“Decoding the Dots”: The ImageStream system (ISS) as a novel and powerful tool for flow cytometric analysis

The aim of this article is to provide a brief review of the ImageStream system (ISS). The ISS technology was developed as a novel method for multiparameter cell analysis and subsequently as a supportive tool for flow cytometry (FC). ISS integrates the features of FC and fluorescent microscopy collecting images of acquired cells for offline digital image analysis. The article presents an overview of the main characteristics of ISS and a comparison between ISS, FC and the laser scanning cytometer (LSC). We reviewed ISS applications focusing on those involved in cellular phenotyping and provide our own experience with using ISS as a supportive tool to classical FC and demonstrate the compatibility between FC and ISS photometric analysis as well as the advantages of using ISS to confirm FC results.     

Influence of dam removal on trichopteran assemblages in the lowland Drzewiczka River,Poland

The aim of this study is to assess the response of Trichoptera in a patchy tailwater stretch of the Drzewiczka River (Poland) to dam removal as compared to the previous pulse discharge disturbance. The study was carried out in the river at the end of a whitewater slalom canoeing track located downstream of the dam reservoir called Drzewieckie Lake (area 0.84 km²). Between February and April 2002, the dam reservoir was gradually emptied before its dredging, and the Drzewiczka River recovered its natural discharge (1.7–4.5 m³ s⁻¹) for several years (temporary renaturisation). Altogether, 120 monthly samples of macroinvertebrates and their environments were collected in two sampling cycles, S1 in 2000–2001 during high discharge fluctuations (2.1–12.0 m³ s⁻¹ daily) and S2 in 2002–2003 during renaturisation after emptying the reservoir, from the following five habitats: H_P—pool habitat, H_S—stagnant habitat, H_M—macrophyte habitat, H_B—bank habitat and H_R—riffle habitat. On the basis of trichopteran abundance, the patterns in their assemblages were recognised with use of a self-organising map that was a Kohonen artificial neural network. The obtained classification of trichopteran samples was found to be based in general on the spatial criterion, i.e. dependent on habitats, irrespective of which sampling cycle the samples originated from, which clearly showed that the habitat mosaic of the river bed was observed both in S1 and S2. In addition, a very important function was noted for riparian and land plants, which developed intensively at the bottom of the Drzewieckie Reservoir immediately after it was emptied. They restricted extensive transport of reservoir sediments to the downstream river reach, thus reducing differences between S1 and S2 in the tailwater. It is also worth noting that in S1, in comparison to S2, Cyrnus trimaculatus, Mystacides azurea and Lype reducta flowing downstream from the reservoir were observed more frequently in the river, which confirmed that impoundments can be conducive to the presence of certain species downstream of dams. Summing up, although artificial short-term flow fluctuations usually diminish the quality and quantity of benthos, the article presents a case in which they were small and short enough to allow the formation of a mosaic of bed patches and positively affected certain parameters of macrobenthic communities according to the intermediate disturbance hypothesis. Handling editor: David Dudgeon     

Evaluation of the potential role of water in spread of conidia of the Neotyphodium endophyte of Poa ampla

Neotyphodium endophytes are asexual, filamentous fungi, mutualistically associated with diverse cool season grasses. Infected seeds and vegetative organs of infected host plants are the only known modes of propagation of the asexual endophytes. In the last decade certain Epichloë and Neotyphodium-infected grass species have been shown to have epiphyllous structures of the endophytes, hyphae, conidiophores, and conidia, growing on leaf blades. The production of epiphyllous conidia suggests the possibility that some of these endophytes may have the ability for plant-to-plant spread. The objective of this study was to determine the possible mechanisms involved in liberation and dispersal of asexual spores of Neotyphodium growing in vitro and epiphyllously on leaves of Poa ampla. Our results indicate that water dispersal is the most likely means of dissemination of conidia of the Neotyphodium sp. Wind generated by dry compressed air does not dislodge the conidia from slide cultures or from P. ampla leaves.     

Sex-specific fledgling success and brood sex ratio in the Great Reed Warbler (Acrocephalus arundinaceus)

We examine sex ratio variation and sex-specific probability of successful fledgling in relation to hatching date across 376 broods of Great Reed Warblers (Acrocephalus arundinaceus). The sex ratio of complete broods as well as broods with partial mortality did not deviate significantly from parity (0.5 and 0.53, respectively). Variation in sex ratio between broods was not larger than expected from binomial distribution, thus females seem not to manipulate the sex ratio of their broods in the studied population. As a consequence, sex ratio did not vary in relation to hatching date, years and fishponds. Female offspring showed lower fledgling success than their brothers, but the relationship between probability of successful fledgling and hatching date differed between sexes. Fledgling success of female offspring declined with hatching date more strongly than the success of male offspring. Thus, our study shows that Great Reed Warblers do not adjust offspring sex to match observed seasonal sex-specific variation in survival.     

The hydromechanics of hydrocephalus: Steady-state solutions for cylindrical geometry

Hydrocephalus is a state in which the circulation of cerebrospinal fluid is disturbed. This fluid, produced within the brain at a constant rate, moves through internal cavities in it (ventricles), then exits through passages so that it may be absorbed by the surrounding membranes (meninges). Failure of fluid to move properly through these passages results in the distention of the passages and the ventricles. Ultimately, this distention causes large displacements and distortion of brain tissue as well as an increase of fluid in the extracellular space of the brain (edema). We use a two-phase model of fluid-saturated material to simulate the steady state of the hydrocephalic brain. Analytic solutions for the displacement of brain tissue and the distribution of edema for the annular regions of an idealized cylindrical geometry and small-strain theory are found. The solutions are used for a large-deformation analysis by superposition of the responses obtained for incrementally increasing loading. The effects of structural and hydraulic differences of white and gray brain matter, and the ependymal lining surrounding the venticles, are examined. The results reproduce the characteristic steady-state distribution of edema seen in hydrocephalus, and are compared with experiment.     

Triple immunofluorescence staining with antibodies raised in the same species to study the complex innervation pattern of intrapulmonary chemoreceptors.

A general problem in immunocytochemistry is the development of a reliable multiple immunolabeling method when primary antibodies must be used that originate in the same species. We have developed a protocol for the immunodetection of three antigens in a single tissue preparation, using unconjugated primary antibodies raised in the same species. Immunocytochemical detection of neuronal nitric oxide synthase, calcitonin gene-related peptide, and calbindin D28k in the lung of rats demonstrated that part of the pulmonary neuroepithelial bodies are selectively contacted by at least three different nerve fiber populations. The first antigen was detected using tyramide signal amplification, a very sensitive method allowing a dilution of the first primary antibody far beyond the detection limit of fluorescently labeled secondary antibodies. The second antigen was visualized by a fluorophore-conjugated secondary monovalent Fab antibody that at the same time blocks the access of the third secondary antibody to the second primary antibody. Moreover, the monovalence of the Fab fragment prevents the third primary antibody from binding with the second-step secondary antibody. The triple staining technique described here is generally applicable, uses commercially available products only, and allows the detection of three antigens in the same preparation with primary antibodies that are raised in the same species.     

Chronobiologic Evaluation of Angiotensin-Converting Enzyme Activity in Serum and Lung Tissue from Normotensive and Spontaneously Hypertensive Rats

Angiotensin-converting enzyme (ACE) activity in serum and lung tissue from both normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR) was determined at six different circadian times. In WKY rats serum ACE varied significantly within 24 h, mainly due to reduced enzyme activity at 12:00 h. In SHR the 24-h profile of serum ACE did not exhibit time-dependent differences. Mean serum ACE activity over 24 h was significantly higher in WKY than in SHR. In lung tissue ACE activity did not depend on the circadian time in either strain. Mean enzyme activity in lung tissue was not different between WKY and SHR. We conclude that circadian changes in the activity of serum and tissue ACE are unlikely to play an important role in the regulation of the circadian blood pressure profile in both normotensive and spontaneously hypertensive rats.     

Presence of cna, emp and pls genes and pathogenicity of methicillin-resistant Staphylococcus aureus strains

Methicillin-resistant strains of Staphylococcus aureus (MRSA) are important etiological factors responsible for hospital-acquired infections. The aim of this study was to analyze the influence of the presence of emp, pls and cna genes on the pathogenicity of MRSA strains. The presence of these genes was tested by PCR in 302 MRSA strains isolated from hospitalized patients and from carriers. For each tested gene, proportions of positive and negative strains were similar among the infected patients and carriers. We did not find any obvious correlation between the presence of the three tested genes and the infectivity of strains. Our results may also suggest that a lack of emp and presence of pls may correlate with reduced virulence of these strains.