scholarly journals Labeling of Monilinia fructicola with GFP and Its Validation for Studies on Host-Pathogen Interactions in Stone and Pome Fruit

Genes ◽  
2019 ◽  
Vol 10 (12) ◽  
pp. 1033
Author(s):  
Silvia Rodríguez-Pires ◽  
Eduardo Antonio Espeso ◽  
Nuria Baró-Montel ◽  
Rosario Torres ◽  
Paloma Melgarejo ◽  
...  

To compare in vivo the infection process of Monilinia fructicola on nectarines and apples using confocal microscopy it is necessary to transform a pathogenic strain with a construct expressing a fluorescent chromophore such as GFP. Thus, germinated conidia of the pathogen were transformed with Agrobacterium tumefaciens carrying the plasmid pPK2-hphgfp that allowed the expression of a fluorescent Hph-GFP chimera. The transformants were selected according to their resistance to hygromycin B, provided by the constitutive expression of the hph-gfp gene driven by the glyceraldehyde 3P dehydrogenase promoter of Aspergillus nidulans. The presence of T-DNA construct in the genomic DNA was confirmed by PCR using a range of specific primers. Subsequent PCR-mediated analyses proved integration of the transgene at a different genomic location in each transformant and the existence of structural reorganizations at these insertion points. The expression of Hph-GFP in three independent M. fructicola transformants was monitored by immunodetection and epifluorescence and confocal microscopy. The Atd9-M. fructicola transformant displayed no morphological defects and showed growth and pathogenic characteristics similar to the wild type. Microscopy analysis of the Atd9 transformant evidenced that nectarine infection by M. fructicola was at least three times faster than on apples.

1991 ◽  
Vol 112 (3) ◽  
pp. 385-395 ◽  
Author(s):  
R M Lynch ◽  
K E Fogarty ◽  
F S Fay

Hexokinase isozyme I is proposed to be associated with mitochondria in vivo. Moreover, it has been suggested that this association is modulated in coordination with changes in cell metabolic state. To test these hypotheses, we analyzed the subcellular distribution of hexokinase relative to mitochondria in paraformaldehyde-fixed astrocytes using immunocytochemistry and quantitative three-dimensional confocal microscopy. Analysis of the extent of colocalization between hexokinase and mitochondria revealed that approximately 70% of cellular hexokinase is associated with mitochondria under basal metabolic conditions. In contrast to the immunocytochemical studies, between 15 to 40% of cellular hexokinase was found to be associated with mitochondria after fractionation of astrocyte cultures depending on the exact fractionation conditions. The discrepancy between fractionation studies and those based on imaging of distributions in fixed cells indicates the usefulness of using techniques that can evaluate the distributions of "cytosolic" enzymes in cells whose subcellular ultrastructure is not severely disrupted. To determine if hexokinase distribution is modulated in concert with changes in cell metabolism, the localization of hexokinase with mitochondria was evaluated after inhibition of glucose metabolism with 2-deoxyglucose. After incubation with 2-deoxyglucose there was an approximate 35% decrease in the amount of hexokinase associated with mitochondria. These findings support the hypothesis that hexokinase is bound to mitochondria in rat brain astrocytes in vivo, and that this association is sensitive to cell metabolic state.


2019 ◽  
Vol 2019 ◽  
pp. 1-15
Author(s):  
Zi-cong Wu ◽  
Qiang Xue ◽  
Zhen-ling Zhao ◽  
Peng-jun Zhou ◽  
Qun Zhou ◽  
...  

Background. Huzhentongfeng (HZTF) is an extract from four Chinese medical herbs for treating gout. This study aims to evaluate its antigout activity and preliminary explore its mechanism in vivo and in vitro. Methods. The rats were intragastrically administered with HZTF for 5 days and then injected 0.1 ml (10 mg) of MSU crystals to their joints for generating a gout model to analyze the paw volume and histopathology of joint synovial tissues of rats with different doses. We also investigated the antioxidant capacity of HZTF in vitro using indication including lipid peroxidation, DPPH·, and ABTS+ radical-scavenging capacity; besides, we used qRT-PCR to measure the effect of HZTF on interleukin (IL)-1β, caspase-1, NLRP3, and NQO1 expression in hydrogen peroxide-stimulated RAW264.7 macrophages and IL-1β, IL-6, and tumor necrosis factor (TNF)-α in MSU crystal-induced THP-1 monocytes. Confocal microscopy analysis was used to observe the dimerization of ASC adapter proteins. In addition, we also established quality standard of HZTF by using the high-performance liquid chromatography (HPLC) method. Results. HZTF could significantly suppress the paw swelling and neutrophil infiltration induced by MSU intra-articular injection in rats compared with the control group. HZTF also showed inhibition effects of inflammatory cytokines (IL-1β, IL-6, and TNF-α) secretion at 25.00 and 50.00 μg/ml in MSU-induced THP-1 cells but showed no effects of IL-1β, IL-6, and TNF-α mRNA expression in MSU-induced THP-1 cells. Furthermore, confocal microscopy analysis showed that HZTF could prevent the oligomerization of ASC. Moreover, HZTF also showed effects in cell-free and cell-base tests of antioxidant capacity. Conclusion. The results prove that HZTF possessed the potential preventive effect against gout arthritis, and the effect may be attributed to its preventing effect on neutrophil infiltration and proinflammatory cytokines secretion such as IL-1β, IL-6, and TNF-α which were caused by the activation of inflammasome.


2020 ◽  
Vol 17 (4) ◽  
pp. 725-732
Author(s):  
S. V. Trufanov ◽  
A. V. Zaitsev ◽  
N. P. Shakhbazyan

Purpose: to study the combined Photo-Activated Chromophore for Keratitis — Corneal Cross-Linking (PACK-CXL) in combination with fulguration of the infiltration zone in the treatment of medically refractive acanthamoebic keratitis.Patients and methods. The study included 9 patients (10 eyes) with medically refractive acanthamoebic keratitis. The diagnosis was confirmed by confocal microscopy data from a microbiological study of scraping of corneal tissue from the lesion site with Romanovsky-Giemsa stain. All patients underwent combined surgical treatment of PACK-CXL with pre-fulguration. Optical coherence tomography (OCT) of the anterior segment of the eye was also performed using an RTVue-100 apparatus (Optovue USA), determination of visual acuity, photographing before and after surgery.Results. In 6 cases (60 %), a positive effect was noted, relief of the symptoms of the disease and the formation of turbidity within a month after the procedure, as well as an increase in the maximum corrected visual acuity. According in vivo confocal microscopy, 6 months after the intervention, no signs of infection were detected. In 4 cases, the therapeutic effect was absent. Subsequently, 3 patients (3 eyes) underwent therapeutic keratoplasty. In one eye, the infectious process was stopped medically for 6 months.Conclusion. The combined PACK-CXL method together with fulguration can be effective and safe in the treatment of medically refractive acanthamoebic keratitis, allowing keratoplasty to be performed with an optical goal if necessary, after stopping the infection process in a distant period. 


2007 ◽  
Vol 35 (6) ◽  
pp. 582-585 ◽  
Author(s):  
Gianluca Martone ◽  
Fabrizio Casprini ◽  
Claudio Traversi ◽  
Francesca Lepri ◽  
Patrizia Pichierri ◽  
...  

2016 ◽  
Vol 40 (6) ◽  
pp. 317-323 ◽  
Author(s):  
Vincenzo Grassia ◽  
Enrica Gentile ◽  
Dario Di Stasio ◽  
Abdolreza Jamilian ◽  
Giovanni Matarese ◽  
...  

2009 ◽  
Vol 147 (4) ◽  
pp. 725-735.e1 ◽  
Author(s):  
Gianluca Martone ◽  
Paolo Frezzotti ◽  
Gian Marco Tosi ◽  
Claudio Traversi ◽  
Vincenzo Mittica ◽  
...  

2018 ◽  
Vol 48 (6) ◽  
pp. 276-280 ◽  
Author(s):  
Sevgi Subaşı ◽  
Nurşen Yüksel ◽  
Müge Toprak ◽  
Büşra Yılmaz Tuğan

Sign in / Sign up

Export Citation Format

Share Document