Search Results (22)

A de novo missense variant in KCNH3 has been identified in a patient with neurological symptoms including seizures. Here, we confirm the previously reported loss-of-function features for the associated Kv12.2 mutant A371V and investigate the underlying mechanism. Loss of function was not rescued by low temperature during channel biogenesis. Elevated external K⁺ reduced the rectification of Kv12.2 conductance as predicted by the GHK current equation, allowing the detection of currents mediated by homomeric A371V Kv12.2 channels and a detailed biophysical analysis of the mutant. Compared to wild-type, the voltage dependences of activation and deactivation of A371V Kv12.2 channels were shifted in the positive direction by 15 to 20 mV. Moreover, A371V Kv12.2 channels exhibited accelerated inactivation kinetics combined with a dramatic negative shift in the voltage dependence of inactivation by more than 100 mV. Even in heteromeric wild-type + A371V Kv12.2 channels, inactivation was enhanced, leading to a significant current reduction at physiological potentials. Our Kv12.2 data show similarities to Kv11 channels regarding C-type inactivation and differences regarding the sensitivity to external K⁺ and pharmacological inhibition of inactivation. The gating modification caused by the A371V amino acid substitution in Kv12.2 renders loss of function voltage-dependent, with a possible impact on neuronal excitability and firing behavior. Full article

In this study, vesicular lipid systems and semi-solid formulations for the skin application of Palmitoyl-GHK were formulated and characterized. Palmitoyl-GHK is a cosmetic peptide with anti-aging action, capable of treating the signs of skin aging by mainly stimulating collagen synthesis in the dermis. The so-called “ethosomes” were evaluated as nanovesicular systems constituted of phosphatidylcholine, organized in vesicles, ethanol, and water. In addition, semi-solid systems were produced and characterized, namely an organogel based on phosphatidylcholine, isopropyl palmitate, and water, a gel based on Poloxamer 407, and the poloxamer organogel, created by combining organogel and Poloxamer gel. To make the ethosomal dispersions suitable for skin application, xanthan gum was added as a gelling agent. Studies were therefore carried out on semi-solid formulations to determine (i) the spreadability, a key factor that influences various aspects of a topical/transdermal formulation, (ii) the occlusive factor, important to guarantee good effectiveness of a dermocosmetic product, and finally, (iii) the hydrating power, to study the effect of a formulation applied to the skin. A formulation study enabled the selection of the most suitable formulations for the incorporation of the active ingredient of interest. Palmitoyl-GHK was found to be soluble both in ethosomes and in the poloxamer organogel. In vitro studies were therefore conducted to evaluate the release kinetics of Palmitoyl-GHK from the formulations, via Franz cells. The qualitative–quantitative analysis, through analytical HPLC, highlighted that the active ingredient is released more slowly from semi-solid formulations compared to vesicular systems; in particular, the presence of poloxamer allows a controlled release of the peptide. Further studies will be necessary to verify the anti-aging efficacy of formulations containing the peptide. Full article

Cosmetically active compounds (CACs), both of lipophilic and hydrophilic origin, have difficulty reaching the deeper layers of the skin, and this shortcoming significantly reduces their efficacy. One such CAC that occurs naturally in the human body and displays many beneficial properties (via reducing fine lines and wrinkles, tightening skin, improving its elasticity, etc.) is the glycyl-L-histidyl-L-lysine tripeptide complex of copper (GHK–Cu). GHK–Cu is a fairly hydrophilic compound with limited permeation through the lipophilic stratum corneum. On the other hand, liposomes capable of encapsulating GHK–Cu may improve its permeation potential. The present review discusses various issues related to obtaining insight into the permeation of CACs through the skin. Methods for studying the transport of CACs encapsulated by liposomes and free GHK–Cu across the skin barrier are summarized. An analysis of the literature data reveals that the transport of liposomes containing GHK–Cu received little attention. This research gap gives an impetus to the methodological developments for assessing the effect of liposomes on GHK–Cu transportation and trafficking. Full article

Siting an offshore wind project is considered a complex planning problem with multiple interrelated objectives and constraints. Hence, compactness and contiguity are indispensable properties in spatial modeling for Renewable Energy Sources (RES) planning processes. The proposed methodology demonstrates the development of a raster-based spatial optimization model for future Offshore Wind Farm (OWF) multi-objective site-prospecting in terms of the simulated Annual Energy Production (AEP), Wind Power Variability (WPV) and the Depth Profile (DP) towards an integer mathematical programming approach. Geographic Information Systems (GIS), statistical modeling, and spatial optimization techniques are fused as a unified framework that allows exploring rigorously and systematically multiple alternatives for OWF planning. The stochastic generation scheme uses a Generalized Hurst-Kolmogorov (GHK) process embedded in a Symmetric-Moving-Average (SMA) model, which is used for the simulation of a wind process, as extracted from the UERRA (MESCAN-SURFEX) reanalysis data. The generated AEP and WPV, along with the bathymetry raster surfaces, are then transferred into the multi-objective spatial optimization algorithm via the Gurobi optimizer. Using a weighted spatial optimization approach, considering and guaranteeing compactness and continuity of the optimal solutions, the final optimal areas (clusters) are extracted for the North and Central Aegean Sea. The optimal OWF clusters, show increased AEP and minimum WPV, particularly across offshore areas from the North-East Aegean (around Lemnos Island) to the Central Aegean Sea (Cyclades Islands). All areas have a Hurst parameter in the range of 0.55–0.63, indicating greater long-term positive autocorrelation in specific areas of the North Aegean Sea. Full article

Based on the Hodgkin–Huxley theory, this paper establishes several nonlinear system models, analyzes the models’ stability, and studies the conditions for repetitive discharge of neuronal membrane potential. Our dynamic analysis showed that the main channel currents (the fast transient sodium current, the potassium delayed rectifier current, and the fixed leak current) of a neuron determine its dynamic properties and that the GHK formula will greatly widen the stimulation current range of the repetitive discharge condition compared with the Nernst equation. The model including the change in ion concentration will lead to spreading depression (SD)-like depolarization, and the inclusion of a Na-K pump will weaken the current stimulation effect by decreasing the extracellular K accumulation. The results indicate that the Hodgkin–Huxley model is suitable for describing the response to initial stimuli, but due to changes in ion concentration, it is not suitable for describing the response to long-term stimuli. Full article

Liposomes are self-assembled spherical systems composed of amphiphilic phospholipids. They can be used as carriers of both hydrophobic and hydrophilic substances, such as the anti-aging and wound-healing copper-binding peptide, GHK-Cu (glycyl-L-histidyl-L-lysine). Anionic (AL) and cationic (CL) hydrogenated lecithin-based liposomes were obtained as GHK-Cu skin delivery systems using the thin-film hydration method combined with freeze–thaw cycles and the extrusion process. The influence of total lipid content, lipid composition and GHK-Cu concentration on the physicochemical properties of liposomes was studied. The lipid bilayer fluidity and the peptide encapsulation efficiency (EE) were also determined. Moreover, in vitro assays of tyrosinase and elastase inhibition were performed. Stable GHK-Cu-loaded liposome systems of small sizes (approx. 100 nm) were obtained. The bilayer fluidity was higher in the case of cationic liposomes. As the best carriers, 25 mg/cm³ CL and AL hydrated with 0.5 mg/cm³ GHK-Cu were selected with EE of 31.7 ± 0.9% and 20.0 ± 2.8%, respectively. The obtained results confirmed that the liposomes can be used as carriers for biomimetic peptides such as copper-binding peptide and that the GHK-Cu did not significantly affect the tyrosinase activity but led to 48.90 ± 2.50% elastase inhibition, thus reducing the rate of elastin degeneration and supporting the structural integrity of the skin. Full article

Neurodegenerative diseases affect millions of people worldwide. The failure of the enzymatic degradation, the oxidative stress, the dyshomeostasis of metal ions, among many other biochemical events, might trigger the pathological route, but the onset of these pathologies is unknown. Multi-target and multifunctional molecules could address several biomolecular issues of the pathologies. The tripeptide GHK, a bioactive fragment of several proteins, and the related copper(II) complex have been largely used for many purposes, from cosmetic to therapeutic applications. GHK derivatives were synthesized to increase the peptide stability and improve the target delivery. Herein we report the synthesis of a new biotin–GHK conjugate (BioGHK) through orthogonal reactions. BioGHK is still capable of coordinating copper(II), as observed by spectroscopic and spectrometric measurements. The spectroscopic monitoring of the copper-induced ascorbate oxidation was used to measure the antioxidant activity Cu(II)-BioGHK complex, whereas antiglycant activity of the ligand towards harmful reactive species was investigated using MALDI-TOF. The affinity of BioGHK for streptavidin was evaluated using a spectrophotometric assay and compared to that of biotin. Finally, the antiaggregant activity towards amyloid-β was evaluated using a turn-on fluorescent dye. BioGHK could treat and/or prevent several adverse biochemical reactions that characterize neurodegenerative disorders, such as Alzheimer’s disease. Full article

Metal chelators are used for various industrial and medical purposes based on their physicochemical properties and biological activities. In biological systems, copper ions bind to certain enzymes as cofactors to confer catalytic activity or bind to specific proteins for safe storage and transport. However, unbound free copper ions can catalyze the production of reactive oxygen species (ROS), causing oxidative stress and cell death. The present study aims to identify amino acids with copper chelation activities that might mitigate oxidative stress and toxicity in skin cells exposed to copper ions. A total of 20 free amino acids and 20 amidated amino acids were compared for their copper chelation activities in vitro and the cytoprotective effects in cultured HaCaT keratinocytes exposed to CuSO₄. Among the free amino acids, cysteine showed the highest copper chelation activity, followed by histidine and glutamic acid. Among the amidated amino acids, cysteinamide showed the highest copper chelation activity, followed by histidinamide and aspartic acid. CuSO₄ (0.4–1.0 mM) caused cell death in a concentration-dependent manner. Among the free and amidated amino acids (1.0 mM), only histidine and histidinamide prevented the HaCaT cell death induced by CuSO₄ (1.0 mM). Cysteine and cysteinamide had no cytoprotective effects despite their potent copper-chelating activities. EDTA and GHK-Cu, which were used as reference compounds, had no cytoprotective effects either. Histidine and histidinamide suppressed the CuSO₄-induced ROS production, glutathione oxidation, lipid peroxidation, and protein carbonylation in HaCaT cells, whereas cysteine and cysteinamide had no such effects. Bovine serum albumin (BSA) showed copper-chelating activity at 0.5–1.0 mM (34–68 mg mL⁻¹). Histidine, histidinamide, and BSA at 0.5–1.0 mM enhanced the viability of cells exposed to CuCl₂ or CuSO₄ (0.5 mM or 1.0 mM) whereas cysteine and cysteinamide had no such effects. The results of this study suggest that histidine and histidinamide have more advantageous properties than cysteine and cysteinamide in terms of alleviating copper ion-induced toxic effects in the skin. Full article

Several years have passed since the Zika virus (ZIKV) pandemic reoccurred in 2015–2016. However, there is still a lack of proved protective vaccines or effective drugs against ZIKV. The peptide brevinin-2GHk (BR2GK), pertaining to the brevinin-2 family of antimicrobial peptides, has been reported to exhibit only weak antibacterial activity, and its antiviral effects have not been investigated. Thus, we analyzed the effect of BR2GK on ZIKV infection. BR2GK showed significant inhibitory activity in the early and middle stages of ZIKV infection, with negligible cytotoxicity. Furthermore, BR2GK was suggested to bind with ZIKV E protein and disrupt the integrity of the envelope, thus directly inactivating ZIKV. In addition, BR2GK can also penetrate the cell membrane, which may contribute to inhibition of the middle stage of ZIKV infection. BR2GK blocked ZIKV E protein expression with an IC₅₀ of 3.408 ± 0.738 μΜ. In summary, BR2GK was found to be a multi-functional candidate and a potential lead compound for further development of anti-ZIKV drugs. Full article

Two Pore Channels (TPCs) are cation-selective voltage- and ligand-gated ion channels in membranes of intracellular organelles of eukaryotic cells. In plants, the TPC1 subtype forms the slowly activating vacuolar (SV) channel, the most dominant ion channel in the vacuolar membrane. Controversial reports about the permeability properties of plant SV channels fueled speculations about the physiological roles of this channel type. TPC1 is thought to have high Ca²⁺ permeability, a conclusion derived from relative permeability analyses using the Goldman–Hodgkin–Katz (GHK) equation. Here, we investigated in computational analyses the properties of the permeation pathway of TPC1 from Arabidopsis thaliana. Using the crystal structure of AtTPC1, protein modeling, molecular dynamics (MD) simulations, and free energy calculations, we identified a free energy minimum for Ca²⁺, but not for K⁺, at the luminal side next to the selectivity filter. Residues D269 and E637 coordinate in particular Ca²⁺ as demonstrated in in silico mutagenesis experiments. Such a Ca²⁺-specific coordination site in the pore explains contradicting data for the relative Ca²⁺/K⁺ permeability and strongly suggests that the Ca²⁺ permeability of SV channels is largely overestimated from relative permeability analyses. This conclusion was further supported by in silico electrophysiological studies showing a remarkable permeation of K⁺ but not Ca²⁺ through the open channel. Full article

Acidosis and its associated pathologies predispose patients to develop cardiac arrhythmias and even cardiac arrest. These arrhythmias are assumed to be the result of membrane depolarization, however, the exact mechanism of depolarization during acidosis is not well defined. In our study, the model of quantum tunneling of protons is used to explain the membrane depolarization that occurs during acidosis. It is found that protons can tunnel through closed activation and inactivation gates of voltage-gated sodium channels Nav1.5 that are present in the membrane of cardiac cells. The quantum tunneling of protons results in quantum conductance, which is evaluated to assess its effect on membrane potential. The quantum conductance of extracellular protons is higher than that of intracellular protons. This predicts an inward quantum current of protons through the closed sodium channels. Additionally, the values of quantum conductance are influential and can depolarize the membrane potential according to the quantum version of the GHK equation. The quantum mechanism of depolarization is distinct from other mechanisms because the quantum model suggests that protons can directly depolarize the membrane potential, and not only through indirect effects as proposed by other mechanisms in the literature. Understanding the pathophysiology of arrhythmias mediated by depolarization during acidosis is crucial to treat and control them and to improve the overall clinical outcomes of patients. Full article

The tripeptide NH₂–Gly–His–Lys–COOH (GHK), cis-urocanic acid (cis-UCA) and Cu(II) ions are physiological constituents of the human body and they co-occur (e.g., in the skin and the plasma). While GHK is known as Cu(II)-binding molecule, we found that urocanic acid also coordinates Cu(II) ions. Furthermore, both ligands create ternary Cu(II) complex being probably physiologically functional species. Regarding the natural concentrations of the studied molecules in some human tissues, together with the affinities reported here, we conclude that the ternary complex [GHK][Cu(II)][cis-urocanic acid] may be partly responsible for biological effects of GHK and urocanic acid described in the literature. Full article

In this study, an alternative method is developed to replace chemical synthesis to produce glycyl-histidyl-lysine (GHK) tripeptides with a bacterial fermentation system. The target GHK tripeptides are cloned into expression plasmids carrying histidine-glutathione-S-transferase (GST) double tags and TEV (tobacco etch virus) cleavage sites at the N-terminus. After overexpression in Escherichia coli (E. coli) BL21 cells, the recombinant proteins are purified and recovered by high-pressure liquid chromatography (HPLC). UV-vis absorption spectroscopy was used to investigate the chemical and biological properties of the recombinant GHK tripeptides. The results demonstrated that one recombinant GHK tripeptide can bind one copper ion to form a GHK-Cu complex with high affinity, and the recombinant GHK peptide to copper ion ratio is 1:1. X-ray absorption near-edge spectroscopy (XANES) of the copper ions indicated that the oxidation state of copper in the recombinant GHK-Cu complexes here was Cu(II). All of the optical spectrum evidence suggests that the recombinant GHK tripeptide appears to possess the same biophysical and biochemical features as the GHK tripeptide isolated from human plasma. Due to the high binding affinity of GHK tripeptides to copper ions, we used zebrafish as an in vivo model to elucidate whether recombinant GHK tripeptides possess detoxification potential against the cardiotoxicity raised by waterborne Cu(II) exposure. Here, exposure to Cu(II) induced bradycardia and heartbeat irregularity in zebrafish larvae; however, the administration of GHK tripeptides could rescue those experiencing cardiotoxicity, even at the lowest concentration of 1 nM, where the GHK-Cu complex minimized CuSO₄-induced cardiotoxicity effects at a GHK:Cu ratio of 1:10. On the other hand, copper and the combination with the GHK tripeptide did not significantly alter other cardiovascular parameters, including stroke volume, ejection fraction, and fractional shortening. Meanwhile, the heart rate and cardiac output were boosted after exposure with 1 nM of GHK peptides. In this study, recombinant GHK tripeptide expression was performed, along with purification and chemical property characterization, which revealed a potent cardiotoxicity protection function in vivo with zebrafish for the first time. Full article

Identifying the determinants of firms’ investment in knowledge, this study first explores the heterogeneous impacts of research and development (R&D) on product, process, organization, and marketing innovation. Second, it examines if there exists a complementary (substitute) relation in terms of firms’ preference between four types of innovation. Studying 1500 firms of seven developing economies of the Association of Southeast Asian Nations (ASEAN), we applied the least absolute shrinkage and selection operator (LASSO), a machine learning-based regression, to identify key predictors likely to influence firms’ R&D propensity and intensity. Estimating the knowledge function, we found—in line with LASSO—that medium-sized firms, human capital (training) and credit facilities favorably affect firms’ decision to invest in R&D. Contrarily, the impact is adverse if the first or main product generates firms’ large share of revenue, a unique finding not captured by previous studies. The marginal effects of four univariate probit models indicate that firms’ investment in R&D translates into innovation. However, the application of the Geweke–Hajivassiliour–Keane (GHK)-simulator based multivariate probit, which considers simultaneity of firms’ innovation decisions that univariate probit ignores, suggests that the relationship between different types of innovation is complementary. Firms’ strategy to adopt a particular type of innovation is influenced by other types. This led to the estimation of R&D’s impact on technological and nontechnological innovation, which shows that while firms innovate both types, there is a skewed link between nontechnological innovation and the services sector. Full article

Brevinins are an important antimicrobial peptide (AMP) family discovered in the skin secretions of Ranidae frogs. The members demonstrate a typical C-terminal ranabox, as well as a diverse range of other structural characteristics. In this study, we identified a novel brevinin-2 peptide from the skin secretion of Sylvirana guentheri, via cloning transcripts, and identifying the expressed mature peptide, in the skin secretion. The confirmed amino acid sequence of the mature peptide was designated brevinin-2GHk (BR2GK). Moreover, as a previous study had demonstrated that the N-terminus of brevinin-2 is responsible for exerting antimicrobial activity, we also designed a series of truncated derivatives of BR2GK. The results show that the truncated derivatives exhibit significantly improved antimicrobial activity and cytotoxicity compared to the parent peptide, except a Pro¹⁴ substituted analog. The circular dichroism (CD) analysis of this analog revealed that it did not fold into a helical conformation in the presence of either lipopolysaccharides (LPS) or TFE, indicating that position 14 is involved in the formation of the α-helix. Furthermore, three more analogs with the substitutions of Ala, Lys and Arg at the position 14, respectively, revealed the influence on the membrane disruption potency on bacteria and mammalian cells by the structural changes at this position. Overall, the N-terminal 25-mer truncates demonstrated the potent antimicrobial activity with low cytotoxicity. Full article