scholarly journals Interactions of Metarhizium brunneum-7 with Phytophagous Mites Following Different Application Strategies

Insects ◽  
2020 ◽  
Vol 11 (6) ◽  
pp. 330
Author(s):  
Dana Ment ◽  
Sukirtha Raman ◽  
Shira Gal ◽  
David Ezra ◽  
Eric Palevsky
Keyword(s):  
Common Bean ◽  
Fluorescent Protein ◽  
Mite Population ◽  
Confocal Imaging ◽  
Endophytic Colonization ◽  
Metarhizium Brunneum ◽  
Phytophagous Mites ◽  
Volkamer Lemon ◽  
Bulb Mite ◽  
Citrus Leaves

Metarhizium brunneum is a generalist entomopathogenic fungus known to be virulent against Acari. We investigated Metarhizium brunneum-7 (Mb7) interactions in three systems of phytophagous mites and their respective plant hosts: Volkamer lemon (Citrus volkameriana) and the citrus rust mite Phyllocoptruta oleivora; common bean (Phaseolus vulgaris) and the two-spotted spider mite Tetranychus urticae; and spring onion (Allium cepa) and the bulb mite Rhizoglyphus robini. All three mite species were susceptible to directly applied Mb7 conidia. Results obtained using the standard method for studying endophytic colonization vs. live confocal imaging of plant tissues using the green fluorescent protein (GFP)-transformed fungus differed markedly, demonstrating that microscopy validation was more definite than the standard process of recovery from plant tissue. Endophytic colonization was observed in conidium-infiltrated citrus leaves and in roots of onion plants treated with soil-drenched conidia, but not in common bean treated by either spray or drench of conidia. Endophytic colonization of citrus leaves did not affect the citrus mite population. Drench application in common bean reduced two-spotted mite population. Similarly, drench application in onion reduced bulb mite population. This study emphasizes the importance of the host plant effects on Mb7 control efficacy of mite pests, and the merits of live-imaging techniques in studying endophytic interaction.

scholarly journals Fluorescence Cross-Correlation Spectroscopy Yields True Affinity and Binding Kinetics of Plasmodium Lactate Transport Inhibitors

2021 ◽  
Vol 14 (8) ◽  
pp. 757
Author(s):  
Iga Jakobowska ◽  
Frank Becker ◽  
Stefano Minguzzi ◽  
Kerrin Hansen ◽  
Björn Henke ◽  
...  
Keyword(s):  
Rate Constants ◽  
Cross Correlation ◽  
Fluorescent Protein ◽  
Correlation Spectroscopy ◽  
Confocal Imaging ◽  
Binding Kinetics ◽  
Acceptor Site ◽  
Lactate Transport ◽  
Entry Site ◽  
Hydrogen Bond Acceptor

Blocking lactate export in the parasitic protozoan Plasmodium falciparum is a novel strategy to combat malaria. We discovered small drug-like molecules that inhibit the sole plasmodial lactate transporter, PfFNT, and kill parasites in culture. The pentafluoro-3-hydroxy-pent-2-en-1-one BH296 blocks PfFNT with nanomolar efficiency but an in vitro selected PfFNT G107S mutation confers resistance against the drug. We circumvented the mutation by introducing a nitrogen atom as a hydrogen bond acceptor site into the aromatic ring of the inhibitor yielding BH267.meta. The current PfFNT inhibitor efficiency values were derived from yeast-based lactate transport assays, yet direct affinity and binding kinetics data are missing. Here, we expressed PfFNT fused with a green fluorescent protein in human embryonic kidney cells and generated fluorescent derivatives of the inhibitors, BH296 and BH267.meta. Using confocal imaging, we confirmed the location of the proposed binding site at the cytosolic transporter entry site. We then carried out fluorescence cross-correlation spectroscopy measurements to assign true Ki-values, as well as kon and koff rate constants for inhibitor binding to PfFNT wildtype and the G107S mutant. BH296 and BH267.meta gave similar rate constants for binding to PfFNT wildtype. BH296 was inactive on PfFNT G107S, whereas BH267.meta bound the mutant protein albeit with weaker affinity than to PfFNT wildtype. Eventually, using a set of PfFNT inhibitor compounds, we found a robust correlation of the results from the biophysical FCCS binding assay to inhibition data of the functional transport assay.

scholarly journals Optical reconstruction of murine colorectal mucosa at cellular resolution

2015 ◽  
Vol 308 (9) ◽  
pp. G721-G735 ◽  
Author(s):  
Cambrian Y. Liu ◽  
Philip E. Dubé ◽  
Nandini Girish ◽  
Ajay T. Reddy ◽  
D. Brent Polk
Keyword(s):  
Cell Function ◽  
Fluorescent Protein ◽  
Confocal Imaging ◽  
Host Cells ◽  
Cellular Mechanisms ◽  
Colorectal Mucosa ◽  
Cellular Resolution ◽  
Volume Imaging ◽  
Mucosal Layer ◽  
Optical Reconstruction

The mucosal layer of the colon is a unique and dynamic site where host cells interface with one another and the microbiome, with major implications for physiology and disease. However, the cellular mechanisms mediating colonic regeneration, inflammation, dysplasia, and dysbiosis remain undercharacterized, partly because the use of thin tissue sections in many studies removes important volumetric context. To address these challenges in visualization, we have developed the deep mucosal imaging (DMI) method to reconstruct continuous extended volumes of mouse colorectal mucosa at cellular resolution. Use of ScaleA2 and SeeDB clearing agents enabled full visualization of the colonic crypt, the fundamental unit of adult colon. Confocal imaging of large colorectal expanses revealed epithelial structures involved in repair, inflammation, tumorigenesis, and stem cell function, in fluorescent protein-labeled, immunostained, paraffin-embedded, or human biopsy samples. We provide freely available software to reconstruct and explore on computers with standard memory allocations the large DMI datasets containing in toto representations of distal colonic mucosal volume. Extended-volume imaging of colonic mucosa through the novel, extensible, and readily adopted DMI approach will expedite mechanistic investigations of intestinal physiology and pathophysiology at intracrypt to multicrypt length scales.

Confocal Imaging of Subcellular Ca2+ Concentrations Using a Dual-Excitation Ratiometric Indicator Based on Green Fluorescent Protein

2002 ◽  
Vol 2002 (125) ◽  
pp. pl4-pl4 ◽  
Author(s):  
S. Shimozono ◽  
T. Fukano ◽  
T. Nagai ◽  
Y. Kirino ◽  
H. Mizuno ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Confocal Imaging ◽  
Green Fluorescent

scholarly journals Quantitation of mitochondrial dynamics by photolabeling of individual organelles shows that mitochondrial fusion is blocked during the Bax activation phase of apoptosis

2004 ◽  
Vol 164 (4) ◽  
pp. 493-499 ◽  
Author(s):  
Mariusz Karbowski ◽  
Damien Arnoult ◽  
Hsiuchen Chen ◽  
David C. Chan ◽  
Carolyn L. Smith ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Mitochondrial Dynamics ◽  
Dynamic Balance ◽  
Mitochondrial Fusion ◽  
Confocal Imaging ◽  
Time Range ◽  
Mitochondrial Morphology ◽  
Green Fluorescent ◽  
Mitochondrial Membrane Permeabilization ◽  
Activation Phase

A dynamic balance of organelle fusion and fission regulates mitochondrial morphology. During apoptosis this balance is altered, leading to an extensive fragmentation of the mitochondria. Here, we describe a novel assay of mitochondrial dynamics based on confocal imaging of cells expressing a mitochondrial matrix–targeted photoactivable green fluorescent protein that enables detection and quantification of organelle fusion in living cells. Using this assay, we visualize and quantitate mitochondrial fusion rates in healthy and apoptotic cells. During apoptosis, mitochondrial fusion is blocked independently of caspase activation. The block in mitochondrial fusion occurs within the same time range as Bax coalescence on the mitochondria and outer mitochondrial membrane permeabilization, and it may be a consequence of Bax/Bak activation during apoptosis.

Resting Microglial Motility Is Independent of Synaptic Plasticity in Mammalian Brain

2008 ◽  
Vol 99 (4) ◽  
pp. 2026-2032 ◽  
Author(s):  
Long-Jun Wu ◽  
Min Zhuo
Keyword(s):  
Synaptic Plasticity ◽  
Brain Injuries ◽  
Fluorescent Protein ◽  
Hippocampal Slices ◽  
Time Lapse ◽  
Long Term Potentiation ◽  
Confocal Imaging ◽  
Mammalian Brain ◽  
Protective Function ◽  
Green Fluorescent

Microglia are well known for their roles in brain injuries and infections. However, there is no function attributes to resting microglia thus far. Here we performed a combination of simultaneous electrophysiology and time-lapse confocal imaging in green fluorescent protein–labeled microglia in acute hippocampal slices. In contrast to CA1 neurons, microglia showed no spontaneous or evoked synaptic currents. Neither glutamate- nor GABA-induced current/chemotaxis of microglia was detected. Strong tetanic stimulation of Schaffer-collateral pathways that induce CA1 long-term potentiation did not affect microglial motilities. Our results suggest that microglia are highly reserved for neuronal protective function but not synaptic plasticity in the brain.

Nerve-mast cell (RBL) interaction: RBL membrane ruffling occurs at the contact site with an activated neurite

2002 ◽  
Vol 283 (6) ◽  
pp. C1738-C1744 ◽  
Author(s):  
N. Mori ◽  
R. Suzuki ◽  
T. Furuno ◽  
D. M. McKay ◽  
M. Wada ◽  
...  
Keyword(s):  
Mast Cell ◽  
Cell Activation ◽  
Fluorescent Protein ◽  
Cell Communication ◽  
Scorpion Venom ◽  
Confocal Imaging ◽  
Mucosal Mast Cell ◽  
Contact Site ◽  
Membrane Ruffling

Mast cell-neurite interaction serves as a model for neuroimmune interaction. We have shown that neurite-mast cell communication can occur via substance P interacting with neurokinin (NK)-1 receptors on the mucosal mast cell-like cell, the rat basophilic leukemia (RBL) cell. Neurite (murine superior cervical ganglia) and RBL cell [expressing the granule-associated antigen CD63-green fluorescent protein (GFP) conjugate] cocultures were established and stimulated with bradykinin (BK; 10 nM) or scorpion venom (SV; 10 pg/ml), both of which activate only neurites. Cell activation was assessed by confocal imaging of Ca2+ (cells preloaded with fluo 3), and analyses of RBL CD63-GFP+ granule movement were conducted. Neurite activation by BK or SV was followed by RBL Ca2+mobilization, which was inhibited by an NK-1 receptor antagonist (NK-1 RA). Moreover, membrane ruffling was observed on RBL pseudopodial extensions in contact with the activated neurite, but not on noncontacting pseudopodia. RBL membrane ruffling was inhibited by NK-1 RA, but not NK-2 RA, and was accompanied by a significant increase in granule movement (0.13 ± 0.04 vs. 0.05 ± 0.01 μm/s) that was most evident at the point of neurite contact: many of the granules moved toward the plasmalemma. This is the first documentation of such precise (restricted to the membrane's contact site) transfer of information between nerves and mast cells that could allow for very subtle in vivo communication between these two cell types.

scholarly journals Kinetics and Strain Specificity of Rhizosphere and Endophytic Colonization by Enteric Bacteria on Seedlings of Medicago sativa and Medicago truncatula

2003 ◽  
Vol 69 (3) ◽  
pp. 1783-1790 ◽  
Author(s):  
Yuemei Dong ◽  
A. Leonardo Iniguez ◽  
Brian M. M. Ahmer ◽  
Eric W. Triplett
Keyword(s):  
Medicago Sativa ◽  
Medicago Truncatula ◽  
Mycorrhizal Fungi ◽  
Fluorescent Protein ◽  
Enteric Bacteria ◽  
Health Concern ◽  
Strain Specificity ◽  
E Coli ◽  
Endophytic Colonization ◽  
K 12

ABSTRACT The presence of human-pathogenic, enteric bacteria on the surface and in the interior of raw produce is a significant health concern. Several aspects of the biology of the interaction between these bacteria and alfalfa (Medicago sativa) seedlings are addressed here. A collection of enteric bacteria associated with alfalfa sprout contaminations, along with Escherichia coli K-12, Salmonella enterica serotype Typhimurium strain ATCC 14028, and an endophyte of maize, Klebsiella pneumoniae 342, were labeled with green fluorescent protein, and their abilities to colonize the rhizosphere and the interior of the plant were compared. These strains differed widely in their endophytic colonization abilities, with K. pneumoniae 342 and E. coli K-12 being the best and worst colonizers, respectively. The abilities of the pathogens were between those of K. pneumoniae 342 and E. coli K-12. All Salmonella bacteria colonized the interiors of the seedlings in high numbers with an inoculum of 102 CFU, although infection characteristics were different for each strain. For most strains, a strong correlation between endophytic colonization and rhizosphere colonization was observed. These results show significant strain specificity for plant entry by these strains. Significant colonization of lateral root cracks was observed, suggesting that this may be the site of entry into the plant for these bacteria. At low inoculum levels, a symbiosis mutant of Medicago truncatula, dmi1, was colonized in higher numbers on the rhizosphere and in the interior by a Salmonella endophyte than was the wild-type host. Endophytic entry of M. truncatula appears to occur by a mechanism independent of the symbiotic infections by Sinorhizobium meliloti or mycorrhizal fungi.

Effect of salmonella typhimurium A1-R on quiescent cancer cells that do not respond to standard chemotherapy.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. e13576-e13576
Author(s):  
Shuya Yano ◽  
Yong Zhang ◽  
Fuminari Uehara ◽  
Yukihiko Hiroshima ◽  
Shinji Miwa ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Salmonella Typhimurium ◽  
Cancer Cells ◽  
Nude Mice ◽  
Fluorescent Protein ◽  
Confocal Imaging ◽  
Cytotoxic Agents ◽  
Cycle Phase ◽  
Solid Cancer ◽  
Proliferating Cells

e13576 Background: Quiescent cancer cells are a major impediment to treating solid cancer with chemotherapy, since in most tumors the majority of the cells are quiescent. Methods: The cell-cycle phase of each human MKN45 gastric cancer cell was imaged using a fluorescence ubiquitination cell cycle indicator (FUCCI). With FUCCI, quiescent cancer cells express mKusabira-Orange fluorescent protein (red) and proliferating cells express mAzami-Green fluorescent protein (green). FUCCI-labeled cancer cells in tumor spheres and subcutaneous tumor in nude mice were treated with Salmonella typhimurium A1-R. Results: Time-lapse confocal imaging showed that cancer cells in tumor spheres in serum-free culture become and remained quiescent. S. typhimurium A1-R infected and killed quiescent cancer cells in tumor spheres. In contrast, cytotoxic agents did not kill the quiescent cancer cells in the tumor spheres. S. typhimurium A1-R targeting of FUCCI-expressing subcutaneous tumors growing in nude mice resulted in the killing of quiescent cancer cells resistant to cytotoxic agents. Conclusions: S. typhimurium A1-R can kill quiescent cancer cells which suggests a new therapeutic paradigm potentially more effective than current therapeutics which are ineffective against quiescent cancer cells.

scholarly journals Plasticity of Lh cells caused by cell proliferation and recruitment of existing cells

2019 ◽  
Vol 240 (2) ◽  
pp. 361-377 ◽  
Author(s):  
Romain Fontaine ◽  
Eirill Ager-Wick ◽  
Kjetil Hodne ◽  
Finn-Arne Weltzien
Keyword(s):  
Cell Proliferation ◽  
Fluorescent Protein ◽  
Pituitary Cell ◽  
Confocal Imaging ◽  
Nuclear Antigen ◽  
Classical Type ◽  
Pituitary Cells ◽  
Cell Hyperplasia ◽  
Lh Cells

Luteinizing hormone (Lh) and follicle-stimulating hormone (Fsh) control reproduction in vertebrates. Using a transgenic line of medaka, in which green fluorescent protein expression is controlled by the endogenous lhb promotor, we studied development and plasticity of Lh cells, comparing juveniles and adults of both genders. Confocal imaging and 3D reconstruction revealed hypertrophy and hyperplasia of Lh cells in both genders from juvenile to adult stages. We show that Lh cell hyperplasia may be caused by recruitment of existing pituitary cells that start to produce lhb, as evidenced by time lapse recordings of primary pituitary cell cultures, and/or through Lh cell proliferation, demonstrated through a combination of 5-bromo-2′-deoxyuridine incubation experiments and proliferating cell nuclear antigen staining. Proliferating Lh cells do not belong to the classical type of multipotent stem cells, as they do not stain with anti-sox2. Estradiol exposure in vivo increased pituitary cell proliferation, particularly Lh cells, whereas pituitary lhb and gpa expression levels decreased. RNA-seq and in situ hybridization showed that Lh cells express two estrogen receptors, esr1 and esr2b, and the aromatase gene cyp19a1b, suggesting a direct effect of estradiol, and possibly androgens, on Lh cell proliferation. In conclusion, our study reveals a high degree of plasticity in the medaka Lh cell population, resulting from a combination of recruitment and cell proliferation.

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