Characterization of NGF, trkANGFR, and p75NTRin Retina of Mice Lacking Reelin Glycoprotein

Both Reelin and Nerve Growth Factor (NGF) exert crucial roles in retinal development. Retinogenesis is severely impaired inE-reelermice, a model of Reelin deficiency showing specific Green Fluorescent Protein expression in Rod Bipolar Cells (RBCs). Since no data are available on Reelin and NGF cross-talk, NGF andtrkANGFR/p75NTRexpression was investigated in retinas fromE-reelerversus control mice, by confocal microscopy, Western blotting, and real time PCR analysis. A scattered increase of NGF protein was observed in the Ganglion Cell Layer and more pronounced in the Inner Nuclear Layer (INL). A selective increase ofp75NTRwas detected in most of RBCs and in other cell subtypes of INL. On the contrary, a slight trend towards a decrease was detected fortrkANGFR, albeit not significant. Confocal data were validated by Western blot and real time PCR. Finally, the decreasedtrkANGFR/p75NTRratio, representative ofp75NTRincrease, significantly correlated withE-reelerversus E-control. These data indicate that NGF-trkANGFR/p75NTRis affected inE-reelerretina and thatp75NTRmight represent the main NGF receptor involved in the process. This first NGF-trkANGFR/p75NTRcharacterization suggests thatE-reelermight be suitable for exploring Reelin-NGF cross-talk, representing an additional information source in those pathologies characterized by retinal degeneration.

Download Full-text

Parasite Burden Measurement in the Leishmania major Infected Mice by Using the Direct Fluorescent Microscopy, Limiting Dilu-tion Assay, and Real-Time PCR Analysis

Iranian Journal of Parasitology ◽

10.18502/ijpa.v15i4.4867 ◽

2020 ◽

Author(s):

Sepideh HAGHDOUST ◽

Mahdieh AZIZI ◽

Mostafa HAJI MOLLA HOSEINI ◽

Mojgan BANDEHPOUR ◽

Mandana MOHSENI MASOOLEH ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Leishmania Major ◽

Fluorescent Protein ◽

Fluorescent Microscopy ◽

Parasite Burden ◽

Pcr Analysis ◽

Green Fluorescent ◽

Limiting Dilution

Background: We aimed to compare parasite burden in BALB/c mice, using three methods including the direct fluorescent microscopic using recombinant Leishmania major expressing an enhanced green fluorescent protein, limiting dilution assay, and real-time PCR technique. Methods: The current study was carried out in 2018, to induce stable enhanced green fluorescent protein (EGFP) production. Initially, the linearized DNA expression cassette (pLEXSY-egfp-sat2) was integrated into the ssu locus of L. major. The expression of EGFP in recombinant parasite was analyzed using direct fluorescent microscopy. Afterward, BALB/c mice were infected with the L. majorEGFP, and the infection was evaluated in the foot-pads and inguinal lymph-nodes using an in vivo imaging system. Subsequently, eight BALB/c mice were infected with L. majorEGFP, and the results of evaluating parasite burden by a SYBR-Green based real-time PCR analysis and the limiting dilution assays were compared to the results obtained from the direct fluorescent microscopy. Results: The results of the direct fluorescent microscopy showed that EGFP gene stably was expressed in parasites. Moreover, the in vivo imaging analysis of foot-pad lesions revealed that the infection caused by L. majorEGFP was progressing over time. Additionally, significant correlations were observed between the results of parasite burden assay using the direct fluorescent microscopy and either limiting dilution assay (r=0.976, P<0.0001) or quantitative real-time PCR assay (r=0.857, P<0.001). Conclusion: Ultimately, the utilization of the direct fluorescent microscopy by employing a stable EGFP-expressing L. major is a suitable substitution for the existing methods to quantify parasite burden.

Download Full-text

Quantification of green fluorescent protein fluorescence using real-time PCR thermal cycler

BioTechniques ◽

10.2144/000112221 ◽

2006 ◽

Vol 41 (2) ◽

pp. 150-154 ◽

Cited By ~ 5

Author(s):

Jan Utermark ◽

Petr Karlovsky

Keyword(s):

Green Fluorescent Protein ◽

Real Time ◽

Real Time Pcr ◽

Fluorescent Protein ◽

Green Fluorescent Protein Fluorescence ◽

Protein Fluorescence ◽

Thermal Cycler ◽

Green Fluorescent

Download Full-text

Rapid Screening Method for Compounds That Affect the Growth and Germination of Candida albicans, Using a Real-Time PCR Thermocycler

Applied and Environmental Microbiology ◽

10.1128/aem.06227-11 ◽

2011 ◽

Vol 77 (22) ◽

pp. 8193-8196 ◽

Cited By ~ 6

Author(s):

Lucja M. Jarosz ◽

Bastiaan P. Krom

Keyword(s):

Candida Albicans ◽

Real Time ◽

Real Time Pcr ◽

Fluorescent Protein ◽

Screening Method ◽

Rapid Screening ◽

Content Type ◽

Wide Range ◽

Hyphal Formation ◽

Green Fluorescent

ABSTRACTWe propose a screening method for compounds affecting growth and germination inCandida albicansusing a real-time PCR thermocycler to quantify green fluorescent protein (GFP) fluorescence. Using PACT1-GFPand PHWP1-GFPreporter strains, the effects of a wide range of compounds on growth and hyphal formation were quantitatively assessed within 3 h after inoculation.

Download Full-text

Real-time PCR quantification of a green fluorescent protein-labeled, genetically engineeredPseudomonas putidastrain during 2-chlorobenzoate degradation in soil

FEMS Microbiology Letters ◽

10.1111/j.1574-6968.2004.tb09497.x ◽

2004 ◽

Vol 233 (2) ◽

pp. 307-314 ◽

Cited By ~ 27

Author(s):

Gejiao Wang ◽

Terry J Gentry ◽

Gregor Grass ◽

Karen Josephson ◽

Christopher Rensing ◽

...

Keyword(s):

Green Fluorescent Protein ◽

Real Time ◽

Real Time Pcr ◽

Fluorescent Protein ◽

Green Fluorescent ◽

Degradation In Soil

Download Full-text

Vibrio harveyi Adheres to and Penetrates Tissues of the European Abalone Haliotis tuberculata within the First Hours of Contact

Applied and Environmental Microbiology ◽

10.1128/aem.01036-14 ◽

2014 ◽

Vol 80 (20) ◽

pp. 6328-6333 ◽

Cited By ~ 18

Author(s):

Marion Cardinaud ◽

Annaïck Barbou ◽

Carole Capitaine ◽

Adeline Bidault ◽

Antoine Marie Dujon ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Fluorescent Protein ◽

Vibrio Harveyi ◽

Bacterial Pathogen ◽

Content Type ◽

Infection Dynamics ◽

Haliotis Tuberculata ◽

Green Fluorescent ◽

Portal Of Entry

ABSTRACTVibrio harveyiis a marine bacterial pathogen responsible for episodic epidemics generally associated with massive mortalities in many marine organisms, including the European abaloneHaliotis tuberculata. The aim of this study was to identify the portal of entry and the dynamics of infection ofV. harveyiin the European abalone. The results indicate that the duration of contact betweenV. harveyiand the European abalone influences the mortality rate and precocity. Immediately after contact, the epithelial and mucosal area situated between the gills and the hypobranchial gland was colonized byV. harveyi. Real-time PCR analyses and culture quantification of a green fluorescent protein-tagged strain ofV. harveyiin abalone tissues revealed a high density of bacteria adhering to and then penetrating the whole gill-hypobranchial gland tissue after 1 h of contact.V. harveyiwas also detected in the hemolymph of a significant number of European abalones after 3 h of contact. In conclusion, this article shows that a TaqMan real-time PCR assay is a powerful and useful technique for the detection of a marine pathogen such asV. harveyiin mollusk tissue and for the study of its infection dynamics. Thus, we have revealed that the adhesion and then the penetration ofV. harveyiin European abalone organs begin in the first hours of contact. We also hypothesize that the portal of entry ofV. harveyiin the European abalone is the area situated between the gills and the hypobranchial gland.

Download Full-text