Search Results (94)

Heterotetramerization of Kv1.1 and Kv1.2 α-subunits expands the functional diversity of voltage-gated potassium Kv1 channels in the central nervous system (CNS), thus necessitating the study of the properties of these heterochannels, including their interactions with ligands. We report on the expression, electrophysiological, and ligand-binding properties of human heterochannels Kv(1.1-1.2)₂ formed by dimeric concatemers Kv1.1-Kv1.2 fused with fluorescent protein mKate2 in Neuro-2a cells. Kv(1.1-1.2)₂ is a low-voltage-activated, highly active, non-inactivating channel with a fast activation rate. Its activation rate and half-maximum activation voltage are similar to that of the Kv1.1 channel, but differ from that of Kv1.2. This suggests that the membrane expression of Kv(1.1-1.2)₂ may functionally compensate for the absence of membrane presentation of homotetrameric Kv1.1 channels in CNS. Hongotoxin 1 fused with fluorescent protein GFP (HgTx-G) is shown to be a pore-blocking ligand of Kv(1.1-1.2)₂ with a dissociation constant of 100 pM. Using confocal microscopy and competitive binding assay, HgTx-G and cells expressing Kv(1.1-1.2)₂, the apparent dissociation constants of the complexes between Kv(1.1-1.2)₂ and peptides Ce1, Ce4, hongotoxin 1, MeKTx11-1, agitoxin 2, charybdotoxin, and scyllatoxin were evaluated to be 14, 33, 40, 250, 800, and >>3300 pM, respectively. Heterotetramerization of α-subunits has a different effect on the affinity of ligands compared to those for Kv1.1 and Kv1.2 channels. Full article

Voltage-gated Na⁺ channels (Nav) are the molecular determinants of action potential initiation and propagation. Among the nine voltage-gated Na⁺ channel isoforms (Nav1.1–Nav1.9), Nav1.2 and Nav1.6 are of particular interest because of their developmental expression profile throughout the central nervous system (CNS) and their association with channelopathies. Although the α-subunit coded by each of the nine isoforms can sufficiently confer transient Na⁺ currents (I_Na), in vivo these channels are modulated by auxiliary proteins like intracellular fibroblast growth factor (iFGFs) through protein–protein interaction (PPI), and probes developed from iFGF/Nav PPI complexes have been shown to precisely modulate Nav channels. Previous studies identified ZL0177, a peptidomimetic derived from a short peptide sequence at the FGF14/Nav1.6 PPI interface, as a functional modulator of Nav1.6-mediated I_Na⁺. However, the isoform specificity, binding sites, and putative physiological impact of ZL0177 on neuronal excitability remain unexplored. Here, we used automated planar patch-clamp electrophysiology to assess ZL0177’s functional activity in cells stably expressing Nav1.2 or Nav1.6. While ZL0177 was found to suppress I_Na in both Nav1.2- and Nav1.6-expressing cells, ZL0177 elicited functionally divergent effects on channel kinetics that were isoform-specific and supported by differential docking of the compound to AlphaFold structures of the two channel isoforms. Computational modeling predicts that ZL0177 modulates Nav1.2 and Nav1.6 in an isoform-specific manner, eliciting phenotypically divergent effects on action potential discharge. Taken together, these results highlight the potential of PPI derivatives for isoform-specific regulation of Nav channels and the development of therapeutics for channelopathies. Full article

Background/Objectives: Diabetes mellitus (DM) is an increasingly prevalent endocrine disorder affecting humans and companion animals. Type 1 DM (T1DM) and type 2 DM (T2DM) are well characterized in humans, and canine DM most often resembles T1DM, marked by insulin dependence and β-cell destruction. Conversely, feline DM shares key features with human T2DM, including insulin resistance, obesity-related inflammation, and islet amyloidosis. This review provides a comprehensive comparative analysis of antidiabetic therapies in humans, dogs, and cats, focusing on three core areas: disease pathophysiology, pharmacological and delivery strategies, and translational implications. In human medicine, a wide array of insulin analogs, oral hypoglycemic agents, and incretin-based therapies, including glucagon-like peptide-1 receptor agonists (liraglutide) and sodium-glucose cotransporter-2 inhibitors (empagliflozin), are available. Veterinary treatments remain limited to species-adapted insulin formulations and off-label use of human drugs. Interspecies differences in gastrointestinal physiology, drug metabolism, and behavioral compliance influence therapeutic efficacy and pharmacokinetics. Recent innovations, such as microneedle patches for insulin delivery and continuous glucose monitoring systems, show promise in humans and animals. Companion animals with naturally occurring diabetes serve as valuable models for preclinical testing of novel delivery platforms and long-acting formulations under real-world settings. While these technologies show potential, challenges remain in regulatory approval and behavioral adaptation in animals. Conclusions: Future research should prioritize pharmacokinetic bridging studies, veterinary-specific formulation trials, and device validation in animal models. By highlighting shared and species-specific characteristics of DM pathogenesis and treatment, this review advocates a One Health approach toward optimized antidiabetic therapies that benefit human and veterinary medicine. Full article

Low-molecular-weight polypeptides (<3 kDa) were prepared from Periplaneta americana via enzymatic hydrolysis and ultrafiltration, yielding 3.53 ± 0.01 mg/g of peptide-rich extract. The extract was primarily composed of peptides, proteins, polysaccharides, phenolics, and flavonoids. HPLC-MS analysis identified 1402 peptide sequences, 80.51% of which were below 1000 Da, predominantly consisting of tri-, tetra-, and octapeptides. Monosaccharide profiling detected D-(+)-galactose, and quantitative assays determined the contents of total phenolics (12.28 mg/g), flavonoids (15.50 mg/g), proteins (85.84 mg/g), and total sugars (17.62 mg/g). The biological activities of the extract were systematically evaluated. The peptide fraction inhibited hyaluronidase activity by 58% at 5 mg/mL, suggesting protection of extracellular matrix integrity. In HaCaT keratinocytes, it promoted cell proliferation by 62.6%, accelerated scratch wound closure by 54%, upregulated Wnt-10b and β-catenin expression, and reduced intracellular ROS levels under oxidative stress. In LPS-stimulated RAW 264.7 macrophages, the extract decreased TNF-α, IL-6, and IL-1β production by 30%, 25%, and 28%, respectively, reduced MDA levels by 35.2%, and enhanced CAT and SOD activities by 12.3% and 60.3%. In vivo, complete closure of full-thickness skin wounds in mice was achieved by day 14. Safety evaluations using the chick chorioallantoic membrane assay and human patch tests confirmed the extract to be non-irritating and non-toxic. These findings highlight Periplaneta americana extract as a promising multifunctional bioactive ingredient for cosmetic and dermatological applications. Further studies on its active components, mechanisms of action, and clinical efficacy are warranted to support its development in skin health and aesthetic medicine. Full article

TRPA1 (Transient Receptor Potential Ankyrin 1), a cation channel predominantly expressed in sensory neurons, plays a critical role in detecting noxious stimuli and mediating pain signal transmission. As a key player in nociceptive signaling pathways, TRPA1 has emerged as a promising therapeutic target for the development of novel analgesics. Crotalphine (CRP), a 14-amino acid peptide, has been demonstrated to specifically activate TRPA1 and elicit potent analgesic effects. Previous cryo-EM (cryo-electron microscopy) studies have elucidated the structural mechanisms of TRPA1 activation by small-molecule agonists, such as iodoacetamide (IA), through covalent modification of N-terminal cysteine residues. However, the molecular interactions between TRPA1 and peptide ligands, including crotalphine, remain unclear. Here, we present the cryo-EM structure of ligand-free human TRPA1 consistent with the literature, as well as TRPA1 complexed with crotalphine, with resolutions of 3.1 Å and 3.8 Å, respectively. Through a combination of single-particle cryo-EM studies, patch-clamp electrophysiology, and microscale thermophoresis (MST), we have identified the cysteine residue at position 621 (Cys621) within the TRPA1 ion channel as the primary binding site for crotalphine. Upon binding to the reactive pocket containing C621, crotalphine induces rotational and translational movements of the transmembrane domain. This allosteric modulation coordinately dilates both the upper and lower gates, facilitating ion permeation. Full article

Scorpion venoms are a rich source of peptides that modulate the activity of ion channels and can serve as a new drug for channelopathies. Cvill6 and Cvill7 are two new peptides isolated from the venom of Centruroides villegasi with MW of 4277 Da and 4287 Da and they consist of 38 and 39 amino acids, respectively, including six cysteines. Sequence alignment revealed high similarity with members of the α-KTx2 subfamily of potassium channel toxins. In electrophysiology, Cvill7 potently inhibited Kv1.2 ion channels with an IC₅₀ of 16 pM and Kv1.3 with an IC₅₀ of 7.2 nM. In addition, it exhibited partial activity on KCa3.1 and Kv1.1, with ~16% and ~34% inhibition at 100 nM, respectively. In contrast, Cvill6 blocked Kv1.2 with low affinity (IC₅₀ of 3.9 nM) and showed modest inhibition of Kv1.3 (~11%) and KCa3.1 (~27%) at 100 nM concentration. Neither peptide showed any activity against other K⁺ channels tested in this study (Kv1.5, Kv11.1, KCa1.1, and KCa2.2). Notably, Cvill7 has a remarkable affinity for Kv1.2 and high selectivity of 450-fold over Kv1.3 and 12,000-fold over Kv1.1. These pharmacological properties make Cvill7 a potential candidate to target Kv1.2 gain of function (GOF)-related channelopathies such as epilepsy. Full article

Chrono Control Penta is a novel plant derived multifunctional bioactive peptide, which offer a tailored targeted approach to skin health by addressing both pigmentation and aging. Chrono Control Penta inhibits tyrosinase with an IC₅₀ value of 202.8 µM. Additionally, it significantly increased collagen (+87.53%) and elastin (+61.29%) production and secretion (+66.29% and +69.74%, respectively) and decreased the Matrix metalloproteinase-9 (MMP-9) and MMP-2 secretion in aged human dermal fibroblasts, vs. aging condition. At the clinical level, Chrono Control Penta was demonstrated to be already active after 2 weeks, promoting a 9.3% reduction in pigmentation after 6 weeks of use, showing its efficacy in promoting skin complexion. Furthermore, it exhibited significant moisturizing (13.05%), anti-wrinkle (11.55%), and purifying effects (12.45%), as well as firming effects (6.35%), after 6 weeks. The peptide was also well tolerated, with no adverse effects reported in clinical patch tests. This timely study presents novel research on a plant derived peptide, Chrono Control Penta, a significantly contribution to the burgeoning cosmetic peptide market. Our rigorous findings make it a new powerful ingredient, offering a comprehensive solution for skin health, and establishing a strong foundation for future research and application. Full article

The occurrence of antibiotic-resistant bacteria is a serious global health issue, and it emphasizes the need for novel antimicrobial agents. This review explores the potential of snake venom as another alternative strategy against antimicrobial resistance. Snake venoms are complex combinations of bioactive peptides and proteins, including metalloproteases (MPs), serine proteases (SPs), phospholipase A₂ (PLA₂) enzymes, three-finger toxins (3FTXs), cysteine-rich secretory proteins (CRISPs), L-amino acid oxidases (LAAOs), and antimicrobial peptides (AMPs). The antibacterial products possess wide-spectrum antibacterial activity against resistant microbes via diverse mechanisms such as cell membrane disruption, enzymatic hydrolysis of microbial structures, generation of oxidative stress, inhibition of biofilms, and immunomodulation. Strong antimicrobial activity is reported by most studies, but these are mostly restricted to in vitro testing with low translational use. Although preliminary insights into molecular targets and physiological effects exist, further studies are needed to clarify long-term safety and therapeutic potential. Special attention is given to snake venom-derived extracellular vesicles (SVEVs), which enhance the therapeutic potential of venom toxins by protecting them from degradation, improving bioavailability, and facilitating targeted delivery. Furthermore, innovative delivery strategies such as PEGylation, liposomes, hydrogels, microneedle patches, biopolymer films, and nanoparticles are discussed for their role in reducing systemic toxicity and enhancing antimicrobial efficacy. The rational modification of venom-derived peptides further expands their therapeutic utility by improving pharmacokinetics and minimizing off-target effects. Together, these approaches highlight the translational potential of snake venom-based therapies as next-generation antimicrobials in the fight against resistant infections. By outlining these challenges and directions, this review positions snake venom as an overlooked but fertile resource in the battle against antibiotic resistance. Full article

Background/Objectives: COVID-19 vaccines effectively prevent severe disease, but unequal distribution, especially in low- and middle-income countries, has led to vaccine-resistant strains. This highlights the urgent need for alternative vaccine platforms that are safe, thermostable, and easy to distribute. This study evaluates the immunogenicity, stability, and scalability of a dissolved microneedle array patch (MAP) delivering the rS1RS09 subunit vaccine, comprising the SARS-CoV-2 S1 monomer and RS09, a TLR-4 agonist peptide. Methods: The rS1RS09 vaccine was administered via MAP or intramuscular injection in murine models. The immune responses of the MAP with and without gamma irradiation as terminal sterilization were assessed at doses of 5, 15, and 45 µg, alongside neutralizing antibody responses to Wuhan, Delta, and Omicron variants. The long-term storage stability was also evaluated through protein degradation analyses at varying temperatures. Results: The rS1RS09 vaccine elicited stronger immune responses and ACE2-binding inhibition than S1 monomer alone or trimer. The MAP delivery induced sgnificantly higher and longer-lasting S1-specific IgG responses for up to 70 weeks compared to intramuscular injections. Robust Th2-prevalent immune responses were generated in all the groups vaccinated via the MAP and significant neutralizing antibodies were elicited at 15 and 45 µg, showing dose-sparing potential. The rS1RS09 in MAP has remained stable with minimal protein degradation for 19 months at room temperature or under refrigeration, regardless of gamma-irradiation. After an additional month of storage at 42 °C, cit showed less than 3% degradation, ompared to over 23% in liquid vaccines Conclusions: Gamma-irradiated MAP-rS1RS09 is a promising platform for stable, scalable vaccine production and distribution, eliminating cold chain logistics. These findings support its potential for mass vaccination efforts, particularly in resource-limited settings. Full article

Lactococcus lactis is a potential bacterial cell factory to develop delivery systems for vaccines and therapeutic proteins. Much progress has been made in applications using engineered L. lactis against, e.g., inflammatory bowel disease and cervical cancer, but the improvement of secretion and cell anchoring efficacy is still desirable. A double-labeling method based on biarsenical hairpin binding and nickel–polyhistidine affinity was used for visualization of protein trafficking and the quantification of targeted proteins on the cell surface and in the cytoplasm. To investigate the importance of mature domain targeting signals (MTSs), we generated truncated constructs encoding 126, 66, and 26 amino acid residues from the N-terminus of the basic membrane protein A (BmpA) and fused those with the gene for the human papillomavirus serotype 16 (HPV16) E7 oncoprotein. Overexpression of fusion proteins was observed to come at the cost of cell proliferation. L. lactis cells produced and displayed the shortest fusion protein only with difficulty, suggesting that the entire absence of a homologous sequence containing MTSs significantly impedes the export and surface anchoring of fusion proteins. With 40 amino acids following the signal peptide and containing one MTS, effective translocation was possible. Mutations of MTSs towards increased hydrophobicity resulted in increased secreted and surface-displayed fusion protein, suggesting the potential to design rationally improved constructs. Full article

Advanced molecular probes are required to study the functional activity of the Kv1.2 potassium channel in normal and pathological conditions. To address this, a fully active Kv1.2 channel fused with fluorescent protein mKate2 (K-Kv1.2) was engineered that has high plasma membrane presentation due to the S371T substitution, and hongotoxin 1 (HgTx1) fused with eGFP at the C-terminus (HgTx-G) was produced. HgTx-G and HgTx1 N-terminally labeled with Atto488 fluorophore were shown to be fluorescent probes of Kv1.2 in cells with dissociation constants (K_d) of 120 and 80 pM, respectively. K-Kv1.2 and HgTx-G were used as components of an analytical system to study peptide blockers of the channel and helped to find out that Ce1 and Ce4 peptides from Centruroides elegans venom possess high affinity (K_d of 10 and 30 pM) and selectivity for Kv1.2. Using molecular docking and molecular modeling techniques, the complexes of Kv1.2 with HgTx1, Ce1, and Ce4 were modeled, and determinants of the high affinity binding were proposed. New fluorescent probes and selective blockers of Kv1.2 can be used to resolve Kv1.2-related challenges in neuroscience and neuropharmacology. Full article

Background/Objective: Indwelling intrauterine contraceptive devices (IUDs) have surfaces that facilitate the attachment of Candida spp., creating a suitable environment for biofilm formation. Due to this, vulvovaginal candidiasis (VVC) is frequently linked to IUD usage, necessitating the prompt removal of these devices for effective treatment. In this study, we evaluated the susceptibility of antimicrobial peptides in vitro against biofilm forming, Amphotericin B (MIC50 > 2 mg L⁻¹) resistant Candida krusei and Candida tropicalis isolated from IUD users who had signs of vaginal candidiasis (hemorrhage, pelvic pain, inflammation, itching, and vaginal discharge). Three antimicrobial peptides, namely, epinecidin-1 (epi-1) and its two variants, namely, variant-1 (Var-1) and variant-2 (Var-2), which were reported to have enhanced antibacterial activity were tested against IUD isolates (C. krusei and C. tropicalis) with pathogenic form of Candida albicans as control. Variants of epi-1, namely, Var-1 and Var-2 were created by substituting lysine in the place of histidine and alanine. Methods: The antimicrobial activity was measured using the microbroth dilution method to determine the minimum inhibitory concentration (MIC) of peptides against C. albicans, C. krusei and C. tropicalis. The MIC of each peptide was used for biofilm assay by Crystal violet staining, Scanning Electron Microscopy, and Reactive Oxygen Species (ROS) assay. To find the possible mechanism of anti-biofilm activity by the peptides, their ability to interact with Candida spp. cell membrane proteins such as Exo-β-(1,3)-Glucanase, Secreted Aspartic Proteinase (Sap) 1, and N-terminal Domain Adhesin: Als 9-2 were determined through PatchDock. Results: The MIC values of peptides: epi-1, var-1 and var-2 against C. albicans are 128 μg mL⁻¹, 64 μg mL⁻¹ and 32 μg mL⁻¹, C. tropicalis are 256 μg mL⁻¹, 64 μg mL^−1, and 32 μg mL⁻¹ and C. krusei are 128 µg mL⁻¹, 128 µg mL⁻¹ and 64 µg mL⁻¹, respectively. Both the variants outperformed epi-1. Specifically for tested Candida spp., var-1 showed two- to four-fold enhancements and var-2 showed two- to eight-fold enhancements compared to epi-1. Electron microscopy confirmed that the mechanism of action involves pore formation thus inducing reactive oxygen species in Candida spp. cell membrane. Computational analysis showed that the peptides have a high tendency to interact with Candida spp. cell membrane proteins such as Exo-β-(1,3)-Glucanase, Secreted Aspartic Proteinase (Sap) 1, and N-terminal Domain Adhesin: Als 9-2, thereby preventing biofilm formation. Conclusions: The in vitro evidence supports the potential use of epi-1 and its variants to be used as an anti-biofilm agent to coat IUDs in the future for therapeutic purposes. Full article

Background: Natural products are gaining attention for their potential benefits in gastrointestinal health. Plant-derived polysaccharides are essential for boosting intestinal immunity and maintaining gut homeostasis. This study investigated the effects of Korean red ginseng polysaccharides (KRG-P) on intestinal homeostasis including IgA and SCFA production and mucosal barrier integrity. Methods: Mice were orally administered KRG-P at doses of 50 mg/kg or 200 mg/kg for 10 days. Fecal IgA levels were measured on days 3, 5, and 11 and IgA from cultured Peyer’s patch cells from KRG-P-treated mice were analyzed. Additionally, mRNA and protein expression levels of α-defensin, lysozyme, and E-cadherin in the small intestine were examined. Short-chain fatty acids (SCFAs) content in the cecum was also assessed. Results: KRG-P-treated groups showed a significant increase in fecal IgA levels on days 5 and 11, with no notable change on day 3. Cultured Peyer’s patch cells from mice demonstrated heightened IgA production. Additionally, KRG-P administration upregulated α-defensin and lysozyme mRNA expression, along with elevated protein expression of E-cadherin, α-defensin, and lysozyme, in the small intestine. KRG-P treatment also led to increased cecal SCFA levels, including acetate, butyrate, and propionate. Conclusions: KRG-P may promote intestinal homeostasis and host defense mechanisms by activating immune cells in Peyer’s patches, stimulating IgA production, enhancing antimicrobial peptide expression, and modulating gut microbiota metabolism through increased SCFA production. Full article

A possible molecular mechanism of the ligand–receptor binding of Ac-Lys-Lys-Lys-NH₂ (Ac-KKK-NH₂) to the Na_V1.8 channel that is responsible for nociceptive signal coding in the peripheral nervous system is investigated by a number of experimental and theoretical techniques. Upon Ac-KKK-NH₂ application at 100 nM, a significant decrease in the effective charge carried by the Na_V1.8 channel activation gating system Z_eff is demonstrated in the patch-clamp experiments. A strong Ac-KKK-NH₂ analgesic effect at both the spinal and supraspinal levels is detected in vivo in the formalin test. The distances between the positively charged amino groups in the Ac-KKK-NH₂ molecule upon binding to the Na_V1.8 channel are 11–12 Å, as revealed by the conformational analysis. The blind docking with the Na_V1.8 channel has made it possible to locate the Ac-KKK-NH₂ binding site on the extracellular side of the voltage-sensing domain VSD_I. The Ac-KKK-NH₂ amino groups are shown to form ionic bonds with Asp151 and Glu157 and a hydrogen bond with Thr161, which affects the coordinated movement of the voltage sensor up and down, thus modulating the Z_eff value. According to the results presented, Ac-KKK-NH₂ is a promising candidate for the role of an analgesic medicinal substance that can be applied for pain relief in humans. Full article

Skin and soft-tissue infections require significant consideration because of their prolonged treatment duration and propensity to rapidly progress, resulting in severe complications. The primary challenge in their treatment stems from the involvement of drug-resistant microorganisms that can form impermeable biofilms, as well as the possibility of infection extending deep into tissues, thereby complicating drug delivery. Dissolving microneedle patches are an innovative transdermal drug-delivery system that effectively enhances drug penetration through the stratum corneum barrier, thereby increasing drug concentration at the site of infection. They offer highly efficient, safe, and patient-friendly alternatives to conventional topical formulations. This comprehensive review focuses on recent advances and emerging trends in dissolving-microneedle technology for antimicrobial skin-infection therapy. Conventional antibiotic microneedles are compared with those based on emerging antimicrobial agents, such as quorum-sensing inhibitors, antimicrobial peptides, and antimicrobial-matrix materials. The review also highlights the potential of innovative microneedles incorporating chemodynamic, nanoenzyme antimicrobial, photodynamic, and photothermal antibacterial therapies. This review explores the advantages of various antimicrobial therapies and emphasizes the potential of their combined application to improve the efficacy of microneedles. Finally, this review analyzes the druggability of different antimicrobial microneedles and discusses possible future developments. Full article