scholarly journals Monitoring Tritrophic Biocontrol Interactions Between Bacillus spp., Fusarium oxysporum f. sp. cubense, Tropical Race 4, and Banana Plants in vivo Based on Fluorescent Transformation System

2021 ◽  
Vol 12 ◽  
Author(s):  
Ping He ◽  
Shu Li ◽  
Shengtao Xu ◽  
Huacai Fan ◽  
Yongfen Wang ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Fluorescent Protein ◽  
Laser Scanning Confocal Microscopy ◽  
Transformation System ◽  
Scanning Confocal Microscopy ◽  
Bacillus Spp ◽  
Green Fluorescent ◽  
Race 4

Bacillus spp. is effective biocontrol agents for Fusarium wilt of banana (FWB), tropical race 4 (TR4). This study explores the colonization by Bacillus subtilis, Bacillus velezensis, and Bacillus amyloliquefaciens of host banana plants and elucidates the mechanism of antagonistic TR4 biocontrol. The authors selected one B. subtilis strain, three B. velezensis strains, and three B. amyloliquefaciens strains that are proven to significantly inhibit TR4 in vitro, optimized the genetic transformation conditions and explored their colonization process in banana plants. The results showed that we successfully constructed an optimized fluorescent electro-transformation system (OD600 of bacteria concentration=0.7, plasmid concentration=50ng/μl, plasmid volume=2μl, transformation voltage=1.8kV, and transformation capacitance=400Ω) of TR4-inhibitory Bacillus spp. strains. The red fluorescent protein (RFP)-labeled strains were shown to have high stability with a plasmid-retention frequency above 98%, where bacterial growth rates and TR4 inhibition are unaffected by fluorescent plasmid insertion. In vivo colonizing observation by Laser Scanning Confocal Microscopy (LSCM) and Scanning Electron Microscopy (SEM) showed that Bacillus spp. can colonize the internal cells of banana plantlets roots. Further, fluorescent observation by LSCM showed these RFP-labeled bacteria exhibit chemotaxis (chemotaxis ratio was 1.85±0.04) toward green fluorescent protein (GFP)-labeled TR4 hyphae in banana plants. We conclude that B. subtilis, B. velezensis, and B. amyloliquefaciens can successfully colonize banana plants and interact with TR4. Monitoring its dynamic interaction with TR4 and its biocontrol mechanism is under further study.

scholarly journals Peanut Clump Virus RNA-1-Encoded P15 Regulates Viral RNA Accumulation but Is Not Abundant at Viral RNA Replication Sites

2001 ◽  
Vol 75 (4) ◽  
pp. 1941-1948 ◽  
Author(s):  
Patrice Dunoyer ◽  
Etienne Herzog ◽  
Odile Hemmer ◽  
Christophe Ritzenthaler ◽  
Christiane Fritsch
Keyword(s):  
Laser Scanning ◽  
Fluorescent Protein ◽  
Laser Scanning Confocal Microscopy ◽  
Viral Rna ◽  
Epifluorescence Microscopy ◽  
Scanning Confocal Microscopy ◽  
Virus Rna ◽  
Green Fluorescent ◽  
Rna Accumulation

ABSTRACT RNA-1 of peanut clump pecluvirus (PCV) encodes N-terminally overlapping proteins which contain helicase-like (P131) and polymerase-like (P191) domains and is able to replicate in the absence of RNA-2 in protoplasts of tobacco BY-2 cells. RNA-1 also encodes P15, which is expressed via a subgenomic RNA. To investigate the role of P15, we analyzed RNA accumulation in tobacco BY-2 protoplasts inoculated with RNA-1 containing mutations in P15. For all the mutants, the amount of progeny RNA-1 produced was significantly lower than that obtained for wild-type RNA-1. If RNA-2 was included in the inoculum, the accumulation of both progeny RNAs was diminished, but near-normal yields of both could be recovered if the inoculum was supplemented with a small, chimeric viral replicon expressing P15, demonstrating that P15 has an effect on viral RNA accumulation. To further analyze the role of P15, transcripts were produced expressing P15 fused to enhanced green fluorescent protein (EGFP). Following inoculation to protoplasts, epifluorescence microscopy revealed that P15 accumulated as spots around the nucleus and in the cytoplasm. Intracellular sites of viral RNA synthesis were visualized by laser scanning confocal microscopy of infected protoplasts labeled with 5-bromouridine 5′-triphosphate (BrUTP). BrUTP labeling also occured in spots distributed within the cytoplasm and around the nucleus. However, the BrUTP-labeled RNA and EGFP/P15 very rarely colocalized, suggesting that P15 does not act primarily at sites of viral replication but intervenes indirectly to control viral accumulation levels.

Penetration of Surface-Inoculated Bacteria as a Result of Hydrodynamic Shock Wave Treatment of Beef Steaks

2002 ◽  
Vol 65 (4) ◽  
pp. 616-620 ◽  
Author(s):  
T. A. LORCA ◽  
M. D. PIERSON ◽  
J. R. CLAUS ◽  
J. D. EIFERT ◽  
J. E. MARCY ◽  
...  
Keyword(s):  
Shock Wave ◽  
Laser Scanning ◽  
Fluorescent Protein ◽  
Laser Scanning Confocal Microscopy ◽  
Exterior Surface ◽  
Safety Hazard ◽  
E Coli ◽  
Scanning Confocal Microscopy ◽  
Beef Steaks ◽  
Green Fluorescent

The top surface of the raw eye of round steaks was inoculated with either green fluorescent protein (GFP)-labeled Escherichia coli (E. coli-GFP) or rifampin-resistant E. coli (E. coli-rif). Cryostat sampling in concert with laser scanning confocal microscopy (LSCM) or plating onto antibiotic selective agar was used to determine if hydrodynamic shock wave (HSW) treatment resulted in the movement of the inoculated bacteria from the outer inoculated surface to the interior of intact beef steaks. HSW treatment induced the movement of both marker bacteria into the steaks to a maximum depth of 300 μm (0.3 mm). Because popular steak-cooking techniques involve the application of heat from the exterior surface of the steak to achieve internal temperatures ranging from 55 to 82°C, the extent of bacterial penetration observed in HSW-treated steaks does not appear to pose a safety hazard to consumers.

scholarly journals Studying the Neurovascular Unit: An Improved Blood–Brain Barrier Model

2009 ◽  
Vol 29 (12) ◽  
pp. 1879-1884 ◽  
Author(s):  
Christoph M Zehendner ◽  
Heiko J Luhmann ◽  
Christoph RW Kuhlmann
Keyword(s):  
Blood Brain Barrier ◽  
Laser Scanning ◽  
Neurovascular Unit ◽  
Cell Types ◽  
Laser Scanning Confocal Microscopy ◽  
Brain Barrier ◽  
Scanning Confocal Microscopy ◽  
Blood Brain

The blood–brain barrier (BBB) closely interacts with the neuronal parenchyma in vivo. To replicate this interdependence in vitro, we established a murine coculture model composed of brain endothelial cell (BEC) monolayers with cortical organotypic slice cultures. The morphology of cell types, expression of tight junctions, formation of reactive oxygen species, caspase-3 activity in BECs, and alterations of electrical resistance under physiologic and pathophysiological conditions were investigated. This new BBB model allows the application of techniques such as laser scanning confocal microscopy, immunohistochemistry, fluorescent live cell imaging, and electrical cell substrate impedance sensing in real time for studying the dynamics of BBB function under defined conditions.

scholarly journals The 25-kDa Movement Protein of PVX Elicits Nb-Mediated Hypersensitive Cell Death in Potato

1999 ◽  
Vol 12 (6) ◽  
pp. 536-543 ◽  
Author(s):  
Isabelle Malcuit ◽  
María Rosa Marano ◽  
Tony A. Kavanagh ◽  
Walter De Jong ◽  
Alec Forsyth ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Fluorescent Protein ◽  
Movement Protein ◽  
Potato Virus X ◽  
Laser Scanning Confocal Microscopy ◽  
Hypersensitive Resistance ◽  
Hypersensitive Cell Death ◽  
Scanning Confocal Microscopy ◽  
Gus Reporter ◽  
Green Fluorescent

The potato gene Nb confers hypersensitive resistance to potato virus X (PVX). To characterize the viral elicitor of this resistance we introduced modifications into the genome of avirulent strains (ROTH1 and CP2) and virulent strains (UK3 and CP4) of PVX. From the analysis of the modified viral genomes, the Nb avirulence determinant was mapped in the PVX 25K gene coding for the 25-kDa movement protein. Furthermore, we showed that the isoleucine residue at position 6 of this protein was required for activation of the Nb response. Transient co-expression of the avirulent 25K gene with the β-glucuronidase (GUS) reporter gene introduced by particle bombardment in resistant and susceptible potato cells confirmed that the elicitor activity provided by the 25-kDa protein did not require other PVX-encoded proteins. To study cellular events associated with the Nb response, the 25-kDa proteins of PVX strains ROTH1 and UK3 were tagged with the green fluorescent protein (GFP) so that the dynamics of the subcellular distribution of the 25KGFP fusion proteins could be followed in living potato epidermal cells by laser scanning confocal microscopy. Using this method, we showed that the Nb-mediated response is associated with degradation of subcellular structures.

Characterization and imaging of A6 epithelial cell clones expressing fluorescently labeled ENaC subunits

2001 ◽  
Vol 281 (2) ◽  
pp. C624-C632 ◽  
Author(s):  
Bonnie L. Blazer-Yost ◽  
Michael Butterworth ◽  
Amy D. Hartman ◽  
Gretchen E. Parker ◽  
Carla J. Faletti ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Fluorescent Protein ◽  
Laser Scanning Confocal Microscopy ◽  
Scanning Confocal Microscopy ◽  
Parent Line ◽  
Rate Limiting Step ◽  
Cell Clones ◽  
Clonal Lines ◽  
Green Fluorescent ◽  
Rate Limiting

A6 model renal epithelial cells were stably transfected with enhanced green fluorescent protein (EGFP)-tagged α- or β-subunits of the epithelial Na+channel (ENaC). Transfected RNA and proteins were both expressed in low abundance, similar to the endogenous levels of ENaC in native cells. In living cells, laser scanning confocal microscopy revealed a predominately subapical distribution of EGFP-labeled subunits, suggesting a readily accessible pool of subunits available to participate in Na+ transport. The basal level of Na+ transport in the clonal lines was enhanced two- to fourfold relative to the parent line. Natriferic responses to insulin or aldosterone were similar in magnitude to the parent line, while forskolin-stimulated Na+ transport was 64% greater than control in both the α- and β-transfected lines. In response to forskolin, EGFP-labeled channel subunits traffic to the apical membrane. These data suggest that channel regulators, not the channel per se, form the rate-limiting step in response to insulin or aldosterone stimulation, while the number of channel subunits is important for basal as well as cAMP-stimulated Na+transport.

scholarly journals Integrin αvβ6-targeted MR molecular imaging of breast cancer in a xenograft mouse model

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Dengfeng Li ◽  
Chengyan Dong ◽  
Xiaohong Ma ◽  
Xinming Zhao
Keyword(s):  
Breast Cancer ◽  
Molecular Imaging ◽  
Magnetic Resonance ◽  
Laser Scanning ◽  
Laser Scanning Confocal Microscopy ◽  
Hek293 Cells ◽  
Targeted Nanoparticles ◽  
Scanning Confocal Microscopy

Abstract Background The motif RXDLXXL-based nanoprobes allow specific imaging of integrin αvβ6, a protein overexpressed during tumorigenesis and tumor progression of various tumors. We applied a novel RXDLXXL-coupled cyclic arginine-glycine-aspartate (RGD) nonapeptide conjugated with ultrasmall superparamagnetic iron oxide nanoparticles (referred to as cFK-9-USPIO) for the application of integrin αvβ6-targeted magnetic resonance (MR) molecular imaging for breast cancer. Methods A novel MR-targeted nanoprobe, cFK-9-USPIO, was synthesized by conjugating integrin αvβ6-targeted peptide cFK-9 to N-amino (−NH2)-modified USPIO nanoparticles via a dehydration esterification reaction. Integrin αvβ6-positive mouse breast cancer (4 T1) and integrin αvβ6 negative human embryonic kidney 293 (HEK293) cell lines were incubated with cFK-9-AbFlour 647 (blocking group) or cFK-9-USPIO (experimental group), and subsequently imaged using laser scanning confocal microscopy (LSCM) and 3.0 Tesla magnetic resonance imaging (MRI) system. The affinity of cFK-9 targeting αvβ6 was analyzed by calculating the mean fluorescent intensity in cells, and the nanoparticle targeting effect was measured by the reduction of T2 values in an in vitro MRI. The in vivo MRI capability of cFK-9-USPIO was investigated in 4 T1 xenograft mouse models. Binding of the targeted nanoparticles to αvβ6-positive 4 T1 tumors was determined by ex vivo histopathology. Results In vitro laser scanning confocal microscopy (LSCM) imaging showed that the difference in fluorescence intensity between the targeting and blocking groups of 4 T1 cells was significantly greater than that in HEK293 cells (P < 0.05). The in vitro MRI demonstrated a more remarkable T2 reduction in 4 T1 cells than in HEK293 cells (P < 0.001). The in vivo MRI of 4 T1 xenograft tumor-bearing nude mice showed significant T2 reduction in tumors compared to controls. Prussian blue staining further confirmed that αvβ6 integrin-targeted nanoparticles were specifically accumulated in 4 T1 tumors and notably fewer nanoparticles were detected in 4 T1 tumors of mice injected with control USPIO and HEK293 tumors of mice administered cFK-9-USPIO. Conclusions Integrin αvβ6-targeted nanoparticles have great potential for use in the detection of αvβ6-overexpressed breast cancer with MR molecular imaging.

Morphological assessment of the development of multinucleated giant cells in glioma by using mitosis-specific phosphorylated antibodies

2003 ◽  
Vol 98 (4) ◽  
pp. 854-859 ◽  
Author(s):  
Kenkou Maeda ◽  
Masaaki Mizuno ◽  
Toshihiko Wakabayashi ◽  
Syuntarou Takasu ◽  
Tetsurou Nagasaka ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Giant Cells ◽  
Laser Scanning Confocal Microscopy ◽  
Multinucleated Giant Cells ◽  
Content Type ◽  
Scanning Confocal Microscopy ◽  
Drug Treatments ◽  
Fine Print

Object. The nature and origin of multinucleated giant cells in glioma have not been made clear. To investigate the phosphorylation of intermediate filaments, the authors studied multinucleated giant cells in vitro and in vivo by using mitosis-specific phosphorylated antibodies. Methods. Cultured human glioma cells were immunostained with monoclonal antibodies (mAbs) 4A4, KT13, and TM71, which recognized the phosphorylation of vimentin at Ser55, glial fibrillary acidic protein at Ser13, and vimentin at Ser71, respectively. Subsequently, the nature of multinucleated giant cells was investigated using laser scanning confocal microscopy. In addition, paraffin-embedded tissue sections obtained in three patients with giant cell glioblastoma were also investigated. Multinucleated giant cells were immunoreacted with the mAb 4A4 and not with KT13 and TM71 in vitro and in vivo. In addition, the authors obtained these results in multinucleated giant cells under natural conditions, without drug treatments. Conclusions. Findings in this investigation indicated that multinucleated giant cells are those remaining in mitosis between metaphase and telophase, undergoing neither fusion nor degeneration.

scholarly journals Dynamics of Actin-Based Movement byRickettsia rickettsii in Vero Cells

1999 ◽  
Vol 67 (8) ◽  
pp. 4201-4207 ◽  
Author(s):  
Robert A. Heinzen ◽  
Scott S. Grieshaber ◽  
Levi S. Van Kirk ◽  
Clinton J. Devin
Keyword(s):  
Laser Scanning ◽  
Fluorescent Protein ◽  
Average Rate ◽  
Time Lapse ◽  
Vero Cells ◽  
Laser Scanning Confocal Microscopy ◽  
Phenotypic Differences ◽  
Rickettsia Rickettsii ◽  
Scanning Confocal Microscopy ◽  
Green Fluorescent

ABSTRACT Actin-based motility (ABM) is a virulence mechanism exploited by invasive bacterial pathogens in the genera Listeria,Shigella, and Rickettsia. Due to experimental constraints imposed by the lack of genetic tools and their obligate intracellular nature, little is known about rickettsial ABM relative toListeria and Shigella ABM systems. In this study, we directly compared the dynamics and behavior of ABM ofRickettsia rickettsii and Listeria monocytogenes. A time-lapse video of moving intracellular bacteria was obtained by laser-scanning confocal microscopy of infected Vero cells synthesizing β-actin coupled to green fluorescent protein (GFP). Analysis of time-lapse images demonstrated that R. rickettsii organisms move through the cell cytoplasm at an average rate of 4.8 ± 0.6 μm/min (mean ± standard deviation). This speed was 2.5 times slower than that of L. monocytogenes, which moved at an average rate of 12.0 ± 3.1 μm/min. Although rickettsiae moved more slowly, the actin filaments comprising the actin comet tail were significantly more stable, with an average half-life approximately three times that of L. monocytogenes (100.6 ± 19.2 s versus 33.0 ± 7.6 s, respectively). The actin tail associated with intracytoplasmic rickettsiae remained stationary in the cytoplasm as the organism moved forward. In contrast, actin tails of rickettsiae trapped within the nucleus displayed dramatic movements. The observed phenotypic differences between the ABM of Listeria andRickettsia may indicate fundamental differences in the mechanisms of actin recruitment and polymerization.

scholarly journals Streamlined Embedding of Cell Monolayers on Gridded Glass-Bottom Imaging Dishes for Correlative Light and Electron Microscopy

2010 ◽  
Vol 16 (6) ◽  
pp. 747-754 ◽  
Author(s):  
Hugo H. Hanson ◽  
James E. Reilly ◽  
Rebecca Lee ◽  
William G. Janssen ◽  
Greg R. Phillips
Keyword(s):  
Electron Microscopy ◽  
Laser Scanning ◽  
Fluorescent Protein ◽  
Light And Electron Microscopy ◽  
Laser Scanning Confocal Microscopy ◽  
Step Method ◽  
Cell Monolayers ◽  
Scanning Confocal Microscopy ◽  
Transmission Electron ◽  
Green Fluorescent

AbstractCorrelative light and electron microscopy (CLEM) has facilitated study of intracellular trafficking. Routine application of CLEM would be advantageous for many laboratories but previously described techniques are particularly demanding, even for those with access to laser scanning confocal microscopy (LSCM) and transmission electron microscopy (TEM). We describe streamlined methods for TEM of green fluorescent protein (GFP)-labeled organelles after imaging by LSCM using gridded glass bottom imaging dishes. GFP-MAP 1A/1B LC3 (GFP-LC3) transfected cells were treated with rapamycin, fixed and imaged by LSCM. Confocal image stacks were acquired enabling full visualization of each GFP-LC3 labeled organelle. After LSCM, cells were embedded for TEM using a simplified two step method that stabilizes the glass bottom such that the block can be separated from the glass by mild heating. All imaging and TEM processing are performed in the same dish. The LSCM imaged cells were relocated on the block and serial sectioned. Correlation of LSCM, DIC, and TEM images was facilitated by cellular landmarks. All GFP labeled structures were successfully reidentified and imaged by serial section TEM. This method could make CLEM more accessible to nonspecialized laboratories with basic electron microscopy expertise and could be used routinely to confirm organelle localization of fluorescent puncta.

scholarly journals Quantitative confocal microscopy and calibration for measuring differences in cyclic-di-GMP signalling by bacteria on biomedical hydrogels

2021 ◽  
Vol 8 (1) ◽  
pp. 201453
Author(s):  
Jacob Blacutt ◽  
Ziyang Lan ◽  
Elizabeth M. Cosgriff-Hernandez ◽  
Vernita D. Gordon
Keyword(s):  
Confocal Microscopy ◽  
Laser Scanning ◽  
Fluorescent Protein ◽  
Laser Scanning Confocal Microscopy ◽  
Glycol Diacrylate ◽  
Scanning Confocal Microscopy ◽  
Light Passing ◽  
Gfp Reporter ◽  
Green Fluorescent ◽  
Biofilm Development

The growth of bacterial biofilms on implanted medical devices causes harmful infections and device failure. Biofilm development initiates when bacteria attach to and sense a surface. For the common nosocomial pathogen Pseudomonas aeruginosa and many others, the transition to the biofilm phenotype is controlled by the intracellular signal and second messenger cyclic-di-GMP (c-di-GMP). It is not known how biomedical materials might be adjusted to impede c-di-GMP signalling, and there are few extant methods for conducting such studies. Here, we develop such a method. We allowed P. aeruginosa to attach to the surfaces of poly(ethylene glycol) diacrylate (PEGDA) hydrogels. These bacteria contained a plasmid for a green fluorescent protein (GFP) reporter for c-di-GMP. We used laser-scanning confocal microscopy to measure the dynamics of the GFP reporter for 3 h, beginning 1 h after introducing bacteria to the hydrogel. We controlled for the effects of changes in bacterial metabolism using a promoterless plasmid for GFP, and for the effects of light passing through different hydrogels being differently attenuated by using fluorescent plastic beads as ‘standard candles’ for calibration. We demonstrate that this method can measure statistically significant differences in c-di-GMP signalling associated with different PEGDA gel types and with the surface-exposed protein PilY1.

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