Mechanisms of equinatoxin II-induced transport through the membrane of a giant phospholipid vesicle

Protein equinatoxin II from sea anemone Actinia equina L. was used to form pores in phospholipid membranes. We studied the effect of these pores on the net transmembrane transport of sucrose and glucose by observing single giant (cell-size) vesicles under the phase contrast microscope. Sugar composition in the vesicle was determined by measuring the width of the halo, which appears around the vesicle in the phase contrast image. The transport of sugars was induced when a vesicle, filled with the sucrose solution, was transferred into the isomolar environment of a glucose solution with added equinatoxin II. Typically, a vesicle grew to a critical size, then the membrane broke by bursting and the vesicle shrank, started to grow again, and the whole process was repeated. The consecutive membrane breaks occurred in the same spot. The observed behavior was interpreted by the diffusion flow of the glucose molecules through the equinatoxin II-induced pores and the consequent increase of the vesicle water content. The burst relaxed the critically strained membrane, which then apparently resealed. A mathematical model of the described behavior was developed and was used to obtain the equinatoxin II-induced membrane permeability for the glucose molecules. Its dependence on the equinatoxin II concentration is in agreement with the previous reports.     

Nonlinear Dispersive Model of Electroporation for Irregular Nucleated Cells

In this work, the electroporation phenomenon induced by pulsed electric field on different nucleated biological cells is studied. A nonlinear, non‐local, dispersive, and space–time multiphysics model based on Maxwell’s and asymptotic Smoluchowski’s equations has been developed to calculate the transmembrane voltage and pore density on both plasma and nuclear membrane perimeters. The irregular cell shape has been modeled by incorporating in the numerical algorithm the analytical functions pertaining to Gielis curves. The dielectric dispersion of the cell media has been modeled considering the multi‐relaxation Debye‐based relationship. Two different irregular nucleated cells have been investigated and their response has been studied applying both the dispersive and non‐dispersive models. By a comparison of the obtained results, differences can be highlighted confirming the need to make use of the dispersive model to effectively investigate the cell response in terms of transmembrane voltages, pore densities, and electroporation opening angle, especially when irregular cell shapes and short electric pulses are considered. Bioelectromagnetics. 2019;40:331–342. © 2019 Wiley Periodicals, Inc.     

Do multiple experimenters improve the reproducibility of animal studies?

The credibility of scientific research has been seriously questioned by the widely claimed “reproducibility crisis”. In light of this crisis, there is a growing awareness that the rigorous standardisation of experimental conditions may contribute to poor reproducibility of animal studies. Instead, systematic heterogenisation has been proposed as a tool to enhance reproducibility, but a real-life test across multiple independent laboratories is still pending. The aim of this study was therefore to test whether heterogenisation of experimental conditions by using multiple experimenters improves the reproducibility of research findings compared to standardised conditions with only one experimenter. To this end, we replicated the same animal experiment in 3 independent laboratories, each employing both a heterogenised and a standardised design. Whereas in the standardised design, all animals were tested by a single experimenter; in the heterogenised design, 3 different experimenters were involved in testing the animals. In contrast to our expectation, the inclusion of multiple experimenters in the heterogenised design did not improve the reproducibility of the results across the 3 laboratories. Interestingly, however, a variance component analysis indicated that the variation introduced by the different experimenters was not as high as the variation introduced by the laboratories, probably explaining why this heterogenisation strategy did not bring the anticipated success. Even more interestingly, for the majority of outcome measures, the remaining residual variation was identified as an important source of variance accounting for 41% (CI₉₅ [34%, 49%]) to 72% (CI₉₅ [58%, 88%]) of the observed total variance. Despite some uncertainty surrounding the estimated numbers, these findings argue for systematically including biological variation rather than eliminating it in animal studies and call for future research on effective improvement strategies.

An experimenter heterogenisation was not sufficient to prevent idiosyncratic results in a multi-laboratory setting. Astonishingly, neither the experimenter nor the laboratory accounted for the main portion of the observed variation, but a high amount of residual variation in fact remained unexplained despite strict standardisation regimes.     

Gluconacetobacter maltaceti sp. nov., a novel vinegar producing acetic acid bacterium

Comparison of HaeIII- and HpaII-restriction profiles of PCR-amplified 16S-23S rDNA ITS regions of Gluconacetobacter sp. LMG 1529^T and SKU 1109 with restriction profiles of reference strains of acetic acid bacteria described by Tr?ek and Teuber [34] revealed the same but unique restriction profiles for LMG 1529^T and SKU 1109. Further analyses of nearly complete 16S rRNA gene sequences, nearly complete 16S-23S rDNA ITS sequences, as well as concatenated partial sequences of the housekeeping genes dnaK, groEL and rpoB, allocated both strains to a single phylogenetic cluster well separated from the other species of the genus Gluconacetobacter. DNA–DNA hybridizations confirmed their novel species identity by 73% DNA–DNA relatedness between both strains, and values below the species level (<70%) between SKU 1109 and the type strains of the closest phylogenetic neighbors. The classification of strains LMG 1529^T and SKU 1109 into a single novel species was confirmed also by AFLP and (GTG)₅-PCR DNA fingerprinting data, as well as by phenotypic data. Strains LMG 1529^T and SKU 1109 can be differentiated from their closely related Gluconacetobacter species, Gluconacetobacter entanii and Gluconacetobacter hansenii, by their ability to form 2-keto-d-gluconic acid from d-glucose, their ability to use d-mannitol, d-gluconate and glycerol as carbon source and form acid from d-fructose, and their ability to grow without acetic acid. The major fatty acid of LMG 1529^T and SKU 1109 is C_18:1ω7c (60.2–64.8%). The DNA G + C content of LMG 1529^T and SKU 1109 is 62.5 and 63.3 mol% respectively. The name Gluconacetobacter maltaceti sp. nov. is proposed. The type strain is LMG 1529^T (= NBRC 14815^T = NCIMB 8752^T).     

Study of the efficacy of Aureobasidium strains belonging to three different species: A. pullulans,A. subglaciale and A. melanogenum against Botrytis cinerea of tomato

The difference in antagonistic activity against the causal agent of grey mould (Botrytis cinerea) of tomato between Aureobasidium strains belonging to three different species, namely A. pullulans, A. melanogenum and A. subglaciale, was evaluated by in vitro and in vivo assays. In the yeast–pathogen direct interaction experiment, all the strains significantly reduced B. cinerea growth, with A. melanogenum the least efficient species (17.8% of reduction) compared to A. pullulans and subglaciale (22 and 27.8%). The non-volatile metabolites produced by all three species reduced mycelial growth between 95 and 100%. These metabolites were characterised by FT-IR spectroscopy as polysaccharides, lytic enzymes, siderophores and antibiotics. The inhibitory effect of Aureobasidium strains on pathogenic enzymes such as xylanase, polygalacturonase and pectinase was measured showing A. pullulans strains as capable of strong inhibition of xylanase, an enzyme directly related to the virulence of necrotrophic pathogens such as B. cinerea. Our data demonstrate that the different species of Aureobasidium isolated from a range of non-conventional environments exerted variable efficacy against B. cinerea, with A. pullulans as the most active species followed by A. subglaciale and A. melanogenum as ineffective and not suitable for biocontrol applications.     

The relationship between osteoclastogenic and anti-osteoclastogenic pro-inflammatory cytokines differs in human osteoporotic and osteoarthritic bone tissues

Background

Pro-inflammatory cytokines possess osteoclastogenic or anti-osteoclastogenic activities. They influence osteoclasts directly or via the receptor activator of nuclear factor κB (RANK), RANK ligand (RANKL) and osteoprotegerin (OPG) system. Recent evidence suggests that inflammation may play a role in osteoporosis (OP) and osteoarthritis (OA). We aimed therefore to determine whether there is a difference between both groups: first, in the expression of the osteoclastogenic and anti-osteoclastogenic cytokines, second, in correlation of these cytokines with bone mineral density (BMD) and levels of bone turnover markers (BTM) and third, in correlation between the expression of these cytokines and osteoclast specific genes and RANK/RANKL/OPG genes.

Methods

Human bone samples from 54 age and sex matched patients with OP or OA were collected during hip arthroplasty surgery. The expression of 25 genes encoding pro-inflammatory cytokines, their receptors, osteoclast specific genes and RANK/RANKL/OPG genes was measured using quantitative real-time PCR. Total hip, femoral neck and lumbar spine BMD and BTM in blood samples were measured. The comparison between OP and OA was assessed using Student''s t-test or Mann-Whitney U test and correlations between gene expression, BMD and BTM were determined using nonparametric correlation.

Results

The results demonstrated a higher expression of interleukin (IL)-6 and IL-1α in OP, and interferon (IFN)-γ in OA (p < 0.0005). Negative correlations of total hip BMD with tumor necrosis factor-α (TNF-α) in OA and with RANKL/RANK in OP were found (p < 0.05). Significant correlations with BTM were shown for IL-1α and IFN-γ in OP (rho = 0.608 and -0.634) and for TNF-α, IL-6 and transforming growth factor-β1 (TGF-β1) in OA (rho = 0.591, -0.521 and 0.636). Results showed OP specific negative correlations (IFN-γ with ITGB3, IFN-β1 with CTSK, tartrate resistant acid phosphatase (TRAP), CALCR, RANK, RANKL, IL-1α with CTSK, OPG, IL-17A with CALCR) and positive (TGF-β1 with CTSK, TRAP, RANK), and OA specific negative (IL-1α with osteoclast associated immunoglobulin-like receptor (OSCAR), TNF-α with RANK, RANKL, OPG) and positive (IL-6 with RANK, RANKL, OPG) correlations.

Conclusions

Our results demonstrate that the relationship between osteoclastogenic and anti-osteoclastogenic pro-inflammatory cytokines differs in human OP and OA bone and could present an important factor for characteristics of OP and OA bone phenotypes.     

Osmoadaptation Strategy of the Most Halophilic Fungus,Wallemia ichthyophaga,Growing Optimally at Salinities above 15% NaCl

Wallemia ichthyophaga is a fungus from the ancient basidiomycetous genus Wallemia (Wallemiales, Wallemiomycetes) that grows only at salinities between 10% (wt/vol) NaCl and saturated NaCl solution. This obligate halophily is unique among fungi. The main goal of this study was to determine the optimal salinity range for growth of the halophilic W. ichthyophaga and to unravel its osmoadaptation strategy. Our results showed that growth on solid growth media was extremely slow and resulted in small colonies. On the other hand, in the liquid batch cultures, the specific growth rates of W. ichthyophaga were higher, and the biomass production increased with increasing salinities. The optimum salinity range for growth of W. ichthyophaga was between 15 and 20% (wt/vol) NaCl. At 10% NaCl, the biomass production and the growth rate were by far the lowest among all tested salinities. Furthermore, the cell wall content in the dry biomass was extremely high at salinities above 10%. Our results also showed that glycerol was the major osmotically regulated solute, since its accumulation increased with salinity and was diminished by hypo-osmotic shock. Besides glycerol, smaller amounts of arabitol and trace amounts of mannitol were also detected. In addition, W. ichthyophaga maintained relatively small intracellular amounts of potassium and sodium at constant salinities, but during hyperosmotic shock, the amounts of both cations increased significantly. Given our results and the recent availability of the genome sequence, W. ichthyophaga should become well established as a novel model organism for studies of halophily in eukaryotes.     

Plasmid analysis of high acetic acid-resistant bacterial strains by two-dimensional agarose gel electrophoresis and insights into the phenotype of plasmid pJK2-1

Plasmid profiling can be used for quick molecular characterization of bacteria. In the study reported here, this method was used to compare the plasmid profiles of strains of Gluconacetobacter europaeus, one of the dominant species in industrial vinegar production. Further analysis of three selected strains by two-dimensional agarose gel electrophoresis showed that the plasmid profiles are composed of different forms of plasmids of the same size. One of these plasmids, pJK2-1, was introduced into Gluconacetobacter oboediens JK3 as a chimeric plasmid (pJT2) with pUC18. The recombinant strain showed a shorter lag phase in a medium with 3 and 5 % (v/v) acetic acid. Deletion of a part of plasmid pJK2-1 allowed a region that contributes to this novel phenotype of G. oboediens JK3 pJT2 to be identified. Non-problematic handling of G. oboediens JK3 warrants further study in elucidating the function of plasmids involved in the production of vinegar.