scholarly journals Towards Determining Biosignature Retention in Icy World Plumes

Life ◽  
2020 ◽  
Vol 10 (4) ◽  
pp. 40
Author(s):  
Kathryn Bywaters ◽  
Carol R. Stoker ◽  
Nelio Batista Do Nascimento ◽  
Lawrence Lemke
Keyword(s):  
Vacuum Chamber ◽  
Dynamic Range ◽  
Cell Types ◽  
Space Environment ◽  
Epifluorescence Microscopy ◽  
Experimental Conditions ◽  
E Coli ◽  
Life Detection ◽  
Cellular Integrity ◽  
Sem Imaging

With the discovery of the persistent jets of water being ejected to space from Enceladus, an understanding of the effect of the space environment on potential organisms and biosignatures in them is necessary for planning life detection missions. We experimentally determine the survivability of microbial cells in liquid medium when ejected into vacuum. Epifluorescence microscopy, using a lipid stain, and SEM imaging were used to interrogate the cellular integrity of E. coli after ejected through a pressurized nozzle into a vacuum chamber. The experimental samples showed a 94% decrease in visible intact E. coli cells but showed a fluorescence residue in the shape of the sublimated droplets that indicated the presence of lipids. The differences in the experimental conditions versus those expected on Enceladus should not change the analog value because the process a sample would undergo when ejected into space was representative. E. coli was selected for testing although other cell types could vary physiologically which would affect their response to a vacuum environment. More testing is needed to determine the dynamic range in concentration of cells expected to survive the plume environment. However, these results suggest that lipids may be directly detectable evidence of life in icy world plumes.

scholarly journals The Inflammatory Response to Enterotoxigenic E. coli and Probiotic E. faecium in a Coculture Model of Porcine Intestinal Epithelial and Dendritic Cells

2018 ◽  
Vol 2018 ◽  
pp. 1-16 ◽  
Author(s):  
Henriette Loss ◽  
Jörg R. Aschenbach ◽  
Karsten Tedin ◽  
Friederike Ebner ◽  
Ulrike Lodemann
Keyword(s):  
Dendritic Cells ◽  
Immune Responses ◽  
Transforming Growth Factor ◽  
Cell Types ◽  
Inflammasome Activation ◽  
Intestinal Epithelial ◽  
Experimental Conditions ◽  
E Coli ◽  
Coculture Model

The gut epithelium constitutes an interface between the intestinal contents and the underlying gut-associated lymphoid tissue (GALT) including dendritic cells (DC). Interactions of intestinal epithelial cells (IEC) and resident DC are characterized by bidirectional crosstalk mediated by various factors, such as transforming growth factor-β (TGF-β) and thymic stromal lymphopoietin (TSLP). In the present study, we aimed (1) to model the interplay of both cell types in a porcine in vitro coculture consisting of IEC (cell line IPEC-J2) and monocyte-derived DC (MoDC) and (2) to assess whether immune responses to bacteria are altered because of the interplay between IPEC-J2 cells and MoDC. With regard to the latter, we focused on the inflammasome pathway. Here, we propose caspase-13 as a promising candidate for the noncanonical inflammasome activation in pigs. We conducted challenge experiments with enterotoxigenic Escherichia coli (ETEC) and probiotic Enterococcus faecium (E. faecium) NCIMB 10415. As potential mediators of IEC/DC interactions, TGF-β and TSLP were selected for analyses. Cocultured MoDC showed attenuated ETEC-induced inflammasome-related and proinflammatory interleukin (IL)-8 reactions compared with MoDC monocultures. Caspase-13 was more strongly expressed in IPEC-J2 cells cocultured with MoDC and upon ETEC incubation. We found that IPEC-J2 cells and MoDC were capable of releasing TSLP. The latter cells secreted greater amounts of TSLP when cocultured with IPEC-J2 cells. TGF-β was not modulated under the present experimental conditions in either cell types. We conclude that, in the presence of IPEC-J2 cells, porcine MoDC exhibited a more tolerogenic phenotype, which might be partially regulated by autocrine TSLP production. Noncanonical inflammasome signaling appeared to be modulated in IPEC-J2 cells. Our results indicate that the reciprocal interplay of the intestinal epithelium and GALT is essential for promoting balanced immune responses.

scholarly journals A practical solution to pseudoreplication bias in single-cell studies

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Kip D. Zimmerman ◽  
Mark A. Espeland ◽  
Carl D. Langefeld
Keyword(s):  
Single Cell ◽  
Mixed Models ◽  
Random Effect ◽  
Cell Types ◽  
Error Rates ◽  
Specific Cell ◽  
Experimental Conditions ◽  
Type 1 Error ◽  
Sample Correlation ◽  
Inflated Type

AbstractCells from the same individual share common genetic and environmental backgrounds and are not statistically independent; therefore, they are subsamples or pseudoreplicates. Thus, single-cell data have a hierarchical structure that many current single-cell methods do not address, leading to biased inference, highly inflated type 1 error rates, and reduced robustness and reproducibility. This includes methods that use a batch effect correction for individual as a means of accounting for within-sample correlation. Here, we document this dependence across a range of cell types and show that pseudo-bulk aggregation methods are conservative and underpowered relative to mixed models. To compute differential expression within a specific cell type across treatment groups, we propose applying generalized linear mixed models with a random effect for individual, to properly account for both zero inflation and the correlation structure among measures from cells within an individual. Finally, we provide power estimates across a range of experimental conditions to assist researchers in designing appropriately powered studies.

scholarly journals Relative versus absolute RNA quantification: a comparative analysis based on the example of endothelial expression of vasoactive receptors

2021 ◽  
Vol 23 (1) ◽  
Author(s):  
Kevin Kuhlmann ◽  
Melanie Cieselski ◽  
Julia Schumann
Keyword(s):  
Dynamic Range ◽  
Genetic Material ◽  
Housekeeping Genes ◽  
Digital Pcr ◽  
Absolute Quantification ◽  
Test Procedures ◽  
Experimental Conditions ◽  
Rna Quantification ◽  
The Stability ◽  
Inflammatory Milieu

Abstract Background In the present study, two distinct PCR methods were used for the quantification of genetic material and their results were compared: real-time-PCR (qPCR; relative quantification) and droplet digital PCR (ddPCR; absolute quantification). The comparison of the qPCR and the ddPCR was based on a stimulation approach of microvascular endothelial cells in which the effect of a pro-inflammatory milieu on the expression of vasoactive receptors was investigated. Results There was consistency in directions of effects for the majority of genes tested. With regard to the indicated dimension of the effects, the overall picture was more differentiated. It was striking that deviations were more pronounced if the measured values were on the extreme edges of the dynamic range of the test procedures. Conclusions To obtain valid and reliable results, dilution series are recommended, which should be carried out initially. In case of ddPCR the number of copies per µl should be adjusted to the low three-digit range. With regard to qPCR it is essential that the stability and reliability of the reference genes used is guaranteed. Here, ddPCR offers the advantage that housekeeping genes are not required. Furthermore, an absolute quantification of the sample can be easily performed by means of ddPCR. Before using ddPCR, however, care should be taken to optimize the experimental conditions. Strict indications for this methodology should also be made with regard to economic and timing factors.

The Flavonols Quercetin, Myricetin, Kaempferol, and Galangin Inhibit the Net Oxygen Consumption by Immune Complex- Stimulated Human and Rabbit Neutrophils

2014 ◽  
Vol 69 (7-8) ◽  
pp. 346-356 ◽  
Author(s):  
Andréa S. G. Figueiredo-Rinhel ◽  
Everton O. L. Santos ◽  
Luciana M. Kabeya ◽  
Ana Elisa C. S. Azzolini ◽  
Livia M. C. Simões-Ambrosio ◽  
...  
Keyword(s):  
Oxygen Consumption ◽  
Immune Complex ◽  
Respiratory Burst ◽  
Human Neutrophil ◽  
Inflammatory Diseases ◽  
Cell Types ◽  
Oxygen Electrode ◽  
Experimental Models ◽  
Human Neutrophils ◽  
Experimental Conditions

Stimulated human neutrophils exhibit increased net oxygen consumption (NOC) due to the conversion of O2 into the superoxide anion by the NADPH oxidase enzymatic complex during the respiratory burst. In several inflammatory diseases, overproduction of these oxidants causes tissue damage. The present study aims to: (a) optimize the experimental conditions used to measure the NOC in serum-opsonized zymosan (OZ)-and insoluble immune complex (i-IC)-stimulated human and rabbit neutrophils; and (b) compare the effect of four flavonols (quercetin, myricetin, kaempferol, and galangin) on this activity. We used a Clark-type oxygen electrode to measure the NOC of stimulated neutrophils. Eliciting the neutrophil respiratory burst with OZ and i-IC yielded similar maximum O2 uptake levels within the same species, but the human neutrophil NOC was almost four times higher than the rabbit neutrophil NOC. The optimal experimental conditions established for both cell types were 4·106 neutrophils mL-1, 2 mg mL-1 OZ, and 240 µg mL-1 i-IC. Upon stimulation with OZ or i-IC, the tested flavonols reduced the human and rabbit neutrophil NOC in the same order of potency - quercetin and galangin were the most and the least potent, respectively. These compounds were around four times more effective in inhibiting the rabbit as compared to the human neutrophil NOC, respectively. The four flavonols were not toxic to human or rabbit neutrophils. The experimental conditions used are suitable for both the determination of human and rabbit neutrophil NOC and for the assessment of the modulatory effects of natural compounds on these activities. The relationship between the level of NOC and the inhibitory potency of the flavonols suggests that rabbit neutrophils can be useful experimental models to predict the effect of drugs on immune complexstimulated human neutrophils.

scholarly journals An inexpensive, customizable microscopy system for the automated quantification and characterization of multiple adherent cell types

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e4937 ◽  
Author(s):  
Vishwaratn Asthana ◽  
Yuqi Tang ◽  
Adam Ferguson ◽  
Pallavi Bugga ◽  
Anantratn Asthana ◽  
...  
Keyword(s):  
Tumor Cells ◽  
Dynamic Range ◽  
Low Cost ◽  
Cell Types ◽  
Cell Counting ◽  
Adherent Cell ◽  
Essential Components ◽  
Automated Quantification ◽  
Automated Microscopy ◽  
Multiple Cell

Cell quantification assays are essential components of most biological and clinical labs. However, many currently available quantification assays, including flow cytometry and commercial cell counting systems, suffer from unique drawbacks that limit their overall efficacy. In order to address the shortcomings of traditional quantification assays, we have designed a robust, low-cost, automated microscopy-based cytometer that quantifies individual cells in a multiwell plate using tools readily available in most labs. Plating and subsequent quantification of various dilution series using the automated microscopy-based cytometer demonstrates the single-cell sensitivity, near-perfect R2 accuracy, and greater than 5-log dynamic range of our system. Further, the microscopy-based cytometer is capable of obtaining absolute counts of multiple cell types in one well as part of a co-culture setup. To demonstrate this ability, we recreated an experiment that assesses the tumoricidal properties of primed macrophages on co-cultured tumor cells as a proof-of-principle test. The results of the experiment reveal that primed macrophages display enhanced cytotoxicity toward tumor cells while simultaneously losing the ability to proliferate, an example of a dynamic interplay between two cell populations that our microscopy-based cytometer is successfully able to elucidate.

Ultra-Wideband Chaos Life-Detection Radar with Sinusoidal Wave Modulation

2017 ◽  
Vol 27 (13) ◽  
pp. 1730046 ◽  
Author(s):  
Hang Xu ◽  
Ying Li ◽  
Jianguo Zhang ◽  
Hong Han ◽  
Bing Zhang ◽  
...  
Keyword(s):  
Dynamic Range ◽  
Vital Signs ◽  
Radar System ◽  
Ultra Wideband ◽  
Thick Wall ◽  
Sinusoidal Wave ◽  
Echo Signal ◽  
Range Resolution ◽  
Life Detection ◽  
Lock In

We propose and experimentally demonstrate an ultra-wideband (UWB) chaos life-detection radar. The proposed radar transmits a wideband chaotic-pulse-position modulation (CPPM) signal modulated by a single-tone sinusoidal wave. A narrow-band split ring sensor is used to collect the reflected sinusoidal wave, and a lock-in amplifier is utilized to identify frequencies of respiration and heartbeat by detecting the phase change of the sinusoidal echo signal. Meanwhile, human location is realized by correlating the CPPM echo signal with its delayed duplicate and combining the synthetic aperture technology. Experimental results demonstrate that the human target can be located accurately and his vital signs can be detected in a large dynamic range through a 20-cm-thick wall using our radar system. The down-range resolution is 15[Formula: see text]cm, benefiting from the 1-GHz bandwidth of the CPPM signal. The dynamic range for human location is 50[Formula: see text]dB, and the dynamic ranges for heartbeat and respiration detection respectively are 20[Formula: see text]dB and 60[Formula: see text]dB in our radar system. In addition, the bandwidth of the CPPM signal can be adjusted from 620[Formula: see text]MHz to 1.56[Formula: see text]GHz to adapt to different requirements.

Fluorescent phospholipids preferentially accumulate in sub-tegumental cells of schistosomula of Schistosoma mansoni

1992 ◽  
Vol 103 (3) ◽  
pp. 823-830 ◽  
Author(s):  
S.T. Furlong ◽  
K.S. Thibault ◽  
R.A. Rogers
Keyword(s):  
Cellular Network ◽  
Biochemical Analysis ◽  
Phosphatidyl Ethanolamine ◽  
De Novo ◽  
Cell Types ◽  
Host Immunity ◽  
Epifluorescence Microscopy ◽  
Specific Cell ◽  
Back Exchange ◽  
Intact Molecule

Schistosomes do not make sterols or fatty acids de novo and thus require host lipids for survival. The acquisition of host lipids may also be an important factor in the schistosome's defense from host immunity; however, little is known about the regulation of this process. Here we have examined binding of radiolabeled and fluorescently labeled liposomes to schistosomula, and followed incorporation of fluorescent phospholipids into the worm by both morphological and biochemical methods. Saturable binding of radiolabeled phosphatidylcholine containing liposomes was observed and epifluorescence microscopy showed binding of C6-NBD-phosphatidylcholine (C6-NBD-PC), C12-NBD-phosphatidylcholine (C12-NBD-PC) and C6-NBD-phosphatidyl-ethanolamine (C6-NBD-PE) containing liposomes on the surface of the parasite. Following back-exchange with unlabeled liposomes, NBD-PC and NBD-PE were observed to be preferentially incorporated into specific cell types within the worm. Furthermore, cells which had accumulated the fluorescent lipid formed an interconnecting cellular network immediately below the tegument, identified as cytons. By contrast, fluorescein-PE was found only on the surface of the parasite and in the gut but not in the cytons. Biochemical analysis demonstrated that > 90% of the C6-NBD-PC and C12-NBD-PC remained as the intact molecule after a one hour incubation with the parasite, but that greater than 70% of the NBD-PE was converted to other lipids. These studies demonstrate that incorporation of phospholipid analogs into schistosomula can be followed morphologically and biochemically. As there was little localization of NBD-PE or NBD-PC in the gut, these analogs must be assimilated by crossing the tegument.(ABSTRACT TRUNCATED AT 250 WORDS)

Haematotoxicology: Scientific Basis and Regulatory Aspects

2002 ◽  
Vol 30 (2_suppl) ◽  
pp. 111-113 ◽  
Author(s):  
Laura Gribaldo
Keyword(s):  
Target Genes ◽  
Cell Types ◽  
Scientific Basis ◽  
Cancer Drugs ◽  
Experimental Conditions ◽  
Clinical Drug Development ◽  
Anti Cancer ◽  
Starting Dose ◽  
Life Threatening

Haematopoietic tissues are the targets of numerous xenobiotics. The purpose of in vitro haematotoxicology is the prediction of adverse haematological effects from toxicants on human haematopoietic targets under controlled experimental conditions in the laboratory. Building on its foundations in experimental haematology and the wealth of haematotoxicological data found in experimental oncology, this field of alternatives toxicology has developed rapidly during the past decade. Preclinical and clinical drug development for anti-cancer drugs differs from that for other pharmaceuticals, because of the life-threatening nature of the disease. Treatment with anti-cancer drugs at clinically efficacious doses usually induces serious side-effects. The design of preclinical toxicology studies for anti-cancer drugs is intended to identify a safe clinical starting dose, characterise toxicities that could be encountered in human clinical trials, and determine whether these toxicities are reversible, manageable, and predictable. Although the myeloid colony-forming unit (CFU-GM) progenitor is most frequently evaluated, other defined progenitors and stem cells, as well as cell types found in the bone-marrow stroma, can now be evaluated in vitro. Genetic damage to haematopoietic cells can occur in the absence of any overt haematological signs. The development of tissue-specific screening systems that are able to give information about the toxic effects of chemicals, drugs and environmental hazards on target genes is needed, in order to make preliminary decisions or to set priorities for selection among large groups of chemicals and possible drugs.

scholarly journals Elucidation and Characterization of New Chlorinated By-Products after Electrochemical Degradation of Hydrochlorothiazide Using Graphite–Poly Vinyl Chloride Electrode

Catalysts ◽  
2018 ◽  
Vol 8 (11) ◽  
pp. 540
Author(s):  
Zainab Mussa ◽  
Fouad Al-Qaim ◽  
Ali Yuzir ◽  
Hirofumi Hara ◽  
Shamila Azman ◽  
...  
Keyword(s):  
Electrochemical Oxidation ◽  
Treatment Process ◽  
Oxidation Process ◽  
Electrochemical Treatment ◽  
Electrochemical Process ◽  
Experimental Conditions ◽  
E Coli ◽  
Flight Mass Spectrometry ◽  
Negative Ionization ◽  
By Products

This paper describes an electrochemical treatment process of hydrochlorothiazide (HDZ) under different conditions such as initial concentration, sodium chloride and applied voltage. In this present study, HDZ was treated by electrochemical oxidation process using graphite-PVC composite electrode as anode and Platinum (Pt) as cathode. All results were analyzed using liquid chromatography-time of flight/mass spectrometry (LC-TOF/MS). It was found that at high applied voltages, and high amounts of NaCl, the electrochemical treatment process was more efficient. The removal% of HDZ was 92% at 5 V after 60 min. From the obtained results, the electrochemical oxidation process of HDZ followed pseudo first order with rate constant values ranged between 0.0009 and 0.0502 min−1, depending on the experimental conditions. Energy consumption was also considered in this study, it was ranged between 0.9058 and 5.56 Wh/mg using 0.5, 0.3 and 0.1 g NaCl within interval times of (10, 20, 30, 40, 50, 60, 70, and 80 min). Five chlorinated and one non-chlorinated by-products were formed and analyzed in negative ionization (NI) mode during the electrochemical process. Due to the strong oxidizing potential of the chlorine (Cl2) and hypochlorite ion (ClO−), HDZ and its by-products were removed after 140 min. Furthermore, a novel synthesis of chlorothiaizde as one of the new by-products was reported in this present study. Toxicity was impacted by the formation of the by-products, especially at 20 min. The inhibition percentage (I%) of E. coli bacteria was decreased to be the lowest value after 140 min.

scholarly journals Endothelial-to-Mesenchymal Transition (EndoMT): Roles in Tumorigenesis, Metastatic Extravasation and Therapy Resistance

2019 ◽  
Vol 2019 ◽  
pp. 1-13 ◽  
Author(s):  
Valentin Platel ◽  
Sébastien Faure ◽  
Isabelle Corre ◽  
Nicolas Clere
Keyword(s):  
Endothelial Cells ◽  
Growth Factor ◽  
Transforming Growth Factor ◽  
Cell Types ◽  
Therapy Resistance ◽  
Experimental Conditions ◽  
Potential Implication ◽  
Mesenchymal Transition ◽  
Endothelial To Mesenchymal Transition

Cancer cells evolve in a very complex tumor microenvironment, composed of several cell types, among which the endothelial cells are the major actors of the tumor angiogenesis. Today, these cells are also characterized for their plasticity, as endothelial cells have demonstrated their potential to modify their phenotype to differentiate into mesenchymal cells through the endothelial-to-mesenchymal transition (EndoMT). This cellular plasticity is mediated by various stimuli including transforming growth factor-β (TGF-β) and is modulated dependently of experimental conditions. Recently, emerging evidences have shown that EndoMT is involved in the development and dissemination of cancer and also in cancer cell to escape from therapeutic treatment. In this review, we summarize current updates on EndoMT and its main induction pathways. In addition, we discuss the role of EndoMT in tumorigenesis, metastasis, and its potential implication in cancer therapy resistance.

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