Carbon isotope variability and sedimentology of the Upper Permian carbonate rocks and changes across the Permian-Triassic boundary in the Masore section (Western Slovenia)

Carbonate and total organic carbon stable isotope analyses of the Upper Permian and Lower Triassic succession in the Masore section in western Slovenia indicate a high storage of organic matter during the Upper Permian, as well as the well known worldwide light carbon isotope event across the P/Tr boundary. The perturbations in the global carbon cycle observed in the investigated section span an approximately 50 cm thick interval (from –11 cm below to +41 cm above the lithostratigraphically determined P/Tr boundary), and coincide more or less with changes in lithology, as well as with an abrupt disappearance of Upper Permian marine fauna. In this section changes in the sedimentary environment are most probably related to Upper Permian—Lower Triassic sea level changes. The carbonate and organic carbon negative peak anomaly could be explained by accelerated changes in the end Permian carbon cycle, due to some co-occurring events, such as pronounced erosion and oxidation of organic carbon, a possible release of methane from stored hydrates, and volcanic activity, as well as by a sudden reduction in primary productivity triggered by not yet completely satisfactorily explained mechanisms.     

A novel mechanism of pore formation: membrane penetration by the N-terminal amphipathic region of equinatoxin

Equinatoxin II is a representative of actinoporins, eukaryotic pore-forming toxins from sea anemones. It creates pores in natural and artificial lipid membranes by an association of three or four monomers. Cysteine-scanning mutagenesis was used to study the structure of the N terminus, which is proposed to be crucial in transmembrane pore formation. We provide data for two steps of pore formation: a lipid-bound monomeric intermediate state and a final oligomeric pore. Results show that residues 10-28 are organized as an alpha-helix in both steps. In the first step, the whole region is transferred to a lipid-water interface, laying flat on the membrane. In the pore-forming state, the hydrophilic side of the amphipathic helix lines the pore lumen. The pore has a restriction around Asp-10, according to the permeabilization ratio of ions flowing through pores formed by chemically modified mutants. A general model was introduced to derive the tilt angle of the helix from the ion current data. This study reveals that actinoporins use a unique single helix insertion mechanism for pore formation.     

Cell-mediated immune response to high-passage Borrelia spirochetes in C57bl/6 mice is strictly dependent on antigen specificity

Inbred C57bl/6 mice were challenged with high-passage Borrelia afzelii, Borrelia garinii and Borrelia burgdorferi sensu stricto and tested for antigen specific T-cell response in vitro. Sonicated preparations of washed spirochetes were potent cell activators, capable of stimulating polyclonal proliferation after 72h of culture while increasing the incubation time up to 120h provoked specific cell-mediated response. Isolated murine spleocytes previously sensitized to B. burgdorferi sensu lato but not those from control mice could be induced for antigen-specific proliferation in vitro, as revealed by [3H]thymidine incorporation assay, Moreover, in mice presensitized to B. burgdorferi sensu lato, detectable cell-mediated response could be induced only with antigen preparations derived from a corresponding strain but not with those obtained from other Borrelia genospecies. The current study emphasises that the B. burgdorferi antigen-specific response may also be expected in different genospecies infections in men.     

The biomechanics of standing and balancing in paralyzed people

Prolonged immobilization results in several physiological problems. It has been demonstrated that standing exercises can ameliorate many of these problems. Standing exercises can be performed efficiently with the help of functional electrical stimulation (FES). A novel robotic mechanism which aids the unsupported standing of paraplegics, providing balancing exercise has been developed. The balancing strategy is based on voluntary activity of the paraplegic's upper body and artificially controlled stiffness in the ankles.     

Cytotoxic activity of a tumor protease-activated pore-forming toxin

Equinatoxin II is a pore forming toxin produced by the sea anemone Actinia equina. It is able to kill very unspecifically most cell types by the membrane-perturbing action of an amphiphilic alpha-helix located at its N-terminal. A normally active N-terminal mutant, containing one single cys in the amphiphilic alpha-helix, becomes totally inactive when it is bound to avidin via a biotinylated linker. By choosing, as a linker, a peptide containing a tumor protease cleavage site, we were able to construct an enzymatically activable conjugate which should be selective for tumor cells. The introduced cleavage site was designed in order to be digested by both cathepsin B and matrix metalloproteases (MMPs). We confirmed that this conjugate could be activated in vitro by cathepsin B and MMPs. After having measured the enzymatic activity of fibrosarcoma and breast carcinoma cells, we analyzed the cytotoxic effect of the conjugate on the same lines and on human red blood cells (HRBC) as controls. We found that the conjugate was activated, at least in part, by the tumor cell lines used, whereas it was inactive on HRBC. That the activation process was dependent on the enzymatic action of cathepsin B and MMPs, was indicated by three lines of evidence: (1) binding occurred normally on all type of cells including HRBC which however were insensitive being devoid of enzymes; (2) the cytotoxic effect correlated with the amount of cathepsin B activity expressed by the cells; (3) conjugate activation was reduced by specific inhibitors of cathepsin B and MMPs. These results demonstrate the possibility of tumor cell killing by a pore-forming toxin conjugate specifically activated by tumor proteases.     

Biomechanical characterization and clinical implications of artificially induced toe-walking: differences between pure soleus, pure gastrocnemius and combination of soleus and gastrocnemius contractures

The purpose of this study was to characterize biomechanically three different toe-walking gait patterns, artificially induced in six neurologically intact subjects and to compare them to selected cases of pathological toe-walking. The subjects, equipped with lightweight mechanical exoskeleton with elastic ropes attached to the left leg's heel on one end and on shank and thigh on the other end in a similar anatomical locations where soleus and gastrocnemius muscles attach to skeleton, walked at speed of approximately 1m/s along the walkway under four experimental conditions: normal walking (NW), soleus contracture emulation (SOL), gastrocnemius contracture emulation (GAS) and emulation of both soleus and gastrocnemius contractures (SOLGAS). Reflective markers and force platform data were collected and ankle, knee and hip joint angles, moments and powers were calculated using inverse dynamic model for both legs. Characteristic peaks of averaged kinematic and kinetic patterns were compared among all four experimental conditions in one-way ANOVA. In the left leg SOL contracture mainly influenced the ankle angle trajectory, while GAS and SOLGAS contractures influenced the ankle and knee angle trajectories. GAS and SOLGAS contractures significantly increased ankle moment during midstance as compared to SOL contracture and NW. All three toe-walking experimental conditions exhibited significant power absorption in the ankle during loading response, which was absent in the NW condition, while during preswing significant decrease in power absorption as compared to NW was seen. In the knee joint SOL contracture diminished, GAS contracture increased while SOLGAS contracture approximately halved knee extensor moment during midstance as compared to NW. All three toe-walking experimental conditions decreased hip range of motion, hip flexor moment and power requirements during stance phase. Main difference in the right leg kinematic and kinetic patterns was seen in the knee moment trajectory, where significant increase in the knee extensor moment took place in terminal stance for GAS and SOLGAS experimental conditions as compared to SOL and NW. The kinetic trajectories under SOL and GAS experimental conditions were qualitatively compared to two selected clinical cases showing considerable similarity. This implies that distinct differences in kinetics between SOL, GAS and SOLGAS experimental conditions, as described in this paper, may be clinically relevant in determining the relative contribution of soleus and gastrocnemius muscles contractures to toe-walking in particular pathological gait.     

Measuring the Induced Membrane Voltage with Di-8-ANEPPS

Placement of a cell into an external electric field causes a local charge redistribution inside and outside of the cell in the vicinity of the cell membrane, resulting in a voltage across the membrane. This voltage, termed the induced membrane voltage (also induced transmembrane voltage, or induced transmembrane potential difference) and denoted by ΔΦ, exists only as long as the external field is present. If the resting voltage is present on the membrane, the induced voltage superimposes (adds) onto it. By using one of the potentiometric fluorescent dyes, such as di-8-ANEPPS, it is possible to observe the variations of ΔΦ on the cell membrane and to measure its value noninvasively. di-8-ANEPPS becomes strongly fluorescent when bound to the lipid bilayer of the cell membrane, with the change of the fluorescence intensity proportional to the change of ΔΦ. This video shows the protocol for measuring ΔΦ using di-8-ANEPPS and also demonstrates the influence of cell shape on the amplitude and spatial distribution of ΔΦ.     

Effect of fasting on hypogean (<Emphasis Type="Italic">Niphargus stygius</Emphasis>) and epigean (<Emphasis Type="Italic">Gammarus fossarum</Emphasis>) amphipods: a laboratory study

Two amphipods, the hypogean Niphargus stygius and epigean Gammarus fossarum, were analyzed for fatty acid (FA) composition, electron transport system (ETS) activity and respiration (R) during a laboratory fasting experiment. In agreement with ETS and R measurements (and the ETS/R ratio), the hypogean N. stygius utilized FA more slowly than the epigean G. fossarum. Inter-specific differences in the utilization of certain FA during fasting were also revealed. While N. stygius tended to preserve all of its FA during the experimental fasting period, G. fossarum showed a tendency to utilize MUFA (monounsaturated FA) and SAFA (saturated FA) and preferentially retain PUFA (polyunsaturated FA). The significant correlations between ETS activity and composition of specific FA during fasting can be linked to R. During the fasting, both ETS activity and respiration rate of G. fossarum decreased, however, ETS/R ratio increased. In contrast, N. stygius did not show significant changes in these parameters. This is the first report, which connects ETS activity with changes in concentrations of specific FA during fasting. Such evolutionary adaptations of hypogean species enables them to better survive chronically low and/or discontinuous food supplies compared to epigean species, which live in environments where food shortages are much less frequent.     

Robustness of Treatment Planning for Electrochemotherapy of Deep-Seated Tumors

Treatment of cutaneous and subcutaneous tumors with electrochemotherapy has become a regular clinical method, while treatment of deep-seated tumors is still at an early stage of development. We present a method for preparing a dedicated patient-specific, computer-optimized treatment plan for electrochemotherapy of deep-seated tumors based on medical images. The treatment plan takes into account the patient’s anatomy, tissue conductivity changes during electroporation and the constraints of the pulse generator. Analysis of the robustness of a treatment plan made with this method shows that the effectiveness of the treatment is not affected significantly by small single errors in electrode positioning. However, when many errors occur simultaneously, the resulting drop in effectiveness is larger, which means that it is necessary to be as accurate as possible in electrode positioning. The largest effect on treatment effectiveness stems from uncertainties in dielectric properties and electroporation thresholds of treated tumors and surrounding tissues, which emphasizes the need for more accurate measurements and more research. The presented methods for treatment planning and robustness analysis allow quantification of the treatment reproducibility and enable the setting of suitable safety margins to improve the likelihood of successful treatment of deep-seated tumors by electrochemotherapy.     

Two-step membrane binding by Equinatoxin II,a pore-forming toxin from the sea anemone,involves an exposed aromatic cluster and a flexible helix

Equinatoxin II (EqtII) belongs to a unique family of 20-kDa pore-forming toxins from sea anemones. These toxins preferentially bind to membranes containing sphingomyelin and create cation-selective pores by oligomerization of 3-4 monomers. In this work we have studied the binding of EqtII to lipid membranes by the use of lipid monolayers and surface plasmon resonance (SPR). The binding is a two-step process, separately mediated by two regions of the molecule. An exposed aromatic cluster involving tryptophans 112 and 116 mediates the initial attachment that is prerequisite for the next step. Steric shielding of the aromatic cluster or mutation of Trp-112 and -116 to phenylalanine significantly reduces the toxin-lipid interaction. The second step is promoted by the N-terminal amphiphilic helix, which translocates into the lipid phase. The two steps were distinguished by the use of a double cysteine mutant having the N-terminal helix fixed to the protein core by a disulfide bond. The kinetics of membrane binding derived from the SPR experiments could be fitted to a two-stage binding model. Finally, by using membrane-embedded quenchers, we showed that EqtII does not insert deeply in the membrane. The first step of the EqtII binding is reminiscent of the binding of the evolutionarily distant cholesterol-dependant cytolysins, which share a similar structural motif in the membrane attachment domain.