Impact of pre-transplantation minimal residual disease determined by multiparameter flow cytometry on the outcome of AML patients with FLT3-ITD after allogeneic stem cell transplantation.

PubMed

Zhao, Xiaosu; Wang, Zhidong; Ruan, Guorui; Liu, Yanrong; Wang, Yu; Zhang, Xiaohui; Xu, Lanping; Huang, Xiaojun; Chang, Yingjun

2018-06-01

In this study, using multiparameter flow cytometry (FCM), we investigate the impact of minimal residual disease prior to transplantation (pre-MRD) on the transplant outcomes of AML patients with fms-related tyrosine kinase 3 (FLT3)-internal tandem duplication (ITD) mutation. A total of 20 patients who received HLA-matched sibling donor transplantation (MSDT) and 63 patients who received unmanipulated haploidentical hematopoietic stem cell transplantation (haplo-HSCT) were enrolled. Patients were classified into four groups based on the status of pre-FCM: group 1 with positive pre-FCM before MSDT, group 2 with negative pre-FCM before MSDT, group 3 with positive pre-FCM before haplo-HSCT, and group 4 with positive pre-FCM before haplo-HSCT. The results showed that patients in group 1 had the highest cumulative incidence of relapse (2-year CIR, 75.0%), the lowest leukemia-free survival (2-year LFS, 33.3%), and the overall survival (2-year OS, 25.0%) among all four groups. The other three groups of patients had comparable CIR (2-year CIR: group 2 vs. 3 vs. 4, 12.5% vs. 31.3% vs. 22.2%, P > 0.05) and LFS (2-year LFS: group 2 vs. 3 vs. 4, 87.5% vs. 62.5% vs. 66.5%, P > 0.05). Multivariate analysis indicated that disease status (> CR) and pre-MRD were associated with a higher CIR and a lower LFS when patients were classified by pre-MRD and transplant type. Our results suggested that AML patients with FLT3-ITD were able to be separated into high-risk and low-risk relapse groups based on pre-MRD, as determined by multiparameter FCM. Haplo-HSCT might overcome the negative impact of pre-MRD on patient outcomes compared to MSDT. These results require further investigation in prospective study with large numbers of cases.

Application of cryopreservation to genetic analyses of a photosynthetic picoeukaryote community.

PubMed

Kawachi, Masanobu; Kataoka, Takafumi; Sato, Mayumi; Noël, Mary-Hélène; Kuwata, Akira; Demura, Mikihide; Yamaguchi, Haruyo

2016-02-01

Cryopreservation is useful for long-term maintenance of living strains in microbial culture collections. We applied this technique to environmental specimens from two monitoring sites at Sendai Bay, Japan and compared the microbial diversity of photosynthetic picoeukaryotes in samples before and after cryopreservation. Flow cytometry (FCM) showed no considerable differences between specimens. We used 2500 cells sorted with FCM for next-generation sequencing of 18S rRNA gene amplicons and after removing low-quality sequences obtained 10,088-37,454 reads. Cluster analysis and comparative correlation analysis of observed high-level operational taxonomic units indicated similarity between specimens before and after cryopreservation. The effects of cryopreservation on cells were assessed with representative culture strains, including fragile cryptophyte cells. We confirmed the usefulness of cryopreservation for genetic studies on environmental specimens, and found that small changes in FCM cytograms after cryopreservation may affect biodiversity estimation. Copyright © 2015 Elsevier B.V. All rights reserved.

Screening of carcinoma metastasis by flow cytometry: A study of 238 cases.

PubMed

Acosta, Maria; Pereira, José; Arroz, Maria

2016-05-01

Malignant epithelial cells may be detected in different specimens, by immunophenotyping using flow cytometry (FCM). CD326 (epithelial-specific antigen, clone Ber-Ep4) was used to identify epithelial cells, CD45 to discriminate between leucocytes (positive for this antigen) and non-hematological cells (negative for this antigen), and CD33 to identify monocytes/macrophages. This combination is particularly useful in effusions to characterize large cells and distinguish between monocyte/macrophages (CD45+ CD33+ CD326-), mesothelial cells (CD45 ± (dim) CD33 - CD326-) and epithelial cells (CD45 - CD33 - CD326 +). We evaluated the efficiency of flow cytometry to detect malignant epithelial cells in 238 fresh samples, including effusions, lymph node biopsies, fine needle aspirates, bone marrow aspirates, cerebrospinal fluid, among others. These are specimens expected to lack epithelial cells. FCM results were then compared to the results of smear and cell block morphology, as well as immunocytochemistry on paraffin wax embedded cell blocks, when available. Final diagnosis was the gold standard and a very good sensitivity (96.7%) and specificity (99.3%) were obtained. We concluded that the detection of CD326 positive cells using FCM is strongly indicative of the presence of carcinoma cells. © 2015 International Clinical Cytometry Society. © 2015 International Clinical Cytometry Society.

Cell-production rates estimated by the use of vincristine sulphate and flow cytometry. II. Correlation between the cell-production rates of ageing ascites tumours and the number of S phase tumour cells.

PubMed

Barfod, I H; Barfod, N M

1980-01-01

A new method for the evaluation of cell production rates combining flow cytometry (FCM) and the stathmokinetic method using vincristine sulphate (VS) has been used for the analysis of three aneuploid ascites tumours at different stages of growth. Using this technique it was possible to estimate the well-known decrease in cell production rates of ageing ascites tumours. The percentage of normal host cells in the aneuploid tumours studied was easily determined by FCM prior to the calculation of the tumour cell-production rates. A correlation was found between the percentage of tumour cells in the S phase and the tumour cell-production rate. This correlation is probably explained by the gradual transfer of proliferating cells in S phase to resting G1 and G2 phases with increasing tumour age.

Flow Cytometry Method Analysis of Apoptosis: No Significant Difference Between EDTA and EDTA-free Trypsin Treatment Procedure.

PubMed

Xu, Xiao-yan; Nie, Xiao-cui; Ma, Hai-ying; Song, Guo-qing; Zhang, Xiao-tong; Jin, Yu-nan; Yu, Yan-qiu

2015-04-01

Flow cytometry method (FCM) is a generally accepted tool to analyze apoptosis. Although apoptosis assay kit was applied by many companies, the manufacturers were not consistent with whether using Trypsin with EDTA to collect the adherent cells. In another words, the influence of EDTA on apoptotic ratio is not clear. In this work, we compared the proportion of apoptotic cells with EDTA or EDTA-free Trypsin treatment by FCM. We concluded that Trypsin with or without EDTA has little influence on the proportion of apoptotic cells. In addition, we found that the ratio of necrosis and apoptosis was different in cells collected by scraping. WAVE2 protein was analyzed as a typical example for movement related protein. WAVE2 expression is elevated in the EDTA Trypsin treated group, compared with EDTA-free Trypsin treatment and scrapping group. © The Author(s) 2014.

Diagnostic Flow Cytometry and the AIDS Pandemic.

PubMed

Clift, Ian C

2015-01-01

The onset of the AIDS pandemic in the early 1980s coincided with the convergence of technologies now collectively known as flow cytometry (FCM). Major advances in FCM led significantly toward our understanding of the pathogenicity of the disease, which in turn led to wider adoption of the technology, including using it effectively in a variety of diagnostics. CD4+ T lymphocyte population counts, along with human immunodeficiency virus (HIV) viral load, remain the gold standard in diagnosis and continue to play a major role in the monitoring of advanced retroviral therapies. Arguably, the spread of AIDS (acquired immunodeficiency syndrome), the HIV virus, and the toll of the virus on humanity have been considerably altered by the concurrent development of FCM, the details of which are presented herein. Copyright© by the American Society for Clinical Pathology (ASCP).

Features of free software packages in flow cytometry: a comparison between four non-commercial software sources.

PubMed

Sahraneshin Samani, Fazel; Moore, Jodene K; Khosravani, Pardis; Ebrahimi, Marzieh

2014-08-01

Flow cytometers designed to analyze large particles are enabling new applications in biology. Data analysis is a critical component of the process FCM. In this article we compare features of four free software packages including WinMDI, Cyflogic, Flowing software, and Cytobank.

Modifications of haematology analyzers to improve cell counting and leukocyte differentiating in cerebrospinal fluid controls of the Joint German Society for Clinical Chemistry and Laboratory Medicine.

PubMed

Kleine, Tilmann O; Nebe, C Thomas; Löwer, Christa; Lehmitz, Reinhard; Kruse, Rolf; Geilenkeuser, Wolf-Jochen; Dorn-Beineke, Alexandra

2009-08-01

Flow cytometry (FCM) is used with haematology analyzers (HAs) to count cells and differentiate leukocytes in cerebrospinal fluid (CSF). To evaluate the FCM techniques of HAs, 10 external DGKL trials with CSF controls were carried out in 2004 to 2008. Eight single platform HAs with and without CSF equipment were evaluated with living blood leukocytes and erythrocytes in CSF like DGKL controls: Coulter (LH750,755), Abbott CD3200, CD3500, CD3700, CD4000, Sapphire, ADVIA 120(R) CSF assay, and Sysmex XE-2100(R). Results were compared with visual counting of native cells in Fuchs-Rosenthal chamber, unstained, and absolute values of leukocyte differentiation, assayed by dual platform analysis with immune-FCM (FACSCalibur, CD45, CD14) and the chamber counts. Reference values X were compared with HA values Y by statistical evaluation with Passing/Bablock (P/B) linear regression analysis to reveal conformity of both methods. The HAs, studied, produced no valid results with DGKL CSF controls, because P/B regression revealed no conformity with the reference values due to:-blank problems with impedance analysis,-leukocyte loss with preanalytical erythrocyte lysis procedures, especially of monocytes,-inaccurate results with ADVIA cell sphering and cell differentiation with algorithms and enzyme activities (e.g., peroxidase). HA techniques have to be improved, e.g., using no erythrocyte lysis and CSF adequate techniques, to examine CSF samples precise and accurate. Copyright 2009 International Society for Advancement of Cytometry.

flowAI: automatic and interactive anomaly discerning tools for flow cytometry data.

PubMed

Monaco, Gianni; Chen, Hao; Poidinger, Michael; Chen, Jinmiao; de Magalhães, João Pedro; Larbi, Anis

2016-08-15

Flow cytometry (FCM) is widely used in both clinical and basic research to characterize cell phenotypes and functions. The latest FCM instruments analyze up to 20 markers of individual cells, producing high-dimensional data. This requires the use of the latest clustering and dimensionality reduction techniques to automatically segregate cell sub-populations in an unbiased manner. However, automated analyses may lead to false discoveries due to inter-sample differences in quality and properties. We present an R package, flowAI, containing two methods to clean FCM files from unwanted events: (i) an automatic method that adopts algorithms for the detection of anomalies and (ii) an interactive method with a graphical user interface implemented into an R shiny application. The general approach behind the two methods consists of three key steps to check and remove suspected anomalies that derive from (i) abrupt changes in the flow rate, (ii) instability of signal acquisition and (iii) outliers in the lower limit and margin events in the upper limit of the dynamic range. For each file analyzed our software generates a summary of the quality assessment from the aforementioned steps. The software presented is an intuitive solution seeking to improve the results not only of manual but also and in particular of automatic analysis on FCM data. R source code available through Bioconductor: http://bioconductor.org/packages/flowAI/ CONTACTS: mongianni1@gmail.com or Anis_Larbi@immunol.a-star.edu.sg Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Time-gated flow cytometry: an ultra-high selectivity method to recover ultra-rare-event μ-targets in high-background biosamples

NASA Astrophysics Data System (ADS)

Jin, Dayong; Piper, James A.; Leif, Robert C.; Yang, Sean; Ferrari, Belinda C.; Yuan, Jingli; Wang, Guilan; Vallarino, Lidia M.; Williams, John W.

2009-03-01

A fundamental problem for rare-event cell analysis is auto-fluorescence from nontarget particles and cells. Time-gated flow cytometry is based on the temporal-domain discrimination of long-lifetime (>1 μs) luminescence-stained cells and can render invisible all nontarget cell and particles. We aim to further evaluate the technique, focusing on detection of ultra-rare-event 5-μm calibration beads in environmental water dirt samples. Europium-labeled 5-μm calibration beads with improved luminescence homogeneity and reduced aggregation were evaluated using the prototype UV LED excited time-gated luminescence (TGL) flow cytometer (FCM). A BD FACSAria flow cytometer was used to sort accurately a very low number of beads (<100 events), which were then spiked into concentrated samples of environmental water. The use of europium-labeled beads permitted the demonstration of specific detection rates of 100%+/-30% and 91%+/-3% with 10 and 100 target beads, respectively, that were mixed with over one million nontarget autofluorescent background particles. Under the same conditions, a conventional FCM was unable to recover rare-event fluorescein isothiocyanate (FITC) calibration beads. Preliminary results on Giardia detection are also reported. We have demonstrated the scientific value of lanthanide-complex biolabels in flow cytometry. This approach may augment the current method that uses multifluorescence-channel flow cytometry gating.

Cell-production rates estimated by the use of vincristine sulphate and flow cytometry. I. An in vitro study using murine tumour cell lines.

PubMed

Barfod, I H; Barfod, N M

1980-01-01

A method for the evaluation of cell-production rates is described which combines flow cytometry (FCM) and the stathmokinetic method. By means of FCM it is possible to estimate the distribution of cells with G1, S and (G2 + M) DNA content in a population. As this method gives the relative (G2 + M) DNA content of cells within the cell cycle, it may be possible to evaluate cell-production rates by this technique. In the present study it was found that administration of a metaphase-arresting (stathmokinetic) agent, vincristine sulphate (VS), to asynchronous cell populations of three different murine tumour cell lines in vitro increased the peak representing cells with (G2 + M) DNA content as the number of mitotic (M) cells increased during the period of treatment. The accumulation of mitotic cells was determined by cell counts on smears under the microscope and compared with increase in the (G2 + M) DNA peak measured by FCM as a function of time after the administration of VS. Good agreement was obtained between the cell-production rates as estimated by FCM and by mitotic counts in all three cell lines investigated.

Comparison between Flow Cytometry and Traditional Culture Methods for Efficacy Assessment of Six Disinfectant Agents against Nosocomial Bacterial Species

PubMed Central

Massicotte, Richard; Mafu, Akier A.; Ahmad, Darakhshan; Deshaies, Francis; Pichette, Gilbert; Belhumeur, Pierre

2017-01-01

The present study was undertaken to compare the use of flow cytometry (FCM) and traditional culture methods for efficacy assessment of six disinfectants used in Quebec hospitals including: two quaternary ammonium-based, two activated hydrogen peroxide-based, one phenol-based, and one sodium hypochlorite-based. Four nosocomial bacterial species, Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Vancomycin-resistant Enterococci faecalis, were exposed to minimum lethal concentrations (MLCs) and sublethal concentrations (1/2 MLCs) of disinfectants under study. The results showed a strong correlation between the two techniques for the presence of dead and live cell populations, as well as, evidence of injured populations with the FCM. The only exception was observed with sodium hypochlorite at higher concentrations where fluorescence was diminished and underestimating dead cell population. The results also showed that FCM can replace traditional microbiological methods to study disinfectant efficacy on bacteria. Furthermore, FCM profiles for E. coli and E. faecalis cells exposed to sublethal concentrations exhibited distinct populations of injured cells, opening a new aspect for future research and investigation to elucidate the role of injured, cultural/noncuturable/resuscitable cell populations in infection control. PMID:28217115

Detection of activated basophils using flow cytometry for diagnosis in atopic patients.

PubMed

Cozon, G; Ferrándiz, J; Peyramond, D; Brunet, J

1999-01-01

human basophils release mediators of allergy after cross-linking of IgE receptors by allergens. Specific activation of basophils is detectable through flow cytometry (FCM) using an anti-CD63 fluorescein-conjugated monoclonal antibody. this study evaluate the detection of activated basophils by FCM in routine diagnosis of atopic diseases as regard to skin prick tests and specific immunoglobulin E antibodies. whole blood from twenty patients suspected of atopy was preincubated with interleukin-3 (IL-3), then incubated with specific allergens. After staining using anti-CD63 antibodies, activated basophils were detected through FCM. IL-3-preincubation increases the spontaneous expression of CD63 even at low concentrations (0.1 ng/ml) on the basophils of 2 patients out of 20. The sensitivity and specificity of FCM were respectively 0.56 +/- 0.17 (m +/- SD) and 1.0 +/- 0.0 for the detection of dust mite-activated basophils without IL-3 preincubation, and 0.73 +/- 0.13 and 1.0 +/- 0.0 for the detention of grass pollen-activated basophils. IL-3-preincubation increased the sensitivity in a dose-dependent manner but decreased the specificity fo FCM for detecting dust mite hypersensitivity. this method allow for rapid and easy detection of activated basophils from whole blood, and could be of interest for detecting allergies to non-conventional allergens such as pharmaceutical drugs.

Correlation between ploidy status using flow cytometry and nucleolar organizer regions in benign and malignant epithelial odontogenic tumors.

PubMed

Mohamed Mahmoud, Sarah Ahmed; El-Rouby, Dalia Hussein; El-Ghani, Safa Fathy Abd; Badawy, Omnia Mohamed

2017-06-01

Differentiation between the aggressive benign odontogenic tumors and their malignant counterparts is controversial and difficult. While flow cytometry (FCM) allowed DNA analysis in neoplasia, argyrophilic organizer regions (AgNORs) number and/or size in a nucleus are correlated with the ribosomal gene activity and therefore with cellular proliferation. The aim of this research was to study the diagnostic accuracy of FCM and AgNORs staining in differentiating between benign and malignant epithelial odontogenic tumors and to correlate between these two interventions. Sixteen benign cases [8 cases of ameloblastoma (AB) and 8 cases of keratocystic odontogenic tumor (KCOT)] and 13 malignant epithelial odontogenic tumors [8 cases of ameloblastic carcinoma (ABC) and 5 cases of clear cell odontogenic carcinoma(CCOC)] were included in the current study. For FCM analysis, a single cell suspension from Formalin fixed paraffin-embedded (FFPE) tumors was prepared according to a modified method described by Hedley (1989) and AgNORs staining were performed in accordance to the Ploton protocol (1986). Analysis of AgNORs was performed using both quantitative and qualitative methods. The work revealed that all the examined tumors were diploid, except for 40% of CCOC cases. The S-phase fraction (SPF) value, AgNORs count and AgNORs area/cell showed statistically significant difference on comparing benign and malignant groups. A weak positive correlation was observed between SPF and AgNORs count. The SPF value was considered to be more sensitive and specific in differentiation between aggressive benign and malignant epithelial odontogenic tumors in comparison to AgNORs counting. Copyright © 2017 Elsevier Ltd. All rights reserved.

Cytometric methods for measuring bacteria in water: advantages, pitfalls and applications.

PubMed

Hammes, Frederik; Egli, Thomas

2010-06-01

Rapid detection of microbial cells is a challenge in microbiology, particularly when complex indigenous communities or subpopulations varying in viability, activity and physiological state are investigated. Flow cytometry (FCM) has developed during the last 30 years into a multidisciplinary technique for analysing bacteria. When used correctly, FCM can provide a broad range of information at the single-cell level, including (but not limited to) total counts, size measurements, nucleic acid content, cell viability and activity, and detection of specific bacterial groups or species. The main advantage of FCM is that it is fast and easy to perform. It is a robust technique, which is adaptable to different types of samples and methods, and has much potential for automation. Hence, numerous FCM applications have emerged in industrial biotechnology, food and pharmaceutical quality control, routine monitoring of drinking water and wastewater systems, and microbial ecological research in soils and natural aquatic habitats. This review focuses on the information that can be gained from the analysis of bacteria in water, highlighting some of the main advantages, pitfalls and applications.

Laser Scanning Cytometry: Principles and Applications—An Update

PubMed Central

Pozarowski, Piotr; Holden, Elena; Darzynkiewicz, Zbigniew

2012-01-01

Laser scanning cytometer (LSC) is the microscope-based cytofluorometer that offers a plethora of unique analytical capabilities, not provided by flow cytometry (FCM). This review describes attributes of LSC and covers its numerous applications derived from plentitude of the parameters that can be measured. Among many LSC applications the following are emphasized: (a) assessment of chromatin condensation to identify mitotic, apoptotic cells, or senescent cells; (b) detection of nuclear or mitochondrial translocation of critical factors such as NF-κB, p53, or Bax; (c) semi-automatic scoring of micronuclei in mutagenicity assays; (d) analysis of fluorescence in situ hybridization (FISH) and use of the FISH analysis attribute to measure other punctuate fluorescence patterns such as γH2AX foci or receptor clustering; (e) enumeration and morphometry of nucleoli and other cell organelles; (f) analysis of progeny of individual cells in clonogenicity assay; (g) cell immunophenotyping; (h) imaging, visual examination, or sequential analysis using different probes of the same cells upon their relocation; (i) in situ enzyme kinetics, drug uptake, and other time-resolved processes; (j) analysis of tissue section architecture using fluorescent and chromogenic probes; (k) application for hypocellular samples (needle aspirate, spinal fluid, etc.); and (l) other clinical applications. Advantages and limitations of LSC are discussed and compared with FCM. PMID:23027005

Combining flow cytometry and 16S rRNA gene pyrosequencing: a promising approach for drinking water monitoring and characterization.

PubMed

Prest, E I; El-Chakhtoura, J; Hammes, F; Saikaly, P E; van Loosdrecht, M C M; Vrouwenvelder, J S

2014-10-15

The combination of flow cytometry (FCM) and 16S rRNA gene pyrosequencing data was investigated for the purpose of monitoring and characterizing microbial changes in drinking water distribution systems. High frequency sampling (5 min intervals for 1 h) was performed at the outlet of a treatment plant and at one location in the full-scale distribution network. In total, 52 bulk water samples were analysed with FCM, pyrosequencing and conventional methods (adenosine-triphosphate, ATP; heterotrophic plate count, HPC). FCM and pyrosequencing results individually showed that changes in the microbial community occurred in the water distribution system, which was not detected with conventional monitoring. FCM data showed an increase in the total bacterial cell concentrations (from 345 ± 15 × 10(3) to 425 ± 35 × 10(3) cells mL(-1)) and in the percentage of intact bacterial cells (from 39 ± 3.5% to 53 ± 4.4%) during water distribution. This shift was also observed in the FCM fluorescence fingerprints, which are characteristic of each water sample. A similar shift was detected in the microbial community composition as characterized with pyrosequencing, showing that FCM and genetic fingerprints are congruent. FCM and pyrosequencing data were subsequently combined for the calculation of cell concentration changes for each bacterial phylum. The results revealed an increase in cell concentrations of specific bacterial phyla (e.g., Proteobacteria), along with a decrease in other phyla (e.g., Actinobacteria), which could not be concluded from the two methods individually. The combination of FCM and pyrosequencing methods is a promising approach for future drinking water quality monitoring and for advanced studies on drinking water distribution pipeline ecology. Copyright © 2014 Elsevier Ltd. All rights reserved.

Rapid Estimation of Astaxanthin and the Carotenoid-to-Chlorophyll Ratio in the Green Microalga Chromochloris zofingiensis Using Flow Cytometry.

PubMed

Chen, Junhui; Wei, Dong; Pohnert, Georg

2017-07-19

The green microalga Chromochloris zofingiensis can accumulate significant amounts of valuable carotenoids, mainly natural astaxanthin, a product with applications in functional food, cosmetics, nutraceuticals, and with potential therapeutic value in cardiovascular and neurological diseases. To optimize the production of astaxanthin, it is essential to monitor the content of astaxanthin in algal cells during cultivation. The widely used HPLC (high-performance liquid chromatography) method for quantitative astaxanthin determination is time-consuming and laborious. In the present work, we present a method using flow cytometry (FCM) for in vivo determination of the astaxanthin content and the carotenoid-to-chlorophyll ratio (Car/Chl) in mixotrophic C. zofingiensis . The method is based on the assessment of fluorescent characteristics of cellular pigments. The mean fluorescence intensity (MFI) of living cells was determined by FCM to monitor pigment formation based on the correlation between MFI detected in particular channels (FL1: 533 ± 15 nm; FL2: 585 ± 20 nm; FL3: >670 nm) and pigment content in algal cells. Through correlation and regression analysis, a linear relationship was observed between MFI in FL2 (band-pass filter, emission at 585 nm in FCM) and astaxanthin content (in HPLC) and applied for predicting astaxanthin content. With similar procedures, the relationships between MFI in different channels and Car/Chl ratio in mixotrophic C. zofingiensis were also determined. Car/Chl ratios could be estimated by the ratios of MFI (FL1/FL3, FL2/FL3). FCM is thus a highly efficient and feasible method for rapid estimation of astaxanthin content in the green microalga C. zofingiensis . The rapid FCM method is complementary to the current HPLC method, especially for rapid evaluation and prediction of astaxanthin formation as it is required during the high-throughput culture in the laboratory and mass cultivation in industry.

Rapid Estimation of Astaxanthin and the Carotenoid-to-Chlorophyll Ratio in the Green Microalga Chromochloris zofingiensis Using Flow Cytometry

PubMed Central

Chen, Junhui; Pohnert, Georg

2017-01-01

The green microalga Chromochloris zofingiensis can accumulate significant amounts of valuable carotenoids, mainly natural astaxanthin, a product with applications in functional food, cosmetics, nutraceuticals, and with potential therapeutic value in cardiovascular and neurological diseases. To optimize the production of astaxanthin, it is essential to monitor the content of astaxanthin in algal cells during cultivation. The widely used HPLC (high-performance liquid chromatography) method for quantitative astaxanthin determination is time-consuming and laborious. In the present work, we present a method using flow cytometry (FCM) for in vivo determination of the astaxanthin content and the carotenoid-to-chlorophyll ratio (Car/Chl) in mixotrophic C. zofingiensis. The method is based on the assessment of fluorescent characteristics of cellular pigments. The mean fluorescence intensity (MFI) of living cells was determined by FCM to monitor pigment formation based on the correlation between MFI detected in particular channels (FL1: 533 ± 15 nm; FL2: 585 ± 20 nm; FL3: >670 nm) and pigment content in algal cells. Through correlation and regression analysis, a linear relationship was observed between MFI in FL2 (band-pass filter, emission at 585 nm in FCM) and astaxanthin content (in HPLC) and applied for predicting astaxanthin content. With similar procedures, the relationships between MFI in different channels and Car/Chl ratio in mixotrophic C. zofingiensis were also determined. Car/Chl ratios could be estimated by the ratios of MFI (FL1/FL3, FL2/FL3). FCM is thus a highly efficient and feasible method for rapid estimation of astaxanthin content in the green microalga C. zofingiensis. The rapid FCM method is complementary to the current HPLC method, especially for rapid evaluation and prediction of astaxanthin formation as it is required during the high-throughput culture in the laboratory and mass cultivation in industry. PMID:28753934

Absolute counting of neutrophils in whole blood using flow cytometry.

PubMed

Brunck, Marion E G; Andersen, Stacey B; Timmins, Nicholas E; Osborne, Geoffrey W; Nielsen, Lars K

2014-12-01

Absolute neutrophil count (ANC) is used clinically to monitor physiological dysfunctions such as myelosuppression or infection. In the research laboratory, ANC is a valuable measure to monitor the evolution of a wide range of disease states in disease models. Flow cytometry (FCM) is a fast, widely used approach to confidently identify thousands of cells within minutes. FCM can be optimised for absolute counting using spiked-in beads or by measuring the sample volume analysed. Here we combine the 1A8 antibody, specific for the mouse granulocyte protein Ly6G, with flow cytometric counting in straightforward FCM assays for mouse ANC, easily implementable in the research laboratory. Volumetric and Trucount™ bead assays were optimized for mouse neutrophils, and ANC values obtained with these protocols were compared to ANC measured by a dual-platform assay using the Orphee Mythic 18 veterinary haematology analyser. The single platform assays were more precise with decreased intra-assay variability compared with ANC obtained using the dual protocol. Defining ANC based on Ly6G expression produces a 15% higher estimate than the dual protocol. Allowing for this difference in ANC definition, the flow cytometry counting assays using Ly6G can be used reliably in the research laboratory to quantify mouse ANC from a small volume of blood. We demonstrate the utility of the volumetric protocol in a time-course study of chemotherapy induced neutropenia using four drug regimens. © 2014 International Society for Advancement of Cytometry.

Multi-parameter flow cytometry as a process analytical technology (PAT) approach for the assessment of bacterial ghost production.

PubMed

Langemann, Timo; Mayr, Ulrike Beate; Meitz, Andrea; Lubitz, Werner; Herwig, Christoph

2016-01-01

Flow cytometry (FCM) is a tool for the analysis of single-cell properties in a cell suspension. In this contribution, we present an improved FCM method for the assessment of E-lysis in Enterobacteriaceae. The result of the E-lysis process is empty bacterial envelopes-called bacterial ghosts (BGs)-that constitute potential products in the pharmaceutical field. BGs have reduced light scattering properties when compared with intact cells. In combination with viability information obtained from staining samples with the membrane potential-sensitive fluorescent dye bis-(1,3-dibutylarbituric acid) trimethine oxonol (DiBAC4(3)), the presented method allows to differentiate between populations of viable cells, dead cells, and BGs. Using a second fluorescent dye RH414 as a membrane marker, non-cellular background was excluded from the data which greatly improved the quality of the results. Using true volumetric absolute counting, the FCM data correlated well with cell count data obtained from colony-forming units (CFU) for viable populations. Applicability of the method to several Enterobacteriaceae (different Escherichia coli strains, Salmonella typhimurium, Shigella flexneri 2a) could be shown. The method was validated as a resilient process analytical technology (PAT) tool for the assessment of E-lysis and for particle counting during 20-l batch processes for the production of Escherichia coli Nissle 1917 BGs.

Mapping cell populations in flow cytometry data for cross-sample comparison using the Friedman-Rafsky test statistic as a distance measure.

PubMed

Hsiao, Chiaowen; Liu, Mengya; Stanton, Rick; McGee, Monnie; Qian, Yu; Scheuermann, Richard H

2016-01-01

Flow cytometry (FCM) is a fluorescence-based single-cell experimental technology that is routinely applied in biomedical research for identifying cellular biomarkers of normal physiological responses and abnormal disease states. While many computational methods have been developed that focus on identifying cell populations in individual FCM samples, very few have addressed how the identified cell populations can be matched across samples for comparative analysis. This article presents FlowMap-FR, a novel method for cell population mapping across FCM samples. FlowMap-FR is based on the Friedman-Rafsky nonparametric test statistic (FR statistic), which quantifies the equivalence of multivariate distributions. As applied to FCM data by FlowMap-FR, the FR statistic objectively quantifies the similarity between cell populations based on the shapes, sizes, and positions of fluorescence data distributions in the multidimensional feature space. To test and evaluate the performance of FlowMap-FR, we simulated the kinds of biological and technical sample variations that are commonly observed in FCM data. The results show that FlowMap-FR is able to effectively identify equivalent cell populations between samples under scenarios of proportion differences and modest position shifts. As a statistical test, FlowMap-FR can be used to determine whether the expression of a cellular marker is statistically different between two cell populations, suggesting candidates for new cellular phenotypes by providing an objective statistical measure. In addition, FlowMap-FR can indicate situations in which inappropriate splitting or merging of cell populations has occurred during gating procedures. We compared the FR statistic with the symmetric version of Kullback-Leibler divergence measure used in a previous population matching method with both simulated and real data. The FR statistic outperforms the symmetric version of KL-distance in distinguishing equivalent from nonequivalent cell populations. FlowMap-FR was also employed as a distance metric to match cell populations delineated by manual gating across 30 FCM samples from a benchmark FlowCAP data set. An F-measure of 0.88 was obtained, indicating high precision and recall of the FR-based population matching results. FlowMap-FR has been implemented as a standalone R/Bioconductor package so that it can be easily incorporated into current FCM data analytical workflows. © The Authors. Published by Wiley Periodicals, Inc. on behalf of ISAC.

Three-dimensional DNA image cytometry by optical projection tomographic microscopy for early cancer diagnosis.

PubMed

Agarwal, Nitin; Biancardi, Alberto M; Patten, Florence W; Reeves, Anthony P; Seibel, Eric J

2014-04-01

Aneuploidy is typically assessed by flow cytometry (FCM) and image cytometry (ICM). We used optical projection tomographic microscopy (OPTM) for assessing cellular DNA content using absorption and fluorescence stains. OPTM combines some of the attributes of both FCM and ICM and generates isometric high-resolution three-dimensional (3-D) images of single cells. Although the depth of field of the microscope objective was in the submicron range, it was extended by scanning the objective's focal plane. The extended depth of field image is similar to a projection in a conventional x-ray computed tomography. These projections were later reconstructed using computed tomography methods to form a 3-D image. We also present an automated method for 3-D nuclear segmentation. Nuclei of chicken, trout, and triploid trout erythrocyte were used to calibrate OPTM. Ratios of integrated optical densities extracted from 50 images of each standard were compared to ratios of DNA indices from FCM. A comparison of mean square errors with thionin, hematoxylin, Feulgen, and SYTOX green was done. Feulgen technique was preferred as it showed highest stoichiometry, least variance, and preserved nuclear morphology in 3-D. The addition of this quantitative biomarker could further strengthen existing classifiers and improve early diagnosis of cancer using 3-D microscopy.

Immuno-analysis of microparticles: probing at the limits of detection

PubMed Central

Latham, Sharissa L.; Tiberti, Natalia; Gokoolparsadh, Naveena; Holdaway, Karen; Olivier Couraud, Pierre; Grau, Georges E. R.; Combes, Valery

2015-01-01

Microparticle (MP) research is clouded by debate regarding the accuracy and validity of flow cytometry (FCM) as an analytical methodology, as it is influenced by many variables including the pre-analytical conditions, instruments physical capabilities and detection parameters. This study utilises a simplistic in vitro system for generating MP, and through comparative analysis with immuno-electron microscopy (Immuno-EM) assesses the strengths and limitations of probe selection and high-sensitivity FCM. Of the markers examined, MP were most specifically labelled with phosphatidylserine ligands, annexin V and lactadherin, although only ~60% MP are PS positive. Whilst these two ligands detect comparable absolute MP numbers, they interact with the same population in distinct manners; annexin V binding is enhanced on TNF induced MP. CD105 and CD54 expression were, as expected, consistent and enhanced following TNF activation respectively. Their labelling however accounted for as few as 30–40% of MP. The greatest discrepancies between FCM and I-EM were observed in the population solely labelled for the surface antigen. These findings demonstrate that despite significant improvements in resolution, high-sensitivity FCM remains limited in detecting small-size MP expressing low antigen levels. This study highlights factors to consider when selecting endothelial MP probes, as well as interpreting and representing data. PMID:26553743

The feasibility of automated online flow cytometry for in-situ monitoring of microbial dynamics in aquatic ecosystems

PubMed Central

Besmer, Michael D.; Weissbrodt, David G.; Kratochvil, Bradley E.; Sigrist, Jürg A.; Weyland, Mathias S.; Hammes, Frederik

2014-01-01

Fluorescent staining coupled with flow cytometry (FCM) is often used for the monitoring, quantification and characterization of bacteria in engineered and environmental aquatic ecosystems including seawater, freshwater, drinking water, wastewater, and industrial bioreactors. However, infrequent grab sampling hampers accurate characterization and subsequent understanding of microbial dynamics in all of these ecosystems. A logic technological progression is high throughput and full automation of the sampling, staining, measurement, and data analysis steps. Here we assess the feasibility and applicability of automated FCM by means of actual data sets produced with prototype instrumentation. As proof-of-concept we demonstrate examples of microbial dynamics in (i) flowing tap water from a municipal drinking water supply network and (ii) river water from a small creek subject to two rainfall events. In both cases, automated measurements were done at 15-min intervals during 12–14 consecutive days, yielding more than 1000 individual data points for each ecosystem. The extensive data sets derived from the automated measurements allowed for the establishment of baseline data for each ecosystem, as well as for the recognition of daily variations and specific events that would most likely be missed (or miss-characterized) by infrequent sampling. In addition, the online FCM data from the river water was combined and correlated with online measurements of abiotic parameters, showing considerable potential for a better understanding of cause-and-effect relationships in aquatic ecosystems. Although several challenges remain, the successful operation of an automated online FCM system and the basic interpretation of the resulting data sets represent a breakthrough toward the eventual establishment of fully automated online microbiological monitoring technologies. PMID:24917858

Flow cytometric bacterial cell counts challenge conventional heterotrophic plate counts for routine microbiological drinking water monitoring.

PubMed

Van Nevel, S; Koetzsch, S; Proctor, C R; Besmer, M D; Prest, E I; Vrouwenvelder, J S; Knezev, A; Boon, N; Hammes, F

2017-04-15

Drinking water utilities and researchers continue to rely on the century-old heterotrophic plate counts (HPC) method for routine assessment of general microbiological water quality. Bacterial cell counting with flow cytometry (FCM) is one of a number of alternative methods that challenge this status quo and provide an opportunity for improved water quality monitoring. After more than a decade of application in drinking water research, FCM methodology is optimised and established for routine application, supported by a considerable amount of data from multiple full-scale studies. Bacterial cell concentrations obtained by FCM enable quantification of the entire bacterial community instead of the minute fraction of cultivable bacteria detected with HPC (typically < 1% of all bacteria). FCM measurements are reproducible with relative standard deviations below 3% and can be available within 15 min of samples arriving in the laboratory. High throughput sample processing and complete automation are feasible and FCM analysis is arguably less expensive than HPC when measuring more than 15 water samples per day, depending on the laboratory and selected staining procedure(s). Moreover, many studies have shown FCM total (TCC) and intact (ICC) cell concentrations to be reliable and robust process variables, responsive to changes in the bacterial abundance and relevant for characterising and monitoring drinking water treatment and distribution systems. The purpose of this critical review is to initiate a constructive discussion on whether FCM could replace HPC in routine water quality monitoring. We argue that FCM provides a faster, more descriptive and more representative quantification of bacterial abundance in drinking water. Copyright © 2017 Elsevier Ltd. All rights reserved.

Full color emitting fluorescent carbon material as reversible pH sensor with multicolor live cell imaging.

PubMed

Sharma, Vinay; Kaur, Navpreet; Tiwari, Pranav; Mobin, Shaikh M

2018-05-01

Carbon-based nano materials are developed as a cytocompatible alternative to semiconducting quantum dots for bioimaging and fluorescence-based sensing. The green alternatives for the synthesis of carbon materials are imminent. The present study demonstrates microwave based one step quick synthesis of fluorescent carbon material (FCM) having three variants: (i) un-doped fluorescent carbon material (UFCM) (ii) nitrogen doped FCM (N@FCM), and (iii) nitrogen & phosphorus co-doped FCM (N-P@FCM) using sugarcane extract as a carbon source. The N doping was performed using ethylenediamine and phosphoric acid was used for P doping. The heteroatom doped FCM were synthesized due to insolubility of UFCM in water. Unlike, UFCM, the N@FCM and N-P@FCM were found to be highly soluble in water. The N-P@FCM shows highest quantum yield among the three. The N-P@FCM was explored for alkaline pH sensing and it shows a quenching of fluorescence in the pH range 09-14. The sensing behaviour shows reversibility and high selectivity. Further, the sensor was also investigated for their biocompatibility and hence employed as a promising multicolour probe for cancer cell imaging. The generality in cell imaging was investigated by flow cytometry. The hetero-atom doped green carbon-dots may open new avenues for sensing and selective cellular targeting. Copyright © 2018 Elsevier B.V. All rights reserved.

Usefullness of IGH/TCR PCR studies in lymphoproliferative disorders with inconclusive clonality by flow cytometry.

PubMed

Ribera, Jordi; Zamora, Lurdes; Juncà, Jordi; Rodríguez, Inés; Marcé, Silvia; Cabezón, Marta; Millá, Fuensanta

2013-07-25

In up to 5-15% of studies of lymphoproliferative disorders (LPD) flow cytometry (FCM) or immunomorphologic methods cannot discriminate malignant from reactive processes. The aim of this work was to determine the usefulness of PCR for solving these diagnostic uncertainties. We analyzed IGH and TCRγ genes by PCR in 106 samples with inconclusive FCM results. A clonal result was registered in 36/106 studies, with a LPD being confirmed in 27 (75%) of these cases. Specifically, 9/9 IGH clonal and 16/25 TCRγ clonal results were finally diagnosed with LPD. Additionally, 2 clonal TCRγ samples with suspicion of undefined LPD were finally diagnosed with T LPD. Although polyclonal results were obtained in 47 of the cases studied (38 IGH and 9 TCRγ), hematologic neoplasms were diagnosed in 4/38 IGH polyclonal and in 1/9 TCRγ polyclonal studies. There were also 14 PCR polyclonal results (4 IGH, 10 TCRγ), albeit non-conclusive. Of these, 2/4 were eventually diagnosed with B-cell lymphoma and 3/10 with T-cell LPD. In 8 IGH samples the results of PCR techniques were non-informative but in 3/8 cases a B lymphoma was finally confirmed. We concluded that PCR is a useful technique to identify LPD when FCM is inconclusive. A PCR clonal B result is indicative of malignancy but IGH polyclonal and non-conclusive results do not exclude lymphoid neoplasms. Interpretation of T-cell clonality should be based on all the available clinical and analytical data. © 2013 Clinical Cytometry Society. Copyright © 2013 Clinical Cytometry Society.

Development of a flow cytometric method to analyze subpopulations of bacteria in probiotic products and dairy starters.

PubMed

Bunthof, Christine J; Abee, Tjakko

2002-06-01

Flow cytometry (FCM) is a rapid and sensitive technique that can determine cell numbers and measure various physiological characteristics of individual cells by using appropriate fluorescent probes. Previously, we developed an FCM assay with the viability probes carboxyfluorescein diacetate (cFDA) and TOTO-1 [1'-(4,4,7,7-tetramethyl-4,7-diazaundecamethylene)-bis-4-[3-methyl-2,3dihydro(benzo-1,3-oxazole)-2-methylidene]-1-(3'-trimethylammoniumpropyl)-pyridinium tetraiodide] for (stressed) lactic acid bacteria (C. J. Bunthof, K. Bloemen, P. Breeuwer, F. M. Rombouts, and T. Abee, Appl. Environ. Microbiol. 67:2326-2335, 2001). cFDA stains intact cells with enzymatic activity, and TOTO-1 stains membrane-permeabilized cells. Here we used this assay to study the viability of bacterial suspensions in milk, dairy fermentation starters, and probiotic products. To facilitate FCM analysis of bacteria in milk, a commercially available milk-clearing solution was used. The procedure was optimized to increase the signal-to-noise ratio. FCM enumerations were accurate down to a concentration of 10(5) cells ml(-1). The level of retrieval of Lactobacillus plantarum WCFS 1 suspended in milk was high, and viability was not affected by the procedure. The plate counts for cleared samples of untreated cell suspensions were nearly as high as the total FCM counts, and the correlation was strong (r > 0.99). In dairy fermentation starters and in probiotic products the FCM total cell counts were substantially higher than the numbers of CFU. Three functional populations could be distinguished: culturable cells, cells that are intact and metabolically active but not culturable, and permeabilized cells. The proportions of the populations differed in the products tested. This FCM method provides tools to assess the functionality of different populations in fermentation starters and probiotic products.

Development of a Flow Cytometric Method To Analyze Subpopulations of Bacteria in Probiotic Products and Dairy Starters

PubMed Central

Bunthof, Christine J.; Abee, Tjakko

2002-01-01

Flow cytometry (FCM) is a rapid and sensitive technique that can determine cell numbers and measure various physiological characteristics of individual cells by using appropriate fluorescent probes. Previously, we developed an FCM assay with the viability probes carboxyfluorescein diacetate (cFDA) and TOTO-1 {1′-(4,4,7,7-tetramethyl-4,7-diazaundecamethylene)-bis-4-[3-methyl-2,3dihydro(benzo-1,3-oxazole)-2-methylidene]-1-(3′-trimethylammoniumpropyl)-pyridinium tetraiodide} for (stressed) lactic acid bacteria (C. J. Bunthof, K. Bloemen, P. Breeuwer, F. M. Rombouts, and T. Abee, Appl. Environ. Microbiol. 67:2326-2335, 2001). cFDA stains intact cells with enzymatic activity, and TOTO-1 stains membrane-permeabilized cells. Here we used this assay to study the viability of bacterial suspensions in milk, dairy fermentation starters, and probiotic products. To facilitate FCM analysis of bacteria in milk, a commercially available milk-clearing solution was used. The procedure was optimized to increase the signal-to-noise ratio. FCM enumerations were accurate down to a concentration of 105 cells ml−1. The level of retrieval of Lactobacillus plantarum WCFS 1 suspended in milk was high, and viability was not affected by the procedure. The plate counts for cleared samples of untreated cell suspensions were nearly as high as the total FCM counts, and the correlation was strong (r > 0.99). In dairy fermentation starters and in probiotic products the FCM total cell counts were substantially higher than the numbers of CFU. Three functional populations could be distinguished: culturable cells, cells that are intact and metabolically active but not culturable, and permeabilized cells. The proportions of the populations differed in the products tested. This FCM method provides tools to assess the functionality of different populations in fermentation starters and probiotic products. PMID:12039752

Performance standard-based validation study for local lymph node assay: 5-bromo-2-deoxyuridine-flow cytometry method.

PubMed

Ahn, Ilyoung; Kim, Tae-Sung; Jung, Eun-Sun; Yi, Jung-Sun; Jang, Won-Hee; Jung, Kyoung-Mi; Park, Miyoung; Jung, Mi-Sook; Jeon, Eun-Young; Yeo, Kyeong-Uk; Jo, Ji-Hoon; Park, Jung-Eun; Kim, Chang-Yul; Park, Yeong-Chul; Seong, Won-Keun; Lee, Ai-Young; Chun, Young Jin; Jeong, Tae Cheon; Jeung, Eui Bae; Lim, Kyung-Min; Bae, SeungJin; Sohn, Soojung; Heo, Yong

2016-10-01

Local lymph node assay: 5-bromo-2-deoxyuridine-flow cytometry method (LLNA: BrdU-FCM) is a modified non-radioisotopic technique with the additional advantages of accommodating multiple endpoints with the introduction of FCM, and refinement and reduction of animal use by using a sophisticated prescreening scheme. Reliability and accuracy of the LLNA: BrdU-FCM was determined according to OECD Test Guideline (TG) No. 429 (Skin Sensitization: Local Lymph Node Assay) performance standards (PS), with the participation of four laboratories. Transferability was demonstrated through successfully producing stimulation index (SI) values for 25% hexyl cinnamic aldehyde (HCA) consistently greater than 3, a predetermined threshold, by all participating laboratories. Within- and between-laboratory reproducibility was shown using HCA and 2,4-dinitrochlorobenzene, in which EC2.7 values (the estimated concentrations eliciting an SI of 2.7, the threshold for LLNA: BrdU-FCM) fell consistently within the acceptance ranges, 0.025-0.1% and 5-20%, respectively. Predictive capacity was tested using the final protocol version 1.3 for the 18 reference chemicals listed in OECD TG 429, of which results showed 84.6% sensitivity, 100% specificity, and 88.9% accuracy compared with the original LLNA. The data presented are considered to meet the performance criteria for the PS, and its predictive capacity was also sufficiently validated. Copyright © 2016 Elsevier Inc. All rights reserved.

Automated four color CD4/CD8 analysis of leukocytes by scanning fluorescence microscopy using Quantum dots

NASA Astrophysics Data System (ADS)

Bocsi, Jozsef; Mittag, Anja; Varga, Viktor S.; Molnar, Bela; Tulassay, Zsolt; Sack, Ulrich; Lenz, Dominik; Tarnok, Attila

2006-02-01

Scanning Fluorescence Microscope (SFM) is a new technique for automated motorized microscopes to measure multiple fluorochrome labeled cells (Bocsi et al. Cytometry 2004, 61A:1). The ratio of CD4+/CD8+ cells is an important in immune diagnostics in immunodeficiency and HIV. Therefor a four-color staining protocol (DNA, CD3, CD4 and CD8) for automated SFM analysis of lymphocytes was developed. EDTA uncoagulated blood was stained with organic and inorganic (Quantum dots) fluorochromes in different combinations. Aliquots of samples were measured by Flow Cytometry (FCM) and SFM. By SFM specimens were scanned and digitized using four fluorescence filter sets. Automated cell detection (based on Hoechst 33342 fluorescence), CD3, CD4 and CD8 detection were performed, CD4/CD8 ratio was calculated. Fluorescence signals were well separable on SFM and FCM. Passing and Bablok regression of all CD4/CD8 ratios obtained by FCM and SFM (F(X)=0.0577+0.9378x) are in the 95% confidence interval. Cusum test did not show significant deviation from linearity (P>0.10). This comparison indicates that there is no systemic bias between the two different methods. In SFM analyses the inorganic Quantum dot staining was very stable in PBS in contrast to the organic fluorescent dyes, but bleached shortly after mounting with antioxidant and free radical scavenger mounting media. This shows the difficulty of combinations of organic dyes and Quantum dots. Slide based multi-fluorescence labeling system and automated SFM are applicable tools for the CD4/CD8 ratio determination in peripheral blood samples. Quantum Dots are stable inorganic fluorescence labels that may be used as reliable high resolution dyes for cell labeling.

Bleaching response of Symbiodinium (zooxanthellae): determination by flow cytometry.

PubMed

Lee, Co Sin; Yeo, Yin Sheng Wilson; Sin, Tsai Min

2012-10-01

Coral bleaching is of increasing concern to reef management and stakeholders. Thus far, quantification of coral bleaching tends to be heavily reliant on the enumeration of zooxanthellae, with less emphasis on assessment of photosynthetic or physiological condition, these being often assessed separately by techniques such as liquid chromatography. Traditional methods of enumeration using microscopy are time consuming, subjected to low precision and great observer error. In this study, we presented a method for the distinction of physoiological condition and rapid enumeration of zooxanthellae using flow cytometry (FCM). Microscopy verified that healthy looking/live versus damaged/dead zooxanthellae could be reliably and objectively distinguished and counted by FCM on the basis of red and green fluorescence and light scatter. Excellent correlations were also determined between FCM and microscopy estimates of cell concentrations of fresh zooxanthellae isolates from Pocillopora damicornis. The relative intensities of chlorophyll and β-carotene fluorescences were shown to be important in understanding the results of increased cell counts in freshly isolated zooxanthellae experimentally exposed to high temperatures (34, 36, and 38°C) over 24 h, with ambient temperature (29°C) used as controls. The ability to simultaneously identify and enumerate subpopulations of different physiological states in the same sample provides an enormous advantage in not just determining bleaching responses, but elucidating adaptive response and mechanisms for tolerance. Therefore, this approach might provide a rapid, convenient, and reproducible methodology for climate change studies and reef management programs. Copyright © 2012 International Society for Advancement of Cytometry.

Properties of human blood monocytes. I. CD91 expression and log orthogonal light scatter provide a robust method to identify monocytes that is more accurate than CD14 expression.

PubMed

Hudig, Dorothy; Hunter, Kenneth W; Diamond, W John; Redelman, Doug

2014-03-01

This study was designed to improve identification of human blood monocytes by using antibodies to molecules that occur consistently on all stages of monocyte development and differentiation. We examined blood samples from 200 healthy adults without clinically diagnosed immunological abnormalities by flow cytometry (FCM) with multiple combinations of antibodies and with a hematology analyzer (Beckman LH750). CD91 (α2 -macroglobulin receptor) was expressed only by monocytes and to a consistent level among subjects [mean median fluorescence intensity (MFI) = 16.2 ± 3.2]. Notably, only 85.7 ± 5.82% of the CD91(+) monocytes expressed high levels of the classical monocyte marker CD14, with some CD91(+) CD16(+) cells having negligible CD14, indicating that substantial FCM under-counts will occur when monocytes are identified by high CD14. CD33 (receptor for sialyl conjugates) was co-expressed with CD91 on monocytes but CD33 expression varied by nearly ten-fold among subjects (mean MFI = 17.4 ± 7.7). In comparison to FCM analyses, the hematology analyzer systematically over-counted monocytes and eosinophils while lymphocyte and neutrophil differential values generally agreed with FCM methods. CD91 is a better marker to identify monocytes than CD14 or CD33. Furthermore, FCM (with anti-CD91) identifies monocytes better than a currently used clinical CBC instrument. Use of anti-CD91 together with anti-CD14 and anti-CD16 supports the identification of the diagnostically significant monocyte populations with variable expression of CD14 and CD16. Copyright © 2013 Clinical Cytometry Society.

Direct quantification of bacterial biomass in influent, effluent and activated sludge of wastewater treatment plants by using flow cytometry.

PubMed

Foladori, P; Bruni, L; Tamburini, S; Ziglio, G

2010-07-01

A rapid multi-step procedure, potentially amenable to automation, was proposed for quantifying viable and active bacterial cells, estimating their biovolume using flow cytometry (FCM) and to calculate their biomass within the main stages of a wastewater treatment plant: raw wastewater, settled wastewater, activated sludge and effluent. Fluorescent staining of bacteria using SYBR-Green I + Propidium Iodide (to discriminate cell integrity or permeabilisation) and BCECF-AM (to identify enzymatic activity) was applied to count bacterial cells by FCM. A recently developed specific procedure was applied to convert Forward Angle Light Scatter measured by FCM into the corresponding bacterial biovolume. This conversion permits the calculation of the viable and active bacterial biomass in wastewater, activated sludge and effluent, expressed as Volatile Suspended Solids (VSS) or particulate Chemical Oxygen Demand (COD). Viable bacterial biomass represented only a small part of particulate COD in raw wastewater (4.8 +/- 2.4%), settled wastewater (10.7 +/- 3.1%), activated sludge (11.1 +/- 2.1%) and effluent (3.2 +/- 2.2%). Active bacterial biomass counted for a percentage of 30-47% of the viable bacterial biomass within the stages of the wastewater treatment plant. Copyright 2010 Elsevier Ltd. All rights reserved.

Applications of Flow Cytometry to Clinical Microbiology†

PubMed Central

Álvarez-Barrientos, Alberto; Arroyo, Javier; Cantón, Rafael; Nombela, César; Sánchez-Pérez, Miguel

2000-01-01

Classical microbiology techniques are relatively slow in comparison to other analytical techniques, in many cases due to the need to culture the microorganisms. Furthermore, classical approaches are difficult with unculturable microorganisms. More recently, the emergence of molecular biology techniques, particularly those on antibodies and nucleic acid probes combined with amplification techniques, has provided speediness and specificity to microbiological diagnosis. Flow cytometry (FCM) allows single- or multiple-microbe detection in clinical samples in an easy, reliable, and fast way. Microbes can be identified on the basis of their peculiar cytometric parameters or by means of certain fluorochromes that can be used either independently or bound to specific antibodies or oligonucleotides. FCM has permitted the development of quantitative procedures to assess antimicrobial susceptibility and drug cytotoxicity in a rapid, accurate, and highly reproducible way. Furthermore, this technique allows the monitoring of in vitro antimicrobial activity and of antimicrobial treatments ex vivo. The most outstanding contribution of FCM is the possibility of detecting the presence of heterogeneous populations with different responses to antimicrobial treatments. Despite these advantages, the application of FCM in clinical microbiology is not yet widespread, probably due to the lack of access to flow cytometers or the lack of knowledge about the potential of this technique. One of the goals of this review is to attempt to mitigate this latter circumstance. We are convinced that in the near future, the availability of commercial kits should increase the use of this technique in the clinical microbiology laboratory. PMID:10755996

Functional analysis and classification of phytoplankton based on data from an automated flow cytometer.

PubMed

Malkassian, Anthony; Nerini, David; van Dijk, Mark A; Thyssen, Melilotus; Mante, Claude; Gregori, Gerald

2011-04-01

Analytical flow cytometry (FCM) is well suited for the analysis of phytoplankton communities in fresh and sea waters. The measurement of light scatter and autofluorescence properties of particles by FCM provides optical fingerprints, which enables different phytoplankton groups to be separated. A submersible version of the CytoSense flow cytometer (the CytoSub) has been designed for in situ autonomous sampling and analysis, making it possible to monitor phytoplankton at a short temporal scale and obtain accurate information about its dynamics. For data analysis, a manual clustering is usually performed a posteriori: data are displayed on histograms and scatterplots, and group discrimination is made by drawing and combining regions (gating). The purpose of this study is to provide greater objectivity in the data analysis by applying a nonmanual and consistent method to automatically discriminate clusters of particles. In other words, we seek for partitioning methods based on the optical fingerprints of each particle. As the CytoSense is able to record the full pulse shape for each variable, it quickly generates a large and complex dataset to analyze. The shape, length, and area of each curve were chosen as descriptors for the analysis. To test the developed method, numerical experiments were performed on simulated curves. Then, the method was applied and validated on phytoplankton cultures data. Promising results have been obtained with a mixture of various species whose optical fingerprints overlapped considerably and could not be accurately separated using manual gating. Copyright © 2011 International Society for Advancement of Cytometry.

Rapid detection of total and viable Legionella pneumophila in tap water by immunomagnetic separation, double fluorescent staining and flow cytometry.

PubMed

Keserue, Hans-Anton; Baumgartner, Andreas; Felleisen, Richard; Egli, Thomas

2012-11-01

We developed a rapid detection method for Legionella pneumophila (Lp) by filtration, immunomagnetic separation, double fluorescent staining, and flow cytometry (IMS-FCM method). The method requires 120 min and can discriminate 'viable' and 'membrane-damaged' cells. The recovery is over 85% of spiked Lp SG 1 cells in 1 l of tap water and detection limits are around 50 and 15 cells per litre for total and viable Lp, respectively. The method was compared using water samples from house installations in a blind study with three environmental laboratories performing the ISO 11731 plating method. In 53% of the water samples from different taps and showers significantly higher concentrations of Lp were detected by flow cytometry. No correlation to the plate culture method was found. Since also 'viable but not culturable' (VNBC) cells are detected by our method, this result was expected. The IMS-FCM method is limited by the specificity of the used antibodies; in the presented case they target Lp serogroups 1-12. This and the fact that no Lp-containing amoebae are detected may explain why in 21% of all samples higher counts were observed using the plate culture method. Though the IMS-FCM method is not yet fit to completely displace the established plating method (ISO 11731) for routine Lp monitoring, it has major advantages to plating and can quickly provide important insights into the ecology of this pathogen in water distribution systems. © 2012 The Authors. Microbial Biotechnology © 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.

The relative value of cytometry and cytology in the management of bladder cancer: the Memorial Sloan-Kettering Cancer Center experience.

PubMed

Badalament, R A; Fair, W R; Whitmore, W F; Melamed, M R

1988-02-01

The flow cytometric studies presented herein are based almost entirely on DNA measurements and represent an early application of this diagnostic test. Nevertheless, the MSKCC experience with FCM has demonstrated that it is technically feasible and clinically useful. The sensitivity of FCM is in the range of 80% to 85% overall, and is superior to that of conventional voided or bladder wash cytology. In the absence of inflammation secondary to infection, calculi, or intravesical agents such as BCG, the specificity is greater than 90%. In the presence of inflammation, FCM appears to be less specific than conventional cytology. A potential advantage of FCM over cytology is the quantitative nature of the examination permitting comparisons of sequential examinations. Also, there are refinements in technique that hold promise of increasing the accuracy or clinical usefulness of FCM, eg, the joint measurements of DNA and differentiation antigens defined by monoclonal antibodies. Yet, despite the current and potential advantages of FCM, voided urinary cytology continues to be the procedure of choice for detection and monitoring urothelial carcinoma, not only because of our long experience and better understanding of this test, but because of its proven high specificity, because it is noninvasive, widely available, and may help to detect upper tract or urethral tumors.

Rapid assessment of viable but non-culturable Bacillus coagulans MTCC 5856 in commercial formulations using Flow cytometry.

PubMed

Majeed, Muhammed; Majeed, Shaheen; Nagabhushanam, Kalyanam; Punnapuzha, Ardra; Philip, Sheena; Mundkur, Lakshmi

2018-01-01

Accurate enumeration of bacterial count in probiotic formulation is imperative to ensure that the product adheres to regulatory standards and citation in consumer product label. Standard methods like plate count, can enumerate only replicating bacterial population under selected culture conditions. Viable but non culturable bacteria (VBNC) retain characteristics of living cells and can regain cultivability by a process known as resuscitation. This is a protective mechanism adapted by bacteria to evade stressful environmental conditions. B. coagulans MTCC 5856(LactoSpore®) is a probiotic endospore which can survive for decades in hostile environments without dividing. In the present study, we explored the use of flow cytometry to enumerate the viable count of B. coagulans MTCC 5856 under acidic and alkaline conditions, high temperature and in commercial formulations like compressed tablets and capsules. Flow cytometry (FCM) was comparable to plate count method when the spores were counted at physiological conditions. We show that VBNC state is induced in B. coagulans MTCC 5856by high temperature and acidic pH. The cells get resuscitated under physiological conditions and FCM was sensitive to detect the VBNC spores. Flow cytometry showed excellent ability to assess the viable spore count in commercial probiotic formulations of B. coagulans MTCC 5856. The results establish Flow cytometry as a reliable method to count viable bacteria in commercial probiotic preparations. Sporulation as well as existence as VBNC could contribute to the extreme stability of B. coagulans MTCC 5856.

Rapid assessment of viable but non-culturable Bacillus coagulans MTCC 5856 in commercial formulations using Flow cytometry

PubMed Central

Majeed, Muhammed; Majeed, Shaheen; Nagabhushanam, Kalyanam; Punnapuzha, Ardra; Philip, Sheena

2018-01-01

Accurate enumeration of bacterial count in probiotic formulation is imperative to ensure that the product adheres to regulatory standards and citation in consumer product label. Standard methods like plate count, can enumerate only replicating bacterial population under selected culture conditions. Viable but non culturable bacteria (VBNC) retain characteristics of living cells and can regain cultivability by a process known as resuscitation. This is a protective mechanism adapted by bacteria to evade stressful environmental conditions. B. coagulans MTCC 5856(LactoSpore®) is a probiotic endospore which can survive for decades in hostile environments without dividing. In the present study, we explored the use of flow cytometry to enumerate the viable count of B. coagulans MTCC 5856 under acidic and alkaline conditions, high temperature and in commercial formulations like compressed tablets and capsules. Flow cytometry (FCM) was comparable to plate count method when the spores were counted at physiological conditions. We show that VBNC state is induced in B. coagulans MTCC 5856by high temperature and acidic pH. The cells get resuscitated under physiological conditions and FCM was sensitive to detect the VBNC spores. Flow cytometry showed excellent ability to assess the viable spore count in commercial probiotic formulations of B. coagulans MTCC 5856. The results establish Flow cytometry as a reliable method to count viable bacteria in commercial probiotic preparations. Sporulation as well as existence as VBNC could contribute to the extreme stability of B. coagulans MTCC 5856. PMID:29474436

Monitoring microbiological changes in drinking water systems using a fast and reproducible flow cytometric method.

PubMed

Prest, E I; Hammes, F; Kötzsch, S; van Loosdrecht, M C M; Vrouwenvelder, J S

2013-12-01

Flow cytometry (FCM) is a rapid, cultivation-independent tool to assess and evaluate bacteriological quality and biological stability of water. Here we demonstrate that a stringent, reproducible staining protocol combined with fixed FCM operational and gating settings is essential for reliable quantification of bacteria and detection of changes in aquatic bacterial communities. Triplicate measurements of diverse water samples with this protocol typically showed relative standard deviation values and 95% confidence interval values below 2.5% on all the main FCM parameters. We propose a straightforward and instrument-independent method for the characterization of water samples based on the combination of bacterial cell concentration and fluorescence distribution. Analysis of the fluorescence distribution (or so-called fluorescence fingerprint) was accomplished firstly through a direct comparison of the raw FCM data and subsequently simplified by quantifying the percentage of large and brightly fluorescent high nucleic acid (HNA) content bacteria in each sample. Our approach enables fast differentiation of dissimilar bacterial communities (less than 15 min from sampling to final result), and allows accurate detection of even small changes in aquatic environments (detection above 3% change). Demonstrative studies on (a) indigenous bacterial growth in water, (b) contamination of drinking water with wastewater, (c) household drinking water stagnation and (d) mixing of two drinking water types, univocally showed that this FCM approach enables detection and quantification of relevant bacterial water quality changes with high sensitivity. This approach has the potential to be used as a new tool for application in the drinking water field, e.g. for rapid screening of the microbial water quality and stability during water treatment and distribution in networks and premise plumbing. Copyright © 2013 Elsevier Ltd. All rights reserved.

Cytochemistry and C-values: The Less-well-known World of Nuclear DNA Amounts

PubMed Central

Greilhuber, J.

2008-01-01

Background In the plant sciences there are two widely applied technologies for measuring nuclear DNA content: Feulgen absorbance cytophotometry and flow cytometry (FCM). While FCM is, with good reasons, increasingly popular among plant scientists, absorbance-cytophotometric techniques lose ground. This results in a narrowing of the methodological repertoire, which is neither desirable nor beneficial. Both approaches have their advantages, but static cytophotometry seems to pose more instrumental difficulties and material-based problems than FCM, so that Feulgen-based data in the literature are often less reliable than one would expect. Scope The purpose of this article is to present a selective overview of the field of nuclear DNA content measurement, and C-values in particular, with a focus on the technical difficulties imposed by the characteristics of the biological material and with some comments on the photometrical aspects of the work. For over 20 years it has been known that plant polyphenols cause problems in Feulgen DNA cytophotometry, since they act as major staining inhibitors leading to unreliable results. However, little information is available about the chemical classes of plant metabolites capable of DNA staining interference and the mechanisms of their inhibition. Plant slimes are another source of concern. Conclusions In FCM research to uncover the effects of secondary metabolites on measurement results has begun only recently. In particular, the analysis of intraspecific genome size variation demands a stringent methodology which accounts for inhibitors. FCM tests for inhibitory effects of endogenous metabolites should become obligatory. The use of dry seeds for harvesting embryo and endosperm nuclei for FCM and Feulgen densitometry may often provide a means of circumventing staining inhibitors. The importance of internal standardization is highlighted. Our goal is a better understanding of phytochemical/cytochemical interactions in plant DNA photometry for the benefit of an ever-growing list of plant genome sizes. PMID:17951594

Practical way to develop 10-color flow cytometry protocols for the clinical laboratory

NASA Astrophysics Data System (ADS)

Tárnok, Attila; Bocsi, Jozsef

2010-02-01

The latest development of commercial routine flow cytometers (FCM) is that they are equipped with three (blue, red, violet) or more lasers and many PMT detectors. Nowadays routine clinical instruments are capable of detecting 10 or more fluorescence colors simultaneously. Thereby, presenting opportunities for getting detailed information on the single cell level for cytomics and systems biology for improve diagnostics and monitoring of patients. The University Leipzig, Germany) recently started a cluster of excellence to study the molecular background of life style and environment associated diseases, enrolling 25000 individuals (LIFE). To this end the most comprehensive FCM protocol has to be developed for this study. We aimed to optimize fluorochrome and antibody combinations to the characteristics of the instrument for successful 10-color FCM. Systematic review of issues related to sampling, preparation, instrument settings, spillover and compensation matrix, reagent performance, and general principles of panel construction was performed. 10-color FCM enables for increased accuracy in cell subpopulation identification, the ability to obtain detailed information from blood specimens, improved laboratory efficiency, and the means to consistently detect major and rare cell populations. Careful attention to details of instrument and reagent performance allows for the development of panels suitable for screening of samples from healthy and diseased donors. The characteristics of this technique are particularly well suited for the analysis of broad human population cohorts and have the potential to reach the everyday practice in a standardized way for the clinical laboratory.

Rapid differentiation of methicillin-resistant and methicillin-susceptible Staphylococcus aureus by flow cytometry after brief antibiotic exposure.

PubMed

Shrestha, Nabin K; Scalera, Nikole M; Wilson, Deborah A; Procop, Gary W

2011-06-01

We noticed that methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) isolates yielded side-scatter (SSC) and fluorescence intensity (FI) differences on flow cytometry (FCM) following incubation in oxacillin broth. The purpose of this study was to determine whether MRSA and MSSA could be reliably differentiated by FCM. S. aureus isolates were incubated in oxacillin-containing Mueller-Hinton broth, stained using the FASTEST total viable organisms kit, and analyzed by FCM in the MicroPRO instrument. SSC versus FI were examined, and gates 1 and 2 were defined to encompass the majority of MSSA and MRSA signal events, respectively. A count ratio (CR) was defined as the ratio of counts in gate 2 to those in gate 1. Initially, 33 isolates were tested after 4 h of incubation for proof-of-concept. Twenty others were then tested after incubation intervals ranging from 30 min to 4 h to determine the earliest possible time for differentiation. Next, 100 separate isolates were tested to determine the best CR cutoff value. Finally, the CR was validated by using an independent cohort of 121 isolates. We noted that MRSA isolates had higher SSC and FI readings than did MSSA isolates after 2 h of incubation. The receiver-operator characteristics curve showed that a CR cutoff of 0.0445 reliably differentiated MRSA from MSSA. In the validation cohort, this cutoff had a sensitivity of 100% and a specificity of 98.7% for identifying MRSA from among S. aureus isolates, following 2 h of incubation. This study demonstrates that MRSA and MSSA can be accurately differentiated by FCM after 2 h of incubation in an oxacillin-containing liquid culture medium.

Rapid Differentiation of Methicillin-Resistant and Methicillin-Susceptible Staphylococcus aureus by Flow Cytometry after Brief Antibiotic Exposure▿

PubMed Central

Shrestha, Nabin K.; Scalera, Nikole M.; Wilson, Deborah A.; Procop, Gary W.

2011-01-01

We noticed that methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) isolates yielded side-scatter (SSC) and fluorescence intensity (FI) differences on flow cytometry (FCM) following incubation in oxacillin broth. The purpose of this study was to determine whether MRSA and MSSA could be reliably differentiated by FCM. S. aureus isolates were incubated in oxacillin-containing Mueller-Hinton broth, stained using the FASTEST total viable organisms kit, and analyzed by FCM in the MicroPRO instrument. SSC versus FI were examined, and gates 1 and 2 were defined to encompass the majority of MSSA and MRSA signal events, respectively. A count ratio (CR) was defined as the ratio of counts in gate 2 to those in gate 1. Initially, 33 isolates were tested after 4 h of incubation for proof-of-concept. Twenty others were then tested after incubation intervals ranging from 30 min to 4 h to determine the earliest possible time for differentiation. Next, 100 separate isolates were tested to determine the best CR cutoff value. Finally, the CR was validated by using an independent cohort of 121 isolates. We noted that MRSA isolates had higher SSC and FI readings than did MSSA isolates after 2 h of incubation. The receiver-operator characteristics curve showed that a CR cutoff of 0.0445 reliably differentiated MRSA from MSSA. In the validation cohort, this cutoff had a sensitivity of 100% and a specificity of 98.7% for identifying MRSA from among S. aureus isolates, following 2 h of incubation. This study demonstrates that MRSA and MSSA can be accurately differentiated by FCM after 2 h of incubation in an oxacillin-containing liquid culture medium. PMID:21471343

IP-FCM measures physiologic protein-protein interactions modulated by signal transduction and small-molecule drug inhibition.

PubMed

Smith, Stephen E P; Bida, Anya T; Davis, Tessa R; Sicotte, Hugues; Patterson, Steven E; Gil, Diana; Schrum, Adam G

2012-01-01

Protein-protein interactions (PPI) mediate the formation of intermolecular networks that control biological signaling. For this reason, PPIs are of outstanding interest in pharmacology, as they display high specificity and may represent a vast pool of potentially druggable targets. However, the study of physiologic PPIs can be limited by conventional assays that often have large sample requirements and relatively low sensitivity. Here, we build on a novel method, immunoprecipitation detected by flow cytometry (IP-FCM), to assess PPI modulation during either signal transduction or pharmacologic inhibition by two different classes of small-molecule compounds. First, we showed that IP-FCM can detect statistically significant differences in samples possessing a defined PPI change as low as 10%. This sensitivity allowed IP-FCM to detect a PPI that increases transiently during T cell signaling, the antigen-inducible interaction between ZAP70 and the T cell antigen receptor (TCR)/CD3 complex. In contrast, IP-FCM detected no ZAP70 recruitment when T cells were stimulated with antigen in the presence of the src-family kinase inhibitor, PP2. Further, we tested whether IP-FCM possessed sufficient sensitivity to detect the effect of a second, rare class of compounds called SMIPPI (small-molecule inhibitor of PPI). We found that the first-generation non-optimized SMIPPI, Ro-26-4550, inhibited the IL-2:CD25 interaction detected by IP-FCM. This inhibition was detectable using either a recombinant CD25-Fc chimera or physiologic full-length CD25 captured from T cell lysates. Thus, we demonstrate that IP-FCM is a sensitive tool for measuring physiologic PPIs that are modulated by signal transduction and pharmacologic inhibition.

Comparison of quantitative flow cytometric data provided by panels with lower and increased color number

NASA Astrophysics Data System (ADS)

Bocsi, József; Mittag, Anja; Pierzchalski, Arkadiusz; Baumgartner, Adolf; Dähnert, Ingo; Tárnok, Attila

2012-03-01

To date the flow cytometry (FCM) industry is booming with new generations of commercial clinical instruments. Long-term clinical studies have the dilemma that moving to new instruments being capable of more complex cell-analysis makes it difficult to compare new data with those obtained on older instruments with less complex analysis panels. Since 15 years we conduct follow-up studies on children with congenital heart diseases. In this period we moved from 2- to 3- and now to 10-color FCM immunophenotyping panels. Questions arise how to compare and transfer data from lower to higher level of complexity. Two comparable antibody panels for leukocyte immunophenotyping (12-tube 2-colors, and 9-tube 4-colors) were measured on a BD FACScalibur FCM (calibration: Spherotech beads) in 19 blood samples from children with congenital heart disease. This increase of colors was accompanied by moving antibodies that were in the 2-color panel either FITC or PE labeled to red dyes such as PerCP or APC. Algorithms were developed for bridging data for quantitative characterization of antigen expression (mean fluorescence intensity) and frequency of different cell subpopulations in combination with rainbow bead standard data. This approach worked for the most relevant antibodies (CD3, CD4, CD8 etc.) well, but rendered substantial uncertainty for activation markers (CD69 etc.). Our techniques are particularly well suited to the analysis in long-term studies and have the potential to compare older and recent results in a standardized way.

Interphase cytogenetics of prostatic carcinoma in fine needle aspirate smears of radical prostatectomy specimens: A practical screening tool?

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

Wang, R.Y.; Troncoso, P.; El-Naggar, A.K.

1994-09-01

Identification of chromosomal aberrations that may be used for diagnostic or prognostic evaluation of prostatic adenocarcinoma has been the subject of great interest. In a previous study, we applied the fluorescence in situ hybridization (FISH) method on paraffin-embedded material to show that trisomy 7 was associated with the progression of human prostate cancer. In this study, we attempted to assess the utility of the FISH technique in detecting aneuploidy in fine needle aspirate (FNA) smears of prostatic tissues and to compare FISH results with that of DNA flow cytometry (FCM). Paired samples of normal and tumor FNA smears were obtainedmore » from 10 radical prostatectomy specimens. Dual-color chromosomes 7 and 9-specific centromeric DNA probes were used for FISH. FISH analysis demonstrated increased frequencies of trisomy 7 cells in all 10 tumors studied when compared with the paired normals. In contrast, 6 of 10 tumors were determined to be diploid by FCM. Our results show that FNA of radical prostatectomy specimens is a practical method for obtaining suitable material for both FISH and FCM analyses of prostate carcinoma. Thus, interphase FISH may be a practical screening tool to determine aneuploidy in FNA smears of prostatic carcinoma.« less

Characterization of the Population of the Sulfur-Oxidizing Symbiont of Codakia orbicularis (Bivalvia, Lucinidae) by Single-Cell Analyses▿ †

PubMed Central

Caro, Audrey; Gros, Olivier; Got, Patrice; De Wit, Rutger; Troussellier, Marc

2007-01-01

We investigated the characteristics of the sulfur-oxidizing symbiont hosted in the gills of Codakia orbicularis, a bivalve living in shallow marine tropical environments. Special attention was paid to describing the heterogeneity of the population by using single-cell approaches including flow cytometry (FCM) and different microscopic techniques and by analyzing a cell size fractionation experiment. Up to seven different subpopulations were distinguished by FCM based on nucleic acid content and light side scattering of the cells. The cell size analysis of symbionts showed that the symbiotic population was very heterogeneous in size, i.e., ranging from 0.5 to 5 μm in length, with variable amounts of intracellular sulfur. The side-scatter signal analyzed by FCM, which is often taken as a proxy of cell size, was greatly influenced by the sulfur content of the symbionts. FCM revealed an important heterogeneity in the relative nucleic acid content among the subclasses. The larger cells contained exceptionally high levels of nucleic acids, suggesting that these cells contained multiple copies of their genome, i.e., ranging from one copy for the smaller cells to more than four copies for the larger cells. The proportion of respiring symbionts (5-cyano-2,3-ditolyl-terazolium chloride positive) in the bacteriocytes of Codakia revealed that around 80% of the symbionts hosted by Codakia maintain respiratory activity throughout the year. These data allowed us to gain insight into the functioning of the symbionts within the host and to propose some hypotheses on how the growth of the symbionts is controlled by the host. PMID:17259363

Characterisation of a major phytoplankton bloom in the River Thames (UK) using flow cytometry and high performance liquid chromatography.

PubMed

Moorhouse, H L; Read, D S; McGowan, S; Wagner, M; Roberts, C; Armstrong, L K; Nicholls, D J E; Wickham, H D; Hutchins, M G; Bowes, M J

2018-05-15

Recent river studies have observed rapid phytoplankton dynamics, driven by diurnal cycling and short-term responses to storm events, highlighting the need to adopt new high-frequency characterisation methods to understand these complex ecological systems. This study utilised two such analytical methods; pigment analysis by high performance liquid chromatography (HPLC) and cell counting by flow cytometry (FCM), alongside traditional chlorophyll spectrophotometry and light microscopy screening, to characterise the major phytoplankton bloom of 2015 in the River Thames, UK. All analytical techniques observed a rapid increase in chlorophyll a concentration and cell abundances from March to early June, caused primarily by a diatom bloom. Light microscopy identified a shift from pennate to centric diatoms during this period. The initial diatom bloom coincided with increased HPLC peridinin concentrations, indicating the presence of dinoflagellates which were likely to be consuming the diatom population. The diatom bloom declined rapidly in early June, coinciding with a storm event. There were low chlorophyll a concentrations (by both HPLC and spectrophotometric methods) throughout July and August, implying low biomass and phytoplankton activity. However, FCM revealed high abundances of pico-chlorophytes and cyanobacteria through July and August, showing that phytoplankton communities remain active and abundant throughout the summer period. In combination, these techniques are able to simultaneously characterise a wider range of phytoplankton groups, with greater certainty, and provide improved understanding of phytoplankton functioning (e.g. production of UV inhibiting pigments by cyanobacteria in response to high light levels) and ecological status (through examination of pigment degradation products). Combined HPLC and FCM analyses offer rapid and cost-effective characterisation of phytoplankton communities at appropriate timescales. This will allow a more-targeted use of light microscopy to capture phytoplankton peaks or to investigate periods of rapid community succession. This will lead to greater system understanding of phytoplankton succession in response to biogeochemical drivers. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

Microparticle Analysis in Disorders of Hemostasis and Thrombosis

PubMed Central

Mooberry, Micah J.; Key, Nigel S.

2015-01-01

Microparticles (MPs) are submicron vesicles released from the plasma membrane of eukaryotic cells in response to activation or apoptosis. MPs are known to be involved in numerous biologic processes, including inflammation, the immune response, cancer metastasis, and angiogenesis. Their earliest recognized and most widely accepted role, however, is the ability to promote and support the process of blood coagulation. Consequently, there is ongoing interest in studying MPs in disorders of hemostasis and thrombosis. Both phosphatidylserine (PS) exposure and the presence of tissue factor (TF) in the MP membrane may account for their procoagulant properties, and elevated numbers of MPs in plasma have been reported in numerous prothrombotic conditions. To date, however, there are few data on true causality linking MPs to the genesis of thrombosis. A variety of methodologies have been employed to characterize and quantify MPs, although detection is challenging due to their submicron size. Flow cytometry (FCM) remains the most frequently utilized strategy for MP detection; however, it is associated with significant technological limitations. Additionally, pre-analytical and analytical variables can influence the detection of MPs by FCM, rendering data interpretation difficult. Lack of methodologic standardization in MP analysis by FCM confounds the issue further, although efforts are currently underway to address this limitation. Moving forward, it will be important to address these technical challenges as a scientific community if we are to better understand the role that MPs play in disorders of hemostasis and thrombosis. PMID:25704723

Genome size of Alexandrium catenella and Gracilariopsis lemaneiformis estimated by flow cytometry

NASA Astrophysics Data System (ADS)

Du, Qingwei; Sui, Zhenghong; Chang, Lianpeng; Wei, Huihui; Liu, Yuan; Mi, Ping; Shang, Erlei; Zeeshan, Niaz; Que, Zhou

2016-08-01

Flow cytometry (FCM) technique has been widely applied to estimating the genome size of various higher plants. However, there is few report about its application in algae. In this study, an optimized procedure of FCM was exploited to estimate the genome size of two eukaryotic algae. For analyzing Alexandrium catenella, an important red tide species, the whole cell instead of isolated nucleus was studied, and chicken erythrocytes were used as an internal reference. The genome size of A. catenella was estimated to be 56.48 ± 4.14 Gb (1C), approximately nineteen times larger than that of human genome. For analyzing Gracilariopsis lemaneiformis, an important economical red alga, the purified nucleus was employed, and Arabidopsis thaliana and Chondrus crispus were used as internal references, respectively. The genome size of Gp. lemaneiformis was 97.35 ± 2.58 Mb (1C) and 112.73 ± 14.00 Mb (1C), respectively, depending on the different internal references. The results of this research will promote the related studies on the genomics and evolution of these two species.

Evaluation of slide based cytometry (SBC) for concentration measurements of fluorescent dyes in solution

NASA Astrophysics Data System (ADS)

Pierzchalski, Arkadiusz; Marecka, Monika; Müller, Hans-Willy; Bocsi, József; Tárnok, Attila

2009-02-01

Flow cytometers (FCM) are built for particle measurements. In principle, concentration measurement of a homogeneous solution is not possible with FCM due to the lack of a trigger signal. In contrast to FCM slide based cytometry systems could act as tools for the measurement of concentrations using volume defined cell counting chambers. These chambers enable to analyze a well defined volume. Sensovation AG (Stockach, Germany) introduced an automated imaging system that combines imaging with cytometric features analysis. Aim of this study was to apply this imaging system to quantify the fluorescent molecule concentrations. The Lumisens (Sensovation AG) slide-based technology based on fluorescence digital imaging microscopy was used. The instrument is equipped with an inverted microscope, blue and red LEDs, double band-pass filters and a high-resolution cooled 16-bit digital camera. The instrument was focussed on the bottom of 400μm deep 6 chamber slides (IBIDI GmbH, Martinsried, Germany) or flat bottom 96 well plates (Greiner Bio One GmbH, Frickenhausen, Germany). Fluorescent solutions were imaged under 90% pixel saturation in a broad concentration range (FITC: 0.0002-250 μg/ml, methylene blue (MethB): 0.0002-250 μg/ml). Exposition times were recorded. Images were analysed by the iCys (CompuCyte Corp., Cambridge, MA, USA) image analysis software with the phantom contour function. Relative fluorescence intensities were calculated from mean fluorescence intensities per phantom contours divided by the exposition time. Solution concentrations could be distinguished over a broad dynamic range of 3.5 to 5.5 decades log (range FITC: 0.0002-31.25μg/ml, MethB: 0.0076-31.25μg/ml) with a good linear relationship between dye concentration and relative fluorescence intensity. The minimal number of fluorescent molecules per pixel as determined by the mean fluorescence intensity and the molecular weight of the fluorochrome were about 800 molecules FITC and ~2.000 MethB. The novel slide-based imaging system is suitable for detection of fluorescence differences over a broad range of concentrations. This approach may lead to novel assays for measuring concentration differences in cell free solutions and cell cultures e.g. in secretion assays.

Image and flow cytometric analysis of gold nanoparticle uptake by macrophages

NASA Astrophysics Data System (ADS)

Fixler, Dror; Ankri, Rinat; Weiss, Ronald; Grahnert, Anja; Melzer, Susanne; Tárnok, Attila

2016-03-01

Background/Aim: In atherosclerosis stable and vulnerable atherosclerotic plaque types are distinguished that behave differently concerning rupture, thrombosis and clinical events. The stable are rich in M2 macrophages. The unstable are rich in inflammatory M1 macrophages and are highly susceptible to rupture, setting patients at risk for thrombotic events when they undergo invasive diagnosis such as coronary angiography. Therefore, novel approaches for non-invasive detection and classification of vulnerable plaques in vivo are needed. Whereas classical approaches fail to differentiate between both plaque types, a new biophotonic method (combination of the diffusion reflection (DR) method with flow cytometry (FCM) or image cytometry (IC)) to analyze gold nanoparticle (GNP) loading of plaques could overcome this limitation. Methods: Two types of GNP were used three variants of gold nanorods (GNRI with 40x18 nm, II 65x25 nm and III 52x13 nm in size) and gold nanospheres (GNS with an average diameter of 18.5 nm). The GNS had an absorption peak at 520 nm and the GNR at 630 nm. Monocytes were isolated from human buffy blood samples, differentiated into macrophages and their subtypes and labelled with GNR and GNS for 3 and 24 h. GNS and GNR loading were determined by FCM and/or IC. Macrophages within tissue-like phantoms were analyzed by the DR system. Results: After GNR labelling of macrophages the FCM light scatter values increased up to 3.7 fold and the DR slope changed from an average slope of 0.196 (macrophages only) to an average slope of 0.827 (macrophages labelled with GNR). But, GNRIII did not present much higher DR slopes than the control phantoms, indicating that macrophages take up GNRIII in a lower amount than GNRI or II. IC and microscopy showed that all particle variants were taken up by the cells in a heterogeneous fashion. Conclusion and outlook: The combination of FCM and DR measurements provides a potential novel, highly sensitive and non-invasive method for the identification of atherosclerotic vulnerable plaques, aimed to develop a potential tool for in vivo tracking. Further experiments will show, if different macrophage subtypes (M1 or M2) take up the particles differently and may thereby serve to distinguish stable from vulnerable plaques.

Flow cytometry for immediate follow-up of drinking water networks after maintenance.

PubMed

Van Nevel, Sam; Buysschaert, Benjamin; De Roy, Karen; De Gusseme, Bart; Clement, Lieven; Boon, Nico

2017-03-15

Drinking water networks need maintenance every once in a while, either planned interventions or emergency repairs. When this involves opening of the water pipes, precautionary measures need to be taken to avoid contamination of the drinking water at all time. Drinking water suppliers routinely apply plating for faecal indicator organisms as quality control in such a situation. However, this takes at least 21 h of waiting time, which can be crucial when dealing with major supply pipes. A combination of flow cytometric (FCM) bacterial cell counts with FCM fingerprinting techniques is proposed in this study as a fast and sensitive additional technique. In three full scale situations, major supply pipes with 400-1050 mm diameter were emptied for maintenance, shock-chlorinated and flushed with large amounts of clean drinking water before taking back in operation. FCM measurements of the discharged flushing water revealed fast lowering and stabilizing bacterial concentrations once flushing is initiated. Immediate comparison with clean reference drinking water used for flushing was done, and the moment when both waters had similar bacterial concentrations was considered as the endpoint of the necessary flushing works. This was usually after 2-4 h of flushing. FCM fingerprinting, based on both bacteria and FCM background, was used as additional method to verify how similar flushing and reference samples were and yielded similar results. The FCM approved samples were several hours later approved as well by the drinking water supplier after plating and incubation for total Coliforms and Enterococci. These were used as decisive control to set the pipes back in operation. FCM proved to be a more conservative test than plating, yet it yielded immediate results. Application of these FCM methods can therefore avoid long unnecessary waiting times and large drinking water losses. Copyright © 2016 Elsevier Ltd. All rights reserved.

Host stress physiology and Trypanosoma haemoparasite infection influence innate immunity in the woylie (Bettongia penicillata).

PubMed

Hing, Stephanie; Currie, Andrew; Broomfield, Steven; Keatley, Sarah; Jones, Krista; Thompson, R C Andrew; Narayan, Edward; Godfrey, Stephanie S

2016-06-01

Understanding immune function is critical to conserving wildlife in view of infectious disease threats, particularly in threatened species vulnerable to stress, immunocompromise and infection. However, few studies examine stress, immune function and infection in wildlife. We used a flow cytometry protocol developed for human infants to assess phagocytosis, a key component of innate immunity, in a critically endangered marsupial, the woylie (Bettongia penicillata). The effects of stress physiology and Trypanosoma infection on phagocytosis were investigated. Blood and faecal samples were collected from woylies in a captive facility over three months. Trypanosoma status was determined using PCR. Faecal cortisol metabolites (FCM) were quantified by enzyme-immunoassay. Mean phagocytosis measured was >90%. An interaction between sex and FCM influenced the percentage of phagocytosing leukocytes, possibly reflecting the influence of sex hormones and glucocorticoids. An interaction between Trypanosoma status and FCM influenced phagocytosis index, suggesting that stress physiology and infection status influence innate immunity. Copyright © 2016 Elsevier Ltd. All rights reserved.

[Application and usefulness of flowcytometry in the haematology laboratory].

PubMed

Kubota, K; Makino, M

1991-02-01

Recent technological advances, in both hardware and software, and availability of various monoclonal antibodies (MoAb) for membrane antigens of blood cells have expanded the application of flow cytometry (FCM) in medicine. In the haematology laboratory, FCM has been used mainly for assessment of leukemia and lymphoma and for determination of lymphocyte subsets. In acute leukemia, FCM is useful to classify ALL accurately, particularly for bi phenotypic or mixed lineage leukemia. In lymphocyte subset determination, we found that the use of magnetic beads to remove contaminating monocytes and some granulocytes to purify the lymphocyte-population is helpful in clarify the subsets. We present data describing the age dependent variation in lymphocyte subsets in the pediatric population. In early life (up to 2 years old), CD4 (+) 2H4 (+) lymphocyte overwhelmed CD4 (+) 2H4 (-) cells, implying predominance of suppressor-inducer activity. We also presented some cases of markedly increased double negative T cells (gamma/delta TCR) and a rare case of double positive (CD4+, CD8+) T cells.

Visual selection and maintenance of the cell lines with high plant regeneration ability and low ploidy level in Dianthus acicularis by monitoring with flow cytometry analysis.

PubMed

Shiba, Tomonori; Mii, Masahiro

2005-12-01

Efficient plant regeneration system from cell suspension cultures was established in D. acicularis (2n=90) by monitoring ploidy level and visual selection of the cultures. The ploidy level of the cell cultures closely related to the shoot regeneration ability. The cell lines comprising original ploidy levels (2C+4C cells corresponding to DNA contents of G1 and G2 cells of diploid plant, respectively) showed high regeneration ability, whereas those containing the cells with 8C or higher DNA C-values showed low or no regeneration ability. The highly regenerable cell lines thus selected consisted of compact cell clumps with yellowish color and relatively moderate growth, suggesting that it is possible to select visually the highly regenerable cell lines with the original ploidy level. All the regenerated plantlets from the highly regenerable cell cultures exhibited normal phenotypes and no variations in ploidy level were observed by flow cytometry (FCM) analysis.

B cell increases and ex vivo IL-2 production as secondary endpoints for the detection of sensitizers in non-radioisotopic local lymph node assay using flow cytometry.

PubMed

Jung, Kyoung-Mi; Jang, Won-Hee; Lee, Yong-Kyoung; Yum, Young Na; Sohn, Soojung; Kim, Bae-Hwan; Chung, Jin-Ho; Park, Young-Ho; Lim, Kyung-Min

2012-03-25

Non-radioisotopic local lymph node assay (LLNA) using 5-bromo-2'-deoxyuridine (BrdU) with flow cytometry (FCM) is gaining attention since it is free from the regulatory issues in traditional LLNA (tLLNA) accompanying in vivo uses of radioisotope, (3)H-thymidine. However, there is also concern over compromised performance of non-radioisotopic LLNA, raising needs for additional endpoints to improve the accuracy. With the full 22 reference substances enlisted in OECD Test Guideline No. 429, we evaluated the performance of LLNA:BrdU-FCM along with the concomitant measurements of B/T cell ratio and ex vivo cytokine production from isolated lymph node cells (LNCs) to examine the utility of these markers as secondary endpoints. Mice (Balb/c, female) were topically treated with substances on both ears for 3 days and then, BrdU was intraperitoneally injected on day 5. After a day, lymph nodes were isolated and undergone FCM to determine BrdU incorporation and B/T cell sub-typing with B220+ and CD3e+. Ex vivo cytokine production by LNCs was measured such as IL-2, IL-4, IL-6, IL-12, IFN-γ, MCP-1, GM-CSF and TNFα. Mice treated with sensitizers showed preferential increases in B cell population and the selective production of IL-2, which matched well with the increases in BrdU incorporation. When compared with guinea pig or human data, BrdU incorporation, B cell increase and IL-2 production ex vivo could successfully identify sensitizers with the accuracy comparable to tLLNA, suggesting that these markers may be useful for improving the accuracy of LLNA:BrdU-FCM or as stand-alone non-radioisotopic endpoints. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

A multicolour flow cytometric assay for c-MYC protein in B-cell lymphoma.

PubMed

Alayed, Khaled; Schweitzer, Karen; Awadallah, Amad; Shetty, Shashirekha; Turakhia, Samir; Meyerson, Howard

2018-05-16

Develop an objective assay to detect c-MYC protein expression using multiparametric flow cytometry (FCM) as an alternative to immunohistochemistry (IHC). 57 patient samples and 11 cell line samples were evaluated. Cell suspensions were obtained and c-MYC staining was performed in combination with CD45 and CD19 and, in some samples, CD10. The percentage of c-MYC+ cells by FCM was correlated with the percentage determined by IHC. The relationship between c-MYC protein expression and the presence of a c-MYC gene rearrangement in aggressive and high-grade lymphomas was also assessed. c-MYC expression by FCM and IHC demonstrated a high degree of correlation in a training set of 33 patient cases, r=0.92, 11 cell line samples, r=0.81 and in a validation set of 24 aggressive and high-grade B-cell lymphomas, r=0.85. c-MYC gene was rearranged by fluorescence in situ hybridisation in 6/9 samples with high c-MYC expression (>40%) by FCM and 6/14 by IHC. We have developed a reliable multicolour FCM assay to detect c-MYC expression suitable for clinical laboratories that should be helpful to accurately quantify c-MYC expression in B-cell lymphomas. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

High-purity flow sorting of early meiocytes based on DNA analysis of guinea pig spermatogenic cells.

PubMed

Rodríguez-Casuriaga, Rosana; Geisinger, Adriana; Santiñaque, Federico F; López-Carro, Beatriz; Folle, Gustavo A

2011-08-01

Mammalian spermatogenesis is still nowadays poorly understood at the molecular level. Testis cellular heterogeneity is a major drawback for spermatogenic gene expression studies, especially when research is focused on stages that are usually very short and poorly represented at the cellular level such as initial meiotic prophase I (i.e., leptotene [L] and zygotene [Z]). Presumably, genes whose products are involved in critical meiotic events such as alignment, pairing and recombination of homologous chromosomes are expressed during the short stages of early meiotic prophase. Aiming to characterize mammalian early meiotic gene expression, we have found the guinea pig (Cavia porcellus) as an especially attractive model. A detailed analysis of its first spermatogenic wave by flow cytometry (FCM) and optical microscopy showed that guinea pig testes exhibit a higher representation of early meiotic stages compared to other studied rodents, partly because of their longer span, and also as a result of the increased number of cells entering meiosis. Moreover, we have found that adult guinea pig testes exhibit a peculiar 4C DNA content profile, with a bimodal peak for L/Z and P spermatocytes that is absent in other rodents. Besides, we show that this unusual 4C peak allows the separation by FCM of highly pure L/Z spermatocyte populations aside from pachytene ones, even from adult individuals. To our knowledge, this is the first report on an accurate and suitable method for highly pure early meiotic prophase cell isolation from adult mammals, and thus sets an interesting approach for gene expression studies aiming at a deeper understanding of the molecular groundwork underlying male gamete production. Copyright © 2011 International Society for Advancement of Cytometry.

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

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.

Endothelial Progenitor Cells (EPC) Count by Multicolor Flow Cytometry in Healthy Individuals and Diabetes Mellitus (DM) Patients.

PubMed

Falay, Mesude; Aktas, Server

2016-11-01

The present study aimed to determine circulating Endothelial Progenitor Cell (EPC) counts by multicolor flow cytometry in healthy individuals and diabetic subjects by means of forming an analysis procedure using a combination of monoclonal antibodies (moAbs), which would correctly detect the circulating EPC count. The circulating EPC count was detected in 40 healthy individuals (20 Female, 20 Male; age range: 26 - 50 years) and 30 Diabetes Mellitus (DM) patients (15 Female, 15 Male; age range: 42 - 55) by multicolor flow cytometry (FCM) in a single-tube panel consisting of 5 CD45/CD31/CD34/CD309/ SYTO® and 16 monoclonal antibodies. Circulating EPC count was 11.33 (7.89 - 15.25) cells/µL in the healthy control group and 4.80 (0.70 - 10.85) cells/µL in the DM group. EPC counts were significantly lower in DM cases that developed coronary artery disease (53.3%) as compared to those that did not (p < 0.001). In the present study, we describe a method that identifies circulating EPC counts by multicolor flow cytometry in a single tube and determines the circulating EPC count in healthy individuals. This is the first study conducted on EPC count in Turkish population. We think that the EPC count found in the present study will be a guide for future studies.

Metastatic pattern and DNA ploidy in stage IV breast cancer at initial diagnosis. Relation to response and survival.

PubMed

De Lena, M; Romero, A; Rabinovich, M; Leone, B; Vallejo, C; Machiavelli, M; Cuevas, M; Rodriguez, R; Lacava, J; Perez, J

1993-06-01

Sixty-nine patients with metastatic breast cancer (MBC) at initial diagnosis were analyzed to verify if metastatic pattern and clinical outcome are related to DNA ploidy determined by flow cytometry (FCM). Characteristics of 55 fully evaluable patients were as follows: median age: 61 years; postmenopausal: 75%; bone-only metastases (BM): 60%; extraosseous-only metastases (EM): 40%. Overall response rates (CR + PR) obtained with different chemotherapies and/or hormonal therapies were 58% and 68% for patients with BM and EM, respectively. Sixty percent of specimens resulted aneuploid, and the mean coefficient of variation of the complete series was 5.1%. In the whole group of patients DNA ploidy of primary tumor did not predict the metastatic pattern and had no influence upon response to treatment, duration of response, time to progression, and overall survival. When analyses were carried out according to metastatic pattern, those patients with BM showed similar results. However, within the group with EM, those with diploid tumors presented a significantly better survival (median 18 vs 13 months, p = .04). FCM-DNA analysis seems to identify a subgroup of patients with poor prognosis constituted by those who had aneuploid primary tumors and metastases to extraosseous sites.

Aneuploidy in benign tumors and nonneoplastic lesions of musculoskeletal tissues.

PubMed

Alho, A; Skjeldal, S; Pettersen, E O; Melvik, J E; Larsen, T E

1994-02-15

Aneuploidy in DNA flow cytometry (FCM) of musculoskeletal tumors is generally considered to be a sign of malignancy. Previously, giant cell tumor of the bone has been reported to contain aneuploid (near-diploid) DNA stemlines. Otherwise, only spordic cases have been reported. The authors wanted to study the relationships among DNA FCM, histology, and clinical course of nonmalignant musculoskeletal lesions. Twenty-eight histologically benign tumors and seven nonneoplastic lesions were subjected to DNA FCM: After tissue preparation mechanically and with ribonuclease and trypsin, the isolated nuclei were stained with propidium iodine using chicken and rainbow trout erythrocytes as controls. In the DNA FCM histograms, ploidy and cell cycle fractions were determined using a computerized mathematical model. The histologic diagnoses were made without knowledge of the DNA FCM results. Aneuploidy was found in eight lesions. A shoulder in the diploid peak, suggesting a diploid and a near-diploid population, was found in DNA histograms of a condensing osteitis of the clavicle (a benign inflammatory process) and of a giant cell tumor of bone. The latter lesion also had a tetraploid population. Six benign tumors--two enchondromas, one osteochondroma, one subcutaneous and one intramuscular lipoma, and a calcifying aponeurotic fibroma--showed clear aneuploidy with separate peaks. The S-phase fraction was less than 10% in all cases. The highest aneuploid population, DNA index = 1.70, in a subcutaneous lipoma, was small, with an undetectable S phase. Despite nonradical operations in seven lesions, no recurrences were observed during a median follow-up of 49 months (range, 28-73 months). Small aneuploid populations with low DNA synthetic activity may be compatible with a benign histologic picture and uneventful clinical course of the musculoskeletal lesion.

Assessing microbiological water quality in drinking water distribution systems with disinfectant residual using flow cytometry.

PubMed

Gillespie, Simon; Lipphaus, Patrick; Green, James; Parsons, Simon; Weir, Paul; Juskowiak, Kes; Jefferson, Bruce; Jarvis, Peter; Nocker, Andreas

2014-11-15

Flow cytometry (FCM) as a diagnostic tool for enumeration and characterization of microorganisms is rapidly gaining popularity and is increasingly applied in the water industry. In this study we applied the method to obtain a better understanding of total and intact cell concentrations in three different drinking water distribution systems (one using chlorine and two using chloramines as secondary disinfectants). Chloramine tended to result in lower proportions of intact cells than chlorine over a wider residual range, in agreement with existing knowledge that chloramine suppresses regrowth more efficiently. For chlorinated systems, free chlorine concentrations above 0.5 mg L(-1) were found to be associated with relatively low proportions of intact cells, whereas lower disinfectant levels could result in substantially higher percentages of intact cells. The threshold for chlorinated systems is in good agreement with guidelines from the World Health Organization. The fact that the vast majority of samples failing the regulatory coliform standard also showed elevated proportions of intact cells suggests that this parameter might be useful for evaluating risk of failure. Another interesting parameter for judging the microbiological status of water, the biological regrowth potential, greatly varied among different finished waters providing potential help for investment decisions. For its measurement, a simple method was introduced that can easily be performed by water utilities with FCM capability. Copyright © 2014 Elsevier Ltd. All rights reserved.

Appraisal of within- and between-laboratory reproducibility of non-radioisotopic local lymph node assay using flow cytometry, LLNA:BrdU-FCM: comparison of OECD TG429 performance standard and statistical evaluation.

PubMed

Yang, Hyeri; Na, Jihye; Jang, Won-Hee; Jung, Mi-Sook; Jeon, Jun-Young; Heo, Yong; Yeo, Kyung-Wook; Jo, Ji-Hoon; Lim, Kyung-Min; Bae, SeungJin

2015-05-05

Mouse local lymph node assay (LLNA, OECD TG429) is an alternative test replacing conventional guinea pig tests (OECD TG406) for the skin sensitization test but the use of a radioisotopic agent, (3)H-thymidine, deters its active dissemination. New non-radioisotopic LLNA, LLNA:BrdU-FCM employs a non-radioisotopic analog, 5-bromo-2'-deoxyuridine (BrdU) and flow cytometry. For an analogous method, OECD TG429 performance standard (PS) advises that two reference compounds be tested repeatedly and ECt(threshold) values obtained must fall within acceptable ranges to prove within- and between-laboratory reproducibility. However, this criteria is somewhat arbitrary and sample size of ECt is less than 5, raising concerns about insufficient reliability. Here, we explored various statistical methods to evaluate the reproducibility of LLNA:BrdU-FCM with stimulation index (SI), the raw data for ECt calculation, produced from 3 laboratories. Descriptive statistics along with graphical representation of SI was presented. For inferential statistics, parametric and non-parametric methods were applied to test the reproducibility of SI of a concurrent positive control and the robustness of results were investigated. Descriptive statistics and graphical representation of SI alone could illustrate the within- and between-laboratory reproducibility. Inferential statistics employing parametric and nonparametric methods drew similar conclusion. While all labs passed within- and between-laboratory reproducibility criteria given by OECD TG429 PS based on ECt values, statistical evaluation based on SI values showed that only two labs succeeded in achieving within-laboratory reproducibility. For those two labs that satisfied the within-lab reproducibility, between-laboratory reproducibility could be also attained based on inferential as well as descriptive statistics. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Generation of Monoclonal Antibodies against Immunoglobulin Proteins of the Domestic Ferret (Mustela putorius furo)

PubMed Central

2017-01-01

The domestic ferret (Mustela putorius furo) serves as an animal model for the study of several viruses that cause human disease, most notably influenza. Despite the importance of this animal model, characterization of the immune response by flow cytometry (FCM) is severely hampered due to the limited number of commercially available reagents. To begin to address this unmet need and to facilitate more in-depth study of ferret B cells including the identification of antibody-secreting cells, eight unique murine monoclonal antibodies (mAb) with specificity for ferret immunoglobulin (Ig) were generated using conventional B cell hybridoma technology. These mAb were screened for reactivity against ferret peripheral blood mononuclear cells by FCM and demonstrate specificity for CD79β+ B cells. Several of these mAb are specific for the light chain of surface B cell receptor (BCR) and enable segregation of kappa and lambda B cells. Additionally, a mAb that yielded surface staining of nearly all surface BCR positive cells (i.e., pan ferret Ig) was generated. Collectively, these MαF-Ig mAb offer advancement compared to the existing portfolio of polyclonal anti-ferret Ig detection reagents and should be applicable to a wide array of immunologic assays including the identification of antibody-secreting cells by FCM. PMID:28286781

Chemokine CCL28 induces apoptosis of decidual stromal cells via binding CCR3/CCR10 in human spontaneous abortion.

PubMed

Sun, Chan; Zhang, Yuan-Yuan; Tang, Chuan-Ling; Wang, Song-Cun; Piao, Hai-Lan; Tao, Yu; Zhu, Rui; Du, Mei-Rong; Li, Da-Jin

2013-10-01

Spontaneous abortion is the most common complication of pregnancy. Immune activation and the subsequent inflammation-induced tissue injury are often observed at the maternal-fetal interface as the final pathological assault in recurrent spontaneous abortion. However, the precise mechanisms responsible for spontaneous abortion involving inflammation are not fully understood. Chemokine CCL28 and its receptors CCR3 and CCR10 are important regulators in inflammatory process. Here, we examined the expression of CCL28 and its receptors in decidual stromal cells (DSCs) by immunochemistry and flow cytometry (FCM), and compared their expression level in DSCs from normal pregnancy versus spontaneous abortion, and their relationship to inflammatory cytokines production by DSCs. We further analyzed regulation of the pro-inflammatory cytokines on CCL28 expression in DSCs by real-time polymerase chain reaction, In-cell Western and FCM. The effects of CCL28-CCR3/CCR10 interaction on DSC apoptosis was investigated by Annexin V staining and FCM analysis or DAPI staining and nuclear morphology. Higher levels of the inflammatory cytokines interleukin (IL)-1β, IL-17A and tumor necrosis factor-α, and increased CCR3/CCR10 expression were observed in DSCs from spontaneous abortion compared with normal pregnancy. Treatment with inflammatory cytokines differently affected CCL28 and CCR3/CCR10 expression in DSCs. Human recombinant CCL28 promoted DSC apoptosis, which was eliminated by pretreatment with neutralizing antibodies against CCR3/CCR10 and CCL28. However, CCL28 did not affect DSC growth. These results suggest that the inflammation-promoted up-regulation of CCL28 and its receptors interaction in DSCs is involved in human spontaneous abortion via inducing DSC apoptosis.

Relationship between DNA ploidy and proliferative cell nuclear antigen index in canine hemangiopericytoma.

PubMed

Kang, Seong-Kwi; Park, Nam-Yong; Cho, Ho-Sung; Shin, Sung-Shik; Kang, Mun-Il; Kim, Sang-Ki; Hyun, Changbaig; Park, In-Chul; Kim, Jong-Tack; Jeong, Cheol; Park, Sung-Hee; Park, Su-Jin; Jeong, Jae-Ho; Kim, You-Jung; Ochiai, Kenji; Umemura, Takashi; Cho, Kyoung-Oh

2006-03-01

The mitotic index is reported to be correlated with recurrence, mean patient survival, and metastasis of canine hemangiopericytoma (CHP). However, to the authors' knowledge, studies investigating the parameters that can predict recurrence or metastasis of CHP with low mitotic index have not been done. To evaluate growth kinetics of CHP with low mitotic index, a retrospective analysis of the proliferative activity by antiproliferative cell nuclear antigen monoclonal antibody and DNA contents by flow cytometry (FCM) was performed with 21 formalin-fixed and paraffin-embedded CHP samples. Of the 21 tumors evaluated by FCM, 6 (26.6%) were aneuploid tumors, and 15 (71.4%) were diploid tumors. There was significant correlation between the PCNA index and ploidy pattern. The diploid group had 39.1 +/- 9.2 PCNA index, whereas the aneuploid group's proliferative cell nuclear antigen (PCNA) index was 63.1 +/- 8.2. The diploid group had mean mitotic index value of 1.140 +/- 0.855, and the aneuploid group had a mean value of 1.067 +/- 0.767. From these results, the CHP samples with low mitotic index were classified into either the aneuploid group with higher PCNA index or the diploid group with lower PCNA index, suggesting that DNA ploidy and proliferative activity may give an indication about malignancy of CHPs with a low mitotic index.

A new bias field correction method combining N3 and FCM for improved segmentation of breast density on MRI.

PubMed

Lin, Muqing; Chan, Siwa; Chen, Jeon-Hor; Chang, Daniel; Nie, Ke; Chen, Shih-Ting; Lin, Cheng-Ju; Shih, Tzu-Ching; Nalcioglu, Orhan; Su, Min-Ying

2011-01-01

Quantitative breast density is known as a strong risk factor associated with the development of breast cancer. Measurement of breast density based on three-dimensional breast MRI may provide very useful information. One important step for quantitative analysis of breast density on MRI is the correction of field inhomogeneity to allow an accurate segmentation of the fibroglandular tissue (dense tissue). A new bias field correction method by combining the nonparametric nonuniformity normalization (N3) algorithm and fuzzy-C-means (FCM)-based inhomogeneity correction algorithm is developed in this work. The analysis is performed on non-fat-sat T1-weighted images acquired using a 1.5 T MRI scanner. A total of 60 breasts from 30 healthy volunteers was analyzed. N3 is known as a robust correction method, but it cannot correct a strong bias field on a large area. FCM-based algorithm can correct the bias field on a large area, but it may change the tissue contrast and affect the segmentation quality. The proposed algorithm applies N3 first, followed by FCM, and then the generated bias field is smoothed using Gaussian kernal and B-spline surface fitting to minimize the problem of mistakenly changed tissue contrast. The segmentation results based on the N3+FCM corrected images were compared to the N3 and FCM alone corrected images and another method, coherent local intensity clustering (CLIC), corrected images. The segmentation quality based on different correction methods were evaluated by a radiologist and ranked. The authors demonstrated that the iterative N3+FCM correction method brightens the signal intensity of fatty tissues and that separates the histogram peaks between the fibroglandular and fatty tissues to allow an accurate segmentation between them. In the first reading session, the radiologist found (N3+FCM > N3 > FCM) ranking in 17 breasts, (N3+FCM > N3 = FCM) ranking in 7 breasts, (N3+FCM = N3 > FCM) in 32 breasts, (N3+FCM = N3 = FCM) in 2 breasts, and (N3 > N3+FCM > FCM) in 2 breasts. The results of the second reading session were similar. The performance in each pairwise Wilcoxon signed-rank test is significant, showing N3+FCM superior to both N3 and FCM, and N3 superior to FCM. The performance of the new N3+FCM algorithm was comparable to that of CLIC, showing equivalent quality in 57/60 breasts. Choosing an appropriate bias field correction method is a very important preprocessing step to allow an accurate segmentation of fibroglandular tissues based on breast MRI for quantitative measurement of breast density. The proposed algorithm combining N3+FCM and CLIC both yield satisfactory results.

Recombinant scFv antibodies against infectious pancreatic necrosis virus isolated by flow cytometry.

PubMed

Xu, Li-Ming; Zhao, Jing-Zhuang; Liu, Miao; Cao, Yong-Sheng; Yin, Jia-Sheng; Liu, Hong-Bai; Lu, Tongyan

2016-11-01

Infectious pancreatic necrosis is a significant disease of farmed salmonids in China. In this study, a single chain variable fragment (scFv) antibody library derived from rainbow trout (Oncorhynchus mykiss) and viral protein VP2 of a Chinese infectious pancreatic necrosis virus (IPNV) isolate ChRtm213 were co-expressed by a bacterial display technology. The library was subjected to three rounds of screening by flow cytometry (FCM) to select IPNV specific antibodies. Six antibody clones with different mean fluorescence intensities (MFI) were obtained by picking colonies at random. The antibody clones were expressed and purified. The purified IPNV-specific scFv antibodies were used successfully in Western blotting, enzyme linked immunosorbent assay (ELISA) and an immunofluorescence antibody test (IFAT). This method provides a high throughput means to screen an antibody library by flow cytometry, and isolate a panel of antibody that can be used as potential reagents for the detection and study of IPNV that are prevalent in China. Copyright © 2016 Elsevier B.V. All rights reserved.

Hyperspectral Image Classification for Land Cover Based on an Improved Interval Type-II Fuzzy C-Means Approach

PubMed Central

Li, Zhao-Liang

2018-01-01

Few studies have examined hyperspectral remote-sensing image classification with type-II fuzzy sets. This paper addresses image classification based on a hyperspectral remote-sensing technique using an improved interval type-II fuzzy c-means (IT2FCM*) approach. In this study, in contrast to other traditional fuzzy c-means-based approaches, the IT2FCM* algorithm considers the ranking of interval numbers and the spectral uncertainty. The classification results based on a hyperspectral dataset using the FCM, IT2FCM, and the proposed improved IT2FCM* algorithms show that the IT2FCM* method plays the best performance according to the clustering accuracy. In this paper, in order to validate and demonstrate the separability of the IT2FCM*, four type-I fuzzy validity indexes are employed, and a comparative analysis of these fuzzy validity indexes also applied in FCM and IT2FCM methods are made. These four indexes are also applied into different spatial and spectral resolution datasets to analyze the effects of spectral and spatial scaling factors on the separability of FCM, IT2FCM, and IT2FCM* methods. The results of these validity indexes from the hyperspectral datasets show that the improved IT2FCM* algorithm have the best values among these three algorithms in general. The results demonstrate that the IT2FCM* exhibits good performance in hyperspectral remote-sensing image classification because of its ability to handle hyperspectral uncertainty. PMID:29373548

The CD4+/CD8+ Ratio in Pulmonary Tuberculosis: Systematic and Meta-Analysis Article.

PubMed

Yin, Yongmei; Qin, Jie; Dai, Yaping; Zeng, Fanwei; Pei, Hao; Wang, Jun

2015-02-01

The ratio of CD4+/CD8+ has been used as a clinically index to evaluate patients' immunity. Numerous researchers have studied CD4+/CD8+ ratio in pulmonary tuberculosis (PTB) patients. However, the change of CD4+/CD8+ ratio remains controversial. We present a meta-analysis of 15 case-control studies to identify the change of CD4+/CD8+ ratio in PTB patients. We assessed heterogeneity of effect estimates within each group using I(2) test. Subgroup analysis was performed to explore the potential source of heterogeneity. To investigate further the potential publication bias, we visually examined the funnel plots. For robustness of results, we performed sensitivity analysis by removing studies. Data entry and analyses were carried out with RevMan 5.2 (The Nordic Cochrane Centre). Twelve peripheral blood studies were categorized into two subgroups. Eight studies presented a significant decrease of CD4+/CD8+ ratio in PTB cases compared to healthy subjects (SMD: -0.45; 95% CI -0.65--0.25; I(2) = 7%). Other four studies researched on the newly diagnosed patients presented a more seriously and significantly decrease (SMD: -2.17; 95% CI -2.61--1.74; I(2) = 37%). The pooled analysis of bronchoalveolar lavage fluid (BALF) studies showed a significant increase of CD4+/CD8+ ratio using Flow Cytometry (FCM) (SMD: 4.75; 95% CI 3.44-6.05; I(2) =0%). The present meta-analysis indicated that there was a synthetic evidence for the reduced CD4+/CD8+ ratio in peripheral blood of PTB patients, especially newly diagnosed cases. However, the CD4+/CD8+ ratio in BALF was increased using method of FCM.

Predictive capacity of a non-radioisotopic local lymph node assay using flow cytometry, LLNA:BrdU-FCM: Comparison of a cutoff approach and inferential statistics.

PubMed

Kim, Da-Eun; Yang, Hyeri; Jang, Won-Hee; Jung, Kyoung-Mi; Park, Miyoung; Choi, Jin Kyu; Jung, Mi-Sook; Jeon, Eun-Young; Heo, Yong; Yeo, Kyung-Wook; Jo, Ji-Hoon; Park, Jung Eun; Sohn, Soo Jung; Kim, Tae Sung; Ahn, Il Young; Jeong, Tae-Cheon; Lim, Kyung-Min; Bae, SeungJin

2016-01-01

In order for a novel test method to be applied for regulatory purposes, its reliability and relevance, i.e., reproducibility and predictive capacity, must be demonstrated. Here, we examine the predictive capacity of a novel non-radioisotopic local lymph node assay, LLNA:BrdU-FCM (5-bromo-2'-deoxyuridine-flow cytometry), with a cutoff approach and inferential statistics as a prediction model. 22 reference substances in OECD TG429 were tested with a concurrent positive control, hexylcinnamaldehyde 25%(PC), and the stimulation index (SI) representing the fold increase in lymph node cells over the vehicle control was obtained. The optimal cutoff SI (2.7≤cutoff <3.5), with respect to predictive capacity, was obtained by a receiver operating characteristic curve, which produced 90.9% accuracy for the 22 substances. To address the inter-test variability in responsiveness, SI values standardized with PC were employed to obtain the optimal percentage cutoff (42.6≤cutoff <57.3% of PC), which produced 86.4% accuracy. A test substance may be diagnosed as a sensitizer if a statistically significant increase in SI is elicited. The parametric one-sided t-test and non-parametric Wilcoxon rank-sum test produced 77.3% accuracy. Similarly, a test substance could be defined as a sensitizer if the SI means of the vehicle control, and of the low, middle, and high concentrations were statistically significantly different, which was tested using ANOVA or Kruskal-Wallis, with post hoc analysis, Dunnett, or DSCF (Dwass-Steel-Critchlow-Fligner), respectively, depending on the equal variance test, producing 81.8% accuracy. The absolute SI-based cutoff approach produced the best predictive capacity, however the discordant decisions between prediction models need to be examined further. Copyright © 2015 Elsevier Inc. All rights reserved.

Fluorescence triggering: A general strategy for enumerating and phenotyping extracellular vesicles by flow cytometry.

PubMed

Arraud, Nicolas; Gounou, Céline; Turpin, Delphine; Brisson, Alain R

2016-02-01

Plasma contains cell-derived extracellular vesicles (EVs) which participate in various physiopathological processes and have potential biomedical applications. Despite intense research activity, knowledge on EVs is limited mainly due to the difficulty of isolating and characterizing sub-micrometer particles like EVs. We have recently reported that a simple flow cytometry (FCM) approach based on triggering the detection on a fluorescence signal enabled the detection of 50× more Annexin-A5 binding EVs (Anx5+ EVs) in plasma than the conventional FCM approach based on light scattering triggering. Here, we present the application of the fluorescence triggering approach to the enumeration and phenotyping of EVs from platelet free plasma (PFP), focusing on CD41+ and CD235a+ EVs, as well as their sub-populations which bind or do not bind Anx5. Higher EV concentrations were detected by fluorescence triggering as compared to light scattering triggering, namely 40× for Anx5+ EVs, 75× for CD41+ EVs, and 15× for CD235a+ EVs. We found that about 30% of Anx5+ EVs were of platelet origin while only 3% of them were of erythrocyte origin. In addition, a majority of EVs from platelet and erythrocyte origin do not expose PS, in contrast to the classical theory of EV formation. Furthermore, the same PFP samples were analyzed fresh and after freeze-thawing, showing that freeze-thawing processes induce an increase, of about 35%, in the amount of Anx5+ EVs, while the other EV phenotypes remain unchanged. The method of EV detection and phenotyping by fluorescence triggering is simple, sensitive and reliable. We foresee that its application to EV studies will improve our understanding on the formation mechanisms and functions of EVs in health and disease and help the development of EV-based biomarkers. © 2015 International Society for Advancement of Cytometry.

Application of flow cytometry with a fluorescent dye to measurement of intracellular nitric oxide in plant cells.

PubMed

Kępczyński, Jan; Cembrowska-Lech, Danuta

2018-04-27

A simple and rapid method involving flow cytometry and NO-specific probe (DAF-FM DA) proved useful for detection and determination of intracellular NO production in Medicago truncatula suspension cells and leaves as well as in cells of Avena fatua, Amaranthus retroflexus embryos and leaves. The measurement of nitric oxide (NO) in plant material is important for examining the regulatory roles of endogenous NO in various physiological processes. The possibility of detecting and determining intracellular NO production by flow cytometry (FCM) with 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM DA), an NO-specific probe in Medicago truncatula cells in suspension and leaves as well as in cells of embryos and leaves of Avena fatua L. or Amaranthus retroflexus L. was explored. To detect and measure NO production by cell suspension or embryos and leaves, the recommended DAF-FM DA concentration is 5 or 10 µM, respectively, applied for 30 min. Exogenous NO increased the intensity of the fluorescent signal in embryos and leaves of both plants, while carboxy-PTIO (cPTIO), an NO scavenger, decreased it. Thus, these results demonstrate that NO can be detected and an increase and a decrease of its intracellular level can be estimated. Wounding was observed to increase the fluorescence signal, indicating an increase in the intracellular NO level. In addition, the levels of exogenous and endogenous ascorbic acid were demonstrated to have no effect on the NO-related fluorescence signal, indicating the signal's specificity only in relation with NO. The applicability of the proposed method for detection and determination of NO was confirmed (1) by in situ NO imaging in cell suspensions and (2) by determining the NO concentration in embryos and leaves using the Griess reagent. In view of the data obtained, FCM is recommended as a rapid and simple method with which to detect and determine intracellular NO production in plant cells.

Flow cytometric monitoring of bacterioplankton phenotypic diversity predicts high population-specific feeding rates by invasive dreissenid mussels.

PubMed

Props, Ruben; Schmidt, Marian L; Heyse, Jasmine; Vanderploeg, Henry A; Boon, Nico; Denef, Vincent J

2018-02-01

Species invasion is an important disturbance to ecosystems worldwide, yet knowledge about the impacts of invasive species on bacterial communities remains sparse. Using a novel approach, we simultaneously detected phenotypic and derived taxonomic change in a natural bacterioplankton community when subjected to feeding pressure by quagga mussels, a widespread aquatic invasive species. We detected a significant decrease in diversity within 1 h of feeding and a total diversity loss of 11.6 ± 4.1% after 3 h. This loss of microbial diversity was caused by the selective removal of high nucleic acid populations (29 ± 5% after 3 h). We were able to track the community diversity at high temporal resolution by calculating phenotypic diversity estimates from flow cytometry (FCM) data of minute amounts of sample. Through parallel FCM and 16S rRNA gene amplicon sequencing analysis of environments spanning a broad diversity range, we showed that the two approaches resulted in highly correlated diversity measures and captured the same seasonal and lake-specific patterns in community composition. Based on our results, we predict that selective feeding by invasive dreissenid mussels directly impacts the microbial component of the carbon cycle, as it may drive bacterioplankton communities toward less diverse and potentially less productive states. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

Quantification of green fluorescent protein by in vivo imaging, PCR, and flow cytometry: comparison of transgenic strains and relevance for fetal cell microchimerism.

PubMed

Fujiki, Yutaka; Tao, Kai; Bianchi, Diana W; Giel-Moloney, Maryann; Leiter, Andrew B; Johnson, Kirby L

2008-02-01

Animal models are increasingly being used for the assessment of fetal cell microchimerism in maternal tissue. We wished to determine the optimal transgenic mouse strain and analytic technique to facilitate the detection of rare transgenic microchimeric fetal cells amongst a large number of maternal wild-type cells. We evaluated two strains of mice transgenic for the enhanced green fluorescent protein (EGFP): a commercially available, commonly used strain (C57BL/6-Tg(ACTB-EGFP)10sb/J) (CAG) and a newly created strain (ROSA26-EGFP) using three different techniques: in vivo and ex vivo fluorescent imaging (for whole body and dissected organs, respectively), PCR amplification of gfp, and flow cytometry (FCM). By fluorescent imaging, organs from CAG mice were 10-fold brighter than organs from ROSA26-EGFP mice (P < 0.0001). By PCR, more transgene from CAG mice was detected compared to ROSA26-EGFP mice (P = 0.04). By FCM, ROSA26-EGFP cell fluorescence was more uniform than CAG cells. A greater proportion of cells from ROSA26-EGFP organs were positive for EGFP than cells from CAG organs, but CAG mice had a greater proportion of cells with the brightest fluorescent intensity. Each transgenic strain possesses characteristics that make it useful under specific experimental circumstances. The CAG mouse model is preferable when experiments require brighter cells, whereas ROSA26-EGFP is more appropriate when uniform or ubiquitous expression is more important than brightness. Investigators must carefully select the transgenic strain most suited to the experimental design to obtain the most consistent and reproducible data. In vivo imaging allows for phenotypic evaluation of whole animals and intact organs; however, we did not evaluate its utility for the detection of rare, fetal microchimeric cells in the maternal organs. Finally, while PCR amplification of a paternally inherited transgene does allow for the quantitative determination of rare microchimeric cells, FCM allows for both quantitative and qualitative evaluations of fetal cells at very high sensitivity in a plethora of maternal organs. (c) 2008 International Society for Analytical Cytology

Cost-minimization analysis favours intravenous ferric carboxymaltose over ferric sucrose or oral iron as preoperative treatment in patients with colon cancer and iron deficiency anaemia.

PubMed

Calvet, Xavier; Gené, Emili; ÀngelRuíz, Miquel; Figuerola, Ariadna; Villoria, Albert; Cucala, Mercedes; Mearin, Fermín; Delgado, Salvadora; Calleja, Jose Luis

2016-01-01

Ferric Carboxymaltose (FCM), Iron Sucrose (IS) and Oral Iron (OI) are alternative treatments for preoperative anaemia. To compare the cost implications, using a cost-minimization analysis, of three alternatives: FCM vs. IS vs. OI for treating iron-deficient anaemia before surgery in patients with colon cancer. Data from 282 patients with colorectal cancer and anaemia were obtained from a previous study. One hundred and eleven received FCS, 16 IS and 155 OI. Costs of intravenous iron drugs were obtained from the Spanish Regulatory Agency. Direct and indirect costs were obtained from the analytical accounting unit of the Hospital. In the base case mean costs per patient were calculated. Sensitivity analysis and probabilistic Monte Carlo simulation were performed. Total costs per patient were 1827® in the FCM group, 2312® in the IS group and 2101® in the OI group. Cost savings per patient for FCM treatment were 485® compared to IS and 274® compared to OI. A Monte Carlo simulation favoured the use of FCM in 84.7% and 84.4% of simulations when compared to IS and OI, respectively. FCM infusion before surgery reduced costs in patients with colon cancer and iron-deficiency anaemia when compared with OI and IS.

Systems biology and clinical cytomics: The 10th Leipziger Workshop and the 3rd International Workshop on Slide-Based Cytometry, Leipzig, Germany, April 2005.

PubMed

Tárnok, Attila; Valet, Günther K; Emmrich, Frank

2006-01-01

Despite very significant technical and software improvements in flow cytometry (FCM) since the 1980's, the demand for a cytometric technology combining both quantitative cell analysis and morphological documentation in Cytomics became evident. Improvements in microtechnology and computing permit nowadays similar quantitative and stoichiometric single cell-based high-throughput analyses by microscopic instruments, like Slide-Based Cytometry (SBC). SBC and related techniques offer unique tools to perform complex immunophenotyping, thereby enabling diagnostic procedures during early disease stages. Multicolor or polychromatic analysis of cells by SBC is of special importance not only as a cytomics technology platform but also because of low quantities of required reagents and biological material. The exact knowledge of the location of each cell on the slide permits repetitive restaining and reanalysis of specimens. Various separate measurements of the same specimen can be ultimately fused to one database increasing the information obtained per cell. Relocation and optical evaluation of cells as typical SBC feature, can be of integral importance for cytometric analysis, since artifacts can be excluded and morphology of measured cells can be documented. Progress in cell analytic: In the SBC, new horizons can be opened by the new techniques of structural and functional analysis with the high resolution from intracellular and membrane (confocal microscopy, nanoscopy, total internal fluorescence microscopy (TIRFM), and tissue level (tissomics), to organ and organism level (in vivo cytometry, optical whole body imaging). Predictive medicine aims at the detection of changes in patient's state prior to the manifestation of the disease or the complication. Such instances concern immune consequences of surgeries or noninfectious posttraumatic shock in intensive care patients or the pretherapeutic identification of high risk patients in cancer cytostatic therapy. Preventive anti-infectious or anti-shock therapy as well as curative chemotherapy in combination with stem cell transplantation may provide better survival chances for patient at concomitant cost containment. Predictive medicine-guided optimization of therapy could lead to individualized medicine that gives significant therapeutic effect and may lower or abrogate potential therapeutic side effects. The 10th Leipziger Workshop combined with the 3rd International Workshop on SBC aimed to offer new methods in Image- and Slide-Based Cytometry for solutions in clinical research. It moved towards practical applications in the clinics and the clinical laboratory. This development will be continued in 2006 at the upcoming Leipziger Workshop and the International Workshop on Slide-Based Cytometry.

Chromosomal aberrations and aneuploidy in oral potentially malignant lesions: distinctive features for tongue

PubMed Central

2011-01-01

Background The mucosae of the oral cavity are different at the histological level but appear all equally exposed to common genotoxic agents. As a result of this exposure, changes in the mucosal epithelia may develop giving rise to Oral Potentially Malignant Lesions (OPMLs), which with time may in turn progress to Oral Squamous Cell Carcinomas (OSCCs). Therefore, much effort should be devoted to identify features able to predict the likeliness of progression associated with an OPML. Such features may be helpful in assisting the clinician to establish both appropriate therapies and follow-up schedules. Here, we report a pilot study that compared the occurrence of DNA aneuploidy and chromosomal copy number aberrations (CNAs) in the OPMLs from different oral anatomical subsites. Methods Samples from histologically diagnosed OPMLs were processed for high resolution DNA flow cytometry (hr DNA-FCM) in order to determine the relative DNA content expressed by the DNA index (DI). Additionally, array-Comparative Genomic Hybridization (a-CGH) analysis was performed on DNA obtained from diploid nuclei suspensions directly. When aneuploid nuclei were detected, these were physically separated from diploid nuclei on the base of their DI values by means of a DNA-FCM-Sorter in order to improve the a-CGH analysis. Results Tongue OPMLs were more frequently associated with DNA aneuploidy and CNAs than OPMLs arising from all the other mucosal subsites. Conclusions We suggest that the follow-up and the management of the patients with tongue OPMLs should receive a distinctive special attention. Clearly, this hypothesis should be validated in a prospective clinical study. PMID:21995418

Synergetic effects of ultrasound and slightly acidic electrolyzed water against Staphylococcus aureus evaluated by flow cytometry and electron microscopy.

PubMed

Li, Jiao; Ding, Tian; Liao, Xinyu; Chen, Shiguo; Ye, Xingqian; Liu, Donghong

2017-09-01

This study evaluated the synergetic effects of ultrasound and slightly acidic electrolyzed water (SAEW) on the inactivation of Staphylococcus aureus using flow cytometry and electron microscopy. The individual ultrasound treatment for 10min only resulted in 0.36logCFU/mL reductions of S. aureus, while the SAEW treatment alone for 10min resulted in 3.06logCFU/mL reductions. The log reductions caused by combined treatment were enhanced to 3.68logCFU/mL, which were greater than the sum of individual treatments. This phenomenon was referred to as synergistic effects. FCM analysis distinguished live and dead cells as well as revealed dynamic changes in the physiological states of S. aureus after different treatments. The combined treatment greatly reduced the number of viable but nonculturable (VBNC) bacteria to 0.07%; in contrast, a single ultrasound treatment for 10min induced the formation of VBNC cells to 45.75%. Scanning and transmission electron microscopy analysis revealed that greater damage to the appearance and ultrastructure of S. aureus were achieved after combined ultrasound-SAEW treatment compared to either treatment alone. These results indicated that combining ultrasound with SAEW is a promising sterilization technology with potential uses for environmental remediation and food preservation. Copyright © 2016 Elsevier B.V. All rights reserved.

Fluorescence lifetime as a new parameter in analytical cytology measurements

NASA Astrophysics Data System (ADS)

Steinkamp, John A.; Deka, Chiranjit; Lehnert, Bruce E.; Crissman, Harry A.

1996-05-01

A phase-sensitive flow cytometer has been developed to quantify fluorescence decay lifetimes on fluorochrome-labeled cells/particles. This instrument combines flow cytometry (FCM) and frequency-domain fluorescence spectroscopy measurement principles to provide unique capabilities for making phase-resolved lifetime measurements, while preserving conventional FCM capabilities. Cells are analyzed as they intersect a high-frequency, intensity-modulated (sine wave) laser excitation beam. Fluorescence signals are processed by conventional and phase-sensitive signal detection electronics and displayed as frequency distribution histograms. In this study we describe results of fluorescence intensity and lifetime measurements on fluorescently labeled particles, cells, and chromosomes. Examples of measurements on intrinsic cellular autofluorescence, cells labeled with immunofluorescence markers for cell- surface antigens, mitochondria stains, and on cellular DNA and protein binding fluorochromes will be presented to illustrate unique differences in measured lifetimes and changes caused by fluorescence quenching. This innovative technology will be used to probe fluorochrome/molecular interactions in the microenvironment of cells/chromosomes as a new parameter and thus expand the researchers' understanding of biochemical processes and structural features at the cellular and molecular level.

Effect of serotonin on the expression of antigens and DNA levels in Yersinia pestis cells with different plasmid content

NASA Astrophysics Data System (ADS)

Klueva, Svetlana N.; Korsukov, Vladimir N.; Schukovskaya, Tatyana N.; Kravtsov, Alexander L.

2004-08-01

Using flow cytometry (FCM) the influence of exogenous serotonin on culture growth, DNA content and fluorescence intensity of cells binding FITC-labelled plague polyclonal immunoglobulins was studied in Yersinia pestis EV (pFra+, pCad+, pPst+), Yersinia pestis KM218 (pFra-, pCad-, pPst-), Yersinia pestis KM 216 (pFra-, pCad-, pPst+). The results have been obtained by FCM showed serotonin accelerated Yersinia pestis EV (pFra+, pCad+, pPst+), Yersinia pestis KM218 (pFra-, pCad-, pPst-) culture growth during cultivation in Hottinger broth pH 7.2 at 28°C at concentration of 10-5 M. The presence of 10-5 M serotonin in nutrient broth could modulate DNA content in 37°C growing population of plague microbe independently of their plasmid content. Serotonin have been an impact on the distribution pattern of the cells according to their phenotypical characteristics, which was reflected in the levels of population heterogeneity in the intensity of specific immunofluorescence determined by FMC.

Antifouling Coatings Influence both Abundance and Community Structure of Colonizing Biofilms: a Case Study in the Northwestern Mediterranean Sea

PubMed Central

Camps, Mercedes; Barani, Aude; Gregori, Gérald; Bouchez, Agnès; Le Berre, Brigitte; Bressy, Christine; Blache, Yves

2014-01-01

When immersed in seawater, substrates are rapidly colonized by both micro- and macroorganisms. This process is responsible for important economic and ecological prejudices, particularly when related to ship hulls or aquaculture nets. Commercial antifouling coatings are supposed to reduce biofouling, i.e., micro- and macrofoulers. In this study, biofilms that primarily settled on seven different coatings (polyvinyl chloride [PVC], a fouling release coating [FRC], and five self-polishing copolymer coatings [SPC], including four commercial ones) were quantitatively studied, after 1 month of immersion in summer in the Toulon Bay (Northwestern Mediterranean Sea, France), by using flow cytometry (FCM), microscopy, and denaturing gradient gel electrophoresis. FCM was used after a pretreatment to separate cells from the biofilm matrix, in order to determine densities of heterotrophic bacteria, picocyanobacteria, and pico- and nanoeukaryotes on these coatings. Among diatoms, the only microphytobenthic class identified by microscopy, Licmophora, Navicula, and Nitzschia were determined to be the dominant taxa. Overall, biocide-free coatings showed higher densities than all other coatings, except for one biocidal coating, whatever the group of microorganisms. Heterotrophic bacteria always showed the highest densities, and diatoms showed the lowest, but the relative abundances of these groups varied depending on the coating. In particular, the copper-free SPC failed to prevent diatom settlement, whereas the pyrithione-free SPC exhibited high picocyanobacterial density. These results highlight the interest in FCM for antifouling coating assessment as well as specific selection among microbial communities by antifouling coatings. PMID:24907329

Understanding the link between meteorology and speciated abundance of bioaerosols in an urban environment using colocated flow cytometry and real-time autofluorescence measurements

NASA Astrophysics Data System (ADS)

Negron Marty, Arnaldo; DeLeon-Rodriguez, Natasha; Waters, Samantha; Ziemba, Luke; Anderson, Bruce; Bergin, Michael; Konstantinidis, Kostas; Nenes, Athanasios

2017-04-01

The abundance and speciation of primary biological atmospheric particles (PBAP) has been of great interest due to their potential impact on human health, cloud formation and contribution to atmospheric nutrient deposition [1, 2]. During this study state-of-the-art sampling techniques and protocols have been developed and combined with the speciation of PBAP by flow cytometry (FCM). An effective FCM protocol has been developed to identify and quantify speciated bioaerosols populations. In addition, a Wideband Integrated Bioaerosol Sensor (WIBS) has been used to understand the temporal variability of the PBAP, by measuring the autofluorescence of the atmospheric particles [3]. The techniques developed here have been applied to understand the PBAP variability and abundance in downtown Atlanta under different meteorological conditions. FCM results show the presence of a low nucleic acid (LNA) and a high nucleic acid (HNA) content subpopulation. The contribution of each subpopulation to the total biological atmospheric particles (TBAP) varies depending on the predominant meteorological conditions. Results suggest the HNA subpopulation, named fungal spores, dominates the composition of the TBAP during humid and warm days after rain events. However, during dry episodes the HNA subpopulation is diminished and the LNA subpopulation dominates the composition of the TBAP in downtown Atlanta. WIBS size distribution shifts between dry periods and humid and warm periods agreed well with the LNA and HNA subpopulations behavior. Our finding suggests Atlanta average PBAP concentration is around 1-8 x 104 part. /m3during Spring, where WIBS represents the lower bound and FCM the upper bound of the quantification. Additional experiments performed with different types of pollen, fungi and bacteria were used to better understand the scattering and fluorescence properties of them under different growing phases. References: [1] Morris, C. E., F. Conen, J. Alex Huffman, V. Phillips, U. Poschl and D. C. Sands (2014). Bioprecipitation: a feedback cycle linking earth history, ecosystem dynamics and land use through biological ice nucleators in the atmosphere. Glob Chang Biol 20(2): 341-351. [2] Després, V. R., J. Alex Huffman, S. M. Burrows, C. Hoose, A. S. Safatov, G. Buryak, J., Fröhlich-Nowoisky, W. Elbert, M. O. Andreae, U. Pöschl and R. Jaenicke (2012). Primary biological aerosol particles in the atmosphere: a review. Tellus B 64(0). [3] Gabey, A. M., M. W. Gallagher, J. Whitehead, J. R. Dorsey, P. H. Kaye and W. R. Stanley (2010). Measurements and comparison of primary biological aerosol above and below a tropical forest canopy using a dual channel fluorescence spectrometer. Atmospheric Chemistry and Physics 10(10): 4453-4466.

The detailed analysis of the changes of murine dendritic cells (DCs) induced by thymic peptide

PubMed Central

Hu, Xiaofang; Zheng, Wei; Wang, Lu; Wan, Nan; Wang, Bing; Li, Weiwei; Hua, Hui; Hu, Xu; Shan, Fengping

2012-01-01

The aim of present research is to analyze the detailed changes of dendritic cells (DCs) induced by pidotimod(PTD). These impacts on DCs of both bone marrow derived DCs and established DC2.4 cell line were assessed with use of conventional scanning electron microscopy (SEM), flow cytometry (FCM), transmission electron microscopy (TEM), cytochemistry assay FITC-dextran, bio-assay and enzyme linked immunosorbent assay (ELISA). We demonstrated the ability of PTD to induce DC phynotypic and functional maturation as evidenced by higher expression of key surface molecules such as MHC II, CD80 and CD86. The functional tests proved the downregulation of ACP inside the DCs, occurred when phagocytosis of DCs decreased, with simultaneously antigen presentation increased toward maturation. Finally, PTD also stimulated production of more cytokine IL-12 and less TNF-α. Therefore it is concluded that PTD can markedly exert positive induction to murine DCs. PMID:22863756

Sperm quality assessments for endangered razorback suckers Xyrauchen Texanus

USGS Publications Warehouse

Jenkins, Jill A.; Eilts, Bruce E.; Guitreau, Amy M.; Figiel, Chester R.; Draugelis-Dale, Rassa O.; Tiersch, Terrence R.

2011-01-01

Flow cytometry (FCM) and computer-assisted sperm motion analysis (CASA) methods were developed and validated for use with endangered razorback suckers Xyrauchen texanus collected (n=64) during the 2006 spawning season. Sperm motility could be activated within osmolality ranges noted during milt collections (here 167–343 mOsm/kg). We hypothesized that sperm quality of milt collected into isoosmotic (302 mOsm/kg) or hyperosmotic (500 mOsm/kg) Hanks' balanced salt solution would not differ. Pre-freeze viabilities were similar between osmolalities (79%±6 (S.E.M.) and 76%±7); however, post-thaw values were greater in hyperosmotic buffer (27%±3 and 12%±2; P=0.0065), as was mitochondrial membrane potential (33%±4 and 13%±2; P=0.0048). Visual estimates of pre-freeze motility correlated with total (r=0.7589; range 23–82%) and progressive motility (r=0.7449) by CASA and were associated with greater viability (r=0.5985; Pr=-0.83; P=0.0116) and mitochondrial function (r=-0.91; P=0.0016). By FCM-based assessments of DNA integrity, whereby increased fluorochrome binding indicated more fragmentation, higher levels were negatively correlated with count (r=-0.77; Pr=-0.66; P=0.0004). Fragmentation was higher in isotonic buffer (P=0.0234). To increase reproductive capacity of natural populations, the strategy and protocols developed can serve as a template for use with other imperiled fish species, biomonitoring, and genome banking.

Qualitative and quantitative analysis of monomers in polyesters for food contact materials.

PubMed

Brenz, Fabrian; Linke, Susanne; Simat, Thomas

2017-02-01

Polyesters (PESs) are gaining more importance on the food contact material (FCM) market and the variety of properties and applications is expected to be wide. In order to acquire the desired properties manufacturers can combine several FCM-approved polyvalent carboxylic acids (PCAs) and polyols as monomers. However, information about the qualitative and quantitative composition of FCM articles is often limited. The method presented here describes the analysis of PESs with the identification and quantification of 25 PES monomers (10 PCA, 15 polyols) by HPLC with diode array detection (HPLC-DAD) and GC-MS after alkaline hydrolysis. Accurate identification and quantification were demonstrated by the analysis of seven different FCM articles made of PESs. The results explained between 97.2% and 103.4% w/w of the polymer composition whilst showing equal molar amounts of PCA and polyols. Quantification proved to be precise and sensitive with coefficients of variation (CVs) below 6.0% for PES samples with monomer concentrations typically ranging from 0.02% to 75% w/w. The analysis of 15 PES samples for the FCM market revealed the presence of five different PCAs and 11 different polyols (main monomers, co-monomers, non-intentionally added substances (NIAS)) showing the wide variety of monomers in modern PESs. The presented method provides a useful tool for commercial, state and research laboratories as well as for producers and distributors facing the task of FCM risk assessment. It can be applied for the identification and quantification of migrating monomers and the prediction of oligomer compositions from the identified monomers, respectively.

An image segmentation method based on fuzzy C-means clustering and Cuckoo search algorithm

NASA Astrophysics Data System (ADS)

Wang, Mingwei; Wan, Youchuan; Gao, Xianjun; Ye, Zhiwei; Chen, Maolin

2018-04-01

Image segmentation is a significant step in image analysis and machine vision. Many approaches have been presented in this topic; among them, fuzzy C-means (FCM) clustering is one of the most widely used methods for its high efficiency and ambiguity of images. However, the success of FCM could not be guaranteed because it easily traps into local optimal solution. Cuckoo search (CS) is a novel evolutionary algorithm, which has been tested on some optimization problems and proved to be high-efficiency. Therefore, a new segmentation technique using FCM and blending of CS algorithm is put forward in the paper. Further, the proposed method has been measured on several images and compared with other existing FCM techniques such as genetic algorithm (GA) based FCM and particle swarm optimization (PSO) based FCM in terms of fitness value. Experimental results indicate that the proposed method is robust, adaptive and exhibits the better performance than other methods involved in the paper.

An analysis of the health service efficiency and patient experience with two different intravenous iron preparations in a UK anaemia clinic.

PubMed

Wilson, Paul D; Hutchings, Adam; Jeans, Aruna; Macdougall, Iain C

2013-01-01

Historically, the Renal Unit at King's College Hospital used intravenous (IV) iron sucrose (IS) to treat iron deficiency anaemia in patients with chronic kidney disease who were not on dialysis (CKD-ND). As part of a service initiative to improve patient experience, new products were considered as alternatives. This study investigated the potential impact on patient experience and service costs by switching from IS to ferric carboxymaltose (FCM). A decision analytical model was used to calculate the impact of switching from IS to FCM for a cohort of CKD-ND patients. Service provision data were collected for 365 patients who received 600 mg IS within a 12 month period, creating the IS data set. The service provision data, along with a clinically relevant FCM administration protocol (stipulating total doses of 500 mg FCM), were used to calculate a corresponding theoretical data set for FCM for the same cohort of patients. The FCM protocol saved each patient two hospital visits and 2.66 hours of time (equating to approximately a saving of £36.21 in loss of earnings) and £19 in travel costs. Direct attributable costs for iron administration (which included drug, disposables, nursing staff, and hospital-provided patient transport costs) were £58,646 for IS vs £46,473 for FCM. Direct overhead costs (which included nursing preparation time, administration staff, clinic space, and consultant time costs) were £40,172 for the IS service vs £15,174 for the FCM service. Based on clinical experience with the products, this analysis assumes that 500 mg FCM is therapeutically equivalent to 600 mg IS. Consultant time costs are assumed to be the same between the two treatment groups. IV iron administration protocols and data are specific to King's College Hospital. The design is retrospective and changes to the management of the clinic, including service delivery optimization, may also affect real costs. FCM was associated with fewer hospital visits and reduced transport costs for CKD-ND patients receiving IV iron and has the potential to save 19-37% in service costs. Owing to increased administration efficiency, FCM can improve the overall patient experience while reducing the total cost of the King's College Hospital IV iron service for CKD-ND patients, compared with treatment with IS.

Flow cytometry and real-time quantitative PCR as tools for assessing plasmid persistence.

PubMed

Loftie-Eaton, Wesley; Tucker, Allison; Norton, Ann; Top, Eva M

2014-09-01

The maintenance of a plasmid in the absence of selection for plasmid-borne genes is not guaranteed. However, plasmid persistence can evolve under selective conditions. Studying the molecular mechanisms behind the evolution of plasmid persistence is key to understanding how plasmids are maintained under nonselective conditions. Given the current crisis of rapid antibiotic resistance spread by multidrug resistance plasmids, this insight is of high medical relevance. The conventional method for monitoring plasmid persistence (i.e., the fraction of plasmid-containing cells in a population over time) is based on cultivation and involves differentiating colonies of plasmid-containing and plasmid-free cells on agar plates. However, this technique is time-consuming and does not easily lend itself to high-throughput applications. Here, we present flow cytometry (FCM) and real-time quantitative PCR (qPCR) as alternative tools for monitoring plasmid persistence. For this, we measured the persistence of a model plasmid, pB10::gfp, in three Pseudomonas hosts and in known mixtures of plasmid-containing and -free cells. We also compared three performance criteria: dynamic range, resolution, and variance. Although not without exceptions, both techniques generated estimates of overall plasmid loss rates that were rather similar to those generated by the conventional plate count (PC) method. They also were able to resolve differences in loss rates between artificial plasmid persistence assays. Finally, we briefly discuss the advantages and disadvantages for each technique and conclude that, overall, both FCM and real-time qPCR are suitable alternatives to cultivation-based methods for routine measurement of plasmid persistence, thereby opening avenues for high-throughput analyses. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Effect of Ferric Carboxymaltose on Exercise Capacity in Patients With Chronic Heart Failure and Iron Deficiency.

PubMed

van Veldhuisen, Dirk J; Ponikowski, Piotr; van der Meer, Peter; Metra, Marco; Böhm, Michael; Doletsky, Artem; Voors, Adriaan A; Macdougall, Iain C; Anker, Stefan D; Roubert, Bernard; Zakin, Lorraine; Cohen-Solal, Alain

2017-10-10

Iron deficiency is common in patients with heart failure (HF) and is associated with reduced exercise capacity and poor outcomes. Whether correction of iron deficiency with (intravenous) ferric carboxymaltose (FCM) affects peak oxygen consumption [peak VO 2 ], an objective measure of exercise intolerance in HF, has not been examined. We studied patients with systolic HF (left ventricular ejection fraction ≤45%) and mild to moderate symptoms despite optimal HF medication. Patients were randomized 1:1 to treatment with FCM for 24 weeks or standard of care. The primary end point was the change in peak VO 2 from baseline to 24 weeks. Secondary end points included the effect on hematinic and cardiac biomarkers, quality of life, and safety. For the primary analysis, patients who died had a value of 0 imputed for 24-week peak VO 2 . Additional sensitivity analyses were performed to determine the impact of imputation of missing peak VO 2 data. A total of 172 patients with HF were studied and received FCM (n=86) or standard of care (control group, n=86). At baseline, the groups were well matched; mean age was 64 years, 75% were male, mean left ventricular ejection fraction was 32%, and peak VO 2 was 13.5 mL/min/kg. FCM significantly increased serum ferritin and transferrin saturation. At 24 weeks, peak VO 2 had decreased in the control group (least square means -1.19±0.389 mL/min/kg) but was maintained on FCM (-0.16±0.387 mL/min/kg; P =0.020 between groups). In a sensitivity analysis, in which missing data were not imputed, peak VO 2 at 24 weeks decreased by -0.63±0.375 mL/min/kg in the control group and by -0.16±0.373 mL/min/kg in the FCM group; P =0.23 between groups). Patients' global assessment and functional class as assessed by the New York Heart Association improved on FCM versus standard of care. Treatment with intravenous FCM in patients with HF and iron deficiency improves iron stores. Although a favorable effect on peak VO 2 was observed on FCM, compared with standard of care in the primary analysis, this effect was highly sensitive to the imputation strategy for peak VO 2 among patients who died. Whether FCM is associated with an improved outcome in these high-risk patients needs further study. URL: http://www.clinicaltrials.gov. Unique identifier: NCT01394562. © 2017 The Authors.

Effect of Ferric Carboxymaltose on Exercise Capacity in Patients With Chronic Heart Failure and Iron Deficiency

PubMed Central

Ponikowski, Piotr; van der Meer, Peter; Metra, Marco; Böhm, Michael; Doletsky, Artem; Voors, Adriaan A.; Macdougall, Iain C.; Anker, Stefan D.; Roubert, Bernard; Zakin, Lorraine; Cohen-Solal, Alain

2017-01-01

Background: Iron deficiency is common in patients with heart failure (HF) and is associated with reduced exercise capacity and poor outcomes. Whether correction of iron deficiency with (intravenous) ferric carboxymaltose (FCM) affects peak oxygen consumption [peak VO2], an objective measure of exercise intolerance in HF, has not been examined. Methods: We studied patients with systolic HF (left ventricular ejection fraction ≤45%) and mild to moderate symptoms despite optimal HF medication. Patients were randomized 1:1 to treatment with FCM for 24 weeks or standard of care. The primary end point was the change in peak VO2 from baseline to 24 weeks. Secondary end points included the effect on hematinic and cardiac biomarkers, quality of life, and safety. For the primary analysis, patients who died had a value of 0 imputed for 24-week peak VO2. Additional sensitivity analyses were performed to determine the impact of imputation of missing peak VO2 data. Results: A total of 172 patients with HF were studied and received FCM (n=86) or standard of care (control group, n=86). At baseline, the groups were well matched; mean age was 64 years, 75% were male, mean left ventricular ejection fraction was 32%, and peak VO2 was 13.5 mL/min/kg. FCM significantly increased serum ferritin and transferrin saturation. At 24 weeks, peak VO2 had decreased in the control group (least square means −1.19±0.389 mL/min/kg) but was maintained on FCM (−0.16±0.387 mL/min/kg; P=0.020 between groups). In a sensitivity analysis, in which missing data were not imputed, peak VO2 at 24 weeks decreased by −0.63±0.375 mL/min/kg in the control group and by −0.16±0.373 mL/min/kg in the FCM group; P=0.23 between groups). Patients’ global assessment and functional class as assessed by the New York Heart Association improved on FCM versus standard of care. Conclusions: Treatment with intravenous FCM in patients with HF and iron deficiency improves iron stores. Although a favorable effect on peak VO2 was observed on FCM, compared with standard of care in the primary analysis, this effect was highly sensitive to the imputation strategy for peak VO2 among patients who died. Whether FCM is associated with an improved outcome in these high-risk patients needs further study. Clinical Trial Registration: URL: http://www.clinicaltrials.gov. Unique identifier: NCT01394562. PMID:28701470

Eigenspace-based fuzzy c-means for sensing trending topics in Twitter

NASA Astrophysics Data System (ADS)

Muliawati, T.; Murfi, H.

2017-07-01

As the information and communication technology are developed, the fulfillment of information can be obtained through social media, like Twitter. The enormous number of internet users has triggered fast and large data flow, thus making the manual analysis is difficult or even impossible. An automated methods for data analysis is needed, one of which is the topic detection and tracking. An alternative method other than latent Dirichlet allocation (LDA) is a soft clustering approach using Fuzzy C-Means (FCM). FCM meets the assumption that a document may consist of several topics. However, FCM works well in low-dimensional data but fails in high-dimensional data. Therefore, we propose an approach where FCM works on low-dimensional data by reducing the data using singular value decomposition (SVD). Our simulations show that this approach gives better accuracies in term of topic recall than LDA for sensing trending topic in Twitter about an event.

Results of the randomized phase IIB ARCTIC trial of low-dose rituximab in previously untreated CLL.

PubMed

Howard, D R; Munir, T; McParland, L; Rawstron, A C; Milligan, D; Schuh, A; Hockaday, A; Allsup, D J; Marshall, S; Duncombe, A S; O'Dwyer, J L; Smith, A F; Longo, R; Varghese, A; Hillmen, P

2017-11-01

ARCTIC was a multicenter, randomized-controlled, open, phase IIB non-inferiority trial in previously untreated chronic lymphocytic leukemia (CLL). Conventional frontline therapy in fit patients is fludarabine, cyclophosphamide and rituximab (FCR). The trial hypothesized that including mitoxantrone with low-dose rituximab (FCM-miniR) would be non-inferior to FCR. A total of 200 patients were recruited to assess the primary end point of complete remission (CR) rates according to IWCLL criteria. Secondary end points were progression-free survival (PFS), overall survival (OS), overall response rate, minimal residual disease (MRD) negativity, safety and cost-effectiveness. The trial closed following a pre-planned interim analysis. At final analysis, CR rates were 76 FCR vs 55% FCM-miniR (adjusted odds ratio: 0.37; 95% confidence interval: 0.19-0.73). MRD-negativity rates were 54 FCR vs 44% FCM-miniR. More participants experienced serious adverse reactions with FCM-miniR (49%) compared to FCR (41%). There are no significant differences between the treatment groups for PFS and OS. FCM-miniR is not expected to be cost-effective over a lifetime horizon. In summary, FCM-miniR is less well tolerated than FCR with an inferior response and MRD-negativity rate and increased toxicity, and will not be taken forward into a confirmatory trial. The trial demonstrated that oral FCR yields high response rates compared to historical series with intravenous chemotherapy.

Development of a novel flow cytometric approach to evaluate fish sperm chromatin using fixed samples

USGS Publications Warehouse

Jenkins, Jill A.

2013-01-01

The integrity of the paternal DNA is essential for the accurate transmission of genetic information, yet fertilization is not inhibited by chromatin breakage. Some methods are available for the sensitive detection of DNA damage and can be applied in studies of environmental toxicology, carcinogenesis, aging, and assisted reproduction techniques in both clinical and experimental settings. Because semen samples obtained from remote locations undergo chromatin damage prior to laboratory assessment, the present study was undertaken to evaluate treatments for effective chromatin staining in the development of a DNA fragmentation assay using fixed milt from yellow perch (Perca flavescens). Similar to the sperm chromatin structure assay (SCSA), susceptibility of nuclear DNA to acid-induced denaturation was measured by flow cytometry (FCM). Use of 10% buffered formalin for milt fixation allowed easier peak discrimination than 4% paraformaldehyde. The effects of time and temperature of incubation in 0.08 N HCl were evaluated in order to determine the ideal conditions for promoting DNA decondensation and making strand breaks more available for staining and detection by FCM. The best results were obtained with incubation at 37°C for 1 minute, followed by cold propidium iodide staining for 30 minutes.

Clinical experience with ferric carboxymaltose in the treatment of cancer- and chemotherapy-associated anaemia

PubMed Central

Steinmetz, T.; Tschechne, B.; Harlin, O.; Klement, B.; Franzem, M.; Wamhoff, J.; Tesch, H.; Rohrberg, R.; Marschner, N.

2013-01-01

Background Intravenous (i.v.) iron can improve anaemia of chronic disease and response to erythropoiesis-stimulating agents (ESAs), but data on its use in practice and without ESAs are limited. This study evaluated effectiveness and tolerability of ferric carboxymaltose (FCM) in routine treatment of anaemic cancer patients. Patients and methods Of 639 patients enrolled in 68 haematology/oncology practices in Germany, 619 received FCM at the oncologist's discretion, 420 had eligible baseline haemoglobin (Hb) measurements, and 364 at least one follow-up Hb measurement. Data of transfused patients were censored from analysis before transfusion. Results The median total iron dose was 1000 mg per patient (interquartile range 600–1500 mg). The median Hb increase was comparable in patients receiving FCM alone (1.4 g/dl [0.2–2.3 g/dl; N = 233]) or FCM + ESA (1.6 g/dl [0.7–2.4 g/dl; N = 46]). Patients with baseline Hb up to 11.0 g/dl and serum ferritin up to 500 ng/ml benefited from FCM treatment (stable Hb ≥11.0 g/dl). Also patients with ferritin >500 ng/ml but low transferrin saturation benefited from FCM treatment. FCM was well tolerated, 2.3% of patients reported putative drug-related adverse events. Conclusions The substantial Hb increase and stabilisation at 11–12 g/dl in FCM-treated patients suggest a role for i.v. iron alone in anaemia correction in cancer patients. PMID:23071262

Antiproliferative effects of cinobufacini on human hepatocellular carcinoma HepG2 cells detected by atomic force microscopy

PubMed Central

Wu, Qing; Lin, Wei-Dong; Liao, Guan-Qun; Zhang, Li-Guo; Wen, Shun-Qian; Lin, Jia-Ying

2015-01-01

AIM: To investigate the antiproliferative activity of cinobufacini on human hepatocellular carcinoma HepG2 cells and the possible mechanism of its action. METHODS: HepG2 cells were treated with different concentrations of cinobufacini. Cell viability was measured by methylthiazolyl tetrazolium (MTT) assay. Cell cycle distribution was analyzed by flow cytometry (FCM). Cytoskeletal and nuclear alterations were observed by fluorescein isothiocyanate-phalloidin and DAPI staining under a laser scanning confocal microscope. Changes in morphology and ultrastructure of cells were detected by atomic force microscopy (AFM) at the nanoscale level. RESULTS: MTT assay indicated that cinobufacini significantly inhibited the viability of HepG2 cells in a dose-dependent manner. With the concentration of cinobufacini increasing from 0 to 0.10 mg/mL, the cell viability decreased from 74.9% ± 2.7% to 49.41% ± 2.2% and 39.24% ± 2.1% (P < 0.05). FCM analysis demonstrated cell cycle arrest at S phase induced by cinobufacini. The immunofluorescence studies of cytoskeletal and nuclear morphology showed that after cinobufacini treatment, the regular reorganization of actin filaments in HepG2 cells become chaotic, while the nuclei were not damaged seriously. Additionally, high-resolution AFM imaging revealed that cell morphology and ultrastructure changed a lot after treatment with cinobufacini. It appeared as significant shrinkage and deep pores in the cell membrane, with larger particles and a rougher cell surface. CONCLUSION: Cinobufacini inhibits the viability of HepG2 cells via cytoskeletal destruction and cell membrane toxicity. PMID:25624718

Scanning fluorescent microscopy is an alternative for quantitative fluorescent cell analysis.

PubMed

Varga, Viktor Sebestyén; Bocsi, József; Sipos, Ferenc; Csendes, Gábor; Tulassay, Zsolt; Molnár, Béla

2004-07-01

Fluorescent measurements on cells are performed today with FCM and laser scanning cytometry. The scientific community dealing with quantitative cell analysis would benefit from the development of a new digital multichannel and virtual microscopy based scanning fluorescent microscopy technology and from its evaluation on routine standardized fluorescent beads and clinical specimens. We applied a commercial motorized fluorescent microscope system. The scanning was done at 20 x (0.5 NA) magnification, on three channels (Rhodamine, FITC, Hoechst). The SFM (scanning fluorescent microscopy) software included the following features: scanning area, exposure time, and channel definition, autofocused scanning, densitometric and morphometric cellular feature determination, gating on scatterplots and frequency histograms, and preparation of galleries of the gated cells. For the calibration and standardization Immuno-Brite beads were used. With application of shading compensation, the CV of fluorescence of the beads decreased from 24.3% to 3.9%. Standard JPEG image compression until 1:150 resulted in no significant change. The change of focus influenced the CV significantly only after +/-5 microm error. SFM is a valuable method for the evaluation of fluorescently labeled cells. Copyright 2004 Wiley-Liss, Inc.

Fluorescence Confocal Microscopy for Ex Vivo Diagnosis of Conjunctival Tumors: A Pilot Study.

PubMed

Iovieno, Alfonso; Longo, Caterina; De Luca, Mariacarla; Piana, Simonetta; Fontana, Luigi; Ragazzi, Moira

2016-08-01

To evaluate the potential use of fluorescence confocal microscopy (FCM) for ex vivo diagnosis and excision margin assessment of conjunctival neoplasms. Validity study. setting: Single institution. Consecutive patients with clinically suspicious conjunctival lesions. Conjunctival lesions were excised in toto using a standard "no-touch technique" by a single surgeon (A.I.). Collected specimens were examined with a commercially available laser scanning fluorescence confocal microscope after immersion in a 0.6 mM solution of acridine orange dye for 10-20 seconds. Specimens were subsequently processed with standard histologic analysis. FCM diagnosis of the nature and extension of conjunctival lesions. Sixteen consecutive patients were included in the study (11 male, 5 female; mean age 58.1 ± 26.1 years, range 10-90 years). The median time needed to process and analyze a sample with FCM was 15 minutes. Eleven of 16 lesions were identified by FCM as squamous (2 benign papillomas, 2 grade 2 conjunctival intraepithelial neoplasias, 7 in situ squamous carcinomas) and 5 as nonsquamous (1 pingueculum, 1 dermolipoma, 2 melanocytic nevi, 1 melanoma). In all cases FCM was able to detect horizontal and vertical extension of the lesion. All FCM findings were confirmed by corresponding subsequent histologic examination. FCM provides a fast ex vivo preliminary diagnosis of suspicious conjunctival lesions with good histologic details and margin assessment, and may represent a novel tool for intraoperative and postsurgical management of conjunctival tumors. This is the first study to investigate ex vivo FCM application in ophthalmology. Copyright © 2016 Elsevier Inc. All rights reserved.

Effects of assimilable organic carbon and free chlorine on bacterial growth in drinking water.

PubMed

Liu, Xiaolu; Wang, Jingqi; Liu, Tingting; Kong, Weiwen; He, Xiaoqing; Jin, Yi; Zhang, Bolin

2015-01-01

Assimilable organic carbon (AOC) is one of the most important factors affecting the re-growth of microorganisms in drinking water. High AOC concentrations result in biological instability, but disinfection kills microbes to ensure the safety of drinking water. Free chlorine is an important oxidizing agent used during the disinfection process. Therefore, we explored the combined effects of AOC and free chlorine on bacterial growth in drinking water using flow cytometry (FCM). The initial AOC concentration was 168 μg.L(-1) in all water samples. Without free chlorine, the concentrations of intact bacteria increased but the level of AOC decreased. The addition of sodium hypochlorite caused an increase and fluctuation in AOC due to the oxidation of organic carbon. The concentrations of intact bacteria decreased from 1.1 × 10(5) cells.mL(-1) to 2.6 × 10(4) cells.mL(-1) at an initial free chlorine dose of 0.6 mg.L(-1) to 4.8 × 10(4) cells.mL(-1) at an initial free chlorine dose of 0.3 mg.L(-1) due to free chlorine originating from sodium hypochlorite. Additionally, free chlorine might be more obviously affected AOC concentrations than microbial growth did. These results suggested that AOC and free chlorine might have combined effects on microbial growth. In this study, our results showed concentrations determined by FCM were higher than those by HPC, which indicated that some E. coli detected by FCM might not be detected using HPC in drinking water. The level of free chlorine might restrain the consumption of AOC by inhibiting the growth of E. coli; on the other hand, chlorination might increase the level of AOC, thereby increase the potential for microbial growth in the drinking water network.

Effects of Assimilable Organic Carbon and Free Chlorine on Bacterial Growth in Drinking Water

PubMed Central

Liu, Tingting; Kong, Weiwen; He, Xiaoqing; Jin, Yi; Zhang, Bolin

2015-01-01

Assimilable organic carbon (AOC) is one of the most important factors affecting the re-growth of microorganisms in drinking water. High AOC concentrations result in biological instability, but disinfection kills microbes to ensure the safety of drinking water. Free chlorine is an important oxidizing agent used during the disinfection process. Therefore, we explored the combined effects of AOC and free chlorine on bacterial growth in drinking water using flow cytometry (FCM). The initial AOC concentration was 168 μg.L-1 in all water samples. Without free chlorine, the concentrations of intact bacteria increased but the level of AOC decreased. The addition of sodium hypochlorite caused an increase and fluctuation in AOC due to the oxidation of organic carbon. The concentrations of intact bacteria decreased from 1.1×105 cells.mL-1 to 2.6×104 cells.mL-1 at an initial free chlorine dose of 0.6 mg.L-1 to 4.8×104 cells.mL-1 at an initial free chlorine dose of 0.3 mg.L-1 due to free chlorine originating from sodium hypochlorite. Additionally, free chlorine might be more obviously affected AOC concentrations than microbial growth did. These results suggested that AOC and free chlorine might have combined effects on microbial growth. In this study, our results showed concentrations determined by FCM were higher than those by HPC, which indicated that some E. coli detected by FCM might not be detected using HPC in drinking water. The level of free chlorine might restrain the consumption of AOC by inhibiting the growth of E. coli; on the other hand, chlorination might increase the level of AOC, thereby increase the potential for microbial growth in the drinking water network. PMID:26034988

Long-Term Effects of Residual Chlorine on Pseudomonas aeruginosa in Simulated Drinking Water Fed With Low AOC Medium

PubMed Central

Mao, Guannan; Song, Yuhao; Bartlam, Mark; Wang, Yingying

2018-01-01

Residual chlorine is often required to remain present in public drinking water supplies during distribution to ensure water quality. It is essential to understand how bacteria respond to long-term chlorine exposure, especially with the presence of assimilable organic carbon (AOC). This study aimed to investigate the effects of chlorination on Pseudomonas aeruginosa in low AOC medium by both conventional plating and culture-independent methods including flow cytometry (FCM) and quantitative PCR (qPCR). In a simulated chlorinated system using a bioreactor, membrane damage and DNA damage were measured by FCM fluorescence fingerprint. The results indicated membrane permeability occurred prior to DNA damage in response to chlorination. A regrowth of P. aeruginosa was observed when the free chlorine concentration was below 0.3 mg/L. The bacterial response to long-term exposure to a constant low level of free chlorine (0.3 mg/L) was subsequently studied in detail. Both FCM and qPCR data showed a substantial reduction during initial exposure (0–16 h), followed by a plateau where the cell concentration remained stable (16–76 h), until finally all bacteria were inactivated with subsequent continuous chlorine exposure (76–124 h). The results showed three-stage inactivation kinetics for P. aeruginosa at a low chlorine level with extended exposure time: an initial fast inactivation stage, a relatively stable middle stage, and a final stage with a slower rate than the initial stage. A series of antibiotic resistance tests suggested long-term exposure to low chlorine level led to the selection of antibiotic-resistant P. aeruginosa. The combined results suggest that depletion of residual chlorine in low AOC medium systems could reactivate P. aeruginosa, leading to a possible threat to drinking water safety. PMID:29774019

[The Enhanceing effect of IL-12 on phagocytosis and killing of Mycobacterium tuberculosis by neutrophils in tuberculosis patients].

PubMed

Jiang, Li-na; Yao, Chun-yan; Jin, Qi-li; He, Wen-xin; Li, Bai-qing

2011-11-01

To explore the effects of IL-12 on phagocytosis and killing of Mycobacterium tuberculosis by neutrophils or polymorphonuclear cells (PMNs) in tuberculosis patients. The fresh peripheral blood samples from TB patients and healthy adults were incubated with M.tb labeled with FITC, and the percentages of phagocytosis of M.tb by PMNs was measured by flow cytometry (FCM). The fresh peripheral blood samples were incubated with DCFH-DA, and with or without M.tb for different times, the percentage of activation and the ROS production of PMNs were measured by FCM. Whole blood samples were pretreated with IL-12, the changes of phagocytosis, activation and ROS production of PMNs were measured by FCM. The percentages of phagocytosis by PMNs, activation and ROS production of PMNs in both TB patients and healthy adults increased dependent on the time of incubation with M.tb. Only the phagocytosis of M.tb by PMNs at 5 min in TB patients of tuberculosis patients (51.82±6.93)% was obviously higher than that in healthy adults (47.20±4.26)%, (P<0.05). Pretreatment of whole blood with IL-12 before incubation with M.tb, the percentages of phagocytosis, activation and ROS production of PMNs in both TB patients and healthy adults increased in dose dependent manner, but no significant difference was found between both groups. The results indicated that the phagocytosis of M.tb and ROS production by PMNs in TB patients were almost the same as that in healthy controls, except for phagocytosis is higher at early stage. Furthermore, IL-12 can enhance the responsiveness to the phagocytosis and ROS production of PMNs.

Handling and storage of human body fluids for analysis of extracellular vesicles

PubMed Central

Yuana, Yuana; Böing, Anita N.; Grootemaat, Anita E.; van der Pol, Edwin; Hau, Chi M.; Cizmar, Petr; Buhr, Egbert; Sturk, Auguste; Nieuwland, Rienk

2015-01-01

Because procedures of handling and storage of body fluids affect numbers and composition of extracellular vesicles (EVs), standardization is important to ensure reliable and comparable measurements of EVs in a clinical environment. We aimed to develop standard protocols for handling and storage of human body fluids for EV analysis. Conditions such as centrifugation, single freeze–thaw cycle, effect of time delay between blood collection and plasma preparation and storage were investigated. Plasma is the most commonly studied body fluid in EV research. We mainly focused on EVs originating from platelets and erythrocytes and investigated the behaviour of these 2 types of EVs independently as well as in plasma samples of healthy subjects. EVs in urine and saliva were also studied for comparison. All samples were analysed simultaneously before and after freeze–thawing by resistive pulse sensing, nanoparticle tracking analysis, conventional flow cytometry (FCM) and transmission (scanning) electron microscopy. Our main finding is that the effect of centrifugation markedly depends on the cellular origin of EVs. Whereas erythrocyte EVs remain present as single EVs after centrifugation, platelet EVs form aggregates, which affect their measured concentration in plasma. Single erythrocyte and platelet EVs are present mainly in the range of 100–200 nm, far below the lower limit of what can be measured by conventional FCM. Furthermore, the effects of single freeze–thaw cycle, time delay between blood collection and plasma preparation up to 1 hour and storage up to 1 year are insignificant (p>0.05) on the measured concentration and diameter of EVs from erythrocyte and platelet concentrates and EVs in plasma, urine and saliva. In conclusion, in standard protocols for EV studies, centrifugation to isolate EVs from collected body fluids should be avoided. Freezing and storage of collected body fluids, albeit their insignificant effects, should be performed identically for comparative EV studies and to create reliable biorepositories. PMID:26563735

Handling and storage of human body fluids for analysis of extracellular vesicles.

PubMed

Yuana, Yuana; Böing, Anita N; Grootemaat, Anita E; van der Pol, Edwin; Hau, Chi M; Cizmar, Petr; Buhr, Egbert; Sturk, Auguste; Nieuwland, Rienk

2015-01-01

Because procedures of handling and storage of body fluids affect numbers and composition of extracellular vesicles (EVs), standardization is important to ensure reliable and comparable measurements of EVs in a clinical environment. We aimed to develop standard protocols for handling and storage of human body fluids for EV analysis. Conditions such as centrifugation, single freeze-thaw cycle, effect of time delay between blood collection and plasma preparation and storage were investigated. Plasma is the most commonly studied body fluid in EV research. We mainly focused on EVs originating from platelets and erythrocytes and investigated the behaviour of these 2 types of EVs independently as well as in plasma samples of healthy subjects. EVs in urine and saliva were also studied for comparison. All samples were analysed simultaneously before and after freeze-thawing by resistive pulse sensing, nanoparticle tracking analysis, conventional flow cytometry (FCM) and transmission (scanning) electron microscopy. Our main finding is that the effect of centrifugation markedly depends on the cellular origin of EVs. Whereas erythrocyte EVs remain present as single EVs after centrifugation, platelet EVs form aggregates, which affect their measured concentration in plasma. Single erythrocyte and platelet EVs are present mainly in the range of 100-200 nm, far below the lower limit of what can be measured by conventional FCM. Furthermore, the effects of single freeze-thaw cycle, time delay between blood collection and plasma preparation up to 1 hour and storage up to 1 year are insignificant (p>0.05) on the measured concentration and diameter of EVs from erythrocyte and platelet concentrates and EVs in plasma, urine and saliva. In conclusion, in standard protocols for EV studies, centrifugation to isolate EVs from collected body fluids should be avoided. Freezing and storage of collected body fluids, albeit their insignificant effects, should be performed identically for comparative EV studies and to create reliable biorepositories.

[Intravenous ferric carboxymaltose-associated hypophosphatemia in patients with iron deficiency anemia. A common side effect].

PubMed

Sánchez González, Rebeca; Ternavasio-de la Vega, Hugo Guillermo; Moralejo Alonso, Leticia; Inés Revuelta, Sandra; Fuertes Martín, Aurelio

2015-08-07

To determine the frequency, severity, time of onset and factors associated with the development of hypophosphatemia (HF) in patients with iron deficiency anemia treated with intravenous ferric carboxymatose (ivFCM). Retrospective cohort study in patients iron deficiency anemia who received ivFCM and had an a prior and subsequent determination of serum phosphate. We carried out a comparative analysis between baseline and post-ivFCM levels of serum phosphate. In order to identify variables independently associated with HF a logistic regression analysis was also performed. One hundred twenty-five patients were included. HF frequency was 58%. The median time to onset of HF was 18 days. Age, baseline ferritin levels and baseline phosphate levels were independently associated with the development of HF. The risk of HF in patients with baseline phosphate levels ≤ 3.1mg/dl was 67% higher than patients with ≥ 3.7 mg/dl. ivFCM-associated HF is a frequent, early and, sometimes, prolonged effect in patients with iron deficiency anemia. Serum phosphate levels should be monitored after ivFCM administration, especially in older patients and in those with lower baseline phosphate or ferritin levels. Copyright © 2014 Elsevier España, S.L.U. All rights reserved.

A Ranking Analysis/An Interlinking Approach of New Triangular Fuzzy Cognitive Maps and Combined Effective Time Dependent Matrix

NASA Astrophysics Data System (ADS)

Adiga, Shreemathi; Saraswathi, A.; Praveen Prakash, A.

2018-04-01

This paper aims an interlinking approach of new Triangular Fuzzy Cognitive Maps (TrFCM) and Combined Effective Time Dependent (CETD) matrix to find the ranking of the problems of Transgenders. Section one begins with an introduction that briefly describes the scope of Triangular Fuzzy Cognitive Maps (TrFCM) and CETD Matrix. Section two provides the process of causes of problems faced by Transgenders using Fuzzy Triangular Fuzzy Cognitive Maps (TrFCM) method and performs the calculations using the collected data among the Transgender. In Section 3, the reasons for the main causes for the problems of the Transgenders. Section 4 describes the Charles Spearmans coefficients of rank correlation method by interlinking of Triangular Fuzzy Cognitive Maps (TrFCM) Method and CETD Matrix. Section 5 shows the results based on our study.

Detecting endotoxin with a flow cytometry-based magnetic aptasensor.

PubMed

Zuo, Ming-Yan; Chen, Li-Juan; Jiang, Hao; Tan, Lin; Luo, Zhao-Feng; Wang, Yan-Mei

2014-12-01

Endotoxin, which is also known as lipopolysaccharide (LPS), is a marker for intruding gram-negative pathogens. It is essential to detect endotoxin quickly and sensitively in a complex milieu. A new flow cytometry (FCM)-based magnetic aptasensor assay that employs two endotoxin-binding aptamers and magnetic beads has been developed to detect endotoxin. The endotoxin-conjugated sandwich complex on magnetic beads was observed by scanning confocal laser microscopy. The resulting magnetic aptasensor rapidly detected (<1 min) endotoxin within a broad dynamic detection range of 10(-8) to 10(0)mg/ml in the presence of bovine serum albumin (BSA), RNA, sucrose, and glucose, which are most likely to coexist with endotoxin in the majority of biological liquids. Only 2 μl of magnetic aptasensor was required to quantify the endotoxin solution. Furthermore, the magnetic aptasensor could be regenerated seven times and still presented an outstanding response to the endotoxin solution. Therefore, the magnetic aptasensor exhibited high sensitivity, selectivity, and reproducibility, thereby serving as a powerful tool for the quality control and high-throughput detection of endotoxin in the food and pharmaceutical industries. Copyright © 2014 Elsevier Inc. All rights reserved.

Hydrophysical correlation and water mass indication of optical physiological parameters of picophytoplankton in Prydz Bay during autumn 2008.

PubMed

Zhang, Fang; Ma, Yuxin; Lin, Ling; He, Jianfeng

2012-12-01

Flow cytometry (FCM) is efficient in detecting both abundance and optical physiological parameters including cell size and cellular carbon content-side scatter (SSC), carotenoids-green and orange fluorescence (FL1 and FL2), and red fluorescence-chlorophylls (FL3) can be obtained by FCM. The utilization of these physiological parameters in indicating water masses in Prydz Bay was investigated for the first time. Picophytoplankton were very sensitive to hydrophysical changes and present distinct characteristics of water masses: Picophytoplankton in water closer to the Amery Ice Shelf were more affected by salinity than by temperature, while temperature became more important than salinity the nearer the picophytoplankton were to the deep sea. The picophytoplankton dealt with declines in light by increasing the size of cells, which increase the fixation of carbon. This can also be increased by high temperature and salinity. Pure water masses can increase the content of chlorophylls and cellular carbon. Generally, the distributions of all the five parameters at upper water depths were less affected by temperature and salinity than by water masses; and these parameters can be as indicators to Summer Surface Water (SSW), Winter Water (WW) and Continental Shelf Water (CSW). Copyright © 2012 Elsevier B.V. All rights reserved.

[Effect of Recombinant Adenovirus AdE-SH2-Caspase 8 on the Apoptosis of Imatinib-resistant K562/G01 Cell Line].

PubMed

Wang, Lin; Fei, Chang; Huang, Zheng-Lan; Li, Hui; Liu, Zhang-Lin; Feng, Wen-Li

2015-08-01

To investigate the effect of SH2-Caspase 8 fusion protein expressed by recombinant adenovirus AdE-SH2-Caspase8-HA-GFP (SC) on the apoptosis of K562/G01 cell line, which is a BCR/ABL positive chronic myeloid leukemia cell line and resistant to imatinib. The K562/G01 cell line was infected with AdE-SH2-Caspase 8-HA-GFP adenovirus (SC), then the cells were divided into 3 groups: AdE-SH2m-Caspase 8-HA-GFP (SmC) group, AdE-GFP (CMV) group and PBS group as control. The infection efficiency was observed under fluorescent microscopy and by flow cytometry. The expression of fusion protein SH2-Caspase 8-HA was measured by Western blot. The morphology of the cells detected by Wright's staining. The apoptosis of the cells were detected by flow cytometry and DNA ladder. The expression of Caspase 3 and PARP were detected by Western blot. The infection efficiency of SC on K562/G01 cells was high which was confirmed by fluorescent microscopy and FCM. SH2-Caspase 8-HA fusion protein were expressed correctly in K562/G01 cells. After treatment with SC the apoptosis of K562/G01 cells could be observed by microscopy. The result of FCM showed that early apoptosis of K562/G01 cells increased significantly as compared with control groups (P < 0.05). DNA ladder showed that the classic DNA ladders appeared in K562/G01 cells after treatment with SC. The wester blot detection showed that the expression level of apoptosis-related protein Caspase 3 and PARP increased. The recombinant adenovirus SC expressing SH2-Caspase 8 fusion protein can induces the apoptosis of K562/G01 cells.

Endoreduplication intensity as a marker of seed developmental stage in the Fabaceae.

PubMed

Rewers, Monika; Sliwinska, Elwira

2012-12-01

Flow cytometry (FCM) can be used to study cell cycle activity in developing, mature and germinating seeds. It provides information about a seed's physiological state and therefore can be used by seed growers for assessing optimal harvest times and presowing treatments. Because an augmented proportion of 4C nuclei usually is indicative of high mitotic activity, the 4C/2C ratio is commonly used to follow the progress of seed development and germination. However, its usefulness for polysomatic (i.e., containing cells with different DNA content) seeds is questioned. Changes in cell cycle/endoreduplication activity in developing seeds of five members of the Fabaceae were studied to determine a more suitable marker of seed developmental stages for polysomatic species based on FCM measurements. Seeds of Phaseolus vulgaris, Medicago sativa, Pisum sativum, Vicia sativa, and Vicia faba var. minor were collected 20, 30, 40, 50, and 60 days after flowering (DAF), embryos were isolated and the proportion of nuclei with different DNA contents in the embryo axis and cotyledon was established. The ratios 4C/2C and (Σ>2C)/2C were calculated. Dried seeds were subjected to laboratory germination tests following international seed testing association (ISTA) rules. Additionally, the absolute nuclear DNA content was estimated in the leaves of the studied species. During seed development nuclei with DNA contents from 2C to 128C were detected; the endopolyploidy pattern depended on the species, seed organ and developmental stage. The cell cycle/endoreduplication parameters correlated negatively with genome size. The (Σ>2C)/2C ratio in the cotyledons reflected the seed developmental stage and corresponded with seed germinability. Therefore, this ratio is recommended as a marker in polysomatic seed research and production instead of the 4C/2C ratio, which does not consider the occurrence of endopolyploid cells. Copyright © 2012 International Society for Advancement of Cytometry.

A budget impact analysis of parenteral iron treatments for iron deficiency anemia in the UK: reduced resource utilization with iron isomaltoside 1000.

PubMed

Pollock, Richard F; Muduma, Gorden

2017-01-01

The reported prevalence of iron deficiency anemia (IDA) varies widely but estimates suggest that 3% of men and 8% of women have IDA in the UK. Parenteral iron is indicated for patients intolerant or unresponsive to oral iron or requiring rapid iron replenishment. This study evaluated differences in the cost of treating these patients with iron isomaltoside (Monofer ® , IIM) relative to other intravenous iron formulations. A budget impact model was developed to evaluate the cost of using IIM relative to ferric carboxymaltose (Ferinject ® , FCM), low molecular weight iron dextran (Cosmofer ® , LMWID), and iron sucrose (Venofer ® , IS) in patients with IDA. To establish iron need, iron deficits were modeled using a simplified dosing table. The base case analysis was conducted over 1 year in patients with IDA with mean bodyweight of 82.4 kg (SD 22.5 kg) and hemoglobin levels of 9.99 g/dL (SD 1.03 g/dL) based on an analysis of patient characteristics in IDA trials. Costs were modeled using UK health care resource groups. Using IIM required 1.3 infusions to correct the mean iron deficit, compared with 1.3, 1.8, and 7.7 with LMWID, FCM, and IS, respectively. Patients using IIM required multiple infusions in 35% of cases, compared with 35%, 77%, and 100% of patients with LMWID, FCM, and IS, respectively. Total costs were estimated to be GBP 451 per patient with IIM or LMWID, relative to GBP 594 with FCM (a GBP 143 or 24% saving with IIM) or GBP 2,600 with IS (a GBP 2,149 or 83% saving with IIM). Using IIM or LMWID in place of FCM or IS resulted in a marked reduction in the number of infusions required to correct iron deficits in patients with IDA. The reduction in infusions was accompanied by substantial reductions in cost relative to FCM and IS over 1 year.

Quantitative evaluation of morphological changes in activated platelets in vitro using digital holographic microscopy.

PubMed

Kitamura, Yutaka; Isobe, Kazushige; Kawabata, Hideo; Tsujino, Tetsuhiro; Watanabe, Taisuke; Nakamura, Masayuki; Toyoda, Toshihisa; Okudera, Hajime; Okuda, Kazuhiro; Nakata, Koh; Kawase, Tomoyuki

2018-06-18

Platelet activation and aggregation have been conventionally evaluated using an aggregometer. However, this method is suitable for short-term but not long-term quantitative evaluation of platelet aggregation, morphological changes, and/or adhesion to specific materials. The recently developed digital holographic microscopy (DHM) has enabled the quantitative evaluation of cell size and morphology without labeling or destruction. Thus, we aim to validate its applicability in quantitatively evaluating changes in cell morphology, especially in the aggregation and spreading of activated platelets, thus modifying typical image analysis procedures to suit aggregated platelets. Freshly prepared platelet-rich plasma was washed with phosphate-buffered saline and treated with 0.1% CaCl 2 . Platelets were then fixed and subjected to DHM, scanning electron microscopy (SEM), atomic force microscopy, optical microscopy, and flow cytometry (FCM). Tightly aggregated platelets were identified as single cells. Data obtained from time-course experiments were plotted two-dimensionally according to the average optical thickness versus attachment area and divided into four regions. The majority of the control platelets, which supposedly contained small and round platelets, were distributed in the lower left region. As activation time increased, however, this population dispersed toward the upper right region. The distribution shift demonstrated by DHM was essentially consistent with data obtained from SEM and FCM. Therefore, DHM was validated as a promising device for testing platelet function given that it allows for the quantitative evaluation of activation-dependent morphological changes in platelets. DHM technology will be applicable to the quality assurance of platelet concentrates, as well as diagnosis and drug discovery related to platelet functions. Copyright © 2018 Elsevier Ltd. All rights reserved.

Plastic fiber optics for micro-imaging of fluorescence signals in living cells

NASA Astrophysics Data System (ADS)

Sakurai, Takashi; Natsume, Mitsuo; Koida, Kowa

2015-03-01

The fiber-coupled microscope (FCM) enables in vivo imaging at deep sites in the tissues or organs that other optical techniques are unable to reach. To develop FCM-based intravital imaging, we employed a plastic optical fiber (POF) bundle that included more than 10,000-units of polystyrene core and polymethyl methacrylate cladding. Each POF had a diameter of less than 5 μm the tip of the bundle was less than 0.5 mm wide, and the flexible wire had a length of 1,000 mm. The optical performance of the plastic FCM was sufficient for detection of significant signal changes in an acinus of rat pancreas labeled with a calcium ion-sensitive fluorescent dye. In the future, the potential power of plastic FCM is expected to increase, enabling analysis of structure and organization of specific functions in live cells within vulnerable organs.

Biological characteristics of HCC by ultrasound-guided aspiration biopsy and its clinical application

PubMed Central

Lin, Li-Wu; Lin, Xue-Ying; He, Yi-Mi; Gao, Shang-Da; Lin, Xiao-Dong

2003-01-01

AIM: To probe the pathological biological characteristics of hepatocellular carcinoma (HCC) by the ultrasound-guided aspiration biopsy and assess the clinical application value of this method. METHODS: The biopsy and DNA analysis by flow cytometry (FCM) were taken in 46 cases with HCC nodules, including 26 cases and 20 cases with nodules ≤ 3 cm and > 3 cm in diameters respectively, and 12 cases with intrahepatic benign hyperplastic nodules. They were taken in 22 cases of 46 cases with HCC before and after the therapy. Fine-needles and automatic histological incised biopsy needles were used. The fresh biopsy tissue was produced into the single cell suspension, which was sent for DNA detection and ratio analysis of cell period. The ratio of each DNA period of cell proliferation of each group was calculated and compared with each other. The DNA aneuploid (AN) and apoptosis cell peak were observed and their percentages were calculated. RESULTS: The ratios of S and G2/M periods of DNA, which reflect cell hyperproliferation, in the group with HCC tumors > 3 cm in diameter were markedly higher than those of the group with HCC nodules ≤ 3 cm in diameter and the group with the benign hyperplastic nodules (P < 0.01 except A:B of S period, P < 0.05). The ratios of the middle group were also apparently higher than those of the latter group (P < 0.01). The ratio of DNA AN of 46 cases with HCC nodules was 34.8% (16/46). None of the cases with the intrahepatic hyperplastic nodules appeared AN. The DNA AN appeared more apparently with the growth of the tumors. The AN ratio of the group with tumors > 3 cm in diameter was 55% (11/20), markedly higher than that of the group with tumors ≤ 3 cm in diameter which was 19.2% (5/26) (P < 0.01). The FCM DNA analysis of 22 specimens of hepatic carcinoma tissue before therapy showed that the aneuploid peaks appeared in 5 cases (22.7%). The ratio of G1 period rose after therapy while the S period and G2/M ratios fell (P < 0.01). The aneuploid peak disappeared in the 5 cases after the therapy, while the apoptosis peaks in 12 cases (54.5%) appeared. CONCLUSION: Addition to supply the information of the pathological morphology of the tumor, the ultrasound-guided fine-needle aspiration tissue could be sent for FCM DNA analysis to comprehend its pathological biological characteristics. This can not only provide the clinic the reliable information about the occurrence, development, diagnosis, curative effect and prognosis of tumors but also supply biological information for clinic to choose therapeutic schemes. PMID:12717834

Evaluating physiological stress in Sumatran tigers (Panthera tigris ssp. sumatrae) managed in Australian zoos.

PubMed

Parnell, Tempe; Narayan, Edward J; Magrath, Michael J L; Roe, Sheila; Clark, Giles; Nicolson, Vere; Martin-Vegue, Patrick; Mucci, Al; Hero, Jean-Marc

2014-01-01

Glucocorticoid quantification using non-invasive methods provides a powerful tool for assessing the health and welfare of wildlife in zoo-based programmes. In this study, we provide baseline data on faecal-based glucocorticoid (cortisol) monitoring of Sumatran tigers (Panthera tigris ssp. sumatrae) managed at the Melbourne Zoo in Victoria, Australia. We sampled five tigers daily for 60 days. Faecal cortisol metabolites (FCMs) in tiger faecal extracts were quantified using enzyme immunoassays that were successfully validated using parallelism and accuracy recovery checks. Two female tigers had significantly higher mean FCM levels than the two males and another female, suggesting that females may have higher FCM levels. A significant elevation was noted in the FCM levels for one female 2 days after she was darted and anaesthetized; however, the FCM levels returned to baseline levels within 3 days after the event. Comparative analysis of FCM levels of tigers sampled at Melbourne Zoo with tigers sampled earlier at two other Australian Zoos (Dreamworld Themepark and Australia Zoo) showed that FCM levels varied between zoos. Differences in the enclosure characteristics, timing of sampling, size and composition of groupings and training procedures could all contribute to this variation. Overall, we recommend the use of non-invasive sampling for the assessment of adrenocortical activity of felids managed in zoos in Australia and internationally in order to improve the welfare of these charismatic big cats.

Evaluating physiological stress in Sumatran tigers (Panthera tigris ssp. sumatrae) managed in Australian zoos

PubMed Central

Parnell, Tempe; Narayan, Edward J.; Magrath, Michael J. L.; Roe, Sheila; Clark, Giles; Nicolson, Vere; Martin-Vegue, Patrick; Mucci, Al; Hero, Jean-Marc

2014-01-01

Glucocorticoid quantification using non-invasive methods provides a powerful tool for assessing the health and welfare of wildlife in zoo-based programmes. In this study, we provide baseline data on faecal-based glucocorticoid (cortisol) monitoring of Sumatran tigers (Panthera tigris ssp. sumatrae) managed at the Melbourne Zoo in Victoria, Australia. We sampled five tigers daily for 60 days. Faecal cortisol metabolites (FCMs) in tiger faecal extracts were quantified using enzyme immunoassays that were successfully validated using parallelism and accuracy recovery checks. Two female tigers had significantly higher mean FCM levels than the two males and another female, suggesting that females may have higher FCM levels. A significant elevation was noted in the FCM levels for one female 2 days after she was darted and anaesthetized; however, the FCM levels returned to baseline levels within 3 days after the event. Comparative analysis of FCM levels of tigers sampled at Melbourne Zoo with tigers sampled earlier at two other Australian Zoos (Dreamworld Themepark and Australia Zoo) showed that FCM levels varied between zoos. Differences in the enclosure characteristics, timing of sampling, size and composition of groupings and training procedures could all contribute to this variation. Overall, we recommend the use of non-invasive sampling for the assessment of adrenocortical activity of felids managed in zoos in Australia and internationally in order to improve the welfare of these charismatic big cats. PMID:27293659

Sensitive and Specific Fluorescent Probes for Functional Analysis of the Three Major Types of Mammalian ABC Transporters

PubMed Central

Lebedeva, Irina V.; Pande, Praveen; Patton, Wayne F.

2011-01-01

An underlying mechanism for multi drug resistance (MDR) is up-regulation of the transmembrane ATP-binding cassette (ABC) transporter proteins. ABC transporters also determine the general fate and effect of pharmaceutical agents in the body. The three major types of ABC transporters are MDR1 (P-gp, P-glycoprotein, ABCB1), MRP1/2 (ABCC1/2) and BCRP/MXR (ABCG2) proteins. Flow cytometry (FCM) allows determination of the functional expression levels of ABC transporters in live cells, but most dyes used as indicators (rhodamine 123, DiOC2(3), calcein-AM) have limited applicability as they do not detect all three major types of ABC transporters. Dyes with broad coverage (such as doxorubicin, daunorubicin and mitoxantrone) lack sensitivity due to overall dimness and thus may yield a significant percentage of false negative results. We describe two novel fluorescent probes that are substrates for all three common types of ABC transporters and can serve as indicators of MDR in flow cytometry assays using live cells. The probes exhibit fast internalization, favorable uptake/efflux kinetics and high sensitivity of MDR detection, as established by multidrug resistance activity factor (MAF) values and Kolmogorov-Smirnov statistical analysis. Used in combination with general or specific inhibitors of ABC transporters, both dyes readily identify functional efflux and are capable of detecting small levels of efflux as well as defining the type of multidrug resistance. The assay can be applied to the screening of putative modulators of ABC transporters, facilitating rapid, reproducible, specific and relatively simple functional detection of ABC transporter activity, and ready implementation on widely available instruments. PMID:21799851

Cloud classification from satellite data using a fuzzy sets algorithm: A polar example

NASA Technical Reports Server (NTRS)

Key, J. R.; Maslanik, J. A.; Barry, R. G.

1988-01-01

Where spatial boundaries between phenomena are diffuse, classification methods which construct mutually exclusive clusters seem inappropriate. The Fuzzy c-means (FCM) algorithm assigns each observation to all clusters, with membership values as a function of distance to the cluster center. The FCM algorithm is applied to AVHRR data for the purpose of classifying polar clouds and surfaces. Careful analysis of the fuzzy sets can provide information on which spectral channels are best suited to the classification of particular features, and can help determine likely areas of misclassification. General agreement in the resulting classes and cloud fraction was found between the FCM algorithm, a manual classification, and an unsupervised maximum likelihood classifier.

Fuzzy C-mean clustering on kinetic parameter estimation with generalized linear least square algorithm in SPECT

NASA Astrophysics Data System (ADS)

Choi, Hon-Chit; Wen, Lingfeng; Eberl, Stefan; Feng, Dagan

2006-03-01

Dynamic Single Photon Emission Computed Tomography (SPECT) has the potential to quantitatively estimate physiological parameters by fitting compartment models to the tracer kinetics. The generalized linear least square method (GLLS) is an efficient method to estimate unbiased kinetic parameters and parametric images. However, due to the low sensitivity of SPECT, noisy data can cause voxel-wise parameter estimation by GLLS to fail. Fuzzy C-Mean (FCM) clustering and modified FCM, which also utilizes information from the immediate neighboring voxels, are proposed to improve the voxel-wise parameter estimation of GLLS. Monte Carlo simulations were performed to generate dynamic SPECT data with different noise levels and processed by general and modified FCM clustering. Parametric images were estimated by Logan and Yokoi graphical analysis and GLLS. The influx rate (K I), volume of distribution (V d) were estimated for the cerebellum, thalamus and frontal cortex. Our results show that (1) FCM reduces the bias and improves the reliability of parameter estimates for noisy data, (2) GLLS provides estimates of micro parameters (K I-k 4) as well as macro parameters, such as volume of distribution (Vd) and binding potential (BP I & BP II) and (3) FCM clustering incorporating neighboring voxel information does not improve the parameter estimates, but improves noise in the parametric images. These findings indicated that it is desirable for pre-segmentation with traditional FCM clustering to generate voxel-wise parametric images with GLLS from dynamic SPECT data.

Classification of arrhythmia using hybrid networks.

PubMed

Haseena, Hassan H; Joseph, Paul K; Mathew, Abraham T

2011-12-01

Reliable detection of arrhythmias based on digital processing of Electrocardiogram (ECG) signals is vital in providing suitable and timely treatment to a cardiac patient. Due to corruption of ECG signals with multiple frequency noise and presence of multiple arrhythmic events in a cardiac rhythm, computerized interpretation of abnormal ECG rhythms is a challenging task. This paper focuses a Fuzzy C- Mean (FCM) clustered Probabilistic Neural Network (PNN) and Multi Layered Feed Forward Network (MLFFN) for the discrimination of eight types of ECG beats. Parameters such as fourth order Auto Regressive (AR) coefficients along with Spectral Entropy (SE) are extracted from each ECG beat and feature reduction has been carried out using FCM clustering. The cluster centers form the input of neural network classifiers. The extensive analysis of Massachusetts Institute of Technology- Beth Israel Hospital (MIT-BIH) arrhythmia database shows that FCM clustered PNNs is superior in cardiac arrhythmia classification than FCM clustered MLFFN with an overall accuracy of 99.05%, 97.14%, respectively.

Conjunctival and corneal reactions in rabbits following short- and repeated exposure to preservative-free tafluprost, commercially available latanoprost and 0.02% benzalkonium chloride

PubMed Central

Liang, H; Baudouin, C; Pauly, A; Brignole-Baudouin, F

2008-01-01

Aim: To compare the conjunctival and corneal reactions of commercially available solution of latanoprost (Xalatan) and preservative-free (PF) tafluprost in rabbits. Methods: The rabbits received 50 μl of phosphate-buffered saline (PBS), PF-tafluprost 0.0015%, latanoprost 0.005% or benzalkonium chloride (BAK) 0.02%; all solutions were applied at 5 min intervals for a total of 15 times. The ocular surface toxicity was investigated using slit-lamp biomicroscopy examination, flow cytometry (FCM) and on imprints for CD45 and tumour necrosis factor-receptor 1 (TNFR1) conjunctival impression cytology (CIC) and corneal in vivo confocal microscopy (IVCM). Standard immunohistology also assessed inflammatory/apoptotic cells. Results: Clinical observation and IVCM images showed the highest ocular surface toxicity with latanoprost and BAK, while PF-tafluprost and PBS eyes presented almost normal corneoconjunctival aspects. FCM showed a higher expression of CD45+ and TNFR1+ in latanoprost- or BAK-instilled groups, compared with PF-tafluprost and PBS groups. Latanoprost induced fewer positive cells for inflammatory marker expressions in CIC specimens compared with BAK-alone, both of which were higher than with PF-tafluprost or PBS. Immunohistology showed the same tendency of toxic ranking. Conclusion: The authors confirm that rabbit corneoconjunctival surfaces presented a better tolerance when treated with PF-tafluprost compared with commercially available latanoprost or BAK solution. PMID:18723745

[Lymphocytic Clonal Expansion in Adult Patients with Epstein-Barr Virus-Associated Lymphoproliferative Disease].

PubMed

Zhong, Feng-Luan; Zhang, Hong-Yu; Zhang, Qian; Feng, Jia; Zhang, Wen-Li; Xu, Lei; Xu, Hai-Chan; Wen, Juan-Juan; Meng, Qing-Xiang

2017-12-01

To explore the lymphocytic clonal expansion in adult patients with Epstein-Barr virus-associated lymphoproliferative diseases (EBV+LPD), and to investigate the experimental methods for EBV+LPD cells so as to provide a more objective measure for the diagnosis, classification and prognosis in the early stage of this disease. Peripheral blood samples from 5 patients with EBV+LPD, 4 patients with adult infectious mononucleosis(IM) as negative control and 3 patients with acute NK-cell leukemia(ANKL) as positive control were collected. Prior to immunochemotherapy, viral loads and clonality were analysed by flow cytometry (FCM), T cell receptor gene rearrangement (TCR) was detected by real-time polymerase chain reaction (RT-PCR), and diversity of EB virus terminal repeat (EBV-TR) was detected by Southern blot. FCM showed only 1 case with clonal TCRVβ in 5 patients with EBV+LPD, TCR clonal expansion could be detected both in patients with IM(4 of 4) and 4 patients with EBV+LPD(4 of 5), Out of patients with EBV+LPD, 1 patient displayed a monoclonal band and 2 patients showed oligoclonal bands when detecting EBV-TR by southen blot. Detecting the diversity of EBV-TR by Southern blot may be the most objective way to reflex clonal transformation of EBV+LPD, which is of great benefit to the diagnosis, classification and prognosis in the early stage of this disease.

Quantification of chemotaxis during pediatric cardiac surgery by flow and laser scanning cytometry

NASA Astrophysics Data System (ADS)

Tarnok, Attila; Schmid, Joerg W.; Osmancik, Pavel; Lenz, Dominik; Pipek, Michal; Hambsch, Joerg; Gerstner, Andreas O.; Schneider, Peter

2002-05-01

Cardiac surgery with cardiopulmonary bypass (CPB) alters the leukocyte composition of the peripheral blood (PB). This response contributes to the sometimes adverse outcome with capillary leakage. Migration of activated cells to sites of inflammation, driven by chemokines is part of this response. In order to determine the chemotactic activity of patients serum during and after surgery we established an assay for PB leukocytes (PBL). PBL from healthy donors were isolated and 250,000 cells were placed into a migration chamber separated by a filter from a second lower chamber filled with patient serum. After incubation cells from top and bottom chamber were removed and stained with a cocktail of monoclonal antibodies for leukocyte subsets and analyzed on a flow cytometer (FCM). Cells at the bottom of the filter belong to the migrating compartment and were quantified by LSC after staining of nucleated cells. Increased chemotactic activity started at onset of anaesthesia followed by a phase of low activity immediately after surgery and a second phase of a high post-operative activity. The in vitro results correlated with results obtained by immunopenotyping of circulating PBL. Manipulation of the chemokine pattern might prove beneficial to prevent extravasation of cells leading to tissue damage. In chemotaxis assays with low amount of available serum the combined use of FCM and Laser Scanning LSC proved as an appropriate analytical tool.

High efficacy and safety of low-dose CD19-directed CAR-T cell therapy in 51 refractory or relapsed B acute lymphoblastic leukemia patients.

PubMed

Pan, J; Yang, J F; Deng, B P; Zhao, X J; Zhang, X; Lin, Y H; Wu, Y N; Deng, Z L; Zhang, Y L; Liu, S H; Wu, T; Lu, P H; Lu, D P; Chang, A H; Tong, C R

2017-12-01

Refractory or relapsed B lymphoblastic leukemia (B-ALL) patients have a dismal outcome with current therapy. We treated 42 primary refractory/hematological relapsed (R/R) and 9 refractory minimal residual disease by flow cytometry (FCM-MRD + ) B-ALL patients with optimized second generation CD19-directed CAR-T cells. The CAR-T-cell infusion dosages were initially ranged from 0.05 to 14 × 10 5 /kg and were eventually settled at 1 × 10 5 /kg for the most recent 20 cases. 36/40 (90%) evaluated R/R patients achieved complete remission (CR) or CR with incomplete count recovery (CRi), and 9/9 (100%) FCM-MRD + patients achieved MRD - . All of the most recent 20 patients achieved CR/CRi. Most cases only experienced mild to moderate CRS. 8/51 cases had seizures that were relieved by early intervention. Twenty three of twenty seven CR/CRi patients bridged to allogeneic hematopoietic stem cell transplantation (allo-HCT) remained in MRD - with a median follow-up time of 206 (45-427) days, whereas 9 of 18 CR/CRi patients without allo-HCT relapsed. Our results indicate that a low CAR-T-cell dosage of 1 × 10 5 /kg, is effective and safe for treating refractory or relapsed B-ALL, and subsequent allo-HCT could further reduce the relapse rate.

A budget impact analysis of parenteral iron treatments for iron deficiency anemia in the UK: reduced resource utilization with iron isomaltoside 1000

PubMed Central

Pollock, Richard F; Muduma, Gorden

2017-01-01

Background and aims The reported prevalence of iron deficiency anemia (IDA) varies widely but estimates suggest that 3% of men and 8% of women have IDA in the UK. Parenteral iron is indicated for patients intolerant or unresponsive to oral iron or requiring rapid iron replenishment. This study evaluated differences in the cost of treating these patients with iron isomaltoside (Monofer®, IIM) relative to other intravenous iron formulations. Methods A budget impact model was developed to evaluate the cost of using IIM relative to ferric carboxymaltose (Ferinject®, FCM), low molecular weight iron dextran (Cosmofer®, LMWID), and iron sucrose (Venofer®, IS) in patients with IDA. To establish iron need, iron deficits were modeled using a simplified dosing table. The base case analysis was conducted over 1 year in patients with IDA with mean bodyweight of 82.4 kg (SD 22.5 kg) and hemoglobin levels of 9.99 g/dL (SD 1.03 g/dL) based on an analysis of patient characteristics in IDA trials. Costs were modeled using UK health care resource groups. Results Using IIM required 1.3 infusions to correct the mean iron deficit, compared with 1.3, 1.8, and 7.7 with LMWID, FCM, and IS, respectively. Patients using IIM required multiple infusions in 35% of cases, compared with 35%, 77%, and 100% of patients with LMWID, FCM, and IS, respectively. Total costs were estimated to be GBP 451 per patient with IIM or LMWID, relative to GBP 594 with FCM (a GBP 143 or 24% saving with IIM) or GBP 2,600 with IS (a GBP 2,149 or 83% saving with IIM). Conclusion Using IIM or LMWID in place of FCM or IS resulted in a marked reduction in the number of infusions required to correct iron deficits in patients with IDA. The reduction in infusions was accompanied by substantial reductions in cost relative to FCM and IS over 1 year. PMID:28848355

Flow cytometric analysis of red-eared slider turtles (Trachemys scripta) from Tar Creek Superfund Site.

PubMed

Hays, Kimberly A; McBee, Karen

2007-05-01

Tar Creek Superfund Site (TCSFS) was heavily mined from the 1890s to 1970 and currently is contaminated with lead, zinc, and cadmium. Flow cytometry (FCM) was used to measure variation in nuclear DNA content of red blood cells collected from Trachemys scripta living within TCSFS and reference sites, Lake Carl Blackwell (LCB) and Sequoyah National Wildlife Refuge (SNWR). We also used atomic absorption spectrometry to measure Pb in blood and carapace and Cd in blood samples of turtles from TCSFS and SNWR. Mean coefficients of variation around the G(1) peak ranged from 5.33 to 5.48 and showed no significant difference between contaminated and reference populations; however, there was a significantly higher frequency of aneuploidy at TCSFS when compared with both reference populations. Blood Pb levels were not significantly different between TCSFS and SNWR populations. Pb levels in carapace samples did not differ significantly between sites; however, Pb levels were higher in carapace than blood for both populations. Blood Cd was significantly higher in animals at TCSFS than SNWR.

Improved resolution in extracellular vesicle populations using 405 instead of 488 nm side scatter

PubMed Central

McVey, Mark J.; Spring, Christopher M.; Kuebler, Wolfgang M.

2018-01-01

ABSTRACT Improvements in identification and assessment of extracellular vesicles (EVs) have fuelled a recent surge in EV publications investigating their roles as biomarkers and mediators of disease. Meaningful scientific comparisons are, however, hampered by difficulties in accurate, reproducible enumeration and characterization of EVs in biological fluids. High-sensitivity flow cytometry (FCM) is presently the most commonly applied strategy to assess EVs, yet its utility is limited by variant ability to resolve smaller EVs. Here, we propose the use of 405 nm (violet) wavelength lasers in place of 488 nm (blue) for side scatter (SSC) detection to obtain greater resolution of EVs using high-sensitivity FCM. To test this hypothesis, we modelled EV resolution by violet versus blue SSC in silico and compared resolution of reference beads and biological EVs from plasma and bronchoalveolar lavage (BAL) fluid using either violet or blue wavelength SSC EV detection. Mie scatter modelling predicted that violet as compared to blue SSC increases resolution of small (100–500 nm) spherical particles with refractive indices (1.34–1.46) similar to EVs by approximately twofold in terms of light intensity and by nearly 20% in SSC signal quantum efficiency. Resolution of reference beads was improved by violet instead of blue SSC with two- and fivefold decreases in coefficients of variation for particles of 300–500 nm and 180–240 nm size, respectively. Resolution was similarly improved for detection of EVs from plasma or BAL fluid. Violet SSC detection for high-sensitivity FCM allows for significantly greater resolution of EVs in plasma and BAL compared to conventional blue SSC and particularly improves resolution of smaller EVs. Notably, the proposed strategy is readily implementable and inexpensive for machines already equipped with 405 nm SSC or the ability to accommodate 405/10 nm bandpass filters in their violet detector arrays. PMID:29696076

[Isolation and purification of primary Kupffer cells from mouse liver].

PubMed

Sun, Chao; Luo, Qingbo; Lu, Xiuxian; Zheng, Daofeng; He, Diao; Wu, Zhongjun

2016-08-01

Objective To isolate and purify Kupffer cells (KCs) from BALB/c mice by an efficient method of low-speed centrifugation and rapid adherence. Methods The mouse liver tissue was perfused in situ and digested with 0.5 g/L collagenase type IV in vitro by water bath. Then, through the low-speed centrifugation, KCs were separated from the mixed hepatocytes, and purified by rapid adherent characteristics. Finally, the production and activity of KCs obtained by this modified method were compared with those isolated by Percoll density gradient centrifugation. We used F4/80 antibody immunofluorescence technique to observe morphological features of KCs, flow cytometry (FCM) to detect the expression of F4/80 antibody and the ink uptake test to observe the phagocytic activity. Moreover, using FCM, we evaluated the expressions of molecules associated with antigen presentation, including major histocompatibility complex class II (MHC II), CD40, CD86 and CD68 on the surface of KCs subjected to hypoxia/reoxygenation (H/R) modeling. And, ELISA was conducted to measure tumor necrosis factor-α (TNF-α) production of the cultured KCs following H/R. Results The yield of KCs was (5.83±0.54)×10(6) per mouse liver and the survival rate of KCs was up to 92% by low-speed centrifugation and rapid adherent method. Compared with Percoll density gradient centrifugation [the yield of KCs was (2.19±0.43)×10(6) per liver], this new method significantly improved the yield of KCs. F4/80 immunofluorescence showed typical morphologic features of KCs such as spindle or polygon shapes and FCM identified nearly 90% F4/80 positive cells. The phagocytic assay showed that lots of ink particles were phagocytosed into the isolated cells. KC H/R models expressed more MHC II, CD40 and CD86 and produced more TNF-α participating in inflammation. Conclusion The efficient method to isolate and purify KCs from BALB /c mice has been successfully established.

Fast evaluation of 69 basal cell carcinomas with ex vivo fluorescence confocal microscopy: criteria description, histopathological correlation, and interobserver agreement.

PubMed

Bennàssar, Antoni; Carrera, Cristina; Puig, Susana; Vilalta, Antoni; Malvehy, Josep

2013-07-01

Fluorescence confocal microscopy (FCM) represents a first step toward a rapid "bedside pathology" in the Mohs surgery setting and in other fields of general pathology. To describe and validate FCM criteria for the main basal cell carcinoma (BCC) subtypes and to demonstrate the overall agreement with classic pathologic analysis of hematoxylin-eosin-stained samples. DESIGN A total of 69 BCCs from 66 patients were prospectively imaged using ex vivo FCM. Confocal mosaics were evaluated in real time and compared with classic pathologic analysis. Department of Dermatology, Hospital Clínic of Barcelona, Barcelona, Spain, between November 2010 and July 2011. Patients with BCC attending the Mohs Surgery Unit. Presence or absence of BCC and histological subtype (superficial, nodular, and infiltrating) in the confocal mosaics. Eight criteria for BCC were described, evaluated, and validated. Although there were minor differences among BCC subtypes, the most BCC-defining criteria were peripheral palisading, clefting, nuclear pleomorphism, and presence of stroma. These criteria were validated with independent observers (κ values >0.7 [corrected] for most criteria). We herein propose, describe, and validate FCM criteria for BCC diagnosis. Fluorescence confocal microscopy is an attractive alternative to histopathologic analysis of frozen sections during Mohs surgery because large areas of freshly excised tissue can be assessed in real time without the need for tissue processing while minimizing labor and costs.

Intravenous iron treatments for iron deficiency anemia in inflammatory bowel disease: a budget impact analysis of iron isomaltoside 1000 (Monofer) in the UK.

PubMed

Pollock, R F; Muduma, G

2017-12-01

Iron deficiency is the leading cause of anemia in patients with inflammatory bowel disease (IBD). Intravenous iron is the first-line treatment for clinically active IBD or previous oral iron intolerance. The aim of the present study was to develop a comparative model of iron deficiency and delivery for iron isomaltoside (IIM), ferric carboxymaltose (FCM), low molecular weight iron dextran (LMWID), and iron sucrose (IS) in the treatment of iron deficiency anemia associated with IBD. Areas covered: A model was developed to evaluate iron delivery characteristics, resource use and costs associated with IIM, FCM, LMWID and IS. Iron deficiency was modeled using dosing tables and retreatments were modeled based on a pooled retrospective analysis. The analyses were conducted over 5 years in patients with IBD with mean bodyweight of 75.4 kg and hemoglobin levels of 10.77 g/dL based on observational data. Expert opinion: The modeling analysis showed that using IIM required 1.2 infusions (per treatment) to correct the mean iron deficit, compared with 1.6, 1.2, and 7.1 with FCM, LMWID and IS, respectively. Costs were estimated to be 2,518 pounds sterling (GBP) per patient with IIM or LMWID, relative to GBP 3,309 with FCM or GBP 14,382 with IS.

Participatory Development and Analysis of a Fuzzy Cognitive Map of the Establishment of a Bio-Based Economy in the Humber Region

PubMed Central

Penn, Alexandra S.; Knight, Christopher J. K.; Lloyd, David J. B.; Avitabile, Daniele; Kok, Kasper; Schiller, Frank; Woodward, Amy; Druckman, Angela; Basson, Lauren

2013-01-01

Fuzzy Cognitive Mapping (FCM) is a widely used participatory modelling methodology in which stakeholders collaboratively develop a ‘cognitive map’ (a weighted, directed graph), representing the perceived causal structure of their system. This can be directly transformed by a workshop facilitator into simple mathematical models to be interrogated by participants by the end of the session. Such simple models provide thinking tools which can be used for discussion and exploration of complex issues, as well as sense checking the implications of suggested causal links. They increase stakeholder motivation and understanding of whole systems approaches, but cannot be separated from an intersubjective participatory context. Standard FCM methodologies make simplifying assumptions, which may strongly influence results, presenting particular challenges and opportunities. We report on a participatory process, involving local companies and organisations, focussing on the development of a bio-based economy in the Humber region. The initial cognitive map generated consisted of factors considered key for the development of the regional bio-based economy and their directional, weighted, causal interconnections. A verification and scenario generation procedure, to check the structure of the map and suggest modifications, was carried out with a second session. Participants agreed on updates to the original map and described two alternate potential causal structures. In a novel analysis all map structures were tested using two standard methodologies usually used independently: linear and sigmoidal FCMs, demonstrating some significantly different results alongside some broad similarities. We suggest a development of FCM methodology involving a sensitivity analysis with different mappings and discuss the use of this technique in the context of our case study. Using the results and analysis of our process, we discuss the limitations and benefits of the FCM methodology in this case and in general. We conclude by proposing an extended FCM methodology, including multiple functional mappings within one participant-constructed graph. PMID:24244303

Safety of intravenous ferric carboxymaltose versus oral iron in patients with nondialysis-dependent CKD: an analysis of the 1-year FIND-CKD trial.

PubMed

Roger, Simon D; Gaillard, Carlo A; Bock, Andreas H; Carrera, Fernando; Eckardt, Kai-Uwe; Van Wyck, David B; Cronin, Maureen; Meier, Yvonne; Larroque, Sylvain; Macdougall, Iain C

2017-09-01

The evidence base regarding the safety of intravenous (IV) iron therapy in patients with chronic kidney disease (CKD) is incomplete and largely based on small studies of relatively short duration. FIND-CKD (ClinicalTrials.gov number NCT00994318) was a 1-year, open-label, multicenter, prospective study of patients with nondialysis-dependent CKD, anemia and iron deficiency randomized (1:1:2) to IV ferric carboxymaltose (FCM), targeting higher (400-600 µg/L) or lower (100-200 µg/L) ferritin, or oral iron. A post hoc analysis of adverse event rates per 100 patient-years was performed to assess the safety of FCM versus oral iron over an extended period. The safety population included 616 patients. The incidence of one or more adverse events was 91.0, 100.0 and 105.0 per 100 patient-years in the high ferritin FCM, low ferritin FCM and oral iron groups, respectively. The incidence of adverse events with a suspected relation to study drug was 15.9, 17.8 and 36.7 per 100 patient-years in the three groups; for serious adverse events, the incidence was 28.2, 27.9 and 24.3 per 100 patient-years. The incidence of cardiac disorders and infections was similar between groups. At least one ferritin level ≥800 µg/L occurred in 26.6% of high ferritin FCM patients, with no associated increase in adverse events. No patient with ferritin ≥800 µg/L discontinued the study drug due to adverse events. Estimated glomerular filtration rate remained the stable in all groups. These results further support the conclusion that correction of iron deficiency anemia with IV FCM is safe in patients with nondialysis-dependent CKD. © The Author 2017. Published by Oxford University Press on behalf of ERA-EDTA.

Phthalates and food-contact materials: enforcing the 2008 European Union plastics legislation.

PubMed

Petersen, J H; Jensen, L K

2010-11-01

The migration of phthalates into foodstuffs from food-contact materials (FCM) is a well-known source of food contamination. In 2005, the European Food Safety Authority finalized its risk assessment for several of the classical phthalate plasticizers. In their risk management procedure the European Commission transformed the tolerable daily intakes established by the Authority into legislative limits for phthalates in both plastic and food simulants, while taking exposure from other sources into consideration. These limits have been into force since 1 July 2008. A detailed interpretation of the regulation of these substances was agreed upon in the European network of FCM reference laboratories. This paper reports results from a Danish control campaign of samples collected by official food inspectors and analysed by a newly validated analytical method run under accreditation. Samples were from FCM producers, FCM importers and importers of packed foodstuffs from third-party countries. Products containing phthalates above the current limits were found in several categories of FCM: conveyor belts (six of six), lids from packed foodstuffs in glasses (eight of 28), tubes for liquid foodstuffs (four of five) and gloves (five of 14). More than 20% of the samples analysed contained dibutylphthalate (DBP) or di-(2-ethylhexyl)phthalate (DEHP) above the compositional limits of 0.05% and 0.1%, respectively. Analysis of residual phthalates in metal lid gaskets instead of analysis of phthalates in the food when controlling foodstuffs packed outside the European Union proved to be an efficient and simple control method. All findings of phthalates were associated with the use of plasticized polyvinylchloride (PVC).

Renal function in patients with non-dialysis chronic kidney disease receiving intravenous ferric carboxymaltose: an analysis of the randomized FIND-CKD trial.

PubMed

Macdougall, Iain C; Bock, Andreas H; Carrera, Fernando; Eckardt, Kai-Uwe; Gaillard, Carlo; Van Wyck, David; Meier, Yvonne; Larroque, Sylvain; Roger, Simon D

2017-01-17

Preclinical studies demonstrate renal proximal tubular injury after administration of some intravenous iron preparations but clinical data on renal effects of intravenous iron are sparse. FIND-CKD was a 56-week, randomized, open-label, multicenter study in which patients with non-dialysis dependent chronic kidney disease (ND-CKD), anemia and iron deficiency without erythropoiesis-stimulating agent therapy received intravenous ferric carboxymaltose (FCM), targeting either higher (400-600 μg/L) or lower (100-200 μg/L) ferritin values, or oral iron. Mean (SD) eGFR at baseline was 34.9 (11.3), 32.8 (10.8) and 34.2 (12.3) mL/min/1.73 m 2 in the high ferritin FCM (n = 97), low ferritin FCM (n = 89) and oral iron (n = 167) groups, respectively. Corresponding values at month 12 were 35.6 (13.8), 32.1 (12.7) and 33.4 (14.5) mL/min/1.73 m 2 . The pre-specified endpoint of mean (SE) change in eGFR from baseline to month 12 was +0.7 (0.9) mL/min/1.73 m 2 with high ferritin FCM (p = 0.15 versus oral iron), -0.9 (0.9) mL/min/1.73 m 2 with low ferritin FCM (p = 0.99 versus oral iron) and -0.9 (0.7) mL/min/1.73 m 2 with oral iron. No significant association was detected between quartiles of FCM dose, change in ferritin or change in TSAT versus change in eGFR. Dialysis initiation was similar between groups. Renal adverse events were rare, with no indication of between-group differences. Intravenous FCM at doses that maintained ferritin levels of 100-200 μg/L or 400-600 μg/L did not negatively impact renal function (eGFR) in patients with ND-CKD over 12 months versus oral iron, and eGFR remained stable. These findings show no evidence of renal toxicity following intravenous FCM over a 1-year period. ClinicalTrials.gov NCT00994318 (first registration 12 October 2009).

Identification of cryovolcanism on Titan using fuzzy cognitive maps

NASA Astrophysics Data System (ADS)

Furfaro, Roberto; Kargel, Jeffrey S.; Lunine, Jonathan I.; Fink, Wolfgang; Bishop, Michael P.

2010-04-01

Future planetary exploration of Titan will require higher degrees of on-board automation, including autonomous determination of sites where the probability of significant scientific findings is the highest. In this paper, a novel Artificial Intelligence (AI) method for the identification and interpretation of sites that yield the highest potential of cryovolcanic activity is presented. We introduce the theory of fuzzy cognitive maps (FCM) as a tool for the analysis of remotely collected data in planetary exploration. A cognitive model embedded in a fuzzy logic framework is constructed via the synergistic interaction of planetary scientists and AI experts. As an application example, we show how FCM can be employed to solve the challenging problem of recognizing cryovolcanism from Synthetic Aperture Radar (SAR) Cassini data. The fuzzy cognitive map is constructed using what is currently known about cryovolcanism on Titan and relies on geological mapping performed by planetary scientists to interpret different locales as cryovolcanic in nature. The system is not conceived to replace the human scientific interpretation, but to enhance the scientists' ability to deal with large amounts of data, and it is a first step in designing AI systems that will be able, in the future, to autonomously make decisions in situations where human analysis and interpretation is not readily available or could not be sufficiently timely. The proposed FCM is tested on Cassini radar data to show the effectiveness of the system in reaching conclusions put forward by human experts and published in the literature. Four tests are performed using the Ta SAR image (October 2004 fly-by). Two regions (i.e. Ganesa Macula and the lobate high backscattering region East of Ganesa) are interpreted by the designed FCM as exhibiting cryovolcanism in agreement with the initial interpretation of the regions by Stofan et al. (2006). Importantly, the proposed FCM is shown to be flexible and adaptive as new data and knowledge are acquired during the course of exploration. Subsequently, the FCM has been modified to include topographic information derived from SAR stereo data. With this additional information, the map concludes that Ganesa Macula is not a cryovolcanic region. In conclusion, the FCM methodology is shown to be a critical and powerful component of future autonomous robotic spacecraft (e.g., orbiter(s), balloon(s), surface/lake lander(s), rover(s)) that will be deployed for the exploration of Titan.

Application of an enhanced fuzzy algorithm for MR brain tumor image segmentation

NASA Astrophysics Data System (ADS)

Hemanth, D. Jude; Vijila, C. Kezi Selva; Anitha, J.

2010-02-01

Image segmentation is one of the significant digital image processing techniques commonly used in the medical field. One of the specific applications is tumor detection in abnormal Magnetic Resonance (MR) brain images. Fuzzy approaches are widely preferred for tumor segmentation which generally yields superior results in terms of accuracy. But most of the fuzzy algorithms suffer from the drawback of slow convergence rate which makes the system practically non-feasible. In this work, the application of modified Fuzzy C-means (FCM) algorithm to tackle the convergence problem is explored in the context of brain image segmentation. This modified FCM algorithm employs the concept of quantization to improve the convergence rate besides yielding excellent segmentation efficiency. This algorithm is experimented on real time abnormal MR brain images collected from the radiologists. A comprehensive feature vector is extracted from these images and used for the segmentation technique. An extensive feature selection process is performed which reduces the convergence time period and improve the segmentation efficiency. After segmentation, the tumor portion is extracted from the segmented image. Comparative analysis in terms of segmentation efficiency and convergence rate is performed between the conventional FCM and the modified FCM. Experimental results show superior results for the modified FCM algorithm in terms of the performance measures. Thus, this work highlights the application of the modified algorithm for brain tumor detection in abnormal MR brain images.

Prospective iterative trial of proteasome inhibitor-based desensitization.

PubMed

Woodle, E S; Shields, A R; Ejaz, N S; Sadaka, B; Girnita, A; Walsh, R C; Alloway, R R; Brailey, P; Cardi, M A; Abu Jawdeh, B G; Roy-Chaudhury, P; Govil, A; Mogilishetty, G

2015-01-01

A prospective iterative trial of proteasome inhibitor (PI)-based therapy for reducing HLA antibody (Ab) levels was conducted in five phases differing in bortezomib dosing density and plasmapheresis timing. Phases included 1 or 2 bortezomib cycles (1.3 mg/m(2) × 6-8 doses), one rituximab dose and plasmapheresis. HLA Abs were measured by solid phase and flow cytometry (FCM) assays. Immunodominant Ab (iAb) was defined as highest HLA Ab level. Forty-four patients received 52 desensitization courses (7 patients enrolled in multiple phases): Phase 1 (n = 20), Phase 2 (n = 12), Phase 3 (n = 10), Phase 4 (n = 5), Phase 5 (n = 5). iAb reductions were observed in 38 of 44 (86%) patients and persisted up to 10 months. In Phase 1, a 51.5% iAb reduction was observed at 28 days with bortezomib alone. iAb reductions increased with higher bortezomib dosing densities and included class I, II, and public antigens (HLA DRβ3, HLA DRβ4 and HLA DRβ5). FCM median channel shifts decreased in 11/11 (100%) patients by a mean of 103 ± 54 mean channel shifts (log scale). Nineteen out of 44 patients (43.2%) were transplanted with low acute rejection rates (18.8%) and de novo DSA formation (12.5%). In conclusion, PI-based desensitization consistently and durably reduces HLA Ab levels providing an alternative to intravenous immune globulin-based desensitization. © Copyright 2014 The American Society of Transplantation and the American Society of Transplant Surgeons.

GEM-loaded magnetic albumin nanospheres modified with cetuximab for simultaneous targeting, magnetic resonance imaging, and double-targeted thermochemotherapy of pancreatic cancer cells.

PubMed

Wang, Ling; An, Yanli; Yuan, Chenyan; Zhang, Hao; Liang, Chen; Ding, Fengan; Gao, Qi; Zhang, Dongsheng

2015-01-01

Targeted delivery is a promising strategy to improve the diagnostic imaging and therapeutic effect of cancers. In this paper, novel cetuximab (C225)-conjugated, gemcitabine (GEM)-containing magnetic albumin nanospheres (C225-GEM/MANs) were fabricated and applied as a theranostic nanocarrier to conduct simultaneous targeting, magnetic resonance imaging (MRI), and double-targeted thermochemotherapy against pancreatic cancer cells. Fe3O4 nanoparticles (NPs) and GEM co-loaded albumin nanospheres (GEM/MANs) were prepared, and then C225 was further conjugated to synthesize C225-GEM/MANs. Their morphology, mean particle size, GEM encapsulation ratio, specific cell-binding ability, and thermal dynamic profiles were characterized. The effects of discriminating different EGFR-expressing pancreatic cancer cells (AsPC-1 and MIA PaCa-2) and monitoring cellular targeting effects were assessed by targeted MRI. Lastly, the antitumor efficiency of double/C225/magnetic-targeted and nontargeted thermochemotherapy was compared with chemotherapy alone using 3-(4, 5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and flow cytometry (FCM) assay. When treated with targeted nanospheres, AsPC-1 cells showed a significantly less intense MRI T2 signal than MIA PaCa-2 cells, while both cells had similar signal strength when incubated with nontargeted nanospheres. T2 signal intensity was significantly lower when magnetic and C225 targeting were combined, rather than used alone. The inhibitory and apoptotic rates of each thermochemotherapy group were significantly higher than those of the chemotherapy-alone groups. Additionally, both MTT and FCM analysis verified that double-targeted thermochemotherapy had the highest targeted killing efficiency among all groups. The C225-GEM/MANs can distinguish various EGFR-expressing live pancreatic cancer cells, monitor diverse cellular targeting effects using targeted MRI imaging, and efficiently mediate double-targeted thermochemotherapy against pancreatic cancer cells.

Cooperative therapeutic effects of herpes simplex virus thymidine kinase gene/ganciclovir system and chemotherapeutic agents on prostate cancer in vitro.

PubMed

Xing, Yifei; Xiao, Yajun; Lu, Gongcheng; Zeng, Fuqing; Zhao, Jun; Xiong, Ping; Feng, Wei

2006-01-01

The killing effects of herpes simplex virus thymidine kinase gene/ganciclovir (HSV-tk/GCV) approach by the addition of several commonly clinical chemotherapeutic agents on hormone refractory prostate cancer (HRPC) cells PC-3m were investigated. After transferring of the HSV-tk gene into PC-3m cells, mRNA and protein expression of HSV-tk was detected by reverse-transcript polymerase chain reaction (RT-PCR) and strept avidin-biotin complex (SABC) immunohistochemical method. The killing effect of GCV, cisplatin (CDDP), etoposide (VP-16), vincristine (VCR), methotrexate (MTX), 5-fluorouracil (5-Fu), and suramin on PC-3m cells was evaluated by morphological assessment analysis, trypan blue exclusion assay and MTT assay respectively. Additionally, the cooperative effect of HSV-tk/GCV system combined with the above agents on the target cancer cells was determined by MTT. Furthermore, apoptosis and necrosis induced by GCV plus 5-Fu or suramin was analyzed by flow cytometry (FCM). The results showed that that there was HSV-tk mRNA and protein expression in pDR2-tk plasmid transduced PC-3m cell. Combination of GCV with VP-16, VCR, 5-Fu or suramin led to an enhanced cellular killing effect, but with CDDP resulted in a reduced one and with MTX in an approximate one. FCM revealed that synergistic use of GCV and 5-fu or suramin resulted in a rather large proportion of apoptosis and necrosis with the apoptosis index being 36.38% and 35.51%, and the proportion of necrosis being 33.05% and 28.87%, respectively. In conclusion, HSV-tk/CGV approach by addition of certain clinical available chemotherapeutic drugs brings on statistically significant enhanced cell killing over single-agent treatment. Our results highlight the potential for such new combination therapies for future treatments of HRPC.

Ferric carboxymaltose-induced hypophosphataemia after kidney transplantation.

PubMed

Sari, V; Atiqi, R; Hoorn, E J; Heijboer, A C; van Gelder, T; Hesselink, D A

2017-03-01

Ferric carboxymaltose (FCM) can induce hypophosphataemia in the general population and patients with chronic kidney disease (CKD). Less is known about the effect of FCM in the kidney transplant population. It has been suggested that fibroblast growth factor 23 (FGF-23)-mediated renal phosphate wasting may be the most likely cause of this phenomenon. In the current study, the effects of FCM on phosphate metabolism were studied in a cohort of kidney transplant recipients. Two index patients receiving FCM are described. Additionally, data of 23 kidney transplant recipients who received a single dose of FCM intravenously between 1 January 2014 and 1 July 2015 were collected. Changes in the serum phosphate concentration were analysed in all subjects. Change in plasma FGF-23 concentrations was analysed in the index patients. In the two index patients an increase in FGF-23 and a decrease in phosphate concentrations were observed after FCM administration. In the 23 kidney transplant patients, median estimated glomerular filtration rate was 42 ml/min/1.73 m2 ( range 10-90 ml/ min/1.73 m2). Mean phosphate concentration before and after FCM administration was 1.05 ±; 0.35 mmol/l and 0.78 ±; 0.41 mmol/l, respectively (average decrease of 0.27 mmol/l; p = 0.003). In the total population, 13 (56.5%) patients showed a transient decline in phosphate concentration after FCM administration. Hypophosphataemia following FCM administration was severe (i.e. < 0.5 mmol/l) in 8 (34.8%) patients. Administration of a single dose of FCM may induce transient and mostly asymptomatic renal phosphate wasting and hypophosphataemia in kidney transplant recipients. This appears to be explained by an increase in FGF-23 concentration.

The combination of kinetic and flow cytometric semen parameters as a tool to predict fertility in cryopreserved bull semen.

PubMed

Gliozzi, T M; Turri, F; Manes, S; Cassinelli, C; Pizzi, F

2017-11-01

Within recent years, there has been growing interest in the prediction of bull fertility through in vitro assessment of semen quality. A model for fertility prediction based on early evaluation of semen quality parameters, to exclude sires with potentially low fertility from breeding programs, would therefore be useful. The aim of the present study was to identify the most suitable parameters that would provide reliable prediction of fertility. Frozen semen from 18 Italian Holstein-Friesian proven bulls was analyzed using computer-assisted semen analysis (CASA) (motility and kinetic parameters) and flow cytometry (FCM) (viability, acrosomal integrity, mitochondrial function, lipid peroxidation, plasma membrane stability and DNA integrity). Bulls were divided into two groups (low and high fertility) based on the estimated relative conception rate (ERCR). Significant differences were found between fertility groups for total motility, active cells, straightness, linearity, viability and percentage of DNA fragmented sperm. Correlations were observed between ERCR and some kinetic parameters, and membrane instability and some DNA integrity indicators. In order to define a model with high relation between semen quality parameters and ERCR, backward stepwise multiple regression analysis was applied. Thus, we obtained a prediction model that explained almost half (R 2=0.47, P<0.05) of the variation in the conception rate and included nine variables: five kinetic parameters measured by CASA (total motility, active cells, beat cross frequency, curvilinear velocity and amplitude of lateral head displacement) and four parameters related to DNA integrity evaluated by FCM (degree of chromatin structure abnormality Alpha-T, extent of chromatin structure abnormality (Alpha-T standard deviation), percentage of DNA fragmented sperm and percentage of sperm with high green fluorescence representative of immature cells). A significant relationship (R 2=0.84, P<0.05) was observed between real and predicted fertility. Once the accuracy of fertility prediction has been confirmed, the model developed in the present study could be used by artificial insemination centers for bull selection or for elimination of poor fertility ejaculates.

Microbial-sized, carboxylate-modified microspheres as surrogate tracers in a variety of subsurface environments: An overview

USGS Publications Warehouse

Harvey, Ronald W.; Metge, David W.; LeBlanc, Denis R.

2017-01-01

Since 1986, fluorescent carboxylate-modified polystyrene/latex microspheres (FCM) have been co-injected into aquifers along with conservative tracers and viruses, bacteria, and (or) protozoa. Use of FCM has resulted in new information about subsurface transport behaviors of microorganisms in fractured crystalline rock, karst limestone, soils, and granular aquifers. FCM have been used as surrogates for oocysts of the pathogenic protist Cryptosporidium parvum in karst limestone and granular drinking-water aquifers. The advantages of FCM in subsurface transport studies are that they are safe in tracer applications, negatively charged, easy to detect, chemically inert, and available in wide range of sizes. The limitations of FCM are that the quantities needed for some field transport studies can be prohibitively expensive and that their surface characteristics may not match the microorganisms of interest. These limitations may be ameliorated, in part by using chemically modified FCM so that their surface characteristics are a better match to that of the organisms. Also, more sensitive methods of detection may allow using smaller quantities of FCM. To assess how the transport behaviors of FCM and pathogens might compare at the field scale, it is helpful to conduct side-by-side comparisons of their transport behaviors using the geologic media and site-specific conditions that characterize the field site.

Generalized fuzzy C-means clustering algorithm with improved fuzzy partitions.

PubMed

Zhu, Lin; Chung, Fu-Lai; Wang, Shitong

2009-06-01

The fuzziness index m has important influence on the clustering result of fuzzy clustering algorithms, and it should not be forced to fix at the usual value m = 2. In view of its distinctive features in applications and its limitation in having m = 2 only, a recent advance of fuzzy clustering called fuzzy c-means clustering with improved fuzzy partitions (IFP-FCM) is extended in this paper, and a generalized algorithm called GIFP-FCM for more effective clustering is proposed. By introducing a novel membership constraint function, a new objective function is constructed, and furthermore, GIFP-FCM clustering is derived. Meanwhile, from the viewpoints of L(p) norm distance measure and competitive learning, the robustness and convergence of the proposed algorithm are analyzed. Furthermore, the classical fuzzy c-means algorithm (FCM) and IFP-FCM can be taken as two special cases of the proposed algorithm. Several experimental results including its application to noisy image texture segmentation are presented to demonstrate its average advantage over FCM and IFP-FCM in both clustering and robustness capabilities.

Neutronics Studies of Uranium-bearing Fully Ceramic Micro-encapsulated Fuel for PWRs

DOE PAGES

George, Nathan M.; Maldonado, G. Ivan; Terrani, Kurt A.; ...

2014-12-01

Our study evaluated the neutronics and some of the fuel cycle characteristics of using uranium-based fully ceramic microencapsulated (FCM) fuel in a pressurized water reactor (PWR). Specific PWR lattice designs with FCM fuel have been developed that are expected to achieve higher specific burnup levels in the fuel while also increasing the tolerance to reactor accidents. The SCALE software system was the primary analysis tool used to model the lattice designs. A parametric study was performed by varying tristructural isotropic particle design features (e.g., kernel diameter, coating layer thicknesses, and packing fraction) to understand the impact on reactivity and resultingmore » operating cycle length. Moreover, to match the lifetime of an 18-month PWR cycle, the FCM particle fuel design required roughly 10% additional fissile material at beginning of life compared with that of a standard uranium dioxide (UO 2) rod. Uranium mononitride proved to be a favorable fuel for the fuel kernel due to its higher heavy metal loading density compared with UO 2. The FCM fuel designs evaluated maintain acceptable neutronics design features for fuel lifetime, lattice peaking factors, and nonproliferation figure of merit.« less

Effect of low power laser irradiation on macrophage phagocytic capacity

NASA Astrophysics Data System (ADS)

Lu, Cuixia; Song, Sheng; Tang, Yu; Zhou, Feifan

2011-03-01

Phagocytosis and subsequent degradation of pathogens by macrophages play a pivotal role in host innate immunity in mammals. Laser irradiation has been found to produce photobiological effects with evidence of interference with immunological functions. However, the effects of laser on the immune response have not been extensively characterized. In this study, we focused our attention on the effects of He-Ne laser on the phagocytic activity of macrophages by using flow cytometry (FCM). After irradiating at fluence of 0, 1, 2 J/cm2 with He-Ne laser (632.8 nm, 3mw), the cells were incubated with microsphere and then subjected to FACS analysis. The results showed that Low-power laser irradiation (LPLI) leads to an increase in phagocytosis on both mouse peritoneal macrophages and the murine macrophage-like cell line RAW264.7. In addition, we demonstrated that LPLI increased phagocytosis of microsphere in a dose-dependent manner, reaching a maximum at fluence of 2 J/cm2. Taken together, our results indicated that Low-power laser irradiation with appropriate dosage can enhance the phagocytosis of macrophage, and provided a theoretical base for the clinical use of the He-Ne laser.

Drug‐specific hypophosphatemia and hypersensitivity reactions following different intravenous iron infusions

PubMed Central

Hvas, Christian L.; Dahlerup, Jens F.

2017-01-01

Aims Intravenous (IV) iron infusions have been associated with hypophosphataemia (HP) and hypersensitivity reactions (HSRs). No studies have compared the side effects of ferric carboxymaltose (FCM) with those of isomaltoside 1000 (ISM). This study aimed to describe the occurrence of HP and HSRs following the administration of either FCM or ISM. Methods Data on 231 outpatients treated with IV iron infusions, between November 2011 and April 2014, were collected. During that period, the department made a switch from FCM to ISM and then back to FCM. Of the 231 patients, 39 received both FCM and ISM during the period. The prevalences of HP and HSRs were compared between the two drugs. Results We found more HP events when FCM was given (64 vs. 9; P < 0.01). In contrast, more patients had mild HSRs when ISM was given (2.5% vs. 10.7%; P < 0.01). A comparison of the two drugs in the subpopulation who received both drug types (n = 39) revealed a difference in phosphate decrease (P < 0.01), with the most marked decrease occurring with FCM. Nine patients who had HSRs were exposed to both drugs. No potential HSR crossover between the two drugs was found. Conclusion We found a higher risk of HP with FCM administration when compared to ISM administration. Conversely, we found a higher risk of mild HSRs with ISM administration when compared to FCM administration. The impacts of the two types of side effects should be considered when choosing an IV iron drug. PMID:27859495

Drug-specific hypophosphatemia and hypersensitivity reactions following different intravenous iron infusions.

PubMed

Bager, Palle; Hvas, Christian L; Dahlerup, Jens F

2017-05-01

Intravenous (IV) iron infusions have been associated with hypophosphataemia (HP) and hypersensitivity reactions (HSRs). No studies have compared the side effects of ferric carboxymaltose (FCM) with those of isomaltoside 1000 (ISM). This study aimed to describe the occurrence of HP and HSRs following the administration of either FCM or ISM. Data on 231 outpatients treated with IV iron infusions, between November 2011 and April 2014, were collected. During that period, the department made a switch from FCM to ISM and then back to FCM. Of the 231 patients, 39 received both FCM and ISM during the period. The prevalences of HP and HSRs were compared between the two drugs. We found more HP events when FCM was given (64 vs. 9; P < 0.01). In contrast, more patients had mild HSRs when ISM was given (2.5% vs. 10.7%; P < 0.01). A comparison of the two drugs in the subpopulation who received both drug types (n = 39) revealed a difference in phosphate decrease (P < 0.01), with the most marked decrease occurring with FCM. Nine patients who had HSRs were exposed to both drugs. No potential HSR crossover between the two drugs was found. We found a higher risk of HP with FCM administration when compared to ISM administration. Conversely, we found a higher risk of mild HSRs with ISM administration when compared to FCM administration. The impacts of the two types of side effects should be considered when choosing an IV iron drug. © 2016 The British Pharmacological Society.

A uniform bacterial growth potential assay for different water types.

PubMed

Farhat, Nadia; Hammes, Frederik; Prest, Emmanuelle; Vrouwenvelder, Johannes

2018-06-06

The bacterial growth potential is important to understand and manage bacterial regrowth-related water quality concerns. Bacterial growth potential depends on growth promoting/limiting compounds, therefore, nutrient availability is the key factor governing bacterial growth potential. Selecting proper tools for bacterial growth measurement is essential for routine implementation of the growth potential measurement. This study proposes a growth potential assay that is universal and can be used for different water types and soil extract without restrictions of pure culture or cultivability of the bacterial strain. The proposed assay measures the sample bacterial growth potential by using the indigenous community as inocula. Flow cytometry (FCM) and adenosine tri-phosphate (ATP) were used to evaluate the growth potential of six different microbial communities indigenous to the sample being analyzed, with increasing carbon concentrations. Bottled mineral water, non-chlorinated tap water, seawater, river water, wastewater effluent and a soil organic carbon extract were analyzed. Results showed that indigenous bacterial communities followed normal batch growth kinetics when grown on naturally present organic carbon. Indigenous bacterial growth could detect spiked organic carbon concentrations as low as 10 μg/L. The indigenous community in all samples responded proportionally to the increase in acetate-carbon and proportional growth could be measured with both FCM and ATP. Bacterial growth was proportional to the carbon concentration but not the same proportion factor for the different water samples tested. The effect of inoculating the same water with different indigenous microbial communities on the growth potential was also examined. The FCM results showed that the highest increase in total bacterial cell concentration was obtained with bacteria indigenous to the water sample. The growth potential assay using indigenous bacterial community revealed consistent results of bacterial growth in all the different samples tested and therefore providing a fast, more stable, and accurate approach for monitoring the biological stability of waters compared to the previously developed assays. The growth potential assay can be used to aid in detecting growth limitations by compounds other than organic carbon. Copyright © 2018 Elsevier Ltd. All rights reserved.

Unsupervised seismic facies analysis with spatial constraints using regularized fuzzy c-means

NASA Astrophysics Data System (ADS)

Song, Chengyun; Liu, Zhining; Cai, Hanpeng; Wang, Yaojun; Li, Xingming; Hu, Guangmin

2017-12-01

Seismic facies analysis techniques combine classification algorithms and seismic attributes to generate a map that describes main reservoir heterogeneities. However, most of the current classification algorithms only view the seismic attributes as isolated data regardless of their spatial locations, and the resulting map is generally sensitive to noise. In this paper, a regularized fuzzy c-means (RegFCM) algorithm is used for unsupervised seismic facies analysis. Due to the regularized term of the RegFCM algorithm, the data whose adjacent locations belong to same classification will play a more important role in the iterative process than other data. Therefore, this method can reduce the effect of seismic data noise presented in discontinuous regions. The synthetic data with different signal/noise values are used to demonstrate the noise tolerance ability of the RegFCM algorithm. Meanwhile, the fuzzy factor, the neighbour window size and the regularized weight are tested using various values, to provide a reference of how to set these parameters. The new approach is also applied to a real seismic data set from the F3 block of the Netherlands. The results show improved spatial continuity, with clear facies boundaries and channel morphology, which reveals that the method is an effective seismic facies analysis tool.

Local delivery of fluorescent dye for fiber-optics confocal microscopy of the living heart.

PubMed

Huang, Chao; Kaza, Aditya K; Hitchcock, Robert W; Sachse, Frank B

2014-01-01

Fiber-optics confocal microscopy (FCM) is an emerging imaging technology with various applications in basic research and clinical diagnosis. FCM allows for real-time in situ microscopy of tissue at sub-cellular scale. Recently FCM has been investigated for cardiac imaging, in particular, for discrimination of cardiac tissue during pediatric open-heart surgery. FCM relies on fluorescent dyes. The current clinical approach of dye delivery is based on systemic injection, which is associated with high dye consumption, and adverse clinical events. In this study, we investigated approaches for local dye delivery during FCM imaging based on dye carriers attached to the imaging probe. Using three-dimensional confocal microscopy, automated bench tests, and FCM imaging we quantitatively characterized dye release of carriers composed of open-pore foam only and foam loaded with agarose hydrogel. In addition, we compared local dye delivery with a model of systemic dye delivery in the isolated perfused rodent heart. We measured the signal-to-noise ratio (SNR) of images acquired in various regions of the heart. Our evaluations showed that foam-agarose dye carriers exhibited a prolonged dye release vs. foam-only carriers. Foam-agarose dye carriers allowed reliable imaging of 5-9 lines, which is comparable to 4-8 min of continuous dye release. Our study in the living heart revealed that the SNR of FCM images using local and systemic dye delivery is not different. However, we observed differences in the imaged tissue microstructure with the two approaches. Structural features characteristic of microvasculature were solely observed for systemic dye delivery. Our findings suggest that local dye delivery approach for FCM imaging constitutes an important alternative to systemic dye delivery. We suggest that the approach for local dye delivery will facilitate clinical translation of FCM, for instance, for FCM imaging during pediatric heart surgery.

Genome-wide association study of the four-constitution medicine.

PubMed

Yin, Chang Shik; Park, Hi Joon; Chung, Joo-Ho; Lee, Hye-Jung; Lee, Byung-Cheol

2009-12-01

Four-constitution medicine (FCM), also known as Sasang constitutional medicine, and the heritage of the long history of individualized acupuncture medicine tradition, is one of the holistic and traditional systems of constitution to appraise and categorize individual differences into four major types. This study first reports a genome-wide association study on FCM, to explore the genetic basis of FCM and facilitate the integration of FCM with conventional individual differences research. Healthy individuals of the Korean population were classified into the four constitutional types (FCTs). A total of 353,202 single nucleotide polymorphisms (SNPs) were typed using whole genome amplified samples, and six-way comparison of FCM types provided lists of significantly differential SNPs. In one-to-one FCT comparisons, 15,944 SNPs were significantly differential, and 5 SNPs were commonly significant in all of the three comparisons. In one-to-two FCT comparisons, 22,616 SNPs were significantly differential, and 20 SNPs were commonly significant in all of the three comparison groups. This study presents the association between genome-wide SNP profiles and the categorization of the FCM, and it could further provide a starting point of genome-based identification and research of the constitutions of FCM.

Optimized multiparametric flow cytometric analysis of circulating endothelial cells and their subpopulations in peripheral blood of patients with solid tumors: a technical analysis.

PubMed

Zhou, Fangbin; Zhou, Yaying; Yang, Ming; Wen, Jinli; Dong, Jun; Tan, Wenyong

2018-01-01

Circulating endothelial cells (CECs) and their subpopulations could be potential novel biomarkers for various malignancies. However, reliable enumerable methods are warranted to further improve their clinical utility. This study aimed to optimize a flow cytometric method (FCM) assay for CECs and subpopulations in peripheral blood for patients with solid cancers. An FCM assay was used to detect and identify CECs. A panel of 60 blood samples, including 44 metastatic cancer patients and 16 healthy controls, were used in this study. Some key issues of CEC enumeration, including sample material and anticoagulant selection, optimal titration of antibodies, lysis/wash procedures of blood sample preparation, conditions of sample storage, sufficient cell events to enhance the signal, fluorescence-minus-one controls instead of isotype controls to reduce background noise, optimal selection of cell surface markers, and evaluating the reproducibility of our method, were integrated and investigated. Wilcoxon and Mann-Whitney U tests were used to determine statistically significant differences. In this validation study, we refined a five-color FCM method to detect CECs and their subpopulations in peripheral blood of patients with solid tumors. Several key technical issues regarding preanalytical elements, FCM data acquisition, and analysis were addressed. Furthermore, we clinically validated the utility of our method. The baseline levels of mature CECs, endothelial progenitor cells, and activated CECs were higher in cancer patients than healthy subjects ( P <0.01). However, there was no significant difference in resting CEC levels between healthy subjects and cancer patients ( P =0.193). We integrated and comprehensively addressed significant technical issues found in previously published assays and validated the reproducibility and sensitivity of our proposed method. Future work is required to explore the potential of our optimized method in clinical oncologic applications.

Non-invasive evaluation of physiological stress in an iconic Australian marsupial: the Koala (Phascolarctos cinereus).

PubMed

Narayan, Edward J; Webster, Koa; Nicolson, Vere; Mucci, Al; Hero, Jean-Marc

2013-06-15

Koalas (Phascolarctos cinereus) are the only extant representatives of Australia's unique marsupial family Phascolarctidae and were listed as nationally Vulnerable in 2012. Causes of mortality are diverse, although the disease chlamydiosis, dog attacks, collisions with cars, and loss of habitat represent the principal reasons for the continued species decline. Koala breeding facilities in Queensland and New South Wales, Australia have been established for conservation and tourism. Non-invasive monitoring of physiological stress is important for determining the sub-lethal effects of environmental stressors on the well-being, reproduction and survival of Koalas in Zoos and also in the wild. In this study, we developed a faecal cortisol metabolite (FCM) enzyme-immunoassay (EIA) for monitoring physiological stress in Koalas from two established Zoos in Australia and also within a free-living sub-population from Queensland. Biological validation of the FCM EIA was done using an adrenocorticotropic hormone (ACTH) challenge. We discovered excretory lag-times of FCM of 24 h in females (n=2) and 48 h in male (n=2) Koalas in response to the ACTH challenge. FCM levels showed an episodic and delayed peak response lasting up to 9 days post ACTH challenge. This finding should be taken into consideration when designing future experiments to study the impacts of short-term (acute) and chronic stressors on the Koalas. Laboratory validations were done using parallelism and recovery checks (extraction efficiency) of the cortisol standard against pooled Koala faecal extracts. Greater than 99% recovery of the cortisol standard was obtained as well as a parallel displacement curve against Koala faecal extracts. FCM levels of the captive Koalas (n=10 males and 13 females) significantly differed by sex, reproductive condition (lactating versus non-lactating Koalas) and the handling groups. Handled male Koalas had 200% higher FCM levels than their non-handled counterparts, while females were not affected by handling as long they were not undergoing lactation. There was no significant difference in FCM levels between the captive and wild Koalas (n=9 males and 7 females). Overall, these results provide foundation knowledge on non-invasive FCM analysis in this iconic Australian marsupial. Non-invasive stress endocrinology opens up opportunities for evaluating the sub-lethal physiological effects of management activities (including caging, translocation) on the nutritional status, reproductive behaviors and disease status of captive and managed in situ Koala populations. Copyright © 2013 Elsevier Inc. All rights reserved.

Current and Prospective Methods for Plant Disease Detection

PubMed Central

Fang, Yi; Ramasamy, Ramaraja P.

2015-01-01

Food losses due to crop infections from pathogens such as bacteria, viruses and fungi are persistent issues in agriculture for centuries across the globe. In order to minimize the disease induced damage in crops during growth, harvest and postharvest processing, as well as to maximize productivity and ensure agricultural sustainability, advanced disease detection and prevention in crops are imperative. This paper reviews the direct and indirect disease identification methods currently used in agriculture. Laboratory-based techniques such as polymerase chain reaction (PCR), immunofluorescence (IF), fluorescence in-situ hybridization (FISH), enzyme-linked immunosorbent assay (ELISA), flow cytometry (FCM) and gas chromatography-mass spectrometry (GC-MS) are some of the direct detection methods. Indirect methods include thermography, fluorescence imaging and hyperspectral techniques. Finally, the review also provides a comprehensive overview of biosensors based on highly selective bio-recognition elements such as enzyme, antibody, DNA/RNA and bacteriophage as a new tool for the early identification of crop diseases. PMID:26287253

Financial impact of intravenous iron treatments on the management of anaemia inpatients: a 1 year observational study.

PubMed

Delpeuch, Amina; Ruivard, Marc; Abergel, Armand; Aumaitre, Olivier; Boisgard, Stéphane; Bagel, Sandrine; Sautou, Valérie

2018-03-08

Background Intravenous (IV) iron preparations bypass the difficulties (malabsorption and side effects) associated with oral iron for the treatment of iron deficiency anaemia (IDA). Ferric carboxymaltose (FCM) can be administered as a single infusion over short periods of time but is more expensive than iron sucrose (IS) when the patients are hospitalized. Objectives To evaluate the appropriateness of FCM prescriptions and to establish the economic impact of this management (including disease coding) compared to the use of IV IS. Setting This study was conducted for inpatients in all departments (orthopaedic department, gastroenterology department and two units of the internal medicine department) where FCM was widely prescribed. Method We retrospectively identified 224 patients, diagnosed with IDA using laboratory parameters and/or disease coding, who received FCM between January and December 2014. Main outcome measure The primary outcome was the rate of appropriateness of FCM prescriptions and the financial impact compared to IV IS. Results 89 Patients were included. The total additional cost for an inappropriate prescription of IV FCM (68% of cases) was of 6053 €. The total incremental cost of unsuitable disease coding was estimated at 31,688 €. Indications for IV FCM were categorized: intestinal bleeding (31%), malabsorption (17%), intolerance (9%) and refractory to oral iron (7%). The majority of patients (62%) received 1000 mg of FCM per week. The average length of hospital stay was of 10 days. Conclusion The prescription of IV iron was appropriate in most cases but did not necessarily require FCM. The use of IV IS, in many cases, could present a cost-saving option for inpatients with IDA. The lack of an IDA coding generated incremental costs.

Choice of High-Dose Intravenous Iron Preparation Determines Hypophosphatemia Risk

PubMed Central

Schaefer, Benedikt; Würtinger, Philipp; Finkenstedt, Armin; Braithwaite, Vickie; Viveiros, André; Effenberger, Maria; Sulzbacher, Irene; Moschen, Alexander; Griesmacher, Andrea; Tilg, Herbert; Vogel, Wolfgang; Zoller, Heinz

2016-01-01

Background Ferric carboxymaltose (FCM) and iron isomaltoside 1000 (IIM) are increasingly used because they allow correction of severe iron deficiency in a single infusion. A transient decrease in serum phosphate concentrations is a frequent side effect of FCM. Aim To characterize this adverse event and search for its predictors in a gastroenterology clinic patient cohort. Methods Electronic medical records of patients attending the University Hospital of Innsbruck were searched for the keywords ferric carboxymaltose or iron isomaltoside. Eighty-one patients with documented administration of FCM or IIM with plasma phosphate concentrations before and after treatment were included. Results The prevalence of hypophosphatemia (<0.8 mmol/L) increased from 11% to 32.1% after treatment with i.v. iron. The hypophosphatemia risk was greater after FCM (45.5%) compared with IIM (4%). Severe hypophosphatemia (<0.6 mmol/L) occurred exclusively after FCM (32.7%). The odds for hypophosphatemia after i.v. iron treatment were independently determined by baseline phosphate and the choice of i.v. iron preparation (FCM vs. IIM—OR = 20.8; 95% CI, 2.6–166; p = 0.004). The median time with hypophosphatemia was 41 days, but prolonged hypophosphatemia of ≥ 2 months was documented in 13 of 17 patients in whom follow-up was available. A significant increase in the phosphaturic hormone intact FGF-23 in hypophosphatemic patients shows that this adverse event is caused by FCM-induced hormone dysregulation. Conclusion Treatment with FCM is associated with a high risk of developing severe and prolonged hypophosphatemia and should therefore be monitored. Hypophosphatemia risk appears to be substantially lower with IIM. PMID:27907058

Choice of High-Dose Intravenous Iron Preparation Determines Hypophosphatemia Risk.

PubMed

Schaefer, Benedikt; Würtinger, Philipp; Finkenstedt, Armin; Braithwaite, Vickie; Viveiros, André; Effenberger, Maria; Sulzbacher, Irene; Moschen, Alexander; Griesmacher, Andrea; Tilg, Herbert; Vogel, Wolfgang; Zoller, Heinz

2016-01-01

Ferric carboxymaltose (FCM) and iron isomaltoside 1000 (IIM) are increasingly used because they allow correction of severe iron deficiency in a single infusion. A transient decrease in serum phosphate concentrations is a frequent side effect of FCM. To characterize this adverse event and search for its predictors in a gastroenterology clinic patient cohort. Electronic medical records of patients attending the University Hospital of Innsbruck were searched for the keywords ferric carboxymaltose or iron isomaltoside. Eighty-one patients with documented administration of FCM or IIM with plasma phosphate concentrations before and after treatment were included. The prevalence of hypophosphatemia (<0.8 mmol/L) increased from 11% to 32.1% after treatment with i.v. iron. The hypophosphatemia risk was greater after FCM (45.5%) compared with IIM (4%). Severe hypophosphatemia (<0.6 mmol/L) occurred exclusively after FCM (32.7%). The odds for hypophosphatemia after i.v. iron treatment were independently determined by baseline phosphate and the choice of i.v. iron preparation (FCM vs. IIM-OR = 20.8; 95% CI, 2.6-166; p = 0.004). The median time with hypophosphatemia was 41 days, but prolonged hypophosphatemia of ≥ 2 months was documented in 13 of 17 patients in whom follow-up was available. A significant increase in the phosphaturic hormone intact FGF-23 in hypophosphatemic patients shows that this adverse event is caused by FCM-induced hormone dysregulation. Treatment with FCM is associated with a high risk of developing severe and prolonged hypophosphatemia and should therefore be monitored. Hypophosphatemia risk appears to be substantially lower with IIM.

Isolation and purification of oligopeptides from Ruditapes philippinarum and its inhibition on the growth of DU‑145 cells in vitro.

PubMed

Yang, Zuisu; Zhao, Yuqin; Yan, Haiqiang; Xu, Lv; Ding, Guofang; Yu, Di; Sun, Yu

2015-02-01

Ruditapes philippinarum is a member of the Veneridae family of marine bivalve molluscs. RPOI‑1 (Ruditapes philippinarum oligopeptide) is a tetrapeptide that can be extracted from Ruditapes philippinarum by means of enzymolysis. This study showed that RPOI‑1 strongly inhibits proliferation and induces apoptosis in DU‑145 human prostate cancer cells. When cells were treated with varying concentrations of RPOI‑1, significant inhibition of proliferation was detected by an MTT assay, and sub‑G1 and G2/M phase cell cycle arrest was observed using flow cytometric (FCM) analysis. Furthermore, morphological changes characteristic of apoptosis and an increase in the proportion of apoptotic cells were observed using double sequential acridine orange/ethidium bromide staining, FCM analysis and transmission election microscopy. FCM studies showed that exposing DU‑145 cells to 10, 20 and 30 mg/ml RPOI‑1 for 24 h increased the percentage of cells in the early‑stages of apoptotis in a dose‑dependent manner, with the numbers rising from 3.01% in the control group to 13.40% in the group treated with the highest dose.

Ma Huang Tang ameliorates asthma though modulation of Th1/Th2 cytokines and inhibition of Th17 cells in ovalbumin-sensitized mice.

PubMed

Ma, Chun-Hua; Ma, Zhan-Qiang; Fu, Qiang; Ma, Shi-Ping

2014-05-01

Ma Huang Tang (Ephedra decoction, MHT) is a famous classical formula from Shang Han Lun by Zhang Zhongjing in the Han Dynasty. The anti-asthmatic effects of MHT and the possible mechanisms were tested. An asthma model was established by ovalbumin (OVA)-induction in mice. A total of forty-eight mice were randomly assigned to six experimental groups: control, model, dexamethasone (2 mg·kg(-1)) and MHT (5, 10, and 20 mg·kg(-1)). Airway resistance (Raw) was measured by the forced oscillation technique, histological studies were evaluated by hematoxylin and eosin (HE) staining, Th1/Th2 and Th17 cytokines were evaluated by enzyme-linked immunosorbent assay (ELISA), and Th17 cells were evaluated by flow cytometry (FCM). This study demonstrated that MHT inhibited OVA-induced increases in Raw and eosinophil count; interleukin (IL)-4 and IL-17 levels were recovered in bronchoalveolar lavage fluid, increased IFN-γ level in bronchoalveolar lavage fluid. Histological studies demonstrated that MHT substantially inhibited OVA-induced eosinophilia in lung tissue. Flow cytometry studies demonstrated that MHT substantially inhibited Th17 cells. These findings suggest that MHT may effectively ameliorate the progression of asthma, and could be further investigated for potential use as a therapy for patients with allergic asthma. Copyright © 2014 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.

[Important differences between faculties of medicine. Implications for family and community medicine].

PubMed

González Lopez-Valcarcel, Beatriz; Ortún, Vicente; Barber, Patricia; Harris, Jeffrey E

2014-03-01

To determine if there are significant differences between universities in the proclivity to choose Family and Community Medicine (FCM), given the constraints imposed by the number of choice. To test the hypothesis that the Schools of Medicine that have the FCM as a compulsory subject in the degree (3 of 27) had the highest preference for this specialty. Observational study on the data file of all the individuals taking the MIR examination between 2003 and 2011. Spain. All those who sat the examinations called by MIR 2003-2011. Position in the ranking of each candidate, elected position (specialty and center), post code of residence, sex, nationality and university in which they studied, and post code location for the residence chosen. The percentage electing FCM is highly correlated with the position in the ranking: 8% of graduates for the 'best' college, 46% for the worst. Very noticeable and consistent differences in the preparation for the MIR among the 27 medical schools. Ranking in the exam, female and foreigner, help predict the choice of FCM. The FCM compulsory curriculum from three universities does not seem to exert any influence. The convenient yardstick competition between the schools of medicine, FCM in their curriculum and the emphasis on the most attractive attributes of the specialty can contribute to the necessary renewal of FCM. Copyright © 2013 Elsevier España, S.L. All rights reserved.

Comparison of K-means and fuzzy c-means algorithm performance for automated determination of the arterial input function.

PubMed

Yin, Jiandong; Sun, Hongzan; Yang, Jiawen; Guo, Qiyong

2014-01-01

The arterial input function (AIF) plays a crucial role in the quantification of cerebral perfusion parameters. The traditional method for AIF detection is based on manual operation, which is time-consuming and subjective. Two automatic methods have been reported that are based on two frequently used clustering algorithms: fuzzy c-means (FCM) and K-means. However, it is still not clear which is better for AIF detection. Hence, we compared the performance of these two clustering methods using both simulated and clinical data. The results demonstrate that K-means analysis can yield more accurate and robust AIF results, although it takes longer to execute than the FCM method. We consider that this longer execution time is trivial relative to the total time required for image manipulation in a PACS setting, and is acceptable if an ideal AIF is obtained. Therefore, the K-means method is preferable to FCM in AIF detection.

Comparison of K-Means and Fuzzy c-Means Algorithm Performance for Automated Determination of the Arterial Input Function

PubMed Central

Yin, Jiandong; Sun, Hongzan; Yang, Jiawen; Guo, Qiyong

2014-01-01

The arterial input function (AIF) plays a crucial role in the quantification of cerebral perfusion parameters. The traditional method for AIF detection is based on manual operation, which is time-consuming and subjective. Two automatic methods have been reported that are based on two frequently used clustering algorithms: fuzzy c-means (FCM) and K-means. However, it is still not clear which is better for AIF detection. Hence, we compared the performance of these two clustering methods using both simulated and clinical data. The results demonstrate that K-means analysis can yield more accurate and robust AIF results, although it takes longer to execute than the FCM method. We consider that this longer execution time is trivial relative to the total time required for image manipulation in a PACS setting, and is acceptable if an ideal AIF is obtained. Therefore, the K-means method is preferable to FCM in AIF detection. PMID:24503700

An interactive method based on the live wire for segmentation of the breast in mammography images.

PubMed

Zewei, Zhang; Tianyue, Wang; Li, Guo; Tingting, Wang; Lu, Xu

2014-01-01

In order to improve accuracy of computer-aided diagnosis of breast lumps, the authors introduce an improved interactive segmentation method based on Live Wire. This paper presents the Gabor filters and FCM clustering algorithm is introduced to the Live Wire cost function definition. According to the image FCM analysis for image edge enhancement, we eliminate the interference of weak edge and access external features clear segmentation results of breast lumps through improving Live Wire on two cases of breast segmentation data. Compared with the traditional method of image segmentation, experimental results show that the method achieves more accurate segmentation of breast lumps and provides more accurate objective basis on quantitative and qualitative analysis of breast lumps.

Infrared Ship Target Segmentation Based on Spatial Information Improved FCM.

PubMed

Bai, Xiangzhi; Chen, Zhiguo; Zhang, Yu; Liu, Zhaoying; Lu, Yi

2016-12-01

Segmentation of infrared (IR) ship images is always a challenging task, because of the intensity inhomogeneity and noise. The fuzzy C-means (FCM) clustering is a classical method widely used in image segmentation. However, it has some shortcomings, like not considering the spatial information or being sensitive to noise. In this paper, an improved FCM method based on the spatial information is proposed for IR ship target segmentation. The improvements include two parts: 1) adding the nonlocal spatial information based on the ship target and 2) using the spatial shape information of the contour of the ship target to refine the local spatial constraint by Markov random field. In addition, the results of K -means are used to initialize the improved FCM method. Experimental results show that the improved method is effective and performs better than the existing methods, including the existing FCM methods, for segmentation of the IR ship images.

Finite cover method with mortar elements for elastoplasticity problems

NASA Astrophysics Data System (ADS)

Kurumatani, M.; Terada, K.

2005-06-01

Finite cover method (FCM) is extended to elastoplasticity problems. The FCM, which was originally developed under the name of manifold method, has recently been recognized as one of the generalized versions of finite element methods (FEM). Since the mesh for the FCM can be regular and squared regardless of the geometry of structures to be analyzed, structural analysts are released from a burdensome task of generating meshes conforming to physical boundaries. Numerical experiments are carried out to assess the performance of the FCM with such discretization in elastoplasticity problems. Particularly to achieve this accurately, the so-called mortar elements are introduced to impose displacement boundary conditions on the essential boundaries, and displacement compatibility conditions on material interfaces of two-phase materials or on joint surfaces between mutually incompatible meshes. The validity of the mortar approximation is also demonstrated in the elastic-plastic FCM.

Numerical modeling of flow and transport in the far-field of a generic nuclear waste repository in fractured crystalline rock using updated fracture continuum model

NASA Astrophysics Data System (ADS)

Hadgu, T.; Kalinina, E.; Klise, K. A.; Wang, Y.

2016-12-01

Disposal of high-level radioactive waste in a deep geological repository in crystalline host rock is one of the potential options for long term isolation. Characterization of the natural barrier system is an important component of the disposal option. In this study we present numerical modeling of flow and transport in fractured crystalline rock using an updated fracture continuum model (FCM). The FCM is a stochastic method that maps the permeability of discrete fractures onto a regular grid. The original method by McKenna and Reeves (2005) has been updated to provide capabilities that enhance representation of fractured rock. As reported in Hadgu et al. (2015) the method was first modified to include fully three-dimensional representations of anisotropic permeability, multiple independent fracture sets, and arbitrary fracture dips and orientations, and spatial correlation. More recently the FCM has been extended to include three different methods. (1) The Sequential Gaussian Simulation (SGSIM) method uses spatial correlation to generate fractures and define their properties for FCM (2) The ELLIPSIM method randomly generates a specified number of ellipses with properties defined by probability distributions. Each ellipse represents a single fracture. (3) Direct conversion of discrete fracture network (DFN) output. Test simulations were conducted to simulate flow and transport using ELLIPSIM and direct conversion of DFN methods. The simulations used a 1 km x 1km x 1km model domain and a structured with grid block of size of 10 m x 10m x 10m, resulting in a total of 106 grid blocks. Distributions of fracture parameters were used to generate a selected number of realizations. For each realization, the different methods were applied to generate representative permeability fields. The PFLOTRAN (Hammond et al., 2014) code was used to simulate flow and transport in the domain. Simulation results and analysis are presented. The results indicate that the FCM approach is a viable method to model fractured crystalline rocks. The FCM is a computationally efficient way to generate realistic representation of complex fracture systems. This approach is of interest for nuclear waste disposal models applied over large domains. SAND2016-7509 A

Segmentation of pomegranate MR images using spatial fuzzy c-means (SFCM) algorithm

NASA Astrophysics Data System (ADS)

Moradi, Ghobad; Shamsi, Mousa; Sedaaghi, M. H.; Alsharif, M. R.

2011-10-01

Segmentation is one of the fundamental issues of image processing and machine vision. It plays a prominent role in a variety of image processing applications. In this paper, one of the most important applications of image processing in MRI segmentation of pomegranate is explored. Pomegranate is a fruit with pharmacological properties such as being anti-viral and anti-cancer. Having a high quality product in hand would be critical factor in its marketing. The internal quality of the product is comprehensively important in the sorting process. The determination of qualitative features cannot be manually made. Therefore, the segmentation of the internal structures of the fruit needs to be performed as accurately as possible in presence of noise. Fuzzy c-means (FCM) algorithm is noise-sensitive and pixels with noise are classified inversely. As a solution, in this paper, the spatial FCM algorithm in pomegranate MR images' segmentation is proposed. The algorithm is performed with setting the spatial neighborhood information in FCM and modification of fuzzy membership function for each class. The segmentation algorithm results on the original and the corrupted Pomegranate MR images by Gaussian, Salt Pepper and Speckle noises show that the SFCM algorithm operates much more significantly than FCM algorithm. Also, after diverse steps of qualitative and quantitative analysis, we have concluded that the SFCM algorithm with 5×5 window size is better than the other windows.

Modeling Uncertainties in EEG Microstates: Analysis of Real and Imagined Motor Movements Using Probabilistic Clustering-Driven Training of Probabilistic Neural Networks.

PubMed

Dinov, Martin; Leech, Robert

2017-01-01

Part of the process of EEG microstate estimation involves clustering EEG channel data at the global field power (GFP) maxima, very commonly using a modified K-means approach. Clustering has also been done deterministically, despite there being uncertainties in multiple stages of the microstate analysis, including the GFP peak definition, the clustering itself and in the post-clustering assignment of microstates back onto the EEG timecourse of interest. We perform a fully probabilistic microstate clustering and labeling, to account for these sources of uncertainty using the closest probabilistic analog to KM called Fuzzy C-means (FCM). We train softmax multi-layer perceptrons (MLPs) using the KM and FCM-inferred cluster assignments as target labels, to then allow for probabilistic labeling of the full EEG data instead of the usual correlation-based deterministic microstate label assignment typically used. We assess the merits of the probabilistic analysis vs. the deterministic approaches in EEG data recorded while participants perform real or imagined motor movements from a publicly available data set of 109 subjects. Though FCM group template maps that are almost topographically identical to KM were found, there is considerable uncertainty in the subsequent assignment of microstate labels. In general, imagined motor movements are less predictable on a time point-by-time point basis, possibly reflecting the more exploratory nature of the brain state during imagined, compared to during real motor movements. We find that some relationships may be more evident using FCM than using KM and propose that future microstate analysis should preferably be performed probabilistically rather than deterministically, especially in situations such as with brain computer interfaces, where both training and applying models of microstates need to account for uncertainty. Probabilistic neural network-driven microstate assignment has a number of advantages that we have discussed, which are likely to be further developed and exploited in future studies. In conclusion, probabilistic clustering and a probabilistic neural network-driven approach to microstate analysis is likely to better model and reveal details and the variability hidden in current deterministic and binarized microstate assignment and analyses.

Modeling Uncertainties in EEG Microstates: Analysis of Real and Imagined Motor Movements Using Probabilistic Clustering-Driven Training of Probabilistic Neural Networks

PubMed Central

Dinov, Martin; Leech, Robert

2017-01-01

Part of the process of EEG microstate estimation involves clustering EEG channel data at the global field power (GFP) maxima, very commonly using a modified K-means approach. Clustering has also been done deterministically, despite there being uncertainties in multiple stages of the microstate analysis, including the GFP peak definition, the clustering itself and in the post-clustering assignment of microstates back onto the EEG timecourse of interest. We perform a fully probabilistic microstate clustering and labeling, to account for these sources of uncertainty using the closest probabilistic analog to KM called Fuzzy C-means (FCM). We train softmax multi-layer perceptrons (MLPs) using the KM and FCM-inferred cluster assignments as target labels, to then allow for probabilistic labeling of the full EEG data instead of the usual correlation-based deterministic microstate label assignment typically used. We assess the merits of the probabilistic analysis vs. the deterministic approaches in EEG data recorded while participants perform real or imagined motor movements from a publicly available data set of 109 subjects. Though FCM group template maps that are almost topographically identical to KM were found, there is considerable uncertainty in the subsequent assignment of microstate labels. In general, imagined motor movements are less predictable on a time point-by-time point basis, possibly reflecting the more exploratory nature of the brain state during imagined, compared to during real motor movements. We find that some relationships may be more evident using FCM than using KM and propose that future microstate analysis should preferably be performed probabilistically rather than deterministically, especially in situations such as with brain computer interfaces, where both training and applying models of microstates need to account for uncertainty. Probabilistic neural network-driven microstate assignment has a number of advantages that we have discussed, which are likely to be further developed and exploited in future studies. In conclusion, probabilistic clustering and a probabilistic neural network-driven approach to microstate analysis is likely to better model and reveal details and the variability hidden in current deterministic and binarized microstate assignment and analyses. PMID:29163110

Comparative safety of intravenous ferumoxytol versus ferric carboxymaltose in iron deficiency anemia: A randomized trial.

PubMed

Adkinson, N Franklin; Strauss, William E; Macdougall, Iain C; Bernard, Kristine E; Auerbach, Michael; Kaper, Robert F; Chertow, Glenn M; Krop, Julie S

2018-05-01

Few trials have examined rates of hypersensitivity reactions (HSRs) with intravenous iron formulations used to treat iron deficiency anemia (IDA). This randomized, multicenter, double-blind clinical trial compared the safety, and efficacy of ferumoxytol versus ferric carboxymaltose (FCM), focusing on rates of HSRs and hypotension as the primary end point. Patients with IDA of any etiology in whom oral iron was unsatisfactory or intolerable received ferumoxytol (n = 997) or FCM (n = 1000) intravenously over ≥15 minutes on days 1 and 8 or 9 for total respective doses of 1.02 g and 1.50 g. Composite incidences of moderate-to-severe HSRs, including anaphylaxis, or moderate-to-severe hypotension from baseline to week 5 (primary safety end point) were 0.6% and 0.7% in the ferumoxytol and FCM groups, respectively, with ferumoxytol noninferior to FCM. No anaphylaxis was reported in either group. The secondary safety end point of incidences of moderate-to-severe HSRs, including anaphylaxis, serious cardiovascular events, and death from baseline to week 5 were 1.3% and 2.0% in the ferumoxytol and FCM groups, respectively (noninferiority test P < .0001). Least-squares mean changes in hemoglobin at week 5 were 1.4 g/dL and 1.6 g/dL in the ferumoxytol and FCM groups, respectively (noninferiority test P < .0001). Incidence of hypophosphatemia was 0.4% for ferumoxytol and 38.7% for FCM. © 2018 The Authors American Journal of Hematology Published by Wiley Periodicals, Inc.

Integrating dynamic fuzzy C-means, data envelopment analysis and artificial neural network to online prediction performance of companies in stock exchange

NASA Astrophysics Data System (ADS)

Jahangoshai Rezaee, Mustafa; Jozmaleki, Mehrdad; Valipour, Mahsa

2018-01-01

One of the main features to invest in stock exchange companies is their financial performance. On the other hand, conventional evaluation methods such as data envelopment analysis are not only a retrospective process, but are also a process, which are incomplete and ineffective approaches to evaluate the companies in the future. To remove this problem, it is required to plan an expert system for evaluating organizations when the online data are received from stock exchange market. This paper deals with an approach for predicting the online financial performance of companies when data are received in different time's intervals. The proposed approach is based on integrating fuzzy C-means (FCM), data envelopment analysis (DEA) and artificial neural network (ANN). The classical FCM method is unable to update the number of clusters and their members when the data are changed or the new data are received. Hence, this method is developed in order to make dynamic features for the number of clusters and clusters members in classical FCM. Then, DEA is used to evaluate DMUs by using financial ratios to provide targets in neural network. Finally, the designed network is trained and prepared for predicting companies' future performance. The data on Tehran Stock Market companies for six consecutive years (2007-2012) are used to show the abilities of the proposed approach.

Reliable and accurate CD4+ T cell count and percent by the portable flow cytometer CyFlow MiniPOC and "CD4 Easy Count Kit-Dry", as revealed by the comparison with the gold standard dual platform technology.

PubMed

Nasi, Milena; De Biasi, Sara; Bianchini, Elena; Gibellini, Lara; Pinti, Marcello; Scacchetti, Tiziana; Trenti, Tommaso; Borghi, Vanni; Mussini, Cristina; Cossarizza, Andrea

2015-01-01

An accurate and affordable CD4+ T cells count is an essential tool in the fight against HIV/AIDS. Flow cytometry (FCM) is the "gold standard" for counting such cells, but this technique is expensive and requires sophisticated equipment, temperature-sensitive monoclonal antibodies (mAbs) and trained personnel. The lack of access to technical support and quality assurance programs thus limits the use of FCM in resource-constrained countries. We have tested the accuracy, the precision and the carry-over contamination of Partec CyFlow MiniPOC, a portable and economically affordable flow cytometer designed for CD4+ count and percentage, used along with the "CD4% Count Kit-Dry". Venous blood from 59 adult HIV+ patients (age: 25-58 years; 43 males and 16 females) was collected and stained with the "MiniPOC CD4% Count Kit-Dry". CD4+ count and percentage were then determined in triplicate by the CyFlow MiniPOC. In parallel, CD4 count was performed using mAbs and a CyFlow Counter, or by a dual platform system (from Beckman Coulter) based upon Cytomic FC500 ("Cytostat tetrachrome kit" for mAbs) and Coulter HmX Hematology Analyzer (for absolute cell count). The accuracy of CyFlow MiniPOC against Cytomic FC500 showed a correlation coefficient (CC) of 0.98 and 0.97 for CD4+ count and percentage, respectively. The accuracy of CyFlow MiniPOC against CyFlow Counter showed a CC of 0.99 and 0.99 for CD4 T cell count and percentage, respectively. CyFlow MiniPOC showed an excellent repeatability: CD4+ cell count and percentage were analyzed on two instruments, with an intra-assay precision below ± 5% deviation. Finally, there was no carry-over contamination for samples at all CD4 values, regardless of their position in the sequence of analysis. The cost-effective CyFlow MiniPOC produces rapid, reliable and accurate results that are fully comparable with those from highly expensive dual platform systems.

[EFFECT OF RECOMBINANT ADENOVIRUS-BONE MORPHOGENETIC PROTEIN 12 TRANSFECTION ON DIFFERENTIATION OF PERIPHERAL BLOOD MESENCHYMAL STEM CELLS INTO TENDON/LIGAMENT CELLS].

PubMed

Fu, Weili; Chen, Gang; Tang, Xin; Li, Qi; Ll, Jian

2015-04-01

To research the effect of recombinant adenovirus-bone morphogenetic protein 12 (Ad-BMP-12) transfection on the differentiation of peripheral blood mesenchymal stem cells (MSCs) into tendon/ligament cells. Peripheral blood MSCs were isolated from New Zealand rabbits (3-4 months old) and cultured in vitro until passage 3. The recombinant adenoviral vector system was prepared using AdEasy system, then transfected into MSCs at passage 3 (transfected group); untransfected MSCs served as control (untransfected group). The morphological characteristics and growth of transfected cells were observed under inverted phase contrast microscope. The transfection efficiency and green fluorescent protein (GFP) expression were detected by flow cytometry (FCM) and fluorescence microscopy. After cultured for 14 days in vitro, the expressions of tendon/ligament-specific markers were determined by immunohistochemistry and real-time fluorescent quantitative PCR. GFP expression could be observed in peripheral blood MSCs at 8 hours after transfection. At 24 hours after transfection, the cells had clear morphology and grew slowly under inverted phase contrast microscope and almost all expressed GFP at the same field under fluorescence microscopy. FCM analysis showed that the transfection efficiency of the transfected group was 99.57%, while it was 2.46% in the untransfected group. The immunohistochemistry showed that the expression of collagen type I gradually increased with culture time in vitro. Real-time fluorescent quantitative PCR results showed that the mRNA expressions of the tendon/ligament-specific genes (Tenomodulin, Tenascin-C, and Decorin) in the transfected group were significantly higher than those in untransfected group (0.061+/- 0.013 vs. 0.004 +/- 0.002, t = -7.700, P=0.031; 0.029 +/- 0.008 vs. 0.003 +/- 0.001, t = -5.741, P=0.020; 0.679 +/- 0.067 vs. 0.142 +/- 0.024, t = -12.998, P=0.000). Ad-BMP-12 can significantly promote differentiation of peripheral blood MSCs into tendon/ligament fibroblasts and enhance the expressions of tendon/ligament-specific phenotypic differentiation, which would provide the evidence for peripheral blood MSCs applied for tendon/ligament regeneration.

Radio-sensitization by Piper longumine of human breast adenoma MDA-MB-231 cells in vitro.

PubMed

Yao, Jian-Xin; Yao, Zhi-Feng; Li, Zhan-Feng; Liu, Yong-Biao

2014-01-01

The current study investigated the effects of Piper longumine on radio-sensitization of human breast cancer MDA-MB-231 cells and underlying mechanisms. Human breast cancer MDA-MB-231 cells were cultured in vitro and those in logarithmic growth phase were selected for experiments divided into four groups: control, X-ray exposed, Piper longumine, and Piper longumine combined with X-rays. Conogenic assays were performed to determine the radio-sensitizing effects. Cell survival curves were fitted by single-hit multi-target model and then the survival fraction (SF), average lethal dose (D0), quasi-threshold dose (Dq) and sensitive enhancement ratio (SER) were calculated. Cell apoptosis was analyzed by flow cytometry (FCM).Western blot assays were employed for expression of apoptosis-related proteins (Bc1-2 and Bax) after treatment with Piper longumine and/or X-ray radiation. The intracellular reactive oxygen species (ROS) level was detected by FCM with a DCFH-DA probe. The cloning formation capacity was decreased in the group of piperlongumine plus radiation, which displayed the values of SF2, D0, Dq significantly lower than those of radiation alone group and the sensitive enhancement ratio (SER) of D0 was1.22 and 1.29, respectively. The cell apoptosis rate was increased by the combination treatment of Piper longumine and radiation. Piper longumine increased the radiation-induced intracellular levels of ROS. Compared with the control group and individual group, the combination group demonstrated significantly decreased expression of Bcl-2 with increased Bax. Piper longumine at a non-cytotoxic concentration can enhance the radio-sensitivity of MDA- MB-231cells, which may be related to its regulation of apoptosis-related protein expression and the increase of intracellular ROS level, thus increasing radiation-induced apoptosis.

Change detection of bitemporal multispectral images based on FCM and D-S theory

NASA Astrophysics Data System (ADS)

Shi, Aiye; Gao, Guirong; Shen, Shaohong

2016-12-01

In this paper, we propose a change detection method of bitemporal multispectral images based on the D-S theory and fuzzy c-means (FCM) algorithm. Firstly, the uncertainty and certainty regions are determined by thresholding method applied to the magnitudes of difference image (MDI) and spectral angle information (SAI) of bitemporal images. Secondly, the FCM algorithm is applied to the MDI and SAI in the uncertainty region, respectively. Then, the basic probability assignment (BPA) functions of changed and unchanged classes are obtained by the fuzzy membership values from the FCM algorithm. In addition, the optimal value of fuzzy exponent of FCM is adaptively determined by conflict degree between the MDI and SAI in uncertainty region. Finally, the D-S theory is applied to obtain the new fuzzy partition matrix for uncertainty region and further the change map is obtained. Experiments on bitemporal Landsat TM images and bitemporal SPOT images validate that the proposed method is effective.

Use of bioassays to assess hazard of food contact material extracts: State of the art.

PubMed

Severin, Isabelle; Souton, Emilie; Dahbi, Laurence; Chagnon, Marie Christine

2017-07-01

This review focuses on the use of in vitro bioassays for the hazard assessment of food contact materials (FCM) as a relevant strategy, in complement to analytical methods. FCM may transfer constituents to foods, not always detected by analytical chemistry, resulting in low but measurable human exposures. Testing FCM extracts with bioassays represents the biological response of a combination of substances, able to be released from the finished materials. Furthermore, this approach is particularly useful regarding the current risk assessment challenges with unpredicted/unidentified non-intentionally added substances (NIAS) that can be leached from the FCM in the food. Bioassays applied to assess hazard of different FCM types are described for, to date, the toxicological endpoints able to be expressed at low levels; cytotoxicity, genotoxicity and endocrine disruption potential. The bioassay strengths and relative key points needed to correctly use and improve the performance of bioassays for an additional FCM risk assessment is developed. This review compiles studies showing that combining both chemical and toxicological analyses presents a very promising and pragmatic tool for identifying new undesirable NIAS (not predicted) which can represent a great part of the migrating substances and/or "cocktail effect". Copyright © 2017 Elsevier Ltd. All rights reserved.

In vivo fluorescence confocal microscopy: indocyanine green enhances the contrast of epidermal and dermal structures

NASA Astrophysics Data System (ADS)

Skvara, Hans; Kittler, Harald; Schmid, Johannes A.; Plut, Ulrike; Jonak, Constanze

2011-09-01

In recent years, in vivo skin imaging devices have been successfully implemented in skin research as well as in clinical routine. Of particular importance is the use of reflectance confocal microscopy (RCM) and fluorescence confocal microscopy (FCM) that enable visualization of the tissue with a resolution comparable to histology. A newly developed commercially available multi-laser device in which both technologies are integrated now offers the possibility to directly compare RCM with FCM. The fluorophore indocyanine green (ICG) was intradermally injected into healthy forearm skin of 10 volunteers followed by in vivo imaging at various time points. In the epidermis, accurate assessment of cell morphology with FCM was supplemented by identification of pigmented cells and structures with RCM. In dermal layers, only with FCM connective tissue fibers were clearly contoured down to a depth of more than 100 μm. The fluorescent signal still provided a favorable image contrast 24 and 48 hours after injection. Subsequently, ICG was applied to different types of skin diseases (basal cell carcinoma, actinic keratosis, seborrhoeic keratosis, and psoriasis) in order to demonstrate the diagnostic benefit of FCM when directly compared with RCM. Our data suggest a great impact of FCM in combination with ICG on clinical and experimental dermatology in the future.

Adrenal activity in maned wolves is higher on farmlands and park boundaries than within protected areas.

PubMed

Spercoski, Katherinne M; Morais, Rosana N; Morato, Ronaldo G; de Paula, Rogério C; Azevedo, Fernanda C; May-Júnior, Joares A; Santos, Jean P; Reghelin, Angela L; Wildt, David E; Songsasen, Nucharin

2012-11-01

In this study we measured excreted fecal corticoid metabolites (FCM) in maned wolves (Chrysocyon brachyurus) living within a protected reserve, on farmlands or in a boundary zone between the two habitats, and determined the impacts of season and reproductive status on adrenal activity. Feces were collected within a national park (n=191 samples), a park boundary zone (n=39) and on nearby farmlands (n=27), processed and analyzed by enzyme immunoassay. FCM amounts from samples collected on farmlands were higher (P<0.05) than in those collected inside the reserve and from the boundary zone. In relation to seasonality, FCM were elevated (P<0.05) in spring (September-November) when wolf pairs were raising young. We then divided the samples collected during breeding season (March-August) into cycling females and male/non-cycling females based on fecal progesterone: fecal testosterone ratio. FCM concentrations of the former collected inside the park were higher than (P<0.05) than the latter group. However, there were no differences in FCM levels between the two groups for samples collected in the boundary zone and on farmlands. Furthermore, FCM concentrations of male/non-cycling females samples collected on farmlands were 2- to 5-fold higher (P<0.05) than in counterparts collected inside the park. The consistently high FCM concentrations in samples collected on farmlands indicate that, in addition to seasonality, gender and reproductive status, anthropogenic pressures also contribute to elevating adrenal steroid for individuals living in altered habitat. Copyright © 2012 Elsevier Inc. All rights reserved.

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

Berndt, Alexander, E-mail: alexander.berndt@med.uni-jena.de; Büttner, Robert, E-mail: Robert-Buettner@gmx.net; Gühne, Stefanie, E-mail: stefanie_guehne@gmx.net

Crosstalk between carcinoma associated fibroblasts (CAFs) and oral squamous cell carcinoma (OSCC) cells is suggested to mediate phenotype transition of cancer cells as a prerequisite for tumour progression, to predict patients’ outcome, and to influence the efficacy of EGFR inhibitor therapies. Here we investigate the influence of activated fibroblasts as a model for CAFs on phenotype and EGFR signalling in OSCC cells in vitro. For this, immortalised hTERT-BJ1 fibroblasts were activated with TGFβ1 and PDGFAB to generate a myofibroblast or proliferative phenotype, respectively. Conditioned media (FCM{sub TGF}, FCM{sub PDGF}) were used to stimulate PE/CA-PJ15 OSCC cells. Results were compared tomore » the effect of conditioned media of non-stimulated fibroblasts (FCM{sub B}). FCM{sub TGF} stimulation leads to an up-regulation of vimentin in the OSCC cells and an enhancement of invasive behaviour, indicating EMT-like effects. Similarly, FCM{sub TGF}≫FCM{sub PDGF} induced up-regulation of EGFR, but not of ErbB2/ErbB3. In addition, we detected an increase in basal activities of ERK, PI3K/Akt and Stat3 (FCM{sub TGF}>FCM{sub PDGF}) accompanied by protein interaction of vimentin with pERK. These effects are correlated with an increased proliferation. In summary, our results suggest that the activated myofibroblast phenotype provides soluble factors which are able to induce EMT-like phenomena and to increase EGFR signalling as well as cell proliferation in OSCC cells. Our results indicate a possible influence of activated myofibroblasts on EGFR-inhibitor therapy. Therefore, CAFs may serve as promising novel targets for combined therapy strategies. - Highlights: • A cell culture model for cancer associated fibroblasts is described. • The mutual interaction with OSCC cells leads to up-regulation of EGFR in tumour cells. • mCAF induces EGFR downstream signalling with increased proliferation in OSCC. • Erk activation is associated with protein interaction with vimentin as sign of EMT. • Results qualify CAF as promising new therapeutic targets in OSCC.« less

Use of the Fracture Continuum Model for Numerical Modeling of Flow and Transport of Deep Geologic Disposal of Nuclear Waste in Crystalline Rock

NASA Astrophysics Data System (ADS)

Hadgu, T.; Kalinina, E.; Klise, K. A.; Wang, Y.

2015-12-01

Numerical modeling of disposal of nuclear waste in a deep geologic repository in fractured crystalline rock requires robust characterization of fractures. Various methods for fracture representation in granitic rocks exist. In this study we used the fracture continuum model (FCM) to characterize fractured rock for use in the simulation of flow and transport in the far field of a generic nuclear waste repository located at 500 m depth. The FCM approach is a stochastic method that maps the permeability of discrete fractures onto a regular grid. The method generates permeability fields using field observations of fracture sets. The original method described in McKenna and Reeves (2005) was designed for vertical fractures. The method has since then been extended to incorporate fully three-dimensional representations of anisotropic permeability, multiple independent fracture sets, and arbitrary fracture dips and orientations, and spatial correlation (Kalinina et al. 20012, 2014). For this study the numerical code PFLOTRAN (Lichtner et al., 2015) has been used to model flow and transport. PFLOTRAN solves a system of generally nonlinear partial differential equations describing multiphase, multicomponent and multiscale reactive flow and transport in porous materials. The code is designed to run on massively parallel computing architectures as well as workstations and laptops (e.g. Hammond et al., 2011). Benchmark tests were conducted to simulate flow and transport in a specified model domain. Distributions of fracture parameters were used to generate a selected number of realizations. For each realization, the FCM method was used to generate a permeability field of the fractured rock. The PFLOTRAN code was then used to simulate flow and transport in the domain. Simulation results and analysis are presented. The results indicate that the FCM approach is a viable method to model fractured crystalline rocks. The FCM is a computationally efficient way to generate realistic representation of complex fracture systems. This approach is of interest for nuclear waste disposal models applied over large domains.

Single‐dose intravenous iron in Southeast Asian heart failure patients: A pilot randomized placebo‐controlled study (PRACTICE‐ASIA‐HF)

PubMed Central

Yeo, Poh Shuan Daniel; Hadi, Farid Abdul; Cushway, Timothy; Lee, Kim Yee; Yin, Fang Fang; Ching, Anne; Li, Ruili; Loh, Seet Yoong; Lim, Shir Lynn; Wong, Raymond Ching‐Chiew; Tai, Bee Choo; Richards, Arthur Mark; Lam, Carolyn S.P.

2018-01-01

Abstract Aims Iron deficiency is highly prevalent in Southeast Asians with heart failure (HF) and associated with worse outcomes. This trial aimed to assess the effect of intravenous iron in Southeast Asians hospitalized with decompensated HF. Methods and results Fifty patients hospitalized for acute decompensated HF, regardless of ejection fraction, with iron deficiency (defined as serum ferritin <300 ng/mL if transferrin saturation is <20%) were randomized to receive either one dose of intravenous ferric carboxymaltose (FCM) 1000 mg or placebo (0.9% saline) following HF stabilization and before discharge in two Singapore tertiary centres. The primary endpoint was difference in 6‐min walk test (6MWT) distance over 12 weeks, while secondary endpoints were quality of life assessed using validated Kansas City Cardiomyopathy Questionnaire (KCCQ) and Visual Analogue Scale (VAS). Improvement in 6MWT distance at Week 12 was observed in both FCM and placebo groups (from 252 ± 123 to 334 ± 128 m and from 243 ± 67 to 301 ± 83 m, respectively). Unadjusted analysis showed 6MWT distance for FCM exceeded that for placebo, but adjustment for baseline covariates and time attenuated this effect {adjusted mean difference between groups: 0.88 m [95% confidence interval (CI) −30.2 to 32.0, P = 0.956]}. KCCQ overall summary and VAS were similar in both groups [adjusted mean difference: KCCQ −1.48 (95% CI −8.27 to 5.31, P = 0.670) and VAS 0.26 (95% CI −0.33 to 0.86, P = 0.386)]. FCM was well tolerated with no serious treatment‐related adverse events. Conclusions Intravenous FCM administered pre‐discharge in Southeast Asians hospitalized with decompensated HF is clinically feasible. Changes in 6MWT distance should be measured beyond Week 12 to account for background therapy effects. PMID:29345426

Naringin Protects Ovalbumin-Induced Airway Inflammation in a Mouse Model of Asthma.

PubMed

Guihua, Xiong; Shuyin, Liu; Jinliang, Gao; Wang, Shumin

2016-04-01

Many plant species containing flavonoids have been widely used in traditional Chinese medicine. Naringin, a well-known flavanone glycoside of citrus fruits, possesses antioxidant, anti-inflammatory, anti-apoptotic, anti-ulcer, anti-osteoporosis, and anti-carcinogenic properties. The aim of the study was to investigate the anti-asthmatic effects of naringin and the possible mechanisms. Asthma model was established by ovalbumin. A total of 50 mice were randomly assigned to five experimental groups: control, model, and dexamethasone (2 mg/kg, orally) and naringin (5 mg/kg, 10 mg/kg, orally). Airway resistance (Raw) were measured, histological studies were evaluated by the hematoxylin and eosin (HE) staining, OVA-specific serum and BALF IgE levels and Th1/Th2 cytokines were evaluated by enzyme-linked immunosorbent assay (ELISA), and Th1/Th2 cells was evaluated by flow cytometry (FCM). T-bet and GABA3 in the lung were evaluated by Western blot. Our study demonstrated that naringin inhibited OVA-induced increases in Raw and eosinophil count; OVA-induced effects on interleukin (IL)-4 and INF-gamma levels were blunted with naringin administration. Histological studies demonstrated that naringin substantially inhibited OVA-induced eosinophilia in lung tissue and airway tissue. Flow cytometry studies demonstrated that naringin substantially inhibited Th2 cells and enhanced Th1 cells. Naringin substantially inhibited GABA3 and increased T-bet. These findings suggest that naringin may effectively ameliorate the progression of asthma and could be used as a therapy for patients with allergic asthma.

Methionine enkephalin (MENK) inhibits tumor growth through regulating CD4+Foxp3+ regulatory T cells (Tregs) in mice.

PubMed

Li, Xuan; Meng, Yiming; Plotnikoff, Nicolas P; Youkilis, Gene; Griffin, Noreen; Wang, Enhua; Lu, Changlong; Shan, Fengping

2015-01-01

Methionine enkephalin (MENK), an endogenous neuropeptide, plays an crucial role in both neuroendocrine and immune systems. CD4+Foxp3+ regulatory T cells (Tregs) are identified as a major subpopulation of T lymphocytes in suppressing immune system to keep balanced immunity. The aim of this research work was to elucidate the mechanisms via which MENK interacts with Tregs in cancer situation. The influence of MENK on transforming growth factor-β (TGF-β) mediated conversion from naïve CD4+CD25- T cells to CD4+CD25+ Tregs was determined and the data from flow cytometry (FCM) analysis indicated that MENK effectively inhibited the expression of Foxp3 during the process of TGF-βinduction. Furthermore, this inhibiting process was accompanied by diminishing phosphorylation and nuclear translocation of Smad2/3, confirmed by western blot (WB) analysis and immunofluorescence (IF) at molecular level. We established sarcoma mice model with S180 to investigate whether MENK could modulate Tregs in tumor circumstance. Our findings showed that MENK delayed the development of tumor in S180 tumor bearing mice and down-regulated level of Tregs. Together, these novel findings reached a conclusion that MENK could inhibit Tregs activity directly and retard tumor development through down-regulating Tregs in mice. This work advances the deepening understanding of the influence of MENK on Tregs in cancer situation, and relation of MENK with immune system, supporting the implication of MENK as a new strategy for cancer immunotherapy.

Breast density quantification with cone-beam CT: A post-mortem study

PubMed Central

Johnson, Travis; Ding, Huanjun; Le, Huy Q.; Ducote, Justin L.; Molloi, Sabee

2014-01-01

Forty post-mortem breasts were imaged with a flat-panel based cone-beam x-ray CT system at 50 kVp. The feasibility of breast density quantification has been investigated using standard histogram thresholding and an automatic segmentation method based on the fuzzy c-means algorithm (FCM). The breasts were chemically decomposed into water, lipid, and protein immediately after image acquisition was completed. The percent fibroglandular volume (%FGV) from chemical analysis was used as the gold standard for breast density comparison. Both image-based segmentation techniques showed good precision in breast density quantification with high linear coefficients between the right and left breast of each pair. When comparing with the gold standard using %FGV from chemical analysis, Pearson’s r-values were estimated to be 0.983 and 0.968 for the FCM clustering and the histogram thresholding techniques, respectively. The standard error of the estimate (SEE) was also reduced from 3.92% to 2.45% by applying the automatic clustering technique. The results of the postmortem study suggested that breast tissue can be characterized in terms of water, lipid and protein contents with high accuracy by using chemical analysis, which offers a gold standard for breast density studies comparing different techniques. In the investigated image segmentation techniques, the FCM algorithm had high precision and accuracy in breast density quantification. In comparison to conventional histogram thresholding, it was more efficient and reduced inter-observer variation. PMID:24254317

Automatic quantitative analysis of in-stent restenosis using FD-OCT in vivo intra-arterial imaging.

PubMed

Mandelias, Kostas; Tsantis, Stavros; Spiliopoulos, Stavros; Katsakiori, Paraskevi F; Karnabatidis, Dimitris; Nikiforidis, George C; Kagadis, George C

2013-06-01

A new segmentation technique is implemented for automatic lumen area extraction and stent strut detection in intravascular optical coherence tomography (OCT) images for the purpose of quantitative analysis of in-stent restenosis (ISR). In addition, a user-friendly graphical user interface (GUI) is developed based on the employed algorithm toward clinical use. Four clinical datasets of frequency-domain OCT scans of the human femoral artery were analyzed. First, a segmentation method based on fuzzy C means (FCM) clustering and wavelet transform (WT) was applied toward inner luminal contour extraction. Subsequently, stent strut positions were detected by utilizing metrics derived from the local maxima of the wavelet transform into the FCM membership function. The inner lumen contour and the position of stent strut were extracted with high precision. Compared to manual segmentation by an expert physician, the automatic lumen contour delineation had an average overlap value of 0.917 ± 0.065 for all OCT images included in the study. The strut detection procedure achieved an overall accuracy of 93.80% and successfully identified 9.57 ± 0.5 struts for every OCT image. Processing time was confined to approximately 2.5 s per OCT frame. A new fast and robust automatic segmentation technique combining FCM and WT for lumen border extraction and strut detection in intravascular OCT images was designed and implemented. The proposed algorithm integrated in a GUI represents a step forward toward the employment of automated quantitative analysis of ISR in clinical practice.

Reactor physics behavior of transuranic-bearing TRISO-particle fuel in a pressurized water reactor

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

Pope, M. A.; Sen, R. S.; Ougouag, A. M.

2012-07-01

Calculations have been performed to assess the neutronic behavior of pins of Fully-Ceramic Micro-encapsulated (FCM) fuel in otherwise-conventional Pressurized Water Reactor (PWR) fuel pins. The FCM fuel contains transuranic (TRU) - only oxide fuel in tri-isotropic (TRISO) particles with the TRU loading coming from the spent fuel of a conventional LWR after 5 years of cooling. Use of the TRISO particle fuel would provide an additional barrier to fission product release in the event of cladding failure. Depletion calculations were performed to evaluate reactivity-limited burnup of the TRU-only FCM fuel. These calculations showed that due to relatively little space availablemore » for fuel, the achievable burnup with these pins alone is quite small. Various reactivity parameters were also evaluated at each burnup step including moderator temperature coefficient (MTC), Doppler, and soluble boron worth. These were compared to reference UO{sub 2} and MOX unit cells. The TRU-only FCM fuel exhibits degraded MTC and Doppler coefficients relative to UO{sub 2} and MOX. Also, the reactivity effects of coolant voiding suggest that the behavior of this fuel would be similar to a MOX fuel of very high plutonium fraction, which are known to have positive void reactivity. In general, loading of TRU-only FCM fuel into an assembly without significant quantities of uranium presents challenges to the reactor design. However, if such FCM fuel pins are included in a heterogeneous assembly alongside LEU fuel pins, the overall reactivity behavior would be dominated by the uranium pins while attractive TRU destruction performance levels in the TRU-only FCM fuel pins is retained. From this work, it is concluded that use of heterogeneous assemblies such as these appears feasible from a preliminary reactor physics standpoint. (authors)« less

Reactor Physics Behavior of Transuranic-Bearing TRISO-Particle Fuel in a Pressurized Water Reactor

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

Michael A. Pope; R. Sonat Sen; Abderrafi M. Ougouag

2012-04-01

Calculations have been performed to assess the neutronic behavior of pins of Fully-Ceramic Micro-encapsulated (FCM) fuel in otherwise-conventional Pressurized Water Reactor (PWR) fuel pins. The FCM fuel contains transuranic (TRU)-only oxide fuel in tri-isotropic (TRISO) particles with the TRU loading coming from the spent fuel of a conventional LWR after 5 years of cooling. Use of the TRISO particle fuel would provide an additional barrier to fission product release in the event of cladding failure. Depletion calculations were performed to evaluate reactivity-limited burnup of the TRU-only FCM fuel. These calculations showed that due to relatively little space available for fuel,more » the achievable burnup with these pins alone is quite small. Various reactivity parameters were also evaluated at each burnup step including moderator temperature coefficient (MTC), Doppler, and soluble boron worth. These were compared to reference UO{sub 2} and MOX unit cells. The TRU-only FCM fuel exhibits degraded MTC and Doppler coefficients relative to UO{sub 2} and MOX. Also, the reactivity effects of coolant voiding suggest that the behavior of this fuel would be similar to a MOX fuel of very high plutonium fraction, which are known to have positive void reactivity. In general, loading of TRU-only FCM fuel into an assembly without significant quantities of uranium presents challenges to the reactor design. However, if such FCM fuel pins are included in a heterogeneous assembly alongside LEU fuel pins, the overall reactivity behavior would be dominated by the uranium pins while attractive TRU destruction performance levels in the TRU-only FCM fuel pins is. From this work, it is concluded that use of heterogeneous assemblies such as these appears feasible from a preliminary reactor physics standpoint.« less

Evaluating ex vivo fluorescence confocal microscopy images of basal cell carcinomas in Mohs excised tissue.

PubMed

Longo, C; Rajadhyaksha, M; Ragazzi, M; Nehal, K; Gardini, S; Moscarella, E; Lallas, A; Zalaudek, I; Piana, S; Argenziano, G; Pellacani, G

2014-09-01

Fluorescence confocal microscopy (FCM) is an emerging technology for rapid imaging of excised tissue, without the need for frozen- or fixed-section processing. Basal cell carcinomas (BCCs) can be detected in Mohs excisions although few studies have described the major BCC findings as seen on FCM. To describe the major BCC findings of excised tissue during Mohs surgery and to correlate them with histopathology. Freshly excised tumours and frozen-thawed discarded tissue of BCC during Mohs surgery were analysed by means of FCM. A side-by-side correlation between FCM images and histological sections was performed. The FCM features of overlying skin and adnexal structures were also described. Sixty-four BCC cases were analysed. Distinct BCC types appeared unique in terms of shape and size of tumour islands [bigger in nodular (18/25), smaller and rounded in micronodular (7/7) and tiny cords for infiltrative ones (24/30)] and for the presence of clefting, palisading and increased nucleus/cytoplasm ratio. An excellent correlation was found between FCM and histological findings (Cohen's κ statistics = 0·9). In six cases, the presence of sebaceous glands and intense stroma reaction represented possible confounders. Fluorescence confocal microscopy is a fast and new imaging technique that allows an excellent visualization of skin structures and BCC findings during Mohs surgery. © 2014 British Association of Dermatologists.

Fuzzy Cognitive Maps for Glacier Hazards Assessment: Application to Predicting the Potential for Glacier Lake Outbursts

NASA Astrophysics Data System (ADS)

Furfaro, R.; Kargel, J. S.; Fink, W.; Bishop, M. P.

2010-12-01

Glaciers and ice sheets are among the largest unstable parts of the solid Earth. Generally, glaciers are devoid of resources (other than water), are dangerous, are unstable and no infrastructure is normally built directly on their surfaces. Areas down valley from large alpine glaciers are also commonly unstable due to landslide potential of moraines, debris flows, snow avalanches, outburst floods from glacier lakes, and other dynamical alpine processes; yet there exists much development and human occupation of some disaster-prone areas. Satellite remote sensing can be extremely effective in providing cost-effective and time- critical information. Space-based imagery can be used to monitor glacier outlines and their lakes, including processes such as iceberg calving and debris accumulation, as well as changing thicknesses and flow speeds. Such images can also be used to make preliminary identifications of specific hazardous spots and allows preliminary assessment of possible modes of future disaster occurrence. Autonomous assessment of glacier conditions and their potential for hazards would present a major advance and permit systematized analysis of more data than humans can assess. This technical leap will require the design and implementation of Artificial Intelligence (AI) algorithms specifically designed to mimic glacier experts’ reasoning. Here, we introduce the theory of Fuzzy Cognitive Maps (FCM) as an AI tool for predicting and assessing natural hazards in alpine glacier environments. FCM techniques are employed to represent expert knowledge of glaciers physical processes. A cognitive model embedded in a fuzzy logic framework is constructed via the synergistic interaction between glaciologists and AI experts. To verify the effectiveness of the proposed AI methodology as applied to predicting hazards in glacier environments, we designed and implemented a FCM that addresses the challenging problem of autonomously assessing the Glacier Lake Outburst Flow Potential and Impound Water Upstream Flow Potential. The FCM is constructed using what is currently our understanding of how glacier lake outbursts occur, whereas the causal connection between concepts is defined to capture the expertise of glacier scientists. The proposed graph contains 27 nodes and a network of connections that represent the causal link between concepts. To test the developed FCM, we defined three scenarios representing glacier lake environmental conditions that either occurred or that are likely to occur in such highly dynamic environments. For each case, the FCM has been initialized using observables extracted from hypothesized remote sensing imagery. The map, which converges to a fixed point for all of the test scenarios within 15 iterations, shows reasoning consistent with that of glacier experts. The FCM-based cognitive approach has the potential to be the AI core of real-time operational hazards assessment and detection systems.

Research on the precise positioning of customers in large data environment

NASA Astrophysics Data System (ADS)

Zhou, Xu; He, Lili

2018-04-01

Customer positioning has always been a problem that enterprises focus on. In this paper, FCM clustering algorithm is used to cluster customer groups. However, due to the traditional FCM clustering algorithm, which is susceptible to the influence of the initial clustering center and easy to fall into the local optimal problem, the short board of FCM is solved by the gray optimization algorithm (GWO) to achieve efficient and accurate handling of a large number of retailer data.

The new generation of intravenous iron: chemistry, pharmacology, and toxicology of ferric carboxymaltose.

PubMed

Funk, Felix; Ryle, Peter; Canclini, Camillo; Neiser, Susann; Geisser, Peter

2010-01-01

An ideal preparation for intravenous iron replacement therapy should balance effectiveness and safety. Compounds that release iron rapidly tend to cause toxicity, while large molecules can induce antibody formation and cause anaphylactic reactions. There is therefore a need for an intravenous iron preparation that delivers appropriate amounts of iron in a readily available form but with minimal side effects and thus with an excellent safety profile. In this paper, a review is given on the chemistry, pharmacology, and toxicology of ferric carboxymaltose (FCM, Ferinject), a stable and robust complex formulated as a colloidal solution with a physiological pH. The complex is gradually taken up mainly from the hepatic reticulo-endothelial system (RES), followed by effective delivery of iron to the endogeneous transport system for the haem synthesis in new erythrocytes, as shown in studies on the pharmacodynamics and pharmacokinetics with radio-labelled FCM. Studies with radio-labelled FCM also demonstrated a barrier function of the placenta and a low transfer of iron into the milk of lactating rats. Safety pharmacology studies indicated a favourable profile with regard to cardiovascular, central nervous, respiratory, and renal toxicity. A high maximum non-lethal dose was demonstrated in the single-dose toxicity studies. Furthermore, based on the No-Observed-Adverse-Effect-Levels (NOAELs) found in repeated-dose toxicity studies and on the cumulative doses administered, FCM has good safety margins. Reproductive and developmental toxicity studies did not reveal any direct or indirect harmful effects. No genotoxic potential was found in in vitro or in vivo studies. Moreover, antigenicity studies showed no cross-reactivity of FMC with anti-dextran antibodies and also suggested that FCM does not possess sensitizing potential. Lastly, no evidence of irritation was found in local tolerance studies with FCM. This excellent toxicity profile and the high effectiveness of FCM allow the administration of high doses as a single infusion or bolus injection, which will enhance the cost-effectiveness and convenience of iron replacement therapy. In conclusion, FCM has many of the characteristics of an ideal intravenous iron preparation.

The finite cell method for polygonal meshes: poly-FCM

NASA Astrophysics Data System (ADS)

Duczek, Sascha; Gabbert, Ulrich

2016-10-01

In the current article, we extend the two-dimensional version of the finite cell method (FCM), which has so far only been used for structured quadrilateral meshes, to unstructured polygonal discretizations. Therefore, the adaptive quadtree-based numerical integration technique is reformulated and the notion of generalized barycentric coordinates is introduced. We show that the resulting polygonal (poly-)FCM approach retains the optimal rates of convergence if and only if the geometry of the structure is adequately resolved. The main advantage of the proposed method is that it inherits the ability of polygonal finite elements for local mesh refinement and for the construction of transition elements (e.g. conforming quadtree meshes without hanging nodes). These properties along with the performance of the poly-FCM are illustrated by means of several benchmark problems for both static and dynamic cases.

Force Measurement Services at Kebs: AN Overview of Equipment, Procedures and Uncertainty

NASA Astrophysics Data System (ADS)

Bangi, J. O.; Maranga, S. M.; Nganga, S. P.; Mutuli, S. M.

This paper describes the facilities, instrumentation and procedures currently used in the force laboratory at the Kenya Bureau of Standards (KEBS) for force measurement services. The laboratory uses the Force Calibration Machine (FCM) to calibrate force-measuring instruments. The FCM derives its traceability via comparisons using reference transfer force transducers calibrated by the Force Standard Machines (FSM) of a National Metrology Institute (NMI). The force laboratory is accredited to ISO/IEC 17025 by the Germany Accreditation Body (DAkkS). The accredited measurement scope of the laboratory is 1 MN to calibrate force transducers in both compression and tension modes. ISO 376 procedures are used while calibrating force transducers. The KEBS reference transfer standards have capacities of 10, 50, 300 and 1000 kN to cover the full range of the FCM. The uncertainty in the forces measured by the FCM were reviewed and determined in accordance to the new EURAMET calibration guide. The relative expanded uncertainty of force W realized by FCM was evaluated in a range from 10 kN-1 MN, and was found to be 5.0 × 10-4 with the coverage factor k being equal to 2. The overall normalized error (En) of the comparison results was also found to be less than 1. The accredited Calibration and Measurement Capability (CMC) of the KEBS force laboratory was based on the results of those intercomparisons. The FCM enables KEBS to provide traceability for the calibration of class ‘1’ force instruments as per the ISO 376.

Faecal corticosterone metabolite concentrations are not a good predictor of habitat suitability for common gartersnakes.

PubMed

Halliday, William D; Gilmour, Kathleen M; Blouin-Demers, Gabriel

2015-01-01

Measuring habitat suitability is important in conservation and in wildlife management. Measuring the abundance or presence-absence of a species in various habitats is not sufficient to measure habitat suitability because these metrics can be poor predictors of population success. Therefore, having some measure of population success is essential in assessing habitat suitability, but estimating population success is difficult. Identifying suitable proxies for population success could thus be beneficial. We examined whether faecal corticosterone metabolite (fCM) concentrations could be used as a proxy for habitat suitability in common gartersnakes (Thamnophis sirtalis). We conducted a validation study and confirmed that fCM concentrations indeed reflect circulating corticosterone concentrations. We estimated abundance, reproductive output and growth rate of gartersnakes in field and in forest habitat and we also measured fCM concentrations of gartersnakes from these same habitats. Common gartersnakes were more abundant and had higher reproductive outputs and higher growth rates in field habitat than in forest habitat, but fCM concentrations did not differ between the same two habitats. Our results suggest either that fCM concentrations are not a useful metric of habitat suitability in common gartersnakes or that the difference in suitability between the two habitats was too small to induce changes in fCM concentrations. Incorporating fitness metrics in estimates of habitat suitability is important, but these metrics of fitness have to be sensitive enough to vary between habitats.

Faecal cortisol metabolites in Bengal (Panthera tigris tigris) and Sumatran tigers (Panthera tigris sumatrae).

PubMed

Narayan, Edward J; Parnell, Tempe; Clark, Giles; Martin-Vegue, Patrick; Mucci, Al; Hero, Jean-Marc

2013-12-01

The tiger (Panthera tigris) faces a great risk of extinction as its wild numbers have plummeted due to poaching and habitat destruction so ex-situ conservation programs are becoming ever more necessary. Reliable non-invasive biomarkers of the stress hormone (cortisol) are necessary for assessing the health and welfare of tigers in captivity. To our knowledge, non-invasive stress endocrinology methods have not been tested as widely in tigers. The first aim of this study was to describe and validate a faecal cortisol metabolite enzyme-immmunoassay (FCM EIA) for two tiger sub-species, the Bengal tiger (Panthera tigris tigris) and the Sumatran tiger (Panthera tigris sumatrae). Individual tigers (n=22) were studied in two large Zoos in Queensland, Australia (Dreamworld Theme Park and Australia Zoo). Fresh faecal samples (<12 h old) were collected each morning from both Zoos over a study period of 21 days. Biological validation was conducted separately by collecting feces 5 days before and 5 days after blood was taken from four male and five female tigers. Results showed that mean FCM levels increased by 138% and 285% in the male and female tigers within 1 day after bloods were taken, returning to baseline in 5 days. Laboratory validations of the FCM EIA were done using an extraction efficiency test and parallelism. Results showed >89% recovery of the cortisol standard that was added to tiger faecal extract. We also obtained parallel displacement of the serially diluted cortisol standard against serially diluted tiger faecal extract. Our second aim was to determine whether the FCM levels were significantly different between tiger sub-species and sex. Results showed no significant difference in mean FCM levels between the Bengal and Sumatran tiger sub-species. Mean levels of FCMs were significantly higher in females than in male tigers. Those male and female tigers with reported health issues during the study period expressed higher FCM levels than the reportedly healthy tigers. Interestingly, those tigers that took part in some activity (such as walks, photos, presentations and guest feeds) expressed moderately higher FCM levels at Dreamworld and lower FCM levels at Australia Zoo in comparison to those tigers that did not take part in such activities. These results indicate potential habituation in some tigers for routine activity through specialized training and pre-conditioning. In conclusion, the FCM EIA described in this study provides a reliable non-invasive method for evaluating the stress status of tigers in Zoos. Copyright © 2013 Elsevier Inc. All rights reserved.

[Zinc-dependent metalloprotease 1 promotes apoptosis of RAW264.7 macrophages].

PubMed

Li, Peng; He, Yonglin; Zhang, Jiming; Fang, Chencheng

2015-12-01

To construct the eukaryotic expression vector of zinc-dependent metalloprotease 1 (zmp1) gene from Bacillus Calmette-Guerin (BCG) and investigate its impact on the apoptosis of RAW264.7 macrophages. Zmp1 gene was amplified from the genome of BCG by PCR. The zmp1 gene fragment was inserted into multiple cloning sites of pEGFP-N1 to construct the eukaryotic expression vector pEGFP-N1-zmp1. The constructed pEGFP-N1-zmp1 was transfected into RAW264.7 cells by Lipofectamine(TM) 2000. The expression of green fluorescent protein (GFP) was observed by fluorescence microscopy. The zmp1 mRNA was detected by quantitative real-time PCR (qR-PCR). The effect of Zmp1 protein on the apoptosis of RAW264.7 macrophages was detected by flow cytometry (FCM). With zmp1 gene amplified by PCR, we successfully constructed the recombinant vector pEGFP-N1-zmp1 as demonstrated by restriction enzyme analysis and sequencing. GFP was seen in RAW264.7 cells 24 hours after transfected with the recombinant plasmid. As qRT-PCR showed, the expression level of zmp1 mRNA was up-regulated. The early apoptotic rate increased 48 hours after transfection. The increased expression of Zmp1 in RAW264.7 cells promotes the apoptosis of RAW264.7 cells.

Expression and Significance of Neuroligins in Myenteric Cells of Cajal in Hirschsprung's Disease

PubMed Central

Wang, Jian; Mou, Yaru; Zhang, Qiangye; Zhang, Fan; Yang, Hongchao; Zhang, Wentong; Li, Aiwu

2013-01-01

Purpose The aim of this study was to investigate the expression and significance of neuroligins in myenteric cells of Cajal (ICC-MY) in Hirschsprung’s disease (HSCR). Methods Longitudinal muscle with adherent myenteric plexus (LMMP) from surgical excision waste colon of HSCR children were prepared by peeling off the mucous layer, sub-mucosal layer and circular muscle. Neuroligins, c-Kit (c-Kit-immunoreactivity representing ICC) and their relationship were assessed by double labeling immunofluorescence staining. ICC-MY were dissociated and cultured from LMMP by enzymolysis method, and were purified and analyzed using a combination of magnetic-activated cell sorting (MACS) and flow cytometry (FCM). Western-blot analysis was applied to compare and evaluate the expression levels of neuroligins in ICC-MY which were dissociated from different segments of HSCR (ganglionic colonic segment, transitional colonic segment and aganglionic colonic segment). Results Neuroligins and c-Kit were expressed on the same cells (ICC-MY); ICC-MY were dissociated, cultured and purified. For HSCR, neuroligins were expressed significantly in ICC-MY from ganglionic colonic segments, moderately in those from transitional colonic segments and down-regulated significantly in those from aganglionic colonic segments. Conclusions Neuroligins were expressed in ICC-MY of human beings, and the expression varies from different segments of HSCR. This abnormal expression might play an important role in the pathogenesis of this disease through affecting the synaptic function of ICC-MY. PMID:23840625

Ozone levels in the Empty Quarter of Saudi Arabia--application of adaptive neuro-fuzzy model.

PubMed

Rahman, Syed Masiur; Khondaker, A N; Khan, Rouf Ahmad

2013-05-01

In arid regions, primary pollutants may contribute to the increase of ozone levels and cause negative effects on biotic health. This study investigates the use of adaptive neuro-fuzzy inference system (ANFIS) for ozone prediction. The initial fuzzy inference system is developed by using fuzzy C-means (FCM) and subtractive clustering (SC) algorithms, which determines the important rules, increases generalization capability of the fuzzy inference system, reduces computational needs, and ensures speedy model development. The study area is located in the Empty Quarter of Saudi Arabia, which is considered as a source of huge potential for oil and gas field development. The developed clustering algorithm-based ANFIS model used meteorological data and derived meteorological data, along with NO and NO₂ concentrations and their transformations, as inputs. The root mean square error and Willmott's index of agreement of the FCM- and SC-based ANFIS models are 3.5 ppbv and 0.99, and 8.9 ppbv and 0.95, respectively. Based on the analysis of the performance measures and regression error characteristic curves, it is concluded that the FCM-based ANFIS model outperforms the SC-based ANFIS model.

FIND-CKD: a randomized trial of intravenous ferric carboxymaltose versus oral iron in patients with chronic kidney disease and iron deficiency anaemia.

PubMed

Macdougall, Iain C; Bock, Andreas H; Carrera, Fernando; Eckardt, Kai-Uwe; Gaillard, Carlo; Van Wyck, David; Roubert, Bernard; Nolen, Jacqueline G; Roger, Simon D

2014-11-01

The optimal iron therapy regimen in patients with non-dialysis-dependent chronic kidney disease (CKD) is unknown. Ferinject® assessment in patients with Iron deficiency anaemia and Non-Dialysis-dependent Chronic Kidney Disease (FIND-CKD) was a 56-week, open-label, multicentre, prospective and randomized study of 626 patients with non-dialysis-dependent CKD, anaemia and iron deficiency not receiving erythropoiesis-stimulating agents (ESAs). Patients were randomized (1:1:2) to intravenous (IV) ferric carboxymaltose (FCM), targeting a higher (400-600 µg/L) or lower (100-200 µg/L) ferritin or oral iron therapy. The primary end point was time to initiation of other anaemia management (ESA, other iron therapy or blood transfusion) or haemoglobin (Hb) trigger of two consecutive values <10 g/dL during Weeks 8-52. The primary end point occurred in 36 patients (23.5%), 49 patients (32.2%) and 98 patients (31.8%) in the high-ferritin FCM, low-ferritin FCM and oral iron groups, respectively [hazard ratio (HR): 0.65; 95% confidence interval (CI): 0.44-0.95; P = 0.026 for high-ferritin FCM versus oral iron]. The increase in Hb was greater with high-ferritin FCM versus oral iron (P = 0.014) and a greater proportion of patients achieved an Hb increase ≥1 g/dL with high-ferritin FCM versus oral iron (HR: 2.04; 95% CI: 1.52-2.72; P < 0.001). Rates of adverse events and serious adverse events were similar in all groups. Compared with oral iron, IV FCM targeting a ferritin of 400-600 µg/L quickly reached and maintained Hb level, and delayed and/or reduced the need for other anaemia management including ESAs. Within the limitations of this trial, no renal toxicity was observed, with no difference in cardiovascular or infectious events. NCT00994318. © The Author 2014. Published by Oxford University Press on behalf of ERA-EDTA.

Long Term Uncertainty Investigations of 1 MN Force Calibration Machine at NPL, India (NPLI)

NASA Astrophysics Data System (ADS)

Kumar, Rajesh; Kumar, Harish; Kumar, Anil; Vikram

2012-01-01

The present paper is an attempt to study the long term uncertainty of 1 MN hydraulic multiplication system (HMS) force calibration machine (FCM) at the National Physical Laboratory, India (NPLI), which is used for calibration of the force measuring instruments in the range of 100 kN - 1 MN. The 1 MN HMS FCM was installed at NPLI in 1993 and was built on the principle of hydraulic amplifications of dead weights. The best measurement capability (BMC) of the machine is ± 0.025% (k = 2) and it is traceable to national standards by means of precision force transfer standards (FTS). The present study discusses the uncertainty variations of the 1 MN HMS FCM over the years and describes the other parameters in detail, too. The 1 MN HMS FCM was calibrated in the years 2004, 2006, 2007, 2008, 2009 and 2010 and the results have been reported.

TGF-β1, released by myofibroblasts, differentially regulates transcription and function of sodium and potassium channels in adult rat ventricular myocytes.

PubMed

Kaur, Kuljeet; Zarzoso, Manuel; Ponce-Balbuena, Daniela; Guerrero-Serna, Guadalupe; Hou, Luqia; Musa, Hassan; Jalife, José

2013-01-01

Cardiac injury promotes fibroblasts activation and differentiation into myofibroblasts, which are hypersecretory of multiple cytokines. It is unknown whether any of such cytokines are involved in the electrophysiological remodeling of adult cardiomyocytes. We cultured adult cardiomyocytes for 3 days in cardiac fibroblast conditioned medium (FCM) from adult rats. In whole-cell voltage-clamp experiments, FCM-treated myocytes had 41% more peak inward sodium current (I(Na)) density at -40 mV than myocytes in control medium (p<0.01). In contrast, peak transient outward current (I(to)) was decreased by ∼55% at 60 mV (p<0.001). Protein analysis of FCM demonstrated that the concentration of TGF-β1 was >3 fold greater in FCM than control, which suggested that FCM effects could be mediated by TGF-β1. This was confirmed by pre-treatment with TGF-β1 neutralizing antibody, which abolished the FCM-induced changes in both I(Na) and I(to). In current-clamp experiments TGF-β1 (10 ng/ml) prolonged the action potential duration at 30, 50, and 90 repolarization (p<0.05); at 50 ng/ml it gave rise to early afterdepolarizations. In voltage-clamp experiments, TGF-β1 increased I(Na) density in a dose-dependent manner without affecting voltage dependence of activation or inactivation. I(Na) density was -36.25±2.8 pA/pF in control, -59.17±6.2 pA/pF at 0.1 ng/ml (p<0.01), and -58.22±6.6 pA/pF at 1 ng/ml (p<0.01). In sharp contrast, I(to) density decreased from 22.2±1.2 pA/pF to 12.7±0.98 pA/pF (p<0.001) at 10 ng/ml. At 1 ng/ml TGF-β1 significantly increased SCN5A (Na(V)1.5) (+73%; p<0.01), while reducing KCNIP2 (Kchip2; -77%; p<0.01) and KCND2 (K(V)4.2; -50% p<0.05) mRNA levels. Further, the TGF-β1-induced increase in I(Na) was mediated through activation of the PI3K-AKT pathway via phosphorylation of FOXO1 (a negative regulator of SCN5A). TGF-β1 released by myofibroblasts differentially regulates transcription and function of the main cardiac sodium channel and of the channel responsible for the transient outward current. The results provide new mechanistic insight into the electrical remodeling associated with myocardial injury.

Automated fibroglandular tissue segmentation and volumetric density estimation in breast MRI using an atlas-aided fuzzy C-means method

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

Wu, Shandong; Weinstein, Susan P.; Conant, Emily F.

Purpose: Breast magnetic resonance imaging (MRI) plays an important role in the clinical management of breast cancer. Studies suggest that the relative amount of fibroglandular (i.e., dense) tissue in the breast as quantified in MR images can be predictive of the risk for developing breast cancer, especially for high-risk women. Automated segmentation of the fibroglandular tissue and volumetric density estimation in breast MRI could therefore be useful for breast cancer risk assessment. Methods: In this work the authors develop and validate a fully automated segmentation algorithm, namely, an atlas-aided fuzzy C-means (FCM-Atlas) method, to estimate the volumetric amount of fibroglandularmore » tissue in breast MRI. The FCM-Atlas is a 2D segmentation method working on a slice-by-slice basis. FCM clustering is first applied to the intensity space of each 2D MR slice to produce an initial voxelwise likelihood map of fibroglandular tissue. Then a prior learned fibroglandular tissue likelihood atlas is incorporated to refine the initial FCM likelihood map to achieve enhanced segmentation, from which the absolute volume of the fibroglandular tissue (|FGT|) and the relative amount (i.e., percentage) of the |FGT| relative to the whole breast volume (FGT%) are computed. The authors' method is evaluated by a representative dataset of 60 3D bilateral breast MRI scans (120 breasts) that span the full breast density range of the American College of Radiology Breast Imaging Reporting and Data System. The automated segmentation is compared to manual segmentation obtained by two experienced breast imaging radiologists. Segmentation performance is assessed by linear regression, Pearson's correlation coefficients, Student's pairedt-test, and Dice's similarity coefficients (DSC). Results: The inter-reader correlation is 0.97 for FGT% and 0.95 for |FGT|. When compared to the average of the two readers’ manual segmentation, the proposed FCM-Atlas method achieves a correlation ofr = 0.92 for FGT% and r = 0.93 for |FGT|, and the automated segmentation is not statistically significantly different (p = 0.46 for FGT% and p = 0.55 for |FGT|). The bilateral correlation between left breasts and right breasts for the FGT% is 0.94, 0.92, and 0.95 for reader 1, reader 2, and the FCM-Atlas, respectively; likewise, for the |FGT|, it is 0.92, 0.92, and 0.93, respectively. For the spatial segmentation agreement, the automated algorithm achieves a DSC of 0.69 ± 0.1 when compared to reader 1 and 0.61 ± 0.1 for reader 2, respectively, while the DSC between the two readers’ manual segmentation is 0.67 ± 0.15. Additional robustness analysis shows that the segmentation performance of the authors' method is stable both with respect to selecting different cases and to varying the number of cases needed to construct the prior probability atlas. The authors' results also show that the proposed FCM-Atlas method outperforms the commonly used two-cluster FCM-alone method. The authors' method runs at ∼5 min for each 3D bilateral MR scan (56 slices) for computing the FGT% and |FGT|, compared to ∼55 min needed for manual segmentation for the same purpose. Conclusions: The authors' method achieves robust segmentation and can serve as an efficient tool for processing large clinical datasets for quantifying the fibroglandular tissue content in breast MRI. It holds a great potential to support clinical applications in the future including breast cancer risk assessment.« less

Automated fibroglandular tissue segmentation and volumetric density estimation in breast MRI using an atlas-aided fuzzy C-means method

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

Wu, Shandong; Weinstein, Susan P.; Conant, Emily F.

2013-12-15

Purpose: Breast magnetic resonance imaging (MRI) plays an important role in the clinical management of breast cancer. Studies suggest that the relative amount of fibroglandular (i.e., dense) tissue in the breast as quantified in MR images can be predictive of the risk for developing breast cancer, especially for high-risk women. Automated segmentation of the fibroglandular tissue and volumetric density estimation in breast MRI could therefore be useful for breast cancer risk assessment. Methods: In this work the authors develop and validate a fully automated segmentation algorithm, namely, an atlas-aided fuzzy C-means (FCM-Atlas) method, to estimate the volumetric amount of fibroglandularmore » tissue in breast MRI. The FCM-Atlas is a 2D segmentation method working on a slice-by-slice basis. FCM clustering is first applied to the intensity space of each 2D MR slice to produce an initial voxelwise likelihood map of fibroglandular tissue. Then a prior learned fibroglandular tissue likelihood atlas is incorporated to refine the initial FCM likelihood map to achieve enhanced segmentation, from which the absolute volume of the fibroglandular tissue (|FGT|) and the relative amount (i.e., percentage) of the |FGT| relative to the whole breast volume (FGT%) are computed. The authors' method is evaluated by a representative dataset of 60 3D bilateral breast MRI scans (120 breasts) that span the full breast density range of the American College of Radiology Breast Imaging Reporting and Data System. The automated segmentation is compared to manual segmentation obtained by two experienced breast imaging radiologists. Segmentation performance is assessed by linear regression, Pearson's correlation coefficients, Student's pairedt-test, and Dice's similarity coefficients (DSC). Results: The inter-reader correlation is 0.97 for FGT% and 0.95 for |FGT|. When compared to the average of the two readers’ manual segmentation, the proposed FCM-Atlas method achieves a correlation ofr = 0.92 for FGT% and r = 0.93 for |FGT|, and the automated segmentation is not statistically significantly different (p = 0.46 for FGT% and p = 0.55 for |FGT|). The bilateral correlation between left breasts and right breasts for the FGT% is 0.94, 0.92, and 0.95 for reader 1, reader 2, and the FCM-Atlas, respectively; likewise, for the |FGT|, it is 0.92, 0.92, and 0.93, respectively. For the spatial segmentation agreement, the automated algorithm achieves a DSC of 0.69 ± 0.1 when compared to reader 1 and 0.61 ± 0.1 for reader 2, respectively, while the DSC between the two readers’ manual segmentation is 0.67 ± 0.15. Additional robustness analysis shows that the segmentation performance of the authors' method is stable both with respect to selecting different cases and to varying the number of cases needed to construct the prior probability atlas. The authors' results also show that the proposed FCM-Atlas method outperforms the commonly used two-cluster FCM-alone method. The authors' method runs at ∼5 min for each 3D bilateral MR scan (56 slices) for computing the FGT% and |FGT|, compared to ∼55 min needed for manual segmentation for the same purpose. Conclusions: The authors' method achieves robust segmentation and can serve as an efficient tool for processing large clinical datasets for quantifying the fibroglandular tissue content in breast MRI. It holds a great potential to support clinical applications in the future including breast cancer risk assessment.« less

Prevalence and determinants of stereotypic behaviours and physiological stress among tigers and leopards in Indian zoos

PubMed Central

Vaz, Janice; Narayan, Edward J.; Dileep Kumar, R.; Thenmozhi, K.; Thiyagesan, Krishnamoorthy

2017-01-01

India’s charismatic wildlife species are facing immense pressure from anthropogenic-induced environmental perturbations. Zoos play a major role in the conservation of threatened species, but their adaptation in captivity is posing a major challenge globally. Stress from inadequate adaptation could lead to suppression of cognitive functioning and increased display of stereotypic behaviour. It is thus necessary to measure biological traits like behaviour, stress physiology, and contextual factors driving the animals maintained at zoos. In this study, we assessed stereotypic behaviour and stress physiology employing standard behaviour scoring, non-invasive stress monitoring, and their contextual drivers in a sub-population of two large felid species managed in six Indian zoos. The prevalence and intensity of stereotypic behaviours and levels of faecal corticosterone metabolites (FCM) were ascertained among 41 Royal Bengal tigers Panthera tigris tigris and 21 Indian leopards Panthera pardus fusca between April 2014 and March 2015. Behavioural observations showed that tigers spent more time stereotyping (12%) than leopards (7%) during daylight hours. Stress levels assessed using FCM revealed that tigers (23.6 ± 1.62 ng/g) had marginally lower level of corticosterone metabolites than leopards (27.2 ±1.36 ng/g). Stereotypic behaviour increased significantly with FCM level when the effect of heath status was controlled in tigers, and the effects tree cover, stone, den and keeper attitude controlled in leopards. Comparison of stereotypes of tigers with various biological and environmental factors using binary logistic regression revealed that stereotypic prevalence decreased with increased enclosure size, and enclosure enrichments like presence of pools and stones, when managed socially with conspecifics, and with positive keeper attitude, these factors accounting for 43% of variations in stereotypic prevalence among tigers. Stereotype among leopards was significantly absent when associated with increased tree cover and presence of pool, and den in the enclosure, age and among zoo-born than wild-born ones. These factors explain 81% of variations in stereotypic prevalence in them. A comparison of FCM levels with context-dependent factors revealed that stress levels among tigers decreased significantly with enclosure size and with individuals from nil to low, and severity of health issues. These factors explain 64% of variations in FCM levels. In leopards, the presence of stones in the enclosure and keepers with positive attitude resulted in significant decrease in FCM levels, these factors together accounting for 94% of variations. Multiple regressions on selected variables based on Factor Analysis of Mixed Data showed that in tigers the intensity of stereotype decreased significantly with enclosure size, sociality and positive keeper attitude and FCM level with health problems. Similarly, analyses in leopards revealed that intensity of stereotype decreased significantly with tree cover, age and FCM level with positive keeper attitude. Overall, our study suggests that to reduce stereotypes and stress level, tigers in captivity should be managed in larger enclosures enriched with pool, and stones, and in appropriate social conditions with adequate veterinary care. Leopards should be managed in enclosures with dense tree cover, pool, stones and den. Positive keeper attitude plays a crucial role in the welfare of both the species in captivity. Our study is promising and is comparable with their natural behaviour in the wild; for example, tigers require larger natural habitats, while leopards can manage even with smaller isolated patches but with dense vegetation cover. PMID:28414723

Prevalence and determinants of stereotypic behaviours and physiological stress among tigers and leopards in Indian zoos.

PubMed

Vaz, Janice; Narayan, Edward J; Dileep Kumar, R; Thenmozhi, K; Thiyagesan, Krishnamoorthy; Baskaran, Nagarajan

2017-01-01

India's charismatic wildlife species are facing immense pressure from anthropogenic-induced environmental perturbations. Zoos play a major role in the conservation of threatened species, but their adaptation in captivity is posing a major challenge globally. Stress from inadequate adaptation could lead to suppression of cognitive functioning and increased display of stereotypic behaviour. It is thus necessary to measure biological traits like behaviour, stress physiology, and contextual factors driving the animals maintained at zoos. In this study, we assessed stereotypic behaviour and stress physiology employing standard behaviour scoring, non-invasive stress monitoring, and their contextual drivers in a sub-population of two large felid species managed in six Indian zoos. The prevalence and intensity of stereotypic behaviours and levels of faecal corticosterone metabolites (FCM) were ascertained among 41 Royal Bengal tigers Panthera tigris tigris and 21 Indian leopards Panthera pardus fusca between April 2014 and March 2015. Behavioural observations showed that tigers spent more time stereotyping (12%) than leopards (7%) during daylight hours. Stress levels assessed using FCM revealed that tigers (23.6 ± 1.62 ng/g) had marginally lower level of corticosterone metabolites than leopards (27.2 ±1.36 ng/g). Stereotypic behaviour increased significantly with FCM level when the effect of heath status was controlled in tigers, and the effects tree cover, stone, den and keeper attitude controlled in leopards. Comparison of stereotypes of tigers with various biological and environmental factors using binary logistic regression revealed that stereotypic prevalence decreased with increased enclosure size, and enclosure enrichments like presence of pools and stones, when managed socially with conspecifics, and with positive keeper attitude, these factors accounting for 43% of variations in stereotypic prevalence among tigers. Stereotype among leopards was significantly absent when associated with increased tree cover and presence of pool, and den in the enclosure, age and among zoo-born than wild-born ones. These factors explain 81% of variations in stereotypic prevalence in them. A comparison of FCM levels with context-dependent factors revealed that stress levels among tigers decreased significantly with enclosure size and with individuals from nil to low, and severity of health issues. These factors explain 64% of variations in FCM levels. In leopards, the presence of stones in the enclosure and keepers with positive attitude resulted in significant decrease in FCM levels, these factors together accounting for 94% of variations. Multiple regressions on selected variables based on Factor Analysis of Mixed Data showed that in tigers the intensity of stereotype decreased significantly with enclosure size, sociality and positive keeper attitude and FCM level with health problems. Similarly, analyses in leopards revealed that intensity of stereotype decreased significantly with tree cover, age and FCM level with positive keeper attitude. Overall, our study suggests that to reduce stereotypes and stress level, tigers in captivity should be managed in larger enclosures enriched with pool, and stones, and in appropriate social conditions with adequate veterinary care. Leopards should be managed in enclosures with dense tree cover, pool, stones and den. Positive keeper attitude plays a crucial role in the welfare of both the species in captivity. Our study is promising and is comparable with their natural behaviour in the wild; for example, tigers require larger natural habitats, while leopards can manage even with smaller isolated patches but with dense vegetation cover.

Dendritic Cells in the Context of Human Tumors: Biology and Experimental Tools.

PubMed

Volovitz, Ilan; Melzer, Susanne; Amar, Sarah; Bocsi, József; Bloch, Merav; Efroni, Sol; Ram, Zvi; Tárnok, Attila

2016-01-01

Dendritic cells (DC) are the most potent and versatile antigen-presenting cells (APC) in the immune system. DC have an exceptional ability to comprehend the immune context of a captured antigen based on molecular signals identified from its vicinity. The analyzed information is then conveyed to other immune effector cells. Such capability enables DC to play a pivotal role in mediating either an immunogenic response or immune tolerance towards an acquired antigen. This review summarizes current knowledge on DC in the context of human tumors. It covers the basics of human DC biology, elaborating on the different markers, morphology and function of the different subsets of human DC. Human blood-borne DC are comprised of at least three subsets consisting of one plasmacytoid DC (pDC) and two to three myeloid DC (mDC) subsets. Some tissues have unique DC. Each subset has a different phenotype and function and may induce pro-tumoral or anti-tumoral effects. The review also discusses two methods fundamental to the research of DC on the single-cell level: multicolor flow cytometry (FCM) and image-based cytometry (IC). These methods, along with new genomics and proteomics tools, can provide high-resolution information on specific DC subsets and on immune and tumor cells with which they interact. The different layers of collected biological data may then be integrated using Immune-Cytomics modeling approaches. Such novel integrated approaches may help unravel the complex network of cellular interactions that DC carry out within tumors, and may help harness this complex immunological information into the development of more effective treatments for cancer.

UN TRISO Compaction in SiC for FCM Fuel Irradiations

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

Terrani, Kurt A.; Trammell, Michael P.; Kiggans, James O.

2016-11-01

The U.S. Department of Energy Office of Nuclear Energy (DOE-NE) Advanced Fuels Campaign (AFC) is conducting research and development to elevate the technology readiness level of Fully Ceramic Microencapsulated (FCM) fuels, a candidate nuclear fuel with potentially enhanced accident tolerance due to very high fission product retention. One of the early activities in FY17 was to demonstrate production of FCM pellets with uranium nitride TRISO particles. This was carried out in preparation of the larger pellet production campaign in support of the upcoming irradiation testing of this fuel form at INL’s Advanced Test Reactor.

Classification of posture maintenance data with fuzzy clustering algorithms

NASA Technical Reports Server (NTRS)

Bezdek, James C.

1991-01-01

Sensory inputs from the visual, vestibular, and proprioreceptive systems are integrated by the central nervous system to maintain postural equilibrium. Sustained exposure to microgravity causes neurosensory adaptation during spaceflight, which results in decreased postural stability until readaptation occurs upon return to the terrestrial environment. Data which simulate sensory inputs under various conditions were collected in conjunction with JSC postural control studies using a Tilt-Translation Device (TTD). The University of West Florida proposed applying the Fuzzy C-Means Clustering (FCM) Algorithms to this data with a view towards identifying various states and stages. Data supplied by NASA/JSC were submitted to the FCM algorithms in an attempt to identify and characterize cluster substructure in a mixed ensemble of pre- and post-adaptational TTD data. Following several unsuccessful trials with FCM using a full 11 dimensional data set, a set of two channels (features) were found to enable FCM to separate pre- from post-adaptational TTD data. The main conclusions are that: (1) FCM seems able to separate pre- from post-TTD subject no. 2 on the one trial that was used, but only in certain subintervals of time; and (2) Channels 2 (right rear transducer force) and 8 (hip sway bar) contain better discrimination information than other supersets and combinations of the data that were tried so far.

Printed paper and board food contact materials as a potential source of food contamination.

PubMed

Van Bossuyt, Melissa; Van Hoeck, Els; Vanhaecke, Tamara; Rogiers, Vera; Mertens, Birgit

2016-11-01

Food contact materials (FCM) are estimated to be the largest source of food contamination. Apart from plastics, the most commonly used FCM are made of printed paper and board. Unlike their plastic counterparts, these are not covered by a specific European regulation. Several contamination issues have raised concerns towards potential adverse health effects caused by exposure to substances migrating from printed paper and board FCM. In the current study, an inventory combining the substances which may be used in printed paper and board FCM, was created. More than 6000 unique compounds were identified, the majority (77%) considered non-evaluated in terms of potential toxicity. Based on a preliminary study of their physicochemical properties, it is estimated that most of the non-evaluated single substances have the potential to migrate into the food and become bioavailable after oral intake. Almost all are included in the FACET tool, indicating that their use in primary food packaging has been confirmed by industry. Importantly, 19 substances are also present in one of the lists with substances of concern compiled by the European Chemicals Agency (ECHA). To ensure consumer safety, the actual use of these substances in printed paper and board FCM should be investigated urgently. Copyright © 2016 Elsevier Inc. All rights reserved.

First identification of the herpes simplex virus by skin-dedicated ex vivo fluorescence confocal microscopy during herpetic skin infections.

PubMed

Cinotti, E; Perrot, J L; Labeille, B; Campolmi, N; Thuret, G; Naigeon, N; Bourlet, T; Pillet, S; Cambazard, F

2015-06-01

Skin-dedicated ex vivo fluorescence confocal microscopy (FCM) has so far been used to identify cutaneous tumours on freshly excised samples using acridine orange as fluorochrome. To use FCM for a new indication, namely, the identification of the herpes simplex virus (HSV) in skin lesions, using fluorescent antibodies. Six roof samples from skin vesicles suspicious for HSV lesions were incubated with anti-HSV-1 and anti-HSV-2 antibodies coupled with fluorescein isothiocyanate, and examined under skin-dedicated ex vivo FCM. The positive controls were swabs taken from the floor of each vesicle and observed under conventional direct fluorescence assay (DFA) and by viral cultures. Roof samples from three bullae of bullous pemphigoid were the negative controls. Using ex vivo FCM, the samples from the lesions clinically suspicious for HSV infection were seen to be fluorescent after incubation with anti-HSV-1, and were negative after incubation with anti-HSV-2 antibodies. Conventional DFA with an optical microscope and cultures confirmed the presence of HSV-1 infection. By using fluorescent antibodies to identify precise structures, ex vivo FCM can be used for indications other than tumour identification. More specifically, it can be an additional diagnostic tool for HSV infection. © 2014 British Association of Dermatologists.

Evaluating Stress Physiology and Parasite Infection Parameters in the Translocation of Critically Endangered Woylies (Bettongia penicillata).

PubMed

Hing, Stephanie; Northover, Amy S; Narayan, Edward J; Wayne, Adrian F; Jones, Krista L; Keatley, Sarah; Thompson, R C Andrew; Godfrey, Stephanie S

2017-03-01

Translocation can be stressful for wildlife. Stress may be important in fauna translocation because it has been suggested that it can exacerbate the impact of infectious disease on translocated wildlife. However, few studies explore this hypothesis by measuring stress physiology and infection indices in parallel during wildlife translocations. We analysed faecal cortisol metabolite (FCM) concentration and endoparasite parameters (nematodes, coccidians and haemoparasites) in a critically endangered marsupial, the woylie (Bettongia penicillata), 1-3 months prior to translocation, at translocation, and 6 months later. FCM for both translocated and resident woylies was significantly higher after translocation compared to before or at translocation. In addition, body condition decreased with increasing FCM after translocation. These patterns in host condition and physiology may be indicative of translocation stress or stress associated with factors independent of the translocation. Parasite factors also influenced FCM in translocated woylies. When haemoparasites were detected, there was a significant negative relationship between strongyle egg count and FCM. This may reflect the influence of glucocorticoids on the immune response to micro- and macro-parasites. Our results indicate that host physiology and infection patterns can change significantly during translocation, but further investigation is required to determine how these patterns influence translocation success.

Reliable and Accurate CD4+ T Cell Count and Percent by the Portable Flow Cytometer CyFlow MiniPOC and “CD4 Easy Count Kit-Dry”, as Revealed by the Comparison with the Gold Standard Dual Platform Technology

PubMed Central

Nasi, Milena; De Biasi, Sara; Bianchini, Elena; Gibellini, Lara; Pinti, Marcello; Scacchetti, Tiziana; Trenti, Tommaso; Borghi, Vanni; Mussini, Cristina; Cossarizza, Andrea

2015-01-01

Background An accurate and affordable CD4+ T cells count is an essential tool in the fight against HIV/AIDS. Flow cytometry (FCM) is the “gold standard” for counting such cells, but this technique is expensive and requires sophisticated equipment, temperature-sensitive monoclonal antibodies (mAbs) and trained personnel. The lack of access to technical support and quality assurance programs thus limits the use of FCM in resource-constrained countries. We have tested the accuracy, the precision and the carry-over contamination of Partec CyFlow MiniPOC, a portable and economically affordable flow cytometer designed for CD4+ count and percentage, used along with the “CD4% Count Kit-Dry”. Materials and Methods Venous blood from 59 adult HIV+ patients (age: 25–58 years; 43 males and 16 females) was collected and stained with the “MiniPOC CD4% Count Kit-Dry”. CD4+ count and percentage were then determined in triplicate by the CyFlow MiniPOC. In parallel, CD4 count was performed using mAbs and a CyFlow Counter, or by a dual platform system (from Beckman Coulter) based upon Cytomic FC500 (“Cytostat tetrachrome kit” for mAbs) and Coulter HmX Hematology Analyzer (for absolute cell count). Results The accuracy of CyFlow MiniPOC against Cytomic FC500 showed a correlation coefficient (CC) of 0.98 and 0.97 for CD4+ count and percentage, respectively. The accuracy of CyFlow MiniPOC against CyFlow Counter showed a CC of 0.99 and 0.99 for CD4 T cell count and percentage, respectively. CyFlow MiniPOC showed an excellent repeatability: CD4+ cell count and percentage were analyzed on two instruments, with an intra-assay precision below ±5% deviation. Finally, there was no carry-over contamination for samples at all CD4 values, regardless of their position in the sequence of analysis. Conclusion The cost-effective CyFlow MiniPOC produces rapid, reliable and accurate results that are fully comparable with those from highly expensive dual platform systems. PMID:25622041

TMPYP4 exerted antitumor effects in human cervical cancer cells through activation of p38 mitogen-activated protein kinase.

PubMed

Cheng, Ming-Jun; Cao, Yun-Gui

2017-07-03

The aim of the present study was to investigate the potential effects of the 5,10,15,20-tetrakis (1-methylpyridinium-4-yl) porphyrin (TMPyP4) on the proliferation and apoptosis of human cervical cancer cells and the underlying mechanisms by which TMPyP4 exerted its actions. After human cervical cancer cells were treated with different doses of TMPyP4, cell viability was determined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) method, the apoptosis was observed by flow cytometry (FCM), and the expression of p38 mitogen-activated protein kinase (MAPK), phosphated p38 MAPK (p-p38 MAPK), capase-3, MAPKAPK2 (MK-2) and poly ADP-ribose polymerase (PARP) was measured by Western blot analysis. The analysis revealed that TMPyP4 potently suppressed cell viability and induced the apoptosis of human cervical cancer cells in a dose-dependent manner. In addition, the up-regulation of p-p38 MAPK expression levels was detected in TMPyP4-treated human cervical cancer cells. However, followed by the block of p38 MAPK signaling pathway using the inhibitor SB203580, the effects of TMPyP4 on proliferation and apoptosis of human cervical cancer cells were significantly changed. It was indicated that TMPyP4-inhibited proliferation and -induced apoptosis in human cervical cancer cells was accompanied by activating the p38 MAPK signaling pathway. Taken together, our study demonstrates that TMPyP4 may represent a potential therapeutic method for the treatment of cervical carcinoma.

[Study on the relationship between renal apoptosis and expression of caspase protein in fluoride induced rat].

PubMed

Gao, Jiping; Song, Guohua; Liu, Maolin; Wang, Yu; Yang, Xia

2014-01-01

To study the relationship between death receptor pathway, mitochondrion pathway and fluoride-induced apoptosis of renal cell. Male Sprague-Dawley rats were divided randomly into four groups (control, low-fluoride, medium-fluoride,and high-fluoride) and administered 0, 50, 100, and 200 mg/L of sodium fluoride, respectively, via drinking water for 120 days. The incidence of dental fluorosis were observed, the body weights and urine fluoride levels were measured. Apoptosis was detected by the Flow Cytometry (FCM). The expressions of protein of Caspase-3, Caspase-8, Caspase-9, Cyt C were detectedby immunohistoehemistry. The apoptosis rate in the fluoride exposed low does group,middle dose group and high dose group increased significantly as compared with control group. The average optical density value of Caspase-3, Caspase-8, Caspase-9 and Cyt C were higher in the fluoride exposed middle dose group and high dose group than those in the control group (P < 0.05). Death receptor pathway and mitochondrion pathway may participate in the process of fluoride-induced apoptosis of renal cell.

Simian virus 40 inhibits differentiation and maturation of rhesus macaque DC-SIGN(+) dendritic cells.

PubMed

Changyong, C; Sun, M; Li, H; Brockmeyer, N; Wu, Nan Ping

2010-09-24

Dendritic cells (DC) are the initiators and modulators of the immune responses. Some species of pathogenic microorganisms have developed immune evasion strategies by controlling antigen presentation function of DC. Simian virus 40 (SV40) is a DNA tumor virus of rhesus monkey origin. It can induce cell transformation and tumorigenesis in many vertebrate species, but often causes no visible effects and persists as a latent infection in rhesus monkeys under natural conditions. To investigate the interaction between SV40 and rhesus monkey DC, rhesus monkey peripheral blood monocyte-derived DC were induced using recombinant human Interleukin-4 (rhIL-4) and infective SV40, the phenotype and function of DC-specific intracellular adhesion molecule-3 grabbing nonintegrin (DC-SIGN)(+) DC were analyzed by flow cytometry (FCM) and mixed lymphocyte reaction (MLR). Results showed that SV40 can down-regulate the expression of CD83 and CD86 on DC and impair DC-induced activation of T cell proliferation. These findings suggest that SV40 might also cause immune suppression by influencing differentiation and maturation of DC.

Utility of a Fluorescence Microscopy Imaging System for Analyzing the DNA Ploidy of Pathological Megakaryocytes Including 5q- Syndrome.

PubMed

Nakahara, Takako; Suemori, Shinichiro; Tsujioka, Takayuki; Kataoka, Mikio; Kataoka, Hiromi; Shibakura, Misako; Tohyama, Kaoru

2018-06-01

To investigate megakaryocyte (MK) DNA ploidy in various hematological diseases, fluorescence microscopy imaging system (FMI) can be used to analyze DNA ploidy with cell morphology at the single-cell level by using specialized image-processing software. Here we compared DNA ploidy obtained by FMI measured with that obtained flow cytometry (FCM). With FMI, we could evaluate the DNA ploidy in long-term preserved bone marrow smear samples after staining. We next analyzed the MK DNA ploidy in 42 bone marrow smear samples including 26 myeloid neoplasm cases, and we compared the DNA ploidy and platelet counts in the patients' peripheral blood; the production of platelets was significantly high compared to DNA ploidy in the myeloproliferative neoplasms group. The FMI method revealed that the patients with 5q- syndrome exhibited relatively low DNA ploidy despite high platelet counts, and this result suggested that increased DNA ploidy is not indispensable to abundant platelet production. The FMI method for DNA ploidy will be a useful tool to clarify the relationship between DNA ploidy and platelet production by MKs.

Preparation of Deep Sea Fish Oil-Based Nanostructured Lipid Carriers with Enhanced Cellular Uptake.

PubMed

Zhu, Qiu-Yun; Guissi, Fida; Yang, Ru-Ya; Wang, Qian; Wang, Ke; Chen, Dan; Han, Zhi-Hao; Ma, Yi; Zhang, Min; Gu, Yue-Qing

2015-12-01

Nanostructured lipid carriers (NLC) are a promising pharmaceutical delivery system with mean diameter less than 200 nm which are dispersed in an aqueous phase containing emulsifier(s), to increase the water solubility, stability and bioavailability of oil compounds. Herein we prepared a promising NLC with glyceryl monostearate (GMS) as the solid lipid template and deep sea fish oil as the liquid lipid template using melted-ultrasonic method. Fish oil-NLC had a mean size of 84.7 ± 2.6 nm and a zeta potential that ranged from -17.87 mV to -32.91 mV. The nanoparticles exhibited good stability for four weeks with a high encapsulation efficiency of 87.5 ± 5.2%. Afterwards, confocal laser scanning microscopy (CLSM) and flow cytometry (FCM) were used to investigate the contribution of Fish oil-NLC in enhancing fluorescein isothiocyanate (FITC) cellular uptake in comparison with free FITC. The results of this study indicated the possibility of this carrier to overcome the shortcomings of deep sea fish oil and to provide a novel bifunctional carrier with nutritional potential and drug delivery ability.

Efficient killing effect of osteosarcoma cells by cinobufacini and cisplatin in combination.

PubMed

Huang, Tao; Gong, Wei-Hua; Li, Xiu-Cheng; Zou, Chun-Ping; Jiang, Guang-Jian; Li, Xu-Hui; Qian, Hao

2012-01-01

To study the killing effects on osteosarcoma cells of cinobufacini and cisplatin in combination and the related mechanisms so as to explore the chemotherapeutic method with integrated traditional Chinese and Western medicines. Cinobufacini and cisplatin were applied to OS732 cells singly or jointly and survival rates were measured by MTT assay. Changes in cellular shape were observed with inverted phase contrast and fluorescence microscopy and apoptosis rates were analyzed with flow cytometry (FCM). Immunocytochemistry were used to examine the Fas expression of OS732 cells. The combination of cinobufacini and cisplatin had the effect of up-regulating Fas expression and inducing apoptosis. The survival rate of combined application of 100 μg/ml cinobufacini and 1 μg/ml cisplatin on OS-732 cells was significantly lower than with either of the agents alone (p<0.01). Changes in cellular shape and apoptotic rates also indicated the apoptosis-inducing effects of combined application were much enhanced. The combination of cinobufacini and cisplatin demonstrated strong killing effects on OS-732 cells which might be related to up-regulation of Fas expression.

Cellular Apoptosis of Hemocytes from Dendrolimus tabulaeformis Tsai et Liu Larvae Induced with the Secondary Metabolites of Beauveria brongniartii (Sacc.) Petch

PubMed Central

Fan, Jinhua; Xie, Yingping; Xue, Jiaoliang; Zhang, Yingling; Yang, Qian

2013-01-01

To investigate the effect of the secondary metabolites of entomopathogenic fungus on the hemocyte immunity of host insect, the secondary metabolite complex (SMC) of Beauveria brongniartii was used in three concentrations (5.5, 55, and 550 µg/mL), and the 4th instar larvae of the pine caterpillar Dendrolimus tabulaeformis were employed as host insects. The larvae were inoculated with the SMC solutions by injection in bioassays. Apoptosis of the larval hemocytes was observed using fluorescence microscopy (FM), transmission electron microscopy (TEM), and flow cytometry (FCM). The FM results showed that in the treated groups, larval hemocytes exhibited symptoms of early apoptosis at 6 h post-treatment by radiating a non-uniform kelly fluorescence and exhibited symptoms of late apoptosis at 12 h post-treatment by radiating a non-uniform orange fluorescence. Under TEM, the following ultra-structural changes associated with apoptosis of the larval hemocytes were observed in the treated groups: the nuclei were hypertrophied, slight folds were on the nuclear envelope, the chromatin became concentrated, the mitochondrial cristae disappeared or were disorderly, most cells developed blebs, and fibrillar aggregation appeared and accumulated in the cytoplasm. Apoptosis of the larval hemocytes was detected by FCM at 6 h post-treatment; the percentage of early apoptotic cells in the SMC 5.5, 55, and 550 µg/mL treatment groups were 11.93%, 13.10%, and 18.42%, respectively. Late apoptosis first occurred at 12 h post-treatment; the highest rate of apoptosis was 36.54 ± 4.37% at 24 h post-treatment in the SMC 55 µg/mL treatment group. In general, the cellular apoptosis rate was positively correlated with the SMC concentration and the time post-treatment. These results indicate that secondary metabolites of B . brongniartii are able to attack the hemocytes of D . tabulaeformis larvae and induce cellular apoptosis, thereby providing new evidence that secondary metabolites of mycopathogens can act on host immune systems. PMID:23940771

Upregulation of CC Chemokine Receptor 7 (CCR7) Enables Migration of Xenogeneic Human Adipose-Derived Mesenchymal Stem Cells to Rat Secondary Lymphoid Organs.

PubMed

Ma, Tian; Luan, Shao-Liang; Huang, Hong; Sun, Xing-Kun; Yang, Yan-Mei; Zhang, Hui; Han, Wei-Dong; Li, Hong; Han, Yan

2016-12-30

BACKGROUND CC chemokine receptor 7 (CCR7) expression is vital for cell migration to secondary lymphoid organs (SLOs). Our previous work showed that inducing CCR7 expression enabled syngeneic mesenchymal stem cells (MSCs) to migrate into SLOs, resulting in enhanced immunosuppressive performance in mice. Given that human adipose-derived stem cells (hASCs) are widely used in clinical therapy, we further investigated whether upregulation of CCR7 enables xenogeneic hASCs to migrate to rat SLOs. MATERIAL AND METHODS hASCs rarely express CCR7; therefore, hASCs were transfected with lentivirus encoding rat CCR7 (rCCR7) plus green fluorescence protein (GFP) or GFP alone. CCR7 mRNA and cell surface expression of rCCR7-hASCs and GFP-hASCs were examined by reverse transcription-polymerase chain reaction (RT-PCR) and flow cytometry (FCM), respectively. The phenotype, differentiation, and proliferation capacity of each cell type was also determined. To examine migration, rCCR7-hASCs and GFP-hASCs were injected intravenously into Lewis rats, and the proportion of GFP-positive cells in the spleen and lymph nodes was determined with FCM. RESULTS mRNA and cell surface protein expression of CCR7 was essentially undetectable in hASCs and GFP-ASCs; however, CCR7 was highly expressed in rCCR7-ASCs. rCCR7-hASCs, GFP-hASCs, and hASCs shared a similar immunophenotype, and maintained the ability of multilineage differentiation and proliferation. In addition, the average proportion of GFP-positive cells was significantly higher following transplantation of rCCR7-hASCs compared with GFP-hASCs (p<0.01). CONCLUSIONS These results suggest that upregulation of rat CCR7 expression does not change the phenotype, differentiation, or proliferation capacity of hASCs, but does enable efficient migration of hASCs to rat SLOs.

Research on the lesion segmentation of breast tumor MR images based on FCM-DS theory

NASA Astrophysics Data System (ADS)

Zhang, Liangbin; Ma, Wenjun; Shen, Xing; Li, Yuehua; Zhu, Yuemin; Chen, Li; Zhang, Su

2017-03-01

Magnetic resonance imaging (MRI) plays an important role in the treatment of breast tumor by high intensity focused ultrasound (HIFU). The doctors evaluate the scale, distribution and the statement of benign or malignancy of breast tumor by analyzing variety modalities of MRI, such as the T2, DWI and DCE images for making accurate preoperative treatment plan and evaluating the effect of the operation. This paper presents a method of lesion segmentation of breast tumor based on FCM-DS theory. Fuzzy c-means clustering (FCM) algorithm combined with Dempster-Shafer (DS) theory is used to process the uncertainty of information, segmenting the lesion areas on DWI and DCE modalities of MRI and reducing the scale of the uncertain parts. Experiment results show that FCM-DS can fuse the DWI and DCE images to achieve accurate segmentation and display the statement of benign or malignancy of lesion area by Time-Intensity Curve (TIC), which could be beneficial in making preoperative treatment plan and evaluating the effect of the therapy.

Older mothers follow conservative strategies under predator pressure: the adaptive role of maternal glucocorticoids in yellow-bellied marmots.

PubMed

Monclús, Raquel; Tiulim, Justin; Blumstein, Daniel T

2011-11-01

When the maternal environment is a good predictor of the offspring environment, maternal glucocorticoid (GC) levels might serve to pre-program offspring to express certain phenotypes or life-history characteristics that will increase their fitness. We conducted a field study to assess the effects of naturally occurring maternal GC levels on their offspring in yellow-bellied marmots (Marmota flaviventris) subjected to different predator pressures. Maternal fecal corticosteroid metabolites (FCM) were positively correlated with predator pressure. Predators had both direct and indirect effects on pups. We found that older mothers with higher FCM levels had smaller and female-biased litters. Moreover, sons from older mothers with high FCM levels dispersed significantly more than those from older mothers with low FCM levels, whereas the opposite pattern was found in pups from younger mothers. These age-related effects may permit females to make adaptive decisions that increase their pups' fitness according to their current situation. Copyright © 2011 Elsevier Inc. All rights reserved.

Correlative cryogenic tomography of cells using light and soft x-rays

PubMed Central

Smith, Elizabeth A.; Cinquin, Bertrand P.; Do, Myan; McDermott, Gerry; Le Gros, Mark A.; Larabell, Carolyn A.

2013-01-01

Correlated imaging is the process of imaging a specimen with two complementary modalities, and then combining the two data sets to create a highly informative, composite view. A recent implementation of this concept has been the combination of soft x-ray tomography (SXT) with fluorescence cryogenic microscopy (FCM). SXT-FCM is used to visualize cells that are held in a near-native, cryo-preserved state. The resultant images are, therefore, highly representative of both the cellular architecture and molecular organization in vivo. SXT quantitatively visualizes the cell and sub-cellular structures; FCM images the spatial distribution of fluorescently labeled molecules. Here, we review the characteristics of SXT-FCM, and briefly discuss how this method compares with existing correlative imaging techniques. We also describe how the incorporation of a cryo-rotation stage into a cryogenic fluorescence microscope allows acquisition of fluorescence cryogenic tomography (FCT) data. FCT is optimally suited to correlation with SXT, since both techniques image the specimen in 3-D, potentially with similar, isotropic spatial resolution. PMID:24355261

[Chromosome study on chronic lymphocytic leukemia using CpG-oligodeoxynucleotide as immunostimulant agent].

PubMed

Wu, Yafang; Xue, Yongquan; Chen, Suning; Yao, Li; Jiang, Hui; Zhang, Jun; Shen, Juan; Pan, Jinlan; Wang, Yong; Bai, Shuxiao

2010-02-01

To investigate whether CpG-oligodeoxynucleotide (CpG-ODN) can improve the detection rate of the karyotypic abnormalities in chronic lymphocytic leukemia (CLL). The bone marrow (BM) or peripheral blood (PB) cells from 57 cases of CLL were collected and cultured with CpG-ODN DSP30+interleukin-2 (IL-2), phytohemagglutinin (PHA), pokeweed (PWM) or IL-2, respectively. Five days later cells were harvested for chromosome preparation. Karyotypic analysis was done using R banding technique. Panel fluorescence in situ hybridization (FISH) was carried out on 19 cases of CLL with normal karyotypes using the following probes: Cen12, D13S25, Rb1, ATM, p53, MYB and IgH. Genomic DNA from 21 cases of them was extracted from BM or PB leukocytes. The immunoglobulin variable heavy chain (IgVH) was amplified by polymerase chain reaction (PCR) and sequenced. CD38 and ZAP70 expressions in the leukemic cells were determined by flow cytometry (FCM). The detection rate of karyotypic abnormalities in the CpG-ODN+IL-2 group (43.85%) was obviously higher than that in the PHA (15.09%), PWM (17.31%) and IL-2 (3.13%) groups (P<0.01). Fifty-two types of karyotypic abnormalities were found. Among them, trisomy12 (+12) or +12 with other abnormalities were the most common, while translocations were the most frequent structural abnormalities including 3 unbalanced and 11 balanced translocations, among them 7 had rearrangements involving 14q32. Thirteen cases showed one or more abnormalities on FISH including trisomy 12 and p53 deletion each in one case, IgH rearrangement and partial deletion each in one case, 13q14.3 deletion in 11 cases of which 5 cases also had Rb1 deletion, 1 case had Rb1 partial deletion. No case with ATM or MYB deletions was found. PCR detected IgVH mutations in 10/21 cases. FCM showed 10/45 cases were CD38 positive, but 35 /45 were CD38 negative, 11/27 cases expressed ZAP70, but 16/27 did not. Among the 26 cases examined for CD38 and ZAP70 expressions simultaneously, 5 cases were CD38+ZAP70+, 13 were CD38-ZAP70-, 6 were CD38-ZAP70+, and 2 were CD38+ZAP70-, respectively. Statistic analysis showed a correlation between complex karyotype and IgVH without mutation, but no association between karyotype and CD38 or ZAP70 expression was observed. CpG-ODN immunostimulation can obviously raise the detection rate of abnormal karyotypes, especially translocations in CLL. FISH is an important complement to conventional karyotypic analysis. The combination of both methods can provide more comprehensive genetic information for CLL.

Photosynthetic inhibition and oxidative stress to the toxic Phaeocystis globosa caused by a diketopiperazine isolated from products of algicidal bacterium metabolism.

PubMed

Tan, Shuo; Hu, Xiaoli; Yin, Pinghe; Zhao, Ling

2016-05-01

Algicidal bacteria have been turned out to be available for inhibiting Phaeocystis globosa which frequently caused harmful algal blooms and threatened to economic development and ecological balance. A marine bacterium Bacillus sp. Ts-12 exhibited significant algicidal activity against P. globosa by indirect attack. In present study, an algicidal compound was isolated by silica gel column, Sephadex G-15 column and HPLC, further identified as hexahydropyrrolo[1,2-a]pyrazine-1,4-dione, cyclo-(Pro-Gly), by GC-MS and (1)H-NMR. Cyclo-(Pro-Gly) significantly increased the level of reactive oxygen species (ROS) within P. globosa cells, further activating the enzymatic and non-enzymatic antioxidant systems, including superoxide dismutase (SOD), catalase (CAT), glutathione (GSH) and ascorbic acid (AsA). The increase in methane dicarboxylic aldehyde (MDA) content showed that the surplus ROS induced lipid peroxidation on membrane system. Transmission electron microscope (TEM) and flow cytometry (FCM) analysis revealed that cyclo-(Pro-Gly) caused reduction of Chl-a content, destruction of cell membrane integrity, chloroplasts and nuclear structure. Real-time PCR assay showed that the transcriptions of photosynthesis related genes (psbA, psbD, rbcL) were significantly inhibited. This study indicated that cyclo-(Pro-Gly) from marine Bacillus sp. Ts-12 exerted photosynthetic inhibition and oxidative stress to P. globosa and eventually led to the algal cells lysis. This algicidal compound might be potential bio-agent for controlling P. globosa red tide.

Gold Nanoparticles of Diameter 13 nm Induce Apoptosis in Rabbit Articular Chondrocytes

NASA Astrophysics Data System (ADS)

Huang, Hao; Quan, Ying-yao; Wang, Xiao-ping; Chen, Tong-sheng

2016-05-01

Gold nanoparticles (AuNPs) have been widely used in biomedical science including antiarthritic agents, drug loading, and photothermal therapy. In this report, we studied the effects of AuNPs with diameters of 3, 13, and 45 nm, respectively, on rabbit articular chondrocytes. AuNPs were capped with citrate and their diameter and zeta potential were measured by dynamic light scattering (DLS). Cell viability was evaluated by Cell Counting Kit-8 (CCK-8) assay after the rabbit articular chondrocytes were pre-incubated with 3, 13, and 45 nm AuNPs, respectively, for 24 h. Flow cytometry (FCM) analysis with annexin V/propidium iodide (PI) double staining and fluorescence imaging with Hoechst 33258 staining were used to determine the fashion of AuNPs-induced chondrocyte death. Further, 13 nm AuNPs (2 nM) significantly induced chondrocyte death accompanying apoptotic characteristics including mitochondrial damage, externalization of phosphatidylserine and nuclear concentration. However, 3 nm AuNPs (2 nM) and 45 nm (0.02 nM) AuNPs did not induce cytotoxicity in chondrocytes. Although 13 nm AuNPs (2 nM) increased the intracellular reactive oxygen species (ROS) level, pretreatment with Nacetyl cysteine (NAC), a ROS scavenger, did not prevent the cytotoxicity induced by 13 nm AuNPs, indicating that 13 nm AuNPs (2 nM) induced ROS-independent apoptosis in chondrocytes. These results demonstrate the size-dependent cytotoxicity of AuNPs in chondrocytes, which must be seriously considered when using AuNPs for treatment of osteoarthritis (OA).

Production, characterization and application of monoclonal antibodies to the coelomocytes of sea urchin Strongylocentrotus intermedius.

PubMed

Wang, Yinan; Meng, Shaodong; Zhang, Jialin; Ding, Jun; Li, Qiang

2018-04-01

Sea urchin is one of marine animals with high economic and great scientific research values. Axial organ is a glandular organ that has been presumed as coelomocytes origin site. In this paper, two monoclonal antibodies (3G10 and 6B3) against coelomocytes of sea urchin Strongylocentrotus intermedius were developed by hybridoma technique. The mAbs were characterized by indirect immunofluorescence assay test (IIFAT), flow cytometry (FCM) and western blot assay. Results showed that mAb 3G10 recognized a protein of a molecular weight of 17 kDa in the spherule cells, while mAb 6B3 reacted with a protein of a molecular weight of 35 kDa in the phagocytes. Furthermore, specificity analysis revealed that the two mAbs could react with the coelomocytes of sea urchin S. nudus and Hemicentrotus pulcherrimus, but not with those of other common echinoderms including sea cucumber Apostichopus japonicus and starfish Asterias rollestoni. To determine whether the coelomocytes exist in the axial organ of sea urchin, the IIFAT assays were carried out based on the two mAbs. Result showed that positive fluorescence signals were distributed in the organ. It was revealed that the axial organ was rich in coelomocytes, which suggests that the organ may play as a producing source or reservoir in the ontogenesis of coelomocytes of sea urchin. Copyright © 2018 Elsevier Ltd. All rights reserved.

[Inhibition effects of black rice pericarp extracts on cell proliferation of PC-3 cells].

PubMed

Jiang, Weiwei; Yu, Xudong; Ren, Guofeng

2013-05-01

To observe the inhibitive effects of black rice pericarp extracts on cell proliferation of human prostate cancer cell PC-3 and to explore its effecting mechanism. The black rice pericarp extract was used to treat the PC-3 cells. The inhibitory effect of black rice pericarp extract on cells proliferation of PC-3 was tested by MTT method. Cell apoptosis rates and cell cycle were measured by flow cytometric assay (FCM). Western blot was used to study the protein expression levels of p38, p-p38, JNK, p-JNK. A dose-dependent and time-dependent proliferation inhibition of black rice pericarp extract was demonstrated in PC-3. The most prominent experiment condition was inhibitory concentration with 300microg/ml and treated for 72 h. The experiment result of flow cytometry analysis demonstrates that the apoptosis rate of PC-3 cells increased along with the increasing of black rice pericarp extract concentration, and a G1-S cell cycle arrest was induced in a dose-dependent manner. After PC-3 cell was treated with black rice pericarp extract for 72 h, the expressions of p-p38, p-JNK protein increased. Black rice pericarp extract could inhibit proliferation, change the cell cycle distributions and induce apoptosis in human prostatic cancer cell PC-3. Its inhibitory effect may be through promoting activation of the JNK, p38 signaling pathway. These results suggest that black rice pericarp extract maybe has an inhibitory effect on prostatic cancer.

Analytical strategies for organic food packaging contaminants.

PubMed

Sanchis, Yovana; Yusà, Vicent; Coscollà, Clara

2017-03-24

In this review, we present current approaches in the analysis of food-packaging contaminants. Gas and liquid chromatography coupled to mass spectrometry detection have been widely used in the analysis of some relevant families of these compounds such as primary aromatic amines, bisphenol A, bisphenol A diglycidyl ether and related compounds, UV-ink photoinitiators, perfluorinated compounds, phthalates and non-intentionally added substances. Main applications for sample treatment and different types of food-contact material migration studies have been also discussed. Pressurized Liquid Extraction, Solid-Phase Microextraction, Focused Ultrasound Solid-Liquid Extraction and Quechers have been mainly used in the extraction of food contact material (FCM) contaminants, due to the trend of minimising solvent consumption, automatization of sample preparation and integration of extraction and clean-up steps. Recent advances in analytical methodologies have allowed unequivocal identification and confirmation of these contaminants using Liquid Chromatography coupled to High Resolution Mass Spectrometry (LC-HRMS) through mass accuracy and isotopic pattern applying. LC-HRMS has been used in the target analysis of primary aromatic amines in different plastic materials, but few studies have been carried out applying this technique in post-target and non-target analysis of FCM contaminants. Copyright © 2017 Elsevier B.V. All rights reserved.

Inserting ex vivo fluorescence confocal microscopy perioperatively in Mohs micrographic surgery expedites bedside assessment of excision margins in recurrent basal cell carcinoma.

PubMed

Longo, Caterina; Ragazzi, Moira; Castagnetti, Fabio; Gardini, Stefano; Palmieri, Tamara; Lallas, Aimilios; Moscarella, Elvira; Piana, Simonetta; Pellacani, Giovanni; Zalaudek, Iris; Argenziano, Giuseppe

2013-01-01

Mohs micrographic surgery can be employed in recurrent basal cell carcinoma, although it is a time-consuming technique. Recently, ex vivo fluorescence confocal microscopy (FCM) has been employed to obtain a fast assessment of tumor margins at the bedside. In our case we successfully employed ex vivo FCM to assess the tumor margins and we treated the persistent tumor with intensity-modulated radiation therapy. Our case demonstrates that a multidisciplinary approach is very efficient in managing complex and recurrent tumors and highlights the benefits of FCM as a new technique that can be used in the surgical theater to speed up the entire procedure.

Analyzing the impact of social factors on homelessness: a Fuzzy Cognitive Map approach

PubMed Central

2013-01-01

Background The forces which affect homelessness are complex and often interactive in nature. Social forces such as addictions, family breakdown, and mental illness are compounded by structural forces such as lack of available low-cost housing, poor economic conditions, and insufficient mental health services. Together these factors impact levels of homelessness through their dynamic relations. Historic models, which are static in nature, have only been marginally successful in capturing these relationships. Methods Fuzzy Logic (FL) and fuzzy cognitive maps (FCMs) are particularly suited to the modeling of complex social problems, such as homelessness, due to their inherent ability to model intricate, interactive systems often described in vague conceptual terms and then organize them into a specific, concrete form (i.e., the FCM) which can be readily understood by social scientists and others. Using FL we converted information, taken from recently published, peer reviewed articles, for a select group of factors related to homelessness and then calculated the strength of influence (weights) for pairs of factors. We then used these weighted relationships in a FCM to test the effects of increasing or decreasing individual or groups of factors. Results of these trials were explainable according to current empirical knowledge related to homelessness. Results Prior graphic maps of homelessness have been of limited use due to the dynamic nature of the concepts related to homelessness. The FCM technique captures greater degrees of dynamism and complexity than static models, allowing relevant concepts to be manipulated and interacted. This, in turn, allows for a much more realistic picture of homelessness. Through network analysis of the FCM we determined that Education exerts the greatest force in the model and hence impacts the dynamism and complexity of a social problem such as homelessness. Conclusions The FCM built to model the complex social system of homelessness reasonably represented reality for the sample scenarios created. This confirmed that the model worked and that a search of peer reviewed, academic literature is a reasonable foundation upon which to build the model. Further, it was determined that the direction and strengths of relationships between concepts included in this map are a reasonable approximation of their action in reality. However, dynamic models are not without their limitations and must be acknowledged as inherently exploratory. PMID:23971944

Hepcidin Response to Iron Therapy in Patients with Non-Dialysis Dependent CKD: An Analysis of the FIND-CKD Trial.

PubMed

Gaillard, Carlo A; Bock, Andreas H; Carrera, Fernando; Eckardt, Kai-Uwe; Van Wyck, David B; Bansal, Sukhvinder S; Cronin, Maureen; Meier, Yvonne; Larroque, Sylvain; Roger, Simon D; Macdougall, Iain C

2016-01-01

Hepcidin is the key regulator of iron homeostasis but data are limited regarding its temporal response to iron therapy, and response to intravenous versus oral iron. In the 56-week, open-label, multicenter, prospective, randomized FIND-CKD study, 626 anemic patients with non-dialysis dependent chronic kidney disease (ND-CKD) and iron deficiency not receiving an erythropoiesis stimulating agent were randomized (1:1:2) to intravenous ferric carboxymaltose (FCM), targeting higher (400-600μg/L) or lower (100-200μg/L) ferritin, or to oral iron. Serum hepcidin levels were measured centrally in a subset of 61 patients. Mean (SD) baseline hepcidin level was 4.0(3.5), 7.3(6.4) and 6.5(5.6) ng/mL in the high ferritin FCM (n = 17), low ferritin FCM (n = 16) and oral iron group (n = 28). The mean (SD) endpoint value (i.e. the last post-baseline value) was 26.0(9.1),15.7(7.7) and 16.3(11.0) ng/mL, respectively. The increase in hepcidin from baseline was significantly smaller with low ferritin FCM or oral iron vs high ferritin FCM at all time points up to week 52. Significant correlations were found between absolute hepcidin and ferritin values (r = 0.65, p<0.001) and between final post-baseline increases in both parameters (r = 0.70, p<0.001). The increase in hepcidin levels over the 12-month study generally mirrored the cumulative iron dose in each group. Hepcidin and transferrin saturation (TSAT) absolute values showed no correlation, although there was an association between final post-baseline increases (r = 0.42, p<0.001). Absolute values (r = 0.36, p = 0.004) and final post-baseline increases of hepcidin and hemoglobin (p = 0.30, p = 0.030) correlated weakly. Baseline hepcidin levels were not predictive of a hematopoietic response to iron therapy. In conclusion, hepcidin levels rose in response to either intravenous or oral iron therapy, but the speed and extent of the rise was greatest with intravenous iron targeting a higher ferritin level. However neither the baseline level nor the change in hepcidin was able to predict response to therapy in this cohort.

Correlative cryogenic tomography of cells using light and soft x-rays.

PubMed

Smith, Elizabeth A; Cinquin, Bertrand P; Do, Myan; McDermott, Gerry; Le Gros, Mark A; Larabell, Carolyn A

2014-08-01

Correlated imaging is the process of imaging a specimen with two complementary modalities, and then combining the two data sets to create a highly informative, composite view. A recent implementation of this concept has been the combination of soft x-ray tomography (SXT) with fluorescence cryogenic microscopy (FCM). SXT-FCM is used to visualize cells that are held in a near-native, cryopreserved. The resultant images are, therefore, highly representative of both the cellular architecture and molecular organization in vivo. SXT quantitatively visualizes the cell and sub-cellular structures; FCM images the spatial distribution of fluorescently labeled molecules. Here, we review the characteristics of SXT-FCM, and briefly discuss how this method compares with existing correlative imaging techniques. We also describe how the incorporation of a cryo-rotation stage into a cryogenic fluorescence microscope allows acquisition of fluorescence cryogenic tomography (FCT) data. FCT is optimally suited for correlation with SXT, since both techniques image the specimen in 3-D, potentially with similar, isotropic spatial resolution. © 2013 Elsevier B.V. All rights reserved.

Lack of anodic capacitance causes power overshoot in microbial fuel cells.

PubMed

Peng, Xinhong; Yu, Han; Yu, Hongbing; Wang, Xin

2013-06-01

Power overshoot commonly makes the performance evaluation of microbial fuel cells (MFCs) inaccurate. Here, three types of carbon with different capacitance (ultracapacitor activated carbon (UAC), plain activated carbon (PAC) and carbon black (CB)) rolled on stainless steel mesh (SSM) as anodes to investigate the relationship between overshoot and anodic capacitance. It was not observed in all cycles of UAC-MFCs (from Cycle 2 to 4) due to the largest abiotic capacitance (Cm(abiotic)) of 2.1F/cm(2), while this phenomenon was eliminated in PAC-MFCs (Cm(abiotic)=1.6 F/cm(2)) from Cycle 3 and in CB-MFCs (Cm(abiotic)=0.5F/cm(2)) from Cycle 4, indicated that the Cm(abiotic) of the anode stored charges and functioned as electron shuttle to overcome the power overshoot. With bacterial colonization, the transient charge storage in biofilm resulted in a 0.1-0.4F/cm(2) increase in total capacitance for anodes, which was the possible reason for the elimination of power overshoot in PAC/CB-MFCs after multi cycle acclimation. Copyright © 2013 Elsevier Ltd. All rights reserved.

Bacteria permeabilization and disruption caused by sludge reduction technologies evaluated by flow cytometry.

PubMed

Foladori, P; Tamburini, S; Bruni, L

2010-09-01

Technologies proposed in the last decades for the reduction of the sludge production in wastewater treatment plants and based on the mechanism of cell lysis-cryptic growth (physical, mechanical, thermal, chemical, oxidative treatments) have been widely investigated at lab-, pilot- and, in some cases, at full-scale but the effects on cellular lysis have not always been demonstrated in depth. The research presented in this paper aims to investigate how these sludge reduction technologies affect the integrity and permeabilization of bacterial cells in sludge using flow cytometry (FCM), which permits the rapid and statistically accurate quantification of intact, permeabilised or disrupted bacteria in the sludge using a double fluorescent DNA-staining instead of using conventional methods like plate counts and microscope. Physical/mechanical treatments (ultrasonication and high pressure homogenisation) caused moderate effects on cell integrity and caused significant cell disruption only at high specific energy levels. Conversely, thermal treatment caused significant damage of bacterial membranes even at moderate temperatures (45-55 °C). Ozonation significantly affected cell integrity, even at low ozone dosages, below 10 mgO(3)/gTSS, causing an increase of permeabilised and disrupted cells. At higher ozone dosages the compounds solubilised after cell lysis act as scavengers in the competition between soluble compounds and (particulate) bacterial cells. An original aspect of this paper, not yet reported in the literature, is the comparison of the effects of these sludge reduction technologies on bacterial cell integrity and permeabilization by converting pressure, temperature and ozone dosage to an equivalent value of specific energy. Among these technologies, comparison of the applied specific energy demonstrates that achieving the complete disruption of bacterial cells is not always economically advantageous because excessive energy levels may be required. Copyright © 2010 Elsevier Ltd. All rights reserved.

Advanced soft computing diagnosis method for tumour grading.

PubMed

Papageorgiou, E I; Spyridonos, P P; Stylios, C D; Ravazoula, P; Groumpos, P P; Nikiforidis, G N

2006-01-01

To develop an advanced diagnostic method for urinary bladder tumour grading. A novel soft computing modelling methodology based on the augmentation of fuzzy cognitive maps (FCMs) with the unsupervised active Hebbian learning (AHL) algorithm is applied. One hundred and twenty-eight cases of urinary bladder cancer were retrieved from the archives of the Department of Histopathology, University Hospital of Patras, Greece. All tumours had been characterized according to the classical World Health Organization (WHO) grading system. To design the FCM model for tumour grading, three experts histopathologists defined the main histopathological features (concepts) and their impact on grade characterization. The resulted FCM model consisted of nine concepts. Eight concepts represented the main histopathological features for tumour grading. The ninth concept represented the tumour grade. To increase the classification ability of the FCM model, the AHL algorithm was applied to adjust the weights of the FCM. The proposed FCM grading model achieved a classification accuracy of 72.5%, 74.42% and 95.55% for tumours of grades I, II and III, respectively. An advanced computerized method to support tumour grade diagnosis decision was proposed and developed. The novelty of the method is based on employing the soft computing method of FCMs to represent specialized knowledge on histopathology and on augmenting FCMs ability using an unsupervised learning algorithm, the AHL. The proposed method performs with reasonably high accuracy compared to other existing methods and at the same time meets the physicians' requirements for transparency and explicability.

ggCyto: Next Generation Open-Source Visualization Software for Cytometry.

PubMed

Van, Phu; Jiang, Wenxin; Gottardo, Raphael; Finak, Greg

2018-06-01

Open source software for computational cytometry has gained in popularity over the past few years. Efforts such as FlowCAP, the Lyoplate and Euroflow projects have highlighted the importance of efforts to standardize both experimental and computational aspects of cytometry data analysis. The R/BioConductor platform hosts the largest collection of open source cytometry software covering all aspects of data analysis and providing infrastructure to represent and analyze cytometry data with all relevant experimental, gating, and cell population annotations enabling fully reproducible data analysis. Data visualization frameworks to support this infrastructure have lagged behind. ggCyto is a new open-source BioConductor software package for cytometry data visualization built on ggplot2 that enables ggplot-like functionality with the core BioConductor flow cytometry data structures. Amongst its features are the ability to transform data and axes on-the-fly using cytometry-specific transformations, plot faceting by experimental meta-data variables, and partial matching of channel, marker and cell populations names to the contents of the BioConductor cytometry data structures. We demonstrate the salient features of the package using publicly available cytometry data with complete reproducible examples in a supplementary material vignette. https://bioconductor.org/packages/devel/bioc/html/ggcyto.html. gfinak@fredhutch.org. Supplementary data are available at Bioinformatics online and at http://rglab.org/ggcyto/.

Investigating the Potential of the Flipped Classroom Model in K-12 Mathematics Teaching and Learning

ERIC Educational Resources Information Center

Katsa, Maria; Sergis, Stylianos; Sampson, Demetrios G.

2016-01-01

The Flipped Classroom model (FCM) is a promising blended educational innovation aiming to improve the teaching and learning practice in various subject domains and educational levels. However, despite this encouraging evidence, research on the explicit benefits of the FCM on K-12 Mathematics education is still scarce and, in some cases, even…

Dissecting the Role of IGFBP-2 in Development of Acute Myeloid Leukemia

DTIC Science & Technology

2011-06-01

surface proteins on freshly isolated and cultured cells, as determined by flow cytometry ... Surface Immune Molecules on Phenotypic HSCs during Culture (A and B) A summary of the result of flow cytometry analysis of surface expression of indicated...from the distant implanted tumor were counted by flow cytometry analysis. The flow cytometry result was confirmed by counting GFP+ surface foci of

Spectral embedding based active contour (SEAC) for lesion segmentation on breast dynamic contrast enhanced magnetic resonance imaging.

PubMed

Agner, Shannon C; Xu, Jun; Madabhushi, Anant

2013-03-01

Segmentation of breast lesions on dynamic contrast enhanced (DCE) magnetic resonance imaging (MRI) is the first step in lesion diagnosis in a computer-aided diagnosis framework. Because manual segmentation of such lesions is both time consuming and highly susceptible to human error and issues of reproducibility, an automated lesion segmentation method is highly desirable. Traditional automated image segmentation methods such as boundary-based active contour (AC) models require a strong gradient at the lesion boundary. Even when region-based terms are introduced to an AC model, grayscale image intensities often do not allow for clear definition of foreground and background region statistics. Thus, there is a need to find alternative image representations that might provide (1) strong gradients at the margin of the object of interest (OOI); and (2) larger separation between intensity distributions and region statistics for the foreground and background, which are necessary to halt evolution of the AC model upon reaching the border of the OOI. In this paper, the authors introduce a spectral embedding (SE) based AC (SEAC) for lesion segmentation on breast DCE-MRI. SE, a nonlinear dimensionality reduction scheme, is applied to the DCE time series in a voxelwise fashion to reduce several time point images to a single parametric image where every voxel is characterized by the three dominant eigenvectors. This parametric eigenvector image (PrEIm) representation allows for better capture of image region statistics and stronger gradients for use with a hybrid AC model, which is driven by both boundary and region information. They compare SEAC to ACs that employ fuzzy c-means (FCM) and principal component analysis (PCA) as alternative image representations. Segmentation performance was evaluated by boundary and region metrics as well as comparing lesion classification using morphological features from SEAC, PCA+AC, and FCM+AC. On a cohort of 50 breast DCE-MRI studies, PrEIm yielded overall better region and boundary-based statistics compared to the original DCE-MR image, FCM, and PCA based image representations. Additionally, SEAC outperformed a hybrid AC applied to both PCA and FCM image representations. Mean dice similarity coefficient (DSC) for SEAC was significantly better (DSC = 0.74 ± 0.21) than FCM+AC (DSC = 0.50 ± 0.32) and similar to PCA+AC (DSC = 0.73 ± 0.22). Boundary-based metrics of mean absolute difference and Hausdorff distance followed the same trends. Of the automated segmentation methods, breast lesion classification based on morphologic features derived from SEAC segmentation using a support vector machine classifier also performed better (AUC = 0.67 ± 0.05; p < 0.05) than FCM+AC (AUC = 0.50 ± 0.07), and PCA+AC (AUC = 0.49 ± 0.07). In this work, we presented SEAC, an accurate, general purpose AC segmentation tool that could be applied to any imaging domain that employs time series data. SE allows for projection of time series data into a PrEIm representation so that every voxel is characterized by the dominant eigenvectors, capturing the global and local time-intensity curve similarities in the data. This PrEIm allows for the calculation of strong tensor gradients and better region statistics than the original image intensities or alternative image representations such as PCA and FCM. The PrEIm also allows for building a more accurate hybrid AC scheme.

Utilization of physicochemical variables developed from changes in sensory attributes and consumer acceptability to predict the shelf life of fresh-cut mango fruit.

PubMed

Salinas-Hernández, Rosa María; González-Aguilar, Gustavo A; Tiznado-Hernández, Martín Ernesto

2015-01-01

Sensory evaluation is the ideal tool for shelf-life determination. With the objective to develop an easy shelf-life indicator, color (L*, a*, b*, chroma and hue angle), total soluble solids (TSS), firmness (F), pH, acidity, and the sensory attributes of appearance, brightness, browning, odor, flavor, texture, color, acidity and sweetness were evaluated in fresh cut mangoes (FCM) stored at 5, 10, 15 and 20 °C. Overall acceptability was evaluated by consumers. Correlation analysis between sensory attributes and physicochemical variables was carried out. Physicochemical cut-off points based on sensory attributes and consumer acceptability was obtained by regression analysis and utilized to estimate FCM shelf-life by kinetic models fitted to each variable. The validation of the model was done by comparing the shelf life estimated by kinetic models and consumers. It was recorded large correlations between appearance, brightness, and color with L*; appearance and color with chroma and hue angle; sweetness and flavor with TSS, and between F and texture. The shelf life estimated based on consumer using a 9 point hedonic scale was in the range of 10-12, 2.3-2.6, 1.3-1.5 and 1.0-1.1 days for 5, 10, 15 and 20 °C. It was recorded large correlation coefficients between the shelf life estimated by consumer acceptability scores and physicochemical variables. Kinetic models based on physicochemical variables showed a tendency to overestimate the shelf life as compared with the models bases on the sensory attributes. It was concluded that physicochemical variables can be used as a tool to estimate the FCM shelf life.

Sensitivity and specificity of univariate MRI analysis of experimentally degraded cartilage

PubMed Central

Lin, Ping-Chang; Reiter, David A.; Spencer, Richard G.

2010-01-01

MRI is increasingly used to evaluate cartilage in tissue constructs, explants, and animal and patient studies. However, while mean values of MR parameters, including T1, T2, magnetization transfer rate km, apparent diffusion coefficient ADC, and the dGEMRIC-derived fixed charge density, correlate with tissue status, the ability to classify tissue according to these parameters has not been explored. Therefore, the sensitivity and specificity with which each of these parameters was able to distinguish between normal and trypsin- degraded, and between normal and collagenase-degraded, cartilage explants were determined. Initial analysis was performed using a training set to determine simple group means to which parameters obtained from a validation set were compared. T1 and ADC showed the greatest ability to discriminate between normal and degraded cartilage. Further analysis with k-means clustering, which eliminates the need for a priori identification of sample status, generally performed comparably. Use of fuzzy c-means (FCM) clustering to define centroids likewise did not result in improvement in discrimination. Finally, a FCM clustering approach in which validation samples were assigned in a probabilistic fashion to control and degraded groups was implemented, reflecting the range of tissue characteristics seen with cartilage degradation. PMID:19705467

Comparative exploration of multidimensional flow cytometry software: a model approach evaluating T cell polyfunctional behavior.

PubMed

Spear, Timothy T; Nishimura, Michael I; Simms, Patricia E

2017-08-01

Advancement in flow cytometry reagents and instrumentation has allowed for simultaneous analysis of large numbers of lineage/functional immune cell markers. Highly complex datasets generated by polychromatic flow cytometry require proper analytical software to answer investigators' questions. A problem among many investigators and flow cytometry Shared Resource Laboratories (SRLs), including our own, is a lack of access to a flow cytometry-knowledgeable bioinformatics team, making it difficult to learn and choose appropriate analysis tool(s). Here, we comparatively assess various multidimensional flow cytometry software packages for their ability to answer a specific biologic question and provide graphical representation output suitable for publication, as well as their ease of use and cost. We assessed polyfunctional potential of TCR-transduced T cells, serving as a model evaluation, using multidimensional flow cytometry to analyze 6 intracellular cytokines and degranulation on a per-cell basis. Analysis of 7 parameters resulted in 128 possible combinations of positivity/negativity, far too complex for basic flow cytometry software to analyze fully. Various software packages were used, analysis methods used in each described, and representative output displayed. Of the tools investigated, automated classification of cellular expression by nonlinear stochastic embedding (ACCENSE) and coupled analysis in Pestle/simplified presentation of incredibly complex evaluations (SPICE) provided the most user-friendly manipulations and readable output, evaluating effects of altered antigen-specific stimulation on T cell polyfunctionality. This detailed approach may serve as a model for other investigators/SRLs in selecting the most appropriate software to analyze complex flow cytometry datasets. Further development and awareness of available tools will help guide proper data analysis to answer difficult biologic questions arising from incredibly complex datasets. © Society for Leukocyte Biology.

Monitoring stress in captive and free-ranging African wild dogs (Lycaon pictus) using faecal glucocorticoid metabolites.

PubMed

Van der Weyde, L K; Martin, G B; Paris, M C J

2016-01-15

An understanding of stress physiology is important for species management because high levels of stress can hamper reproduction and affect an individual's ability to cope with threats to their survival, such as disease and human-wildlife conflict. A commonly used indicator of stress, faecal concentrations of cortisol metabolites (FCM), can be used to assess the impact of social, biological and environmental factors. Measurements of FCM are particularly valuable for endangered species that are logistically challenging to study and where non-invasive techniques are preferred. As the second most endangered canid in Africa, the African wild dog (Lycaon pictus) has been the focus of considerable conservation research, yet there is still little understanding of factors associated with stress, in either captive or free-ranging populations. The present study therefore aimed to determine whether stress levels differ between captive and free-ranging populations, and to detect social, biological and environmental factors that are stressful in these populations. Faecal samples were collected from 20 captive and 62 free-ranging animals. Within free-ranging populations, the sexes differed significantly, but there was no effect of social status, age or breeding period for either sex. Captive females had higher FCM concentrations than free-ranging females. In captive populations, FCM concentrations differed among zoos and with reproductive status in females, but were not related to age class or group-housing structure. In conclusion, FCM is a useful indicator of stress and should be considered an integrative aspect of management, for both in situ and ex situ African wild dog populations. Copyright © 2015 Elsevier Inc. All rights reserved.

Ex vivo fluorescence confocal microscopy for fast evaluation of tumour margins during Mohs surgery.

PubMed

Bennàssar, A; Vilata, A; Puig, S; Malvehy, J

2014-02-01

Ex vivo fluorescence confocal microscopy (FCM) enables real-time imaging of skin morphology directly in freshly excised tissue. FCM displays wide field-of-view mosaics with cellular resolution, thus enabling a rapid bedside pathology. An application of interest is rapid detection of residual basal cell carcinoma (BCC) in skin excisions during Mohs surgery. We sought to evaluate the sensitivity and specificity of ex vivo imaging with FCM for the detection of residual BCC in Mohs tissue excisions, and to calculate the time invested up to the diagnosis for both FCM and frozen sections. Eighty consecutive BCCs were prospectively collected and the margins scanned with ex vivo FCM, including excisions with and without residual BCC of all major subtypes. Each mosaic was divided into two or four, resulting in 480 submosaics for study. Every confocal submosaic was assessed for the presence or absence of BCC and compared with standard frozen sections as the gold standard. Furthermore, the time spent for each technique was calculated and compared. The overall sensitivity and specificity of detecting residual BCC were 88% and 99%, respectively. Moreover, the new technique reduced by almost two-thirds the time invested when compared with the processing of a frozen section (P < 0·001). The results demonstrate the feasibility of confocal mosaicing microscopy in fresh tissue for rapid surgical pathology, potentially to expedite and guide Mohs surgery with high accuracy. This observation is an important step towards the goal of using real-time surgical pathology for skin tumours. © 2013 British Association of Dermatologists.

Integrating bioassays and analytical chemistry as an improved approach to support safety assessment of food contact materials.

PubMed

Veyrand, Julien; Marin-Kuan, Maricel; Bezencon, Claudine; Frank, Nancy; Guérin, Violaine; Koster, Sander; Latado, Hélia; Mollergues, Julie; Patin, Amaury; Piguet, Dominique; Serrant, Patrick; Varela, Jesus; Schilter, Benoît

2017-10-01

Food contact materials (FCM) contain chemicals which can migrate into food and result in human exposure. Although it is mandatory to ensure that migration does not endanger human health, there is still no consensus on how to pragmatically assess the safety of FCM since traditional approaches would require extensive toxicological and analytical testing which are expensive and time consuming. Recently, the combination of bioassays, analytical chemistry and risk assessment has been promoted as a new paradigm to identify toxicologically relevant molecules and address safety issues. However, there has been debate on the actual value of bioassays in that framework. In the present work, a FCM anticipated to release the endocrine active chemical 4-nonyphenol (4NP) was used as a model. In a migration study, the leaching of 4NP was confirmed by LC-MS/MS and GC-MS. This was correlated with an increase in both estrogenic and anti-androgenic activities as measured with bioassays. A standard risk assessment indicated that according to the food intake scenario applied, the level of 4NP measured was lower, close or slightly above the acceptable daily intake. Altogether these results show that bioassays could reveal the presence of an endocrine active chemical in a real-case FCM migration study. The levels reported were relevant for safety assessment. In addition, this work also highlighted that bioactivity measured in migrate does not necessarily represent a safety issue. In conclusion, together with analytics, bioassays contribute to identify toxicologically relevant molecules leaching from FCM and enable improved safety assessment.

Intravenous Iron Administration and Hypophosphatemia in Clinical Practice

PubMed Central

Hardy, S.; Vandemergel, X.

2015-01-01

Introduction. Parenteral iron formulations are frequently used to correct iron deficiency anemia (IDA) and iron deficiency (ID). Intravenous formulation efficacy on ferritin and hemoglobin level improvement is greater than that of oral formulations while they are associated with lower gastrointestinal side effects. Ferric carboxymaltose- (FCM-) related hypophosphatemia is frequent and appears without clinical significance. The aim of this study was to assess the prevalence, duration, and potential consequences of hypophosphatemia after iron injection. Patients and Methods. The medical records of all patients who underwent parenteral iron injection between 2012 and 2014 were retrospectively reviewed. Pre- and postinjection hemoglobin, ferritin, plasma phosphate, creatinine, and vitamin D levels were assessed. Patients who developed moderate (range: 0.32–0.80 mmol/L) or severe (<0.32 mmol/L) hypophosphatemia were questioned for symptoms. Results. During the study period, 234 patients received iron preparations but 104 were excluded because of missing data. Among the 130 patients included, 52 received iron sucrose (FS) and 78 FCM formulations. Among FS-treated patients, 22% developed hypophosphatemia versus 51% of FCM-treated patients, including 13% who developed profound hypophosphatemia. Hypophosphatemia severity correlated with the dose of FCM (p = 0.04) but not with the initial ferritin, hemoglobin, or vitamin D level. Mean hypophosphatemia duration was 6 months. No immediate clinical consequence was found except for persistent fatigue despite anemia correction in some patients. Conclusions. Hypophosphatemia is frequent after parenteral FCM injection and may have clinical consequences, including persistent fatigue. Further studies of chronic hypophosphatemia long-term consequences, especially bone assessments, are needed. PMID:26000018

Physiological response to etho-ecological stressors in male Alpine chamois: timescale matters!

PubMed

Corlatti, Luca; Palme, Rupert; Lovari, Sandro

2014-07-01

From a life history perspective, glucocorticoids secreted by the neuroendocrine system, integrating different sources of stress through an adaptive feedback mechanism, may have important consequences on individual fitness. Although stress responses have been the object of several investigations, few studies have explored the role of proximate mechanisms responsible for the potential trade-offs between physiological stress and life history traits integrating social and environmental stressors. In 2011 and 2012, we collected data on faecal cortisol metabolites (FCM) in a marked male population of Alpine chamois, within the Gran Paradiso National Park (Italy). Using a model selection approach we analysed the effect of potential etho-ecological stressors such as age, social status (territorial vs. non-territorial males), minimum temperature, snow depth and precipitation on FCM variation. To correctly interpret environmentally and socially induced stress responses, we conducted model selections over multiple temporal scales defined a priori: year, cold months, spring, warm months, mating season. Over the year, FCM levels showed a negative relationship with minimum temperature, but altogether, climatic stressors had negligible effects on glucocorticoid secretion, possibly owing to good adaptations of chamois to severe weather conditions. Age was negatively related to FCM during the rut, possibly due to greater experience of older males in agonistic contests. Social status was an important determinant of FCM excretion: while both the 'stress of subordination' and the 'stress of domination' hypotheses received some support in spring and during the mating season, respectively, previous data suggest that only the latter may have detrimental fitness consequences on male chamois.

Physiological response to etho-ecological stressors in male Alpine chamois: timescale matters!

NASA Astrophysics Data System (ADS)

Corlatti, Luca; Palme, Rupert; Lovari, Sandro

2014-07-01

From a life history perspective, glucocorticoids secreted by the neuroendocrine system, integrating different sources of stress through an adaptive feedback mechanism, may have important consequences on individual fitness. Although stress responses have been the object of several investigations, few studies have explored the role of proximate mechanisms responsible for the potential trade-offs between physiological stress and life history traits integrating social and environmental stressors. In 2011 and 2012, we collected data on faecal cortisol metabolites (FCM) in a marked male population of Alpine chamois, within the Gran Paradiso National Park (Italy). Using a model selection approach we analysed the effect of potential etho-ecological stressors such as age, social status (territorial vs. non-territorial males), minimum temperature, snow depth and precipitation on FCM variation. To correctly interpret environmentally and socially induced stress responses, we conducted model selections over multiple temporal scales defined a priori: year, cold months, spring, warm months, mating season. Over the year, FCM levels showed a negative relationship with minimum temperature, but altogether, climatic stressors had negligible effects on glucocorticoid secretion, possibly owing to good adaptations of chamois to severe weather conditions. Age was negatively related to FCM during the rut, possibly due to greater experience of older males in agonistic contests. Social status was an important determinant of FCM excretion: while both the `stress of subordination' and the `stress of domination' hypotheses received some support in spring and during the mating season, respectively, previous data suggest that only the latter may have detrimental fitness consequences on male chamois.

Cumulative asbestos exposure for US automobile mechanics involved in brake repair (circa 1950s-2000).

PubMed

Finley, Brent L; Richter, Richard O; Mowat, Fionna S; Mlynarek, Steve; Paustenbach, Dennis J; Warmerdam, John M; Sheehan, Patrick J

2007-11-01

We analyzed cumulative lifetime exposure to chrysotile asbestos experienced by brake mechanics in the US during the period 1950-2000. Using Monte Carlo methods, cumulative exposures were calculated using the distribution of 8-h time-weighted average exposure concentrations for brake mechanics and the distribution of job tenure data for automobile mechanics. The median estimated cumulative exposures for these mechanics, as predicted by three probabilistic models, ranged from 0.16 to 0.41 fibers per cubic centimeter (f/cm(3)) year for facilities with no dust-control procedures (1970s), and from 0.010 to 0.012 f/cm(3) year for those employing engineering controls (1980s). Upper-bound (95%) estimates for the 1970s and 1980s were 1.96 to 2.79 and 0.07-0.10 f/cm(3) year, respectively. These estimates for US brake mechanics are consistent with, but generally slightly lower than, those reported for European mechanics. The values are all substantially lower than the cumulative exposure of 4.5 f/cm(3) year associated with occupational exposure to 0.1 f/cm(3) of asbestos for 45 years that is currently permitted under the current occupational exposure limits in the US. Cumulative exposures were usually about 100- to 1,000-fold less than those of other occupational groups with asbestos exposure for similar time periods. The cumulative lifetime exposure estimates presented here, combined with the negative epidemiology data for brake mechanics, could be used to refine the risk assessments for chrysotile-exposed populations.

[Flowcytometry DNA analysis of oral and maxillofacial non-Hodgkin's lymphoma].

PubMed

Ma, Li; He, Zhixiu; Wu, Lanyan; Cai, Yixin; Huang, Hechang; Lei, Song

2002-06-01

The purpose of this study was to investigate the relationship between the results of flowcytometry analyses of different clinical stage, location, pathologic grade and cell origin of oral and maxillofacial non-Hodgkin's lymphoma (NHL), and the diagnostic value of flowcytometry analysis in lymphoma. This study analyzed 50 oral and maxillofacial NHL cases and 10 reactive lymph nodes (formalin fixed and paraffin embedded) by flowcytometry (FCM). Reactive lymph nodes were all diploid. The diploid rate of NHL was 54%, and aneuploidy rate was 46%. There was statistically significant difference between reactive lymph nodes and NHL in the DNA ploidy status and cell cycle data (SPF, CV, S + G2/M, DI). The S phase fraction (SPF) and S + G2/M had close relationship with the grade of NHL. SPF value and DNA ploidy status had no obvious relationship with the prognosis. The results suggested that the FCM had diagnostic value in NHL, especially when the morphological diagnosis was difficult. Although the cell cycle data had no prognostic value, SPF and SPF + G2/M can show the proliferative status of NHL, which can help clinical doctor select therapeutic method.

Investigation of the accuracy of breast tissue segmentation methods for the purpose of developing breast deformation models for use in adaptive radiotherapy

NASA Astrophysics Data System (ADS)

Juneja, P.; Harris, E. J.; Evans, P. M.

2014-03-01

Realistic modelling of breast deformation requires the breast tissue to be segmented into fibroglandular and fatty tissue and assigned suitable material properties. There are a number of breast tissue segmentation methods proposed and used in the literature. The purpose of this study was to validate and compare the accuracy of various segmentation methods and to investigate the effect of the tissue distribution on the segmentation accuracy. Computed tomography (CT) data for 24 patients, both in supine and prone positions were segmented into fibroglandular and fatty tissue. The segmentation methods explored were: physical density thresholding; interactive thresholding; fuzzy c-means clustering (FCM) with three classes (FCM3) and four classes (FCM4); and k-means clustering. Validation was done in two-stages: firstly, a new approach, supine-prone validation based on the assumption that the breast composition should appear the same in the supine and prone scans was used. Secondly, outlines from three experts were used for validation. This study found that FCM3 gave the most accurate segmentation of breast tissue from CT data and that the segmentation accuracy is adversely affected by the sparseness of the fibroglandular tissue distribution.

Efficient architecture for spike sorting in reconfigurable hardware.

PubMed

Hwang, Wen-Jyi; Lee, Wei-Hao; Lin, Shiow-Jyu; Lai, Sheng-Ying

2013-11-01

This paper presents a novel hardware architecture for fast spike sorting. The architecture is able to perform both the feature extraction and clustering in hardware. The generalized Hebbian algorithm (GHA) and fuzzy C-means (FCM) algorithm are used for feature extraction and clustering, respectively. The employment of GHA allows efficient computation of principal components for subsequent clustering operations. The FCM is able to achieve near optimal clustering for spike sorting. Its performance is insensitive to the selection of initial cluster centers. The hardware implementations of GHA and FCM feature low area costs and high throughput. In the GHA architecture, the computation of different weight vectors share the same circuit for lowering the area costs. Moreover, in the FCM hardware implementation, the usual iterative operations for updating the membership matrix and cluster centroid are merged into one single updating process to evade the large storage requirement. To show the effectiveness of the circuit, the proposed architecture is physically implemented by field programmable gate array (FPGA). It is embedded in a System-on-Chip (SOC) platform for performance measurement. Experimental results show that the proposed architecture is an efficient spike sorting design for attaining high classification correct rate and high speed computation.

Functional modulation on macrophage by low dose naltrexone (LDN).

PubMed

Yi, Zhe; Guo, Shengnan; Hu, Xu; Wang, Xiaonan; Zhang, Xiaoqing; Griffin, Noreen; Shan, Fengping

2016-10-01

Previously it was confirmed that naltrexone, a non-peptide δ-opioid receptor selective antagonist is mainly used for alcoholic dependence and opioid addiction treatment. However, there is increasing data on immune regulation of low dose naltrexone (LDN). The aim of this work was to explore the effect of LDN on the phenotype and function of macrophage. The changes of macrophage after treatment with LDN were examined using flow cytometry (FCM); FITC-dextran phagocytosis and enzyme-linked immunosorbent assay (ELISA). We have found that LDN enhances function of macrophage as confirmed by up-regulating MHC II molecule and CD64 on macrophage while down-regulating CD206 expression. Furthermore the productions of TNF-α, IL-6, IL-1β, increased significantly. Macrophages in LDN treated group performed the enhanced phagocytosis. Therefore it is concluded that LDN could promote function of macrophage and this work has provided concrete data of impact on immune system by LDN. Especially the data would support interaction between CD4+T cell and macrophage in AIDS treatment with LDN in Africa (LDN has already been approved in Nigeria for the use in AIDS treatment). Copyright © 2016. Published by Elsevier B.V.

A novel toolbox for E. coli lysis monitoring.

PubMed

Rajamanickam, Vignesh; Wurm, David; Slouka, Christoph; Herwig, Christoph; Spadiut, Oliver

2017-01-01

The bacterium Escherichia coli is a well-studied recombinant host organism with a plethora of applications in biotechnology. Highly valuable biopharmaceuticals, such as antibody fragments and growth factors, are currently being produced in E. coli. However, the high metabolic burden during recombinant protein production can lead to cell death, consequent lysis, and undesired product loss. Thus, fast and precise analyzers to monitor E. coli bioprocesses and to retrieve key process information, such as the optimal time point of harvest, are needed. However, such reliable monitoring tools are still scarce to date. In this study, we cultivated an E. coli strain producing a recombinant single-chain antibody fragment in the cytoplasm. In bioreactor cultivations, we purposely triggered cell lysis by pH ramps. We developed a novel toolbox using UV chromatograms as fingerprints and chemometric techniques to monitor these lysis events and used flow cytometry (FCM) as reference method to quantify viability offline. Summarizing, we were able to show that a novel toolbox comprising HPLC chromatogram fingerprinting and data science tools allowed the identification of E. coli lysis in a fast and reliable manner. We are convinced that this toolbox will not only facilitate E. coli bioprocess monitoring but will also allow enhanced process control in the future.

Metal ions induced heat shock protein response by elevating superoxide anion level in HeLa cells transformed by HSE-SEAP reporter gene.

PubMed

Yu, Zhanjiang; Yang, Xiaoda; Wang, Kui

2006-06-01

The aim of this work is to define the relationship between heat shock protein (HSP) and reactive oxygen species (ROS) in the cells exposed to different concentrations of metal ions, and to evaluate a new method for tracing the dynamic levels of cellular reactive oxygen species using a HSE-SEAP reporter gene. The expression of heat shock protein was measured using a secreted alkaline phosphatase (SEAP) reporter gene transformed into HeLa cell strain, the levels of superoxide anion (O(2)(-)) and hydrogen peroxide (H(2)O(2)) were determined by NBT reduction assay and DCFH staining flow cytometry (FCM), respectively. The experimental results demonstrated that the expression of heat shock protein induced by metal ions was linearly related to the cellular superoxide anion level before cytotoxic effects were observed, but not related to the cellular hydrogen peroxide level. The experimental results suggested that metal ions might induce heat shock protein by elevating cellular superoxide anion level, and thus the expression of heat shock protein indicated by the HSE-SEAP reporter gene can be an effective model for monitoring the dynamic level of superoxide anion and early metal-induced oxidative stress/cytotoxicity.

Phase-sensitive flow cytometer

DOEpatents

Steinkamp, John A.

1993-01-01

A phase-sensitive flow cytometer (FCM) provides additional FCM capability to use the fluorescence lifetime of one or more fluorochromes bound to single cells to provide additional information regarding the cells. The resulting fluorescence emission can be resolved into individual fluorescence signals if two fluorochromes are present or can be converted directly to a decay lifetime from a single fluorochrome. The excitation light for the fluorochromes is modulated to produce an amplitude modulated fluorescence pulse as the fluorochrome is excited in the FCM. The modulation signal also forms a reference signal that is phase-shifted a selected amount for subsequent mixing with the output modulated fluorescence intensity signal in phase-sensitive detection circuitry. The output from the phase-sensitive circuitry is then an individual resolved fluorochrome signal or a single fluorochrome decay lifetime, depending on the applied phase shifts.

Weak imposition of frictionless contact constraints on automatically recovered high-order, embedded interfaces using the finite cell method

NASA Astrophysics Data System (ADS)

Bog, Tino; Zander, Nils; Kollmannsberger, Stefan; Rank, Ernst

2018-04-01

The finite cell method (FCM) is a fictitious domain approach that greatly simplifies simulations involving complex structures. Recently, the FCM has been applied to contact problems. The current study continues in this field by extending the concept of weakly enforced boundary conditions to inequality constraints for frictionless contact. Furthermore, it formalizes an approach that automatically recovers high-order contact surfaces of (implicitly defined) embedded geometries by means of an extended Marching Cubes algorithm. To further improve the accuracy of the discretization, irregularities at the boundary of contact zones are treated with multi-level hp-refinements. Numerical results and a systematic study of h-, p- and hp-refinements show that the FCM can efficiently provide accurate results for problems involving contact.

Hyperspectral cytometry.

PubMed

Grégori, Gérald; Rajwa, Bartek; Patsekin, Valery; Jones, James; Furuki, Motohiro; Yamamoto, Masanobu; Paul Robinson, J

2014-01-01

Hyperspectral cytometry is an emerging technology for single-cell analysis that combines ultrafast optical spectroscopy and flow cytometry. Spectral cytometry systems utilize diffraction gratings or prism-based monochromators to disperse fluorescence signals from multiple labels (organic dyes, nanoparticles, or fluorescent proteins) present in each analyzed bioparticle onto linear detector arrays such as multianode photomultipliers or charge-coupled device sensors. The resultant data, consisting of a series of characterizing every analyzed cell, are not compensated by employing the traditional cytometry approach, but rather are spectrally unmixed utilizing algorithms such as constrained Poisson regression or non-negative matrix factorization. Although implementations of spectral cytometry were envisioned as early as the 1980s, only recently has the development of highly sensitive photomultiplier tube arrays led to design and construction of functional prototypes and subsequently to introduction of commercially available systems. This chapter summarizes the historical efforts and work in the field of spectral cytometry performed at Purdue University Cytometry Laboratories and describes the technology developed by Sony Corporation that resulted in release of the first commercial spectral cytometry system-the Sony SP6800. A brief introduction to spectral data analysis is also provided, with emphasis on the differences between traditional polychromatic and spectral cytometry approaches.

Mass cytometry: a highly multiplexed single-cell technology for advancing drug development.

PubMed

Atkuri, Kondala R; Stevens, Jeffrey C; Neubert, Hendrik

2015-02-01

Advanced single-cell analysis technologies (e.g., mass cytometry) that help in multiplexing cellular measurements in limited-volume primary samples are critical in bridging discovery efforts to successful drug approval. Mass cytometry is the state-of-the-art technology in multiparametric single-cell analysis. Mass cytometers (also known as cytometry by time-of-flight or CyTOF) combine the cellular analysis principles of traditional fluorescence-based flow cytometry with the selectivity and quantitative power of inductively coupled plasma-mass spectrometry. Standard flow cytometry is limited in the number of parameters that can be measured owing to the overlap in signal when detecting fluorescently labeled antibodies. Mass cytometry uses antibodies tagged to stable isotopes of rare earth metals, which requires minimal signal compensation between the different metal tags. This unique feature enables researchers to seamlessly multiplex up to 40 independent measurements on single cells. In this overview we first present an overview of mass cytometry and compare it with traditional flow cytometry. We then discuss the emerging and potential applications of CyTOF technology in the pharmaceutical industry, including quantitative and qualitative deep profiling of immune cells and their applications in assessing drug immunogenicity, extensive mapping of signaling networks in single cells, cell surface receptor quantification and multiplexed internalization kinetics, multiplexing sample analysis by barcoding, and establishing cell ontologies on the basis of phenotype and/or function. We end with a discussion of the anticipated impact of this technology on drug development lifecycle with special emphasis on the utility of mass cytometry in deciphering a drug's pharmacokinetics and pharmacodynamics relationship. Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.

Web-based analysis and publication of flow cytometry experiments.

PubMed

Kotecha, Nikesh; Krutzik, Peter O; Irish, Jonathan M

2010-07-01

Cytobank is a Web-based application for storage, analysis, and sharing of flow cytometry experiments. Researchers use a Web browser to log in and use a wide range of tools developed for basic and advanced flow cytometry. In addition to providing access to standard cytometry tools from any computer, Cytobank creates a platform and community for developing new analysis and publication tools. Figure layouts created on Cytobank are designed to allow transparent access to the underlying experiment annotation and data processing steps. Since all flow cytometry files and analysis data are stored on a central server, experiments and figures can be viewed or edited by anyone with the proper permission, from any computer with Internet access. Once a primary researcher has performed the initial analysis of the data, collaborators can engage in experiment analysis and make their own figure layouts using the gated, compensated experiment files. Cytobank is available to the scientific community at http://www.cytobank.org. (c) 2010 by John Wiley & Sons, Inc.

Web-Based Analysis and Publication of Flow Cytometry Experiments

PubMed Central

Kotecha, Nikesh; Krutzik, Peter O.; Irish, Jonathan M.

2014-01-01

Cytobank is a web-based application for storage, analysis, and sharing of flow cytometry experiments. Researchers use a web browser to log in and use a wide range of tools developed for basic and advanced flow cytometry. In addition to providing access to standard cytometry tools from any computer, Cytobank creates a platform and community for developing new analysis and publication tools. Figure layouts created on Cytobank are designed to allow transparent access to the underlying experiment annotation and data processing steps. Since all flow cytometry files and analysis data are stored on a central server, experiments and figures can be viewed or edited by anyone with the proper permissions from any computer with Internet access. Once a primary researcher has performed the initial analysis of the data, collaborators can engage in experiment analysis and make their own figure layouts using the gated, compensated experiment files. Cytobank is available to the scientific community at www.cytobank.org PMID:20578106

Burn Injuries and Their Impact on Cognitive-Communication Skills in the Inpatient Rehabilitation Setting.

PubMed

Hendricks, Carla Tierney; Camara, Kristin; Violick Boole, Kathryn; Napoli, Maureen F; Goldstein, Richard; Ryan, Colleen M; Schneider, Jeffrey C

The prevalence and extent of cognitive-communication disorders and factors that have impact on outcomes are examined in the burn population within an inpatient rehabilitation facility. A retrospective data analysis was conducted on adults diagnosed with burn injury (n = 144). Descriptive statistics were used to identify the prevalence of cognitive-communication deficits on admission and discharge. The main outcomes were cognitive-communication ratings on discharge from inpatient rehabilitation as measured by the memory and problem-solving domains of the Functional Independence Measure (FIM) and composite score of the Functional Communication Measure (FCM). Medical, demographic and rehabilitation predictors of the main outcomes were assessed using regression analyses. On admission to inpatient rehabilitation, 79% of the total population presented with cognitive-communication impairments, and of them, 27% presented with persistent deficits on discharge. Admission FIM memory score, marital status, and age were significant predictors of discharge FIM memory score. Admission FIM problem-solving score, age, marital status, and prehospital living-with were significant predictors of discharge FIM problem-solving score. Admission FCM score and age were significant predictors of discharge FCM cognitive score. Persons with burn injuries are at risk for cognitive-communication impairments, which may persist after inpatient rehabilitation. FIM data obtained on admission can be used as a screening tool to identify these at-risk patients. Future work is needed to assess the efficacy of speech-language pathologist intervention for cognitive-communication deficits within the burn injury population.

Modeling and Analysis of FCM UN TRISO Fuel Using the PARFUME Code

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

Blaise Collin

2013-09-01

The PARFUME (PARticle Fuel ModEl) modeling code was used to assess the overall fuel performance of uranium nitride (UN) tri-structural isotropic (TRISO) ceramic fuel in the frame of the design and development of Fully Ceramic Matrix (FCM) fuel. A specific modeling of a TRISO particle with UN kernel was developed with PARFUME, and its behavior was assessed in irradiation conditions typical of a Light Water Reactor (LWR). The calculations were used to access the dimensional changes of the fuel particle layers and kernel, including the formation of an internal gap. The survivability of the UN TRISO particle was estimated dependingmore » on the strain behavior of the constituent materials at high fast fluence and burn-up. For nominal cases, internal gas pressure and representative thermal profiles across the kernel and layers were determined along with stress levels in the pyrolytic carbon (PyC) and silicon carbide (SiC) layers. These parameters were then used to evaluate fuel particle failure probabilities. Results of the study show that the survivability of UN TRISO fuel under LWR irradiation conditions might only be guaranteed if the kernel and PyC swelling rates are limited at high fast fluence and burn-up. These material properties are unknown at the irradiation levels expected to be reached by UN TRISO fuel in LWRs. Therefore, more effort is needed to determine them and positively conclude on the applicability of FCM fuel to LWRs.« less

Impact of pixel-based machine-learning techniques on automated frameworks for delineation of gross tumor volume regions for stereotactic body radiation therapy.

PubMed

Kawata, Yasuo; Arimura, Hidetaka; Ikushima, Koujirou; Jin, Ze; Morita, Kento; Tokunaga, Chiaki; Yabu-Uchi, Hidetake; Shioyama, Yoshiyuki; Sasaki, Tomonari; Honda, Hiroshi; Sasaki, Masayuki

2017-10-01

The aim of this study was to investigate the impact of pixel-based machine learning (ML) techniques, i.e., fuzzy-c-means clustering method (FCM), and the artificial neural network (ANN) and support vector machine (SVM), on an automated framework for delineation of gross tumor volume (GTV) regions of lung cancer for stereotactic body radiation therapy. The morphological and metabolic features for GTV regions, which were determined based on the knowledge of radiation oncologists, were fed on a pixel-by-pixel basis into the respective FCM, ANN, and SVM ML techniques. Then, the ML techniques were incorporated into the automated delineation framework of GTVs followed by an optimum contour selection (OCS) method, which we proposed in a previous study. The three-ML-based frameworks were evaluated for 16 lung cancer cases (six solid, four ground glass opacity (GGO), six part-solid GGO) with the datasets of planning computed tomography (CT) and 18 F-fluorodeoxyglucose (FDG) positron emission tomography (PET)/CT images using the three-dimensional Dice similarity coefficient (DSC). DSC denotes the degree of region similarity between the GTVs contoured by radiation oncologists and those estimated using the automated framework. The FCM-based framework achieved the highest DSCs of 0.79±0.06, whereas DSCs of the ANN-based and SVM-based frameworks were 0.76±0.14 and 0.73±0.14, respectively. The FCM-based framework provided the highest segmentation accuracy and precision without a learning process (lowest calculation cost). Therefore, the FCM-based framework can be useful for delineation of tumor regions in practical treatment planning. Copyright © 2017 Associazione Italiana di Fisica Medica. Published by Elsevier Ltd. All rights reserved.

Atmospheric Energy Deposition Modeling and Inference for Varied Meteoroid Structures

NASA Technical Reports Server (NTRS)

Wheeler, Lorien; Mathias, Donovan; Stokan, Edward; Brown, Peter

2018-01-01

Asteroids populations are highly diverse, ranging from coherent monoliths to loosely-bound rubble piles with a broad range of material and compositional properties. These different structures and properties could significantly affect how an asteroid breaks up and deposits energy in the atmosphere, and how much ground damage may occur from resulting blast waves. We have previously developed a fragment-cloud model (FCM) for assessing the atmospheric breakup and energy deposition of asteroids striking Earth. The approach represents ranges of breakup characteristics by combining progressive fragmentation with releases of variable fractions of debris and larger discrete fragments. In this work, we have extended the FCM to also represent asteroids with varied initial structures, such as rubble piles or fractured bodies. We have used the extended FCM to model the Chelyabinsk, Benesov, Kosice, and Tagish Lake meteors, and have obtained excellent matches to energy deposition profiles derived from their light curves. These matches provide validation for the FCM approach, help guide further model refinements, and enable inferences about pre-entry structure and breakup behavior. Results highlight differences in the amount of small debris vs. discrete fragments in matching the various flare characteristics of each meteor. The Chelyabinsk flares were best represented using relatively high debris fractions, while Kosice and Benesov cases were more notably driven by their discrete fragmentation characteristics, perhaps indicating more cohesive initial structures. Tagish Lake exhibited a combination of these characteristics, with lower-debris fragmentation at high altitudes followed by sudden disintegration into small debris in the lower flares. Results from all cases also suggest that lower ablation coefficients and debris spread rates may be more appropriate for the way in which debris clouds are represented in FCM, offering an avenue for future model refinement.

Anti-EGFR-Conjugated Hollow Gold Nanospheres Enhance Radiocytotoxic Targeting of Cervical Cancer at Megavoltage Radiation Energies

NASA Astrophysics Data System (ADS)

Liu, Jiao; Liang, Ying; Liu, Ting; Li, Dengke; Yang, Xingsheng

2015-05-01

The study aimed to confirm that anti-epidermal growth factor receptor (EGFR) monoclonal antibody-conjugated hollow gold nanospheres (anti-EGFR/HGNs) can be selectively uptaken by cervical cancer cells and induce its apoptosis when combined with radiotherapy, as a result enhancing radiosensitivity of cervical cancer cells. HGNs with a mean diameter of 54.6 ± 7.11 nm and wall thickness of 5.01 ± 2.23 nm were viewed by transmission electron microscopy (TEM). Cell uptake was assayed by inductively coupled plasma atomic emission spectroscopy (ICP-AES). The cytotoxicity on HeLa cells, which were used in our experiment, was assessed by CCK-8 assay. Cell cycle and apoptosis were examined by an Annexin V-FITC/propidium iodide (PI) kit with flow cytometry (FCM). The expression of several critical apoptosis-related proteins, including Bcl-2, Bax, Bad, and active caspase 3, was tested by western blot analysis. Cells treated by anti-EGFR/HGNs showed an obvious increase in nanoparticle uptake compared to naked HGNs. Anti-EGFR/HGNs combined with radiation resulted in a significant growth inhibition, compared with radiation combined with naked HGNs. Anti-EGFR/HGNs remarkably increased the ratio of HeLa cells in the G2/M phase and induced more apoptosis by an obvious deregulation of Bcl-2 and upregulation of Bax, Bad, and caspase 3 when combined with radiation. Therefore, anti-EGFR/HGNs can increase the targeted uptake of HGNs by HeLa cells and enhance radiocytotoxic targeting of cervical cancer at megavoltage radiation energies.

Phase-sensitive flow cytometer

DOEpatents

Steinkamp, J.A.

1993-12-14

A phase-sensitive flow cytometer (FCM) provides additional FCM capability to use the fluorescence lifetime of one or more fluorochromes bound to single cells to provide additional information regarding the cells. The resulting fluorescence emission can be resolved into individual fluorescence signals if two fluorochromes are present or can be converted directly to a decay lifetime from a single fluorochrome. The excitation light for the fluorochromes is modulated to produce an amplitude modulated fluorescence pulse as the fluorochrome is excited in the FCM. The modulation signal also forms a reference signal that is phase-shifted a selected amount for subsequent mixing with the output modulated fluorescence intensity signal in phase-sensitive detection circuitry. The output from the phase-sensitive circuitry is then an individual resolved fluorochrome signal or a single fluorochrome decay lifetime, depending on the applied phase shifts. 15 figures.

Cytobank: providing an analytics platform for community cytometry data analysis and collaboration.

PubMed

Chen, Tiffany J; Kotecha, Nikesh

2014-01-01

Cytometry is used extensively in clinical and laboratory settings to diagnose and track cell subsets in blood and tissue. High-throughput, single-cell approaches leveraging cytometry are developed and applied in the computational and systems biology communities by researchers, who seek to improve the diagnosis of human diseases, map the structures of cell signaling networks, and identify new cell types. Data analysis and management present a bottleneck in the flow of knowledge from bench to clinic. Multi-parameter flow and mass cytometry enable identification of signaling profiles of patient cell samples. Currently, this process is manual, requiring hours of work to summarize multi-dimensional data and translate these data for input into other analysis programs. In addition, the increase in the number and size of collaborative cytometry studies as well as the computational complexity of analytical tools require the ability to assemble sufficient and appropriately configured computing capacity on demand. There is a critical need for platforms that can be used by both clinical and basic researchers who routinely rely on cytometry. Recent advances provide a unique opportunity to facilitate collaboration and analysis and management of cytometry data. Specifically, advances in cloud computing and virtualization are enabling efficient use of large computing resources for analysis and backup. An example is Cytobank, a platform that allows researchers to annotate, analyze, and share results along with the underlying single-cell data.

BioMig--A Method to Evaluate the Potential Release of Compounds from and the Formation of Biofilms on Polymeric Materials in Contact with Drinking Water.

PubMed

Wen, Gang; Kötzsch, Stefan; Vital, Marius; Egli, Thomas; Ma, Jun

2015-10-06

In contact with water, polymeric materials (plastics) release compounds that can support suspended microbial growth and/or biofilm formation. The different methods presently used in the European Union to test plastics take 7-16 weeks to obtain a result. In industry, this delays material and product development as well as quality testing. Therefore, we developed a method package (BioMig) that allows testing of plastic materials with high reproducibility in 2 weeks for their potential biofilm (or biomass) formation and release of carbonaceous migration products when in contact with water. BioMig consists of (i) an extended migration potential test (seven times for 24 h at 60 °C), based on the European norm EN 12873-1 and the German UBA (Umweltbundesamt) guideline, and (ii) a biomass formation potential (BFP) test (14 days at 30 °C), which is a modified version of the Dutch biofilm production potential test. In the migration potential test, the amount of carbon released into water by the specimen is quantified by monitoring total and assimilable organic carbon over time; furthermore, the modular design of the test also allows one to assess additional parameters such as pathogen growth potential on the migration water or toxic effects on microbial growth. Flow cytometry (FCM)-based total cell counting (TCC) is used to quantify microbial growth in suspension and on surfaces after removal with mild sonication without affecting cell integrity. The BFP test allows one to determine both the planktonic (pBFP) and the sessile (sBFP) cell fractions. The sBFP consists of surface-attached cells after removal (>90% efficiency). Results for four standard test materials (PE-Xa, PE-Xc, EPDM 2%, and EPDM 20%), plus positive (PVC-P) and negative (glass) controls are presented. FCM-based TCC demonstrates that the release of growth-supporting carbon and proliferation of surface-attached cells stops increasing and stabilizes after 14 days of incubation; this allows for faster assessment of growth-supporting properties of plastics with BioMig compared to established tests.

[Effects of traditional Tibetan drug Liu Tea on proliferation and chemotherapeutic sensitivity of drug-resistant human gastric cancer cell BGC823/5-FU].

PubMed

Cheng, Yan; Hasiqi, Mei-Ge; Qin, Xiao-Zhen; Tang, Xiang-You; Chen, Jian-Nan; Wang, Hui-Yin; Gao, Ao

2016-02-01

To investigate the effects of Liu Tea extracts(LTE) on proliferation, apoptosis and drug sensitivity of drug-resistant gastric cancer cell BGC823/5-FU. MTT assay was used to analyze effect of LTE on cell growth and sensitivity chemotherapeutic drugs, and synergistic effect of the combination of LTE with 5-FU on BGC823/5-FU cells. Combination index (CI) was calculated by CompuSyn. Cell apoptosis was measured by flow cytometry (FCM). Protein expressions of P-gp, Bcl-2, Bax and Caspase-3 (17KD) were detected by Western blot at different concentrations of LTE in BGC823/5-FU cells (100, 200, 400 mg•L⁻¹). The results showed that LTE had an inhibitory effect on growth of BGC823/5-FU cell in a dose-time-dependent manner and significantly reduced IC₅₀ of 5-FU, CDDP, PTX and ADM to BGC823/5-FU cells(P<0.05), indicating it could reverse tolerance of drug resistant cells to chemotherapeutic drugs, with reversion multiples of 2.35, 1.68, 1.96, 0.52. The combination of LTE with 5-FU had positive synergistic effect on the BGC-823 cell line. FCM assay suggested that LTE could induce BGC823/5-FU apoptosis. The apoptosis rate was up to 46.2% when the cells were treated with 800 mg•L⁻¹ LTE after 24 h(P<0.01). According to the protein detection results, with the increase in concentration of LTE, the protein expression of Bcl-2 was gradually decreased(P<0.01), the expression of Bax and Caspase-3 were extremely increased(P<0.01), with statistical significance in difference(P<0.01) but no difference in the expression of P-gp between experiment group and control group. LTE can inhibit the growth of drug-resistant human gastric cancer cell BGC823/5-FU and reverse its chemotherapeutic tolerance to some extent. Inhibition of antiapoptotic proteins, activation of proapoptotic proteins and induction of apoptosis of resistant cells may be its main mechanisms. Copyright© by the Chinese Pharmaceutical Association.

Expression of gap junction genes connexin 32 and connexin 43 mRNAs and proteins, and their role in hepatocarcinogenesis

PubMed Central

Ma, Xiang-Dong; Ma, Xing; Sui, Yan-Fang; Wang, Wen-Liang

2002-01-01

AIM: To investigate the relationship between hepatocarcinogenesis and the expression of connexin32 (cx32), connexin43 (cx43) mRNAs and proteins in vitro. METHODS: Gap junction genes cx32 and cx43 mRNA in hepatocellular carcinoma cell lines HHCC, SMMC-7721 and normal liver cell line QZG were detected by in situ hybridization (ISH) with digoxin-labeled cx32, and cx43 cDNA probes. Expression of Cx32 and Cx43 proteins in the cell lines was revealed by indirect immuno-fluorescence and flow cytometry (FCM). RESULTS: Blue positive hybridization signals of cx32 and cx43 mRNAs detected by ISH with cx32 and cx43 cDNA probes respectively were located in cytoplasm of cells of HHCC, SMMC-7721 and QZG. No significant difference of either cx32 mRNA or cx43 mRNA was tested among HHCC, SMMC-7721 and QZG (P = 2.673, HHCC vs QZG; P = 1.375, SMMC-7721 vs QZG). FCM assay showed that the positive rates of Cx32 protein in HHCC, SMMC-7721 and QZG were 0.7%, 1.7% and 99.0%, and the positive rates of Cx43 protein in HHCC, SMMC-7721 and QZG were 7.3%, 26.5% and 99.1% respectively. Significant differences of both Cx32 and Cx43 protein expression existed between hepatocellular carcinoma cell lines and normal liver cell line (P = 0.0069, HHCC vs QZG; P = 0.0087, SMMC-7721 vs QZG). Moreover, the fluorescent intensities of Cx32 and Cx43 proteins in HHCC, SMMC-7721 were lower than that in QZG. CONCLUSION: Hepatocellular carcinoma cell lines HHCC and SMMC-7721 exhibited lower positive rates and fluorescent intensities of Cx32, Cx43 proteins compared with that of normal liver cell line QZG. It is suggested that lower expression of both Cx32 and Cx43 proteins in hepatocellular carcinoma cells could play pivotal roles in the hepatocarcinogenesis. Besides, genetic defects of cx32 and cx43 in post-translational processing should be considered. PMID:11833073

Downregulation of ROCK2 through nanocomplex sensitizes the cytotoxic effect of temozolomide in U251 glioma cells.

PubMed

Wen, Xiaojun; Huang, Amin; Liu, Zhonglin; Liu, Yunyun; Hu, Jingyang; Liu, Jun; Shuai, Xintao

2014-01-01

Rho-associated coiled-coil kinase 2 (ROCK2) is an attractive therapeutic target because it is overexpressed in many malignancies, including glioma. Therefore, we designed the current study to determine whether the downregulation of ROCK2 would sensitize the cytotoxic effect of temozolomide (TMZ) in U251 cells. Glycol-polyethyleneimine (PEG-PEI) was used to deliver siROCK2 to U251 cells, and the physical characteristics of the PEG-PEI/siROCK2 complex (referred to as the siROCK2 complex) were investigated. The transfection efficiency and cell uptake were determined by flow cytometry (FCM) and confocal laser microscopy (CLSM), respectively. U251 cells were then treated with 100 μM TMZ, siROCK2 complexes or their combination. The apoptosis rate and cell migration were measured by FCM and wound-healing assay, respectively. The levels of Bax, Bcl-2, cleaved caspase-3, MMP-2, and MMP-9 were detected to analyze the degrees of apoptosis and migration. Our results revealed that the characteristics of the siROCK2 complexes depended closely on the N/P ratios. PEG-PEI served as a good vector for siROCK2 and exhibited low cytotoxicity toward U251 cells. The CLSM assay showed that the siROCK2 complexes were successfully uptaken and that both the protein and mRNA levels of ROCK2 were significantly suppressed. Furthermore, the combination treatment induced a higher apoptosis rate and markedly increased the gap distance of U251 cells in the wound-healing assay. Levels of the proapoptotic proteins Bax and cleaved caspase-3 were significantly increased, whereas levels of the antiapoptotic protein Bcl-2 and the migration-related proteins MMP-2 and MMP-9 were significantly reduced by the combination treatment compared with either treatment alone. In conclusion, our results demonstrate that the combination of TMZ and siROCK2 effectively induces apoptosis and inhibits the migration of U251 cells. Therefore, the combination of TMZ and siROCK2 complex is a potential therapeutic approach for human glioma.

Downregulation of ROCK2 through Nanocomplex Sensitizes the Cytotoxic Effect of Temozolomide in U251 Glioma Cells

PubMed Central

Liu, Yunyun; Hu, Jingyang; Liu, Jun; Shuai, Xintao

2014-01-01

Objective Rho-associated coiled-coil kinase 2 (ROCK2) is an attractive therapeutic target because it is overexpressed in many malignancies, including glioma. Therefore, we designed the current study to determine whether the downregulation of ROCK2 would sensitize the cytotoxic effect of temozolomide (TMZ) in U251 cells. Methods Glycol-polyethyleneimine (PEG-PEI) was used to deliver siROCK2 to U251 cells, and the physical characteristics of the PEG-PEI/siROCK2 complex (referred to as the siROCK2 complex) were investigated. The transfection efficiency and cell uptake were determined by flow cytometry (FCM) and confocal laser microscopy (CLSM), respectively. U251 cells were then treated with 100 μM TMZ, siROCK2 complexes or their combination. The apoptosis rate and cell migration were measured by FCM and wound-healing assay, respectively. The levels of Bax, Bcl-2, cleaved caspase-3, MMP-2, and MMP-9 were detected to analyze the degrees of apoptosis and migration. Results Our results revealed that the characteristics of the siROCK2 complexes depended closely on the N/P ratios. PEG-PEI served as a good vector for siROCK2 and exhibited low cytotoxicity toward U251 cells. The CLSM assay showed that the siROCK2 complexes were successfully uptaken and that both the protein and mRNA levels of ROCK2 were significantly suppressed. Furthermore, the combination treatment induced a higher apoptosis rate and markedly increased the gap distance of U251 cells in the wound-healing assay. Levels of the proapoptotic proteins Bax and cleaved caspase-3 were significantly increased, whereas levels of the antiapoptotic protein Bcl-2 and the migration-related proteins MMP-2 and MMP-9 were significantly reduced by the combination treatment compared with either treatment alone. Conclusions In conclusion, our results demonstrate that the combination of TMZ and siROCK2 effectively induces apoptosis and inhibits the migration of U251 cells. Therefore, the combination of TMZ and siROCK2 complex is a potential therapeutic approach for human glioma. PMID:24642531

The Heterotrophic Bacterial Response During the Meso-scale Southern Ocean Iron Experiment (SOFeX)

NASA Astrophysics Data System (ADS)

Oliver, J. L.; Barber, R. T.; Ducklow, H. W.

2002-12-01

Previous meso-scale iron enrichments have demonstrated the stimulatory effect of iron on primary productivity and the accelerated flow of carbon into the surface ocean foodweb. In stratified waters, heterotrophic activity can work against carbon export by remineralizing POC and/or DOC back to CO2, effectively slowing the biological pump. To assess the response of heterotrophic activity to iron enrichment, we measured heterotrophic bacterial production and abundance during the Southern Ocean Iron Experiment (SOFeX). Heterotrophic bacterial processes primarily affect the latter of the two carbon export mechanisms, removal of DOC to the deep ocean. Heterotrophic bacterial production (BP), measured via tritiated thymidine (3H-TdR) and leucine (3H-Leu) incorporation, increased ~40% over the 18-d observation period in iron fertilized waters south of the Polar Front (South Patch). Also, South Patch BP was 61% higher than in the surrounding unfertilized waters. Abundance, measured by flow cytometry (FCM) and acridine orange direct counts (AODC), also increased in the South Patch from 3 to 5 x 108 cells liter-1, a 70% increase. Bacterial biomass increased from ~3.6 to 6.3 μg C liter-1, a clear indication that production rates exceeded removal rates (bactivory, viral lysis) over the course of 18 days. Biomass within the fertilized patch was 11% higher than in surrounding unfertilized waters reflecting a similar trend. This pattern is in contrast to SOIREE where no accumulation of biomass was observed. High DNA-containing (HDNA) cells detected by FCM also increased over time in iron fertilized waters from 20% to 46% relative to the total population suggesting an active subpopulation of cells that were growing faster than the removal rates. In iron fertilized waters north of the Polar Front (North Patch), BP and abundance were ~90% and 80% higher, respectively, than in unfertilized waters. Our results suggest an active bacterial population that responded to iron fertilization by utilizing newly produced DOC and/or iron and which grew at rates that exceeded removal rates. Differences in the microbial response between SOFeX and SOIREE are subtle, and may be related to differences in foodweb structure prior to and during the response to iron enrichment.

In Vivo and In Vitro Detection of Luminescent and Fluorescent Lactobacillus reuteri and Application of Red Fluorescent mCherry for Assessing Plasmid Persistence

PubMed Central

Karimi, Shokoufeh; Ahl, David; Vågesjö, Evelina; Holm, Lena; Phillipson, Mia; Jonsson, Hans; Roos, Stefan

2016-01-01

Lactobacillus reuteri is a symbiont that inhabits the gastrointestinal (GI) tract of mammals, and several strains are used as probiotics. After introduction of probiotic strains in a complex ecosystem like the GI tract, keeping track of them is a challenge. The main objectives of this study were to introduce reporter proteins that would enable in vivo and in vitro detection of L. reuteri and increase knowledge about its interactions with the host. We describe for the first time cloning of codon-optimized reporter genes encoding click beetle red luciferase (CBRluc) and red fluorescent protein mCherry in L. reuteri strains ATCC PTA 6475 and R2LC. The plasmid persistence of mCherry-expressing lactobacilli was evaluated by both flow cytometry (FCM) and conventional plate count (PC), and the plasmid loss rates measured by FCM were lower overall than those determined by PC. Neutralization of pH and longer induction duration significantly improved the mCherry signal. The persistency, dose-dependent signal intensity and localization of the recombinant bacteria in the GI tract of mice were studied with an in vivo imaging system (IVIS), which allowed us to detect fluorescence from 6475-CBRluc-mCherry given at a dose of 1×1010 CFU and luminescence signals at doses ranging from 1×105 to 1×1010 CFU. Both 6475-CBRluc-mCherry and R2LC-CBRluc were localized in the colon 1 and 2 h after ingestion, but the majority of the latter were still found in the stomach, possibly reflecting niche specificity for R2LC. Finally, an in vitro experiment showed that mCherry-producing R2LC adhered efficiently to the intra cellular junctions of cultured IPEC-J2 cells. In conclusion, the two reporter genes CBRluc and mCherry were shown to be suitable markers for biophotonic imaging (BPI) of L. reuteri and may provide useful tools for future studies of in vivo and in vitro interactions between the bacteria and the host. PMID:27002525

Performance of Transuranic-Loaded Fully Ceramic Micro-Encapsulated Fuel in LWRs Final Report, Including Void Reactivity Evaluation

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

Michael A. Pope; R. Sonat Sen; Brian Boer

2011-09-01

The current focus of the Deep Burn Project is on once-through burning of transuranics (TRU) in light-water reactors (LWRs). The fuel form is called Fully-Ceramic Micro-encapsulated (FCM) fuel, a concept that borrows the tri-isotropic (TRISO) fuel particle design from high-temperature reactor technology. In the Deep Burn LWR (DB-LWR) concept, these fuel particles are pressed into compacts using SiC matrix material and loaded into fuel pins for use in conventional LWRs. The TRU loading comes from the spent fuel of a conventional LWR after 5 years of cooling. Unit cell and assembly calculations have been performed using the DRAGON-4 code tomore » assess the physics attributes of TRU-only FCM fuel in an LWR lattice. Depletion calculations assuming an infinite lattice condition were performed with calculations of various reactivity coefficients performed at each step. Unit cells and assemblies containing typical UO2 and mixed oxide (MOX) fuel were analyzed in the same way to provide a baseline against which to compare the TRU-only FCM fuel. Then, assembly calculations were performed evaluating the performance of heterogeneous arrangements of TRU-only FCM fuel pins along with UO2 pins.« less

New proposal of mechanical reinforcement structures to annular REBaCuO bulk magnet for compact and cryogen-free NMR spectrometer

NASA Astrophysics Data System (ADS)

Fujishiro, H.; Takahashi, K.; Naito, T.; Yanagi, Y.; Itoh, Y.; Nakamura, T.

2018-07-01

We have proposed new reinforcement structures using an aluminum alloy ring to the annular REBaCuO bulks applicable to compact and cryogen-free 400 MHz (9.4 T) nuclear magnetic resonance (NMR) spectrometer using a numerical simulation of mechanical stress. The thermal compressive stress, σθcool, which was applied to the annular bulks during cooling due to the difference of thermal expansion coefficient between bulk and aluminum alloy, became fairly enhanced at the surface of the uppermost bulk for the new reinforcement structures, compared to the conventional reinforcement with the same height as the annular bulk, in which the compressive σθcool value was reduced. During field-cooled magnetization (FCM), the electromagnetic hoop stress, σθFCM, became the maximum at the innermost edge of the uppermost ring bulk at intermediate time step. The actual total hoop stress, σθ (= σθcool + σθFCM), due to both cooling and FCM processes was also analyzed and the new ring structures are fairly effective to reduce the σθ value and became lower than the fracture strength of the bulk. The new reinforcement structures have a possibility to avoid the fracture of the bulks and to realize a 400 MHz NMR spectrometer.

Organizing the Cellular and Molecular Heterogeneity in High-Grade Serous Ovarian Cancer by Mass Cytometry

DTIC Science & Technology

2014-10-01

Bendall SC, Sung P, Nolan GP, Arvin AM. Single-cell mass cytometry analysis of human tonsil T cell remodeling by varicella zoster virus. Cell Rep...Perspectives on Flow Cytometry 2013, September 20, 2013, Mass Cytometry and Cell Cycle, Mexico City, Mexico (by Web Conference) Nolan: Nuclear

Electric potential calculation in molecular simulation of electric double layer capacitors

NASA Astrophysics Data System (ADS)

Wang, Zhenxing; Olmsted, David L.; Asta, Mark; Laird, Brian B.

2016-11-01

For the molecular simulation of electric double layer capacitors (EDLCs), a number of methods have been proposed and implemented to determine the one-dimensional electric potential profile between the two electrodes at a fixed potential difference. In this work, we compare several of these methods for a model LiClO4-acetonitrile/graphite EDLC simulated using both the traditional fixed-charged method (FCM), in which a fixed charge is assigned a priori to the electrode atoms, or the recently developed constant potential method (CPM) (2007 J. Chem. Phys. 126 084704), where the electrode charges are allowed to fluctuate to keep the potential fixed. Based on an analysis of the full three-dimensional electric potential field, we suggest a method for determining the averaged one-dimensional electric potential profile that can be applied to both the FCM and CPM simulations. Compared to traditional methods based on numerically solving the one-dimensional Poisson’s equation, this method yields better accuracy and no supplemental assumptions.

A Verification Method for MASOES.

PubMed

Perozo, N; Aguilar Perozo, J; Terán, O; Molina, H

2013-02-01

MASOES is a 3agent architecture for designing and modeling self-organizing and emergent systems. This architecture describes the elements, relationships, and mechanisms, both at the individual and the collective levels, that favor the analysis of the self-organizing and emergent phenomenon without mathematically modeling the system. In this paper, a method is proposed for verifying MASOES from the point of view of design in order to study the self-organizing and emergent behaviors of the modeled systems. The verification criteria are set according to what is proposed in MASOES for modeling self-organizing and emerging systems and the principles of the wisdom of crowd paradigm and the fuzzy cognitive map (FCM) theory. The verification method for MASOES has been implemented in a tool called FCM Designer and has been tested to model a community of free software developers that works under the bazaar style as well as a Wikipedia community in order to study their behavior and determine their self-organizing and emergent capacities.

Theorem Proving In Higher Order Logics

NASA Technical Reports Server (NTRS)

Carreno, Victor A. (Editor); Munoz, Cesar A.; Tahar, Sofiene

2002-01-01

The TPHOLs International Conference serves as a venue for the presentation of work in theorem proving in higher-order logics and related areas in deduction, formal specification, software and hardware verification, and other applications. Fourteen papers were submitted to Track B (Work in Progress), which are included in this volume. Authors of Track B papers gave short introductory talks that were followed by an open poster session. The FCM 2002 Workshop aimed to bring together researchers working on the formalisation of continuous mathematics in theorem proving systems with those needing such libraries for their applications. Many of the major higher order theorem proving systems now have a formalisation of the real numbers and various levels of real analysis support. This work is of interest in a number of application areas, such as formal methods development for hardware and software application and computer supported mathematics. The FCM 2002 consisted of three papers, presented by their authors at the workshop venue, and one invited talk.

Unsupervised fuzzy segmentation of 3D magnetic resonance brain images

NASA Astrophysics Data System (ADS)

Velthuizen, Robert P.; Hall, Lawrence O.; Clarke, Laurence P.; Bensaid, Amine M.; Arrington, J. A.; Silbiger, Martin L.

1993-07-01

Unsupervised fuzzy methods are proposed for segmentation of 3D Magnetic Resonance images of the brain. Fuzzy c-means (FCM) has shown promising results for segmentation of single slices. FCM has been investigated for volume segmentations, both by combining results of single slices and by segmenting the full volume. Different strategies and initializations have been tried. In particular, two approaches have been used: (1) a method by which, iteratively, the furthest sample is split off to form a new cluster center, and (2) the traditional FCM in which the membership grade matrix is initialized in some way. Results have been compared with volume segmentations by k-means and with two supervised methods, k-nearest neighbors and region growing. Results of individual segmentations are presented as well as comparisons on the application of the different methods to a number of tumor patient data sets.

A robust fuzzy local Information c-means clustering algorithm with noise detection

NASA Astrophysics Data System (ADS)

Shang, Jiayu; Li, Shiren; Huang, Junwei

2018-04-01

Fuzzy c-means clustering (FCM), especially with spatial constraints (FCM_S), is an effective algorithm suitable for image segmentation. Its reliability contributes not only to the presentation of fuzziness for belongingness of every pixel but also to exploitation of spatial contextual information. But these algorithms still remain some problems when processing the image with noise, they are sensitive to the parameters which have to be tuned according to prior knowledge of the noise. In this paper, we propose a new FCM algorithm, combining the gray constraints and spatial constraints, called spatial and gray-level denoised fuzzy c-means (SGDFCM) algorithm. This new algorithm conquers the parameter disadvantages mentioned above by considering the possibility of noise of each pixel, which aims to improve the robustness and obtain more detail information. Furthermore, the possibility of noise can be calculated in advance, which means the algorithm is effective and efficient.

Hyperchromatic laser scanning cytometry

NASA Astrophysics Data System (ADS)

Tárnok, Attila; Mittag, Anja

2007-02-01

In the emerging fields of high-content and high-throughput single cell analysis for Systems Biology and Cytomics multi- and polychromatic analysis of biological specimens has become increasingly important. Combining different technologies and staining methods polychromatic analysis (i.e. using 8 or more fluorescent colors at a time) can be pushed forward to measure anything stainable in a cell, an approach termed hyperchromatic cytometry. For cytometric cell analysis microscope based Slide Based Cytometry (SBC) technologies are ideal as, unlike flow cytometry, they are non-consumptive, i.e. the analyzed sample is fixed on the slide. Based on the feature of relocation identical cells can be subsequently reanalyzed. In this manner data on the single cell level after manipulation steps can be collected. In this overview various components for hyperchromatic cytometry are demonstrated for a SBC instrument, the Laser Scanning Cytometer (Compucyte Corp., Cambridge, MA): 1) polychromatic cytometry, 2) iterative restaining (using the same fluorochrome for restaining and subsequent reanalysis), 3) differential photobleaching (differentiating fluorochromes by their different photostability), 4) photoactivation (activating fluorescent nanoparticles or photocaged dyes), and 5) photodestruction (destruction of FRET dyes). With the intelligent combination of several of these techniques hyperchromatic cytometry allows to quantify and analyze virtually all components of relevance on the identical cell. The combination of high-throughput and high-content SBC analysis with high-resolution confocal imaging allows clear verification of phenotypically distinct subpopulations of cells with structural information. The information gained per specimen is only limited by the number of available antibodies and by sterical hindrance.

An Intelligent Hierarchical Decision Architecture for Operational Test and Evaluation

DTIC Science & Technology

1996-05-01

Results .......................................... 60 3.4 CONTRIBUTION...FCM Fuzzy Cognitive Map FMEA Failure Modes and Effects Analysis HWIL Hardware-in-the-Loop IBL Increase in Break Locks xiv IDA Institute for Defense... 60 .5 .40 3 .25 0.21 0.26 Figure 8 PROD-ALL COMMFFY Compositional Method .65 . 7 5 . 60 M 0.67 Figure 9 PROD-MAX COMMFFY Compositional Method 49

Design of hybrid radial basis function neural networks (HRBFNNs) realized with the aid of hybridization of fuzzy clustering method (FCM) and polynomial neural networks (PNNs).

PubMed

Huang, Wei; Oh, Sung-Kwun; Pedrycz, Witold

2014-12-01

In this study, we propose Hybrid Radial Basis Function Neural Networks (HRBFNNs) realized with the aid of fuzzy clustering method (Fuzzy C-Means, FCM) and polynomial neural networks. Fuzzy clustering used to form information granulation is employed to overcome a possible curse of dimensionality, while the polynomial neural network is utilized to build local models. Furthermore, genetic algorithm (GA) is exploited here to optimize the essential design parameters of the model (including fuzzification coefficient, the number of input polynomial fuzzy neurons (PFNs), and a collection of the specific subset of input PFNs) of the network. To reduce dimensionality of the input space, principal component analysis (PCA) is considered as a sound preprocessing vehicle. The performance of the HRBFNNs is quantified through a series of experiments, in which we use several modeling benchmarks of different levels of complexity (different number of input variables and the number of available data). A comparative analysis reveals that the proposed HRBFNNs exhibit higher accuracy in comparison to the accuracy produced by some models reported previously in the literature. Copyright © 2014 Elsevier Ltd. All rights reserved.

Alternatives to current flow cytometry data analysis for clinical and research studies.

PubMed

Gondhalekar, Carmen; Rajwa, Bartek; Patsekin, Valery; Ragheb, Kathy; Sturgis, Jennifer; Robinson, J Paul

2018-02-01

Flow cytometry has well-established methods for data analysis based on traditional data collection techniques. These techniques typically involved manual insertion of tube samples into an instrument that, historically, could only measure 1-3 colors. The field has since evolved to incorporate new technologies for faster and highly automated sample preparation and data collection. For example, the use of microwell plates on benchtop instruments is now a standard on virtually every new instrument, and so users can easily accumulate multiple data sets quickly. Further, because the user must carefully define the layout of the plate, this information is already defined when considering the analytical process, expanding the opportunities for automated analysis. Advances in multi-parametric data collection, as demonstrated by the development of hyperspectral flow-cytometry, 20-40 color polychromatic flow cytometry, and mass cytometry (CyTOF), are game-changing. As data and assay complexity increase, so too does the complexity of data analysis. Complex data analysis is already a challenge to traditional flow cytometry software. New methods for reviewing large and complex data sets can provide rapid insight into processes difficult to define without more advanced analytical tools. In settings such as clinical labs where rapid and accurate data analysis is a priority, rapid, efficient and intuitive software is needed. This paper outlines opportunities for analysis of complex data sets using examples of multiplexed bead-based assays, drug screens and cell cycle analysis - any of which could become integrated into the clinical environment. Copyright © 2017. Published by Elsevier Inc.

OpenCyto: An Open Source Infrastructure for Scalable, Robust, Reproducible, and Automated, End-to-End Flow Cytometry Data Analysis

PubMed Central

Finak, Greg; Frelinger, Jacob; Jiang, Wenxin; Newell, Evan W.; Ramey, John; Davis, Mark M.; Kalams, Spyros A.; De Rosa, Stephen C.; Gottardo, Raphael

2014-01-01

Flow cytometry is used increasingly in clinical research for cancer, immunology and vaccines. Technological advances in cytometry instrumentation are increasing the size and dimensionality of data sets, posing a challenge for traditional data management and analysis. Automated analysis methods, despite a general consensus of their importance to the future of the field, have been slow to gain widespread adoption. Here we present OpenCyto, a new BioConductor infrastructure and data analysis framework designed to lower the barrier of entry to automated flow data analysis algorithms by addressing key areas that we believe have held back wider adoption of automated approaches. OpenCyto supports end-to-end data analysis that is robust and reproducible while generating results that are easy to interpret. We have improved the existing, widely used core BioConductor flow cytometry infrastructure by allowing analysis to scale in a memory efficient manner to the large flow data sets that arise in clinical trials, and integrating domain-specific knowledge as part of the pipeline through the hierarchical relationships among cell populations. Pipelines are defined through a text-based csv file, limiting the need to write data-specific code, and are data agnostic to simplify repetitive analysis for core facilities. We demonstrate how to analyze two large cytometry data sets: an intracellular cytokine staining (ICS) data set from a published HIV vaccine trial focused on detecting rare, antigen-specific T-cell populations, where we identify a new subset of CD8 T-cells with a vaccine-regimen specific response that could not be identified through manual analysis, and a CyTOF T-cell phenotyping data set where a large staining panel and many cell populations are a challenge for traditional analysis. The substantial improvements to the core BioConductor flow cytometry packages give OpenCyto the potential for wide adoption. It can rapidly leverage new developments in computational cytometry and facilitate reproducible analysis in a unified environment. PMID:25167361

OpenCyto: an open source infrastructure for scalable, robust, reproducible, and automated, end-to-end flow cytometry data analysis.

PubMed

Finak, Greg; Frelinger, Jacob; Jiang, Wenxin; Newell, Evan W; Ramey, John; Davis, Mark M; Kalams, Spyros A; De Rosa, Stephen C; Gottardo, Raphael

2014-08-01

Flow cytometry is used increasingly in clinical research for cancer, immunology and vaccines. Technological advances in cytometry instrumentation are increasing the size and dimensionality of data sets, posing a challenge for traditional data management and analysis. Automated analysis methods, despite a general consensus of their importance to the future of the field, have been slow to gain widespread adoption. Here we present OpenCyto, a new BioConductor infrastructure and data analysis framework designed to lower the barrier of entry to automated flow data analysis algorithms by addressing key areas that we believe have held back wider adoption of automated approaches. OpenCyto supports end-to-end data analysis that is robust and reproducible while generating results that are easy to interpret. We have improved the existing, widely used core BioConductor flow cytometry infrastructure by allowing analysis to scale in a memory efficient manner to the large flow data sets that arise in clinical trials, and integrating domain-specific knowledge as part of the pipeline through the hierarchical relationships among cell populations. Pipelines are defined through a text-based csv file, limiting the need to write data-specific code, and are data agnostic to simplify repetitive analysis for core facilities. We demonstrate how to analyze two large cytometry data sets: an intracellular cytokine staining (ICS) data set from a published HIV vaccine trial focused on detecting rare, antigen-specific T-cell populations, where we identify a new subset of CD8 T-cells with a vaccine-regimen specific response that could not be identified through manual analysis, and a CyTOF T-cell phenotyping data set where a large staining panel and many cell populations are a challenge for traditional analysis. The substantial improvements to the core BioConductor flow cytometry packages give OpenCyto the potential for wide adoption. It can rapidly leverage new developments in computational cytometry and facilitate reproducible analysis in a unified environment.

Imaging flow cytometry for phytoplankton analysis.

PubMed

Dashkova, Veronika; Malashenkov, Dmitry; Poulton, Nicole; Vorobjev, Ivan; Barteneva, Natasha S

2017-01-01

This review highlights the concepts and instrumentation of imaging flow cytometry technology and in particular its use for phytoplankton analysis. Imaging flow cytometry, a hybrid technology combining speed and statistical capabilities of flow cytometry with imaging features of microscopy, is rapidly advancing as a cell imaging platform that overcomes many of the limitations of current techniques and contributed significantly to the advancement of phytoplankton analysis in recent years. This review presents the various instrumentation relevant to the field and currently used for assessment of complex phytoplankton communities' composition and abundance, size structure determination, biovolume estimation, detection of harmful algal bloom species, evaluation of viability and metabolic activity and other applications. Also we present our data on viability and metabolic assessment of Aphanizomenon sp. cyanobacteria using Imagestream X Mark II imaging cytometer. Herein, we highlight the immense potential of imaging flow cytometry for microalgal research, but also discuss limitations and future developments. Copyright © 2016 Elsevier Inc. All rights reserved.

Flow cytometry: basic principles and applications.

PubMed

Adan, Aysun; Alizada, Günel; Kiraz, Yağmur; Baran, Yusuf; Nalbant, Ayten

2017-03-01

Flow cytometry is a sophisticated instrument measuring multiple physical characteristics of a single cell such as size and granularity simultaneously as the cell flows in suspension through a measuring device. Its working depends on the light scattering features of the cells under investigation, which may be derived from dyes or monoclonal antibodies targeting either extracellular molecules located on the surface or intracellular molecules inside the cell. This approach makes flow cytometry a powerful tool for detailed analysis of complex populations in a short period of time. This review covers the general principles and selected applications of flow cytometry such as immunophenotyping of peripheral blood cells, analysis of apoptosis and detection of cytokines. Additionally, this report provides a basic understanding of flow cytometry technology essential for all users as well as the methods used to analyze and interpret the data. Moreover, recent progresses in flow cytometry have been discussed in order to give an opinion about the future importance of this technology.

A Self-Adaptive Fuzzy c-Means Algorithm for Determining the Optimal Number of Clusters

PubMed Central

Wang, Zhihao; Yi, Jing

2016-01-01

For the shortcoming of fuzzy c-means algorithm (FCM) needing to know the number of clusters in advance, this paper proposed a new self-adaptive method to determine the optimal number of clusters. Firstly, a density-based algorithm was put forward. The algorithm, according to the characteristics of the dataset, automatically determined the possible maximum number of clusters instead of using the empirical rule n and obtained the optimal initial cluster centroids, improving the limitation of FCM that randomly selected cluster centroids lead the convergence result to the local minimum. Secondly, this paper, by introducing a penalty function, proposed a new fuzzy clustering validity index based on fuzzy compactness and separation, which ensured that when the number of clusters verged on that of objects in the dataset, the value of clustering validity index did not monotonically decrease and was close to zero, so that the optimal number of clusters lost robustness and decision function. Then, based on these studies, a self-adaptive FCM algorithm was put forward to estimate the optimal number of clusters by the iterative trial-and-error process. At last, experiments were done on the UCI, KDD Cup 1999, and synthetic datasets, which showed that the method not only effectively determined the optimal number of clusters, but also reduced the iteration of FCM with the stable clustering result. PMID:28042291

Long-term safety and outcome of fludarabine, cyclophosphamide and mitoxantrone (FCM) regimen in previously untreated patients with advanced follicular lymphoma: 12 years follow-up of a phase 2 trial.

PubMed

Magnano, Laura; Montoto, Silvia; González-Barca, Eva; Briones, Javier; Sancho, Juan Manuel; Muntañola, Ana; Salar, Antonio; Besalduch, Joan; Escoda, Lourdes; Moreno, Carol; Domingo-Domenech, Eva; Estany, Cristina; Oriol, Albert; Altés, Albert; Pedro, Carmen; Gardella, Santiago; Asensio, Antoni; Vivancos, Pilar; Fernández de Sevilla, Alberto; Ribera, Josep María; Colomer, Dolors; Campo, Elias; López-Guillermo, Armando

2017-04-01

Fludarabine combinations are very affective in follicular lymphoma (FL) with high rates of complete response and prolonged survival. However, late toxicities could be a concern. The aim of the present study was to analyze the long-term impact on survival, relapse and late toxicities of a trial of treatment with fludarabine, mitoxantrone and cyclophosphamide (FCM regimen) for untreated patients with advanced stage FL. One hundred and twenty patients enrolled in a phase 2 trial of treatment with FCM regimen between 2000 and 2003 were evaluated. After a median follow-up of 12 years, 52 patients eventually relapsed/progressed with 10 year progression-free survival (PFS) of 46 %. Ten patients showed histological transformation to aggressive lymphoma with a risk of transformation of 2 and 9 % at 5 and 10 years, respectively. Three patients developed therapy-related myelodysplastic syndrome/acute myeloid leukaemia (MDS/AML) and seven solid neoplasms with an overall risk of 3 and 8 % at 5 and 10 years, respectively. Twenty-six patients eventually died during the follow-up. Overall survival at 10 years was 83 %. In conclusion, FCM regimen allows excellent long-lasting response in previously untreated patients with FL. The incidence of late events including histological transformation and secondary neoplasia is low but not negligible.

Microfluidic Impedance Flow Cytometry Enabling High-Throughput Single-Cell Electrical Property Characterization

PubMed Central

Chen, Jian; Xue, Chengcheng; Zhao, Yang; Chen, Deyong; Wu, Min-Hsien; Wang, Junbo

2015-01-01

This article reviews recent developments in microfluidic impedance flow cytometry for high-throughput electrical property characterization of single cells. Four major perspectives of microfluidic impedance flow cytometry for single-cell characterization are included in this review: (1) early developments of microfluidic impedance flow cytometry for single-cell electrical property characterization; (2) microfluidic impedance flow cytometry with enhanced sensitivity; (3) microfluidic impedance and optical flow cytometry for single-cell analysis and (4) integrated point of care system based on microfluidic impedance flow cytometry. We examine the advantages and limitations of each technique and discuss future research opportunities from the perspectives of both technical innovation and clinical applications. PMID:25938973

Flow cytometry shows added value in diagnosing lymphoma in brain biopsies.

PubMed

van der Meulen, Matthijs; Bromberg, Jacoline E C; Lam, King H; Dammers, Ruben; Langerak, Anton W; Doorduijn, Jeanette K; Kros, Johan M; van den Bent, Martin J; van der Velden, Vincent H J

2018-05-10

To assess the sensitivity, specificity and turnaround time of flow cytometric analysis on brain biopsies compared to histology plus immunohistochemistry analysis in tumors with clinical suspicion of lymphoma. All brain biopsies performed between 2010 and 2015 at our institution and analyzed by both immunohistochemistry and flow cytometry were included in this retrospective study. Immunohistochemistry was considered the gold standard. In a total of 77 biopsies from 71 patients, 49 lymphomas were diagnosed by immunohistochemistry, flow cytometry results were concordant in 71 biopsies (92,2%). We found a specificity and sensitivity of flow cytometry of 100% and 87,8%, respectively. The time between the biopsy and reporting the result (turnaround time) was significantly shorter for flow cytometry, compared to immunohistochemistry (median: 1 versus 5 days). Flow cytometry has a high specificity and can confirm the diagnosis of a lymphoma significantly faster than immunohistochemistry. This allows for rapid initiation of treatment in this highly aggressive tumor. However, since its sensitivity is less than 100%, we recommend to perform histology plus immunohistochemistry in parallel to flow cytometry. This article is protected by copyright. All rights reserved. © 2018 International Clinical Cytometry Society.

Constructing Ultrahigh-Capacity Zinc-Nickel-Cobalt Oxide@Ni(OH)2 Core-Shell Nanowire Arrays for High-Performance Coaxial Fiber-Shaped Asymmetric Supercapacitors.

PubMed

Zhang, Qichong; Xu, Weiwei; Sun, Juan; Pan, Zhenghui; Zhao, Jingxin; Wang, Xiaona; Zhang, Jun; Man, Ping; Guo, Jiabin; Zhou, Zhenyu; He, Bing; Zhang, Zengxing; Li, Qingwen; Zhang, Yuegang; Xu, Lai; Yao, Yagang

2017-12-13

Increased efforts have recently been devoted to developing high-energy-density flexible supercapacitors for their practical applications in portable and wearable electronics. Although high operating voltages have been achieved in fiber-shaped asymmetric supercapacitors (FASCs), low specific capacitance still restricts the further enhancement of their energy density. This article specifies a facile and cost-effective method to directly grow three-dimensionally well-aligned zinc-nickel-cobalt oxide (ZNCO)@Ni(OH) 2 nanowire arrays (NWAs) on a carbon nanotube fiber (CNTF) with an ultrahigh specific capacitance of 2847.5 F/cm 3 (10.678 F/cm 2 ) at a current density of 1 mA/cm 2 , These levels are approximately five times higher than those of ZNCO NWAs/CNTF electrodes (2.10 F/cm 2 ) and four times higher than Ni(OH) 2 /CNTF electrodes (2.55 F/cm 2 ). Benefiting from their unique features, we successfully fabricated a prototype coaxial FASC (CFASC) with a maximum operating voltage of 1.6 V, which was assembled by adopting ZNCO@Ni(OH) 2 NWAs/CNTF as the core electrode and a thin layer of carbon coated vanadium nitride (VN@C) NWAs on a carbon nanotube strip (CNTS) as the outer electrode with KOH poly(vinyl alcohol) (PVA) as the gel electrolyte. A high specific capacitance of 94.67 F/cm 3 (573.75 mF/cm 2 ) and an exceptional energy density of 33.66 mWh/cm 3 (204.02 μWh/cm 2 ) were achieved for our CFASC device, which represent the highest levels of fiber-shaped supercapacitors to date. More importantly, the fiber-shaped ZnO-based photodetector is powered by the integrated CFASC, and it demonstrates excellent sensitivity in detecting UV light. Thus, this work paves the way to the construction of ultrahigh-capacity electrode materials for next-generation wearable energy-storage devices.

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

Effects of allicin on both telomerase activity and apoptosis in gastric cancer SGC-7901 cells.

PubMed

Sun, Li; Wang, Xu

2003-09-01

To investigate the effects of allicin on both telomerase activity and apoptosis in gastric cancer SGC-7901 cells. The gastric cancer SGC-7901 adenocarcinoma cells were treated with allicin and the cell cycle, inhibitory rate, apoptosis, telomerase activity and morphologic changes were studied by MTT assay, flow cytometry (FCM), TRAP-PCR-ELISA assay, light microscope, electron microscope respectively. Results were compared with that of AZT (3'-Azido-3'-deoxythymidine). SGC-7901 cells were suppressed after exposure to allicin of 0.016 mg/ml, 0.05 mg/ml, and 0.1 mg/ml for 48 h. Compared with the control, the difference was significant (P<0.05). Allicin could induce apoptosis of the cells in a dose-dependent and non-linear manner and increase the proportion of cells in the G(2)/M phase. Compared with the control, the difference was significant in terms of the percentage of cells in the G2/M phase (P<0.05). Allicin could inhibit telomerase activity in a time-dependent and dose-dependent pattern. After exposure to allicin at 0.016 mg/ml for 24 hours, SGC-7901 cells showed typical morphologic change. Allicin can inhibit telomerase activity and induce apoptosis of gastric cancer SGC-7901 cells. Allicin may be more effective than AZT.

Toxic effect of NiCl2 on development of the bursa of Fabricius in broiler chickens

PubMed Central

Yin, Shuang; Cui, Hengmin; Peng, Xi; Fang, Jing; Zuo, Zhicai; Deng, Junliang; Wang, Xun; Wu, Bangyuan; Guo, Hongrui

2016-01-01

This study was conducted with objective of evaluating the toxic effects of nickel chloride (NiCl2) on development of bursa of Fabricius in broilers fed on diets supplemented with 0, 300, 600 and 900 mg/kg of NiCl2 for 42 days by using the methods of experimental pathology, flow cytometry (FCM), and quantitative real-time polymerase chain reaction (qRT-PCR). The results showed that dietary NiCl2 in 300 mg/kg and over induced toxic suppression in the bursal development, which was characterized by decreasing lymphocytes histopathologically and relative weight, increasing G0/G1 phase (a prolonged nondividing state), reducing S phase (DNA replication) and proliferating index, and increasing percentages of apoptotic cells. Concurrently, the mRNA expression levels of bax, cytochrome c (cyt c), apoptotic peptidase activating factor 1 (Apaf-1), caspase-3, caspase-6, caspase-7 and caspase-9 were increased and the bcl-2 mRNA expression levels were decreased. The toxic suppression of bursal development finally impaired humoral immunity duo to the reduction of B lymphocyte population and B lymphocyte activity in the broiler chicken. This study provides new evidences for further studying the effect mechanism of Ni and Ni compoundson B-cell or bursa of Fabricius. PMID:26683707

PCV2 vaccination induces IFN-γ/TNF-α co-producing T cells with a potential role in protection.

PubMed

Koinig, Hanna C; Talker, Stephanie C; Stadler, Maria; Ladinig, Andrea; Graage, Robert; Ritzmann, Mathias; Hennig-Pauka, Isabel; Gerner, Wilhelm; Saalmüller, Armin

2015-03-03

Porcine circovirus type 2 (PCV2) is one of the economically most important pathogens for swine production worldwide. Vaccination is a powerful tool to control porcine circovirus diseases (PCVD). However, it is not fully understood how PCV2 vaccination interacts with the porcine immune system. Especially knowledge on the cellular immune response against PCV2 is sparse. In this study we analysed antigen-specific T cell responses against PCV2 in a controlled vaccination and infection experiment. We focused on the ability of CD4(+) T cells to produce cytokines using multicolour flow cytometry (FCM). Vaccination with a PCV2 subunit vaccine (Ingelvac CircoFLEX®) induced PCV2-specific antibodies only in five out of 12 animals. Conversely, vaccine-antigen specific CD4(+) T cells which simultaneously produced IFN-γ and TNF-α and had a phenotype of central and effector memory T cells were detected in all vaccinated piglets. After challenge, seroconversion occurred earlier in vaccinated and infected pigs compared to the non-vaccinated, infected group. Vaccinated pigs were fully protected against viremia after subsequent challenge. Therefore, our data suggests that the induction of IFN-γ/TNF-α co-producing T cells by PCV2 vaccination may serve as a potential correlate of protection for this type of vaccine.

Production a monoclonal antibody specific to granulocytes of swimming crab (Portunus trituberculatus) and its cross reactivity with other crustaceans.

PubMed

Cheng, Shun-Feng; Wu, Xiao-Chun; Zhang, Min

2016-10-01

In this study, a monoclonal antibody (mAb) 3F4 specific to granulocytes of swimming crab, Portunus trituberculatus, was obtained by immunizing mice with whole haemocytes. mAb 3F4 showed strong immunofluorescent reaction with granulocytes, but no reaction with hyalinocytes. The positive cell percentage of granulocytes was 86.3% detected by Flow cytometry (FCM). A special antigen with molecular weight of about 26kDa was further recognized by mAb 3F4 in haemocytes of P. trituberculatus. mAb 3F4 also showed strong cross-reactivity with haemocytes of Eriocheir sinensis and Petalomera japonica, but no reaction with other crustaceans tested. In E. sinensis, the positive cell percentage was 73.4% for granulocytes and 59.8% for hyalinocytes; while in P. japonica, the positive cell percentage was 81.2% for granulocytes and 7.1% for hyalinocytes. There was also a special antigen with molecular weight of about 31kDa identified by mAb 3F4 in haemocytes of E.sinensis, but no corresponding protein band in P. japonica haemocytes. These results demonstrated that mAb 3F4 can be used as a marker for granulocytes of crabs. Copyright © 2016 Elsevier B.V. All rights reserved.

Garlic-derived compound S-allylmercaptocysteine (SAMC) is active against anaplastic thyroid cancer cell line 8305C (HPACC).

PubMed

Liu, Yuexin; Yan, Jinyin; Han, Xiaochen; Hu, Wanning

2015-01-01

Epidemiological and experimental carcinogenesis studies provide evidence that components of garlic have anticancer activity. In this study, the apoptotic effects of Garlic-derived compound S-allylmercaptocysteine (SAMC) were investigated in 8305C human anaplastic thyroid carcinoma cells. The cell line 8305C (HPACC) were treated with SAMC and the MTT assay, flow cytometry (FCM), electron microscope method were used to test cell cycle, inhibitory rate and morphologic changes respectively. HPACC-8305C cells were suppressed after exposure to SAMC of 0.02 mg/ml, 0.06 mg/ml, and 0.1 mg/ml for 48 h. Compared with the control, the difference was significant (P< 0.05). SAMC could induce apoptosis of the cells in a dose-dependent and non-linear manner and increase the proportion of cells in the G2/M phase. Compared with the control, the difference was significant in terms of the percentage of cells in the G2/M phase (P< 0.05). After exposure to SAMC at 0.02 mg/ml for 24 hours, HPACC-8305C cells showed typical morphologic change. SAMC inhibits the growth of HPACC-8305C cells by induction of apoptotic cell death and inhibit telomerase activity, which appears to account for its anti-cancer activity.

Quercetin conjugated with silica nanoparticles inhibits tumor growth in MCF-7 breast cancer cell lines.

PubMed

Aghapour, Fahimeh; Moghadamnia, Ali Akbar; Nicolini, Andrea; Kani, Seydeh Narges Mousavi; Barari, Ladan; Morakabati, Payam; Rezazadeh, Leyla; Kazemi, Sohrab

2018-06-12

Quercetin is a plant polyphenol from the flavonoid group that plays a fundamental role in controlling homeostasis due to its potent antioxidant properties. However, quercetin has extremely low water solubility, which is a major challenge in drug absorption. In this study, we described a simple method for the synthesis of quercetin nanoparticles. The quercetin nanoparticles had an average diameter of 82 nm and prominent yellow emission under UV irradiation. Therefore, we used an in vitro model treated with quercetin and quercetin nanoparticles to investigate the effects of quercetin nanoparticles on MCF-7 breast cancer cell line. MCF-7 cells were cultured with different concentrations (1-100 μM) of quercetin nanoparticles at the 24th, 48th and 72 nd hours, and cell cycle and apoptosis assays were detected by flow cytometry (FCM). In this study, we found that quercetin nanoparticles (1-100 μM) could significantly reduce cell vitality, growth rate and colony formation of MCF-7 cells. Quercetin nanoparticles can inhibit cell growth by blocking the cell cycle and promoting apoptosis in MCF-7 cells more than quercetin. As a result, quercetin nanoparticles may be useful therapy or prevention on breast cancer. Copyright © 2018 Elsevier Inc. All rights reserved.

[The attitudes and behavior of the general primary-care physician towards the neurological patient].

PubMed

Casabella Abril, B; Pérez Sánchez, J

1995-04-15

1) To find the opinion of general practitioners working in primary care (GP in PC) regarding how they deal with neurological patients. 2) To find the effect on this question of intern training in family and community medicine (FCM). A survey filled out by a representative sample of GP in PC working at PC public clinics in 1991 in a health region in Catalonia. 56 GP in PC. A self-administered selection questionnaire (multiple choice and scale of 5 points). MEASUREMENTS, MAIN RESULTS AND CONCLUSIONS: Less confidence handling neurological patients than patients with other common medical conditions. Greater need for recycling in neurology than in other basic areas of medicine. Positive impact of FCM intern training on doctors' approach to the examination of neurological patients and application of basic exploratory techniques (ophthalmoscope, reflex hammer, diapason and phonendoscope). The GP intern-trained in FCM lacks confidence in present out-patient specialised support (the area neuropsychiatrist).

The Semen pH Affects Sperm Motility and Capacitation.

PubMed

Zhou, Ji; Chen, Li; Li, Jie; Li, Hongjun; Hong, Zhiwei; Xie, Min; Chen, Shengrong; Yao, Bing

2015-01-01

As the chemical environment of semen can have a profound effect on sperm quality, we examined the effect of pH on the motility, viability and capacitation of human sperm. The sperm in this study was collected from healthy males to avoid interference from other factors. The spermatozoa cultured in sperm nutrition solution at pH 5.2, 6.2, 7.2 and 8.2 were analyzed for sperm total motility, progressive motility (PR), hypo-osmotic swelling (HOS) rate, and sperm penetration. Our results showed that these parameters were similar in pH 7.2 and 8.2 sperm nutrition solutions, but decreased in pH 5.2 and 6.2 solutions. The HOS rate exhibited positive correlation with the sperm total motility and PR. In addition, the sperm Na(+)/K(+)-ATPase activity at different pHs was measured, and the enzyme activity was significantly lower in pH 5.2 and 6.2 media, comparing with that in pH 8.2 and pH 7.2 solutions. Using flow cytometry (FCM) and laser confocal scanning microscopy (LCSM) analysis, the intracellular Ca2(+ )concentrations of sperm cultured in sperm capacitation solution at pH 5.2, 6.2, 7.2 and 8.2 were determined. Compared with that at pH 7.2, the mean fluorescence intensity of sperm in pH 5.2 and 6.2 media decreased significantly, while that of pH 8.2 group showed no difference. Our results suggested that the declined Na(+)/K(+)-ATPase activity at acidic pHs result in decreased sperm movement and capacitation, which could be one of the mechanisms of male infertility.

Cross-species antiviral activity of goose interferon lambda against duck plague virus is related to its positive self-regulatory feedback loop.

PubMed

Chen, Shun; Zhang, Wei; Zhou, Qin; Wang, Anqi; Sun, Lipei; Wang, Mingshu; Jia, Renyong; Zhu, Dekang; Liu, Mafeng; Sun, Kunfeng; Yang, Qiao; Wu, Ying; Chen, Xiaoyue; Cheng, Anchun

2017-06-01

Duck plague virus (DPV) is a virus of the Herpesviridae family that leads to acute disease with a high mortality rate in ducks. Control of the disease contributes to the development of poultry breeding. Type III IFN family (IFN-λs) is a novel member of the IFN family, and goose IFN-λ (goIFN-λ) is a newly identified gene whose antiviral function has only been investigated to a limited extent. Here, the cross-species antiviral activity of goIFN-λ against DPV in duck embryo fibroblasts (DEFs) was studied. We found that pre-treatment with goIFN-λ greatly increased the expression of IFN-λ in both heterologous DEFs and homologous goose embryo fibroblasts (GEFs), while differentially inducing IFNα- and IFN-stimulated genes. Additionally, a positive self-regulatory feedback loop of goIFN-λ was blocked by a mouse anti-goIFN-λ polyclonal antibody, which was confirmed in both homologous GEFs and goose peripheral blood mononuclear cells (PBMCs). The suppression of the BAC-DPV-EGFP by goIFN-λ in DEFs was confirmed by fluorescence microscopy, flow cytometry (FCM) analysis, viral copies and titre detection, which can be rescued by mouse anti-goIFN-λ polyclonal antibody incubation. Finally, reporter gene assays indicated that the cross-species antiviral activity of goIFN-λ against BAC-DPV-EGFP is related to its positive self-regulatory feedback loop and subsequent ISG induction. Our data shed light on the fundamental mechanisms of goIFN-λ antiviral function in vitro and extend the considerable range of therapeutic applications in multiple-poultry disease.

Evaluating Monitoring Strategies to Detect Precipitation-Induced Microbial Contamination Events in Karstic Springs Used for Drinking Water

PubMed Central

Besmer, Michael D.; Hammes, Frederik; Sigrist, Jürg A.; Ort, Christoph

2017-01-01

Monitoring of microbial drinking water quality is a key component for ensuring safety and understanding risk, but conventional monitoring strategies are typically based on low sampling frequencies (e.g., quarterly or monthly). This is of concern because many drinking water sources, such as karstic springs are often subject to changes in bacterial concentrations on much shorter time scales (e.g., hours to days), for example after precipitation events. Microbial contamination events are crucial from a risk assessment perspective and should therefore be targeted by monitoring strategies to establish both the frequency of their occurrence and the magnitude of bacterial peak concentrations. In this study we used monitoring data from two specific karstic springs. We assessed the performance of conventional monitoring based on historical records and tested a number of alternative strategies based on a high-resolution data set of bacterial concentrations in spring water collected with online flow cytometry (FCM). We quantified the effect of increasing sampling frequency and found that for the specific case studied, at least bi-weekly sampling would be needed to detect precipitation events with a probability of >90%. We then proposed an optimized monitoring strategy with three targeted samples per event, triggered by precipitation measurements. This approach is more effective and efficient than simply increasing overall sampling frequency. It would enable the water utility to (1) analyze any relevant event and (2) limit median underestimation of peak concentrations to approximately 10%. We conclude with a generalized perspective on sampling optimization and argue that the assessment of short-term dynamics causing microbial peak loads initially requires increased sampling/analysis efforts, but can be optimized subsequently to account for limited resources. This offers water utilities and public health authorities systematic ways to evaluate and optimize their current monitoring strategies. PMID:29213255

Different Roles of GRP78 on Cell Proliferation and Apoptosis in Cartilage Development.

PubMed

Xiong, Zhangyuan; Jiang, Rong; Li, Xiangzhu; Liu, Yanna; Guo, Fengjin

2015-09-07

Eukaryotic cells possess several mechanisms to adapt to endoplasmic reticulum (ER) stress and thereby survive. ER stress activates a set of signaling pathways collectively termed as the unfolded protein response (UPR). We previously reported that Bone morphogenetic protein 2 (BMP2) mediates mild ER stress and activates UPR signal molecules in chondrogenesis. The mammalian UPR protects the cell against the stress of misfolded proteins in the endoplasmic reticulum. Failure to adapt to ER stress causes the UPR to trigger apoptosis. Glucose regulated protein 78 (GRP78), as an important molecular chaperone in UPR signaling pathways, is responsible for binding to misfolded or unfolded protein during ER stress. However the influence on GRP78 in BMP2-induced chondrocyte differentiation has not yet been elucidated and the molecular mechanism underlyng these processes remain unexplored. Herein we demonstrate that overexpression of GRP78 enhanced cell proliferation in chondrocyte development with G1 phase advance, S phase increasing and G2-M phase transition. Furthermore, overexpression of GRP78 inhibited ER stress-mediated apoptosis and then reduced apoptosis in chondrogenesis induced by BMP2, as assayed by cleaved caspase3, caspase12, C/EBP homologous protein (CHOP/DDIT3/GADD153), p-JNK (phosphorylated c-Jun N-terminal kinase) expression during the course of chondrocyte differentiation by Western blot. In addition, flow cytometry (FCM) assay, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling (TUNEL) assay and immune-histochemistry analysis also proved this result in vitro and in vivo. It was demonstrated that GRP78 knockdown via siRNA activated the ER stress-specific caspase cascade in developing chondrocyte tissue. Collectively, these findings reveal a novel critical role of GRP78 in regulating ER stress-mediated apoptosis in cartilage development and the molecular mechanisms involved.

Antibacterial activity and mode of action of ε-polylysine against Escherichia coli O157:H7.

PubMed

Zhang, Xiaowei; Shi, Ce; Liu, Zuojia; Pan, Fengguang; Meng, Rizeng; Bu, Xiujuan; Xing, Heqin; Deng, Yanhong; Guo, Na; Yu, Lu

2018-04-10

Gram-negative Escherichia coli O157:H7 were chosen as model bacteria to evaluate the antimicrobial mechanism of ε-polylysine (ε-PL). The antibacterial activity of ε-PL was detected by measuring the minimum inhibitory concentration values as well as the time-kill curve. The membrane integrity was determined by ultraviolet (UV) absorption, membrane potential (MP) assay and flow cytometry (FCM) experiments. The permeability of the inner membrane was detected by β-galactosidase activity assay. Furthermore, electron microscopy [scanning electron microscopy (SEM) and transmission electron microscopy (TEM)] was utilized to observe bacterial morphology. These results demonstrated that ε-PL showed its antibacterial activity by changing the integrity and permeability of cell membranes, leading to rapid cell death. The electron microscopy analysis (SEM and TEM) results indicated that the bacterial cell morphology, membrane integrity and permeability were spoiled when the E. coli O157:H7 cells were exposed to minimum inhibitory concentrations of ε-PL (16 µg ml -1 ). In addition, the bacterial membrane was damaged more severely when the concentration of ε-PL was increased. The present study investigated the antimicrobial mechanism of ε-PL by measuring the content of cytoplasmic β-galactosidase, proteins and DNA. In addition, SEM and TEM were carried out to assess the mechanism. These results show that ε-PL has the ability to decrease the content of large molecules, cellular soluble proteins and nucleic acids associated with increasing the content of cytoplasmic β-galactosidase in supernatant by causing damage to the cell membranes. Consequently, the use of ε-PL as a natural antimicrobial agent should eventually become an appealing method in the field of food preservation.

Hydrogen protects against hyperoxia-induced apoptosis in type II alveolar epithelial cells via activation of PI3K/Akt/Foxo3a signaling pathway.

PubMed

Wu, Dan; Liang, Mulin; Dang, Hongxing; Fang, Fang; Xu, Feng; Liu, Chengjun

2018-01-08

Oxidative stress is regarded as a key regulator in the pathogenesis of prolonged hyperoxia-induced lung injury, which causes injury to alveolar epithelial cells and eventually leads to development of bronchopulmonary dysplasia (BPD). Many studies have shown that hydrogen has a protective effect in a variety of cells. However, the mechanisms by which hydrogen rescues cells from damage due to oxidative stress in BPD remains to be fully elucidated. This study sought to evaluate the effects of hydrogen on hyperoxia-induced lung injury and to investigate the underlying mechanism. Primary type II alveolar epithelial cells (AECIIs) were divided into four groups: control (21% oxygen), hyperoxia (95% oxygen), hyperoxia + hydrogen, and hyperoxia + hydrogen + LY294002 (a PI3K/Akt inhibitor). Proliferation and apoptosis of AECIIs were assessed using MTS assay and flow cytometry (FCM), respectively. Gene and protein expression were detected by quantitative polymerase chain reaction (q-PCR) and western blot analysis. Stimulation with hyperoxia decreased the expression of P-Akt, P- FoxO3a, cyclinD1 and Bcl-2. Hyperoxic conditions increased levels of Bim, Bax, and Foxo3a, which induced proliferation restriction and apoptosis of AECIIs. These effects of hyperoxia were reversed with hydrogen pretreatment. Furthermore, the protective effects of hydrogen were abrogated by PI3K/Akt inhibitor LY294002. The results indicate that hydrogen protects AECIIs from hyperoxia-induced apoptosis by inhibiting apoptosis factors and promoting the expression of anti-apoptosis factors. These effects were associated with activation of the PI3K/Akt/FoxO3a pathway. Copyright © 2017 Elsevier Inc. All rights reserved.

Evaluation of optimal DNA staining for triggering by scanning fluorescence microscopy (SFM)

NASA Astrophysics Data System (ADS)

Mittag, Anja; Marecka, Monika; Pierzchalski, Arkadiusz; Malkusch, Wolf; Bocsi, József; Tárnok, Attila

2009-02-01

In imaging and flow cytometry, DNA staining is a common trigger signal for cell identification. Selection of the proper DNA dye is restricted by the hardware configuration of the instrument. The Zeiss Imaging Solutions GmbH (München, Germany) introduced a new automated scanning fluorescence microscope - SFM (Axio Imager.Z1) which combines fluorescence imaging with cytometric parameters measurement. The aim of the study was to select optimal DNA dyes as trigger signal in leukocyte detection and subsequent cytometric analysis of double-labeled leukocytes by SFM. Seven DNA dyes (DAPI, Hoechst 33258, Hoechst 33342, POPO-3, PI, 7-AAD, and TOPRO-3) were tested and found to be suitable for the implemented filtersets (fs) of the SFM (fs: 49, fs: 44, fs: 20). EDTA blood was stained after erythrocyte lysis with DNA dye. Cells were transferred on microscopic slides and embedded in fluorescent mounting medium. Quality of DNA fluorescence signal as well as spillover signals were analyzed by SFM. CD45-APC and CD3-PE as well as CD4-FITC and CD8-APC were selected for immunophenotyping and used in combination with Hoechst. Within the tested DNA dyes DAPI showed relatively low spillover and the best CV value. Due to the low spillover of UV DNA dyes a triple staining of Hoechst and APC and PE (or APC and FITC, respectively) could be analyzed without difficulty. These results were confirmed by FCM measurements. DNA fluorescence is applicable for identifying and triggering leukocytes in SFM analyses. Although some DNA dyes exhibit strong spillover in other fluorescence channels, it was possible to immunophenotype leukocytes. DAPI seems to be best suitable for use in the SFM system and will be used in protocol setups as primary parameter.

Behavior and stability of adenosine triphosphate (ATP) during chlorine disinfection.

PubMed

Nescerecka, Alina; Juhna, Talis; Hammes, Frederik

2016-09-15

Adenosine triphosphate (ATP) analysis is a cultivation-independent alternative method for the determination of bacterial viability in both chlorinated and non-chlorinated water. Here we investigated the behavior and stability of ATP during chlorination in detail. Different sodium hypochlorite doses (0-22.4 mg-Cl2 L(-1); 5 min exposure) were applied to an Escherichia coli pure culture suspended in filtered river water. We observed decreasing intracellular ATP with increasing chlorine concentrations, but extracellular ATP concentrations only increased when the chlorine dose exceeded 0.35 mg L(-1). The release of ATP from chlorine-damaged bacteria coincided with severe membrane damage detected with flow cytometry (FCM). The stability of extracellular ATP was subsequently studied in different water matrixes, and we found that extracellular ATP was stable in sterile deionized water and also in chlorinated water until extremely high chlorine doses (≤11.2 mg-Cl2 L(-1); 5 min exposure). In contrast, ATP decreased relatively slowly (k = 0.145 h(-1)) in 0.1 μm filtered river water, presumably due to degradation by either extracellular enzymes or the fraction of bacteria that were able to pass through the filter. Extracellular ATP decreased considerably faster (k = 0.368 h(-1)) during batch growth of a river water bacterial community. A series of growth potential tests showed that extracellular ATP molecules were utilized as a phosphorus source during bacteria proliferation. From the combined data we conclude that ATP released from bacteria at high chlorine doses could promote bacteria regrowth, contributing to biological instability in drinking water distribution systems. Copyright © 2016 Elsevier Ltd. All rights reserved.

Interleukin-15-transferred cytokine-induced killer cells elevated anti-tumor activity in a gastric tumor-bearing nude mice model.

PubMed

Peng, Zheng; Liang, Wentao; Li, Zexue; Xu, Yingxin; Chen, Lin

2016-02-01

Gastric cancer is the second leading cause of cancer-related mortality worldwide. Adoptive cell therapy (ACT) for gastric cancer is a novel therapy modality. However, the therapeutic effectiveness in vivo is still limited. The objective of this study was to assess the value of interleukin-15 (IL-15)-transferred cytokine-induced killer (CIK) cells in ACT for gastric cancer. IL-15-IRES-TK retroviral vector was constructed and transferred into the CIK cells. A gastric tumor-bearing nude mice model was constructed by subcutaneously injecting gastric cancer cells, BGC-823. Gastric tumor-bearing nude mice were randomly divided into three groups (five mice each group) and injected with physiological saline, CIK cells, and IL-15-IRES-TK-transfected CIK cells for 2 weeks, respectively. IL-15-IRES-TK-transferred CIK cells were prepared successfully and flow cytometry (FCM) analysis indicated that the transfection rate reached 85.7% after 5 days culture. In vivo experiment, we found that CIK cells retarded tumor growth by reducing tumor volume and tumor weight, as well as increasing tumor inhibition rate. Furthermore, IL-15-IRES-TK-transferred CIK cells showed a much stronger inhibition on tumor growth than CIK cells alone. Tumor morphology observation and growth indexes also showed that IL-15-transfected CIK cells had stronger cytotoxicity to tumor tissue than CIK cells. IL-15-IRES-TK transfection could elevate the effects of CIK cells to gastric carcinoma. The engineered CIK cells carrying IL-15-IRES-TK may be used in the ACT for gastric carcinoma, but prudent clinical trial is still indispensable. © 2015 International Federation for Cell Biology.

Evaluating Monitoring Strategies to Detect Precipitation-Induced Microbial Contamination Events in Karstic Springs Used for Drinking Water.

PubMed

Besmer, Michael D; Hammes, Frederik; Sigrist, Jürg A; Ort, Christoph

2017-01-01

Monitoring of microbial drinking water quality is a key component for ensuring safety and understanding risk, but conventional monitoring strategies are typically based on low sampling frequencies (e.g., quarterly or monthly). This is of concern because many drinking water sources, such as karstic springs are often subject to changes in bacterial concentrations on much shorter time scales (e.g., hours to days), for example after precipitation events. Microbial contamination events are crucial from a risk assessment perspective and should therefore be targeted by monitoring strategies to establish both the frequency of their occurrence and the magnitude of bacterial peak concentrations. In this study we used monitoring data from two specific karstic springs. We assessed the performance of conventional monitoring based on historical records and tested a number of alternative strategies based on a high-resolution data set of bacterial concentrations in spring water collected with online flow cytometry (FCM). We quantified the effect of increasing sampling frequency and found that for the specific case studied, at least bi-weekly sampling would be needed to detect precipitation events with a probability of >90%. We then proposed an optimized monitoring strategy with three targeted samples per event, triggered by precipitation measurements. This approach is more effective and efficient than simply increasing overall sampling frequency. It would enable the water utility to (1) analyze any relevant event and (2) limit median underestimation of peak concentrations to approximately 10%. We conclude with a generalized perspective on sampling optimization and argue that the assessment of short-term dynamics causing microbial peak loads initially requires increased sampling/analysis efforts, but can be optimized subsequently to account for limited resources. This offers water utilities and public health authorities systematic ways to evaluate and optimize their current monitoring strategies.

Intraoperative diagnosis of nonpigmented nail tumours with ex vivo fluorescence confocal microscopy: 10 cases.

PubMed

Debarbieux, S; Gaspar, R; Depaepe, L; Dalle, S; Balme, B; Thomas, L

2015-04-01

Ex vivo fluorescence confocal microscopy (FCM) permits real-time imaging of freshly excised skin tissues. Its usefulness as a time-sparing alternative to frozen sections in Mohs surgery of basal cell carcinoma is well documented. The purpose of this study was to describe the ex vivo FCM features of a series of benign and malignant nonpigmented tumours of the nail unit, and to correlate them with conventional histopathology. Nail apparatus tumours from 10 patients were imaged during surgical exploration using ex vivo FCM after immersion in acridine orange. Confocal mosaics of the freshly performed biopsies were evaluated in real time and retrospectively compared with haematoxylin and eosin sections. Our series included two invasive epithelial tumours (Group 1), four in situ or minimally invasive squamous cell carcinomas (SCC) (Group 2), three benign epithelial tumours (Group 3) and one nodular melanoma (Group 4). The correlation was excellent for malignant epithelial tumours exhibiting marked cytological and architectural atypias (Bowen disease, invasive SCC and onycholemmal carcinoma). Onychomatricomas exhibited a very peculiar aspect with densely cellular papillae. The correlation was less favourable for minimally invasive well-differentiated SCCs with slight cytological atypias. The correlation was poor for our case of amelanotic invasive subungual melanoma. Ex vivo FCM could be a useful tool to shorten management of nonpigmented nail tumours: in the case of a malignant tumour, it could indeed lead to performing wide excision during the same surgical procedure and possibly assessing the surgical margins. © 2014 British Association of Dermatologists.

Production of LEU Fully Ceramic Microencapsulated Fuel for Irradiation Testing

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

Terrani, Kurt A; Kiggans Jr, James O; McMurray, Jake W

2016-01-01

Fully Ceramic Microencapsulated (FCM) fuel consists of tristructural isotropic (TRISO) fuel particles embedded inside a SiC matrix. This fuel inherently possesses multiple barriers to fission product release, namely the various coating layers in the TRISO fuel particle as well as the dense SiC matrix that hosts these particles. This coupled with the excellent oxidation resistance of the SiC matrix and the SiC coating layer in the TRISO particle designate this concept as an accident tolerant fuel (ATF). The FCM fuel takes advantage of uranium nitride kernels instead of oxide or oxide-carbide kernels used in high temperature gas reactors to enhancemore » heavy metal loading in the highly moderated LWRs. Production of these kernels with appropriate density, coating layer development to produce UN TRISO particles, and consolidation of these particles inside a SiC matrix have been codified thanks to significant R&D supported by US DOE Fuel Cycle R&D program. Also, surrogate FCM pellets (pellets with zirconia instead of uranium-bearing kernels) have been neutron irradiated and the stability of the matrix and coating layer under LWR irradiation conditions have been established. Currently the focus is on production of LEU (7.3% U-235 enrichment) FCM pellets to be utilized for irradiation testing. The irradiation is planned at INL s Advanced Test Reactor (ATR). This is a critical step in development of this fuel concept to establish the ability of this fuel to retain fission products under prototypical irradiation conditions.« less

An introduction to mass cytometry: fundamentals and applications.

PubMed

Tanner, Scott D; Baranov, Vladimir I; Ornatsky, Olga I; Bandura, Dmitry R; George, Thaddeus C

2013-05-01

Mass cytometry addresses the analytical challenges of polychromatic flow cytometry by using metal atoms as tags rather than fluorophores and atomic mass spectrometry as the detector rather than photon optics. The many available enriched stable isotopes of the transition elements can provide up to 100 distinguishable reporting tags, which can be measured simultaneously because of the essential independence of detection provided by the mass spectrometer. We discuss the adaptation of traditional inductively coupled plasma mass spectrometry to cytometry applications. We focus on the generation of cytometry-compatible data and on approaches to unsupervised multivariate clustering analysis. Finally, we provide a high-level review of some recent benchmark reports that highlight the potential for massively multi-parameter mass cytometry.

Deposition, clearance, and shortening of Kevlar para-aramid fibrils in acute, subchronic, and chronic inhalation studies in rats.

PubMed

Kelly, D P; Merriman, E A; Kennedy, G L; Lee, K P

1993-10-01

The deposition and clearance of lung-deposited Kevlar para-aramid fibrils (subfibers) have been investigated as part of a subchronic and chronic inhalation toxicity testing program. Fibrils recovered from lung tissue in para-aramid-exposed Sprague-Dawley rats were microscopically counted and measured after exposures to airborne fibrils which were about 12 microns median length (ML) and < 0.3 micron median diameter. In each of three studies lung-recovered fibrils were progressively shorter with increasing residence time in the lungs. Twenty-eight days after a single 6-hr exposure at 400 respirable fibrils per cubic centimeter (f/cm3) the ML of recovered fibrils decreased to about 5 microns. Twenty-four months after a 3-week exposure to 25 or 400 f/cm3, fibrils reached about 2 microns ML. After 2 years of continuous exposure at 2.5, 25, or 100 f/cm3 or 1 year exposure plus 1 year recovery at 400 f/cm3, fibril ML approached 4 microns. In the 2-year study, the lung-fiber accumulation rate/exposure concentration was similar for the three highest concentrations and was about 3 x greater than that seen at 2.5 f/cm3, indicating that concentrations of about 25 f/cm3 or more may overwhelm clearance mechanisms. Time required for fibrils to be reduced to < 5 microns in the lung was markedly less at lower exposure concentration and shorter exposure time. The primary shortening mechanism is proposed to be long fibril cutting by enzymatic attack at fibril defects. However, length-selective fibril deposition and clearance may contribute to shortening in the first few days after exposure. The enzymatic cutting hypothesis is supported by measured increases in numbers of short fibers following cessation of exposures, continued shortening of the fibril length distribution up to 2 years following exposure, and in vitro fibril shortening after 3 months in a proteolytic enzyme preparation. The conclusion is that para-aramid fibrils are less durable in the lungs of rats than expected from the known chemical resistance of commercial yarn. These data suggest that at the low para-aramid fibril exposures found in the workplace, this fibril-shortening mechanism may limit the residence time of long fibers in the lungs of exposed workers. In addition, associated cascade impactor aerodynamic measurements indicate that due to their ribbon shape and curly nature, para-aramid fibrils behave aerodynamically larger than straight fibers.

Cr(VI) induces DNA damage, cell cycle arrest and polyploidization: a flow cytometric and comet assay study in Pisum sativum.

PubMed

Rodriguez, Eleazar; Azevedo, Raquel; Fernandes, Pedro; Santos, Conceição

2011-07-18

Chromium(VI) is recognized as the most toxic valency of Cr, but its genotoxicity and cytostaticity in plants is still poorly studied. In order to analyze Cr(VI) cyto- and gentotoxicity, Pisum sativum L. plants were grown in soil and watered with solutions with different concentrations of Cr up to 2000 mg/L. After 28 days of exposure, leaves showed no significant variations in either cell cycle dynamics or ploidy level. As for DNA damage, flow cytometric (FCM) histograms showed significant differences in full peak coefficient of variation (FPCV) values, suggesting clastogenicity. This is paralleled by the Comet assay results, showing an increase in DNA damage for 1000 and 2000 mg/L. In roots, exposure to 2000 mg/L resulted in cell cycle arrest at the G(2)/M checkpoint. It was also verified that under the same conditions 40% of the individuals analyzed suffered polyploidization having both 2C and 4C levels. DNA damage analysis by the Comet assay and FCM revealed dose-dependent increases in DNA damage and FPCV. Through this, we have unequivocally demonstrated for the first time in plants that Cr exposure can result in DNA damage, cell cycle arrest, and polyploidization. Moreover, we critically compare the validity of the Comet assay and FCM in evaluating cytogenetic toxicity tests in plants and demonstrate that the data provided by both techniques complement each other and present high correlation levels. In conclusion, the data presented provides new insight on Cr effects in plants in general and supports the use of the parameters tested in this study as reliable endpoints for this metal toxicity in plants. © 2011 American Chemical Society

Blockade of Tumor Cell TGF-Betas: A Strategy to Reverse Antiestrogen Resistance in Human Breast Cancer

DTIC Science & Technology

2002-01-01

the TM- FKHRL1 construct exhibited exclusive nuclear localization Cell Cycle Analysis by Flow Cytometry of the HA-tagged mutant under any experimental...distribution as measured by flow cytometry (Figure 8A). ALS AND METHODS. Consistent with its antiapoptotic effect, these results, addi- tion of TGFI3... flow cytometry . Under these conditions more than 95% of selected cells expressed GFP at the time of experiments. Immunoblot Analysis. Cells were

A Study of the Effects of High Power Pulsed 2450 MHz Microwaves, ELF modulated Microwaves, and ELF Fields on Human Lymphocytes and Selected Cell Lines

DTIC Science & Technology

1993-01-27

Considerable effect was expended in investigating shifts in intercellular calcium of one particular cell line, Jurket, using flow cytometry methods. No...culture. The following analysis were used to characterize the immortalized cell lines: flow cytometry , electron microscopy, two-dimensional protein gel...further characterized by flow cytometry , electron microscopy, two dimensional protein electrophoresis and nuclear run-off assay. Flow cytometric analysis of

Hypoxia and Prx1 in Malignant Progression of Prostate Cancer

DTIC Science & Technology

2006-09-01

Species (ROS) Formation The rate of ROS formation was determined by flow cytometry analysis using the probe 20,70-dichlorofluorescin diacetate (DCFH-DA...DA were subjected to 4-h hypoxia treatment. After the indicated time, fluorescent cells were analyzed by flow cytometry . Western Blot Analysis Equal...species (ROS) generation was measured by flow cytometry at 0.5, 1, 2, 3, 6, 12, or 24 h after hypoxia treatment. The rate of ROS generation increased

Genetic Algorithm-Based Optimization to Match Asteroid Energy Deposition Curves

NASA Technical Reports Server (NTRS)

Tarano, Ana; Mathias, Donovan; Wheeler, Lorien; Close, Sigrid

2018-01-01

An asteroid entering Earth's atmosphere deposits energy along its path due to thermal ablation and dissipative forces that can be measured by ground-based and spaceborne instruments. Inference of pre-entry asteroid properties and characterization of the atmospheric breakup is facilitated by using an analytic fragment-cloud model (FCM) in conjunction with a Genetic Algorithm (GA). This optimization technique is used to inversely solve for the asteroid's entry properties, such as diameter, density, strength, velocity, entry angle, and strength scaling, from simulations using FCM. The previous parameters' fitness evaluation involves minimizing error to ascertain the best match between the physics-based calculated energy deposition and the observed meteors. This steady-state GA provided sets of solutions agreeing with literature, such as the meteor from Chelyabinsk, Russia in 2013 and Tagish Lake, Canada in 2000, which were used as case studies in order to validate the optimization routine. The assisted exploration and exploitation of this multi-dimensional search space enables inference and uncertainty analysis that can inform studies of near-Earth asteroids and consequently improve risk assessment.

Effects of matrine on the proliferation of HT29 human colon cancer cells and its antitumor mechanism

PubMed Central

CHANG, CHENG; LIU, SHAO-PING; FANG, CHUN-HUA; HE, REN-SHENG; WANG, ZHEN; ZHU, YOU-QING; JIANG, SHAO-WEI

2013-01-01

Matrine is one of the main active components that is extracted from the dry roots of Sophora flavescens. The compound has potent antitumor activity in various cancer cell lines. However, the anticancer activity of matrine in colon cancer cells remains unclear. The purpose of the present study was to investigate the effects of matrine on the growth of human colon cancer cells and the expression of the associated proteins. Cancer cell proliferation was measured by 3-(4,5-dimethylthiazolyl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. The cell cycle distribution and apoptosis were analyzed by flow cytometry (FCM). The activation of the caspases and the expression of pro-apoptotic and anti-apoptotic factors were examined using western blot analysis. Matrine was shown to significantly inhibit the proliferation of HT29 cells in a dose- and time-dependent manner, and also to reduce the percentage of cells in the G2/M phase, which was most frequently associated with an increase of cells arrested in the G0/G1 phase of the cell cycle. Western blot analysis revealed that matrine induced the activation of caspase-3 and -9 and the release of cytochrome C (Cyto C) from the mitochondria to the cytosol. Furthermore, the pro-apoptotic factor, Bax, was upregulated and the anti-apoptotic factor, Bcl-2, was downregulated, eventually leading to a reduction in the ratio of Bcl-2/Bax proteins. The results demonstrated that matrine inhibits proliferation and induces apoptosis of HT29 human cells in vitro. The induction of apoptosis appears to occur through the upregulation of Bax, the downregulation of Bcl-2, the release of Cyto C from the mitochondria to the cytosol and the activation of caspase-3 and caspase-9, which subsequently trigger major apoptotic cascades. Matrine has potent antitumor activity in HT29 cells and may be used as a novel effective reagent in the treatment of colon cancer. PMID:24137393

Technical advances in flow cytometry-based diagnosis and monitoring of paroxysmal nocturnal hemoglobinuria

PubMed Central

Correia, Rodolfo Patussi; Bento, Laiz Cameirão; Bortolucci, Ana Carolina Apelle; Alexandre, Anderson Marega; Vaz, Andressa da Costa; Schimidell, Daniela; Pedro, Eduardo de Carvalho; Perin, Fabricio Simões; Nozawa, Sonia Tsukasa; Mendes, Cláudio Ernesto Albers; Barroso, Rodrigo de Souza; Bacal, Nydia Strachman

2016-01-01

ABSTRACT Objective: To discuss the implementation of technical advances in laboratory diagnosis and monitoring of paroxysmal nocturnal hemoglobinuria for validation of high-sensitivity flow cytometry protocols. Methods: A retrospective study based on analysis of laboratory data from 745 patient samples submitted to flow cytometry for diagnosis and/or monitoring of paroxysmal nocturnal hemoglobinuria. Results: Implementation of technical advances reduced test costs and improved flow cytometry resolution for paroxysmal nocturnal hemoglobinuria clone detection. Conclusion: High-sensitivity flow cytometry allowed more sensitive determination of paroxysmal nocturnal hemoglobinuria clone type and size, particularly in samples with small clones. PMID:27759825

Visual analysis of mass cytometry data by hierarchical stochastic neighbour embedding reveals rare cell types.

PubMed

van Unen, Vincent; Höllt, Thomas; Pezzotti, Nicola; Li, Na; Reinders, Marcel J T; Eisemann, Elmar; Koning, Frits; Vilanova, Anna; Lelieveldt, Boudewijn P F

2017-11-23

Mass cytometry allows high-resolution dissection of the cellular composition of the immune system. However, the high-dimensionality, large size, and non-linear structure of the data poses considerable challenges for the data analysis. In particular, dimensionality reduction-based techniques like t-SNE offer single-cell resolution but are limited in the number of cells that can be analyzed. Here we introduce Hierarchical Stochastic Neighbor Embedding (HSNE) for the analysis of mass cytometry data sets. HSNE constructs a hierarchy of non-linear similarities that can be interactively explored with a stepwise increase in detail up to the single-cell level. We apply HSNE to a study on gastrointestinal disorders and three other available mass cytometry data sets. We find that HSNE efficiently replicates previous observations and identifies rare cell populations that were previously missed due to downsampling. Thus, HSNE removes the scalability limit of conventional t-SNE analysis, a feature that makes it highly suitable for the analysis of massive high-dimensional data sets.

The sensitive hare: sublethal effects of predator stress on reproduction in snowshoe hares.

PubMed

Sheriff, Michael J; Krebs, Charles J; Boonstra, Rudy

2009-11-01

1. Prey responses to high predation risk can be morphological or behavioural and ultimately come at the cost of survival, growth, body condition, or reproduction. These sub-lethal predator effects have been shown to be mediated by physiological stress. We tested the hypothesis that elevated glucocorticoid concentrations directly cause a decline in reproduction in individual free-ranging female snowshoe hares, Lepus americanus. We measured the cortisol concentration from each dam (using a faecal analysis enzyme immunoassay) and her reproductive output (litter size, offspring birth mass, offspring right hind foot (RHF) length) 30 h after birth. 2. In a natural monitoring study, we monitored hares during the first and second litter from the population peak (2006) to the second year of the decline (2008). We found that faecal cortisol metabolite (FCM) concentration in dams decreased 52% from the first to the second litter. From the first to the second litter, litter size increased 122%, offspring body mass increased 130%, and offspring RHF length increased 112%. Dam FCM concentrations were inversely related to litter size (r(2) = 0.19), to offspring birth mass (r(2) = 0.32), and to offspring RHF length (r(2) = 0.64). 3. In an experimental manipulation, we assigned wild-caught, pregnant hares to a control and a stressed group and held them in pens. Hares in the stressed group were exposed to a dog 1-2 min every other day before parturition to simulate high predation risk. At parturition, unsuccessful-stressed dams (those that failed to give birth to live young) and stressed dams had 837% and 214%, respectively, higher FCM concentrations than control dams. Of those females that gave birth, litter size was similar between control and stressed dams. However, offspring from stressed dams were 37% lighter and 16% smaller than offspring from control dams. Increasing FCM concentration in dams caused the decline of offspring body mass (r(2) = 0.57) and RHF (r(2) = 0.52). 4. This is the first study in a free-ranging population of mammals to show that elevated, predator-induced, glucocorticoid concentrations in individual dams caused a decline in their reproductive output measured both by number and quality of offspring. Thus, we provide evidence that any stressor, not just predation, which increases glucocorticoid concentrations will result in a decrease in reproductive output.

[Clinical usefulness of urine-formed elements' information obtained from bacteria detection by flow cytometry method that uses nucleic acid staining].

PubMed

Nakagawa, Hiroko; Yuno, Tomoji; Itho, Kiichi

2009-03-01

Recently, specific detection method for Bacteria, by flow cytometry method using nucleic acid staining, was developed as a function of automated urine formed elements analyzer for routine urine testing. Here, we performed a basic study on this bacteria analysis method. In addition, we also have a comparison among urine sediment analysis, urine Gram staining and urine quantitative cultivation, the conventional methods performed up to now. As a result, the bacteria analysis with flow cytometry method that uses nucleic acid staining was excellent in reproducibility, and higher sensitivity compared with microscopic urinary sediment analysis. Based on the ROC curve analysis, which settled urine culture method as standard, cut-off level of 120/microL was defined and its sensitivity = 85.7%, specificity = 88.2%. In the analysis of scattergram, accompanied with urine culture method, among 90% of rod positive samples, 80% of dots were appeared in the area of 30 degrees from axis X. In addition, one case even indicated that analysis of bacteria by flow cytometry and scattergram of time series analysis might be helpful to trace the progress of causative bacteria therefore the information supposed to be clinically significant. Reporting bacteria information with nucleic acid staining flow cytometry method is expected to contribute to a rapid diagnostics and treatment of urinary tract infections. Besides, the contribution to screening examination of microbiology and clinical chemistry, will deliver a more efficient solution to urine analysis.

Image segmentation using fuzzy LVQ clustering networks

NASA Technical Reports Server (NTRS)

Tsao, Eric Chen-Kuo; Bezdek, James C.; Pal, Nikhil R.

1992-01-01

In this note we formulate image segmentation as a clustering problem. Feature vectors extracted from a raw image are clustered into subregions, thereby segmenting the image. A fuzzy generalization of a Kohonen learning vector quantization (LVQ) which integrates the Fuzzy c-Means (FCM) model with the learning rate and updating strategies of the LVQ is used for this task. This network, which segments images in an unsupervised manner, is thus related to the FCM optimization problem. Numerical examples on photographic and magnetic resonance images are given to illustrate this approach to image segmentation.

In Vivo Myeloperoxidase Imaging and Flow Cytometry Analysis of Intestinal Myeloid Cells.

PubMed

Hülsdünker, Jan; Zeiser, Robert

2016-01-01

Myeloperoxidase (MPO) imaging is a non-invasive method to detect cells that produce the enzyme MPO that is most abundant in neutrophils, macrophages, and inflammatory monocytes. While lacking specificity for any of these three cell types, MPO imaging can provide guidance for further flow cytometry-based analysis of tissues where these cell types reside. Isolation of leukocytes from the intestinal tract is an error-prone procedure. Here, we describe a protocol for intestinal leukocyte isolation that works reliable in our hands and allows for flow cytometry-based analysis, in particular of neutrophils.

CytoSPADE: high-performance analysis and visualization of high-dimensional cytometry data

PubMed Central

Linderman, Michael D.; Simonds, Erin F.; Qiu, Peng; Bruggner, Robert V.; Sheode, Ketaki; Meng, Teresa H.; Plevritis, Sylvia K.; Nolan, Garry P.

2012-01-01

Motivation: Recent advances in flow cytometry enable simultaneous single-cell measurement of 30+ surface and intracellular proteins. CytoSPADE is a high-performance implementation of an interface for the Spanning-tree Progression Analysis of Density-normalized Events algorithm for tree-based analysis and visualization of this high-dimensional cytometry data. Availability: Source code and binaries are freely available at http://cytospade.org and via Bioconductor version 2.10 onwards for Linux, OSX and Windows. CytoSPADE is implemented in R, C++ and Java. Contact: michael.linderman@mssm.edu Supplementary Information: Additional documentation available at http://cytospade.org. PMID:22782546

Trapped magnetic-field properties of prototype for Gd-Ba-Cu-O/MgB2 hybrid-type superconducting bulk magnet

NASA Astrophysics Data System (ADS)

Naito, Tomoyuki; Mochizuki, Hidehiko; Fujishiro, Hiroyuki; Teshima, Hidekazu

2016-03-01

We have studied experimentally and numerically the trapped magnetic-field properties of a hybrid-type superconducting bulk magnet, which comprised an inner Gd-Ba-Cu-O (GdBCO) disk-bulk and an outer MgB2 ring-bulk, under field-cooled magnetization (FCM) and pulsed-field magnetization (PFM). The trapped field by FCM at the center of the hybrid bulk was 4.5 T at 20 K, which was 0.2 T higher than that of the inner GdBCO disk-bulk without MgB2 ring-bulk. The experimental results by FCM were quantitatively reproduced by the numerical estimations for a model, which makes it possible to understand the trapped field properties of the hybrid bulk. The total magnetic flux by FCM, which was estimated numerically, was enhanced by about 1.7 times from 0.91 mWb of the single GdBCO bulk to 1.53 mWb of the hybrid bulk. We also succeeded in magnetizing the whole hybrid bulk by applying multi-pulsed-fields. The central trapped field of 1.88 T was not enhanced, but the total magnetic flux, which was obtained experimentally, was evidently increased by 2.5 times (0.25 \\to 0.62 mWb) for the hybrid bulk. The obtained results suggest that the hybridization is effective to enhance the total magnetic flux. To confirm the reinforcing effect of the MgB2 ring to the GdBCO disk during the cooling and magnetization processes, we have measured the thermal dilatation, {\\text{}}{dL}({\\text{}}T)/{\\text{}}L(300 K), of the GdBCO, MgB2 and stainless steel. As a result, the thermal dilatation of MgB2 was smaller than that of GdBCO. MgB2 ring-bulk shows no compression effect to resist the hoop stress of the GdBCO disk-bulk during the FCM process. The reinforcing material such as the stainless steel ring must be set outside the GdBCO disk-bulk.

An adaptive Fuzzy C-means method utilizing neighboring information for breast tumor segmentation in ultrasound images.

PubMed

Feng, Yuan; Dong, Fenglin; Xia, Xiaolong; Hu, Chun-Hong; Fan, Qianmin; Hu, Yanle; Gao, Mingyuan; Mutic, Sasa

2017-07-01

Ultrasound (US) imaging has been widely used in breast tumor diagnosis and treatment intervention. Automatic delineation of the tumor is a crucial first step, especially for the computer-aided diagnosis (CAD) and US-guided breast procedure. However, the intrinsic properties of US images such as low contrast and blurry boundaries pose challenges to the automatic segmentation of the breast tumor. Therefore, the purpose of this study is to propose a segmentation algorithm that can contour the breast tumor in US images. To utilize the neighbor information of each pixel, a Hausdorff distance based fuzzy c-means (FCM) method was adopted. The size of the neighbor region was adaptively updated by comparing the mutual information between them. The objective function of the clustering process was updated by a combination of Euclid distance and the adaptively calculated Hausdorff distance. Segmentation results were evaluated by comparing with three experts' manual segmentations. The results were also compared with a kernel-induced distance based FCM with spatial constraints, the method without adaptive region selection, and conventional FCM. Results from segmenting 30 patient images showed the adaptive method had a value of sensitivity, specificity, Jaccard similarity, and Dice coefficient of 93.60 ± 5.33%, 97.83 ± 2.17%, 86.38 ± 5.80%, and 92.58 ± 3.68%, respectively. The region-based metrics of average symmetric surface distance (ASSD), root mean square symmetric distance (RMSD), and maximum symmetric surface distance (MSSD) were 0.03 ± 0.04 mm, 0.04 ± 0.03 mm, and 1.18 ± 1.01 mm, respectively. All the metrics except sensitivity were better than that of the non-adaptive algorithm and the conventional FCM. Only three region-based metrics were better than that of the kernel-induced distance based FCM with spatial constraints. Inclusion of the pixel neighbor information adaptively in segmenting US images improved the segmentation performance. The results demonstrate the potential application of the method in breast tumor CAD and other US-guided procedures. © 2017 American Association of Physicists in Medicine.

Adenovirus expressing IFN-λ (Ad/hIFN-λ) produced anti-tumor effects through inducing apoptosis in human tongue squamous cell carcinoma cell.

PubMed

Song, Bing; Yang, Yong; Wang, Yan-Li; Fan, Xiao-Hui; Huang, Yu-Mei; Ci, Hao-Su; Zuo, Jin-Hua

2015-01-01

To investigate the potential therapeutic effects of adenovirus expressing IFN-λ1 and IFN-λ2 (Ad/hIFN-λ) in treating squamous cell carcinoma of the oral tongue (SCCOT) and to explore the underlying mechanisms. Two SCCOT cell lines HSC-3 and Tca8113 were adopted as study objects. Cell Counting Kit-8 (CCK-8) cell proliferation and viability assay was performed to evaluate the antiproliferative effects of Ad/hIFN-λ and IFN-λ treatments at different dosages. Flow cytometry (FCM) was performed to investigate the apoptosis rate induced by Ad/hIFN-λ. In vivo study was performed through evaluating tumorigenicity and tumor volume on BALB/c nu/nu mice inoculated with HSC-3 cells with or without infection of Ad/hIFN-λ. qPCR was used to screen important apoptosis related genes expression and western blot (WB) was performed to verify the results. WB was also used to test the phosphorylation of STATs protein in the JAK/STAT signaling pathways. Our results indicated an obvious antiproliferative effect of Ad/hIFN-λ in vitro on infected HSC-3 and Tca8113 cells. The antiproliferative effects started to appear at 48 h (day 2) after infection. IFN-λs alone treating HSC-3 and Tca8113 cells also showed a dose-dependent inhibitory manner. Though the antiproliferative effects did not show on 24 h (day 1), early apoptosis rate already increased significantly in cells infected with Ad/hIFN-λ (P<0.05) detected by FCM. The underlying mechanisms of antiproliferative activity rely on the IFN-λ signaling by phosphorylation of STATs protein. Expression of Bax, Bcl-2 and Caspase-3 were promoted by Ad/hIFN-λ leading to higher apoptosis rate. Upper stream of p21 and Rb dephosphorylation explained the Caspase-3 activation. Animal study showed that HSC-3 cells infected with Ad/hIFN-λ significantly promoted the survival rate and decreased mean tumor volume comparing to HSC-3 cells group. Ad/hIFN-λ injection had obvious antiproliferative effects on HSC-3 and Tca8113 cells. Ad/hIFN-λ induced apoptosis in SCCOT cells through increasing Bcl-2, Bax and Caspase-3 expression. Ad/hIFN-λ is a potential therapeutic strategy in treating oral tongue carcinoma.

Levels of circulating CD45dimCD34+VEGFR2+ progenitor cells correlate with outcome in metastatic renal cell carcinoma patients treated with tyrosine kinase inhibitors

PubMed Central

Farace, F; Gross-Goupil, M; Tournay, E; Taylor, M; Vimond, N; Jacques, N; Billiot, F; Mauguen, A; Hill, C; Escudier, B

2011-01-01

Background: Predicting the efficacy of antiangiogenic therapy would be of clinical value in patients (pts) with metastatic renal cell carcinoma (mRCC). We tested the hypothesis that circulating endothelial cell (CEC), bone marrow-derived CD45dimCD34+VEGFR2+ progenitor cell or plasma angiogenic factor levels are associated with clinical outcome in mRCC pts undergoing treatment with tyrosine kinase inhibitors (TKI). Methods: Fifty-five mRCC pts were prospectively monitored at baseline (day 1) and day 14 during treatment (46 pts received sunitinib and 9 pts received sorafenib). Circulating endothelial cells (CD45−CD31+CD146+7-amino-actinomycin (7AAD)− cells) were measured in 1 ml whole blood using four-color flow cytometry (FCM). Circulating CD45dimCD34+VEGFR2+7AAD− progenitor cells were measured in progenitor-enriched fractions by four-color FCM. Plasma VEGF, sVEGFR2, SDF-1α and sVCAM-1 levels were determined by ELISA. Correlations between baseline CEC, CD45dimCD34+VEGFR2+7AAD− progenitor cells, plasma factors, as well as day 1–day 14 changes in CEC, CD45dimCD34+VEGFR2+7AAD− progenitor, plasma factor levels, and response to TKI, progression-free survival (PFS) and overall survival (OS) were examined. Results: No significant correlation between markers and response to TKI was observed. No association between baseline CEC, plasma VEGF, sVEGFR-2, SDF-1α, sVCAM-1 levels with PFS and OS was observed. However, baseline CD45dimCD34+VEGFR2+7AAD− progenitor cell levels were associated with PFS (P=0.01) and OS (P=0.006). Changes in this population and in SDF-1α levels between day 1 and day 14 were associated with PFS (P=0.03, P=0.002). Changes in VEGF and SDF-1α levels were associated with OS (P=0.02, P=0.007). Conclusion: Monitoring CD45dimCD34+VEGFR2+ progenitor cells, plasma VEGF and SDF-1α levels could be of clinical interest in TKI-treated mRCC pts to predict outcome. PMID:21386843

Reduction of quaternary ammonium-induced ocular surface toxicity by emulsions: an in vivo study in rabbits

PubMed Central

Liang, H.; Brignole-Baudouin, F.; Rabinovich-Guilatt, L.; Mao, Z.; Riancho, L.; Faure, M.O.; Warnet, J.M.; Lambert, G.

2008-01-01

Purpose To evaluate and compare the toxicological profiles of two quaternary ammonium compounds (QAC), benzalkonium chloride (BAK), and cetalkonium chloride (CKC), in standard solution or cationic emulsion formulations in rabbit eyes using newly developed in vivo and ex vivo experimental approaches. Methods Seventy eyes of 35 adult male New Zealand albino rabbits were used in this study. They were randomly divided into five groups: 50 µl of phosphate-buffered saline (PBS), PBS containing 0.02% BAK or 0.002% CKC (BAK Sol and CKC Sol, respectively), and emulsion containing 0.02% BAK or 0.002% CKC (BAK Em and CKC Em, respectively) were applied to rabbit eyes 15 times at 5-min intervals. The ocular surface changes induced by these eye drops were investigated using slit-lamp examination, flow cytometry (FCM), impression cytology (IC) on conjunctiva, and corneal in vivo confocal microscopy (IVCM). Standard immunohistology in cryosections was also examined for cluster of differentiation (CD) 45+ infiltrating and terminal deoxynucleotidyl transferase-mediated dUTP-nick end labeling (TUNEL)+ apoptotic cells. Results Clinical observations and IVCM showed that the highest toxicity was induced by BAK Sol, characterized by damaged corneal epithelium and a high level of inflammatory infiltration. BAK Em and CKC Sol presented moderate effects, and CKC Em showed the lowest toxicity with results similar to those of PBS. Conjunctival imprints analyzed by FCM showed a higher expression of RLA-DR and TNFR1 markers in BAK Sol-instilled eyes than in all other groups, especially at 4 h. Immunohistology was correlated with in vivo and ex vivo findings and confirmed this toxicity profile. A high level of infiltration of CD45+ inflammatory cells and TUNEL+ apoptotic cells was observed in limbus and conjunctiva, especially in QAC solution-receiving eyes compared to QAC emulsion-instilled eyes. Conclusions The acute administration of 15 instillations at 5 min intervals was a rapid and efficient model to assess quaternary ammonium toxicity profiles. This model showed the highest toxicity, induced by the BAK solution, and the lowest level of toxicity, induced by the CKC emulsion. These in vivo and ex vivo experimental approaches demonstrated that ocular surface toxicity was reduced by using an emulsion instead of a traditional solution and that a CKC emulsion was safe for future ocular administration. PMID:18347566

Reduction of quaternary ammonium-induced ocular surface toxicity by emulsions: an in vivo study in rabbits.

PubMed

Liang, H; Brignole-Baudouin, F; Rabinovich-Guilatt, L; Mao, Z; Riancho, L; Faure, M O; Warnet, J M; Lambert, G; Baudouin, C

2008-01-31

To evaluate and compare the toxicological profiles of two quaternary ammonium compounds (QAC), benzalkonium chloride (BAK), and cetalkonium chloride (CKC), in standard solution or cationic emulsion formulations in rabbit eyes using newly developed in vivo and ex vivo experimental approaches. Seventy eyes of 35 adult male New Zealand albino rabbits were used in this study. They were randomly divided into five groups: 50 microl of phosphate-buffered saline (PBS), PBS containing 0.02% BAK or 0.002% CKC (BAK Sol and CKC Sol, respectively), and emulsion containing 0.02% BAK or 0.002% CKC (BAK Em and CKC Em, respectively) were applied to rabbit eyes 15 times at 5-min intervals. The ocular surface changes induced by these eye drops were investigated using slit-lamp examination, flow cytometry (FCM), impression cytology (IC) on conjunctiva, and corneal in vivo confocal microscopy (IVCM). Standard immunohistology in cryosections was also examined for cluster of differentiation (CD) 45+ infiltrating and terminal deoxynucleotidyl transferase-mediated dUTP-nick end labeling (TUNEL)+ apoptotic cells. Clinical observations and IVCM showed that the highest toxicity was induced by BAK Sol, characterized by damaged corneal epithelium and a high level of inflammatory infiltration. BAK Em and CKC Sol presented moderate effects, and CKC Em showed the lowest toxicity with results similar to those of PBS. Conjunctival imprints analyzed by FCM showed a higher expression of RLA-DR and TNFR1 markers in BAK Sol-instilled eyes than in all other groups, especially at 4 h. Immunohistology was correlated with in vivo and ex vivo findings and confirmed this toxicity profile. A high level of infiltration of CD45+ inflammatory cells and TUNEL+ apoptotic cells was observed in limbus and conjunctiva, especially in QAC solution-receiving eyes compared to QAC emulsion-instilled eyes. The acute administration of 15 instillations at 5 min intervals was a rapid and efficient model to assess quaternary ammonium toxicity profiles. This model showed the highest toxicity, induced by the BAK solution, and the lowest level of toxicity, induced by the CKC emulsion. These in vivo and ex vivo experimental approaches demonstrated that ocular surface toxicity was reduced by using an emulsion instead of a traditional solution and that a CKC emulsion was safe for future ocular administration.

An active, collaborative approach to learning skills in flow cytometry.

PubMed

Fuller, Kathryn; Linden, Matthew D; Lee-Pullen, Tracey; Fragall, Clayton; Erber, Wendy N; Röhrig, Kimberley J

2016-06-01

Advances in science education research have the potential to improve the way students learn to perform scientific interpretations and understand science concepts. We developed active, collaborative activities to teach skills in manipulating flow cytometry data using FlowJo software. Undergraduate students were given compensated clinical flow cytometry listmode output (FCS) files and asked to design a gating strategy to diagnose patients with different hematological malignancies on the basis of their immunophenotype. A separate cohort of research trainees was given uncompensated data files on which they performed their own compensation, calculated the antibody staining index, designed a sequential gating strategy, and quantified rare immune cell subsets. Student engagement, confidence, and perceptions of flow cytometry were assessed using a survey. Competency against the learning outcomes was assessed by asking students to undertake tasks that required understanding of flow cytometry dot plot data and gating sequences. The active, collaborative approach allowed students to achieve learning outcomes not previously possible with traditional teaching formats, for example, having students design their own gating strategy, without forgoing essential outcomes such as the interpretation of dot plots. In undergraduate students, favorable perceptions of flow cytometry as a field and as a potential career choice were correlated with student confidence but not the ability to perform flow cytometry data analysis. We demonstrate that this new pedagogical approach to teaching flow cytometry is beneficial for student understanding and interpretation of complex concepts. It should be considered as a useful new method for incorporating complex data analysis tasks such as flow cytometry into curricula. Copyright © 2016 The American Physiological Society.

TEM characterization of irradiated U-7Mo/Mg dispersion fuel

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

Gan, J.; Keiser, D. D.; Miller, B. D.

This paper presents the results of transmission electron microscopy (TEM) characterization on neutron-irradiated samples taken from the low-flux and high-flux sides of the same fuel plate with U-7Mo fuel particles dispersed in Mg matrix with aluminum alloy Al6061 as cladding material that was irradiated edge-on to the core in the Advanced Test Reactor. The corresponding local fission density and fission rate of the fuel particles and the average fuel-plate centerline temperature for the low-flux and high-flux samples are estimated to be 3.7 × 10 21 f/cm 3, 7.4 × 10 14 f/cm 3/s and 123 °C, and 5.5 × 10more » 21 f/cm3, 11.0 × 10 14 f/cm 3/s and 158 °C, respectively. Complex interaction layers developed at the Al-Mg interface, consisting of Al 3Mg 2 and Al 12Mg 17 along with precipitates of MgO, Mg 2Si and FeAl 5.3. No interaction between Mg matrix and U-Mo fuel particle was identified. For the U-Mo fuel particles, at low fission density, small elongated bubbles wrapped around the clean areas with a fission gas bubble superlattice, which suggests that bubble coalescence is an important mechanism for converting the fission gas bubble superlattice to large bubbles. At high fission density, no bubbles or porosity were observed in the Mg matrix, and pockets of residual fission gas bubble superlattice were observed in the U-Mo fuel particle interior.« less

Reference set design for relational modeling of fuzzy systems

NASA Astrophysics Data System (ADS)

Lapohos, Tibor; Buchal, Ralph O.

1994-10-01

One of the keys to the successful relational modeling of fuzzy systems is the proper design of fuzzy reference sets. This has been discussed throughout the literature. In the frame of modeling a stochastic system, we analyze the problem numerically. First, we briefly describe the relational model and present the performance of the modeling in the most trivial case: the reference sets are triangle shaped. Next, we present a known fuzzy reference set generator algorithm (FRSGA) which is based on the fuzzy c-means (Fc-M) clustering algorithm. In the second section of this chapter we improve the previous FRSGA by adding a constraint to the Fc-M algorithm (modified Fc-M or MFc-M): two cluster centers are forced to coincide with the domain limits. This is needed to obtain properly shaped extreme linguistic reference values. We apply this algorithm to uniformly discretized domains of the variables involved. The fuzziness of the reference sets produced by both Fc-M and MFc-M is determined by a parameter, which in our experiments is modified iteratively. Each time, a new model is created and its performance analyzed. For certain algorithm parameter values both of these two algorithms have shortcomings. To eliminate the drawbacks of these two approaches, we develop a completely new generator algorithm for reference sets which we call Polyline. This algorithm and its performance are described in the last section. In all three cases, the modeling is performed for a variety of operators used in the inference engine and two defuzzification methods. Therefore our results depend neither on the system model order nor the experimental setup.

TEM characterization of irradiated U-7Mo/Mg dispersion fuel

DOE PAGES

Gan, J.; Keiser, D. D.; Miller, B. D.; ...

2017-07-15

This paper presents the results of transmission electron microscopy (TEM) characterization on neutron-irradiated samples taken from the low-flux and high-flux sides of the same fuel plate with U-7Mo fuel particles dispersed in Mg matrix with aluminum alloy Al6061 as cladding material that was irradiated edge-on to the core in the Advanced Test Reactor. The corresponding local fission density and fission rate of the fuel particles and the average fuel-plate centerline temperature for the low-flux and high-flux samples are estimated to be 3.7 × 10 21 f/cm 3, 7.4 × 10 14 f/cm 3/s and 123 °C, and 5.5 × 10more » 21 f/cm3, 11.0 × 10 14 f/cm 3/s and 158 °C, respectively. Complex interaction layers developed at the Al-Mg interface, consisting of Al 3Mg 2 and Al 12Mg 17 along with precipitates of MgO, Mg 2Si and FeAl 5.3. No interaction between Mg matrix and U-Mo fuel particle was identified. For the U-Mo fuel particles, at low fission density, small elongated bubbles wrapped around the clean areas with a fission gas bubble superlattice, which suggests that bubble coalescence is an important mechanism for converting the fission gas bubble superlattice to large bubbles. At high fission density, no bubbles or porosity were observed in the Mg matrix, and pockets of residual fission gas bubble superlattice were observed in the U-Mo fuel particle interior.« less

Integration of lyoplate based flow cytometry and computational analysis for standardized immunological biomarker discovery.

PubMed

Villanova, Federica; Di Meglio, Paola; Inokuma, Margaret; Aghaeepour, Nima; Perucha, Esperanza; Mollon, Jennifer; Nomura, Laurel; Hernandez-Fuentes, Maria; Cope, Andrew; Prevost, A Toby; Heck, Susanne; Maino, Vernon; Lord, Graham; Brinkman, Ryan R; Nestle, Frank O

2013-01-01

Discovery of novel immune biomarkers for monitoring of disease prognosis and response to therapy in immune-mediated inflammatory diseases is an important unmet clinical need. Here, we establish a novel framework for immunological biomarker discovery, comparing a conventional (liquid) flow cytometry platform (CFP) and a unique lyoplate-based flow cytometry platform (LFP) in combination with advanced computational data analysis. We demonstrate that LFP had higher sensitivity compared to CFP, with increased detection of cytokines (IFN-γ and IL-10) and activation markers (Foxp3 and CD25). Fluorescent intensity of cells stained with lyophilized antibodies was increased compared to cells stained with liquid antibodies. LFP, using a plate loader, allowed medium-throughput processing of samples with comparable intra- and inter-assay variability between platforms. Automated computational analysis identified novel immunophenotypes that were not detected with manual analysis. Our results establish a new flow cytometry platform for standardized and rapid immunological biomarker discovery with wide application to immune-mediated diseases.

Integration of Lyoplate Based Flow Cytometry and Computational Analysis for Standardized Immunological Biomarker Discovery

PubMed Central

Villanova, Federica; Di Meglio, Paola; Inokuma, Margaret; Aghaeepour, Nima; Perucha, Esperanza; Mollon, Jennifer; Nomura, Laurel; Hernandez-Fuentes, Maria; Cope, Andrew; Prevost, A. Toby; Heck, Susanne; Maino, Vernon; Lord, Graham; Brinkman, Ryan R.; Nestle, Frank O.

2013-01-01

Discovery of novel immune biomarkers for monitoring of disease prognosis and response to therapy in immune-mediated inflammatory diseases is an important unmet clinical need. Here, we establish a novel framework for immunological biomarker discovery, comparing a conventional (liquid) flow cytometry platform (CFP) and a unique lyoplate-based flow cytometry platform (LFP) in combination with advanced computational data analysis. We demonstrate that LFP had higher sensitivity compared to CFP, with increased detection of cytokines (IFN-γ and IL-10) and activation markers (Foxp3 and CD25). Fluorescent intensity of cells stained with lyophilized antibodies was increased compared to cells stained with liquid antibodies. LFP, using a plate loader, allowed medium-throughput processing of samples with comparable intra- and inter-assay variability between platforms. Automated computational analysis identified novel immunophenotypes that were not detected with manual analysis. Our results establish a new flow cytometry platform for standardized and rapid immunological biomarker discovery with wide application to immune-mediated diseases. PMID:23843942

Data transmission and acquisition in NEMO

NASA Astrophysics Data System (ADS)

Bunkheila, G.

2006-11-01

A comprehensive system for data transmission and acquisition has been developed for an "à la NEMO" underwater neutrino telescope based on Čerenkov light detection using photomultipliers (PMTs) as sensors. Signals generated by each sensor are triggered, sampled and tagged by an electronics board, called Front End Module (FEM). Data streams from up to eight FEMs located on one tower floor are collected by a concentration board called Floor Control Module (FCM) and sent to a twin FCM board—located at the onshore station and plugged into an interface machine (FCM Interface, or FCMI) via a PCI bus—through a DWDM-compliant optical fiber and using a self-synchronous serial protocol. All sensor data reach the onshore lab through FCMI where they are made available to subsequent elaboration processes, such as time-wise alignment and muon track event-triggering. To meet requirements of the latter, onshore data unpacking is carried out with respect to their topological origin. The system promised, and keeps on showing, very light charges on power consumption and infrastructure complexity, while having recently proved to behave at high performance levels in its optical part.

NaNet3: The on-shore readout and slow-control board for the KM3NeT-Italia underwater neutrino telescope

NASA Astrophysics Data System (ADS)

Ammendola, R.; Biagioni, A.; Frezza, O.; Lo Cicero, F.; Martinelli, M.; Paolucci, P. S.; Pontisso, L.; Simula, F.; Vicini, P.; Ameli, F.; Nicolau, C. A.; Pastorelli, E.; Simeone, F.; Tosoratto, L.; Lonardo, A.

2016-04-01

The KM3NeT-Italia underwater neutrino detection unit, the tower, consists of 14 floors. Each floor supports 6 Optical Modules containing front-end electronics needed to digitize the PMT signal, format and transmit the data and 2 hydrophones that reconstruct in real-time the position of Optical Modules, for a maximum tower throughput of more than 600 MB/s. All floor data are collected by the Floor Control Module (FCM) board and transmitted by optical bidirectional virtual point-to-point connections to the on-shore laboratory, each FCM needing an on-shore counterpart as communication endpoint. In this contribution we present NaNet3, an on-shore readout board based on Altera Stratix V GX FPGA able to manage multiple FCM data channels with a capability of 800 Mbps each. The design is a NaNet customization for the KM3NeT-Italia experiment, adding support in its I/O interface for a synchronous link protocol with deterministic latency at physical level and for a Time Division Multiplexing protocol at data level.

Breast density quantification using magnetic resonance imaging (MRI) with bias field correction: A postmortem study

PubMed Central

Ding, Huanjun; Johnson, Travis; Lin, Muqing; Le, Huy Q.; Ducote, Justin L.; Su, Min-Ying; Molloi, Sabee

2013-01-01

Purpose: Quantification of breast density based on three-dimensional breast MRI may provide useful information for the early detection of breast cancer. However, the field inhomogeneity can severely challenge the computerized image segmentation process. In this work, the effect of the bias field in breast density quantification has been investigated with a postmortem study. Methods: T1-weighted images of 20 pairs of postmortem breasts were acquired on a 1.5 T breast MRI scanner. Two computer-assisted algorithms were used to quantify the volumetric breast density. First, standard fuzzy c-means (FCM) clustering was used on raw images with the bias field present. Then, the coherent local intensity clustering (CLIC) method estimated and corrected the bias field during the iterative tissue segmentation process. Finally, FCM clustering was performed on the bias-field-corrected images produced by CLIC method. The left–right correlation for breasts in the same pair was studied for both segmentation algorithms to evaluate the precision of the tissue classification. Finally, the breast densities measured with the three methods were compared to the gold standard tissue compositions obtained from chemical analysis. The linear correlation coefficient, Pearson's r, was used to evaluate the two image segmentation algorithms and the effect of bias field. Results: The CLIC method successfully corrected the intensity inhomogeneity induced by the bias field. In left–right comparisons, the CLIC method significantly improved the slope and the correlation coefficient of the linear fitting for the glandular volume estimation. The left–right breast density correlation was also increased from 0.93 to 0.98. When compared with the percent fibroglandular volume (%FGV) from chemical analysis, results after bias field correction from both the CLIC the FCM algorithms showed improved linear correlation. As a result, the Pearson's r increased from 0.86 to 0.92 with the bias field correction. Conclusions: The investigated CLIC method significantly increased the precision and accuracy of breast density quantification using breast MRI images by effectively correcting the bias field. It is expected that a fully automated computerized algorithm for breast density quantification may have great potential in clinical MRI applications. PMID:24320536

Breast density quantification using magnetic resonance imaging (MRI) with bias field correction: a postmortem study.

PubMed

Ding, Huanjun; Johnson, Travis; Lin, Muqing; Le, Huy Q; Ducote, Justin L; Su, Min-Ying; Molloi, Sabee

2013-12-01

Quantification of breast density based on three-dimensional breast MRI may provide useful information for the early detection of breast cancer. However, the field inhomogeneity can severely challenge the computerized image segmentation process. In this work, the effect of the bias field in breast density quantification has been investigated with a postmortem study. T1-weighted images of 20 pairs of postmortem breasts were acquired on a 1.5 T breast MRI scanner. Two computer-assisted algorithms were used to quantify the volumetric breast density. First, standard fuzzy c-means (FCM) clustering was used on raw images with the bias field present. Then, the coherent local intensity clustering (CLIC) method estimated and corrected the bias field during the iterative tissue segmentation process. Finally, FCM clustering was performed on the bias-field-corrected images produced by CLIC method. The left-right correlation for breasts in the same pair was studied for both segmentation algorithms to evaluate the precision of the tissue classification. Finally, the breast densities measured with the three methods were compared to the gold standard tissue compositions obtained from chemical analysis. The linear correlation coefficient, Pearson's r, was used to evaluate the two image segmentation algorithms and the effect of bias field. The CLIC method successfully corrected the intensity inhomogeneity induced by the bias field. In left-right comparisons, the CLIC method significantly improved the slope and the correlation coefficient of the linear fitting for the glandular volume estimation. The left-right breast density correlation was also increased from 0.93 to 0.98. When compared with the percent fibroglandular volume (%FGV) from chemical analysis, results after bias field correction from both the CLIC the FCM algorithms showed improved linear correlation. As a result, the Pearson's r increased from 0.86 to 0.92 with the bias field correction. The investigated CLIC method significantly increased the precision and accuracy of breast density quantification using breast MRI images by effectively correcting the bias field. It is expected that a fully automated computerized algorithm for breast density quantification may have great potential in clinical MRI applications.

Aptamer-facilitated mass cytometry.

PubMed

Mironov, Gleb G; Bouzekri, Alexandre; Watson, Jessica; Loboda, Olga; Ornatsky, Olga; Berezovski, Maxim V

2018-05-01

Mass cytometry is a novel cell-by-cell analysis technique, which uses elemental tags instead of fluorophores. Sample cells undergo rapid ionization in inductively coupled plasma and the ionized elemental tags are then analyzed by means of time-of-flight mass spectrometry. Benefits of the mass cytometry approach are in no need for compensation, the high number of detection channels (up to 100) and low background noise. In this work, we applied a biotinylated aptamer against human PTK7 receptor for characterization of positive (human acute lymphoblastic leukemia) and negative (human Burkitt's lymphoma) cells by a mass cytometry instrument. Our proof of principal experiments showed that biotinylated aptamers in conjunction with metal-labeled neutravidin can be successfully utilized for mass cytometry experiments at par with commercially available antibodies. Graphical abstract Biotinylated aptamers in conjunction with metal-labeled neutravidin bind to cell biomarkers, and then injected into the inductively coupled plasma (ICP) source, where cells are vaporized, atomized, and ionized in the plasma for subsequent mass spectrometry (MS) analysis of lanthanide metals.

A Method for the Interpretation of Flow Cytometry Data Using Genetic Algorithms.

PubMed

Angeletti, Cesar

2018-01-01

Flow cytometry analysis is the method of choice for the differential diagnosis of hematologic disorders. It is typically performed by a trained hematopathologist through visual examination of bidimensional plots, making the analysis time-consuming and sometimes too subjective. Here, a pilot study applying genetic algorithms to flow cytometry data from normal and acute myeloid leukemia subjects is described. Initially, Flow Cytometry Standard files from 316 normal and 43 acute myeloid leukemia subjects were transformed into multidimensional FITS image metafiles. Training was performed through introduction of FITS metafiles from 4 normal and 4 acute myeloid leukemia in the artificial intelligence system. Two mathematical algorithms termed 018330 and 025886 were generated. When tested against a cohort of 312 normal and 39 acute myeloid leukemia subjects, both algorithms combined showed high discriminatory power with a receiver operating characteristic (ROC) curve of 0.912. The present results suggest that machine learning systems hold a great promise in the interpretation of hematological flow cytometry data.

Membrane microparticles mediate transfer of P-glycoprotein to drug sensitive cancer cells.

PubMed

Bebawy, M; Combes, V; Lee, E; Jaiswal, R; Gong, J; Bonhoure, A; Grau, G E R

2009-09-01

Multidrug resistance (MDR), a significant impediment to the successful treatment of cancer clinically, has been attributed to the overexpression of P-glycoprotein (P-gp), a plasma membrane multidrug efflux transporter. P-gp maintains sublethal intracellular drug concentrations by virtue of its drug efflux capacity. The cellular regulation of P-gp expression is currently known to occur at either pre- or post-transcriptional levels. In this study, we identify a 'non-genetic' mechanism whereby microparticles (MPs) serve as vectors in the acquisition and spread of MDR. MPs isolated from drug-resistant cancer cells (VLB(100)) were co-cultured with drug sensitive cells (CCRF-CEM) over a 4 h period to allow for MP binding and P-gp transfer. Presence of P-gp on MPs was established using flow cytometry (FCM) and western blotting. Whole-cell drug accumulation assays using rhodamine 123 and daunorubicin (DNR) were carried out to validate the transfer of functional P-gp after co-culture. We establish that MPs shed in vitro from drug-resistant cancer cells incorporate cell surface P-gp from their donor cells, effectively bind to drug-sensitive recipient cells and transfer functional P-gp to the latter. These findings serve to substantially advance our understanding of the molecular basis for the emergence of MDR in cancer clinically and lead to new treatment strategies which target and inhibit MP mediated transfer of P-gp during the course of treatment.

Magnetic iron oxide nanoparticles mediated gene therapy for breast cancer--an in vitro study.

PubMed

Wei, Weizhong; Xu, Chunfang; Wu, Hua

2006-01-01

The aim of this study was to evaluate the feasibility and efficacy of using TRAIL gene to treat breast cancer mediated with a novel carrier - magnetic iron oxide nanoparticles (poly-MAG-1000) coated with PEI. The magnetic iron oxide nanoparticles were used as gene carrier to transfect TRAIL gene into MCF-7 cells. The polyMAG-1000 without TRAIL gene was transfected into the tumor cells as negative control. TRAIL gene transfection with liposome as carrier served as positive control. The apoptosis of cells was detected with TUNEL method. The apoptosis ratio of tumor cells was measured with flow cytometry (FCM). It was found that the apoptosis occurred in the tumor cells after transfection of TRAIL gene mediated by both polyMAG-1000 and liposome. The apoptosis ratio in the group with polyMAG-1000 as gene carrier was (25.11+/-2.85) %, whereas it was (5.06+/- 1.05) % in the control group with polyMAG-1000 (P<0.01). The apoptosis ratio was as low as (18.31+/-2.44) % in the group with liposome as gene carrier (P<0.05, as compared with the group with polyMAG-1000 as gene carrier). It is suggested that TRAIL gene may induce apoptosis in MCF-7 breast cancer cells. The magnetic iron oxide nanoparticles coated with PEI may be a potential gene carrier with high transfection efficacy for cancer gene therapy..

A novel modulation of structural and functional changes of mouse bone marrow derived dendritic cells (BMDCs) by interleukin-2(IL-2).

PubMed

Hu, Xiaofang; Cao, Yan; Meng, Yiming; Hou, Mingxiao

2015-01-01

IL-2 is a pleiotropic cytokine produced by T cell after antigen activation of T cell and it is so called T cell growth factor. A large number of documents suggest that Il-2 plays pivotal roles in the immune response and now Il-2 is an approved drug being used for various kinds of diseases such as cancer and dermatitis. (1) The aim of present exploration was to look at effect of IL-2 on structural, phenotypic and functional maturation of murine BMDCs. The structural and phenotypic maturation of BMDCs under influence of IL-2 were evaluated by light microscope and flow cytometry (FCM). The functional maturation of BMDCs was confirmed by cytochemistry assay, FITC-dextran, acid phosphatase (ACP) activity, bio-assay and enzyme linked immunosorbent assay (ELISA).We elucidated that IL-2 up-regulated the expression of key surface markers such as: CD80, CD83, CD86, CD40 and MHC II molecules on BMDCs, down-regulated phagocytosis activity, induced more production of IL-12 and TNF-α secreted by BMDCs. Therefore it can be concluded that IL-2 effectively enhance the maturation of BMDCs. Our results provide direct evidence to support IL-2 would be used as a potent adjuvant in preparation of DC-based vaccines, as well as an immune remedy for cancer situation.

Using Gelatin Nanoparticle Mediated Intranasal Delivery of Neuropeptide Substance P to Enhance Neuro-Recovery in Hemiparkinsonian Rats.

PubMed

Zhao, Ying-Zheng; Jin, Rong-Rong; Yang, Wei; Xiang, Qi; Yu, Wen-Ze; Lin, Qian; Tian, Fu-Rong; Mao, Kai-Li; Lv, Chuan-Zhu; Wáng, Yi-Xiáng J; Lu, Cui-Tao

2016-01-01

Intranasal administration of phospholipid-based gelatin nanoparticles (GNP) was prepared to investigate the neuro-recovery effects of neuropeptide Substance P (SP) on hemiparkinsonian rats. The SP-loaded gelatin nanoparticles (SP-GNP) were prepared by a water-in-water emulsion method and possessed high stability, encapsulating efficiency and loading capacity. PC-12 cells were used to examine the growth enhancement of SP-GNP in vitro by MTT assays and flow cytometry (FCM). The therapeutic effects of SP-GNP on 6-hydroxydopamine (6-OHDA) induced hemiparkinsonian rats were assessed by quantifying rotational behavior and the levels of tyrosine hydroxylase (TH), phosphorylated c-Jun protein (p-c-Jun) and Caspase-3 (Cas-3) expressed in substantia nigra (SN) region of hemiparkinsonian rats. PC-12 cells under SP-GNP treatment showed better cell viability and lower degree of apoptosis than those under SP solution treatment. Hemiparkinsonian rats under intranasal SP-GNP administration demonstrated better behavioral improvement, higher level of TH in SN along with much lower extent of p-c-Jun and Cas-3 than those under intranasal SP solution administration and intravenous SP-GNP administration. With the advantages of GNP and nose-to-brain pathway, SP can be effectively delivered into the damaged SN region and exhibit its neuro-recovery function through the inhibition on JNK pathway and dopaminergic neuron apoptosis.

The cascaded moving k-means and fuzzy c-means clustering algorithms for unsupervised segmentation of malaria images

NASA Astrophysics Data System (ADS)

Abdul-Nasir, Aimi Salihah; Mashor, Mohd Yusoff; Halim, Nurul Hazwani Abd; Mohamed, Zeehaida

2015-05-01

Malaria is a life-threatening parasitic infectious disease that corresponds for nearly one million deaths each year. Due to the requirement of prompt and accurate diagnosis of malaria, the current study has proposed an unsupervised pixel segmentation based on clustering algorithm in order to obtain the fully segmented red blood cells (RBCs) infected with malaria parasites based on the thin blood smear images of P. vivax species. In order to obtain the segmented infected cell, the malaria images are first enhanced by using modified global contrast stretching technique. Then, an unsupervised segmentation technique based on clustering algorithm has been applied on the intensity component of malaria image in order to segment the infected cell from its blood cells background. In this study, cascaded moving k-means (MKM) and fuzzy c-means (FCM) clustering algorithms has been proposed for malaria slide image segmentation. After that, median filter algorithm has been applied to smooth the image as well as to remove any unwanted regions such as small background pixels from the image. Finally, seeded region growing area extraction algorithm has been applied in order to remove large unwanted regions that are still appeared on the image due to their size in which cannot be cleaned by using median filter. The effectiveness of the proposed cascaded MKM and FCM clustering algorithms has been analyzed qualitatively and quantitatively by comparing the proposed cascaded clustering algorithm with MKM and FCM clustering algorithms. Overall, the results indicate that segmentation using the proposed cascaded clustering algorithm has produced the best segmentation performances by achieving acceptable sensitivity as well as high specificity and accuracy values compared to the segmentation results provided by MKM and FCM algorithms.

A review of fuzzy cognitive maps in medicine: Taxonomy, methods, and applications.

PubMed

Amirkhani, Abdollah; Papageorgiou, Elpiniki I; Mohseni, Akram; Mosavi, Mohammad R

2017-04-01

A high percentage of medical errors, committed because of physician's lack of experience, huge volume of data to be analyzed, and inaccessibility to medical records of previous patients, can be reduced using computer-aided techniques. Therefore, designing more efficient medical decision-support systems (MDSSs) to assist physicians in decision-making is crucially important. Through combining the properties of fuzzy logic and neural networks, fuzzy cognitive maps (FCMs) are among the latest, most efficient, and strongest artificial intelligence techniques for modeling complex systems. This review study is conducted to identify different FCM structures used in MDSS designs. The best structure for each medical application can be introduced by studying the properties of FCM structures. This paper surveys the most important decision- making methods and applications of FCMs in the medical field in recent years. To investigate the efficiency and capability of different FCM models in designing MDSSs, medical applications are categorized into four key areas: decision-making, diagnosis, prediction, and classification. Also, various diagnosis and decision support problems addressed by FCMs in recent years are reviewed with the goal of introducing different types of FCMs and determining their contribution to the improvements made in the fields of medical diagnosis and treatment. In this survey, a general trend for future studies in this field is provided by analyzing various FCM structures used for medical purposes, and the results from each category. Due to the unique specifications of FCMs in integrating human knowledge and experience with computer-aided techniques, they are among practical instruments for MDSS design. In the not too distant future, they will have a significant role in medical sciences. Copyright © 2017 Elsevier B.V. All rights reserved.

Environmental and Intrinsic Correlates of Stress in Free-Ranging Wolves

PubMed Central

Molnar, Barbara; Fattebert, Julien; Palme, Rupert; Ciucci, Paolo; Betschart, Bruno; Smith, Douglas W.; Diehl, Peter-Allan

2015-01-01

Background When confronted with a stressor, animals react with several physiological and behavioral responses. Although sustained or repeated stress can result in severe deleterious physiological effects, the causes of stress in free-ranging animals are yet poorly documented. In our study, we aimed at identifying the main factors affecting stress levels in free-ranging wolves (Canis lupus). Methodology/Principal Findings We used fecal cortisol metabolites (FCM) as an index of stress, after validating the method for its application in wolves. We analyzed a total of 450 fecal samples from eleven wolf packs belonging to three protected populations, in Italy (Abruzzo), France (Mercantour), and the United States (Yellowstone). We collected samples during two consecutive winters in each study area. We found no relationship between FCM concentrations and age, sex or social status of individuals. At the group level, our results suggest that breeding pair permanency and the loss of pack members through processes different from dispersal may importantly impact stress levels in wolves. We measured higher FCM levels in comparatively small packs living in sympatry with a population of free-ranging dogs. Lastly, our results indicate that FCM concentrations are associated with endoparasitic infections of individuals. Conclusions/Significance In social mammals sharing strong bonds among group members, the death of one or several members of the group most likely induces important stress in the remainder of the social unit. The potential impact of social and territorial stability on stress levels should be further investigated in free-ranging populations, especially in highly social and in territorial species. As persistent or repeated stressors may facilitate or induce pathologies and physiological alterations that can affect survival and fitness, we advocate considering the potential impact of anthropogenic causes of stress in management and conservation programs regarding wolves and other wildlife. PMID:26398784

Environmental and Intrinsic Correlates of Stress in Free-Ranging Wolves.

PubMed

Molnar, Barbara; Fattebert, Julien; Palme, Rupert; Ciucci, Paolo; Betschart, Bruno; Smith, Douglas W; Diehl, Peter-Allan

2015-01-01

When confronted with a stressor, animals react with several physiological and behavioral responses. Although sustained or repeated stress can result in severe deleterious physiological effects, the causes of stress in free-ranging animals are yet poorly documented. In our study, we aimed at identifying the main factors affecting stress levels in free-ranging wolves (Canis lupus). We used fecal cortisol metabolites (FCM) as an index of stress, after validating the method for its application in wolves. We analyzed a total of 450 fecal samples from eleven wolf packs belonging to three protected populations, in Italy (Abruzzo), France (Mercantour), and the United States (Yellowstone). We collected samples during two consecutive winters in each study area. We found no relationship between FCM concentrations and age, sex or social status of individuals. At the group level, our results suggest that breeding pair permanency and the loss of pack members through processes different from dispersal may importantly impact stress levels in wolves. We measured higher FCM levels in comparatively small packs living in sympatry with a population of free-ranging dogs. Lastly, our results indicate that FCM concentrations are associated with endoparasitic infections of individuals. In social mammals sharing strong bonds among group members, the death of one or several members of the group most likely induces important stress in the remainder of the social unit. The potential impact of social and territorial stability on stress levels should be further investigated in free-ranging populations, especially in highly social and in territorial species. As persistent or repeated stressors may facilitate or induce pathologies and physiological alterations that can affect survival and fitness, we advocate considering the potential impact of anthropogenic causes of stress in management and conservation programs regarding wolves and other wildlife.

Evaluation of radioisotopic and non-radioisotopic versions of local lymph node assays for subcategorization of skin sensitizers compliant to UN GHS rev 4.

PubMed

Ha, Soojin; Ahn, Il Young; Kim, Da-Eun; Lee, Jong Kwon; Sohn, Soojung; Jung, Mi-Sook; Heo, Yong; Omori, Takashi; Bae, SeungJin; Lim, Kyung-Min

2017-04-01

Recently UN GHS has introduced the sub-categorization of skin sensitizers for which ECt (concentration estimated to induce stimulation index above threshold) of the murine local lymph node assay (LLNA) is used as criteria. Non-radioisotopic variants of LLNA, LLNA: DA, LLNA: BrdU-ELISA, LNCC and LLNA: BrdU-FCM were developed yet their utilities for potency sub-categorization are not established. Here we assessed the agreement of LLNA variants with LLNA or human data in potency sub-categorization for 22 reference substances of OECD TG429. Concordance of sub-categorization with LLNA was highest for LLNA: BrdU-FCM(91%, κ = 0.833, weighted kappa) followed by LLNA: BrdU-ELISA (82%, κ = 0.744) and LLNA: DA (73%, κ = 0.656) whereas LNCC only showed a modest association (64%, κ = 0.441). With human data, LLNA agreed best (77%) followed by LLNA: DA and LLNA: BrdU-FCM(73%), LLNA: BrdU-ELISA (68%) and LNCC(55%). Bland-Altman plot revealed that ECt's of LLNA variants largely agreed with LLNA where most values fell within 95% limit of agreement. Correlation between ECt's of LLNA and LLNA variants were high except for LNCC(pair-wise with LLNA, LLNA: DA, r = 0.848, LLNA: BrdU-ELISA, r = 0.744, LLNA: BrdU-FCM, r=0.786, and LNCC, r = 0.561 by Pearson). Collectively, these results demonstrated that LLNA variants exhibit performance comparable to LLNA in the potency sub-categorization although additional substances shall be analyzed in the future. Copyright © 2017 Elsevier Inc. All rights reserved.

Excretion and measurement of corticosterone and testosterone metabolites in bank voles (Myodes glareolus).

PubMed

Sipari, Saana; Ylönen, Hannu; Palme, Rupert

2017-03-01

The bank vole is a commonly used model species in behavioral and ecophysiological studies. Thus, presenting a validated method for noninvasive monitoring of corticosterone and testosterone secretion is of high relevance. Here, we evaluated the effect of time of day and an ACTH challenge test on measured fecal corticosterone (FCM) and testosterone (FTM) metabolites in both sexes. Furthermore, we performed radiometabolism experiments for both steroids and sexes to study metabolism and excretion of 3 H-corticosterone and 3 H-testosterone. FCM and FTM were analysed with a 5α-pregnane-3β,11β,21-triol-20-one enzyme immunoassay (EIA) and a testosterone (measuring 17β-hydroxyandrostanes) EIA, respectively. Males had significantly higher FCM levels than females and their main excretion route was via the feces (∼72%), whereas females excreted nearly equal portions in both feces and urine. For testosterone the main excretion route was via the feces in both sexes (∼80%). The time course of excretion was similar in both sexes, but for the first time a significant difference between injected steroids was found: Corticosterone was excreted faster than testosterone, both in urine (median of peak levels: 4h vs 6h) and feces (6h vs 8h). Several metabolites were present in the feces and the tested EIAs reacted with some of them. Time of day had a significant effect on measured fecal steroid metabolites. As expected, males had significantly higher FTM levels than females. ACTH administration significantly increased FCM values; peaks were observed 4-8h after injection. In conclusion, both tested EIAs proved suited for a noninvasive measurement of glucocorticoids and androgens in bank voles. Copyright © 2016 Elsevier Inc. All rights reserved.

Critical Role of CD8 T Cells in Mediating Sex-Based Differences in a Murine Model of Lupus

DTIC Science & Technology

2009-08-21

into  female  transfers  (fF)  mice  that  was  reduced  in  CD8  depleted  fF  mice.  Flow   cytometry   analysis  showed increased numbers of splenic...splenocytes were first analyzed by flow cytometry for CD4 and CD8 T cells and F1 mice received either: a) unfractionated splenocytes (CD8 intactF1...using magnetic beads purchased from Invitrogen (Carlsbad, CA) according to the manufacturer’s instructions. Flow cytometry analysis before cell

Multinode acoustic focusing for parallel flow cytometry

PubMed Central

Piyasena, Menake E.; Suthanthiraraj, Pearlson P. Austin; Applegate, Robert W.; Goumas, Andrew M.; Woods, Travis A.; López, Gabriel P.; Graves, Steven W.

2012-01-01

Flow cytometry can simultaneously measure and analyze multiple properties of single cells or particles with high sensitivity and precision. Yet, conventional flow cytometers have fundamental limitations with regards to analyzing particles larger than about 70 microns, analyzing at flow rates greater than a few hundred microliters per minute, and providing analysis rates greater than 50,000 per second. To overcome these limits, we have developed multi-node acoustic focusing flow cells that can position particles (as small as a red blood cell and as large as 107 microns in diameter) into as many as 37 parallel flow streams. We demonstrate the potential of such flow cells for the development of high throughput, parallel flow cytometers by precision focusing of flow cytometry alignment microspheres, red blood cells, and the analysis of CD4+ cellular immunophenotyping assay. This approach will have significant impact towards the creation of high throughput flow cytometers for rare cell detection applications (e.g. circulating tumor cells), applications requiring large particle analysis, and high volume flow cytometry. PMID:22239072

Masks in Imaging Flow Cytometry

PubMed Central

Dominical, Venina; Samsel, Leigh; McCoy, J. Philip

2016-01-01

Data analysis in imaging flow cytometry incorporates elements of flow cytometry together with other aspects of morphological analysis of images. A crucial early step in this analysis is the creation of a mask to distinguish the portion of the image upon which further examination of specified features can be performed. Default masks are provided by the manufacturer of the imaging flow cytometer but additional custom masks can be created by the individual user for specific applications. Flawed or inaccurate masks can have a substantial negative impact on the overall analysis of a sample, thus great care must be taken to ensure the accuracy of masks. Here we discuss various types of masks and cite examples of their use. Furthermore we provide our insight for how to approach selecting and assessing the optimal mask for a specific analysis. PMID:27461256

Hybrid approach for detection of dental caries based on the methods FCM and level sets

NASA Astrophysics Data System (ADS)

Chaabene, Marwa; Ben Ali, Ramzi; Ejbali, Ridha; Zaied, Mourad

2017-03-01

This paper presents a new technique for detection of dental caries that is a bacterial disease that destroys the tooth structure. In our approach, we have achieved a new segmentation method that combines the advantages of fuzzy C mean algorithm and level set method. The results obtained by the FCM algorithm will be used by Level sets algorithm to reduce the influence of the noise effect on the working of each of these algorithms, to facilitate level sets manipulation and to lead to more robust segmentation. The sensitivity and specificity confirm the effectiveness of proposed method for caries detection.

Query by example video based on fuzzy c-means initialized by fixed clustering center

NASA Astrophysics Data System (ADS)

Hou, Sujuan; Zhou, Shangbo; Siddique, Muhammad Abubakar

2012-04-01

Currently, the high complexity of video contents has posed the following major challenges for fast retrieval: (1) efficient similarity measurements, and (2) efficient indexing on the compact representations. A video-retrieval strategy based on fuzzy c-means (FCM) is presented for querying by example. Initially, the query video is segmented and represented by a set of shots, each shot can be represented by a key frame, and then we used video processing techniques to find visual cues to represent the key frame. Next, because the FCM algorithm is sensitive to the initializations, here we initialized the cluster center by the shots of query video so that users could achieve appropriate convergence. After an FCM cluster was initialized by the query video, each shot of query video was considered a benchmark point in the aforesaid cluster, and each shot in the database possessed a class label. The similarity between the shots in the database with the same class label and benchmark point can be transformed into the distance between them. Finally, the similarity between the query video and the video in database was transformed into the number of similar shots. Our experimental results demonstrated the performance of this proposed approach.

Exposure to airborne asbestos in thermal power plants in Mongolia

PubMed Central

Damiran, Naransukh; Silbergeld, Ellen K; Frank, Arthur L; Lkhasuren, Oyuntogos; Ochir, Chimedsuren; Breysse, Patrick N

2015-01-01

Background: Coal-fired thermal power plants (TPPs) in Mongolia use various types of asbestos-containing materials (ACMs) in thermal insulation of piping systems, furnaces, and other products. Objective: To investigate the occupational exposure of insulation workers to airborne asbestos in Mongolian power plants. Methods: Forty-seven air samples were collected from four power plants in Mongolia during the progress of insulation work. The samples were analyzed by phase contrast microscopy (PCM) and transmission electron microscopy (TEM). Results: The average phase contrast microscopy equivalent (PCME) asbestos fiber concentration was 0.93 f/cm3. Sixteen of the 41 personal and one of the area samples exceeded the United States Occupational Safety and Health Administration (US OSHA) short-term exposure limit of 1.0 f/cm3. If it is assumed that the short-term samples collected are representative of full-shift exposure, then the exposures are approximately 10 times higher than the US OSHA 8-hour permissible exposure limit of 0.1 f/cm3. Conclusion: Power plant insulation workers are exposed to airborne asbestos at concentrations that exceed the US OSHA Permissible Exposure Limit. Action to mitigate the risks should be taken in Mongolia. PMID:25730489

Exposure to airborne asbestos in thermal power plants in Mongolia.

PubMed

Damiran, Naransukh; Silbergeld, Ellen K; Frank, Arthur L; Lkhasuren, Oyuntogos; Ochir, Chimedsuren; Breysse, Patrick N

2015-01-01

Coal-fired thermal power plants (TPPs) in Mongolia use various types of asbestos-containing materials (ACMs) in thermal insulation of piping systems, furnaces, and other products. To investigate the occupational exposure of insulation workers to airborne asbestos in Mongolian power plants. Forty-seven air samples were collected from four power plants in Mongolia during the progress of insulation work. The samples were analyzed by phase contrast microscopy (PCM) and transmission electron microscopy (TEM). The average phase contrast microscopy equivalent (PCME) asbestos fiber concentration was 0·93 f/cm(3). Sixteen of the 41 personal and one of the area samples exceeded the United States Occupational Safety and Health Administration (US OSHA) short-term exposure limit of 1·0 f/cm(3). If it is assumed that the short-term samples collected are representative of full-shift exposure, then the exposures are approximately 10 times higher than the US OSHA 8-hour permissible exposure limit of 0·1 f/cm(3). Power plant insulation workers are exposed to airborne asbestos at concentrations that exceed the US OSHA Permissible Exposure Limit. Action to mitigate the risks should be taken in Mongolia.

Assessing framing assumptions in quantitative health impact assessments: a housing intervention example.

PubMed

Mesa-Frias, Marco; Chalabi, Zaid; Foss, Anna M

2013-09-01

Health impact assessment (HIA) is often used to determine ex ante the health impact of an environmental policy or an environmental intervention. Underpinning any HIA is the framing assumption, which defines the causal pathways mapping environmental exposures to health outcomes. The sensitivity of the HIA to the framing assumptions is often ignored. A novel method based on fuzzy cognitive map (FCM) is developed to quantify the framing assumptions in the assessment stage of a HIA, and is then applied to a housing intervention (tightening insulation) as a case-study. Framing assumptions of the case-study were identified through a literature search of Ovid Medline (1948-2011). The FCM approach was used to identify the key variables that have the most influence in a HIA. Changes in air-tightness, ventilation, indoor air quality and mould/humidity have been identified as having the most influence on health. The FCM approach is widely applicable and can be used to inform the formulation of the framing assumptions in any quantitative HIA of environmental interventions. We argue that it is necessary to explore and quantify framing assumptions prior to conducting a detailed quantitative HIA during the assessment stage. Copyright © 2013 Elsevier Ltd. All rights reserved.

Metabolic and endocrine profiles and reproductive parameters in dairy cows under grazing conditions: effect of polymorphisms in somatotropic axis genes

PubMed Central

2011-01-01

Background The present study hypothesized that GH-AluI and IGF-I-SnabI polymorphisms do change the metabolic/endocrine profiles in Holstein cows during the transition period, which in turn are associated with productive and reproductive parameters. Methods Holstein cows (Farm 1, primiparous cows, n = 110, and Farm 2, multiparous cows, n = 76) under grazing conditions were selected and GH and IGF-I genotypes were determined. Blood samples for metabolic/endocrine determinations were taken during the transition period and early lactation in both farms. Data was analyzed by farm using a repeated measures analyses including GH and IGF-I genotypes, days and interactions as fixed effects, sire and cow as random effects and calving date as covariate. Results and Discussion Frequencies of GH and IGF-I alleles were L:0.84, V:0.16 and A:0.60, B:0.40, respectively. The GH genotype was not associated with productive or reproductive variables, but interaction with days affected FCM yield in multiparous (farm 2) cows (LL yielded more than LV cows) in early lactation. The GH genotype affected NEFA and IGF-I concentrations in farm 1 (LV had higher NEFA and lower IGF-I than LL cows) suggesting a better energy status of LL cows. There was no effect of IGF-I genotype on productive variables, but a trend was found for FCM in farm 2 (AB cows yielded more than AA cows). IGF-I genotype affected calving first service interval in farm 1, and the interaction with days tended to affect FCM yield (AB cows had a shorter interval and yielded more FCM than BB cows). IGF-I genotype affected BHB, NEFA, and insulin concentrations in farm 1: primiparous BB cows had lower NEFA and BHB and higher insulin concentrations. In farm 2, there was no effect of IGF-I genotype, but there was an interaction with days on IGF-I concentration, suggesting a greater uncoupling somatropic axis in AB and BB than AA cows, being in accordance with greater FCM yield in AB cows. Conclusion The GH and IGF-I genotypes had no substantial effect on productive parameters, although IGF-I genotype affected calving-first service interval in primiparous cows. Besides, these genotypes may modify the endocrine/metabolic profiles of the transition dairy cow under grazing conditions. PMID:21635772

Design, fabrication, and evaluation of on-chip micro-supercapacitors

NASA Astrophysics Data System (ADS)

Beidaghi, Majid

Due to the increasing demand for high power and reliable miniaturized energy storage devices, the development of micro-supercapacitors or electrochemical micro-capacitors have attracted much attention in recent years. This dissertation investigates several strategies to develop on-chip micro-supercapacitors with high power and energy density. Micro-supercapacitors based on interdigitated carbon micro-electrode arrays are fabricated through carbon microelectromechanical systems (C-MEMS) technique which is based on carbonization of patterned photoresist. To improve the capacitive behavior, electrochemical activation is performed on carbon micro-electrode arrays. The developed micro-supercapacitors show specific capacitances as high as 75 mFcm-2 at a scan rate of 5 mVs -1 after electrochemical activation for 30 minutes. The capacitance loss is less than 13% after 1000 cyclic voltammetry (CV) cycles. These results indicate that electrochemically activated C-MEMS micro-electrode arrays are promising candidates for on-chip electrochemical micro-capacitor applications. The energy density of micro-supercapacitors was further improved by conformal coating of polypyrrole (PPy) on C-MEMS structures. In these types of micro-devices the three dimensional (3D) carbon microstructures serve as current collectors for high energy density PPy electrodes. The electrochemical characterizations of these micro-supercapacitors show that they can deliver a specific capacitance of about 162.07 mFcm-2 and a specific power of 1.62mWcm -2 at a 20 mVs-1 scan rate. Addressing the need for high power micro-supercapacitors, the application of graphene as electrode materials for micro-supercapacitor was also investigated. The present study suggests a novel method to fabricate graphene-based micro-supercapacitors with thin film or in-plane interdigital electrodes. The fabricated micro-supercapacitors show exceptional frequency response and power handling performance and could effectively charge and discharge at rates as high as 50 Vs-1. CV measurements show that the specific capacitance of the micro-supercapacitor based on reduced graphene oxide and carbon nanotube composites is 6.1 mFcm -2 at scan rate of 0.01Vs-1. At a very high scan rate of 50 Vs-1, a specific capacitance of 2.8 mFcm-2 (stack capacitance of 3.1 Fcm-3) is recorded. This unprecedented performance can potentially broaden the future applications of micro-supercapacitors.

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

One-dimensional acoustic standing waves in rectangular channels for flow cytometry.

PubMed

Austin Suthanthiraraj, Pearlson P; Piyasena, Menake E; Woods, Travis A; Naivar, Mark A; Lόpez, Gabriel P; Graves, Steven W

2012-07-01

Flow cytometry has become a powerful analytical tool for applications ranging from blood diagnostics to high throughput screening of molecular assemblies on microsphere arrays. However, instrument size, expense, throughput, and consumable use limit its use in resource poor areas of the world, as a component in environmental monitoring, and for detection of very rare cell populations. For these reasons, new technologies to improve the size and cost-to-performance ratio of flow cytometry are required. One such technology is the use of acoustic standing waves that efficiently concentrate cells and particles to the center of flow channels for analysis. The simplest form of this method uses one-dimensional acoustic standing waves to focus particles in rectangular channels. We have developed one-dimensional acoustic focusing flow channels that can be fabricated in simple capillary devices or easily microfabricated using photolithography and deep reactive ion etching. Image and video analysis demonstrates that these channels precisely focus single flowing streams of particles and cells for traditional flow cytometry analysis. Additionally, use of standing waves with increasing harmonics and in parallel microfabricated channels is shown to effectively create many parallel focused streams. Furthermore, we present the fabrication of an inexpensive optical platform for flow cytometry in rectangular channels and use of the system to provide precise analysis. The simplicity and low-cost of the acoustic focusing devices developed here promise to be effective for flow cytometers that have reduced size, cost, and consumable use. Finally, the straightforward path to parallel flow streams using one-dimensional multinode acoustic focusing, indicates that simple acoustic focusing in rectangular channels may also have a prominent role in high-throughput flow cytometry. Copyright © 2012 Elsevier Inc. All rights reserved.

qFlow Cytometry-Based Receptoromic Screening: A High-Throughput Quantification Approach Informing Biomarker Selection and Nanosensor Development.

PubMed

Chen, Si; Weddell, Jared; Gupta, Pavan; Conard, Grace; Parkin, James; Imoukhuede, Princess I

2017-01-01

Nanosensor-based detection of biomarkers can improve medical diagnosis; however, a critical factor in nanosensor development is deciding which biomarker to target, as most diseases present several biomarkers. Biomarker-targeting decisions can be informed via an understanding of biomarker expression. Currently, immunohistochemistry (IHC) is the accepted standard for profiling biomarker expression. While IHC provides a relative mapping of biomarker expression, it does not provide cell-by-cell readouts of biomarker expression or absolute biomarker quantification. Flow cytometry overcomes both these IHC challenges by offering biomarker expression on a cell-by-cell basis, and when combined with calibration standards, providing quantitation of biomarker concentrations: this is known as qFlow cytometry. Here, we outline the key components for applying qFlow cytometry to detect biomarkers within the angiogenic vascular endothelial growth factor receptor family. The key aspects of the qFlow cytometry methodology include: antibody specificity testing, immunofluorescent cell labeling, saturation analysis, fluorescent microsphere calibration, and quantitative analysis of both ensemble and cell-by-cell data. Together, these methods enable high-throughput quantification of biomarker expression.

SCENERY: a web application for (causal) network reconstruction from cytometry data

PubMed Central

Papoutsoglou, Georgios; Athineou, Giorgos; Lagani, Vincenzo; Xanthopoulos, Iordanis; Schmidt, Angelika; Éliás, Szabolcs; Tegnér, Jesper

2017-01-01

Abstract Flow and mass cytometry technologies can probe proteins as biological markers in thousands of individual cells simultaneously, providing unprecedented opportunities for reconstructing networks of protein interactions through machine learning algorithms. The network reconstruction (NR) problem has been well-studied by the machine learning community. However, the potentials of available methods remain largely unknown to the cytometry community, mainly due to their intrinsic complexity and the lack of comprehensive, powerful and easy-to-use NR software implementations specific for cytometry data. To bridge this gap, we present Single CEll NEtwork Reconstruction sYstem (SCENERY), a web server featuring several standard and advanced cytometry data analysis methods coupled with NR algorithms in a user-friendly, on-line environment. In SCENERY, users may upload their data and set their own study design. The server offers several data analysis options categorized into three classes of methods: data (pre)processing, statistical analysis and NR. The server also provides interactive visualization and download of results as ready-to-publish images or multimedia reports. Its core is modular and based on the widely-used and robust R platform allowing power users to extend its functionalities by submitting their own NR methods. SCENERY is available at scenery.csd.uoc.gr or http://mensxmachina.org/en/software/. PMID:28525568

Impact of ferric carboxymaltose on the evolution of hemoglobin and ECOG performance status in iron-deficient patients with solid tumors: a 3-month follow-up retrospective study.

PubMed

Coussirou, J; Debourdeau, A; Stancu, A; Jean, C; Azouza, W; Chanet, B; De Crozals, F; Boustany, R; Debourdeau, P

2018-05-24

Anemia is often associated with a lower quality of life and less tolerance to treatments in cancer patients. The aims of this retrospective study were to assess the biological (hemoglobin, Hb) and clinical (ECOG index) impact of ferric carboxymaltose (FCM) and to identify predictive factors of response in cancer patients with iron deficiency. We included 133 patients with solid tumors who received at least one dose of FCM in 2015. At baseline, most patients had metastatic cancer (70%), were undergoing chemotherapy (82%), suffered from anemia (90%), and 72% had an ECOG 0-1 index. Mean Hb level was statistically higher at M1 (108.3 g/L ± 13.9), M2 (110.3 g/L ± 16.1), and M3 (111.7 g/L ± 12.6) than M0 (99.2 g/L ± 13.9). Mean ECOG score increased significantly at M1 (1.31 ± 0.80) and M2 (1.31 ± 0.87) compared to M0 (1.13 ± 0.80). Variations of ECOG index between M0 and M1 were independent of levels of Hb and ferritin at inclusion and pretreatment use of transfusion and ESAs. Increase of Hb level was higher in patients with Hb < 100 g/L, ferritinemia < 800 ng/ml, or transfused before inclusion. In multivariate analysis, an ECOG index of 0 was the only predictive factor of an increase of ECOG index and Hb level < 100 g/L and ferritinemia < 800 ng/ml were predictive of an increase in Hb. Even though there was no improvement in ECOG index, this study did identify an increase of Hb for patients receiving FCM, indicating its potential benefit in iron-deficient cancer patients.

Induction of apoptosis in human liver carcinoma HepG2 cell line by 5-allyl-7-gen-difluoromethylenechrysin.

PubMed

Tan, Xiang-Wen; Xia, Hong; Xu, Jin-Hua; Cao, Jian-Guo

2009-05-14

To investigate the effect of 5-allyl-7-gen-difluoromethylenechrysin (ADFMChR) on apoptosis of human liver carcinoma HepG2 cell line and the molecular mechanisms involved. HepG2 cells and L-02 cells were cultured in vitro and the inhibitory effect of ADFMChR on their proliferation was measured by MTT assay. The apoptosis of HepG2 cells was determined by flow cytometry (FCM) using propidium iodide (PI) fluorescence staining. DNA ladder bands were observed by DNA agarose gel electrophoresis. The influence of ADFMChR on the proxisome proliferator-activated receptor gamma (PPARgamma), NF-kappaB, Bcl-2 and Bax protein expression of HepG2 cells were analyzed by Western blotting. MTT assay showed that ADFMChR significantly inhibited proliferation of HepG2 cells in a dose-dependent manner, with little effect on growth of L-02 cells, and when IC(50) was measured as 8.45 micromol/L and 191.55 micromol/L respectively, the potency of ADFMChR to HepG2 cells, was found to be similar to 5-fluorouracil (5-FU, IC(50) was 9.27 micromol/L). The selective index of ADFMChR cytotoxicity to HepG2 cells was 22.67 (191.55/8.45), higher than 5-FU (SI was 7.05 (65.37/9.27). FCM with PI staining demonstrated that the apoptosis rates of HepG2 cells treated with 3.0, 10.0 and 30.0 micromol/L ADFMChR for 48 h were 5.79%, 9.29% and 37.8%, respectively, and were significantly higher when treated with 30.0 micromol/L ADFMChR than when treated with 30.0 micromol/L ChR (16.0%) (P < 0.05) and were similar to those obtained with 30.0 micromol/L 5-FU (41.0%). DNA agarose gel electrophoresis showed that treatment of HepG2 cells with 10.0 micromol/L ADFMChR for 48 h and 72 h resulted in typical DNA ladders which could be reversed by 10.00 micromol/L GW9662, a blocker of PPARgamma. Western blotting analysis revealed that after 24 h of treatment with 3.0, 10.0, 30.0 micromol/L ADFMChR, PPARgamma and Bax protein expression in HepG2 cells increased but Bcl-2 and NF-kappaB expression decreased; however, pre-incubation with 10.0 micromol/L GW9662 could efficiently antagonize and weaken the regulatory effect of 3.0, 30.0 micromol/L ADFMChR on PPARgamma and NF-kappaB protein expression in HepG2 cells. ADFMChR induces apoptosis of HepG2 cell lines by activating PPARgamma, inhibiting protein expression of Bcl-2 and NF-kappaB, and increasing Bax expression.

Single Cell Mass Cytometry for Analysis of Immune System Functional States

PubMed Central

Bjornson, Zach B.; Nolan, Garry P.; Fantl, Wendy J.

2013-01-01

Single cell mass cytometry facilitates high-dimensional, quantitative analysis of the effects of bioactive molecules on cell populations at single-cell resolution. Datasets are generated with antibody panels (upwards of 40) in which each antibody is conjugated to a polymer chelated with a stable metal isotope, usually in the Lanthanide series of the periodic table. Isotope labelled antibodies recognize surface markers to delineate cell types and intracellular signaling molecules to provide a measure of the network state—and thereby demarcating multiple cell state functions such as apoptosis, DNA damage and cell cycle. By measuring all these parameters simultaneously, the signaling state of an individual cell can be measured at its network state. This review will cover the basics of mass cytometry as well as outline steps already taken to allow it to stand aside traditional fluorescence based cytometry in the immunologist’s analytical arsenal in their study of immune states during infection. PMID:23999316

Genomic Instability at Premalignant and Early Stages of Breast Cancer Development

DTIC Science & Technology

1999-08-01

by routine DNA flow cytometry vation. ERBB2 expression was detected with a to determine DNA index (DI). commercially available antibody (Oncogene Sci...supplements the information gained from ic microsatellite primers. We observed that the ploidy analysis by DNA flow cytometry alone. In DNA so obtained...preserved the proportionality of many cases where flow cytometry could not be per- the different alleles as found in the original sample. formed because the

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

Multivariate data analysis methods for the interpretation of microbial flow cytometric data.

PubMed

Davey, Hazel M; Davey, Christopher L

2011-01-01

Flow cytometry is an important technique in cell biology and immunology and has been applied by many groups to the analysis of microorganisms. This has been made possible by developments in hardware that is now sensitive enough to be used routinely for analysis of microbes. However, in contrast to advances in the technology that underpin flow cytometry, there has not been concomitant progress in the software tools required to analyse, display and disseminate the data and manual analysis, of individual samples remains a limiting aspect of the technology. We present two new data sets that illustrate common applications of flow cytometry in microbiology and demonstrate the application of manual data analysis, automated visualisation (including the first description of a new piece of software we are developing to facilitate this), genetic programming, principal components analysis and artificial neural nets to these data. The data analysis methods described here are equally applicable to flow cytometric applications with other cell types.

In vivo integrated photoacoustic and confocal microscopy of hemoglobin oxygen saturation and oxygen partial pressure.

PubMed

Wang, Yu; Hu, Song; Maslov, Konstantin; Zhang, Yu; Xia, Younan; Wang, Lihong V

2011-04-01

We developed dual-modality microscope integrating photoacoustic microscopy (PAM) and fluorescence confocal microscopy (FCM) to noninvasively image hemoglobin oxygen saturation (sO₂) and oxygen partial pressure (pO₂) in vivo in single blood vessels with high spatial resolution. While PAM measures sO₂ by imaging hemoglobin optical absorption at two wavelengths, FCM quantifies pO₂ using phosphorescence quenching. The variations of sO₂ and pO₂ values in multiple orders of vessel branches under hyperoxic (100% oxygen) and normoxic (21% oxygen) conditions correlate well with the oxygen-hemoglobin dissociation curve. In addition, the total concentration of hemoglobin is imaged by PAM at an isosbestic wavelength.

Immunological tools: engaging students in the use and analysis of flow cytometry and enzyme-linked immunosorbent assay (ELISA).

PubMed

Ott, Laura E; Carson, Susan

2014-01-01

Flow cytometry and enzyme-linked immunosorbent assay (ELISA) are commonly used techniques associated with clinical and research applications within the immunology and medical fields. The use of these techniques is becoming increasingly valuable in many life science and engineering disciplines as well. Herein, we report the development and evaluation of a novel half-semester course that focused on introducing undergraduate and graduate students to advance conceptual and technical skills associated with flow cytometry and ELISA, with emphasis on applications, experimental design, and data analysis. This course was offered in the North Carolina State University Biotechnology Program over three semesters and consisted of weekly lectures and laboratories. Students performed and/or analyzed flow cytometry and ELISA in three separate laboratory exercises: (1) identification of transgenic zebrafish hematopoietic cells, (2) analysis of transfection efficiency, and (3) analysis of cytokine production upon lipopolysaccharide stimulation. Student learning outcomes were achieved as demonstrated by multiple means of assessment, including three laboratory reports, a data analysis laboratory practicum, and a cumulative final exam. Further, anonymous student self-assessment revealed increased student confidence in the knowledge and skill sets defined in the learning outcomes. Copyright © 2014 The International Union of Biochemistry and Molecular Biology.

Artificial intelligence techniques coupled with seasonality measures for hydrological regionalization of Q90 under Brazilian conditions

NASA Astrophysics Data System (ADS)

Beskow, Samuel; de Mello, Carlos Rogério; Vargas, Marcelle M.; Corrêa, Leonardo de L.; Caldeira, Tamara L.; Durães, Matheus F.; de Aguiar, Marilton S.

2016-10-01

Information on stream flows is essential for water resources management. The stream flow that is equaled or exceeded 90% of the time (Q90) is one the most used low stream flow indicators in many countries, and its determination is made from the frequency analysis of stream flows considering a historical series. However, stream flow gauging network is generally not spatially sufficient to meet the necessary demands of technicians, thus the most plausible alternative is the use of hydrological regionalization. The objective of this study was to couple the artificial intelligence techniques (AI) K-means, Partitioning Around Medoids (PAM), K-harmonic means (KHM), Fuzzy C-means (FCM) and Genetic K-means (GKA), with measures of low stream flow seasonality, for verification of its potential to delineate hydrologically homogeneous regions for the regionalization of Q90. For the performance analysis of the proposed methodology, location attributes from 108 watersheds situated in southern Brazil, and attributes associated with their seasonality of low stream flows were considered in this study. It was concluded that: (i) AI techniques have the potential to delineate hydrologically homogeneous regions in the context of Q90 in the study region, especially the FCM method based on fuzzy logic, and GKA, based on genetic algorithms; (ii) the attributes related to seasonality of low stream flows added important information that increased the accuracy of the grouping; and (iii) the adjusted mathematical models have excellent performance and can be used to estimate Q90 in locations lacking monitoring.

SCENERY: a web application for (causal) network reconstruction from cytometry data.

PubMed

Papoutsoglou, Georgios; Athineou, Giorgos; Lagani, Vincenzo; Xanthopoulos, Iordanis; Schmidt, Angelika; Éliás, Szabolcs; Tegnér, Jesper; Tsamardinos, Ioannis

2017-07-03

Flow and mass cytometry technologies can probe proteins as biological markers in thousands of individual cells simultaneously, providing unprecedented opportunities for reconstructing networks of protein interactions through machine learning algorithms. The network reconstruction (NR) problem has been well-studied by the machine learning community. However, the potentials of available methods remain largely unknown to the cytometry community, mainly due to their intrinsic complexity and the lack of comprehensive, powerful and easy-to-use NR software implementations specific for cytometry data. To bridge this gap, we present Single CEll NEtwork Reconstruction sYstem (SCENERY), a web server featuring several standard and advanced cytometry data analysis methods coupled with NR algorithms in a user-friendly, on-line environment. In SCENERY, users may upload their data and set their own study design. The server offers several data analysis options categorized into three classes of methods: data (pre)processing, statistical analysis and NR. The server also provides interactive visualization and download of results as ready-to-publish images or multimedia reports. Its core is modular and based on the widely-used and robust R platform allowing power users to extend its functionalities by submitting their own NR methods. SCENERY is available at scenery.csd.uoc.gr or http://mensxmachina.org/en/software/. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

R-HPDC Process with Forced Convection Mixing Device for Automotive Part of A380 Aluminum Alloy

PubMed Central

Zhou, Bing; Kang, Yonglin; Qi, Mingfan; Zhang, Huanhuan; Zhu, Guoming

2014-01-01

The continuing quest for cost-effective and complex shaped aluminum castings with fewer defects for applications in the automotive industries has aroused the interest in rheological high pressure die casting (R-HPDC). A new machine, forced convection mixing (FCM) device, based on the mechanical stirring and convection mixing theory for the preparation of semisolid slurry in convenience and functionality was proposed to produce the automotive shock absorber part by R-HPDC process. The effect of barrel temperature and rotational speed of the device on the grain size and morphology of semi-solid slurry were extensively studied. In addition, flow behavior and temperature field of the melt in the FCM process was investigated combining computational fluid dynamics simulation. The results indicate that the microstructure and pore defects at different locations of R-HPDC casting have been greatly improved. The vigorous fluid convection in FCM process has changed the temperature field and composition distribution of conventional solidification. Appropriately increasing the rotational speed can lead to a uniform temperature filed sooner. The lower barrel temperature leads to a larger uniform degree of supercooling of the melt that benefits the promotion of nucleation rate. Both of them contribute to the decrease of the grain size and the roundness of grain morphology. PMID:28788608

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

Hu, Nantao; Zhang, Liling; Yang, Chao

Thin, robust, lightweight, and flexible supercapacitors (SCs) have aroused growing attentions nowadays due to the rapid development of flexible electronics. Graphene-polyaniline (PANI) hybrids are attractive candidates for high performance SCs. In order to utilize them in real devices, it is necessary to improve the capacitance and the structure stability of PANI. Here we report a hierarchical three-dimensional structure, in which all of PANI nanofibers (NFs) are tightly wrapped inside reduced graphene oxide (rGO) nanosheet skeletons, for high-performance flexible SCs. The as-fabricated film electrodes with this unique structure showed a highest gravimetric specific capacitance of 921 F/g and volumetric capacitance ofmore » 391 F/cm 3. The assembled solid-state SCs gave a high specific capacitance of 211 F/g (1 A/g), a high area capacitance of 0.9 F/cm 2, and a competitive volumetric capacitance of 25.6 F/cm 3. The SCs also exhibited outstanding rate capability (~75% retention at 20 A/g) as well as excellent cycling stability (100% retention at 10 A/g for 2000 cycles). Additionally, no structural failure and loss of performance were observed under the bending state. Lastly, this structure design paves a new avenue for engineering rGO/PANI or other similar hybrids for high performance flexible energy storage devices.« less

Appearance Constrained Semi-Automatic Segmentation from DCE-MRI is Reproducible and Feasible for Breast Cancer Radiomics: A Feasibility Study.

PubMed

Veeraraghavan, Harini; Dashevsky, Brittany Z; Onishi, Natsuko; Sadinski, Meredith; Morris, Elizabeth; Deasy, Joseph O; Sutton, Elizabeth J

2018-03-19

We present a segmentation approach that combines GrowCut (GC) with cancer-specific multi-parametric Gaussian Mixture Model (GCGMM) to produce accurate and reproducible segmentations. We evaluated GCGMM using a retrospectively collected 75 invasive ductal carcinoma with ERPR+ HER2- (n = 15), triple negative (TN) (n = 9), and ER-HER2+ (n = 57) cancers with variable presentation (mass and non-mass enhancement) and background parenchymal enhancement (mild and marked). Expert delineated manual contours were used to assess the segmentation performance using Dice coefficient (DSC), mean surface distance (mSD), Hausdorff distance, and volume ratio (VR). GCGMM segmentations were significantly more accurate than GrowCut (GC) and fuzzy c-means clustering (FCM). GCGMM's segmentations and the texture features computed from those segmentations were the most reproducible compared with manual delineations and other analyzed segmentation methods. Finally, random forest (RF) classifier trained with leave-one-out cross-validation using features extracted from GCGMM segmentation resulted in the best accuracy for ER-HER2+ vs. ERPR+/TN (GCGMM 0.95, expert 0.95, GC 0.90, FCM 0.92) and for ERPR + HER2- vs. TN (GCGMM 0.92, expert 0.91, GC 0.77, FCM 0.83).

Three dimensional two-photon brain imaging in freely moving mice using a miniature fiber coupled microscope with active axial-scanning.

PubMed

Ozbay, Baris N; Futia, Gregory L; Ma, Ming; Bright, Victor M; Gopinath, Juliet T; Hughes, Ethan G; Restrepo, Diego; Gibson, Emily A

2018-05-25

We present a miniature head mounted two-photon fiber-coupled microscope (2P-FCM) for neuronal imaging with active axial focusing enabled using a miniature electrowetting lens. We show three-dimensional two-photon imaging of neuronal structure and record neuronal activity from GCaMP6s fluorescence from multiple focal planes in a freely-moving mouse. Two-color simultaneous imaging of GFP and tdTomato fluorescence is also demonstrated. Additionally, dynamic control of the axial scanning of the electrowetting lens allows tilting of the focal plane enabling neurons in multiple depths to be imaged in a single plane. Two-photon imaging allows increased penetration depth in tissue yielding a working distance of 450 μm with an additional 180 μm of active axial focusing. The objective NA is 0.45 with a lateral resolution of 1.8 μm, an axial resolution of 10 μm, and a field-of-view of 240 μm diameter. The 2P-FCM has a weight of only ~2.5 g and is capable of repeatable and stable head-attachment. The 2P-FCM with dynamic axial scanning provides a new capability to record from functionally distinct neuronal layers, opening new opportunities in neuroscience research.

Flow-cytometric determination of genotoxic effects of exposure to petroleum in mink and sea otters

USGS Publications Warehouse

Bickham, J.W.; Mazet, J.A.; Blake, J.; Smolen, M.J.; Lou, Y.; Ballachey, Brenda E.

1998-01-01

Three experiments were conducted to investigate the genotoxic effects of crude oil on mink and sea otters, In the first experiment, the effects on mink of chronic exposure to weathered Prudhoe Bay crude oil were studied, Female mink were fed a diet that included weathered crude oil for a period of 3 weeks prior to mating, during pregnancy and until weaning. Kits were exposed through lactation and by diet after weaning until 4 months of age. Kidney and liver tissues of the kits were examined using flow cytometry (FCM) and it was found that the genome size was increased in kidney samples from the experimental group compared to the control group. This effect was probably due to some type of DNA amplification and it could have been inherited from the exposed mothers or have been a somatic response to oil exposure in the pups, No evidence of clastogenic effects, as measured by the coefficient of variation (CV) of the G(1) peak, was found in kidney or liver tissue. In the second experiment, yearling female mink were exposed either by diet or externally to crude oil or bunker C fuel oil. Evidence for clastogenic damage was found in spleen tissue for the exposure groups, but not in kidney tissue. No evidence of increased genome size was observed. In the third experiment, blood was obtained from wild-caught sea otters in Prince William Sound. The sea otters represented two populations: one from western Prince William Sound that was potentially exposed to oil from the Exxon Valdez oil spill and a reference population from eastern Prince William Sound that did not receive oil from the spill. The spill had occurred 1.5 years prior to obtaining the blood samples. Although the mean CVs did not differ between the populations, the exposed population had a significantly higher variance of CV measurements and five out of 15 animals from the exposed population had CVs higher than the 95% confidence limits of the reference population, It is concluded that FCM is a sensitive indicator of the clastogenic effects of oil exposure and that haematopoietic tissues and blood are best for detecting clastogenic damage. Moreover, the observed differences in the genome size of the kidney cells mere possibly heritable effects, but this needs further investigation. Lastly, sea otters exposed to spilled oil 1.5 years earlier showed evidence of clastogenic damage in one-third of the individuals sampled.

NLS-RARα promotes proliferation and inhibits differentiation in HL-60 cells.

PubMed

Hu, Xiu-Xiu; Zhong, Liang; Zhang, Xi; Gao, Yuan-Mei; Liu, Bei-Zhong

2014-01-01

A unique mRNA produced in leukemic cells from a t(15;17) acute promyelocytic leukemia (APL) patient encodes a fusion protein between the retinoic acid receptor α (RARα) and a myeloid gene product called PML. Studies have reported that neutrophil elastase (NE) cleaves bcr-1-derived PML-RARα in early myeloid cells, leaving only the nuclear localization signal (NLS) of PML attached to RARα. The resultant NLS-RARα fusion protein mainly localizes to, and functions within, the cell nucleus. It is speculated that NLS-RARα may act in different ways from the wild-type RARα, but its biological characteristics have not been reported. This study takes two approaches. Firstly, the NLS-RARα was silenced with pNLS-RARα-shRNA. The mRNA and protein expression of NLS-RARα were detected by RT-PCR and Western blot respectively. Cell proliferation in vitro was assessed by MTT assay. Flow cytometry (FCM) was used to detect the differentiation of cells. Secondly, the NLS-RARα was over-expressed by preparation of recombinant adenovirus HL-60/pAd-NLS-RARα. The assays of mRNA and protein expression of NLS-RARα, and cell proliferation, were as above. By contrast, cell differentiation was stimulated by all trans retinoic acid (ATRA) (2.5µmol/L) at 24h after virus infection of pAd-NLS-RARα, and then detected by CD11b labeling two days later. The transcription and translation of C-MYC was detected in HL-60/pAd-NLS-RARα cells which treated by ATRA. Our results showed that compared to the control groups, the expression of NLS-RARα was significantly reduced in the HL-60/pNLS-RARα-shRNA cells, and increased dramatically in the HL-60/pAd-NLS-RARα cells. The proliferation was remarkably inhibited in the HL-60/pNLS-RARα-shRNA cells in a time-dependent manner, but markedly promoted in the HL-60/pAd-NLS-RARα cells. FCM outcome revealed the differentiation increased in HL-60/pNLS-RARα-shRNA cells, and decreased in the HL-60/pAd-NLS-RARα cells treated with 2.5µmol/L ATRA. The expression of C-MYC increased strikingly in HL-60/pAd-NLS-RARα cells treated with 2.5µmol/L ATRA. Down-regulation of NLS-RARα expression inhibited the proliferation and induced the differentiation of HL-60 cells. On the contrary, over-expression of NLS-RARα promoted proliferation and reduced the ATRA-induced differentiation of HL-60 cells.

Diagnosis of Plasma Cell Dyscrasias and Monitoring of Minimal Residual Disease by Multiparametric Flow Cytometry

PubMed Central

Soh, Kah Teong; Tario, Joseph D.; Wallace, Paul K.

2018-01-01

Synopsis Plasma cell dyscrasia (PCD) is a heterogeneous disease which has seen a tremendous change in outcomes due to improved therapies. Over the last few decades, multiparametric flow cytometry has played an important role in the detection and monitoring of PCDs. Flow cytometry is a high sensitivity assay for early detection of minimal residual disease (MRD) that correlates well with progression-free survival and overall survival. Before flow cytometry can be effectively implemented in the clinical setting sample preparation, panel configuration, analysis, and gating strategies must be optimized to ensure accurate results. Current consensus methods and reporting guidelines for MRD testing are discussed. PMID:29128071

Automatic classification of atypical lymphoid B cells using digital blood image processing.

PubMed

Alférez, S; Merino, A; Mujica, L E; Ruiz, M; Bigorra, L; Rodellar, J

2014-08-01

There are automated systems for digital peripheral blood (PB) cell analysis, but they operate most effectively in nonpathological blood samples. The objective of this work was to design a methodology to improve the automatic classification of abnormal lymphoid cells. We analyzed 340 digital images of individual lymphoid cells from PB films obtained in the CellaVision DM96:150 chronic lymphocytic leukemia (CLL) cells, 100 hairy cell leukemia (HCL) cells, and 90 normal lymphocytes (N). We implemented the Watershed Transformation to segment the nucleus, the cytoplasm, and the peripheral cell region. We extracted 44 features and then the clustering Fuzzy C-Means (FCM) was applied in two steps for the lymphocyte classification. The images were automatically clustered in three groups, one of them with 98% of the HCL cells. The set of the remaining cells was clustered again using FCM and texture features. The two new groups contained 83.3% of the N cells and 71.3% of the CLL cells, respectively. The approach has been able to automatically classify with high precision three types of lymphoid cells. The addition of more descriptors and other classification techniques will allow extending the classification to other classes of atypical lymphoid cells. © 2013 John Wiley & Sons Ltd.

Adaptive fuzzy system for 3-D vision

NASA Technical Reports Server (NTRS)

Mitra, Sunanda

1993-01-01

An adaptive fuzzy system using the concept of the Adaptive Resonance Theory (ART) type neural network architecture and incorporating fuzzy c-means (FCM) system equations for reclassification of cluster centers was developed. The Adaptive Fuzzy Leader Clustering (AFLC) architecture is a hybrid neural-fuzzy system which learns on-line in a stable and efficient manner. The system uses a control structure similar to that found in the Adaptive Resonance Theory (ART-1) network to identify the cluster centers initially. The initial classification of an input takes place in a two stage process; a simple competitive stage and a distance metric comparison stage. The cluster prototypes are then incrementally updated by relocating the centroid positions from Fuzzy c-Means (FCM) system equations for the centroids and the membership values. The operational characteristics of AFLC and the critical parameters involved in its operation are discussed. The performance of the AFLC algorithm is presented through application of the algorithm to the Anderson Iris data, and laser-luminescent fingerprint image data. The AFLC algorithm successfully classifies features extracted from real data, discrete or continuous, indicating the potential strength of this new clustering algorithm in analyzing complex data sets. The hybrid neuro-fuzzy AFLC algorithm will enhance analysis of a number of difficult recognition and control problems involved with Tethered Satellite Systems and on-orbit space shuttle attitude controller.

Isolation and identification of entomopathogenic nematodes from citrus orchards in South Africa and their biocontrol potential against false codling moth.

PubMed

Malan, Antoinette P; Knoetze, Rinus; Moore, Sean D

2011-10-01

A survey was conducted to determine the diversity and frequency of endemic entomopathogenic nematodes (EPN) in citrus orchards in the Western Cape, Eastern Cape and Mpumalanga provinces of South Africa. The main aim of the survey was to obtain nematodes as biological control agents against false codling moth (FCM), Thaumatotibia leucotreta, a key pest of citrus in South Africa. From a total of 202 samples, 35 (17%) tested positive for the presence of EPN. Of these, four isolates (11%) were found to be steinernematids, while 31 (89%) were heterorhabditids. Sequencing and characterisation of the internal transcribed spacer (ITS) region was used to identify all nematode isolates to species level. Morphometrics, morphology and biology of the infective juvenile (IJ) and the first-generation male were used to support molecular identification and characterisation. The Steinernema spp. identified were Steinernema khoisanae, Steinernema yirgalemense and Steinernema citrae. This is the first report of S. yirgalemense in South Africa, while for S. citrae it is the second new steinernematid to be identified from South Africa. Heterorhabditis species identified include Heterorhabditis bacteriophora, Heterorhabditis zealandica and an unknown species of Heterorhabditis. Laboratory bioassays, using 24-well bioassay disks, have shown isolates of all six species found during the survey, to be highly virulent against the last instar of FCM larvae. S. yirgalemense, at a concentration of 50IJs/FCM larva caused 100% mortality and 74% at a concentration of 200IJs/pupa. Using a sand bioassay, S. yirgalemense gave 93% control of cocooned pupae and emerging moths at a concentration of 20IJs/cm(2). This is the first report on the potential use of EPN to control the soil-borne life stages of FCM, which includes larvae, pupae and emerging moths. It was shown that emerging moths were infected with nematodes, which may aid in control and dispersal. Copyright © 2011 Elsevier Inc. All rights reserved.

Therapy of Prostate Cancer Using a Human Antibody Targeting the Type 1 Insulin-Like Growth Factor Receptor (IGF-IR)

DTIC Science & Technology

2009-09-01

euthanized, tumors harvested and portions processed for IHC, Western blot, flow cytometry , culture, and RNA analysis. If not enough tissue is available...temperature for 60 minutes. Samples were analyzed by flow cytometry using a BD FACScan. Data were analyzed with CellQuestPRO software. Evaluation of BrdUrd...were approved by the University of Washington Institutional Animal Care and Use Committee (IACUC). Flow cytometry . To measure tumor IGF-IR expression

Dose-Dependent Thresholds of 10-ns Electric Pulse Induced Plasma Membrane Disruption and Cytotoxicity in Multiple Cell Lines

DTIC Science & Technology

2011-01-01

normalized to parallel controls. Flow Cytometry and Confocal Microscopy Upon exposure to 10-ns EP, aliquots of the cellular suspension were added to a tube...Survival data was processed and plotted using GrapherH software (Golden Software, Golden, Colorado). Flow cytometry results were processed in C6 software...Accuri Cytometers, Inc., Ann Arbor, MI) and FCSExpress software (DeNovo Software, Los Angeles, CA). Final analysis and presentation of flow cytometry

The crosstalk: Tumor-infiltrating lymphocytes rich in regulatory T cells suppressed cancer-associated fibroblasts

PubMed Central

2013-01-01

Background. The interactions between cancer-associated fibroblasts (CAFs) and cancer cells or tumor-infiltrating lymphocytes (TILs) and cancer cells play important roles in cancer progression and metastasis. However, studies related to the crosstalk between CAFs and TILs in tumor microenvironment (TME) are still lacking. In this study, we mainly investigated the interactions between CAFs and TILs. Material and methods. The distribution of TILs rich in regulatory T cells (Tregs) in breast cancer tissues was evaluated using hematoxylin-eosin staining and immunohistochemistry with anti-CD3, anti-Foxp3, and anti-α-smooth muscle actin antibodies. Homologous CAFs/normal fibroblasts (NFs) and TILs cultured in vitro were identified and detected using immunocytochemistry and flow cytometry (FCM). The direct interaction among these cell types was studied via a factorial design in a co-cultured system. Their indirect interaction was assayed using Transwell plates. The cell cycle and apoptosis of CAFs/NFs co-cultured with TILs was analyzed using propidium iodide staining. Results. Histochemistry demonstrated most of the TILs including Tregs, were distributed in the cancer stroma, adjoining to CAFs. This finding implies that both cell types interact closely in the TME. Identification of the cultured cells showed that CAFs maintained their activated phenotype within limited passages in vitro, and that the TILs population contained a high percentage of Tregs. Data analysis of the factorial design suggests significant interactions among CAFs, NFs, and TILs in both direct and indirect contact ways. The CAFs and NFs were suppressed signally by TILs, which are probably induced by the secretory cytokines derived from TILs or Tregs. Although apoptosis was not detected in CAFs/NFs, the cell cycle assay suggested that the CAFs/NFs were arrested in the G2/M phase by the TILs and their secretory cytokines. Conclusion. CAFs and NFs were dramatically suppressed by Tregs-rich TILs. This suggests the interaction between TILs and CAFs might modify the TME in an unknown manner. PMID:23336253

Antimicrobial Activity of New Materials Based on Lavender and Basil Essential Oils and Hydroxyapatite.

PubMed

Predoi, Daniela; Iconaru, Simona Liliana; Buton, Nicolas; Badea, Monica Luminita; Marutescu, Luminita

2018-04-30

This study presents, for the first-time, the results of a study on the hydrodynamic diameter of essential oils (EOs) of basil and lavender in water, and solutions of EOs of basil (B) and lavender (L) and hydroxyapatite (HAp). The possible influence of basil and lavender EOs on the size of hydroxyapatite nanoparticles was analyzed by Scanning Electron Microscopy (SEM). We also investigated the in vitro antimicrobial activity of plant EOs and plant EOs hydroxyapatite respectively, against Gram-positive bacteria (methicillin-resistant Staphylococcus aureus 1144 (MRSA 1144) and S. aureus 1426) and Gram-negative bacteria ( Escherichia coli ATCC 25922 and Escherichia coli ESBL 4493). From the autocorrelation function, obtained by Dynamic Light Scattering (DLS) measurements it was observed that basil yielded one peak at an average hydrodynamic diameter of 354.16 nm, while lavender yielded one peak at an average hydrodynamic diameter of 259.76 nm. In the case of HAp nanoparticles coated with basil (HApB) and lavender (HApL) essential oil, the aggregation was minimal. We found that the lavender EO exhibited a very good inhibitory growth activity (MIC values ranging from <0.1% for E. coli reference strain to 0.78% for S. aureus strains). The biological studies indicated that HapL material displayed an enhanced antimicrobial activity, indicating the potential use of HAp as vehicle for low concentrations of lavender EO with antibacterial properties. Flow cytometry analysis (FCM) allowed us to determine some of the potential mechanisms of the antimicrobial activities of EOs, suggesting that lavender EO was active against E. coli by interfering with membrane potential, the membrane depolarization effect being increased by incorporation of the EOs into the microporous structure of HAp. These findings could contribute to the development of new antimicrobial agents that are urgently needed for combating the antibiotic resistance phenomena.

Imaging Flow Cytometry Analysis to Identify Differences of Survival Motor Neuron Protein Expression in Patients With Spinal Muscular Atrophy.

PubMed

Arakawa, Reiko; Arakawa, Masayuki; Kaneko, Kaori; Otsuki, Noriko; Aoki, Ryoko; Saito, Kayoko

2016-08-01

Spinal muscular atrophy is a neurodegenerative disorder caused by the deficient expression of survival motor neuron protein in motor neurons. A major goal of disease-modifying therapy is to increase survival motor neuron expression. Changes in survival motor neuron protein expression can be monitored via peripheral blood cells in patients; therefore we tested the sensitivity and utility of imaging flow cytometry for this purpose. After the immortalization of peripheral blood lymphocytes from a human healthy control subject and two patients with spinal muscular atrophy type 1 with two and three copies of SMN2 gene, respectively, we used imaging flow cytometry analysis to identify significant differences in survival motor neuron expression. A bright detail intensity analysis was used to investigate differences in the cellular localization of survival motor neuron protein. Survival motor neuron expression was significantly decreased in cells derived from patients with spinal muscular atrophy relative to those derived from a healthy control subject. Moreover, survival motor neuron expression correlated with the clinical severity of spinal muscular atrophy according to SMN2 copy number. The cellular accumulation of survival motor neuron protein was also significantly decreased in cells derived from patients with spinal muscular atrophy relative to those derived from a healthy control subject. The benefits of imaging flow cytometry for peripheral blood analysis include its capacities for analyzing heterogeneous cell populations; visualizing cell morphology; and evaluating the accumulation, localization, and expression of a target protein. Imaging flow cytometry analysis should be implemented in future studies to optimize its application as a tool for spinal muscular atrophy clinical trials. Copyright © 2016 Elsevier Inc. All rights reserved.

Assessment of Possible Cycle Lengths for Fully-Ceramic Micro-Encapsulated Fuel-Based Light Water Reactor Concepts

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

R. Sonat Sen; Michael A. Pope; Abderrafi M. Ougouag

2012-04-01

The tri-isotropic (TRISO) fuel developed for High Temperature reactors is known for its extraordinary fission product retention capabilities [1]. Recently, the possibility of extending the use of TRISO particle fuel to Light Water Reactor (LWR) technology, and perhaps other reactor concepts, has received significant attention [2]. The Deep Burn project [3] currently focuses on once-through burning of transuranic fissile and fissionable isotopes (TRU) in LWRs. The fuel form for this purpose is called Fully-Ceramic Micro-encapsulated (FCM) fuel, a concept that borrows the TRISO fuel particle design from high temperature reactor technology, but uses SiC as a matrix material rather thanmore » graphite. In addition, FCM fuel may also use a cladding made of a variety of possible material, again including SiC as an admissible choice. The FCM fuel used in the Deep Burn (DB) project showed promising results in terms of fission product retention at high burnup values and during high-temperature transients. In the case of DB applications, the fuel loading within a TRISO particle is constituted entirely of fissile or fissionable isotopes. Consequently, the fuel was shown to be capable of achieving reasonable burnup levels and cycle lengths, especially in the case of mixed cores (with coexisting DB and regular LWR UO2 fuels). In contrast, as shown below, the use of UO2-only FCM fuel in a LWR results in considerably shorter cycle length when compared to current-generation ordinary LWR designs. Indeed, the constraint of limited space availability for heavy metal loading within the TRISO particles of FCM fuel and the constraint of low (i.e., below 20 w/0) 235U enrichment combine to result in shorter cycle lengths compared to ordinary LWRs if typical LWR power densities are also assumed and if typical TRISO particle dimensions and UO2 kernels are specified. The primary focus of this summary is on using TRISO particles with up to 20 w/0 enriched uranium kernels loaded in Pressurized Water Reactor (PWR) assemblies. In addition to consideration of this 'naive' use of TRISO fuel in LWRs, several refined options are briefly examined and others are identified for further consideration including the use of advanced, high density fuel forms and larger kernel diameters and TRISO packing fractions. The combination of 800 {micro}m diameter kernels of 20% enriched UN and 50% TRISO packing fraction yielded reactivity sufficient to achieve comparable burnup to present-day PWR fuel.« less

Towards the harmonization between National Forest Inventory and Forest Condition Monitoring. Consistency of plot allocation and effect of tree selection methods on sample statistics in Italy.

PubMed

Gasparini, Patrizia; Di Cosmo, Lucio; Cenni, Enrico; Pompei, Enrico; Ferretti, Marco

2013-07-01

In the frame of a process aiming at harmonizing National Forest Inventory (NFI) and ICP Forests Level I Forest Condition Monitoring (FCM) in Italy, we investigated (a) the long-term consistency between FCM sample points (a subsample of the first NFI, 1985, NFI_1) and recent forest area estimates (after the second NFI, 2005, NFI_2) and (b) the effect of tree selection method (tree-based or plot-based) on sample composition and defoliation statistics. The two investigations were carried out on 261 and 252 FCM sites, respectively. Results show that some individual forest categories (larch and stone pine, Norway spruce, other coniferous, beech, temperate oaks and cork oak forests) are over-represented and others (hornbeam and hophornbeam, other deciduous broadleaved and holm oak forests) are under-represented in the FCM sample. This is probably due to a change in forest cover, which has increased by 1,559,200 ha from 1985 to 2005. In case of shift from a tree-based to a plot-based selection method, 3,130 (46.7%) of the original 6,703 sample trees will be abandoned, and 1,473 new trees will be selected. The balance between exclusion of former sample trees and inclusion of new ones will be particularly unfavourable for conifers (with only 16.4% of excluded trees replaced by new ones) and less for deciduous broadleaves (with 63.5% of excluded trees replaced). The total number of tree species surveyed will not be impacted, while the number of trees per species will, and the resulting (plot-based) sample composition will have a much larger frequency of deciduous broadleaved trees. The newly selected trees have-in general-smaller diameter at breast height (DBH) and defoliation scores. Given the larger rate of turnover, the deciduous broadleaved part of the sample will be more impacted. Our results suggest that both a revision of FCM network to account for forest area change and a plot-based approach to permit statistical inference and avoid bias in the tree sample composition in terms of DBH (and likely age and structure) are desirable in Italy. As the adoption of a plot-based approach will keep a large share of the trees formerly selected, direct tree-by-tree comparison will remain possible, thus limiting the impact on the time series comparability. In addition, the plot-based design will favour the integration with NFI_2.

Scorpion venom peptide SPVII promotes irradiated cells proliferation and increases the expression of the IL-3 receptor

PubMed Central

2013-01-01

Background The previous investigation demonstrated the radioprotective efficacy of peptides isolated from the venom of Buthus Martti Karsch. In this study, the effect of isolated scorpion venom peptide II (SVPII) on irradiated M-NFS-60 cells and mouse bone marrow mononuclear cells (BM-MNCs) was observed. The AlamarBlue cell viability assay, a colony-forming unit (CFU) assay, flow cytometry (FCM), immunofluorescence, and Western blotting were used to evaluate cell proliferation, cell cycle progression, and the expression of the IL-3 receptor (IL-3R) protein in non-irradiated and irradiated cells. Results Proliferation of irradiated M-NFS-60 cells was significantly accelerated by SPVII, and this effect was further enhanced by co-application of IL-3. Similarly, SPVII increased the number of BM-MNC CFUs and this proliferative effect was greater in the presence of SVPII plus IL-3. In addition, SPVII significantly altered cell cycle progression; SVPII enhanced the fraction of unirradiated M-NFS-60 cells in S phase and the fraction of irradiated M-NFS-60 cells arrested in G2/M. The expression of IL-3R protein by unirradiated M-NFS-60 cells was enhanced significantly by SVPII, and SVPII-induced IL-3R overexpression was 10-fold greater in irradiated M-NFS-60 cells. Conclusions These results indicated the hematopoietic growth factor (HGF)-like effects of SVPII on irradiated cells, possibly mediated by upregulation of IL-3R. PMID:23835458

Low dose naltrexone (LDN) enhances maturation of bone marrow dendritic cells (BMDCs).

PubMed

Meng, Jingjuan; Meng, Yiming; Plotnikoff, Nicholas P; Youkilis, Gene; Griffin, Noreen; Shan, Fengping

2013-12-01

It has been demonstrated previously that immune cell activation and proliferation were sensitive to the effects of naltrexone, a non-peptidic δ-opioid receptor selective antagonist and opioid receptors on BMDCs have been detected [1]. However, there is little prior data published on naltrexone and DCs. Therefore, we hypothesized that LDN could exert modulating effect on BMDCs. In present study, we studied influence of LDN on both phenotypic and functional maturation of BMDCs. Changes of BMDC post-treatment with LDN were evaluated using conventional light microscope and transmission electron microscopy (TEM); flow cytometry(FCM); cytochemistry; acid phosphatase activity(ACP) test; FITC-dextran bio-assay; mixed lymphocytes and enzyme-linked immunosorbent assay (ELISA). We have found that LDN enhances maturation of BMDCs as evidenced by 1) up-regulating the expression of MHC II, CD40, CD83, CD80 and CD86 molecules on BMDCs; 2) down-regulating the rates of pinocytosis and phagocytosis accompanied by the results of decreased ACP, and FITC-dextran bio-assay; 3) mounting potential of BMDCs to drive T cell; and 4) inducing secretion of higher levels of IL-12 and TNF-α. It is therefore concluded that LDN can efficiently promote the maturation of BMDCs via precise modulation inside and outside BMDCs. Our study has provided meaningful mode of action on the role of LDN in immunoregulation, and rationale on future application of LDN for enhancing host immunity in cancer therapy and potent use in the design of DC-based vaccines for a number of diseases.

Enhanced 4T1 breast carcinoma anticancer activity by co-delivery of doxorubicin and curcumin with core-shell drug-carrier based on heparin modified poly(L-lactide) grafted polyethylenimine cationic nanoparticles.

PubMed

Guo, Oingfa; Li, Xiaolu; Yang, Yi; Wei, Jing; Zhao, Qian; Luo, Feng; Qian, Zhiyong

2014-02-01

Use of single chemotherapy agents has shown some limitations in anti-tumor treatment, such as development of drug resistance, severe adverse reactions and limited regime for therapeutic use. Combination of two or more therapeutic drugs is a feasible strategy to overcome these limitations. This paper reports study of co-delivery by core-shell nanoparticles (NPs) with hydrophobic PLLA core loaded with curcumin (Cur) and hydrophilic heparin shell adsorbing Doxorubicin (DOX). Characterizations of Cur-PEA NPs, Cur-PEA/heparin NPs and DOX adsorbing into Cur-PEA/heparin NPs (DOX-Cur NPs) were also investigated by transmission electron microscope (TEM) and Malvern Zetasizer. Studies on cellular uptake of DOX-Cur NPs demonstrated that both drugs were effectively taken up by 4T1 tumor cells. Furthermore, DOX-Cur NPs suppressed 4T1 tumor cells growth more efficiently than either DOX or Cur alone at the same concentrations, as measured by flow cytometry (FCM). We found out that intravenous injection of DOX-Cur NPs efficiently inhibited growth of subcutaneous 4T1 breast carcinoma in vivo (p < 0.01) and prolonged survival of the treated 4T1 breast carcinoma mice. Moreover, the pathological damage to the cardiac tissue in mice treated with DOX-Cur NPs was significantly less severe than that of mice treated with free DOX. This study suggested that DOX-Cur NPs may have promising applications in breast carcinoma therapy.

Effects of canonical NF-κB signaling pathway on the proliferation and odonto/osteogenic differentiation of human stem cells from apical papilla.

PubMed

Li, Junjun; Yan, Ming; Wang, Zilu; Jing, Shuanglin; Li, Yao; Liu, Genxia; Yu, Jinhua; Fan, Zhipeng

2014-01-01

NF-κB signaling pathway plays a complicated role in the biological functions of mesenchymal stem cells. However, the effects of NF-κB pathway on the odonto/osteogenic differentiation of stem cells from apical papilla (SCAPs) remain unclear. The present study was designed to evaluate the effects of canonical NF-κB pathway on the osteo/odontogenic capacity of SCAPs in vitro. Western blot results demonstrated that NF-κB pathway in SCAPs was successfully activated by TNF-α or blocked by BMS-345541. NF-κB pathway-activated SCAPs presented a higher proliferation activity compared with control groups, as indicated by dimethyl-thiazol-diphenyl tetrazolium bromide assay (MTT) and flow cytometry assay (FCM). Wound scratch assay revealed that NF-κB pathway-activated SCAPs presented an improved migration capacity, enhanced alkaline phosphatase (ALP) activity, and upregulated mineralization capacity of SCAPs, as compared with control groups. Meanwhile, the odonto/osteogenic markers (ALP/ALP, RUNX2/RUNX2, OSX/OSX, OCN/OCN, OPN/OPN, BSP/BSP, DSPP/DSP, and DMP-1/DMP-1) in NF-κB pathway-activated SCAPs were also significantly upregulated as compared with control groups at both protein and mRNA levels. However, NF-κB pathway-inhibited SCAPs exhibited a lower proliferation/migration capacity, and decreased odonto/osteogenic ability in comparison with control groups. Our findings suggest that classical NF-κB pathway plays a paramount role in the proliferation and committed differentiation of SCAPs.

Methionine enkephalin (MENK) improves lymphocyte subpopulations in human peripheral blood of 50 cancer patients by inhibiting regulatory T cells (Tregs)

PubMed Central

Wang, Qiushi; Gao, Xinghua; Yuan, Zhe; Wang, Zhe; Meng, Yiming; Cao, Yan; Plotnikoff, Nicolas P; Griffin, Noreen; Shan, Fengping

2014-01-01

MENK, a penta-peptide is considered as being involved in the regulatory feedback loop between the immune and neuroendocrine systems, with marked modulation of various functions of human immune cells. The aim of the present work was to investigate change of lymphocyte subpopulations in peripheral blood of 50 cancer patients before and after treatment with MENK. Peripheral blood mononuclear cells (PBMCs) of peripheral blood from 50 cancer patients were isolated by density gradient centrifugation using Ficoll-Paque solution and cultured with MENK. We measured proliferation of total nucleated cells, subpopulations of individual CD4+T cells, CD8+T cells, CD4+CD25+ regulatory T cells (Treg), natural killer cells (NK) before and after treatment with 10-12M MENK in cell culture by flow cytometry (FCM). Our results indicated that MENK showed a strong inhibiting effect on Treg cells while it stimulated marked proliferation of other lymphocyte subpopulations. All data obtained were of significance statistically. It was therefore concluded that MENK could work as a strong immune booster with great potential in restoring damaged human immune system and we could consider MENK as a drug to treat cancer patients, whose immune systems are damaged by chemotherapy or radiotherapy. Furthermore we could consider MENK as a chemotherapy additive, which would sustain immune system of cancer patients during the process of chemotherapy to get maximized efficacy with minimized side effect. PMID:25424790

Chemical composition of the SFE-CO extracts from Cajanus cajan (L.) Huth and their antimicrobial activity in vitro and in vivo.

PubMed

Zu, Yuan-gang; Liu, Xiao-lei; Fu, Yu-jie; Wu, Nan; Kong, Yu; Wink, Michael

2010-12-01

The in vitro and in vivo antimicrobial activities of SFE-CO₂(supercritical fluid extraction) extracts and ethanol extracts from Cajanus cajan (L.) Huth were investigated. The flavonoid compounds orientin, vitexin, isovitexin, pinostrobin and the stilbene cajaninstilbene acid were detected in SFE-CO₂ extracts by HPLC-DAD. In vitro antimicrobial activities of the extracts were evaluated against eight microbial strains (the bacteria Staphylococcus epidermidis, Staphylococcus aureus, Bacillus subtilis, Proteus vulgaris, Pseudomonas aeruginosa, Escherichia coli; and the fungi Aspergillus niger and Candida albicans). A marked inhibitory effect of the SFE extracts was observed against Staphylococcus epidermidis, Staphylococcus aureus and Bacillus subtilis. The IC(50) of SFE-CO₂ extracts ranged from 0.0557 mg/ml to 0.0689 mg/ml consisting of cancer (MCF-7 (0.0557 mg/ml)) as well as non-cancer (BHK-21 (0.0641 mg/ml), RAW264.7 (0.0689 mg/ml) and Vero (0.0625 mg/ml)) cells. Flow cytometry (FCM) was used to analyze death rate of the most sensitive strain (Staphylococcus aureus) caused by the SFE extracts. Additionally, the whole cell proteins of Staphylococcus aureus were analyzed by SDS-PAGE to detect if there were changes in protein patterns. In vivo antimicrobial activity was studies in mice that had been inoculated with Staphylococcus aureus. The potential mechanism of antimicrobial activity in vivo was studied by histopathology. Copyright © 2010 Elsevier GmbH. All rights reserved.

Inflammation promotes oral squamous carcinoma immune evasion via induced programmed death ligand-1 surface expression.

PubMed

Lu, Wanlu; Lu, Libing; Feng, Yun; Chen, Jiao; Li, Yan; Kong, Xiangli; Chen, Sixiu; Li, Xiaoyu; Chen, Qianming; Zhang, Ping

2013-05-01

The association between inflammation and cancer provides a new target for tumor biotherapy. The inflammatory cells and molecules within the tumor microenvironment have decisive dual roles in antitumor immunity and immune evasion. In the present study, phytohemagglutinin (PHA) was used to stimulate peripheral blood mononuclear cells (PBMCs) to simulate the tumor inflammatory microenvironment. The effect of immune cells and inflammatory cytokines on the surface expression of programmed cell death-1 ligand 1 (PD-L1) and tumor immune evasion was investigated using flow cytometry (FCM) and an in vivo xenotransplantation model. Based on the data, PHA-activated, but not resting, immune cells were able to promote the surface expression of PD-L1 in Tca8113 oral squamous carcinoma cells via the secretion of inflammatory cytokines, but not by cell-cell contact. The majority of the inflammatory cytokines had no significant effect on the proliferation, cell cycle progression and apoptosis of the Tca8113 cells, although they each induced the expression of PD-L1 in a dose-dependent manner. In total, 99% of the Tca8113 cells expressed PD-L1 following treatment with the supernatant of PHA-stimulated PBMCs. The PHA-supernatant pretreated Tca8113 cells unusually induced Tca8113 antigen-specific CD8 + T cell apoptosis in vitro and the evasion of antigen-specific T cell attraction in a nude mouse tumor-bearing model. These results indicate a new mechanism for the promotion of tumor immune evasion by the tumor inflammatory microenvironment.

Inflammation promotes oral squamous carcinoma immune evasion via induced programmed death ligand-1 surface expression

PubMed Central

LU, WANLU; LU, LIBING; FENG, YUN; CHEN, JIAO; LI, YAN; KONG, XIANGLI; CHEN, SIXIU; LI, XIAOYU; CHEN, QIANMING; ZHANG, PING

2013-01-01

The association between inflammation and cancer provides a new target for tumor biotherapy. The inflammatory cells and molecules within the tumor microenvironment have decisive dual roles in antitumor immunity and immune evasion. In the present study, phytohemagglutinin (PHA) was used to stimulate peripheral blood mononuclear cells (PBMCs) to simulate the tumor inflammatory microenvironment. The effect of immune cells and inflammatory cytokines on the surface expression of programmed cell death-1 ligand 1 (PD-L1) and tumor immune evasion was investigated using flow cytometry (FCM) and an in vivo xenotransplantation model. Based on the data, PHA-activated, but not resting, immune cells were able to promote the surface expression of PD-L1 in Tca8113 oral squamous carcinoma cells via the secretion of inflammatory cytokines, but not by cell-cell contact. The majority of the inflammatory cytokines had no significant effect on the proliferation, cell cycle progression and apoptosis of the Tca8113 cells, although they each induced the expression of PD-L1 in a dose-dependent manner. In total, 99% of the Tca8113 cells expressed PD-L1 following treatment with the supernatant of PHA-stimulated PBMCs. The PHA-supernatant pretreated Tca8113 cells unusually induced Tca8113 antigen-specific CD8+ T cell apoptosis in vitro and the evasion of antigen-specific T cell attraction in a nude mouse tumor-bearing model. These results indicate a new mechanism for the promotion of tumor immune evasion by the tumor inflammatory microenvironment PMID:23761816

Evaluation of the damage of cell wall and cell membrane for various extracellular polymeric substance extractions of activated sludge.

PubMed

Guo, Xuesong; Liu, Junxin; Xiao, Benyi

2014-10-20

Extracellular polymeric substances (EPS) are susceptible to contamination by intracellular substances released during the extraction of EPS owing to the damage caused to microbial cell structures. The damage to cell walls and cell membranes in nine EPS extraction processes of activated sludge was evaluated in this study. The extraction of EPS (including proteins, carbohydrates and DNA) was the highest using the NaOH extraction method and the lowest using formaldehyde extraction. All nine EPS extraction methods in this study resulted in cell wall and membrane damage. The damage to cell walls, evaluated by 2-keto-3-deoxyoctonate (KDO) and N-acetylglucosamine content changes in extracted EPS, was the most significant in the NaOH extraction process. Formaldehyde extraction showed a similar extent of damage to cell walls to those detected in the control method (centrifugation), while those in the formaldehyde-NaOH and cation exchange resin extractions were slightly higher than those detected in the control. N-acetylglucosamine was more suitable than KDO for the evaluation of cell wall damage in the EPS extraction of activated sludge. The damage to cell membranes was characterized by two fluorochromes (propidium iodide and FITC Annexin V) with flow cytometry (FCM) measurement. The highest proportion of membrane-damaged cells was detected in NaOH extraction (26.54% of total cells) while membrane-damaged cells comprised 8.19% of total cells in the control. Copyright © 2014 Elsevier B.V. All rights reserved.

[Effects of Rhizoma kaempferiae volatile oil on tumor growth and cell cycle of MKN-45 human gastric cancer cells orthotopically transplanted in nude mice].

PubMed

Xiao, Yan; Wei, Pin-Kang; Li, Jun; Shi, Jun; Yu, Zhi-Hong; Lin, Hui-Ming

2006-07-01

To evaluate the effects of Rhizoma kaempferiae volatile oil on tumor growth and cell cycle of MKN-45 human gastric cancer cells orthotopically transplanted in nude mice. One hundred and five nude mice orthotopically transplanted with MKN-45 human gastric cancer cells were randomly divided into seven groups: untreated group, normal saline-treated group, dissolvant-treated group, cyclophosphamide (CTX)-treated group and high-, medium-, and low-dose Rhizoma kaempferiae volatile oil-treated groups. Corresponding interventions were implemented in each group except the untreated group. The antitumor effects in vivo were evaluated. Cell cycle distribution and apoptosis of MKN-45 human gastric cancer cells were determined by using flow cytometry (FCM). The ultrastructure of MKN-45 gastric cancer cells was observed by a transmission electron microscope. In the high-, medium-, and low-dose Rhizoma kaempferiae volatile oil-treated groups, the growth inhibition rates of gastric cancer were 57.2%, 28.0% and 5.0% respectively, and the gastric cancer cells were arrested at G(0)/G(1) phase. This antitumor effect was dose-dependent. The apoptotic cells occurred more frequently in the high-dose Rhizoma kaempferiae volatile oil-treated group and the CTX-treated group than those in the medium- and low-dose Rhizoma kaempferiae volatile oil-treated groups. The Rhizoma kaempferiae volatile oil is an effective composition for growth inhibition of gastric cancer, and its mechanism may be related to regulating the cell cycle and inducing apoptosis.

Mifepristone sensitizing cisplatin for cervical adenocarcinoma HeLa cell sensitivity to chemotherapy and its mechanism.

PubMed

Li, Caihong; Ye, Hong

2013-01-01

The study was designed to investigate proliferation inhibition for cervical adenocarcinoma HeLa cell treated with cisplatin combined with mifepristone and access its possible mechanism. HeLa cell was processed by different concentrations of mifepristone, cisplatin, and their combination respectively. Cell's proliferation inhibition rate and induction apoptosis ability were detected by MTT assay, FCM; the expression of P53, survivin and HPV E6 protein were measured by Western Blot. The results showed that cisplatin inhibits proliferation of HeLa cells in different concentrations (p <0.01). Mifepristone had no effect on HeLa cell proliferation inhibition rate during 24 and 48 hours (p > 0.05). Mifepristone at low concentrations (< or = 10 micromol/l) combined with cisplatin can significantly enhance the inhibitory effect of cisplatin on HeLa cell line. Flow cytometry showed that mifepristone at low concentrations (< or = 10 micromol/l) combined with cisplatin can induce apparent apoptosis of HeLa cell line in concentration dependent manner. Western blotting demonstrated that the expression of P53 protein increased and the expression of HPV E6 survivin protein decreased in HeLa cells treated with MIF at low concentrations (< or = 10 micromol/l) combined with cisplatin. Mifepristone at low concentrations (< or = 10 micromol/1) can enhance chemosensitivity and capability of inducing apoptosis of cisplatin to HeLa cells. The strengthening effect of growth inhibition and chemosensitivity to cisplatin of mifepristone are associated with down-regulating HPV E6 survivin protein and upregulating p53 protein.

[Establishment of a new human glioma cell line and analysis of its biological characteristics].

PubMed

Chen, Guilin; Li, Yanyan; Xie, Xueshun; Chen, Jinming; Wu, Tingfeng; Li, Xuetao; Wang, Hangzhou; Zhou, Youxin; Du, Ziwei

2015-02-01

To establish a new glioma cell line and analyze its biological characteristics, and to provide a useful cellular tool with new features for cancer research. Glioma tissue was taken from surgical specimen clinical of a clinical patient. Primary culture was carried out, and a cell line (SHG139) was established after 10 passages. Immunofluorescence staining was performed to detect the expression of proteins, and cell proliferation and cycle were detected by flow cytometry method (FCM). The biological characteristics of SHG139 cells were detected by chromosome karyotype analysis. SHG139s glioma cells derived from SHG139 glioma cell line were cultured with neural stem cell medium. Then stem cell markers were determined. SHG139s cells were induced with serum-containing medium, and their expression of A2B5, GFAP, β-III tubulin, and GalC was detected. Intracranial xenograft tumor of both SHG139 glioma cells and SHG139s glioma stem cell spheres was generated in rats. The expressions of A2B5, GalC, GFAP, S-100, and vimentin in the 20 and 60 passages of SHG139 cells were positive, consistent with the immunohistochemical results and pathological features. SHG139 cells proliferated significantly within 24 h after subculture, and their total number of chromosomes was 68 and mostly multiploid. They were positive for A2B5 (84.12±9.96)%, nestin (73.86±5.01)%, and NG2 (73.37±2.09)%. SHG139s cells were induced, and the ratio of positive cells of GFAP, β-III tubulin and GalC was (92.89±2.24)%, (64.85±4.09)% and (33.57±4.14)%, respectively. SHG139 is an astroglioma cell line, from which SHG139s cells can be successfully obtained by culture with NSCM. SHG139s cells are of A2B5(+)/CD133(-) GSCs subgroup cells, with potentials of self-renewal and multi-directional differentiation. Compared with the intracranial SHG139 xenograft tumor, the intracranial SHG139s xenograft tumor is more malignant and aggressive.

Automation of sample preparation for mass cytometry barcoding in support of clinical research: protocol optimization.

PubMed

Nassar, Ala F; Wisnewski, Adam V; Raddassi, Khadir

2017-03-01

Analysis of multiplexed assays is highly important for clinical diagnostics and other analytical applications. Mass cytometry enables multi-dimensional, single-cell analysis of cell type and state. In mass cytometry, the rare earth metals used as reporters on antibodies allow determination of marker expression in individual cells. Barcode-based bioassays for CyTOF are able to encode and decode for different experimental conditions or samples within the same experiment, facilitating progress in producing straightforward and consistent results. Herein, an integrated protocol for automated sample preparation for barcoding used in conjunction with mass cytometry for clinical bioanalysis samples is described; we offer results of our work with barcoding protocol optimization. In addition, we present some points to be considered in order to minimize the variability of quantitative mass cytometry measurements. For example, we discuss the importance of having multiple populations during titration of the antibodies and effect of storage and shipping of labelled samples on the stability of staining for purposes of CyTOF analysis. Data quality is not affected when labelled samples are stored either frozen or at 4 °C and used within 10 days; we observed that cell loss is greater if cells are washed with deionized water prior to shipment or are shipped in lower concentration. Once the labelled samples for CyTOF are suspended in deionized water, the analysis should be performed expeditiously, preferably within the first hour. Damage can be minimized if the cells are resuspended in phosphate-buffered saline (PBS) rather than deionized water while waiting for data acquisition.

Meta-analysis of two computer-assisted screening methods for diagnosing oral precancer and cancer.

PubMed

Ye, Xiaojing; Zhang, Jing; Tan, Yaqin; Chen, Guanying; Zhou, Gang

2015-11-01

The early diagnosis of oral precancer and cancer is crucial and could have the highest impact on improving survival rates. A meta-analysis was conducted to compare the accuracy between the OralCDx brush biopsy and DNA-image cytometry in diagnosing both conditions. Bibliographic databases were systematically searched for original relevant studies on the early diagnosis of oral precancer and oral cancer. Study characteristics were evaluated to determine the accuracy of the two screening strategies. Thirteen studies (eight of OralCDx brush biopsy and five of DNA-image cytometry) were identified as having reported on 1981 oral mucosa lesions. The meta-analysis found that the area under the summary receiver operating characteristic curves of the OralCDx brush biopsy and DNA-image cytometry were 0.8879 and 0.9885, respectively. The pooled sensitivity, specificity, and diagnostic odds ratio of the OralCDx brush biopsy were 86% (95% CI 81-90), 81% (95% CI 78-85), and 20.36 (95% CI 2.72-152.67), respectively, while these modalities of DNA-image cytometry were 89% (95% CI 83-94), 99% (95% CI 97-100), and 446.08 (95% CI 73.36-2712.43), respectively. Results of a pairwise comparison between each modality demonstrated that specificity, area under the curve (AUC), and Q(∗) index of DNA-image cytometry was significantly higher than that of the OralCDx brush biopsy (Z=2.821, p<0.05; Z=1.711, p<0.05; Z=1.727, p<0.05), but no significant difference in sensitivity was found (Z=1.520, p>0.05). In conclusion, the meta-analysis of the published studies indicated that DNA-image cytometry is more accurate than the OralCDx brush biopsy in diagnosing oral precancer and oral cancer. Copyright © 2015 Elsevier Ltd. All rights reserved.

Single-cell mass cytometry and transcriptome profiling reveal the impact of graphene on human immune cells.

PubMed

Orecchioni, Marco; Bedognetti, Davide; Newman, Leon; Fuoco, Claudia; Spada, Filomena; Hendrickx, Wouter; Marincola, Francesco M; Sgarrella, Francesco; Rodrigues, Artur Filipe; Ménard-Moyon, Cécilia; Cesareni, Gianni; Kostarelos, Kostas; Bianco, Alberto; Delogu, Lucia G

2017-10-24

Understanding the biomolecular interactions between graphene and human immune cells is a prerequisite for its utilization as a diagnostic or therapeutic tool. To characterize the complex interactions between graphene and immune cells, we propose an integrative analytical pipeline encompassing the evaluation of molecular and cellular parameters. Herein, we use single-cell mass cytometry to dissect the effects of graphene oxide (GO) and GO functionalized with amino groups (GONH 2 ) on 15 immune cell populations, interrogating 30 markers at the single-cell level. Next, the integration of single-cell mass cytometry with genome-wide transcriptome analysis shows that the amine groups reduce the perturbations caused by GO on cell metabolism and increase biocompatibility. Moreover, GONH 2 polarizes T-cell and monocyte activation toward a T helper-1/M1 immune response. This study describes an innovative approach for the analysis of the effects of nanomaterials on distinct immune cells, laying the foundation for the incorporation of single-cell mass cytometry on the experimental pipeline.

Magnetic fingerprints of rolling cells for quantitative flow cytometry in whole blood

NASA Astrophysics Data System (ADS)

Reisbeck, Mathias; Helou, Michael Johannes; Richter, Lukas; Kappes, Barbara; Friedrich, Oliver; Hayden, Oliver

2016-09-01

Over the past 50 years, flow cytometry has had a profound impact on preclinical and clinical applications requiring single cell function information for counting, sub-typing and quantification of epitope expression. At the same time, the workflow complexity and high costs of such optical systems still limit flow cytometry applications to specialized laboratories. Here, we present a quantitative magnetic flow cytometer that incorporates in situ magnetophoretic cell focusing for highly accurate and reproducible rolling of the cellular targets over giant magnetoresistance sensing elements. Time-of-flight analysis is used to unveil quantitative single cell information contained in its magnetic fingerprint. Furthermore, we used erythrocytes as a biological model to validate our methodology with respect to precise analysis of the hydrodynamic cell diameter, quantification of binding capacity of immunomagnetic labels, and discrimination of cell morphology. The extracted time-of-flight information should enable point-of-care quantitative flow cytometry in whole blood for clinical applications, such as immunology and primary hemostasis.

Ultrasensitive automated RNA in situ hybridization for kappa and lambda light chain mRNA detects B-cell clonality in tissue biopsies with performance comparable or superior to flow cytometry.

PubMed

Guo, Ling; Wang, Zhen; Anderson, Courtney M; Doolittle, Emerald; Kernag, Siobhan; Cotta, Claudiu V; Ondrejka, Sarah L; Ma, Xiao-Jun; Cook, James R

2018-03-01

The assessment of B-cell clonality is a critical component of the evaluation of suspected lymphoproliferative disorders, but analysis from formalin-fixed, paraffin-embedded tissues can be challenging if fresh tissue is not available for flow cytometry. Immunohistochemical and conventional bright field in situ hybridization stains for kappa and lambda are effective for evaluation of plasma cells but are often insufficiently sensitive to detect the much lower abundance of light chains present in B-cells. We describe an ultrasensitive RNA in situ hybridization assay that has been adapted for use on an automated immunohistochemistry platform and compare results with flow cytometry in 203 consecutive tissues and 104 consecutive bone marrows. Overall, in 203 tissue biopsies, RNA in situ hybridization identified light chain-restricted B-cells in 85 (42%) vs 58 (29%) by flow cytometry. Within 83 B-cell non-Hodgkin lymphomas, RNA in situ hybridization identified restricted B-cells in 74 (89%) vs 56 (67%) by flow cytometry. B-cell clonality could be evaluated in only 23/104 (22%) bone marrow cases owing to poor RNA preservation, but evaluable cases showed 91% concordance with flow cytometry. RNA in situ hybridization allowed for recognition of biclonal/composite lymphomas not identified by flow cytometry and highlighted unexpected findings, such as coexpression of kappa and lambda RNA in 2 cases and the presence of lambda light chain RNA in a T lymphoblastic lymphoma. Automated RNA in situ hybridization showed excellent interobserver reproducibility for manual evaluation (average K=0.92), and an automated image analysis system showed high concordance (97%) with manual evaluation. Automated RNA in situ hybridization staining, which can be adopted on commonly utilized immunohistochemistry instruments, allows for the interpretation of clonality in the context of the morphological features in formalin-fixed, paraffin-embedded tissues with a clinical sensitivity similar or superior to flow cytometry.

Ultrasensitive Automated RNA in situ Hybridization for Kappa and Lambda Light Chain mRNA Detects B-cell Clonality in Tissue Biopsies with Performance Comparable or Superior to Flow Cytometry

PubMed Central

Guo, Ling; Wang, Zhen; Anderson, Courtney M.; Doolittle, Emerald; Kernag, Siobhan; Cotta, Claudiu V.; Ondrejka, Sarah L.; Ma, Xiao-Jun; Cook, James R.

2017-01-01

The assessment of B-cell clonality is a critical component of the evaluation of suspected lymphoproliferative disorders, but analysis from formalin fixed paraffin embedded tissues can be challenging if fresh tissue is not available for flow cytometry. Immunohistochemical and conventional bright field in situ hybridization stains for kappa and lambda are effective for evaluation of plasma cells, but are often insufficiently sensitive to detect the much lower abundance of light chains present in B cells. We describe an ultrasensitive RNA in situ hybridization assay which has been adapted for use on an automated immunohistochemistry platform and compare results with flow cytometry in 203 consecutive tissues and 104 consecutive bone marrows. Overall, in 203 tissue biopsies, RNA in situ hybridization identified light chain restricted B-cells in 85 (42%) vs. 58 (29%) by flow cytometry. Within 83 B-cell non-Hodgkin lymphomas, RNA in situ hybridization identified a restricted B-cells in 74 (89%) vs. 56 (67%) by flow cytometry. B-cell clonality could be evaluated in only 23/104 (22%) bone marrow cases due to poor RNA preservation, but evaluable cases showed 91% concordance with flow cytometry. RNA in situ hybridization allowed for recognition of biclonal/composite lymphomas not identified by flow cytometry, and highlighted unexpected findings, such as coexpression of kappa and lambda RNA in 2 cases and the presence of lambda light chain RNA in a T lymphoblastic lymphoma. Automated RNA in situ hybridization showed excellent interobserver reproducibility for manual evaluation (average K=0.92), and an automated image analysis system showed high concordance (97%) with manual evaluation. Automated RNA in situ hybridization staining, which can be adopted on commonly utilized immunohistochemistry instruments, allows for the interpretation of clonality in the context of the morphologic features in formalin fixed, paraffin embedded tissues with a clinical sensitivity similar or superior to flow cytometry. PMID:29052600

CytometryML and other data formats

NASA Astrophysics Data System (ADS)

Leif, Robert C.

2006-02-01

Cytology automation and research will be enhanced by the creation of a common data format. This data format would provide the pathology and research communities with a uniform way for annotating and exchanging images, flow cytometry, and associated data. This specification and/or standard will include descriptions of the acquisition device, staining, the binary representations of the image and list-mode data, the measurements derived from the image and/or the list-mode data, and descriptors for clinical/pathology and research. An international, vendor-supported, non-proprietary specification will allow pathologists, researchers, and companies to develop and use image capture/analysis software, as well as list-mode analysis software, without worrying about incompatibilities between proprietary vendor formats. Presently, efforts to create specifications and/or descriptions of these formats include the Laboratory Digital Imaging Project (LDIP) Data Exchange Specification; extensions to the Digital Imaging and Communications in Medicine (DICOM); Open Microscopy Environment (OME); Flowcyt, an extension to the present Flow Cytometry Standard (FCS); and CytometryML. The feasibility of creating a common data specification for digital microscopy and flow cytometry in a manner consistent with its use for medical devices and interoperability with both hospital information and picture archiving systems has been demonstrated by the creation of the CytometryML schemas. The feasibility of creating a software system for digital microscopy has been demonstrated by the OME. CytometryML consists of schemas that describe instruments and their measurements. These instruments include digital microscopes and flow cytometers. Optical components including the instruments' excitation and emission parts are described. The description of the measurements made by these instruments includes the tagged molecule, data acquisition subsystem, and the format of the list-mode and/or image data. Many of the CytometryML data-types are based on the Digital Imaging and Communications in Medicine (DICOM). Binary files for images and list-mode data have been created and read.

A CLIPS expert system for clinical flow cytometry data analysis

NASA Technical Reports Server (NTRS)

Salzman, G. C.; Duque, R. E.; Braylan, R. C.; Stewart, C. C.

1990-01-01

An expert system is being developed using CLIPS to assist clinicians in the analysis of multivariate flow cytometry data from cancer patients. Cluster analysis is used to find subpopulations representing various cell types in multiple datasets each consisting of four to five measurements on each of 5000 cells. CLIPS facts are derived from results of the clustering. CLIPS rules are based on the expertise of Drs. Stewart, Duque, and Braylan. The rules incorporate certainty factors based on case histories.

Research on a pulmonary nodule segmentation method combining fast self-adaptive FCM and classification.

PubMed

Liu, Hui; Zhang, Cai-Ming; Su, Zhi-Yuan; Wang, Kai; Deng, Kai

2015-01-01

The key problem of computer-aided diagnosis (CAD) of lung cancer is to segment pathologically changed tissues fast and accurately. As pulmonary nodules are potential manifestation of lung cancer, we propose a fast and self-adaptive pulmonary nodules segmentation method based on a combination of FCM clustering and classification learning. The enhanced spatial function considers contributions to fuzzy membership from both the grayscale similarity between central pixels and single neighboring pixels and the spatial similarity between central pixels and neighborhood and improves effectively the convergence rate and self-adaptivity of the algorithm. Experimental results show that the proposed method can achieve more accurate segmentation of vascular adhesion, pleural adhesion, and ground glass opacity (GGO) pulmonary nodules than other typical algorithms.

High porous bio-nanocarbons prepared by carbonization and NaOH activation of polysaccharides for electrode material of EDLC

NASA Astrophysics Data System (ADS)

Takeuchi, Kenji; Fujishige, Masatsugu; Ishida, Nobuaki; Kunieda, Yoshihiro; Kato, Yosuke; Tanaka, Yusuke; Ochi, Toshiyuki; Shirotori, Hisashi; Uzuhashi, Yuji; Ito, Suguru; Oshida, Kyo-ichi; Endo, Morinobu

2018-07-01

Carbonization and post-activation of polysaccharides (utilized as food residue) created new bio-nanocarbons for the electrode of electric double layer capacitors (EDLC). Large specific capacitance (46.1 F/g, 26.4 F/cm3) and high rate performance was confirmed under optimized conditions of carbonization temperature (600 °C) and supplied amount of sodium hydroxide in NaOH-activation process (250 wt %). The capacitance and rate performance were larger than the reported values, 42.9 F/g, 19.7 F/cm3 of currently used activated carbon MSP-20. The feature that NaOH is usable as the activation agent, instead of KOH, is advantageous for reducing the cost of EDLC.

High content analysis of differentiation and cell death in human adipocytes.

PubMed

Doan-Xuan, Quang Minh; Sarvari, Anitta K; Fischer-Posovszky, Pamela; Wabitsch, Martin; Balajthy, Zoltan; Fesus, Laszlo; Bacso, Zsolt

2013-10-01

Understanding adipocyte biology and its homeostasis is in the focus of current obesity research. We aimed to introduce a high-content analysis procedure for directly visualizing and quantifying adipogenesis and adipoapoptosis by laser scanning cytometry (LSC) in a large population of cell. Slide-based image cytometry and image processing algorithms were used and optimized for high-throughput analysis of differentiating cells and apoptotic processes in cell culture at high confluence. Both preadipocytes and adipocytes were simultaneously scrutinized for lipid accumulation, texture properties, nuclear condensation, and DNA fragmentation. Adipocyte commitment was found after incubation in adipogenic medium for 3 days identified by lipid droplet formation and increased light absorption, while terminal differentiation of adipocytes occurred throughout day 9-14 with characteristic nuclear shrinkage, eccentric nuclei localization, chromatin condensation, and massive lipid deposition. Preadipocytes were shown to be more prone to tumor necrosis factor alpha (TNFα)-induced apoptosis compared to mature adipocytes. Importantly, spontaneous DNA fragmentation was observed at early stage when adipocyte commitment occurs. This DNA damage was independent from either spontaneous or induced apoptosis and probably was part of the differentiation program. © 2013 International Society for Advancement of Cytometry. Copyright © 2013 International Society for Advancement of Cytometry.

Highly multiparametric analysis by mass cytometry.

PubMed

Ornatsky, Olga; Bandura, Dmitry; Baranov, Vladimir; Nitz, Mark; Winnik, Mitchell A; Tanner, Scott

2010-09-30

This review paper describes a new technology, mass cytometry, that addresses applications typically run by flow cytometer analyzers, but extends the capability to highly multiparametric analysis. The detection technology is based on atomic mass spectrometry. It offers quantitation, specificity and dynamic range of mass spectrometry in a format that is familiar to flow cytometry practitioners. The mass cytometer does not require compensation, allowing the application of statistical techniques; this has been impossible given the constraints of fluorescence noise with traditional cytometry instruments. Instead of "colors" the mass cytometer "reads" the stable isotope tags attached to antibodies using metal-chelating labeling reagents. Because there are many available stable isotopes, and the mass spectrometer provides exquisite resolution between detection channels, many parameters can be measured as easily as one. For example, in a single tube the technique allows for the ready detection and characterization of the major cell subsets in blood or bone marrow. Here we describe mass cytometric immunophenotyping of human leukemia cell lines and leukemia patient samples, differential cell analysis of normal peripheral and umbilical cord blood; intracellular protein identification and metal-encoded bead arrays. Copyright © 2010 Elsevier B.V. All rights reserved.

Flow cytometry and conventional enumeration of microorganisms in ships' ballast water and marine samples.

PubMed

Joachimsthal, Eva L; Ivanov, Volodymyr; Tay, Joo-Hwa; Tay, Stephen T-L

2003-03-01

Conventional methods for bacteriological testing of water quality take long periods of time to complete. This makes them inappropriate for a shipping industry that is attempting to comply with the International Maritime Organization's anticipated regulations for ballast water discharge. Flow cytometry for the analysis of marine and ship's ballast water is a comparatively fast and accurate method. Compared to a 5% standard error for flow cytometry analysis the standard methods of culturing and epifluorescence analysis have errors of 2-58% and 10-30%, respectively. Also, unlike culturing methods, flow cytometry is capable of detecting both non-viable and viable but non-culturable microorganisms which can still pose health risks. The great variability in both cell concentrations and microbial content for the samples tested is an indication of the difficulties facing microbial monitoring programmes. The concentration of microorganisms in the ballast tank was generally lower than in local seawater. The proportion of aerobic, microaerophilic, and facultative anaerobic microorganisms present appeared to be influenced by conditions in the ballast tank. The gradual creation of anaerobic conditions in a ballast tank could lead to the accumulation of facultative anaerobic microorganisms, which might represent a potential source of pathogenic species.

Flow cytometry in the post fluorescence era.

PubMed

Nolan, Garry P

2011-12-01

While flow cytometry once enabled researchers to examine 10--15 cell surface parameters, new mass flow cytometry technology enables interrogation of up to 45 parameters on a single cell. This new technology has increased understanding of cell expression and how cells differentiate during hematopoiesis. Using this information, knowledge of leukemia cell biology has also increased. Other new technologies, such as SPADE analysis and single cell network profiling (SCNP), are enabling researchers to put different cancers into more biologically similar categories and have the potential to enable more personalized medicine. Copyright © 2011. Published by Elsevier Ltd.

Small lasers in flow cytometry.

PubMed

Telford, William G

2004-01-01

Laser technology has made tremendous advances in recent years, particularly in the area of diode and diode-pumped solid state sources. Flow cytometry has been a direct beneficiary of these advances, as these small, low-maintenance, inexpensive lasers with reasonable power outputs are integrated into flow cytometers. In this chapter we review the contribution and potential of solid-state lasers to flow cytometry, and show several examples of these novel sources integrated into production flow cytometers. Technical details and critical parameters for successful application of these lasers for biomedical analysis are reviewed.

Examination pf Potential Anti-Tumor Activity of N-Thiolated B-Lactam Antibiotics in Nude Mice Bearing Human Breast Tumors

DTIC Science & Technology

2005-08-01

temperature in the dark, and then analyzed by flow cytometry within 3 hr of staining. 2.7. Caspase-3/-7 activity assay To measure cell-free caspase-3/-7...were treated with 50 mM of lactam 12 for the indicated hours. (A) Measurement of sub-G1 DNA content by flow cytometry analysis. The percentage of sub...Daniel for critical reading of the manuscript. We also appreciate the assistance of the Flow Cytometry Core at H. Lee Moffitt Cancer Center

Non-linear optical flow cytometry using a scanned, Bessel beam light-sheet.

PubMed

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

2015-05-29

Modern flow cytometry instruments have become vital tools for high-throughput analysis of single cells. However, as issues with the cellular labeling techniques often used in flow cytometry have become more of a concern, the development of label-free modalities for cellular analysis is increasingly desired. Non-linear optical phenomena (NLO) are of growing interest for label-free analysis because of the ability to measure the intrinsic optical response of biomolecules found in cells. We demonstrate that a light-sheet consisting of a scanned Bessel beam is an optimal excitation geometry for efficiently generating NLO signals in a microfluidic environment. The balance of photon density and cross-sectional area provided by the light-sheet allowed significantly larger two-photon fluorescence intensities to be measured in a model polystyrene microparticle system compared to measurements made using other excitation focal geometries, including a relaxed Gaussian excitation beam often used in conventional flow cytometers.

Managing Multi-center Flow Cytometry Data for Immune Monitoring

PubMed Central

White, Scott; Laske, Karoline; Welters, Marij JP; Bidmon, Nicole; van der Burg, Sjoerd H; Britten, Cedrik M; Enzor, Jennifer; Staats, Janet; Weinhold, Kent J; Gouttefangeas, Cécile; Chan, Cliburn

2014-01-01

With the recent results of promising cancer vaccines and immunotherapy1–5, immune monitoring has become increasingly relevant for measuring treatment-induced effects on T cells, and an essential tool for shedding light on the mechanisms responsible for a successful treatment. Flow cytometry is the canonical multi-parameter assay for the fine characterization of single cells in solution, and is ubiquitously used in pre-clinical tumor immunology and in cancer immunotherapy trials. Current state-of-the-art polychromatic flow cytometry involves multi-step, multi-reagent assays followed by sample acquisition on sophisticated instruments capable of capturing up to 20 parameters per cell at a rate of tens of thousands of cells per second. Given the complexity of flow cytometry assays, reproducibility is a major concern, especially for multi-center studies. A promising approach for improving reproducibility is the use of automated analysis borrowing from statistics, machine learning and information visualization21–23, as these methods directly address the subjectivity, operator-dependence, labor-intensive and low fidelity of manual analysis. However, it is quite time-consuming to investigate and test new automated analysis techniques on large data sets without some centralized information management system. For large-scale automated analysis to be practical, the presence of consistent and high-quality data linked to the raw FCS files is indispensable. In particular, the use of machine-readable standard vocabularies to characterize channel metadata is essential when constructing analytic pipelines to avoid errors in processing, analysis and interpretation of results. For automation, this high-quality metadata needs to be programmatically accessible, implying the need for a consistent Application Programming Interface (API). In this manuscript, we propose that upfront time spent normalizing flow cytometry data to conform to carefully designed data models enables automated analysis, potentially saving time in the long run. The ReFlow informatics framework was developed to address these data management challenges. PMID:26085786

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

Vasdekis, Andreas E.; Stephanopoulos, Gregory

The sampling and manipulation of cells down to the individual has been of substantial interest since the very beginning of Life Sciences. Herein, our objective is to highlight the most recent developments in single cell manipulation, as well as pioneering ones. First, flow-through methods will be discussed, namely methods in which the single cells flow continuously in an ordered manner during their analysis. This section will be followed by confinement techniques that enable cell isolation and confinement in one, two- or three-dimensions. Flow cytometry and droplet microfluidics are the two most common methods of flow-through analysis. While both are high-throughputmore » techniques, their difference lays in the fact that the droplet encapsulated cells experience a restricted and personal microenvironment, while in flow cytometry cells experience similar nutrient and stimuli initial concentrations. These methods are rather well established; however, they recently enabled immense strides in single cell phenotypic analysis, namely the identification and analysis of metabolically distinct individuals from an isogenic population using both droplet microfluidics and flow cytometry.« less

Three-dimensional skeleton networks of graphene wrapped polyaniline nanofibers: an excellent structure for high-performance flexible solid-state supercapacitors

PubMed Central

Hu, Nantao; Zhang, Liling; Yang, Chao; Zhao, Jian; Yang, Zhi; Wei, Hao; Liao, Hanbin; Feng, Zhenxing; Fisher, Adrian; Zhang, Yafei; Xu, Zhichuan J.

2016-01-01

Thin, robust, lightweight, and flexible supercapacitors (SCs) have aroused growing attentions nowadays due to the rapid development of flexible electronics. Graphene-polyaniline (PANI) hybrids are attractive candidates for high performance SCs. In order to utilize them in real devices, it is necessary to improve the capacitance and the structure stability of PANI. Here we report a hierarchical three-dimensional structure, in which all of PANI nanofibers (NFs) are tightly wrapped inside reduced graphene oxide (rGO) nanosheet skeletons, for high-performance flexible SCs. The as-fabricated film electrodes with this unique structure showed a highest gravimetric specific capacitance of 921 F/g and volumetric capacitance of 391 F/cm3. The assembled solid-state SCs gave a high specific capacitance of 211 F/g (1 A/g), a high area capacitance of 0.9 F/cm2, and a competitive volumetric capacitance of 25.6 F/cm3. The SCs also exhibited outstanding rate capability (~75% retention at 20 A/g) as well as excellent cycling stability (100% retention at 10 A/g for 2000 cycles). Additionally, no structural failure and loss of performance were observed under the bending state. This structure design paves a new avenue for engineering rGO/PANI or other similar hybrids for high performance flexible energy storage devices. PMID:26795067

Three-dimensional skeleton networks of graphene wrapped polyaniline nanofibers: An excellent structure for high-performance flexible solid-state supercapacitors

DOE PAGES

Hu, Nantao; Zhang, Liling; Yang, Chao; ...

2016-01-22

Thin, robust, lightweight, and flexible supercapacitors (SCs) have aroused growing attentions nowadays due to the rapid development of flexible electronics. Graphene-polyaniline (PANI) hybrids are attractive candidates for high performance SCs. In order to utilize them in real devices, it is necessary to improve the capacitance and the structure stability of PANI. Here we report a hierarchical three-dimensional structure, in which all of PANI nanofibers (NFs) are tightly wrapped inside reduced graphene oxide (rGO) nanosheet skeletons, for high-performance flexible SCs. The as-fabricated film electrodes with this unique structure showed a highest gravimetric specific capacitance of 921 F/g and volumetric capacitance ofmore » 391 F/cm 3. The assembled solid-state SCs gave a high specific capacitance of 211 F/g (1 A/g), a high area capacitance of 0.9 F/cm 2, and a competitive volumetric capacitance of 25.6 F/cm 3. The SCs also exhibited outstanding rate capability (~75% retention at 20 A/g) as well as excellent cycling stability (100% retention at 10 A/g for 2000 cycles). Additionally, no structural failure and loss of performance were observed under the bending state. Lastly, this structure design paves a new avenue for engineering rGO/PANI or other similar hybrids for high performance flexible energy storage devices.« less

Studies on processing, consumer survey and storage stability of a ready-to-reconstitute fish cutlet mix.

PubMed

Shaviklo, G Reza; Thorkelsson, Gudjon; Sveinsdottir, Kolbrun; Pourreza, Fatemeh

2013-10-01

A convenience ready-to-reconstitute cutlet mix containing 30% fish protein powder was developed to improve the nutritional quality of the product. Consumer survey was based on the home use test (HUT) method. The acceptance of the fish cutlet mix (FCM) was studied using a 9-point hedonic scale ranging from 1 (extremely dislike) to 9 (extremely like). Product's characteristics and stability were studied during 6 months of storage at 27 ± 2 °C. The FCM packed in a polyethylene bag and cardboard box was stable during the storage period. There were no changes in colour, moisture gain and water activity, and TBARS values remained low. The FCM was accepted by the consumers in the study (n = 85). The average liking was high (7.5 ± 1.3) and it was influenced by frequency of fish and chicken consumption, educational level and household size. People who ate fish once a week liked the product more than other consumers. Also those with higher educational level and bigger household size. The results in this paper are important information for companies planning to develop ready-to-eat products fortified with fish proteins. The products could be means of increasing fish consumption in countries/areas where there is no tradition of consuming fresh or frozen fish.

Effects of siRNA-mediated knockdown of jumonji domain containing 2A on proliferation, migration and invasion of the human breast cancer cell line MCF-7

PubMed Central

LI, BEI-XU; LUO, CHENG-LIANG; LI, HUI; YANG, PENG; ZHANG, MING-CHANG; XU, HONG-MEI; XU, HONG-FEI; SHEN, YI-WEN; XUE, AI-MIN; ZHAO, ZI-QIN

2012-01-01

Jumonji domain containing 2A (JMJD2A) is a potential cancer-associated gene that may be involved in human breast cancer. The present study aimed to investigate suppressive effects on the MCF-7 human breast cancer cell line by transfection with JMJD2A-specific siRNA. Quantitative real-time PCR and western blot analysis were used to detect the expression levels of JMJD2A. Flow cytometric (FCM) analysis and WST-8 assay were used to evaluate cell proliferation. Boyden chambers were used in cell migration and invasion assays to evaluate the cell exercise capacity. Expression levels of JMJD2A mRNA and protein in the siRNA group were both downregulated successfully by transfection. FCM results showed that the percentage of cells in the G0/G1 phase in the siRNA group was significantly greater than that in the blank (P<0.05) and negative control groups (P<0.05). Additionally, the mean absorbance in the siRNA group was significantly lower (P<0.05), as observed by WST-8 assay. Moreover, a decreased number of migrated cells in the siRNA group was observed (P<0.05) using a cell migration and invasion assay. These data indicated that knockdown of JMJD2A may cause inhibition of proliferation, migration and invasion of MCF-7 cells. This study provides a new perspective in understanding the molecular mechanisms underlying the progression of breast cancer and offers a potential therapeutic target for breast cancer. PMID:23170139

DAFi: A directed recursive data filtering and clustering approach for improving and interpreting data clustering identification of cell populations from polychromatic flow cytometry data.

PubMed

Lee, Alexandra J; Chang, Ivan; Burel, Julie G; Lindestam Arlehamn, Cecilia S; Mandava, Aishwarya; Weiskopf, Daniela; Peters, Bjoern; Sette, Alessandro; Scheuermann, Richard H; Qian, Yu

2018-04-17

Computational methods for identification of cell populations from polychromatic flow cytometry data are changing the paradigm of cytometry bioinformatics. Data clustering is the most common computational approach to unsupervised identification of cell populations from multidimensional cytometry data. However, interpretation of the identified data clusters is labor-intensive. Certain types of user-defined cell populations are also difficult to identify by fully automated data clustering analysis. Both are roadblocks before a cytometry lab can adopt the data clustering approach for cell population identification in routine use. We found that combining recursive data filtering and clustering with constraints converted from the user manual gating strategy can effectively address these two issues. We named this new approach DAFi: Directed Automated Filtering and Identification of cell populations. Design of DAFi preserves the data-driven characteristics of unsupervised clustering for identifying novel cell subsets, but also makes the results interpretable to experimental scientists through mapping and merging the multidimensional data clusters into the user-defined two-dimensional gating hierarchy. The recursive data filtering process in DAFi helped identify small data clusters which are otherwise difficult to resolve by a single run of the data clustering method due to the statistical interference of the irrelevant major clusters. Our experiment results showed that the proportions of the cell populations identified by DAFi, while being consistent with those by expert centralized manual gating, have smaller technical variances across samples than those from individual manual gating analysis and the nonrecursive data clustering analysis. Compared with manual gating segregation, DAFi-identified cell populations avoided the abrupt cut-offs on the boundaries. DAFi has been implemented to be used with multiple data clustering methods including K-means, FLOCK, FlowSOM, and the ClusterR package. For cell population identification, DAFi supports multiple options including clustering, bisecting, slope-based gating, and reversed filtering to meet various autogating needs from different scientific use cases. © 2018 International Society for Advancement of Cytometry. © 2018 International Society for Advancement of Cytometry.

Minimal Residual Disease Evaluation in Childhood Acute Lymphoblastic Leukemia: An Economic Analysis

PubMed Central

Gajic-Veljanoski, O.; Pham, B.; Pechlivanoglou, P.; Krahn, M.; Higgins, Caroline; Bielecki, Joanna

2016-01-01

Background Minimal residual disease (MRD) testing by higher performance techniques such as flow cytometry and polymerase chain reaction (PCR) can be used to detect the proportion of remaining leukemic cells in bone marrow or peripheral blood during and after the first phases of chemotherapy in children with acute lymphoblastic leukemia (ALL). The results of MRD testing are used to reclassify these patients and guide changes in treatment according to their future risk of relapse. We conducted a systematic review of the economic literature, cost-effectiveness analysis, and budget-impact analysis to ascertain the cost-effectiveness and economic impact of MRD testing by flow cytometry for management of childhood precursor B-cell ALL in Ontario. Methods A systematic literature search (1998–2014) identified studies that examined the incremental cost-effectiveness of MRD testing by either flow cytometry or PCR. We developed a lifetime state-transition (Markov) microsimulation model to quantify the cost-effectiveness of MRD testing followed by risk-directed therapy to no MRD testing and to estimate its marginal effect on health outcomes and on costs. Model input parameters were based on the literature, expert opinion, and data from the Pediatric Oncology Group of Ontario Networked Information System. Using predictions from our Markov model, we estimated the 1-year cost burden of MRD testing versus no testing and forecasted its economic impact over 3 and 5 years. Results In a base-case cost-effectiveness analysis, compared with no testing, MRD testing by flow cytometry at the end of induction and consolidation was associated with an increased discounted survival of 0.0958 quality-adjusted life-years (QALYs) and increased discounted costs of $4,180, yielding an incremental cost-effectiveness ratio (ICER) of $43,613/QALY gained. After accounting for parameter uncertainty, incremental cost-effectiveness of MRD testing was associated with an ICER of $50,249/QALY gained. In the budget-impact analysis, the 1-year cost expenditure for MRD testing by flow cytometry in newly diagnosed patients with precursor B-cell ALL was estimated at $340,760. We forecasted that the province would have to pay approximately $1.3 million over 3 years and $2.4 million over 5 years for MRD testing by flow cytometry in this population. Conclusions Compared with no testing, MRD testing by flow cytometry in newly diagnosed patients with precursor B-cell ALL represents good value for money at commonly used willingness-to-pay thresholds of $50,000/QALY and $100,000/QALY. PMID:27099644

Minimal Residual Disease Evaluation in Childhood Acute Lymphoblastic Leukemia: An Economic Analysis.

PubMed

2016-01-01

Minimal residual disease (MRD) testing by higher performance techniques such as flow cytometry and polymerase chain reaction (PCR) can be used to detect the proportion of remaining leukemic cells in bone marrow or peripheral blood during and after the first phases of chemotherapy in children with acute lymphoblastic leukemia (ALL). The results of MRD testing are used to reclassify these patients and guide changes in treatment according to their future risk of relapse. We conducted a systematic review of the economic literature, cost-effectiveness analysis, and budget-impact analysis to ascertain the cost-effectiveness and economic impact of MRD testing by flow cytometry for management of childhood precursor B-cell ALL in Ontario. A systematic literature search (1998-2014) identified studies that examined the incremental cost-effectiveness of MRD testing by either flow cytometry or PCR. We developed a lifetime state-transition (Markov) microsimulation model to quantify the cost-effectiveness of MRD testing followed by risk-directed therapy to no MRD testing and to estimate its marginal effect on health outcomes and on costs. Model input parameters were based on the literature, expert opinion, and data from the Pediatric Oncology Group of Ontario Networked Information System. Using predictions from our Markov model, we estimated the 1-year cost burden of MRD testing versus no testing and forecasted its economic impact over 3 and 5 years. In a base-case cost-effectiveness analysis, compared with no testing, MRD testing by flow cytometry at the end of induction and consolidation was associated with an increased discounted survival of 0.0958 quality-adjusted life-years (QALYs) and increased discounted costs of $4,180, yielding an incremental cost-effectiveness ratio (ICER) of $43,613/QALY gained. After accounting for parameter uncertainty, incremental cost-effectiveness of MRD testing was associated with an ICER of $50,249/QALY gained. In the budget-impact analysis, the 1-year cost expenditure for MRD testing by flow cytometry in newly diagnosed patients with precursor B-cell ALL was estimated at $340,760. We forecasted that the province would have to pay approximately $1.3 million over 3 years and $2.4 million over 5 years for MRD testing by flow cytometry in this population. Compared with no testing, MRD testing by flow cytometry in newly diagnosed patients with precursor B-cell ALL represents good value for money at commonly used willingness-to-pay thresholds of $50,000/QALY and $100,000/QALY.

Prediction of outcome in neurogenic oropharyngeal dysphagia within 72 hours of acute stroke.

PubMed

Ickenstein, Guntram W; Höhlig, Carolin; Prosiegel, Mario; Koch, Horst; Dziewas, Rainer; Bodechtel, Ulf; Müller, Rainer; Reichmann, Heinz; Riecker, Axel

2012-10-01

Stroke is the most frequent cause of neurogenic oropharyngeal dysphagia (NOD). In the acute phase of stroke, the frequency of NOD is greater than 50% and, half of this patient population return to good swallowing within 14 days while the other half develop chronic dysphagia. Because dysphagia leads to aspiration pneumonia, malnutrition, and in-hospital mortality, it is important to pay attention to swallowing problems. The question arises if a prediction of severe chronic dysphagia is possible within the first 72 hours of acute stroke. On admission to the stroke unit, all stroke patients were screened for swallowing problems by the nursing staff within 2 hours. Patients showing signs of aspiration were included in the study (n = 114) and were given a clinical swallowing examination (CSE) by the swallowing/speech therapist within 24 hours and a swallowing endoscopy within 72 hours by the physician. The primary outcome of the study was the functional communication measure (FCM) of swallowing (score 1-3, tube feeding dependency) on day 90. The grading system with the FCM swallowing and the penetration-aspiration scale (PAS) in the first 72 hours was tested in a multivariate analysis for its predictive value for tube feeding-dependency on day 90. For the FCM level 1 to 3 (P < .0022) and PAS level 5 to 8 (P < .00001), the area under the curve (AUC) was 72.8% and showed an odds ratio of 11.8 (P < .00001; 95% confidence interval 0.036-0.096), achieving for the patient a 12 times less chance of being orally fed on day 90 and therefore still being tube feeding-dependent. We conclude that signs of aspiration in the first 72 hours of acute stroke can predict severe swallowing problems on day 90. Consequently, patients should be tested on admission to a stroke unit and evaluated with established dysphagia scales to prevent aspiration pneumonia and malnutrition. A dysphagia program can lead to better communication within the stroke unit team to initiate the appropriate diagnostics and swallowing therapy as soon as possible. Copyright © 2012 National Stroke Association. Published by Elsevier Inc. All rights reserved.

CytometryML binary data standards

NASA Astrophysics Data System (ADS)

Leif, Robert C.

2005-03-01

CytometryML is a proposed new Analytical Cytology (Cytomics) data standard, which is based on a common set of XML schemas for encoding flow cytometry and digital microscopy text based data types (metadata). CytometryML schemas reference both DICOM (Digital Imaging and Communications in Medicine) codes and FCS keywords. Flow Cytometry Standard (FCS) list-mode has been mapped to the DICOM Waveform Information Object. The separation of the large binary data objects (list mode and image data) from the XML description of the metadata permits the metadata to be directly displayed, analyzed, and reported with standard commercial software packages; the direct use of XML languages; and direct interfacing with clinical information systems. The separation of the binary data into its own files simplifies parsing because all extraneous header data has been eliminated. The storage of images as two-dimensional arrays without any extraneous data, such as in the Adobe Photoshop RAW format, facilitates the development by scientists of their own analysis and visualization software. Adobe Photoshop provided the display infrastructure and the translation facility to interconvert between the image data from commercial formats and RAW format. Similarly, the storage and parsing of list mode binary data type with a group of parameters that are specified at compilation time is straight forward. However when the user is permitted at run-time to select a subset of the parameters and/or specify results of mathematical manipulations, the development of special software was required. The use of CytometryML will permit investigators to be able to create their own interoperable data analysis software and to employ commercially available software to disseminate their data.

Research on a Pulmonary Nodule Segmentation Method Combining Fast Self-Adaptive FCM and Classification

PubMed Central

Liu, Hui; Zhang, Cai-Ming; Su, Zhi-Yuan; Wang, Kai; Deng, Kai

2015-01-01

The key problem of computer-aided diagnosis (CAD) of lung cancer is to segment pathologically changed tissues fast and accurately. As pulmonary nodules are potential manifestation of lung cancer, we propose a fast and self-adaptive pulmonary nodules segmentation method based on a combination of FCM clustering and classification learning. The enhanced spatial function considers contributions to fuzzy membership from both the grayscale similarity between central pixels and single neighboring pixels and the spatial similarity between central pixels and neighborhood and improves effectively the convergence rate and self-adaptivity of the algorithm. Experimental results show that the proposed method can achieve more accurate segmentation of vascular adhesion, pleural adhesion, and ground glass opacity (GGO) pulmonary nodules than other typical algorithms. PMID:25945120

Annual Progress Report FY-92. Volume 1

DTIC Science & Technology

1993-01-21

Billups, L Flow Cytom Resh Psychologist 12 0180 CS Hamm, C DCI 7 DESCRIPTION GRADE MOS BRANCH NAME ACTIVITY Kyle Metabolic Unit Nursing Service Supv...3349 Salata, Kalman PhD. Mitogen-Inducible T Suppressor Cell 13 Assay by Flow Cytometry (12/89) 3350 Salata, Kalman PhD. Flow Cytometric Analysis of...17 Immunotherapy (3/90) 3354 Salata, Kalman PhD. Two Way Mixed Lymphocyte Culture: 18 Analysis by Two Color Flow Cytometry (4/90) 3355 Salata, Kalman

78 FR 5186 - Clinical Flow Cytometry in Hematologic Malignancies; Public Workshop; Request for Comments

Federal Register 2010, 2011, 2012, 2013, 2014

2013-01-24

... in the diagnosis of leukemia and lymphoma and more recently in the detection of minimal residual... chronic lymphocytic leukemia (CLL); (3) Third-party flow cytometry data analysis software; and (4... held February 27, 2013 (77 FR 76051, December 26, 2012). An FDA workshop for acute lymphocytic leukemia...

Applications of flow cytometry to toxicological mycotoxin effects in cultured mammalian cells: a review.

PubMed

Juan-García, Ana; Manyes, Lara; Ruiz, María-José; Font, Guillermina

2013-06-01

This review gives an overview of flow cytometry applications to toxicological studies of several physiological target sites of mycotoxins on different mammalian cell lines. Mycotoxins are secondary metabolites of fungi that may be present in food, feed, air and water. The increasing presence of mycotoxins in crops, their wide distribution in the food chain, and their potential for toxicity demonstrate the need for further knowledge. Flow cytometry has become a valuable tool in mycotoxin studies in recent years for the rapid analysis of single cells in a mixture. In toxicology, the power of these methods lies in the possibility of determining a wide range of cell parameters, providing valuable information to elucidate cell growth and viability, metabolic activity, mitochondrial membrane potential and membrane integrity mechanisms. There are studies using flow cytometry technique on Alternaria, Aspergillus, Fusarium and Penicillium mycotoxins including information about cell type, assay conditions and functional parameters. Most of the studies collected in the literature are on deoxynivalenol and zearalenone mycotoxins. Cell cycle analysis and apoptosis are the processes more widely investigated. Copyright © 2013 Elsevier Ltd. All rights reserved.

Development of an unbiased, semi-automated approach for classifying plasma cell immunophenotype following multicolor flow cytometry of bone marrow aspirates.

PubMed

Post, Steven R; Post, Ginell R; Nikolic, Dejan; Owens, Rebecca; Insuasti-Beltran, Giovanni

2018-03-24

Despite increased usage of multiparameter flow cytometry (MFC) to assess diagnosis, prognosis, and therapeutic efficacy (minimal residual disease, MRD) in plasma cell neoplasms (PCNs), standardization of methodology and data analysis is suboptimal. We investigated the utility of using the mean and median fluorescence intensities (FI) obtained from MFC to objectively describe parameters that distinguish plasma cell (PC) phenotypes. In this retrospective study, flow cytometry results from bone marrow aspirate specimens from 570 patients referred to the Myeloma Institute at UAMS were evaluated. Mean and median FI data were obtained from 8-color MFC of non-neoplastic, malignant, and mixed PC populations using antibodies to CD38, CD138, CD19, CD20, CD27, CD45, CD56, and CD81. Of 570 cases, 252 cases showed only non-neoplastic PCs, 168 showed only malignant PCs, and 150 showed mixed PC populations. Statistical analysis of median FI data for each CD marker showed no difference in expression intensity on non-neoplastic and malignant PCs, between pure and mixed PC populations. ROC analysis of the median FI of CD expression in non-neoplastic and malignant PCs was used to develop an algorithm to convert quantitative FI values to qualitative assessments including "negative," "positive," "dim," and "heterogeneous" expression. FI data derived from 8-color MFC can be used to define marker expression on PCs. Translation of FI data from Infinicyt software to an Excel worksheet streamlines workflow and eliminates transcriptional errors when generating flow reports. © 2018 International Clinical Cytometry Society. © 2018 International Clinical Cytometry Society.

Nutrient digestibility and milk production responses to increasing levels of palmitic acid supplementation vary in cows receiving diets with or without whole cottonseed.

PubMed

Rico, J E; de Souza, J; Allen, M S; Lock, A L

2017-01-01

Our study evaluated the dose-dependent effects of a palmitic acid-enriched supplement in basal diets with or without the inclusion of whole cottonseed on nutrient digestibility and production responses of dairy cows. Sixteen Holstein cows (149 ± 56 days in milk) were used in a split plot Latin square design experiment. Cows were blocked by 3.5% fat-corrected milk (FCM) and allocated to a main plot receiving either a basal diet with soyhulls (SH, = 8) or a basal diet with whole cottonseed (CS, = 8) that was fed throughout the experiment. A palmitic acid-enriched supplement (PA 88.5% C16:0) was fed at 0, 0.75, 1.50, or 2.25% of ration DM in a replicated 4 × 4 Latin Square design within each basal diet group. Periods were 14 d with the final 4 d used for data collection. PA dose increased milk fat content linearly, and cubically affected yields of milk fat and 3.5% FCM. The PA dose did not affect milk protein and lactose contents, BW, and BCS, but tended to increase yields of milk, milk protein, and milk lactose. Also, PA dose reduced DMI and 16-carbon fatty acid digestibility quadratically, and increased 18-carbon fatty acid digestibility quadratically. There were no effects of basal diet on the yield of milk or milk components, but DMI tended to decrease in CS compared with SH, increasing feed efficiency (3.5% FCM/DMI). Compared with SH, CS diets increased yield of preformed milk fatty acids and 16-carbon fatty acid digestibility, and tended to decrease 18-carbon fatty acid digestibility. We observed basal diet × PA dose interactions for yields of milk and milk protein and for 16-carbon and total fatty acid digestibility, as well as tendency for yields of milk fat and 3.5% FCM. Also, there was a tendency for an interaction between basal diet and PA dose for NDF digestibility, which increased more for CS with increasing PA than for SH. PA dose linearly decreased digestibility of total fatty acids in SH diets but did not affect it in CS diets Results demonstrate that responses to PA dose are affected by the dietary basal diet. Additionally, the decrease in fatty acid digestibility only in the SH diets suggests that digestibility is impacted mainly by the profile of 16- and 18-carbon fatty acids reaching the duodenum. Under the dietary conditions evaluated, the yield of 3.5% FCM and milk fat were optimal when PA was fed at 1.5% of ration DM.

Drug testing and flow cytometry analysis on a large number of uniform sized tumor spheroids using a microfluidic device

NASA Astrophysics Data System (ADS)

Patra, Bishnubrata; Peng, Chien-Chung; Liao, Wei-Hao; Lee, Chau-Hwang; Tung, Yi-Chung

2016-02-01

Three-dimensional (3D) tumor spheroid possesses great potential as an in vitro model to improve predictive capacity for pre-clinical drug testing. In this paper, we combine advantages of flow cytometry and microfluidics to perform drug testing and analysis on a large number (5000) of uniform sized tumor spheroids. The spheroids are formed, cultured, and treated with drugs inside a microfluidic device. The spheroids can then be harvested from the device without tedious operation. Due to the ample cell numbers, the spheroids can be dissociated into single cells for flow cytometry analysis. Flow cytometry provides statistical information in single cell resolution that makes it feasible to better investigate drug functions on the cells in more in vivo-like 3D formation. In the experiments, human hepatocellular carcinoma cells (HepG2) are exploited to form tumor spheroids within the microfluidic device, and three anti-cancer drugs: Cisplatin, Resveratrol, and Tirapazamine (TPZ), and their combinations are tested on the tumor spheroids with two different sizes. The experimental results suggest the cell culture format (2D monolayer vs. 3D spheroid) and spheroid size play critical roles in drug responses, and also demonstrate the advantages of bridging the two techniques in pharmaceutical drug screening applications.

Application of a Short Intracellular pH Method to Flow Cytometry for Determining Saccharomyces cerevisiae Vitality ▿

PubMed Central

Weigert, Claudia; Steffler, Fabian; Kurz, Tomas; Shellhammer, Thomas H.; Methner, Frank-Jürgen

2009-01-01

The measurement of yeast's intracellular pH (ICP) is a proven method for determining yeast vitality. Vitality describes the condition or health of viable cells as opposed to viability, which defines living versus dead cells. In contrast to fluorescence photometric measurements, which show only average ICP values of a population, flow cytometry allows the presentation of an ICP distribution. By examining six repeated propagations with three separate growth phases (lag, exponential, and stationary), the ICP method previously established for photometry was transferred successfully to flow cytometry by using the pH-dependent fluorescent probe 5,6-carboxyfluorescein. The correlation between the two methods was good (r2 = 0.898, n = 18). With both methods it is possible to track the course of growth phases. Although photometry did not yield significant differences between exponentially and stationary phases (P = 0.433), ICP via flow cytometry did (P = 0.012). Yeast in an exponential phase has a unimodal ICP distribution, reflective of a homogeneous population; however, yeast in a stationary phase displays a broader ICP distribution, and subpopulations could be defined by using the flow cytometry method. In conclusion, flow cytometry yielded specific evidence of the heterogeneity in vitality of a yeast population as measured via ICP. In contrast to photometry, flow cytometry increases information about the yeast population's vitality via a short measurement, which is suitable for routine analysis. PMID:19581482

Recognition of Naegleriae ameba surface protein epitopes by anti-human CD45 antibodies.

PubMed

Ravine, Terrence J; Polski, Jacek M; Jenkins, James

2010-04-01

Phagocytosis is a highly conserved mechanism exhibited by both free-living amebas and mammalian blood cells. Similarities demonstrated by either cell type during engulfment of the same bacterial species may imply analogous surface proteins involved in receptor-mediated endocytosis. The increased availability of anti-human leukocyte antibodies or clusters of differentiation (CD) markers used in conjunction with flow cytometric (FCM) and/or immunohistochemical (IHC) analysis provides investigators with a relatively easy method to screen different cell populations for comparable plasma membrane proteins. In this study, we incubated Naegleria and Acanthamoeba amebas with several directly conjugated anti-human leukocyte monoclonal antibodies (mAb) for similarly recognized amebic epitopes. CD marker selection was based upon a recognized role of each mAb in phagocyte activation and/or uptake of bacteria. These included CD14, CD45, and CD206. In FCM, only one CD45 antibody demonstrated strong reactivity with both Naegleria fowleri and Naegleria gruberi that was not expressed in similarly tested Acanthamoeba species. Additional testing of N. gruberi by IHC demonstrated reactivity to a different CD45 antibody. Our results suggest a possible utility of using anti-human leukocyte antibodies to screen amebic cells for similarly expressed protein epitopes. In doing so, several important items must be considered when selecting potential mAbs for testing to increase the probability of a positive result.

Textile Supercapacitors

NASA Astrophysics Data System (ADS)

Jost, Kristy Alana

Innovative and interdisciplinary solutions to wearable textile energy storage are explored as power sources for wearable electronics and smart textiles. Due to their long cycle life, non-toxic and inexpensive materials, supercapacitors were converted into textiles. Textile supercapacitors were developed using scalable fabrication methods including screen-printing, yarn making, and 3D computerized knitting. The electrode materials reported in this thesis undergo thorough electrochemical analysis, and are capable of storing up to 0.5 F/cm2 which is on par with conventionally solid supercapacitors (0.6 F/cm2). Capacitive yarns are capable of storing up to 37 mF/cm and are shown to be knittable on industrial knitting equipment. Both are some of the highest reported capacitance for all-carbon systems in the field. Yet both are the only systems composed of inexpensive and non-toxic activated carbon, the most commonly used electrode material used in supercapacitors, opposed to carbon nanotubes or graphene, which are typically more 10-100 times more expensive. However, all of the fabrication techniques reported here are also capable of incorporating a wide variety of materials, ultimately broadening the applications of textile energy storage as a whole. Fully machine knitted supercapacitors are also explored and electrochemically characterized in order to determine how the textile structure affects the capacitance. In conclusion, a wide variety of fabrication techniques for making textile supercapacitors were successfully explored.

Pomegranate MR images analysis using ACM and FCM algorithms

NASA Astrophysics Data System (ADS)

Morad, Ghobad; Shamsi, Mousa; Sedaaghi, M. H.; Alsharif, M. R.

2011-10-01

Segmentation of an image plays an important role in image processing applications. In this paper segmentation of pomegranate magnetic resonance (MR) images has been explored. Pomegranate has healthy nutritional and medicinal properties for which the maturity indices and quality of internal tissues play an important role in the sorting process in which the admissible determination of features mentioned above cannot be easily achieved by human operator. Seeds and soft tissues are the main internal components of pomegranate. For research purposes, such as non-destructive investigation, in order to determine the ripening index and the percentage of seeds in growth period, segmentation of the internal structures should be performed as exactly as possible. In this paper, we present an automatic algorithm to segment the internal structure of pomegranate. Since its intensity of stem and calyx is close to the internal tissues, the stem and calyx pixels are usually labeled to the internal tissues by segmentation algorithm. To solve this problem, first, the fruit shape is extracted from its background using active contour model (ACM). Then stem and calyx are removed using morphological filters. Finally the image is segmented by fuzzy c-means (FCM). The experimental results represent an accuracy of 95.91% in the presence of stem and calyx, while the accuracy of segmentation increases to 97.53% when stem and calyx are first removed by morphological filters.

Spaceflight Flow Cytometry: Design Challenges and Applications

NASA Technical Reports Server (NTRS)

Pappas, Dimitri; Kao, Shih-Hsin; Jeevarajan, Antony S.

2004-01-01

Future space exploration missions will require analytical technology capable of providing both autonomous medical care to the crew and investigative capabilities to researchers. While several promising candidate technologies exist for further development, flow cytometry is an attractive technology as it offers both crew health and a wide array of biochemistry and immunology assays. While flow cytometry has been widely used for cellular analysis in both clinical and research settings, the requirements for proper operation in spaceflight impose constraints on any instrument designs. The challenges of designing a spaceflight-ready flow cytometer are discussed, as well as some preliminary results using a prototype system.

Novel Methods of Determining Urinary Calculi Composition: Petrographic Thin Sectioning of Calculi and Nanoscale Flow Cytometry Urinalysis

PubMed Central

Gavin, Carson T; Ali, Sohrab N; Tailly, Thomas; Olvera-Posada, Daniel; Alenezi, Husain; Power, Nicholas E; Hou, Jinqiang; St. Amant, Andre H; Luyt, Leonard G; Wood, Stephen; Wu, Charles; Razvi, Hassan; Leong, Hon S

2016-01-01

Accurate determination of urinary stone composition has significant bearing on understanding pathophysiology, choosing treatment modalities and preventing recurrence. A need exists for improved methods to determine stone composition. Urine of 31 patients with known renal calculi was examined with nanoscale flow cytometry and the calculi collected during surgery subsequently underwent petrographic thin sectioning with polarized and fluorescent microscopy. Fluorescently labeled bisphosphonate probes (Alendronate-fluorescein/Alendronate-Cy5) were developed for nanoscale flow cytometry to enumerate nanocrystals that bound the fluorescent probes. Petrographic sections of stones were also imaged by fluorescent and polarized light microscopy with composition analysis correlated to alendronate +ve nanocrystal counts in corresponding urine samples. Urine samples from patients with Ca2+ and Mg2+ based calculi exhibited the highest alendronate +ve nanocrystal counts, ranging from 100–1000 nm in diameter. This novel urine based assay was in agreement with composition determined by petrographic thin sections with Alendronate probes. In some cases, high alendronate +ve nanocrystal counts indicated a Ca2+ or Mg2+ composition, as confirmed by petrographic analysis, overturning initial spectrophotometric diagnosis of stone composition. The combination of nanoscale flow cytometry and petrographic thin sections offer an alternative means for determining stone composition. Nanoscale flow cytometry of alendronate +ve nanocrystals alone may provide a high-throughput means of evaluating stone burden. PMID:26771074

Immunological Tools: Engaging Students in the Use and Analysis of Flow Cytometry and Enzyme-linked Immunosorbent Assay (ELISA)

ERIC Educational Resources Information Center

Ott, Laura E.; Carson, Susan

2014-01-01

Flow cytometry and enzyme-linked immunosorbent assay (ELISA) are commonly used techniques associated with clinical and research applications within the immunology and medical fields. The use of these techniques is becoming increasingly valuable in many life science and engineering disciplines as well. Herein, we report the development and…

A benchmark for evaluation of algorithms for identification of cellular correlates of clinical outcomes.

PubMed

Aghaeepour, Nima; Chattopadhyay, Pratip; Chikina, Maria; Dhaene, Tom; Van Gassen, Sofie; Kursa, Miron; Lambrecht, Bart N; Malek, Mehrnoush; McLachlan, G J; Qian, Yu; Qiu, Peng; Saeys, Yvan; Stanton, Rick; Tong, Dong; Vens, Celine; Walkowiak, Sławomir; Wang, Kui; Finak, Greg; Gottardo, Raphael; Mosmann, Tim; Nolan, Garry P; Scheuermann, Richard H; Brinkman, Ryan R

2016-01-01

The Flow Cytometry: Critical Assessment of Population Identification Methods (FlowCAP) challenges were established to compare the performance of computational methods for identifying cell populations in multidimensional flow cytometry data. Here we report the results of FlowCAP-IV where algorithms from seven different research groups predicted the time to progression to AIDS among a cohort of 384 HIV+ subjects, using antigen-stimulated peripheral blood mononuclear cell (PBMC) samples analyzed with a 14-color staining panel. Two approaches (FlowReMi.1 and flowDensity-flowType-RchyOptimyx) provided statistically significant predictive value in the blinded test set. Manual validation of submitted results indicated that unbiased analysis of single cell phenotypes could reveal unexpected cell types that correlated with outcomes of interest in high dimensional flow cytometry datasets. © 2015 International Society for Advancement of Cytometry.

Opto-fluidics based microscopy and flow cytometry on a cell phone for blood analysis.

PubMed

Zhu, Hongying; Ozcan, Aydogan

2015-01-01

Blood analysis is one of the most important clinical tests for medical diagnosis. Flow cytometry and optical microscopy are widely used techniques to perform blood analysis and therefore cost-effective translation of these technologies to resource limited settings is critical for various global health as well as telemedicine applications. In this chapter, we review our recent progress on the integration of imaging flow cytometry and fluorescent microscopy on a cell phone using compact, light-weight and cost-effective opto-fluidic attachments integrated onto the camera module of a smartphone. In our cell-phone based opto-fluidic imaging cytometry design, fluorescently labeled cells are delivered into the imaging area using a disposable micro-fluidic chip that is positioned above the existing camera unit of the cell phone. Battery powered light-emitting diodes (LEDs) are butt-coupled to the sides of this micro-fluidic chip without any lenses, which effectively acts as a multimode slab waveguide, where the excitation light is guided to excite the fluorescent targets within the micro-fluidic chip. Since the excitation light propagates perpendicular to the detection path, an inexpensive plastic absorption filter is able to reject most of the scattered light and create a decent dark-field background for fluorescent imaging. With this excitation geometry, the cell-phone camera can record fluorescent movies of the particles/cells as they are flowing through the microchannel. The digital frames of these fluorescent movies are then rapidly processed to quantify the count and the density of the labeled particles/cells within the solution under test. With a similar opto-fluidic design, we have recently demonstrated imaging and automated counting of stationary blood cells (e.g., labeled white blood cells or unlabeled red blood cells) loaded within a disposable cell counting chamber. We tested the performance of this cell-phone based imaging cytometry and blood analysis platform by measuring the density of red and white blood cells as well as hemoglobin concentration in human blood samples, which showed a good match to our measurement results obtained using a commercially available hematology analyzer. Such a cell-phone enabled opto-fluidics microscopy, flow cytometry, and blood analysis platform could be especially useful for various telemedicine applications in remote and resource-limited settings.

Line-Focused Optical Excitation of Parallel Acoustic Focused Sample Streams for High Volumetric and Analytical Rate Flow Cytometry.

PubMed

Kalb, Daniel M; Fencl, Frank A; Woods, Travis A; Swanson, August; Maestas, Gian C; Juárez, Jaime J; Edwards, Bruce S; Shreve, Andrew P; Graves, Steven W

2017-09-19

Flow cytometry provides highly sensitive multiparameter analysis of cells and particles but has been largely limited to the use of a single focused sample stream. This limits the analytical rate to ∼50K particles/s and the volumetric rate to ∼250 μL/min. Despite the analytical prowess of flow cytometry, there are applications where these rates are insufficient, such as rare cell analysis in high cellular backgrounds (e.g., circulating tumor cells and fetal cells in maternal blood), detection of cells/particles in large dilute samples (e.g., water quality, urine analysis), or high-throughput screening applications. Here we report a highly parallel acoustic flow cytometer that uses an acoustic standing wave to focus particles into 16 parallel analysis points across a 2.3 mm wide optical flow cell. A line-focused laser and wide-field collection optics are used to excite and collect the fluorescence emission of these parallel streams onto a high-speed camera for analysis. With this instrument format and fluorescent microsphere standards, we obtain analysis rates of 100K/s and flow rates of 10 mL/min, while maintaining optical performance comparable to that of a commercial flow cytometer. The results with our initial prototype instrument demonstrate that the integration of key parallelizable components, including the line-focused laser, particle focusing using multinode acoustic standing waves, and a spatially arrayed detector, can increase analytical and volumetric throughputs by orders of magnitude in a compact, simple, and cost-effective platform. Such instruments will be of great value to applications in need of high-throughput yet sensitive flow cytometry analysis.

Elucidation of the critical epitope of an anti-EGFR monoclonal antibody EMab-134.

PubMed

Kaneko, Mika K; Yamada, Shinji; Itai, Shunsuke; Chang, Yao-Wen; Nakamura, Takuro; Yanaka, Miyuki; Kato, Yukinari

2018-07-01

The epidermal growth factor receptor (EGFR) is a type-1 transmembrane receptor tyrosine kinase, which activates the downstream signaling cascades in many tumors, such as oral and lung cancers. We previously developed EMab-134, a novel anti-EGFR monoclonal antibody (mAb), which reacts with endogenous EGFR-expressing cancer cell lines and normal cells independent of glycosylation in Western blotting, flow cytometry, and immunohistochemical analysis. EMab-134 showed very high sensitivity (94.7%) to oral squamous cell carcinomas in immunohistochemical analysis. In this study, we performed enzyme-linked immunosorbent assay (ELISA), flow cytometry, and immunohistochemical analysis to determine the epitope of EMab-134. A blocking peptide (375-394 amino acids of EGFR) neutralized the EMab-134 reaction against oral cancer cells in flow cytometry and immunohistochemistry. The minimum epitope of EMab-134 was found to be the 377- RGDSFTHTPP -386 sequence. Our findings can be applied for the production of more functional anti-EGFR mAbs that in turn can be used for antitumor treatments.

Determination of critical epitope of PcMab-47 against human podocalyxin.

PubMed

Itai, Shunsuke; Yamada, Shinji; Kaneko, Mika K; Kato, Yukinari

2018-07-01

Podocalyxin (PODXL) is a type I transmembrane protein, which is highly glycosylated. PODXL is expressed in some types of human cancer tissues including oral, breast, and lung cancer tissues and may promote tumor growth, invasion, and metastasis. We previously produced PcMab-47, a novel anti-PODXL monoclonal antibody (mAb) which reacts with endogenous PODXL-expressing cancer cell lines and normal cells independently of glycosylation in Western blot, flow cytometry, and immunohistochemical analysis. In this study, we used enzyme-linked immunosorbent assay (ELISA), flow cytometry, and immunohistochemical analysis to determine the epitope of PcMab-47. The minimum epitope of PcMab-47 was found to be Asp207, His208, Leu209, and Met210. A blocking peptide containing this minimum epitope completely neutralized PcMab-47 reaction against oral cancer cells by flow cytometry and immunohistochemical analysis. These findings could lead to the production of more functional anti-PODXL mAbs, which are advantageous for antitumor activities.

[Experimental study of IFN-alpha and IFN-gamma on reversing ATRA-resistance in MR2 cell line].

PubMed

He, Peng-Cheng; Zhang, Mei; Li, Jing; Cao, Yun-Xin; Cai, Rui-Bo; Liu, Ya-Lin

2007-03-01

To explore the possibility and the possible mechanism of reversing ATRA-resistance in MR2 cells by using IFN-alpha and IFN-gamma in combination with all-trans retinoic acid (ATRA). After MR2 cells(ATRA-resistance cell line) were treated with IFN-alpha, IFN-gamma and ATRA alone or IFN-alpha and IFN-gamma in combination with ATRA respectively, the cell proliferation was tested by MTT colorimetry, the cell differentiation was tested through light microscope, by NBT test and flow cytometry (FCM). The expression of promyelocytic leukemia (PML) protein was observed by indirect immunofluorescence staining. Both IFN-alpha and IFN-gamma could inhibit the proliferation of MR2 cells. The effects were more obviously in both IFN-alpha+ATRA group and IFN-gamma+ATRA group. But there were no significant difference between either IFN-alpha group and IFN-gamma group or IFN-alpha+ATRA group and IFN-gamma+ATRA group (P>0.05). Both IFN could also induce the differentiation of MR2 cells. The effects of IFN-alpha+ATRA group and IFN-gamma+ATRA group were more obvious. However, the differentiation of MR2 cells induced by IFN-gamma+ATRA group was more higher than that by IFN-alpha+ATRA group (P<0.05). Both IFN could induce the expression of PML protein. The reversing effcet of IFN-gamma+ATRA group on ATRA-resistence in MR2 cells are more powerful than that of IFN-alpha+ATRA group, which may be related to the different signal transduction pathway of IFN-alpha and IFN-gamma.

Construction and cellular immune response induction of HA-based alphavirus replicon vaccines against human-avian influenza (H5N1).

PubMed

Yang, Shi-gui; Wo, Jian-er; Li, Min-wei; Mi, Fen-fang; Yu, Cheng-bo; Lv, Guo-liang; Cao, Hong-Cui; Lu, Hai-feng; Wang, Bao-hong; Zhu, Hanping; Li, Lan-Juan

2009-12-09

Several approaches are being taken worldwide to develop vaccines against H5N1 viruses; most of them, however, pose both practical and immunological challenges. One potential strategy for improving the immunogenicity of vaccines involves the use of alphavirus replicons and VP22, a herpes simplex type 1 (HSV-1) protein. In this study, we analysed the antigenic peptides and homogeneity of the HA sequences (human isolates of the H5N1 subtype, from 1997 to 2003) and explored a novel alphavirus replicon system of VP22 fused with HA, to assess whether the immunogenicity of an HA-based replicon vaccine could be induced and augmented via fusion with VP22. Further, replicon particles expressing VP22, and enhanced green fluorescent protein (EGFP) were individually used as controls. Cellular immune responses in mice immunised with replicons were evaluated by identifying specific intracellular cytokine production with flow cytometry (FCM). Animal-based experimentation indicated that both the IL-4 expression of CD4(+) T cells and the IFN-gamma expression of CD8(+) T cells were significantly increased in mice immunised with VPR-HA and VPR-VP22/HA. A dose titration effect vis-à-vis both IL-4 expression and IFN-gamma expression were observed in VPR-HA- and VPR-VP22/HA-vaccinated mice. Our results revealed that both VPR-VP22/HA and VPR-HA replicon particles presented a promising approach for developing vaccines against human-avian influenza, and VP22 could enhance the immunogenicity of the HA antigens to which it is fused.

Systemic mesenchymal stem cells reduce growth rate of cisplatin-resistant ovarian cancer.

PubMed

Zhu, Pengfei; Chen, Mo; Wang, Li; Ning, Yanxia; Liang, Jie; Zhang, Hao; Xu, Congjian; Chen, Sifeng; Yao, Liangqing

2013-01-01

Epithelial ovarian cancer is one of the most malignant cancers in women and resistant to chemotherapy is the major obstacle for the five-year survival rate. Cisplatin is one of the effective anticancer drug used in the ovarian cancer. To find a good strategy to cure the tumors which is resistant to cisplatin, the cisplatin-resistant 3SKOV3 cells were selected from SKOV-3 ovarian cancer cells. Furthermore, the isolated mesenchymal stem cells were infused systemically to try to cure the transplanted tumor induced by 3SKOV3 cells in nude mice. The morphology and cell membrane CD44 expression were investigated by microscope and flow cytometry. The biological behaviors of resistant 3SKOV3 and its parental SKOV3 cells, including proliferation, adhesion, and cell cycle were determined by CCK8, absorbance assay and FCM methods. The transplanted tumors were set up in nude mice with 3SKOV3 cells injection. The growth rate of transplanted tumors was detected following with MSCs injection. The 3SKOV3 cells have different morphologic manifestation and expressed high level of CD44 molecule. At the same time, 3SKOV3 cells have less adhesion ability and less S-phase ratio. The isolated MSCs from bone marrow could inhibit the growth of transplanted tumor via systemic injection. The cisplatin-resistant 3SKOV3 cells have the different biological behaviors as its parental SKOV3 cells. The present study indicated that systemic MSCs have the therapeutic role on ovarian cancer. However, further investigations are in progress to elucidate the underlying mechanism.

Role of HLA-G1 in trophoblast cell proliferation, adhesion and invasion

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

Jiang, Feng, E-mail: jiangfeng1161@163.com; Zhao, Hongxi; Wang, Li

Trophoblast cells are important in embryo implantation and fetomaternal tolerance. HLA-G is specifically expressed at the maternal–fetal interface and is a regulator in pregnancy. The aim of the present study was to detect the effect of HLA-G1 on trophoblast cell proliferation, adhesion, and invasion. Human trophoblast cell lines (JAR and HTR-8/SVneo cells) were infected with HLA-G1-expressing lentivirus. After infection, HLA-G1 expression of the cells was detected by western blotting. Cell proliferation was detected by the BrdU assay. The cell cycle and apoptosis of JAR and HTR-8/SVneo cells was measured by flow cytometry (FCM). The invasion of the cells under different conditionsmore » was detected by the transwell invasion chamber assay. HLA-G1 didn't show any significant influence on the proliferation, apoptosis, adhesion, and invasion of trophocytes in normal culture conditions. However, HLA-G1 inhibited JAR and HTR-8/SVneo cells invasion induced by hepatocyte growth factor (HGF) under normal oxygen conditions. In conditions of hypoxia, HLA-G1 couldn't inhibit the induction of cell invasion by HGF. HLA-G1 is not an independent factor for regulating the trophocytes. It may play an indirect role in embryo implantation and formation of the placenta. - Highlights: • HLA-G1 could not influence trophocytes under normal conditions. • HLA-G1 inhibited cell invasion induced by HGF under normal oxygen condition. • HLA-G1 could not influence cell invasion under hypoxia conditions.« less

Thermal and Nonthermal Effects of Discontinuous Microwave Exposure (2.45 Gigahertz) on the Cell Membrane of Escherichia coli

PubMed Central

Rougier, Carole; Chazal, Philippe; Leveque, Philippe; Leprat, Patrick

2014-01-01

The aim of this study was to investigate the effects on the cell membranes of Escherichia coli of 2.45-GHz microwave (MW) treatment under various conditions with an average temperature of the cell suspension maintained at 37°C in order to examine the possible thermal versus nonthermal effects of short-duration MW exposure. To this purpose, microwave irradiation of bacteria was performed under carefully defined and controlled parameters, resulting in a discontinuous MW exposure in order to maintain the average temperature of the bacterial cell suspensions at 37°C. Escherichia coli cells were exposed to 200- to 2,000-W discontinuous microwave (DW) treatments for different periods of time. For each experiment, conventional heating (CH) in a water bath at 37°C was performed as a control. The effects of DW exposure on cell membranes was investigated using flow cytometry (FCM), after propidium iodide (PI) staining of cells, in addition to the assessment of intracellular protein release in bacterial suspensions. No effect was detected when bacteria were exposed to conventional heating or 200 W, whereas cell membrane integrity was slightly altered when cell suspensions were subjected to powers ranging from 400 to 2,000 W. Thermal characterization suggested that the temperature reached by the microwave-exposed samples for the contact time studied was not high enough to explain the measured modifications of cell membrane integrity. Because the results indicated that the cell response is power dependent, the hypothesis of a specific electromagnetic threshold effect, probably related to the temperature increase, can be advanced. PMID:24907330

The Association between Splenocyte Apoptosis and Alterations of Bax, Bcl-2 and Caspase-3 mRNA Expression, and Oxidative Stress Induced by Dietary Nickel Chloride in Broilers

PubMed Central

Huang, Jianying; Cui, Hengmin; Peng, Xi; Fang, Jing; Zuo, Zhicai; Deng, Junliang; Wu, Bangyuan

2013-01-01

Two hundred and forty avian broilers were equally divided into four groups, and raised with a corn-soybean basal diet or the same diet supplemented with 300, 600, 900 mg/kg NiCl2 for 42 days. Numbers or percentages of apoptotic splenocytes by flow cytometry (FCM) and TUNEL were higher (p < 0.05 or p < 0.01) in the 300, 600 and 900 mg/kg groups than those in the control group. Results measured by qRT-PCR and ELISA showed that mRNA expression and contents were significantly higher (p < 0.05 or p < 0.01) in Bax and Caspase-3, and were significantly lower (p < 0.05 or p < 0.01) in Bcl-2 of the 300, 600 and 900 mg/kg groups. Also, the SOD, CAT and GSH-Px activities, and the ability to inhibit hydroxyl radical, and GSH contents were significantly decreased (p < 0.05 or p < 0.01), and MDA contents were increased (p < 0.05 or p < 0.01) in all groups. In conclusion, dietary NiCl2 in excess of 300 mg/kg caused apoptosis, altered Bax, Bcl-2 and Caspase-3 mRNA expression levels and contents, and induced oxidative stress in the spleen. Also, splenocyte apoptosis was closely related to the alternations of Bax, Bcl-2 and Caspase-3 mRNA expression, and oxidative damage. The splenic immunity and blood filtration functions were impaired in broilers. PMID:24351749

Effects of Canonical NF-κB Signaling Pathway on the Proliferation and Odonto/Osteogenic Differentiation of Human Stem Cells from Apical Papilla

PubMed Central

Li, Junjun; Yan, Ming; Wang, Zilu; Jing, Shuanglin; Li, Yao; Liu, Genxia; Yu, Jinhua; Fan, Zhipeng

2014-01-01

Background Information. NF-κB signaling pathway plays a complicated role in the biological functions of mesenchymal stem cells. However, the effects of NF-κB pathway on the odonto/osteogenic differentiation of stem cells from apical papilla (SCAPs) remain unclear. The present study was designed to evaluate the effects of canonical NF-κB pathway on the osteo/odontogenic capacity of SCAPs in vitro. Results. Western blot results demonstrated that NF-κB pathway in SCAPs was successfully activated by TNF-α or blocked by BMS-345541. NF-κB pathway-activated SCAPs presented a higher proliferation activity compared with control groups, as indicated by dimethyl-thiazol-diphenyl tetrazolium bromide assay (MTT) and flow cytometry assay (FCM). Wound scratch assay revealed that NF-κB pathway-activated SCAPs presented an improved migration capacity, enhanced alkaline phosphatase (ALP) activity, and upregulated mineralization capacity of SCAPs, as compared with control groups. Meanwhile, the odonto/osteogenic markers (ALP/ALP, RUNX2/RUNX2, OSX/OSX, OCN/OCN, OPN/OPN, BSP/BSP, DSPP/DSP, and DMP-1/DMP-1) in NF-κB pathway-activated SCAPs were also significantly upregulated as compared with control groups at both protein and mRNA levels. However, NF-κB pathway-inhibited SCAPs exhibited a lower proliferation/migration capacity, and decreased odonto/osteogenic ability in comparison with control groups. Conclusion. Our findings suggest that classical NF-κB pathway plays a paramount role in the proliferation and committed differentiation of SCAPs. PMID:24864235

[Regulatory T cells inhibit proliferation of mouse lymphoma cell line EL4 in vitro].

PubMed

Zhang, Chen; Kong, Yan; Guo, Jun; Ying, Zhi-Tao; Yuan, Zhi-Hong; Zhang, Yun-Tao; Zheng, Wen; Song, Yu-Qin; Li, Ping-Ping; Zhu, Jun

2010-10-01

This study was aimed to investigate the effect of regulatory T (Treg) cells on the T cell lymphoma EL4 cells and its mechanism in vitro. C57BL/6 mouse Treg cells were isolated by magnetic cell sorting (MACS). The purity of Treg cells and their expression of Foxp3 were identified by flow cytometry (FCM) and PT-PCR respectively. The suppression of Treg cells on EL4 cells was detected by 3H-TdR method. At the same time, enzyme-linked immunosorbent assay (ELISA) was used to detect the secretion of cytokine TGF-β1 and IL-10. The results showed that CD4+CD25+ T cells could be successfully isolated by MACS with the purity reaching 94.52% and the expression of Foxp3 reaching 84.72%. After sorting, the expression of Foxp3 mRNA could be detected by RT-PCR. 3H-TdR assay confirmed that regulatory T cells could suppress the proliferation of EL4 cells with or without antigen presenting cells (APC) or dendritic cells (DC), APC or DC might effectively enhance the suppression. In addition, DC alone also suppressed the proliferation. TGF-β1 and IL-10 could be detected in the supernatant by ELISA. It is concluded that the Treg cells can obviously suppress the proliferation of T cell lymphoma cells in vitro, APC or DC can enhance this suppressive effect, while the DC alone also can suppress the proliferation of EL4 cells, the TGF-β1 and IL-10 cytokine pathway may be one of the mechanisms of suppression.

Nitrogen Starvation Induced Oxidative Stress in an Oil-Producing Green Alga Chlorella sorokiniana C3

PubMed Central

He, Chen-Liu; Wang, Qiang

2013-01-01

Microalgal lipid is one of the most promising feedstocks for biodiesel production. Chlorella appears to be a particularly good option, and nitrogen (N) starvation is an efficient environmental pressure used to increase lipid accumulation in Chlorella cells. The effects of N starvation of an oil-producing wild microalga, Chlorella sorokiniana C3, on lipid accumulation were investigated using thin layer chromatography (TLC), confocal laser scanning microscopy (CLSM) and flow cytometry (FCM). The results showed that N starvation resulted in lipid accumulation in C. sorokiniana C3 cells, oil droplet (OD) formation and significant lipid accumulation in cells were detected after 2 d and 8 d of N starvation, respectively. During OD formation, reduced photosynthetic rate, respiration rate and photochemistry efficiency accompanied by increased damage to PSII were observed, demonstrated by chlorophyll (Chl) fluorescence, 77K fluorescence and oxygen evolution tests. In the mean time the rate of cyclic electron transportation increased correspondingly to produce more ATP for triacylglycerols (TAGs) synthesis. And 0.5 d was found to be the turning point for the early stress response and acclimation of cells to N starvation. Increased level of membrane peroxidation was also observed during OD formation, and superoxide dismutase (SOD), peroxide dismutase (POD) and catalase (CAT) enzyme activity assays suggested impaired reactive oxygen species (ROS) scavenging ability. Significant neutral lipid accumulation was also observed by artificial oxidative stress induced by H2O2 treatment. These results suggested coupled neutral lipid accumulation and oxidative stress during N starvation in C. sorokiniana C3. PMID:23874918

Facile and green reduction of covalently PEGylated nanographene oxide via a `water-only' route for high-efficiency photothermal therapy

NASA Astrophysics Data System (ADS)

Chen, Jingqin; Wang, Xiaoping; Chen, Tongsheng

2014-02-01

A facile and green strategy is reported for the fabrication of nanosized and reduced covalently PEGylated graphene oxide (nrGO-PEG) with great biocompatibility and high near-infrared (NIR) absorbance. Covalently PEGylated nGO (nGO-PEG) was synthesized by the reaction of nGO-COOH and methoxypolyethylene glycol amine (mPEG-NH2). The neutral and purified nGO-PEG solution was then directly bathed in water at 90°C for 24 h without any additive to obtain nrGO-PEG. Covalent PEGylation not only prevented the aggregation of nGO but also dramatically promoted the reduction extent of nGO during this reduction process. The resulting single-layered nrGO-PEG sheets were approximately 50 nm in average lateral dimension and exhibited great biocompatibility and approximately 7.6-fold increment in NIR absorption. Moreover, this facile reduction process repaired the aromatic structure of GO. CCK-8 and flow cytometry (FCM) assays showed that exposure of A549 cells to 100 μg/mL of nrGO-PEG for 2 h, exhibiting 71.5% of uptake ratio, did not induce significant cytotoxicity. However, after irradiation with 808 nm laser (0.6 W/cm2) for 5 min, the cells incubated with 6 μg/mL of nrGO-PEG solution showed approximately 90% decrease of cell viability, demonstrating the high-efficiency photothermal therapy of nrGO-PEG to tumor cells in vitro. This work established nrGO-PEG as a promising photothermal agent due to its small size, great biocompatibility, high photothermal efficiency, and low cost.

Categorizing document by fuzzy C-Means and K-nearest neighbors approach

NASA Astrophysics Data System (ADS)

Priandini, Novita; Zaman, Badrus; Purwanti, Endah

2017-08-01

Increasing of technology had made categorizing documents become important. It caused by increasing of number of documents itself. Managing some documents by categorizing is one of Information Retrieval application, because it involve text mining on its process. Whereas, categorization technique could be done both Fuzzy C-Means (FCM) and K-Nearest Neighbors (KNN) method. This experiment would consolidate both methods. The aim of the experiment is increasing performance of document categorize. First, FCM is in order to clustering training documents. Second, KNN is in order to categorize testing document until the output of categorization is shown. Result of the experiment is 14 testing documents retrieve relevantly to its category. Meanwhile 6 of 20 testing documents retrieve irrelevant to its category. Result of system evaluation shows that both precision and recall are 0,7.

Possibilistic clustering for shape recognition

NASA Technical Reports Server (NTRS)

Keller, James M.; Krishnapuram, Raghu

1993-01-01

Clustering methods have been used extensively in computer vision and pattern recognition. Fuzzy clustering has been shown to be advantageous over crisp (or traditional) clustering in that total commitment of a vector to a given class is not required at each iteration. Recently fuzzy clustering methods have shown spectacular ability to detect not only hypervolume clusters, but also clusters which are actually 'thin shells', i.e., curves and surfaces. Most analytic fuzzy clustering approaches are derived from Bezdek's Fuzzy C-Means (FCM) algorithm. The FCM uses the probabilistic constraint that the memberships of a data point across classes sum to one. This constraint was used to generate the membership update equations for an iterative algorithm. Unfortunately, the memberships resulting from FCM and its derivatives do not correspond to the intuitive concept of degree of belonging, and moreover, the algorithms have considerable trouble in noisy environments. Recently, the clustering problem was cast into the framework of possibility theory. Our approach was radically different from the existing clustering methods in that the resulting partition of the data can be interpreted as a possibilistic partition, and the membership values may be interpreted as degrees of possibility of the points belonging to the classes. An appropriate objective function whose minimum will characterize a good possibilistic partition of the data was constructed, and the membership and prototype update equations from necessary conditions for minimization of our criterion function were derived. The ability of this approach to detect linear and quartic curves in the presence of considerable noise is shown.

A risk management model for familial breast cancer: A new application using Fuzzy Cognitive Map method.

PubMed

Papageorgiou, Elpiniki I; Jayashree Subramanian; Karmegam, Akila; Papandrianos, Nikolaos

2015-11-01

Breast cancer is the most deadly disease affecting women and thus it is natural for women aged 40-49 years (who have a family history of breast cancer or other related cancers) to assess their personal risk for developing familial breast cancer (FBC). Besides, as each individual woman possesses different levels of risk of developing breast cancer depending on their family history, genetic predispositions and personal medical history, individualized care setting mechanism needs to be identified so that appropriate risk assessment, counseling, screening, and prevention options can be determined by the health care professionals. The presented work aims at developing a soft computing based medical decision support system using Fuzzy Cognitive Map (FCM) that assists health care professionals in deciding the individualized care setting mechanisms based on the FBC risk level of the given women. The FCM based FBC risk management system uses NHL to learn causal weights from 40 patient records and achieves a 95% diagnostic accuracy. The results obtained from the proposed model are in concurrence with the comprehensive risk evaluation tool based on Tyrer-Cuzick model for 38/40 patient cases (95%). Besides, the proposed model identifies high risk women by calculating higher accuracy of prediction than the standard Gail and NSAPB models. The testing accuracy of the proposed model using 10-fold cross validation technique outperforms other standard machine learning based inference engines as well as previous FCM-based risk prediction methods for BC. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Does hierarchy stability influence testosterone and cortisol levels of bearded capuchin monkeys (Sapajus libidinosus) adult males? A comparison between two wild groups.

PubMed

Mendonça-Furtado, Olívia; Edaes, Mariana; Palme, Rupert; Rodrigues, Agatha; Siqueira, José; Izar, Patrícia

2014-11-01

Testosterone and cortisol are hormones expected to play a major role in competitive behaviours (i.e. aggression), and are related to rank and hierarchical stability. Through a non-invasive technique, we analyzed faecal testosterone (FTM(1)) and cortisol (FCM(2)) metabolites of dominant and subordinate males from two wild groups of bearded capuchin monkeys. One group had a stable dominance hierarchy while the other had an unstable hierarchy, with a marked conflict period related to a male take-over. In the unstable hierarchy group (1) the dominant male had higher FTM peaks than subordinates, and (2) basal FTM levels were higher than in the stable group. These findings are in accordance with the Challenge Hypothesis and rank-based predictions, and confirm that in Sapajus libidinosus hierarchy stability, social status, aggression rates and testosterone are closely related. Dominants of both groups had higher basal and peak FCM levels, suggesting that in S. libidinosus the dominant male has a higher allostatic load than subordinates, related to his role in protection against predators, intragroup appeasement, and control of food sources. Finally, we suggest that males of S. libidinosus are resistant to testosterone suppression by cortisol, because in the unstable group in spite of an increase in FCM there was also an increase in FTM during the conflict period. This article is part of a Special Issue entitled: Neotropical Behaviour. Copyright © 2014 Elsevier B.V. All rights reserved.