Differential Cellular Effects of Electroporation and Electrochemotherapy in Monolayers of Human Microvascular Endothelial Cells

In vivo electroporation of tumours shows disruption of blood flow and creates a vascular effect with an initial rapid and transient vasoconstriction phase and a much longer lasting phase with changed microvascular endothelium. These changes are not well understood but are presumed to involve the cytoskeleton. The paper presents for the first time differential in vitro effects describing cytoskeleton changes and monolayer integrity changes by both electroporation and electrochemotherapy of monolayers of human microvascular endothelial cells (HMEC-1). After the application of electric field pulses, the morphology of cells, and both the F-actin and Beta-tubulin cytoskeleton proteins were affected. During both electroporation and electrochemotherapy, the initial phase of cellular damage was noticed at 10 min as swollen cells and honeycomb-like actin bundles. The electroporation-induced cellular effects, observed from electric pulses >150 V, were voltage-dependent and within 24 hrs partly recoverable. The electrochemotherapy-induced cellular effects developed at 2 hrs in spindle-like cells, and more densely packed F-actin and Beta-tubulin were observed, which were dependent on the amount of bleomycin and the voltages applied (>50 V). In addition, for electrochemotherapy with electric pulses >150 V cellular changes were not recoverable within 24 hrs. The effects on monolayer integrity were reflected in the enhanced monolayer permeability, with the electrochemotherapy showing an earlier onset and synergy. We conclude that electrochemotherapy as compared to electroporation leads within 24 hrs to a quicker and more pronounced monolayer integrity damage and endothelial cell death, which together provide further insight into the cellular changes of the vascular disruption of electrochemotherapy.     

Nonlinear dynamics of the blood flow studied by Lyapunov exponents

In order to gain an insight into the dynamics of the cardiovascular system throughout which the blood circulates, the signals measured from peripheral blood flow in humans were analyzed by calculating the Lyapunov exponents. Over a wide range of algorithm parameters, paired values of both the global and the local Lyapunov exponents were obtained, and at least one exponent equaled zero within the calculation error. This may be an indication of the deterministic nature and finite number of degrees of freedom of the cardiovascular system governing the blood-flow dynamics on a time scale of minutes. A difference was observed in the Lyapunov dimension of controls and athletes.     

A Characterization of Scale Invariant Responses in Enzymatic Networks

An ubiquitous property of biological sensory systems is adaptation: a step increase in stimulus triggers an initial change in a biochemical or physiological response, followed by a more gradual relaxation toward a basal, pre-stimulus level. Adaptation helps maintain essential variables within acceptable bounds and allows organisms to readjust themselves to an optimum and non-saturating sensitivity range when faced with a prolonged change in their environment. Recently, it was shown theoretically and experimentally that many adapting systems, both at the organism and single-cell level, enjoy a remarkable additional feature: scale invariance, meaning that the initial, transient behavior remains (approximately) the same even when the background signal level is scaled. In this work, we set out to investigate under what conditions a broadly used model of biochemical enzymatic networks will exhibit scale-invariant behavior. An exhaustive computational study led us to discover a new property of surprising simplicity and generality, uniform linearizations with fast output (ULFO), whose validity we show is both necessary and sufficient for scale invariance of three-node enzymatic networks (and sufficient for any number of nodes). Based on this study, we go on to develop a mathematical explanation of how ULFO results in scale invariance. Our work provides a surprisingly consistent, simple, and general framework for understanding this phenomenon, and results in concrete experimental predictions.     

Microcystins: Synthesis and structure–activity relationship studies toward PP1 and PP2A

Microcystins are highly toxic cyanotoxins responsible for plant, animal and human poisoning. Exposure to microcystins, mainly through drinkable water and contaminated food, is a current world health concern. Although it is quite challenging, the synthesis of these potent cyanotoxins, analogs and derivatives helps to evaluate their toxicological properties and to elucidate their binding mechanisms to their main targets Protein Phosphatase-1 (PP1) and -2A (PP2A). This review focuses on synthetic approaches to prepare microcystins and analogs and compiles structure–activity relationship (SAR) studies that describe the unique features of microcystins that make them so potent.     

Explaining variability in the production of seed and allergenic pollen by invasive <Emphasis Type="Italic">Ambrosia artemisiifolia</Emphasis> across Europe

To better manage invasive populations, it is vital to understand the environmental drivers underlying spatial variation in demographic performance of invasive individuals and populations. The invasive common ragweed, Ambrosia artemisiifolia, has severe adverse effects on agriculture and human health, due to its vast production of seeds and allergenic pollen. Here, we identify the scale and nature of environmental factors driving individual performance of A. artemisiifolia, and assess their relative importance. We studied 39 populations across the European continent, covering different climatic and habitat conditions. We found that plant size is the most important determinant in variation of per-capita seed and pollen production. Using plant volume as a measure of individual performance, we found that the local environment (i.e. the site) is far more influential for plant volume (explaining 25% of all spatial variation) than geographic position (regional level; 8%) or the neighbouring vegetation (at the plot level; 4%). An overall model including environmental factors at all scales performed better (27%), including the weather (bigger plants in warm and wet conditions), soil type (smaller plants on soils with more sand), and highlighting the negative effects of altitude, neighbouring vegetation and bare soil. Pollen and seed densities varied more than 200-fold between sites, with highest estimates in Croatia, Romania and Hungary. Pollen densities were highest on arable fields, while highest seed densities were found along infrastructure, both significantly higher than on ruderal sites. We discuss implications of these findings for the spatial scale of management interventions against A. artemisiifolia.     

Functional Characterization of Novel Phenylalanine Hydroxylase p.Gln226Lys Mutation Revealed Its Non-responsiveness to Tetrahydrobiopterin Treatment in Hepatoma Cellular Model

Treatment with tetrahydrobiopterin (BH4) is the latest therapeutic option approved for patients with phenylketonuria (PKU)—one of the most frequent inborn metabolic diseases. PKU or phenylalanine hydroxylase (PAH) deficiency is caused by mutations in the PAH gene. Given that some PAH mutations are responsive to BH4 treatment while others are non-responsive, for every novel mutation that is discovered it is essential to confirm its pathogenic effect and to assess its responsiveness to a BH4 treatment in vitro, before the drug is administered to patients. We found a c.676C>A (p.Gln226Lys) mutation in the PAH gene in two unrelated patients with PKU. The corresponding aberrant protein has never been functionally characterized in vitro and its response to BH4 treatment is unknown. Computational analyses proposed that glutamine at position 226 is an important, evolutionary conserved amino acid while the substitution with lysine probably disturbs tertiary protein structure and impacts posttranslational PAH modifications. Using hepatoma cellular model, we demonstrated that the amount of mutant p.Gln226Lys PAH detected by Western blot was only 1.2% in comparison to wild-type PAH. The addition of sepiapterin, intracellular precursor of BH4, did not increase PAH protein yield thus marking p.Gln226Lys as BH4-non-responsive mutation. Therefore, computational, experimental, and clinical data were all in accordance showing that p.Gln226Lys is a severe pathogenic PAH mutation. Its non-responsiveness to BH4 treatment in hepatoma cellular model should be considered when deciding treatment options for PKU patients carrying this mutation. Consequently, our study will facilitate clinical genetic practice, particularly genotype-based stratification of PKU treatment.