New equations for redox and nano-signal transduction

Cells maintain redox potentials (Eh) in intracellular compartments, sometimes referred to as redox environments. These potentials are often very reducing, for example in the cytoplasm, but throughout the cell different potentials are maintained, commensurate with the functionality of that particular part of the cell. Furthermore, within a simple cellular compartment, "hot-spots" of redox poise may be maintained. However, despite this complexity, the quantification of such redox potentials has been attempted, and there is indeed a need to accurately assess such potentials, and to monitor how they might change with time. Changes in intracellular potentials may control the oxidation or reduction of protein residues, such as cysteine, which would alter the conformation of those proteins and so modulate their function. Although there are several methods for estimating the intracellular redox potential, the most accessible technique is the measurement of intracellular concentrations of GSH and GSSG, and the calculation of Eh using the Nernst equation. However, using this equation shows that the Eh imposed by the glutathione couple is dependent on the total concentration of glutathione present, and therefore values of Eh obtained may be erroneous. Here, we suggest new equations that can be used to calculate the redox environments of cells.     

Hydrogen peroxide-induced gene expression in Arabidopsis thaliana

Hydrogen peroxide (H(2)O(2)) is generated in plants after exposure to a variety of biotic and abiotic stresses, and has been shown to induce a number of cellular responses. Previously, we showed that H(2)O(2) generated during plant-elicitor interactions acts as a signaling molecule to induce the expression of defense genes and initiate programmed cell death in Arabidopsis thaliana suspension cultures. Here, we report for the first time the identification by RNA differential display of four genes whose expression is induced by H(2)O(2). These include genes that have sequence homology to previously identified Arabidopsis genes encoding a late embryogenesis-abundant protein, a DNA-damage repair protein, and a serine/threonine kinase. Their putative roles in H(2)O(2)-induced defense responses are discussed.     

Signaling on the Stigma: Potential New Roles for ROS and NO in Plant Cell Signaling

Reactive oxygen species (ROS) and reactive nitrogen species, particularly NO, are key components of diverse signaling networks in animals and plants. We have recently shown that epidermal cells of stigmas from a range of different angiosperms accumulate relatively large amounts of ROS, principally H₂O₂, whereas pollen produces NO. Importantly, ROS/H₂O₂ levels appeared reduced in stigma cells supporting developing pollen grains compared to cells without pollen grains attached. To explore a possible link between pollen NO production and reduced levels of stigmatic ROS/H₂O₂, we supplied stigmas with NO and observed an overall reduction in levels of stigmatic ROS/H₂O₂. These new and unexpected data suggest a potential new signaling role for ROS/H₂O₂ and NO in pollen-stigma recognition processes.Key Words: stigma, pollen, reactive oxygen species, hydrogen peroxide, nitric oxide, signaling, defense     

Effects of a Novel Pharmacologic Inhibitor of Myeloperoxidase in a Mouse Atherosclerosis Model

Inflammation and oxidative stress play fundamental roles in the pathogenesis of atherosclerosis. Myeloperoxidase has been extensively implicated as a key mediator of inflammatory and redox-dependent processes in atherosclerosis. However, the effect of synthetic myeloperoxidase inhibitors on atherosclerosis has been insufficiently studied. In this study, ApoE^−/− mice were randomized to low- and high-dose INV-315 groups for 16 weeks on high-fat diet. INV-315 resulted in reduced plaque burden and improved endothelial function in response to acetylcholine. These effects occurred without adverse events or changes in body weight or blood pressure. INV-315 treatment resulted in a decrease in iNOS gene expression, superoxide production and nitrotyrosine content in the aorta. Circulating IL-6 and inflammatory CD11b⁺/Ly6G^low/7/4^hi monocytes were significantly decreased in response to INV-315 treatment. Acute pretreatment with INV-315 blocked TNFα-mediated leukocyte adhesion in cremasteric venules and inhibited myeloperoxidase activity. Cholesterol efflux was significantly increased by high-dose INV-315 via ex-vivo reverse cholesterol transport assays. Our results suggest that myeloperoxidase inhibition may exert anti-atherosclerotic effects via inhibition of oxidative stress and enhancement of cholesterol efflux. These findings demonstrate a role for pharmacologic modulation of myeloperoxidase in atherosclerosis.     

Targeting TMPRSS2 and Cathepsin B/L together may be synergistic against SARS-CoV-2 infection

The entry of SARS-CoV-2 into target cells requires the activation of its surface spike protein, S, by host proteases. The host serine protease TMPRSS2 and cysteine proteases Cathepsin B/L can activate S, making two independent entry pathways accessible to SARS-CoV-2. Blocking the proteases prevents SARS-CoV-2 entry in vitro. This blockade may be achieved in vivo through ‘repurposing’ drugs, a potential treatment option for COVID-19 that is now in clinical trials. Here, we found, surprisingly, that drugs targeting the two pathways, although independent, could display strong synergy in blocking virus entry. We predicted this synergy first using a mathematical model of SARS-CoV-2 entry and dynamics in vitro. The model considered the two pathways explicitly, let the entry efficiency through a pathway depend on the corresponding protease expression level, which varied across cells, and let inhibitors compromise the efficiency in a dose-dependent manner. The synergy predicted was novel and arose from effects of the drugs at both the single cell and the cell population levels. Validating our predictions, available in vitro data on SARS-CoV-2 and SARS-CoV entry displayed this synergy. Further, analysing the data using our model, we estimated the relative usage of the two pathways and found it to vary widely across cell lines, suggesting that targeting both pathways in vivo may be important and synergistic given the broad tissue tropism of SARS-CoV-2. Our findings provide insights into SARS-CoV-2 entry into target cells and may help improve the deployability of drug combinations targeting host proteases required for the entry.     

Speech facilitation by left inferior frontal cortex stimulation

Electrophysiological studies in humans and animals suggest that noninvasive neurostimulation methods such as transcranial direct current stimulation (tDCS) can elicit long-lasting [1], polarity-dependent [2] changes in neocortical excitability. Application of tDCS can have significant and selective behavioral consequences that are associated with the cortical location of the stimulation electrodes and the task engaged during stimulation [3-8]. However, the mechanism by which tDCS affects human behavior is unclear. Recently, functional magnetic resonance imaging (fMRI) has been used to determine the spatial topography of tDCS effects [9-13], but no behavioral data were collected during stimulation. The present study is unique in this regard, in that both neural and behavioral responses were recorded using a novel combination of left frontal anodal tDCS during an overt picture-naming fMRI study. We found that tDCS had significant behavioral and regionally specific neural facilitation effects. Furthermore, faster naming responses correlated with decreased blood oxygen level-dependent (BOLD) signal in Broca's area. Our data support the importance of Broca's area within the normal naming network and as such indicate that Broca's area may be a suitable candidate site for tDCS in neurorehabilitation of anomic patients, whose brain damage spares this region.     

Spontaneously Fluctuating Motor Cortex Excitability in Alternating Hemiplegia of Childhood: A Transcranial Magnetic Stimulation Study

Background

Alternating hemiplegia of childhood is a very rare and serious neurodevelopmental syndrome; its genetic basis has recently been established. Its characteristic features include typically-unprovoked episodes of hemiplegia and other transient or more persistent neurological abnormalities.

Methods

We used transcranial magnetic stimulation to assess the effect of the condition on motor cortex neurophysiology both during and between attacks of hemiplegia. Nine people with alternating hemiplegia of childhood were recruited; eight were successfully tested using transcranial magnetic stimulation to study motor cortex excitability, using single and paired pulse paradigms. For comparison, data from ten people with epilepsy but not alternating hemiplegia, and ten healthy controls, were used.

Results

One person with alternating hemiplegia tested during the onset of a hemiplegic attack showed progressively diminishing motor cortex excitability until no response could be evoked; a second person tested during a prolonged bilateral hemiplegic attack showed unusually low excitability. Three people tested between attacks showed asymptomatic variation in cortical excitability, not seen in controls. Paired pulse paradigms, which probe intracortical inhibitory and excitatory circuits, gave results similar to controls.

Conclusions

We report symptomatic and asymptomatic fluctuations in motor cortex excitability in people with alternating hemiplegia of childhood, not seen in controls. We propose that such fluctuations underlie hemiplegic attacks, and speculate that the asymptomatic fluctuation we detected may be useful as a biomarker for disease activity.