Refining Vision Videos

[Context and motivation] Complex software-based systems involve several stakeholders, their activities and interactions with the system. Vision videos are used during the early phases of a project to complement textual representations. They visualize previously abstract visions of the product and its use. By creating, elaborating, and discussing vision videos, stakeholders and developers gain an improved shared understanding of how those abstract visions could translate into concrete scenarios and requirements to which individuals can relate. [Question/problem] In this paper, we investigate two aspects of refining vision videos: (1) Refining the vision by providing alternative answers to previously open issues about the system to be built. (2) A refined understanding of the camera perspective in vision videos. The impact of using a subjective (or "ego") perspective is compared to the usual third-person perspective. [Methodology] We use shopping in rural areas as a real-world application domain for refining vision videos. Both aspects of refining vision videos were investigated in an experiment with 20 participants. [Contribution] Subjects made a significant number of additional contributions when they had received not only video or text but also both - even with very short text and short video clips. Subjective video elements were rated as positive. However, there was no significant preference for either subjective or non-subjective videos in general.Comment: 15 pages, 25th International Working Conference on Requirements Engineering: Foundation for Software Quality 201

Isolation of Uncultured Bacteria from Antarctica Using Long Incubation Periods and Low Nutritional Media

Uncultured microorganisms comprise most of the microbial diversity existing on our planet. Despite advances in environmental sequencing and single-cell genomics, in-depth studies about bacterial metabolism and screening of novel bioproducts can only be assessed by culturing microbes in the laboratory. Here we report uncultured, or recalcitrant, microorganisms from an Antarctic soil sample, using relatively simple methods: oligotrophic media, extended incubation periods, observation under stereo microscopy, and selection of slow-growing bacteria. We managed to isolate several rare microorganisms belonging to infrequently isolated or recently described genera, for example Lapillicoccus, Flavitalea, Quadrisphaera, Motilibacter, and Polymorphobacter. Additionally, we obtained isolates presenting 16S rRNA sequence similarity ranging from 92.08 to 94.46% with any other known cultured species, including two distinct isolates from the class Thermoleophilia, that although common in Antarctic soils (as identified by metagenomics), was never reported to be isolated from such samples. Our data indicates that simple methods are still useful for cultivating recalcitrant microorganisms, even when dealing with samples from extreme environments

Cannabidiol causes activated hepatic stellate cell death through a mechanism of endoplasmic reticulum stress-induced apoptosis

The major cellular event in the development and progression of liver fibrosis is the activation of hepatic stellate cells (HSCs). Activated HSCs proliferate and produce excess collagen, leading to accumulation of scar matrix and fibrotic liver. As such, the induction of activated HSC death has been proposed as a means to achieve resolution of liver fibrosis. Here we demonstrate that cannabidiol (CBD), a major non-psychoactive component of the plant Cannabis sativa, induces apoptosis in activated HSCs through a cannabinoid receptor-independent mechanism. CBD elicits an endoplasmic reticulum (ER) stress response, characterized by changes in ER morphology and the initiation of RNA-dependent protein kinase-like ER kinase-, activating transcription factor-6-, and inositol-requiring ER-to-nucleus signal kinase-1 (IRE1)-mediated signaling cascades. Furthermore, CBD induces downstream activation of the pro-apoptotic IRE1/ASK1/c-Jun N-terminal kinase pathway, leading to HSC death. Importantly, we show that this mechanism of CBD-induced ER stress-mediated apoptosis is specific to activated HSCs, as it occurs in activated human and rat HSC lines, and in primary in vivo-activated mouse HSCs, but not in quiescent HSCs or primary hepatocytes from rat. Finally, we provide evidence that the elevated basal level of ER stress in activated HSCs has a role in their susceptibility to the pro-apoptotic effect of CBD. We propose that CBD, by selectively inducing death of activated HSCs, represents a potential therapeutic agent for the treatment of liver fibrosis

A Threshold Equation for Action Potential Initiation

In central neurons, the threshold for spike initiation can depend on the stimulus and varies between cells and between recording sites in a given cell, but it is unclear what mechanisms underlie this variability. Properties of ionic channels are likely to play a role in threshold modulation. We examined in models the influence of Na channel activation, inactivation, slow voltage-gated channels and synaptic conductances on spike threshold. We propose a threshold equation which quantifies the contribution of all these mechanisms. It provides an instantaneous time-varying value of the threshold, which applies to neurons with fluctuating inputs. We deduce a differential equation for the threshold, similar to the equations of gating variables in the Hodgkin-Huxley formalism, which describes how the spike threshold varies with the membrane potential, depending on channel properties. We find that spike threshold depends logarithmically on Na channel density, and that Na channel inactivation and K channels can dynamically modulate it in an adaptive way: the threshold increases with membrane potential and after every action potential. Our equation was validated with simulations of a previously published multicompartemental model of spike initiation. Finally, we observed that threshold variability in models depends crucially on the shape of the Na activation function near spike initiation (about −55 mV), while its parameters are adjusted near half-activation voltage (about −30 mV), which might explain why many models exhibit little threshold variability, contrary to experimental observations. We conclude that ionic channels can account for large variations in spike threshold

Subcellular optogenetic inhibition of G proteins generates signaling gradients and cell migration

Cells sense gradients of extracellular cues and generate polarized responses such as cell migration and neurite initiation. There is static information on the intracellular signaling molecules involved in these responses, but how they dynamically orchestrate polarized cell behaviors is not well understood. A limitation has been the lack of methods to exert spatial and temporal control over specific signaling molecules inside a living cell. Here we introduce optogenetic tools that act downstream of native G protein–coupled receptor (GPCRs) and provide direct control over the activity of endogenous heterotrimeric G protein subunits. Light-triggered recruitment of a truncated regulator of G protein signaling (RGS) protein or a Gβγ-sequestering domain to a selected region on the plasma membrane results in localized inhibition of G protein signaling. In immune cells exposed to spatially uniform chemoattractants, these optogenetic tools allow us to create reversible gradients of signaling activity. Migratory responses generated by this approach show that a gradient of active G protein αi and βγ subunits is sufficient to generate directed cell migration. They also provide the most direct evidence so for a global inhibition pathway triggered by Gi signaling in directional sensing and adaptation. These optogenetic tools can be applied to interrogate the mechanistic basis of other GPCR-modulated cellular functions

Warm Body Temperature Facilitates Energy Efficient Cortical Action Potentials

The energy efficiency of neural signal transmission is important not only as a limiting factor in brain architecture, but it also influences the interpretation of functional brain imaging signals. Action potential generation in mammalian, versus invertebrate, axons is remarkably energy efficient. Here we demonstrate that this increase in energy efficiency is due largely to a warmer body temperature. Increases in temperature result in an exponential increase in energy efficiency for single action potentials by increasing the rate of Na+ channel inactivation, resulting in a marked reduction in overlap of the inward Na+, and outward K+, currents and a shortening of action potential duration. This increase in single spike efficiency is, however, counterbalanced by a temperature-dependent decrease in the amplitude and duration of the spike afterhyperpolarization, resulting in a nonlinear increase in the spike firing rate, particularly at temperatures above approximately 35°C. Interestingly, the total energy cost, as measured by the multiplication of total Na+ entry per spike and average firing rate in response to a constant input, reaches a global minimum between 37–42°C. Our results indicate that increases in temperature result in an unexpected increase in energy efficiency, especially near normal body temperature, thus allowing the brain to utilize an energy efficient neural code

Protection of Visual Functions by Human Neural Progenitors in a Rat Model of Retinal Disease

BACKGROUND: A promising clinical application for stem and progenitor cell transplantation is in rescue therapy for degenerative diseases. This strategy seeks to preserve rather than restore host tissue function by taking advantage of unique properties often displayed by these versatile cells. In studies using different neurodegenerative disease models, transplanted human neural progenitor cells (hNPC) protected dying host neurons within both the brain and spinal cord. Based on these reports, we explored the potential of hNPC transplantation to rescue visual function in an animal model of retinal degeneration, the Royal College of Surgeons rat. METHODOLOGY/PRINCIPAL FINDINGS: Animals received unilateral subretinal injections of hNPC or medium alone at an age preceding major photoreceptor loss. Principal outcomes were quantified using electroretinography, visual acuity measurements and luminance threshold recordings from the superior colliculus. At 90–100 days postnatal, a time point when untreated rats exhibit little or no retinal or visual function, hNPC-treated eyes retained substantial retinal electrical activity and visual field with near-normal visual acuity. Functional efficacy was further enhanced when hNPC were genetically engineered to secrete glial cell line-derived neurotrophic factor. Histological examination at 150 days postnatal showed hNPC had formed a nearly continuous pigmented layer between the neural retina and retinal pigment epithelium, as well as distributed within the inner retina. A concomitant preservation of host cone photoreceptors was also observed. CONCLUSIONS/SIGNIFICANCE: Wild type and genetically modified human neural progenitor cells survive for prolonged periods, migrate extensively, secrete growth factors and rescue visual functions following subretinal transplantation in the Royal College of Surgeons rat. These results underscore the potential therapeutic utility of hNPC in the treatment of retinal degenerative diseases and suggest potential mechanisms underlying their effect in vivo

Characterisation of a Peripheral Neuropathic Component of the Rat Monoiodoacetate Model of Osteoarthritis

Joint degeneration observed in the rat monoiodoacetate (MIA) model of osteoarthritis shares many histological features with the clinical condition. The accompanying pain phenotype has seen the model widely used to investigate the pathophysiology of osteoarthritis pain, and for preclinical screening of analgesic compounds. We have investigated the pathophysiological sequellae of MIA used at low (1 mg) or high (2 mg) dose. Intra-articular 2 mg MIA induced expression of ATF-3, a sensitive marker for peripheral neuron stress/injury, in small and large diameter DRG cell profiles principally at levels L4 and 5 (levels predominated by neurones innervating the hindpaw) rather than L3. At the 7 day timepoint, ATF-3 signal was significantly smaller in 1 mg MIA treated animals than in the 2 mg treated group. 2 mg, but not 1 mg, intra-articular MIA was also associated with a significant reduction in intra-epidermal nerve fibre density in plantar hindpaw skin, and produced spinal cord dorsal and ventral horn microgliosis. The 2 mg treatment evoked mechanical pain-related hypersensitivity of the hindpaw that was significantly greater than the 1 mg treatment. MIA treatment produced weight bearing asymmetry and cold hypersensitivity which was similar at both doses. Additionally, while pregabalin significantly reduced deep dorsal horn evoked neuronal responses in animals treated with 2 mg MIA, this effect was much reduced or absent in the 1 mg or sham treated groups. These data demonstrate that intra-articular 2 mg MIA not only produces joint degeneration, but also evokes significant axonal injury to DRG cells including those innervating targets outside of the knee joint such as hindpaw skin. This significant neuropathic component needs to be taken into account when interpreting studies using this model, particularly at doses greater than 1 mg MIA

Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018.

Over the past decade, the Nomenclature Committee on Cell Death (NCCD) has formulated guidelines for the definition and interpretation of cell death from morphological, biochemical, and functional perspectives. Since the field continues to expand and novel mechanisms that orchestrate multiple cell death pathways are unveiled, we propose an updated classification of cell death subroutines focusing on mechanistic and essential (as opposed to correlative and dispensable) aspects of the process. As we provide molecularly oriented definitions of terms including intrinsic apoptosis, extrinsic apoptosis, mitochondrial permeability transition (MPT)-driven necrosis, necroptosis, ferroptosis, pyroptosis, parthanatos, entotic cell death, NETotic cell death, lysosome-dependent cell death, autophagy-dependent cell death, immunogenic cell death, cellular senescence, and mitotic catastrophe, we discuss the utility of neologisms that refer to highly specialized instances of these processes. The mission of the NCCD is to provide a widely accepted nomenclature on cell death in support of the continued development of the field