Gating elements for carvacrol activation of the OTOP1 proton channel.

Otopetrin 1 (OTOP1) is a proton-activated channel crucial for animals' perception of sour taste. Despite its significance, the gating mechanism of OTOP1 remains poorly understood. Here, we demonstrate that carvacrol activates the mouse OTOP1 (mOTOP1) channel under neutral and acidic conditions. Functional analysis showed that carvacrol enhances pH fluorescence signals in OTOP1-expressing cells, with reduced efficacy at lower pH levels. Carvacrol selectively activates mOTOP1, while mOTOP2, mOTOP3, and Chelonia mydas OTOP1 (CmOTOP1) are insensitive to carvacrol activation under neutral pH. Through chimera and point mutation experiments, swapping S134 in transmembrane segment 3 (TM3) and T247 in the TM5-6 linker abolished carvacrol activation of mOTOP1 and conferred activation on CmOTOP1, suggesting these two residues are critical for carvacrol sensitivity. These findings highlight TM3 and TM5-6 linker as pivotal gating apparatus of OTOP1 channels and potential docking sites for drug design.

Bat-derived oligopeptide LE6 inhibits the contact-kinin pathway and harbors anti-thromboinflammation and stroke potential.

Thrombosis and inflammation are primary contributors to the onset and progression of ischemic stroke. The contact-kinin pathway, initiated by plasma kallikrein (PK) and activated factor XII (FXIIa), functions bidirectionally with the coagulation and inflammation cascades, providing a novel target for therapeutic drug development in ischemic stroke. In this study, we identified a bat-derived oligopeptide from Myotis myotis (Borkhausen, 1797), designated LE6 (Leu-Ser-Glu-Glu-Pro-Glu, 702 Da), with considerable potential in stroke therapy due to its effects on the contact kinin pathway. Notably, LE6 demonstrated significant inhibitory effects on PK and FXIIa, with inhibition constants of 43.97 µmol/L and 6.37 µmol/L, respectively. In vitro analyses revealed that LE6 prolonged plasma recalcification time and activated partial thromboplastin time. In murine models, LE6 effectively inhibited carrageenan-induced mouse tail thrombosis, FeCl 3-induced carotid artery thrombosis, and photochemically induced intracerebral thrombosis. Furthermore, LE6 significantly decreased inflammation and stroke injury in transient middle cerebral artery occlusion models. Notably, the low toxicity, hemolytic activity, and bleeding risk of LE6, along with its synthetic simplicity, underscore its clinical applicability. In conclusion, as an inhibitor of FXIIa and PK, LE6 offers potential therapeutic benefits in stroke treatment by mitigating inflammation and preventing thrombus formation.

Novel GPIb-independent platelet aggregation induced by botrocetin: implications for diagnosis and antithrombotic therapy.

BACKGROUND: Snake venom botrocetin facilitates von Willebrand factor (VWF) binding to platelet GPIbα and has been widely used for the diagnosis of von Willebrand disease and GPIb-related disorders. Botrocetin is also commonly employed for the development/characterization of antithrombotics targeting the GPIb-VWF axis. OBJECTIVES: To explore the alternative receptor(s)/mechanisms that participate in botrocetin-induced platelet aggregation. METHODS: The effects of botrocetin on platelet aggregation were examined using platelets from wild-type, VWF- and fibrinogen-deficient, GPIbα-deficient, IL4Rα/GPIbα-transgenic, ITGA2B and ITGB3-deficient mice, and Bernard-Soulier syndrome and healthy human samples. Platelet-fibrinogen and platelet-VWF interaction were measured using flow cytometry. GPIbα-VWF binding was evaluated utilizing enzyme-linked immunosorbent assay. Botrocetin-αIIbß3 and botrocetin-GPIbα interactions were measured using enzyme-linked immunosorbent assay and fluorescence anisotropy assays. Heparinized whole blood from healthy donors was examined for thrombus formation and growth in a perfusion chamber. RESULTS: Botrocetin could induce aggregation of platelets from a Bernard-Soulier syndrome patient and GPIbα-deficient mice as well as platelets lacking the N-terminal extracellular domain of GPIbα. Botrocetin could interact with αIIbß3 and facilitated αIIbß3-VWF interaction independent of GPIb. Botrocetin competitively bound to the ligand-binding domain of activated rather than resting αIIbß3. Although botrocetin-induced platelet aggregation requires VWF, strikingly, in the absence of VWF, botrocetin blocked fibrinogen and other ligand binding to αIIbß3 and inhibited platelet aggregation and thrombus formation. Consistently, recombinant botrocetin defective in VWF binding inhibited αIIbß3- and GPIb-mediated platelet aggregation, spreading, and thrombus formation. CONCLUSION: Our study provides insights into avoiding the misdiagnosis of GPIb-related disorders and developing botrocetin mutants as potential new antithrombotics that may simultaneously target both αIIbß3 and GPIbα.

Unlocking the potential of chicken liver byproducts: Identification of antioxidant peptides through in silico approaches and anti-aging effects of a selected peptide in Caenorhabditis elegans.

Chicken meat processing generates a substantial number of byproducts, which are either underutilized or improperly disposed. In this study, we employed in silico approaches to identify antioxidant peptides in chicken liver byproducts. Notably, the peptide WYR exhibited remarkable 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) radical scavenging activity with an IC50 of 0.13 ± 0.01 mg/mL and demonstrated stability under various conditions, including thermal, pH, NaCl, and simulated gastrointestinal digestion. Molecular docking analysis revealed significant hydrogen bonding interactions, while molecular dynamics showed differential stability with ABTS and 2,2-Diphenyl-1-picrylhydrazyl (DPPH). WYR exhibited improved stress resistance, decreased levels of reactive oxygen species (ROS), elevated the activities of superoxide dismutase (SOD) and catalase (CAT), and modulated the expression of crucial genes through the insulin/insulin-like growth factor (IIS) signaling pathway, mitogen-activated protein kinase (MAPK), and heat shock transcription factor-1 (HSF-1) pathways. These effects collectively contributed to the extension of Caenorhabditis elegans' lifespan. This study not only provides an effective method for antioxidant peptide analysis but also highlights the potential for enhancing the utilization of poultry byproducts.

Endophilin A2 controls touch and mechanical allodynia via kinesin-mediated Piezo2 trafficking.

BACKGROUND: Tactile and mechanical pain are crucial to our interaction with the environment, yet the underpinning molecular mechanism is still elusive. Endophilin A2 (EndoA2) is an evolutionarily conserved protein that is documented in the endocytosis pathway. However, the role of EndoA2 in the regulation of mechanical sensitivity and its underlying mechanisms are currently unclear. METHODS: Male and female C57BL/6 mice (8-12 weeks) and male cynomolgus monkeys (7-10 years old) were used in our experiments. Nerve injury-, inflammatory-, and chemotherapy-induced pathological pain models were established for this study. Behavioral tests of touch, mechanical pain, heat pain, and cold pain were performed in mice and nonhuman primates. Western blotting, immunostaining, co-immunoprecipitation, proximity ligation and patch-clamp recordings were performed to gain insight into the mechanisms. RESULTS: The results showed that EndoA2 was primarily distributed in neurofilament-200-positive (NF200+) medium-to-large diameter dorsal root ganglion (DRG) neurons of mice and humans. Loss of EndoA2 in mouse NF200+ DRG neurons selectively impaired the tactile and mechanical allodynia. Furthermore, EndoA2 interacted with the mechanically sensitive ion channel Piezo2 and promoted the membrane trafficking of Piezo2 in DRG neurons. Moreover, as an adaptor protein, EndoA2 also bound to kinesin family member 5B (KIF5B), which was involved in the EndoA2-mediated membrane trafficking process of Piezo2. Loss of EndoA2 in mouse DRG neurons damaged Piezo2-mediated rapidly adapting mechanically activated currents, and re-expression of EndoA2 rescued the MA currents. In addition, interference with EndoA2 also suppressed touch sensitivity and mechanical hypersensitivity in nonhuman primates. CONCLUSIONS: Our data reveal that the KIF5B/EndoA2/Piezo2 complex is essential for Piezo2 trafficking and for sustaining transmission of touch and mechanical hypersensitivity signals. EndoA2 regulates touch and mechanical allodynia via kinesin-mediated Piezo2 trafficking in sensory neurons. Our findings identify a potential new target for the treatment of mechanical pain.

The price of P2X7R freedom is neuroinflammation.

Exploring the mechanisms of microglia activation has revealed insights into the interconnections of the immune system and brain. Huang et al. demonstrate that the complex of sodium/potassium-transporting ATPase subunit alpha (NKAα1) and purinergic P2X7 receptor (P2X7R) maintains the resting state of microglial membranes. Stress increases free P2X7R that then binds to ATP to activate microglia, which may promote anxious behaviors.

Human transferrin receptor can mediate SARS-CoV-2 infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has been detected in almost all organs of coronavirus disease-19 patients, although some organs do not express angiotensin-converting enzyme-2 (ACE2), a known receptor of SARS-CoV-2, implying the presence of alternative receptors and/or co-receptors. Here, we show that the ubiquitously distributed human transferrin receptor (TfR), which binds to diferric transferrin to traffic between membrane and endosome for the iron delivery cycle, can ACE2-independently mediate SARS-CoV-2 infection. Human, not mouse TfR, interacts with Spike protein with a high affinity (KD ~2.95 nM) to mediate SARS-CoV-2 endocytosis. TfR knock-down (TfR-deficiency is lethal) and overexpression inhibit and promote SARS-CoV-2 infection, respectively. Humanized TfR expression enables SARS-CoV-2 infection in baby hamster kidney cells and C57 mice, which are known to be insusceptible to the virus infection. Soluble TfR, Tf, designed peptides blocking TfR-Spike interaction and anti-TfR antibody show significant anti-COVID-19 effects in cell and monkey models. Collectively, this report indicates that TfR is a receptor/co-receptor of SARS-CoV-2 mediating SARS-CoV-2 entry and infectivity by likely using the TfR trafficking pathway.

Transferrin Is Up-Regulated by Microbes and Acts as a Negative Regulator of Immunity to Induce Intestinal Immunotolerance.

Cross-talks (e.g., host-driven iron withdrawal and microbial iron uptake between host gastrointestinal tract and commensal microbes) regulate immunotolerance and intestinal homeostasis. However, underlying mechanisms that regulate the cross-talks remain poorly understood. Here, we show that bacterial products up-regulate iron-transporter transferrin and transferrin acts as an immunosuppressor by interacting with cluster of differentiation 14 (CD14) to inhibit pattern recognition receptor (PRR) signaling and induce host immunotolerance. Decreased intestinal transferrin is found in germ-free mice and human patients with ulcerative colitis, which are characterized by impaired intestinal immunotolerance. Intestinal transferrin and host immunotolerance are returned to normal when germ-free mice get normal microbial commensalism, suggesting an association between microbial commensalism, transferrin, and host immunotolerance. Mouse colitis models show that transferrin shortage impairs host's tolerogenic responses, while its supplementation promotes immunotolerance. Designed peptide blocking transferrin-CD14 interaction inhibits immunosuppressive effects of transferrin. In monkeys with idiopathic chronic diarrhea, transferrin shows comparable or even better therapeutic effects than hydrocortisone. Our findings reveal that by up-regulating host transferrin to silence PRR signaling, commensal bacteria counteract immune activation induced by themselves to shape host immunity and contribute for intestinal tolerance.

Gastroesophageal Reflux Disease and Rhinosinusitis: A Bidirectional Mendelian Randomization Study.

INTRODUCTION: Comorbidities, such as gastroesophageal reflux disease (GERD), are common in patients with rhinosinusitis (RS). However, the link between RS and GERD has not been fully understood. This study aimed to investigate the causal relationship between GERD and acute (ARS) or chronic RS (CRS), providing references for the pathogenesis and management of RS. METHODS: The data were obtained from the Integrative Epidemiology Unit Open GWAS project and FinnGen. A total of 972,838 individuals were included. The inverse variance-weighted (IVW) method was applied to obtain the primary results of the study. Weighted median, MR-Egger, and mode-based methods were used to determine the robustness of the results. Cochran's Q statistic and MR-Egger method were applied to detect heterogeneity and pleiotrophy in instrumental variables (IVs). Other sensitivity analyses included MR-PRESSO and leave-one-out analysis. RESULTS: The MR study showed that GERD was associated with an increased risk of CRS (OR: 1.36, 95% CI: 1.18-1.57, p < 0.001). The results of other analysis methods were broadly consistent with the IVW estimate. No heterogeneity was detected by Cochran's Q test (p = 0.061) and MR-PRESSO (p = 0.074). No horizontal pleiotropy was shown in IVs (p = 0.700). GERD was also associated with an increased risk of ARS (OR: 1.31, 95% CI: 1.17-1.48, p < 0.001). Some analytical results were inconsistent with the IVW estimate. No heterogeneity and pleiotropy were observed. There was no sufficient evidence for a reverse causal effect of RS on GERD. CONCLUSION: Our study supported that GERD promoted the risk of CRS and may be a potential risk factor for ARS. This provides additional support for further investigation into the mechanisms of GERD on RS.

Orientational Nanoconjugation with Gold Endows Marked Antimicrobial Potential and Drugability of Ultrashort Dipeptides.

Antibiotic resistance is a global threat. Antimicrobial peptides (AMPs) are highly desirable to treat multidrug-resistant pathogen infection. However, few AMPs are clinically available, due to high cost, instability, and poor selectivity. Here, ultrashort AMPs (2-3 residues with an N-terminal cysteine) are designed and assembled as gold nanoparticles. Au-S conjugation and ultrashort size restrict nonspecific reactions and peptide orientation, thus concentrating positively charged residues on the surface. The nanostructured assemblies enormously enhance antimicrobial abilities by 1000-6000-fold and stability. One representative (Au-Cys-Arg-NH2, Au_CR) shows selective antibacterial activity against Staphylococcus aureus with 10 nM minimal inhibitory concentration. Au_CR has comparable or better in vivo antimicrobial potency than vancomycin and methicillin, with low propensity to induce resistance, little side effects, and high stability (17.5 h plasma half-life). Au_CR acts by inducing collapse of membrane potential and rupture of the bacterial membrane. The report provides insights for developing AMP-metal nanohybrids, particularly tethering nonspecific reactions and AMP orientation on the metal surface.

Design methods for antimicrobial peptides with improved performance.

The recalcitrance of pathogens to traditional antibiotics has made treating and eradicating bacterial infections more difficult. In this regard, developing new antimicrobial agents to combat antibiotic-resistant strains has become a top priority. Antimicrobial peptides (AMPs), a ubiquitous class of naturally occurring compounds with broad-spectrum antipathogenic activity, hold significant promise as an effective solution to the current antimicrobial resistance (AMR) crisis. Several AMPs have been identified and evaluated for their therapeutic application, with many already in the drug development pipeline. Their distinct properties, such as high target specificity, potency, and ability to bypass microbial resistance mechanisms, make AMPs a promising alternative to traditional antibiotics. Nonetheless, several challenges, such as high toxicity, lability to proteolytic degradation, low stability, poor pharmacokinetics, and high production costs, continue to hamper their clinical applicability. Therefore, recent research has focused on optimizing the properties of AMPs to improve their performance. By understanding the physicochemical properties of AMPs that correspond to their activity, such as amphipathicity, hydrophobicity, structural conformation, amino acid distribution, and composition, researchers can design AMPs with desired and improved performance. In this review, we highlight some of the key strategies used to optimize the performance of AMPs, including rational design and de novo synthesis. We also discuss the growing role of predictive computational tools, utilizing artificial intelligence and machine learning, in the design and synthesis of highly efficacious lead drug candidates.

Functional Analyses of Three Targeted DNA Antimicrobial Peptides Derived from Goats.

With the increase in drug-resistant bacteria, new antibacterial drugs have emerged as a prominent area of research and development. Antimicrobial peptides (AMPs), as innate immune agents, have garnered significant attention due to their potent, rapid, and broad-spectrum antibacterial activity. This study focused on investigating the functionality of three AMPs (CATH 1, CATH 2, and MAP34-B) derived from goat submandibular glands. Among these AMPs, CATH 2 and MAP34-B exhibited direct antibacterial activity against both Gram-negative and Gram-positive bacteria, primarily targeting the bacterial membrane. Additionally, these two AMPs were found to have the potential to induce reactive oxygen species (ROS) production in bacterial cells and interact with bacterial genome DNA, which may play a crucial role in their mechanisms of action. Furthermore, both CATH 1 and CATH 2 demonstrated significant antioxidant activity, and all three AMPs exhibited potential anti-inflammatory activity. Importantly, the cytotoxic activity of these AMPs against mammalian cells was found to be weak, and their hemolytic activity was extremely low. Overall, the characteristics of these three AMPs found in goat submandibular glands offer new insights for the study of host protection from an immunological perspective. They hold promise as potential candidates for the development of novel antibacterial agents, particularly in the context of combating drug-resistant bacteria.

Bioactive Peptides from Barnacles and Their Potential for Antifouling Development.

Barnacles, a prevalent fouler organism in intertidal zones, has long been a source of annoyance due to significant economic losses and ecological impacts. Numerous antifouling approaches have been explored, including extensive research on antifouling chemicals. However, the excessive utilization of small-molecule chemicals appears to give rise to novel environmental concerns. Therefore, it is imperative to develop new strategies. Barnacles exhibit appropriate responses to environmental challenges with complex physiological processes and unique sensory systems. Given the assumed crucial role of bioactive peptides, an increasing number of peptides with diverse activities are being discovered in barnacles. Fouling-related processes have been identified as potential targets for antifouling strategies. In this paper, we present a comprehensive review of peptides derived from barnacles, aiming to underscore their significant potential in the quest for innovative solutions in biofouling prevention and drug discovery.

Identification and Characterization of RK22, a Novel Antimicrobial Peptide from Hirudinaria manillensis against Methicillin Resistant Staphylococcus aureus.

Staphylococcus aureus (S. aureus) infections are a leading cause of morbidity and mortality, which are compounded by drug resistance. By manipulating the coagulation system, S. aureus gains a significant advantage over host defense mechanisms, with hypercoagulation induced by S. aureus potentially aggravating infectious diseases. Recently, we and other researchers identified that a higher level of LL-37, one endogenous antimicrobial peptide with a significant killing effect on S. aureus infection, resulted in thrombosis formation through the induction of platelet activation and potentiation of the coagulation factor enzymatic activity. In the current study, we identified a novel antimicrobial peptide (RK22) from the salivary gland transcriptome of Hirudinaria manillensis (H. manillensis) through bioinformatic analysis, and then synthesized it, which exhibited good antimicrobial activity against S. aureus, including a clinically resistant strain with a minimal inhibitory concentration (MIC) of 6.25 µg/mL. The RK22 peptide rapidly killed S. aureus by inhibiting biofilm formation and promoting biofilm eradication, with good plasma stability, negligible cytotoxicity, minimal hemolytic activity, and no significant promotion of the coagulation system. Notably, administration of RK22 significantly inhibited S. aureus infection and the clinically resistant strain in vivo. Thus, these findings highlight the potential of RK22 as an ideal treatment candidate against S. aureus infection.

A target-based discovery from a parasitic helminth as a novel therapeutic approach for autoimmune diseases.

BACKGROUND: Regulatory T cells (Tregs) can alleviate the development of autoimmune and inflammatory diseases, thereby proposing their role as a new therapeutic strategy. Parasitic helminths have co-evolved with hosts to generate immunological privilege and immune tolerance through inducing Tregs. Thus, constructing a "Tregs-induction"-based discovery pipeline from parasitic helminth is a promising strategy to control autoimmune and inflammatory diseases. METHODS: The gel filtration chromatography and reverse-phase high-performance liquid chromatography (RP-HPLC) were used to isolate immunomodulatory components from the egg extracts of Schistosoma japonicum. The extracted peptides were evaluated for their effects on Tregs suppressive functions using flow cytometry, ELISA and T cell suppression assay. Finally, we carried out colitis and psoriasis models to evaluate the function of Tregs induced by helminth-derived peptide in vivo. FINDINGS: Here, based on target-driven discovery strategy, we successfully identified a small 3 kDa peptide (SjDX5-53) from egg extracts of schistosome, which promoted both human and murine Tregs production. SjDX5-53 presented immunosuppressive function by arresting dendritic cells (DCs) at an immature state and augmenting the proportion and suppressive capacity of Tregs. In mouse models, SjDX5-53 protected mice against autoimmune-related colitis and psoriasis through inducing Tregs and inhibiting inflammatory T-helper (Th) 1 and Th17 responses. INTERPRETATION: SjDX5-53 exhibited the promising therapeutic effects in alleviating the phenotype of immune-related colitis and psoriasis. This study displayed a screening and validation pipeline of the inducer of Tregs from helminth eggs, highlighting the discovery of new biologics inspired by co-evolution of hosts and their parasites. FUNDING: This study was supported by the Natural Science Foundation of China (82272368) and Natural Science Foundation of Jiangsu Province (BK20211586).

Isolation and Characterization of Poeciguamerin, a Peptide with Dual Analgesic and Anti-Thrombotic Activity from the Poecilobdella manillensis Leech.

When Poecilobdella manillensis attacks its prey, the prey bleeds profusely but feels little pain. We and other research teams have identified several anticoagulant molecules in the saliva of P. manillensis, but the substance that produces the paralyzing effect in P. manillensis is not known. In this study, we successfully isolated, purified, and identified a serine protease inhibitor containing an antistasin-like domain from the salivary secretions of P. manillensis. This peptide (named poeciguamerin) significantly inhibited elastase activity and slightly inhibited FXIIa and kallikrein activity, but had no effect on FXa, trypsin, or thrombin activity. Furthermore, poeciguamerin exhibited analgesic activity in the foot-licking and tail-withdrawal mouse models and anticoagulant activity in the FeCl3-induced carotid artery thrombosis mouse model. In this study, poeciguamerin was found to be a promising elastase inhibitor with potent analgesic and antithrombotic activity for the inhibition of pain and thrombosis after surgery or in inflammatory conditions.

Blood-brain barrier injury and neuroinflammation induced by SARS-CoV-2 in a lung-brain microphysiological system.

In some patients, COVID-19 can trigger neurological symptoms with unclear pathogenesis. Here we describe a microphysiological system integrating alveolus and blood-brain barrier (BBB) tissue chips that recapitulates neuropathogenesis associated with infection by SARS-CoV-2. Direct exposure of the BBB chip to SARS-CoV-2 caused mild changes to the BBB, and infusion of medium from the infected alveolus chip led to more severe injuries on the BBB chip, including endothelial dysfunction, pericyte detachment and neuroinflammation. Transcriptomic analyses indicated downregulated expression of the actin cytoskeleton in brain endothelium and upregulated expression of inflammatory genes in glial cells. We also observed early cerebral microvascular damage following lung infection with a low viral load in the brains of transgenic mice expressing human angiotensin-converting enzyme 2. Our findings suggest that systemic inflammation is probably contributing to neuropathogenesis following SARS-CoV-2 infection, and that direct viral neural invasion might not be a prerequisite for this neuropathogenesis. Lung-brain microphysiological systems should aid the further understanding of the systemic effects and neurological complications of viral infection.

Advanced Research on Animal Venoms in China.

For millennia, scientists, researchers, and the general public have been intrigued by animal venoms due to their potent effects and paradoxical ability to both harm and heal [...].