Flow cytometry and FACS applied to filamentous fungi

Flow cytometry is an automated, laser- or impedance-based, high throughput method that allows very rapid analysis of multiple chemical and physical characteristics of single cells within a cell population. It is an extremely powerful technology that has been used for over four decades with filamentous fungi. Although single cells within a cell population are normally analysed rapidly on a cell-by-cell basis using the technique, flow cytometry can also be used to analyse cell (e.g. spore) aggregates or entire microcolonies. Living or fixed cells can be stained with a wide range of fluorescent reporters to label different cell components or measure different physiological processes. Flow cytometry is also suited for measurements of cell size, interaction, aggregation or shape using non-labelled cells by means of analysing their light scattering characteristics. Fluorescence-activated cell sorting (FACS) is a specialized form of flow cytometry that provides a method for sorting a heterogeneous mixture of cells into two or more containers based upon the fluorescence and/or light scattering properties of each cell. The major advantage of analysing cells by flow cytometry over microscopy is the speed of analysis: thousands of cells can be analysed per second or sorted in minutes. Drawbacks of flow cytometry are that specific cells cannot be followed in time and normally spatial information relating to individual cells is lacking. A big advantage over microscopy is when using FACS, cells with desired characteristics can be sorted for downstream experimentation (e.g. for growth, infection, enzyme production, gene expression assays or ‘omics’ approaches). In this review, we explain the basic concepts of flow cytometry and FACS, define its advantages and disadvantages in comparison with microscopy, and describe the wide range of applications in which these powerful technologies have been used with filamentous fungi.     

Smoking in Relation to Coronary Atherosclerotic Plaque Burden,Volume and Composition on Intravascular Ultrasound

Background

This study aimed to evaluate the relationship between cigarette smoking and coronary atherosclerotic burden, volume and composition as determined in-vivo by grayscale and virtual histology (VH) intravascular ultrasound (IVUS).

Methods and Results

Between 2008 and 2011, (VH-)IVUS of a non-culprit coronary artery was performed in 581 patients undergoing coronary angiography. To account for differences in baseline characteristics, current smokers were matched to never smokers by age, gender and indication for catheterization, resulting in 280 patients available for further analysis. Coronary atherosclerotic plaque volume, burden, composition (fibrous, fibro-fatty, dense calcium and necrotic core) and high-risk lesions (VH-IVUS derived thin-cap fibroatheroma (TCFA), plaque burden ≥70%, minimal luminal area ≤4.0 mm²) were assessed. Cigarette smoking showed a tendency towards higher coronary plaque burden (mean±SD, 38.6±12.5% in current versus 36.4±11.0% in never smokers, p = 0.080; and odds ratio (OR) of current smoking for plaque burden above versus below the median 1.69 (1.04–2.75), p = 0.033). This effect was driven by an association in patients presenting with an acute coronary syndrome (ACS) (current smokers, plaque burden 38.3±12.8% versus never smokers, plaque burden 35.0±11.2%, p = 0.049; OR 1.88 (1.02–3.44), p = 0.042). Fibrous tissue tended to be lower in current smokers (mean±SD, 57.7±10.5% versus 60.4±12.6%, p = 0.050) and fibro-fatty tissue was higher in current smokers (median[IQR], 9.6[6.0–13.7]% versus 8.6[5.8–12.2]%, p = 0.039). However, differences in percentage necrotic core and dense calcium could not be demonstrated. Also, no differences were found with regard to high-risk lesions.

Conclusions

An association between smoking and degree of coronary atherosclerosis was present in patients undergoing coronary angiography who presented with ACS. Although smoking was associated with higher fibro-fatty percentage, no associations could be demonstrated with percentage necrotic core, nor with VH-IVUS derived TCFA lesions. Since the magnitude of the differences in both degree and composition of atherosclerosis was modest, clinical relevance of the findings may be questioned.     

Rethinking counselling in prenatal screening: An ethical analysis of informed consent in the context of non-invasive prenatal testing (NIPT)

Informed consent is a key condition for prenatal screening programmes to reach their aim of promoting reproductive autonomy. Reaching this aim is currently being challenged with the introduction of non-invasive prenatal testing (NIPT) in first-trimester prenatal screening programmes: amongst others its procedural ease—it only requires a blood draw and reaches high levels of reliability—might hinder women’s understanding that they should make a personal, informed decision about screening. We offer arguments for a renewed recognition and use of informed consent compared to informed choice, and for a focus on value-consistent choices and personalized informational preferences. We argue for a three-step counselling model in which three decision moments are distinguished and differently addressed: (1) professionals explore women’s values concerning whether and why they wish to know whether their baby has a genetic disorder; (2) women receive layered medical-technical information and are asked to make a decision about screening; (3) during post-test counselling, women are supported in decision-making about the continuation or termination of their pregnancy. This model might also be applicable in other fields of genetic (pre-test) counselling, where techniques for expanding genome analysis and burdensome test-outcomes challenge counselling of patients.     

Binding of radiation-induced phenylalanine radicals to DNA: influence on the biological activity of the DNA and on its sensitivity to the induction of breaks by gamma-rays.

When an aqueous solution of double-stranded DNA of bacteriophage PM2 containing phenylalanine and saturated with N2O is irradiated with gamma-rays, radiation-induced phenylalanine radicals are bound covalently. Under the conditions used, about 25 phenylalanine molecules may be bound per lethal hit. For single-stranded PM2 DNA, most of the phenylalanine radicals bound are non-lethal. Evidence is presented that, in double-stranded DNA, an appreciable fraction of the single-strand breaks is induced by phenylalanine radicals. Radiation products of phenylalanine and the phenylalanine bound to the DNA decrease the sensitivity of the DNA to the induction of single-strand breaks. There are indications that the high efficiency of protection by radiation products of phenylalanine is due to their positive charge, which will result in a relatively high concentrations fo these compounds in the vicinity of the negatively-charged DNA molecules.     

Bacterial luciferase reporters: the Swiss army knife of molecular biology

Bioluminescence is a process during which light in the visible spectrum is emitted as a consequence of an enzymatic reaction catalyzed by luciferases. Luciferases have been identified mainly in marine organisms and are used for several biological purposes include camouflage, repulsion, attraction, communication and illumination. Some of the currently known luciferases have become indispensible tools in modern molecular biology and are used for diverse applications such as autoinducer-1 activity assays, promoter test assays in both prokaryotes and eukaryotes, imaging of bacterial infections in live animals, in vivo activity assays genes involved in host response and disease and monitoring of bacterial contaminations of food products. With the present review, the authors intend to give an overview on the currently used bacterial luciferase reporter systems, their methodologies and applications and compare them to other reporter systems.     

Influence of SOS repair on the specificity of radiation mutagenesis in bacteriophage ?X174

Summary The ochre mutant oc9 of bacteriophage X174 was irradiated with -rays and the revertants were assayed on unirradiated and UV-irradiated host bacteria carrying an amber suppressor. The yield of revertants (amber+wild type) was higher on UV-irradiated than on unirradiated bacteria, showing that -irradiated X174 was subjected to W-mutagenesis.For oc9 -irradiated in the presence of oxygen the fraction of amber mutants among the revertants was lower when mutants were scored on UV-irradiated bacteria than when assayed on unirradiated indicator cells. The same fraction of ambers was obtained when mutants were assayed on unirradiated and UV-irradiated samples of a recA indicator strain. UV-irradiated X174 showed a similar phenomenon. These results suggest that the specificity with regard to insertion of bases opposite radiation damage in X174 DNA is different for host cells in which SOS repair has been induced and cells in which SOS repair is not operative.     

18F-2-deoxy-2-fluoro-D-glucose positron emission tomography, computed tomography, and magnetic resonance imaging for the detection of experimental colorectal liver metastases

During the treatment of colorectal liver metastases, evaluation of treatment efficacy is of the utmost importance for decision making. The aim of the present study was to explore the ability of preclinical imaging modalities to detect experimental liver metastases. Nine male Wag/Rij rats underwent a laparotomy with intraportal injection of CC531 tumor cells. On days 7, 10, and 14 after tumor induction, sequential positron emission tomography (PET), computed tomography (CT), and magnetic resonance imaging (MRI) scans were acquired of each rat. At each time point, three rats were euthanized and the metastases in the liver were documented histologically. Topographically, the liver was divided into eight segments and the image findings were compared on a segment-by-segment basis with the histopathologic findings. Sixty-four liver segments were analyzed, 20 of which contained tumor deposits. The overall sensitivity of PET, CT, and MRI was 30%, 25%, and 20%, respectively. For the detection of tumors with a histologic diameter exceeding 1 mm (n = 8), the sensitivity of PET, CT, and MRI was 63%, 38%, and 38%, respectively. The overall specificity of PET, CT, and MRI was 98%, 100%, and 93%, respectively. This study showed encouraging detectability and sensitivity for preclinical imaging of small liver tumors and provides valuable information on the imaging techniques for designing future protocols.