62 research outputs found
New quick method for isolating RNA from laser captured cells stained by immunofluorescent immunohistochemistry; RNA suitable for direct use in fluorogenic TaqMan one-step real-time RT-PCR
We describe a new approach for reliably isolating one-step real-time quantitative RT-PCR-quality RNA from laser captured cells retrieved from frozen sections previously subjected to immunofluorescent immunohistochemistry (IF-IHC) and subsequently subjected to fluorogenic one-step real-time RT-PCR analysis without the need for costly, time-consuming linear amplification. One cell’s worth of RNA can now be interrogated with confidence. This approach represents an amalgam of technologies already offered commercially by Applied Biosystems, Arcturus and Invitrogen. It is the primary focus of this communication to expose the details and execution of an important new LCM RNA isolation technique, but also provide a detailed account of the IF-IHC procedure preceding RNA isolation, and provide information regarding our approach to fluorogenic one-step real-time RT-PCR in general. Experimental results shown here are meant to supplement the primary aim and are not intended to represent a complete scientific study. It is important to mention, that since LCM-RT-PCR is still far less expensive than micro-array analysis, we feel this approach to isolating RNA from LCM samples will be of continuing use to many researchers with limited budgets in the years ahead
High-resolution imaging of human atherosclerotic carotid plaques with micro18F-FDG PET scanning exploring plaque vulnerability
FDG-PET can be used to identify vulnerable plaques in atherosclerotic disease. Clinical FDG-PET camera systems are restricted in terms of resolution for the visualization of detailed inflammation patterns in smaller vascular structures. The aim of the study is to evaluate the possible added value of a high-resolution microPET system in excised carotid plaques using FDG. In this study, 17 patients with planned carotid endarterectomy were included. Excised plaques were incubated in FDG and subsequently imaged with microPET. Macrophage presence in plaques was evaluated semi-quantitatively by immunohistochemistry. Plaque calcification was assessed additionally with CT and correlated to FDG uptake. Finally, FDG uptake and macrophage infiltration were compared with patient symptomatology. Heterogeneous distributions and variable intensities of FDG uptake were found within the plaques. A positive correlation between the distribution of macrophages and the FDG uptake (r = 0.68, P <.01) was found. A negative correlation was found between areas of calcifications and FDG uptake (r = -0.84, P <.001). Ratio FDG(max) values as well as degree of CD68 accumulation were significantly higher in CVA patients compared with TIA or amaurosis fugax patients (P <.05) and CVA patients compared with asymptomatic patients (P <.05). This ex vivo study demonstrates that excised carotid plaques can be visualized in detail using FDG microPET. Enhancement of clinical PET/CT resolution for similar imaging results in patients is needed
Conidiation Color Mutants of Aspergillus fumigatus Are Highly Pathogenic to the Heterologous Insect Host Galleria mellonella
The greater wax moth Galleria mellonella has been widely used as
a heterologous host for a number of fungal pathogens including Candida
albicans and Cryptococcus neoformans. A positive
correlation in pathogenicity of these yeasts in this insect model and animal
models has been observed. However, very few studies have evaluated the
possibility of applying this heterologous insect model to investigate virulence
traits of the filamentous fungal pathogen Aspergillus
fumigatus, the leading cause of invasive aspergillosis. Here, we have
examined the impact of mutations in genes involved in melanin biosynthesis on
the pathogenicity of A. fumigatus in the G.
mellonella model. Melanization in A. fumigatus confers
bluish-grey color to conidia and is a known virulence factor in mammal models.
Surprisingly, conidial color mutants in B5233 background that have deletions in
the defined six-gene cluster required for DHN-melanin biosynthesis caused
enhanced insect mortality compared to the parent strain. To further examine and
confirm the relationship between melanization defects and enhanced virulence in
the wax moth model, we performed random insertional mutagenesis in the Af293
genetic background to isolate mutants producing altered conidia colors. Strains
producing conidia of previously identified colors and of novel colors were
isolated. Interestingly, these color mutants displayed a higher level of
pathogenicity in the insect model compared to the wild type. Although some of
the more virulent color mutants showed increased resistance to hydrogen
peroxide, overall phenotypic characterizations including secondary metabolite
production, metalloproteinase activity, and germination rate did not reveal a
general mechanism accountable for the enhanced virulence of these color mutants
observed in the insect model. Our observations indicate instead, that
exacerbated immune response of the wax moth induced by increased exposure of
PAMPs (pathogen-associated molecular patterns) may cause self-damage that
results in increased mortality of larvae infected with the color mutants. The
current study underscores the limitations of using this insect model for
inferring the pathogenic potential of A. fumigatus strains in
mammals, but also points to the importance of understanding the innate immunity
of the insect host in providing insights into the pathogenicity level of
different fungal strains in this model. Additionally, our observations that
melanization defective color mutants demonstrate increased virulence in the
insect wax moth, suggest the potential of using melanization defective mutants
of native insect fungal pathogens in the biological control of insect
populations
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