Cross Pharmacological, Biochemical and Computational Studies of a Human Kv3.1b Inhibitor from Androctonus australis Venom

The voltage-gated K+ channels Kv3.1 display fast activation and deactivation kinetics and are known to have a crucial contribution to the fast-spiking phenotype of certain neurons. AahG50, as a natural product extracted from Androctonus australis hector venom, inhibits selectively Kv3.1 channels. In the present study, we focused on the biochemical and pharmacological characterization of the component in AahG50 scorpion venom that potently and selectively blocks the Kv3.1 channels. We used a combined optimization through advanced biochemical purification and patch-clamp screening steps to characterize the peptide in AahG50 active on Kv3.1 channels. We described the inhibitory effect of a toxin on Kv3.1 unitary current in black lipid bilayers. In silico, docking experiments are used to study the molecular details of the binding. We identified the first scorpion venom peptide inhibiting Kv3.1 current at 170 nM. This toxin is the alpha-KTx 15.1, which occludes the Kv3.1 channel pore by means of the lysine 27 lateral chain. This study highlights, for the first time, the modulation of the Kv3.1 by alpha-KTx 15.1, which could be an interesting starting compound for developing therapeutic biomolecules against Kv3.1-associated diseases.

Fast activation maximization for molecular sequence design

Background Optimization of DNA and protein sequences based on Machine Learning models is becoming a powerful tool for molecular design. Activation maximization offers a simple design strategy for differentiable models: one-hot coded sequences are first approximated by a continuous representation, which is then iteratively optimized with respect to the predictor oracle by gradient ascent. While elegant, the current version of the method suffers from vanishing gradients and may cause predictor pathologies leading to poor convergence. Results Here, we introduce Fast SeqProp, an improved activation maximization method that combines straight-through approximation with normalization across the parameters of the input sequence distribution. Fast SeqProp overcomes bottlenecks in earlier methods arising from input parameters becoming skewed during optimization. Compared to prior methods, Fast SeqProp results in up to 100-fold faster convergence while also finding improved fitness optima for many applications. We demonstrate Fast SeqProp’s capabilities by designing DNA and protein sequences for six deep learning predictors, including a protein structure predictor. Conclusions Fast SeqProp offers a reliable and efficient method for general-purpose sequence optimization through a differentiable fitness predictor. As demonstrated on a variety of deep learning models, the method is widely applicable, and can incorporate various regularization techniques to maintain confidence in the sequence designs. As a design tool, Fast SeqProp may aid in the development of novel molecules, drug therapies and vaccines.

Cryo-EM structure of the human Kv3.1 channel reveals gating control by the cytoplasmic T1 domain

Kv3 channels have distinctive gating kinetics tailored for rapid repolarization in fast-spiking neurons. Malfunction of this process due to genetic variants in the KCNC1 gene causes severe epileptic disorders, yet the structural determinants for the unusual gating properties remain elusive. Here, we present cryo-EM structures of the human Kv3.1a channel, revealing a unique arrangement of the cytoplasmic T1 domain which facilitates interactions with C-terminal axonal targeting motif and key components of the gating machinery. Additional interactions between S1/S2 linker and turret domain strengthen the VSD/PD interface. Supported by MD simulations and electrophysiological and mutational analyses, we identify close communication between α6 helix of T1 domain, S4/S5 linker and S6T helix as responsible for the ultra-fast activation/deactivation and open state stabilisation that are unique to Kv3 channels. These findings provide fundamentally new insights into gating control and disease mechanisms and guide strategies for the design of pharmaceutical drugs targeting Kv3 channels.

Integrated Underreamer Technology with Real-Time Communication Helped Eliminate Rathole in Exploratory Operation Offshore Nigeria

This paper describes the successful deployment of integrated underreamer technology with real-time communication through mud-pulse telemetry system, to drill and eliminate rathole in 17 1/2-in × 20-in successfully in one run and helped set casing as close as possible to the depth of suspected pressure ramp on an exploratory well offshore Nigeria. This technology uses the same communication system (actuator bypass) as Measurement While Drilling tools (MWD), Logging While Drilling tools (LWD) and Rotary Steerable System (RSS). Integrated underreamers broadly used in the drilling operations support optimized casing and completion programs and helps reduce operational risks such as wellbore instability. The ball drop and hydraulically activated reamer technologies available today comes with limitations and HSE risks. The distinctive functionalities of the integrated underreamer technology described here, such as unlimited and fast activation and deactivation via downlinking and real time downhole feedback, reduce uncertainties and operational costs in the complex and challenging deep offshore drilling operations. The real-time communication through mud-pulse telemetry system enabled the placement of integrated underreamer 6 meters from the bit thereby reducing rathole length to approximately 9 meters compared to 80 meters for conventional underreamer application. The integrated underreamer is compatible with existing RSS and provide unlimited activation cycles. The integrated underreamer offers flexibility in placement in the bottom hole assembly (BHA) and it can be used as a near bit reamer, or as main reamer or as both. In this case, the integrated near bit underreamer eliminated the need for a dedicated rathole removal run. It also offered a feedback confirmation of the cutter blades activation status and provided hole opening log thereby reducing the operational uncertainties for the under reaming, saving rig time up to 16 hours for shoulder test. The underreamer was successfully deployed to drill and ream the challenging 14 ¾" × 17 ½" and ream 17 ½" × 20" section offshore Nigeria. Both sections were drilled and reamed to section Total Depth (TD) in one run with all directional reuirements and Measuring While Drilling (MWD)/Logging While Drilling (LWD) met saving client approximately 4 days of rig spread cost. The reamer appeared to provide an in-gauge borehole allowing for successful running and cementing of liners without any issues, demonstrating superior borehole quality. The new Technology proved to be a reliable and flexible hole enlargement while drilling solution that help to improve drilling performance, reduce operational risks and save cost.

A proximal–distal difference in bat wing muscle thermal sensitivity parallels a difference in operating temperatures along the wing

Flight is a demanding form of locomotion, requiring fast activation and relaxation in wing muscles to produce the necessary wingbeat frequencies. Bats maintain high body temperatures during flight, but their wing muscles cool under typical environmental conditions. Because distal wing muscles are colder during flight than proximal muscles, we hypothesized that they would be less temperature sensitive to compensate for temperature effects, resulting in proximal–distal differences in temperature sensitivity that match differences in muscle operating temperature. We measured contractile rates across temperatures in the proximal pectoralis muscle and an interosseous in the handwing of Carollia perspicillata , a small neotropical fruit bat, and compared their thermal dependence with that of a forearm muscle measured in a previous study. We found that the contractile properties of the pectoralis were significantly more temperature sensitive than those of the distal muscles. This suggests that cooling of the distal wing muscles imposes a selective pressure on muscle contractile function which has led to shifts in temperature sensitivity. This study is the first to demonstrate differences in temperature sensitivity along the length of a single limb in an endotherm and suggests that temperature variation may be underappreciated as a determinant of locomotor performance in endotherms generally.

Why Ablation of Sites With Purkinje Activation Is Antiarrhythmic: The Interplay Between Fast Activation and Arrhythmogenesis

Ablation of sites showing Purkinje activity is antiarrhythmic in some patients with idiopathic ventricular fibrillation (iVF). The mechanism for the therapeutic success of ablation is not fully understood. We propose that deeper penetrance of the Purkinje network allows faster activation of the ventricles and is proarrhythmic in the presence of steep repolarization gradients. Reduction of Purkinje penetrance, or its indirect reducing effect on apparent propagation velocity may be a therapeutic target in patients with iVF.

Chaperone-Mediated Stress Sensing in Mycobacterium tuberculosis Enables Fast Activation and Sustained Response

ABSTRACT Dynamical properties of gene regulatory networks are tuned to ensure bacterial survival. In mycobacteria, the MprAB-σE network responds to the presence of stressors, such as surfactants that cause surface stress. Positive feedback loops in this network were previously predicted to cause hysteresis, i.e., different responses to identical stressor levels for prestressed and unstressed cells. Here, we show that hysteresis does not occur in nonpathogenic Mycobacterium smegmatis but does occur in Mycobacterium tuberculosis. However, the observed rapid temporal response in M. tuberculosis is inconsistent with the model predictions. To reconcile these observations, we implement a recently proposed mechanism for stress sensing, namely, the release of MprB from the inhibitory complex with the chaperone DnaK upon the stress exposure. Using modeling and parameter fitting, we demonstrate that this mechanism can accurately describe the experimental observations. Furthermore, we predict perturbations in DnaK expression that can strongly affect dynamical properties. Experiments with these perturbations agree with model predictions, confirming the role of DnaK in fast and sustained response. IMPORTANCE Gene regulatory networks controlling stress response in mycobacterial species have been linked to persistence switches that enable bacterial dormancy within a host. However, the mechanistic basis of switching and stress sensing is not fully understood. In this paper, combining quantitative experiments and mathematical modeling, we uncover how interactions between two master regulators of stress response—the MprAB two-component system (TCS) and the alternative sigma factor σE—shape the dynamical properties of the surface stress network. The result show hysteresis (history dependence) in the response of the pathogenic bacterium M. tuberculosis to surface stress and lack of hysteresis in nonpathogenic M. smegmatis. Furthermore, to resolve the apparent contradiction between the existence of hysteresis and fast activation of the response, we utilize a recently proposed role of chaperone DnaK in stress sensing. These result leads to a novel system-level understanding of bacterial stress response dynamics.

Elicitins' Oligomeric States Affect The Hypersensitive Response And Resistance In Tobacco

Successful plant defence against microbial pathogens is based on early recognition and fast activation of inducible responses. Key mechanisms include detection of microbe-associated molecular patterns by membrane-localized Pattern Recognition Receptors that induce a basal resistance response. A well-described model of such responses to pathogens involves interaction between Solanaceae plants with proteinaceous elicitors secreted by oomycetes, called elicitins. It has been hypothesised that elicitins' formation of oligomeric structures could be involved in their recognition and activation of defensive transduction cascades. In tests of this hypothesis reported here, using several approaches, we observed differences in tobacco plant responses induced by the elicitin β-cryptogein (β-CRY) and its homodimer (β-CRY DIM). We also found that the C-terminal domain of elicitins of other ELI clades plays a significant role in stabilization of their oligomeric structure and restraint in the cell wall. In addition, covalently crosslinking β-CRY DIM impaired formation of signalling complexes, thereby reducing its capacity to elicit the hypersensitive response and resistance in the host plant, with no significant changes in pathogenesis-related protein expression. The results illuminate the poorly understood role of elicitins' oligomeric structures in oomycetes' interaction with plants, by revealing details of effects of β-CRY dimerization on tobacco plants' recognition and defence responses.