Treatment of a single plant cell with chemicals

1992 ◽  
Vol 70 (1) ◽  
pp. 225-227 ◽  
Author(s):  
Kazuhiro Toyoda ◽  
Tomonori Shiraishi ◽  
Issei Kobayashi ◽  
Naoto Yamaoka ◽  
Hitoshi Kunoh
Keyword(s):  
Single Cell ◽  
Acridine Orange ◽  
Plant Cell ◽  
Cytoplasmic Streaming ◽  
Target Cells ◽  
Physiological Effects ◽  
Single Plant ◽  
Gel Beads ◽  
Cytochalasin A ◽  
Gel Bead

A technique to treat a single plant cell with chemicals was devised using gel beads and epidermal cells of barley coleoptile. Acridine orange (a fluorescent dye) and cytochalasin A (an inhibitor of cytoplasmic streaming) were separately infiltrated into gel beads that were then individually transferred onto a single cell of the coleoptile using a micromanipulator. Coleoptile cells on which a gel bead infiltrated with acridine orange had been placed gave a characteristic greenish fluorescence when observed with a fluorescence microscope 24 h after the onset of incubation. These results indicate that acridine orange diffused from the gel bead and permeated into the coleoptile cell. When coleoptile cells were treated with gel beads infiltrated with cytochalasin A, cytoplasmic streaming ceased in more than 90% of the cells by 6 h after the onset of treatment. These results demonstrate that cytochalasin A diffused from the gel bead and permeated into the symplast of the target cells. The technique could be applied to test physiological effects of fungal products on a single plant cell. Key words: single cell chemical treatment, cytoplasmic streaming, barley coleoptile, cytochalasin A, acridine orange.

Extraction of RNA from an intact single plant cell with cell wall using focused electric field

2019 ◽  
Author(s):  
Sangamithirai Subramanian Parimalam ◽  
Naoyuki Sotta ◽  
Takashi Kuromori ◽  
Toru Fujiwara ◽  
Masami Yokota Hirai ◽  
...  
Keyword(s):  
Cell Wall ◽  
Electric Field ◽  
Plant Cell ◽  
Single Plant ◽  
Focused Electric Field

scholarly journals An on-chip imaging droplet-sorting system: a real-time shape recognition method to screen target cells in droplets with single cell resolution

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Mathias Girault ◽  
Hyonchol Kim ◽  
Hisayuki Arakawa ◽  
Kenji Matsuura ◽  
Masao Odaka ◽  
...  
Keyword(s):  
Single Cell ◽  
Real Time ◽  
Shape Recognition ◽  
Target Cells ◽  
Recognition Method ◽  
System A ◽  
Time Shape ◽  
Droplet Sorting ◽  
On Chip ◽  
Sorting System

scholarly journals Transcriptome Based Projection of Single Cells to Uncover Development and Heterogeneity of Abnormal Hematopoietic Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2520-2520
Author(s):  
Parashar Dhapola ◽  
Mikael Sommarin ◽  
Mohamed Eldeeb ◽  
Amol Ugale ◽  
David Bryder ◽  
...  
Keyword(s):  
Single Cell ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Single Cells ◽  
Hematopoietic Cells ◽  
Leukemic Cells ◽  
Target Cells ◽  
Cell Identity ◽  
Large Patient ◽  
Low Dimensional

Single-cell transcriptomics (scRNA-Seq) has accelerated the investigation of hematopoietic differentiation. Based on scRNA-Seq data, more refined models of lineage determination in stem- and progenitor cells are now available. Despite such advances, characterizing leukemic cells using single-cell approaches remains challenging. The conventional strategies of scRNA-Seq analysis map all cells on the same low dimensional space using approaches like tSNE and UMAP. However, when used for comparing normal and leukemic cells, such methods are often inadequate as the transcriptome of the leukemic cells has systematically diverged, resulting in irrelevant separation of leukemic subpopulations from their healthy counterpart. Here, we have developed a new computational approach bundled into a tool called Nabo (nabo.readthedocs.io) that has the capacity to directly compare cells that are otherwise unalignable. First, Nabo creates a shared nearest neighbor graph of the reference population, and the heterogeneity of this population is subsequently defined by performing clustering on the graph and calculating a low dimensional representation using t-SNE or UMAP. Nabo then calculates the similarity of incoming cells from a target population to each cell in the reference graph using a modified Canberra metric. The reference cells with higher similarity to the target cells obtain higher mapping scores. The built-in classifier is used to assign each target cell a reference cluster identity. We tested Nabo's accuracy on control datasets and found that Nabo's performance in terms of accuracy and robustness of projection is comparable to state-of-art methods. Moreover, Nabo is a generalized domain adaptation algorithm and hence can perform classification of target cells that are arbitrarily dissimilar to reference cells. Nabo could identify the cell-identity of sorted CD19+ B cells, CD14+ monocytes and CD56+ by projecting these unlabeled cells onto labelled peripheral blood mononuclear cells with an average specificity higher than 0.98. The general applicability of Nabo was demonstrated by successfully integrating pancreatic cells, sequenced in three different studies using different sequencing chemistries with comparable or better accuracy than existing methods. Also, it was conclusively demonstrated that Nabo can predict the identity of human HSPC subpopulations to the same accuracy as can be achieved by established cell-surface markers. Having Nabo at hand, we aimed to uncover the heterogeneity of hematopoietic cells from different stages of AML. Nabo showed that AML cells lacked the heterogeneity of normal CD34+ cells and were devoid of cells with HSC gene signature. A large patient-to-patient variability was found where leukemic cells mapped to distinct stages of myeloid progenitors. To ask whether this variability could reflect differences in leukemia-initiating cell identity, we induced leukemia in murine granulocyte-monocyte-lymphoid progenitors (GMLPs) using an inducible model for MLL-ENL-driven AML. On projection, more than 70% of MLL-ENL-activated cells mapped to a distinct Flt3+ subpopulation present within healthy GMLPs. Statistical validity of this projection was verified using two novel null models for testing cell projections: 1) ablated node model, wherein the mapping strength of target cells are evaluated after removal of high mapping score source nodes, and 2) high entropy features model, which rules out the background noise effect. By separating Flt3+ and Flt3- cells prior to activation of the fusion gene and performing in vitro replating assays, we could demonstrate that Flt3+ GMLPs contained 3-4 fold more leukemia-initiating cells (1/1.34 cells) than Flt3- GMLPs (1/4.89 cells), indicating that leukemia-initiating cells within GMLPs express Flt3. Taken together, Nabo represents a robust cell projection strategy for relevant analysis of scRNA-Seq data that permits an interpretable inference of cross-population relationships. Nabo is designed to compare disparate cellular populations by using the heterogeneity of one population as a point of reference allowing for cell-type specification even following perturbations that have resulted in large molecular changes to the cells of interest. As such, Nabo has critical implementation for delineation of leukemia heterogeneity and identification of leukemia-initiating cell population. Disclosures No relevant conflicts of interest to declare.

scholarly journals 894 The bispecific innate cell engagers AFM13 (CD30/CD16A) and AFM24 (EGFR/CD16A) increase the fraction of tumor target-responsive NK cells and boost serial killing

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A938-A938
Author(s):  
Chiara Zambarda ◽  
Karolin Guldevall ◽  
Chiara Zambarda ◽  
Karolin Guldevall ◽  
Christian Breunig ◽  
...  
Keyword(s):  
Nk Cells ◽  
Single Cell ◽  
Tumor Cells ◽  
Cell Biology ◽  
Nk Cell ◽  
Biological Evaluation ◽  
Target Cells ◽  
List Type ◽  
Tumor Target ◽  
Serial Killing

BackgroundThe use of bispecific natural killer (NK) cell engagers has emerged as a successful strategy for immune cell activation and killing of tumor cells through antibody-dependent cellular cytotoxicity (ADCC). Among these, tetravalent, bispecific innate cell engagers (ICE®) with specificity for the activating receptor CD16A selectively triggering innate responses from NK cells or macrophages represent the most clinically advanced concept. The CD30/CD16A specific ICE® AFM13, has shown efficacy in patients with CD30+ lymphomas as monotherapy1 and combination therapy with check-point inhibitors2 and most recently in combination with adoptive NK cell therapy.3 The EGFR/CD16A specific ICE® AFM24, targeting a variety of solid tumors like colorectal, or lung cancer with a unique mode of action independent of EGFR signaling inhibition, is currently evaluated in an ongoing Ph1/2a clinical study.MethodsWe used a microchip-based screening with single cell resolution4 to elucidate the dynamic responses of individual NK cells towards tumor target cells upon treatment with AFM13 or AFM24.ResultsWe found that AFM13 and AFM24 mediated potent activation of NK cells, leading to increased responsive cytotoxic NK cells and significantly increased the number of NK cells that exerted engagement with multiple target cells rendering these NK cells serial killers. Strikingly, bispecific ICE® molecules triggered stronger cytotoxic responses compared to monoclonal antibodies. One suggested strategy to boost killing by NK cells is to use molecular inhibitors or protein constructs that prevent shedding of CD16.5 However, previous results have shown that this can lead to impaired detachment from target cells, reducing the capacity for an individual NK cell to form serial contacts to target cells.6 We observed that the elevated NK cell killing induced by ICE® molecules was largely conserved when cells were treated with the shedding inhibitor Batimastat. Analysis of the functional dynamics of NK cells revealed that inhibition of CD16 shedding prevented NK cell detachment from target cells, resulting in cell cluster formation. This might strongly impact targeting of distant tumor cells by an individual NK cell thus limiting its anti-tumoral activity.ConclusionsIn conclusion, we show that both AFM13 and AFM24 increase the fraction of tumor-target responsive NK cells and boost serial killing of target cells by individual NK cells. Based on these data, ICE® molecules can be characterized as potent anti-tumoral agents leveraging the enormous potential of NK cells while maintaining crucial features of NK cell biology.AcknowledgementsWe thank members of the Önfelt lab for their valuable help and feedback.ReferencesSawas A, Elgedawe H, Vlad G, Lipschitz M, Chen P-H, Rodig SJ, et al. Clinical and biological evaluation of the novel CD30/CD16A tetravalent bispecific antibody (AFM13) in relapsed or refractory CD30-positive lymphoma with cutaneous presentation: a biomarker phase Ib/IIa study (NCT03192202). Blood 2018;132(Supplement 1):2908–2908.Bartlett NL, Herrera AF, Domingo-Domenech E, Mehta A, Forero-Torres A, Garcia-Sanz R, et al. A phase 1b study of AFM13 in combination with pembrolizumab in patients with relapsed or refractory Hodgkin lymphoma. Blood 2020. Blood 2020;136(21):2401–2409.Kerbauy LN, Marin ND, Kaplan M, Banerjee PP, Berrien-Elliott MM, Becker-Hapak M, et al. Combining AFM13, a bispecific CD30/CD16 antibody, with cytokine-activated blood and cord blood–derived NK cells facilitates CAR-like responses against CD30 + malignancies. Clin Cancer Res Epub 2021.Guldevall K, Brandt L, Forslund E, Olofsson K, Frisk TW, Olofsson PE, et al. Microchip screening platform for single cell assessment of NK cell cytotoxicity. Front Immunol 2016;7:119.Romee R, Foley B, Lenvik T, Wang Y, Zhang B, Ankarlo D, et al. NK cell CD16 surface expression and function is regulated by a disintegrin and metalloprotease-17 (ADAM17). Blood 2013;121(18):3599–608.Srpan K, Ambrose A, Karampatzakis A, Saeed M, Cartwright ANR, Guldevall K, et al. Shedding of CD16 disassembles the NK cell immune synapse and boosts serial engagement of target cells. J Cell Biol 2018;217(9):3267–83.Ethics ApprovalThis work was performed with NK cells from healthy anonymous blood donors, which requires no ethical permit according to local regulations.

scholarly journals Pasting Behavior and Viscoelastic Properties of Fresh, Chilled, and Rehydrated Freeze-Dried Gel Beads from Blends of Tapioca Flour, Soy Flour, and Cane Sugar

2021 ◽  
Vol 20 (4) ◽  
Author(s):  
Nattagan Chantagith ◽  
◽  
Natnaree Katkaew ◽  
Panida Rattanapitigorn ◽  
◽  
...  
Keyword(s):  
Cane Sugar ◽  
Soy Flour ◽  
Significant Relationships ◽  
Gel Beads ◽  
Freeze Dried ◽  
Mass Fractions ◽  
Starch Retrogradation ◽  
Tapioca Flour ◽  
Pasting Behavior ◽  
Gel Bead

Abstract An extreme vertices design for a mixture of three components was used to establish the proportions of tapioca flour (50% to 100% w/w), soy flour (0% to 50% w/w), and cane sugar (0% to 10% w/w) mass fractions in a food gel bead system. Thus, nine compositions were prepared and analyzed. The pasting profiles of the mixtures were studied using a Rapid Visco Analyzer. The texture profiles of fresh, chilled, and rehydrated freeze-dried gel beads were studied using a texture analyzer. Increasing the proportion of soy flour in the range of 11.25% to 50.00% w/w decreased the peak viscosity, breakdown, final viscosity, and setback of mixed flour. Tapioca flour in the proportion of 81.25% to 100.00% w/w recorded the lowest hardness of fresh gel beads (92.00 to 283.00 g). Soy flour in the proportion of 11.25% to 50.00% w/w exhibited lower texture profiles (hardness, chewiness, and gumminess) than tapioca flour in gel beads for both chilled and rehydrated freeze-dried gel beads. Significant relationships were found among pasting profiles of the flour mixtures and texture profiles of fresh, chilled, and rehydrated freeze-dried gel beads, implying a functional role for soy flour in food gel beads. In conclusion, soy flour can act as an anti-retrogradation agent for the gel beads both in chilled (stored at 4°C for 7 days) and freeze-dried conditions. A small amount of cane sugar does not affect the inhibition of starch retrogradation in the gel bead system. Keywords: Anti-retrogradation, Food gel bead, Pasting profile, Soy flour, Tapioca flour, Texture profiles

scholarly journals High-Throughput Single-Cell Transcriptome Profiling of Plant Cell Types

Cell Reports ◽  
2019 ◽  
Vol 27 (7) ◽  
pp. 2241-2247.e4 ◽  
Author(s):  
Christine N. Shulse ◽  
Benjamin J. Cole ◽  
Doina Ciobanu ◽  
Junyan Lin ◽  
Yuko Yoshinaga ◽  
...  
Keyword(s):  
Single Cell ◽  
Plant Cell ◽  
High Throughput ◽  
Transcriptome Profiling ◽  
Cell Types ◽  
Cell Transcriptome ◽  
Single Cell Transcriptome

scholarly journals Single cell census of human kidney organoids shows reproducibility and diminished off-target cells after transplantation

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Ayshwarya Subramanian ◽  
Eriene-Heidi Sidhom ◽  
Maheswarareddy Emani ◽  
Katherine Vernon ◽  
Nareh Sahakian ◽  
...  
Keyword(s):  
Single Cell ◽  
Single Cells ◽  
Human Kidney ◽  
Mouse Kidney ◽  
Target Cells ◽  
Rna Seq ◽  
Kidney Capsule ◽  
Time Points ◽  
Human Ipsc ◽  
Kidney Organoids

AbstractHuman iPSC-derived kidney organoids have the potential to revolutionize discovery, but assessing their consistency and reproducibility across iPSC lines, and reducing the generation of off-target cells remain an open challenge. Here, we profile four human iPSC lines for a total of 450,118 single cells to show how organoid composition and development are comparable to human fetal and adult kidneys. Although cell classes are largely reproducible across time points, protocols, and replicates, we detect variability in cell proportions between different iPSC lines, largely due to off-target cells. To address this, we analyze organoids transplanted under the mouse kidney capsule and find diminished off-target cells. Our work shows how single cell RNA-seq (scRNA-seq) can score organoids for reproducibility, faithfulness and quality, that kidney organoids derived from different iPSC lines are comparable surrogates for human kidney, and that transplantation enhances their formation by diminishing off-target cells.

Determination of oxygen profiles and diffusivity in encapsulated biomass k-carrageenan gel beads

1997 ◽  
Vol 36 (1) ◽  
pp. 271-277 ◽  
Author(s):  
Qin Zhou ◽  
Paul L. Bishop
Keyword(s):  
Gas Flow ◽  
Consumption Rate ◽  
Thin Liquid Film ◽  
Reaction Diffusion ◽  
Gel Beads ◽  
Reaction Diffusion Model ◽  
Volatile Organic ◽  
Oxygen Profiles ◽  
Gel Bead

Biofiltration is a popular method for removing volatile organic compounds (VOCs). One promising medium for biofilters is biomass encapsulated gel beads. Like any other biodegradation system, oxygen concentration is an important factor affecting microbial activities in gel beads and thus the VOC removal efficiency. This paper summarizes the studies on oxygen distribution and diffusivity in k-carrageenan gel beads using oxygen microelectrodes to measure oxygen profiles. By using a reaction-diffusion model and the concentration measurements obtained, a homogeneous diffusivity constant and an oxygen consumption rate constant in k-carrageenan gel beads were estimated. The estimated oxygen diffusivity in the gel bead is 46.3% of the value in water when the bead is immersed in water and 53.9% that of water when the bead is in air with a thin liquid film surrounding it. To provide more information for the design and operation of biofilters using biomass-loaded gel beads, we also investigated and report on effects of biomass immobilization time, TCE influent concentration and TCE gas flow rate on oxygen concentrations in the gel bead.

scholarly journals Single-cell transcriptomics identifies multiple pathways underlying antitumor function of TCR- and CD8αβ-engineered human CD4+ T cells

2020 ◽  
Vol 6 (27) ◽  
pp. eaaz7809 ◽  
Author(s):  
Jan A. Rath ◽  
Gagan Bajwa ◽  
Benoit Carreres ◽  
Elisabeth Hoyer ◽  
Isabelle Gruber ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Single Cell ◽  
Cell Function ◽  
Xenograft Model ◽  
Cell Receptor ◽  
Target Cells ◽  
Antigen Specificity ◽  
Molecular Features ◽  
Mouse Xenograft Model

Transgenic coexpression of a class I–restricted tumor antigen–specific T cell receptor (TCR) and CD8αβ (TCR8) redirects antigen specificity of CD4+ T cells. Reinforcement of biophysical properties and early TCR signaling explain how redirected CD4+ T cells recognize target cells, but the transcriptional basis for their acquired antitumor function remains elusive. We, therefore, interrogated redirected human CD4+ and CD8+ T cells by single-cell RNA sequencing and characterized them experimentally in bulk and single-cell assays and a mouse xenograft model. TCR8 expression enhanced CD8+ T cell function and preserved less differentiated CD4+ and CD8+ T cells after tumor challenge. TCR8+CD4+ T cells were most potent by activating multiple transcriptional programs associated with enhanced antitumor function. We found sustained activation of cytotoxicity, costimulation, oxidative phosphorylation– and proliferation-related genes, and simultaneously reduced differentiation and exhaustion. Our study identifies molecular features of TCR8 expression that can guide the development of enhanced immunotherapies.

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