Fly into the light: eliminating Drosophila melanogaster with chlorophyllin-based Photodynamic Inactivation.

Fruit flies spoil crops in agricultural settings. As conventional pesticides may generate negative off-target effects on humans or the environment, existing treatment methods need eco-friendly and safe alternatives. Photodynamic Inactivation (PDI) is based on the photosensitizer-mediated and light-induced overproduction of reactive oxygen species in targets. We here explore the potential of PDI for the control of fruit fly pests. Drosophila melanogaster serves as well-established model organism in this study. Two distinct experimental approaches are presented: the feed assay, in which fruit flies are provided with sodium magnesium chlorophyllin (Chl, approved as food additive E140) along with sucrose (3%) as their food, and the spray assay, where the photosensitizer is sprayed onto the insects. We show that PDI based on Chl can induce moribundity rates of Drosophila melanogaster of more than 99% with 5 mM Chl and LED illumination (395 nm, 8 h incubation in the dark, radiant exposure 78.9 J/cm2) with the feed assay. If the radiant exposure is doubled to 157.8 J/cm2, 88% of insects are killed by PDI based on 1 mM Chl. The photoactive compound is also effective if presented on strawberries without addition of sucrose with somewhat lower moribundity (71% at 5 mM Chl). Spraying Chl onto insects is less effective than feeding the photosensitizer: 5 mM Chl resulted in 79.5% moribundity (drug to light interval 8 h, radiant exposure 78.9 J/cm2), but if 5 h of sun light (532 J/cm2) and overnight (14 h) dark incubation is used for activation of Chl, more than 95% of insects are killed. As conclusion, Chl serves as effective photoinsecticide against Drosophila melanogaster if a drug to light interval of 8 h is maintained. Feeding the photoactive compound together with sucrose is more effective than spraying it onto insects and increasing the radiant exposure allows for lowering the photosensitizer concentration. Photodynamic Inactivation might therefore represent an eco-friendly addition to the farmers armamentarium against (semi-transparent) insects.

A finite element method framework to model extracellular neural stimulation.

Objective.Increasing complexity in extracellular stimulation experiments and neural implant design also requires realistic computer simulations capable of modeling the neural activity of nerve cells under the influence of an electrical stimulus. Classical model approaches are often based on simplifications, are not able to correctly calculate the electric field generated by complex electrode designs, and do not consider electrical effects of the cell on its surrounding. A more accurate approach is the finite element method (FEM), which provides necessary techniques to solve the Poisson equation for complex geometries under consideration of electrical tissue properties. Especially in situations where neurons experience large and non-symmetric extracellular potential gradients, a FEM solution that implements the cell membrane model can improve the computer simulation results. To investigate the response of neurons in an electric field generated by complex electrode designs, a FEM framework for extracellular stimulation was developed in COMSOL.Approach.Methods to implement morphologically- and biophysically-detailed neurons including active Hodgkin-Huxley (HH) cell membrane dynamics as well as the stimulation setup are described in detail. Covered methods are (a) development of cell and electrode geometries including meshing strategies, (b) assignment of physics for the conducting spaces and the realization of active electrodes, (c) implementation of the HH model, and (d) coupling of the physics to get a fully described model.Main results.Several implementation examples are briefly presented: (a) a full FEM implementation of a HH model cell stimulated with a honeycomb electrode, (b) the electric field of a cochlear electrode placed inside the cochlea, and (c) a proof of concept implementation of a detailed double-cable cell membrane model for myelinated nerve fibers.Significance.The presented concepts and methods provide basic and advanced techniques to realize a full FEM framework for innovative studies of neural excitation in response to extracellular stimulation.

Block Phenomena During Electric Micro-Stimulation of Pyramidal Cells and Retinal Ganglion Cells.

Electric micro-stimulation of the nervous system is a means to restore various body functions. The stimulus amplitude necessary to generate action potentials, the lower threshold (LT), is well characterized for many neuronal populations. However, electric overstimulation above an upper threshold (UT) prevents action potential generation and therefore hinders optimal neuro-rehabilitation. Previous studies demonstrated the impact of the UT in micro-stimulation of retinal ganglion cells (RGCs). The observed phenomenon is mostly explained by (i) reversed sodium ion flow in the soma membrane, and (ii) anodal surround block that hinders spike conduction in strongly hyperpolarized regions of the axon at high stimulus intensities. However, up to now, no detailed study of the nature of these phenomena has been presented, particularly for different cell types. Here, we present computational analyses of LT and UT for layer 5 pyramidal cells (PCs) as well as alpha RGCs. Model neurons were stimulated in close vicinity to the cell body and LTs and UTs as well as the ratio UT/LT were compared. Aside from a simple point source electrode and monophasic stimuli also realistic electrode and pulse configurations were examined. The analysis showed: (i) in RGCs, the soma contributed to action potential initiation and block for small electrode distances, whereas in PCs the soma played no role in LTs or UTs. (ii) In both cell types, action potential always initiated within the axon initial segment at LT. (iii) In contrast to a complete block of spike conductance at UT that occurred in RGCs, an incomplete block of spiking appeared in PC axon collaterals. (iv) PC axon collateral arrangement influenced UTs but had small impact on LTs. (v) Population responses of RGCs change from circular regions of activation to ring-shaped patterns for increasing stimulus amplitude. A better understanding of the stimulation window that can reliably activate target neurons will benefit the future development of neuroprostheses.

Mutation testing with hyperproperties.

We present a new method for model-based mutation-driven test case generation. Mutants are generated by making small syntactical modifications to the model or source code of the system under test. A test case kills a mutant if the behavior of the mutant deviates from the original system when running the test. In this work, we use hyperproperties-which allow to express relations between multiple executions-to formalize different notions of killing for both deterministic as well as non-deterministic models. The resulting hyperproperties are universal in the sense that they apply to arbitrary reactive models and mutants. Moreover, an off-the-shelf model checking tool for hyperproperties can be used to generate test cases. Furthermore, we propose solutions to overcome the limitations of current model checking tools via a model transformation and a bounded SMT encoding. We evaluate our approach on a number of models expressed in two different modeling languages by generating tests using a state-of-the-art mutation testing tool.

Blood Management in Revision Total Hip Arthroplasty for Metal-on-Metal Devices: The Efficiency of an Intraoperative Cell Salvage System.

BACKGROUND: The aim of this series was to investigate the efficiency of an intraoperative cell salvage system (ICS) removing metal ions during revision of metal-on-metal (MoM) devices to proof the possibility of re-transfusion of the collected blood. MATERIALS AND METHODS: Between 2014 and 2018, five consecutive patients underwent revision surgery of their MoM total hip arthroplasty due to wear of the polyethylene-metal sandwich inlay or local massive metallosis with aseptic loosing of the cup. Aspiration of joint fluid of all hip prostheses was done and blood was taken to measure the metal ion concentrations, preoperatively. Perioperatively, blood was collected using an ICS before and after filtration and analyzed for Co and Cr concentrations. At that time, there was no re-transfusion of the collected and filtrated blood due to unknown metal ion concentrations. RESULTS: The mean preoperative serum Co and Cr concentrations in the blood were 31.28 µg/L (range 0.22-77.47) and 17.33 µg/L (range 0.59-51.31), whereas the mean local concentrations in the aspiration fluid were 728-fold and 822-fold higher. The Co and Cr concentrations measured in the collected blood before filtration were 70.61 µg/L (range 9.40-173.00) and 337.21 µg/L (range 8.76-1383.0) and decreased markedly to average concentrations of 15.49 µg/L and 41.88 µg/L, respectively. These differences were statistically not significant (Co: p = 0.117, Cr: p = 0.175), although the mean reduction rates were 78% and 88% for Co and Cr, respectively. CONCLUSION: The current series showed that in case of revision of MoM hip devices, metal ions are still contained in the collected blood following filtration using a modern high-level ICS. Therefore, we would only recommend blood re-transfusion in case of low preoperative Co and Cr concentrations and sufficient renal function to warrant patients' safety.

Analysis of upper threshold mechanisms of spherical neurons during extracellular stimulation.

Exceeding a certain stimulation strength can prevent the generation of somatic action potentials, as has been demonstrated in vitro with extracellularly stimulated dorsal root ganglion cells as well as retinal ganglion cells. This phenomenon, termed upper threshold, is currently thought to be a consequence of sodium current reversal in strongly depolarized regions. Here we analyze the contribution of membrane kinetics, using spherical model neurons that are stimulated externally with a microelectrode, in more detail. During extracellular pulse application, the electric field depolarizes one part and hyperpolarizes the other part of the cell. Strong transmembrane currents are generated only in the active depolarized region, changing the overall polarization level. The asymmetric membrane voltage distribution caused by the stimulus strongly influences the cell's behavior during and even after the stimulus. Effects on membrane voltage and transmembrane currents during and after the stimulus are shown and discussed in detail. Aside from the sodium current reversal, two more key mechanisms were identified in causing the upper threshold: strong potassium currents and inactivation of sodium channels. The contributions of the mechanisms involved strongly depend on cell properties, stimulus parameters, and other factors such as temperature. The conclusions presented here are based on several retinal ganglion cell models of the Fohlmeister group, a model with original Hodgkin-Huxley membrane, and a pyramidal cell model. NEW & NOTEWORTHY The upper threshold phenomenon in extracellular stimulation is analyzed in detail for spherical cells. Three main mechanisms were identified that prevent the generation of action potentials at high stimulation strengths: 1) strong potassium currents, 2) inactivating sodium ion channels, and 3) sodium current reversal. Ion channel kinetics in retinal ganglion cells, pyramidal cells, and the original Hodgkin-Huxley model were investigated under the influence of an extracellular stimulus.

Upper stimulation threshold for retinal ganglion cell activation.

OBJECTIVE: The existence of an upper threshold in electrically stimulated retinal ganglion cells (RGCs) is of interest because of its relevance to the development of visual prosthetic devices, which are designed to restore partial sight to blind patients. The upper threshold is defined as the stimulation level above which no action potentials (direct spikes) can be elicited in electrically stimulated retina. APPROACH: We collected and analyzed in vitro recordings from rat RGCs in response to extracellular biphasic (anodic-cathodic) pulse stimulation of varying amplitudes and pulse durations. Such responses were also simulated using a multicompartment model. MAIN RESULTS: We identified the individual cell variability in response to stimulation and the phenomenon known as upper threshold in all but one of the recorded cells (n = 20/21). We found that the latencies of spike responses relative to stimulus amplitude had a characteristic U-shape. In silico, we showed that the upper threshold phenomenon was observed only in the soma. For all tested biphasic pulse durations, electrode positions, and pulse amplitudes above lower threshold, a propagating action potential was observed in the distal axon. For amplitudes above the somatic upper threshold, the axonal action potential back-propagated in the direction of the soma, but the soma's low level of hyperpolarization prevented action potential generation in the soma itself. SIGNIFICANCE: An upper threshold observed in the soma does not prevent spike conductance in the axon.

Impact of Electrode Position on the Elicitation of Sodium Spikes in Retinal Bipolar Cells.

Bipolar cells of the magnocellular pathway in the primate retina can generate action potentials because they have an axonal segment with high sodium channel density, comparable to the sodium channel band in retinal ganglion cells or pyramidal cells. The similarity between the non-human primate and the human retina is of interest for the research on retinal implants for the blind, and especially, the conditions to elicit sodium spikes in bipolar cells using extracellular stimulation. A comparison of excitation characteristics of three model neurons, a bipolar cell, a retinal ganglion cell, and a cortical pyramidal cell, demonstrates the similarities and differences regarding stimulation with microelectrodes. Moving a microelectrode parallel to the axon of a neuron commonly allows to generate spikes for every position - and this rule holds both for cathodic and anodic pulses. However, for the simulated bipolar cell anodic pulses cannot generate sodium spikes directly. Further, there is only a small region for electrode placing where extracellular cathodic stimulation causes direct spike initiation in the sodium channel band. For all other positions, a sodium spike can only be generated by antidromic current flow originating from strongly depolarized terminals.