Synthetic Analogues of Huwentoxin-IV Spider Peptide With Altered Human NaV1.7/NaV1.6 Selectivity Ratios

Huwentoxin-IV (HwTx-IV), a peptide discovered in the venom of the Chinese bird spider Cyriopagopus schmidti, has been reported to be a potent antinociceptive compound due to its action on the genetically-validated NaV1.7 pain target. Using this peptide for antinociceptive applications in vivo suffers from one major drawback, namely its negative impact on the neuromuscular system. Although studied only recently, this effect appears to be due to an interaction between the peptide and the NaV1.6 channel subtype located at the presynaptic level. The aim of this work was to investigate how HwTx-IV could be modified in order to alter the original human (h) NaV1.7/NaV1.6 selectivity ratio of 23. Nineteen HwTx-IV analogues were chemically synthesized and tested for their blocking effects on the Na+ currents flowing through these two channel subtypes stably expressed in cell lines. Dose-response curves for these analogues were generated, thanks to the use of an automated patch-clamp system. Several key amino acid positions were targeted owing to the information provided by earlier structure-activity relationship (SAR) studies. Among the analogues tested, the potency of HwTx-IV E4K was significantly improved for hNaV1.6, leading to a decreased hNaV1.7/hNaV1.6 selectivity ratio (close to 1). Similar decreased selectivity ratios, but with increased potency for both subtypes, were observed for HwTx-IV analogues that combine a substitution at position 4 with a modification of amino acid 1 or 26 (HwTx-IV E1G/E4G and HwTx-IV E4K/R26Q). In contrast, increased selectivity ratios (>46) were obtained if the E4K mutation was combined to an additional double substitution (R26A/Y33W) or simply by further substituting the C-terminal amidation of the peptide by a carboxylated motif, linked to a marked loss of potency on hNaV1.6 in this latter case. These results demonstrate that it is possible to significantly modulate the selectivity ratio for these two channel subtypes in order to improve the potency of a given analogue for hNaV1.6 and/or hNaV1.7 subtypes. In addition, selective analogues for hNaV1.7, possessing better safety profiles, were produced to limit neuromuscular impairments.

Paradoxical Potentiation of Acid-Sensing Ion Channel 3 (ASIC3) by Amiloride via Multiple Mechanisms and Sites Within the Channel

Acid-Sensing Ion Channels (ASICs) are proton-gated sodium-selective cation channels that have emerged as metabolic and pain sensors in peripheral sensory neurons and contribute to neurotransmission in the CNS. These channels and their related degenerin/epithelial sodium channel (DEG/ENaC) family are often characterized by their sensitivity to amiloride inhibition. However, amiloride can also cause paradoxical potentiation of ASIC currents under certain conditions. Here we characterized and investigated the determinants of paradoxical potentiation by amiloride on ASIC3 channels. While inhibiting currents induced by acidic pH, amiloride potentiated sustained currents at neutral pH activation. These effects were accompanied by alterations in gating properties including (1) an alkaline shift of pH-dependent activation, (2) inhibition of pH-dependent steady-state desensitization (SSD), (3) prolongation of desensitization kinetics, and (4) speeding of recovery from desensitization. Interestingly, extracellular Ca2+ was required for paradoxical potentiation and it diminishes the amiloride-induced inhibition of SSD. Site-directed mutagenesis within the extracellular non-proton ligand-sensing domain (E79A, E423A) demonstrated that these residues were critical in mediating the amiloride-induced inhibition of SSD. However, disruption of the purported amiloride binding site (G445C) within the channel pore blunted both the inhibition and potentiation of amiloride. Together, our results suggest that the myriad of modulatory and blocking effects of amiloride are the result of a complex competitive interaction between amiloride, Ca2+, and protons at probably more than one site in the channel.

Characterization of Functional Components in Bovine Colostrum That Inhibit Norovirus Capsid Protruding Domains Interacting with HBGA Ligands

Human noroviruses (huNoVs) cause epidemic acute gastroenteritis with significant mortality and morbidity worldwide. However, there are no commercial vaccines or antivirals against these important pathogens so far. In this study, we found that bovine colostrum (bCM) inhibited huNoV VLPs and their capsid-protruding (P) domains binding to histo-blood group antigens (HBGAs) that are huNoV receptor or attachment factors for infection, suggesting that bCM may function as a natural antiviral against huNoVs. We then characterized the bCM for the functional inhibition components by sequentially separating bCM into multiple fractions through various chromatography approaches, followed by determining their inhibitory abilities against huNoV receptor-binding P protein interacting with HBGAs. The protein components of bCM functional fractions were examined by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Our data suggested that some milk proteins, likely in the form of glycoproteins, contribute to the observed blocking effects of bCM. Our findings lay an important foundation to further develop bCM into a potential natural antiviral against huNoVs.

Small molecule inhibitors against PD-1/PD-L1 immune checkpoints and current methodologies for their development: a review

Programmed death-1/programmed death ligand-1 (PD-1/PD-L1) based immunotherapy is a revolutionary cancer therapy with great clinical success. The majority of clinically used PD-1/PD-L1 inhibitors are monoclonal antibodies but their applications are limited due to their poor oral bioavailability and immune-related adverse effects (irAEs). In contrast, several small molecule inhibitors against PD-1/PD-L1 immune checkpoints show promising blockage effects on PD-1/PD-L1 interactions without irAEs. However, proper analytical methods and bioassays are required to effectively screen small molecule derived PD-1/PD-L1 inhibitors. Herein, we summarize the biophysical and biochemical assays currently employed for the measurements of binding capacities, molecular interactions, and blocking effects of small molecule inhibitors on PD-1/PD-L1. In addition, the discovery of natural products based PD-1/PD-L1 antagonists utilizing these screening assays are reviewed. Potential pitfalls for obtaining false leading compounds as PD-1/PD-L1 inhibitors by using certain binding bioassays are also discussed in this review.

Biogas as a Transport Fuel—A System Analysis of Value Chain Development in a Swedish Context

Biofuels policy instruments are important in the development and diffusion of biogas as a transport fuel in Sweden. Their effectiveness with links to geodemographic conditions has not been analysed systematically in studying biogas development in a less urbanised regions, with high potential and primitive gas infrastructure. One such region identified is Gävleborg in Sweden. By using value chain statistics, interviews with related actors, and studying biofuels policy instruments and implications for biogas development, it is found that the policy measures have not been as effective in the region as in the rest of Sweden due to different geodemographic characteristics of the region, which has resulted in impeded biogas development. In addition to factors found in previous studies, the less-developed biogas value chain in this region can be attributed particularly to undefined rules of the game, which is lack of consensus on trade-off of resources and services, unnecessary competition among several fuel alternatives, as well as the ambiguity of municipalities’ prioritization, and regional cultural differences. To strengthen the regional biogas sector, system actors need a strategy to eliminate blocking effects of identified local factors, and national policy instruments should provide mechanisms to process geographical conditions in regulatory, economic support, and market formation.

RhoA/ROCK2 signalling is enhanced by PDGF-AA in fibro-adipogenic progenitor cells in DMD

ABSTRACTThe lack of dystrophin expression in Duchenne muscular dystrophy (DMD) leads to muscle necrosis and replacement of muscle tissue by fibro-adipose tissue. Although the role of some growth factors in the process of fibrogenesis has been previously studied, the pathways that are activated by PDGF-AA in muscular dystrophies have not been described so far. Herein we report the effects of PDGF-AA on the fibrotic process in muscular dystrophies by performing a quantitative proteomic study in DMD isolated fibro-adipogenic precursor cells (FAPs) treated with PDGF-AA. In vitro studies showed that RhoA/ROCK2 pathway is activated by PDGF-AA and induces the activation of FAPs. The inhibition of RhoA/ROCK signalling pathway by C3-exoenzyme or fasudil attenuated the effects of PDGF-AA. The blocking effects of RhoA/ROCK pathway were analysed in the dba/2J-mdx murine model with fasudil. Grip strength test showed an improvement in the muscle function and histological studies demonstrated reduction of the fibrotic area. Our results suggest that blockade of RhoA/ROCK could attenuate the activation of FAPs and could be considered a potential therapeutic approach for muscular dystrophies.

Elastic Downsampling: An Adaptive Downsampling Technique to Preserve Image Quality

This paper presents a new adaptive downsampling technique called elastic downsampling, which enables high compression rates while preserving the image quality. Adaptive downsampling techniques are based on the idea that image tiles can use different sampling rates depending on the amount of information conveyed by each block. However, current approaches suffer from blocking effects and artifacts that hinder the user experience. To bridge this gap, elastic downsampling relies on a Perceptual Relevance analysis that assigns sampling rates to the corners of blocks. The novel metric used for this analysis is based on the luminance fluctuations of an image region. This allows a gradual transition of the sampling rate within tiles, both horizontally and vertically. As a result, the block artifacts are removed and fine details are preserved. Experimental results (using the Kodak and USC Miscelanea image datasets) show a PSNR improvement of up to 15 dB and a superior SSIM (Structural Similarity) when compared with other techniques. More importantly, the algorithms involved are computationally cheap, so it is feasible to implement them in low-cost devices. The proposed technique has been successfully implemented using graphics processors (GPU) and low-power embedded systems (Raspberry Pi) as target platforms.