Biomechanics of invasive growth by Armillaria rhizomorphs

Rhizomorphs of wood-decay basidiomycetes are root-like structures produced by the coordinated growth of thousands of hyphae. Very little is known about their development nor the way that they penetrate soils and rotting wood. In this study, we applied techniques used in previous studies on hyphae to explore the mechanics of the invasive growth process in Armillaria gallica. Growth rate measurements were made in media with different gel strengths. The osmolyte composition of rhizomorph sap was determined spectroscopically and the forces exerted by growing tips were measured using a force transducer. Cultured rhizomorphs extended at much faster rates than unbundled hyphae (3.5 mm d⁻¹ versus 1.5 mm d⁻¹) and their growth accelerated in response to increased medium gel strength (to 7.4 mm d⁻¹). Measurements of rhizomorph osmolality provided a turgor pressure estimate of 760 kPa (7.5 atm.), and spectroscopic analysis showed that this pressure was generated by the accumulation of erythritol, mannitol, and KCl. Forces exerted by growing tips ranged from 1 to 6 mN, corresponding to pressures of 40–300 kPa (0.4–3.0 atm.). Pressures exerted by extending rhizomorphs are comparable to those produced by individual vegetative hyphae. This suggests that the mechanical behavior of hyphae is similar whether they grow as unbundled cells or aggregate to form macroscopic rhizomorphs.     

Integrin β1 regulates leiomyoma cytoskeletal integrity and growth

Uterine leiomyomas are characterized by an excessive extracellular matrix, increased mechanical stress, and increased active RhoA. Previously, we observed that mechanical signaling was attenuated in leiomyoma, but the mechanisms responsible remain unclear. Integrins, especially integrin β1, are transmembrane adhesion receptors that couple extracellular matrix stresses to the intracellular cytoskeleton to influence cell proliferation and differentiation. Here we characterized integrin and laminin to signaling in leiomyoma cells. We observed a 2.25 ± 0.32 fold increased expression of integrin β1 in leiomyoma cells, compared to myometrial cells. Antibody-mediated inhibition of integrin β1 led to significant growth inhibition in leiomyoma cells and a loss of cytoskeletal integrity. Specifically, polymerization of actin filaments and formation of focal adhesions were reduced by inhibition of integrin β1. Inhibition of integrin β1 in leiomyoma cells led to 0.81 ± 0.02 fold decrease in active RhoA, and resembled levels found in serum-starved cells. Likewise, inhibition of integrin β1 was accompanied by a decrease in phospho-ERK. Compared to myometrial cells, leiomyoma cells demonstrated increased expression of integrin α6 subunit to laminin receptor (1.91 ± 0.11 fold), and increased expression of laminin 5α (1.52 ± 0.02), laminin 5β (3.06 ± 0.92), and laminin 5γ (1.66 ± 0.06). Of note, leiomyoma cells grown on laminin matrix appear to realign themselves. Taken together, the findings reveal that the attenuated mechanical signaling in leiomyoma cells is accompanied by an increased expression and a dependence on integrin β1 signaling in leiomyoma cells, compared to myometrial cells.     

Anticancer activities of an antimicrobial peptide derivative of Ixosin-B amide

In nature, antimicrobial peptides (AMPs) represent the first line of defense against infection by pathogens; thus, they are generally good candidates for the development of antimicrobial agents. Recently, we reported two potent antimicrobial peptides, KWLRRVWRWWR-amide (MAP-04-03) and KRLRRVWRRWR-amide (MAP-04-04), which were derived from a fragment of Ixosin-B-amide (KSDVRRWRSRY). Since some cationic AMPs exhibited cytotoxic activity against cancer cells, in the current study, we further investigated the anticancer activity of these potent antimicrobial peptides by antiproliferative assays and wound-healing assays, and the effect of peptide on the cytoskeleton alteration and cell morphology were analyzed by confocal microscopy. Results indicated that MAP-04-03 not only exhibited inhibitory effects on the proliferation (IC₅₀ = 61.5 μM) and on the cell migration of MCF-7 breast cancer cells (at a concentration of 5 μM), but also affected the cytoskeleton at the concentration of 25 μM. These results demonstrated that MAP-04-03 can serve as a lead peptide analog for developing potent anticancer agents.     

The role of tryptophans on the cellular uptake and membrane interaction of arginine-rich cell penetrating peptides

Cell-penetrating peptides (CPP) are able to efficiently transport cargos across cell membranes without being cytotoxic to cells, thus present a great potential in drug delivery and diagnosis. While the role of cationic residues in CPPs has been well studied, that of Trp is still not clear. Herein 7 peptide analogs of RW9 (RRWWRRWRR, an efficient CPP) were synthesized in which Trp were systematically replaced by Phe residues. Quantification of cellular uptake reveals that substitution of Trp by Phe strongly reduces the internalization of all peptides despite the fact that they strongly accumulate in the cell membrane. Cellular internalization and biophysical studies show that not only the number of Trp residues but also their positioning in the helix and the size of the hydrophobic face they form are important for their internalization efficacy, the highest uptake occurring for the analog with 3 Trp residues. Using CD and ATR-FTIR spectroscopy we observe that all peptides became structured in contact with lipids, mainly in α-helix. Intrinsic tryptophan fluorescence studies indicate that all peptides partition in the membrane in about the same manner (Kp ~ 10⁵) and that they are located just below the lipid headgroups (~ 10 Å) with slightly different insertion depths for the different analogs. Plasmon Waveguide Resonance studies reveal a direct correlation between the number of Trp residues and the reversibility of the interaction following membrane washing. Thus a more interfacial location of the CPP renders the interaction with the membrane more adjustable and transitory enhancing its internalization ability.     

Gravitaxis of Bursaria truncatella: Electrophysiological and behavioural analyses of a large ciliate cell

Bursaria truncatella is a giant ciliate. Its volume of 3×10⁷ μm³ and a sedimentation rate of 923 μm s^?1 would induce the cell to rapidly sink to the bottom of a pond unless compensating mechanisms exist. The upward swimming behaviour of a cell population (negative gravitaxis) may be either a result of reorientations of the cells (graviorientation) and/or direction-dependent changes in propulsion rate (gravikinesis). The special statocyst hypothesis assumes a stimulation of mechanosensitive ion channels by forces of the cytoplasmic mass acting on the lower membrane. Here, we present basic electrophysiological data on B. truncatella. Investigation of the mechanosensitivity reveals a polar distribution of depolarising and hyperpolarising mechanosensitive channels at least on the dorsal membrane of the cell. Analysis of swimming behaviour demonstrates that Bursaria orients against the gravity vector (r_Oc=0.34) and performs a negative gravikinesis (?633 μm s^?1) compensating the sedimentation rate by 70%. Under hypergravity conditions gravitaxis in Bursaria is enhanced. Microgravity experiments indicate an incomplete relaxation of graviresponses during 4 s of weightlessness. Experimental data are in accordance with the special statocyst hypothesis of graviperception, as was demonstrated in other ciliates.     

Force platform for rats measures fore and hind forces concurrently

Animal models are commonly used to test the efficacy of impact loading regimens on bone strength. We designed an inexpensive force platform to concurrently measure the separate peak vertical impact forces produced by the fore and hindfeet of immature F-344 rats when dropped onto the platform. The force platform consisted of three load cells placed in a triangular pattern under a flat plate. Rats were dropped from heights of 30, 45 and 60 cm onto the platform so that they landed on all four feet concurrently. The peak vertical impact forces produced by the feet of the rats were measured using a sampling frequency of 100 kHz. The location of each foot at landing relative to the load cells, and the force received by each load cell were combined in a series of static equations to solve for the vertical impact forces produced by the fore and hindfeet. The forces produced by feet when rats stood on the single platform were similarly determined. The forces exerted separately by the fore and hindfeet of young rats when landing on the plate as a ratio to standing forces were then calculated. Rats when standing bore more weight on their hindfeet but landed with more weight on their forefeet, which provides rationale for the greater response to landing forces of bones in the forelimbs than those in the hindlimbs. This system provided a useful method to simultaneously measure peak vertical impact forces in fore and hindfeet in rats.     

Collision of two action potentials in a single excitable cell

BackgroundIt is a common incident in nature, that two waves or pulses run into each other head-on. The outcome of such an event is of special interest, because it allows conclusions about the underlying physical nature of the pulses. The present experimental study dealt with the head-on meeting of two action potentials (AP) in a single excitable plant cell (Chara braunii internode).MethodsThe membrane potential was monitored with multiple sensors along a single excitable cell. In control experiments, an AP was excited electrically at either end of the cell cylinder. Subsequently, stimuli were applied simultaneously at both ends of the cell in order to generate two APs that met each other head-on.ResultsWhen two action potentials propagated into each other, the pulses did not penetrate but annihilated (N = 26 experiments in n = 10 cells).ConclusionsAPs in excitable plant cells did not penetrate upon meeting head-on. In the classical electrical model, this behavior is specifically attributed to relaxation of ion channel proteins. From an acoustic point of view, annihilation can be viewed as a result of nonlinear material properties (e.g. a phase change).General significanceThe present results suggest that APs in excitable animal and plant cells belong to a similar class of nonlinear phenomena. Intriguingly, other excitation waves in biology (intracellular waves, cortical spreading depression, etc.) also annihilate upon collision and are thus expected to follow the same underlying principles as the observed action potentials.     

Cyclic strain dominates over microtopography in regulating cytoskeletal and focal adhesion remodeling of human mesenchymal stem cells

Human bone marrow-derived mesenchymal stem cell (hMSCs) function depends on chemical factors and also on the physical cues of the microenvironmental niche. Here, this physical microenvironment is recapitulated with controlled modes of mechanical strain applied to substrata containing three-dimensional features in order to analyze the effects on cell morphology, focal adhesion distribution, and gene expression. Ten percentage of strain at 1 Hz is delivered for 48 h to hMSCs cultured on flat surfaces, or on substrata with 15 μm-high microtopographic posts spaced 75 μm apart. Adding strain to microtopography produced stable semicircular focal adhesions, and actin spanning from post to post. Strain dominated over microtopography for expression of genes for the cytoskeleton (caldesmon-1 and calponin 3), cell adhesion (integrin-α2, vinculin, and paxillin), and extracellular matrix remodeling (MMP13) (p < 0.05). Overall, attention to external mechanical stimuli is necessary for optimizing the stem cell niche for regenerative medicine.     

HDAC inhibitor-induced activation of NF-κB prevents apoptotic response of E1A + Ras-transformed cells to proapoptotic stimuli

HDAC inhibitors (HDACIs) are capable of suppressing the cell growth of tumour cells due to the induction of apoptosis and/or cell cycle arrest. This allows of considering HDACIs as promising agents for tumour therapy. The final outcome – apoptotic cell death or cell cycle arrest – depends on the type of tumour and cellular context. In this report, we addressed the issue by analysing effects produced in E1A + Ras-transformed MEF cells by HDAC inhibitors sodium butyrate (NaB), Trichostatin A (TSA) and some others. It has been shown that the HDACIs induced cell cycle arrest in E1A + Ras-transformed cells but not apoptosis. The antiapoptotic effect of HDACIs is likely to be a result of NF-κB-dependent signaling pathway activation. HDACI-induced activation of NF-κB takes place in spite of a deregulated PI3K/Akt pathway in E1A + Ras cells, suggesting an alternative mechanism for the activation of NF-κB based on acetylation. HDACI-dependent activation of NF-κB prevents the induction of apoptosis by cytostatic agent adriamycin and serum deprivation. Accordingly, suppression of NF-κB activity in HDACI-arrested cells by the chemical inhibitor CAPE or RelA-siRNA resulted in the induction of an apoptotic programme. Thus, our findings suggest that the activation of the NF-κB pathway in HDACI-treated E1A + Ras-transformed cells blocks apoptosis and may thereby play a role in triggering the programme of cell cycle arrest and cellular senescence.     

Statin therapy influences endothelial cell morphology and F-actin cytoskeleton structure when exposed to static and laminar shear stress conditions

AimsTo determine how statin drugs (3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors) affect endothelial cell (EC) shape and F-actin cytoskeleton arrangement in the presence of physiologically relevant wall shear stress (WSS) of 12.5 dyn/cm².Main methodsHuman abdominal aortic endothelial cells (HAAECs) were cultured to a confluent monolayer within three dimensional tissue culture models and presheared for 6 h at 12.5 dyn/cm² within a continuous flow loop. Statins were added to the perfusion media and the perfusion was continued for a further 24 h. ECs were then analyzed for morphology and F-actin cytoskeleton arrangement using light microscopy and laser scanning confocal microscopy.Key findingsECs became rounded with a significantly higher shape index with the addition of 10 μM simvastatin under both static and flow conditions. F-actin cytoskeleton structure was disorganized and fragmented with statin treatment under static and flow conditions. Neither of these findings were observed with the addition of both simvastatin and 200 μM mevalonate, confirming regulation through the cholesterol biosynthesis pathway.SignificanceEC morphology and F-actin cytoskeleton arrangement are regulated through the cholesterol biosynthesis pathway and are therefore impacted by statin treatment. ECs treated with statins became rounded, which is usually associated with unhealthy cells in regions of the vasculature prone to developing atherosclerotic plaques.     

Role of AKAP 149–PKA–PDE4A complex in cell survival and cell differentiation processes

The cellular localization of A-kinase anchoring proteins (AKAPs), protein kinase A (PKAs) and phosphodiesterases (PDEs) is a key step to the spatiotemporal regulation of the second messenger adenosine 3′,5′-cyclic monophosphate (cAMP). In this paper the cellular distribution of the mitochondrial AKAP 149–PKA–PDE4A complex and its implications in the cell death induced by YTX treatment, a known PDE modulator, was studied. K-562 cell line was incubated with YTX for 24 or 48 h. Under these conditions AKAP 149, PKA and type-4A PDE (PDE4A) levels were measured in the cytosol, in the plasma membrane and in the nucleus. Apoptotic hallmarks were also measured after the same conditions. In addition, YTX effect on cell viability was checked after AKAP 149 and PDE4A silencing. The results obtained show a decrease in AKAP 149–PKA–PDE4A levels in cytosol after YTX exposure. 24 h after the toxin addition, the complex expression increased in the plasma membrane and after 48 h in the nucleus domain. Furthermore Bcl-2 levels were decreased and the expression of caspase 3 together with caspase 8 activity were increased after 24 h of toxin incubation but not after 48 h. These results suggest apoptotic cell death at 24 h and a non-apoptotic cell death after 48 h. When AKAP 149 and PDE4A were silenced YTX did not induce cellular death. In summary, AKAP 149–PKA–PDE4A complex localization is related with YTX effect in K-562 cell line. When this complex is mainly located in the plasma membrane apoptosis is activated while when the complex is in the nuclear domain non-apoptotic cellular death or cellular differentiation is activated. Therefore AKAP 149–PKA–PDE4A distribution and integrity have a key role in cellular survival.     

Electroporation by subnanosecond pulses

Electropermeabilization of cell membranes by micro- and nanosecond-duration stimuli has been studied extensively, whereas effects of picosecond electric pulses (psEP) remain essentially unexplored. We utilized whole-cell patch clamp and Di-8-ANEPPS voltage-sensitive dye measurements to characterize plasma membrane effects of 500 ps stimuli in rat hippocampal neurons (RHN), NG108, and CHO cells. Even a single 500-ps pulse at 190 kV/cm increased membrane conductance and depolarized cells. These effects were augmented by applying brief psEP bursts (5–125 pulses), whereas the rate of pulse delivery (8 Hz–1 kHz) played little role. psEP-treated cells displayed large inward current at negative membrane potentials but modest or no conductance changes at positive potentials. A 1-kHz burst of 25 pulses increased the whole-cell conductance in the range (?100)–(?60) mV to 22–26 nS in RHN and NG108 cells (from 3 and 0.7 nS, respectively), but only to 5 nS in CHO (from 0.3 nS). The conductance increase was reversible within about 2 min. Such pattern of cell permeabilization, with characteristic inward rectification and slow recovery, was similar to earlier reported effects of 60- and 600-ns pulses, pointing to the similarity of structural membrane rearrangements in spite of a different membrane charging mechanism.     

The influence of saponins on cell membrane cholesterol

We studied the influence of structurally different saponins on the cholesterol content of cellular membranes. Therefore a cell culture model using ECV-304 urinary bladder carcinoma cells was developed. To measure the cholesterol content we used radiolabeled ³H-cholesterol which is chemically and physiologically identical to natural cholesterol. The cells were pre-incubated with ³H-cholesterol and after a medium change, they were treated with saponins to assess a saponin-induced cholesterol liberation from the cell membrane. In another experiment the cells were pre-incubated with saponins and after a medium change, they were treated with ³H-cholesterol to assess a saponin-induced inhibition of cholesterol uptake into the cell membrane. Furthermore, the membrane toxicity of all applied saponins was analyzed using extracellular LDH quantification and the general cytotoxicity was analyzed using a colorimetric MTT-assay and DNA quantification. Our results revealed a correlation between membrane toxicity and general cytotoxicity. We also compared the results from the experiments on the saponin-induced cholesterol liberation as well as the saponin-induced inhibition of cholesterol uptake with the membrane toxicity. A significant reduction in the cell membrane cholesterol content was noted for those saponins who showed membrane toxicity (IC₅₀ <60 μM). These potent membrane toxic saponins either liberated ³H-cholesterol from intact cell membranes or blocked the integration of supplemented ³H-cholesterol into the cell membrane. Saponins with little influence on the cell membrane (IC₅₀ >100 μM) insignificantly altered the cell membrane cholesterol content. The results suggested that the general cytotoxicity of saponins is mainly dependent on their membrane toxicity and that the membrane toxicity might be caused by the loss of cholesterol from the cell membrane.We also analyzed the influence of a significantly membrane toxic saponin on the cholesterol content of intracellular membranes such as those of endosomes and lysosomes. In these experiments ECV-304 cells were either incubated with ³H-cholesterol or with ³H-cholesterol and 5 μM saponin. After isolation of the endosomes/lysosomes their ³H-cholesterol content was measured. A significant influence of the saponins on the cholesterol content of endosomal/lysosomal membranes was not detected.     

Effects of bisphenol A on the microtubule arrays in root meristematic cells of Pisum sativum L.

Bisphenol A (BPA), a widely used chemical in the plastics industry that displays weak oestrogenic properties, is an emerging environmental pollutant, potentially harmful to living organisms. The presumed cytotoxicity of BPA to plant cells has been poorly studied. To understand how BPA might influence plant cell division and affect the underlying cytoskeleton, the effects of BPA on the microtubule (MT) arrays of meristematic root-tip cells of Pisum sativum L. were investigated. Root tips of young seedlings were exposed to 20, 50 and 100 mg/L BPA for 1, 3, 6, 12 and 24 h. The effects of each treatment were determined by means of confocal laser scanning microscopy after immunolabelling of tubulin and counterstaining of DNA, and by use of light and transmission electron microscopy. It was found that BPA affected normal chromosome segregation, hampered the completion of cytokinesis and deranged interphase and mitotic MT arrays. BPA effects were dependent on the stage of each cell at the time of BPA entrance. Moreover, BPA induced the formation of macrotubules with a mean diameter of 32 ± 0.14 nm, compared with 23 ± 0.70 nm for the MT arrays in untreated cells. Finally, all MT arrays and macrotubules were depolymerised upon longer treatment. Taken together, the data suggest that BPA exerts acute anti-mitotic effects on meristematic root-tip cells of P. sativum, MT arrays constitute a primary sub-cellular target of BPA toxicity, and the manifested chromosomal abnormalities could be attributed to the disruption of the MT cytoskeleton.     

Radiofrequency radiations induced genotoxic and carcinogenic effects on chickpea (Cicer arietinum L.) root tip cells

Present study was under taken to predict the possible DNA damages (genotoxicity) and carcinogenicity caused by radiofrequency radiations (RF) to living tissue. Dry seeds of chickpea were treated with GSM cell phone (900 MHz) and laptop (3.31 GHz) as RF source for 24 and 48 h. Untreated seeds were used as (0 h) negative control and Gamma rays (250 Gray) as positive control. Plant chromosomal aberration assay was used as genotoxicity marker. All the treatment of RF inhibits seed germination percentage. 48 h laptop treatment has the most negative effect as compared to untreated control. A decrease was observed in mitotic index (M.I) and increase in abnormality index (A.I) with the increase in exposure duration and frequency in (Hz). Cell membrane damages were also observed only in 48 h exposure of cell phone and laptop (RF). Maximum nuclear membrane damages and ghost cells were again recorded in 48 h exposure of cell phone and laptop. The radiofrequency radiations (900 MHz and 3.31 GHz) are only genotoxic as they induce micronuclei, bi-nuclei, multi-nuclei and scattered nuclei but could be carcinogenic as 48 h incubation of RF induced fragmentation and ghost cells. Therefore cell phones and laptop should not be used unnecessarily to avoid possible genotoxic and carcinogenic effects.     

Cation selectivity of the plasma membrane of tobacco protoplasts in the electroporated state

Cation selectivity of the cellular membrane of tobacco culture cells (cell line ‘bright yellow-2’) exposed to pulsed electric fields in the millisecond range was investigated. The whole cell configuration of the patch clamp technique was established on protoplasts prepared from these cells. Ion selectivity of the electroporated membrane was investigated by measuring the reversal potential of currents passing through field-induced pores. To this end the membrane was hyper- or depolarized for 10 ms (prepulse); subsequently the voltage was driven to opposite polarity at a constant rate (+ 40 or ? 40 mV/ms, respectively). The experiment was started by polarizing the membrane to moderately negative or positive voltages (prepulse potential ± 150 mV) that would not induce pore formation. Subsequently, an extended voltage range was scanned in the porated state of the membrane (prepulse potential ± 600 mV). IV curves in the porated and the non-porated state (obtained at the same prepulse polarity) were superimposed to determine the voltage at which both curves intersected (‘Intersection potential’). Using a modified version of the Goldmann–Hodgkin–Katz equation relative permeabilities to Ca^2 + and various monovalent alkali and organic cations were calculated. Pores were found to be fairly cation selective, with a selectivity sequence determined to be Ca^2 + > Li⁺ > Rb⁺ ≈ K⁺ ≈ Na⁺ > TEA⁺ ≈ TBA⁺ > Cl^?. Relative permeability to monovalent cations was inversely related to the ionic diameter. By fitting a formalism suggested by Dwyer at al. (J. Gen. Physiol. 75 (1980), 469–492) the effective average diameter of field induced pores was estimated to be about 1.8 nm. Implications of these results for biotechnology and electroporation theory are discussed.     

Variations in hemocyte counts in the mussel,Mytilus edulis: Similar reaction patterns occur in disappearance and return of molluscan hemocytes and vertebrate leukocytes

It was asked whether variations in hemocyte counts in a mussel can be explained by mechanisms known to govern the leukocyte number in vertebrates. Hemolymph of 25 freshly collected Mytilus edulis contained (4.2 ± 1.75) × 10⁶ cells/mL including basophilic and eosinophilic granulocytes and 6.6 ± 5.5% hyalinocytes (15 animals). After 12 or 30 days under optimal laboratory conditions, hemocytes in circulation decreased to less than 1 × 10⁶/mL, the lowest number observed being 5 × 10⁵ cells/mL. Within 2 min of a stressful stimulus, cell numbers doubled or increased by a factor of 3 or 4. After stressing mussels by keeping them out of water for 1 h, cell counts were as high as 1.2 × 10⁷ cells/mL. The quick rate of increase in cell counts is not due to hemocyte proliferation. In mussels, returned to optimal water conditions, cell numbers dropped following an exponential decay curve (y = 5.6865 · (0.9936^X). Not all hemocyte types decreased in number to the same extent. After a strong decrease in the total cell count induced by injection of LPS, the remaining hemocyte population contained a larger percentage of basophils. This indicated the disappearance of eosinophilic cells from the circulation. Stress situations caused their return. Hemocytopenia or stress-induced hemocytosis in M. edulis, both in conjunction with changes in the percentage of granulocytes present, resembles margination/demargination processes in mammals where the concentration of circulating leukocyte subsets depends on the expression of adhesive receptor–ligand molecules on the surface of specific leukocyte types and vascular endothelial cells. In Mytilus edulis, variations in the concentration of distinct cell groups excluded heart activity to explain cell fluctuations. Furthermore, in this mussel, where hemocyte proliferation is not the reason for rapid hemocytosis, cell divisions were nevertheless demonstrated; they seem to be important in maintaining hemocyte homeostasis as 10–20% of cells in circulation possess the capacity to proliferate. They belong to the group of basophilic granulocytes.     

Partial permeabilisation and depolarization of Salmonella enterica Typhimurium cells after treatment with pulsed electric fields and high pressure carbon dioxide

The aim of the present study is to investigate the efficiency of the combined pulsed electric fields and high pressure carbon dioxide (PEF + HPCD) treatment on the Gram-negative pathogen Salmonella Typhimurium in a liquid medium, by means of both plate count technique and flow cytometry (FCM). PEF was applied at two conditions: (1) 1 single pulse of 1 ms length at 30 kV/cm and (2) 12 pulses of 4 ms length at 30 kV/cm, while HPCD at 12 MPa, 22 °C and 35 °C for different treating times (0–45 min). At both temperatures, the application of PEF as HPCD pre-treatment was demonstrated to enhance the inactivation kinetics and to decrease the treatment time to inactivate S. Typhimurium if compared to HPCD alone. Further, the approach based on FCM permitted to investigate the functional status of bacterial cells after PEF and HPCD treatments distinguishing among viable bacteria (considered as intact cells), permeabilised cells and depolarised cells simultaneously. It has been demonstrated that the synergistic effect is due to the electroporation effect of PEF which lead to changes in the cell membrane potential but also in a partial structural damage, favoring the subsequent CO₂ penetration into the cells and increasing the inactivation kinetics, thus improving the efficiency of the entire process.     

Magnetic susceptibility of iron in malaria-infected red blood cells

During intra-erythrocytic maturation, malaria parasites catabolize up to 80% of cellular haemoglobin. Haem is liberated inside the parasite and converted to haemozoin, preventing haem iron from participating in cell-damaging reactions. Several experimental techniques exploit the relatively large paramagnetic susceptibility of malaria-infected cells as a means of sorting cells or investigating haemoglobin degradation, but the source of the dramatic increase in cellular magnetic susceptibility during parasite growth has not been unequivocally determined. Plasmodium falciparum cultures were enriched using high-gradient magnetic fractionation columns and the magnetic susceptibility of cell contents was directly measured. The forms of haem iron in the erythrocytes were quantified spectroscopically. In the 3D7 laboratory strain, the parasites converted approximately 60% of host cell haemoglobin to haemozoin and this product was the primary source of the increase in cell magnetic susceptibility. Haemozoin iron was found to have a magnetic susceptibility of (11.0 ± 0.9) × 10^? 3 mL mol^? 1. The calculated volumetric magnetic susceptibility (SI units) of the magnetically enriched cells was (1.88 ± 0.60) × 10^? 6 relative to water while that of uninfected cells was not significantly different from water. Magnetic enrichment of parasitised cells can therefore be considered dependent primarily on the magnetic susceptibility of the parasitised cells.     

Live-cell FRET imaging reveals clustering of the prion protein at the cell surface induced by infectious prions

Prion diseases are associated to the conversion of the prion protein into a misfolded pathological isoform. The mechanism of propagation of protein misfolding by protein templating remains largely unknown. Neuroblastoma cells were transfected with constructs of the prion protein fused to both CFP-GPI-anchored and to YFP-GPI-anchored and directed to its cell membrane location. Live-cell FRET imaging between the prion protein fused to CFP or YFP was measured giving consistent values of 10 ± 2%. This result was confirmed by fluorescence lifetime imaging microscopy and indicates intermolecular interactions between neighbor prion proteins. In particular, considering that a maximum FRET efficiency of 17 ± 2% was determined from a positive control consisting of a fusion CFP-YFP-GPI-anchored. A stable cell clone expressing the two fusions containing the prion protein was also selected to minimize cell-to-cell variability. In both, stable and transiently transfected cells, the FRET efficiency consistently increased in the presence of infectious prions — from 4 ± 1% to 7 ± 1% in the stable clone and from 10 ± 2% to 16 ± 1% in transiently transfected cells. These results clearly reflect an increased clustering of the prion protein on the membrane in the presence of infectious prions, which was not observed in negative control using constructs without the prion protein and upon addition of non-infected brain. Our data corroborates the recent view that the primary site for prion conversion is the cell membrane. Since our fluorescent cell clone is not susceptible to propagate infectivity, we hypothesize that the initial event of prion infectivity might be the clustering of the GPI-anchored prion protein.