Variation in heat-shock proteins among species of desert fishes (Poeciliidae, Poeciliopsis)

Analysis of the heat-shock proteins (hsps) of six closely related species of Poeciliopsis demonstrated the existence of biochemical diversity in the hsp100, hsp70, hsp60, and hsp30 protein families among species. Each species expressed five to seven hsp70-related isoforms. Constitutive 70-kD isoforms were identical among species, but four different patterns of heat-inducible isoforms were seen in these six species. Members of the hsp70 family of molecular chaperones are included among the most highly conserved proteins known, and the possibility of variation in hsp70 among closely related species has rarely been addressed. The hsp30 family is known to be less conserved than the hsp70 family, and, as expected, the Poeciliopsis hsp30 patterns showed more variation. Most of the hsp30 isoforms characteristic of a particular species were unique to that species. Hsp100 and hsp60 were identical in five of the species, but alternate isoforms were found in P. monacha. The small size and limited geographical distribution of the P. monacha population have probably contributed to the uniqueness of the monacha pattern. Two of the species were shown to acquire thermotolerance, the ability to withstand normally lethal temperatures when subjected to a gradual temperature increase. Rapid-heating protocols commonly used to establish critical thermal maxima of organisms do not include this inducible component of thermoresistance and therefore do not adequately assess an organism's capacity to withstand thermal stress.      

Ciliate–adenovirus interactions in experimental co-cultures of Euplotes octocarinatus and in wastewater environment

The aim of the present work was to study the dynamics of the interactions between human adenovirus and ciliates under both experimental and field conditions. Experimental co-cultures of the ciliated protozoan Euplotes octocarinatus and human adenovirus (HAdV) type 2 were established and virus internalization was investigated using nested PCR and direct immunofluorescence (IF). In addition, to study protozoa-virus interactions in the field, wild ciliates were isolated from active sludges of a wastewater treatment plant and analyzed for the presence of adenovirus using direct IF. In vitro experiments revealed HAdV type 2 inside Euplotes cells after 15 min of contact and its persistence until at least 35 days post infection. In addition, our results showed the adsorption of adenovirus on the surface of wild ciliates. We conclude that HAdV is taken up by ciliates, however more studies are necessary in order to better investigate the mechanisms, the infectivity of internalized virus and the protective effects of internalization against disinfection.     

Link Prediction in Criminal Networks: A Tool for Criminal Intelligence Analysis

The problem of link prediction has recently received increasing attention from scholars in network science. In social network analysis, one of its aims is to recover missing links, namely connections among actors which are likely to exist but have not been reported because data are incomplete or subject to various types of uncertainty. In the field of criminal investigations, problems of incomplete information are encountered almost by definition, given the obvious anti-detection strategies set up by criminals and the limited investigative resources. In this paper, we work on a specific dataset obtained from a real investigation, and we propose a strategy to identify missing links in a criminal network on the basis of the topological analysis of the links classified as marginal, i.e. removed during the investigation procedure. The main assumption is that missing links should have opposite features with respect to marginal ones. Measures of node similarity turn out to provide the best characterization in this sense. The inspection of the judicial source documents confirms that the predicted links, in most instances, do relate actors with large likelihood of co-participation in illicit activities.     

Actin filaments during terminal differentiation of urothelial cells in the rat urinary bladder

The localisation of actin filaments was studied in rat urothelial cells during differentiation which accompanied regeneration after cell damage induced by cyclophosphamide (CP). By immunofluorescence it was established that actin filaments equally stained along the cell circumference in basal and intermediate cells, while basolateral cell membrane expression was found in terminally differentiated superficial cells. During regeneration, after CP treatment, simple urothelial hyperplasia developed with smaller cuboidal superficial cells, in which actin filaments were equally distributed under the apical and basolateral plasma membranes. As demonstrated by immunoelectron microscopy, the apical surface of these superficial cells was covered with microvilli containing bundles of actin filaments. Within 1 week, the urothelium reverted to its normal three-layer thickness. Superficial cells became larger and flattened and the unthickened apical plasma membrane matured into a thick asymmetric unit membrane. Concomitantly actin filaments disappeared from apical areas of superficial cells while remaining abundant at basolateral areas. Our results indicate that in the urothelium subcellular distribution of actin filaments can be considered as a marker of cell differentiation. Accepted: 16 September 1999     

Effects of different detachment procedures on viability,nitroxide reduction kinetics and plasma membrane heterogeneity of V‐79 cells

Cell detachment procedures can cause severe damage to cells. Many studies require cells to be detached before measurements; therefore, research on cells that have been grown attached to the bottom of the culture dish and later detached represents a special problem with respect to the experimental results when the properties of cell membranes undergo small changes such as in spectroscopic studies of membrane permeability. We characterized the influence of three different detachment procedures: cell scraping by rubber policeman, trypsinization and a citrate buffer treatment on V‐79 cells in the plateau phase of growth (arrested in G₁). We have measured cell viability by a dye‐exclusion test; nitroxide reduction kinetics and membrane fluidity by EPR (electron paramagnetic resonance) method using the lipophilic spin‐probe MeFASL(10,3) (5‐doxylpalmitoyl‐methylester), which partitions mainly in cell membranes and the hydrophilic spin‐probe TEMPONE (4‐oxo‐2,2,6,6‐tetramethylpiperidine‐1‐oxyl). The resulting cell damage due to the detachment process was observed with SEM (scanning electron microscopy). We found out that cell viability was 91% for trypsin treatment, 85% for citrate treatment and 70% for cell scraping. Though the plasma membrane was mechanically damaged by scraping, the membrane domain structure was not significantly altered compared with other detachment methods. On the other hand, the spin‐probe reduction rate, which depends both on the transport across plasma membrane as well as on metabolic properties of cells, was the highest for trypsin method, suggesting that metabolic rate was the least influenced. Only the reduction rate of trypsin‐treated cells stayed unchanged after 4 h of stirring in suspension. These results suggest that, compared with scraping cells or using citrate buffer, the most suitable detachment method for V‐79 cells is detachment by trypsin and keeping cells in the stirred cell suspension until measurement. This method provides the highest cell viability, less visible damage on SEM micrographs and leaves the metabolic rate of cells unchanged.     

Protruding membrane nanotubes: attachment of tubular protrusions to adjacent cells by several anchoring junctions

Membrane nanotubes are a morphologically versatile group of membrane structures (some resembling filopodia), usually connecting two closely positioned cells. In this article, we set morphological criteria that distinguish the membrane nanotubes from filopodia, as there is no specific molecular marker known to date that unequivocally differentiates between filopodia and protruding nanotubes. Membrane nanotubes have been extensively studied from the morphological point of view and the transport that can be conducted through them, but little is known about the way they connect to the adjacent cell. Our results show that the nanotubes may connect to a neighboring cell by anchoring junctions. Among cell adhesion proteins, N-cadherin, β-catenin, nectin-2, afadin and the desmosomal protein desmoplakin-2 were immune-labeled. We found that N-cadherin and β-catenin are concentrated in nanotubes, while the concentrations of other junction-involved proteins are not increased in these structures. On the basis of data from transmission electron microscopy, we propose a model of the nanotube attachment where the connection of nanotubes is stabilized by several anchoring junctions, most likely adherens junctions that are formed when the nanotube is sliding along the target cell membrane.     

Improving the clinical assessment of consciousness with advances in electrophysiological and neuroimaging techniques

In clinical neurology, a comprehensive understanding of consciousness has been regarded as an abstract concept - best left to philosophers. However, times are changing and the need to clinically assess consciousness is increasingly becoming a real-world, practical challenge. Current methods for evaluating altered levels of consciousness are highly reliant on either behavioural measures or anatomical imaging. While these methods have some utility, estimates of misdiagnosis are worrisome (as high as 43%) - clearly this is a major clinical problem. The solution must involve objective, physiologically based measures that do not rely on behaviour. This paper reviews recent advances in physiologically based measures that enable better evaluation of consciousness states (coma, vegetative state, minimally conscious state, and locked in syndrome). Based on the evidence to-date, electroencephalographic and neuroimaging based assessments of consciousness provide valuable information for evaluation of residual function, formation of differential diagnoses, and estimation of prognosis.     

Identification and characterization of putative osmosensors, HwSho1A and HwSho1B, from the extremely halotolerant black yeast Hortaea werneckii

In Saccharomyces cerevisiae, the Sho1 protein is one of two potential osmosensors that can activate the kinase cascade of the HOG pathway in response to increased extracellular osmolarity. Two novel SHO1-like genes, HwSHO1A and HwSHO1B, have been cloned from the saltern-inhabiting, extremely halotolerant black yeast Hortaea werneckii. The HwSho1 protein isoforms are 93.8% identical in their amino-acid sequences, and have a conserved SH3 domain. When the HwSHO1 genes were transferred into S. cerevisae cells lacking the SHO1 gene, both of the HwSho1 isoforms fully complemented the function of the native S. cerevisiae Sho1 protein. Through microscopic and biochemical validation, we demonstrate that in S. cerevisiae, both of the HwSho1 proteins have characteristic subcellular localizations similar to the S. cerevisiae Sho1 protein, and they can both activate the HOG pathway under conditions of osmotic stress. To a lower extent, crosstalk to the mating pathway expressing HwSho1 proteins is conserved in the PBS2 deleted S. cerevisiae strain. These data show that the HwSho1 proteins from H. werneckii are true functional homologs of the Sho1 protein of S. cerevisiae.