immersed boundary and lattice Boltzmann method (IB-LBM). The fluid dynamics is obtained by solving the discrete lattice Boltzmann equation, and the cells motion and deformation are handled by the immersed boundary method. A micro-fence consists of two parallel slope post rows which are adopted to separate red blood cells (RBCs) from white blood cells (WBCs), in which the cells to be separated are transported one after another by the flow into the passageway between the two post rows. Effected by the cross flow, RBCs are schemed to get through the pores of the nether post row since they are smaller and more deformable compared with WBCs. WBCs are required to move along the nether post row till they get out the micro-fence. Simulation results indicate that for a fix width of pores, the slope angle of the post row plays an important role in cell sorting. The cells mixture can not be separated properly in a small slope angle, while obvious blockages by WBCs will take place to disturb the continuous cell sorting in a big slope angle. As an optimal result, an adaptive slope angle is found to sort RBCs form WBCs correctly and continuously.

Flow sorting of C-genome chromosomes from wild relatives of wheat Aegilops markgrafii, Ae. triuncialis and Ae. cylindrica, and their molecular organization.

PubMed

Molnár, István; Vrána, Jan; Farkas, András; Kubaláková, Marie; Cseh, András; Molnár-Láng, Márta; Doležel, Jaroslav

2015-08-01

Aegilops markgrafii (CC) and its natural hybrids Ae. triuncialis (U(t)U(t)C(t)C(t)) and Ae. cylindrica (D(c)D(c)C(c)C(c)) represent a rich reservoir of useful genes for improvement of bread wheat (Triticum aestivum), but the limited information available on their genome structure and the shortage of molecular (cyto-) genetic tools hamper the utilization of the extant genetic diversity. This study provides the complete karyotypes in the three species obtained after fluorescent in situ hybridization (FISH) with repetitive DNA probes, and evaluates the potential of flow cytometric chromosome sorting. The flow karyotypes obtained after the analysis of 4',6-diamidino-2-phenylindole (DAPI)-stained chromosomes were characterized and the chromosome content of the peaks on the flow karyotypes was determined by FISH. Twenty-nine conserved orthologous set (COS) markers covering all seven wheat homoeologous chromosome groups were used for PCR with DNA amplified from flow-sorted chromosomes and genomic DNA. FISH with repetitive DNA probes revealed that chromosomes 4C, 5C, 7C(t), T6U(t)S.6U(t)L-5C(t)L, 1C(c) and 5D(c) could be sorted with purities ranging from 66 to 91 %, while the remaining chromosomes could be sorted in groups of 2-5. This identified a partial wheat-C-genome homology for group 4 and 5 chromosomes. In addition, 1C chromosomes were homologous with group 1 of wheat; a small segment from group 2 indicated 1C-2C rearrangement. An extensively rearranged structure of chromosome 7C relative to wheat was also detected. The possibility of purifying Aegilops chromosomes provides an attractive opportunity to investigate the structure and evolution of the Aegilops C genome and to develop molecular tools to facilitate the identification of alien chromatin and support alien introgression breeding in bread wheat. © The Author 2015. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Flow cytometry sorting of nuclei enables the first global characterization of Paramecium germline DNA and transposable elements.

PubMed

Guérin, Frédéric; Arnaiz, Olivier; Boggetto, Nicole; Denby Wilkes, Cyril; Meyer, Eric; Sperling, Linda; Duharcourt, Sandra

2017-04-26

DNA elimination is developmentally programmed in a wide variety of eukaryotes, including unicellular ciliates, and leads to the generation of distinct germline and somatic genomes. The ciliate Paramecium tetraurelia harbors two types of nuclei with different functions and genome structures. The transcriptionally inactive micronucleus contains the complete germline genome, while the somatic macronucleus contains a reduced genome streamlined for gene expression. During development of the somatic macronucleus, the germline genome undergoes massive and reproducible DNA elimination events. Availability of both the somatic and germline genomes is essential to examine the genome changes that occur during programmed DNA elimination and ultimately decipher the mechanisms underlying the specific removal of germline-limited sequences. We developed a novel experimental approach that uses flow cell imaging and flow cytometry to sort subpopulations of nuclei to high purity. We sorted vegetative micronuclei and macronuclei during development of P. tetraurelia. We validated the method by flow cell imaging and by high throughput DNA sequencing. Our work establishes the proof of principle that developing somatic macronuclei can be sorted from a complex biological sample to high purity based on their size, shape and DNA content. This method enabled us to sequence, for the first time, the germline DNA from pure micronuclei and to identify novel transposable elements. Sequencing the germline DNA confirms that the Pgm domesticated transposase is required for the excision of all ~45,000 Internal Eliminated Sequences. Comparison of the germline DNA and unrearranged DNA obtained from PGM-silenced cells reveals that the latter does not provide a faithful representation of the germline genome. We developed a flow cytometry-based method to purify P. tetraurelia nuclei to high purity and provided quality control with flow cell imaging and high throughput DNA sequencing. We identified 61 germline transposable elements including the first Paramecium retrotransposons. This approach paves the way to sequence the germline genomes of P. aurelia sibling species for future comparative genomic studies.

A real-time traffic control method for the intersection with pre-signals under the phase swap sorting strategy

PubMed Central

Bie, Yiming; Wang, Yinhai

2017-01-01

To deal with the conflicts between left-turn and through traffic streams and increase the discharge capacity, this paper addresses the pre-signal which is implemented at a signalized intersection. Such an intersection with pre-signal is termed as a tandem intersection. For the tandem intersection, phase swap sorting strategy is deemed as the most effective phasing scheme in view of some exclusive merits, such as easier compliance of drivers, and shorter sorting area. However, a major limitation of the phase swap sorting strategy is not considered in previous studies: if one or more vehicle is left at the sorting area after the signal light turns to red, the capacity of the approach would be dramatically dropped. Besides, previous signal control studies deal with a fixed timing plan that is not adaptive with the fluctuation of traffic flows. Therefore, to cope with these two gaps, this paper firstly takes an in-depth analysis of the traffic flow operations at the tandem intersection. Secondly, three groups of loop detectors are placed to obtain the real-time vehicle information for adaptive signalization. The lane selection behavior in the sorting area is considered to set the green time for intersection signals. With the objective of minimizing the vehicle delay, the signal control parameters are then optimized based on a dynamic programming method. Finally, numerical experiments show that average vehicle delay and maximum queue length can be reduced under all scenarios. PMID:28531198

A real-time traffic control method for the intersection with pre-signals under the phase swap sorting strategy.

PubMed

Bie, Yiming; Liu, Zhiyuan; Wang, Yinhai

2017-01-01

To deal with the conflicts between left-turn and through traffic streams and increase the discharge capacity, this paper addresses the pre-signal which is implemented at a signalized intersection. Such an intersection with pre-signal is termed as a tandem intersection. For the tandem intersection, phase swap sorting strategy is deemed as the most effective phasing scheme in view of some exclusive merits, such as easier compliance of drivers, and shorter sorting area. However, a major limitation of the phase swap sorting strategy is not considered in previous studies: if one or more vehicle is left at the sorting area after the signal light turns to red, the capacity of the approach would be dramatically dropped. Besides, previous signal control studies deal with a fixed timing plan that is not adaptive with the fluctuation of traffic flows. Therefore, to cope with these two gaps, this paper firstly takes an in-depth analysis of the traffic flow operations at the tandem intersection. Secondly, three groups of loop detectors are placed to obtain the real-time vehicle information for adaptive signalization. The lane selection behavior in the sorting area is considered to set the green time for intersection signals. With the objective of minimizing the vehicle delay, the signal control parameters are then optimized based on a dynamic programming method. Finally, numerical experiments show that average vehicle delay and maximum queue length can be reduced under all scenarios.

High-resolution FISH on super-stretched flow-sorted plant chromosomes.

PubMed

Valárik, M; Bartos, J; Kovárová, P; Kubaláková, M; de Jong, J H; Dolezel, J

2004-03-01

A novel high-resolution fluorescence in situ hybridisation (FISH) strategy, using super-stretched flow-sorted plant chromosomes as targets, is described. The technique that allows longitudinal extension of chromosomes of more than 100 times their original metaphase size is especially attractive for plant species with large chromosomes, whose pachytene chromosomes are generally too long and heterochromatin patterns too complex for FISH analysis. The protocol involves flow cytometric sorting of metaphase chromosomes, mild proteinase-K digestion of air-dried chromosomes on microscopic slides, followed by stretching with ethanol:acetic acid (3 : 1). Stretching ratios were assessed in a number of FISH experiments with super-stretched chromosomes from barley, wheat, rye and chickpea, hybridised with 45S and 5S ribosomal DNAs and the [GAA]n microsatellite, the [TTTAGGG]n telomeric repeat and a bacterial artificial chromosome (BAC) clone as probes. FISH signals on stretched chromosomes were brighter than those on the untreated control, resulting from better accessibility of the stretched chromatin and maximum observed sensitivity of 1 kbp. Spatial resolution of neighbouring loci was improved down to 70 kbp as compared to 5-10 Mbp after FISH on mitotic chromosomes, revealing details of adjacent DNA sequences hitherto not obtained with any other method. Stretched chromosomes are advantageous over extended DNA fibres from interphase nuclei as targets for FISH studies because they still retain chromosomal integrity. Although the method is confined to species for which chromosome flow sorting has been developed, it provides a unique system for controlling stretching degree of mitotic chromosomes and high-resolution bar-code FISH.

In vitro experimental investigation of voice production

PubMed Central

Horáčcek, Jaromír; Brücker, Christoph; Becker, Stefan

2012-01-01

The process of human phonation involves a complex interaction between the physical domains of structural dynamics, fluid flow, and acoustic sound production and radiation. Given the high degree of nonlinearity of these processes, even small anatomical or physiological disturbances can significantly affect the voice signal. In the worst cases, patients can lose their voice and hence the normal mode of speech communication. To improve medical therapies and surgical techniques it is very important to understand better the physics of the human phonation process. Due to the limited experimental access to the human larynx, alternative strategies, including artificial vocal folds, have been developed. The following review gives an overview of experimental investigations of artificial vocal folds within the last 30 years. The models are sorted into three groups: static models, externally driven models, and self-oscillating models. The focus is on the different models of the human vocal folds and on the ways in which they have been applied. PMID:23181007

ADP and brucellosis indemnity systems development

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sanders, W.M.; Harlan, B.L.

1976-01-01

Our initial study of the USDA/TAHC Brucellosis Indemnity Program in Texas has shown that both the efficiency and rate of claim payments can be increased by the application of present day computer technologies. Two main factors contribute to these increases: the number of discrepancies that are caused by poor penmanship, transposition of numbers, and other human errors can be monitored and minimized; and the documented information can be indexed, sorted, and searched faster, more efficiently, and without human error. The overall flow of documentation that is used to control the movement of infected or exposed animals through commerce should bemore » studied. A new system should be designed that fully utilizes present day computer and electronic technologies.« less

Geomorphological map and preliminary analysis of Quaternary sediments in the Planica-Tamar valley (Julian Alps, NW Slovenia)

NASA Astrophysics Data System (ADS)

Novak, Andrej; Šmuc, Andrej

2016-04-01

The Planica-Tamar valley is located in the Julian Alps in north-west Slovenia. The Planica-Tamar valley represents typical mountain glacial valley bounded by steep, mainly carbonate cliffs with some glacial deposits still preserved. The valley is currently being filled with numerous Holocene sediments deposited by rock falls, landslides, mass gravity flows and fluvial flows. These deposits are forming active or inactive interfingering talus slopes, alluvial and debris-flow fans, all of them with a complex history of sedimentation and erosion forming unconformity bounded sedimentary units. In order to make a thorough analysis of these deposits a detailed geomorphological map in a scale of 1:10 000 has been made. Six different types of sedimentary deposits were defined and mapped. These are moraines, lacustrine sediments, fluvio-glacial deposits, talus slopes, debris fans and alluvial fans. Other mapped features also include shape of ravines, their depths, ridges and direction of sedimentary flow. Additionally areas of active, semi-active and inactive sedimentation were marked. Moraines forms a ridge in the bottom of the valleys and are composed of unconsolidated, poorly sorted, subangular grains ranging from clay size to a few cubic meters big blocks. Lacustrine sediments are represented by laminated well sorted sand and silt, while fluvio-glacial deposits are composed of washed out subrounded sands and gravels. Talus slope deposits are characterised by clast-supported poorly sorted very angular gravel. Debris flow fans are represented by extremely poorly sorted matrix-supported gravels with grain size ranging from clay to few cubic meters big blocks. Alluvial fans are composed by variety of sedimentary textures. Sediments at the fan apex are clast-supported poorly sorted very angular gravels with up to a few cubic meters big block. In the middle part of the fan the sieve deposits are common, while in the distal parts a few centimeters thick layers of sand and moderately sorted clast or sandy matrix-supported angular gravels occur. In cross-sections of alluvial fans distinct palaeosoil horizons are present indicating longer inactivity of that part of the fan. The geomorphological map forms a base for further research and thorough analysis of Quaternary deposits in order to reconstruct the Holocene dynamic of triggering and sedimentation of different types of slope deposits and relate them to base rock geology, tectonic and local/regional climate events. Key words: geomorphological mapping, Holocene slope deposits, alluvial fans, debris fans, Alpine geomorphology.

IAP-Based Cell Sorting Results in Homogeneous Transplantable Dopaminergic Precursor Cells Derived from Human Pluripotent Stem Cells.

PubMed

Lehnen, Daniela; Barral, Serena; Cardoso, Tiago; Grealish, Shane; Heuer, Andreas; Smiyakin, Andrej; Kirkeby, Agnete; Kollet, Jutta; Cremer, Harold; Parmar, Malin; Bosio, Andreas; Knöbel, Sebastian

2017-10-10

Human pluripotent stem cell (hPSC)-derived mesencephalic dopaminergic (mesDA) neurons can relieve motor deficits in animal models of Parkinson's disease (PD). Clinical translation of differentiation protocols requires standardization of production procedures, and surface-marker-based cell sorting is considered instrumental for reproducible generation of defined cell products. Here, we demonstrate that integrin-associated protein (IAP) is a cell surface marker suitable for enrichment of hPSC-derived mesDA progenitor cells. Immunomagnetically sorted IAP + mesDA progenitors showed increased expression of ventral midbrain floor plate markers, lacked expression of pluripotency markers, and differentiated into mature dopaminergic (DA) neurons in vitro. Intrastriatal transplantation of IAP + cells sorted at day 16 of differentiation in a rat model of PD resulted in functional recovery. Grafts from sorted IAP + mesDA progenitors were more homogeneous in size and DA neuron density. Thus, we suggest IAP-based sorting for reproducible prospective enrichment of mesDA progenitor cells in clinical cell replacement strategies. Copyright © 2017 Miltenyi Biotec GmbH. Published by Elsevier Inc. All rights reserved.

Sperm sexing in sheep and cattle: the exception and the rule.

PubMed

de Graaf, S P; Beilby, K H; Underwood, S L; Evans, G; Maxwell, W M C

2009-01-01

Flow cytometric sorting for the preselection of sex has progressed considerably in the 20 years since its inception. This technique has allowed the production of pre-sexed offspring in a multitude of species and become a commercial success in cattle around the world. However, due to the stress inherent to the sex-sorting process, sex-sorted spermatozoa are widely recognized as functionally compromised in terms of their fertilizing lifespan within the female reproductive tract as a result of reduced motility and viability and changed functional state. These characteristics, when compared to non-sorted controls, are manifest in vivo as lower fertility. However, improvements to the technology and a greater understanding of its biological impact have facilitated recent developments in sheep, showing sex-sorting is capable of selecting a functionally superior population in terms of both in vitro and in vivo function. These results are reviewed in the context of recent developments in other species and the reasons for success after artificial insemination with sex-sorted ram spermatozoa are discussed.

Sperm sorting procedure induces a redistribution of Hsp70 but not Hsp60 and Hsp90 in boar spermatozoa.

PubMed

Spinaci, Marcella; Volpe, Sara; Bernardini, Chiara; de Ambrogi, Marco; Tamanini, Carlo; Seren, Eraldo; Galeati, Giovanna

2006-01-01

Heat shock proteins, besides their protective function against stresses, have been recently indicated as key factors for sperm fertilizing ability. Since sexing sperm by high-speed flow-cytometry subjects them to different physical, mechanical, and chemical stresses, the present study was designed to verify, by immunofluorescence and Western blot, whether the sorting procedure induces any modification in the amount and cellular distribution of heat shock proteins 60, 70, and 90 (Hsp60, Hsp70, Hsp90). Immunolocalization and Western blot quantification of both Hsp60 and Hsp90 did not reveal differences between unsorted and sorted semen. On the contrary, a redistribution of Hsp70 immunoreactivity from the equatorial subsegment toward the equator of sperm cells was recorded after sorting; this relocation suggests capacitation-like changes of sperm membrane. This modification seems to be caused mainly by incubation with Hoechst 33342, while both passage of sperm through flow cytometer and laser beam represent only minor stimuli. A further Hsp70 redistribution seems to be due to the final steps of sperm sorting, charging, and deflection of drops, and to the dilution during collection. On the other hand, staining procedure and mechanical stress seem to be the factors most injurious to sperm viability. Moreover, Hsp70 relocation was deeply influenced by the storage method. In fact, storing sexed spermatozoa, after centrifugation, in a small volume in presence of seminal plasma induced a reversion of Hsp70 redistribution, while storage in the diluted catch fluid of collection tubes caused Hsp70 relocation in most sorted spermatozoa.

A sorting mechanism for a riffle-pool sequence

Treesearch

Thomas Lisle

1979-01-01

Transport of coarse, heterogeneous debris in a natural stream under a wide range of flows usually results in a remarkably stable, undulatory bed profile, which manifests an in transit sorting process of the bed material. In general, finer material representative of the bulk of the normal bed load resides in the deep sections, or pools, below flood stages. At high...

Pinched flow fractionation of microbubbles for ultrasound contrast agent enrichment

NASA Astrophysics Data System (ADS)

Versluis, Michel; Kok, Maarten; Segers, Tim

2014-11-01

An ultrasound contrast agent (UCA) suspension contains a wide size distribution of encapsulated microbubbles (typically 1-10 μm in diameter) that resonate to the driving ultrasound field by the intrinsic relationship between bubble size and ultrasound frequency. Medical transducers, however, operate in a narrow frequency range, which severely limits the number of bubbles that contribute to the echo signal. Thus, the sensitivity can be improved by narrowing down the size distribution of the bubble suspension. Here, we present a novel, low-cost, lab-on-a-chip method for the sorting of contrast microbubbles by size, based on a microfluidic separation technique known as pinched flow fractionation (PFF). We show by experimental and numerical investigation that the inclusion of particle rotation is essential for an accurate physical description of the sorting behavior of the larger bubbles. Successful sorting of a bubble suspension with a narrow size distribution (3.0 +/- 0.6 μm) has been achieved with a PFF microdevice. This sorting technique can be easily parallelized, and may lead to a significant improvement in the sensitivity of contrast-enhanced medical ultrasound. This work is supported by NanoNextNL, a micro and nanotechnology consortium of the Government of the Netherlands and 130 partners.

Microfluidic sorting and multimodal typing of cancer cells in self-assembled magnetic arrays.

PubMed

Saliba, Antoine-Emmanuel; Saias, Laure; Psychari, Eleni; Minc, Nicolas; Simon, Damien; Bidard, François-Clément; Mathiot, Claire; Pierga, Jean-Yves; Fraisier, Vincent; Salamero, Jean; Saada, Véronique; Farace, Françoise; Vielh, Philippe; Malaquin, Laurent; Viovy, Jean-Louis

2010-08-17

We propose a unique method for cell sorting, "Ephesia," using columns of biofunctionalized superparamagnetic beads self-assembled in a microfluidic channel onto an array of magnetic traps prepared by microcontact printing. It combines the advantages of microfluidic cell sorting, notably the application of a well controlled, flow-activated interaction between cells and beads, and those of immunomagnetic sorting, notably the use of batch-prepared, well characterized antibody-bearing beads. On cell lines mixtures, we demonstrated a capture yield better than 94%, and the possibility to cultivate in situ the captured cells. A second series of experiments involved clinical samples--blood, pleural effusion, and fine needle aspirates--issued from healthy donors and patients with B-cell hematological malignant tumors (leukemia and lymphoma). The immunophenotype and morphology of B-lymphocytes were analyzed directly in the microfluidic chamber, and compared with conventional flow cytometry and visual cytology data, in a blind test. Immunophenotyping results using Ephesia were fully consistent with those obtained by flow cytometry. We obtained in situ high resolution confocal three-dimensional images of the cell nuclei, showing intranuclear details consistent with conventional cytological staining. Ephesia thus provides a powerful approach to cell capture and typing allowing fully automated high resolution and quantitative immunophenotyping and morphological analysis. It requires at least 10 times smaller sample volume and cell numbers than cytometry, potentially increasing the range of indications and the success rate of microbiopsy-based diagnosis, and reducing analysis time and cost.

Microfluidic sorting and multimodal typing of cancer cells in self-assembled magnetic arrays

PubMed Central

Saliba, Antoine-Emmanuel; Saias, Laure; Psychari, Eleni; Minc, Nicolas; Simon, Damien; Bidard, François-Clément; Mathiot, Claire; Pierga, Jean-Yves; Fraisier, Vincent; Salamero, Jean; Saada, Véronique; Farace, Françoise; Vielh, Philippe; Malaquin, Laurent; Viovy, Jean-Louis

2010-01-01

We propose a unique method for cell sorting, “Ephesia,” using columns of biofunctionalized superparamagnetic beads self-assembled in a microfluidic channel onto an array of magnetic traps prepared by microcontact printing. It combines the advantages of microfluidic cell sorting, notably the application of a well controlled, flow-activated interaction between cells and beads, and those of immunomagnetic sorting, notably the use of batch-prepared, well characterized antibody-bearing beads. On cell lines mixtures, we demonstrated a capture yield better than 94%, and the possibility to cultivate in situ the captured cells. A second series of experiments involved clinical samples—blood, pleural effusion, and fine needle aspirates— issued from healthy donors and patients with B-cell hematological malignant tumors (leukemia and lymphoma). The immunophenotype and morphology of B-lymphocytes were analyzed directly in the microfluidic chamber, and compared with conventional flow cytometry and visual cytology data, in a blind test. Immunophenotyping results using Ephesia were fully consistent with those obtained by flow cytometry. We obtained in situ high resolution confocal three-dimensional images of the cell nuclei, showing intranuclear details consistent with conventional cytological staining. Ephesia thus provides a powerful approach to cell capture and typing allowing fully automated high resolution and quantitative immunophenotyping and morphological analysis. It requires at least 10 times smaller sample volume and cell numbers than cytometry, potentially increasing the range of indications and the success rate of microbiopsy-based diagnosis, and reducing analysis time and cost. PMID:20679245

Size-sensitive particle trajectories in three-dimensional micro-bubble acoustic streaming flows

NASA Astrophysics Data System (ADS)

Volk, Andreas; Rossi, Massimiliano; Hilgenfeldt, Sascha; Rallabandi, Bhargav; Kähler, Christian; Marin, Alvaro

2015-11-01

Oscillating microbubbles generate steady streaming flows with interesting features and promising applications for microparticle manipulation. The flow around oscillating semi-cylindrical bubbles has been typically assumed to be independent of the axial coordinate. However, it has been recently revealed that particle motion is strongly three-dimensional: Small tracer particles follow vortical trajectories with pronounced axial displacements near the bubble, weaving a toroidal stream-surface. A well-known consequence of bubble streaming flows is size-dependent particle migration, which can be exploited for sorting and trapping of microparticles in microfluidic devices. In this talk, we will show how the three-dimensional toroidal topology found for small tracer particles is modified as the particle size increases up to 1/3 of the bubble radius. Our results show size-sensitive particle positioning along the axis of the semi-cylindrical bubble. In order to analyze the three-dimensional sorting and trapping capabilities of the system, experiments with an imposed flow and polydisperse particle solutions are also shown.

Localization of male-specifically expressed MROS genes of Silene latifolia by PCR on flow-sorted sex chromosomes and autosomes.

PubMed

Kejnovský, E; Vrána, J; Matsunaga, S; Soucek, P; Siroký, J; Dolezel, J; Vyskot, B

2001-07-01

The dioecious white campion Silene latifolia (syn. Melandrium album) has heteromorphic sex chromosomes, XX in females and XY in males, that are larger than the autosomes and enable their separation by flow sorting. The group of MROS genes, the first male-specifically expressed genes in dioecious plants, was recently identified in S. latifolia. To localize the MROS genes, we used the flow-sorted X chromosomes and autosomes as a template for PCR with internal primers. Our results indicate that the MROS3 gene is located in at least two copies tandemly arranged on the X chromosome with additional copy(ies) on the autosome(s), while MROS1, MROS2, and MROS4 are exclusively autosomal. The specificity of PCR products was checked by digestion with a restriction enzyme or reamplification using nested primers. Homology search of databases has shown the presence of five MROS3 homologues in A. thaliana, four of them arranged in two tandems, each consisting of two copies. We conclude that MROS3 is a low-copy gene family, connected with the proper pollen development, which is present not only in dioecious but also in other dicot plant species.

Hydrodynamic lift of vesicles and red blood cells in flow--from Fåhræus & Lindqvist to microfluidic cell sorting.

PubMed

Geislinger, Thomas M; Franke, Thomas

2014-06-01

Hydrodynamic lift forces acting on cells and particles in fluid flow receive ongoing attention from medicine, mathematics, physics and engineering. The early findings of Fåhræus & Lindqvist on the viscosity change of blood with the diameter of capillaries motivated extensive studies both experimentally and theoretically to illuminate the underlying physics. We review this historical development that led to the discovery of the inertial and non-inertial lift forces and elucidate the origins of these forces that are still not entirely clear. Exploiting microfluidic techniques induced a tremendous amount of new insights especially into the more complex interactions between the flow field and deformable objects like vesicles or red blood cells. We trace the way from the investigation of single cell dynamics to the recent developments of microfluidic techniques for particle and cell sorting using hydrodynamic forces. Such continuous and label-free on-chip cell sorting devices promise to revolutionize medical analyses for personalized point-of-care diagnosis. We present the state-of-the-art of different hydrodynamic lift-based techniques and discuss their advantages and limitations. Copyright © 2014 Elsevier B.V. All rights reserved.

Continuous high throughput molecular adhesion based cell sorting using ridged microchannels

NASA Astrophysics Data System (ADS)

Tasadduq, Bushra; Wang, Gonghao; Alexeev, Alexander; Sarioglu, Ali Fatih; Sulchek, Todd

2016-11-01

Cell molecular interactions govern important physiological processes such as stem cell homing, inflammation and cancer metastasis. But due to a lack of effective separation technologies selective to these interactions it is challenging to specifically sort cells. Other label free separation techniques based on size, stiffness and shape do not provide enough specificity to cell type, and correlation to clinical condition. We propose a novel microfluidic device capable of high throughput molecule dependent separation of cells by flowing them through a microchannel decorated with molecule specific coated ridges. The unique aspect of this sorting design is the use of optimized gap size which is small enough to lightly squeeze the cells while flowing under the ridged part of the channel to increase the surface area for interaction between the ligand on cell surface and coated receptor molecule but large enough so that biomechanical markers, stiffness and viscoelasticity, do not dominate the cell separation mechanism. We are able to separate Jurkat cells based on its expression of PSGL-1ligand using ridged channel coated with P selectin at a flow rate of 0.045ml/min and achieve 2-fold and 5-fold enrichment of PSGL-1 positive and negative Jurkat cells respectively.

Characterization of pancreatic stem cells derived from adult human pancreas ducts by fluorescence activated cell sorting.

PubMed

Lin, Han-Tso; Chiou, Shih-Hwa; Kao, Chung-Lan; Shyr, Yi-Ming; Hsu, Chien-Jen; Tarng, Yih-Wen; Ho, Larry L-T; Kwok, Ching-Fai; Ku, Hung-Hai

2006-07-28

To isolate putative pancreatic stem cells (PSCs) from human adult tissues of pancreas duct using serum-free, conditioned medium. The characterization of surface phenotype of these PSCs was analyzed by flow cytometry. The potential for pancreatic lineage and the capability of beta-cell differentiation in these PSCs were evaluated as well. By using serum-free medium supplemented with essential growth factors, we attempted to isolate the putative PSCs which has been reported to express nestin and pdx-1. The Matrigel(TM) was employed to evaluate the differential capacity of isolated cells. Dithizone staining, insulin content/secretion measurement, and immunohistochemistry staining were used to monitor the differentiation. Fluorescence activated cell sorting (FACS) was used to detect the phenotypic markers of putative PSCs. A monolayer of spindle-like cells was cultivated. The putative PSCs expressed pdx-1 and nestin. They were also able to differentiate into insulin-, glucagon-, and somatostatin-positive cells. The spectrum of phenotypic markers in PSCs was investigated; a similarity was revealed when using human bone marrow-derived stem cells as the comparative experiment, such as CD29, CD44, CD49, CD50, CD51, CD62E, PDGFR-alpha, CD73 (SH2), CD81, CD105(SH3). In this study, we successfully isolated PSCs from adult human pancreatic duct by using serum-free medium. These PSCs not only expressed nestin and pdx-1 but also exhibited markers attributable to mesenchymal stem cells. Although work is needed to elucidate the role of these cells, the application of these PSCs might be therapeutic strategies for diabetes mellitus.

Heterogeneity in the growth hormone pituitary gland system of rats and humans: Implications to microgravity based research

NASA Technical Reports Server (NTRS)

Hymer, W. C.; Grindeland, R.; Hayes, C.; Lanham, J. W.; Cleveland, C.; Todd, P.; Morrison, Dennis R.

1988-01-01

The cell separation techniques of velocity sedimentation, flow cytometry and continuous flow electrophoresis were used to obtain enriched populations of growth hormone (GH) cells. The goal was to isolate a GH cell subpopulation which releases GH molecules which are very high in biological activity, it was important to use a method which was effective in processing large numbers of cells over a short time span. The techniques based on sedimentation are limited by cell density overlaps and streaming. While flow cytometry is useful in the analytical mode for objectively establishing cell purity, the numbers of cells which can be processed in the sort mode are so small as to make this approach ineffective in terms of the long term goals. It was shown that continuous flow electrophoresis systems (CFES) can separate GH cells from other cell types on the basis of differences in surface charge. The bioreactive producers appear to be more electrophoretically mobile than the low producers. Current ground based CFES efforts are hampered by cell clumping in low ionic strength buffers and poor cell recoveries from the CFES device.

Expression of a functional asialoglycoprotein receptor in human renal proximal tubular epithelial cells.

PubMed

Seow, Ying-ying T; Tan, Michelle G K; Woo, Keng Thye

2002-07-01

The asialoglycoprotein receptor (ASGPR) is a C lectin which binds and endocytoses serum glycoproteins. In humans, the ASGPR is shown mainly to occur in hepatocytes, but does occur extrahepatically in thyroid, in small and large intestines, and in the testis. In the kidney, there has been evidence both for and against its existence in mesangial cells. Standard light microscopy examination of renal tissue stained with an antibody against the ASGPR was performed. The mRNA expression for the ASGPR H1 and H2 subunits in primary human renal proximal tubular epithelial cells (RPTEC), in the human proximal tubular epithelial cell line HK2, and in human renal cortex was investigated using reverse-transcribed nested polymerase chain reaction. ASGPR protein expression as well as ligand binding and uptake were also examined using confocal microscopy and flow cytometry (fluorescence-activated cell sorting). Light microscopy of paraffin renal biopsy sections stained with a polyclonal antibody against the ASGPR showed proximal tubular epithelial cell staining of the cytoplasm and particularly in the basolateral region. Renal cortex and RPTEC specifically have mRNA for both H1 and H2 subunits of the ASGPR, but HK2 only expresses mRNA for H1. Using a monoclonal antibody, the presence of the ASGPR in RPTEC was shown by fluorescence-activated cell sorting and immunofluorescent staining. Specific binding and uptake of fluorescein isothiocyanate labelled asialofetuin which is a specific ASGPR ligand was also demonstrated in RPTEC. Primary renal proximal tubular epithelial cells have a functional ASGPR, consisting of the H1 and H2 subunits, that is capable of specific ligand binding and uptake. Copyright 2002 S. Karger AG, Basel

[Lentivirus-mediated RNA interference of CD133 inhibits the proliferation of CD133(+) liver cancer stem cells and increases their cisplatin chemosensitivity].

PubMed

Lan, Xi; Wang, Yong; Cao, Shu; Zou, Dongling; Li, Fang; Li, Shaolin

2012-12-01

To study the effects of CD133 suppression by lentivirus-mediated RNA interference (RNAi) on the proliferation and chemosensitivity of CD133(+) cancer stem cells (CSCs) sorted from HepG2 cell line. CD133(+) and CD133- cells were sorted from HepG2 cell line by flow cytometry, and the expression of CD133 before and after cell sorting were detected. The stem cell property of sorted CD133(+) cells were validated by sphere-forming assay in vitro and xenograft experiments in vivo. Lentivirus-mediated short hairpin RNA (shRNA) targeting CD133 were transfected into CD133(+) cells, and CD133 mRNA and protein expressions of the transfected cells were detected by RT-PCR and Western blotting, respectively. Before and after the transfection, the proliferative ability of CD133(+) cells was evaluated by colony formation assay, and the cell growth inhibition rate and apoptosis following cisplatin exposure were detected using CCK-8 assay and flow cytometry. The sorted CD133(+) cells showed a high purity of (88.74∓3.19)%, as compared with the purity of (3.36∓1.80)% before cell sorting. CD133(+) cells showed a high tumor sphere formation ability and tumorigenesis capacity compared with CD133- cells. CD133 shRNA transfection significantly inhibited CD133 mRNA and protein expressions in CD133(+) cells (P<0.01), resulting also in a significantly lowered cell proliferative ability (P<0.01) and an increased growth inhibition rate (P<0.01) and obviously increased cell apoptosis (P<0.05) after cisplatin exposure. Lentivirus-mediated RNAi for CD133 suppression inhibits the proliferation of CD133(+) liver cancer stem cells and increases their chemosensitivity to cisplatin.

Effect of sex sorting on stallion spermatozoa: Heterologous oocyte binding, tyrosine phosphorylation and acrosome reaction assay.

PubMed

Balao da Silva, C M; Spinaci, M; Bucci, D; Giaretta, E; Peña, F J; Mari, G; Galeati, G

2013-09-01

The interest on sex sorting by flow cytometry on the equine industry has been increasing over the years. In this work, three different tests were performed in order to evaluate the membrane status of sorted stallion spermatozoa: assessment of binding ability to porcine oocytes, evaluation of acrosome integrity after stimulation with A23187, and detection of tyrosine phosphorylation. These evaluations were made after incubation for 0h, 1.5h and 3h in a capacitating medium. Sorted stallion spermatozoa attached similarly to the porcine oocytes, when compared with control samples. Sorted spermatozoa were more prone to undergo acrosome reaction (P<0.05), at the beginning and after 1.5h and 3h of incubation, and also had higher tyrosine phosphorylation of the tail (P<0.001), only at the beginning of the incubation period. Apparently sex sorted stallion spermatozoa are in a more advanced status of membrane destabilization, which could be associated with capacitation, although similar binding ability to porcine oocytes is maintained. Copyright © 2013 Elsevier B.V. All rights reserved.

Automated single cell sorting and deposition in submicroliter drops

NASA Astrophysics Data System (ADS)

Salánki, Rita; Gerecsei, Tamás; Orgovan, Norbert; Sándor, Noémi; Péter, Beatrix; Bajtay, Zsuzsa; Erdei, Anna; Horvath, Robert; Szabó, Bálint

2014-08-01

Automated manipulation and sorting of single cells are challenging, when intact cells are needed for further investigations, e.g., RNA or DNA sequencing. We applied a computer controlled micropipette on a microscope admitting 80 PCR (Polymerase Chain Reaction) tubes to be filled with single cells in a cycle. Due to the Laplace pressure, fluid starts to flow out from the micropipette only above a critical pressure preventing the precise control of drop volume in the submicroliter range. We found an anomalous pressure additive to the Laplace pressure that we attribute to the evaporation of the drop. We have overcome the problem of the critical dropping pressure with sequentially operated fast fluidic valves timed with a millisecond precision. Minimum drop volume was 0.4-0.7 μl with a sorting speed of 15-20 s per cell. After picking NE-4C neuroectodermal mouse stem cells and human primary monocytes from a standard plastic Petri dish we could gently deposit single cells inside tiny drops. 94 ± 3% and 54 ± 7% of the deposited drops contained single cells for NE-4C and monocytes, respectively. 7.5 ± 4% of the drops contained multiple cells in case of monocytes. Remaining drops were empty. Number of cells deposited in a drop could be documented by imaging the Petri dish before and after sorting. We tuned the adhesion force of cells to make the manipulation successful without the application of microstructures for trapping cells on the surface. We propose that our straightforward and flexible setup opens an avenue for single cell isolation, critically needed for the rapidly growing field of single cell biology.

High speed flow cytometric separation of viable cells

DOEpatents

Sasaki, D.T.; Van den Engh, G.J.; Buckie, A.M.

1995-11-14

Hematopoietic cell populations are separated to provide cell sets and subsets as viable cells with high purity and high yields, based on the number of original cells present in the mixture. High-speed flow cytometry is employed using light characteristics of the cells to separate the cells, where high flow speeds are used to reduce the sorting time.

High speed flow cytometric separation of viable cells

DOEpatents

Sasaki, Dennis T.; Van den Engh, Gerrit J.; Buckie, Anne-Marie

1995-01-01

Hematopoietic cell populations are separated to provide cell sets and subsets as viable cells with high purity and high yields, based on the number of original cells present in the mixture. High-speed flow cytometry is employed using light characteristics of the cells to separate the cells, where high flow speeds are used to reduce the sorting time.

Optically enhanced acoustophoresis

NASA Astrophysics Data System (ADS)

McDougall, Craig; O'Mahoney, Paul; McGuinn, Alan; Willoughby, Nicholas A.; Qiu, Yongqiang; Demore, Christine E. M.; MacDonald, Michael P.

2017-08-01

Regenerative medicine has the capability to revolutionise many aspects of medical care, but for it to make the step from small scale autologous treatments to larger scale allogeneic approaches, robust and scalable label free cell sorting technologies are needed as part of a cell therapy bioprocessing pipeline. In this proceedings we describe several strategies for addressing the requirements for high throughput without labeling via: dimensional scaling, rare species targeting and sorting from a stable state. These three approaches are demonstrated through a combination of optical and ultrasonic forces. By combining mostly conservative and non-conservative forces from two different modalities it is possible to reduce the influence of flow velocity on sorting efficiency, hence increasing robustness and scalability. One such approach can be termed "optically enhanced acoustophoresis" which combines the ability of acoustics to handle large volumes of analyte with the high specificity of optical sorting.

Microfluidic droplet sorting using integrated bilayer micro-valves

NASA Astrophysics Data System (ADS)

Chen, Yuncong; Tian, Yang; Xu, Zhen; Wang, Xinran; Yu, Sicong; Dong, Liang

2016-10-01

This paper reports on a microfluidic device capable of sorting microfluidic droplets utilizing conventional bilayer pneumatic micro-valves as sorting controllers. The device consists of two micro-valves placed symmetrically on two sides of a sorting area, each on top of a branching channel at an inclined angle with respect to the main channel. Changes in transmitted light intensity, induced by varying light absorbance by each droplet, are used to divert the droplet from the sorting area into one of the three outlet channels. When no valve is activated, the droplet flows into the outlet channel in the direction of the main channel. When one of the valves is triggered, the flexible membrane of valve will first be deflected. Once the droplet leaves the detection point, the deflected membrane will immediately return to its default flattened position, thereby exerting a drawing pressure on the droplet and deviating it from its original streamline to the outlet on the same side as the valve. This sorting method will be particularly suitable for numerous large-scale integrated microfluidic systems, where pneumatic micro-valves are already used. Only few structural modifications are needed to achieve droplet sorting capabilities in these systems. Due to the mechanical nature of diverting energy applied to droplets, the proposed sorting method may induce only minimal interference to biological species or microorganisms encapsulated inside the droplets that may accompany electrical, optical and magnetic-based techniques.

Sorting waves and associated eigenvalues

NASA Astrophysics Data System (ADS)

Carbonari, Costanza; Colombini, Marco; Solari, Luca

2017-04-01

The presence of mixed sediment always characterizes gravel bed rivers. Sorting processes take place during bed load transport of heterogeneous sediment mixtures. The two main elements necessary to the occurrence of sorting are the heterogeneous character of sediments and the presence of an active sediment transport. When these two key ingredients are simultaneously present, the segregation of bed material is consistently detected both in the field [7] and in laboratory [3] observations. In heterogeneous sediment transport, bed altimetric variations and sorting always coexist and both mechanisms are independently capable of driving the formation of morphological patterns. Indeed, consistent patterns of longitudinal and transverse sorting are identified almost ubiquitously. In some cases, such as bar formation [2] and channel bends [5], sorting acts as a stabilizing effect and therefore the dominant mechanism driving pattern formation is associated with bed altimetric variations. In other cases, such as longitudinal streaks, sorting enhances system instability and can therefore be considered the prevailing mechanism. Bedload sheets, first observed by Khunle and Southard [1], represent another classic example of a morphological pattern essentially triggered by sorting, as theoretical [4] and experimental [3] results suggested. These sorting waves cause strong spatial and temporal fluctuations of bedload transport rate typical observed in gravel bed rivers. The problem of bed load transport of a sediment mixture is formulated in the framework of a 1D linear stability analysis. The base state consists of a uniform flow in an infinitely wide channel with active bed load transport. The behaviour of the eigenvalues associated with fluid motion, bed evolution and sorting processes in the space of the significant flow and sediment parameters is analysed. A comparison is attempted with the results of the theoretical analysis of Seminara Colombini and Parker [4] and Stecca, Siviglia and Blom [6]. [1] Kuhnle, R.A. and Southard, J.B. 1988. Bed Load Transport Fluctuations in a Gravel Bed Laboratory Channel. Water Resources Research, 24(2), 247-260. [2] Lanzoni, S. and Tubino, M. 1999. Grain sorting and bar instability. Journal of Fluid Mechanics. 393, 149-174. [3] Recking, A., Frey, P., Paquier, A. and Belleudy, P. 2009. An experimental investigation of mechanisms involved in bed load sheet production and migration. Journal of Geophysical Research, 114, F03010. [4] Seminara, G., Colombini, M. and Parker, G. 1996. Nearly pure sorting waves and formation of bedload sheets. Journal of Fluid Mechanics. 312, (1996), 253-278. [5] Seminara, G., Solari, L. and Tubino, M. 1997. Finite amplitude scour and grain sorting in wide channel bends. XXVII IAHR Congress, San Francisco, 1445-1450. [6] Stecca, G., Siviglia, A. and Blom, A. 2014. Mathematical analysis of the Saint-Venant-Hirano model for mixed-sediment morphodynamics. Water Resources Research, 50, 7563-7589. [7] Whiting, P.J., Dietrich, W.E., Leopold, L. B., Drake, T. G. and Shreve, R.L. 1988. Bedload sheets in heterogeneous sediment. Geology, 16, 105-108.

Fast "hyperlayer" separation development in sedimentation field flow fractionation.

PubMed

Kassab, James R; Cardot, Philippe J P; Zahoransky, Richard A; Battu, Serge

2005-11-05

Specific prototypes of sedimentation field flow fractionation devices (SdFFF) have been developed with relative success for cell sorting. However, no data are available to compare these apparatus with commercial ones. In order to compare with other devices mainly used for non-biological species, biocompatible systems were used for standard particle (latex: 3-10 microm of different size dispersities) separation development. In order to enhance size dependent separations, channels of reduced thickness were used (80 and 100 microm) and channel/carrier-phase equilibration procedures were necessary. For sample injection, the use of inlet tubing linked to the FFF accumulation wall, common for cell sorting, can be extended to latex species when they are eluted in the Steric Hyperlayer elution mode. It avoids any primary relaxation steps (stop flow injection procedure) simplifying series of elution processing. Mixtures composed of four different monodispersed latex beads can be eluted in 6 min with 100 microm channel thickness.

Flow cytometry for the assessment of animal sperm integrity and functionality: state of the art

PubMed Central

Hossain, Md. Sharoare; Johannisson, Anders; Wallgren, Margareta; Nagy, Szabolcs; Siqueira, Amanda Pimenta; Rodriguez-Martinez, Heriberto

2011-01-01

Flow cytometry is now a recognized methodology within animal spermatology, and has moved from being a research tool to become routine in the assessment of animal semen destined to breeding. The availability of ‘bench-top' flow cytometers and of newer and versatile markers for cell structure and function had allowed the instrumentation to measure more sperm parameters, from viability to reactiveness when exposed to exogenous stimuli, and to increase our capabilities to sort spermatozoa for potential fertilizing capacity, or chromosomal sex. The present review summarizes the state of the art regarding flow cytometry applied to animal andrology, albeit keeping an open comparative intent. It critically evaluates the present and future capabilities of flow cytometry for the diagnostics of potential fertility and for the development of current reproductive technologies such as sperm freezing, sperm selection and sperm sorting. The flow cytometry methods will probably further revolutionize our understanding of the sperm physiology and their functionality, and will undoubtedly extend its application in isolating many uncharacterized features of spermatozoa. However, continuous follow-up of the methods is a necessity owing to technical developments and the complexity of mapping spermatozoa. PMID:21478895

Size-selective sorting in bubble streaming flows: Particle migration on fast time scales

NASA Astrophysics Data System (ADS)

Thameem, Raqeeb; Rallabandi, Bhargav; Hilgenfeldt, Sascha

2015-11-01

Steady streaming from ultrasonically driven microbubbles is an increasingly popular technique in microfluidics because such devices are easily manufactured and generate powerful and highly controllable flows. Combining streaming and Poiseuille transport flows allows for passive size-sensitive sorting at particle sizes and selectivities much smaller than the bubble radius. The crucial particle deflection and separation takes place over very small times (milliseconds) and length scales (20-30 microns) and can be rationalized using a simplified geometric mechanism. A quantitative theoretical description is achieved through the application of recent results on three-dimensional streaming flow field contributions. To develop a more fundamental understanding of the particle dynamics, we use high-speed photography of trajectories in polydisperse particle suspensions, recording the particle motion on the time scale of the bubble oscillation. Our data reveal the dependence of particle displacement on driving phase, particle size, oscillatory flow speed, and streaming speed. With this information, the effective repulsive force exerted by the bubble on the particle can be quantified, showing for the first time how fast, selective particle migration is effected in a streaming flow. We acknowledge support by the National Science Foundation under grant number CBET-1236141.

Gravity effects on sediment sorting: limitations of models developed on Earth for Mars

NASA Astrophysics Data System (ADS)

Kuhn, Nikolaus J.; Kuhn, Brigitte; Gartmann, Andres

2015-04-01

Most studies on surface processes on planetary bodies assume that the use of empirical models developed for Earth is possible if the mathematical equations include all the relevant factors, such as gravity, viscosity and the density of water and sediment. However, most models for sediment transport on Earth are at least semi-empirical, using coefficients to link observed sediment movement to controlling factors such as flow velocity, slope and channel dimensions. However, using roughness and drag coefficients, as well as parameters describing incipient motion of particles, observed on Earth on another planet, violates, strictly speaking, the boundary conditions set for their application by fluid dynamics because the coefficienst describe a flow condition, not a particle property. Reduced gravity affects the flow around a settling partcile or over the bed of a watercourse, therefore data and models from Earth do not apply to another planet. Comparing observations from reduced gravity experiments and model results obtained on Earth confirm the significance of this error, e.g. by underestimating settling velocities of sandy particles by 10 to 50% for Mars when using models from Earth. In this study, the relevance of this error is examined by simulating the sorting of sediment deposited from water flowing on Mars. The results indicate that sorting on Mars is less pronounced than models calibrated on Earth suggest. This has implications for the selection of landing sites and, more importantly, the identification of strata potentially bearing traces of past life during rover missions on Mars.

Gravity effects on sediment sorting: limitations of models developed on Earth for Mars

NASA Astrophysics Data System (ADS)

Kuhn, N. J.; Kuhn, B.; Gartmann, A.

2015-10-01

Most studies on surface processes on planetary bodies assume that the use of empirical models developed for Earth is possible if the mathematical equations include all the relevant factors, such as gravity, viscosity and the density of water and sediment. However, most models for sediment transport on Earth are at least semi-empirical, using coefficients to link observed sediment movement to controlling factors such as flow velocity, slope and channel dimensions. However, using roughness and drag coefficients, as well as parameters describing incipient motion of particles, observed on Earth on another planet, violates, strictly speaking, the boundary conditions set for their application by fluid dynamics because the coefficienst describe a flow condition, not a particle property. Reduced gravity affects the flow around a settling partcile or over the bed of a watercourse, therefore data and models from Earth do not apply to another planet. Comparing observations from reduced gravity experiments and model results obtained on Earth confirm the significance of this error, e.g. by underestimating settling velocities of sandy particles by 10 to 50% for Mars when using models from Earth. In this study, the relevance of this error is examined by simulating the sorting of sediment deposited from water flowing on Mars. The results indicate that sorting on Mars is less pronounced than models calibrated on Earth suggest. This has implications for the selection of landing sites and,more importantly, the identification of strata potentially bearing traces of past life during rover missions on Mars. try, 2001

High Speed Size Sorting of Subcellular Organelles by Flow Field-Flow Fractionation.

PubMed

Yang, Joon Seon; Lee, Ju Yong; Moon, Myeong Hee

2015-06-16

Separation/isolation of subcellular species, such as mitochondria, lysosomes, peroxisomes, Golgi apparatus, and others, from cells is important for gaining an understanding of the cellular functions performed by specific organelles. This study introduces a high speed, semipreparative scale, biocompatible size sorting method for the isolation of subcellular organelle species from homogenate mixtures of HEK 293T cells using flow field-flow fractionation (FlFFF). Separation of organelles was achieved using asymmetrical FlFFF (AF4) channel system at the steric/hyperlayer mode in which nuclei, lysosomes, mitochondria, and peroxisomes were separated in a decreasing order of hydrodynamic diameter without complicated preprocessing steps. Fractions in which organelles were not clearly separated were reinjected to AF4 for a finer separation using the normal mode, in which smaller sized species can be well fractionated by an increasing order of diameter. The subcellular species contained in collected AF4 fractions were examined with scanning electron microscopy to evaluate their size and morphology, Western blot analysis using organelle specific markers was used for organelle confirmation, and proteomic analysis was performed with nanoflow liquid chromatography-tandem mass spectrometry (nLC-ESI-MS/MS). Since FlFFF operates with biocompatible buffer solutions, it offers great flexibility in handling subcellular components without relying on a high concentration sucrose solution for centrifugation or affinity- or fluorescence tag-based sorting methods. Consequently, the current study provides an alternative, competitive method for the isolation/purification of subcellular organelle species in their intact states.

Mesenchymal Stem Cells in the Bone Marrow Provide a Supportive Niche for Early Disseminated Breast Tumor-Initiating Cells

DTIC Science & Technology

2011-04-01

Differentiation of mouse embryonic stem cells Immunology: - Flow cytometry - Proliferation Assays - Chromium Release Assays - B...of metastatic cells in close proximation to hepatocytes in the liver. Additionally, re-expression of E-cadherin was observed in the membrane of the...profile CD44+/CD24low/ESA+ using fluorescence- activated cell sorting (FACS) [4]. Subcutaneous injection of low numbers of the sorted cell

Isolation of Human Induced Pluripotent Stem Cell-Derived Dopaminergic Progenitors by Cell Sorting for Successful Transplantation

PubMed Central

Doi, Daisuke; Samata, Bumpei; Katsukawa, Mitsuko; Kikuchi, Tetsuhiro; Morizane, Asuka; Ono, Yuichi; Sekiguchi, Kiyotoshi; Nakagawa, Masato; Parmar, Malin; Takahashi, Jun

2014-01-01

Summary Human induced pluripotent stem cells (iPSCs) can provide a promising source of midbrain dopaminergic (DA) neurons for cell replacement therapy for Parkinson’s disease. However, iPSC-derived donor cells inevitably contain tumorigenic or inappropriate cells. Here, we show that human iPSC-derived DA progenitor cells can be efficiently isolated by cell sorting using a floor plate marker, CORIN. We induced DA neurons using scalable culture conditions on human laminin fragment, and the sorted CORIN+ cells expressed the midbrain DA progenitor markers, FOXA2 and LMX1A. When transplanted into 6-OHDA-lesioned rats, the CORIN+ cells survived and differentiated into midbrain DA neurons in vivo, resulting in significant improvement of the motor behavior, without tumor formation. In particular, the CORIN+ cells in a NURR1+ cell-dominant stage exhibited the best survival and function as DA neurons. Our method is a favorable strategy in terms of scalability, safety, and efficiency and may be advantageous for clinical application. PMID:24672756

Behavioral vs. molecular sources of conflict between nuclear and mitochondrial DNA: The role of male-biased dispersal in a Holarctic sea duck

USGS Publications Warehouse

Peters, Jeffrey L.; Bolender, Kimberly A.; Pearce, John M.

2012-01-01

Genetic studies of waterfowl (Anatidae) have observed the full spectrum of mitochondrial (mt) DNA population divergence, from apparent panmixia to deep, reciprocally monophyletic lineages. Yet, these studies often found weak or no nuclear (nu) DNA structure, which was often attributed to male-biased gene flow, a common behaviour within this family. An alternative explanation for this ‘conflict’ is that the smaller effective population size and faster sorting rate of mtDNA relative to nuDNA lead to different signals of population structure. We tested these alternatives by sequencing 12 nuDNA introns for a Holarctic pair of waterfowl subspecies, the European goosander (Mergus merganser merganser) and the North American common merganser (M. m. americanus), which exhibit strong population structure in mtDNA. We inferred effective population sizes, gene flow and divergence times from published mtDNA sequences and simulated expected differentiation for nuDNA based on those histories. Between Europe and North America, nuDNA ФST was 3.4-fold lower than mtDNA ФST, a result consistent with differences in sorting rates. However, despite geographically structured and monophyletic mtDNA lineages within continents, nuDNA ФST values were generally zero and significantly lower than predicted. This between- and within-continent contrast held when comparing mtDNA and nuDNA among published studies of ducks. Thus, male-mediated gene flow is a better explanation than slower sorting rates for limited nuDNA differentiation within continents, which is also supported by nonmolecular data. This study illustrates the value of quantitatively testing discrepancies between mtDNA and nuDNA to reject the null hypothesis that conflict simply reflects different sorting rates.

Characterization of hepatic progenitors from human fetal liver during second trimester.

PubMed

Rao, Mekala-Subba; Khan, Aleem-Ahmed; Parveen, Nyamath; Habeeb, Mohammed-Aejaz; Habibullah, Chittoor-Mohammed; Pande, Gopal

2008-10-07

To enrich hepatic progenitors using epithelial cell adhesion molecule (EpCAM) as a marker from human fetal liver and investigate the expression of human leukocyte antigen (HLA) and their markers associated with hepatic progenitor cells. EpCAM +ve cells were isolated using magnetic cell sorting (MACS) from human fetuses (n = 10) at 15-25 wk gestation. Expression of markers for hepatic progenitors such as albumin, alpha-fetoprotein (AFP), CD29 (integrin beta1), CD49f (integrin alpha6) and CD90 (Thy 1) was studied by using flow cytometry, immunocytochemistry and RT-PCR; HLA class I (A, B, C) and class II (DR) expression was studied by flow cytometry only. FACS analysis indicated that EpCAM +ve cells were positive for CD29, CD49f, CD90, CD34, HLA class I, albumin and AFP but negative for HLA class II (DR) and CD45. RT PCR showed that EpCAM +ve cells expressed liver epithelial markers (CK18), biliary specific marker (CK19) and hepatic markers (albumin, AFP). On immunocytochemical staining, EpCAM +ve cells were shown positive signals for CK18 and albumin. Our study suggests that these EpCAM +ve cells can be used as hepatic progenitors for cell transplantation with a minimum risk of alloreactivity and these cells may serve as a potential source for enrichment of hepatic progenitor.

Construction of BAC Libraries from Flow-Sorted Chromosomes.

PubMed

Šafář, Jan; Šimková, Hana; Doležel, Jaroslav

2016-01-01

Cloned DNA libraries in bacterial artificial chromosome (BAC) are the most widely used form of large-insert DNA libraries. BAC libraries are typically represented by ordered clones derived from genomic DNA of a particular organism. In the case of large eukaryotic genomes, whole-genome libraries consist of a hundred thousand to a million clones, which make their handling and screening a daunting task. The labor and cost of working with whole-genome libraries can be greatly reduced by constructing a library derived from a smaller part of the genome. Here we describe construction of BAC libraries from mitotic chromosomes purified by flow cytometric sorting. Chromosome-specific BAC libraries facilitate positional gene cloning, physical mapping, and sequencing in complex plant genomes.

A fast sorting algorithm for a hypersonic rarefied flow particle simulation on the connection machine

NASA Technical Reports Server (NTRS)

Dagum, Leonardo

1989-01-01

The data parallel implementation of a particle simulation for hypersonic rarefied flow described by Dagum associates a single parallel data element with each particle in the simulation. The simulated space is divided into discrete regions called cells containing a variable and constantly changing number of particles. The implementation requires a global sort of the parallel data elements so as to arrange them in an order that allows immediate access to the information associated with cells in the simulation. Described here is a very fast algorithm for performing the necessary ranking of the parallel data elements. The performance of the new algorithm is compared with that of the microcoded instruction for ranking on the Connection Machine.

Evaluation of recycling programmes in household waste collection systems.

PubMed

Dahlén, Lisa; Lagerkvist, Anders

2010-07-01

A case study and a literature review have been carried out to address the two questions: how can waste flow data from collection systems be interpreted and compared? and which factors are decisive in the results of recycling programmes in household waste collection systems? The aim is to contribute to the understanding of how recycling programmes affect the quantity of waste and sorting activities. It is shown how the results from various waste sorting systems can be interpreted and made comparable. A set of waste flow indicators is proposed, which together with generic system descriptions can facilitate comparisons of different collections systems. The evaluation of collection systems depends on the system boundaries and will always be site-specific to some degree. Various factors are relevant, e.g. environmental objectives, technical function, operating costs, types of recyclable materials collected separately, property-close collection or drop-off systems, economic incentives, information strategies, residential structure, social codes, etc. Kerbside collection of recyclables and weight-based billing led to increased waste sorting activities in the case study. Forty-three decisive factors are listed and discussed.

The Grain-size Patchiness of Braided Gravel-Bed Streams - example of the Urumqi River (northeast Tian Shan, China)

NASA Astrophysics Data System (ADS)

Guerit, L.; Barrier, L.; Narteau, C.; Métivier, F.; Liu, Y.; Lajeunesse, E.; Gayer, E.; Meunier, P.; Malverti, L.; Ye, B.

2014-02-01

In gravel-bed rivers, sediments are often sorted into patches of different grain-sizes, but in braided streams, the link between this sorting and the channel morpho-sedimentary elements is still unclear. In this study, the size of the bed sediment in the shallow braided gravel-bed Urumqi River is characterized by surface-count and volumetric sampling methods. Three morpho-sedimentary elements are identified in the active threads of the river: chutes at flow constrictions, which pass downstream to anabranches and bars at flow expansions. The surface and surface-layer grain-size distributions of these three elements show that they correspond to only two kinds of grain-size patches: (1) coarse-grained chutes, coarser than the bulk river bed, and (2) finer-grained anabranches and bars, consistent with the bulk river bed. In cross-section, the chute patches are composed of one coarse-grained top layer, which can be interpreted as a local armour layer overlying finer deposits. In contrast, the grain size of the bar-anabranch patches is finer and much more homogeneous in depth than the chute patches. Those patches, which are features of lateral and vertical sorting associated to the transport dynamics that build braided patterns, may be typical of active threads in shallow gravel-bed rivers and should be considered in future works on sorting processes and their geomorphologic and stratigraphic results.

High-throughput microfluidic mixing and multiparametric cell sorting for bioactive compound screening.

PubMed

Young, Susan M; Curry, Mark S; Ransom, John T; Ballesteros, Juan A; Prossnitz, Eric R; Sklar, Larry A; Edwards, Bruce S

2004-03-01

HyperCyt, an automated sample handling system for flow cytometry that uses air bubbles to separate samples sequentially introduced from multiwell plates by an autosampler. In a previously documented HyperCyt configuration, air bubble separated compounds in one sample line and a continuous stream of cells in another are mixed in-line for serial flow cytometric cell response analysis. To expand capabilities for high-throughput bioactive compound screening, the authors investigated using this system configuration in combination with automated cell sorting. Peptide ligands were sampled from a 96-well plate, mixed in-line with fluo-4-loaded, formyl peptide receptor-transfected U937 cells, and screened at a rate of 3 peptide reactions per minute with approximately 10,000 cells analyzed per reaction. Cell Ca(2+) responses were detected to as little as 10(-11) M peptide with no detectable carryover between samples at up to 10(-7) M peptide. After expansion in culture, cells sort-purified from the 10% highest responders exhibited enhanced sensitivity and more sustained responses to peptide. Thus, a highly responsive cell subset was isolated under high-throughput mixing and sorting conditions in which response detection capability spanned a 1000-fold range of peptide concentration. With single-cell readout systems for protein expression libraries, this technology offers the promise of screening millions of discrete compound interactions per day.

Sortilin 1 knockout alters basal adipose glucose metabolism but not diet-induced obesity in mice.

PubMed

Li, Jibiao; Matye, David J; Wang, Yifeng; Li, Tiangang

2017-04-01

Sortilin 1 (Sort1) is a trafficking receptor that has been implicated in the regulation of plasma cholesterol in humans and mice. Here, we use metabolomics and hyperinsulinemic-euglycemic clamp approaches to obtain further understanding of the in vivo effects of Sort1 deletion on diet-induced obesity as well as on adipose lipid and glucose metabolism. Results show that Sort1 knockout (KO) does not affect Western diet-induced obesity nor adipose fatty acid and ceramide concentrations. Under the basal fasting state, chow-fed Sort1 KO mice have decreased adipose glycolytic metabolites, but Sort1 deletion does not affect insulin-stimulated tissue glucose uptake during the insulin clamp. These results suggest that Sort1 loss-of-function in vivo does not affect obesity development, but differentially modulates adipose glucose metabolism under fasting and insulin-stimulated states. © 2017 Federation of European Biochemical Societies.

Sorting cells by their density

PubMed Central

Norouzi, Nazila; Bhakta, Heran C.

2017-01-01

Sorting cells by their type is an important capability in biological research and medical diagnostics. However, most cell sorting techniques rely on labels or tags, which may have limited availability and specificity. Sorting different cell types by their different physical properties is an attractive alternative to labels because all cells intrinsically have these physical properties. But some physical properties, like cell size, vary significantly from cell to cell within a cell type; this makes it difficult to identify and sort cells based on their sizes alone. In this work we continuously sort different cells types by their density, a physical property with much lower cell-to-cell variation within a cell type (and therefore greater potential to discriminate different cell types) than other physical properties. We accomplish this using a 3D-printed microfluidic chip containing a horizontal flowing micron-scale density gradient. As cells flow through the chip, Earth’s gravity makes each cell move vertically to the point where the cell’s density matches the surrounding fluid’s density. When the horizontal channel then splits, cells with different densities are routed to different outlets. As a proof of concept, we use our density sorter chip to sort polymer microbeads by their material (polyethylene and polystyrene) and blood cells by their type (white blood cells and red blood cells). The chip enriches the fraction of white blood cells in a blood sample from 0.1% (in whole blood) to nearly 98% (in the output of the chip), a 1000x enrichment. Any researcher with access to a 3D printer can easily replicate our density sorter chip and use it in their own research using the design files provided as online Supporting Information. Additionally, researchers can simulate the performance of a density sorter chip in their own applications using the Python-based simulation software that accompanies this work. The simplicity, resolution, and throughput of this technique make it suitable for isolating even rare cell types in complex biological samples, in a wide variety of different research and clinical applications. PMID:28723908

Gene Expression Profiling of Liver Cancer Stem Cells by RNA-Sequencing

PubMed Central

Lam, Chi Tat; Ng, Michael N. P.; Yu, Wan Ching; Lau, Joyce; Wan, Timothy; Wang, Xiaoqi; Yan, Zhixiang; Liu, Hang; Fan, Sheung Tat

2012-01-01

Background Accumulating evidence supports that tumor growth and cancer relapse are driven by cancer stem cells. Our previous work has demonstrated the existence of CD90+ liver cancer stem cells (CSCs) in hepatocellular carcinoma (HCC). Nevertheless, the characteristics of these cells are still poorly understood. In this study, we employed a more sensitive RNA-sequencing (RNA-Seq) to compare the gene expression profiling of CD90+ cells sorted from tumor (CD90+CSCs) with parallel non-tumorous liver tissues (CD90+NTSCs) and elucidate the roles of putative target genes in hepatocarcinogenesis. Methodology/Principal Findings CD90+ cells were sorted respectively from tumor and adjacent non-tumorous human liver tissues using fluorescence-activated cell sorting. The amplified RNAs of CD90+ cells from 3 HCC patients were subjected to RNA-Seq analysis. A differential gene expression profile was established between CD90+CSCs and CD90+NTSCs, and validated by quantitative real-time PCR (qRT-PCR) on the same set of amplified RNAs, and further confirmed in an independent cohort of 12 HCC patients. Five hundred genes were differentially expressed (119 up-regulated and 381 down-regulated genes) between CD90+CSCs and CD90+NTSCs. Gene ontology analysis indicated that the over-expressed genes in CD90+CSCs were associated with inflammation, drug resistance and lipid metabolism. Among the differentially expressed genes, glypican-3 (GPC3), a member of glypican family, was markedly elevated in CD90+CSCs compared to CD90+NTSCs. Immunohistochemistry demonstrated that GPC3 was highly expressed in forty-two human liver tumor tissues but absent in adjacent non-tumorous liver tissues. Flow cytometry indicated that GPC3 was highly expressed in liver CD90+CSCs and mature cancer cells in liver cancer cell lines and human liver tumor tissues. Furthermore, GPC3 expression was positively correlated with the number of CD90+CSCs in liver tumor tissues. Conclusions/Significance The identified genes, such as GPC3 that are distinctly expressed in liver CD90+CSCs, may be promising gene candidates for HCC therapy without inducing damages to normal liver stem cells. PMID:22606345

Liver-specific gene expression in cultured human hematopoietic stem cells.

PubMed

Fiegel, Henning C; Lioznov, Michael V; Cortes-Dericks, Lourdes; Lange, Claudia; Kluth, Dietrich; Fehse, Boris; Zander, Axel R

2003-01-01

Hematopoietic and hepatic stem cells share characteristic markers such as CD34, c-kit, and Thy1. Based on the recent observations that hepatocytes may originate from bone marrow, we investigated the potential of CD34(+) bone marrow cells to differentiate into hepatocytic cells in vitro. CD34(+) and CD34(-) human bone marrow cells were separated by magnetic cell sorting. Cells were cultured on a collagen matrix in a defined medium containing hepatocyte growth factor. Cell count and size were measured by flow cytometry, and reverse transcription polymerase chain reaction was carried out for the liver-specific markers CK-19 and albumin. During cell culture, CD34(+) cells showed an increasing cell number and proliferative activity as assessed by Ki-67 staining. Under the specified culture conditions, CD34(+) cells expressed albumin RNA and CK-19 RNA after 28 days, whereas CD34(-) cells did not show liver-specific gene expression. The results indicate that CD34(+) adult human bone marrow stem cells can differentiate into hepatocytic cells in vitro.

Selective flow-induced vesicle rupture to sort by membrane mechanical properties

NASA Astrophysics Data System (ADS)

Pommella, Angelo; Brooks, Nicholas J.; Seddon, John M.; Garbin, Valeria

2015-08-01

Vesicle and cell rupture caused by large viscous stresses in ultrasonication is central to biomedical and bioprocessing applications. The flow-induced opening of lipid membranes can be exploited to deliver drugs into cells, or to recover products from cells, provided that it can be obtained in a controlled fashion. Here we demonstrate that differences in lipid membrane and vesicle properties can enable selective flow-induced vesicle break-up. We obtained vesicle populations with different membrane properties by using different lipids (SOPC, DOPC, or POPC) and lipid:cholesterol mixtures (SOPC:chol and DOPC:chol). We subjected vesicles to large deformations in the acoustic microstreaming flow generated by ultrasound-driven microbubbles. By simultaneously deforming vesicles with different properties in the same flow, we determined the conditions in which rupture is selective with respect to the membrane stretching elasticity. We also investigated the effect of vesicle radius and excess area on the threshold for rupture, and identified conditions for robust selectivity based solely on the mechanical properties of the membrane. Our work should enable new sorting mechanisms based on the difference in membrane composition and mechanical properties between different vesicles, capsules, or cells.

Selective flow-induced vesicle rupture to sort by membrane mechanical properties

PubMed Central

Pommella, Angelo; Brooks, Nicholas J.; Seddon, John M.; Garbin, Valeria

2015-01-01

Vesicle and cell rupture caused by large viscous stresses in ultrasonication is central to biomedical and bioprocessing applications. The flow-induced opening of lipid membranes can be exploited to deliver drugs into cells, or to recover products from cells, provided that it can be obtained in a controlled fashion. Here we demonstrate that differences in lipid membrane and vesicle properties can enable selective flow-induced vesicle break-up. We obtained vesicle populations with different membrane properties by using different lipids (SOPC, DOPC, or POPC) and lipid:cholesterol mixtures (SOPC:chol and DOPC:chol). We subjected vesicles to large deformations in the acoustic microstreaming flow generated by ultrasound-driven microbubbles. By simultaneously deforming vesicles with different properties in the same flow, we determined the conditions in which rupture is selective with respect to the membrane stretching elasticity. We also investigated the effect of vesicle radius and excess area on the threshold for rupture, and identified conditions for robust selectivity based solely on the mechanical properties of the membrane. Our work should enable new sorting mechanisms based on the difference in membrane composition and mechanical properties between different vesicles, capsules, or cells. PMID:26302783

Isolation of human simple repeat loci by hybridization selection.

PubMed

Armour, J A; Neumann, R; Gobert, S; Jeffreys, A J

1994-04-01

We have isolated short tandem repeat arrays from the human genome, using a rapid method involving filter hybridization to enrich for tri- or tetranucleotide tandem repeats. About 30% of clones from the enriched library cross-hybridize with probes containing trimeric or tetrameric tandem arrays, facilitating the rapid isolation of large numbers of clones. In an initial analysis of 54 clones, 46 different tandem arrays were identified. Analysis of these tandem repeat loci by PCR showed that 24 were polymorphic in length; substantially higher levels of polymorphism were displayed by the tetrameric repeat loci isolated than by the trimeric repeats. Primary mapping of these loci by linkage analysis showed that they derive from 17 chromosomes, including the X chromosome. We anticipate the use of this strategy for the efficient isolation of tandem repeats from other sources of genomic DNA, including DNA from flow-sorted chromosomes, and from other species.

Isolation and Characterization of Human Lung Lymphatic Endothelial Cells

PubMed Central

Lorusso, Bruno; Falco, Angela; Madeddu, Denise; Frati, Caterina; Cavalli, Stefano; Graiani, Gallia; Gervasi, Andrea; Rinaldi, Laura; Lagrasta, Costanza; Maselli, Davide; Gnetti, Letizia; Silini, Enrico M.; Quaini, Eugenio; Ampollini, Luca; Carbognani, Paolo; Quaini, Federico

2015-01-01

Characterization of lymphatic endothelial cells from the respiratory system may be crucial to investigate the role of the lymphatic system in the normal and diseased lung. We describe a simple and inexpensive method to harvest, isolate, and expand lymphatic endothelial cells from the human lung (HL-LECs). Fifty-five samples of healthy lung selected from patients undergoing lobectomy were studied. A two-step purification tool, based on paramagnetic sorting with monoclonal antibodies to CD31 and Podoplanin, was employed to select a pure population of HL-LECs. The purity of HL-LECs was assessed by morphologic criteria, immunocytochemistry, flow cytometry, and functional assays. Interestingly, these cells retain in vitro several receptor tyrosine kinases (RTKs) implicated in cell survival and proliferation. HL-LECs represent a clinically relevant cellular substrate to study lymphatic biology, lymphoangiogenesis, interaction with microbial agents, wound healing, and anticancer therapy. PMID:26137493

Application of a novel sorting system for equine mesenchymal stem cells (MSCs)

PubMed Central

Radtke, Catherine L.; Nino-Fong, Rodolfo; Esparza Gonzalez, Blanca P.; McDuffee, Laurie A.

2014-01-01

The objective of this study was to validate non-equilibrium gravitational field-flow fractionation (GrFFF), an immunotag-less method of sorting mesenchymal stem cells (MSCs) into subpopulations, for use with MSCs derived from equine muscle tissue, periosteal tissue, bone marrow, and adipose tissue. Cells were collected from 6 young, adult horses, postmortem. Cells were isolated from left semitendinosus muscle tissue, periosteal tissue from the distomedial aspect of the right tibia, bone marrow aspirates from the fourth and fifth sternebrae, and left supragluteal subcutaneous adipose tissue. Aliquots of 800 × 103 MSCs from each tissue source were separated and injected into a ribbon-like capillary device by continuous flow (GrFFF proprietary system). Cells were sorted into 6 fractions and absorbencies [optical density (OD)] were read. Six fractions from each of the 6 aliquots were then combined to provide pooled fractions that had adequate cell numbers to seed at equal concentrations into assays. Equine muscle tissue-derived, periosteal tissue-derived, bone marrow-derived, and adipose tissue-derived mesenchymal stem cells were consistently sorted into 6 fractions that remained viable for use in further assays. Fraction 1 had more cuboidal morphology in culture when compared to the other fractions. Statistical analysis of the fraction absorbencies (OD) revealed a P-value of < 0.05 when fractions 2 and 3 were compared to fractions 1, 4, 5, and 6. It was concluded that non-equilibrium GrFFF is a valid method for sorting equine muscle tissue-derived, periosteal tissue-derived, bone marrow-derived, and adipose tissue-derived mesenchymal stem cells into subpopulations that remain viable, thus securing its potential for use in equine stem cell applications and veterinary medicine. PMID:25355998

Microfluidic EmbryoSort technology: towards in flow analysis, sorting and dispensing of individual vertebrate embryos

NASA Astrophysics Data System (ADS)

Fuad, Nurul M.; Wlodkowic, Donald

2013-12-01

The demand to reduce the numbers of laboratory animals has facilitated the emergence of surrogate models such as tests performed on zebrafish (Danio rerio) or African clawed frog's (Xenopus levis) eggs, embryos and larvae. Those two model organisms are becoming increasingly popular replacements to current adult animal testing in toxicology, ecotoxicology and also in drug discovery. Zebrafish eggs and embryos are particularly attractive for toxicological analysis due their size (diameter 1.6 mm), optical transparency, large numbers generated per fish and very straightforward husbandry. The current bottleneck in using zebrafish embryos for screening purposes is, however, a tedious manual evaluation to confirm the fertilization status and subsequent dispensing of single developing embryos to multitier plates to perform toxicity analysis. Manual procedures associated with sorting hundreds of embryos are very monotonous and as such prone to significant analytical errors due to operator's fatigue. In this work, we present a proofof- concept design of a continuous flow embryo sorter capable of analyzing, sorting and dispensing objects ranging in size from 1.5 - 2.5 mm. The prototypes were fabricated in polymethyl methacrylate (PMMA) transparent thermoplastic using infrared laser micromachining. The application of additive manufacturing processes to prototype Lab-on-a-Chip sorters using both fused deposition manufacturing (FDM) and stereolithography (SLA) were also explored. The operation of the device was based on a revolving receptacle capable of receiving, holding and positioning single fish embryos for both interrogation and subsequent sorting. The actuation of the revolving receptacle was performed using a DC motor and/or microservo motor. The system was designed to separate between fertilized (LIVE) and non-fertilized (DEAD) eggs, based on optical transparency using infrared (IR) emitters and receivers.

Bears in a Forest of Gene Trees: Phylogenetic Inference Is Complicated by Incomplete Lineage Sorting and Gene Flow

PubMed Central

Kutschera, Verena E.; Bidon, Tobias; Hailer, Frank; Rodi, Julia L.; Fain, Steven R.; Janke, Axel

2014-01-01

Ursine bears are a mammalian subfamily that comprises six morphologically and ecologically distinct extant species. Previous phylogenetic analyses of concatenated nuclear genes could not resolve all relationships among bears, and appeared to conflict with the mitochondrial phylogeny. Evolutionary processes such as incomplete lineage sorting and introgression can cause gene tree discordance and complicate phylogenetic inferences, but are not accounted for in phylogenetic analyses of concatenated data. We generated a high-resolution data set of autosomal introns from several individuals per species and of Y-chromosomal markers. Incorporating intraspecific variability in coalescence-based phylogenetic and gene flow estimation approaches, we traced the genealogical history of individual alleles. Considerable heterogeneity among nuclear loci and discordance between nuclear and mitochondrial phylogenies were found. A species tree with divergence time estimates indicated that ursine bears diversified within less than 2 My. Consistent with a complex branching order within a clade of Asian bear species, we identified unidirectional gene flow from Asian black into sloth bears. Moreover, gene flow detected from brown into American black bears can explain the conflicting placement of the American black bear in mitochondrial and nuclear phylogenies. These results highlight that both incomplete lineage sorting and introgression are prominent evolutionary forces even on time scales up to several million years. Complex evolutionary patterns are not adequately captured by strictly bifurcating models, and can only be fully understood when analyzing multiple independently inherited loci in a coalescence framework. Phylogenetic incongruence among gene trees hence needs to be recognized as a biologically meaningful signal. PMID:24903145

Logistics Lessons Learned in NASA Space Flight

NASA Technical Reports Server (NTRS)

Evans, William A.; DeWeck, Olivier; Laufer, Deanna; Shull, Sarah

2006-01-01

The Vision for Space Exploration sets out a number of goals, involving both strategic and tactical objectives. These include returning the Space Shuttle to flight, completing the International Space Station, and conducting human expeditions to the Moon by 2020. Each of these goals has profound logistics implications. In the consideration of these objectives,a need for a study on NASA logistics lessons learned was recognized. The study endeavors to identify both needs for space exploration and challenges in the development of past logistics architectures, as well as in the design of space systems. This study may also be appropriately applied as guidance in the development of an integrated logistics architecture for future human missions to the Moon and Mars. This report first summarizes current logistics practices for the Space Shuttle Program (SSP) and the International Space Station (ISS) and examines the practices of manifesting, stowage, inventory tracking, waste disposal, and return logistics. The key findings of this examination are that while the current practices do have many positive aspects, there are also several shortcomings. These shortcomings include a high-level of excess complexity, redundancy of information/lack of a common database, and a large human-in-the-loop component. Later sections of this report describe the methodology and results of our work to systematically gather logistics lessons learned from past and current human spaceflight programs as well as validating these lessons through a survey of the opinions of current space logisticians. To consider the perspectives on logistics lessons, we searched several sources within NASA, including organizations with direct and indirect connections with the system flow in mission planning. We utilized crew debriefs, the John Commonsense lessons repository for the JSC Mission Operations Directorate, and the Skylab Lessons Learned. Additionally, we searched the public version of the Lessons Learned Information System (LLIS) and verified that we received the same result using the internal version of LLIS for our logistics lesson searches. In conducting the research, information from multiple databases was consolidated into a single spreadsheet of 300 lessons learned. Keywords were applied for the purpose of sorting and evaluation. Once the lessons had been compiled, an analysis of the resulting data was performed, first sorting it by keyword, then finding duplication and root cause, and finally sorting by root cause. The data was then distilled into the top 7 lessons learned across programs, centers, and activities.

A model for melanosome biogenesis based on the purification and analysis of early melanosomes

PubMed Central

Kushimoto, Tsuneto; Basrur, Venkatesha; Valencia, Julio; Matsunaga, Jun; Vieira, Wilfred D.; Ferrans, Victor J.; Muller, Jacqueline; Appella, Ettore; Hearing, Vincent J.

2001-01-01

Melanosome biogenesis and function were studied after purification of early stage melanosomes and characterization of specific proteins sorted to that organelle. Melanosomes were isolated from highly pigmented human MNT1 melanoma cells after disruption and initial separation by sucrose density gradient centrifugation. Low-density sucrose fractions were found by electron microscopy to be enriched in stage I and stage II melanosomes, and these fractions were further separated and purified by free flow electrophoresis. Tyrosinase and dopachrome tautomerase (DCT) activities were found exclusively in stage II melanosomes, even though DCT (and to some extent tyrosinase) proteins were sorted to stage I melanosomes. Western immunoblotting revealed that these catalytic proteins, as well as TYRP1, MART1, and GP100, were cleaved and inactivated in stage I melanosomes. Proteolytic cleavage was critical for the refolding of GP100 within the melanosomal milieu, and subsequent reorganization of amorphous stage I melanosomes into fibrillar, ovoid, and highly organized stage II melanosomes appears to stabilize the catalytic functions of melanosomal enzymes and allows melanin biosynthesis to begin. These results provide a better understanding of the structural features seen during melanosome biogenesis, and they yield further clues as to the physiological regulation of pigmentation. PMID:11526213

To sort or not to sort: the impact of spike-sorting on neural decoding performance.

PubMed

Todorova, Sonia; Sadtler, Patrick; Batista, Aaron; Chase, Steven; Ventura, Valérie

2014-10-01

Brain-computer interfaces (BCIs) are a promising technology for restoring motor ability to paralyzed patients. Spiking-based BCIs have successfully been used in clinical trials to control multi-degree-of-freedom robotic devices. Current implementations of these devices require a lengthy spike-sorting step, which is an obstacle to moving this technology from the lab to the clinic. A viable alternative is to avoid spike-sorting, treating all threshold crossings of the voltage waveform on an electrode as coming from one putative neuron. It is not known, however, how much decoding information might be lost by ignoring spike identity. We present a full analysis of the effects of spike-sorting schemes on decoding performance. Specifically, we compare how well two common decoders, the optimal linear estimator and the Kalman filter, reconstruct the arm movements of non-human primates performing reaching tasks, when receiving input from various sorting schemes. The schemes we tested included: using threshold crossings without spike-sorting; expert-sorting discarding the noise; expert-sorting, including the noise as if it were another neuron; and automatic spike-sorting using waveform features. We also decoded from a joint statistical model for the waveforms and tuning curves, which does not involve an explicit spike-sorting step. Discarding the threshold crossings that cannot be assigned to neurons degrades decoding: no spikes should be discarded. Decoding based on spike-sorted units outperforms decoding based on electrodes voltage crossings: spike-sorting is useful. The four waveform based spike-sorting methods tested here yield similar decoding efficiencies: a fast and simple method is competitive. Decoding using the joint waveform and tuning model shows promise but is not consistently superior. Our results indicate that simple automated spike-sorting performs as well as the more computationally or manually intensive methods used here. Even basic spike-sorting adds value to the low-threshold waveform-crossing methods often employed in BCI decoding.

To sort or not to sort: the impact of spike-sorting on neural decoding performance

NASA Astrophysics Data System (ADS)

Todorova, Sonia; Sadtler, Patrick; Batista, Aaron; Chase, Steven; Ventura, Valérie

2014-10-01

Objective. Brain-computer interfaces (BCIs) are a promising technology for restoring motor ability to paralyzed patients. Spiking-based BCIs have successfully been used in clinical trials to control multi-degree-of-freedom robotic devices. Current implementations of these devices require a lengthy spike-sorting step, which is an obstacle to moving this technology from the lab to the clinic. A viable alternative is to avoid spike-sorting, treating all threshold crossings of the voltage waveform on an electrode as coming from one putative neuron. It is not known, however, how much decoding information might be lost by ignoring spike identity. Approach. We present a full analysis of the effects of spike-sorting schemes on decoding performance. Specifically, we compare how well two common decoders, the optimal linear estimator and the Kalman filter, reconstruct the arm movements of non-human primates performing reaching tasks, when receiving input from various sorting schemes. The schemes we tested included: using threshold crossings without spike-sorting; expert-sorting discarding the noise; expert-sorting, including the noise as if it were another neuron; and automatic spike-sorting using waveform features. We also decoded from a joint statistical model for the waveforms and tuning curves, which does not involve an explicit spike-sorting step. Main results. Discarding the threshold crossings that cannot be assigned to neurons degrades decoding: no spikes should be discarded. Decoding based on spike-sorted units outperforms decoding based on electrodes voltage crossings: spike-sorting is useful. The four waveform based spike-sorting methods tested here yield similar decoding efficiencies: a fast and simple method is competitive. Decoding using the joint waveform and tuning model shows promise but is not consistently superior. Significance. Our results indicate that simple automated spike-sorting performs as well as the more computationally or manually intensive methods used here. Even basic spike-sorting adds value to the low-threshold waveform-crossing methods often employed in BCI decoding.

Handling of boar spermatozoa during and after flow cytometric sex-sorting process to improve their in vitro fertilizing ability.

PubMed

del Olmo, D; Parrilla, I; Gil, M A; Maside, C; Tarantini, T; Angel, M A; Roca, J; Martinez, E A; Vazquez, J M

2013-09-01

The objective of this study was to develop an adequate sperm handling protocol in order to obtain a sex-sorted sperm population with an optimal fertilizing ability. For this purpose, different aspects of the sorting procedure were examined. The effects of the high dilution rates (experiment 1), type of collection medium used (experiment 2), and sheath fluid composition (experiment 3) on sorted boar sperm quality and function were evaluated. Sperm quality was assessed by motility and viability tests, whereas sperm function was evaluated by an in vitro fertilization assay which determined the penetration and polyspermy rates as well as the mean number of sperm penetrating each oocyte. In experiment 1, the results obtained indicated that the high dilution rates did not cause a decrease either in the sperm quality parameters evaluated or the in vitro fertilization ability of spermatozoa. In experiment 2, although sperm quality was not affected, fertilizing ability was compromised after sorting, regardless of the collection medium that was used. In the experiment 3, all groups displayed adequate sperm quality values, but higher in vitro fertility parameters were obtained for spermatozoa sorted in presence of EDTA in the sheath fluid and egg yolk (EY) in the collection media when compared with those sorted in absence of these protective agents. No differences in penetration rates between unsorted highly diluted (control) and sorted sperm in the presence of EDTA and EY were observed. In conclusion, fertilizing ability was compromised in sex-sorted sperm. The addition of EDTA to sheath fluid and EY to collection medium improved boar sperm fertilizing ability, and both agents should be included as essential media components in future studies. Copyright © 2013 Elsevier Inc. All rights reserved.

Isolation of major pancreatic cell types and long-term culture-initiating cells using novel human surface markers.

PubMed

Dorrell, Craig; Abraham, Stephanie L; Lanxon-Cookson, Kelsea M; Canaday, Pamela S; Streeter, Philip R; Grompe, Markus

2008-09-01

We have developed a novel panel of cell-surface markers for the isolation and study of all major cell types of the human pancreas. Hybridomas were selected after subtractive immunization of Balb/C mice with intact or dissociated human islets and assessed for cell-type specificity and cell-surface reactivity by immunohistochemistry and flow cytometry. Antibodies were identified by specific binding of surface antigens on islet (panendocrine or alpha-specific) and nonislet pancreatic cell subsets (exocrine and duct). These antibodies were used individually or in combination to isolate populations of alpha, beta, exocrine, or duct cells from primary human pancreas by FACS and to characterize the detailed cell composition of human islet preparations. They were also employed to show that human islet expansion cultures originated from nonendocrine cells and that insulin expression levels could be increased to up to 1% of normal islet cells by subpopulation sorting and overexpression of the transcription factors Pdx-1 and ngn3, an improvement over previous results with this culture system. These methods permit the analysis and isolation of functionally distinct pancreatic cell populations with potential for cell therapy.

Preparation of myeloid derived suppressor cells (MDSC) from naive and pancreatic tumor-bearing mice using flow cytometry and automated magnetic activated cell sorting (AutoMACS).

PubMed

Nelson, Nadine; Szekeres, Karoly; Cooper, Denise; Ghansah, Tomar

2012-06-18

MDSC are a heterogeneous population of immature macrophages, dendritic cells and granulocytes that accumulate in lymphoid organs in pathological conditions including parasitic infection, inflammation, traumatic stress, graft-versus-host disease, diabetes and cancer. In mice, MDSC express Mac-1 (CD11b) and Gr-1 (Ly6G and Ly6C) surface antigens. It is important to note that MDSC are well studied in various tumor-bearing hosts where they are significantly expanded and suppress anti-tumor immune responses compared to naïve counterparts. However, depending on the pathological condition, there are different subpopulations of MDSC with distinct mechanisms and targets of suppression. Therefore, effective methods to isolate viable MDSC populations are important in elucidating their different molecular mechanisms of suppression in vitro and in vivo. Recently, the Ghansah group has reported the expansion of MDSC in a murine pancreatic cancer model. Our tumor-bearing MDSC display a loss of homeostasis and increased suppressive function compared to naïve MDSC. MDSC percentages are significantly less in lymphoid compartments of naïve vs. tumor-bearing mice. This is a major caveat, which often hinders accurate comparative analyses of these MDSC. Therefore, enriching Gr-1(+) leukocytes from naïve mice prior to Fluorescence Activated Cell Sorting (FACS) enhances purity, viability and significantly reduces sort time. However, enrichment of Gr-1(+) leukocytes from tumor-bearing mice is optional as these are in abundance for quick FACS sorting. Therefore, in this protocol, we describe a highly efficient method of immunophenotyping MDSC and enriching Gr-1(+) leukocytes from spleens of naïve mice for sorting MDSC in a timely manner. Immunocompetent C57BL/6 mice are inoculated with murine Panc02 cells subcutaneously whereas naïve mice receive 1XPBS. Approximately 30 days post inoculation; spleens are harvested and processed into single-cell suspensions using a cell dissociation sieve. Splenocytes are then Red Blood Cell (RBC) lysed and an aliquot of these leukocytes are stained using fluorochrome-conjugated antibodies against Mac-1 and Gr-1 to immunophenotype MDSC percentages using Flow Cytometry. In a parallel experiment, whole leukocytes from naïve mice are stained with fluorescent-conjugated Gr-1 antibodies, incubated with PE-MicroBeads and positively selected using an automated Magnetic Activated Cell Sorting (autoMACS) Pro Separator. Next, an aliquot of Gr-1(+) leukocytes are stained with Mac-1 antibodies to identify the increase in MDSC percentages using Flow Cytometry. Now, these Gr1(+) enriched leukocytes are ready for FACS sorting of MDSC to be used in comparative analyses (naïve vs. tumor- bearing) in in vivo and in vitro assays.

Computation of subsonic flow around airfoil systems with multiple separation

NASA Technical Reports Server (NTRS)

Jacob, K.

1982-01-01

A numerical method for computing the subsonic flow around multi-element airfoil systems was developed, allowing for flow separation at one or more elements. Besides multiple rear separation also sort bubbles on the upper surface and cove bubbles can approximately be taken into account. Also, compressibility effects for pure subsonic flow are approximately accounted for. After presentation the method is applied to several examples and improved in some details. Finally, the present limitations and desirable extensions are discussed.

The fluid mechanics of continuous flow electrophoresis in perspective

NASA Technical Reports Server (NTRS)

Saville, D. A.

1980-01-01

Buoyancy alters the flow in continuous flow electrophoresis chambers through the mechanism of hydrodynamic instability and, when the instability is supressed by careful cooling of the chamber boundaries, by restructuring the axial flow. The expanded roles of buoyancy follow upon adapting the size of the chamber and the electric field so as to fractionate certain sorts of cell populations. Scale-up problems, hydrodynamic stability and the altered flow fields are discussed to show how phenomena overlooked in the design and operations of narrow-gap devices take on an overwhelming importance in wide-gap chambers

Flow cytometry and sorting

NASA Astrophysics Data System (ADS)

Yentsch, Clarice M.

For the first time oceanographers have a tool, known as a flow cytometer and sorter, which is useful for simultaneous measurement of multiple parameters of individual cells and particles at rapid rates. We are now able to exploit the fluorescent capability of pigments and stains as signals to quantify and separate subpopulations of cells and particles in the 1.0 to 150 μm size range. Analysis rates exceed 1000 cells per second and high sensitivity is attained using laser excitation.The addition of this new technology to the ocean sciences will enable researchers to address problems which were previously intractable. The first unit, funded by the Office of Naval Research and the National Science Foundation, will be at Bigelow Laboratory for Ocean Sciences in West Boothbay Harbor, Maine, in the laboratory of Clarice M. Yentsch and David A. Phinney. In anticipation of this award, a workshop course on flow cytometry (FCM) and sorting techniques was held from October 24 through November 1, 1982, at the Bermuda Biological Station.

Principles and applications of flow cytometry and cell sorting in companion animal medicine.

PubMed

Wilkerson, Melinda J

2012-01-01

Flow cytometry measures multiple characteristic of single cells using light scatter properties and fluorescence properties of fluorescent probes with specificity to cellular constituents. The use of flow cytometry in the veterinary clinical laboratory has become more routine in veterinary diagnostic laboratories and institutions (http://www.vet.k-state.edu/depts/dmp/service/immunology/index.htm), and reference laboratories. The most common applications in small animal medicine includes quantitation of erythrocytes and leukocytes in automated hematology instruments, detection of antibodies to erythrocytes and platelets in cases of immune-mediated diseases, immunophenotyping of leukocytes and lymphocytes in immunodeficiency syndromes, or leukemias and lymphomas. DNA content analysis to identify aneuploidy or replicating cells in tumor preparations has not gained routine acceptance because of the variability of prognostic results. Other applications including cell sorting and multiplexing using microspheres are potential assays of the future once they become validated and the instrumentation footprint becomes more and more compact, less expensive, and easier to use.

Bubble propagation on a rail: a concept for sorting bubbles by size

NASA Astrophysics Data System (ADS)

Franco-Gómez, Andrés; Thompson, Alice B.; Hazel, Andrew L.; Juel, Anne

We demonstrate experimentally that the introduction of a rail, a small height constriction, within the cross-section of a rectangular channel could be used as a robust passive sorting device in two-phase fluid flows. Single air bubbles carried within silicone oil are generally transported on one side of the rail. However, for flow rates marginally larger than a critical value, a narrow band of bubble sizes can propagate (stably) over the rail, while bubbles of other sizes segregate to the side of the rail. The width of this band of bubble sizes increases with flow rate and the size of the most stable bubble can be tuned by varying the rail width. We present a complementary theoretical analysis based on a depth-averaged theory, which is in qualitative agreement with the experiments. The theoretical study reveals that the mechanism relies on a non-trivial interaction between capillary and viscous forces that is fully dynamic, rather than being a simple modification of capillary static solutions.

A cell sorting and trapping microfluidic device with an interdigital channel

NASA Astrophysics Data System (ADS)

Tu, Jing; Qiao, Yi; Xu, Minghua; Li, Junji; Liang, Fupeng; Duan, Mengqin; Ju, An; Lu, Zuhong

2016-12-01

The growing interest in cell sorting and trapping is driving the demand for high performance technologies. Using labeling techniques or external forces, cells can be identified by a series of methods. However, all of these methods require complicated systems with expensive devices. Based on inherent differences in cellular morphology, cells can be sorted by specific structures in microfluidic devices. The weir filter is a basic and efficient cell sorting and trapping structure. However, in some existing weir devices, because of cell deformability and high flow velocity in gaps, trapped cells may become stuck or even pass through the gaps. Here, we designed and fabricated a microfluidic device with interdigital channels for cell sorting and trapping. The chip consisted of a sheet of silicone elastomer polydimethylsiloxane and a sheet of glass. A square-wave-like weir was designed in the middle of the channel, comprising the interdigital channels. The square-wave pattern extended the weir length by three times with the channel width remaining constant. Compared with a straight weir, this structure exhibited a notably higher trapping capacity. Interdigital channels provided more space to slow down the rate of the pressure decrease, which prevented the cells from becoming stuck in the gaps. Sorting a mixture K562 and blood cells to trap cells demonstrated the efficiency of the chip with the interdigital channel to sort and trap large and less deformable cells. With stable and efficient cell sorting and trapping abilities, the chip with an interdigital channel may be widely applied in scientific research fields.

Rapid Isolation of Antibody from a Synthetic Human Antibody Library by Repeated Fluorescence-Activated Cell Sorting (FACS)

PubMed Central

Yim, Sung Sun; Bang, Hyun Bae; Kim, Young Hwan; Lee, Yong Jae; Jeong, Gu Min; Jeong, Ki Jun

2014-01-01

Antibodies and their derivatives are the most important agents in therapeutics and diagnostics. Even after the significant progress in the technology for antibody screening from huge libraries, it takes a long time to isolate an antibody, which prevents a prompt action against the spread of a disease. Here, we report a new strategy for isolating desired antibodies from a combinatorial library in one day by repeated fluorescence-activated cell sorting (FACS). First, we constructed a library of synthetic human antibody in which single-chain variable fragment (scFv) was expressed in the periplasm of Escherichia coli. After labeling the cells with fluorescent antigen probes, the highly fluorescent cells were sorted by using a high-speed cell sorter, and these cells were reused without regeneration in the next round of sorting. After repeating this sorting, the positive clones were completely enriched in several hours. Thus, we screened the library against three viral antigens, including the H1N1 influenza virus, Hepatitis B virus, and Foot-and-mouth disease virus. Finally, the potential antibody candidates, which show KD values between 10 and 100 nM against the target antigens, could be successfully isolated even though the library was relatively small (∼106). These results show that repeated FACS screening without regeneration of the sorted cells can be a powerful method when a rapid response to a spreading disease is required. PMID:25303314

Toxicology Study of Single-walled Carbon Nanotubes and Reduced Graphene Oxide in Human Sperm.

PubMed

Asghar, Waseem; Shafiee, Hadi; Velasco, Vanessa; Sah, Vasu R; Guo, Shirui; El Assal, Rami; Inci, Fatih; Rajagopalan, Adhithi; Jahangir, Muntasir; Anchan, Raymond M; Mutter, George L; Ozkan, Mihrimah; Ozkan, Cengiz S; Demirci, Utkan

2016-08-19

Carbon-based nanomaterials such as single-walled carbon nanotubes and reduced graphene oxide are currently being evaluated for biomedical applications including in vivo drug delivery and tumor imaging. Several reports have studied the toxicity of carbon nanomaterials, but their effects on human male reproduction have not been fully examined. Additionally, it is not clear whether the nanomaterial exposure has any effect on sperm sorting procedures used in clinical settings. Here, we show that the presence of functionalized single walled carbon nanotubes (SWCNT-COOH) and reduced graphene oxide at concentrations of 1-25 μg/mL do not affect sperm viability. However, SWCNT-COOH generate significant reactive superoxide species at a higher concentration (25 μg/mL), while reduced graphene oxide does not initiate reactive species in human sperm. Further, we demonstrate that exposure to these nanomaterials does not hinder the sperm sorting process, and microfluidic sorting systems can select the sperm that show low oxidative stress post-exposure.

Toxicology Study of Single-walled Carbon Nanotubes and Reduced Graphene Oxide in Human Sperm

NASA Astrophysics Data System (ADS)

Asghar, Waseem; Shafiee, Hadi; Velasco, Vanessa; Sah, Vasu R.; Guo, Shirui; El Assal, Rami; Inci, Fatih; Rajagopalan, Adhithi; Jahangir, Muntasir; Anchan, Raymond M.; Mutter, George L.; Ozkan, Mihrimah; Ozkan, Cengiz S.; Demirci, Utkan

2016-08-01

Carbon-based nanomaterials such as single-walled carbon nanotubes and reduced graphene oxide are currently being evaluated for biomedical applications including in vivo drug delivery and tumor imaging. Several reports have studied the toxicity of carbon nanomaterials, but their effects on human male reproduction have not been fully examined. Additionally, it is not clear whether the nanomaterial exposure has any effect on sperm sorting procedures used in clinical settings. Here, we show that the presence of functionalized single walled carbon nanotubes (SWCNT-COOH) and reduced graphene oxide at concentrations of 1-25 μg/mL do not affect sperm viability. However, SWCNT-COOH generate significant reactive superoxide species at a higher concentration (25 μg/mL), while reduced graphene oxide does not initiate reactive species in human sperm. Further, we demonstrate that exposure to these nanomaterials does not hinder the sperm sorting process, and microfluidic sorting systems can select the sperm that show low oxidative stress post-exposure.

Probing Tumor Microenvironment With In Vivo Phage Display

DTIC Science & Technology

2014-10-01

C). (C) Dot plots showing mCherry expression on the X axis and fibroblast activation protein ( FAP ) or rabbit isotype control staining in the Y...by flow cytometry-based cell sorting using an antibody against fibroblast activation protein ( FAP ). During the optimization steps, flow cytometry...expression of αvβ3 and αvβ5 integrins, neuropilin-1 (NRP-1), and fibroblast activation protein ( FAP ) in hb6011 CAFs was analyzed by flow cytometry

Raman spectroscopic instrumentation and plasmonic methods for material characterization

NASA Astrophysics Data System (ADS)

Tanaka, Kazuki

The advent of nanotechnology has led to incredible growth in how we consume, make and approach advanced materials. By exploiting nanoscale material properties, unique control of optical, thermal, mechanical, and electrical characteristics becomes possible. This thesis describes the development of a novel localized surface plasmon resonant (LSPR) color sensitive photosensor, based on functionalization of gold nanoparticles onto tianium dioxide nanowires and sensing by a metal-semiconducting nanowire-metal photodiode structure. This LSPR photosensor has been integrated into a system that incorporates Raman spectroscopy, microfluidics, optical trapping, and sorting flow cytometry into a unique material characterization system called the microfluidic optical fiber trapping Raman sorting flow cytometer (MOFTRSFC). Raman spectroscopy is utilized as a powerful molecular characterization technique used to analyze biological, mineralogical and nanomaterial samples. To combat the inherently weak Raman signal, plasmonic methods have been applied to exploit surface enhanced Raman scattering (SERS) and localized surface plasmon resonance (LSPR), increasing Raman intensity by up to 5 orders of magnitude. The resultant MOFTRSFC system is a prototype instrument that can effectively trap, analyze, and sort micron-sized dielectric particles and biological cells. Raman spectroscopy has been presented in several modalities, including the development of a portable near-infrared Raman spectrometer and other emerging technologies.

Total extract of Korean red ginseng facilitates human bone marrow hematopoietic colony formation in vitro

PubMed Central

Kim, Sang-Gyung; Bae, Sung Hwa; Kim, Seong-Mo; Lee, Ji-Hye; Kim, Min Ji; Jang, Hae-Bong

2014-01-01

Background The number of CD34+ cells in a peripheral blood stem cell collection is the key factor in predicting successful treatment of hematologic malignancies. Korean Red Ginseng (KRG) (Panax ginseng C.A. Meyer) is the most popular medicinal herb in Korea. The objective of this study was to determine the effect of KRG on hematopoietic colony formation. Methods Bone marrow (BM) samples were obtained from 8 human donors after acquiring informed consent. BM mononuclear cells (MNCs) were isolated, and CD34+ cells were sorted using magnetic beads. The sorted CD34+ cells were incubated with or without total extract of KRG (50 µg/mL, 100 µg/mL) or Ginsenoside Rg1 (100 µg/mL), and the hematopoietic colony assay was performed using methylcellulose semisolid medium. The CD34+ cell counts were measured by a single platform assay using flow cytometry. Results The numbers of human BM-MNCs and CD34+ cells obtained after purification were variable among donors (5.6×107 and 1.3-48×107 and 8.9×104 and 1.8-80×104, respectively). The cells expanded 1,944 times after incubation for 12 d. Total extract of KRG added to the hematopoietic stem cell (HSC)-specific medium increased CD34+ cell counts 3.6 times compared to 2.6 times when using HSC medium alone. Total numbers of hematopoietic colonies in KRG medium were more than those observed in conventional medium, especially that of erythroid colonies such as burst forming unit-erythroid. Conclusion Total extract of KRG facilitated CD34+ cell expansion and hematopoietic colony formation, especially of the erythroid lineage. PMID:25325037

Recovery of PET from packaging plastics mixtures by wet shaking table.

PubMed

Carvalho, M T; Agante, E; Durão, F

2007-01-01

Recycling requires the separation of materials appearing in a mass of wastes of heterogeneous composition and characteristics, into single, almost pure, component/material flows. The separation of materials (e.g., some types of plastics) with similar physical properties (e.g., specific gravity) is often accomplished by human sorting. This is the case of the separation of packaging plastics in municipal solid wastes (MSW). The low cost of virgin plastics and low value of recycled plastics necessitate the utilization of low cost techniques and processes in the recycling of packaging plastics. An experimental study was conducted to evaluate the feasibility of production of a PET product, cleaned from PVC and PS, using a wet shaking table. The wet shaking table is an environmentally friendly process, widely used to separate minerals, which has low capital and operational costs. Some operational variables of the equipment, as well as different feed characteristics, were considered. The results show that the separation of these plastics is feasible although, similarly to the mineral field, in somewhat complex flow sheets.

Flow Cytometry Based Detection and Isolation of Plasmodium falciparum Liver Stages In Vitro.

PubMed

Dumoulin, Peter C; Trop, Stefanie A; Ma, Jinxia; Zhang, Hao; Sherman, Matthew A; Levitskaya, Jelena

2015-01-01

Malaria, the disease caused by Plasmodium parasites, remains a major global health burden. The liver stage of Plasmodium falciparum infection is a leading target for immunological and pharmacological interventions. Therefore, novel approaches providing specific detection and isolation of live P. falciparum exoerythrocytic forms (EEFs) are warranted. Utilizing a recently generated parasite strain expressing green fluorescent protein (GFP) we established a method which, allows for detection and isolation of developing live P. falciparum liver stages by flow cytometry. Using this technique we compared the susceptibility of five immortalized human hepatocyte cell lines and primary hepatocyte cultures from three donors to infection by P. falciparum sporozoites. Here, we show that EEFs can be detected and isolated from in vitro infected cultures of the HC-04 cell line and primary human hepatocytes. We confirmed the presence of developing parasites in sorted live human hepatocytes and characterized their morphology by fluorescence microscopy. Finally, we validated the practical applications of our approach by re-examining the importance of host ligand CD81 for hepatocyte infection by P. falciparum sporozoites in vitro and assessment of the inhibitory activity of anti-sporozoite antibodies. This methodology provides us with the tools to study both, the basic biology of the P. falciparum liver stage and the effects of host-derived factors on the development of P. falciparum EEFs.

Flow Cytometry Based Detection and Isolation of Plasmodium falciparum Liver Stages In Vitro

PubMed Central

Dumoulin, Peter C.; Trop, Stefanie A.; Ma, Jinxia; Zhang, Hao; Sherman, Matthew A.; Levitskaya, Jelena

2015-01-01

Malaria, the disease caused by Plasmodium parasites, remains a major global health burden. The liver stage of Plasmodium falciparum infection is a leading target for immunological and pharmacological interventions. Therefore, novel approaches providing specific detection and isolation of live P. falciparum exoerythrocytic forms (EEFs) are warranted. Utilizing a recently generated parasite strain expressing green fluorescent protein (GFP) we established a method which, allows for detection and isolation of developing live P. falciparum liver stages by flow cytometry. Using this technique we compared the susceptibility of five immortalized human hepatocyte cell lines and primary hepatocyte cultures from three donors to infection by P. falciparum sporozoites. Here, we show that EEFs can be detected and isolated from in vitro infected cultures of the HC-04 cell line and primary human hepatocytes. We confirmed the presence of developing parasites in sorted live human hepatocytes and characterized their morphology by fluorescence microscopy. Finally, we validated the practical applications of our approach by re-examining the importance of host ligand CD81 for hepatocyte infection by P. falciparum sporozoites in vitro and assessment of the inhibitory activity of anti-sporozoite antibodies. This methodology provides us with the tools to study both, the basic biology of the P. falciparum liver stage and the effects of host-derived factors on the development of P. falciparum EEFs. PMID:26070149

Bears in a forest of gene trees: phylogenetic inference is complicated by incomplete lineage sorting and gene flow.

PubMed

Kutschera, Verena E; Bidon, Tobias; Hailer, Frank; Rodi, Julia L; Fain, Steven R; Janke, Axel

2014-08-01

Ursine bears are a mammalian subfamily that comprises six morphologically and ecologically distinct extant species. Previous phylogenetic analyses of concatenated nuclear genes could not resolve all relationships among bears, and appeared to conflict with the mitochondrial phylogeny. Evolutionary processes such as incomplete lineage sorting and introgression can cause gene tree discordance and complicate phylogenetic inferences, but are not accounted for in phylogenetic analyses of concatenated data. We generated a high-resolution data set of autosomal introns from several individuals per species and of Y-chromosomal markers. Incorporating intraspecific variability in coalescence-based phylogenetic and gene flow estimation approaches, we traced the genealogical history of individual alleles. Considerable heterogeneity among nuclear loci and discordance between nuclear and mitochondrial phylogenies were found. A species tree with divergence time estimates indicated that ursine bears diversified within less than 2 My. Consistent with a complex branching order within a clade of Asian bear species, we identified unidirectional gene flow from Asian black into sloth bears. Moreover, gene flow detected from brown into American black bears can explain the conflicting placement of the American black bear in mitochondrial and nuclear phylogenies. These results highlight that both incomplete lineage sorting and introgression are prominent evolutionary forces even on time scales up to several million years. Complex evolutionary patterns are not adequately captured by strictly bifurcating models, and can only be fully understood when analyzing multiple independently inherited loci in a coalescence framework. Phylogenetic incongruence among gene trees hence needs to be recognized as a biologically meaningful signal. © The Author 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Substantiation of basic scheme of grain cleaning machine for preparation of agricultural crops seeds

NASA Astrophysics Data System (ADS)

Giyevskiy, A. M.; Orobinsky, V. I.; Tarasenko, A. P.; Chernyshov, A. V.; Kurilov, D. O.

2018-03-01

The article presents data on the feasibility of the concept of a high-efficiency seed cleaner with the consistent use of the air flow in aspiration and the multi-tier placement of the sorting grids in grating mills. As a result of modeling, the directions for further improvement of air-screen seed cleaning machines have been identified: an increase in the proportion of sorting grids in the mills up to 70 ... 80% and an increase in the speed of the air flow in the channel of the pre-filter cleaning up to 8.0 m / s. Experiments have established the competence of using mathematical modeling of airflow in the pneumatic system with the use of a finite-volume method for solving hydrodynamic equations for substantiating the basic parameters of the pneumatic system.

Breast epithelium procurement from stereotactic core biopsy washings: flow cytometry-sorted cell count analysis.

PubMed

Stoler, Daniel L; Stewart, Carleton C; Stomper, Paul C

2002-02-01

Molecular studies of breast lesions have been constrained by difficulties in procuring adequate tissues for analyses. Standard procedures are restricted to larger, palpable masses or the use of paraffin-embedded materials, precluding facile procurement of fresh specimens of early lesions. We describe a study to determine the yield and characteristics of sorted cell populations retrieved in core needle biopsy specimen rinses from a spectrum of breast lesions. Cells from 114 consecutive stereotactic core biopsies of mammographic lesions released into saline washes were submitted for flow cytometric analysis. For each specimen, epithelial cells were separated from stromal and blood tissue based on the presence of cytokeratin 8 and 18 markers. Epithelial cell yields based on pathological diagnoses of the biopsy specimen, patient age, and mammographic appearance of the lesion were determined. Biopsies containing malignant lesions yielded significantly higher numbers of cells than were obtained from benign lesion biopsies. Significantly greater cell counts were observed from lesions from women age 50 or above compared with those of younger women. Mammographic density surrounding the biopsy site, the mammographic appearance of the lesion, and the number of cores taken at the time of biopsy appeared to have little effect on the yield of epithelial cells. We demonstrate the use of flow cytometric sorting of stereotactic core needle biopsy washes from lesions spanning the spectrum of breast pathology to obtain epithelial cells in sufficient numbers to meet the requirements of a variety of molecular and genetic analyses.

Analysis of cellular autofluorescence in touch samples by flow cytometry: implications for front end separation of trace mixture evidence.

PubMed

Katherine Philpott, M; Stanciu, Cristina E; Kwon, Ye Jin; Bustamante, Eduardo E; Greenspoon, Susan A; Ehrhardt, Christopher J

2017-07-01

The goal of this study was to survey optical and biochemical variation in cell populations deposited onto a surface through touch or contact and identify specific features that may be used to distinguish and then sort cell populations from separate contributors in a trace biological mixture. Although we were not able to detect meaningful biochemical variation in touch samples deposited by different contributors through preliminary antibody surveys, we did observe distinct differences in red autofluorescence emissions (650-670 nm), with as much as a tenfold difference in mean fluorescence intensities observed between certain pairs of donors. Results indicate that the level of red autofluorescence in touch samples can be influenced by a donor's contact with specific material prior to handling the substrate from which cells were collected. In particular, we observed increased red autofluorescence in cells deposited subsequent to handling laboratory gloves, plant material, and certain types of marker ink, which could be easily visualized microscopically or using flow cytometry, and persisted after hand washing. To test whether these observed optical differences could potentially be used as the basis for a cell separation workflow, a controlled two-person touch mixture was separated into two fractions via fluorescence-activated cell sorting (FACS) using gating criteria based on intensity of 650-670 nm emissions and then subjected to DNA analysis. Genetic analysis of the sorted fractions provided partial DNA profiles that were consistent with separation of individual contributors from the mixture suggesting that variation in autofluorescence signatures, even if driven by extrinsic factors, may nonetheless be a useful means of isolating contributors to some touch mixtures. Graphical Abstract Conceptual workflow diagram. Trace biological mixtures containing cells from multiple individuals are analyzed by flow cytometry. Cells are then physically separated into two populations based on intensity of red autofluorescence using Fluorescence Activated Cell Sorting. Each isolated cell fraction is subjected to DNA analysis resulting in a DNA profile for each contributor.

Using Educational Games and Simulation Software in a Computer Science Course: Learning Achievements and Student Flow Experiences

ERIC Educational Resources Information Center

Liu, Tsung-Yu

2016-01-01

This study investigates how educational games impact on students' academic performance and multimedia flow experiences in a computer science course. A curriculum consists of five basic learning units, that is, the stack, queue, sort, tree traversal, and binary search tree, was conducted for 110 university students during one semester. Two groups…

Simultaneous material flow analysis of nickel, chromium, and molybdenum used in alloy steel by means of input-output analysis.

PubMed

Nakajima, Kenichi; Ohno, Hajime; Kondo, Yasushi; Matsubae, Kazuyo; Takeda, Osamu; Miki, Takahiro; Nakamura, Shinichiro; Nagasaka, Tetsuya

2013-05-07

Steel is not elemental iron but rather a group of iron-based alloys containing many elements, especially chromium, nickel, and molybdenum. Steel recycling is expected to promote efficient resource use. However, open-loop recycling of steel could result in quality loss of nickel and molybdenum and/or material loss of chromium. Knowledge about alloying element substance flow is needed to avoid such losses. Material flow analyses (MFAs) indicate the importance of steel recycling to recovery of alloying elements. Flows of nickel, chromium, and molybdenum are interconnected, but MFAs have paid little attention to the interconnected flow of materials/substances in supply chains. This study combined a waste input-output material flow model and physical unit input-output analysis to perform a simultaneous MFA for nickel, chromium, and molybdenum in the Japanese economy in 2000. Results indicated the importance of recovery of these elements in recycling policies for end-of-life (EoL) vehicles and constructions. Improvement in EoL sorting technologies and implementation of designs for recycling/disassembly at the manufacturing phase are needed. Possible solutions include development of sorting processes for steel scrap and introduction of easier methods for identifying the composition of secondary resources. Recovery of steel scrap with a high alloy content will reduce primary inputs of alloying elements and contribute to more efficient resource use.

Digital Analysis and Sorting of Fluorescence Lifetime by Flow Cytometry

PubMed Central

Houston, Jessica P.; Naivar, Mark A.; Freyer, James P.

2010-01-01

Frequency-domain flow cytometry techniques are combined with modifications to the digital signal processing capabilities of the Open Reconfigurable Cytometric Acquisition System (ORCAS) to analyze fluorescence decay lifetimes and control sorting. Real-time fluorescence lifetime analysis is accomplished by rapidly digitizing correlated, radiofrequency modulated detector signals, implementing Fourier analysis programming with ORCAS’ digital signal processor (DSP) and converting the processed data into standard cytometric list mode data. To systematically test the capabilities of the ORCAS 50 MS/sec analog-to-digital converter (ADC) and our DSP programming, an error analysis was performed using simulated light scatter and fluorescence waveforms (0.5–25 ns simulated lifetime), pulse widths ranging from 2 to 15 µs, and modulation frequencies from 2.5 to 16.667 MHz. The standard deviations of digitally acquired lifetime values ranged from 0.112 to >2 ns, corresponding to errors in actual phase shifts from 0.0142° to 1.6°. The lowest coefficients of variation (<1%) were found for 10-MHz modulated waveforms having pulse widths of 6 µs and simulated lifetimes of 4 ns. Direct comparison of the digital analysis system to a previous analog phase-sensitive flow cytometer demonstrated similar precision and accuracy on measurements of a range of fluorescent microspheres, unstained cells and cells stained with three common fluorophores. Sorting based on fluorescence lifetime was accomplished by adding analog outputs to ORCAS and interfacing with a commercial cell sorter with a radiofrequency modulated solid-state laser. Two populations of fluorescent microspheres with overlapping fluorescence intensities but different lifetimes (2 and 7 ns) were separated to ~98% purity. Overall, the digital signal acquisition and processing methods we introduce present a simple yet robust approach to phase-sensitive measurements in flow cytometry. The ability to simply and inexpensively implement this system on a commercial flow sorter will both allow better dissemination of this technology and better exploit the traditionally underutilized parameter of fluorescence lifetime. PMID:20662090

Experimental investigation of gravity effects on sediment sorting on Mars

NASA Astrophysics Data System (ADS)

Kuhn, Nikolaus J.; Kuhn, Brigitte; Gartmann, Andres

2016-04-01

Introduction: Sorting of sedimentary rocks is a proxy for the environmental conditions at the time of deposition, in particular the runoff that moved and deposited the material forming the rocks. Settling of sediment in water is strongly influenced by the gravity of a planetary body. As a consequence, sorting of a sedimentary rock varies with gravity for a given depth and velocity of surface runoff. Theoretical considerations for spheres indicate that sorting is more uniform on Mars than on Earth for runoff of identical depth. In reality, such considerations have to be applied with great caution because the shape of a particle strongly influences drag. Drag itself can only be calculated directly for an irregularly shaped particle with great computational effort, if at all. Therefore, even for terrestrial applications, sediment settling velocities are often determined directly, e.g. by measurements using settling tubes. Experiments: In this study the results of settling tube tests conducted under reduced gravity during three Mars Sedimentation Experiment (MarsSedEx I, II and III) flights, conducted between 2012 and 2015, are presented. Ten types of sediment, ranging in size, shape and density were tested in custom-designed settling tubes during parabolas of Martian gravity lasting 20 to 25 seconds. Results: The experiments conducted during the MarsSedEx reduced gravity experiments showed that the violation of fluid dynamics caused by using empirical models and parameter values developed for sediment transport on Earth lead to significant miscalculations for Mars, specifically an underetsimation of settling velcoity because of an overestimation of turbulant drag. The error is caused by the flawed representation of particle drag on Mars. Drag coefficients are not a property of a sediment particle, but a property of the flow around the particle, and thus strongly affected by gravity. Conlcusions: The observed errors in settling velocity when using terrestrial models and parameter values on Mars have implications for sediment movement and sorting, in particular for sandstones and conglomerates, and thus analogies drawn between Earth and Mars. Most significantly, sorting on Mars is less pronounced for given flow conditions than on Earth. References: [1] Kuhn N. J. (2014) Experiments in Reduced Gravity - Sediment Settling on Mars, Elsevier.

THE FLOW OF FUNDS IN SMALL BUSINESSES AND IN LARGE BUSINESSES. PART I.

DTIC Science & Technology

range of variations in the flow of funds in different businesses . The data have at least two possible types of use. The first is of direct concern to...funds in a given business . The study of the cement company is an example of a use of this sort. A second type of use is by research workers who may wish...causes which may lead to differences in the fund flow behavior of different companies, or (c) the effect of general economic conditions on the flow of funds in businesses .

Friction in debris flows: inferences from large-scale flume experiments

USGS Publications Warehouse

Iverson, Richard M.; LaHusen, Richard G.; ,

1993-01-01

A recently constructed flume, 95 m long and 2 m wide, permits systematic experimentation with unsteady, nonuniform flows of poorly sorted geological debris. Preliminary experiments with water-saturated mixtures of sand and gravel show that they flow in a manner consistent with Coulomb frictional behavior. The Coulomb flow model of Savage and Hutter (1989, 1991), modified to include quasi-static pore-pressure effects, predicts flow-front velocities and flow depths reasonably well. Moreover, simple scaling analyses show that grain friction, rather than liquid viscosity or grain collisions, probably dominates shear resistance and momentum transport in the experimental flows. The same scaling indicates that grain friction is also important in many natural debris flows.

The overexpressed human 46-kDa mannose 6-phosphate receptor mediates endocytosis and sorting of. beta. -glucuronidase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Watanabe, H.; Grubb, J.H.; Sly, W.S.

1990-10-01

The authors studied the function of the human small (46-kDa) mannose 6-phosphate receptor (SMPR) in transfected mouse L cells that do not express the larger insulin-like growth factor II/mannose 6-phosphate receptor. Cells overexpressing human SMPR were studied for enzyme binding to cell surface receptors, for binding to intracellular receptors in permeabilized cells, and for receptor-mediated endocytosis of recombinant human {beta}-glucuronidase. Specific binding to human SMPR in permeabilized cells showed a pH optimum between pH 6.0 and pH 6.5. Binding was significant in the present of EDTA but was enhanced by added divalent cations. Up to 2.3{percent} of the total functionalmore » receptor could be detected on the cell surface by enzyme binding. They present experiments showing that at very high levels of overexpression, and at pH 6.5, human SMPR mediated the endocytosis of {beta}-glucuronidase. At pH 7.5, the rate of endocytosis was only 14{percent} the rate seen at pH 6.5. Cells overexpressing human SMPR also showed reduced secretion of newly synthesized {beta}-glucuronidase when compared to cells transfected with vector only, suggesting that overexpressed human SMPR can participate in sorting of newly synthesized {beta}-glucuronidase and partially correct the sorting defect in mouse L cells that do not express the insulin-like growth factor II/mannose 6-phosphate receptor.« less

Continuous separation of breast cancer cells from blood samples using multi-orifice flow fractionation (MOFF) and dielectrophoresis (DEP).

PubMed

Moon, Hui-Sung; Kwon, Kiho; Kim, Seung-Il; Han, Hyunju; Sohn, Joohyuk; Lee, Soohyeon; Jung, Hyo-Il

2011-03-21

Circulating tumor cells (CTCs) are highly correlated with the invasive behavior of cancer, so their isolations and quantifications are important for biomedical applications such as cancer prognosis and measuring the responses to drug treatments. In this paper, we present the development of a microfluidic device for the separation of CTCs from blood cells based on the physical properties of cells. For use as a CTC model, we successfully separated human breast cancer cells (MCF-7) from a spiked blood cell sample by combining multi-orifice flow fractionation (MOFF) and dielectrophoretic (DEP) cell separation technique. Hydrodynamic separation takes advantage of the massive and high-throughput filtration of blood cells as it can accommodate a very high flow rate. DEP separation plays a role in precise post-processing to enhance the efficiency of the separation. The serial combination of these two different sorting techniques enabled high-speed continuous flow-through separation without labeling. We observed up to a 162-fold increase in MCF-7 cells at a 126 µL min(-1) flow rate. Red and white blood cells were efficiently removed with separation efficiencies of 99.24% and 94.23% respectively. Therefore, we suggest that our system could be used for separation and detection of CTCs from blood cells for biomedical applications. This journal is © The Royal Society of Chemistry 2011

Intracellular transport and sorting of mutant human proinsulins that fail to form hexamers

PubMed Central

1991-01-01

Human proinsulin and insulin oligomerize to form dimers and hexamers. It has been suggested that the ability of prohormones to self associate and form aggregates may be responsible for the sorting process at the trans-Golgi. To examine whether insulin oligomerization is required for proper sorting into regulated storage granules, we have constructed point mutations in human insulin B chain that have been previously shown to prevent formation of insulin hexamers (Brange, J., U. Ribel, J. F. Hansen, G. Dodson, M. T. Hansen, S. Havelund, S. G. Melberg, F. Norris, K. Norris, L. Snel, A. R. Sorensen, and H. O. Voight. 1988. Nature [Lond.]. 333:679-682). One mutant (B10His----Asp) allows formation of dimers but not hexamers and the other (B9Ser----Asp) prevents formation of both dimers and hexamers. The mutants were transfected into the mouse pituitary AtT-20 cells, and their ability to be sorted into regulated secretory granules was compared to wild-type insulin. We found that while B10His----Asp is sorted somewhat less efficiently than wild-type insulin as reported previously (Carroll, R. J., R. E. Hammer, S. J. Chan, H. H. Swift, A. H. Rubenstein, and D. F. Steiner. 1988. Proc. Natl. Acad. Sci. USA. 85:8943-8947; Gross, D. J., P. A. Halban, C. R. Kahn, G. C. Weir, and L. Villa-Kumaroff. 1989. Proc. Natl. Acad. Sci. USA. 86:4107-4111). B9Ser----Asp is targeted to granules as efficiently as wild-type insulin. These results indicate that self association of proinsulin into hexamers is not required for its targeting to the regulated secretory pathway. PMID:2040652

Intracellular transport and sorting of mutant human proinsulins that fail to form hexamers.

PubMed

Quinn, D; Orci, L; Ravazzola, M; Moore, H P

1991-06-01

Human proinsulin and insulin oligomerize to form dimers and hexamers. It has been suggested that the ability of prohormones to self associate and form aggregates may be responsible for the sorting process at the trans-Golgi. To examine whether insulin oligomerization is required for proper sorting into regulated storage granules, we have constructed point mutations in human insulin B chain that have been previously shown to prevent formation of insulin hexamers (Brange, J., U. Ribel, J. F. Hansen, G. Dodson, M. T. Hansen, S. Havelund, S. G. Melberg, F. Norris, K. Norris, L. Snel, A. R. Sorensen, and H. O. Voight. 1988. Nature [Lond.]. 333:679-682). One mutant (B10His----Asp) allows formation of dimers but not hexamers and the other (B9Ser----Asp) prevents formation of both dimers and hexamers. The mutants were transfected into the mouse pituitary AtT-20 cells, and their ability to be sorted into regulated secretory granules was compared to wild-type insulin. We found that while B10His----Asp is sorted somewhat less efficiently than wild-type insulin as reported previously (Carroll, R. J., R. E. Hammer, S. J. Chan, H. H. Swift, A. H. Rubenstein, and D. F. Steiner. 1988. Proc. Natl. Acad. Sci. USA. 85:8943-8947; Gross, D. J., P. A. Halban, C. R. Kahn, G. C. Weir, and L. Villa-Kumaroff. 1989. Proc. Natl. Acad. Sci. USA. 86:4107-4111). B9Ser----Asp is targeted to granules as efficiently as wild-type insulin. These results indicate that self association of proinsulin into hexamers is not required for its targeting to the regulated secretory pathway.

That is Cool: the Nature Of Aesthetics in Fluid Physics

NASA Astrophysics Data System (ADS)

Hertzberg, Jean

2013-11-01

Aesthetics has historically been defined as the study of beauty and thus as a metric of art. More recently, psychologists are using the term to describe a spectrum of responses from ``I hate it'' to ``I love it.'' In the context of fluid physics, what is beautiful? What elicits a ``Wow! Awesome! Cool!'' response versus a snore? Can we use aesthetics to deepen or change students' or the public's perceptions of physics and/or the world around them? For example, students seem to appreciate the aesthetics of destruction: environmental fluid dynamics such as storms, tornadoes, floods and wildfires are often responsible for massive destruction, yet humans draw pleasure from watching such physics and the attendant destruction from a safe distance. Can this voyeurism be turned to our advantage in communicating science? Observations of student and Facebook Flow Visualization group choices for fluid physics that draw a positive aesthetic response are sorted into empirical categories; the aesthetics of beauty, power, destruction, and oddness. Each aesthetic will be illustrated with examples drawn from flow visualizations from both the Flow Visualization course (MCEN 4151) taught at the University of Colorado, Boulder, and sources on the web. This work is supported by NSF: EEC 1240294.

EphA2 is a biomarker of hMSCs derived from human placenta and umbilical cord.

PubMed

Shen, Shih-Pei; Liu, Wei-Ting; Lin, Yun; Li, Yuan-Tsung; Chang, Chih-Hao; Chang, Fung-Wei; Wang, Le-Ming; Teng, Sen-Wen; Hsuan, Yogi

2015-12-01

The heterogeneous nature of mesenchymal stem cells (MSCs) and the absence of known MSC-specific biomarkers make it challenging to define MSC phenotypes and characteristics. In this study, we compared the phenotypic and functional features of human placenta-derived MSCs with those of human dermal fibroblasts in vitro in order to identify a biomarker that can be used to increase the purity of MSCs in a primary culture of placenta-derived cells. Liquid chromatography-tandem mass spectrometry analysis was used to analyze and compare the proteome of human placenta-derived MSCs with that of fibroblasts. Quantitative real-time polymerase chain reaction, immunofluorescence, and flow cytometry were used to determine expression levels of EphA2 in placenta-derived MSCs. EphA2-positive cells were enriched by magnetic-activated cell sorting or with a cell sorter. An shRNA-mediated EphA2 knockdown was used to assess the role of EphA2 in MSC response to Tumor necrosis factor (TNF)-α stimulation. Analysis of proteomics data from MSCs and fibroblasts resulted in the identification of the EphA2 surface protein biomarker, which could reliably distinguish MSCs from fibroblasts. EphA2 was significantly upregulated in placenta-derived MSCs when compared to fibroblasts. EphA2 played an important role in MSC migration in response to inflammatory stimuli, such as TNF-α. EphA2-enriched MSCs were also more responsive to inflammatory stimuli in vitro when compared to unsorted MSCs, indicating a role for EphA2 in the immunomodulatory functionality of MSCs. EphA2 can be used to distinguish and isolate MSCs from a primary culture of placenta-derived cells. EphA2-sorted MSCs exhibited superior responsiveness to TNF-α signaling in an inflammatory environment compared with unsorted MSCs or MSC-like cells. Copyright © 2015. Published by Elsevier B.V.

Biological cell controllable patch-clamp microchip

NASA Astrophysics Data System (ADS)

Penmetsa, Siva; Nagrajan, Krithika; Gong, Zhongcheng; Mills, David; Que, Long

2010-12-01

A patch-clamp (PC) microchip with cell sorting and positioning functions is reported, which can avoid drawbacks of random cell selection or positioning for a PC microchip. The cell sorting and positioning are enabled by air bubble (AB) actuators. AB actuators are pneumatic actuators, in which air pressure is generated by microheaters within sealed microchambers. The sorting, positioning, and capturing of 3T3 cells by this type of microchip have been demonstrated. Using human breast cancer cells MDA-MB-231 as the model, experiments have been demonstrated by this microchip as a label-free technical platform for real-time monitoring of the cell viability.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Beer, Neil Reginald; Lee, Abraham P; Hatch, Andrew C

An apparatus for passive sorting of microdroplets including a main flow channel, a flow stream of microdroplets in the main flow channel wherein the microdroplets have substantially the same diameter and wherein the flow stream of microdroplets includes first microdroplets having a first degree of stiffness and second microdroplets having a second degree of stiffness wherein the second degree of stiffness is different than the first degree of stiffness. A second flow channel is connected to the main flow channel for the second microdroplets having a second degree of stiffness. A separator separates the second microdroplets having a second degreemore » of stiffness from the first microdroplets and directs the second microdroplets having a second degree of stiffness into the second flow channel.« less

Method for rapid isolation of sensitive mutants

DOEpatents

Freyer, James P.

1997-01-01

Sensitive mammalian cell mutants are rapidly isolated using flow cytometry. A first population of clonal spheroids is established to contain both normal and mutant cells. The population may be naturally occurring or may arise from mutagenized cells. The first population is then flow sorted by size to obtain a second population of clonal spheroids of a first uniform size. The second population is then exposed to a DNA-damaging agent that is being investigated. The exposed second population is placed in a growth medium to form a third population of clonal spheroids comprising spheroids of increased size from the mammalian cells that are resistant to the DNA-damaging agent and spheroids of substantially the first uniform size formed from the mammalian cells that are sensitive to the DNA-damaging agent. The third population is not flow sorted to differentiate the spheroids formed from resistant mammalian cells from spheroids formed from sensitive mammalian cells. The spheroids formed from sensitive mammalian cells are now treated to recover viable sensitive cells from which a sensitive cell line can be cloned.

Three-dimensional features on oscillating microbubbles streaming flows

NASA Astrophysics Data System (ADS)

Rossi, Massimiliano; Marin, Alvaro G.; Wang, Cheng; Hilgenfeldt, Sascha; Kähler, Christian J.

2013-11-01

Ultrasound-driven oscillating micro-bubbles have been used as active actuators in microfluidic devices to perform manifold tasks such as mixing, sorting and manipulation of microparticles. A common configuration consists in side-bubbles, created by trapping air pockets in blind channels perpendicular to the main channel direction. This configuration results in bubbles with a semi-cylindrical shape that creates a streaming flow generally considered quasi two-dimensional. However, recent experiments performed with three-dimensional velocimetry methods have shown how microparticles can present significant three-dimensional trajectories, especially in regions close to the bubble interface. Several reasons will be discussed such as boundary effects of the bottom/top wall, deformation of the bubble interface leading to more complex vibrational modes, or bubble-particle interactions. In the present investigation, precise measurements of particle trajectories close to the bubble interface will be performed by means of 3D Astigmatic Particle Tracking Velocimetry. The results will allow us to characterize quantitatively the three-dimensional features of the streaming flow and to estimate its implications in practical applications as particle trapping, sorting or mixing.

Method for rapid isolation of sensitive mutants

DOEpatents

Freyer, J.P.

1997-07-29

Sensitive mammalian cell mutants are rapidly isolated using flow cytometry. A first population of clonal spheroids is established to contain both normal and mutant cells. The population may be naturally occurring or may arise from mutagenized cells. The first population is then flow sorted by size to obtain a second population of clonal spheroids of a first uniform size. The second population is then exposed to a DNA-damaging agent that is being investigated. The exposed second population is placed in a growth medium to form a third population of clonal spheroids comprising spheroids of increased size from the mammalian cells that are resistant to the DNA-damaging agent and spheroids of substantially the first uniform size formed from the mammalian cells that are sensitive to the DNA-damaging agent. The third population is not flow sorted to differentiate the spheroids formed from resistant mammalian cells from spheroids formed from sensitive mammalian cells. The spheroids formed from sensitive mammalian cells are now treated to recover viable sensitive cells from which a sensitive cell line can be cloned. 15 figs.

3D pulsed laser-triggered high-speed microfluidic fluorescence-activated cell sorter

PubMed Central

Chen, Yue; Wu, Ting-Hsiang; Kung, Yu-Chun; Teitell, Michael A.; Chiou, Pei-Yu

2014-01-01

We report a 3D microfluidic pulsed laser-triggered fluorescence-activated cell sorter capable of sorting at a throughput of 23,000 cells sec−1 with 90% purity in high-purity mode and at a throughput of 45,000 cells sec−1 with 45% purity in enrichment mode in one stage and in a single channel. This performance is realized by exciting laser-induced cavitation bubbles in a 3D PDMS microfluidic channel to generate high-speed liquid jets that deflect detected fluorescent cells and particles focused by 3D sheath flows. The ultrafast switching mechanism (20 μsec complete on-off cycle), small liquid jet perturbation volume, and three-dimensional sheath flow focusing for accurate timing control of fast (1.5 m sec−1) passing cells and particles are three critical factors enabling high-purity sorting at high-throughput in this sorter. PMID:23844418

Semi-automated sorting using holographic optical tweezers remotely controlled by eye/hand tracking camera

NASA Astrophysics Data System (ADS)

Tomori, Zoltan; Keša, Peter; Nikorovič, Matej; Kaůka, Jan; Zemánek, Pavel

2016-12-01

We proposed the improved control software for the holographic optical tweezers (HOT) proper for simple semi-automated sorting. The controller receives data from both the human interface sensors and the HOT microscope camera and processes them. As a result, the new positions of active laser traps are calculated, packed into the network format and sent to the remote HOT. Using the photo-polymerization technique, we created a sorting container consisting of two parallel horizontal walls where one wall contains "gates" representing a place where the trapped particle enters into the container. The positions of particles and gates are obtained by image analysis technique which can be exploited to achieve the higher level of automation. Sorting is documented on computer game simulation and the real experiment.

Isolation and characterisation of human gingival margin-derived STRO-1/MACS+ and MACS− cell populations

PubMed Central

El-Sayed, Karim M Fawzy; Paris, Sebastian; Graetz, Christian; Kassem, Neemat; Mekhemar, Mohamed; Ungefroren, Hendrick; Fändrich, Fred; Dörfer, Christof

2015-01-01

Recently, gingival margin-derived stem/progenitor cells isolated via STRO-1/magnetic activated cell sorting (MACS) showed remarkable periodontal regenerative potential in vivo. As a second-stage investigation, the present study's aim was to perform in vitro characterisation and comparison of the stem/progenitor cell characteristics of sorted STRO-1-positive (MACS+) and STRO-1-negative (MACS−) cell populations from the human free gingival margin. Cells were isolated from the free gingiva using a minimally invasive technique and were magnetically sorted using anti-STRO-1 antibodies. Subsequently, the MACS+ and MACS− cell fractions were characterized by flow cytometry for expression of CD14, CD34, CD45, CD73, CD90, CD105, CD146/MUC18 and STRO-1. Colony-forming unit (CFU) and multilineage differentiation potential were assayed for both cell fractions. Mineralisation marker expression was examined using real-time polymerase chain reaction (PCR). MACS+ and MACS− cell fractions showed plastic adherence. MACS+ cells, in contrast to MACS− cells, showed all of the predefined mesenchymal stem/progenitor cell characteristics and a significantly higher number of CFUs (P<0.01). More than 95% of MACS+ cells expressed CD105, CD90 and CD73; lacked the haematopoietic markers CD45, CD34 and CD14, and expressed STRO-1 and CD146/MUC18. MACS− cells showed a different surface marker expression profile, with almost no expression of CD14 or STRO-1, and more than 95% of these cells expressed CD73, CD90 and CD146/MUC18, as well as the haematopoietic markers CD34 and CD45 and CD105. MACS+ cells could be differentiated along osteoblastic, adipocytic and chondroblastic lineages. In contrast, MACS− cells demonstrated slight osteogenic potential. Unstimulated MACS+ cells showed significantly higher expression of collagen I (P<0.05) and collagen III (P<0.01), whereas MACS− cells demonstrated higher expression of osteonectin (P<0.05; Mann–Whitney). The present study is the first to compare gingival MACS+ and MACS− cell populations demonstrating that MACS+ cells, in contrast to MACS− cells, harbour stem/progenitor cell characteristics. This study also validates the effectiveness of the STRO-1/MACS+ technique for the isolation of gingival stem/progenitor cells. Human free gingival margin-derived STRO-1/MACS+ cells are a unique renewable source of multipotent stem/progenitor cells. PMID:25257881

Size-based cell sorting with a resistive pulse sensor and an electromagnetic pump in a microfluidic chip.

PubMed

Song, Yongxin; Li, Mengqi; Pan, Xinxiang; Wang, Qi; Li, Dongqing

2015-02-01

An electrokinetic microfluidic chip is developed to detect and sort target cells by size from human blood samples. Target-cell detection is achieved by a differential resistive pulse sensor (RPS) based on the size difference between the target cell and other cells. Once a target cell is detected, the detected RPS signal will automatically actuate an electromagnetic pump built in a microchannel to push the target cell into a collecting channel. This method was applied to automatically detect and sort A549 cells and T-lymphocytes from a peripheral fingertip blood sample. The viability of A549 cells sorted in the collecting well was verified by Hoechst33342 and propidium iodide staining. The results show that as many as 100 target cells per minute can be sorted out from the sample solution and thus is particularly suitable for sorting very rare target cells, such as circulating tumor cells. The actuation of the electromagnetic valve has no influence on RPS cell detection and the consequent cell-sorting process. The viability of the collected A549 cell is not impacted by the applied electric field when the cell passes the RPS detection area. The device described in this article is simple, automatic, and label-free and has wide applications in size-based rare target cell sorting for medical diagnostics. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

CD146 Expression Influences Periapical Cyst Mesenchymal Stem Cell Properties.

PubMed

Paduano, Francesco; Marrelli, Massimo; Palmieri, Francesca; Tatullo, Marco

2016-10-01

Recent studies have identified a new human dental derived progenitor cell population with multi-lineage differentiation potential referred to as human periapical cyst mesenchymal stem cells (hPCy-MSCs). In the present study, we compared two subpopulations of hPCy-MSCs characterised by the low or high expression of CD146 to establish whether this expression can regulate their stem cell properties. Using flow cytometry, we evaluated the stem cell marker profile of hPCy-MSCs during passaging. Furthermore, CD146 Low and CD146 High cells were sorted by magnetic beads and subsequently both cell populations were evaluated for differences in their proliferation, self-renewal, stem cell surface markers, stemness genes expression and osteogenic differentiation potential.We found that hPCy-MSCs possessed a stable expression of several mesenchymal stem cell surface markers, whereas CD146 expression declined during passaging.In addition, sorted CD146 Low cells proliferated significantly faster, displayed higher colony-forming unit-fibroblast capacity and showed higher expression of Klf4 when compared to the CD146 High subset. Significantly, the osteogenic potential of hPCy-MSCs was greater in the CD146 Low than in CD146 High population. These results demonstrate that CD146 is spontaneously downregulated with passaging at both mRNA and protein levels and that the high expression of CD146 reduces the proliferative, self-renewal and osteogenic differentiation potential of hPCy-MSCs. In conclusion, our study demonstrates that changes in the expression of CD146 can influence the stem cell properties of hPCy-MSCs.

Microfluidic devices for label-free separation of cells through transient interaction with asymmetric receptor patterns

NASA Astrophysics Data System (ADS)

Bose, S.; Singh, R.; Hollatz, M. H.; Lee, C.-H.; Karp, J.; Karnik, R.

2012-02-01

Cell sorting serves an important role in clinical diagnosis and biological research. Most of the existing microscale sorting techniques are either non-specific to antigen type or rely on capturing cells making sample recovery difficult. We demonstrate a simple; yet effective technique for isolating cells in an antigen specific manner by using transient interactions of the cell surface antigens with asymmetric receptor patterned surface. Using microfluidic devices incorporating P-selectin patterns we demonstrate separation of HL60 cells from K562 cells. We achieved a sorting purity above 90% and efficiency greater than 85% with this system. We also present a mathematical model incorporating flow mediated and adhesion mediated transport of cells in the microchannel that can be used to predict the performance of these devices. Lastly, we demonstrate the clinical significance of the method by demonstrating single step separation of neutrophils from whole blood. When whole blood is introduced in the device, the granulocyte population gets separated exclusively yielding neutrophils of high purity (<10% RBC contamination). To our knowledge, this is the first ever demonstration of continuous label free sorting of neutrophils from whole blood. We believe this technology will be useful in developing point-of-care diagnostic devices and also for a host of cell sorting applications.

Characterization of aerosols produced by cell sorters and evaluation of containment

PubMed Central

Holmes, Kevin L.

2011-01-01

In spite of the recognition by the flow cytometry community of potential aerosol hazards associated with cell sorting, there has been no previous study that has thoroughly characterized the aerosols that can be produced by cell sorters. In this study an Aerodynamic Particle Sizer was used to determine the concentration and aerodynamic diameter of aerosols produced by a FACS Aria II cell sorter under various conditions. Aerosol containment and evacuation was also evaluated using this novel methodology. The results showed that high concentrations of aerosols in the range of 1–3 μm can be produced in fail mode and that with decreased sheath pressure, aerosol concentration decreased and aerodynamic diameter increased. Although the engineering controls of the FACS Aria II for containment were effective, sort chamber evacuation of aerosols following a simulated nozzle obstruction was ineffective. However, simple modifications to the FACS Aria II are described that greatly improved sort chamber aerosol evacuation. The results of this study will facilitate the risk assessment of cell sorting potentially biohazardous samples by providing much needed data regarding aerosol production and containment. PMID:22052694

Reciprocal chromosome painting shows that the great difference in diploid number between human and African green monkey is mostly due to non-Robertsonian fissions.

PubMed

Finelli, P; Stanyon, R; Plesker, R; Ferguson-Smith, M A; O'Brien, P C; Wienberg, J

1999-07-01

We used reciprocal chromosome painting with both African green monkey (C. aethiops) and human chromosome specific DNA probes to delineate homologous regions in the two species. Probes were derived by fluorescence-activated chromosome flow sorting and then were reciprocally hybridized to metaphase spreads of each species. Segments in the size range of a single chromosome band were identified, demonstrating the sensitivity of the approach when comparing species that diverged more than 20 million years ago. Outgroup analysis shows that the great difference in diploid numbers between the African green monkey (2n = 60) and humans (2n = 46) is mainly owing to fissions, and the direction of change is towards increasing diploid numbers. However, most break points apparently lie outside of the centromere regions, suggesting that the changes were not solely Robertsonian as has been previously assumed. No reciprocal translocations have occurred in the phylogenetic lines leading to humans or African green monkeys. The primate paints established here are a valuable tool to establish interspecies homology, to define rearrangements, and to determine the mechanisms of chromosomal evolution in primate species.

Purification of Cardiomyocytes from Differentiating Pluripotent Stem Cells using Molecular Beacons Targeting Cardiomyocyte-Specific mRNA

PubMed Central

Kim, Sangsung; Park, Hun-Jun; Byun, Jaemin; Cho, Kyu-Won; Saafir, Talib; Song, Ming-Ke; Yu, Shan Ping; Wagner, Mary; Bao, Gang; Yoon, Young-Sup

2013-01-01

Background While methods for generating cardiomyocytes (CMs) from pluripotent stem cells (PSCs) have been reported, current methods produce heterogeneous mixtures of CMs and non-CM cells. Here, we report an entirely novel system in which PSC-derived CMs are purified by CM-specific molecular beacons (MBs). MBs are nano-scale probes that emit a fluorescence signal when hybridized to target mRNAs. Method and Results Five MBs targeting mRNAs of either cardiac troponin T or myosin heavy chain 6/7 were generated. Among five MBs, a MB targeting myosin heavy chain 6/7 mRNA (MHC1-MB) identified up to 99% of HL-1 CMs, a mouse CM cell line, but < 3% of four non-CM cell types in flow cytometry analysis, indicating that MHC1-MB is specific for identifying CMs. We delivered MHC1-MB into cardiomyogenically differentiated PSCs through nucleofection. The detection rate of CMs was similar to the percentages of cardiac troponin T (TNNT2) or cardiac troponin I (TNNI3)-positive CMs, supporting the specificity of MBs. Finally, MHC1-MB-positive cells were FACS-sorted from mouse and human PSC differentiating cultures and ~97% cells expressed TNNT2- or TNNI3 determined by flow cytometry. These MB-based sorted cells maintained their CM characteristics verified by spontaneous beating, electrophysiologic studies, and expression of cardiac proteins. When transplanted in a myocardial infarction model, MB-based purified CMs improved cardiac function and demonstrated significant engraftment for 4 weeks without forming tumors. Conclusions We developed a novel CM selection system that allows production of highly purified CMs. These purified CMs and this system can be valuable for cell therapy and drug discovery. PMID:23995537

Purification of cardiomyocytes from differentiating pluripotent stem cells using molecular beacons that target cardiomyocyte-specific mRNA.

PubMed

Ban, Kiwon; Wile, Brian; Kim, Sangsung; Park, Hun-Jun; Byun, Jaemin; Cho, Kyu-Won; Saafir, Talib; Song, Ming-Ke; Yu, Shan Ping; Wagner, Mary; Bao, Gang; Yoon, Young-Sup

2013-10-22

Although methods for generating cardiomyocytes from pluripotent stem cells have been reported, current methods produce heterogeneous mixtures of cardiomyocytes and noncardiomyocyte cells. Here, we report an entirely novel system in which pluripotent stem cell-derived cardiomyocytes are purified by cardiomyocyte-specific molecular beacons (MBs). MBs are nanoscale probes that emit a fluorescence signal when hybridized to target mRNAs. Five MBs targeting mRNAs of either cardiac troponin T or myosin heavy chain 6/7 were generated. Among 5 MBs, an MB that targeted myosin heavy chain 6/7 mRNA (MHC1-MB) identified up to 99% of HL-1 cardiomyocytes, a mouse cardiomyocyte cell line, but <3% of 4 noncardiomyocyte cell types in flow cytometry analysis, which indicates that MHC1-MB is specific for identifying cardiomyocytes. We delivered MHC1-MB into cardiomyogenically differentiated pluripotent stem cells through nucleofection. The detection rate of cardiomyocytes was similar to the percentages of cardiac troponin T- or cardiac troponin I-positive cardiomyocytes, which supports the specificity of MBs. Finally, MHC1-MB-positive cells were sorted by fluorescence-activated cell sorter from mouse and human pluripotent stem cell differentiating cultures, and ≈97% cells expressed cardiac troponin T or cardiac troponin I as determined by flow cytometry. These MB-based sorted cells maintained their cardiomyocyte characteristics, which was verified by spontaneous beating, electrophysiological studies, and expression of cardiac proteins. When transplanted in a myocardial infarction model, MB-based purified cardiomyocytes improved cardiac function and demonstrated significant engraftment for 4 weeks without forming tumors. We developed a novel cardiomyocyte selection system that allows production of highly purified cardiomyocytes. These purified cardiomyocytes and this system can be valuable for cell therapy and drug discovery.

Selection of transduced CD34+ progenitors and enzymatic correction of cells from Gaucher patients, with bicistronic vectors.

PubMed Central

Migita, M; Medin, J A; Pawliuk, R; Jacobson, S; Nagle, J W; Anderson, S; Amiri, M; Humphries, R K; Karlsson, S

1995-01-01

The gene transfer efficiency of human hematopoietic stem cells is still inadequate for efficient gene therapy of most disorders. To overcome this problem, a selectable retroviral vector system for gene therapy has been developed for gene therapy of Gaucher disease. We constructed a bicistronic retroviral vector containing the human glucocerebrosidase (GC) cDNA and the human small cell surface antigen CD24 (243 bp). Expression of both cDNAs was controlled by the long terminal repeat enhancer/promoter of the Molony murine leukemia virus. The CD24 selectable marker was placed downstream of the GC cDNA and its translation was enhanced by inclusion of the long 5' untranslated region of encephalomyocarditis virus internal ribosomal entry site. Virus-producing GP+envAM12 cells were created by multiple supernatant transductions to create vector producer cells. The vector LGEC has a high titer and can drive expression of GC and the cell surface antigen CD24 simultaneously in transduced NIH 3T3 cells and Gaucher skin fibroblasts. These transduced cells have been successfully separated from untransduced cells by fluorescence-activated cell sorting, based on cell surface expression of CD24. Transduced and sorted NIH 3T3 cells showed higher GC enzyme activity than the unsorted population, demonstrating coordinated expression of both genes. Fibroblasts from Gaucher patients were transduced and sorted for CD24 expression, and GC enzyme activity was measured. The transduced sorted Gaucher fibroblasts had a marked increase in enzyme activity (149%) compared with virgin Gaucher fibroblasts (17% of normal GC enzyme activity). Efficient transduction of CD34+ hematopoietic progenitors (20-40%) was accomplished and fluorescence-activated cell sorted CD24(+)-expressing progenitors generated colonies, all of which (100%) were vector positive. The sorted, CD24-expressing progenitors generated erythroid burst-forming units, colony-forming units (CFU)-granulocyte, CFU-macrophage, CFU-granulocyte/macrophage, and CFU-mix hematopoietic colonies, demonstrating their ability to differentiate into these myeloid lineages in vitro. The transduced, sorted progenitors raised the GC enzyme levels in their progeny cells manyfold compared with untransduced CD34+ progenitors. Collectively, this demonstrates the development of high titer, selectable bicistronic vectors that allow isolation of transduced hematopoietic progenitors and cells that have been metabolically corrected. Images Fig. 2 Fig. 3 PMID:8618847

Interleukin-12 in patients with cancer is synthesized by peripheral helper T lymphocytes.

PubMed

Michelin, Marcia A; Montes, Leticia; Nomelini, Rosekeila S; Abdalla, Douglas R; Aleixo, Andre A R; Murta, Eddie F C

2015-09-01

The production of cytokines by helper T lymphocytes is a critical event in the immune response, as alterations in the regulation of this process may result in an appropriate immune response, persistent infection or the development of autoimmune disease. Previously, this group has used flow cytometry to demonstrate the expression of interleukin-12 (IL-12) in peripheral blood CD4+ T lymphocytes from patients and mice with advanced cancer. The aim of the present study was to investigate whether CD4+ T lymphocytes from the peripheral blood (PB) of patients with cancer produce IL-12, using molecular approaches, flow cytometry and cellular imaging techniques. CD3+ and CD4+ cells, and cells producing IL-12, were isolated from the PB obtained from patients with cancer, using a cell sorting flow cytometry technique. The positivity of cells for CD3, CD4 and IL-12, which were identified by cell sorting, was visualized using immunofluorescent cellular imaging. Total RNA was extracted from the CD3+CD4+IL-12+ cells, obtained by cell sorting, for confirmation of the presence of IL-12 mRNA, using reverse transcription-polymerase chain reaction (RT-PCR). RT-PCR demonstrated the presence of IL-12 mRNA in all patients (n=14), in contrast to the control group, in whom IL-12 expression was not detected. Immunofluorescent analysis of CD4+ T lymphocytes showed positive intracytoplasmatic IL-12 staining. These results demonstrated that CD3+CD4+ T lymphocytes in the PB of patients with cancer have the capacity to synthesize and express IL-12.

Interleukin-12 in patients with cancer is synthesized by peripheral helper T lymphocytes

PubMed Central

MICHELIN, MARCIA A.; MONTES, LETICIA; NOMELINI, ROSEKEILA S.; ABDALLA, DOUGLAS R.; ALEIXO, ANDRE A. R.; MURTA, EDDIE F. C.

2015-01-01

The production of cytokines by helper T lymphocytes is a critical event in the immune response, as alterations in the regulation of this process may result in an appropriate immune response, persistent infection or the development of autoimmune disease. Previously, this group has used flow cytometry to demonstrate the expression of interleukin-12 (IL-12) in peripheral blood CD4+ T lymphocytes from patients and mice with advanced cancer. The aim of the present study was to investigate whether CD4+ T lymphocytes from the peripheral blood (PB) of patients with cancer produce IL-12, using molecular approaches, flow cytometry and cellular imaging techniques. CD3+ and CD4+ cells, and cells producing IL-12, were isolated from the PB obtained from patients with cancer, using a cell sorting flow cytometry technique. The positivity of cells for CD3, CD4 and IL-12, which were identified by cell sorting, was visualized using immunofluorescent cellular imaging. Total RNA was extracted from the CD3+CD4+IL-12+ cells, obtained by cell sorting, for confirmation of the presence of IL-12 mRNA, using reverse transcription-polymerase chain reaction (RT-PCR). RT-PCR demonstrated the presence of IL-12 mRNA in all patients (n=14), in contrast to the control group, in whom IL-12 expression was not detected. Immunofluorescent analysis of CD4+ T lymphocytes showed positive intracytoplasmatic IL-12 staining. These results demonstrated that CD3+CD4+ T lymphocytes in the PB of patients with cancer have the capacity to synthesize and express IL-12. PMID:26622702

The Role of the Clathrin Adaptor AP-1: Polarized Sorting and Beyond

PubMed Central

Nakatsu, Fubito; Hase, Koji; Ohno, Hiroshi

2014-01-01

The selective transport of proteins or lipids by vesicular transport is a fundamental process supporting cellular physiology. The budding process involves cargo sorting and vesicle formation at the donor membrane and constitutes an important process in vesicular transport. This process is particularly important for the polarized sorting in epithelial cells, in which the cargo molecules need to be selectively sorted and transported to two distinct destinations, the apical or basolateral plasma membrane. Adaptor protein (AP)-1, a member of the AP complex family, which includes the ubiquitously expressed AP-1A and the epithelium-specific AP-1B, regulates polarized sorting at the trans-Golgi network and/or at the recycling endosomes. A growing body of evidence, especially from studies using model organisms and animals, demonstrates that the AP-1-mediated polarized sorting supports the development and physiology of multi-cellular units as functional organs and tissues (e.g., cell fate determination, inflammation and gut immune homeostasis). Furthermore, a possible involvement of AP-1B in the pathogenesis of human diseases, such as Crohn’s disease and cancer, is now becoming evident. These data highlight the significant contribution of AP-1 complexes to the physiology of multicellular organisms, as master regulators of polarized sorting in epithelial cells. PMID:25387275

Co-assembly of Viral Envelope Glycoproteins Regulates Their Polarized Sorting in Neurons

PubMed Central

Mardones, Gonzalo A.; Bonifacino, Juan S.

2014-01-01

Newly synthesized envelope glycoproteins of neuroinvasive viruses can be sorted in a polarized manner to the somatodendritic and/or axonal domains of neurons. Although critical for transneuronal spread of viruses, the molecular determinants and interregulation of this process are largely unknown. We studied the polarized sorting of the attachment (NiV-G) and fusion (NiV-F) glycoproteins of Nipah virus (NiV), a paramyxovirus that causes fatal human encephalitis, in rat hippocampal neurons. When expressed individually, NiV-G exhibited a non-polarized distribution, whereas NiV-F was specifically sorted to the somatodendritic domain. Polarized sorting of NiV-F was dependent on interaction of tyrosine-based signals in its cytosolic tail with the clathrin adaptor complex AP-1. Co-expression of NiV-G with NiV-F abolished somatodendritic sorting of NiV-F due to incorporation of NiV-G•NiV-F complexes into axonal transport carriers. We propose that faster biosynthetic transport of unassembled NiV-F allows for its proteolytic activation in the somatodendritic domain prior to association with NiV-G and axonal delivery of NiV-G•NiV-F complexes. Our study reveals how interactions of viral glycoproteins with the host's transport machinery and between themselves regulate their polarized sorting in neurons. PMID:24831812

Automatic online spike sorting with singular value decomposition and fuzzy C-mean clustering

PubMed Central

2012-01-01

Background Understanding how neurons contribute to perception, motor functions and cognition requires the reliable detection of spiking activity of individual neurons during a number of different experimental conditions. An important problem in computational neuroscience is thus to develop algorithms to automatically detect and sort the spiking activity of individual neurons from extracellular recordings. While many algorithms for spike sorting exist, the problem of accurate and fast online sorting still remains a challenging issue. Results Here we present a novel software tool, called FSPS (Fuzzy SPike Sorting), which is designed to optimize: (i) fast and accurate detection, (ii) offline sorting and (iii) online classification of neuronal spikes with very limited or null human intervention. The method is based on a combination of Singular Value Decomposition for fast and highly accurate pre-processing of spike shapes, unsupervised Fuzzy C-mean, high-resolution alignment of extracted spike waveforms, optimal selection of the number of features to retain, automatic identification the number of clusters, and quantitative quality assessment of resulting clusters independent on their size. After being trained on a short testing data stream, the method can reliably perform supervised online classification and monitoring of single neuron activity. The generalized procedure has been implemented in our FSPS spike sorting software (available free for non-commercial academic applications at the address: http://www.spikesorting.com) using LabVIEW (National Instruments, USA). We evaluated the performance of our algorithm both on benchmark simulated datasets with different levels of background noise and on real extracellular recordings from premotor cortex of Macaque monkeys. The results of these tests showed an excellent accuracy in discriminating low-amplitude and overlapping spikes under strong background noise. The performance of our method is competitive with respect to other robust spike sorting algorithms. Conclusions This new software provides neuroscience laboratories with a new tool for fast and robust online classification of single neuron activity. This feature could become crucial in situations when online spike detection from multiple electrodes is paramount, such as in human clinical recordings or in brain-computer interfaces. PMID:22871125

Automatic online spike sorting with singular value decomposition and fuzzy C-mean clustering.

PubMed

Oliynyk, Andriy; Bonifazzi, Claudio; Montani, Fernando; Fadiga, Luciano

2012-08-08

Understanding how neurons contribute to perception, motor functions and cognition requires the reliable detection of spiking activity of individual neurons during a number of different experimental conditions. An important problem in computational neuroscience is thus to develop algorithms to automatically detect and sort the spiking activity of individual neurons from extracellular recordings. While many algorithms for spike sorting exist, the problem of accurate and fast online sorting still remains a challenging issue. Here we present a novel software tool, called FSPS (Fuzzy SPike Sorting), which is designed to optimize: (i) fast and accurate detection, (ii) offline sorting and (iii) online classification of neuronal spikes with very limited or null human intervention. The method is based on a combination of Singular Value Decomposition for fast and highly accurate pre-processing of spike shapes, unsupervised Fuzzy C-mean, high-resolution alignment of extracted spike waveforms, optimal selection of the number of features to retain, automatic identification the number of clusters, and quantitative quality assessment of resulting clusters independent on their size. After being trained on a short testing data stream, the method can reliably perform supervised online classification and monitoring of single neuron activity. The generalized procedure has been implemented in our FSPS spike sorting software (available free for non-commercial academic applications at the address: http://www.spikesorting.com) using LabVIEW (National Instruments, USA). We evaluated the performance of our algorithm both on benchmark simulated datasets with different levels of background noise and on real extracellular recordings from premotor cortex of Macaque monkeys. The results of these tests showed an excellent accuracy in discriminating low-amplitude and overlapping spikes under strong background noise. The performance of our method is competitive with respect to other robust spike sorting algorithms. This new software provides neuroscience laboratories with a new tool for fast and robust online classification of single neuron activity. This feature could become crucial in situations when online spike detection from multiple electrodes is paramount, such as in human clinical recordings or in brain-computer interfaces.

Microfluidic flow rate detection based on integrated optical fiber cantilever.

PubMed

Lien, Victor; Vollmer, Frank

2007-10-01

We demonstrate an integrated microfluidic flow sensor with ultra-wide dynamic range, suitable for high throughput applications such as flow cytometry and particle sorting/counting. A fiber-tip cantilever transduces flow rates to optical signal readout, and we demonstrate a dynamic range from 0 to 1500 microL min(-1) for operation in water. Fiber-optic sensor alignment is guided by preformed microfluidic channels, and the dynamic range can be adjusted in a one-step chemical etch. An overall non-linear response is attributed to the far-field angular distribution of single-mode fiber output.

New views of granular mass flows

USGS Publications Warehouse

Iverson, R.M.; Vallance, J.W.

2001-01-01

Concentrated grain-fluid mixtures in rock avalanches, debris flows, and pyroclastic flows do not behave as simple materials with fixed rheologies. Instead, rheology evolves as mixture agitation, grain concentration, and fluid-pressure change during flow initiation, transit, and deposition. Throughout a flow, however, normal forces on planes parallel to the free upper surface approximately balance the weight of the superincumbent mixture, and the Coulomb friction rule describes bulk intergranular shear stresses on such planes. Pore-fluid pressure can temporarily or locally enhance mixture mobility by reducing Coulomb friction and transferring shear stress to the fluid phase. Initial conditions, boundary conditions, and grain comminution and sorting can influence pore-fluid pressures and cause variations in flow dynamics and deposits.

Hoechst fluorescence intensity can be used to separate viable bromodeoxyuridine-labeled cells from viable non-bromodeoxyuridine-labeled cells

NASA Technical Reports Server (NTRS)

Mozdziak, P. E.; Pulvermacher, P. M.; Schultz, E.; Schell, K.

2000-01-01

BACKGROUND: 5-Bromo-2'-deoxyuridine (BrdU) is a powerful compound to study the mitotic activity of a cell. Most techniques that identify BrdU-labeled cells require conditions that kill the cells. However, the fluorescence intensity of the membrane-permeable Hoechst dyes is reduced by the incorporation of BrdU into DNA, allowing the separation of viable BrdU positive (BrdU+) cells from viable BrdU negative (BrdU-) cells. METHODS: Cultures of proliferating cells were supplemented with BrdU for 48 h and other cultures of proliferating cells were maintained without BrdU. Mixtures of viable BrdU+ and viable BrdU- cells from the two proliferating cultures were stained with Hoechst 33342. The viable BrdU+ and BrdU- cells were sorted into different fractions from a mixture of BrdU+ and BrdU- cells based on Hoechst fluorescence intensity and the ability to exclude the vital dye, propidium iodide. Subsequently, samples from the original mixture, the sorted BrdU+ cell population, and the sorted BrdU- cell population were immunostained using an anti-BrdU monoclonal antibody and evaluated using flow cytometry. RESULTS: Two mixtures consisting of approximately 55% and 69% BrdU+ cells were sorted into fractions consisting of greater than 93% BrdU+ cells and 92% BrdU- cells. The separated cell populations were maintained in vitro after sorting to demonstrate their viability. CONCLUSIONS: Hoechst fluorescence intensity in combination with cell sorting is an effective tool to separate viable BrdU+ from viable BrdU- cells for further study. The separated cell populations were maintained in vitro after sorting to demonstrate their viability. Copyright 2000 Wiley-Liss, Inc.

Multiscale modeling and simulation of microtubule-motor-protein assemblies

NASA Astrophysics Data System (ADS)

Gao, Tong; Blackwell, Robert; Glaser, Matthew A.; Betterton, M. D.; Shelley, Michael J.

2015-12-01

Microtubules and motor proteins self-organize into biologically important assemblies including the mitotic spindle and the centrosomal microtubule array. Outside of cells, microtubule-motor mixtures can form novel active liquid-crystalline materials driven out of equilibrium by adenosine triphosphate-consuming motor proteins. Microscopic motor activity causes polarity-dependent interactions between motor proteins and microtubules, but how these interactions yield larger-scale dynamical behavior such as complex flows and defect dynamics is not well understood. We develop a multiscale theory for microtubule-motor systems in which Brownian dynamics simulations of polar microtubules driven by motors are used to study microscopic organization and stresses created by motor-mediated microtubule interactions. We identify polarity-sorting and crosslink tether relaxation as two polar-specific sources of active destabilizing stress. We then develop a continuum Doi-Onsager model that captures polarity sorting and the hydrodynamic flows generated by these polar-specific active stresses. In simulations of active nematic flows on immersed surfaces, the active stresses drive turbulent flow dynamics and continuous generation and annihilation of disclination defects. The dynamics follow from two instabilities, and accounting for the immersed nature of the experiment yields unambiguous characteristic length and time scales. When turning off the hydrodynamics in the Doi-Onsager model, we capture formation of polar lanes as observed in the Brownian dynamics simulation.

Multiscale modeling and simulation of microtubule-motor-protein assemblies.

PubMed

Gao, Tong; Blackwell, Robert; Glaser, Matthew A; Betterton, M D; Shelley, Michael J

2015-01-01

Microtubules and motor proteins self-organize into biologically important assemblies including the mitotic spindle and the centrosomal microtubule array. Outside of cells, microtubule-motor mixtures can form novel active liquid-crystalline materials driven out of equilibrium by adenosine triphosphate-consuming motor proteins. Microscopic motor activity causes polarity-dependent interactions between motor proteins and microtubules, but how these interactions yield larger-scale dynamical behavior such as complex flows and defect dynamics is not well understood. We develop a multiscale theory for microtubule-motor systems in which Brownian dynamics simulations of polar microtubules driven by motors are used to study microscopic organization and stresses created by motor-mediated microtubule interactions. We identify polarity-sorting and crosslink tether relaxation as two polar-specific sources of active destabilizing stress. We then develop a continuum Doi-Onsager model that captures polarity sorting and the hydrodynamic flows generated by these polar-specific active stresses. In simulations of active nematic flows on immersed surfaces, the active stresses drive turbulent flow dynamics and continuous generation and annihilation of disclination defects. The dynamics follow from two instabilities, and accounting for the immersed nature of the experiment yields unambiguous characteristic length and time scales. When turning off the hydrodynamics in the Doi-Onsager model, we capture formation of polar lanes as observed in the Brownian dynamics simulation.

Multiscale modeling and simulation of microtubule–motor-protein assemblies

PubMed Central

Gao, Tong; Blackwell, Robert; Glaser, Matthew A.; Betterton, M. D.; Shelley, Michael J.

2016-01-01

Microtubules and motor proteins self-organize into biologically important assemblies including the mitotic spindle and the centrosomal microtubule array. Outside of cells, microtubule-motor mixtures can form novel active liquid-crystalline materials driven out of equilibrium by adenosine triphosphate–consuming motor proteins. Microscopic motor activity causes polarity-dependent interactions between motor proteins and microtubules, but how these interactions yield larger-scale dynamical behavior such as complex flows and defect dynamics is not well understood. We develop a multiscale theory for microtubule-motor systems in which Brownian dynamics simulations of polar microtubules driven by motors are used to study microscopic organization and stresses created by motor-mediated microtubule interactions. We identify polarity-sorting and crosslink tether relaxation as two polar-specific sources of active destabilizing stress. We then develop a continuum Doi-Onsager model that captures polarity sorting and the hydrodynamic flows generated by these polar-specific active stresses. In simulations of active nematic flows on immersed surfaces, the active stresses drive turbulent flow dynamics and continuous generation and annihilation of disclination defects. The dynamics follow from two instabilities, and accounting for the immersed nature of the experiment yields unambiguous characteristic length and time scales. When turning off the hydrodynamics in the Doi-Onsager model, we capture formation of polar lanes as observed in the Brownian dynamics simulation. PMID:26764729

Reticulate evolution and incomplete lineage sorting among the ponderosa pines.

PubMed

Willyard, Ann; Cronn, Richard; Liston, Aaron

2009-08-01

Interspecific gene flow via hybridization may play a major role in evolution by creating reticulate rather than hierarchical lineages in plant species. Occasional diploid pine hybrids indicate the potential for introgression, but reticulation is hard to detect because ancestral polymorphism is still shared across many groups of pine species. Nucleotide sequences for 53 accessions from 17 species in subsection Ponderosae (Pinus) provide evidence for reticulate evolution. Two discordant patterns among independent low-copy nuclear gene trees and a chloroplast haplotype are better explained by introgression than incomplete lineage sorting or other causes of incongruence. Conflicting resolution of three monophyletic Pinus coulteri accessions is best explained by ancient introgression followed by a genetic bottleneck. More recent hybridization transferred a chloroplast from P. jeffreyi to a sympatric P. washoensis individual. We conclude that incomplete lineage sorting could account for other examples of non-monophyly, and caution against any analysis based on single-accession or single-locus sampling in Pinus.

Isolation and characterization of anti-SEB peptides using magnetic sorting and bacterial peptide display library technology

NASA Astrophysics Data System (ADS)

Pennington, Joseph M.; Kogot, Joshua M.; Sarkes, Deborah A.; Pellegrino, Paul M.; Stratis-Cullum, Dimitra N.

2012-06-01

Peptide display libraries offer an alternative method to existing antibody development methods enabling rapid isolation of highly stable reagents for detection of new and emerging biological threats. Bacterial display libraries are used to isolate new peptide reagents within 1 week, which is simpler and timelier than using competing display library technology based on phage or yeast. Using magnetic sorting methods, we have isolated peptide reagents with high affinity and specificity to staphylococcal enterotoxin B (SEB), a suspected food pathogen. Flow cytometry methods were used for on-cell characterization and the binding affinity (Kd) of this new peptide reagent was determined to be 56 nm with minimal cross-reactivity to other proteins. These results demonstrated that magnetic sorting for new reagents using bacterial display libraries is a rapid and effective method and has the potential for current and new and emerging food pathogen targets.

Comprehensive two-dimensional gas chromatography/time-of-flight mass spectrometry peak sorting algorithm.

PubMed

Oh, Cheolhwan; Huang, Xiaodong; Regnier, Fred E; Buck, Charles; Zhang, Xiang

2008-02-01

We report a novel peak sorting method for the two-dimensional gas chromatography/time-of-flight mass spectrometry (GC x GC/TOF-MS) system. The objective of peak sorting is to recognize peaks from the same metabolite occurring in different samples from thousands of peaks detected in the analytical procedure. The developed algorithm is based on the fact that the chromatographic peaks for a given analyte have similar retention times in all of the chromatograms. Raw instrument data are first processed by ChromaTOF (Leco) software to provide the peak tables. Our algorithm achieves peak sorting by utilizing the first- and second-dimension retention times in the peak tables and the mass spectra generated during the process of electron impact ionization. The algorithm searches the peak tables for the peaks generated by the same type of metabolite using several search criteria. Our software also includes options to eliminate non-target peaks from the sorting results, e.g., peaks of contaminants. The developed software package has been tested using a mixture of standard metabolites and another mixture of standard metabolites spiked into human serum. Manual validation demonstrates high accuracy of peak sorting with this algorithm.

Regulation of cargo transfer between ESCRT-0 and ESCRT-I complexes by flotillin-1 during endosomal sorting of ubiquitinated cargo

PubMed Central

Meister, M; Bänfer, S; Gärtner, U; Koskimies, J; Amaddii, M; Jacob, R; Tikkanen, R

2017-01-01

Ubiquitin-dependent sorting of membrane proteins in endosomes directs them to lysosomal degradation. In the case of receptors such as the epidermal growth factor receptor (EGFR), lysosomal degradation is important for the regulation of downstream signalling. Ubiquitinated proteins are recognised in endosomes by the endosomal sorting complexes required for transport (ESCRT) complexes, which sequentially interact with the ubiquitinated cargo. Although the role of each ESCRT complex in sorting is well established, it is not clear how the cargo is passed on from one ESCRT to the next. We here show that flotillin-1 is required for EGFR degradation, and that it interacts with the subunits of ESCRT-0 and -I complexes (hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) and Tsg101). Flotillin-1 is required for cargo recognition and sorting by ESCRT-0/Hrs and for its interaction with Tsg101. In addition, flotillin-1 is also required for the sorting of human immunodeficiency virus 1 Gag polyprotein, which mimics ESCRT-0 complex during viral assembly. We propose that flotillin-1 functions in cargo transfer between ESCRT-0 and -I complexes. PMID:28581508

Unpacking boxes: Integration of molecular, morphological and ecological approaches reveals extensive patterns of reticulate evolution in box eucalypts.

PubMed

Flores-Rentería, Lluvia; Rymer, Paul D; Riegler, Markus

2017-03-01

Reticulate evolution by hybridization is considered a common process shaping the evolution of many plant species, however, reticulation could also be due to incomplete lineage sorting in biodiverse systems. For our study we selected a group of closely related plant taxa with contrasting yet partially overlapping geographic distributions and different population sizes, to distinguish between reticulated patterns due to hybridization and incomplete lineage sorting. We predicted that sympatric or proximal populations of different species are more likely to have gene flow than geographically distant populations of the same widespread species. Furthermore, for species with restricted distributions, and therefore, small effective population sizes, we predicted complete lineage sorting. Eastern grey box eucalypt species (Eucalyptus supraspecies Moluccanae) provide an ideal system to explore patterns of reticulate evolution. They form a diverse, recently evolved and phylogenetically undefined group within Eucalyptus, with overlapping morphological features and hybridization in nature. We used a multi-faceted approach, combining analyses of chloroplast and nuclear DNA, as well as seedling morphology, flowering time and ecological spatial differentiation in order to test for species delimitation and reticulate evolution in this group. The multiple layers of results were consistent and suggested a lack of monophyly at different hierarchical levels due to multidirectional gene flow among several species, challenging species delimitation. Chloroplast and nuclear haplotypes were shared among different species in geographic proximity, consistent with hybridization zones. Furthermore, species with restricted distributions appeared better resolved due to lineage sorting in the absence of hybridization. We conclude that a combination of molecular, morphological and ecological approaches is required to disentangle patterns of reticulate evolution in the box eucalypts. Published by Elsevier Inc.

Field fertility of liquid stored and cryopreserved flow cytometrically sex-sorted stallion sperm.

PubMed

Gibb, Z; Grupen, C G; Maxwell, W M C; Morris, L H A

2017-03-01

The fertility of sex-sorted, cryopreserved stallion sperm must be improved for the sex-sorting technology to be applied commercially. To optimise the conditions used to liquid store stallion sperm prior to sex-sorting and assess the fertility of sperm following sex-sorting and cryopreservation. Both in vitro experiment and randomised controlled trial in healthy, client-owned mares. Stallion ejaculates (n = 9) were diluted in either a skimmed milk (KMT) or BSA (I-BSA) based media to 25 × 10 6 sperm/ml directly (+SP25) or washed to remove seminal plasma and diluted to 25 or 111 × 10 6 sperm/ml (-SP25 and -SP111). Sperm were stored for 18 h at 10 to 15°C and -SP25 and +SP25 treatments were centrifuged and resuspended to 111 × 10 6 sperm/ml. Sperm were incubated under H33342 staining conditions and motility, viability and acrosome integrity assessed. Semen was collected from stallions (n = 4), liquid stored at 10-15°C for up to 5 h and sperm either cryopreserved directly, sex-sorted and cryopreserved, or sex-sorted and returned to liquid storage until insemination. Low-dose hysteroscopic insemination was performed in 23 mares randomly allocated to the semen preparation group and pregnancy determined following embryo flushing on Day 9 after ovulation, or via transrectal ultrasonography on Day 14 after ovulation. Skimmed milk was superior to I-BSA in maintaining motility, viability and acrosome integrity. Seminal plasma removal did not affect the parameters measured at the concentrations examined. Conception rates did not differ significantly between the groups, although a high incidence of pregnancy loss was observed in both the cryopreserved groups. While the conception rates achieved are among the highest yet reported for sex-sorted, cryopreserved stallion sperm, the high incidence of pregnancy loss suggests that the development of the resulting embryos was significantly impaired by the sperm processing treatments. © 2016 EVJ Ltd.

Relationship between geomorphology and lithotypes of lahar deposit from Chokai volcano, Japan

NASA Astrophysics Data System (ADS)

Minami, Y.; Ohba, T.; Hayashi, S.; Kataoka, K.

2013-12-01

Chokai volcano, located in the northern Honshu arc in Japan, is an andesitic stratovolcano that collapsed partly at ca. 2500 years ago. A post collapse lahar deposit (Shirayukigawa lahar deposit) is distributed in the northern foot of the volcanic edifice. The deposit consists of 16 units of debris flow, hyperconcentrated flow and streamflow deposits. The Shirayukigawa lahar deposit has a total thickness of 30 m and overlies the 2.5-ka Kisakata debris avalanche deposit. Shirayukigawa lahar deposit forms volcanic fan and volcanic apron. The volcanic fan is subdivided into four areas on the basis of slope angles and of geomorphological features: 1) steeply sloped area, 2) moderately sloped area, 3) gently sloped area and 4) horizontal area. From sedimentary facies and structures, each unit of the Shirayukigawa lahar deposit is classified into one of four lithotypes: clast-supported debris flow deposit (Cc), matrix-supported debris flow deposit (Cm1), hyperconcentrated flow deposit (Cm2) and streamflow deposit (Sl). Each type has the following lithological characteristics. The lithotypes are well correlated with the geomorphology of the volcanic fan. The steeply-sloped and the moderately-sloped areas are dominated by Cc, Cm1, and Cm2, and The horizontal area are dominated by Sl. Debris flow deposit (Cc) is massive, very poorly sorted, partly graded, and clast-supported with polymictic clasts dominated by subrounded to rounded volcanic clasts. Matrix is sandy to muddy. Preferred clast orientation are present. Debris flow deposit (Cm1) is massive, very poorly sorted, and matrix-supported with polymictic clasts dominated by subrounded to rounded volcanic clasts. Matrix is sandy to muddy. Some layers exhibit coarse-tail normal/inverse grading. Most clasts are oriented. Hyperconcentrated flow deposit (Cm2) is massive to diffusely laminated, very poorly sorted and matrix-supported with polymictic clasts dominated by subrounded to rounded volcanic rocks. Matrix is sandy. The clasts are randomly distributed in the sandy matrix except for some clast-concentrated lenticular layers. Clasts smaller than 1cm account for about 10 percent of the deposits. Maximum clast size is about 30 cm. Streamflow deposit (Sl) is weakly parallel/cross-laminated, sorted and partly graded. The deposit contains volcanic clasts smaller than 20cm, which clasts are preferentially oriented and account for about 5% of the deposit. Clasts of the deposits consist of altered andesite, fresh andesite, mudstone and sandstone. The sedimentary clasts were derived from the substrate. The proportion of altered andesite clasts decreases upwards through the units. Matrix components in the lower eight units (C-LHR) are different from those of the upper eight units (S-LHR). In C-LHR units, grayish blue clay is dominant in matrix, whereas in S-LHR units, brownish yellow volcanic sand is dominant in matrix. Hydrothermal clay minerals such as smectite, chlorite, pyrophyllite and kaoline group minerals are rich in C-LHR units, whereas they are poor in S-LHR units. The stratigraphic variation in matrix component reflects temporal variation in supplied materials from source region.

Efficient ecologic and economic operational rules for dammed systems by means of nondominated sorting genetic algorithm II

NASA Astrophysics Data System (ADS)

Niayifar, A.; Perona, P.

2015-12-01

River impoundment by dams is known to strongly affect the natural flow regime and in turn the river attributes and the related ecosystem biodiversity. Making hydropower sustainable implies to seek for innovative operational policies able to generate dynamic environmental flows while maintaining economic efficiency. For dammed systems, we build the ecological and economical efficiency plot for non-proportional flow redistribution operational rules compared to minimal flow operational. As for the case of small hydropower plants (e.g., see the companion paper by Gorla et al., this session), we use a four parameters Fermi-Dirac statistical distribution to mathematically formulate non-proportional redistribution rules. These rules allocate a fraction of water to the riverine environment depending on current reservoir inflows and storage. Riverine ecological benefits associated to dynamic environmental flows are computed by integrating the Weighted Usable Area (WUA) for fishes with Richter's hydrological indicators. Then, we apply nondominated sorting genetic algorithm II (NSGA-II) to an ensemble of non-proportional and minimal flow redistribution rules in order to generate the Pareto frontier showing the system performances in the ecologic and economic space. This fast and elitist multiobjective optimization method is eventually applied to a case study. It is found that non-proportional dynamic flow releases ensure maximal power production on the one hand, while conciliating ecological sustainability on the other hand. Much of the improvement in the environmental indicator is seen to arise from a better use of the reservoir storage dynamics, which allows to capture, and laminate flood events while recovering part of them for energy production. In conclusion, adopting such new operational policies would unravel a spectrum of globally-efficient performances of the dammed system when compared with those resulting from policies based on constant minimum flow releases.

Towards the delineation of the ancestral eutherian genome organization: comparative genome maps of human and the African elephant (Loxodonta africana) generated by chromosome painting.

PubMed Central

Frönicke, Lutz; Wienberg, Johannes; Stone, Gary; Adams, Lisa; Stanyon, Roscoe

2003-01-01

This study presents a whole-genome comparison of human and a representative of the Afrotherian clade, the African elephant, generated by reciprocal Zoo-FISH. An analysis of Afrotheria genomes is of special interest, because recent DNA sequence comparisons identify them as the oldest placental mammalian clade. Complete sets of whole-chromosome specific painting probes for the African elephant and human were constructed by degenerate oligonucleotide-primed PCR amplification of flow-sorted chromosomes. Comparative genome maps are presented based on their hybridization patterns. These maps show that the elephant has a moderately rearranged chromosome complement when compared to humans. The human paint probes identified 53 evolutionary conserved segments on the 27 autosomal elephant chromosomes and the X chromosome. Reciprocal experiments with elephant probes delineated 68 conserved segments in the human genome. The comparison with a recent aardvark and elephant Zoo-FISH study delineates new chromosomal traits which link the two Afrotherian species phylogenetically. In the absence of any morphological evidence the chromosome painting data offer the first non-DNA sequence support for an Afrotherian clade. The comparative human and elephant genome maps provide new insights into the karyotype organization of the proto-afrotherian, the ancestor of extant placental mammals, which most probably consisted of 2n=46 chromosomes. PMID:12965023

Hydrodynamic lift for single cell manipulation in a femtosecond laser fabricated optofluidic chip

NASA Astrophysics Data System (ADS)

Bragheri, Francesca; Osellame, Roberto

2017-08-01

Single cell sorting based either on fluorescence or on mechanical properties has been exploited in the last years in microfluidic devices. Hydrodynamic focusing allows increasing the efficiency of theses devices by improving the matching between the region of optical analysis and that of cell flow. Here we present a very simple solution fabricated by femtosecond laser micromachining that exploits flow laminarity in microfluidic channels to easily lift the sample flowing position to the channel portion illuminated by the optical waveguides used for single cell trapping and analysis.

A Gravity-Driven Microfluidic Particle Sorting Device with Hydrodynamic Separation Amplification

PubMed Central

Huh, Dongeun; Bahng, Joong Hwan; Ling, Yibo; Wei, Hsien-Hung; Kripfgans, Oliver D.; Fowlkes, J. Brian; Grotberg, James B.; Takayama, Shuichi

2008-01-01

This paper describes a simple microfluidic sorting system that can perform size-profiling and continuous mass-dependent separation of particles through combined use of gravity (1g) and hydrodynamic flows capable of rapidly amplifying sedimentation-based separation between particles. Operation of the device relies on two microfluidic transport processes: i) initial hydrodynamic focusing of particles in a microchannel oriented parallel to gravity, ii) subsequent sample separation where positional difference between particles with different mass generated by sedimentation is further amplified by hydrodynamic flows whose streamlines gradually widen out due to the geometry of a widening microchannel oriented perpendicular to gravity. The microfluidic sorting device was fabricated in poly(dimethylsiloxane) (PDMS), and hydrodynamic flows in microchannels were driven by gravity without using external pumps. We conducted theoretical and experimental studies on fluid dynamic characteristics of laminar flows in widening microchannels and hydrodynamic amplification of particle separation. Direct trajectory monitoring, collection, and post-analysis of separated particles were performed using polystyrene microbeads with different sizes to demonstrate rapid (< 1 min) and high-purity (> 99.9 %) separation. Finally, we demonstrated biomedical applications of our system by isolating small-sized (diameter < 6 μm) perfluorocarbon liquid droplets from polydisperse droplet emulsions, which is crucial in preparing contrast agents for safe, reliable ultrasound medical imaging, tracers for magnetic resonance imaging, or transpulmonary droplets used in ultrasound-based occlusion therapy for cancer treatment. Our method enables straightforward, rapid real-time size-monitoring and continuous separation of particles in simple stand-alone microfabricated devices without the need for bulky and complex external power sources. We believe that this system will provide a useful tool o separate colloids and particles for various analytical and preparative applications, and may hold 3 potential for separation of cells or development of diagnostic tools requiring point-of-care sample preparation or testing. PMID:17297936

Effect of sex-sorting and cryopreservation on the post-thaw sperm quality of Iberian red deer spermatozoa.

PubMed

Anel-López, L; García-Álvarez, O; Parrilla, I; Del Olmo, D; Maroto-Morales, A; Fernandez-Santos, M R; Ortiz, J A; Soler, A J; Martínez, E M; Vazquez, J M; Garde, J J

2017-02-01

This study investigated the effect of sex-sorting and cryopreservation on post-thaw characteristics and fertility of red deer (Cervus elaphus) sperm for the first time. Semen was collected by electroejaculation from 10 mature stags during the breeding season, and each ejaculate split into four experimental groups: Bulk sorted spermatozoa, sorted but not sexed (BSS); sorted high purity X-spermatozoa (XSS); sorted high purity Y-spermatozoa (YSS); and, control non-sorted spermatozoa (NS). Following, all samples were frozen over liquid nitrogen. Two straws per stag and sample type were analyzed immediately post-thaw and following a 2-h incubation period at 37 °C. Post-thaw total motility (TM) as assessed by CASA was not different (P < 0.05) among NS, BSS and YSS sperm. For XSS, post-thaw TM was lower (39%, P < 0.05) than that for NS (54%) or BSS (50%), but similar (P > 0.05) to that of YSS (47%) sperm. The percentage of apoptotic spermatozoa as assessed by PI/YO-PRO-1 and flow cytometry analysis, was higher (17%, P ≤ 0.05) for XSS sperm than NS (12%), BSS (13%) and YSS (14%) sperm. Following incubation there were no differences (P > 0.05) in TM or percent apoptosis among treatments. Post-thaw chromatin stability calculated as the DNA fragmentation index (%DFI) was similar among treatments; following incubation %DFI increased in all except YSS, which displayed the lowest value (P < 0.05). Artificial insemination of synchronized hinds yielded 44, 52 and 62% delivery rates for YSS, NS and standard frozen-thawed sperm, respectively (P < 0.05). Notably, 93 and 55% of fawns born were males for the YSS and NS spermatozoa, respectively (P < 0.05). In summary, Y-sorted sperm displayed acceptable post-thaw sperm evaluation parameters and the expected offspring sex ratio. More studies are needed to understand the source of sperm damage that may compromise the fertility of Y-sorted red deer sperm. Copyright © 2016 Elsevier Inc. All rights reserved.

Development of Antibodies Against Novel Cell Surface Proteins In Hormone Refractory Prostate Cancer. Addendum

DTIC Science & Technology

2011-07-01

marker of hormone refractory metastatic prostate cancer. Clin Cancer Res, 2005. 15: 2237- 43 . 3. Tomita K, van Bokhoven A, van Leenders GJ, Ruijter...Santa Cruz Biotechnology), vimentin, Ki-67 (DakoCytomation) and caspase-3 (Cell Signaling Technology). Flow cytometry was performed with N...transduced cells were labeled with N-cadherin–specific antibody and sorted by flow cytometry , gating for a GFP-positive, N-cadherinlow population. The

Comparative environmental evaluation of construction waste management through different waste sorting systems in Hong Kong.

PubMed

Hossain, Md Uzzal; Wu, Zezhou; Poon, Chi Sun

2017-11-01

This study aimed to compare the environmental performance of building construction waste management (CWM) systems in Hong Kong. Life cycle assessment (LCA) approach was applied to evaluate the performance of CWM systems holistically based on primary data collected from two real building construction sites and secondary data obtained from the literature. Different waste recovery rates were applied based on compositions and material flow to assess the influence on the environmental performance of CWM systems. The system boundary includes all stages of the life cycle of building construction waste (including transportation, sorting, public fill or landfill disposal, recovery and reuse, and transformation and valorization into secondary products). A substitutional LCA approach was applied for capturing the environmental gains due to the utilizations of recovered materials. The results showed that the CWM system by using off-site sorting and direct landfilling resulted in significant environmental impacts. However, a considerable net environmental benefit was observed through an on-site sorting system. For example, about 18-30kg CO 2 eq. greenhouse gases (GHGs) emission were induced for managing 1 t of construction waste through off-site sorting and direct landfilling, whereas significant GHGs emission could be potentially avoided (considered as a credit -126 to -182kg CO 2 eq.) for an on-site sorting system due to the higher recycling potential. Although the environmental benefits mainly depend on the waste compositions and their sortability, the analysis conducted in this study can serve as guidelines to design an effective and resource-efficient building CWM system. Copyright © 2017 Elsevier Ltd. All rights reserved.

Children perseverate to a human's actions but not to a robot's actions.

PubMed

Moriguchi, Yusuke; Kanda, Takayuki; Ishiguro, Hiroshi; Itakura, Shoji

2010-01-01

Previous research has shown that young children commit perseverative errors from their observation of another person's actions. The present study examined how social observation would lead children to perseverative tendencies, using a robot. In Experiment 1, preschoolers watched either a human model or a robot sorting cards according to one dimension (e.g. shape), after which they were asked to sort according to a different dimension (e.g. colour). The results showed that children's behaviours in the task were significantly influenced by the human model's actions but not by the robot's actions. Experiment 2 excluded the possibility that children's behaviours were not affected by the robot's actions because they did not observe its actions. We concluded that children's perseverative errors from social observation resulted, in part, from their socio-cognitive ability.

Shrink-induced sorting using integrated nanoscale magnetic traps.

PubMed

Nawarathna, Dharmakeerthi; Norouzi, Nazila; McLane, Jolie; Sharma, Himanshu; Sharac, Nicholas; Grant, Ted; Chen, Aaron; Strayer, Scott; Ragan, Regina; Khine, Michelle

2013-02-11

We present a plastic microfluidic device with integrated nanoscale magnetic traps (NSMTs) that separates magnetic from non-magnetic beads with high purity and throughput, and unprecedented enrichments. Numerical simulations indicate significantly higher localized magnetic field gradients than previously reported. We demonstrated >20 000-fold enrichment for 0.001% magnetic bead mixtures. Since we achieve high purity at all flow-rates tested, this is a robust, rapid, portable, and simple solution to sort target species from small volumes amenable for point-of-care applications. We used the NSMT in a 96 well format to extract DNA from small sample volumes for quantitative polymerase chain reaction (qPCR).

Pulsed laser triggered high speed microfluidic switch

NASA Astrophysics Data System (ADS)

Wu, Ting-Hsiang; Gao, Lanyu; Chen, Yue; Wei, Kenneth; Chiou, Pei-Yu

2008-10-01

We report a high-speed microfluidic switch capable of achieving a switching time of 10 μs. The switching mechanism is realized by exciting dynamic vapor bubbles with focused laser pulses in a microfluidic polydimethylsiloxane (PDMS) channel. The bubble expansion deforms the elastic PDMS channel wall and squeezes the adjacent sample channel to control its fluid and particle flows as captured by the time-resolved imaging system. A switching of polystyrene microspheres in a Y-shaped channel has also been demonstrated. This ultrafast laser triggered switching mechanism has the potential to advance the sorting speed of state-of-the-art microscale fluorescence activated cell sorting devices.

Construction of a DNA library representing 15q11-13 by subtraction of two flow sorted marker chromosome-specific libraries

DOE Office of Scientific and Technical Information (OSTI.GOV)

Blennow, E.; Werelius, B.; Nordenskjoeld, M.

Constitutional extra {open_quotes}marker chromosomes{close_quotes} are found in {approx}0.5/1000 of newborns. Of these, 50% are inverted duplications of the pericentromeric region of chromosome 15, including two variants; (1) inv dup(15)(pter{yields}q11:q11{yields}pter) and (2) inv dup(15) (pter{yields}q12-13::q12-13{yields}pter). Variant (1) is found in phenotypically normal individuals, whereas variant (2) will produce a typical clinical picture including mental retardation, autism, hyperactivity and discrete dysmorphic features. Fluorescence in situ hybridization (FISH) using single copy probes from the Prader-Willi region confirms these observations as well as chromosome painting using a flow-sorted marker chromosome-specific library from a variant (1) marker, hybridized to the chromosomes of a patient withmore » a variant (2) marker chromosome. Followingly, a flow-sorted biotinylated variant (1) library was subtracted from a non-labeled variant (2) library using magnetic beads and subsequent amplification by degenerate oligonucleotide-primed PCR (DOP-PCR). The successful result was demonstrated by using the amplified material for chromosome painting on chromosome slides from variant (1) and variant (2) patients. We have constructed a library from 15q11-13. This region contains genes producing a specific abnormal phenotype when found in a tri- or tetrasomic state. The region also contains the genes responsible for the Prader-Willi and Angelman syndromes when the paternal/maternal copy is missing, respectively. It is therefore a region where parental imprinting plays an important role. The isolated library may be used to isolate single copy clones which will allow further investigations of this region.« less

Flow Sorting and Sequencing Meadow Fescue Chromosome 4F1[C][W

PubMed Central

Kopecký, David; Martis, Mihaela; Číhalíková, Jarmila; Hřibová, Eva; Vrána, Jan; Bartoš, Jan; Kopecká, Jitka; Cattonaro, Federica; Stočes, Štěpán; Novák, Petr; Neumann, Pavel; Macas, Jiří; Šimková, Hana; Studer, Bruno; Asp, Torben; Baird, James H.; Navrátil, Petr; Karafiátová, Miroslava; Kubaláková, Marie; Šafář, Jan; Mayer, Klaus; Doležel, Jaroslav

2013-01-01

The analysis of large genomes is hampered by a high proportion of repetitive DNA, which makes the assembly of short sequence reads difficult. This is also the case in meadow fescue (Festuca pratensis), which is known for good abiotic stress resistance and has been used in intergeneric hybridization with ryegrasses (Lolium spp.) to produce Festulolium cultivars. In this work, we describe a new approach to analyze the large genome of meadow fescue, which involves the reduction of sample complexity without compromising information content. This is achieved by dissecting the genome to smaller parts: individual chromosomes and groups of chromosomes. As the first step, we flow sorted chromosome 4F and sequenced it by Illumina with approximately 50× coverage. This provided, to our knowledge, the first insight into the composition of the fescue genome, enabled the construction of the virtual gene order of the chromosome, and facilitated detailed comparative analysis with the sequenced genomes of rice (Oryza sativa), Brachypodium distachyon, sorghum (Sorghum bicolor), and barley (Hordeum vulgare). Using GenomeZipper, we were able to confirm the collinearity of chromosome 4F with barley chromosome 4H and the long arm of chromosome 5H. Several new tandem repeats were identified and physically mapped using fluorescence in situ hybridization. They were found as robust cytogenetic markers for karyotyping of meadow fescue and ryegrass species and their hybrids. The ability to purify chromosome 4F opens the way for more efficient analysis of genomic loci on this chromosome underlying important traits, including freezing tolerance. Our results confirm that next-generation sequencing of flow-sorted chromosomes enables an overview of chromosome structure and evolution at a resolution never achieved before. PMID:24096412

Composition of the summer photosynthetic pico and nanoplankton communities in the Beaufort Sea assessed by T-RFLP and sequences of the 18S rRNA gene from flow cytometry sorted samples.

PubMed

Balzano, Sergio; Marie, Dominique; Gourvil, Priscillia; Vaulot, Daniel

2012-08-01

The composition of photosynthetic pico and nanoeukaryotes was investigated in the North East Pacific and the Arctic Ocean with special emphasis on the Beaufort Sea during the MALINA cruise in summer 2009. Photosynthetic populations were sorted using flow cytometry based on their size and pigment fluorescence. Diversity of the sorted photosynthetic eukaryotes was determined using terminal-restriction fragment length polymorphism analysis and cloning/sequencing of the 18S ribosomal RNA gene. Picoplankton was dominated by Mamiellophyceae, a class of small green algae previously included in the prasinophytes: in the North East Pacific, the contribution of an Arctic Micromonas ecotype increased steadily northward becoming the only taxon occurring at most stations throughout the Beaufort Sea. In contrast, nanoplankton was more diverse: North Pacific stations were dominated by Pseudo-nitzschia sp. whereas those in the Beaufort Sea were dominated by two distinct Chaetoceros species as well as by Chrysophyceae, Pelagophyceae and Chrysochromulina spp.. This study confirms the importance of Arctic Micromonas within picoplankton throughout the Beaufort Sea and demonstrates that the photosynthetic picoeukaryote community in the Arctic is much less diverse than at lower latitudes. Moreover, in contrast to what occurs in warmer waters, most of the key pico- and nanoplankton species found in the Beaufort Sea could be successfully established in culture.

Single-cell analysis and sorting using droplet-based microfluidics.

PubMed

Mazutis, Linas; Gilbert, John; Ung, W Lloyd; Weitz, David A; Griffiths, Andrew D; Heyman, John A

2013-05-01

We present a droplet-based microfluidics protocol for high-throughput analysis and sorting of single cells. Compartmentalization of single cells in droplets enables the analysis of proteins released from or secreted by cells, thereby overcoming one of the major limitations of traditional flow cytometry and fluorescence-activated cell sorting. As an example of this approach, we detail a binding assay for detecting antibodies secreted from single mouse hybridoma cells. Secreted antibodies are detected after only 15 min by co-compartmentalizing single mouse hybridoma cells, a fluorescent probe and single beads coated with anti-mouse IgG antibodies in 50-pl droplets. The beads capture the secreted antibodies and, when the captured antibodies bind to the probe, the fluorescence becomes localized on the beads, generating a clearly distinguishable fluorescence signal that enables droplet sorting at ∼200 Hz as well as cell enrichment. The microfluidic system described is easily adapted for screening other intracellular, cell-surface or secreted proteins and for quantifying catalytic or regulatory activities. In order to screen ∼1 million cells, the microfluidic operations require 2-6 h; the entire process, including preparation of microfluidic devices and mammalian cells, requires 5-7 d.

Single-cell analysis and sorting using droplet-based microfluidics

PubMed Central

Mazutis, Linas; Gilbert, John; Ung, W Lloyd; Weitz, David A; Griffiths, Andrew D; Heyman, John A

2014-01-01

We present a droplet-based microfluidics protocol for high-throughput analysis and sorting of single cells. compartmentalization of single cells in droplets enables the analysis of proteins released from or secreted by cells, thereby overcoming one of the major limitations of traditional flow cytometry and fluorescence-activated cell sorting. as an example of this approach, we detail a binding assay for detecting antibodies secreted from single mouse hybridoma cells. secreted antibodies are detected after only 15 min by co-compartmentalizing single mouse hybridoma cells, a fluorescent probe and single beads coated with anti-mouse IgG antibodies in 50-pl droplets. the beads capture the secreted antibodies and, when the captured antibodies bind to the probe, the fluorescence becomes localized on the beads, generating a clearly distinguishable fluorescence signal that enables droplet sorting at ~200 Hz as well as cell enrichment. the microfluidic system described is easily adapted for screening other intracellular, cell-surface or secreted proteins and for quantifying catalytic or regulatory activities. In order to screen ~1 million cells, the microfluidic operations require 2–6 h; the entire process, including preparation of microfluidic devices and mammalian cells, requires 5–7 d. PMID:23558786

Towards understanding and managing the learning process in mail sorting.

PubMed

Berglund, M; Karltun, A

2012-01-01

This paper was based on case study research at the Swedish Mail Service Division and it addresses learning time to sort mail at new districts and means to support the learning process on an individual as well as organizational level. The study population consisted of 46 postmen and one team leader in the Swedish Mail Service Division. Data were collected through measurements of time for mail sorting, interviews and a focus group. The study showed that learning to sort mail was a much more complex process and took more time than expected by management. Means to support the learning process included clarification of the relationship between sorting and the topology of the district, a good work environment, increased support from colleagues and management, and a thorough introduction for new postmen. The identified means to support the learning process require an integration of human, technological and organizational aspects. The study further showed that increased operations flexibility cannot be reinforced without a systems perspective and thorough knowledge about real work activities and that ergonomists can aid businesses to acquire this knowledge.

Subscale Ship Airwake Studies Using Novel Vortex Flow Devices with Smoke, Laser-Vapor-Screen and Particle Image Velocimetry

NASA Technical Reports Server (NTRS)

Lamar, John E.; Landman, Drew; Swift, Russell S.; Parikh, Paresh C.

2007-01-01

Ships produce vortices and air-wakes while either underway or stationary in a wind. These flow fields can be detrimental to the conduction of air operations in that they can adversely impact the air vehicles and flight crews. There are potential solutions to these problems for both frigates/destroyers and carriers through the use of novel vortex flow or flow control devices. This appendix highlights several devices which may have application and points out that traditional wind-tunnel testing using smoke, laser-vapor screen, and Particle Image Velocimetry can be useful in sorting out the effectiveness of different devices.

Engineering human ventricular heart muscles based on a highly efficient system for purification of human pluripotent stem cell-derived ventricular cardiomyocytes.

PubMed

Li, Bin; Yang, Hui; Wang, Xiaochen; Zhan, Yongkun; Sheng, Wei; Cai, Huanhuan; Xin, Haoyang; Liang, Qianqian; Zhou, Ping; Lu, Chao; Qian, Ruizhe; Chen, Sifeng; Yang, Pengyuan; Zhang, Jianyi; Shou, Weinian; Huang, Guoying; Liang, Ping; Sun, Ning

2017-09-29

Most infarctions occur in the left anterior descending coronary artery and cause myocardium damage of the left ventricle. Although current pluripotent stem cells (PSCs) and directed cardiac differentiation techniques are able to generate fetal-like human cardiomyocytes, isolation of pure ventricular cardiomyocytes has been challenging. For repairing ventricular damage, we aimed to establish a highly efficient purification system to obtain homogeneous ventricular cardiomyocytes and prepare engineered human ventricular heart muscles in a dish. The purification system used TALEN-mediated genomic editing techniques to insert the neomycin or EGFP selection marker directly after the myosin light chain 2 (MYL2) locus in human pluripotent stem cells. Purified early ventricular cardiomyocytes were estimated by immunofluorescence, fluorescence-activated cell sorting, quantitative PCR, microelectrode array, and patch clamp. In subsequent experiments, the mixture of mature MYL2-positive ventricular cardiomyocytes and mesenchymal cells were cocultured with decellularized natural heart matrix. Histological and electrophysiology analyses of the formed tissues were performed 2 weeks later. Human ventricular cardiomyocytes were efficiently isolated based on the purification system using G418 or flow cytometry selection. When combined with the decellularized natural heart matrix as the scaffold, functional human ventricular heart muscles were prepared in a dish. These engineered human ventricular muscles can be great tools for regenerative therapy of human ventricular damage as well as drug screening and ventricular-specific disease modeling in the future.

Isolation and characterisation of mesenchymal stem/stromal cells in the ovine endometrium.

PubMed

Letouzey, Vincent; Tan, Ker Sin; Deane, James A; Ulrich, Daniela; Gurung, Shanti; Ong, Y Rue; Gargett, Caroline E

2015-01-01

Mesenchymal stem/stromal cells (MSC) were recently discovered in the human endometrium. These cells possess key stem cell properties and show promising results in small animal models when used for preclinical tissue engineering studies. A small number of surface markers have been identified that enrich for MSC from bone marrow and human endometrium, including the Sushi Domain-containing 2 (SUSD2; W5C5) and CD271 markers. In preparation for developing a large animal preclinical model for urological and gynecological tissue engineering applications we aimed to identify and characterise MSC in ovine endometrium and determine surface markers to enable their prospective isolation. Ovine endometrium was obtained from hysterectomised ewes following progesterone synchronisation, dissociated into single cell suspensions and tested for MSC surface markers and key stem cell properties. Purified stromal cells were obtained by flow cytometry sorting with CD49f and CD45 to remove epithelial cells and leukocytes respectively, and MSC properties investigated. There was a small population CD271+ stromal cells (4.5 ± 2.3%) in the ovine endometrium. Double labelling with CD271 and CD49f showed that the sorted CD271+CD49f- stromal cell population possessed significantly higher cloning efficiency, serial cloning capacity and a qualitative increased ability to differentiate into 4 mesodermal lineages (adipocytic, smooth muscle, chondrocytic and osteoblastic) than CD271-CD49f- cells. Immunolabelling studies identified an adventitial perivascular location for ovine endometrial CD271+ cells. This is the first study to characterise MSC in the ovine endometrium and identify a surface marker profile identifying their location and enabling their prospective isolation. This knowledge will allow future preclinical studies with a large animal model that is well established for pelvic organ prolapse research.

The human squamous oesophagus has widespread capacity for clonal expansion from cells at diverse stages of differentiation.

PubMed

Barbera, Mariagnese; di Pietro, Massimiliano; Walker, Elaine; Brierley, Charlotte; MacRae, Shona; Simons, Benjamin D; Jones, Phil H; Stingl, John; Fitzgerald, Rebecca C

2015-01-01

Knowledge of the cellular mechanisms involved in homeostasis of human squamous oesophagus in the steady state and following chronic injury is limited. We aimed to better understand these mechanisms by using a functional 3D approach. Proliferation, mitosis and the expression of progenitor lineage markers were assessed in normal squamous oesophagus from 10 patients by immunofluorescence on 3D epithelial whole mounts. Cells expressing differential levels of epithelial and progenitor markers were isolated using flow cytometry sorting and characterised by qPCR and IF. Their self-renewing potential was investigated by colony forming cells assays and in vitro organotypic culture models. Proliferation and mitotic activity was highest in the interpapillary basal layer and decreased linearly towards the tip of the papilla (p<0.0001). The orientation of mitosis was random throughout the basal layer, and asymmetric divisions were not restricted to specific cell compartments. Cells sorted into distinct populations based on the expression of epithelial and progenitor cell markers (CD34 and EpCAM) showed no difference in self-renewal in 2D culture, either as whole populations or as single cells. In 3D organotypic cultures, all cell subtypes were able to recapitulate the architecture of the tissue of origin and the main factor determining the success of the 3D culture was the number of cells plated, rather than the cell type. Oesophageal epithelial cells demonstrate remarkable plasticity for self-renewal. This situation could be viewed as an ex vivo wounding response and is compatible with recent findings in murine models. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

CD47 is an adverse prognostic factor and a therapeutic target in gastric cancer

PubMed Central

Yoshida, Kazumichi; Tsujimoto, Hironori; Matsumura, Kouji; Kinoshita, Manabu; Takahata, Risa; Matsumoto, Yusuke; Hiraki, Shuichi; Ono, Satoshi; Seki, Shuhji; Yamamoto, Junji; Hase, Kazuo

2015-01-01

CD47 is an antiphagocytic molecule that acts via ligation to signal regulatory protein alpha on phagocytes; its enhanced expression and therapeutic targeting have recently been reported for several malignancies. However, CD47 expression in gastric cancer is not well documented. Immunohistochemical expression of CD47 in surgical specimens was investigated. Expression of CD47 and CD44, a known gastric cancer stem cell marker, were investigated in gastric cancer cell lines by flow cytometry. MKN45 and MKN74 gastric cancer cells were sorted by fluorescence-activated cell sorting according to CD44 and CD47 expression levels, and their in vitro proliferation, spheroid-forming capacity, and in vivo tumorigenicity were studied. In vitro phagocytosis of cancer cells by human macrophages in the presence of a CD47 blocking monoclonal antibody (B6H12) and the survival of immunodeficient mice intraperitoneally engrafted with MKN45 cells and B6H12 were compared to experiments using control antibodies. Immunohistochemistry of the clinical specimens indicated that CD47 was positive in 57 out of 115 cases, and its positivity was an independent adverse prognostic factor. Approximately 90% of the MKN45 and MKN74 cells expressed CD47 and CD44. CD47hi gastric cancer cells showed significantly higher proliferation and spheroid colony formation than CD47lo, and CD44hiCD47hi cells showed the highest proliferation in vitro and tumorigenicity in vivo. B6H12 significantly enhanced in vitro phagocytosis of cancer cells by human macrophages and prolonged the survival of intraperitoneal cancer dissemination in mice compared to control antibodies. In conclusion, CD47 is an adverse prognostic factor and promising therapeutic target in gastric cancer. PMID:26077800

Study of different cross-shaped microchannels affecting thermal-bubble-actuated microparticle manipulation

NASA Astrophysics Data System (ADS)

Li, Weichen; Tsou, Chingfu

2015-10-01

This paper presents a thermal-bubble-actuated microfluidic chip with cross-shaped microchannels for evaluating the effect of different microchannel designs on microparticle manipulation. Four cross-shaped microchannel designs, with orthogonal, misaligned, skewed, and antiskewed types, were proposed in this study. The thermal bubble micropump, which is based on a resistive bulk microheater, was used to drive fluid transportation, and it can be realized using a simple microfabrication process with a silicon-on-isolator wafer. Using commercial COMSOL software, the flow profiles of microfluidics in various cross-shaped microchannels were simulated qualitatively under different pumping pressures. Microbeads, with a diameter of 20 μm, manipulated in four cross-shaped microchannels, were also implemented in this experiment. The results showed that a skewed microchannel design has a higher sorting rate compared with orthogonal, misaligned, and antiskewed microchannels because its flow velocity in the main microchannel is significantly reduced by pumping pressure. Typically, the successful sorting rate for this type of skewed microchannel can reach 30% at a pumping frequency of 100 Hz.

The In Vitro Differentiation of GDNF Gene-Engineered Amniotic Fluid-Derived Stem Cells into Renal Tubular Epithelial-Like Cells.

PubMed

Lu, Ying; Wang, Zhuojun; Chen, Lu; Wang, Jia; Li, Shulin; Liu, Caixia; Sun, Dong

2018-05-01

Amniotic fluid is an alternative source of stem cells, and human amniotic fluid-derived stem cells (AFSCs) obtained from a small amount of amniotic fluid collected during the second trimester represent a novel source for use in regenerative medicine. These AFSCs are characterized by lower diversity, a higher proliferation rate, and a wider differentiation capability than adult mesenchymal stem cells. AFSCs are selected based on the cell surface marker c-kit receptor (CD117) using immunomagnetic sorting. Glial cell line-derived neurotrophic factor (GDNF) is expressed during early kidney development and regulates the proliferation and differentiation of stem cells in vitro. In this study, c-kit-sorted AFSCs were induced toward osteogenic or adipogenic differentiation. AFSCs engineered via the insertion of GDNF were cocultured with mouse renal tubular epithelial cells (mRTECs), which were preconditioned by hypoxia-reoxygenation in vitro. After coculture for 8 days, AFSCs differentiation into epithelial-like cells was evaluated by performing immunofluorescence, flow cytometry, and quantitative real-time polymerase chain reaction to identify cells expressing the renal epithelial markers, cytokeratin 18 (CK18), E-cadherin, aquaporin-1 (AQP1), and paired box 2 gene (Pax2). The GDNF gene enhanced AFSCs differentiation into RTECs. AFSCs possess self-renewal ability and multiple differentiation potential and thus represent a new source of stem cells.

Human-Induced Pluripotent Stem Cell-Derived Mesenchymal Stem Cells as an Individual-Specific and Renewable Source of Adult Stem Cells.

PubMed

Sequiera, Glen Lester; Saravanan, Sekaran; Dhingra, Sanjiv

2017-01-01

This chapter deals with the employment of human-induced pluripotent stem cells (hiPSCs) as a candidate to differentiate into mesenchymal stem cells (MSCs). This would enable to help establish a regular source of human MSCs with the aim of avoiding the problems associated with procuring the MSCs either from different healthy individuals or patients, limited extraction potentials, batch-to-batch variations or from diverse sources such as bone marrow or adipose tissue. The procedures described herein allow for a guided and ensured approach for the regular maintenance of hiPSCs and their subsequent differentiation into MSCs using the prescribed medium. Subsequently, an easy protocol for the successive isolation and purification of the hiPSC-differentiated MSCs is outlined, which is carried out through passaging and can be further sorted through flow cytometry. Further, the maintenance and expansion of the resultant hiPSC-differentiated MSCs using appropriate characterization techniques, i.e., Reverse-transcription PCR and immunostaining is also elaborated. The course of action has been deliberated keeping in mind the awareness and the requisites available to even beginner researchers who mostly have access to regular consumables and medium components found in the general laboratory.

Extremal flows in Wasserstein space

NASA Astrophysics Data System (ADS)

Conforti, Giovanni; Pavon, Michele

2018-06-01

We develop an intrinsic geometric approach to the calculus of variations in the Wasserstein space. We show that the flows associated with the Schrödinger bridge with general prior, with optimal mass transport, and with the Madelung fluid can all be characterized as annihilating the first variation of a suitable action. We then discuss the implications of this unified framework for stochastic mechanics: It entails, in particular, a sort of fluid-dynamic reconciliation between Bohm's and Nelson's stochastic mechanics.

RPAs: Revolution or Retrogression?

DTIC Science & Technology

2010-04-01

be a “never again” syndrome with respect to a Vietnam sort of war. The concept was that somehow the North Atlantic Treaty Organization (NATO) would...Washington, DC: Air Force History and Museums Program, 1998), 58. 64. Michael P. Noonan , “Disrupting the Foreign Fighter Flow,” Foreign Policy Research

Early Detection of NSCLC Using Stromal Markers in Peripheral Blood

DTIC Science & Technology

2015-09-01

post- surgery patients, and COPD patients Subtask 2: Flow cytometry sorting of circulating myeloid cells. Subtask 3: RNA-Sequencing Subtask 4: RNA...recruitment including pre- and post- surgery patients, and COPD patients During this reporting period, we have recruited 23 NSCLC patients and collected

Sorting cells of the microalga Chlorococcum littorale with increased triacylglycerol productivity.

PubMed

Cabanelas, Iago Teles Dominguez; van der Zwart, Mathijs; Kleinegris, Dorinde M M; Wijffels, René H; Barbosa, Maria J

2016-01-01

Despite extensive research in the last decades, microalgae are still only economically feasible for high valued markets. Strain improvement is a strategy to increase productivities, hence reducing costs. In this work, we focus on microalgae selection: taking advantage of the natural biological variability of species to select variations based on desired characteristics. We focused on triacylglycerol (TAG), which have applications ranging from biodiesel to high-value omega-3 fatty-acids. Hence, we demonstrated a strategy to sort microalgae cells with increased TAG productivity. 1. We successfully identified sub-populations of cells with increased TAG productivity using Fluorescence assisted cell sorting (FACS). 2. We sequentially sorted cells after repeated cycles of N-starvation, resulting in five sorted populations (S1-S5). 3. The comparison between sorted and original populations showed that S5 had the highest TAG productivity [0.34 against 0.18 g l(-1) day(-1) (original), continuous light]. 4. Original and S5 were compared in lab-scale reactors under simulated summer conditions confirming the increased TAG productivity of S5 (0.4 against 0.2 g l(-1) day(-1)). Biomass composition analyses showed that S5 produced more biomass under N-starvation because of an increase only in TAG content and, flow cytometry showed that our selection removed cells with lower efficiency in producing TAGs. All combined, our results present a successful strategy to improve the TAG productivity of Chlorococcum littorale, without resourcing to genetic manipulation or random mutagenesis. Additionally, the improved TAG productivity of S5 was confirmed under simulated summer conditions, highlighting the industrial potential of S5 for microalgal TAG production.

Photothermally controlled Marangoni flow around a micro bubble

NASA Astrophysics Data System (ADS)

Namura, Kyoko; Nakajima, Kaoru; Kimura, Kenji; Suzuki, Motofumi

2015-01-01

We have experimentally investigated the control of Marangoni flow around a micro bubble using photothermal conversion. Using a focused laser spot acting as a highly localized heat source on Au nanoparticles/dielectric/Ag mirror thin film enables us to create a micro bubble and to control the temperature gradient around the bubble at a micrometer scale. When we irradiate the laser next to the bubble, a strong main flow towards the bubble and two symmetric rotation flows on either side of it develop. The shape of this rotation flow shows a significant transformation depending on the relative position of the bubble and the laser spot. Using this controllable rotation flow, we have demonstrated sorting of the polystyrene spheres with diameters of 2 μm and 0.75 μm according to their size.

The enormous Chillos Valley Lahar: An ash-flow-generated debris flow from Cotopaxi Volcano, Ecuador

USGS Publications Warehouse

Mothes, P.A.; Hall, M.L.; Janda, R.J.

1998-01-01

The Chillos Valley Lahar (CVL), the largest Holocene debris flow in area and volume as yet recognized in the northern Andes, formed on Cotopaxi volcano's north and northeast slopes and descended river systems that took it 326 km north-northwest to the Pacific Ocean and 130+ km east into the Amazon basin. In the Chillos Valley, 40 km downstream from the volcano, depths of 80-160 m and valley cross sections up to 337000m2 are observed, implying peak flow discharges of 2.6-6.0 million m3/s. The overall volume of the CVL is estimated to be ???3.8 km3. The CVL was generated approximately 4500 years BP by a rhyolitic ash flow that followed a small sector collapse on the north and northeast sides of Cotopaxi, which melted part of the volcano's icecap and transformed rapidly into the debris flow. The ash flow and resulting CVL have identical components, except for foreign fragments picked up along the flow path. Juvenile materials, including vitric ash, crystals, and pumice, comprise 80-90% of the lahar's deposit, whereas rhyolitic, dacitic, and andesitic lithics make up the remainder. The sand-size fraction and the 2- to 10-mm fraction together dominate the deposit, constituting ???63 and ???15 wt.% of the matrix, respectively, whereas the silt-size fraction averages less than ???10 wt.% and the clay-size fraction less than 0.5 wt.%. Along the 326-km runout, these particle-size fractions vary little, as does the sorting coefficient (average = 2.6). There is no tendency toward grading or improved sorting. Limited bulking is recognized. The CVL was an enormous non-cohesive debris flow, notable for its ash-flow origin and immense volume and peak discharge which gave it characteristics and a behavior akin to large cohesive mudflows. Significantly, then, ash-flow-generated debris flows can also achieve large volumes and cover great areas; thus, they can conceivably affect large populated regions far from their source. Especially dangerous, therefore, are snowclad volcanoes with recent silicic ash-flow histories such as those found in the Andes and Alaska.

Unravelling the mystery of stem/progenitor cells in human breast milk.

PubMed

Fan, Yiping; Chong, Yap Seng; Choolani, Mahesh A; Cregan, Mark D; Chan, Jerry K Y

2010-12-28

Mammary stem cells have been extensively studied as a system to delineate the pathogenesis and treatment of breast cancer. However, research on mammary stem cells requires tissue biopsies which limit the quantity of samples available. We have previously identified putative mammary stem cells in human breast milk, and here, we further characterised the cellular component of human breast milk. We identified markers associated with haemopoietic, mesenchymal and neuro-epithelial lineages in the cellular component of human breast milk. We found 2.6 ± 0.8% (mean ± SEM) and 0.7 ± 0.2% of the whole cell population (WCP) were found to be CD133+ and CD34+ respectively, 27.8 ± 9.1% of the WCP to be positive for Stro-1 through flow-cytometry. Expressions of neuro-ectodermal stem cell markers such as nestin and cytokeratin 5 were found through reverse-transcription polymerase chain reaction (RT-PCR), and in 4.17 ± 0.2% and 0.9 ± 0.2% of the WCP on flow-cytometry. We also established the presence of a side-population (SP) (1.8 ± 0.4% of WCP) as well as CD133+ cells (1.7 ± 0.5% of the WCP). Characterisation of the sorted SP and non-SP, CD133+ and CD133- cells carried out showed enrichment of CD326 (EPCAM) in the SP cells (50.6 ± 8.6 vs 18.1 ± 6.0, P-value  = 0.02). However, culture in a wide range of in vitro conditions revealed the atypical behaviour of stem/progenitor cells in human breast milk; in that if they are present, they do not respond to established culture protocols of stem/progenitor cells. The identification of primitive cell types within human breast milk may provide a non-invasive source of relevant mammary cells for a wide-range of applications; even the possibility of banking one's own stem cell for every breastfeeding woman.

Unravelling the Mystery of Stem/Progenitor Cells in Human Breast Milk

PubMed Central

Fan, Yiping; Chong, Yap Seng; Choolani, Mahesh A.; Cregan, Mark D.; Chan, Jerry K. Y.

2010-01-01

Background Mammary stem cells have been extensively studied as a system to delineate the pathogenesis and treatment of breast cancer. However, research on mammary stem cells requires tissue biopsies which limit the quantity of samples available. We have previously identified putative mammary stem cells in human breast milk, and here, we further characterised the cellular component of human breast milk. Methodology/Principal Findings We identified markers associated with haemopoietic, mesenchymal and neuro-epithelial lineages in the cellular component of human breast milk. We found 2.6±0.8% (mean±SEM) and 0.7±0.2% of the whole cell population (WCP) were found to be CD133+ and CD34+ respectively, 27.8±9.1% of the WCP to be positive for Stro-1 through flow-cytometry. Expressions of neuro-ectodermal stem cell markers such as nestin and cytokeratin 5 were found through reverse-transcription polymerase chain reaction (RT-PCR), and in 4.17±0.2% and 0.9±0.2% of the WCP on flow-cytometry. We also established the presence of a side-population (SP) (1.8±0.4% of WCP) as well as CD133+ cells (1.7±0.5% of the WCP). Characterisation of the sorted SP and non-SP, CD133+ and CD133- cells carried out showed enrichment of CD326 (EPCAM) in the SP cells (50.6±8.6 vs 18.1±6.0, P-value  = 0.02). However, culture in a wide range of in vitro conditions revealed the atypical behaviour of stem/progenitor cells in human breast milk; in that if they are present, they do not respond to established culture protocols of stem/progenitor cells. Conclusions/Significance The identification of primitive cell types within human breast milk may provide a non-invasive source of relevant mammary cells for a wide-range of applications; even the possibility of banking one's own stem cell for every breastfeeding woman. PMID:21203434

Monocyte-derived alveolar macrophages drive lung fibrosis and persist in the lung over the life span

PubMed Central

Morales-Nebreda, Luisa; Cuda, Carla M.; Walter, James M.; Chen, Ching-I; Anekalla, Kishore R.; Joshi, Nikita; Williams, Kinola J.N.; Abdala-Valencia, Hiam; Yacoub, Tyrone J.; Chi, Monica; Gates, Khalilah; Homan, Philip J.; Soberanes, Saul; Dominguez, Salina; Saber, Rana; Hinchcliff, Monique; Marshall, Stacy A.; Bharat, Ankit; Berdnikovs, Sergejs; Bhorade, Sangeeta M.; Balch, William E.; Chandel, Navdeep S.; Jain, Manu; Ridge, Karen M.; Bagheri, Neda; Shilatifard, Ali

2017-01-01

Little is known about the relative importance of monocyte and tissue-resident macrophages in the development of lung fibrosis. We show that specific genetic deletion of monocyte-derived alveolar macrophages after their recruitment to the lung ameliorated lung fibrosis, whereas tissue-resident alveolar macrophages did not contribute to fibrosis. Using transcriptomic profiling of flow-sorted cells, we found that monocyte to alveolar macrophage differentiation unfolds continuously over the course of fibrosis and its resolution. During the fibrotic phase, monocyte-derived alveolar macrophages differ significantly from tissue-resident alveolar macrophages in their expression of profibrotic genes. A population of monocyte-derived alveolar macrophages persisted in the lung for one year after the resolution of fibrosis, where they became increasingly similar to tissue-resident alveolar macrophages. Human homologues of profibrotic genes expressed by mouse monocyte-derived alveolar macrophages during fibrosis were up-regulated in human alveolar macrophages from fibrotic compared with normal lungs. Our findings suggest that selectively targeting alveolar macrophage differentiation within the lung may ameliorate fibrosis without the adverse consequences associated with global monocyte or tissue-resident alveolar macrophage depletion. PMID:28694385

IDENTIFICATION AND CHARACTERIZATION OF INFECTIOUS AND NON-INFECTIOUS SUB-POPULATIONS OF ENCEPHALITIZOON INTESTINALIS SPORES PURIFIED FROM IN VITRO CELL CULTURE

EPA Science Inventory

Background: Encephalitizoon intestinalis spores were propagated in rabbit kidney (RK-13) cells and were purified using density gradient (Percoll [registered trademark]) centrifugation. Purified spores were enumeraged and aliquotted using flow cytometry with cell sorting for use...

Perceptions of Communication Patterns in Higher Education.

ERIC Educational Resources Information Center

Johnson, Karen A.

This study examined the perceptions of a cohort of students, faculty, staff, and administrators concerning the vertical and horizontal flow of information at a selected university. Seventeen students, six faculty, five staff, and six administrators participated in the study. Q-methodology served as the data analytic procedure and Q-sorts were…

An improved consensus linkage map of barley based on flow-sorted chromosomes and SNP markers

USDA-ARS?s Scientific Manuscript database

Recent advances in high-throughput genotyping have made it easier to combine information from different mapping populations into consensus genetic maps, which provide increased marker density and genome coverage compared to individual maps. Previously, a SNP-based genotyping platform was developed a...

Successful low dose insemination of flow cytometrically sorted Sika (Cervus nippon) sperm in Wapiti (Cervus elaphus).

PubMed

Gao, Q H; Wei, H J; Han, C M; Du, H Z; Zhang, Z G; Zhao, W G; Zhang, Y; Li, S

2010-03-01

The purpose of this study was to determine a practical method in Wapiti (Cervus elaphus) of using predetermined sexed Sika (Cervus nippon) semen. Semen was collected by electro-ejaculation from one stag of proven fertility and transported to the laboratory where it was retained as unsorted (control) or was separated into X- and Y-chromosome-bearing sperm using a modified high-speed cell sorter. Wapiti hinds (n=81) were inseminated into the uterus by rectum manipulation with 1 x 10(6) (X1 and Y1 group, respectively) or 2 x 10(6) (X2 and Y2 group, respectively) of sorted frozen-thawed and 1 x 10(7) non-sorted frozen-thawed (a commercial dose control) Sika motile sperm 60-66h after removal of intra-vaginal progesterone-impregnated CIDR devices and administration of 700IU of PMSG at the time of CIDR removal. The percentage of hinds calving after insemination was similar for X1 (38.5%), X2 (41.7%), Y1 (44.4%), Y2 (38.9%) groups (P>0.05), but higher for control (75%) treatment (P<0.05). Ultimately 15 out of the 16 Sika and Wapiti-hybrid calves produced by Wapiti hinds inseminated with Y-sorted sperm were male (93.7%) and 10/10 (100%) Sika and Wapiti-hybrid calves from hinds inseminated with X-sorted sperm were female. The sex ratio of the Sika and Wapiti-hybrid calves born to hinds inseminated with sex-sorted sperm deviated significantly (P<0.05) from 50% and 50.0% in the control group. All Sika and Wapiti-hybrid calves were born between 237 and 250d of gestation. Male and female calves in the control group had similar birth weights and weaning weights as calves from hinds inseminated with X- or Y-sorted sperm. In conclusion it can be said that normal Sika and Wapiti-hybrid calves of predicted sex can be produced after artificial insemination of Wapiti does with low numbers of sex-sorted cryopreserved Sika sperm.

Role of adaptor proteins and clathrin in the trafficking of human kidney anion exchanger 1 (kAE1) to the cell surface.

PubMed

Junking, Mutita; Sawasdee, Nunghathai; Duangtum, Natapol; Cheunsuchon, Boonyarit; Limjindaporn, Thawornchai; Yenchitsomanus, Pa-thai

2014-07-01

Kidney anion exchanger 1 (kAE1) plays an important role in acid-base homeostasis by mediating chloride/bicarbornate (Cl-/HCO3-) exchange at the basolateral membrane of α-intercalated cells in the distal nephron. Impaired intracellular trafficking of kAE1 caused by mutations of SLC4A1 encoding kAE1 results in kidney disease - distal renal tubular acidosis (dRTA). However, it is not known how the intracellular sorting and trafficking of kAE1 from trans-Golgi network (TGN) to the basolateral membrane occurs. Here, we studied the role of basolateral-related sorting proteins, including the mu1 subunit of adaptor protein (AP) complexes, clathrin and protein kinase D, on kAE1 trafficking in polarized and non-polarized kidney cells. By using RNA interference, co-immunoprecipitation, yellow fluorescent protein-based protein fragment complementation assays and immunofluorescence staining, we demonstrated that AP-1 mu1A, AP-3 mu1, AP-4 mu1 and clathrin (but not AP-1 mu1B, PKD1 or PKD2) play crucial roles in intracellular sorting and trafficking of kAE1. We also demonstrated colocalization of kAE1 and basolateral-related sorting proteins in human kidney tissues by double immunofluorescence staining. These findings indicate that AP-1 mu1A, AP-3 mu1, AP-4 mu1 and clathrin are required for kAE1 sorting and trafficking from TGN to the basolateral membrane of acid-secreting α-intercalated cells. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Magnetic-Activated Cell Sorting for the Fast and Efficient Separation of Human and Rodent Schwann Cells from Mixed Cell Populations.

PubMed

Ravelo, Kristine M; Andersen, Natalia D; Monje, Paula V

2018-01-01

To date, magnetic-activated cell sorting (MACS) remains a powerful method to isolate distinct cell populations based on differential cell surface labeling. Optimized direct and indirect MACS protocols for cell immunolabeling are presented here as methods to divest Schwann cell (SC) cultures of contaminating cells (specifically, fibroblast cells) and isolate SC populations at different stages of differentiation. This chapter describes (1) the preparation of single-cell suspensions from established human and rat SC cultures, (2) the design and application of cell selection strategies using SC-specific (p75 NGFR , O4, and O1) and fibroblast-specific (Thy-1) markers, and (3) the characterization of both the pre- and post-sorting cell populations. A simple protocol for the growth of hybridoma cell cultures as a source of monoclonal antibodies for cell surface immunolabeling of SCs and fibroblasts is provided as a cost-effective alternative for commercially available products. These steps allow for the timely and efficient recovery of purified SC populations without compromising the viability and biological activity of the cells.

Reliable Analysis of Single-Unit Recordings from the Human Brain under Noisy Conditions: Tracking Neurons over Hours

PubMed Central

Boström, Jan; Elger, Christian E.; Mormann, Florian

2016-01-01

Recording extracellulary from neurons in the brains of animals in vivo is among the most established experimental techniques in neuroscience, and has recently become feasible in humans. Many interesting scientific questions can be addressed only when extracellular recordings last several hours, and when individual neurons are tracked throughout the entire recording. Such questions regard, for example, neuronal mechanisms of learning and memory consolidation, and the generation of epileptic seizures. Several difficulties have so far limited the use of extracellular multi-hour recordings in neuroscience: Datasets become huge, and data are necessarily noisy in clinical recording environments. No methods for spike sorting of such recordings have been available. Spike sorting refers to the process of identifying the contributions of several neurons to the signal recorded in one electrode. To overcome these difficulties, we developed Combinato: a complete data-analysis framework for spike sorting in noisy recordings lasting twelve hours or more. Our framework includes software for artifact rejection, automatic spike sorting, manual optimization, and efficient visualization of results. Our completely automatic framework excels at two tasks: It outperforms existing methods when tested on simulated and real data, and it enables researchers to analyze multi-hour recordings. We evaluated our methods on both short and multi-hour simulated datasets. To evaluate the performance of our methods in an actual neuroscientific experiment, we used data from from neurosurgical patients, recorded in order to identify visually responsive neurons in the medial temporal lobe. These neurons responded to the semantic content, rather than to visual features, of a given stimulus. To test our methods with multi-hour recordings, we made use of neurons in the human medial temporal lobe that respond selectively to the same stimulus in the evening and next morning. PMID:27930664

Axodendritic sorting and pathological missorting of Tau are isoform-specific and determined by axon initial segment architecture.

PubMed

Zempel, Hans; Dennissen, Frank J A; Kumar, Yatender; Luedtke, Julia; Biernat, Jacek; Mandelkow, Eva-Maria; Mandelkow, Eckhard

2017-07-21

Subcellular mislocalization of the microtubule-associated protein Tau is a hallmark of Alzheimer disease (AD) and other tauopathies. Six Tau isoforms, differentiated by the presence or absence of a second repeat or of N-terminal inserts, exist in the human CNS, but their physiological and pathological differences have long remained elusive. Here, we investigated the properties and distributions of human and rodent Tau isoforms in primary forebrain rodent neurons. We found that the Tau diffusion barrier (TDB), located within the axon initial segment (AIS), controls retrograde (axon-to-soma) and anterograde (soma-to-axon) traffic of Tau. Tau isoforms without the N-terminal inserts were sorted efficiently into the axon. However, the longest isoform (2N4R-Tau) was partially retained in cell bodies and dendrites, where it accelerated spine and dendrite growth. The TDB (located within the AIS) was impaired when AIS components (ankyrin G, EB1) were knocked down or when glycogen synthase kinase-3β (GSK3β; an AD-associated kinase tethered to the AIS) was overexpressed. Using superresolution nanoscopy and live-cell imaging, we observed that microtubules within the AIS appeared highly dynamic, a feature essential for the TDB. Pathomechanistically, amyloid-β insult caused cofilin activation and F-actin remodeling and decreased microtubule dynamics in the AIS. Concomitantly with these amyloid-β-induced disruptions, the AIS/TDB sorting function failed, causing AD-like Tau missorting. In summary, we provide evidence that the human and rodent Tau isoforms differ in axodendritic sorting and amyloid-β-induced missorting and that the axodendritic distribution of Tau depends on AIS integrity. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Microfabrication and Test of a Three-Dimensional Polymer Hydro-focusing Unit for Flow Cytometry Applications

NASA Technical Reports Server (NTRS)

Yang, Ren; Feeback, Daniel L.; Wang, Wanjun

2004-01-01

This paper details a novel three-dimensional (3D) hydro-focusing micro cell sorter for micro flow cytometry applications. The unit was microfabricated by means of SU-8 3D lithography. The 3D microstructure for coaxial sheathing was designed, microfabricated, and tested. Three-dimensional hydro-focusing capability was demonstrated with an experiment to sort labeled tanned sheep erythrocytes (red blood cells). This polymer hydro-focusing microstructure is easily microfabricated and integrated with other polymer microfluidic structures.

Microfabrication and Test of a Three-Dimensional Polymer Hydro-Focusing Unit for Flow Cytometry Applications

NASA Technical Reports Server (NTRS)

Yang, Ren; Feedback, Daniel L.; Wang, Wanjun

2004-01-01

This paper details a novel three-dimensional (3D) hydro-focusing micro cell sorter for micro flow cytometry applications. The unit was micro-fabricated by means of SU-8 3D lithography. The 3D microstructure for coaxial sheathing was designed, micro-fabricated, and tested. Three-dimensional hydrofocusing capability was demonstrated with an experiment to sort labeled tanned sheep erythrocytes (red blood cells). This polymer hydro-focusing microstructure is easily micro-fabricated and integrated with other polymer microfluidic structures.

International Society for Analytical Cytology biosafety standard for sorting of unfixed cells.

PubMed

Schmid, Ingrid; Lambert, Claude; Ambrozak, David; Marti, Gerald E; Moss, Delynn M; Perfetto, Stephen P

2007-06-01

Cell sorting of viable biological specimens has become very prevalent in laboratories involved in basic and clinical research. As these samples can contain infectious agents, precautions to protect instrument operators and the environment from hazards arising from the use of sorters are paramount. To this end the International Society of Analytical Cytology (ISAC) took a lead in establishing biosafety guidelines for sorting of unfixed cells (Schmid et al., Cytometry 1997;28:99-117). During the time period these recommendations have been available, they have become recognized worldwide as the standard practices and safety precautions for laboratories performing viable cell sorting experiments. However, the field of cytometry has progressed since 1997, and the document requires an update. Initially, suggestions about the document format and content were discussed among members of the ISAC Biosafety Committee and were incorporated into a draft version that was sent to all committee members for review. Comments were collected, carefully considered, and incorporated as appropriate into a draft document that was posted on the ISAC web site to invite comments from the flow cytometry community at large. The revised document was then submitted to ISAC Council for review. Simultaneously, further comments were sought from newly-appointed ISAC Biosafety committee members. This safety standard for performing viable cell sorting experiments was recently generated. The document contains background information on the biohazard potential of sorting and the hazard classification of infectious agents as well as recommendations on (1) sample handling, (2) operator training and personal protection, (3) laboratory design, (4) cell sorter set-up, maintenance, and decontamination, and (5) testing the instrument for the efficiency of aerosol containment. This standard constitutes an updated and expanded revision of the 1997 biosafety guideline document. It is intended to provide laboratories involved in cell sorting with safety practices that take into account the enhanced hazard potential of high-speed sorting. Most importantly, it states that droplet-based sorting of infectious or hazardous biological material requires a higher level of containment than the one recommended for the risk group classification of the pathogen. The document also provides information on safety features of novel instrumentation, new options for personal protective equipment, and recently developed methods for testing the efficiency of aerosol containment.

Non-linear optical measurements using a scanned, Bessel beam

NASA Astrophysics Data System (ADS)

Collier, Bradley B.; Awasthi, Samir; Lieu, Deborah K.; Chan, James W.

2015-03-01

Oftentimes cells are removed from the body for disease diagnosis or cellular research. This typically requires fluorescent labeling followed by sorting with a flow cytometer; however, possible disruption of cellular function or even cell death due to the presence of the label can occur. This may be acceptable for ex vivo applications, but as cells are more frequently moving from the lab to the body, label-free methods of cell sorting are needed to eliminate these issues. This is especially true of the growing field of stem cell research where specialized cells are needed for treatments. Because differentiation processes are not completely efficient, cells must be sorted to eliminate any unwanted cells (i.e. un-differentiated or differentiated into an unwanted cell type). In order to perform label-free measurements, non-linear optics (NLO) have been increasingly utilized for single cell analysis because of their ability to not disrupt cellular function. An optical system was developed for the measurement of NLO in a microfluidic channel similar to a flow cytometer. In order to improve the excitation efficiency of NLO, a scanned Bessel beam was utilized to create a light-sheet across the channel. The system was tested by monitoring twophoton fluorescence from polystyrene microbeads of different sizes. Fluorescence intensity obtained from light-sheet measurements were significantly greater than measurements made using a static Gaussian beam. In addition, the increase in intensity from larger sized beads was more evident for the light-sheet system.

Evaluating multiple criteria for species delimitation: an empirical example using Hawaiian palms (Arecaceae: Pritchardia)

PubMed Central

2012-01-01

Background Robust species delimitations are fundamental for conservation, evolutionary, and systematic studies, but they can be difficult to estimate, particularly in rapid and recent radiations. The consensus that species concepts aim to identify evolutionarily distinct lineages is clear, but the criteria used to distinguish evolutionary lineages differ based on the perceived importance of the various characteristics of evolving populations. We examined three different species-delimitation criteria (monophyly, absence of genetic intermediates, and diagnosability) to determine whether currently recognized species of Hawaiian Pritchardia are distinct lineages. Results Data from plastid and nuclear genes, microsatellite loci, and morphological characters resulted in various levels of lineage subdivision that were likely caused by differing evolutionary rates between data sources. Additionally, taxonomic entities may be confounded because of the effects of incomplete lineage sorting and/or gene flow. A coalescent species tree was largely congruent with the simultaneous analysis, consistent with the idea that incomplete lineage sorting did not mislead our results. Furthermore, gene flow among populations of sympatric lineages likely explains the admixture and lack of resolution between those groups. Conclusions Delimiting Hawaiian Pritchardia species remains difficult but the ability to understand the influence of the evolutionary processes of incomplete lineage sorting and hybridization allow for mechanisms driving species diversity to be inferred. These processes likely extend to speciation in other Hawaiian angiosperm groups and the biota in general and must be explicitly accounted for in species delimitation. PMID:22353848

Urban adolescent high-risk sexual behavior: corroboration of focus group discussions through pile-sorting. The AIDS Youth Research Team.

PubMed

Stanton, B F; Aronson, R; Borgatti, S; Galbraith, J; Feigelman, S

1993-01-01

Risk activities for acquisition of the human immunodeficiency virus (HIV) remain prevalent among urban adolescents. While interdisciplinary approaches to examine the variables contributing to risk/protective behaviors have been promoted, strategies for such explorations require further formulation. Recently we employed focus group discussions to explore factors placing urban adolescents at risk for engaging in HIV risk behaviors. The focus group format enables substantial interaction on a topic in a limited time period, but does not always provide expression of the full range of behavioral options. In this study we investigated the use of pile-sorts for confirmation of impressions from focus group discussions among 57 urban youths aged 10-14. The pile-sorts revealed some support for most of the views expressed in the group discussions. However, the sorts revealed more variability in views than was expressed in the group discussions. Substantial gender and age-based differences in perceptions were revealed with potentially important intervention implications.

Mathematical modelling of the transport of a poorly sorted granular mixture as a debris-flow. The case of Madeira Island torrential floods in 2010

NASA Astrophysics Data System (ADS)

Ferreira, Rui M. L.; Oliveira, Rodrigo P.; Conde, Daniel

2016-04-01

On the 20th February 2010, heavy rainfall was registered at Madeira Island, North Atlantic. Stony debris flows, mudflows and mudslides ensued causing severe property loss, 1.5 m thick sediment deposits at downtown Funchal including 16th century monuments, and a death toll of 47 lives. Debris-flow fronts propagated downstream while carrying very high concentrations of solid material. These two-phase solid-fluid flows were responsible for most of the infrastructural damage across the island, due to their significantly increased mass and momentum. The objective of the present modelling work is to validate a 2DH model for torrential flows featuring the transport and interaction of several size fractions of a poorly-sorted granular mixture typical of stony debris flow in Madeira. The module for the transport of poorly-sorted material was included in STAV-2D (CERIS-IST), a shallow-water and morphology solver based on a finite-volume method using a flux-splitting technique featuring a reviewed Roe-Riemann solver, with appropriate source-term formulations to ensure full conservativeness. STAV-2D also includes formulations of flow resistance and bedload transport adequate for debris-flows with natural mobile beds (Ferreira et al., 2009) and has been validated with both theoretical solutions and laboratory data (Soares-Frazão et al., 2012; Canelas et al., 2013). The modelling of the existing natural and built environment is fully explicit. All buildings, streets and channels are accurately represented within the mesh geometry. Such detail is relevant for the reliability of the validation using field data, since the major sedimentary deposits within the urban meshwork of Funchal were identified and characterized in terms of volume and grain size distribution during the aftermath of the 20th February of 2010 event. Indeed, the measure of the quality of the numerical results is the agreement between simulated and estimated volume of deposited sediment and between estimated and modelled grain-size distribution of the deposits. The formulations expressing closures for size fraction interaction and active layer dynamics are discussed. The simulation tool resulting from this modelling effort is expected to help the establishment of new methodologies and parameters for hydraulic design and hazard assessment in the Island of Madeira. Acknowledgements This work was partially funded by FEDER, program COMPETE, and by national funds through Portuguese Foundation for Science and Technology (FCT) project RECI/ECM-HID/0371/2012. References Canelas, R.; Murillo, J. & Ferreira, R.M.L. (2013). Two-dimensional depth-averaged modelling of dambreak flows over mobile beds. Journal of Hydraulic Research, 51(4), 392-407. Ferreira, R. M. L., Franca, M. J., Leal, J. G., & Cardoso, A. H. (2009). Mathematical modelling of shallow flows: Closure models drawn from grain-scale mechanics of sediment transport and flow hydrodynamics. Canadian Journal of Civil Engineering, 36(10), 1605-1621. Soares-Frazão and IAHR Working group for Dam-break flows over mobile beds (2012) Dam-break flows over mobile beds: Experiments and benchmark tests for numerical models. Journal of Hydraulic Research, 50(4), 364-375. DOI: 10.1080/00221686.2012.689682

Investigation of zinc oxide particles in cosmetic products by means of centrifugal and asymmetrical flow field-flow fractionation.

PubMed

Sogne, Vanessa; Meier, Florian; Klein, Thorsten; Contado, Catia

2017-09-15

The dimensional characterization of insoluble, inorganic particles, such as zinc oxide ZnO, dispersed in cosmetic or pharmaceutical formulations, is of great interest considering the current need of declaring the possible presence of nanomaterials on the label of commercial products. This work compares the separation abilities of Centrifugal- and Asymmetrical Flow Field-Flow Fractionation techniques (CF3 and AF4, respectively), equipped with UV-vis, MALS and DLS detectors, in size sorting ZnO particles, both as pristine powders and after their extraction from cosmetic matrices. ZnO particles, bare and superficially modified with triethoxycaprylyl silane, were used as test materials. To identify the most suitable procedure necessary to isolate the ZnO particles from the cosmetic matrix, two O/W and two W/O emulsions were formulated on purpose. The suspensions, containing the extracted particles ZnO, were separated by both Field-Flow Fractionation (FFF) techniques to establish a common analysis protocol, applicable for the analysis of ZnO particles extracted from three commercial products, sold in Europe for the baby skin care. Key aspects of this study were the selection of an appropriate dispersing agent enabling the particles to stay in stable suspensions (>24h)and the use of multiple detectors (UV-vis, MALS and DLS) coupled on-line with the FFF channels, to determine the particle dimensions without using the retention parameters. Between the two FFF techniques, CF3 revealed to be the most robust one, able to sort all suspensions created in this work. Copyright © 2017 Elsevier B.V. All rights reserved.

Capture of Fluorescence Decay Times by Flow Cytometry

PubMed Central

Naivar, Mark A.; Jenkins, Patrick; Freyer, James P.

2012-01-01

In flow cytometry, the fluorescence decay time of an excitable species has been largely underutilized and is not likely found as a standard parameter on any imaging cytometer, sorting, or analyzing system. Most cytometers lack fluorescence lifetime hardware mainly owing to two central issues. Foremost, research and development with lifetime techniques has lacked proper exploitation of modern laser systems, data acquisition boards, and signal processing techniques. Secondly, a lack of enthusiasm for fluorescence lifetime applications in cells and with bead-based assays has persisted among the greater cytometry community. In this unit, we describe new approaches that address these issues and demonstrate the simplicity of digitally acquiring fluorescence relaxation rates in flow. The unit is divided into protocol and commentary sections in order to provide a most comprehensive discourse on acquiring the fluorescence lifetime with frequency-domain methods. The unit covers (i) standard fluorescence lifetime acquisition (protocol-based) with frequency-modulated laser excitation, (ii) digital frequency-domain cytometry analyses, and (iii) interfacing fluorescence lifetime measurements onto sorting systems. Within the unit is also a discussion on how digital methods are used for aliasing in order to harness higher frequency ranges. Also, a final discussion is provided on heterodyning and processing of waveforms for multi-exponential decay extraction. PMID:25419263

PcapDB: Search Optimized Packet Capture, Version 0.1.0.0

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ferrell, Paul; Steinfadt, Shannon

PcapDB is a packet capture system designed to optimize the captured data for fast search in the typical (network incident response) use case. The technology involved in this software has been submitted via the IDEAS system and has been filed as a provisional patent. It includes the following primary components: capture: The capture component utilizes existing capture libraries to retrieve packets from network interfaces. Once retrieved the packets are passed to additional threads for sorting into flows and indexing. The sorted flows and indexes are passed to other threads so that they can be written to disk. These components aremore » written in the C programming language. search: The search components provide a means to find relevant flows and the associated packets. A search query is parsed and represented as a search tree. Various search commands, written in C, are then used resolve this tree into a set of search results. The tree generation and search execution management components are written in python. interface: The PcapDB web interface is written in Python on the Django framework. It provides a series of pages, API's, and asynchronous tasks that allow the user to manage the capture system, perform searches, and retrieve results. Web page components are written in HTML,CSS and Javascript.« less

Precision toxicology based on single cell sequencing: an evolving trend in toxicological evaluations and mechanism exploration.

PubMed

Zhang, Boyang; Huang, Kunlun; Zhu, Liye; Luo, Yunbo; Xu, Wentao

2017-07-01

In this review, we introduce a new concept, precision toxicology: the mode of action of chemical- or drug-induced toxicity can be sensitively and specifically investigated by isolating a small group of cells or even a single cell with typical phenotype of interest followed by a single cell sequencing-based analysis. Precision toxicology can contribute to the better detection of subtle intracellular changes in response to exogenous substrates, and thus help researchers find solutions to control or relieve the toxicological effects that are serious threats to human health. We give examples for single cell isolation and recommend laser capture microdissection for in vivo studies and flow cytometric sorting for in vitro studies. In addition, we introduce the procedures for single cell sequencing and describe the expected application of these techniques to toxicological evaluations and mechanism exploration, which we believe will become a trend in toxicology.

Pressure and kinetic energy transport across the cavity mouth in resonating cavities.

PubMed

Bailey, Peter Roger; Abbá, Antonella; Tordella, Daniela

2013-01-01

Basic properties of the incompressible fluid motion in a rectangular cavity located along one wall of a plane channel are considered. For Mach numbers of the order of 1×10(-3) and using the incompressible formulation, we look for observable properties that can be associated with acoustic emission, which is normally observed in this kind of flow beyond a critical value of Reynolds number. The focus is put on the energy dynamics, in particular on the accumulation of energy in the cavity which takes place in the form of pressure and kinetic energy. By increasing the external forcing, we observe that the pressure flow into the cavity increases very rapidly, then peaks. However, the flow of kinetic energy, which is many orders of magnitude lower than that of the pressure, slowly but continuously grows. This leads to the pressure-kinetic energy flows ratio reaching an asymptotic state around the value 1000 for the channel bulk speed Reynolds number. It is interesting to note that beyond this threshold when the channel flow is highly unsteady-a sort of coarse turbulent flow-a sequence of high and low pressure spots is seen to depart from the downward cavity step in the statistically averaged field. The set of spots forms a steady spatial structure, a sort of damped standing wave stretching along the spanwise direction. The line joining the centers of the spots has an inclination similar to the normal to the fronts of density or pressure waves, which are observed to propagate from the downstream cavity edge in compressible cavity flows (at Mach numbers of 1×10(2) to 1×10(3), larger than those considered here). The wavelength of the standing wave is of the order of 1/8 the cavity depth and observed at the channel bulk Reynolds number, Re~2900. In this condition, the measure of the maximum pressure differences in the cavity field shows values of the order of 1×10(-1) Pa. We interpret the presence of this sort of wave as the fingerprint of the noise emission spots which could be observed in simulations where the full compressible formulation is used. The flow is studied by means of a sequence of direct numerical simulations in the Reynolds number range 25-2900. This allows the study to span across the steady laminar regime up to a first coarse turbulent regime. These results are confirmed by the good agreement with a set of laboratory results obtained at a Reynolds number one order of magnitude larger in a different cavity geometry [M. Gharib and A. Roshko, J. Fluid Mech. 177, 501 (1987)]. This leaves room for a certain degree of qualitative universality to be associated with the present findings.

Purification of human induced pluripotent stem cell-derived neural precursors using magnetic activated cell sorting.

PubMed

Rodrigues, Gonçalo M C; Fernandes, Tiago G; Rodrigues, Carlos A V; Cabral, Joaquim M S; Diogo, Maria Margarida

2015-01-01

Neural precursor (NP) cells derived from human induced pluripotent stem cells (hiPSCs), and their neuronal progeny, will play an important role in disease modeling, drug screening tests, central nervous system development studies, and may even become valuable for regenerative medicine treatments. Nonetheless, it is challenging to obtain homogeneous and synchronously differentiated NP populations from hiPSCs, and after neural commitment many pluripotent stem cells remain in the differentiated cultures. Here, we describe an efficient and simple protocol to differentiate hiPSC-derived NPs in 12 days, and we include a final purification stage where Tra-1-60+ pluripotent stem cells (PSCs) are removed using magnetic activated cell sorting (MACS), leaving the NP population nearly free of PSCs.

Tephra Blanket Record of a Violent Strombolian Eruption, Sunset Crater, Arizona

NASA Astrophysics Data System (ADS)

Wagner, K. D.; Ort, M. H.

2015-12-01

New fieldwork provides a detailed description of the widespread tephra of the ~1085 CE Sunset Crater eruption in the San Francisco Volcanic Field, Arizona, and refines interpretation of the eruptive sequence. The basal fine-lapilli tephra-fall-units I-IV are considered in detail. Units I and II are massive, with Unit I composed of angular to spiny clasts and II composed of more equant, oxidized clasts. Units III and IV have inversely graded bases and massive tops and are composed of angular to spiny iridescent and mixed iridescent and oxidized angular clasts, respectively. Xenoliths are rare in all units (<0.1%): sedimentary xenoliths are consistent with the known shallow country rock (Moenkopi and Kaibab Fms); magmatic xenoliths are pumiceous rhyolite mingled with basalt. Unit II is less sideromelane rich (20%) than Units I, III, and IV (60-80%). Above these units are at least two more coarse tephra-fall units. Variably preserved ash and fine-lapilli laminae cap the tephra blanket. This deposit is highly susceptible to reworking, and likely experienced both syn- and post-eruptive aeolian redistribution. It appears as either well sorted, alternating planar-parallel beds of ash and fine lapilli with rare wavy beds, or as cross- or planar-bedded ash. The tephra blanket as a whole is stratigraphically underlain by a fissure-fed lava flow and lapilli-fall units are intercalated with two larger flows. Mean grain size is coarsest in Unit I but coarsens in Units II-IV. Units I, III, and IV are moderately to poorly sorted with no skew. Unit II is better sorted and more coarse-skewed. Units I and III are slightly more platykurtic than II and IV. Without considering possible spatial effects introduced by dispersion patterns, bootstrap ANOVA confidence intervals suggest at least Unit II sorting and skewness are from distinct populations. Isopachs indicate Units I and II were associated with a 10-km-long fissure source. After or during Unit II's deposition, activity localized to Sunset Crater. Units III and IV were emplaced with waxing to sustained activity, and followed by at least two more sustained episodes. Two lava flows began effusing from the cone during this period and remained active after explosive activity ceased. Primary tephra deposition ended with a period of small discrete explosions.

Combining magnetic sorting of mother cells and fluctuation tests to analyze genome instability during mitotic cell aging in Saccharomyces cerevisiae.

PubMed

Patterson, Melissa N; Maxwell, Patrick H

2014-10-16

Saccharomyces cerevisiae has been an excellent model system for examining mechanisms and consequences of genome instability. Information gained from this yeast model is relevant to many organisms, including humans, since DNA repair and DNA damage response factors are well conserved across diverse species. However, S. cerevisiae has not yet been used to fully address whether the rate of accumulating mutations changes with increasing replicative (mitotic) age due to technical constraints. For instance, measurements of yeast replicative lifespan through micromanipulation involve very small populations of cells, which prohibit detection of rare mutations. Genetic methods to enrich for mother cells in populations by inducing death of daughter cells have been developed, but population sizes are still limited by the frequency with which random mutations that compromise the selection systems occur. The current protocol takes advantage of magnetic sorting of surface-labeled yeast mother cells to obtain large enough populations of aging mother cells to quantify rare mutations through phenotypic selections. Mutation rates, measured through fluctuation tests, and mutation frequencies are first established for young cells and used to predict the frequency of mutations in mother cells of various replicative ages. Mutation frequencies are then determined for sorted mother cells, and the age of the mother cells is determined using flow cytometry by staining with a fluorescent reagent that detects bud scars formed on their cell surfaces during cell division. Comparison of predicted mutation frequencies based on the number of cell divisions to the frequencies experimentally observed for mother cells of a given replicative age can then identify whether there are age-related changes in the rate of accumulating mutations. Variations of this basic protocol provide the means to investigate the influence of alterations in specific gene functions or specific environmental conditions on mutation accumulation to address mechanisms underlying genome instability during replicative aging.

Laboratory Experiments of Sand Ripples with Bimodal Size Distributions Under Asymmetric Oscillatory Flows

NASA Astrophysics Data System (ADS)

Calantoni, J.; Landry, B. J.

2010-12-01

The dynamics of sand ripples are vital to understanding numerous coastal processes such as sediment transport, wave attenuation, boundary layer development, and seafloor acoustic properties. Though significant laboratory research has been conducted to elucidate oscillatory flow morphodynamics under various constant and transient forcing conditions, the majority of the previous experiments were conducted only for beds with unimodal size distributions of sediment. Recent oscillatory flow experiments as well as past laboratory observations in uniform flows suggest that the presence of heterogeneous size sand compositions may significantly impact ripple morphology, resulting in a variety of observable effects (e.g., sediment sorting, bed armoring, and altered transport rates). Experimental work was conducted in a small oscillatory flow tunnel at the Sediment Dynamics Laboratory at the Naval Research Laboratory, Stennis Space Center. Three different monochromatic oscillatory forcings having velocity asymmetry were used to study sand ripple dynamics over five bimodal and two unimodal sediment beds. The seven different mixtures were composed using two unimodal sands of different colors (blue/white) and median grain diameters (d=0.31 mm / d=0.65 mm) combined into various mixtures by mass (i.e., 0/100; 10/90; 25/75; 50/50; 75/25; 90/10; and 100/0 which denotes mass percentage of blue/white sand, respectively, within each mixture). High-definition video of the sediment bed profile was acquired in conjunction with sediment trap measurements to resolve differences in ripple geometries, migration and evolution rates due to the different sediment mixtures and flow conditions. Observational findings clearly illustrate sediment stratification within ripple crests and the depth of the active mixing layer in addition to supporting sediment sorting in previous research on symmetric oscillatory flows in which the larger grains collect on top of ripple crests and smaller grains in the troughs. Preliminary quantitative results illuminate variations in equilibrium ripple geometry, ripple migration rates, and transition time scales between equilibrium states, all as functions of the sediment size mixture and flow forcing.

Feasibility study of stain-free classification of cell apoptosis based on diffraction imaging flow cytometry and supervised machine learning techniques.

PubMed

Feng, Jingwen; Feng, Tong; Yang, Chengwen; Wang, Wei; Sa, Yu; Feng, Yuanming

2018-06-01

This study was to explore the feasibility of prediction and classification of cells in different stages of apoptosis with a stain-free method based on diffraction images and supervised machine learning. Apoptosis was induced in human chronic myelogenous leukemia K562 cells by cis-platinum (DDP). A newly developed technique of polarization diffraction imaging flow cytometry (p-DIFC) was performed to acquire diffraction images of the cells in three different statuses (viable, early apoptotic and late apoptotic/necrotic) after cell separation through fluorescence activated cell sorting with Annexin V-PE and SYTOX® Green double staining. The texture features of the diffraction images were extracted with in-house software based on the Gray-level co-occurrence matrix algorithm to generate datasets for cell classification with supervised machine learning method. Therefore, this new method has been verified in hydrogen peroxide induced apoptosis model of HL-60. Results show that accuracy of higher than 90% was achieved respectively in independent test datasets from each cell type based on logistic regression with ridge estimators, which indicated that p-DIFC system has a great potential in predicting and classifying cells in different stages of apoptosis.

Reverse-engineering flow-cytometry gating strategies for phenotypic labelling and high-performance cell sorting.

PubMed

Becht, Etienne; Simoni, Yannick; Coustan-Smith, Elaine; Maximilien, Evrard; Cheng, Yang; Ng, Lai Guan; Campana, Dario; Newell, Evan

2018-06-21

Recent flow and mass cytometers generate datasets of dimensions 20 to 40 and a million single cells. From these, many tools facilitate the discovery of new cell populations associated with diseases or physiology. These new cell populations require the identification of new gating strategies, but gating strategies become exponentially more difficult to optimize when dimensionality increases. To facilitate this step, we developed Hypergate, an algorithm which given a cell population of interest identifies a gating strategy optimized for high yield and purity. Hypergate achieves higher yield and purity than human experts, Support Vector Machines and Random-Forests on public datasets. We use it to revisit some established gating strategies for the identification of innate lymphoid cells, which identifies concise and efficient strategies that allow gating these cells with fewer parameters but higher yield and purity than the current standards. For phenotypic description, Hypergate's outputs are consistent with fields' knowledge and sparser than those from a competing method. Hypergate is implemented in R and available on CRAN. The source code is published at http://github.com/ebecht/hypergate under an Open Source Initiative-compliant licence. Supplementary data are available at Bioinformatics online.

Three-dimensional microbubble streaming flows

NASA Astrophysics Data System (ADS)

Rallabandi, Bhargav; Marin, Alvaro; Rossi, Massimiliano; Kaehler, Christian; Hilgenfeldt, Sascha

2014-11-01

Streaming due to acoustically excited bubbles has been used successfully for applications such as size-sorting, trapping and focusing of particles, as well as fluid mixing. Many of these applications involve the precise control of particle trajectories, typically achieved using cylindrical bubbles, which establish planar flows. Using astigmatic particle tracking velocimetry (APTV), we show that, while this two-dimensional picture is a useful description of the flow over short times, a systematic three-dimensional flow structure is evident over long time scales. We demonstrate that this long-time three-dimensional fluid motion can be understood through asymptotic theory, superimposing secondary axial flows (induced by boundary conditions at the device walls) onto the two-dimensional description. This leads to a general framework that describes three-dimensional flows in confined microstreaming systems, guiding the design of applications that profit from minimizing or maximizing these effects.

Quantitative Detection of Low-Abundance Transcripts at Single-Cell Level in Human Epidermal Keratinocytes by Digital Droplet Reverse Transcription-Polymerase Chain Reaction.

PubMed

Auvré, Frédéric; Coutier, Julien; Martin, Michèle T; Fortunel, Nicolas O

2018-05-08

Genetic and epigenetic characterization of the large cellular diversity observed within tissues is essential to understanding the molecular networks that ensure the regulation of homeostasis, repair, and regeneration, but also pathophysiological processes. Skin is composed of multiple cell lineages and is therefore fully concerned by this complexity. Even within one particular lineage, such as epidermal keratinocytes, different immaturity statuses or differentiation stages are represented, which are still incompletely characterized. Accordingly, there is presently great demand for methods and technologies enabling molecular investigation at single-cell level. Also, most current methods used to analyze gene expression at RNA level, such as RT-qPCR, do not directly provide quantitative data, but rather comparative ratios between two conditions. A second important need in skin biology is thus to determine the number of RNA molecules in a given cell sample. Here, we describe a workflow that we have set up to meet these specific needs, by means of transcript quantification in cellular micro-samples using flow cytometry sorting and reverse transcription-digital droplet polymerase chain reaction. As a proof-of-principle, the workflow was tested for the detection of transcription factor transcripts expressed at low levels in keratinocyte precursor cells. A linear correlation was found between quantification values and keratinocyte input numbers in a low quantity range from 40 cells to 1 cell. Interpretable signals were repeatedly obtained from single-cell samples corresponding to estimated expression levels as low as 10-20 transcript copies per keratinocyte or less. The present workflow may have broad applications for the detection and quantification of low-abundance nucleic acid species in single cells, opening up perspectives for the study of cell-to-cell genetic and molecular heterogeneity. Interestingly, the process described here does not require internal references such as house-keeping gene expression, as it is initiated with defined cell numbers, precisely sorted by flow cytometry.

Alternative fluorescent labeling strategies for characterizing gram-positive pathogenic bacteria: Flow cytometry supported counting, sorting, and proteome analysis of Staphylococcus aureus retrieved from infected host cells.

PubMed

Hildebrandt, Petra; Surmann, Kristin; Salazar, Manuela Gesell; Normann, Nicole; Völker, Uwe; Schmidt, Frank

2016-10-01

Staphylococcus aureus is a Gram-positive opportunistic pathogen that is able to cause a broad range of infectious diseases in humans. Furthermore, S. aureus is able to survive inside nonprofessional phagocytic host cell which serve as a niche for the pathogen to hide from the immune system and antibiotics therapies. Modern OMICs technologies provide valuable tools to investigate host-pathogen interactions upon internalization. However, these experiments are often hampered by limited capabilities to retrieve bacteria from such an experimental setting. Thus, the aim of this study was to develop a labeling strategy allowing fast detection and quantitation of S. aureus in cell lysates or infected cell lines by flow cytometry for subsequent proteome analyses. Therefore, S. aureus cells were labeled with the DNA stain SYTO ® 9, or Vancomycin BODIPY ® FL (VMB), a glycopeptide antibiotic binding to most Gram-positive bacteria which was conjugated to a fluorescent dye. Staining of S. aureus HG001 with SYTO 9 allowed counting of bacteria from pure cultures but not in cell lysates from infection experiments. In contrast, with VMB it was feasible to stain bacteria from pure cultures as well as from samples of infection experiments. VMB can also be applied for histocytochemistry analysis of formaldehyde fixed cell layers grown on coverslips. Proteome analyses of S. aureus labeled with VMB revealed that the labeling procedure provoked only minor changes on proteome level and allowed cell sorting and analysis of S. aureus from infection settings with sensitivity similar to continuous gfp expression. Furthermore, VMB labeling allowed precise counting of internalized bacteria and can be employed for downstream analyses, e.g., proteomics, of strains not easily amendable to genetic manipulation such as clinical isolates. © 2016 International Society for Advancement of Cytometry. © 2016 International Society for Advancement of Cytometry.

A note on coarse-grained gravity-flow deposits within proterozoic lacustrine sedimentary rocks, Transvaal sequence, South Africa

NASA Astrophysics Data System (ADS)

Eriksson, P. G.

A widely developed, thin, coarse-matrix conglomerate occurs within early Proterozoic lacustrine mudrocks in the Transvaal Sequence, South Africa. The poorly sorted tabular chert clasts, alternation of a planar clast fabric with disorientated zones, plus normal and inverse grading in the former rock type suggest deposition by density-modified grain-flow and high density turbidity currents. The lower fan-delta slope palæenvironment inferred for the conglomerate is consistent with the lacustrine interpretation for the enclosing mudrock facies. This intracratonic setting contrasts with the marine environment generally associated with density-modified grain-flow deposits.

A simulation-based approach for evaluating logging residue handling systems.

Treesearch

B. Bruce Bare; Benjamin A. Jayne; Brian F. Anholt

1976-01-01

Describes a computer simulation model for evaluating logging residue handling systems. The flow of resources is traced through a prespecified combination of operations including yarding, chipping, sorting, loading, transporting, and unloading. The model was used to evaluate the feasibility of converting logging residues to chips that could be used, for example, to...

Cell-cycle synchronisation of bloodstream forms of Trypanosoma brucei using Vybrant DyeCycle Violet-based sorting.

PubMed

Kabani, Sarah; Waterfall, Martin; Matthews, Keith R

2010-01-01

Studies on the cell-cycle of Trypanosoma brucei have revealed several unusual characteristics that differ from the model eukaryotic organisms. However, the inability to isolate homogenous populations of parasites in distinct cell-cycle stages has limited the analysis of trypanosome cell division and complicated the understanding of mutant phenotypes with possible impact on cell-cycle related events. Although hydroxyurea-induced cell-cycle arrest in procyclic and bloodstream forms has been applied recently with success, such block-release protocols can complicate the analysis of cell-cycle regulated events and have the potential to disrupt important cell-cycle checkpoints. An alternative approach based on flow cytometry of parasites stained with Vybrant DyeCycle Orange circumvents this problem, but is restricted to procyclic form parasites. Here, we apply Vybrant Dyecycle Violet staining coupled with flow cytometry to effectively select different cell-cycle stages of bloodstream form trypanosomes. Moreover, the sorted parasites remain viable, although synchrony is rapidly lost. This method enables cell-cycle enrichment of populations of trypanosomes in their mammal infective stage, particularly at the G1 phase.

Cell-cycle synchronisation of bloodstream forms of Trypanosoma brucei using Vybrant DyeCycle Violet-based sorting

PubMed Central

Kabani, Sarah; Waterfall, Martin; Matthews, Keith R.

2010-01-01

Studies on the cell-cycle of Trypanosoma brucei have revealed several unusual characteristics that differ from the model eukaryotic organisms. However, the inability to isolate homogenous populations of parasites in distinct cell-cycle stages has limited the analysis of trypanosome cell division and complicated the understanding of mutant phenotypes with possible impact on cell-cycle related events. Although hydroxyurea-induced cell-cycle arrest in procyclic and bloodstream forms has been applied recently with success, such block-release protocols can complicate the analysis of cell-cycle regulated events and have the potential to disrupt important cell-cycle checkpoints. An alternative approach based on flow cytometry of parasites stained with Vybrant DyeCycle Orange circumvents this problem, but is restricted to procyclic form parasites. Here, we apply Vybrant Dyecycle Violet staining coupled with flow cytometry to effectively select different cell-cycle stages of bloodstream form trypanosomes. Moreover, the sorted parasites remain viable, although synchrony is rapidly lost. This method enables cell-cycle enrichment of populations of trypanosomes in their mammal infective stage, particularly at the G1 phase. PMID:19729042

Three-Dimensional Phenomena in Microbubble Acoustic Streaming

NASA Astrophysics Data System (ADS)

Marin, Alvaro; Rossi, Massimiliano; Rallabandi, Bhargav; Wang, Cheng; Hilgenfeldt, Sascha; Kähler, Christian J.

2015-04-01

Ultrasound-driven oscillating microbubbles are used as active actuators in microfluidic devices to perform manifold tasks such as mixing, sorting, and manipulation of microparticles. A common configuration consists of side bubbles created by trapping air pockets in blind channels perpendicular to the main channel direction. This configuration consists of acoustically excited bubbles with a semicylindrical shape that generate significant streaming flow. Because of the geometry of the channels, such flows are generally considered as quasi-two-dimensional. Similar assumptions are often made in many other microfluidic systems based on flat microchannels. However, in this Letter we show that microparticle trajectories actually present a much richer behavior, with particularly strong out-of-plane dynamics in regions close to the microbubble interface. Using astigmatism particle-tracking velocimetry, we reveal that the apparent planar streamlines are actually projections of a stream surface with a pseudotoroidal shape. We, therefore, show that acoustic streaming cannot generally be assumed as a two-dimensional phenomenon in confined systems. The results have crucial consequences for most of the applications involving acoustic streaming such as particle trapping, sorting, and mixing.

Controlling particle trajectories using oscillating microbubbles

NASA Astrophysics Data System (ADS)

Jalikop, Shreyas; Wang, Cheng; Hilgenfeldt, Sascha

2010-11-01

In many applications of microfluidics and biotechnology, such as cytometry and drug delivery, it is vital to manipulate the trajectories of microparticles such as vesicles or cells. On this small scale, inertial or gravitational effects are often too weak to exploit. We propose a mechanism to selectively trap and direct particles based on their size in creeping transport flows (Re1). We employ Rayleigh-Nyborg-Westervelt (RNW) streaming generated by an oscillating microbubble, which in turn generates a streaming flow component around the mobile particles. The result is an attractive interaction that draws the particle closer to the bubble. The impenetrability of the bubble interface destroys time-reversal symmetry and forces the particles onto either narrow trajectory bundles or well-defined closed trajectories, where they are trapped. The effect is dependent on particle size and thus allows for the passive focusing and sorting of selected sizes, on scales much smaller than the geometry of the microfluidic device. The device could eliminate the need for complicated microchannel designs with external magnetic or electric fields in applications such as particle focusing and size-based sorting.

Identification of a distinct population of CD133+CXCR4+ cancer stem cells in ovarian cancer

PubMed Central

Cioffi, Michele; D’Alterio, Crescenzo; Camerlingo, Rosalba; Tirino, Virginia; Consales, Claudia; Riccio, Anna; Ieranò, Caterina; Cecere, Sabrina Chiara; Losito, Nunzia Simona; Greggi, Stefano; Pignata, Sandro; Pirozzi, Giuseppe; Scala, Stefania

2015-01-01

CD133 and CXCR4 were evaluated in the NCI-60 cell lines to identify cancer stem cell rich populations. Screening revealed that, ovarian OVCAR-3, -4 and -5 and colon cancer HT-29, HCT-116 and SW620 over expressed both proteins. We aimed to isolate cells with stem cell features sorting the cells expressing CXCR4+CD133+ within ovarian cancer cell lines. The sorted population CD133+CXCR4+ demonstrated the highest efficiency in sphere formation in OVCAR-3, OVCAR-4 and OVCAR-5 cells. Moreover OCT4, SOX2, KLF4 and NANOG were highly expressed in CD133+CXCR4+ sorted OVCAR-5 cells. Most strikingly CXCR4+CD133+ sorted OVCAR-5 and -4 cells formed the highest number of tumors when inoculated in nude mice compared to CD133−CXCR4−, CD133+CXCR4−, CD133−CXCR4+ cells. CXCR4+CD133+ OVCAR-5 cells were resistant to cisplatin, overexpressed the ABCG2 surface drug transporter and migrated toward the CXCR4 ligand, CXCL12. Moreover, when human ovarian cancer cells were isolated from 37 primary ovarian cancer, an extremely variable level of CXCR4 and CD133 expression was detected. Thus, in human ovarian cancer cells CXCR4 and CD133 expression identified a discrete population with stem cell properties that regulated tumor development and chemo resistance. This cell population represents a potential therapeutic target. PMID:26020117

Responses of Escherichia coli, Listeria monocytogenes, and Staphylococcus aureus to Simulated Food Processing Treatments, Determined Using Fluorescence-Activated Cell Sorting and Plate Counting▿

PubMed Central

Kennedy, Deirdre; Cronin, Ultan P.; Wilkinson, Martin G.

2011-01-01

Three common food pathogenic microorganisms were exposed to treatments simulating those used in food processing. Treated cell suspensions were then analyzed for reduction in growth by plate counting. Flow cytometry (FCM) and fluorescence-activated cell sorting (FACS) were carried out on treated cells stained for membrane integrity (Syto 9/propidium iodide) or the presence of membrane potential [DiOC2(3)]. For each microbial species, representative cells from various subpopulations detected by FCM were sorted onto selective and nonselective agar and evaluated for growth and recovery rates. In general, treatments giving rise to the highest reductions in counts also had the greatest effects on cell membrane integrity and membrane potential. Overall, treatments that impacted cell membrane permeability did not necessarily have a comparable effect on membrane potential. In addition, some bacterial species with extensively damaged membranes, as detected by FCM, appeared to be able to replicate and grow after sorting. Growth of sorted cells from various subpopulations was not always reflected in plate counts, and in some cases the staining protocol may have rendered cells unculturable. Optimized FCM protocols generated a greater insight into the extent of the heterogeneous bacterial population responses to food control measures than did plate counts. This study underlined the requirement to use FACS to relate various cytometric profiles generated by various staining protocols with the ability of cells to grow on microbial agar plates. Such information is a prerequisite for more-widespread adoption of FCM as a routine microbiological analytical technique. PMID:21602370

The glycosphingolipid P₁ is an ovarian cancer-associated carbohydrate antigen involved in migration.

PubMed

Jacob, F; Anugraham, M; Pochechueva, T; Tse, B W C; Alam, S; Guertler, R; Bovin, N V; Fedier, A; Hacker, N F; Huflejt, M E; Packer, N; Heinzelmann-Schwarz, V A

2014-10-14

The level of plasma-derived naturally circulating anti-glycan antibodies (AGA) to P1 trisaccharide has previously been shown to significantly discriminate between ovarian cancer patients and healthy women. Here we aim to identify the Ig class that causes this discrimination, to identify on cancer cells the corresponding P1 antigen recognised by circulating anti-P1 antibodies and to shed light into the possible function of this glycosphingolipid. An independent Australian cohort was assessed for the presence of anti-P1 IgG and IgM class antibodies using suspension array. Monoclonal and human derived anti-glycan antibodies were verified using three independent glycan-based immunoassays and flow cytometry-based inhibition assay. The P1 antigen was detected by LC-MS/MS and flow cytometry. FACS-sorted cell lines were studied on the cellular migration by colorimetric assay and real-time measurement using xCELLigence system. Here we show in a second independent cohort (n=155) that the discrimination of cancer patients is mediated by the IgM class of anti-P1 antibodies (P=0.0002). The presence of corresponding antigen P1 and structurally related epitopes in fresh tissue specimens and cultured cancer cells is demonstrated. We further link the antibody and antigen (P1) by showing that human naturally circulating and affinity-purified anti-P1 IgM isolated from patients ascites can bind to naturally expressed P1 on the cell surface of ovarian cancer cells. Cell-sorted IGROV1 was used to obtain two study subpopulations (P1-high, 66.1%; and P1-low, 33.3%) and observed that cells expressing high P1-levels migrate significantly faster than those with low P1-levels. This is the first report showing that P1 antigen, known to be expressed on erythrocytes only, is also present on ovarian cancer cells. This suggests that P1 is a novel tumour-associated carbohydrate antigen recognised by the immune system in patients and may have a role in cell migration. The clinical value of our data may be both diagnostic and prognostic; patients with low anti-P1 IgM antibodies present with a more aggressive phenotype and earlier relapse.

Mass, energy and material balances of SRF production process. Part 2: SRF produced from construction and demolition waste.

PubMed

Nasrullah, Muhammad; Vainikka, Pasi; Hannula, Janne; Hurme, Markku; Kärki, Janne

2014-11-01

In this work, the fraction of construction and demolition waste (C&D waste) complicated and economically not feasible to sort out for recycling purposes is used to produce solid recovered fuel (SRF) through mechanical treatment (MT). The paper presents the mass, energy and material balances of this SRF production process. All the process streams (input and output) produced in MT waste sorting plant to produce SRF from C&D waste are sampled and treated according to CEN standard methods for SRF. Proximate and ultimate analysis of these streams is performed and their composition is determined. Based on this analysis and composition of process streams their mass, energy and material balances are established for SRF production process. By mass balance means the overall mass flow of input waste material stream in the various output streams and material balances mean the mass flow of components of input waste material stream (such as paper and cardboard, wood, plastic (soft), plastic (hard), textile and rubber) in the various output streams of SRF production process. The results from mass balance of SRF production process showed that of the total input C&D waste material to MT waste sorting plant, 44% was recovered in the form of SRF, 5% as ferrous metal, 1% as non-ferrous metal, and 28% was sorted out as fine fraction, 18% as reject material and 4% as heavy fraction. The energy balance of this SRF production process showed that of the total input energy content of C&D waste material to MT waste sorting plant, 74% was recovered in the form of SRF, 16% belonged to the reject material and rest 10% belonged to the streams of fine fraction and heavy fraction. From the material balances of this process, mass fractions of plastic (soft), paper and cardboard, wood and plastic (hard) recovered in the SRF stream were 84%, 82%, 72% and 68% respectively of their input masses to MT plant. A high mass fraction of plastic (PVC) and rubber material was found in the reject material stream. Streams of heavy fraction and fine fraction mainly contained non-combustible material (such as stone/rock, sand particles and gypsum material). Copyright © 2014 Elsevier Ltd. All rights reserved.

BAD-LAMP controls TLR9 trafficking and signalling in human plasmacytoid dendritic cells.

PubMed

Combes, Alexis; Camosseto, Voahirana; N'Guessan, Prudence; Argüello, Rafael J; Mussard, Julie; Caux, Christophe; Bendriss-Vermare, Nathalie; Pierre, Philippe; Gatti, Evelina

2017-10-13

Toll-like receptors (TLR) are essential components of the innate immune system. Several accessory proteins, such as UNC93B1, are required for transport and activation of nucleic acid sensing Toll-like receptors in endosomes. Here, we show that BAD-LAMP (LAMP5) controls TLR9 trafficking to LAMP1 + late endosomes in human plasmacytoid dendritic cells (pDC), leading to NF-κB activation and TNF production upon DNA detection. An inducible VAMP3 +/ LAMP2 +/ LAMP1 - endolysosome compartment exists in pDCs from which TLR9 activation triggers type I interferon expression. BAD-LAMP-silencing enhances TLR9 retention in this compartment and consequent downstream signalling events. Conversely, sustained BAD-LAMP expression in pDCs contributes to their lack of type I interferon production after exposure to a TGF-β-positive microenvironment or isolation from human breast tumours. Hence, BAD-LAMP limits interferon expression in pDCs indirectly, by promoting TLR9 sorting to late endosome compartments at steady state and in response to immunomodulatory cues.TLR9 is highly expressed by plasmacytoid dendritic cells and detects nucleic acids, but to discriminate between host and microbial nucleic acids TLR9 is sorted into different endosomal compartments. Here the authors show that BAD-LAMP limits type 1 interferon responses by sorting TLR9 to late endosomal compartments.

Improving the layout of recycling centres by use of lean production principles.

PubMed

Sundin, Erik; Björkman, Mats; Eklund, Mats; Eklund, Jörgen; Engkvist, Inga-Lill

2011-06-01

There has been increased focus on recycling in Sweden during recent years. This focus can be attributed to external environmental factors such as tougher legislation, but also to the potential gains for raw materials suppliers. Recycling centres are important components in the Swedish total recycling system. Recycling centres are manned facilities for waste collection where visitors can bring, sort and discard worn products as well as large-sized, hazardous, and electrical waste. The aim of this paper was to identify and describe the main flows and layout types at Swedish recycling centres. The aim was also to adapt and apply production theory for designing and managing recycling centre operations. More specifically, this means using lean production principles to help develop guidelines for recycling centre design and efficient control. Empirical data for this research was primarily collected through interviews and questionnaires among both visitors and employees at 16 Swedish recycling centres. Furthermore, adapted observation protocols have been used in order to explore visitor activities. There was also close collaboration with a local recycling centre company, which shared their layout experiences with the researchers in this project. The recycling centres studied had a variety of problems such as queues of visitors, overloading of material and improper sorting. The study shows that in order to decrease the problems, the recycling centres should be designed and managed according to lean production principles, i.e. through choosing more suitable layout choices with visible and linear flows, providing better visitor information, and providing suitable technical equipment. Improvements can be achieved through proper planning of the layout and control of the flow of vehicles, with the result of increased efficiency and capacity, shorter visits, and cleaner waste fractions. The benefits of a lean production mindset include increased visitor capacity, waste flexibility, improved sorting quality, shorter time for visits and improved working conditions. Copyright © 2011 Elsevier Ltd. All rights reserved.

The Bogus Science.

ERIC Educational Resources Information Center

Anderson, C. C.

1981-01-01

Discusses three sorts of psychological science: Science 1, a natural science with conditional (causal) laws; Science 2, with probabilistic laws; and Science 3, a "human science" trying to capture the complexity of human experience and behavior. Argues that Science 2 and Science 3 should be treated as belief systems which people may find useful for…

Sorting of the Neuroendocrine Secretory Protein Secretogranin II into the Regulated Secretory Pathway

PubMed Central

Courel, Maïté; Vasquez, Michael S.; Hook, Vivian Y.; Mahata, Sushil K.; Taupenot, Laurent

2008-01-01

Secretogranin II (SgII) belongs to the granin family of prohormones widely distributed in dense-core secretory granules (DCGs) of endocrine, neuroendocrine, and neuronal cells, including sympathoadrenal chromaffin cells. The mechanisms by which secretory proteins, and granins in particular, are sorted into the regulated secretory pathway are unsettled. We designed a strategy based on novel chimeric forms of human SgII fused to fluorescent (green fluorescent protein) or chemiluminescent (embryonic alkaline phosphatase) reporters to identify trafficking determinants mediating DCG targeting of SgII in sympathoadrenal cells. Three-dimensional deconvolution fluorescence microscopy and secretagogue-stimulated release studies demonstrate that SgII chimeras are correctly targeted to DCGs and released by exocytosis in PC12 and primary chromaffin cells. Results from a Golgi-retained mutant form of SgII suggest that sorting of SgII into DCGs depends on a saturable sorting machinery at the trans-Golgi/trans-Golgi network. Truncation analyses reveal the presence of DCG-targeting signals within both the N- and C-terminal regions of SgII, with the putative α-helix-containing SgII-(25-41) and SgII-(334-348) acting as sufficient, independent sorting domains. This study defines sequence features of SgII mediating vesicular targeting in sympathoadrenal cells and suggests a mechanism by which discrete domains of the molecule function in sorting, perhaps by virtue of a particular arrangement in tertiary structure and/or interaction with a specific component of the DCG membrane. PMID:18299326

Sorting of the neuroendocrine secretory protein Secretogranin II into the regulated secretory pathway: role of N- and C-terminal alpha-helical domains.

PubMed

Courel, Maïté; Vasquez, Michael S; Hook, Vivian Y; Mahata, Sushil K; Taupenot, Laurent

2008-04-25

Secretogranin II (SgII) belongs to the granin family of prohormones widely distributed in dense-core secretory granules (DCGs) of endocrine, neuroendocrine, and neuronal cells, including sympathoadrenal chromaffin cells. The mechanisms by which secretory proteins, and granins in particular, are sorted into the regulated secretory pathway are unsettled. We designed a strategy based on novel chimeric forms of human SgII fused to fluorescent (green fluorescent protein) or chemiluminescent (embryonic alkaline phosphatase) reporters to identify trafficking determinants mediating DCG targeting of SgII in sympathoadrenal cells. Three-dimensional deconvolution fluorescence microscopy and secretagogue-stimulated release studies demonstrate that SgII chimeras are correctly targeted to DCGs and released by exocytosis in PC12 and primary chromaffin cells. Results from a Golgi-retained mutant form of SgII suggest that sorting of SgII into DCGs depends on a saturable sorting machinery at the trans-Golgi/trans-Golgi network. Truncation analyses reveal the presence of DCG-targeting signals within both the N- and C-terminal regions of SgII, with the putative alpha-helix-containing SgII-(25-41) and SgII-(334-348) acting as sufficient, independent sorting domains. This study defines sequence features of SgII mediating vesicular targeting in sympathoadrenal cells and suggests a mechanism by which discrete domains of the molecule function in sorting, perhaps by virtue of a particular arrangement in tertiary structure and/or interaction with a specific component of the DCG membrane.

Study of an Educational Hand Sorting Intervention for Reducing Aflatoxin B1 in Groundnuts in Rural Gambia.

PubMed

Xu, Y A; Doel, Andrew; Watson, Sinead; Routledge, Michael N; Elliott, Christopher T; Moore, Sophie E; Gong, Yun Yun

2017-01-01

Aflatoxin, a human liver carcinogen, frequently contaminates groundnuts, maize, rice, and other grains, especially in Africa. The aim of this study was to evaluate the effectiveness of an educational intervention that involved training rural Gambian women on how to identify and remove moldy groundnuts to reduce aflatoxin B 1 (AFB 1 ) contamination. In total, 25 women, recruited from the West Kiang region of The Gambia, were trained on how to recognize and remove moldy groundnuts. Market-purchased groundnuts were hand sorted by the women. Groundnuts were sampled at baseline (n =5), after hand sorting ("clean," n =25 and "moldy," n =25), and after roasting (n =5). All samples were analyzed for AFB 1 by enzyme-linked immunosorbent assay. A reduction of 42.9% was achieved based on the median AFB 1 levels at baseline and after hand sorting (clean groundnuts), whereas an alternative estimate, based on the total AFB 1 in moldy and clean groundnuts, indicated a reduction of 96.7%, with a loss of only 2% of the groundnuts. By roasting the already clean sorted groundnuts, the AFB 1 reduction achieved (based on median levels) was 39.3%. This educational intervention on how to identify and remove moldy groundnuts was simple and effective in reducing AFB 1 contamination.

[Research with human embryo stem cells. Foundations and judicial limits].

PubMed

Eser, Albin; Koch, Hans-Georg

2004-01-01

Research with human embryos, and particularly, the use for scientific purposes of human embryonic stem cells has given raise to different sort of problems at the international level. One of the most strict regulation in this field, is this lecture Professors Albin Eser and Hans-Georg Koch analyse the german legal framework in relation with the use of embryos and human embryonic stem cells for scientific purposes.

Improved cell therapy protocol for Parkinson’s disease based on differentiation efficiency and safety of hESC-, hiPSC and non-human primate iPSC-derived DA neurons

PubMed Central

Maria, Sundberg; Helle, Bogetofte; Tristan, Lawson; Gaynor, Smith; Arnar, Astradsson; Michele, Moore; Teresia, Osborn; Oliver, Cooper; Roger, Spealman; Penelope, Hallett; Ole, Isacson

2013-01-01

The main motor symptoms of Parkinson’s disease are due to the loss of dopaminergic (DA) neurons in the ventral midbrain (VM). For the future treatment of Parkinson’s disease with cell transplantation it is important to develop efficient differentiation methods for production of human iPSCs and hESCs-derived midbrain-type DA neurons. Here we describe an efficient differentiation and sorting strategy for DA-neurons from both human ES/iPS cells and non-human primate iPSCs. The use of non-human primate iPSCs for neuronal differentiation and autologous transplantation is important for pre-clinical evaluation of safety and efficacy of stem cell-derived DA neurons. The aim of this study was to improve the safety of human- and non-human primate-iPSC (PiPSC)-derived DA neurons. According to our results, NCAM+/CD29low sorting enriched VM DA-neurons from pluripotent stem cell-derived neural cell populations. NCAM+/CD29low DA-neurons were positive for FOXA2/TH and EN1/TH and this cell population had increased expression levels of FOXA2, LMX1A, TH, GIRK2, PITX3, EN1, NURR1 mRNA compared to unsorted neural cell populations. PiPSC-derived NCAM+/CD29low DA-neurons were able to restore motor function of 6-OHDA lesioned rats 16 weeks after transplantation. The transplanted sorted cells also integrated in the rodent brain tissue, with robust TH+/hNCAM+ neuritic innervation of the host striatum. One year after autologous transplantation, the primate iPSC-derived neural cells survived in the striatum of one primate without any immunosuppression. These neural cell grafts contained FOXA2/TH-positive neurons in the graft site. This is an important proof of concept for the feasibility and safety of iPSC-derived cell transplantation therapies in the future. PMID:23666606

Chromosomal rearrangements and karyotype evolution in carnivores revealed by chromosome painting

PubMed Central

Nie, W; Wang, J; Su, W; Wang, D; Tanomtong, A; Perelman, P L; Graphodatsky, A S; Yang, F

2012-01-01

Chromosomal evolution in carnivores has been revisited extensively using cross-species chromosome painting. Painting probes derived from flow-sorted chromosomes of the domestic dog, which has one of the most rearranged karyotypes in mammals and the highest dipoid number (2n=78) in carnivores, are a powerful tool in detecting both evolutionary intra- and inter-chromosomal rearrangements. However, only a few comparative maps have been established between dog and other non-Canidae species. Here, we extended cross-species painting with dog probes to seven more species representing six carnivore families: Eurasian lynx (Lynx lynx), the stone marten (Martes foina), the small Indian civet (Viverricula indica), the Asian palm civet (Paradoxurus hermaphrodites), Javan mongoose (Hepestes javanicas), the raccoon (Procyon lotor) and the giant panda (Ailuropoda melanoleuca). The numbers and positions of intra-chromosomal rearrangements were found to differ among these carnivore species. A comparative map between human and stone marten, and a map among the Yangtze finless porpoise (Neophocaena phocaenoides asiaeorientalis), stone marten and human were also established to facilitate outgroup comparison and to integrate comparative maps between stone marten and other carnivores with such maps between human and other species. These comparative maps give further insight into genome evolution and karyotype phylogenetic relationships among carnivores, and will facilitate the transfer of gene mapping data from human, domestic dog and cat to other species. PMID:22086079

International Society for the Advancement of Cytometry Cell Sorter Biosafety Standards

PubMed Central

Holmes, Kevin L.; Fontes, Benjamin; Hogarth, Philip; Konz, Richard; Monard, Simon; Pletcher, Charles H.; Wadley, Robert B.; Schmid, Ingrid; Perfetto, Stephen P.

2014-01-01

Flow cytometric cell sorting of biological specimens has become prevalent in basic and clinical research laboratories. These specimens may contain known or unknown infectious agents, necessitating precautions to protect instrument operators and the environment from biohazards arising from the use of sorters. To this end the International Society of Analytical Cytology (ISAC) was proactive in establishing biosafety guidelines in 1997 (Schmid et al., Cytometry 1997;28:99–117) and subsequently published revised biosafety standards for cell sorting of unfixed samples in 2007 (Schmid et al., Cytometry Part A J Int Soc Anal Cytol 2007;71A:414–437). Since their publication, these documents have become recognized worldwide as the standard of practice and safety precautions for laboratories performing cell sorting experiments. However, the field of cytometry has progressed since 2007, and the document requires an update. The new Standards provides guidance: (1) for laboratory design for cell sorter laboratories; (2) for the creation of laboratory or instrument specific Standard Operating Procedures (SOP); and (3) on procedures for the safe operation of cell sorters, including personal protective equipment (PPE) and validation of aerosol containment. PMID:24634405

Accurately tracking single-cell movement trajectories in microfluidic cell sorting devices.

PubMed

Jeong, Jenny; Frohberg, Nicholas J; Zhou, Enlu; Sulchek, Todd; Qiu, Peng

2018-01-01

Microfluidics are routinely used to study cellular properties, including the efficient quantification of single-cell biomechanics and label-free cell sorting based on the biomechanical properties, such as elasticity, viscosity, stiffness, and adhesion. Both quantification and sorting applications require optimal design of the microfluidic devices and mathematical modeling of the interactions between cells, fluid, and the channel of the device. As a first step toward building such a mathematical model, we collected video recordings of cells moving through a ridged microfluidic channel designed to compress and redirect cells according to cell biomechanics. We developed an efficient algorithm that automatically and accurately tracked the cell trajectories in the recordings. We tested the algorithm on recordings of cells with different stiffness, and showed the correlation between cell stiffness and the tracked trajectories. Moreover, the tracking algorithm successfully picked up subtle differences of cell motion when passing through consecutive ridges. The algorithm for accurately tracking cell trajectories paves the way for future efforts of modeling the flow, forces, and dynamics of cell properties in microfluidics applications.

Fast inertial particle manipulation in oscillating flows

NASA Astrophysics Data System (ADS)

Thameem, Raqeeb; Rallabandi, Bhargav; Hilgenfeldt, Sascha

2017-05-01

It is demonstrated that micron-sized particles suspended in fluid near oscillating interfaces experience strong inertial displacements above and beyond the fluid streaming. Experiments with oscillating bubbles show rectified particle lift over extraordinarily short (millisecond) times. A quantitative model on both the oscillatory and the steady time scales describes the particle displacement relative to the fluid motion. The formalism yields analytical predictions confirming the observed scaling behavior with particle size and experimental control parameters. It applies to a large class of oscillatory flows with applications from particle trapping to size sorting.

Microfabrication and Test of a Three-Dimensional Polymer Hydro-focusing Unit for Flow Cytometry Applications

NASA Technical Reports Server (NTRS)

Yang, Ren; Feeback, Daniel L.; Wang, Wan-Jun

2005-01-01

This paper details a novel three-dimensional (3D) hydro-focusing micro cell sorter for micro flow cytometry applications. The unit was microfabricated by means of SU-8 3D lithography. The 3D microstructure for coaxial sheathing was designed, microfabricated, and tested. Three-dimensional hydrofocusing capability was demonstrated with an experiment to sort labeled tanned sheep erythrocytes (red blood cells). This polymer hydro-focusing microstructure is easily microfabricated and integrated with other polymer microfluidic structures. Keywords: SU-8, three-dimensional hydro-focusing, microfluidic, microchannel, cytometer

Isolation and characterization of human mesenchymal stem cells derived from synovial fluid by magnetic-activated cell sorting (MACS).

PubMed

Jia, Zhaofeng; Liang, Yujie; Xu, Xiao; Li, Xingfu; Liu, Qisong; Ou, Yangkan; Duan, Li; Zhu, Weimin; Lu, Wei; Xiong, Jianyi; Wang, Daping

2018-03-01

Mesenchymal stem cells (MSCs) are the primary source of cells used for cell-based therapy in tissue engineering. MSCs are found in synovial fluid, a source that could be conveniently used for cartilage tissue engineering. However, the purification and characterization of SF-MSCs has been poorly documented in the literature. Here, we outline an easy-to-perform approach for the isolation and culture of MSCs derived from human synovial fluid (hSF-MSCs). We have successfully purified hSF-MSCs using magnetic-activated cell sorting (MACS) using the MSC surface marker, CD90. Purified SF-MSCs demonstrate significant renewal capacity following several passages in culture. Furthermore, we demonstrated that MACS-sorted CD90 + cells could differentiated into osteoblasts, adipocytes, and chondrocytes in vitro. In addition, we show that these cells can generate cartilage tissue in micromass culture as well. This study demonstrates that MACS is a useful tool that can be used for the purification of hSF-MSCs from synovial fluid. The proliferation properties and ability to differentiate into chondrocytes make these hSF-MSCs a promising source of stem cells for applications in cartilage repair. © 2017 International Federation for Cell Biology.

The Isolation and Enrichment of Large Numbers of Highly Purified Mouse Spleen Dendritic Cell Populations and Their In Vitro Equivalents.

PubMed

Vremec, David

2016-01-01

Dendritic cells (DCs) form a complex network of cells that initiate and orchestrate immune responses against a vast array of pathogenic challenges. Developmentally and functionally distinct DC subtypes differentially regulate T-cell function. Importantly it is the ability of DC to capture and process antigen, whether from pathogens, vaccines, or self-components, and present it to naive T cells that is the key to their ability to initiate an immune response. Our typical isolation procedure for DC from murine spleen was designed to efficiently extract all DC subtypes, without bias and without alteration to their in vivo phenotype, and involves a short collagenase digestion of the tissue, followed by selection for cells of light density and finally negative selection for DC. The isolation procedure can accommodate DC numbers that have been artificially increased via administration of fms-like tyrosine kinase 3 ligand (Flt3L), either directly through a series of subcutaneous injections or by seeding with an Flt3L secreting murine melanoma. Flt3L may also be added to bone marrow cultures to produce large numbers of in vitro equivalents of the spleen DC subsets. Total DC, or their subsets, may be further purified using immunofluorescent labeling and flow cytometric cell sorting. Cell sorting may be completely bypassed by separating DC subsets using a combination of fluorescent antibody labeling and anti-fluorochrome magnetic beads. Our procedure enables efficient separation of the distinct DC subsets, even in cases where mouse numbers or flow cytometric cell sorting time is limiting.

Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation.

PubMed

Allen, Joselyn N; Dey, Adwitia; Nissly, Ruth; Fraser, James; Yu, Shan; Balandaram, Gayathri; Peters, Jeffrey M; Hankey-Giblin, Pamela A

2017-04-03

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. Diet-induced obesity (DIO) is a valuable model in studying the role of macrophage heterogeneity; however, adequate macrophage isolations are difficult to acquire from inflamed tissues. In this protocol, we outline the isolation steps and necessary troubleshooting guidelines derived from our studies for obtaining a suitable population of tissue-resident macrophages from mice following 18 weeks of high-fat (HFD) or high-fat/high-cholesterol (HFHCD) diet intervention. This protocol focuses on three hallmark tissues studied in obesity and atherosclerosis including the liver, white adipose tissues (WAT), and the aorta. We highlight how dualistic usage of flow cytometry can achieve a new dimension of isolation and characterization of tissue-resident macrophages. A fundamental section of this protocol addresses the intricacies underlying tissue-specific enzymatic digestions and macrophage isolation, and subsequent cell-surface antibody staining for flow cytometric analysis. This protocol addresses existing complexities underlying fluorescent-activated cell sorting (FACS) and presents clarifications to these complexities so as to obtain broad range characterization from adequately sorted cell populations. Alternate enrichment methods are included for sorting cells, such as the dense liver, allowing for flexibility and time management when working with FACS. In brief, this protocol aids the researcher to evaluate macrophage heterogeneity from a multitude of inflamed tissues in a given study and provides insightful troubleshooting tips that have been successful for favorable cellular isolation and characterization of immune cells in DIO-mediated inflammation.

Chronology of Islet Differentiation Revealed By Temporal Cell Labeling

PubMed Central

Miyatsuka, Takeshi; Li, Zhongmei; German, Michael S.

2009-01-01

OBJECTIVE Neurogenin 3 plays a pivotal role in pancreatic endocrine differentiation. Whereas mouse models expressing reporters such as eGFP or LacZ under the control of the Neurog3 gene enable us to label cells in the pancreatic endocrine lineage, the long half-life of most reporter proteins makes it difficult to distinguish cells actively expressing neurogenin 3 from differentiated cells that have stopped transcribing the gene. RESEARCH DESIGN AND METHODS In order to separate the transient neurogenin 3 –expressing endocrine progenitor cells from the differentiating endocrine cells, we developed a mouse model (Ngn3-Timer) in which DsRed-E5, a fluorescent protein that shifts its emission spectrum from green to red over time, was expressed transgenically from the NEUROG3 locus. RESULTS In the Ngn3-Timer embryos, green-dominant cells could be readily detected by microscopy or flow cytometry and distinguished from green/red double-positive cells. When fluorescent cells were sorted into three different populations by a fluorescence-activated cell sorter, placed in culture, and then reanalyzed by flow cytometry, green-dominant cells converted to green/red double-positive cells within 6 h. The sorted cell populations were then used to determine the temporal patterns of expression for 145 transcriptional regulators in the developing pancreas. CONCLUSIONS The precise temporal resolution of this model defines the narrow window of neurogenin 3 expression in islet progenitor cells and permits sequential analyses of sorted cells as well as the testing of gene regulatory models for the differentiation of pancreatic islet cells. PMID:19478145

Multi-locus phylogenetics, lineage sorting, and reticulation in Pinus subsection Australes.

PubMed

Gernandt, David S; Aguirre Dugua, Xitlali; Vázquez-Lobo, Alejandra; Willyard, Ann; Moreno Letelier, Alejandra; Pérez de la Rosa, Jorge A; Piñero, Daniel; Liston, Aaron

2018-04-23

Both incomplete lineage sorting and reticulation have been proposed as causes of phylogenetic incongruence. Disentangling these factors may be most difficult in long-lived, wind-pollinated plants with large population sizes and weak reproductive barriers. We used solution hybridization for targeted enrichment and massive parallel sequencing to characterize low-copy-number nuclear genes and high-copy-number plastomes (Hyb-Seq) in 74 individuals of Pinus subsection Australes, a group of ~30 New World pine species of exceptional ecological and economic importance. We inferred relationships using methods that account for both incomplete lineage sorting and reticulation. Concatenation- and coalescent-based trees inferred from nuclear genes mainly agreed with one another, but they contradicted the plastid DNA tree in recovering the Attenuatae (the California closed-cone pines) and Oocarpae (the egg-cone pines of Mexico and Central America) as monophyletic and the Australes sensu stricto (the southern yellow pines) as paraphyletic to the Oocarpae. The plastid tree featured some relationships that were discordant with morphological and geographic evidence and species limits. Incorporating gene flow into the coalescent analyses better fit the data, but evidence supporting the hypothesis that hybridization explains the non-monophyly of the Attenuatae in the plastid tree was equivocal. Our analyses document cytonuclear discordance in Pinus subsection Australes. We attribute this discordance to ancient and recent introgression and present a phylogenetic hypothesis in which mostly hierarchical relationships are overlain by gene flow. © 2018 The Authors. American Journal of Botany is published by Wiley Periodicals, Inc. on behalf of the Botanical Society of America.

Getting Things Sorted With Lagrangian Coherent Structures

NASA Astrophysics Data System (ADS)

Atis, Severine; Peacock, Thomas; Environmental Dynamics Laboratory Team

2014-11-01

The dispersion of a tracer in a fluid flow is influenced by the Lagrangian motion of fluid elements. Even in laminar regimes, the irregular chaotic behavior of a fluid flow can lead to effective stirring that rapidly redistributes a tracer throughout the domain. For flows with arbitrary time-dependence, the modern approach of Lagrangian Coherent Structures (LCSs) provide a method for identifying the key material lines that organize flow transport. When the advected tracer particles possess a finite size and nontrivial shape, however, their dynamics can differ markedly from passive tracers, thus affecting the dispersion phenomena. We present details of numerical simulations and laboratory experiments that investigate the behavior of finite size particles in 2-dimensional chaotic flows. We show that the shape and the size of the particles alter the underlying LCSs, facilitating segregation between tracers of different shape in the same flow field.

Emerging roles of ARHGAP33 in intracellular trafficking of TrkB and pathophysiology of neuropsychiatric disorders

PubMed Central

Nakazawa, Takanobu; Hashimoto, Ryota; Sakoori, Kazuto; Sugaya, Yuki; Tanimura, Asami; Hashimotodani, Yuki; Ohi, Kazutaka; Yamamori, Hidenaga; Yasuda, Yuka; Umeda-Yano, Satomi; Kiyama, Yuji; Konno, Kohtarou; Inoue, Takeshi; Yokoyama, Kazumasa; Inoue, Takafumi; Numata, Shusuke; Ohnuma, Tohru; Iwata, Nakao; Ozaki, Norio; Hashimoto, Hitoshi; Watanabe, Masahiko; Manabe, Toshiya; Yamamoto, Tadashi; Takeda, Masatoshi; Kano, Masanobu

2016-01-01

Intracellular trafficking of receptor proteins is essential for neurons to detect various extracellular factors during the formation and refinement of neural circuits. However, the precise mechanisms underlying the trafficking of neurotrophin receptors to synapses remain elusive. Here, we demonstrate that a brain-enriched sorting nexin, ARHGAP33, is a new type of regulator for the intracellular trafficking of TrkB, a high-affinity receptor for brain-derived neurotrophic factor. ARHGAP33 knockout (KO) mice exhibit reduced expression of synaptic TrkB, impaired spine development and neuropsychiatric disorder-related behavioural abnormalities. These deficits are rescued by specific pharmacological enhancement of TrkB signalling in ARHGAP33 KO mice. Mechanistically, ARHGAP33 interacts with SORT1 to cooperatively regulate TrkB trafficking. Human ARHGAP33 is associated with brain phenotypes and reduced SORT1 expression is found in patients with schizophrenia. We propose that ARHGAP33/SORT1-mediated TrkB trafficking is essential for synapse development and that the dysfunction of this mechanism may be a new molecular pathology of neuropsychiatric disorders. PMID:26839058

Development and evaluation of an off-the-slide genotyping technique for identifying Giardia cysts and Cryptosporidium oocysts directly from US EPA Method 1623 slides

EPA Science Inventory

ABSTRACT Aims This study developed and systematically evaluated performance and limit of detection of an off-the-slide genotyping procedure for both Cryptosporidium oocysts and Giardia cysts. Methods and Results Slide standards containing flow sorted (oo)cysts were used to e...

Effects of sediment-associated extractable metals, degree of sediment grain sorting, and dissolved organic carbon upon Cryptosporidium parvum removal and transport within riverbank filtration sediments, Sonoma County, California.

PubMed

Metge, David W; Harvey, Ronald W; Aiken, George R; Anders, Robert; Lincoln, George; Jasperse, Jay; Hill, Mary C

2011-07-01

Oocysts of the protozoan pathogen Cryptosporidium parvum are of particular concern for riverbank filtration (RBF) operations because of their persistence, ubiquity, and resistance to chlorine disinfection. At the Russian River RBF site (Sonoma County, CA), transport of C. parvum oocysts and oocyst-sized (3 μm) carboxylate-modified microspheres through poorly sorted (sorting indices, σ(1), up to 3.0) and geochemically heterogeneous sediments collected between 2 and 25 m below land surface (bls) were assessed. Removal was highly sensitive to variations in both the quantity of extractable metals (mainly Fe and Al) and degree of grain sorting. In flow-through columns, there was a log-linear relationship (r(2) = 0.82 at p < 0.002) between collision efficiency (α, the probability that colloidal collisions with grain surfaces would result in attachment) and extractable metals, and a linear relationship (r(2) = 0.99 at p < 0.002) between α and σ(1). Collectively, variability in extractable metals and grain sorting accounted for ∼83% of the variability in α (at p < 0.0002) along the depth profiles. Amendments of 2.2 mg L(-1) of Russian River dissolved organic carbon (DOC) reduced α for oocysts by 4-5 fold. The highly reactive hydrophobic organic acid (HPOA) fraction was particularly effective in re-entraining sediment-attached microspheres. However, the transport-enhancing effects of the riverine DOC did not appear to penetrate very deeply into the underlying sediments, judging from high α values (∼1.0) observed for oocysts being advected through unamended sediments collected at ∼2 m bls. This study suggests that in evaluating the efficacy of RBF operations to remove oocysts, it may be necessary to consider not only the geochemical nature and size distribution of the sediment grains, but also the degrees of sediment sorting and the concentration, reactivity, and penetration of the source water DOC.

Highstand shelf fans: The role of buoyancy reversal in the deposition of a new type of shelf sand body

USGS Publications Warehouse

Steel, Elisabeth; Simms, Alexander R.; Warrick, Jonathan; Yokoyama, Yusuke

2016-01-01

Although sea-level highstands are typically associated with sediment-starved continental shelves, high sea level does not hinder major river floods. Turbidity currents generated by plunging of sediment-laden rivers at the fluvial-marine interface, known as hyperpycnal flows, allow for cross-shelf transport of suspended sand beyond the coastline. Hyperpycnal flows in southern California have deposited six subaqueous fans on the shelf of the northern Santa Barbara Channel in the Holocene. Using eight cores and nine grab samples, we describe the deposits, age, and stratigraphic architecture of two fans in the Santa Barbara Channel. Fan lobes have up to 3 m of relief and are composed of multiple hyperpycnite beds ∼5 cm to 40 cm thick. Deposit architecture and geometry suggest the hyperpycnal flows became positively buoyant and lifted off the seabed, resulting in well-sorted, structureless, elongate sand lobes. Contrary to conventional sequence stratigraphic models, the presence of these features on the continental shelf suggests that active-margin shelves may locally develop high-quality reservoir sand bodies during sea-level highstands, and that such shelves need not be solely the site of sediment bypass. These deposits may provide a Quaternary analogue to many well-sorted sand bodies in the rock record that are interpreted as turbidites but lack typical Bouma-type features.

Increased Cardiac Myocyte Progenitors in Failing Human Hearts

PubMed Central

Kubo, Hajime; Jaleel, Naser; Kumarapeli, Asangi; Berretta, Remus M.; Bratinov, George; Shan, Xiaoyin; Wang, Hongmei; Houser, Steven R.; Margulies, Kenneth B.

2009-01-01

Background Increasing evidence, derived mainly from animal models, supports the existence of endogenous cardiac renewal and repair mechanisms in adult mammalian hearts that could contribute to normal homeostasis and the responses to pathological insults. Methods and Results Translating these results, we isolated small c-kit+ cells from 36 of 37 human hearts using primary cell isolation techniques and magnetic cell sorting techniques. The abundance of these cardiac progenitor cells was increased nearly 4-fold in patients with heart failure requiring transplantation compared with nonfailing controls. Polychromatic flow cytometry of primary cell isolates (<30 μm) without antecedent c-kit enrichment confirmed the increased abundance of c-kit+ cells in failing hearts and demonstrated frequent coexpression of CD45 in these cells. Immunocytochemical characterization of freshly isolated, c-kit–enriched human cardiac progenitor cells confirmed frequent coexpression of c-kit and CD45. Primary cardiac progenitor cells formed new human cardiac myocytes at a relatively high frequency after coculture with neonatal rat ventricular myocytes. These contracting new cardiac myocytes exhibited an immature phenotype and frequent electric coupling with the rat myocytes that induced their myogenic differentiation. Conclusions Despite the increased abundance and cardiac myogenic capacity of cardiac progenitor cells in failing human hearts, the need to replace these organs via transplantation implies that adverse features of the local myocardial environment overwhelm endogenous cardiac repair capacity. Developing strategies to improve the success of endogenous cardiac regenerative processes may permit therapeutic myocardial repair without cell delivery per se. PMID:18645055

Study on the effects of flow in the volute casing on the performance of a sirocco fan

NASA Astrophysics Data System (ADS)

Adachi, Tsutomu; Sugita, Naohiro; Ohomori, Satoshi

2004-08-01

The flow at the exit from the runner blade of a centrifugal fan with forward curved blades (a sirocco fan) sometimes separates and becomes unstable. We have conducted many researches on the impeller shape of a sirocco fan, proper inlet and exit blade angles were considered to obtain optimum performance. In this paper, the casing shape were decided by changing the circumferential angle, magnifying angle and the width, 21 sorts of casings were used. Performance tests, inner flow velocity and pressure distributions were measured as well. Computational fluid dynamic calculations were also made and compared with the experimental results. Finally, the most suitable casing shape for best performance is considered.

Communicative Function Demonstration Induces Kind-Based Artifact Representation in Preverbal Infants

ERIC Educational Resources Information Center

Futo, Judit; Teglas, Erno; Csibra, Gergely; Gergely, Gyorgy

2010-01-01

Human infants grow up in environments populated by artifacts. In order to acquire knowledge about different kinds of human-made objects, children have to be able to focus on the information that is most relevant for sorting artifacts into categories. Traditional theories emphasize the role of superficial, perceptual features in object…

Perception and Production of the Human Figure in Drawings by Children.

ERIC Educational Resources Information Center

Itskowitz, Rivka

This research deals with the development and relative importance of both the conceptual-cognitive and the aesthetic-affective aspect of the perceptual process at various age levels of children. Three tasks were chosen: (1) sorting drawings of human figures--a task that represents a more conceptual function; (2) expressing preferences for those…

"H. Sapiens" as Ecologically Special: What Does Language Contribute?

ERIC Educational Resources Information Center

Ross, Don

2007-01-01

This paper inquires into the extent to which humans are specially constituted relative to other animals by their language. First a principled concept of evolutionary specialness is operationalized. Then it is agreed that humans satisfy the criteria for this sort of specialness in consequence of the kind of cultural evolution in which they have…

Children Perseverate to a Human's Actions but Not to a Robot's Actions

ERIC Educational Resources Information Center

Moriguchi, Yusuke; Kanda, Takayuki; Ishiguro, Hiroshi; Itakura, Shoji

2010-01-01

Previous research has shown that young children commit perseverative errors from their observation of another person's actions. The present study examined how social observation would lead children to perseverative tendencies, using a robot. In Experiment 1, preschoolers watched either a human model or a robot sorting cards according to one…

Education and Disarmament.

ERIC Educational Resources Information Center

Greene, Maxine

1982-01-01

Education should release humans to reach out from their own places in the world so that they can make sense of how they live and what they encounter around themselves. This sort of education, which will "humanize" the world and populate it with "self-understanding" citizens, will allow for the evolution of alternatives to destruction and war. (CJ)

West Antarctic Balance Fluxes: Impact of Smoothing, Algorithm and Topography.

NASA Astrophysics Data System (ADS)

Le Brocq, A.; Payne, A. J.; Siegert, M. J.; Bamber, J. L.

2004-12-01

Grid-based calculations of balance flux and velocity have been widely used to understand the large-scale dynamics of ice masses and as indicators of their state of balance. This research investigates a number of issues relating to their calculation for the West Antarctic Ice Sheet (see below for further details): 1) different topography smoothing techniques; 2) different grid based flow-apportioning algorithms; 3) the source of the flow direction, whether from smoothed topography, or smoothed gravitational driving stress; 4) different flux routing techniques and 5) the impact of different topographic datasets. The different algorithms described below lead to significant differences in both ice stream margins and values of fluxes within them. This encourages caution in the use of grid-based balance flux/velocity distributions and values, especially when considering the state of balance of individual ice streams. 1) Most previous calculations have used the same numerical scheme (Budd and Warner, 1996) applied to a smoothed topography in order to incorporate the longitudinal stresses that smooth ice flow. There are two options to consider when smoothing the topography, the size of the averaging filter and the shape of the averaging function. However, this is not a physically-based approach to incorporating smoothed ice flow and also introduces significant flow artefacts when using a variable weighting function. 2) Different algorithms to apportion flow are investigated; using 4 or 8 neighbours, and apportioning flow to all down-slope cells or only 2 (based on derived flow direction). 3) A theoretically more acceptable approach of incorporating smoothed ice flow is to use the smoothed gravitational driving stress in x and y components to derive a flow direction. The flux can then be apportioned using the flow direction approach used above. 4) The original scheme (Budd and Warner, 1996) uses an elevation sort technique to calculate the balance flux contribution from all cells to each individual cell. However, elevation sort is only successful when ice cannot flow uphill. Other possible techniques include using a recursive call for each neighbour or using a sparse matrix solution. 5) Two digital elevation models are used as input data, which have significant differences in coastal and mountainous areas and therefore lead to different calculations. Of particular interest is the difference in the Rutford Ice Stream/Carlson Inlet and Kamb Ice Stream (Ice Stream C) fluxes.

Assessing the role of detrital zircon sorting on provenance interpretations in an ancient fluvial system using paleohydraulics - Permian Cutler Group, Paradox Basin, Utah and Colorado

NASA Astrophysics Data System (ADS)

Findlay, C. P., III; Ewing, R. C.; Perez, N. D.

2017-12-01

Detrital zircon age signatures used in provenance studies are assumed to be representative of entire catchments from which the sediment was derived, but the extent to which hydraulic sorting can bias provenance interpretations is poorly constrained. Sediment and mineral sorting occurs with changes in hydraulic conditions driven by both allogenic and autogenic processes. Zircon is sorted from less dense minerals due to the difference in density, and any age dependence on zircon size could potentially bias provenance interpretations. In this study, a coupled paleohydraulic and geochemical provenance approach is used to identify changes in paleohydraulic conditions and relate them to spatial variations in provenance signatures from samples collected along an approximately time-correlative source-to-sink pathway in the Permian Cutler Group of the Paradox Basin. Samples proximal to the uplift have a paleoflow direction to the southwest. In the medial basin, paleocurrent direction indicates salt movement caused fluvial pathways divert to the north and northwest on the flanks of anticlines. Channel depth, flow velocity, and discharge calculations were derived from field measurements of grain size and dune and bar cross-stratification indicate that competency of the fluvial system decreased from proximal to the medial basin by up to a factor of 12. Based upon the paleohydraulic calculations, zircon size fractionation would occur along the transect such that the larger zircons are removed from the system prior to reaching the medial basin. Analysis of the size and age distribution of zircons from the proximal and distal fluvial system of the Cutler Group tests if this hydraulic sorting affects the expected Uncompahgre Uplift age distribution.

Self-Sorting of Bidispersed Colloidal Particles Near Contact Line of an Evaporating Sessile Droplet.

PubMed

Patil, Nagesh D; Bhardwaj, Rajneesh; Sharma, Atul

2018-06-13

Here, we investigate deposit patterns and associated morphology formed after the evaporation of an aqueous droplet containing mono- and bidispersed colloidal particles. In particular, the combined effect of substrate heating and particle diameter is investigated. We employ high-speed visualization, optical microscopy, and scanning electron microscopy to characterize the evaporating droplets, particle motion, and deposit morphology, respectively. In the context of monodispersed colloidal particles, an inner deposit and a typical ring form for smaller and larger particles, respectively, on a nonheated surface. The formation of the inner deposit is attributed to early depinning of the contact line, explained by a mechanistic model based on the balance of several forces acting on a particle near the contact line. At larger substrate temperature, a thin ring with inner deposit forms, explained by the self-pinning of the contact line and advection of the particles from the contact line to the center of the droplet due to the Marangoni flow. In the context of bidispersed colloidal particles, self-sorting of the colloidal particles within the ring occurs at larger substrate temperature. The smaller particles deposit at the outermost edge compared to the larger particles, and this preferential deposition in a stagnation region near the contact line is due to the spatially varying height of the liquid-gas interface above the substrate. The sorting occurs at a smaller ratio of the diameters of the smaller and larger particles. At larger substrate temperature and larger ratio, the particles do not get sorted and mix into each other. Our measurements show that there exists a critical substrate temperature as well as a diameter ratio to achieve the sorting. We propose regime maps on substrate temperature-particle diameter and substrate temperature-diameter ratio plane for mono- and bidispersed solutions, respectively.

Identifying genes that extend life span using a high-throughput screening system.

PubMed

Chen, Cuiying; Contreras, Roland

2007-01-01

We developed a high-throughput functional genomic screening system that allows identification of genes prolonging lifespan in the baker's yeast Saccharomyces cerevisiae. The method is based on isolating yeast mother cells with a higher than average number of cell divisions as indicated by the number of bud scars on their surface. Fluorescently labeled wheat germ agglutinin (WGA) was used for specific staining of chitin, a major component of bud scars. The critical new steps in our bud-scar-sorting system are the use of small microbeads, which allows successive rounds of purification and regrowth of the mother cells (M-cell), and utilization of flow cytometry to sort and isolate cells with a longer lifespan based on the number of bud scars specifically labeled with WGA.

Automated Microfluidic Instrument for Label-Free and High-Throughput Cell Separation.

PubMed

Zhang, Xinjie; Zhu, Zhixian; Xiang, Nan; Long, Feifei; Ni, Zhonghua

2018-03-20

Microfluidic technologies for cell separation were reported frequently in recent years. However, a compact microfluidic instrument enabling thoroughly automated cell separation is still rarely reported until today due to the difficult hybrid between the macrosized fluidic control system and the microsized microfluidic device. In this work, we propose a novel and automated microfluidic instrument to realize size-based separation of cancer cells in a label-free and high-throughput manner. Briefly, the instrument is equipped with a fully integrated microfluidic device and a set of robust fluid-driven and control units, and the instrument functions of precise fluid infusion and high-throughput cell separation are guaranteed by a flow regulatory chip and two cell separation chips which are the key components of the microfluidic device. With optimized control programs, the instrument is successfully applied to automatically sort human breast adenocarcinoma cell line MCF-7 from 5 mL of diluted human blood with a high recovery ratio of ∼85% within a rapid processing time of ∼23 min. We envision that our microfluidic instrument will be potentially useful in many biomedical applications, especially cell separation, enrichment, and concentration for the purpose of cell culture and analysis.

Multidirectional cross-species painting illuminates the history of karyotypic evolution in Perissodactyla.

PubMed

Trifonov, Vladimir A; Stanyon, Roscoe; Nesterenko, Anastasia I; Fu, Beiyuan; Perelman, Polina L; O'Brien, Patricia C M; Stone, Gary; Rubtsova, Nadezhda V; Houck, Marlys L; Robinson, Terence J; Ferguson-Smith, Malcolm A; Dobigny, Gauthier; Graphodatsky, Alexander S; Yang, Fengtang

2008-01-01

The order Perissodactyla, the group of odd-toed ungulates, includes three extant families: Equidae, Tapiridae, and Rhinocerotidae. The extremely rapid karyotypic diversification in perissodactyls has so far prevented the establishment of genome-wide homology maps between these three families by traditional cytogenetic approaches. Here we report the first genome-wide comparative chromosome maps of African rhinoceroses, four tapir species, four equine species, and humans. These maps were established by multidirectional chromosome painting, with paint probes derived from flow-sorted chromosomes of Equus grevyi, Tapirus indicus, and Ceratotherium simum as well as painting probes from horse and human. The Malayan tapir (Tapirus indicus), Baird's tapir (T. bairdii), mountain tapir (T. pinchaque), lowland tapir (T. terrestris), and onager (E. hemionus onager), were studied by cross-species chromosome painting for the first time. Our results, when integrated with previously published comparative chromosome maps of the other perissodactyl species, have enabled the reconstruction of perissodactyl, ceratomorph, and equid ancestral karyotypes, and the identification of the defining evolutionary chromosomal rearrangements along each lineage. Our results allow a more reliable estimate of the mode and tempo of evolutionary chromosomal rearrangements, revealing a striking switch between the slowly evolving ceratomorphs and extremely rapidly evolving equids.

A sorting system with automated gates permits individual operant experiments with mice from a social home cage.

PubMed

Winter, York; Schaefers, Andrea T U

2011-03-30

Behavioral experiments based on operant procedures can be time-consuming for small amounts of data. While individual testing and handling of animals can influence attention, emotion, and behavior, and interfere with experimental outcome, many operant protocols require individual testing. We developed an RFID-technology- and transponder-based sorting system that allows removing the human factor for longer-term experiments. Identity detectors and automated gates route mice individually from their social home cage to an adjacent operant compartment with 24/7 operation. CD1-mice learnt quickly to individually pass through the sorting system. At no time did more than a single mouse enter the operant compartment. After 3 days of adjusting to the sorting system, groups of 4 mice completed about 50 experimental trials per day in the operant compartment without experimenter intervention. The automated sorting system eliminates handling, isolation, and disturbance of the animals, eliminates experimenter-induced variability, saves experimenter time, and is financially economical. It makes possible a new approach for high-throughput experimentation, and is a viable tool for increasing quality and efficiency of many behavioral and neurobiological investigations. It can connect a social home cage, through individual sorting automation, to diverse setups including classical operant chambers, mazes, or arenas with video-based behavior classification. Such highly automated systems will permit efficient high-throughput screening even for transgenic animals with only subtle neurological or psychiatric symptoms where elaborate or longer-term protocols are required for behavioral diagnosis. Copyright © 2011 Elsevier B.V. All rights reserved.

Disease-Causing Mutations in BEST1 Gene Are Associated with Altered Sorting of Bestrophin-1 Protein

PubMed Central

Doumanov, Jordan A.; Zeitz, Christina; Gimenez, Paloma Dominguez; Audo, Isabelle; Krishna, Abhay; Alfano, Giovanna; Diaz, Maria Luz Bellido; Moskova-Doumanova, Veselina; Lancelot, Marie-Elise; Sahel, José-Alain; Nandrot, Emeline F.; Bhattacharya, Shomi S.

2013-01-01

Mutations in BEST1 gene, encoding the bestrophin-1 (Best1) protein are associated with macular dystrophies. Best1 is predominantly expressed in the retinal pigment epithelium (RPE), and is inserted in its basolateral membrane. We investigated the cellular localization in polarized MDCKII cells of disease-associated Best1 mutant proteins to study specific sorting motifs of Best1. Real-time PCR and western blots for endogenous expression of BEST1 in MDCK cells were performed. Best1 mutant constructs were generated using site-directed mutagenesis and transfected in MDCK cells. For protein sorting, confocal microscopy studies, biotinylation assays and statistical methods for quantification of mislocalization were used. Analysis of endogenous expression of BEST1 in MDCK cells revealed the presence of BEST1 transcript but no protein. Confocal microscopy and quantitative analyses indicate that transfected normal human Best1 displays a basolateral localization in MDCK cells, while cell sorting of several Best1 mutants (Y85H, Q96R, L100R, Y227N, Y227E) was altered. In contrast to constitutively active Y227E, constitutively inactive Y227F Best1 mutant localized basolaterally similar to the normal Best1 protein. Our data suggest that at least three basolateral sorting motifs might be implicated in proper Best1 basolateral localization. In addition, non-phosphorylated tyrosine 227 could play a role for basolateral delivery. PMID:23880862

Purification of adult hepatic progenitor cells using green fluorescent protein (GFP)-transgenic mice and fluorescence-activated cell sorting.

PubMed

Fujikawa, Takahisa; Hirose, Tetsuro; Fujii, Hideaki; Oe, Shoshiro; Yasuchika, Kentaro; Azuma, Hisaya; Yamaoka, Yoshio

2003-08-01

Recent advances in stem cell research have revealed that hepatic stem/progenitor cells may play an important role in liver development and regeneration. However, a lack of detectable definitive markers in viable cells has hindered their primary culture from adult livers. Enzymatically dissociated liver cells from green fluorescent protein (GFP)-transgenic mice, which express GFP highly in liver endodermal cells, were sorted by GFP expression using a fluorescence-activated cell sorter. Sorted cells were characterized, and also low-density cultured for extended periods to determine their proliferation and clonal differentiation capacities. When CD45(-)TER119(-) side-scatter(low) GFP(high) cells were sorted, alpha-fetoprotein-positive immature endoderm-characterized cells, having high growth potential, were present in this population. Clonal analysis and electron microscopic evaluation revealed that each single cell of this population could differentiate not only into hepatocytes, but also into biliary epithelial cells, showing their bilineage differentiation activity. When surface markers were analyzed, they were positive for Integrin-alpha6 and -beta1, but negative for c-Kit and Thy1.1. Combination of GFP-transgenic mice and fluorescence-activated cell sorting enabled purification of hepatic progenitor cells from adult mouse liver. Further analysis of this population may lead to purification of their human correspondence that would be an ideal cell-source candidate for regenerative medicine.

Characterization of bedload transport in steep-slope streams

NASA Astrophysics Data System (ADS)

Mettra, F.; Heyman, J.; Ancey, C.

2012-04-01

Large fluctuations in the sediment transport rate are observed in rivers, particularly in mountain streams at intermediate flow rates. These fluctuations seem to be, to some degree, correlated to the formation and migration of bedforms. Today the central question is still how to understand and account for the strong bedload variability. Recent experimental studies shed new light on the processes. The objective of this presentation is to show some of our results. To understand the behavior and the origins of sediment transport rate fluctuations in the case of steep-slope streams, we conducted laboratory experiments in a 3-m long, 8-cm wide, transparent flume. The experimental parameters are the flume inclination, flow rate and sediment input rate. Well-sorted natural gravel (8.5 mm mean diameter) were used. We focused on two-dimensional flows and incipient bedforms (i.e., for flow rates just above the threshold of incipient motion). A technique based on accelerometers was developed to record every particle passing through the flume outlet (more specifically, we measured the vibrations of a metallic slab, which was impacted by the falling particles). Analysis of bedload transport rates was then possible on all time scales. Moreover, the bed and flow were monitored using 2 cameras. We computed bed elevation, water depth and erosion/deposition at high temporal and spatial rates from camera shots (one image per second during several hours or days). In our laboratory experiments, the fluctuations of the sediment rate were large even for steady flow conditions involving well-sorted particles. Time series exhibited fluctuations at all scales and displayed long range correlations with a Hurst exponent close to 0.8. The results were compared for different input solid discharges. The main bedforms observed in our flume were anti-dunes migrating upstream. Bedform formation and propagation showed intermittency with pulses (high activity) followed by long sequences of low activity. We tried to interpret our results (bedform behavior, bed scouring) in terms of sediment outflow rate.

Selective Sorting of Cargo Proteins into Bacterial Membrane Vesicles*

PubMed Central

Haurat, M. Florencia; Aduse-Opoku, Joseph; Rangarajan, Minnie; Dorobantu, Loredana; Gray, Murray R.; Curtis, Michael A.; Feldman, Mario F.

2011-01-01

In contrast to the well established multiple cellular roles of membrane vesicles in eukaryotic cell biology, outer membrane vesicles (OMV) produced via blebbing of prokaryotic membranes have frequently been regarded as cell debris or microscopy artifacts. Increasingly, however, bacterial membrane vesicles are thought to play a role in microbial virulence, although it remains to be determined whether OMV result from a directed process or from passive disintegration of the outer membrane. Here we establish that the human oral pathogen Porphyromonas gingivalis has a mechanism to selectively sort proteins into OMV, resulting in the preferential packaging of virulence factors into OMV and the exclusion of abundant outer membrane proteins from the protein cargo. Furthermore, we show a critical role for lipopolysaccharide in directing this sorting mechanism. The existence of a process to package specific virulence factors into OMV may significantly alter our current understanding of host-pathogen interactions. PMID:21056982

Assessment of an Optical Flow Field-Based Polyp Detector for CT Colonography

DTIC Science & Technology

2001-10-25

sort true polyps from false positives based on features extracted from the computed OFFs. II. METHODOLOGY A. Pre-processing The 3D CT data was...subvolume and scrolling direction, as follows:      = ∑ Z ZD y)x,( Smoothy )x,( vv (2) The smoothing filter used is a 3×3 rectangular

Nanoplasmonic lenses for bacteria sorting (Presentation Recording)

NASA Astrophysics Data System (ADS)

Zhu, Xiangchao; Yanik, Ahmet A.

2015-08-01

We demonstrate that patches of two dimensional arrays of circular plasmonic nanoholes patterned on gold-titanium thin film enables subwavelength focusing of visible light in far field region. Efficient coupling of the light with the excited surface plasmon at metal dielectric interface results in strong light transmission. As a result, surface plasmon plays an important role in the far field focusing behavior of the nanohole-aperture patches device. Furthermore, the focal length of the focused beam was found to be predominantly dependent on the overall size of the patch, which is in good agreement with that calculated by Rayleigh-Sommerfield integral formula. The focused light beam can be utilized to separate bio-particles in the dynamic range from 0.1 μm to 1 μm through mainly overcoming the drag force induced by fluid flow. In our proposed model, focused light generated by our plasmonic lenses will push the larger bio-particles in size back to the source of fluid flow and allow the smaller particles to move towards the central aperture of the patch. Such a new kind of plasmonic lenses open up possibility of sorting bacterium-like particles with plasmonic nanolenses, and also represent a promising tool in the field of virology.

Analysis of four microsatellite markers on the long arm of chromosome 9 by meiotic recombination in flow-sorted single sperm

DOE Office of Scientific and Technical Information (OSTI.GOV)

Furlong, R.A.; Goudie, D.R.; Carter, N.P.

1993-06-01

Meiotic recombination in flow-sorted single sperm was used to analyze four highly polymorphic microsatellite markers on the long arm of chromosome 9. The microsatellites comprised three tightly linked markers: 9CMP1 (D9S109), 9CMP2 (D9S127), and D9S53, which map to 9q31, and a reference marker, ASS, which is located in 9q34.1. Haplotypes of single sperm were assessed by using PCR in a single-step multiplex reaction to amplify each locus. Recombinant haplotypes were identified by their relative infrequency and were analyzed using THREELOC, a maximum-likelihood-analysis program, and an adaptation of CRI-MAP. The most likely order of these markers was cen-D9S109-D9S127-D9S53-ASS-tel with D9S109, D9S127,more » and D9S53 being separated by a genetic distance of approximately 3%. The order of the latter three markers did not however achieve statistical significance using the THREELOC program. 21 refs., 2 figs., 4 tabs.« less

Assessing materials handling and storage capacities in port terminals

NASA Astrophysics Data System (ADS)

Dinu, O.; Roşca, E.; Popa, M.; Roşca, M. A.; Rusca, A.

2017-08-01

Terminals constitute the factual interface between different modes and, as a result, buffer stocks are unavoidable whenever transport flows with different discontinuities meet. This is the reason why assessing materials handling and storage capacities is an important issue in the course of attempting to increase operative planning of logistic processes in terminals. Proposed paper starts with a brief review of the compatibilities between different sorts of materials and corresponding transport modes and after, a literature overview of the studies related to ports terminals and their specialization is made. As a methodology, discrete event simulation stands as a feasible technique for assessing handling and storage capacities at the terminal, taking into consideration the multi-flows interaction and the non-uniform arrivals of vessels and inland vehicles. In this context, a simulation model, that integrates the activities of an inland water terminal and describes the essential interactions between the subsystems which influence the terminal capacity, is developed. Different scenarios are simulated for diverse sorts of materials, leading to bottlenecks identification, performance indicators such as average storage occupancy rate, average dwell or transit times estimations, and their evolution is analysed in order to improve the transfer operations in the logistic process

Final technical report

DOE Office of Scientific and Technical Information (OSTI.GOV)

Edward DeLong

2011-10-07

Our overarching goals in this project were to: Develop and improve high-throughput sequencing methods and analytical approaches for quantitative analyses of microbial gene expression at the Hawaii Ocean Time Series Station and the Bermuda Atlantic Time Series Station; Conduct field analyses following gene expression patterns in picoplankton microbial communities in general, and Prochlorococcus flow sorted from that community, as they respond to different environmental variables (light, macronutrients, dissolved organic carbon), that are predicted to influence activity, productivity, and carbon cycling; Use the expression analyses of flow sorted Prochlorococcus to identify horizontally transferred genes and gene products, in particular those thatmore » are located in genomic islands and likely to confer habitat-specific fitness advantages; Use the microbial community gene expression data that we generate to gain insights, and test hypotheses, about the variability, genomic context, activity and function of as yet uncharacterized gene products, that appear highly expressed in the environment. We achieved the above goals, and even more over the course of the project. This includes a number of novel methodological developments, as well as the standardization of microbial community gene expression analyses in both field surveys, and experimental modalities. The availability of these methods, tools and approaches is changing current practice in microbial community analyses.« less

Flow characteristics and longitudinal sorting patterns associated with stable alluvial-bedrock and bedrock-alluvial transitions

NASA Astrophysics Data System (ADS)

Jafarinik, S.; Viparelli, E.

2017-12-01

Recent research recognized the existence of bedrock channels in low-slope rivers, but little is known about the morphodynamics of bedrock-alluvial and alluvial-bedrock transitions in these systems. Bedrock-alluvial and alluvial-bedrock transitions are fluvial features separating bedrock and alluvial reaches. In the bedrock reach the river bed is partially covered with alluvium. An increase in sediment supply to an alluvial reach results in channel bed aggradation. An increase in sediment supply to a bedrock reach, on the other hand, results in a reduction of the exposed bedrock. Mathematical modeling of the alluvial morphodynamics of bedrock reaches reveals that these transitions can characterize transient or equilibrium conditions. Model results show that the magnitude of the alluvial equilibrium slope and the depth of the bedrock surface relative to the downstream water surface base level have a primary control on the equilibrium conditions. Further, numerical results show that when a stable bedrock-alluvial transition forms, the bed material transport capacity in bedrock reach decreases in the flow direction. On the contrary, when a stable alluvial-bedrock transition forms the bed material transport capacity in the bedrock reach increases in the flow direction. These spatial changes in bed material transport capacity are associated with spatial changes in alluvial cover and flow hydrodynamics. Here we present a one-dimensional formulation of alluvial morphodynamics that accounts for the non-uniformity of the bed material and for the spatial change in flow resistances associated with the spatial and temporal changes in flow hydrodynamics of the bedrock reaches. This change in flow resistances can be associated with 1) changes in skin friction due to longitudinal changes in the grain size distribution of the bed surface, and/or 2) changes in bedform geometry associated with the interaction between the alluvial cover and the underlying bedrock. The model has been validated against laboratory experiments with stable alluvial-bedrock transitions and is applied to describe the spatial changes in flow characteristics and sediment sorting patterns upstream of a stable bedrock-alluvial transition.

Electroosmotic flow analysis of a branched U-turn nanofluidic device.

PubMed

Parikesit, Gea O F; Markesteijn, Anton P; Kutchoukov, Vladimir G; Piciu, Oana; Bossche, Andre; Westerweel, Jerry; Garini, Yuval; Young, Ian T

2005-10-01

In this paper, we present the analysis of electroosmotic flow in a branched -turn nanofluidic device, which we developed for detection and sorting of single molecules. The device, where the channel depth is only 150 nm, is designed to optically detect fluorescence from a volume as small as 270 attolitres (al) with a common wide-field fluorescent setup. We use distilled water as the liquid, in which we dilute 110 nm fluorescent beads employed as tracer-particles. Quantitative imaging is used to characterize the pathlines and velocity distribution of the electroosmotic flow in the device. Due to the device's complex geometry, the electroosmotic flow cannot be solved analytically. Therefore we use numerical flow simulation to model our device. Our results show that the deviation between measured and simulated data can be explained by the measured Brownian motion of the tracer-particles, which was not incorporated in the simulation.

IB-LBM study on cell sorting by pinched flow fractionation.

PubMed

Ma, Jingtao; Xu, Yuanqing; Tian, Fangbao; Tang, Xiaoying

2014-01-01

Separation of two categories of cells in pinched flow fractionation(PFF) device is simulated by employing IB-LBM. The separation performances at low Reynolds number (about 1) under different pinched segment widths, flow ratios, cell features, and distances between neighboring cells are studied and the results are compared with those predicted by the empirical formula. The simulation indicates that the diluent flow rate should approximate to or more than the flow rate of particle solution in order to get a relatively ideal separation performance. The discrepancy of outflow position between numerical simulation and the empirical prediction enlarges, when the cells become more flexible. Too short distance between two neighboring cells could lead to cell banding which would result in incomplete separation, and the relative position of two neighboring cells influences the banding of cells. The present study will probably provide some new applications of PFF, and make some suggestions on the design of PFF devices.

The Challenge of Promoting Algorithmic Thinking of Both Sciences- and Humanities-Oriented Learners

ERIC Educational Resources Information Center

Katai, Z.

2015-01-01

The research results we present in this paper reveal that properly calibrated e-learning tools have potential to effectively promote the algorithmic thinking of both science-oriented and humanities-oriented students. After students had watched an illustration (by a folk dance choreography) and an animation of the studied sorting algorithm (bubble…

Isolation of Primary Human Skeletal Muscle Cells

PubMed Central

Spinazzola, Janelle M.; Gussoni, Emanuela

2017-01-01

Primary myoblast culture is a valuable tool in research of muscle disease, pathophysiology, and pharmacology. This protocol describes techniques for dissociation of cells from human skeletal muscle biopsies and enrichment for a highly myogenic population by fluorescence-activated cell sorting (FACS). We also describe methods for assessing myogenicity and population expansion for subsequent in vitro study. PMID:29152538

Chemorheology of reactive systems: Finite element analysis

NASA Technical Reports Server (NTRS)

Douglas, C.; Roylance, D.

1982-01-01

The equations which govern the nonisothermal flow of reactive fluids are outlined, and the means by which finite element analysis is used to solve these equations for the sort of arbitrary boundary conditions encountered in industrial practice are described. The performance of the computer code is illustrated by several trial problems, selected more for their value in providing insight to polymer processing flows than as practical production problems. Although a good deal remains to be learned as to the performance and proper use of this numerical technique, it is undeniably useful in providing better understanding of today's complicated polymer processing problems.

A programmable droplet-based microfluidic device applied to multiparameter analysis of single microbes and microbial communities

PubMed Central

Leung, Kaston; Zahn, Hans; Leaver, Timothy; Konwar, Kishori M.; Hanson, Niels W.; Pagé, Antoine P.; Lo, Chien-Chi; Chain, Patrick S.; Hallam, Steven J.; Hansen, Carl L.

2012-01-01

We present a programmable droplet-based microfluidic device that combines the reconfigurable flow-routing capabilities of integrated microvalve technology with the sample compartmentalization and dispersion-free transport that is inherent to droplets. The device allows for the execution of user-defined multistep reaction protocols in 95 individually addressable nanoliter-volume storage chambers by consecutively merging programmable sequences of picoliter-volume droplets containing reagents or cells. This functionality is enabled by “flow-controlled wetting,” a droplet docking and merging mechanism that exploits the physics of droplet flow through a channel to control the precise location of droplet wetting. The device also allows for automated cross-contamination-free recovery of reaction products from individual chambers into standard microfuge tubes for downstream analysis. The combined features of programmability, addressability, and selective recovery provide a general hardware platform that can be reprogrammed for multiple applications. We demonstrate this versatility by implementing multiple single-cell experiment types with this device: bacterial cell sorting and cultivation, taxonomic gene identification, and high-throughput single-cell whole genome amplification and sequencing using common laboratory strains. Finally, we apply the device to genome analysis of single cells and microbial consortia from diverse environmental samples including a marine enrichment culture, deep-sea sediments, and the human oral cavity. The resulting datasets capture genotypic properties of individual cells and illuminate known and potentially unique partnerships between microbial community members. PMID:22547789

Flow Cytometry Detection of Infectious Rotaviruses in Environmental and Clinical Samples

PubMed Central

Abad, F. Xavier; Pintó, Rosa M.; Bosch, Albert

1998-01-01

A method for the detection of infectious human rotaviruses based on infection of CaCo-2 cells and detection of infected cells by indirect immunofluorescence and flow cytometry (IIF-FC) has been developed. The technique was validated by performing a seminested reverse transcription-PCR assay with sorted cell populations. The efficiency of the procedure has been compared with that of the standard method of infection of MA104 cells and ulterior detection by IIF and optical microscopy (IIF-OM) and with that of infection of MA104 cells and detection by IIF-FC. The limit of sensitivity for the detection of the cell-adapted strain Itor P13, expressed as the most probable number of cytopathogenic units, was established as 200 and 2 for MA104 and CaCo-2 cells, respectively, by the IIF-FC method. The ratio of infectious virus particles to total virus particles for a wild-type rotavirus was determined to be 1/2 × 106 and 1/2 × 104 for IIF-OM with MA104 cells and IIF-FC with CaCo-2 cells, respectively. The use of IIF-FC with CaCo-2 cells was tested with fecal and water samples and proved to be more effective than the standard procedure for rotavirus detection. PMID:9647805

Isolation and characterization of living circulating tumor cells in patients by immunomagnetic negative enrichment coupled with flow cytometry.

PubMed

Lu, Yusheng; Liang, Haiyan; Yu, Ting; Xie, Jingjing; Chen, Shuming; Dong, Haiyan; Sinko, Patrick J; Lian, Shu; Xu, Jianguo; Wang, Jichuang; Yu, Suhong; Shao, Jingwei; Yuan, Bo; Wang, Lie; Jia, Lee

2015-09-01

This study was aimed at establishing a sensitive and specific isolation, characterization, and enumeration method for living circulating tumor cells (CTCs) in patients with colorectal carcinoma. Quantitative isolation and characterization of CTCs were performed through a combination of immunomagnetic negative enrichment and fluorescence-activated cell sorting. Isolated CTCs were identified by immunofluorescence staining. The viability and purity of the sorted cells were determined by flow cytometry. Blood samples spiked with HCT116 cells (range, 3-250 cells) were used to determine specificity, recovery, and sensitivity. The method was used to enumerate, characterize, and isolate living CTCs in 10 mL of blood from patients with colorectal carcinoma. The average recovery of HCT116 cells was 61% or more at each spiking level, and the correlation coefficient was 0.992. An analysis of samples from all 18 patients with colorectal carcinoma revealed that 94.4% were positive for CTCs with an average of 33 ± 24 CTCs per 10 mL of blood and with a diameter of 14 to 20 μm (vs 8-12 μm for lymphoma). All patients were CD47(+) , with only 4.3% to 61.2% being CD44(+) . The number of CTCs was well correlated with the patient TNM stage and could be detected in patients at an early cancer stage. The sorted cells could be recultured, and their viability was preserved. This method provides a novel technique for highly sensitive and specific detection and isolation of CTCs in patients with colorectal carcinoma. This method complements the existing approaches for the de novo functional identification of a wide variety of CTC types. It is likely to help in predicting a patient's disease progression and potentially in selecting the appropriate treatment. © 2015 American Cancer Society.

TH-EF-BRA-06: A Novel Retrospective 3D K-Space Sorting 4D-MRI Technique Using a Radial K-Space Acquisition MRI Sequence

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Y; Subashi, E; Yin, F

Purpose: Current retrospective 4D-MRI provides superior tumor-to-tissue contrast and accurate respiratory motion information for radiotherapy motion management. The developed 4D-MRI techniques based on 2D-MRI image sorting require a high frame-rate of the MR sequences. However, several MRI sequences provide excellent image quality but have low frame-rate. This study aims at developing a novel retrospective 3D k-space sorting 4D-MRI technique using radial k-space acquisition MRI sequences to improve 4D-MRI image quality and temporal-resolution for imaging irregular organ/tumor respiratory motion. Methods: The method is based on a RF-spoiled, steady-state, gradient-recalled sequence with minimal echo time. A 3D radial k-space data acquisition trajectorymore » was used for sampling the datasets. Each radial spoke readout data line starts from the 3D center of Field-of-View. Respiratory signal can be extracted from the k-space center data point of each spoke. The spoke data was sorted based on its self-synchronized respiratory signal using phase sorting. Subsequently, 3D reconstruction was conducted to generate the time-resolved 4D-MRI images. As a feasibility study, this technique was implemented on a digital human phantom XCAT. The respiratory motion was controlled by an irregular motion profile. To validate using k-space center data as a respiratory surrogate, we compared it with the XCAT input controlling breathing profile. Tumor motion trajectories measured on reconstructed 4D-MRI were compared to the average input trajectory. The mean absolute amplitude difference (D) was calculated. Results: The signal extracted from k-space center data matches well with the input controlling respiratory profile of XCAT. The relative amplitude error was 8.6% and the relative phase error was 3.5%. XCAT 4D-MRI demonstrated a clear motion pattern with little serrated artifacts. D of tumor trajectories was 0.21mm, 0.23mm and 0.23mm in SI, AP and ML directions, respectively. Conclusion: A novel retrospective 3D k-space sorting 4D-MRI technique has been developed and evaluated on human digital phantom. NIH (1R21CA165384-01A1)« less

Metal-silicate fractionation in the surface dust layers of accreting planetesimals: Implications for the formation of ordinary chondrites and the nature of asteroid surfaces

NASA Astrophysics Data System (ADS)

Huang, Shaoxiong; Akridge, Glen; Sears, Derek W. G.

Some of the most primitive solar system materials available for study in the laboratory are the ordinary chondrites, the largest meteorite class. The size and distribution of the chondrules (silicate beads) and metal, which leads to the definition of the H, L, and LL classes, suggest sorting before or during aggregation. We suggest that meteorite parent bodies (probably asteroids) had thick dusty surfaces during their early evolution that were easily mobilized by gases evolving from their interiors. Density and size sorting would have occurred in the surface layers as the upward drag forces of the gases (mainly water) acted against the downward force of gravity. The process is analogous to the industrially important process of fluidization and sorting in pyroclastic volcanics. We calculate that gas flow velocities and gas fluxes for the regolith of an asteroid-sized object heated by the impact of accreting objects or by 26Al would have been sufficient for fluidization. It can also explain, quantitatively in some cases, the observed metal-silicate sorting of ordinary chondrites, which has long been ascribed to processes occurring in the primordial solar nebula. Formation of the chondrites in the thick dynamic regolith is consistent with the major properties of chondritic meteorites (i.e., redox state, petrologic type, cooling rate, matrix abundance). These ideas have implications for the nature of asteroid surfaces and the virtual lack of asteroids with ordinary chondrite-like surfaces.

Lake trout in northern Lake Huron spawn on submerged drumlins

USGS Publications Warehouse

Riley, Stephen C.; Binder, Thomas; Wattrus, Nigel J.; Faust, Matthew D.; Janssen, John; Menzies, John; Marsden, J. Ellen; Ebener, Mark P.; Bronte, Charles R.; He, Ji X.; Tucker, Taaja R.; Hansen, Michael J.; Thompson, Henry T.; Muir, Andrew M.; Krueger, Charles C.

2014-01-01

Recent observations of spawning lake trout Salvelinus namaycush near Drummond Island in northern Lake Huron indicate that lake trout use drumlins, landforms created in subglacial environments by the action of ice sheets, as a primary spawning habitat. From these observations, we generated a hypothesis that may in part explain locations chosen by lake trout for spawning. Most salmonines spawn in streams where they rely on streamflows to sort and clean sediments to create good spawning habitat. Flows sufficient to sort larger sediment sizes are generally lacking in lakes, but some glacial bedforms contain large pockets of sorted sediments that can provide the interstitial spaces necessary for lake trout egg incubation, particularly if these bedforms are situated such that lake currents can penetrate these sediments. We hypothesize that sediment inclusions from glacial scavenging and sediment sorting that occurred during the creation of bedforms such as drumlins, end moraines, and eskers create suitable conditions for lake trout egg incubation, particularly where these bedforms interact with lake currents to remove fine sediments. Further, these bedforms may provide high-quality lake trout spawning habitat at many locations in the Great Lakes and may be especially important along the southern edge of the range of the species. A better understanding of the role of glacially-derived bedforms in the creation of lake trout spawning habitat may help develop powerful predictors of lake trout spawning locations, provide insight into the evolution of unique spawning behaviors by lake trout, and aid in lake trout restoration in the Great Lakes.

PACMan to Help Sort Hubble Proposals

NASA Astrophysics Data System (ADS)

Kohler, Susanna

2017-04-01

Every year, astronomers submit over a thousand proposals requesting time on the Hubble Space Telescope (HST). Currently, humans must sort through each of these proposals by hand before sending them off for review. Could this burden be shifted to computers?A Problem of VolumeAstronomer Molly Peeples gathered stats on the HST submissions sent in last week for the upcoming HST Cycle 25 (the deadline was Friday night), relative to previous years. This years proposal round broke the record, with over 1200 proposals submitted in total for Cycle 25. [Molly Peeples]Each proposal cycle for HST time attracts on the order of 1100 proposals accounting for far more HST time than is available. The proposals are therefore carefully reviewed by around 150 international members of the astronomy community during a six-month process to select those with the highest scientific merit.Ideally, each proposal will be read by reviewers that have scientific expertise relevant to the proposal topic: if a proposal requests HST time to study star formation, for instance, then the reviewers assigned to it should have research expertise in star formation.How does this matching of proposals to reviewers occur? The current method relies on self-reported categorization of the submitted proposals. This is unreliable, however; proposals are often mis-categorized by submitters due to misunderstanding or ambiguous cases.As a result, the Science Policies Group at the Space Telescope Science Institute (STScI) which oversees the review of HST proposals must go through each of the proposals by hand and re-categorize them. The proposals are then matched to reviewers with self-declared expertise in the same category.With the number of HST proposals on the rise and the expectation that the upcoming James Webb Space Telescope (JWST) will elicit even more proposals for time than Hubble scientists at STScI and NASA are now asking: could the human hours necessary for this task be spared? Could a computer program conceivably do this matching instead?Comparison of PACMans categorization to the manual sorting for HST Cycle 24 proposals. Green: proposals similarly categorized by both. Yellow: proposals whose manual classifications are within the top 60% of sorted PACMan classifications. Red: proposals categorized differently by each. [Strolger et al. 2017]Introducing PACManLed by Louis-Gregory Strolger (STScI), a team of scientists has developed PACMan: the Proposal Auto-Categorizer and Manager. PACMan is whats known as a Naive Bayesian classifier; its essentially a spam-filtering routine that uses word probabilities to sort proposals into multiple scientific categories and identify people to serve on review panels in those same scientific areas.PACMan works by looking at the words in aproposal and comparing them to those in a training set of proposals in this case, the previous years HST proposals, sorted by humans. By using the training set, PACMan learns how to accurately classify proposals.PACMan then looks up each reviewer on the Astrophysical Data System (ADS) and compiles the abstracts from the reviewers past 10 years worth of scientific publications. This text is then evaluated in a similar way to the text of the proposals, determining each reviewers suitability to evaluate a proposal.How Did It Do?Comparison of PACMan sorting to manual sorting, specifically for the HST Cycle 24 proposals that were recategorized by the Science Policies Group (SPG) from what the submitter (PI) selected. Of these swaps, 48% would have been predicted by PACMan. [Strolger et al. 2017]Strolger and collaborators show that with a training set of one previous cycles proposals, PACMan correctly categorizes the next cycles proposals roughly 87% of the time. By increasing the size of the training set to include more past cycles, PACMans accuracy can be improved up to 95% (though the algorithm will have to be retrained any time the proposal categories change).PACMans results were also consistent for reviewers: it found that nearly all of the reviewers (92%) asked to serve in the last cycle were appropriate reviewers for the subject area based on their ADS publication record.There are still some hiccups in automating this process for instance, finding the reviewers on ADS can require human intervention due to names not being unique. As the scientific community moves toward persistent and distinguishable identifiers (like ORCIDs), however, this problem will be mitigated.Strolger and collaborators believe that PACMan demonstrates a promising means of increasing the efficiency and impartiality of the HST proposal sorting process. This tool will likely be used to assist or replace humans in this processin future HST and JWST cycles.CitationLouis-Gregory Strolger et al 2017 AJ 153 181. doi:10.3847/1538-3881/aa6112

Chinese undergraduates' preferences for altruistic traits in mate selection and personal advertisement: Evidence from Q-sort technique.

PubMed

Guo, Qingke; Feng, Linlin; Wang, Mingming

2017-04-01

Human's preferences for altruistic mates have been confirmed by many researchers. Under the deep influence of Confucianism that authorised more parental control over offspring's mate selection, Chinese people's mating strategies and mate preferences may be different from what the evolutionary psychologists have suggested. This study used the Q-sort technique to assess the roles of altruistic traits in mate selection and personal advertisement. A total of 200 university students participated in the Q-sort procedures and were asked to sort 50 traits (among which altruistic traits were mixed) according to their importance when choosing (or advertising to) a long-term (LT) or a short-term (ST) mate. Our findings were quite different from prior studies. When Chinese participants chose a mate or advertised themselves to a potential mate, kin altruism was considered to be the most important trait; altruistic traits were more preferred by males than by females and females tended to advertise themselves as more altruistic; preferences for altruistic traits showed no difference between LT and ST mate selections (or between personal advertisement to a LT and a ST mate). © 2015 International Union of Psychological Science.

Quadrupole Magnetic Sorting of Porcine Islets of Langerhans

PubMed Central

Shenkman, Rustin M.; Chalmers, Jeffrey J.; Hering, Bernhard J.; Kirchhof, Nicole

2009-01-01

Islet transplantation is emerging as a treatment option for selected patients with type 1 diabetes. Inconsistent isolation, purification, and recovery of large numbers of high-quality islets remain substantial impediments to progress in the field. Removing islets as soon as they are liberated from the pancreas during digestion and circumventing the need for density gradient purification is likely to result in substantially increased viable islet yields by minimizing exposure to proteolytic enzymes, reactive oxygen intermediates, and mechanical stress associated with centrifugation. This study capitalized on the hypervascularity of islets compared with acinar tissue to explore their preferential enrichment with magnetic beads to enable immediate separation in a magnetic field utilizing a quadrupole magnetic sorting. The results demonstrate that (1) preferential enrichment of porcine islets is achievable, but homogeneous bead distribution within the pancreas is difficult to achieve with current protocols; (2) greater than 70% of islets in the dissociated pancreatic tissue were recovered by quadrupole magnetic sorting, but their purity was low; and (3) infused islets purified by density gradients and subsequently passed through quadrupole magnetic sorting had similar potency as uninfused islets. These results demonstrate proof of concept and define the steps for implementation of this technology in pig and human islet isolation. PMID:19505179

Cell separation using tilted-angle standing surface acoustic waves

PubMed Central

Ding, Xiaoyun; Peng, Zhangli; Lin, Sz-Chin Steven; Geri, Michela; Li, Sixing; Li, Peng; Chen, Yuchao; Dao, Ming; Suresh, Subra; Huang, Tony Jun

2014-01-01

Separation of cells is a critical process for studying cell properties, disease diagnostics, and therapeutics. Cell sorting by acoustic waves offers a means to separate cells on the basis of their size and physical properties in a label-free, contactless, and biocompatible manner. The separation sensitivity and efficiency of currently available acoustic-based approaches, however, are limited, thereby restricting their widespread application in research and health diagnostics. In this work, we introduce a unique configuration of tilted-angle standing surface acoustic waves (taSSAW), which are oriented at an optimally designed inclination to the flow direction in the microfluidic channel. We demonstrate that this design significantly improves the efficiency and sensitivity of acoustic separation techniques. To optimize our device design, we carried out systematic simulations of cell trajectories, matching closely with experimental results. Using numerically optimized design of taSSAW, we successfully separated 2- and 10-µm-diameter polystyrene beads with a separation efficiency of ∼99%, and separated 7.3- and 9.9-µm-polystyrene beads with an efficiency of ∼97%. We illustrate that taSSAW is capable of effectively separating particles–cells of approximately the same size and density but different compressibility. Finally, we demonstrate the effectiveness of the present technique for biological–biomedical applications by sorting MCF-7 human breast cancer cells from nonmalignant leukocytes, while preserving the integrity of the separated cells. The method introduced here thus offers a unique route for separating circulating tumor cells, and for label-free cell separation with potential applications in biological research, disease diagnostics, and clinical practice. PMID:25157150

Cell separation using tilted-angle standing surface acoustic waves.

PubMed

Ding, Xiaoyun; Peng, Zhangli; Lin, Sz-Chin Steven; Geri, Michela; Li, Sixing; Li, Peng; Chen, Yuchao; Dao, Ming; Suresh, Subra; Huang, Tony Jun

2014-09-09

Separation of cells is a critical process for studying cell properties, disease diagnostics, and therapeutics. Cell sorting by acoustic waves offers a means to separate cells on the basis of their size and physical properties in a label-free, contactless, and biocompatible manner. The separation sensitivity and efficiency of currently available acoustic-based approaches, however, are limited, thereby restricting their widespread application in research and health diagnostics. In this work, we introduce a unique configuration of tilted-angle standing surface acoustic waves (taSSAW), which are oriented at an optimally designed inclination to the flow direction in the microfluidic channel. We demonstrate that this design significantly improves the efficiency and sensitivity of acoustic separation techniques. To optimize our device design, we carried out systematic simulations of cell trajectories, matching closely with experimental results. Using numerically optimized design of taSSAW, we successfully separated 2- and 10-µm-diameter polystyrene beads with a separation efficiency of ∼ 99%, and separated 7.3- and 9.9-µm-polystyrene beads with an efficiency of ∼ 97%. We illustrate that taSSAW is capable of effectively separating particles-cells of approximately the same size and density but different compressibility. Finally, we demonstrate the effectiveness of the present technique for biological-biomedical applications by sorting MCF-7 human breast cancer cells from nonmalignant leukocytes, while preserving the integrity of the separated cells. The method introduced here thus offers a unique route for separating circulating tumor cells, and for label-free cell separation with potential applications in biological research, disease diagnostics, and clinical practice.

On-chip particle trapping and manipulation

NASA Astrophysics Data System (ADS)

Leake, Kaelyn Danielle

The ability to control and manipulate the world around us is human nature. Humans and our ancestors have used tools for millions of years. Only in recent years have we been able to control objects at such small levels. In order to understand the world around us it is frequently necessary to interact with the biological world. Optical trapping and manipulation offer a non-invasive way to move, sort and interact with particles and cells to see how they react to the world around them. Optical tweezers are ideal in their abilities but they require large, non-portable, and expensive setups limiting how and where we can use them. A cheap portable platform is required in order to have optical manipulation reach its full potential. On-chip technology offers a great solution to this challenge. We focused on the Liquid-Core Anti-Resonant Reflecting Optical Waveguide (liquid-core ARROW) for our work. The ARROW is an ideal platform, which has anti-resonant layers which allow light to be guided in liquids, allowing for particles to easily be manipulated. It is manufactured using standard silicon manufacturing techniques making it easy to produce. The planner design makes it easy to integrate with other technologies. Initially I worked to improve the ARROW chip by reducing the intersection losses and by reducing the fluorescence and background on the ARROW chip. The ARROW chip has already been used to trap and push particles along its channel but here I introduce several new methods of particle trapping and manipulation on the ARROW chip. Traditional two beam traps use two counter propagating beams. A trapping scheme that uses two orthogonal beams which counter to first instinct allow for trapping at their intersection is introduced. This scheme is thoroughly predicted and analyzed using realistic conditions. Simulations of this method were done using a program which looks at both the fluidics and optical sources to model complex situations. These simulations were also used to model and predict a sorting method which combines fluid flow with a single optical source to automatically sort dielectric particles by size in waveguide networks. These simulations were shown to be accurate when repeated on-chip. Lastly I introduce a particle trapping technique that uses Multimode Interference(MMI) patterns in order to trap multiple particles at once. The location of the traps can be adjusted as can the number of trapping location by changing the input wavelength. By changing the wavelength back and forth between two values this MMI can be used to pass a particle down the channel like a conveyor belt.

A mouse model for triple-negative breast cancer tumor-initiating cells (TNBC-TICs) exhibits similar aggressive phenotype to the human disease.

PubMed

Kaur, Punit; Nagaraja, Ganachari M; Zheng, Hongying; Gizachew, Dawit; Galukande, Moses; Krishnan, Sunil; Asea, Alexzander

2012-03-27

Triple-negative breast cancer (TNBC) exhibit characteristics quite distinct from other kinds of breast cancer, presenting as an aggressive disease--recurring and metastasizing more often than other kinds of breast cancer, without tumor-specific treatment options and accounts for 15% of all types of breast cancer with higher percentages in premenopausal African-American and Hispanic women. The reason for this aggressive phenotype is currently the focus of intensive research. However, progress is hampered by the lack of suitable TNBC cell model systems. To understand the mechanistic basis for the aggressiveness of TNBC, we produced a stable TNBC cell line by sorting for 4T1 cells that do not express the estrogen receptor (ER), progesterone receptor (PgR) or the gene for human epidermal growth factor receptor 2 (HER2). As a control, we produced a stable triple-positive breast cancer (TPBC) cell line by transfecting 4T1 cells with rat HER2, ER and PgR genes and sorted for cells with high expression of ER and PgR by flow cytometry and high expression of the HER2 gene by Western blot analysis. We isolated tumor-initiating cells (TICs) by sorting for CD24+/CD44high/ALDH1+ cells from TNBC (TNBC-TICs) and TPBC (TPBC-TICs) stable cell lines. Limiting dilution transplantation experiments revealed that CD24+/CD44high/ALDH1+ cells derived from TNBC (TNBC-TICs) and TPBC (TPBC-TICs) were significantly more effective at repopulating the mammary glands of naïve female BALB/c mice than CD24-/CD44-/ALDH1- cells. Implantation of the TNBC-TICs resulted in significantly larger tumors, which metastasized to the lungs to a significantly greater extent than TNBC, TPBC-TICs, TPBC or parental 4T1 cells. We further demonstrated that the increased aggressiveness of TNBC-TICs correlates with the presence of high levels of mouse twenty-five kDa heat shock protein (Hsp25/mouse HspB1) and seventy-two kDa heat shock protein (Hsp72/HspA1A). Taken together, we have developed a TNBC-TICs model system based on the 4T1 cells which is a very useful metastasis model with the advantage of being able to be transplanted into immune competent recipients. Our data demonstrates that the TNBC-TICs model system could be a useful tool for studies on the pathogenesis and therapeutic treatment for TNBC.

A mouse model for triple-negative breast cancer tumor-initiating cells (TNBC-TICs) exhibits similar aggressive phenotype to the human disease

PubMed Central

2012-01-01

Background Triple-negative breast cancer (TNBC) exhibit characteristics quite distinct from other kinds of breast cancer, presenting as an aggressive disease--recurring and metastasizing more often than other kinds of breast cancer, without tumor-specific treatment options and accounts for 15% of all types of breast cancer with higher percentages in premenopausal African-American and Hispanic women. The reason for this aggressive phenotype is currently the focus of intensive research. However, progress is hampered by the lack of suitable TNBC cell model systems. Methods To understand the mechanistic basis for the aggressiveness of TNBC, we produced a stable TNBC cell line by sorting for 4T1 cells that do not express the estrogen receptor (ER), progesterone receptor (PgR) or the gene for human epidermal growth factor receptor 2 (HER2). As a control, we produced a stable triple-positive breast cancer (TPBC) cell line by transfecting 4T1 cells with rat HER2, ER and PgR genes and sorted for cells with high expression of ER and PgR by flow cytometry and high expression of the HER2 gene by Western blot analysis. Results We isolated tumor-initiating cells (TICs) by sorting for CD24+/CD44high/ALDH1+ cells from TNBC (TNBC-TICs) and TPBC (TPBC-TICs) stable cell lines. Limiting dilution transplantation experiments revealed that CD24+/CD44high/ALDH1+ cells derived from TNBC (TNBC-TICs) and TPBC (TPBC-TICs) were significantly more effective at repopulating the mammary glands of naïve female BALB/c mice than CD24-/CD44-/ALDH1- cells. Implantation of the TNBC-TICs resulted in significantly larger tumors, which metastasized to the lungs to a significantly greater extent than TNBC, TPBC-TICs, TPBC or parental 4T1 cells. We further demonstrated that the increased aggressiveness of TNBC-TICs correlates with the presence of high levels of mouse twenty-five kDa heat shock protein (Hsp25/mouse HspB1) and seventy-two kDa heat shock protein (Hsp72/HspA1A). Conclusions Taken together, we have developed a TNBC-TICs model system based on the 4T1 cells which is a very useful metastasis model with the advantage of being able to be transplanted into immune competent recipients. Our data demonstrates that the TNBC-TICs model system could be a useful tool for studies on the pathogenesis and therapeutic treatment for TNBC. PMID:22452810

International Society for the Advancement of Cytometry cell sorter biosafety standards.

PubMed

Holmes, Kevin L; Fontes, Benjamin; Hogarth, Philip; Konz, Richard; Monard, Simon; Pletcher, Charles H; Wadley, Robert B; Schmid, Ingrid; Perfetto, Stephen P

2014-05-01

Flow cytometric cell sorting of biological specimens has become prevalent in basic and clinical research laboratories. These specimens may contain known or unknown infectious agents, necessitating precautions to protect instrument operators and the environment from biohazards arising from the use of sorters. To this end the International Society of Analytical Cytology (ISAC) was proactive in establishing biosafety guidelines in 1997 (Schmid et al., Cytometry 1997;28:99-117) and subsequently published revised biosafety standards for cell sorting of unfixed samples in 2007 (Schmid et al., Cytometry Part A J Int Soc Anal Cytol 2007;71A:414-437). Since their publication, these documents have become recognized worldwide as the standard of practice and safety precautions for laboratories performing cell sorting experiments. However, the field of cytometry has progressed since 2007, and the document requires an update. The new Standards provides guidance: (1) for laboratory design for cell sorter laboratories; (2) for the creation of laboratory or instrument specific Standard Operating Procedures (SOP); and (3) on procedures for the safe operation of cell sorters, including personal protective equipment (PPE) and validation of aerosol containment. Published © 2014 Wiley Periodicals Inc.

Size-based sorting of micro-particles using microbubble streaming

NASA Astrophysics Data System (ADS)

Wang, Cheng; Jalikop, Shreyas; Hilgenfeldt, Sascha

2009-11-01

Oscillating microbubbles driven by ultrasound have shown great potential in microfluidic applications, such as transporting particles and promoting mixing [1-3]. The oscillations generate secondary steady streaming that can also trap particles. We use the streaming to develop a method of sorting particles of different sizes in an initially well-mixed solution. The solution is fed into a channel consisting of bubbles placed periodically along a side wall. When the bubbles are excited by an ultrasound piezo-electric transducer to produce steady streaming, the flow field is altered by the presence of the particles. This effect is dependent on particle size and results in size-based sorting of the particles. The effectiveness of the separation depends on the dimensions of the bubbles and particles as well as on the ultrasound frequency. Our experimental studies are aimed at a better understanding of the design and control of effective microfluidic separating devices. Ref: [1] P. Marmottant and S. Hilgenfeldt, Nature 423, 153 (2003). [2] P. Marmottant and S. Hilgenfeldt, Proc. Natl. Acad. Science USA, 101, 9523 (2004). [3] P. Marmottant, J.-P. Raven, H. Gardeniers, J. G. Bomer, and S. Hilgenfeldt, J. Fluid Mech., vol.568, 109 (2006).

Sedimentary structures and textures of Rio Orinoco channel sands, Venezuela and Colombia

USGS Publications Warehouse

McKee, Edwin Dinwiddie

1989-01-01

Most sedimentary structures represented in sand bodies of the Rio Orinoco are tabular-planar cross-strata which, together with some wedge-planar cross-strata, are the products of sand-wave deposition. Locally, in areas of river meander where point bars characteristically form, trough structures forming festoon patterns are numerous. At a few localities, sets of nearly horizontal strata occur between tabular-planar sets and are interpreted to be the deposits of very fast currents of the upper flow regime; elsewhere, uncommon lenses and beds of silt, clay, or organic matter consisting of leaves and twigs, seem to be the result of quiet-water settling through gravity. By far the most common grain size represented in the tabular-planar and wedge-planar cross-strata of the sandwave deposits is medium sand (? - ? millimeter) as determined by screen analyses. Many samples, however, also contain moderate quantities of coarse or very coarse sand. Eolian dunes on top of the sand-wave deposits are dominantly fine grained. The river channel sands were determined to be largely moderately well sorted, although in some places they were mostly well sorted, and in others, mostly moderately sorted.

Electropermanent magnet actuation for droplet ferromicrofluidics

PubMed Central

Padovani, José I.; Jeffrey, Stefanie S.; Howe, Roger T.

2016-01-01

Droplet actuation is an essential mechanism for droplet-based microfluidic systems. On-demand electromagnetic actuation is used in a ferrofluid-based microfluidic system for water droplet displacement. Electropermanent magnets (EPMs) are used to induce 50 mT magnetic fields in a ferrofluid filled microchannel with gradients up to 6.4 × 104 kA/m2. Short 50 µs current pulses activate the electropermanent magnets and generate negative magnetophoretic forces that range from 10 to 70 nN on 40 to 80 µm water-in-ferrofluid droplets. Maximum droplet displacement velocities of up to 300 µm/s are obtained under flow and no-flow conditions. Electropermanent magnet-activated droplet sorting under continuous flow is demonstrated using a split-junction microfluidic design. PMID:27583301

Expression of GFP under the control of the RNA helicase VASA permits fluorescence-activated cell sorting isolation of human primordial germ cells.

PubMed

Tilgner, Katarzyna; Atkinson, Stuart P; Yung, Sun; Golebiewska, Anna; Stojkovic, Miodrag; Moreno, Ruben; Lako, Majlinda; Armstrong, Lyle

2010-01-01

The isolation of significant numbers of human primordial germ cells at several developmental stages is important for investigations of the mechanisms by which they are able to undergo epigenetic reprogramming. Only small numbers of these cells can be obtained from embryos of appropriate developmental stages, so the differentiation of human embryonic stem cells is essential to obtain sufficient numbers of primordial germ cells to permit epigenetic examination. Despite progress in the enrichment of human primordial germ cells using fluorescence-activated cell sorting (FACS), there is still no definitive marker of the germ cell phenotype. Expression of the widely conserved RNA helicase VASA is restricted to germline cells, but in contrast to species such as Mus musculus in which reporter constructs expressing green fluorescent protein (GFP) under the control of a Vasa promoter have been developed, such reporter systems are lacking in human in vitro models. We report here the generation and characterization of human embryonic stem cell lines stably carrying a VASA-pEGFP-1 reporter construct that expresses GFP in a population of differentiating human embryonic stem cells that show expression of characteristic markers of primordial germ cells. This population shows a different pattern of chromatin modifications to those obtained by FACS enrichment of Stage Specific Antigen one expressing cells in our previous publication.

Fractalkine (CX3CL1), a new factor protecting β-cells against TNFα.

PubMed

Rutti, Sabine; Arous, Caroline; Schvartz, Domitille; Timper, Katharina; Sanchez, Jean-Charles; Dermitzakis, Emmanouil; Donath, Marc Y; Halban, Philippe A; Bouzakri, Karim

2014-10-01

We have previously shown the existence of a muscle-pancreas intercommunication axis in which CX3CL1 (fractalkine), a CX3C chemokine produced by skeletal muscle cells, could be implicated. It has recently been shown that the fractalkine system modulates murine β-cell function. However, the impact of CX3CL1 on human islet cells especially regarding a protective role against cytokine-induced apoptosis remains to be investigated. Gene expression was determined using RNA sequencing in human islets, sorted β- and non-β-cells. Glucose-stimulated insulin secretion (GSIS) and glucagon secretion from human islets was measured following 24 h exposure to 1-50 ng/ml CX3CL1. GSIS and specific protein phosphorylation were measured in rat sorted β-cells exposed to CX3CL1 for 48 h alone or in the presence of TNFα (20 ng/ml). Rat and human β-cell apoptosis (TUNEL) and rat β-cell proliferation (BrdU incorporation) were assessed after 24 h treatment with increasing concentrations of CX3CL1. Both CX3CL1 and its receptor CX3CR1 are expressed in human islets. However, CX3CL1 is more expressed in non-β-cells than in β-cells while its receptor is more expressed in β-cells. CX3CL1 decreased human (but not rat) β-cell apoptosis. CX3CL1 inhibited human islet glucagon secretion stimulated by low glucose but did not impact human islet and rat sorted β-cell GSIS. However, CX3CL1 completely prevented the adverse effect of TNFα on GSIS and on molecular mechanisms involved in insulin granule trafficking by restoring the phosphorylation (Akt, AS160, paxillin) and expression (IRS2, ICAM-1, Sorcin, PCSK1) of key proteins involved in these processes. We demonstrate for the first time that human islets express and secrete CX3CL1 and CX3CL1 impacts them by decreasing glucagon secretion without affecting insulin secretion. Moreover, CX3CL1 decreases basal apoptosis of human β-cells. We further demonstrate that CX3CL1 protects β-cells from the adverse effects of TNFα on their function by restoring the expression and phosphorylation of key proteins of the insulin secretion pathway.

Activation of ER stress and mTORC1 suppresses hepatic sortilin-1 levels in obese mice

PubMed Central

Ai, Ding; Baez, Juan M.; Jiang, Hongfeng; Conlon, Donna M.; Hernandez-Ono, Antonio; Frank-Kamenetsky, Maria; Milstein, Stuart; Fitzgerald, Kevin; Murphy, Andrew J.; Woo, Connie W.; Strong, Alanna; Ginsberg, Henry N.; Tabas, Ira; Rader, Daniel J.; Tall, Alan R.

2012-01-01

Recent GWAS have identified SNPs at a human chromosom1 locus associated with coronary artery disease risk and LDL cholesterol levels. The SNPs are also associated with altered expression of hepatic sortilin-1 (SORT1), which encodes a protein thought to be involved in apoB trafficking and degradation. Here, we investigated the regulation of Sort1 expression in mouse models of obesity. Sort1 expression was markedly repressed in both genetic (ob/ob) and high-fat diet models of obesity; restoration of hepatic sortilin-1 levels resulted in reduced triglyceride and apoB secretion. Mouse models of obesity also exhibit increased hepatic activity of mammalian target of rapamycin complex 1 (mTORC1) and ER stress, and we found that administration of the mTOR inhibitor rapamycin to ob/ob mice reduced ER stress and increased hepatic sortilin-1 levels. Conversely, genetically increased hepatic mTORC1 activity was associated with repressed Sort1 and increased apoB secretion. Treating WT mice with the ER stressor tunicamycin led to marked repression of hepatic sortilin-1 expression, while administration of the chemical chaperone PBA to ob/ob mice led to amelioration of ER stress, increased sortilin-1 expression, and reduced apoB and triglyceride secretion. Moreover, the ER stress target Atf3 acted at the SORT1 promoter region as a transcriptional repressor, whereas knockdown of Atf3 mRNA in ob/ob mice led to increased hepatic sortilin-1 levels and decreased apoB and triglyceride secretion. Thus, in mouse models of obesity, induction of mTORC1 and ER stress led to repression of hepatic Sort1 and increased VLDL secretion via Atf3. This pathway may contribute to dyslipidemia in metabolic disease. PMID:22466652

Parkin mediates the ubiquitination of VPS35 and modulates retromer-dependent endosomal sorting.

PubMed

Williams, Erin T; Glauser, Liliane; Tsika, Elpida; Jiang, Haisong; Islam, Shariful; Moore, Darren J

2018-06-11

Mutations in a number of genes cause familial forms of Parkinson's disease (PD), including mutations in the vacuolar protein sorting 35 ortholog (VPS35) and parkin genes. In this study, we identify a novel functional interaction between parkin and VPS35. We demonstrate that parkin interacts with and robustly ubiquitinates VPS35 in human neural cells. Familial parkin mutations are impaired in their ability to ubiquitinate VPS35. Parkin mediates the attachment of an atypical poly-ubiquitin chain to VPS35 with three lysine residues identified within the C-terminal region of VPS35 that are covalently modified by ubiquitin. Notably, parkin-mediated VPS35 ubiquitination does not promote the proteasomal degradation of VPS35. Furthermore, parkin does not influence the steady-state levels or turnover of VPS35 in neural cells and VPS35 levels are normal in the brains of parkin knockout mice. These data suggest that ubiquitination of VPS35 by parkin may instead serve a non-degradative cellular function potentially by regulating retromer-dependent sorting. Accordingly, we find that components of the retromer-associated WASH complex are markedly decreased in the brain of parkin knockout mice, suggesting that parkin may modulate WASH complex-dependent retromer sorting. Parkin gene silencing in primary cortical neurons selectively disrupts the vesicular sorting of the autophagy receptor ATG9A, a WASH-dependent retromer cargo. Parkin is not required for dopaminergic neurodegeneration induced by the expression of PD-linked D620N VPS35 in mice, consistent with VPS35 being located downstream of parkin function. Our data reveal a novel functional interaction of parkin with VPS35 that may be important for retromer-mediated endosomal sorting and PD.

Introduction to the session on `Human Space Exploration'

NASA Astrophysics Data System (ADS)

Messidoro, P.

When Schiaparelli tried to interpret the surface of Mars as it appeared from his telescope, in particular with reference to the famous "channels", he formulated the hypothesis that they would have been the product of some intelligent Mars population. Today we know that this interpretation was not correct, but we would like to consider his idea as a sort of vision for a future when the humankind will export our civilization on Mars. In fact the objective of the international plans of "Space Exploration" is exactly to land the humans on Mars to start its colonization. Although a new approach is proposed which includes International Space Station, Moon, Asteroids, etc. in a sort of "flexible path" to look for "new worlds in the Solar System where is possible for the humankind to live and operate", it is confirmed that the "final destination" is Mars.

Enhanced M1 Macrophage Polarization in Human Helicobacter pylori-Associated Atrophic Gastritis and in Vaccinated Mice

PubMed Central

Quiding-Järbrink, Marianne; Raghavan, Sukanya; Sundquist, Malin

2010-01-01

Background Infection with Helicobacter pylori triggers a chronic gastric inflammation that can progress to atrophy and gastric adenocarcinoma. Polarization of macrophages is a characteristic of both cancer and infection, and may promote progression or resolution of disease. However, the role of macrophages and their polarization during H. pylori infection has not been well defined. Methodology/Principal Findings By using a mouse model of infection and gastric biopsies from 29 individuals, we have analyzed macrophage recruitment and polarization during H. pylori infection by flow cytometry and real-time PCR. We found a sequential recruitment of neutrophils, eosinophils and macrophages to the gastric mucosa of infected mice. Gene expression analysis of stomach tissue and sorted macrophages revealed that gastric macrophages were polarized to M1 after H. pylori infection, and this process was substantially accelerated by prior vaccination. Human H. pylori infection was characterized by a mixed M1/M2 polarization of macrophages. However, in H. pylori-associated atrophic gastritis, the expression of inducible nitric oxide synthase was markedly increased compared to uncomplicated gastritis, indicative of an enhanced M1 macrophage polarization in this pre-malignant lesion. Conclusions/Significance These results show that vaccination of mice against H. pylori amplifies M1 polarization of gastric macrophages, and that a similar enhanced M1 polarization is present in human H. pylori-induced atrophic gastritis. PMID:21124899

Low-dose cisplatin protects human neuroblastoma SH-SY5Y cells from paclitaxel-induced apoptosis.

PubMed

Villa, Daniela; Miloso, Mariarosaria; Nicolini, Gabriella; Rigolio, Roberta; Villa, Antonello; Cavaletti, Guido; Tredici, Giovanni

2005-09-01

Combined anticancer therapy using platinum compounds and antitubulins has increased the risk of neurotoxicity. However, the combination of low-dose cisplatin (CDDP) with toxic doses of paclitaxel significantly reduces cellular death in a human neuroblastoma SH-SY5Y cell line. To analyze the mechanisms of this protection, we evaluated various signaling molecules possibly involved in apoptosis and some relevant cell cycle regulatory proteins. CDDP does not interfere with the tubulin-stabilizing action of paclitaxel. The evaluation of molecular pathways involved in apoptosis indicates that the Bcl-2 but not the caspases may be involved in the CDDP protection of paclitaxel-induced apoptosis. The increase in p53 protein and its nuclear accumulation suggests a possible involvement of p53 in CDDP protection. The use of the chemical inhibitor of p53, pifithrin alpha, excluded this possibility. The study of cyclins and the flow cytometric analysis (fluorescence-activated cell sorting) suggest that CDDP exerts a protective action by blocking cells early in the cell cycle. The determination of the mitotic index indicates that CDDP prevents cells from reaching the mitosis. We concluded that low doses of CDDP are protective against toxic doses of paclitaxel and that the possible mechanism of this protection is that the CDDP prevents human neuroblastoma SH-SY5Y cells from achieving mitosis.

Fine-grained bed patch response to near-bankfull flows in a step-pool channel

Treesearch

Daniel A. Marion; Frank Weirich

1999-01-01

Fine-grained bed patches were monitored in a representative step-pool channel in the Arkansas Ouachita Mountains to assess their response to near-bankfull streamflow events. These patches are small, relatively well-sorted bed areas predominantly composed of gravel-size and smaller grains. They occupy 5.2 and 4.1 percent of the active and bankfull channel areas,...

Democratization and Instability in Ukraine, Georgia, and Belarus

DTIC Science & Technology

2014-06-01

Smithsonian “ laissez faire ”35 approaches, whereas autocracies, with their strict control over monetary flows and investments via referential...threat- en profits; hence, they reduce investment and retard growth. Democracy is thus inimical to economic development.41 In laissez faire ...performance of domestic economies. This is the opposite side of laissez - faire , a sort of Keynesian vision on economy44 as influenced by governance

Information Memory Processing and Retrieval: Relationships of Concrete Learning and Concrete and Abstract Cognitions.

ERIC Educational Resources Information Center

Dean, Bonnie L.

Reported is a study related to the Project on an Information Memory Model and designed to encompass the claims of Piaget and Inhelder on differences of kinds of cognition and recall done on figural sorting task cognition at the Project on an Information Memory Model. The work of Piaget and Inhelder has defined learning information flow and related…

Validation of neural spike sorting algorithms without ground-truth information.

PubMed

Barnett, Alex H; Magland, Jeremy F; Greengard, Leslie F

2016-05-01

The throughput of electrophysiological recording is growing rapidly, allowing thousands of simultaneous channels, and there is a growing variety of spike sorting algorithms designed to extract neural firing events from such data. This creates an urgent need for standardized, automatic evaluation of the quality of neural units output by such algorithms. We introduce a suite of validation metrics that assess the credibility of a given automatic spike sorting algorithm applied to a given dataset. By rerunning the spike sorter two or more times, the metrics measure stability under various perturbations consistent with variations in the data itself, making no assumptions about the internal workings of the algorithm, and minimal assumptions about the noise. We illustrate the new metrics on standard sorting algorithms applied to both in vivo and ex vivo recordings, including a time series with overlapping spikes. We compare the metrics to existing quality measures, and to ground-truth accuracy in simulated time series. We provide a software implementation. Metrics have until now relied on ground-truth, simulated data, internal algorithm variables (e.g. cluster separation), or refractory violations. By contrast, by standardizing the interface, our metrics assess the reliability of any automatic algorithm without reference to internal variables (e.g. feature space) or physiological criteria. Stability is a prerequisite for reproducibility of results. Such metrics could reduce the significant human labor currently spent on validation, and should form an essential part of large-scale automated spike sorting and systematic benchmarking of algorithms. Copyright © 2016 Elsevier B.V. All rights reserved.

Effects of sediment-associated extractable metals, degree of sediment grain sorting, and dissolved organic carbon upon Cryptosporidium parvum removal and transport within riverbank filtration sediments, Sonoma County, California

USGS Publications Warehouse

Metge, D.W.; Harvey, R.W.; Aiken, G.R.; Anders, R.; Lincoln, G.; Jasperse, James; Hill, M.C.

2011-01-01

Oocysts of the protozoan pathogen Cryptosporidium parvum are of particular concern for riverbank filtration (RBF) operations because of their persistence, ubiquity, and resistance to chlorine disinfection. At the Russian River RBF site (Sonoma County, CA), transport of C. parvumoocysts and oocyst-sized (3 μm) carboxylate-modified microspheres through poorly sorted (sorting indices, σ1, up to 3.0) and geochemically heterogeneous sediments collected between 2 and 25 m below land surface (bls) were assessed. Removal was highly sensitive to variations in both the quantity of extractable metals (mainly Fe and Al) and degree of grain sorting. In flow-through columns, there was a log–linear relationship (r2 = 0.82 at p < 0.002) between collision efficiency (α, the probability that colloidal collisions with grain surfaces would result in attachment) and extractable metals, and a linear relationship (r2 = 0.99 at p < 0.002) between α and σ1. Collectively, variability in extractable metals and grain sorting accounted for ∼83% of the variability in α (at p < 0.0002) along the depth profiles. Amendments of 2.2 mg L–1 of Russian River dissolved organic carbon (DOC) reduced α for oocysts by 4–5 fold. The highly reactive hydrophobic organic acid (HPOA) fraction was particularly effective in re-entraining sediment-attached microspheres. However, the transport-enhancing effects of the riverine DOC did not appear to penetrate very deeply into the underlying sediments, judging from high α values (∼1.0) observed for oocysts being advected through unamended sediments collected at ∼2 m bls. This study suggests that in evaluating the efficacy of RBF operations to remove oocysts, it may be necessary to consider not only the geochemical nature and size distribution of the sediment grains, but also the degrees of sediment sorting and the concentration, reactivity, and penetration of the source water DOC.

MHC class II/ESO tetramer-based generation of in vitro primed anti-tumor T-helper lines for adoptive cell therapy of cancer.

PubMed

Poli, Caroline; Raffin, Caroline; Dojcinovic, Danijel; Luescher, Immanuel; Ayyoub, Maha; Valmori, Danila

2013-02-01

Generation of tumor-antigen specific CD4(+) T-helper (T(H)) lines through in vitro priming is of interest for adoptive cell therapy of cancer, but the development of this approach has been limited by the lack of appropriate tools to identify and isolate low frequency tumor antigen-specific CD4(+) T cells. Here, we have used recently developed MHC class II/peptide tetramers incorporating an immunodominant peptide from NY-ESO-1 (ESO), a tumor antigen frequently expressed in different human solid and hematologic cancers, to implement an in vitro priming platform allowing the generation of ESO-specific T(H) lines. We isolated phenotypically defined CD4(+) T-cell subpopulations from circulating lymphocytes of DR52b(+) healthy donors by flow cytometry cell sorting and stimulated them in vitro with peptide ESO(119-143), autologous APC and IL-2. We assessed the frequency of ESO-specific cells in the cultures by staining with DR52b/ESO(119-143) tetramers (ESO-tetramers) and TCR repertoire of ESO-tetramer(+) cells by co-staining with TCR variable β chain (BV) specific antibodies. We isolated ESO-tetramer(+) cells by flow cytometry cell sorting and expanded them with PHA, APC and IL-2 to generate ESO-specific T(H) lines. We characterized the lines for antigen recognition, by stimulation with ESO peptide or recombinant protein, cytokine production, by intracellular staining using specific antibodies, and alloreactivity, by stimulation with allo-APC. Using this approach, we could consistently generate ESO-tetramer(+) T(H) lines from conventional CD4(+)CD25(-) naïve and central memory populations, but not from effector memory populations or CD4(+)CD25(+) Treg. In vitro primed T(H) lines recognized ESO with affinities comparable to ESO-tetramer(+) cells from patients immunized with an ESO vaccine and used a similar TCR repertoire. In this study, using MHC class II/ESO tetramers, we have implemented an in vitro priming platform allowing the generation of ESO-monospecific polyclonal T(H) lines from non-immune individuals. This is an approach that is of potential interest for adoptive cell therapy of patients bearing ESO-expressing cancers.

[Effects of handling and processing on deoxynivalenol and zearalenone content of cereals and cereal products].

PubMed

Wolff, J

2005-12-01

Since national limits have been introduced for the content of DON and ZEA in cereals and cereal products designated for human consumption, it is highly important to understand how these toxins are distributed during sorting, cleaning and further processing to bakery products and pasta. Cereals from several crops were analysed before and after sorting and cleaning. After milling, flours, breads, semolinas, pastas and others were analysed. The results show that that the distribution of DON and ZEA was different. ZEA was more effectively removed than DON. The efficacy of the various processes varied markedly from one lot to the other.

Alluvial fan facies of the Yongchong Basin: Implications for tectonic and paleoclimatic changes during Late Cretaceous in SE China

NASA Astrophysics Data System (ADS)

Chen, Liuqin; Steel, Ronald J.; Guo, Fusheng; Olariu, Cornel; Gong, Chenglin

2017-02-01

Late Cretaceous continental redbeds, the Guifeng Group of the Yongchong Basin in SE China have been investigated to conduct detailed fan facies description and interpretation. Tectonic activities determined the alluvial fan development along the basin margin, but the alluvial facies was linked with paleoclimate changes. The Guifeng Group is divided into the Hekou, Tangbian and Lianhe formations in ascending order. The Hekou conglomerates are typically polymict, moderately sorted with erosional bases, cut-and-fill features, normal grading and sieve deposits, representing dominant stream-flows on alluvial fans during the initial opening stage of the basin infill. The Tangbian Formation, however, is characterized by structureless fine-grained sediments with dispersed coarse clasts, and couplets of conglomerate and sandstone or siltstone and mudstone, recording a change to a playa and ephemeral lake environments with occasional stream flooding, thus indicating a basin expanding stage. The hallmark of the Lianhe Formation is disorganized, poorly sorted conglomerates lack of erosional bases, and a wide particle-size range from clay to boulders together reflect mud-rich debris-flows accumulating on fans, likely related to reactivation of faulting along the northwestern mountain fronts during a post-rift stage. The depositional system changes from stream-flows up through playa with ephemeral streams to debris-flows during the accumulation of the three formations are thus attributed to different source rocks and climatic conditions. Therefore, the fluvial-dominated fans of the Hekou Formation recorded a subhumid paleoclimate (Coniacian-Santonian Age). The dominant semiarid climate during the Campanian Age produced abundant fine-grained sediments in the playa and ephemeral lake environments of the Tangbian Formation. A climatic change towards more humidity during the late stage of the Guifeng Group (Maastrichtian Age) probably yielded high deposition rate of coarse clasts in debris-flow dominated fans of the Lianhe Formation. Thus the Late Cretaceous climate changes are inferred to have influenced and preserved signals in the alluvial stratigraphy of the Yongchong Basin.

From Traffic Flow to Economic System

NASA Astrophysics Data System (ADS)

Bando, M.

The optimal velocity model which is applied to traffic flow phenomena explains a spontaneous formation of traffic congestion. We discuss why the model works well in describing both free-flow and congested flow states in a unified way. The essential ingredient is that our model takes account of a sort of time delay in reacting to a given stimulus. This causes instability of many-body system, and yields a kind of phase transition above a certain critical density. Especially there appears a limit cycle on the phase space along which individual vehicle moves, and they show cyclic behavior. Once that we recognize the mechanism the same idea can be applied to a variety of phenomena which show cyclic behavior observed in many-body systems. As an example of such applications, we investigate business cycles commonly observed in economic system. We further discuss a possible origin of a kind of cyclic behavior observed in climate change.

Evidence of photospheric vortex flows at supergranular junctions observed by FG/SOT (Hinode)

NASA Astrophysics Data System (ADS)

Attie, R.; Innes, D. E.; Potts, H. E.

2009-01-01

Context: Twisting motions of different sorts are observed in several layers of the solar atmosphere. Chromospheric sunspot whorls and rotation of sunspots or even higher up in the lower corona sigmoids are examples of the large-scale twisted topology of many solar features. Nevertheless, their occurrence on a large scale in the quiet photosphere has not been investigated yet. Aims: The present study reveals the existence of vortex flows located at the supergranular junctions of the quiet Sun. Methods: We used a 1-h and a 5-h time series of the granulation in blue continuum and G-band images from FG/SOT to derive the photospheric flows. A feature-tracking technique called balltracking was performed to track the granules and reveal the underlying flow fields. Results: In both time series, we identify long lasting vortex flow located at supergranular junctions. The first vortex flow lasts at least 1 h and is ~20´´ wide (~15.5 Mm). The second vortex flow lasts more than 2 h and is ~27´´ wide (~21 Mm).

mAb C19 targets a novel surface marker for the isolation of human cardiac progenitor cells from human heart tissue and differentiated hESCs.

PubMed

Leung, Hau Wan; Moerkamp, Asja T; Padmanabhan, Jayanthi; Ng, Sze-Wai; Goumans, Marie-José; Choo, Andre

2015-05-01

Cardiac progenitor cells (CPCs) have been isolated from adult and developing hearts using an anti-mouse Sca-1 antibody. However, the absence of a human Sca-1 homologue has hampered the clinical application of the CPCs. Therefore, we generated novel monoclonal antibodies (mAbs) specifically raised against surface markers expressed by resident human CPCs. Here, we explored the suitability of one of these mAbs, mAb C19, for the identification, isolation and characterization of CPCs from fetal heart tissue and differentiating cultures of human embryonic stem cells (hESCs). Using whole-cell immunization, mAbs were raised against Sca-1+ CPCs and screened for reactivity to various CPC lines by flow cytometry. mAb C19 was found to be specific for Sca-1+ CPCs, with high cell surface binding capabilities. mAb C19 stained small stem-like cells in cardiac tissue sections. Moreover, during differentiation of hESCs towards cardiomyocytes, a transient population of cells with mAb C19 reactivity was identified and isolated using magnetic-activated cell sorting. Their cell fate was tracked and found to improve cardiomyocyte purity from hESC-derived cultures. mAb C19+ CPCs, from both hESC differentiation and fetal heart tissues, were maintained and expanded in culture, while retaining their CPC-like characteristics and their ability to further differentiate into cardiomyocytes by stimulation with TGFβ1. Finally, gene expression profiling of these mAb C19+ CPCs suggested a highly angiogenic nature, which was further validated by cell-based angiogenesis assays. mAb C19 is a new surface marker for the isolation of multipotent CPCs from both human heart tissues and differentiating hESCs. Copyright © 2015 Elsevier Ltd. All rights reserved.

Attractive and Repulsive Forces on Particles in Oscillatory Flow

NASA Astrophysics Data System (ADS)

Agarwal, Siddhansh; Rallabandi, Bhargav; Raju, David; Thameem, Raqeeb; Hilgenfeldt, Sascha

2016-11-01

A large class of oscillating flows gives rise to rectified streaming motion of the fluid. It has recently been shown that particle transport in such flows, excited by bubbles oscillating at ultrasound frequencies, leads to differential displacement and efficient sorting of microparticles by size. We derive a general expression for the instantaneous radial force experienced by a small spherical particle in the vicinity of an oscillating interface, and generalize the radial projection of the Maxey-Riley equation to include this effect. Varying relevant system parameters, we show that the net effect on the particle can be either an attraction to or a repulsion from the bubble surface, depending in particular on the particle size and the particle/fluid density contrast. We demonstrate that these predictions are in agreement with a variety of experiments.

Microstreaming from Sessile Semicylindrical Bubbles

NASA Astrophysics Data System (ADS)

Hilgenfeldt, Sascha; Rallabandi, Bhargav; Guo, Lin; Wang, Cheng

2014-03-01

Powerful steady streaming flows result from the ultrasonic driving of microbubbles, in particular when these bubbles have semicylindrical cross section and are positioned in contact with a microfluidic channel wall. We have used this streaming in experiment to develop novel methods for trapping and sorting of microparticles by size, as well as for micromixing. Theoretically, we arrive at an analytical description of the streaming flow field through an asymptotic computation that, for the first time, reconciles the boundary layers around the bubble and along the substrate wall, and also takes into account the oscillation modes of the bubble. This approach gives insight into changes in the streaming pattern with bubble size and driving frequency, including a reversal of the flow direction at high frequencies with potentially useful applications. Present address: Mechanical and Aerospace Engineering, Missouri S &T.

Myosin Ib modulates the morphology and the protein transport within multi-vesicular sorting endosomes.

PubMed

Salas-Cortes, Laura; Ye, Fei; Tenza, Danièle; Wilhelm, Claire; Theos, Alexander; Louvard, Daniel; Raposo, Graça; Coudrier, Evelyne

2005-10-15

Members of at least four classes of myosin (I, II, V and VI) have been implicated in the dynamics of a large variety of organelles. Despite their common motor domain structure, some of these myosins, however, are non processive and cannot move organelles along the actin tracks. Here, we demonstrate in the human pigmented MNT-1 cell line that, (1) the overexpression of one of these myosins, myosin 1b, or the addition of cytochalasin D affects the morphology of the sorting multivesicular endosomes; (2) the overexpression of myosin 1b delays the processing of Pmel17 (the product of murine silver locus also named GP100), which occurs in these multivesicular endosomes; (3) myosin 1b associated with endosomes coimmunoprecipitates with Pmel17. All together, these observations suggest that myosin 1b controls the traffic of protein cargo in multivesicular endosomes most probably through its ability to modulate with actin the morphology of these sorting endosomes.

An anti-tumor protein produced by Trichinella spiralis and identified by screening a T7 phage display library, induces apoptosis in human hepatoma H7402 cells

USDA-ARS?s Scientific Manuscript database

Trichinella spiralis infection confers effective resistance to tumor cell expansion. In this study, a T7 phage cDNA display library was constructed to express genes encoded by T. spiralis. Organic phase multi-cell screening was used to sort through candidate proteins in a transfected human chronic m...

7 CFR 996.12 - Outgoing inspection.

Code of Federal Regulations, 2010 CFR

2010-01-01

... STANDARDS FOR DOMESTIC AND IMPORTED PEANUTS MARKETED IN THE UNITED STATES Definitions § 996.12 Outgoing inspection. Outgoing inspection means the sampling, inspection, and certification of either: shelled peanuts... peanuts which have been cleaned, sorted, and otherwise prepared for inshell human consumption markets. ...

7 CFR 996.12 - Outgoing inspection.

Code of Federal Regulations, 2011 CFR

2011-01-01

... STANDARDS FOR DOMESTIC AND IMPORTED PEANUTS MARKETED IN THE UNITED STATES Definitions § 996.12 Outgoing inspection. Outgoing inspection means the sampling, inspection, and certification of either: shelled peanuts... peanuts which have been cleaned, sorted, and otherwise prepared for inshell human consumption markets. ...

Generation of bioaerosols during manual mail unpacking and sorting.

PubMed

Brandl, H; Bachofen, R; Bischoff, M

2005-01-01

The dynamics of bioaerosol generation in specific occupational environments where mail is manually unpacked and sorted was investigated. Total number of airborne particles was determined in four different size classes (0.3-0.5, 0.5-1, 1-5 and >5 microm) by laser particle counting. Time dependent formation of bioaerosols was monitored by culturing methods and by specific staining followed by flow cytometry. Besides handling of regular mail, specially prepared letters ('spiked letters') were added to the mailbags to deliberately release powdered materials from letters and to simulate high impact loads. These letters contained various dry powdered biological and nonbiological materials such as milk powder, mushrooms, herbs and cat litter. Regarding the four size classes, particulate aerosol composition before mail handling was determined as 83.2 +/- 1.0, 15.2 +/- 0.7, 1.7 +/- 0.4 and 0.04 +/- 0.02%, respectively, whereas the composition changed during sorting to 66.8 +/- 7.9, 22.3 +/- 3.6, 10.4 +/- 4.0 and 0.57 +/- 0.27%, respectively. Mail processing resulted in an increase in culturable airborne bacteria and fungi. Maximum concentrations of bacteria reached 450 CFU m(-3), whereas 270 CFU of fungi were detected. Indoor particle concentrations steadily increased during mail handling mostly associated with particles of diameters >1 microm. However, it was not possible to distinguish spiked letters from nonspiked by simple particle counting and CFU determinations. The dynamics of bioaerosol generation have to be addressed when monitoring specific occupational environments (such as mail sorting facilities) regarding the occurrence of biological particles.

Wrinkled alfalfa seeds harbor more aerobic bacteria and are more difficult to sanitize than smooth seeds.

PubMed

Charkowski, A O; Sarreal, C Z; Mandrell, R E

2001-09-01

At least 14 separate outbreaks of food poisoning attributed to either Salmonella enterica or Escherichia coli O157:H7 have been traced to sprouts in the past decade. Seeds contaminated with human pathogens caused most of these outbreaks, thus many sprout growers are now treating alfalfa seeds with the sanitizing agent, calcium hypochlorite (Ca[OCl]2), prior to sprouting. The efficacy of alfalfa seed sanitation varies between seed lots and between seeds within each lot. Alfalfa seeds from different seed lots were sorted by type in an effort to determine if certain seed types carry more aerobic bacteria than other seed types. Seeds with a wrinkled type, characteristic of lygus bug damage, had significantly higher levels of culturable aerobic bacteria and were more difficult to sanitize than smooth, healthy seeds. After sanitation, wrinkled alfalfa seeds that had been inoculated with S. enterica ser. Newport carried significantly higher levels of Salmonella Newport than smooth seeds. If S. enterica is present on wrinkled seeds in naturally contaminated seed lots, it may be difficult to chemically sanitize the seed lot. Removal of the wrinkled alfalfa seeds from the seed lots, perhaps by adapting color sorting equipment similar to that used to sort rice grains and other seeds, should reduce the level of aerobic bacteria in seed lots and may result in lower levels of human pathogens on contaminated alfalfa seeds.

Stochastic Model of Clogging in a Microfluidic Cell Sorter

NASA Astrophysics Data System (ADS)

Fai, Thomas; Rycroft, Chris

2016-11-01

Microfluidic devices for sorting cells by deformability show promise for various medical purposes, e.g. detecting sickle cell anemia and circulating tumor cells. One class of such devices consists of a two-dimensional array of narrow channels, each column containing several identical channels in parallel. Cells are driven through the device by an applied pressure or flow rate. Such devices allows for many cells to be sorted simultaneously, but cells eventually clog individual channels and change the device properties in an unpredictable manner. In this talk, we propose a stochastic model for the failure of such microfluidic devices by clogging and present preliminary theoretical and computational results. The model can be recast as an ODE that exhibits finite time blow-up under certain conditions. The failure time distribution is investigated analytically in certain limiting cases, and more realistic versions of the model are solved by computer simulation.

Integrated fluorescence analysis system

DOEpatents

Buican, Tudor N.; Yoshida, Thomas M.

1992-01-01

An integrated fluorescence analysis system enables a component part of a sample to be virtually sorted within a sample volume after a spectrum of the component part has been identified from a fluorescence spectrum of the entire sample in a flow cytometer. Birefringent optics enables the entire spectrum to be resolved into a set of numbers representing the intensity of spectral components of the spectrum. One or more spectral components are selected to program a scanning laser microscope, preferably a confocal microscope, whereby the spectrum from individual pixels or voxels in the sample can be compared. Individual pixels or voxels containing the selected spectral components are identified and an image may be formed to show the morphology of the sample with respect to only those components having the selected spectral components. There is no need for any physical sorting of the sample components to obtain the morphological information.

In Vivo Rat T-Lymphocyte Pig-a Assay: Detection and Expansion of Cells Deficient in the GPI-Anchored CD48 Surface Marker for Analysis of Mutation in the Endogenous Pig-a Gene.

PubMed

Dobrovolsky, Vasily N; Revollo, Javier; Petibone, Dayton M; Heflich, Robert H

2017-01-01

The Pig-a assay is being developed as an in vivo gene mutation assay for regulatory safety assessments. The assay is based on detecting mutation in the endogenous Pig-a gene of treated rats by using flow cytometry to measure changes in cell surface markers of peripheral blood cells. Here we present a methodology for demonstrating that phenotypically mutant rat T-cells identified by flow cytometry contain mutations in the Pig-a gene, an important step for validating the assay. In our approach, the mutant phenotype T-cells are sorted into individual wells of 96-well plates and expanded into clones. Subsequent sequencing of genomic DNA from the expanded clones confirms that the Pig-a assay detects exactly what it claims to detect-cells with mutations in the endogenous Pig-a gene. In addition, determining the spectra of Pig-a mutations provides information for better understanding the mutational mechanism of compounds of interest. Our methodology of combining phenotypic antibody labeling, magnetic enrichment, sorting, and single-cell clonal expansion can be used in genotoxicity/mutagenicity studies and in other general immunotoxicology research requiring identification, isolation, and expansion of extremely rare subpopulations of T-cells.

Osteometric sorting of skeletal elements from a sample of modern Colombians: a pilot study.

PubMed

Rodríguez, Juan Manuel Guerrero; Hackman, Lucina; Martínez, Wendy; Medina, César Sanabria

2016-03-01

The Colombian armed conflict has been catalogued not only as the longest civil war in the western hemisphere, but also as having one of the highest indexes of missing persons. Among the several challenges faced by forensic practitioners in Colombia, the commingling of human remains has been recognised as one of the most difficult to approach. The method of osteometric sorting described by Byrd and Adams and Byrd (2008) has proven relevant as a powerful tool to aid in the reassociation process of skeletal structures. The aim of this research was to evaluate the three osteometric sorting models developed by Byrd (2008) (paired elements, articulating bone portions and other bone portions) in a sample of modern Colombian individuals. A set of 39 linear measurements was recorded from a sample of 100 individuals (47 females and 53 males aged between 20 and 74 and 18 and 77 years, respectively), which was used to create a reference sample database. A different subset of eight individuals (five females aged between 23 and 48 years, and three males aged between 27 and 43 years) was employed to randomly create six small-scale commingled assemblages for the purposes of testing the osteometric sorting models. Results demonstrate that this method has significant potential for use in the Colombian forensic context.

Downstream lightening and upward heavying, sorting of sediments of uniform grain size but differing in density

NASA Astrophysics Data System (ADS)

Viparelli, E.; Solari, L.; Hill, K. M.

2014-12-01

Downstream fining, i.e. the tendency for a gradual decrease in grain size in the downstream direction, has been observed and studied in alluvial rivers and in laboratory flumes. Laboratory experiments and field observations show that the vertical sorting pattern over a small Gilbert delta front is characterized by an upward fining profile, with preferential deposition of coarse particles in the lowermost part of the deposit. The present work is an attempt to answer the following questions. Are there analogous sorting patterns in mixtures of sediment particles having the same grain size but differing density? To investigate this, we performed experiments at the Hydrosystems Laboratory at the University of Illinois at Urbana-Champaign. During the experiments a Gilbert delta formed and migrated downstream allowing for the study of transport and sorting processes on the surface and within the deposit. The experimental results show 1) preferential deposition of heavy particles in the upstream part of the deposit associated with a pattern of "downstream lightening"; and 2) a vertical sorting pattern over the delta front characterized by a pattern of "upward heavying" with preferential deposition of light particles in the lowermost part of the deposit. The observed downstream lightening is analogous of the downstream fining with preferential deposition of heavy (coarse) particles in the upstream part of the deposit. The observed upward heavying was unexpected because, considering the particle mass alone, the heavy (coarse) particles should have been preferentially deposited in the lowermost part of the deposit. Further, the application of classical fractional bedload transport relations suggests that in the case of mixtures of particles of uniform size and different densities equal mobility is not approached. We hypothesize that granular physics mechanisms traditionally associated with sheared granular flows may be responsible for the observed upward heavying and for the deviation from equal mobility.

New technology for ultrasensitive detection and isolation of rare cells for clinical diagnostics and therapeutics

NASA Astrophysics Data System (ADS)

Leary, James F.; McLaughlin, Scott R.

1995-04-01

A high-speed, 11-parameter, 6-color fluorescence, laser flow cytometer/cell sorter with a number of special and unique features has been built for ultrasensitive detection and isolation of rare cells for clinical diagnostics and therapeutics. The software for real-time data acquisition and sort control, written as C++ programming language modules with a WindowsTM graphical user interface, runs on a 66-MHz 80486 computer joined by an extended bus to 23 sophisticated multi-layered boards of special data acquisition and sorting electronics. Special features include: high-speed (> 100,000 cells/sec) real-time data classification module (U.S. Patent 5,204,884 (1993)); real-time principal component cell sorting; multi-queue signal-processing system with multiple hardware and software event buffers to reduce instrument dead time, LUT charge-pulse definition, high-resolution `flexible' sorting for optimal yield/purity sort strategies (U.S. Patent 5,199,576); pre-focusing optical wavelength correction for a second laser beam; and two trains of three fluorescence detectors-- each adjustable for spatial separation to interrogate only one of two laser beams, syringe- driven or pressure-driven fluidics, and time-windowed parameters. The system has been built to be both expandable and versatile through the use of LUT's and a modular hardware and software design. The instrument is especially useful at detection and isolation of rare cell subpopulations for which our laboratory is well-known. Cell subpopulations at frequencies as small as 10-7 have been successfully studied with this system. Current applications in clinical diagnostics and therapeutics include detection and isolation of (1) fetal cells from material blood for prenatal diagnosis of birth defects, (2) hematopoietic stem and precursor cells for autologous bone marrow transplantation, (3) metastatic breast cancer cells for molecular characterization, and (4) HIV-infected maternal cells in newborn blood to study mother-to-infant vertical transmission of AIDS.

Delineating functional principles of the bow tie structure of a kinase-phosphatase network in the budding yeast.

PubMed

Abd-Rabbo, Diala; Michnick, Stephen W

2017-03-16

Kinases and phosphatases (KP) form complex self-regulating networks essential for cellular signal processing. In spite of having a wealth of data about interactions among KPs and their substrates, we have very limited models of the structures of the directed networks they form and consequently our ability to formulate hypotheses about how their structure determines the flow of information in these networks is restricted. We assembled and studied the largest bona fide kinase-phosphatase network (KP-Net) known to date for the yeast Saccharomyces cerevisiae. Application of the vertex sort (VS) algorithm on the KP-Net allowed us to elucidate its hierarchical structure in which nodes are sorted into top, core and bottom layers, forming a bow tie structure with a strongly connected core layer. Surprisingly, phosphatases tend to sort into the top layer, implying they are less regulated by phosphorylation than kinases. Superposition of the widest range of KP biological properties over the KP-Net hierarchy shows that core layer KPs: (i), receive the largest number of inputs; (ii), form bottlenecks implicated in multiple pathways and in decision-making; (iii), and are among the most regulated KPs both temporally and spatially. Moreover, top layer KPs are more abundant and less noisy than those in the bottom layer. Finally, we showed that the VS algorithm depends on node degrees without biasing the biological results of the sorted network. The VS algorithm is available as an R package ( https://cran.r-project.org/web/packages/VertexSort/index.html ). The KP-Net model we propose possesses a bow tie hierarchical structure in which the top layer appears to ensure highest fidelity and the core layer appears to mediate signal integration and cell state-dependent signal interpretation. Our model of the yeast KP-Net provides both functional insight into its organization as we understand today and a framework for future investigation of information processing in yeast and eukaryotes in general.

Experimental study of the flow over a backward-facing rounded ramp

NASA Astrophysics Data System (ADS)

Duriez, Thomas; Aider, Jean-Luc; Wesfreid, Jose Eduardo

2010-11-01

The backward-facing rounded ramp (BFR) is a very simple geometry leading to boundary layer separation, close to the backward facing step (BFS) flow. The main difference with the BFS flow is that the separation location depends on the incoming flow while it is fixed to the step edge for the BFS flow. Despite the simplicity of the geometry, the flow is complex and the transition process still has to be investigated. In this study we investigate the BFR flow using time-resolved PIV. For Reynolds number ranging between 300 and 12 000 we first study the time averaged properties such as the positions of the separation and reattachment, the recirculation length and the shear layer thickness. The time resolution also gives access to the characteristic frequencies of the time-dependant flow. An appropriate Fourier filtering of the flow field, around each frequency peak in the global spectrum, allows an investigation of each mode in order to extract its wavelength, phase velocity, and spatial distribution. We then sort the spectral content and relate the main frequencies to the most amplified Kelvin-Helmholtz instability mode and its harmonics, the vortex pairing, the low frequency recirculation bubble oscillation and the interactions between all these phenomena.

Identification of innate lymphoid cells in single-cell RNA-Seq data.

PubMed

Suffiotti, Madeleine; Carmona, Santiago J; Jandus, Camilla; Gfeller, David

2017-07-01

Innate lymphoid cells (ILCs) consist of natural killer (NK) cells and non-cytotoxic ILCs that are broadly classified into ILC1, ILC2, and ILC3 subtypes. These cells recently emerged as important early effectors of innate immunity for their roles in tissue homeostasis and inflammation. Over the last few years, ILCs have been extensively studied in mouse and human at the functional and molecular level, including gene expression profiling. However, sorting ILCs with flow cytometry for gene expression analysis is a delicate and time-consuming process. Here we propose and validate a novel framework for studying ILCs at the transcriptomic level using single-cell RNA-Seq data. Our approach combines unsupervised clustering and a new cell type classifier trained on mouse ILC gene expression data. We show that this approach can accurately identify different ILCs, especially ILC2 cells, in human lymphocyte single-cell RNA-Seq data. Our new model relies only on genes conserved across vertebrates, thereby making it in principle applicable in any vertebrate species. Considering the rapid increase in throughput of single-cell RNA-Seq technology, our work provides a computational framework for studying ILC2 cells in single-cell transcriptomic data and may help exploring their conservation in distant vertebrate species.

Physical mapping of the torsion dystonia region of human chromosome 9q34

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ozelius, L.J.; Hewett, J.; Shalish, C.

1994-09-01

Torsion dystonia is a syndrome characterized by loss of voluntary movements appearing as sustained muscle contractions and/or abnormal postures. The DYT1 gene is responsible for a subtype of torsion dystonia in which onset of symptoms tends to occur in a limb at an early age (mean 13 years) and to progress to a generalized state. Expression of the disease gene follows an autosomal dominant mode of inheritance with reduced penetrance. We initially mapped this gene to human chromosome 9q34 and have now defined its location to a < 1 cM region near the ASS locus based on historic recombination eventsmore » around a founder mutation in the Ashkenazic Jewish population. Using the CEPH YAC library and a chromosome 9 flow-sorted YAC library, we have generated a YAC contig spanning about 500 kb of this region. These YACs are being used to identify cosmids by direct hybridization to chromosome 9-specific cosmid libraries. Cosmids are being aligned by restriction digest patterns and by hybridization with oligonucleotide repeat probes. In addition, the cosmids are being {open_quotes}trapped{close_quotes} by exon amplification and these exons used to screen cDNA libraries. Thus far we have identified several candidate transcripts in this region.« less

Flow cytometry of human embryonic kidney cells: A light scattering approach

NASA Technical Reports Server (NTRS)

Kunze, M. E.; Goolsby, C. L.; Todd, P. W.; Morrison, D. R.; Lewis, M. L.

1985-01-01

The mammalian kidney contains cells that transport water, convert vitamin D to active forms, synthesize hormones such a renin and erythropoietin, and produce enzymes such as urokinase, a plasminogen activator. Several of these functions are maintained by human embryonic kidney cells (HEK) cultivated in vitro. Biochemical study of these functions in their individual cell types in vitro requires purified populations of cells. Light-scattering activated cell sorting (LACS) was explored as a means of achieving such purifications. It was found that HEK cells at the first 1 to 5 passages in culture were heterogeneous with respect to 2-parameter light scattering intensity distribution, in which combined measurements included forward angle scattering (2.5 to 19 deg), 90 deg scattering, and time-of-flight size measurements. Size was measured at a resolution of 0.15 microns/channel in 256 channels using pulse-height independent pulse-width measurements. Two-parameter distributions combining these measurements were obtained for HEK cell subpopulations that had been purified by microgravity electrophoresis and subsequently propagated in culture. These distributions contained at least 3 subpopulations in all purified fractions, and results of experiments with prepurified cultured HEK cells indicated that subpopulations of living cells that were high in plasminogen-activator activity also contained the highest per cent of cells with high 90 deg light scatter intensity.

Water Quality Planning in Rivers: Assimilative Capacity and Dilution Flow.

PubMed

Hashemi Monfared, Seyed Arman; Dehghani Darmian, Mohsen; Snyder, Shane A; Azizyan, Gholamreza; Pirzadeh, Bahareh; Azhdary Moghaddam, Mehdi

2017-11-01

Population growth, urbanization and industrial expansion are consequentially linked to increasing pollution around the world. The sources of pollution are so vast and also include point and nonpoint sources, with intrinsic challenge for control and abatement. This paper focuses on pollutant concentrations and also the distance that the pollution is in contact with the river water as objective functions to determine two main necessary characteristics for water quality management in the river. These two necessary characteristics are named assimilative capacity and dilution flow. The mean area of unacceptable concentration [Formula: see text] and affected distance (X) are considered as two objective functions to determine the dilution flow by a non-dominated sorting genetic algorithm II (NSGA-II) optimization algorithm. The results demonstrate that the variation of river flow discharge in different seasons can modify the assimilation capacity up to 97%. Moreover, when using dilution flow as a water quality management tool, results reveal that the content of [Formula: see text] and X change up to 97% and 93%, respectively.

Card sorting to evaluate the robustness of the information architecture of a protocol website.

PubMed

Wentzel, J; Müller, F; Beerlage-de Jong, N; van Gemert-Pijnen, J

2016-02-01

A website on Methicillin-Resistant Staphylococcus Aureus, MRSA-net, was developed for Health Care Workers (HCWs) and the general public, in German and in Dutch. The website's content was based on existing protocols and its structure was based on a card sort study. A Human Centered Design approach was applied to ensure a match between user and technology. In the current study we assess whether the website's structure still matches user needs, again via a card sort study. An open card sort study was conducted. Randomly drawn samples of 100 on-site search queries as they were entered on the MRSA-net website (during one year of use) were used as card input. In individual sessions, the cards were sorted by each participant (18 German and 10 Dutch HCWs, and 10 German and 10 Dutch members of the general public) into piles that were meaningful to them. Each participant provided a label for every pile of cards they created. Cluster analysis was performed on the resulting sorts, creating an overview of clusters of items placed together in one pile most frequently. In addition, pile labels were qualitatively analyzed to identify the participants' mental models. Cluster analysis confirmed existing categories and revealed new themes emerging from the search query samples, such as financial issues and consequences for the patient. Even though MRSA-net addresses these topics, they are not prominently covered in the menu structure. The label analysis shows that 7 of a total of 44 MRSA-net categories were not reproduced by the participants. Additional themes such as information on other pathogens and categories such as legal issues emerged. This study shows that the card sort performed to create MRSA-net resulted in overall long-lasting structure and categories. New categories were identified, indicating that additional information needs emerged. Therefore, evaluating website structure should be a recurrent activity. Card sorting with ecological data as input for the cards is useful to identify changes in needs and mental models. By combining qualitative and quantitative analysis we gained insight into additional information needed by the target group, including their view on the domain and related themes. The results show differences between the four user groups in their sorts, which can mostly be explained by the groups' background. These findings confirm that HCD is a valuable approach to tailor information to the target group. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Sexing mammalian sperm for production of offspring: the state-of-the-art.

PubMed

Johnson, L A

2000-07-02

Predetermination of sex in livestock offspring is in great demand and is of critical importance to providing for the most efficient production of the world's food supply. With the changes that have taken place in animal agriculture over the past generation the application of sex preselection to production systems becomes increasingly necessary. The current technology is based on the well-known difference in X- and Y-sperm in the amount of DNA present. The method has been validated on the basis of live births, laboratory reanalysis of sorted sperm for DNA content and embryo biopsy for sex determination. The technology incorporates modified flow cytometric sorting instrumentation to sort X- and Y-bearing sperm. Resulting populations of X or Y sperm can be used in conjunction with IVF in swine and in cattle for the production of sexed embryos to be transferred to eligible recipients for the duration of gestation. It can also be used for intratubal insemination and for deep-uterine and conventional insemination in cattle. This semipractical sexing method, though currently impractical for some production systems (where large numbers of sperm are required for fertilization) could be used to provide a more flexible progeny-producing option in many livestock operations. Improvements in the production rate of sexed sperm continue as new technology is developed. High-speed sorting is one of the newer technological advances and is being used in our laboratory to increase sorted sperm throughput. With our original technology we sorted 350,000 sperm/h. We now sort 6 million of each sex, under routine conditions. Sorting only the X population results in about 18 million sperm/h. Improvements in the technology will no doubt lead to much greater usage of sexed sperm, depending on the species involved. Insemination of lower sperm numbers in cattle has proven to be an effective means of utilizing the sexing technology. Solving the problems associated with inseminating low sperm numbers in the pig would be advantageous to the utilization of sexed sperm for some type of deep artificial insemination. Such a development would also enhance the economy of using lower sperm numbers with conventional artificial insemination (AI) and aid the swine industry worldwide. The use of sexed sperm for non-ordinary applications such as endangered species, laboratory animals, hobby or pet species is also of interest and will become a part of the move to be more reproductively efficient in the next millennium. Sexed sperm on demand over the next several years will provide livestock producers with many options in seeking to improve efficiency of production and improve quality of products to enhance consumer acceptability.

[Hospital and environment: waste disposal].

PubMed

Faure, P; Rizzo Padoin, N

2003-11-01

Like all production units, hospitals produce waste and are responsible for waste disposal. Hospital waste is particular due to the environmental risks involved, particularly concerning infection, effluents, and radionucleide contamination. Management plans are required to meet environmental, hygiene and regulatory obligations and to define reference waste products. The first step is to optimize waste sorting, with proper definition of the different categories, adequate containers (collection stations, color-coded sacks), waste circuits, intermediate then central storage areas, and finally transfer to an incineration unit. Volume and delay to elimination must be carefully controlled. Elimination of drugs and related products is a second aspect: packaging, perfusion pouches, tubing, radiopharmaceutic agents. These later products are managed with non-sealed sources whose elimination depends on the radioactive period, requiring selective sorting and specific holding areas while radioactivity declines. Elimination of urine and excreta containing anti-cancer drugs or intravesical drugs, particularly coming from protected rooms using radioactive iodine is another aspect. There is also a marginal flow of unused or expired drugs. For a health establishment, elimination of drugs is not included as part of waste disposal. This requires establishing a specific circuit with selective sorting and carefully applied safety regulations. Market orders for collecting and handling hospital wastes must be implemented in compliance with the rules of Public Health Tenders.

Raft-mediated trafficking of apical resident proteins occurs in both direct and transcytotic pathways in polarized hepatic cells: role of distinct lipid microdomains.

PubMed

Slimane, Tounsia Aït; Trugnan, Germain; Van IJzendoorn, Sven C D; Hoekstra, Dick

2003-02-01

In polarized hepatic cells, pathways and molecular principles mediating the flow of resident apical bile canalicular proteins have not yet been resolved. Herein, we have investigated apical trafficking of a glycosylphosphatidylinositol-linked and two single transmembrane domain proteins on the one hand, and two polytopic proteins on the other in polarized HepG2 cells. We demonstrate that the former arrive at the bile canalicular membrane via the indirect transcytotic pathway, whereas the polytopic proteins reach the apical membrane directly, after Golgi exit. Most importantly, cholesterol-based lipid microdomains ("rafts") are operating in either pathway, and protein sorting into such domains occurs in the biosynthetic pathway, largely in the Golgi. Interestingly, rafts involved in the direct pathway are Lubrol WX insoluble but Triton X-100 soluble, whereas rafts in the indirect pathway are both Lubrol WX and Triton X-100 insoluble. Moreover, whereas cholesterol depletion alters raft-detergent insolubility in the indirect pathway without affecting apical sorting, protein missorting occurs in the direct pathway without affecting raft insolubility. The data implicate cholesterol as a traffic direction-determining parameter in the direct apical pathway. Furthermore, raft-cargo likely distinguishing single vs. multispanning membrane anchors, rather than rafts per se (co)determine the sorting pathway.

Raft-mediated Trafficking of Apical Resident Proteins Occurs in Both Direct and Transcytotic Pathways in Polarized Hepatic Cells: Role of Distinct Lipid Microdomains

PubMed Central

Slimane, Tounsia Aït; Trugnan, Germain; van IJzendoorn, Sven C.D.; Hoekstra, Dick

2003-01-01

In polarized hepatic cells, pathways and molecular principles mediating the flow of resident apical bile canalicular proteins have not yet been resolved. Herein, we have investigated apical trafficking of a glycosylphosphatidylinositol-linked and two single transmembrane domain proteins on the one hand, and two polytopic proteins on the other in polarized HepG2 cells. We demonstrate that the former arrive at the bile canalicular membrane via the indirect transcytotic pathway, whereas the polytopic proteins reach the apical membrane directly, after Golgi exit. Most importantly, cholesterol-based lipid microdomains (“rafts”) are operating in either pathway, and protein sorting into such domains occurs in the biosynthetic pathway, largely in the Golgi. Interestingly, rafts involved in the direct pathway are Lubrol WX insoluble but Triton X-100 soluble, whereas rafts in the indirect pathway are both Lubrol WX and Triton X-100 insoluble. Moreover, whereas cholesterol depletion alters raft-detergent insolubility in the indirect pathway without affecting apical sorting, protein missorting occurs in the direct pathway without affecting raft insolubility. The data implicate cholesterol as a traffic direction-determining parameter in the direct apical pathway. Furthermore, raft-cargo likely distinguishing single vs. multispanning membrane anchors, rather than rafts per se (co)determine the sorting pathway. PMID:12589058

Spectrum of benzo[a]pyrene-induced mutations in the Pig-a gene of L5178YTk+/- cells identified with next generation sequencing.

PubMed

Revollo, Javier; Wang, Yiying; McKinzie, Page; Dad, Azra; Pearce, Mason; Heflich, Robert H; Dobrovolsky, Vasily N

2017-12-01

We used Sanger sequencing and next generation sequencing (NGS) for analysis of mutations in the endogenous X-linked Pig-a gene of clonally expanded L5178YTk +/- cells. The clones developed from single cells that were sorted on a flow cytometer based upon the expression pattern of the GPI-anchored marker, CD90, on their surface. CD90-deficient and CD90-proficient cells were sorted from untreated cultures and CD90-deficient cells were sorted from cultures treated with benzo[a]pyrene (B[a]P). Pig-a mutations were identified in all clones developed from CD90-deficient cells; no Pig-a mutations were found in clones of CD90-proficient cells. The spectrum of B[a]P-induced Pig-a mutations was dominated by basepair substitutions, small insertions and deletions at G:C, or at sequences rich in G:C content. We observed high concordance between Pig-a mutations determined by Sanger sequencing and by NGS, but NGS was able to identify mutations in samples that were difficult to analyze by Sanger sequencing (e.g., mixtures of two mutant clones). Overall, the NGS method is a cost and labor efficient high throughput approach for analysis of a large number of mutant clones. Published by Elsevier B.V.

Enrichment and isolation of neurons from adult mouse brain for ex vivo analysis.

PubMed

Berl, Sabina; Karram, Khalad; Scheller, Anja; Jungblut, Melanie; Kirchhoff, Frank; Waisman, Ari

2017-05-01

Isolation of neurons from the adult mouse CNS is important in order to study their gene expression during development or the course of different diseases. Here we present two different methods for the enrichment or isolation of neurons from adult mouse CNS. These methods: are either based on flow cytometry sorting of eYFP expressing neurons, or by depletion of non-neuronal cells by sorting with magnetic-beads. Enrichment by FACS sorting of eYFP positive neurons results in a population of 62.4% NeuN positive living neurons. qPCR data shows a 3-5fold upregulation of neuronal markers. The isolation of neurons based on depletion of non-neuronal cells using the Miltenyi Neuron Isolation Kit, reaches a purity of up to 86.5%. qPCR data of these isolated neurons shows an increase in neuronal markers and an absence of glial markers, proving pure neuronal RNA isolation. Former data related to neuronal gene expression are mainly based on histology, which does not allow for high-throughput transcriptome analysis to examine differential gene expression. These protocols can be used to study cell type specific gene expression of neurons to unravel their function in the process of damage to the CNS. Copyright © 2017 Elsevier B.V. All rights reserved.

Phylogeographic patterns of Lygus pratensis (Hemiptera: Miridae): Evidence for weak genetic structure and recent expansion in northwest China.

PubMed

Zhang, Li-Juan; Cai, Wan-Zhi; Luo, Jun-Yu; Zhang, Shuai; Wang, Chun-Yi; Lv, Li-Min; Zhu, Xiang-Zhen; Wang, Li; Cui, Jin-Jie

2017-01-01

Lygus pratensis (L.) is an important cotton pest in China, especially in the northwest region. Nymphs and adults cause serious quality and yield losses. However, the genetic structure and geographic distribution of L. pratensis is not well known. We analyzed genetic diversity, geographical structure, gene flow, and population dynamics of L. pratensis in northwest China using mitochondrial and nuclear sequence datasets to study phylogeographical patterns and demographic history. L. pratensis (n = 286) were collected at sites across an area spanning 2,180,000 km2, including the Xinjiang and Gansu-Ningxia regions. Populations in the two regions could be distinguished based on mitochondrial criteria but the overall genetic structure was weak. The nuclear dataset revealed a lack of diagnostic genetic structure across sample areas. Phylogenetic analysis indicated a lack of population level monophyly that may have been caused by incomplete lineage sorting. The Mantel test showed a significant correlation between genetic and geographic distances among the populations based on the mtDNA data. However the nuclear dataset did not show significant correlation. A high level of gene flow among populations was indicated by migration analysis; human activities may have also facilitated insect movement. The availability of irrigation water and ample cotton hosts makes the Xinjiang region well suited for L. pratensis reproduction. Bayesian skyline plot analysis, star-shaped network, and neutrality tests all indicated that L. pratensis has experienced recent population expansion. Climatic changes and extensive areas occupied by host plants have led to population expansion of L. pratensis. In conclusion, the present distribution and phylogeographic pattern of L. pratensis was influenced by climate, human activities, and availability of plant hosts.

Striations, Polish, and Related Features from Clasts in Impact-Ejecta Deposits and the "Tillite Problem"

NASA Technical Reports Server (NTRS)

Rampino, M. R.; Ernstson, K.; Anguita, F.; Claudin F.

1997-01-01

Proximal ejecta deposits related to three large terrestrial impacts, the 14.8-Ma Ries impact structure in Germany (the Bunte Breccia), the 65-Ma Chicxulub impact structure in the Yucatan (the Albion and Pook's Hill Diamictites in Belize) and the mid-Tertiary Azuara impact structure in Spain (the Pelarda Fm.) occur in the form of widespread debris-flow deposits most likely originating from ballistic processes. These impact-related diamictites typically are poorly sorted, containing grain sizes from clay to large boulders and blocks, and commonly display evidence of mass flow, including preferred orientation of long axes of clasts, class imbrication, flow noses, plugs and pods of coarse debris, and internal shear planes. Clasts of various lithologies show faceting, various degrees of rounding, striations (including nailhead striae), crescentic chattermarks, mirror-like polish, percussion marks, pitting, and penetration features. Considering the impact history of the Earth, it is surprising that so few ballistic ejecta, deposits have been discovered, unless the preservation potential is extremely low, or such materials exist but have been overlooked or misidentified as other types of geologic deposits . Debris-flow diamictites of various kinds have been reported in the geologic record, but these are commonly attributed to glaciation based on the coarse and poorly sorted nature of the deposits and, in many cases, on the presence of clasts showing features considered diagnostic of glacial action, including striations of various kinds, polish, and pitting. These diamictites are the primary evidence for ancient ice ages. We present evidence of the surface features on clasts from known proximal ejecta debris-flow deposits and compare these features with those reported in diamictites. interpreted as ancient glacial deposits (tillites). Our purpose is to document the types of features seen on clasts in diamictites of ejecta origin in order to help in the interpretation of the origin of ancient diamictites. The recognition of characteristic features in clast populations in ancient diamictites may allow identification and discrimination of debris-flow deposits of various origins (e.g., impact glacial, tectonic) and may shed light on some climatic paradoxes, such as inferred Proterozoic glaciations at low paleolatitudes.

Enigmatic mounds in 'Subglacial Meltwater Corridors' on the Canadian Shield: a record of channelised, subglacial meltwater drainage during Laurentide deglaciation

NASA Astrophysics Data System (ADS)

Haiblen, Anna; Ward, Brent; Normandeau, Philippe; Campbell, Janet

2017-04-01

Esker networks have traditionally been invoked to represent the channelised subglacial drainage system in shield terrains. However, eskers are only one landform found within 'subglacial meltwater corridors' (SMCs) on the Canadian Shield. SMCs are tracts where till has been eroded, bedrock is exposed, and glaciofluvial sediments have been deposited. SMCs are regularly spaced, parallel deglacial ice-flow directions, have undulating longitudinal profiles, and cross modern drainage divides. Our lidar- and field-based mapping near Lac de Gras, Northwest Territories, west of the Keewatin Ice Divide (KID), reveals that eskers are not present in the majority of SMCs. Instead, enigmatic mounds are commonly the dominant landform type. Enigmatic mounds typically occur in groups of 20 to 200. They are commonly composed of sandy diamicton that is coarser grained and better sorted than regional till. This diamicton is occasionally draped with well-sorted, stratified glaciofluvial sediments. Some enigmatic mounds have a single highpoint (individual mounds) while others have a complex, irregular form (complex mounds). Individual mounds have an average long-axis length of 43 m and an average height of < 2 m, however, their size is highly variable: the largest mounds are 170 m long and 15 m high. Complex mounds are typically larger than individual mounds. Our morphometric analysis shows that individual mounds have a mean length-to-width ratio of 1.8. The average mound elongation direction parallels the final ice flow that affected the area. However, where meltwater- and ice-flow directions differ, mound long-axis orientations typically cluster about meltwater flow directions. We have also observed SMCs and enigmatic mounds in the South Rae region of Northwest Territories, 450 km SE of Lac de Gras. Multiple types of enigmatic mounds are present in this area: some are similar to those near Lac de Gras, some are composed of till, and some are composed of sorted and stratified sediments. SMCs likely formed late during deglaciation because the enigmatic mounds and eskers that they contain do not appear to have been significantly affected by ice flow following their deposition. We suggest that transient, sheet-type subglacial meltwater flow events resulted in erosion and transport of basal till. Meltwater was likely sourced from supraglacial lakes that formed and drained catastrophically when the ablation zone of the Laurentide Ice Sheet affected the area. The enigmatic mounds that we have observed near Lac de Gras may have been deposited from a slurry-type flow. Eskers likely formed later, after a channelised drainage system was established. It is possible that SMCs are the Quaternary landscape record of lake-drainage events similar to those that occur in Southwest Greenland today. The hydraulic conditions required to create enigmatic mounds are different to those required for esker formation. Thus, SMCs, not just the eskers that they sometimes contain, should be considered when parameters are developed for numerical models relating to subglacial drainage systems in shield terrains. Determining the genesis of landforms found within SMCs will improve our understanding of hydraulic conditions in the subglacial, channelised drainage system during ice-sheet retreat and decay.

Multiparameter cell affinity chromatography: separation and analysis in a single microfluidic channel.

PubMed

Li, Peng; Gao, Yan; Pappas, Dimitri

2012-10-02

The ability to sort and capture more than one cell type from a complex sample will enable a wide variety of studies of cell proliferation and death and the analysis of disease states. In this work, we integrated a pneumatic actuated control layer to an affinity separation layer to create different antibody-coating regions on the same fluidic channel. The comparison of different antibody capture capabilities to the same cell line was demonstrated by flowing Ramos cells through anti-CD19- and anti-CD71-coated regions in the same channel. It was determined that the cell capture density on the anti-CD19 region was 2.44 ± 0.13 times higher than that on the anti-CD71-coated region. This approach can be used to test different affinity molecules for selectivity and capture efficiency using a single cell line in one separation. Selective capture of Ramos and HuT 78 cells from a mixture was also demonstrated using two antibody regions in the same channel. Greater than 90% purity was obtained on both capture areas in both continuous flow and stop flow separation modes. A four-region antibody-coated device was then fabricated to study the simultaneous, serial capture of three different cell lines. In this case the device showed effective capture of cells in a single separation channel, opening up the possibility of multiple cell sorting. Multiparameter sequential blood sample analysis was also demonstrated with high capture specificity (>97% for both CD19+ and CD4+ leukocytes). The chip can also be used to selectively treat cells after affinity separation.

Purifying, Separating, and Concentrating Cells From a Sample Low in Biomass

NASA Technical Reports Server (NTRS)

Benardini, James N.; LaDuc, Myron T.; Diamond, Rochelle

2012-01-01

Frequently there is an inability to process and analyze samples of low biomass due to limiting amounts of relevant biomaterial in the sample. Furthermore, molecular biological protocols geared towards increasing the density of recovered cells and biomolecules of interest, by their very nature, also concentrate unwanted inhibitory humic acids and other particulates that have an adversarial effect on downstream analysis. A novel and robust fluorescence-activated cell-sorting (FACS)-based technology has been developed for purifying (removing cells from sampling matrices), separating (based on size, density, morphology), and concentrating cells (spores, prokaryotic, eukaryotic) from a sample low in biomass. The technology capitalizes on fluorescent cell-sorting technologies to purify and concentrate bacterial cells from a low-biomass, high-volume sample. Over the past decade, cell-sorting detection systems have undergone enhancements and increased sensitivity, making bacterial cell sorting a feasible concept. Although there are many unknown limitations with regard to the applicability of this technology to environmental samples (smaller cells, few cells, mixed populations), dogmatic principles support the theoretical effectiveness of this technique upon thorough testing and proper optimization. Furthermore, the pilot study from which this report is based proved effective and demonstrated this technology capable of sorting and concentrating bacterial endospore and bacterial cells of varying size and morphology. Two commercial off-the-shelf bacterial counting kits were used to optimize a bacterial stain/dye FACS protocol. A LIVE/DEAD BacLight Viability and Counting Kit was used to distinguish between the live and dead cells. A Bacterial Counting Kit comprising SYTO BC (mixture of SYTO dyes) was employed as a broad-spectrum bacterial counting agent. Optimization using epifluorescence microscopy was performed with these two dye/stains. This refined protocol was further validated using varying ratios and mixtures of cells to ensure homogenous staining compared to that of individual cells, and were utilized for flow analyzer and FACS labeling. This technology focuses on the purification and concentration of cells from low-biomass spacecraft assembly facility samples. Currently, purification and concentration of low-biomass samples plague planetary protection downstream analyses. Having a capability to use flow cytometry to concentrate cells out of low-biomass, high-volume spacecraft/ facility sample extracts will be of extreme benefit to the fields of planetary protection and astrobiology. Successful research and development of this novel methodology will significantly increase the knowledge base for designing more effective cleaning protocols, and ultimately lead to a more empirical and true account of the microbial diversity present on spacecraft surfaces. Refined cleaning and an enhanced ability to resolve microbial diversity may decrease the overall cost of spacecraft assembly and/or provide a means to begin to assess challenging planetary protection missions.

The Cry in the Holy Quran and the Effect on the Human Behavior

ERIC Educational Resources Information Center

al-Domi, Mohammad Mahmoud

2015-01-01

This study aims that cry is the ideal way to release the negative emotions distress, sorrow, and sadness. Which sometimes is also a way to express situations of joy and pleasure of humans. The Almighty Allah also said about cry in The Holy Quran. The prophet pbuh also cry for the expressions of reverence and fear of Allah in perhaps the sort of…

Mechanotransduction Effects on Endothelial Cell Proliferation via CD31 and VEGFR2: Implications for Immunomagnetic Separation.

PubMed

Mahajan, Kalpesh D; Nabar, Gauri M; Xue, Wei; Anghelina, Mirela; Moldovan, Nicanor I; Chalmers, Jeffrey J; Winter, Jessica O

2017-09-01

Immunomagnetic separation is used to isolate circulating endothelial cells (ECs) and endothelial progenitor cells (EPCs) for diagnostics and tissue engineering. However, potentially detrimental changes in cell properties have been observed post-separation. Here, the effect of mechanical force, which is naturally applied during immunomagnetic separation, on proliferation of human umbilical vein endothelial cells (HUVEC), kinase insert domain-positive receptor (KDR) cells, and peripheral blood mononuclear cells (PBMCs). Cells are exposed to CD31 or Vascular Endothelial Growth Factor Receptor-2 (VEGFR2) targeted MACSi beads at varying bead to cell ratios and compared to free antibody and unconjugated beads. A vertical magnetic gradient is applied to static 2D cultures, and a magnetic cell sorter is used to analyze cells in dynamic flow. No significant difference in EC proliferation is observed for controls or VEGFR2-targeting beads, whereas CD31-conjugated beads increase proliferation in a dose dependent manner in static 2-D cultures. This effect occurs in the absence of magnetic field, but is more pronounced with magnetic force. After flow sorting, similar increases in proliferation are seen for CD31 targeting beads. Thus, the effects of targeting antibody and magnetic force applied should be considered when designing immunomagnetic separation protocols for ECs. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Incomplete lineage sorting patterns among human, chimpanzee, and orangutan suggest recent orangutan speciation and widespread selection

PubMed Central

Hobolth, Asger; Dutheil, Julien Y.; Hawks, John; Schierup, Mikkel H.; Mailund, Thomas

2011-01-01

We search the complete orangutan genome for regions where humans are more closely related to orangutans than to chimpanzees due to incomplete lineage sorting (ILS) in the ancestor of human and chimpanzees. The search uses our recently developed coalescent hidden Markov model (HMM) framework. We find ILS present in ∼1% of the genome, and that the ancestral species of human and chimpanzees never experienced a severe population bottleneck. The existence of ILS is validated with simulations, site pattern analysis, and analysis of rare genomic events. The existence of ILS allows us to disentangle the time of isolation of humans and orangutans (the speciation time) from the genetic divergence time, and we find speciation to be as recent as 9–13 million years ago (Mya; contingent on the calibration point). The analyses provide further support for a recent speciation of human and chimpanzee at ∼4 Mya and a diverse ancestor of human and chimpanzee with an effective population size of about 50,000 individuals. Posterior decoding infers ILS for each nucleotide in the genome, and we use this to deduce patterns of selection in the ancestral species. We demonstrate the effect of background selection in the common ancestor of humans and chimpanzees. In agreement with predictions from population genetics, ILS was found to be reduced in exons and gene-dense regions when we control for confounding factors such as GC content and recombination rate. Finally, we find the broad-scale recombination rate to be conserved through the complete ape phylogeny. PMID:21270173

Controlling the trajectories of bubble trains at a microfluidic junction

NASA Astrophysics Data System (ADS)

Parthiban, Pravien; Khan, Saif

2011-11-01

The increasing number of applications facilitated by digital microfluidic flows has resulted in a sustained interest in not only understanding the diverse, interesting and often complex dynamics associated with such flows in microchannel networks but also in developing facile strategies to control them. We find that there are readily accessible flow speeds wherein resistance to flow in microchannels decreases with an increase in the number of confined bubbles present, and exploit this intriguing phenomenon to sort all bubble of a train exclusively into one of the arms of a nominally symmetric microfluidic loop. We also demonstrate how the arm into which the train filters into can be chosen by applying a temporary external stimulus by means of an additional flow of the continuous liquid into one the arms of the loop. Furthermore, we show how by tuning the magnitude and period of this temporary stimulus we can switch controllably, the traffic of bubbles between both arms of the loop even when the loop is asymmetric. The results of this work should aid in developing viable methods to regulate traffic of digital flows in microfluidic networks.

Electrohydrodynamic Flows in Electrochemical Systems

NASA Technical Reports Server (NTRS)

Saville, D. A.

2005-01-01

Recent studies have established a new class of assembly processes with colloidal suspensions. Particles are driven together to form large crystalline structures in both dc and ac fields. The current work centers on this new class of flows in ac fields. In the research carried out under the current award, it was established that: (i) Small colloidal particles crystallize near an electrode due to electrohydrodynamic flows induced by an sinusoidally varying applied potential. (ii) These flows originate due to disturbances in the electrode polarization layer arising from the presence of the particles. Inasmuch as the charge and the field strength both scale on the applied field, the flows are proportional to the square of the applied voltage. (iii) Suspensions of two different sorts of particles can be crystallized and will form well-ordered binary crystals. (iv) At high frequencies the EHD flows die out. Thus, with a homogeneous system the particles become widely spaced due to dipolar repulsion. With a binary suspension, however, the particles may become attractive due to dipolar attraction arising from differences in electrokinetic dipoles. Consequently binary crystals form at both high and low frequencies.

A First Generation Comparative Chromosome Map between Guinea Pig (Cavia porcellus) and Humans.

PubMed

Romanenko, Svetlana A; Perelman, Polina L; Trifonov, Vladimir A; Serdyukova, Natalia A; Li, Tangliang; Fu, Beiyuan; O'Brien, Patricia C M; Ng, Bee L; Nie, Wenhui; Liehr, Thomas; Stanyon, Roscoe; Graphodatsky, Alexander S; Yang, Fengtang

2015-01-01

The domesticated guinea pig, Cavia porcellus (Hystricomorpha, Rodentia), is an important laboratory species and a model for a number of human diseases. Nevertheless, genomic tools for this species are lacking; even its karyotype is poorly characterized. The guinea pig belongs to Hystricomorpha, a widespread and important group of rodents; so far the chromosomes of guinea pigs have not been compared with that of other hystricomorph species or with any other mammals. We generated full sets of chromosome-specific painting probes for the guinea pig by flow sorting and microdissection, and for the first time, mapped the chromosomal homologies between guinea pig and human by reciprocal chromosome painting. Our data demonstrate that the guinea pig karyotype has undergone extensive rearrangements: 78 synteny-conserved human autosomal segments were delimited in the guinea pig genome. The high rate of genome evolution in the guinea pig may explain why the HSA7/16 and HSA16/19 associations presumed ancestral for eutherians and the three syntenic associations (HSA1/10, 3/19, and 9/11) considered ancestral for rodents were not found in C. porcellus. The comparative chromosome map presented here is a starting point for further development of physical and genetic maps of the guinea pig as well as an aid for genome assembly assignment to specific chromosomes. Furthermore, the comparative mapping will allow a transfer of gene map data from other species. The probes developed here provide a genomic toolkit, which will make the guinea pig a key species to unravel the evolutionary biology of the Hystricomorph rodents.

A First Generation Comparative Chromosome Map between Guinea Pig (Cavia porcellus) and Humans

PubMed Central

Romanenko, Svetlana A.; Perelman, Polina L.; Trifonov, Vladimir A.; Serdyukova, Natalia A.; Li, Tangliang; Fu, Beiyuan; O’Brien, Patricia C. M.; Ng, Bee L.; Nie, Wenhui; Liehr, Thomas; Stanyon, Roscoe; Graphodatsky, Alexander S.; Yang, Fengtang

2015-01-01

The domesticated guinea pig, Cavia porcellus (Hystricomorpha, Rodentia), is an important laboratory species and a model for a number of human diseases. Nevertheless, genomic tools for this species are lacking; even its karyotype is poorly characterized. The guinea pig belongs to Hystricomorpha, a widespread and important group of rodents; so far the chromosomes of guinea pigs have not been compared with that of other hystricomorph species or with any other mammals. We generated full sets of chromosome-specific painting probes for the guinea pig by flow sorting and microdissection, and for the first time, mapped the chromosomal homologies between guinea pig and human by reciprocal chromosome painting. Our data demonstrate that the guinea pig karyotype has undergone extensive rearrangements: 78 synteny-conserved human autosomal segments were delimited in the guinea pig genome. The high rate of genome evolution in the guinea pig may explain why the HSA7/16 and HSA16/19 associations presumed ancestral for eutherians and the three syntenic associations (HSA1/10, 3/19, and 9/11) considered ancestral for rodents were not found in C. porcellus. The comparative chromosome map presented here is a starting point for further development of physical and genetic maps of the guinea pig as well as an aid for genome assembly assignment to specific chromosomes. Furthermore, the comparative mapping will allow a transfer of gene map data from other species. The probes developed here provide a genomic toolkit, which will make the guinea pig a key species to unravel the evolutionary biology of the Hystricomorph rodents. PMID:26010445

Identification of key characteristics of male reproductive toxicants as an approach for screening and sorting mechanistic evidence.

EPA Science Inventory

The application of systematic review practices in human health assessment includes integration of multi-disciplinary evidence from epidemiological, experimental, and mechanistic studies. Although mode of action analysis relies on the evaluation of mechanistic and toxicological ou...

Work fluctuations for a Brownian particle between two thermostats

NASA Astrophysics Data System (ADS)

Visco, Paolo

2006-06-01

We explicitly determine the large deviation function of the energy flow of a Brownian particle coupled to two heat baths at different temperatures. This toy model, initially introduced by Derrida and Brunet (2005, Einstein aujourd'hui (Les Ulis: EDP Sciences)), not only allows us to sort out the influence of initial conditions on large deviation functions but also allows us to pinpoint various restrictions bearing upon the range of validity of the Fluctuation Relation.

Principles of transverse flow fractionation of microparticles in superhydrophobic channels.

PubMed

Asmolov, Evgeny S; Dubov, Alexander L; Nizkaya, Tatiana V; Kuehne, Alexander J C; Vinogradova, Olga I

2015-07-07

We propose a concept of fractionation of micron-sized particles in a microfluidic device with a bottom wall decorated by superhydrophobic stripes. The stripes are oriented at an angle α to the direction of a driving force, G, which generally includes an applied pressure gradient and gravity. Separation relies on the initial sedimentation of particles under gravity in the main forward flow, and their subsequent lateral deflection near a superhydrophobic wall due to generation of a secondary flow transverse to G. We provide some theoretical arguments allowing us to quantify the transverse displacement of particles in the microfluidic channel, and confirm the validity of theoretical predictions in test experiments with monodisperse fractions of microparticles. Our results can guide the design of superhydrophobic microfluidic devices for efficient sorting of microparticles with a relatively small difference in size and density.

shRNA-mediated EMMPRIN silencing inhibits human leukemic monocyte lymphoma U937 cell proliferation and increases chemosensitivity to adriamycin.

PubMed

Gao, Hui; Jiang, Qixiao; Han, Yantao; Peng, Jianjun; Wang, Chunbo

2015-03-01

EMMPRIN is a widely distributed cell surface glycoprotein, which plays an important role in tumor progression and confers resistance to some chemotherapeutic drugs. Recent studies have shown that EMMPRIN overexpression indicates poor prognosis in acute myeloid leukemia (AML). However, little was known on the role of EMMPRIN in leukemia. Human leukemia cell line U937 was stably transfected with a EMMPRIN-targeted shRNA-containing vector to investigate the effect of EMMPRIN on cellular functions. EMMPRIN expression was monitored by qRT-PCR and Western blotting. Cell viability and proliferation were determined by trypan blue exclusion and BrdU labeling, respectively. Cell cycle and apoptosis were analyzed by flow cytometry. Cytotoxicity of chemotherapeutic agent adriamycin on cells was assessed by MTT assay. Knockdown of EMMPRIN gene significantly inhibited cell viability and decreased cell proliferation. Fluorescence-activated cell-sorting analysis revealed that the reduced EMMPRIN expression resulted in cell cycle arrest at G1 phase and induced apoptosis. Meanwhile, western blotting analysis showed that EMMPRIN knockdown was associated with downregulation of cell cycle- and apoptosis-related molecules including cyclin D1, cyclin E, as well as increase in cleavage of caspase-3 and PARP. This study also showed that silencing of EMMPRIN sensitized U937 cells to Adriamycin. EMMPRIN is involved in proliferation, growth, and chemosensitivity of human AML line U937, indicating that EMMPRIN may be a promising therapeutic target for AML.

MicroRNA Expression in Alpha and Beta Cells of Human Pancreatic Islets

PubMed Central

Vargas, Nancy; Rosero, Samuel; Piroso, Julieta; Ichii, Hirohito; Umland, Oliver; Zhijie, Jiang; Tsinoremas, Nicholas; Ricordi, Camillo; Inverardi, Luca; Domínguez-Bendala, Juan; Pastori, Ricardo L.

2013-01-01

microRNAs (miRNAs) play an important role in pancreatic development and adult β-cell physiology. Our hypothesis is based on the assumption that each islet cell type has a specific pattern of miRNA expression. We sought to determine the profile of miRNA expression in α-and β-cells, the main components of pancreatic islets, because this analysis may lead to a better understanding of islet gene regulatory pathways. Highly enriched (>98%) subsets of human α-and β-cells were obtained by flow cytometric sorting after intracellular staining with c-peptide and glucagon antibody. The method of sorting based on intracellular staining is possible because miRNAs are stable after fixation. MiRNA expression levels were determined by quantitative high throughput PCR-based miRNA array platform screening. Most of the miRNAs were preferentially expressed in β-cells. From the total of 667 miRNAs screened, the Significant Analysis of Microarray identified 141 miRNAs, of which only 7 were expressed more in α-cells (α-miRNAs) and 134 were expressed more in β-cells (β-miRNAs). Bioinformatic analysis identified potential targets of β-miRNAs analyzing the Beta Cell Gene Atlas, described in the T1Dbase, the web platform, supporting the type 1 diabetes (T1D) community. cMaf, a transcription factor regulating glucagon expression expressed selectively in α-cells (TFα) is targeted by β-miRNAs; miR-200c, miR-125b and miR-182. Min6 cells treated with inhibitors of these miRNAs show an increased expression of cMaf RNA. Conversely, over expression of miR-200c, miR-125b or miR-182 in the mouse alpha cell line αTC6 decreases the level of cMAF mRNA and protein. MiR-200c also inhibits the expression of Zfpm2, a TFα that inhibits the PI3K signaling pathway, at both RNA and protein levels. In conclusion, we identified miRNAs differentially expressed in pancreatic α- and β-cells and their potential transcription factor targets that could add new insights into different aspects of islet biology and pathophysiology. PMID:23383059

MicroRNA expression in alpha and beta cells of human pancreatic islets.

PubMed

Klein, Dagmar; Misawa, Ryosuke; Bravo-Egana, Valia; Vargas, Nancy; Rosero, Samuel; Piroso, Julieta; Ichii, Hirohito; Umland, Oliver; Zhijie, Jiang; Tsinoremas, Nicholas; Ricordi, Camillo; Inverardi, Luca; Domínguez-Bendala, Juan; Pastori, Ricardo L

2013-01-01

microRNAs (miRNAs) play an important role in pancreatic development and adult β-cell physiology. Our hypothesis is based on the assumption that each islet cell type has a specific pattern of miRNA expression. We sought to determine the profile of miRNA expression in α-and β-cells, the main components of pancreatic islets, because this analysis may lead to a better understanding of islet gene regulatory pathways. Highly enriched (>98%) subsets of human α-and β-cells were obtained by flow cytometric sorting after intracellular staining with c-peptide and glucagon antibody. The method of sorting based on intracellular staining is possible because miRNAs are stable after fixation. MiRNA expression levels were determined by quantitative high throughput PCR-based miRNA array platform screening. Most of the miRNAs were preferentially expressed in β-cells. From the total of 667 miRNAs screened, the Significant Analysis of Microarray identified 141 miRNAs, of which only 7 were expressed more in α-cells (α-miRNAs) and 134 were expressed more in β-cells (β-miRNAs). Bioinformatic analysis identified potential targets of β-miRNAs analyzing the Beta Cell Gene Atlas, described in the T1Dbase, the web platform, supporting the type 1 diabetes (T1D) community. cMaf, a transcription factor regulating glucagon expression expressed selectively in α-cells (TFα) is targeted by β-miRNAs; miR-200c, miR-125b and miR-182. Min6 cells treated with inhibitors of these miRNAs show an increased expression of cMaf RNA. Conversely, over expression of miR-200c, miR-125b or miR-182 in the mouse alpha cell line αTC6 decreases the level of cMAF mRNA and protein. MiR-200c also inhibits the expression of Zfpm2, a TFα that inhibits the PI3K signaling pathway, at both RNA and protein levels.In conclusion, we identified miRNAs differentially expressed in pancreatic α- and β-cells and their potential transcription factor targets that could add new insights into different aspects of islet biology and pathophysiology.

Salmon Spawning Effects on Streambed Stability

NASA Astrophysics Data System (ADS)

Buxton, T. H.; Buffington, J. M.; Yager, E.; Fremier, A. K.; Hassan, M. A.

2014-12-01

Female salmon build nests ("redds") in streambeds to protect their eggs from predation and damage by bed scour. During spawning, streambed material is mixed, fine sediment is winnowed downstream, and sediment is moved into a tailspill mound resembling the shape of a dune. Redd surfaces are coarser and better sorted than unspawned beds, which is thought to increase redd stability because larger grains are heavier and harder to move and sorting leads to higher friction angles for grain mobility. However, spawning also loosens sediment and creates topography that accelerates flow, both of which may increase particle mobility. We address factors controlling the relative stability of redds and unspawned beds using simulated salmon redds and water worked ("unspawned") beds composed of mixed-grain surfaces in a laboratory flume. Results show that simulated spawning lowered packing resistance to particle mobility on redds by an average of 32-39% compared to unspawned beds. Reductions in packing were sufficient to counter the higher inherent stability of relatively coarse, well sorted grains on redds, overall reducing critical shear stress by 8-20% relative to unspawned beds. In addition, boundary shear stress was 13-41% higher on redds due to flow convergence over the tailspill structure. Finally, redd instability relative to unspawned beds was observed in visual measurements of grain mobility, where bed-averaged shear stress was 22% lower at incipient motion and 29% lower at the discharge that mobilized all grain sizes on redds. Results of these complementary observations, along with sediment mass transport rates being nearly five times higher on a redd than an unspawned bed, indicate that redds are unstable compared to unspawned beds. Given these findings, further research is needed to investigate linkages between spawning disturbance and streambed mobility that may affect salmon reproduction in streams, and to assess whether a certain level of bed disturbance from spawning is required to restore ecosystem functions in streams with threatened populations of salmon.

A task analysis of the shift from teacher instructions to self-instructions in performing an in-common task.

PubMed

Grote, I; Rosales, J; Baer, D M

1996-11-01

Three preschool children repeatedly did four kinds of sorts with a deck of stimulus cards: a difficult, untaught target sort and three other sorts considered analytic of self-instructing the target performance. The untaught target sort was to find in a deck of cards those matching what two sample cards had in common. Most preschool children must be taught to mediate this problem. The three other kinds of sorts taught skills involved in the target performance or its mediation. As correct self-instructive talk emerged in the target sorts, it was confirmed. The untaught target sorts were interspersed infrequently among the three alternating directly taught skill sorts, to see if accurate target sorts, and accurate self-instructive talk about the target sorts, would emerge as the three skill sorts were mastered. As all the sorts progressed, increasing accuracy was seen first in the skill sorts and then in the untaught target sorts. All three subjects showed subsequent generalization to new target sorts involving other stimulus sets. Correct spontaneous self-instructions about the target sorts increased from near zero at the beginning of the experiment to consistency at its end. Thus the three skill sorts appeared sufficient for the emergence of a self-instructed solution to the previously insoluble target performance.

Parallel sort with a ranged, partitioned key-value store in a high perfomance computing environment

DOEpatents

Bent, John M.; Faibish, Sorin; Grider, Gary; Torres, Aaron; Poole, Stephen W.

2016-01-26

Improved sorting techniques are provided that perform a parallel sort using a ranged, partitioned key-value store in a high performance computing (HPC) environment. A plurality of input data files comprising unsorted key-value data in a partitioned key-value store are sorted. The partitioned key-value store comprises a range server for each of a plurality of ranges. Each input data file has an associated reader thread. Each reader thread reads the unsorted key-value data in the corresponding input data file and performs a local sort of the unsorted key-value data to generate sorted key-value data. A plurality of sorted, ranged subsets of each of the sorted key-value data are generated based on the plurality of ranges. Each sorted, ranged subset corresponds to a given one of the ranges and is provided to one of the range servers corresponding to the range of the sorted, ranged subset. Each range server sorts the received sorted, ranged subsets and provides a sorted range. A plurality of the sorted ranges are concatenated to obtain a globally sorted result.

MarsSedEx I and II: Experimental investigation of gravity effects on sedimentation on Mars

NASA Astrophysics Data System (ADS)

Kuhn, N. J.; Kuhn, B.; Gartmann, A.

2014-12-01

Sorting of sedimentary rocks is a proxy for the environmental conditions at the time of deposition, in particular the runoff that moved and deposited the material forming the rocks. Settling of sediment is strongly influenced by the gravity of a planetary body. As a consequence, sorting of a sedimentary rock varies with gravity for a given depth and velocity of surface runoff. Theoretical considerations for spheres indicate that sorting is less uniform on Mars than on Earth for runoff of identical depth. The effects of gravity on flow hydraulics limit the use of common, semi-empirical models developed to simulate particle settling in terrestrial environments, on Mars. Assessing sedimentation patterns on Mars, aimed at identifying strata potentially hosting traces of life, is potentially affected by such uncertainties. Using first-principle approaches, e.g. through Computational Fluid Dynamics, for calculating settling velocities on other planetary bodies requires a large effort and is limited by the values of boundary conditions, e.g. the shape of the particle. The degree of uncertainty resulting from the differences in gravity on Earth and Mars was therefore tested during three reduced-gravity flights, the MarsSedEx I and II missions, conducted in November 2012 and 2013. Nine types of sediment, ranging in size, shape and density were tested in custom-designed settling tubes during parabolas of Martian gravity lasting 20 to 25 seconds. Based on the observed settling velocities, the uncertainties of empirical relationships developed on Earth to assess particle settling on Mars are discussed. In addition, the potential effects of reduced gravity on patterns of erosion, transport and sorting of sediment, including the implications for identifying strata bearing traces of past life on are examined.

Modular nucleic acid assembled p/MHC microarrays for multiplexed sorting of antigen-specific T cells.

PubMed

Kwong, Gabriel A; Radu, Caius G; Hwang, Kiwook; Shu, Chengyi J; Ma, Chao; Koya, Richard C; Comin-Anduix, Begonya; Hadrup, Sine Reker; Bailey, Ryan C; Witte, Owen N; Schumacher, Ton N; Ribas, Antoni; Heath, James R

2009-07-22

The human immune system consists of a large number of T cells capable of recognizing and responding to antigens derived from various sources. The development of peptide-major histocompatibility (p/MHC) tetrameric complexes has enabled the direct detection of these antigen-specific T cells. With the goal of increasing throughput and multiplexing of T cell detection, protein microarrays spotted with defined p/MHC complexes have been reported, but studies have been limited due to the inherent instability and reproducibility of arrays produced via conventional spotted methods. Herein, we report on a platform for the detection of antigen-specific T cells on glass substrates that offers significant advantages over existing surface-bound schemes. In this approach, called "Nucleic Acid Cell Sorting (NACS)", single-stranded DNA oligomers conjugated site-specifically to p/MHC tetramers are employed to immobilize p/MHC tetramers via hybridization to a complementary-printed substrate. Fully assembled p/MHC arrays are used to detect and enumerate T cells captured from cellular suspensions, including primary human T cells collected from cancer patients. NACS arrays outperform conventional spotted arrays assessed in key criteria such as repeatability and homogeneity. The versatility of employing DNA sequences for cell sorting is exploited to enable the programmed, selective release of target populations of immobilized T cells with restriction endonucleases for downstream analysis. Because of the performance, facile and modular assembly of p/MHC tetramer arrays, NACS holds promise as a versatile platform for multiplexed T cell detection.

Heat Recovery Incinerator-Equipment Selection and Plant Layout for Safety, Human Engineering and Maintainability.

DTIC Science & Technology

1984-10-01

16 4.2 Solid Waste Receiving and Storage Area ........................ 17 4.3 Equipment Location and Spacing...10OROS (Ccr4n-ifl. **,0d. d - -eet -.d tdonfffy by bltk -P-+) HRI, RAM, Human factors, HRI design, IRI safety, solid waste , energy recovery 10 AOSTNACT...health and safety hazards to individuals hand-sorting the conglomerate of solid waste , the potential of dangerous substances and inflammable or ex

Mass, energy and material balances of SRF production process. Part 1: SRF produced from commercial and industrial waste.

PubMed

Nasrullah, Muhammad; Vainikka, Pasi; Hannula, Janne; Hurme, Markku; Kärki, Janne

2014-08-01

This paper presents the mass, energy and material balances of a solid recovered fuel (SRF) production process. The SRF is produced from commercial and industrial waste (C&IW) through mechanical treatment (MT). In this work various streams of material produced in SRF production process are analyzed for their proximate and ultimate analysis. Based on this analysis and composition of process streams their mass, energy and material balances are established for SRF production process. Here mass balance describes the overall mass flow of input waste material in the various output streams, whereas material balance describes the mass flow of components of input waste stream (such as paper and cardboard, wood, plastic (soft), plastic (hard), textile and rubber) in the various output streams of SRF production process. A commercial scale experimental campaign was conducted on an MT waste sorting plant to produce SRF from C&IW. All the process streams (input and output) produced in this MT plant were sampled and treated according to the CEN standard methods for SRF: EN 15442 and EN 15443. The results from the mass balance of SRF production process showed that of the total input C&IW material to MT waste sorting plant, 62% was recovered in the form of SRF, 4% as ferrous metal, 1% as non-ferrous metal and 21% was sorted out as reject material, 11.6% as fine fraction, and 0.4% as heavy fraction. The energy flow balance in various process streams of this SRF production process showed that of the total input energy content of C&IW to MT plant, 75% energy was recovered in the form of SRF, 20% belonged to the reject material stream and rest 5% belonged with the streams of fine fraction and heavy fraction. In the material balances, mass fractions of plastic (soft), plastic (hard), paper and cardboard and wood recovered in the SRF stream were 88%, 70%, 72% and 60% respectively of their input masses to MT plant. A high mass fraction of plastic (PVC), rubber material and non-combustibles (such as stone/rock and glass particles), was found in the reject material stream. Copyright © 2014 Elsevier Ltd. All rights reserved.

Automated processing of shoeprint images based on the Fourier transform for use in forensic science.

PubMed

de Chazal, Philip; Flynn, John; Reilly, Richard B

2005-03-01

The development of a system for automatically sorting a database of shoeprint images based on the outsole pattern in response to a reference shoeprint image is presented. The database images are sorted so that those from the same pattern group as the reference shoeprint are likely to be at the start of the list. A database of 476 complete shoeprint images belonging to 140 pattern groups was established with each group containing two or more examples. A panel of human observers performed the grouping of the images into pattern categories. Tests of the system using the database showed that the first-ranked database image belongs to the same pattern category as the reference image 65 percent of the time and that a correct match appears within the first 5 percent of the sorted images 87 percent of the time. The system has translational and rotational invariance so that the spatial positioning of the reference shoeprint images does not have to correspond with the spatial positioning of the shoeprint images of the database. The performance of the system for matching partial-prints was also determined.

Computer vision-based sorting of Atlantic salmon (Salmo salar) fillets according to their color level.

PubMed

Misimi, E; Mathiassen, J R; Erikson, U

2007-01-01

Computer vision method was used to evaluate the color of Atlantic salmon (Salmo salar) fillets. Computer vision-based sorting of fillets according to their color was studied on 2 separate groups of salmon fillets. The images of fillets were captured using a digital camera of high resolution. Images of salmon fillets were then segmented in the regions of interest and analyzed in red, green, and blue (RGB) and CIE Lightness, redness, and yellowness (Lab) color spaces, and classified according to the Roche color card industrial standard. Comparisons of fillet color between visual evaluations were made by a panel of human inspectors, according to the Roche SalmoFan lineal standard, and the color scores generated from computer vision algorithm showed that there were no significant differences between the methods. Overall, computer vision can be used as a powerful tool to sort fillets by color in a fast and nondestructive manner. The low cost of implementing computer vision solutions creates the potential to replace manual labor in fish processing plants with automation.

Genetic and Functional Heterogeneity of Tumors in Neurofibromatosis 2

DTIC Science & Technology

2016-07-01

mapped to the human genome build 37 (hg19) through BWA v. 0.5.9 [1] with parameters -q 5 -l 32 -k 2 –o 1. The resulting alignments were further sorted...see Table 2). Table 2 – Isogenic human arachnoidal cell (AC) clones with NF2 (exon 8) inactivating mutations generated by CRISPR/Cas genome editing...libraries were aligned to the human genome (GrCH37, Ensembl build 71) using Gsnap [19] version 2014_12_19. Expression levels of genes in the units of count

Installation Restoration Program. Preliminary Assessment: 177th Fighter Interceptor Group, New Jersey Air National Guard, Atlantic City International Airport, Atlantic City, New Jersey

DTIC Science & Technology

1989-11-01

and/or spill sites on Department of Defense (DoD) installations, and " Control hazards to human health, welfare, and the environment that may have...Investigations do not indicate harmful levels of contamination and do not pose a significant threat to human health or the environment. The site does not warrant...Feasibility Study - Investigation confirms the presence of contamination that may pose a threat to human health and/or the environment, and some sort of

A psychoanalytic model for human freedom and rationality.

PubMed

Macklin, R

1976-07-01

The nature and scope of freedom and rationality in man are explored in light of the problems posed by a deterministic framework for understanding and explaining human though, feeling, and behavior. It is argued that the sort of explanation afforded by a psychodynamic theory is fully compatible with attributing freedom and rationality to persons. In particular, psychoanalytic theory is able to account for the existence of causal laws governing all aspects of human behavior, while providing a schema by which we can distinguish rational from irrational behavior, and free acts from those that are unfree.

Berthing and Utilities Requirements Forecasting (BURF) Program of the NAVSHIPS Long Range Workload Planning System (LRPS).

DTIC Science & Technology

1973-02-01

No Deec F3202 B P egcak what(oniud 8 +Yes Fe d . Each phase is discussed in detail in Section 3 of this report. The logic flow of BURFA is shown in...Clas Flowchart for Function INFST D-14 ASSGN Call SORT Initialize Berth Facilities J=1,3 2 ISN I A Ship Yet Assigned E Berth No Determine N1 , N2 3 K N1

Caldron For High-Temperature Alloys

NASA Technical Reports Server (NTRS)

Geringer, Henry J.

1989-01-01

Induction-heated caldron melts high-temperature alloys. Prevents sort of contamination of melts occurring during arc melting in ceramic crucibles. Liquefies 200 grams of solid metal components of alloy like niobium aluminum and makes alloy homogeneous in less than 3 minutes. Plugged sleeve constitutes main body of caldron. Coolant flows through sleeve to prevent it from melting. Mandrel-wound induction coils adjusted to tune source of power. Also serves as mold for casting alloys into such shapes as bars.

A continuous high-throughput bioparticle sorter based on 3D traveling-wave dielectrophoresis.

PubMed

Cheng, I-Fang; Froude, Victoria E; Zhu, Yingxi; Chang, Hsueh-Chia; Chang, Hsien-Chang

2009-11-21

We present a high throughput (maximum flow rate approximately 10 microl/min or linear velocity approximately 3 mm/s) continuous bio-particle sorter based on 3D traveling-wave dielectrophoresis (twDEP) at an optimum AC frequency of 500 kHz. The high throughput sorting is achieved with a sustained twDEP particle force normal to the continuous through-flow, which is applied over the entire chip by a single 3D electrode array. The design allows continuous fractionation of micron-sized particles into different downstream sub-channels based on differences in their twDEP mobility on both sides of the cross-over. Conventional DEP is integrated upstream to focus the particles into a single levitated queue to allow twDEP sorting by mobility difference and to minimize sedimentation and field-induced lysis. The 3D electrode array design minimizes the offsetting effect of nDEP (negative DEP with particle force towards regions with weak fields) on twDEP such that both forces increase monotonically with voltage to further increase the throughput. Effective focusing and separation of red blood cells from debris-filled heterogeneous samples are demonstrated, as well as size-based separation of poly-dispersed liposome suspensions into two distinct bands at 2.3 to 4.6 microm and 1.5 to 2.7 microm, at the highest throughput recorded in hand-held chips of 6 microl/min.

Transposon-mediated generation of BCR-ABL1-expressing transgenic cell lines for unbiased sensitivity testing of tyrosine kinase inhibitors.

PubMed

Byrgazov, Konstantin; Lucini, Chantal Blanche; Berkowitsch, Bettina; Koenig, Margit; Haas, Oskar A; Hoermann, Gregor; Valent, Peter; Lion, Thomas

2016-11-22

Point mutations in the ABL1 kinase domain are an important mechanism of resistance to tyrosine kinase inhibitors (TKI) in BCR-ABL1-positive and, as recently shown, BCR-ABL1-like leukemias. The cell line Ba/F3 lentivirally transduced with mutant BCR-ABL1 constructs is widely used for in vitro sensitivity testing and response prediction to tyrosine kinase inhibitors. The transposon-based Sleeping Beauty system presented offers several advantages over lentiviral transduction including the absence of biosafety issues, faster generation of transgenic cell lines, and greater efficacy in introducing large gene constructs. Nevertheless, both methods can mediate multiple insertions in the genome. Here we show that multiple BCR-ABL1 insertions result in elevated IC50 levels for individual TKIs, thus overestimating the actual resistance of mutant subclones. We have therefore established flow-sorting-based fractionation of BCR-ABL1-transformed Ba/F3 cells facilitating efficient enrichment of cells carrying single-site insertions, as demonstrated by FISH-analysis. Fractions of unselected Ba/F3 cells not only showed a greater number of BCR-ABL1 hybridization signals, but also revealed higher IC50 values for the TKIs tested. The data presented highlight the need to carefully select transfected cells by flow-sorting, and to control the insertion numbers by FISH and real-time PCR to permit unbiased in vitro testing of drug resistance.

Translating Microfluidics: Cell Separation Technologies and their Barriers to Commercialization

PubMed Central

Shields, C. Wyatt; Ohiri, Korine A.; Szott, Lizzy M.; López, Gabriel P.

2016-01-01

Advances in microfluidic cell sorting have revolutionized the ways in which cell-containing fluids are processed, now providing performances comparable to, or exceeding, traditional systems, but in a vastly miniaturized format. These technologies exploit a wide variety of physical phenomena to manipulate cells and fluid flow, such as magnetic traps, sound waves and flow-altering micropatterns, and they can evaluate single cells by immobilizing them onto surfaces for chemotherapeutic assessment, encapsulate cells into picoliter droplets for toxicity screenings and examine the interactions between pairs of cells in response to new, experimental drugs. However, despite the massive surge of innovation in these high-performance lab-on-a-chip devices, few have undergone successful commercialization, and no device has been translated to a widely distributed clinical commodity to date. Persistent challenges such as an increasingly saturated patent landscape as well as complex user interfaces are among several factors that may contribute to their slowed progress. In this article, we identify several of the leading microfluidic technologies for sorting cells that are poised for clinical translation; we examine the principal barriers preventing their routine clinical use; finally, we provide a prospectus to elucidate the key criteria that must be met to overcome those barriers. Once established, these tools may soon transform how clinical labs study various ailments and diseases by separating cells for downstream sequencing and enabling other forms of advanced cellular or sub-cellular analysis. PMID:27282966

How Culture Misdirects Multiculturalism.

ERIC Educational Resources Information Center

Wax, Murray L.

In the ongoing debate over whether or what sort of multiculturalism should be provided by schools, the origin, evolution, and rhetorical function of the basic term "culture" have been unwisely neglected. The 19th century notion of "culture" implied a process of growth and development, of culturing an organism, or of the human organism becoming…

A Three-Pronged Approach for Overcoming Design Fixation

ERIC Educational Resources Information Center

Smith, Steven M.; Linsey, Julie

2011-01-01

Earthquakes, lightning, and history-changing ideas are classic examples of powerful, unpredictable forces of nature. These sorts of phenomena have been difficult to explain and predict, an often frustrating fact as humans try to understand and control the significant influences in our lives. Historically, such phenomena have been attributed to…

Machine Vision Giving Eyes to Robots. Resources in Technology.

ERIC Educational Resources Information Center

Technology Teacher, 1990

1990-01-01

This module introduces machine vision, which can be used for inspection, robot guidance and part sorting. The future for machine vision will include new technology and will bring vision systems closer to the ultimate vision processor, the human eye. Includes a student quiz, outcomes, and activities. (JOW)

Magneto hall effect on unsteady elastico-viscous nanofluid slip flow in a channel in presence of thermal radiation and heat generation with Brownian motion

NASA Astrophysics Data System (ADS)

Karim, M. Enamul; Samad, M. Abdus; Ferdows, M.

2017-06-01

The present note investigates the magneto hall effect on unsteady flow of elastico-viscous nanofluid in a channel with slip boundary considering the presence of thermal radiation and heat generation with Brownian motion. Numerical results are achieved by solving the governing equations by the implicit Finite Difference Method (FDM) obtaining primary and secondary velocities, temperature, nanoparticles volume fraction and concentration distributions within the boundary layer entering into the problem. The influences of several interesting parameters such as elastico-viscous parameter, magnetic field, hall parameter, heat generation, thermal radiation and Brownian motion parameters on velocity, heat and mass transfer characteristics of the fluid flow are discussed with the help of graphs. Also the effects of the pertinent parameters, which are of physical and engineering interest, such as Skin friction parameter, Nusselt number and Sherwood number are sorted out. It is found that the flow field and other quantities of physical concern are significantly influenced by these parameters.

Correlation of ash-flow tuffs.

USGS Publications Warehouse

Hildreth, W.; Mahood, G.

1985-01-01

Discrimination and correlation of ash-flow sheets is important in structurally complex, long-lived volcanic fields where such sheets provide the best keys to the regional stratigraphic framework. Three-dimensional complexities resulting from pulsatory eruptions, sectorial emplacement, mechanical sorting during outflow, thermal and compositional zoning of magmas, the physical zoning of cooling units, and structural and erosional disruption can make such correlation and discrimination difficult. When lithologic, magnetic, petrographic, chemical, and isotopic criteria for correlating ash-flow sheets are critically evaluated, many problems and pitfalls can be identified. Distinctive phenocrysts, pumice clasts, and lithic fragments are among the more reliable criteria, as are high-precision K-Ar ages and thermal remanent magnetization (TRM) directions in unaltered welded tuff. Chemical correlation methods should rely principally upon welded or nonwelded pumice blocks, not upon the ash-flow matrix, which is subject to fractionation, mixing, and contamination during emplacement. Compositional zoning of most large sheets requires that many samples be analyzed before phenocryst, glass or whole-rock chemical trends can be used confidently as correlation criteria.-Authors

Denni Algorithm An Enhanced Of SMS (Scan, Move and Sort) Algorithm

NASA Astrophysics Data System (ADS)

Aprilsyah Lubis, Denni; Salim Sitompul, Opim; Marwan; Tulus; Andri Budiman, M.

2017-12-01

Sorting has been a profound area for the algorithmic researchers, and many resources are invested to suggest a more working sorting algorithm. For this purpose many existing sorting algorithms were observed in terms of the efficiency of the algorithmic complexity. Efficient sorting is important to optimize the use of other algorithms that require sorted lists to work correctly. Sorting has been considered as a fundamental problem in the study of algorithms that due to many reasons namely, the necessary to sort information is inherent in many applications, algorithms often use sorting as a key subroutine, in algorithm design there are many essential techniques represented in the body of sorting algorithms, and many engineering issues come to the fore when implementing sorting algorithms., Many algorithms are very well known for sorting the unordered lists, and one of the well-known algorithms that make the process of sorting to be more economical and efficient is SMS (Scan, Move and Sort) algorithm, an enhancement of Quicksort invented Rami Mansi in 2010. This paper presents a new sorting algorithm called Denni-algorithm. The Denni algorithm is considered as an enhancement on the SMS algorithm in average, and worst cases. The Denni algorithm is compared with the SMS algorithm and the results were promising.

Origin and speciation of Picea schrenkiana and Piceasmithiana in the Center Asian Highlands and Himalayas.

PubMed

Li, Lili; Sun, Yongshuai; Zou, Jiabin; Yue, Wei; Wang, Xi; Liu, Jianquan

Elucidating the evolutionary history of current species diversity, especially trees with large effective population sizes and long generation times, is a complicated exercise confounded by gene flow and incomplete lineage sorting. In the present study, we aim to determine the origin and speciation of Picea schrenkiana and Picea smithiana using population genetic data from chloroplast (cp), mitochondrial (mt), and nuclear (nr) genomes. These two species occur in the Central Asian Highlands and Himalayas, respectively, where they are isolated from other Asian congeneric species by the Qinghai-Tibet Plateau (QTP) or adjacent deserts. Previous studies based on both morphological and molecular evidence suggest that they have contrasting phylogenetic relationships with Picea likiangensis or Picea wilsonii which are closely related and both located in the QTP. We examined genetic variation among 16 loci of three genomes from 30 populations of these four species. At both cpDNA loci and mtDNA loci, P. schrenkiana appeared to be closely related to P. likiangensis , although statistical support for this was weak. However, phylogenetic analyses and speciation tests based on the nuclear data from 11 loci provided evidence that P. schrenkiana and P. smithiana are sister species. These two species diverged around five million years ago (Mya) while the divergence between them and the P. likiangensis - P. wilsonii clade occurred about 18.4 Mya. We also detected gene flow accompanying these speciation events. Our results highlight the complex speciation histories of these alpine conifers due to interspecific gene flow and/or incomplete lineage sorting, and the importance of the early QTP uplifts in promoting the origin of these important conifer species in the Asian highlands.

Comparison of different fertilisation media for an in vitro maturation?fertilisation?culture system using flow-cytometrically sorted X chromosome-bearing spermatozoa for bovine embryo production.

PubMed

Ferré, Luis B; Bogliotti, Yanina; Chitwood, James L; Fresno, Cristóbal; Ortega, Hugo H; Kjelland, Michael E; Ross, Pablo J

2015-05-13

High demand exists among commercial cattle producers for in vitro-derived bovine embryos fertilised with female sex-sorted spermatozoa from high-value breeding stock. The aim of this study was to evaluate three fertilisation media, namely M199, synthetic oviductal fluid (SOF) and Tyrode's albumin-lactate-pyruvate (TALP), on IVF performance using female sex-sorted spermatozoa. In all, 1143, 1220 and 1041 cumulus-oocyte complexes were fertilised in M199, SOF and TALP, respectively. There were significant differences among fertilisation media (P < 0.05) in cleavage rate (M199 = 57%, SOF = 71% and TALP = 72%), blastocyst formation (M199 = 9%, SOF = 20% and TALP = 19%), proportion of Grade 1 blastocysts (M199 = 15%, SOF = 52% and TALP = 51%), proportion of Grade 3 blastocysts (M199 = 58%, SOF = 21% and TALP = 20%) and hatching rates (M199 = 29%, SOF = 60% and TALP = 65%). The inner cell mass (ICM) and trophectoderm (TE) cells of Day 7 blastocysts were also affected by the fertilisation medium. Embryos derived from SOF and TALP fertilisation media had higher numbers of ICM, TE and total cells than those fertilised in M199. In conclusion, fertilisation media affected cleavage rate, as well as subsequent embryo development, quality and hatching ability. SOF and TALP fertilisation media produced significantly more embryos of higher quality than M199.

The Proell Effect: A Macroscopic Maxwell's Demon

NASA Astrophysics Data System (ADS)

Rauen, Kenneth M.

2011-12-01

Maxwell's Demon is a legitimate challenge to the Second Law of Thermodynamics when the "demon" is executed via the Proell effect. Thermal energy transfer according to the Kinetic Theory of Heat and Statistical Mechanics that takes place over distances greater than the mean free path of a gas circumvents the microscopic randomness that leads to macroscopic irreversibility. No information is required to sort the particles as no sorting occurs; the entire volume of gas undergoes the same transition. The Proell effect achieves quasi-spontaneous thermal separation without sorting by the perturbation of a heterogeneous constant volume system with displacement and regeneration. The classical analysis of the constant volume process, such as found in the Stirling Cycle, is incomplete and therefore incorrect. There are extra energy flows that classical thermo does not recognize. When a working fluid is displaced across a regenerator with a temperature gradient in a constant volume system, complimentary compression and expansion work takes place that transfers energy between the regenerator and the bulk gas volumes of the hot and cold sides of the constant volume system. Heat capacity at constant pressure applies instead of heat capacity at constant volume. The resultant increase in calculated, recyclable energy allows the Carnot Limit to be exceeded in certain cycles. Super-Carnot heat engines and heat pumps have been designed and a US patent has been awarded.

Rmax: A systematic approach to evaluate instrument sort performance using center stream catch☆

PubMed Central

Riddell, Andrew; Gardner, Rui; Perez-Gonzalez, Alexis; Lopes, Telma; Martinez, Lola

2015-01-01

Sorting performance can be evaluated with regard to Purity, Yield and/or Recovery of the sorted fraction. Purity is a check on the quality of the sample and the sort decisions made by the instrument. Recovery and Yield definitions vary with some authors regarding both as how efficient the instrument is at sorting the target particles from the original sample, others distinguishing Recovery from Yield, where the former is used to describe the accuracy of the instrument’s sort count. Yield and Recovery are often neglected, mostly due to difficulties in their measurement. Purity of the sort product is often cited alone but is not sufficient to evaluate sorting performance. All of these three performance metrics require re-sampling of the sorted fraction. But, unlike Purity, calculating Yield and/or Recovery calls for the absolute counting of particles in the sorted fraction, which may not be feasible, particularly when dealing with rare populations and precious samples. In addition, the counting process itself involves large errors. Here we describe a new metric for evaluating instrument sort Recovery, defined as the number of particles sorted relative to the number of original particles to be sorted. This calculation requires only measuring the ratios of target and non-target populations in the original pre-sort sample and in the waste stream or center stream catch (CSC), avoiding re-sampling the sorted fraction and absolute counting. We called this new metric Rmax, since it corresponds to the maximum expected Recovery for a particular set of instrument parameters. Rmax is ideal to evaluate and troubleshoot the optimum drop-charge delay of the sorter, or any instrument related failures that will affect sort performance. It can be used as a daily quality control check but can be particularly useful to assess instrument performance before single-cell sorting experiments. Because we do not perturb the sort fraction we can calculate Rmax during the sort process, being especially valuable to check instrument performance during rare population sorts. PMID:25747337

Particle sorting in Filter Porous Media and in Sediment Transport: A Numerical and Experimental Study

NASA Astrophysics Data System (ADS)

Glascoe, L. G.; Ezzedine, S. M.; Kanarska, Y.; Lomov, I. N.; Antoun, T.; Smith, J.; Hall, R.; Woodson, S.

2014-12-01

Understanding the flow of fines, particulate sorting in porous media and fractured media during sediment transport is significant for industrial, environmental, geotechnical and petroleum technologies to name a few. For example, the safety of dam structures requires the characterization of the granular filter ability to capture fine-soil particles and prevent erosion failure in the event of an interfacial dislocation. Granular filters are one of the most important protective design elements of large embankment dams. In case of cracking and erosion, if the filter is capable of retaining the eroded fine particles, then the crack will seal and the dam safety will be ensured. Here we develop and apply a numerical tool to thoroughly investigate the migration of fines in granular filters at the grain scale. The numerical code solves the incompressible Navier-Stokes equations and uses a Lagrange multiplier technique. The numerical code is validated to experiments conducted at the USACE and ERDC. These laboratory experiments on soil transport and trapping in granular media are performed in constant-head flow chamber filled with the filter media. Numerical solutions are compared to experimentally measured flow rates, pressure changes and base particle distributions in the filter layer and show good qualitative and quantitative agreement. To further the understanding of the soil transport in granular filters, we investigated the sensitivity of the particle clogging mechanism to various parameters such as particle size ratio, the magnitude of hydraulic gradient, particle concentration, and grain-to-grain contact properties. We found that for intermediate particle size ratios, the high flow rates and low friction lead to deeper intrusion (or erosion) depths. We also found that the damage tends to be shallower and less severe with decreasing flow rate, increasing friction and concentration of suspended particles. We have extended these results to more realistic heterogeneous population particulates for sediment transport. This work performed under the auspices of the US DOE by LLNL under Contract DE-AC52-07NA27344 and was sponsored by the Department of Homeland Security, Science and Technology Directorate, Homeland Security Advanced Research Projects Agency.

Textural variations and fragmentation processes in peperite formed between felsic lava flow and wet substrate: An example from the Cretaceous Buan Volcanics, southwest Korea

NASA Astrophysics Data System (ADS)

Gihm, Yong Sik; Kwon, Chang Woo

2017-02-01

Multiple exposures of peperite within the Cretaceous Buan Volcanics, southwest Korea, have been examined in order to determine variations in their textural characteristics and to investigate their mode of formation. Along undulating boundaries between rhyolite (lava flow) and deformed host sediment expressed as a series of load and flame structures, exposures commonly contain two distinct types of peperite. Type-1 peperites are composed mostly of rounded juvenile clasts at their base and polyhedral juvenile clasts at their upper levels, interpreted to have formed via a two-stage process. Firstly, abrasion of juvenile clasts occurred after their fragmentation due to shear stress imparted by the overlying and still-moving lava flow, forming rounded juvenile clasts. Subsequent in situ quenching fragmentation of the lava flow produced clasts with platy to polyhedral shapes immediately after emplacement of the lava flow. Type-2 peperites laterally extend into the interior of featureless rhyolite as layers that decrease in thickness with increasing distance away from the flame zone. These layers exhibit horizontal textural variations, ranging from poorly sorted mixtures of ash- to block-sized angular juvenile clasts in the proximal zone, to closely packed polyhedral and tabular juvenile clasts with jigsaw-crack textures in the middle and distal zones. Type-2 peperite are inferred to have formed due to internal steam explosions that resulted from an expansion of heated pore water (leading to an increase in pore fluid pressure) that had been vertically injected into the interior of the rhyolite from the flame zone. The proximal zone, composed mainly of poorly sorted mixtures of juvenile clasts, represents the explosion sites. Juvenile clasts in the middle and distal zones are interpreted to have formed due to three separate processes: the development of fractures in the rhyolite during the internal steam explosions, injection of the host sediment through the fractures, and in situ quenching fragmentation. Deformation of the host sediment exerted an important control on peperite-forming processes, with the internal steam explosions suggested to have formed the closely packed, juvenile clasts with a jigsaw-crack texture rather than the clasts that are widely dispersed.

The Transformative Experience in Engineering Education

NASA Astrophysics Data System (ADS)

Goodman, Katherine Ann

This research evaluates the usefulness of transformative experience (TE) in engineering education. With TE, students 1) apply ideas from coursework to everyday experiences without prompting (motivated use); 2) see everyday situations through the lens of course content (expanded perception); and 3) value course content in new ways because it enriches everyday affective experience (affective value). In a three-part study, we examine how engineering educators can promote student progress toward TE and reliably measure that progress. For the first study, we select a mechanical engineering technical elective, Flow Visualization, that had evidence of promoting expanded perception of fluid physics. Through student surveys and interviews, we compare this elective to the required Fluid Mechanics course. We found student interest in fluids fell into four categories: complexity, application, ubiquity, and aesthetics. Fluid Mechanics promotes interest from application, while Flow Visualization promotes interest based in ubiquity and aesthetics. Coding for expanded perception, we found it associated with students' engineering identity, rather than a specific course. In our second study, we replicate atypical teaching methods from Flow Visualization in a new design course: Aesthetics of Design. Coding of surveys and interviews reveals that open-ended assignments and supportive teams lead to increased ownership of projects, which fuels risk-taking, and produces increased confidence as an engineer. The third study seeks to establish parallels between expanded perception and measurable perceptual expertise. Our visual expertise experiment uses fluid flow images with both novices and experts (students who had passed fluid mechanics). After training, subjects sort images into laminar and turbulent categories. The results demonstrate that novices learned to sort the flow stimuli in ways similar to subjects in prior perceptual expertise studies. In contrast, the experts' significantly better results suggest they are accessing conceptual fluids knowledge to perform this new, visual task. The ability to map concepts onto visual information is likely a necessary step toward expanded perception. Our findings suggest that open-ended aesthetic experiences with engineering content unexpectedly support engineering identity development, and that visual tasks could be developed to measure conceptual understanding, promoting expanded perception. Overall, we find TE a productive theoretical framework for engineering education research.

A cluster analysis method for identification of subpopulations of cells in flow cytometric list-mode arrays

NASA Technical Reports Server (NTRS)

Li, Z. K.

1985-01-01

A specialized program was developed for flow cytometric list-mode data using an heirarchical tree method for identifying and enumerating individual subpopulations, the method of principal components for a two-dimensional display of 6-parameter data array, and a standard sorting algorithm for characterizing subpopulations. The program was tested against a published data set subjected to cluster analysis and experimental data sets from controlled flow cytometry experiments using a Coulter Electronics EPICS V Cell Sorter. A version of the program in compiled BASIC is usable on a 16-bit microcomputer with the MS-DOS operating system. It is specialized for 6 parameters and up to 20,000 cells. Its two-dimensional display of Euclidean distances reveals clusters clearly, as does its 1-dimensional display. The identified subpopulations can, in suitable experiments, be related to functional subpopulations of cells.

Photo-actuation of liquids for light-driven microfluidics: state of the art and perspectives.

PubMed

Baigl, Damien

2012-10-07

Using light to control liquid motion is a new paradigm for the actuation of microfluidic systems. We review here the different principles and strategies to induce or control liquid motion using light, which includes the use of radiation pressure, optical tweezers, light-induced wettability gradients, the thermocapillary effect, photosensitive surfactants, the chromocapillary effect, optoelectrowetting, photocontrolled electroosmotic flows and optical dielectrophoresis. We analyze the performance of these approaches to control using light many kinds of microfluidic operations involving discrete pL- to μL-sized droplets (generation, driving, mixing, reaction, sorting) or fluid flows in microchannels (valve operation, injection, pumping, flow rate control). We show that a complete toolbox is now available to control microfluidic systems by light. We finally discuss the perspectives of digital optofluidics as well as microfluidics based on all optical fluidic chips and optically reconfigurable devices.

Multiscale polar theory of microtubule and motor-protein assemblies

DOE PAGES

Gao, Tong; Blackwell, Robert; Glaser, Matthew A.; ...

2015-01-27

Microtubules and motor proteins are building blocks of self-organized subcellular biological structures such as the mitotic spindle and the centrosomal microtubule array. These same ingredients can form new “bioactive” liquid-crystalline fluids that are intrinsically out of equilibrium and which display complex flows and defect dynamics. It is not yet well understood how microscopic activity, which involves polarity-dependent interactions between motor proteins and microtubules, yields such larger-scale dynamical structures. In our multiscale theory, Brownian dynamics simulations of polar microtubule ensembles driven by cross-linking motors allow us to study microscopic organization and stresses. Polarity sorting and cross-link relaxation emerge as two polar-specificmore » sources of active destabilizing stress. On larger length scales, our continuum Doi-Onsager theory captures the hydrodynamic flows generated by polarity-dependent active stresses. Finally, the results connect local polar structure to flow structures and defect dynamics.« less

Flow cytometry for enrichment and titration in massively parallel DNA sequencing

PubMed Central

Sandberg, Julia; Ståhl, Patrik L.; Ahmadian, Afshin; Bjursell, Magnus K.; Lundeberg, Joakim

2009-01-01

Massively parallel DNA sequencing is revolutionizing genomics research throughout the life sciences. However, the reagent costs and labor requirements in current sequencing protocols are still substantial, although improvements are continuously being made. Here, we demonstrate an effective alternative to existing sample titration protocols for the Roche/454 system using Fluorescence Activated Cell Sorting (FACS) technology to determine the optimal DNA-to-bead ratio prior to large-scale sequencing. Our method, which eliminates the need for the costly pilot sequencing of samples during titration is capable of rapidly providing accurate DNA-to-bead ratios that are not biased by the quantification and sedimentation steps included in current protocols. Moreover, we demonstrate that FACS sorting can be readily used to highly enrich fractions of beads carrying template DNA, with near total elimination of empty beads and no downstream sacrifice of DNA sequencing quality. Automated enrichment by FACS is a simple approach to obtain pure samples for bead-based sequencing systems, and offers an efficient, low-cost alternative to current enrichment protocols. PMID:19304748

Particle sorting by Paramecium cilia arrays.

PubMed

Mayne, Richard; Whiting, James G H; Wheway, Gabrielle; Melhuish, Chris; Adamatzky, Andrew

Motile cilia are cell-surface organelles whose purposes, in ciliated protists and certain ciliated metazoan epithelia, include generating fluid flow, sensing and substance uptake. Certain properties of cilia arrays, such as beating synchronisation and manipulation of external proximate particulate matter, are considered emergent, but remain incompletely characterised despite these phenomena having being the subject of extensive modelling. This study constitutes a laboratory experimental characterisation of one of the emergent properties of motile cilia: manipulation of adjacent particulates. The work demonstrates through automated videomicrographic particle tracking that interactions between microparticles and somatic cilia arrays of the ciliated model organism Paramecium caudatum constitute a form of rudimentary 'sorting'. Small particles are drawn into the organism's proximity by cilia-induced fluid currents at all times, whereas larger particles may be held immobile at a distance from the cell margin when the cell generates characteristic feeding currents in the surrounding media. These findings can contribute to the design and fabrication of biomimetic cilia, with potential applications to the study of ciliopathies. Copyright © 2017 Elsevier B.V. All rights reserved.

Clonogenic colony-forming ability of flow cytometrically isolated hepatic progenitor cells in the murine fetal liver.

PubMed

Taniguchi, H; Kondo, R; Suzuki, A; Zheng, Y W; Takada, Y; Fukunaga, K; Seino, K; Yuzawa, K; Otsuka, M; Fukao, K; Nakauchi, H

2000-01-01

Stem cells are defined as cells having multilineage differentiation potential and self-renewal capability. Hepatic stem cells have aroused considerable interest not only because of their developmental importance but also for their therapeutic potential. However, their presence in the liver has not yet been demonstrated. With the use of a fluorescence-activated cell sorter (FACS) and monoclonal antibodies, we attempted to ascertain whether hepatic stem cells are present in the murine fetal liver. For this purpose, we optimized a cell isolation technique for FACS sorting of fetal liver cells. When isolated CD45 TER119 cells (the non-blood cell fraction in the fetal liver) were tested for their clonogenic colony-forming ability, mechanical dissociation (pipetting) was the most suitable cell isolation technique for FACS sorting. We confirmed that these colonies contained not only cells expressing hepatocyte markers but also cells expressing cholangiocyte markers. To identify hepatic stem cells, studies must focus on CD45TER119- cells in the murine fetal liver.

GIS-based planning system for managing the flow of construction and demolition waste in Brazil.

PubMed

Paz, Diogo Henrique Fernandes da; Lafayette, Kalinny Patrícia Vaz; Sobral, Maria do Carmo

2018-05-01

The objective of this article was to plan a network for municipal management of construction and demolition waste in Brazil with the assistance of a geographic information system, using the city of Recife as a case study. The methodology was carried out in three stages. The first was to map the illegal construction and demolition of waste disposal points across Recife and classify the waste according to its recyclability. In sequence, a method for indicating suitable areas for installation of voluntary delivery points, for small waste generators, are presented. Finally, a method for indicating suitable areas for the installation of trans-shipment and waste sorting areas, developed for large generators, is presented. The results show that a geographic information system is an essential tool in the planning of municipal construction and demolition waste management, in order to facilitate the spatial analysis and control the generation, sorting, collection, transportation, and final destination of construction and demolition waste, increasing the rate of recovery and recycling of materials.

Stem and Progenitor Cell Subsets Are Affected by JAK2 Signaling and Can Be Monitored by Flow Cytometry

PubMed Central

Iida, Ryuji; Welner, Robert S.; Zhao, Wanke; Alberola-lla, José; Medina, Kay L.; Zhao, Zhizhuang Joe; Kincade, Paul W.

2014-01-01

Although extremely rare, hematopoietic stem cells (HSCs) are divisible into subsets that differ with respect to differentiation potential and cell surface marker expression. For example, we recently found that CD86− CD150+ CD48− HSCs have limited potential for lymphocyte production. This could be an important new tool for studying hematological abnormalities. Here, we analyzed HSC subsets with a series of stem cell markers in JAK2V617F transgenic (Tg) mice, where the mutation is sufficient to cause myeloproliferative neoplasia with lymphocyte deficiency. Total numbers of HSC were elevated 3 to 20 fold in bone marrow of JAK2V617F mice. Careful analysis suggested the accumulation involved multiple HSC subsets, but particularly those characterized as CD150HI CD86− CD18L°CD41+ and excluding Hoechst dye. Real-Time PCR analysis of their HSC revealed that the erythropoiesis associated gene transcripts Gata1, Klf1 and Epor were particularly high. Flow cytometry analyses based on two differentiation schemes for multipotent progenitors (MPP) also suggested alteration by JAK2 signals. The low CD86 on HSC and multipotent progenitors paralleled the large reductions we found in lymphoid progenitors, but the few that were produced functioned normally when sorted and placed in culture. Either of two HSC subsets conferred disease when transplanted. Thus, flow cytometry can be used to observe the influence of abnormal JAK2 signaling on stem and progenitor subsets. Markers that similarly distinguish categories of human HSCs might be very valuable for monitoring such conditions. They could also serve as indicators of HSC fitness and suitability for transplantation. PMID:24699465

DIE-RNA: A Reproducible Strategy for the Digestion of Normal and Injured Pancreas, Isolation of Pancreatic Cells from Genetically Engineered Mouse Models and Extraction of High Quality RNA

PubMed Central

Assi, Mohamad; Dauguet, Nicolas; Jacquemin, Patrick

2018-01-01

The isolation of ribonucleic acid (RNA) suitable for gene expression studies is challenging in the pancreas, due to its high ribonuclease activity. This is even more complicated during pancreatitis, a condition associated with inflammation and fibrosis. Our aim was to implement a time-effective and reproducible protocol to isolate high quality RNA from specific pancreatic cell subtypes, in normal and inflammatory conditions. We used two genetically engineered mouse models (GEMM), Ela-CreER/YFP and Sox9-CreER/YFP, to isolate acinar and ductal cells, respectively. To induce pancreatitis, mice received a caerulein treatment (125 μg/kg) for 8 and 72 h. We alternatively used EGTA and calcium buffers that contain collagenase P (0.6 mg/mL) to rapidly digest the pancreas into individual cells. Most of the cells from normal and injured pancreas were single-dissociated, exhibited a round morphology and did not incorporate trypan blue dye. Cell suspensions from Ela- and Sox9-CreER/YFP pancreas were then sorted by flow cytometry to isolate the YFP-positive acinar and ductal cells, respectively. Sorted cells kept a round shape and emitted fluorescence detected by the 38 HE green fluorescence filter. RNA was isolated by column-based purification approach. The RNA integrity number (RIN) was high in sorted acinar cell fractions treated with or without caerulein (8.6 ± 0.17 and 8.4 ± 0.09, respectively), compared to the whole pancreas fraction (4.8 ± 1.1). Given the low number of sorted ductal cells, the RIN value was slightly lower compared to acini (7.4 ± 0.4). Quantitative-PCR experiments indicated that sorted acinar and ductal cells express the specific acinar and ductal markers, respectively. Additionally, RNA preparations from caerulein-treated acinar cells were free from significant contamination with immune cell RNA. We thus validated the DIE (Digestion, Isolation, and Extraction)-RNA tool as a reproducible and efficient protocol to isolate pure acinar and ductal cells in vivo and to extract high quality RNA from these cells. PMID:29535635

DIE-RNA: A Reproducible Strategy for the Digestion of Normal and Injured Pancreas, Isolation of Pancreatic Cells from Genetically Engineered Mouse Models and Extraction of High Quality RNA.

PubMed

Assi, Mohamad; Dauguet, Nicolas; Jacquemin, Patrick

2018-01-01

The isolation of ribonucleic acid (RNA) suitable for gene expression studies is challenging in the pancreas, due to its high ribonuclease activity. This is even more complicated during pancreatitis, a condition associated with inflammation and fibrosis. Our aim was to implement a time-effective and reproducible protocol to isolate high quality RNA from specific pancreatic cell subtypes, in normal and inflammatory conditions. We used two genetically engineered mouse models (GEMM), Ela-CreER/YFP and Sox9-CreER/YFP, to isolate acinar and ductal cells, respectively. To induce pancreatitis, mice received a caerulein treatment (125 μg/kg) for 8 and 72 h. We alternatively used EGTA and calcium buffers that contain collagenase P (0.6 mg/mL) to rapidly digest the pancreas into individual cells. Most of the cells from normal and injured pancreas were single-dissociated, exhibited a round morphology and did not incorporate trypan blue dye. Cell suspensions from Ela- and Sox9-CreER/YFP pancreas were then sorted by flow cytometry to isolate the YFP-positive acinar and ductal cells, respectively. Sorted cells kept a round shape and emitted fluorescence detected by the 38 HE green fluorescence filter. RNA was isolated by column-based purification approach. The RNA integrity number (RIN) was high in sorted acinar cell fractions treated with or without caerulein (8.6 ± 0.17 and 8.4 ± 0.09, respectively), compared to the whole pancreas fraction (4.8 ± 1.1). Given the low number of sorted ductal cells, the RIN value was slightly lower compared to acini (7.4 ± 0.4). Quantitative-PCR experiments indicated that sorted acinar and ductal cells express the specific acinar and ductal markers, respectively. Additionally, RNA preparations from caerulein-treated acinar cells were free from significant contamination with immune cell RNA. We thus validated the DIE (Digestion, Isolation, and Extraction)-RNA tool as a reproducible and efficient protocol to isolate pure acinar and ductal cells in vivo and to extract high quality RNA from these cells.

Effects of debris flow composition on runout, depositional mechanisms, and deposit morphology in laboratory experiments

NASA Astrophysics Data System (ADS)

Haas, Tjalling; Braat, Lisanne; Leuven, Jasper R. F. W.; Lokhorst, Ivar R.; Kleinhans, Maarten G.

2015-09-01

Predicting debris flow runout is of major importance for hazard mitigation. Apart from topography and volume, runout distance and area depends on debris flow composition and rheology, but how is poorly understood. We experimentally investigated effects of composition on debris flow runout, depositional mechanisms, and deposit geometry. The small-scale experimental debris flows were largely similar to natural debris flows in terms of flow behavior, deposit morphology, grain size sorting, channel width-depth ratio, and runout. Deposit geometry (lobe thickness and width) in our experimental debris flows is largely determined by composition, while the effects of initial conditions of topography (i.e., outflow plain slope and channel slope and width) and volume are negligible. We find a clear optimum in the relations of runout with coarse-material fraction and clay fraction. Increasing coarse-material concentration leads to larger runout. However, excess coarse material results in a large accumulation of coarse debris at the flow front and enhances diffusivity, increasing frontal friction and decreasing runout. Increasing clay content initially enhances runout, but too much clay leads to very viscous flows, reducing runout. Runout increases with channel slope and width, outflow plain slope, debris flow volume, and water fraction. These results imply that debris flow runout depends at least as much on composition as on topography. This study improves understanding of the effects of debris flow composition on runout and may aid future debris flow hazard assessments.

Experimental investigation of turbulent flow in smooth and longitudinal grooved tubes

NASA Technical Reports Server (NTRS)

Nitschke, P.

1984-01-01

Turbulent flow in tubes with and without longitudinal grooves is examined. The discovery of fine grooves forming a sort of streamline pattern on the body of sharks led to the expectation that the grooves on a surface reduce the momentum change, and thus the drag. To test this thesis, drag law, velocity profile and the profile of the velocity fluctuation were determined. Results show that for moderate Reynolds numbers the drag coefficient for grooved tubes is about 3 percent smaller than that of the smooth tubes. At higher Reynolds numbers, however, the drag coefficient for grooved tubes becomes larger than that for smooth tubes. No significant differences in the velocity profiles between grooved tubes and smooth tubes are found.

Extrinsic and Intrinsic Apoptotic Responses Induced by Shiitake Culinary-Medicinal Mushroom Lentinus edodes (Agaricomycetes) Aqueous Extract against a Larynx Carcinoma Cell Line.

PubMed

Finimundy, Tiane C; Scola, Gustavo; Scariot, Fernando J; Dillon, Aldo J P; Moura, Sidnei; Echeverrigaray, Sérgio; Henriques, João Pegas; Roesch-Ely, Mariana

2018-01-01

Cumulative evidence from research studies has shown that the shiitake culinary-medicinal mushroom, Lentinus edodes, is an excellent source of natural antitumor agents and is capable of inhibiting cancer cell growth. However, the cell signaling pathway that leads tumor cells to apoptosis is not well understood because many chemical compounds may be acting. This study investigated the chemopreventive effects of an L. edodes aqueous extract on human HEp-2 epithelial larynx carcinoma cells and normal human MRC-5 lung fibroblasts by identifying proliferative and apoptotic pathways. The chemical characterization of the dry powder was assessed by high-performance liquid chromatography. Antiproliferative and proapoptotic effects induced by the extract were evaluated by assessing proliferative markers, cell sorting through flow cytometry, and expression levels of apoptotic proteins with Western blotting. The results suggest that inhibition of cell proliferation was more prominent in HEp-2 than in MRC-5 cells. Cell death analysis showed the appearance of cell populations in the sub-G1 phase, with late apoptotic signal increased in a dose-dependent manner. In addition, the aqueous extract induced depolarization of mitochondria, activating the generation of intracellular reactive oxygen species in HEp-2 cells. These observations suggest that L. edodes extract may exert a chemopreventive effect, regulating mitotic induction of apoptogenic signals. These findings highlight the mushroom's pharmacological potential in cancer treatment.

Parallel integer sorting with medium and fine-scale parallelism

NASA Technical Reports Server (NTRS)

Dagum, Leonardo

1993-01-01

Two new parallel integer sorting algorithms, queue-sort and barrel-sort, are presented and analyzed in detail. These algorithms do not have optimal parallel complexity, yet they show very good performance in practice. Queue-sort designed for fine-scale parallel architectures which allow the queueing of multiple messages to the same destination. Barrel-sort is designed for medium-scale parallel architectures with a high message passing overhead. The performance results from the implementation of queue-sort on a Connection Machine CM-2 and barrel-sort on a 128 processor iPSC/860 are given. The two implementations are found to be comparable in performance but not as good as a fully vectorized bucket sort on the Cray YMP.

Sort computation

NASA Technical Reports Server (NTRS)

Dorband, John E.

1988-01-01

Sorting has long been used to organize data in preparation for further computation, but sort computation allows some types of computation to be performed during the sort. Sort aggregation and sort distribution are the two basic forms of sort computation. Sort aggregation generates an accumulative or aggregate result for each group of records and places this result in one of the records. An aggregate operation can be any operation that is both associative and commutative, i.e., any operation whose result does not depend on the order of the operands or the order in which the operations are performed. Sort distribution copies the value from a field of a specific record in a group into that field in every record of that group.

The Neuroergonomics of Vigilance: Effects of Spatial Uncertainty on Cerebral Blood Flow Velocity and Oculomotor Fatigue

DTIC Science & Technology

2017-02-01

in studies of this sort. One goal for the present study was to test the expectation, based on resource theory, that because of the higher...VDT was mounted on a table 99.10 cm directly in front of the seated observer (visual angle = 23.54°). Ambient illumination in the testing room (5...minimize glare on the VDT. To curb distraction, observers were separated from the TCD equipment by a cubicle wall dividing the width of the testing

Derivation of sorting programs

NASA Technical Reports Server (NTRS)

Varghese, Joseph; Loganantharaj, Rasiah

1990-01-01

Program synthesis for critical applications has become a viable alternative to program verification. Nested resolution and its extension are used to synthesize a set of sorting programs from their first order logic specifications. A set of sorting programs, such as, naive sort, merge sort, and insertion sort, were successfully synthesized starting from the same set of specifications.

Fusion with human lung cancer cells elongates the life span of human umbilical endothelial cells and enhances the anti-tumor immunity.

PubMed

Mu, Xiyan; Fang, Chunju; Zhou, Jing; Xi, Yufeng; Zhang, Li; Wei, Yuquan; Yi, Tao; Wu, Yang; Zhao, Xia

2016-01-01

Human umbilical endothelial cells (HUVECs) have been proved as an effective whole-cell vaccine inhibiting tumor angiogenesis. However, HUVECs divide a very limited number of passages before entering replicative senescence, which limits its application for clinical situation. Here, we fused HUVECs with human pulmonary adenocarcinoma cell line A549s and investigated the anti-tumor immunity of the hybrids against mice Lewis lung cancer. HUVECs were fused with A549s using polyethylene glycol and were sorted by flow cytometry. The fusion cells (HUVEC-A549s) were confirmed by testing the expression of telomerase and VE-cadherin, the senescence-associated β-galactosidase activity, and tube formation ability. HUVEC-A549s were then irradiated and injected into the C57BL/6 mice of protective, therapeutic, and metastatic models. The mechanism of the anti-tumor immunity was explored by analyzing mice sera, spleen T lymphocytes, tumor microenvironment, and histological changes. HUVEC-A549s coexpressed tumor and endothelial markers and maintained the vascular function of tube forming at passage 30 without showing signs of senescence. HUVEC-A549s could induce protective and therapeutic anti-tumor activity for LL(2) model and presented stronger activity against metastasis than HUVECs. Both humoral and cellular immunity were participated in the anti-angiogenic activity, as HUVECs-neutralizing IgG and HUVECs-toxic lymphocytes were increased. Angiogenic mediators (VEGF and TGF-β) and tumor microenvironment cells MDSCs and Tregs were also diminished. Our findings might provide a novel strategy for HUVECs-related immunotherapy, and this vaccine requires lower culture condition than primary HUVECs while enhancing the anti-tumor immunity.

Engineering of Bispecific Affinity Proteins with High Affinity for ERBB2 and Adaptable Binding to Albumin

PubMed Central

Nilvebrant, Johan; Åstrand, Mikael; Georgieva-Kotseva, Maria; Björnmalm, Mattias; Löfblom, John; Hober, Sophia

2014-01-01

The epidermal growth factor receptor 2, ERBB2, is a well-validated target for cancer diagnostics and therapy. Recent studies suggest that the over-expression of this receptor in various cancers might also be exploited for antibody-based payload delivery, e.g. antibody drug conjugates. In such strategies, the full-length antibody format is probably not required for therapeutic effect and smaller tumor-specific affinity proteins might be an alternative. However, small proteins and peptides generally suffer from fast excretion through the kidneys, and thereby require frequent administration in order to maintain a therapeutic concentration. In an attempt aimed at combining ERBB2-targeting with antibody-like pharmacokinetic properties in a small protein format, we have engineered bispecific ERBB2-binding proteins that are based on a small albumin-binding domain. Phage display selection against ERBB2 was used for identification of a lead candidate, followed by affinity maturation using second-generation libraries. Cell surface display and flow-cytometric sorting allowed stringent selection of top candidates from pools pre-enriched by phage display. Several affinity-matured molecules were shown to bind human ERBB2 with sub-nanomolar affinity while retaining the interaction with human serum albumin. Moreover, parallel selections against ERBB2 in the presence of human serum albumin identified several amino acid substitutions that dramatically modulate the albumin affinity, which could provide a convenient means to control the pharmacokinetics. The new affinity proteins competed for ERBB2-binding with the monoclonal antibody trastuzumab and recognized the native receptor on a human cancer cell line. Hence, high affinity tumor targeting and tunable albumin binding were combined in one small adaptable protein. PMID:25089830

Increased choline uptake in macrophages and prostate cancer cells does not allow for differentiation between benign and malignant prostate pathologies.

PubMed

Schwarz, Timo; Seidl, Christof; Schiemann, Matthias; Senekowitsch-Schmidtke, Reingard; Krause, Bernd Joachim

2016-06-01

Inflammatory cells may contribute to the choline uptake in different prostate pathologies. The aim of this study was (i) to assess if inflammatory cells incorporate choline and (ii) to potentially detect differences compared to FDG uptake. Therefore we investigated the uptake of [(3)H]choline and [(18)F]FDG in human prostate carcinoma cells and human inflammatory cells. Macrophages were cultured from isolated mononuclear cells, gained by density gradient centrifugation of human buffy coats. T-lymphocytes, B-lymphocytes and granulocytes were enriched by density gradient centrifugation before cell sorting by means of flow cytometry was performed. [(3)H]choline and [(18)F]FDG uptake of isolated inflammatory cells as well as of LNCaP and PC-3 human prostate carcinoma cells was assessed simultaneously in dual tracer uptake experiments. Macrophages showed highest [(3)H]choline and [(18)F]FDG uptake compared to the tracer uptake rates of leukocytes. [(3)H]choline uptake of macrophages was in the same range as in prostate cancer cells. Lipopolysaccharide stimulation of macrophages resulted in an increase of [(18)F]FDG uptake in macrophages, but not in an increased [(3)H]choline uptake. The high [(3)H]choline uptake in macrophages may be a source of false-positive PET results in diagnosis of prostate cancer by choline-PET/CT. As already known from FDG-PET, discrimination between tumor and inflammation in prostate cancer patients is not possible via choline-PET. The application of choline-PET for reliable primary prostate cancer detection and delineation has to be queried. Copyright © 2016 Elsevier Inc. All rights reserved.

CD146 positive human dental pulp stem cells promote regeneration of dentin/pulp-like structures.

PubMed

Matsui, Mikiko; Kobayashi, Tomoko; Tsutsui, Takeo W

2018-04-01

CD146 and STRO-1 are endothelial biomarkers that are co-expressed on the cellular membranes of blood vessels within human dental pulp tissue. This study characterized the percentage of dentin-like structures produced by CD146-positive (CD146 + ) human dental pulp stem cells (DPSCs), compared with their CD146-negative (CD146 - ) counterparts. DPSC populations were enriched using magnetic-activated cell sorting (MACS), yielding CD146 + and CD146 - cells, as well as mixtures composed of 25% CD146 + cells and 75% CD146 - cells (CD146 +/- ). Cell growth assays indicated that CD146 + cells exhibit an approximate 3-4 h difference in doubling time, compared with CD146 - cells. Cell cycle distributions were determined by flow cytometry analysis. The low percentage of CD146 + cells' DNA content in G 0 /G 1 phase were compared with CD146 - and non-separated cells. In contrast to CD146 - and non-separated cells, prompt mineralization was observed in CD146 + cells. Subsequently, qRT-PCR revealed high mRNA expression of CD146 and Alkaline phosphatase in mineralization-induced CD146 + cells. CD146 + cells were also observed high adipogenic ability by Oil red O staining. Histological examinations revealed an increased area of dentin/pulp-like structures in transplanted CD146 + cells, compared with CD146 - and CD146 +/- cells. Immunohistochemical studies detected dentin matrix protein-1 (DMP1) and dentin sialophosphoprotein (DSPP), as well as human mitochondria, in transplanted DPSCs. Co-expression of CD146 and GFP indicated that CD146 was expressed in transplanted CD146 + cells. CD146 + cells may promote mineralization and generate dentin/pulp-like structures, suggesting a role in self-renewal of stem cells and dental pulp regenerative therapy.

Spin-the-bottle Sort and Annealing Sort: Oblivious Sorting via Round-robin Random Comparisons

PubMed Central

Goodrich, Michael T.

2013-01-01

We study sorting algorithms based on randomized round-robin comparisons. Specifically, we study Spin-the-bottle sort, where comparisons are unrestricted, and Annealing sort, where comparisons are restricted to a distance bounded by a temperature parameter. Both algorithms are simple, randomized, data-oblivious sorting algorithms, which are useful in privacy-preserving computations, but, as we show, Annealing sort is much more efficient. We show that there is an input permutation that causes Spin-the-bottle sort to require Ω(n2 log n) expected time in order to succeed, and that in O(n2 log n) time this algorithm succeeds with high probability for any input. We also show there is a specification of Annealing sort that runs in O(n log n) time and succeeds with very high probability. PMID:24550575

In-Line Sorting of Harumanis Mango Based on External Quality Using Visible Imaging

PubMed Central

Ibrahim, Mohd Firdaus; Ahmad Sa’ad, Fathinul Syahir; Zakaria, Ammar; Md Shakaff, Ali Yeon

2016-01-01

The conventional method of grading Harumanis mango is time-consuming, costly and affected by human bias. In this research, an in-line system was developed to classify Harumanis mango using computer vision. The system was able to identify the irregularity of mango shape and its estimated mass. A group of images of mangoes of different size and shape was used as database set. Some important features such as length, height, centroid and parameter were extracted from each image. Fourier descriptor and size-shape parameters were used to describe the mango shape while the disk method was used to estimate the mass of the mango. Four features have been selected by stepwise discriminant analysis which was effective in sorting regular and misshapen mango. The volume from water displacement method was compared with the volume estimated by image processing using paired t-test and Bland-Altman method. The result between both measurements was not significantly different (P > 0.05). The average correct classification for shape classification was 98% for a training set composed of 180 mangoes. The data was validated with another testing set consist of 140 mangoes which have the success rate of 92%. The same set was used for evaluating the performance of mass estimation. The average success rate of the classification for grading based on its mass was 94%. The results indicate that the in-line sorting system using machine vision has a great potential in automatic fruit sorting according to its shape and mass. PMID:27801799

Tracking heavy water (D2O) incorporation for identifying and sorting active microbial cells

PubMed Central

Berry, David; Mader, Esther; Lee, Tae Kwon; Woebken, Dagmar; Wang, Yun; Zhu, Di; Palatinszky, Marton; Schintlmeister, Arno; Schmid, Markus C.; Hanson, Buck T.; Shterzer, Naama; Mizrahi, Itzhak; Rauch, Isabella; Decker, Thomas; Bocklitz, Thomas; Popp, Jürgen; Gibson, Christopher M.; Fowler, Patrick W.; Huang, Wei E.; Wagner, Michael

2015-01-01

Microbial communities are essential to the function of virtually all ecosystems and eukaryotes, including humans. However, it is still a major challenge to identify microbial cells active under natural conditions in complex systems. In this study, we developed a new method to identify and sort active microbes on the single-cell level in complex samples using stable isotope probing with heavy water (D2O) combined with Raman microspectroscopy. Incorporation of D2O-derived D into the biomass of autotrophic and heterotrophic bacteria and archaea could be unambiguously detected via C-D signature peaks in single-cell Raman spectra, and the obtained labeling pattern was confirmed by nanoscale-resolution secondary ion MS. In fast-growing Escherichia coli cells, label detection was already possible after 20 min. For functional analyses of microbial communities, the detection of D incorporation from D2O in individual microbial cells via Raman microspectroscopy can be directly combined with FISH for the identification of active microbes. Applying this approach to mouse cecal microbiota revealed that the host-compound foragers Akkermansia muciniphila and Bacteroides acidifaciens exhibited distinctive response patterns to amendments of mucin and sugars. By Raman-based cell sorting of active (deuterated) cells with optical tweezers and subsequent multiple displacement amplification and DNA sequencing, novel cecal microbes stimulated by mucin and/or glucosamine were identified, demonstrating the potential of the nondestructive D2O-Raman approach for targeted sorting of microbial cells with defined functional properties for single-cell genomics. PMID:25550518

In-Line Sorting of Harumanis Mango Based on External Quality Using Visible Imaging.

PubMed

Ibrahim, Mohd Firdaus; Ahmad Sa'ad, Fathinul Syahir; Zakaria, Ammar; Md Shakaff, Ali Yeon

2016-10-27

The conventional method of grading Harumanis mango is time-consuming, costly and affected by human bias. In this research, an in-line system was developed to classify Harumanis mango using computer vision. The system was able to identify the irregularity of mango shape and its estimated mass. A group of images of mangoes of different size and shape was used as database set. Some important features such as length, height, centroid and parameter were extracted from each image. Fourier descriptor and size-shape parameters were used to describe the mango shape while the disk method was used to estimate the mass of the mango. Four features have been selected by stepwise discriminant analysis which was effective in sorting regular and misshapen mango. The volume from water displacement method was compared with the volume estimated by image processing using paired t -test and Bland-Altman method. The result between both measurements was not significantly different (P > 0.05). The average correct classification for shape classification was 98% for a training set composed of 180 mangoes. The data was validated with another testing set consist of 140 mangoes which have the success rate of 92%. The same set was used for evaluating the performance of mass estimation. The average success rate of the classification for grading based on its mass was 94%. The results indicate that the in-line sorting system using machine vision has a great potential in automatic fruit sorting according to its shape and mass.

Massive Open Online Courses for Africa by Africa

ERIC Educational Resources Information Center

Oyo, Benedict; Kalema, Billy Mathias

2014-01-01

Africa is known for inadequate access to all sorts of human needs including health, education, food, shelter, transport, security, and energy. Before the emergence of massive open online courses (MOOCs), open access to higher education (HE) was exclusive of Africa. However, as a generally affordable method of post-secondary education delivery,…

Montaigne, Nietzsche, and the Mnemotechnics of Student Agency

ERIC Educational Resources Information Center

Bingham, Charles

2007-01-01

This essay explores the educational implications of the thought of Michel de Montaigne and Friedrich Nietzsche on the subject of memory. It explores the sorts of cultural memory practices that Nietzsche has called "mnemotechnics", that is, the aspects of memory use that allow human beings to live life more fully. Nietzsche and Montaigne's work is…

Designing Realistic Human Behavior into Multi-Agent Systems

DTIC Science & Technology

2001-09-01

different results based on some sort of randomness built into it, a trend can be looked at over time and a success or failure rate can be...simulation remains in that state, very different results can be achieved each simulation run. An analyst can look at success and failure over a long

Managerial Effectiveness in Educational Administration: Concepts and Perspectives

ERIC Educational Resources Information Center

Farahbakhsh, Saeid

2007-01-01

This is the age of leadership and management. Every group activity requires some sort of leadership to organise and direct the group's effort toward some previously decided goals. Although management and leadership are different from each other, they are remarkably close too, especially in the dimension of human relationship. Leadership is the…

Electronic noses and tongues: Applications for the food and pharmaceutical industries

USDA-ARS?s Scientific Manuscript database

The electronic nose (enose) is designed to crudely mimic the human brain in that most contain sensors that non-selectively interact with odor molecules to produce some sort of signal that is then sent to a computer that uses multivariate statistics to determine patterns in the data. This pattern rec...

Cellular uptake and trafficking of polydiacetylene micelles

NASA Astrophysics Data System (ADS)

Gravel, Edmond; Thézé, Benoit; Jacques, Isabelle; Anilkumar, Parambath; Gombert, Karine; Ducongé, Frédéric; Doris, Eric

2013-02-01

Polydiacetylene (PDA) micelles coated with either carboxylate-, ammonium-, or methoxy-polyethyleneglycol (PEG) chains were assembled and loaded with a fluorescent dye (DiO). Their interaction with MCF-7 human breast tumor cells was investigated by epi-fluorescence microscopy and fluorescence-activated cell sorting (FACS) to determine their internalization pathway and intracellular fate. It was found that the ionic character of the micelles influenced their internalization kinetics through a caveolae-mediated pathway and that all micelle types behaved somewhat similarly inside cells.Polydiacetylene (PDA) micelles coated with either carboxylate-, ammonium-, or methoxy-polyethyleneglycol (PEG) chains were assembled and loaded with a fluorescent dye (DiO). Their interaction with MCF-7 human breast tumor cells was investigated by epi-fluorescence microscopy and fluorescence-activated cell sorting (FACS) to determine their internalization pathway and intracellular fate. It was found that the ionic character of the micelles influenced their internalization kinetics through a caveolae-mediated pathway and that all micelle types behaved somewhat similarly inside cells. Electronic supplementary information (ESI) available: Detailed synthetic procedures and supplementary figures. See DOI: 10.1039/c2nr34149b