A Deep Learning Method for Fully Automatic Stomatal Morphometry and Maximal Conductance Estimation

Stomata are integral to plant performance, enabling the exchange of gases between the atmosphere and the plant. The anatomy of stomata influences conductance properties with the maximal conductance rate, gsmax, calculated from density and size. However, current calculations of stomatal dimensions are performed manually, which are time-consuming and error prone. Here, we show how automated morphometry from leaf impressions can predict a functional property: the anatomical gsmax. A deep learning network was derived to preserve stomatal morphometry via semantic segmentation. This forms part of an automated pipeline to measure stomata traits for the estimation of anatomical gsmax. The proposed pipeline achieves accuracy of 100% for the distinction (wheat vs. poplar) and detection of stomata in both datasets. The automated deep learning-based method gave estimates for gsmax within 3.8 and 1.9% of those values manually calculated from an expert for a wheat and poplar dataset, respectively. Semantic segmentation provides a rapid and repeatable method for the estimation of anatomical gsmax from microscopic images of leaf impressions. This advanced method provides a step toward reducing the bottleneck associated with plant phenotyping approaches and will provide a rapid method to assess gas fluxes in plants based on stomata morphometry.

SUMOylation of the Kv4.2 Ternary Complex Increases Surface Expression and Current Amplitude by Reducing Internalization in HEK 293 Cells

Kv4 α-subunits exist as ternary complexes (TC) with potassium channel interacting proteins (KChIP) and dipeptidyl peptidase-like proteins (DPLP); multiple ancillary proteins also interact with the α-subunits throughout the channel’s lifetime. Dynamic regulation of Kv4.2 protein interactions adapts the transient potassium current, IA, mediated by Kv4 α-subunits. Small ubiquitin-like modifier (SUMO) is an 11 kD peptide post-translationally added to lysine (K) residues to regulate protein–protein interactions. We previously demonstrated that when expressed in human embryonic kidney (HEK) cells, Kv4.2 can be SUMOylated at two K residues, K437 and K579. SUMOylation at K437 increased surface expression of electrically silent channels while SUMOylation at K579 reduced IA maximal conductance (Gmax) without altering surface expression. KChIP and DPLP subunits are known to modify the pattern of Kv4.2 post-translational decorations and/or their effects. In this study, co-expressing Kv4.2 with KChIP2a and DPP10c altered the effects of enhanced Kv4.2 SUMOylation. First, the effect of enhanced SUMOylation was the same for a TC containing either the wild-type Kv4.2 or the mutant K437R Kv4.2, suggesting that either the experimental manipulation no longer enhanced K437 SUMOylation or K437 SUMOylation no longer influenced Kv4.2 surface expression. Second, instead of decreasing IA Gmax, enhanced SUMOylation at K579 now produced a significant ∼37–70% increase in IA maximum conductance (Gmax) and a significant ∼30–50% increase in Kv4.2g surface expression that was accompanied by a 65% reduction in TC internalization. Blocking clathrin-mediated endocytosis (CME) in HEK cells expressing the Kv4.2 TC mimicked and occluded the effect of SUMO on IA Gmax; however, the amount of Kv4.2 associated with the major adaptor for constitutive CME, adaptor protein 2 (AP2), was not SUMO dependent. Thus, SUMOylation reduced Kv4.2 internalization by acting downstream of Kv4.2 recruitment into clathrin-coated pits. In sum, the two major findings of this study are: SUMOylation of Kv4.2 at K579 regulates TC internalization most likely by promoting channel recycling. Additionally, there is a reciprocity between Kv4.2 SUMOylation and the Kv4.2 interactome such that SUMOylation regulates the interactome and the interactome influences the pattern and effect of SUMOylation.

Bidirectional flow of the funny current (If) during the pacemaking cycle in murine sinoatrial node myocytes

Sinoatrial node myocytes (SAMs) act as cardiac pacemaker cells by firing spontaneous action potentials (APs) that initiate each heartbeat. The funny current (If) is critical for the generation of these spontaneous APs; however, its precise role during the pacemaking cycle remains unresolved. Here, we used the AP-clamp technique to quantify If during the cardiac cycle in mouse SAMs. We found that If is persistently active throughout the sinoatrial AP, with surprisingly little voltage-dependent gating. As a consequence, it carries both inward and outward current around its reversal potential of −30 mV. Despite operating at only 2 to 5% of its maximal conductance, If carries a substantial fraction of both depolarizing and repolarizing net charge movement during the firing cycle. We also show that β-adrenergic receptor stimulation increases the percentage of net depolarizing charge moved by If, consistent with a contribution of If to the fight-or-flight increase in heart rate. These properties were confirmed by heterologously expressed HCN4 channels and by mathematical models of If. Modeling further suggested that the slow rates of activation and deactivation of the HCN4 isoform underlie the persistent activity of If during the sinoatrial AP. These results establish a new conceptual framework for the role of If in pacemaking, in which it operates at a very small fraction of maximal activation but nevertheless drives membrane potential oscillations in SAMs by providing substantial driving force in both inward and outward directions.

Truncating Tau Reveals Different Pathophysiological Actions of Oligomers in Single Neurons

Tau protein is involved in maintaining the structural integrity of neurons. In tauopathies, including Alzheimer's disease, tau forms oligomers, which can modulate neuronal function. Previously the introduction of oligomeric full-length human tau (aa 1-441; FL-oTau) into pyramidal neurons decreased whole-cell conductance, increased excitability and changed the action potential (AP) waveform. Introducing N-terminally truncated tau (aa 124-441; CFRAG) removed the effects on the AP waveform and input resistance but the increase in excitability remained. A hyperpolarising shift in spike threshold underlies this increase in excitability. The N-terminal fragment (aa 1-123; NFRAG) markedly increased input resistance and changed the AP waveform. Lower concentrations of NFRAG only changed the AP waveform. Thus the two truncations can recapitulate the effects of FL-oTau. To investigate underlying mechanisms, we recorded sodium currents and found that FL-oTau lowers the activation voltage and reduced the maximal conductance, consistent with the lower spike threshold and reduction in AP amplitude.

Deterministic and Stochastic Cellular Mechanisms Contributing to Carbon Monoxide Induced Ventricular Arrhythmias

Chronic exposure to low levels of Carbon Monoxide is associated with an increased risk of cardiac arrhythmia. Microelectrode recordings from rat and guinea pig single isolated ventricular myocytes exposed to CO releasing molecule CORM-2 and excited at 0.2/s show repolarisation changes that develop over hundreds of seconds: action potential prolongation by delayed repolarisation, EADs, multiple EADs and oscillations around the plateau, leading to irreversible repolarisation failure. The measured direct effects of CO on currents in these cells, and ion channels expressed in mammalian systems showed an increase in prolonged late Na+, and a decrease in the maximal T- and L-type Ca++. peak and late Na+, ultra-rapid delayed, delayed rectifier, and the inward rectifier K+ currents. Incorporation of these CO induced changes in maximal currents in ventricular cell models; (Gattoni et al., J. Physiol., 2016, 594, 4193–4224) (rat) and (Luo and Rudy, Circ. Res., 1994, 74, 1071–1096) (guinea-pig) and human endo-, mid-myo- and epi-cardial (O’Hara et al., PLoS Comput. Biol., 2011, 7, e1002061) models, by changes in maximal ionic conductance reproduces these repolarisation abnormalities. Simulations of cell populations with Gaussian distributions of maximal conductance parameters predict a CO induced increase in APD and its variability. Incorporation of these predicted CO induced conductance changes in human ventricular cell electrophysiology into ventricular tissue and wall models give changes in indices for the probability of the initiation of re-entrant arrhythmia.

M-current induced Bogdanov–Takens bifurcation and switching of neuron excitability class

In this work, we consider a general conductance-based neuron model with the inclusion of the acetycholine sensitive, M-current. We study bifurcations in the parameter space consisting of the applied current $I_{app}$ I a p p , the maximal conductance of the M-current $g_{M}$ g M and the conductance of the leak current $g_{L}$ g L . We give precise conditions for the model that ensure the existence of a Bogdanov–Takens (BT) point and show that such a point can occur by varying $I_{app}$ I a p p and $g_{M}$ g M . We discuss the case when the BT point becomes a Bogdanov–Takens–cusp (BTC) point and show that such a point can occur in the three-dimensional parameter space. The results of the bifurcation analysis are applied to different neuronal models and are verified and supplemented by numerical bifurcation diagrams generated using the package . We conclude that there is a transition in the neuronal excitability type organised by the BT point and the neuron switches from Class-I to Class-II as conductance of the M-current increases.

From Biophysical to Integrate-and-Fire Modeling

This article proposes a methodology to extract a low-dimensional integrate-and-fire model from an arbitrarily detailed single-compartment biophysical model. The method aims at relating the modulation of maximal conductance parameters in the biophysical model to the modulation of parameters in the proposed integrate-and-fire model. The approach is illustrated on two well-documented examples of cellular neuromodulation: the transition between type I and type II excitability and the transition between spiking and bursting.

A Potential Mechanism of Sodium Channel Mediating the General Anesthesia Induced by Propofol

General anesthesia has revolutionized healthcare over the past 200 years and continues to show advancements. However, many phenomena induced by general anesthetics including paradoxical excitation are still poorly understood. Voltage-gated sodium channels (NaV) were believed to be one of the proteins targeted during general anesthesia. Based on electrophysiological measurements before and after propofol treatments of different concentrations, we mathematically modified the Hodgkin–Huxley sodium channel formulations and constructed a thalamocortical model to investigate the potential roles of NaV. The ion channels of individual neurons were modeled using the Hodgkin–Huxley type equations. The enhancement of propofol-induced GABAa current was simulated by increasing the maximal conductance and the time-constant of decay. Electroencephalogram (EEG) was evaluated as the post-synaptic potential from pyramidal (PY) cells. We found that a left shift in activation of NaV was induced primarily by a low concentration of propofol (0.3–10 μM), while a left shift in inactivation of NaV was induced by an increasing concentration (0.3–30 μM). Mathematical simulation indicated that a left shift of NaV activation produced a Hopf bifurcation, leading to cell oscillations. Left shift of NaV activation around a value of 5.5 mV in the thalamocortical models suppressed normal bursting of thalamocortical (TC) cells by triggering its chaotic oscillations. This led to irregular spiking of PY cells and an increased frequency in EEG readings. This observation suggests a mechanism leading to paradoxical excitation during general anesthesia. While a left shift in inactivation led to light hyperpolarization in individual cells, it inhibited the activity of the thalamocortical model after a certain depth of anesthesia. This finding implies that high doses of propofol inhibit the network partly by accelerating NaV toward inactivation. Additionally, this result explains why the application of sodium channel blockers decreases the requirement for general anesthetics. Our study provides an insight into the roles that NaV plays in the mechanism of general anesthesia. Since the activation and inactivation of NaV are structurally independent, it should be possible to avoid side effects by state-dependent binding to the NaV to achieve precision medicine in the future.

Exercise Training as a Mediator for Enhancing Coronary Collateral Circulation: A Review of the Evidence

: Coronary collateral vessels supply blood to areas of myocardium at risk after arterial occlusion. Flow through these channels is driven by a pressure gradient between the donor and the occluded artery. Concomitant with increased collateral flow is an increase in shear force, a potent stimulus for collateral development (arteriogenesis). Arteriogenesis is self-limiting, often ceasing prematurely when the pressure gradient is reduced by the expanding lumen of the collateral vessel. After the collateral has reached its self-limited maximal conductance, the only way to drive further increases is to re-establish the pressure gradient. During exercise, the myocardial oxygen demand is increased, subsequently increasing coronary flow. Therefore, exercise may represent a means of driving augmented arteriogenesis in patients with stable coronary artery disease. Studies investigating the ability of exercise to drive collateral development in humans are inconsistent. However, these inconsistencies may be due to the heterogeneity of assessment methods used to quantify change. This article summarises current evidence pertaining to the role of exercise in the development of coronary collaterals, highlighting areas of future research.

Negative Modulation of TRPM8 Channel Function by Protein Kinase C in Trigeminal Cold Thermoreceptor Neurons

TRPM8 is the main molecular entity responsible for cold sensing. This polymodal ion channel is activated by cold, cooling compounds such as menthol, voltage, and rises in osmolality. In corneal cold thermoreceptor neurons (CTNs), TRPM8 expression determines not only their sensitivity to cold, but also their role as neural detectors of ocular surface wetness. Several reports suggest that Protein Kinase C (PKC) activation impacts on TRPM8 function; however, the molecular bases of this functional modulation are still poorly understood. We explored PKC-dependent regulation of TRPM8 using Phorbol 12-Myristate 13-Acetate to activate this kinase. Consistently, recombinant TRPM8 channels, cultured trigeminal neurons, and free nerve endings of corneal CTNs revealed a robust reduction of TRPM8-dependent responses under PKC activation. In corneal CTNs, PKC activation decreased ongoing activity, a key parameter in the role of TRPM8-expressing neurons as humidity detectors, and also the maximal cold-evoked response, which were validated by mathematical modeling. Biophysical analysis indicated that PKC-dependent downregulation of TRPM8 is mainly due to a decreased maximal conductance value, and complementary noise analysis revealed a reduced number of functional channels at the cell surface, providing important clues to understanding the molecular mechanisms of how PKC activity modulates TRPM8 channels in CTNs.