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Background: 211At-radiopharmaceuticals are currently the subject of growing studies for targeted alpha therapy of cancers, which leads to the widening of the scope of the targeting vectors, from small molecules to peptides and proteins. This has prompted, during the past decade, to a renewed interest in developing novel 211At-labelling approaches and novel prosthetic groups to address the diverse scenarios and to reach improved efficiency and robustness of procedures as well as an appropriate in vivo stability of the label. Main body: Translated from the well-known (radio)iodine chemistry, the long preferred electrophilic astatodemetallation using trialkylaryltin precursors is now complemented by new approaches using electrophilic or nucleophilic At. Alternatives to the astatoaryl moiety have been proposed to improve labelling stability, and the range of prosthetic groups available to label proteins has expanded. Conclusion: In this report, we cover the evolution of radiolabelling chemistry, from the initial strategies developed in the late 1970's to the most recent findings. [ABSTRACT FROM AUTHOR]

Mounting evidence has implicated the RNA m6A methylation catalyzed by METTL3 in a wide range of physiological and pathological processes, including tumorigenesis. The detailed m6A landscape and molecular mechanism of METTL3 in prostate cancer (PCa) remains ill-defined. We find that METTL3 is overexpressed in PCa and correlates with worse patient survival. Functional studies establish METTL3 as an oncoprotein dependent on its m6A enzymatic activity in both AR+ and AR− PCa cells. To dissect the regulatory network of m6A pathway in PCa, we map the m6A landscape in clinical tumor samples using m6A-seq and identify genome-wide METTL3-binding transcripts via RIP-seq. Mechanistically, we discover RRBP1 as a direct METTL3 target in which METTL3 stabilizes RRBP1 mRNA in an m6A-dependent manner. RRBP1 positively correlates with METTL3 expression in PCa cohorts and exerts an oncogenic role in aggressive PCa cells. Leveraging the 3D structural protein-protein interaction between METTL3 and METTL14, we successfully develop two potential METTL3 peptide inhibitors (RM3 and RSM3) that effectively suppress cancer cell proliferation in vitro and tumor growth in vivo. Collectively, our study reveals a novel METTL3/m6A/RRBP1 axis in enhancing aggressive traits of PCa, which can be therapeutically targeted by small-peptide METTL3 antagonists. [ABSTRACT FROM AUTHOR]

Objective Background Methods Results Conclusions Reported here are the results of four rimegepant phase I studies, in healthy participants, aimed at determining the in vivo potential of rimegepant (75 mg) for cytochrome P450 (CYP) 3A4‐related drug–drug interactions (DDIs).Rimegepant orally disintegrating tablet (Pfizer Inc., New York, NY, USA) is a calcitonin gene‐related peptide receptor antagonist approved for acute treatment of migraine and preventive treatment of episodic migraine. People with migraine commonly use multiple drug treatments, with the potential for DDIs.Each study was an open‐label, single‐arm, single‐sequence, crossover study. Rimegepant was tested as a victim drug by separate co‐administration of itraconazole (a strong CYP3A4 inhibitor and P‐glycoprotein inhibitor) in Study 1, rifampin (a strong CYP3A4 inducer and moderate CYP2C9 inducer) in Study 2, and fluconazole (a strong CYP2C9 inhibitor and moderate CYP3A4 inhibitor) in Study 3, and as a perpetrator drug by co‐administration with midazolam (a CYP3A4 substrate) in Study 4.Mean values of single‐dose rimegepant maximum concentration (Cmax) and area under the curve from time 0 to infinity (AUC0–inf) increased with itraconazole co‐administration (n = 22) by 1.42‐fold (90% confidence interval [CI] 1.25–1.61) and by 4.14‐fold (90% CI 3.87–4.44), respectively, and decreased with rifampin co‐administration (n = 21) to 36% (90% CI 31.2–41.4%) and to 19% (90% CI 16.3–21.4%), respectively. Co‐administration with fluconazole (n = 23) increased rimegepant mean AUC0–inf by 1.80‐fold (90% CI 1.68–1.93), with no impact on Cmax (1.04‐fold; 90% CI 0.94–1.15). Co‐administration of rimegepant single dose (300 mg; n = 14) or multiple doses (150 mg/day; n = 14) increased the mean Cmax of midazolam by 1.38‐fold (90% CI 1.13–1.67) and 1.53‐fold (90% CI 1.32–1.78), respectively, and the AUC0–inf of midazolam by 1.86‐fold (90% CI 1.58–2.19) and 1.91‐fold (90% CI 1.63–2.25), respectively.Based on the magnitude of DDIs, these studies indicate the following: co‐administration of rimegepant with a strong CYP3A4 inhibitor should be avoided; during co‐administration with a moderate CYP3A4 inhibitor, another dose of rimegepant within 48 h should be avoided; co‐administration of rimegepant with a strong or moderate CYP3A4 inducer should be avoided; CYP2C9 does not play a meaningful role in rimegepant metabolism; and there is no clinically meaningful CYP3A4 inhibition by rimegepant. [ABSTRACT FROM AUTHOR]

The development of new analgesics has been challenging. Candidate drugs often have limited clinical utility due to side effects that arise because many drug targets are involved in signaling pathways other than pain transduction. Here, we explored the potential of targeting protein-protein interactions (PPIs) that mediate pain signaling as an approach to developing drugs to treat chronic pain. We reviewed the approaches used to identify small molecules and peptide modulators of PPIs and their ability to decrease pain-like behaviors in rodent animal models. We analyzed data from rodent and human sensory nerve tissues to build associated signaling networks and assessed both validated and potential interactions and the structures of the interacting domains that could inform the design of synthetic peptides and small molecules. This resource identifies PPIs that could be explored for the development of new analgesics, particularly between scaffolding proteins and receptors for various growth factors and neurotransmitters, as well as ion channels and other enzymes. Targeting the adaptor function of CBL by blocking interactions between its proline-rich carboxyl-terminal domain and its SH3-domain–containing protein partners, such as GRB2, could disrupt endosomal signaling induced by pain-associated growth factors. This approach would leave intact its E3-ligase functions, which are mediated by other domains and are critical for other cellular functions. This potential of PPI modulators to be more selective may mitigate side effects and improve the clinical management of pain. Editor's summary: The limitations of current drugs available for treating chronic pain show that different approaches are needed to find new options. Using data from mouse and human sensory nerve tissues, Nascimento et al. built protein-protein interaction networks according to protein binding domain sequences and structures. In addition to known pain signaling complexes (such as PSD-95 and NMDAR), the networks indicated previously unknown pairs, such as the E3 ligase CBL with the growth factor receptor FGFR and its adaptors. The potential of these pairs to occur and function in pain signaling was supported by their coincident detection in nociceptive neurons. The findings set the groundwork for developing analgesics to target selective protein-protein interactions rather than general enzymatic activities. —Leslie K. Ferrarelli [ABSTRACT FROM AUTHOR]

The proteasome is a proteolytic enzyme complex essential for protein homeostasis in mammalian cells and protozoan parasites like Trichomonas vaginalis (Tv), the cause of the most common, non-viral sexually transmitted disease. Tv and other protozoan 20S proteasomes have been validated as druggable targets for antimicrobials. However, low yields and purity of the native proteasome have hindered studies of the Tv 20S proteasome (Tv20S). We address this challenge by creating a recombinant protozoan proteasome by expressing all seven α and seven β subunits of Tv20S alongside the Ump-1 chaperone in insect cells. The recombinant Tv20S displays biochemical equivalence to its native counterpart, confirmed by various assays. Notably, the marizomib (MZB) inhibits all catalytic subunits of Tv20S, while the peptide inhibitor carmaphycin-17 (CP-17) specifically targets β2 and β5. Cryo-electron microscopy (cryo-EM) unveils the structures of Tv20S bound to MZB and CP-17 at 2.8 Å. These findings explain MZB's low specificity for Tv20S compared to the human proteasome and demonstrate CP-17's higher specificity. Overall, these data provide a structure-based strategy for the development of specific Tv20S inhibitors to treat trichomoniasis. Here the authors report the structure of the proteasome of Trichomonas vaginalis (Tv), a protozoan causing trichomoniasis. It shows how inhibitors target this enzyme and provides basis for developing specific proteasome inhibitors to treat trichomoniasis. [ABSTRACT FROM AUTHOR]

Over the past decade, there has been a growing interest in ferritin-based vaccines due to their enhanced antigen immunogenicity and favorable safety profiles, with several vaccine candidates targeting various pathogens advancing to phase I clinical trials. Nevertheless, challenges associated with particle heterogeneity, improper assembly and unanticipated immunogenicity due to the bulky protein adaptor have impeded further advancement. To overcome these challenges, we devise a universal ferritin-adaptor delivery platform based on structural insights derived from the natural ferritinophagy complex of the human ferritin heavy chain (FTH1) and the nuclear receptor coactivator 4 (NCOA4). The engineered ferritinophagy (Fagy)-tag peptide demonstrate significantly enhanced binding affinity to the 24-mer ferritin nanoparticle, enabling efficient antigen presentation. Subsequently, we construct a self-assembling rabies virus (RABV) vaccine candidate by noncovalently conjugating the Fagy-tagged glycoprotein domain III (GDIII) of RABV to the ferritin nanoparticle, maintaining superior homogeneity, stability and immunogenicity. This vaccine candidate induces potent, rapid, and durable immune responses, and protects female mice against the authentic RABV challenge after single-dose administration. Furthermore, this universal, ferritin-based antigen conjugating strategy offers significant potential for developing vaccine against diverse pathogens and diseases. The study by Fu and colleagues presents a ferritin-adaptor platform for vaccine development, featuring a rabies virus vaccine candidate that enhances antigen stability and provides potent, durable protection after a single-dose administration. [ABSTRACT FROM AUTHOR]

Hypertension significantly impacts the survival and quality of life of animals, often leading to chronic kidney failure. Current clinical drugs used to manage hypertension carry the risk of causing adverse reactions. In contrast, certain natural peptides have demonstrated the ability to safely reduce blood pressure by inhibiting the production of angiotensin. We administered four biologically active peptide solutions to spontaneously hypertensive rats: derived from corn, wheat, egg white, and soybean. The efficacy of these peptides in reducing blood pressure was assessed through regular measurements of systolic pressure. Additionally, we analyzed levels of angiotensin‐converting enzyme and angiotensin 2 using immunohistochemistry and ELISA in vivo. The indicators of oxidative stress and inflammation in hypertensive rats were evaluated using qRT‐PCR and ELISA, respectively. Both wheat (from 182.5 ± 12.26 mmHg at day 0 to 168.86 ± 5.86 mmHg at day 20, p =.0435) and soybean (from 189 ± 2.19 mmHg at day 0 to 178.25 ± 5.14 mmHg at day 20, p =.0017) notably lowered systolic blood pressure compared to their starting systolic blood pressures in spontaneously hypertensive rats. Both wheat and soybean peptides significantly reduced plasma ANG II levels, akin to captopril's effect. Wheat peptides additionally exhibited antioxidant properties. Only the corn peptide showed a significant increase in transcript levels of the proinflammatory factors IL‐6 and TNF‐α. At the protein level, all four kinds of peptides significantly elevated IL‐6 levels while inhibiting TNF‐α secretion. This study demonstrates that wheat peptides and soybean peptides administered as dietary supplements exhibit significant hypotensive and antioxidant effects. [ABSTRACT FROM AUTHOR]

Cysteine-rich peptides play an important role in the plant defense system. The objective of this study was in silico searching for the genes encoding antimicrobial and signaling peptides in the genome of the tall wheatgrass Thinopyrumelongatum (Host) D.R. Dewey (2n = 14, EE), a wild cereal that is highly resistant to pathogens and abiotic stress. Bioinformatic analysis provided information for the identification in the tall wheatgrass genome of 154 novel genes coding for antimicrobial and signaling peptide precursors belonging to nine families. A number of cysteine-rich peptide genes were found to contain introns. The structure of peptide precursors and the location of the peptide genes on the wheatgrass chromosomes were determined. The highest similarity of the wheatgrass peptide sequences with homologous peptides from plants of the genera Triticum and Aegilops was demonstrated, supporting the cytogenetic data on the relatedness between genome E and genome D, as well as the genomes close to it. The data obtained contribute to the characterization of the molecular components of the Th. elongatum immune system and will serve as the basis for further studies of the mechanisms of resistance, as well as for the scientifically based practical use of this species as the resistance donor in wheat breeding. [ABSTRACT FROM AUTHOR]

Cryptococcus neoformans is included in the World Health Organization fungal priority pathogen list, complied to expedite improved research and public-health interventions. The limited number of available antifungal drugs, their associated toxicity, and the emergence of drug-resistant strains make the development of new therapeutic strategies mandatory. Pattern-recognition receptors (PRRs) from the host's innate immune system constitute a potential source of new antimicrobial agents. CD5 and CD6 are lymphoid members of the ancient scavenger receptor cysteine-rich superfamily (SRCR-SF) which bind pathogen-associated molecular patterns (PAMPs) of fungal and bacterial origin. Evidence supports the concept that such binding maps to 11-mer sequences present in each of their three SRCR extracellular domains. Herein, we have designed synthetic peptides containing tandems of such 11-mer sequences (namely CD5-T and CD6-T) and analyzed their C. neoformans-binding properties in vitro. Our results show both inhibitory effects on fungal growth and an ability to impact capsule formation and titanization, two critical virulence factors of C. neoformans involved in immune evasion. These effects hold promise for CD5-T and CD6-T peptides as single or adjuvant therapeutic agents against cryptococcosis. [ABSTRACT FROM AUTHOR]

Changes in the expression of cornified envelope (CE) components are a hallmark of numerous pathological skin conditions and aging, underlying the importance of this stratum corneum structure in the homeostasis of the epidermal barrier. We performed a detailed characterisation of LCE6A, a member of the Late Cornified Envelope protein family. Immunohistochemical and immunoblot experiments confirmed that LCE6A is expressed late during epidermal differentiation. Crosslinking assays of recombinant LCE6A performed either in situ on human skin sections or in vitro demonstrated that LCE6A is indeed a substrate of transglutaminases and crosslinked to CEs. LCE6A‐derived peptides containing a glutamine‐lysine sequence retained these properties of the full‐length protein and reinforced the mechanical resistance of CE submitted to sonication. We designed P26, a LCE6A‐derived biomimetic peptide that similarly reinforced CE in vitro, and evaluated its protective properties ex vivo, on human skin explants, and in two double blind and vehicle‐controlled clinical trials. P26 was able to protect the skin from barrier disruption, to limit the damage resulting from a defective barrier, and could improve the signs of aging such as loss of skin firmness and increased skin roughness. Hence, our detailed characterisation of LCE6A as a component of the CE enabled us to develop a LCE6A‐derived peptide, biologically active with a new and original mode of action that could be of great interest as a cosmetic ingredient and a pharmacologic agent. [ABSTRACT FROM AUTHOR]

Recent studies have revealed that the coagulation system plays a role in mammalian innate defense by entrapping bacteria in clots and generating antibacterial peptides. So, it is very important for the survival of bacteria to defend against the host coagulation system, which suggests that bacterial exotoxins might be a new source of anticoagulants. In this study, we analyzed the genomic sequences of Acinetobacter baumannii and a new bacterial exotoxin protein, F6W77, with five Kunitz-domains, KABP1-5, was identified. Each Kunitz-type domain features a classical six-cysteine framework reticulated by three conserved disulfide bridges, which was obviously similar to animal Kunitz-domain peptides but different from plant Kunitz-domain peptides. Anticoagulation function evaluation showed that towards the intrinsic coagulation pathway, KABP1 and KABP5 had apparently inhibitory activity, KABP4 had weak inhibitory activity, and KBAP2 and KABP3 had no effect even at a high concentration of 20 μg/mL. All five Kunitz-domain peptides, KABP1-5, had no inhibitory activity towards the extrinsic coagulation pathway. Enzyme-inhibitor experiments showed that the high-activity anticoagulant peptide KABP1 had apparently inhibitory activity towards two key coagulation factors, Xa and XIa, which was further confirmed by pull-down experiments that showed that KABP1 can bind to coagulation factors Xa and XIa directly. Structure-function relationship analyses of five Kunitz-type domain peptides showed that the arginine of the P1 site of three new bacterial anticoagulants, KABP1, KABP4 and KABP5, might be the key residue for their anticoagulation activity. In conclusion, with bioinformatics analyses, peptide recombination, and functional evaluation, we firstly found bacterial-exotoxin-derived Kunitz-type serine protease inhibitors with selectively inhibiting activity towards intrinsic coagulation pathways, and highlighted a new interaction between pathogenic bacteria and the human coagulation system. [ABSTRACT FROM AUTHOR]

A comprehensive LC-MS study examined the venom components of the solitary scoliid wasp Scolia oculata. Online mass fingerprinting showed that crude venom contains 25 small molecules (amino acids, biogenic amines, and nucleosides/nucleotides) and 45 peptides with MW 400-2700. The small molecules were identified by elemental composition analysis, and peptide sequences were determined by ESI-MS/MS and MALDI-TOF/TOF MS analyses. As major peptide components, a known peptide, β-scoliidine (DYVTVKGFSPLRKA), and three new peptides, γ-scoliidine (YVTVKGFSPLR), δ-scoliidine (YVTVKGFSPLREP) and ε-scoliidine (DYVTVKGFSPLREP) were identified, all of which are closely homologous to each other. Once the neuroprotective effects of β-scoliidine have already been described, the other three new scoliidine peptides were analyzed against oxidative stress-induced toxicity in PC12 neuronal cells by mitochondrial metabolism assay, and the structure-activity relationship was evaluated. Interestingly, pre-treatment with ε-scoliidine increased the mitochondrial metabolism of PC12 cells (106 ± 3.6%; p = 0.007) exposed to H2O2-induced oxidative stress in contrast to γ- and δ-scoliidines (77.6 ± 4.8 and 68.5 ± 4.1%, respectively) in compared to cells treated only H2O2 (75.8 ± 2.4%). These new peptides were also analyzed for enzyme inhibitor/substrate assays with angiotensin-converting enzyme (ACE), neprilysin (NEP), and acetylcholinesterase (AChE). In these assays, only δ- and ε-scoliidines increased the AChE activity (128.7 ± 3.8%; p = 0.01; and 116.8 ± 3.8% p = 0.03; respectively) in relation to basal activity (100.1 ± 1.6%). In addition, the four peptides were analyzed through in silico analysis, and none of them demonstrated possible hemolytic and toxic activities. In our study, the comprehensive LC-MS and MS/MS analyses of Scolia oculate venom identified four major peptide components of the venom β-, γ-, δ- and ε-scoliidines, and small differences in their primary structures are important to their neuroprotective properties. [ABSTRACT FROM AUTHOR]

The first-line management of cervical dystonia (CD) symptoms is intramuscular injection of botulinum toxin type A (BoNTA). However, a comparison of safety among BoNTAs is difficult because, per regulatory authorities, units of BoNTA activity are not interchangeable. Dysphagia and muscle weakness are widely considered two key adverse events to monitor closely in the treatment of CD. This integrated analysis compared the safety of BoNTAs approved for CD in the US by evaluating relationships between the incidence of dysphagia and muscle weakness in prescribing information and the core neurotoxin content. Coefficients The coefficients of determination (R2) and trendlines were estimated via regression-based lines of best fit. Adverse drug reaction (ADR) rates were strongly correlated with core neurotoxin amounts for conventional BoNTAs (slope coefficients: dysphagia = 0.048, R2 = 0.74; muscle weakness = 0.096, R2 = 0.82). The published ADR rates at approved doses for conventional BoNTAs were higher compared with DaxibotulinumtoxinA (DAXI; DAXXIFY®, Revance Therapeutics, Inc., Nashville, TN, USA) by core neurotoxin content. The use of a core neurotoxin amount was found to be an effective method for comparing the safety of BoNTA products. Current clinical trials suggest that DAXI, a novel BoNTA formulation, provides a potentially wider safety margin compared with other approved BoNTAs for CD. The lower amount of core neurotoxin administered at approved doses compared with conventional BoNTAs may explain low on-target ADRs like muscle weakness, whereas reduced diffusion from the injection site is thought to be responsible for low off-target ADRs like dysphagia. [ABSTRACT FROM AUTHOR]

This study explores the potential of natural bioactive peptides from animal venoms as targeted anti-cancer agents with reduced toxicity. Initially, we screened a broad collection of animal venoms for their antiproliferative activity against cancer cell lines. From this collection, we selected venoms from Naja anchietae and Naja senegalensis due to their promising activity. Utilizing reverse- phase high-performance liquid chromatography (RP HPLC), mass spectrometry (MALDI-TOF MS and MALDI-TOF TOF MSMS), and Edman degradation sequencing, we isolated and characterized three peptides named CTNanc1, CTNanc2, and CTNanc3 from Naja anchietae, and three others named CTNsen1, CTNsen2, and CTNsen3 from Naja senegalensis, each with a molecular weight of around 7 kDa. These purified peptides demonstrated inhibition of U87 glioblastoma cell proliferation, but not of U251 and T98G cells, in cell viability assays. To assess the impact of these treatments on cell viability, apoptosis, and necrosis, flow cytometry assays were conducted on U87 cells at 72 h. The results showed a decrease in cell viability and an increase in dead cells, suggesting that the treatments not only promote apoptosis, but may also lead to increased necrosis or late-stage apoptosis as the exposure time increases. These findings suggest that these peptides could be developed as leads for cancer therapy. [ABSTRACT FROM AUTHOR]

Post-traumatic headache (PTH) is a common and debilitating consequence of traumatic brain injury (TBI), often resembling migraine and tension-type headaches. Despite its prevalence, the optimal treatment for PTH remains unclear, with current strategies largely extrapolated from other headache disorders. This review evaluates the use of onabotulinumtoxin A (ONA) and anti-calcitonin gene-related peptide (CGRP) monoclonal antibodies (mAbs) in the treatment of PTH. A comprehensive literature search was conducted on PubMed, including studies published up to September 2024, focusing on the efficacy, safety, and mechanisms of onabotulinumtoxin A and anti-CGRP mAbs in PTH. Both clinical trials and observational studies were reviewed. ONA, widely recognized for its efficacy in chronic migraine, has shown limited benefits in PTH with only one trial involving abobotulinumtoxin A in a cohort of 40 subjects. A phase 2 trial with fremanezumab, an anti-CGRP monoclonal antibody, failed to demonstrate significant efficacy in PTH, raising questions about the utility of targeting CGRP in this condition. ONA may offer advantages over anti-CGRP mAbs, not only in terms of its broader mechanism of action but also in cost-effectiveness and higher patient adherence. Both ONA and anti-CGRP mAbs are potential options for the management of PTH, but the current evidence is insufficient to establish clear guidelines. The negative results from the fremanezumab trial suggest that CGRP inhibition may not be sufficient for treating PTH, whereas onabotulinumtoxin A's ability to target multiple pain pathways may make it a more promising candidate. [ABSTRACT FROM AUTHOR]

The renin-angiotensin system (RAS) is composed of a series of peptides, receptors, and enzymes that play a pivotal role in maintaining cardiovascular homeostasis. Among the most important players in this system are the angiotensin-II and angiotensin-(1-7) peptides. Our group has recently demonstrated that alamandine (ALA), a peptide with structural and functional similarities to angiotensin-(1-7), interacts with cardiomyocytes, enhancing contractility via the Mas-related G protein-coupled receptor member D (MrgD). It is currently unknown whether this modulation varies along the distinct phases of the day. To address this issue, we assessed the ALA-induced contractility response of cardiomyocytes from mice at four Zeitgeber times (ZTs). At ZT2 (light phase), ALA enhanced cardiomyocyte shortening in an MrgD receptor-dependent manner, which was associated with nitric oxide (NO) production. At ZT14 (dark phase), ALA induced a negative modulation on the cardiomyocyte contraction. β-Alanine, an MrgD agonist, reproduced the time-of-day effects of ALA on myocyte shortening. NG-nitro-L-arginine methyl ester, an NO synthase inhibitor, blocked the increase in fractional shortening induced by ALA at ZT2. No effect of ALA on myocyte shortening was observed at ZT8 and ZT20. Our results show that ALA/MrgD signaling in cardiomyocytes is subject to temporal modulation. This finding has significant implications for pharmacological approaches that combine chronotherapy for cardiac conditions triggered by disruption of circadian rhythms and hormonal signaling. [ABSTRACT FROM AUTHOR]

Objective: To establish a method for detecting the binding affinity of peptides to HLA-B*1301 by HLA molecular reconstitution after weak acid treatment, and its practicality was evaluated. Methods: C1R cells overexpressing HLA-B*1301 were treated with citrate buffer of different pH for different time. After neutralization of pH, cells were resuspended in culture medium containing β2m and brefeldin A. Cells were incubated at 37 °C with peptide (peptide group) or without peptide (blank control), after addition of anti-HLA antibody, the cells were detected by flow cytometry. Ratio of fluorescence intensity between peptide group and blank control group was used as an index to measure the level of HLA molecular remodeling, and then represented the interactions between peptide and HLA-B*1301 molecule. Results: HLA-polypeptide complex was denatalized and dissociated after being treated with pH3.0 buffer for 1 min, and only HLA heavy chain molecules were retained on the cell surface. The addition of peptides with binding force could significantly improve the level of HLA molecular remodeling, and the binding force of peptides with HLA-B*1301 could be evaluated by this method. Conclusion: HLA molecular reconstitution assay is a simple and reliable method to detect the binding of peptides to HLA-B*1301, which can also provide reference for the study of other low frequency HLA molecular antigen presentation patterns. [ABSTRACT FROM AUTHOR]

Objective: To establish a method for detecting the binding affinity of peptides to HLA-B*1301 by HLA molecular reconstitution after weak acid treatment, and its practicality was evaluated. Methods: C1R cells overexpressing HLA-B*1301 were treated with citrate buffer of different pH for different time. After neutralization of pH, cells were resuspended in culture medium containing β2m and brefeldin A. Cells were incubated at 37 °C with peptide (peptide group) or without peptide (blank control), after addition of anti-HLA antibody, the cells were detected by flow cytometry. Ratio of fluorescence intensity between peptide group and blank control group was used as an index to measure the level of HLA molecular remodeling, and then represented the interactions between peptide and HLA-B*1301 molecule. Results: HLA-polypeptide complex was denatalized and dissociated after being treated with pH3.0 buffer for 1 min, and only HLA heavy chain molecules were retained on the cell surface. The addition of peptides with binding force could significantly improve the level of HLA molecular remodeling, and the binding force of peptides with HLA-B*1301 could be evaluated by this method. Conclusion: HLA molecular reconstitution assay is a simple and reliable method to detect the binding of peptides to HLA-B*1301, which can also provide reference for the study of other low frequency HLA molecular antigen presentation patterns. [ABSTRACT FROM AUTHOR]

Background: The incorporation of radionuclides into peptides and larger biomolecules requires efficient and sometimes biorthogonal reaction conditions, to which click chemistry provides a convenient approach. Methods: Traditionally, click-based radiolabeling techniques have focused on classical click chemistry, such as copper(I)-catalyzed alkyne-azide [3+2] cycloaddition (CuAAC), strain-promoted azide-alkyne [3+2] cycloaddition (SPAAC), traceless Staudinger ligation, and inverse electron demand Diels–Alder (IEDDA). Results: However, newly emerging click-based radiolabeling techniques, including tyrosine-click, sulfo-click, sulfur(VI) fluoride exchange (SuFEx), thiol-ene click, azo coupling, hydrazone formations, oxime formations, and RIKEN click offer valuable alternatives to classical click chemistry. Conclusions: This review will discuss the applications of these techniques in peptide radiochemistry. [ABSTRACT FROM AUTHOR]

Problem: Vaccination in pregnancy guards against infection. Maternal antibodies, however, can inhibit antibody production in neonates. We sought to determine the effects of maternal vaccination on neonatal immune response to a SARS‐CoV‐2 mRNA vaccine. Method of Study: We hypothesized that mRNA‐lipid nanoparticles (LNP) vaccination allows for a de novo neonatal antibody response even in the presence of vertically transmitted maternal antibodies. Female mice were vaccinated with SARS‐CoV‐2 spike receptor binding domain (RBD) mRNA‐LNPs. Mice were then bred, and 21‐day‐old pups were inoculated with the same mRNA‐LNPs. Spike‐specific IgG ELISAs were performed using mouse serum. A SARS‐CoV‐2 spike protein peptide library to perform peptide ELISAs characterized high affinity binding domains within the spike protein. Results were analyzed with one‐way ANOVAs with Tukey's multiple comparisons tests. Results: Compared to pups of unvaccinated dams, there were high levels of spike‐specific IgG detected in the pups of vaccinated dams at 3 weeks of life (p < 0.0001). After neonatal vaccination, pups of unvaccinated dams had higher spike‐specific serum IgG than pups of vaccinated dams at 12 weeks of life (p < 0.001). Antibody specificity to peptide moieties within spike RBD were similar when comparing a vaccinated dam to her pup at Week 3 of life, with different binding affinities observed in the pups by Week 15 of life. Conclusions: Pre‐existing maternal antibodies may partially blunt the initial neonatal antibody response to mRNA‐LNPs vaccination. This vaccine strategy, however, does not prohibit the subsequent development of a broad range of RBD antibody specificities that may be protective. [ABSTRACT FROM AUTHOR]

An Arabidopsis (Arabidopsis thaliana) mitogen-activated protein kinase (MAPK) cascade composed of YODA (YDA)-MKK4/MKK5-MPK3/MPK6 plays an essential role downstream of the ERECTA (ER)/ER-LIKE (ERL) receptor complex in regulating stomatal development in the leaf epidermis. STOMAGEN (STO), a peptide ligand produced in mesophyll cells, competes with EPIDERMAL PATTERNING FACTOR2 (EPF2) for binding ER/ERL receptors to promote stomatal formation. In this study, we found that activation of MPK3/MPK6 suppresses STO expression. Using MUTE and STO promoters that confer epidermis- and mesophyll-specific expression, respectively, we generated lines with cell-specific activation and suppression of MPK3/MPK6. The activation or suppression of MPK3/MPK6 in either epidermis or mesophyll cells is sufficient to alter stomatal differentiation. Epistatic analyses demonstrated that STO overexpression can rescue the suppression of stomatal formation conferred by the mesophyll-specific expression of the constitutively active MKK4DD or MKK5DD, but not by the epidermis-specific expression of these constitutively active MKKs. These data suggest that STO is downstream of MPK3/MPK6 in mesophyll cells, but upstream of MPK3/MPK6 in epidermal cells in stomatal development signaling. This function of the MPK3/MPK6 cascade allows it to coordinate plant epidermis development based on its activity in mesophyll cells during leaf development. MPK3/MPK6 cascade functions downstream of ERECTA family receptors in the epidermis and upstream of the STOMAGEN peptide in the mesophyll tissue to control stomatal development. [ABSTRACT FROM AUTHOR]

Plants respond to cold stress at multiple levels, including increasing cytosolic calcium (Ca2+) influx and triggering the expression of cold-responsive genes. In this study, we show that the Ca2+-permeable channel CYCLIC NUCLEOTIDE–GATED CHANNEL20 (CNGC20) positively regulates freezing tolerance in Arabidopsis (Arabidopsis thaliana) by mediating cold-induced Ca2+ influx. Moreover, we demonstrate that the leucine-rich repeat receptor-like kinase PLANT PEPTIDE CONTAINING SULFATED TYROSINE1 RECEPTOR (PSY1R) is activated by cold, phosphorylating and enhancing the activity of CNGC20. The psy1r mutant exhibits decreased cold-evoked Ca2+ influx and freezing tolerance. Conversely, COLD-RESPONSIVE PROTEIN KINASE1 (CRPK1), a protein kinase that negatively regulates cold signaling, phosphorylates and facilitates the degradation of CNGC20 under prolonged periods of cold treatment, thereby attenuating freezing tolerance. This study thus identifies PSY1R and CRPK1 kinases that regulate CNGC20 activity and stability, respectively, thereby antagonistically modulating freezing tolerance in plants. Two kinases phosphorylate distinct sites in a Ca2+-permeable channel to regulate its activity and stability, thereby antagonistically modulating freezing tolerance in plants. [ABSTRACT FROM AUTHOR]

Purpose: The importance of maintaining good mental health with overall well-being has recently drawn attention from various fields. Functional peptides found from various protein sources reportedly reduce mental health problems. We found a new decapeptide (AJI-801) from whey proteins, which can possibly improve mood status and increase blood acetyl-L-carnitine (ALC) and fibroblast growth factor 21 (FGF21) levels. In this study, we assessed the effects of a single intake of whey protein hydrolysate containing a high amount of AJI-801 (WPH) on blood variables and mood status. Methods: A randomized, double-blind, placebo-controlled cross-over trial of two doses of WPH (100 and 500 mg) was conducted. Participants, aged between 20 and 59 years with fatigue were allocated to two groups based on the WPH doses received, and set first test food in each study. The blood ALC and FGF21 levels at baseline and after 60, 120, and 180 min of test food intake were analyzed and the responses to the questionnaire items for mood status were obtained at baseline and after 60 and 180 min of test food intake. Results: There were no significant differences in the blood ALC and FGF21 levels between the two groups. As mood status, intake of 500-mg WPH (including 2.5-mg AJI-801) showed significant improvement in Depression/Dejection of the Profile of Mood States Questionnaire second edition and visual analog scale score for depression, as compared to the placebo. Conclusions: Intake of AJI-801 500-mg WPH (including 2.5-mg AJI-801) contributes to the improvement of feeling down in healthy persons with fatigue. Trial registration: University Hospital Medical Information Network Clinical Trial Registry (UMIN 000046829). [ABSTRACT FROM AUTHOR]

Early life adversity (ELA) is associated with earlier initiation and maintenance of tobacco smoking and with a greater risk of subsequent relapse. There is growing evidence that appetite hormones, including peptide YY (PYY), which modulates craving and satiety responses, play a role in stress and addiction processes. This study employed a quasi‐experimental design to examine the association between ELA and circulating PYY stress responses in smokers and nonsmokers (N = 152, ages 19–73 years) to examine the effects of nicotine addiction. Smokers initiated a quit attempt as part of the study and were classified as either abstinent smokers or relapsed smokers based on their nicotine use during the follow‐up period. PYY levels were measured at five timepoints during three lab sessions and compared between nonsmokers and the two smoking groups (abstainers, relapsers): while smokers were using nicotine ad libitum, 24 h after smokers initiated a quit attempt, and 4 weeks after smokers initiated a quit attempt. Multivariate analyses showed the main effects of time on PYY, which decreased over time within each session. The main effects of ELA during the first (ad libitum smoking) and second (24‐h post‐cessation for smokers) sessions indicated that experiencing ELA was associated with lower PYY. No systematic effect of nicotine addiction or relapse was observed in this study. These findings suggest that adults with higher ELA may experience lower PYY. Additional research is needed to further explore the role of PYY in stress and addiction processes. [ABSTRACT FROM AUTHOR]

Summary: Recently, an antibody which inhibits the glycoprotein A repetitions predominant (GARP)‐mediated release of active transforming growth factor beta (TGFβ) from the TGFβ propeptide latency‐associated peptide (LAP) showed preclinical activity in a murine model of the chronic myeloproliferative neoplasms (MPN). Consequently, we investigated the expression of the immunosuppressive molecules LAP and GARP on peripheral blood lymphocytes from 56 MPN patients and 11 healthy donors (HD). We found that lymphocytes from patients with MPN express higher levels of LAP and GARP with no strong differences found between the different MPN diagnoses. The impact of clinical parameters on the expression of LAP and GARP by lymphocytes showed that patients with calreticulin (CALR)mut MPN have increased expression compared with HD and patients with the Januskinase2 (JAK2) mutation. The fraction of lymphocytes bound to activated platelets (aPLT) strongly correlate to LAP and GARP expression suggesting that it is not the lymphocytes themselves but aPLT, which confer the increased expression of GARP and LAP on MPN patient lymphocytes. Notably, no differences in neither platelet counts nor anti‐thrombotic therapy was identified between patients with JAK2‐ and CALRmut patients. Analysis of platelet gene expression failed to identify differences in expression of relevant genes between JAK2‐ and CALRmut patients. [ABSTRACT FROM AUTHOR]

Amphibians are well-known for their ability to produce and secrete a mixture of bioactive substances in specialized skin glands for the purpose of antibiotic self-protection and defense against predators. Some of these secretions contain various small molecules, such as the highly toxic batrachotoxin, tetrodotoxin, and samandarine. For some time, the presence of peptides in amphibian skin secretions has attracted researchers, consisting of a diverse collection of – to the current state of knowledge – three to 104 amino acid long sequences. From these more than 2000 peptides many are known to exert antimicrobial effects. In addition, there are some reports on amphibian skin peptides that can promote wound healing, regulate immunoreactions, and may serve as antiparasitic and antioxidative substances. So far, the focus has mainly been on skin peptides from frogs and toads (Anura), eclipsing the research on skin peptides of the ca. 700 salamanders and newts (Caudata). Just recently, several novel observations dealing with caudate peptides and their structure-function relationships were reported. This review focuses on the chemistry and bioactivity of caudate amphibian skin peptides and their potential as novel agents for clinical applications. [ABSTRACT FROM AUTHOR]

Rapid and specific diagnosis is necessary for both the treatment and prevention of infectious diseases. Bacteria and viruses that enter the bloodstream can trigger a strong immune response in infected animals and humans. The fluorescence polarization assay (FPA) is a rapid and accurate method for detecting specific antibodies in the blood that are produced in response to infection. One of the first examples of FPA is the non-competitive test for detecting brucellosis in animals, which was followed by the development of other protocols for detecting various infections. Fluorescently labeled polysaccharides (in the case of brucellosis and salmonellosis) or specific peptides (in the case of tuberculosis and salmonellosis, etc.) can be used as biorecognition elements for detecting infections. The availability of new laboratory equipment and mobile devices for fluorescence polarization measurements outside the laboratory has stimulated the development of new fluorescence polarization assays (FPAs) and the emergence of commercial kits on the market for the detection of brucellosis, tuberculosis, and equine infectious anemia viruses. It has been shown that, in addition to antibodies, the FPA method can detect both viruses and nucleic acids. The development of more specific and sensitive biomarkers is essential for the diagnosis of infections and therapy monitoring. This review summarizes studies published between 2003 and 2023 that focus on the detection of infections using FPA. Furthermore, it demonstrates the potential for using new biorecognition elements (e.g., aptamers, proteins, peptides) and the combined use of FPA with new technologies, such as PCR and CRISPR/Cas12a systems, for detecting various infectious agents. [ABSTRACT FROM AUTHOR]

An efficient and versatile glycosylation methodology is crucial for the systematic synthesis of oligosaccharides and glycoconjugates. A direct intermolecular and an indirect intramolecular methodology have been developed, and the former can be applied to the synthesis of medium-to-long-chain glycans like that of nucleotides and peptides. The development of a generally applicable approach for the stereoselective construction of glycosidic bonds remains a major challenge, especially for the synthesis of 1,2-cis glycosides such as β-mannosides, β-L-rhamnosides, and β-D-arabinofuranosides with equatorial glycosidic bonds as well as α-D-glucosides with axial ones. This review introduces the direct formation of cis-glycosides using ZnI2-mediated cis-glycosylations of various constrained glycosyl donors, as well as the recent advances in the development of stereoselective cis-glycosylations. [ABSTRACT FROM AUTHOR]

Amino acids (AAs), serving as the primary monomer of peptides and proteins, are widely present in nature. Benefiting from their inherent advantages, such as chemical diversity, low cost, ease of modification, chirality, biosafety, and bio-absorbability, AAs have been extensively exploited to create self-assembled nanostructures and supramolecular soft materials. In this review article, we systematically describe the recent progress regarding amino acid-derived assembly and functional soft materials. A brief background and several classified assemblies of AAs and their derivatives (chemically modified AAs) are summarized. The key non-covalent interactions to drive the assembly of AAs are emphasized based on the reported systems of self-assembled and co-assembled AAs. We discuss the molecular design of AAs and the general rules behind the hierarchical nanostructures. The resulting soft materials with interesting properties and potential applications are demonstrated. The conclusion and remarks on AA-based supramolecular assemblies are also presented from the viewpoint of chemistry, materials, and bio-applications. [ABSTRACT FROM AUTHOR]

Oriented antibody immobilization has been widely employed in immunoassays and immunodiagnoses due to its efficacy in identifying target antigens. Herein, a heptapeptide ligand, HWRGWVC (HC7), was coupled to poly(glycidyl methacrylate) (PGMA) nanospheres (PGMA-HC7). The antibody immobilization behavior and antigen recognition performance were investigated and compared with those on PGMA nanospheres by nonspecific adsorption and covalent coupling via carbodiimide chemistry. The antibodies tested included bovine, rabbit, and human immunoglobulin G (IgG), while the antigens included horseradish peroxidase (HRP) and β-2-Microglobulin (β2-MG). The nanospheres were characterized using zeta potential and particle size analyzers, scanning electron microscopy, transmission electron microscopy, Fourier transform infrared spectroscopy, and reversed-phase chromatography, proving each synthesis step was succeeded. Isothermal titration calorimetry assay demonstrated the strong affinity interaction between IgG and PGMA-HC7. Notably, PGMA-HC7 achieved rapid and extremely high IgG adsorption capacity (~3 mg/mg) within 5 min via a specific recognition via HC7 without nonspecific interactions. Moreover, the activities of immobilized anti-HRP and anti-β2-MG antibodies obtained via affinity binding were 1.5-fold and 2-fold higher than those of their covalent coupling counterparts. Further, the oriented-immobilized anti-β2-MG antibody on PGMA-HC7 exhibited excellent performance in antigen recognition with a linear detection range of 0–5.3 μg/mL, proving its great potential in immunoassay applications. [ABSTRACT FROM AUTHOR]

The oral cavity is a frequent site for head and neck cancers, which rank as the sixth most common cancer globally, with a 5-year survival rate slightly over 50%. Current treatments are limited, and resistance to therapy remains a significant clinical obstacle. IsCT1, a membrane-active peptide derived from the venom of the scorpion Opisthacanthus madagascariensis, has shown antitumor effects in various cancer cell lines, including breast cancer and chronic myeloid leukemia. However, its hemolytic action limits its potential therapeutic use. This study aims to assess the antitumor and antiproliferative activities of synthetic peptides derived from IsCT1 (IsCT-P, AC-AFPK-IsCT1, AFPK-IsCT1, AC-KKK-IsCT1, and KKK-IsCT1) in the context of oral squamous cell carcinoma. We evaluated the cytotoxic effects of these peptides on tongue squamous cell carcinoma cells and normal cells, as well as their impact on cell cycle phases, the expression of proliferation markers, modulators of cell death pathways, and mitochondrial potential. Our results indicate that the IsCT1 derivatives IsCT-P and AC-AFPK-IsCT1 possess cytotoxic properties towards squamous cell carcinoma cells, reducing mitochondrial membrane potential and the proliferative index. The treatment of cancer cells with AC-AFPK-IsCT1 led to a positive modulation of pro-apoptotic markers p53 and caspases 3 and 8, a decrease in PCNA and Cyclin D1 expression, and cell cycle arrest in the S phase. Notably, contrary to the parental IsCT1 peptide, AC-AFPK-IsCT1 did not exhibit hemolytic activity or cytotoxicity towards normal cells. Therefore, AC-AFPK-IsCT1 might be a viable therapeutic option for head and neck cancer treatment. [ABSTRACT FROM AUTHOR]

Background: Polycystic ovarian syndrome (PCOS) is a metabolic disorder, and its pathogenesis is still unclear, but the changes in the hypothalamicpituitary-gonadal (HPG) axis are considered one of the main causes in the development of PCOS. Kisspeptin is a peptide encoded by the KISS gene that regulates the hypothalamus-pituitary-gonadal (HPG), which may be concerned with the pathogenesis of PCO. Aim: To identify the relation between serum Kisspeptin and sex hormones and metabolic parameters among normal and PCO women. Methods: A prospective case-control study was conducted among 64 subjects (32 with PCO and 32 with the control group). The serum Kisspeptin level was measured using enzyme-linked immunosorbent assay (ELISA) and correlated with other hormones such as LH, FSH, DHEA, testosterone, and prolactin. Results: Serum kisspeptin level was significantly higher in the PCOS group when compared to the control group. There was a statistically significant positive correlation between kisspeptin and each of HOMA IR, LH, FSH, DHEA, testosterone, and prolactin. Conclusions: The increase in kisspeptin level may play a role in pathogenesis of PCOS, and may represent a novel link between metabolic and hormonal disturbance in PCOS females since this peptide was correlated with HOMA-IR, LH, FSH, DHEA, testosterone and prolactin. [ABSTRACT FROM AUTHOR]

The leave-one-out (LOO) green fluorescent protein (GFP) approach to biosensor design combines computational protein design with split protein reconstitution. LOO-GFPs reversibly fold and gain fluorescence upon encountering the target peptide, which can be redefined by computational design of the LOO site. Such an approach can be used to create reusable biosensors for the early detection of emerging biological threats. Enlightening biophysical inferences for nine LOO-GFP biosensor libraries are presented, with target sequences from dengue, influenza, or HIV, replacing beta strands 7, 8, or 11. An initially low hit rate was traced to components of the energy function, manifesting in the over-rewarding of over-tight side chain packing. Also, screening by colony picking required a low library complexity, but designing a biosensor against a peptide of at least 12 residues requires a high-complexity library. This double-bind was solved using a "piecemeal" iterative design strategy. Also, designed LOO-GFPs fluoresced in the unbound state due to unwanted dimerization, but this was solved by fusing a fully functional prototype LOO-GFP to a fiber-forming protein, Drosophila ultrabithorax, creating a biosensor fiber. One influenza hemagglutinin biosensor is characterized here in detail, showing a shifted excitation/emission spectrum, a micromolar affinity for the target peptide, and an unexpected photo-switching ability. [ABSTRACT FROM AUTHOR]

A protected tripeptide Boc-l-Leu-5-AIA-Aib-OMe was synthesized where 5-aminoisophthalic acid (5-AIA), a rigid non-coded aromatic γ-amino acid is incorporated as central residue. The single crystal X-ray diffraction study indicates that the peptide self-assembles into supramolecular sheet through intermolecular hydrogen bonding interaction N–H⋯O and π···π interaction. [ABSTRACT FROM AUTHOR]

Diabetes markedly increases susceptibility to adverse cardiovascular events, including heart failure (HF), leading to heightened morbidity and mortality rates. Elevated levels of natriuretic peptides (NPs), notably B-type natriuretic peptide (BNP) and N-terminal-proBNP (NT-proBNP), correlate with cardiac structural and functional abnormalities, aiding in risk stratification and treatment strategies in individuals with diabetes. This article reviews the intricate relationship between diabetes and HF, emphasizing the role of NPs in risk assessment and guiding therapeutic strategies, particularly in individuals with type 2 diabetes mellitus (T2DM). We also explore the analytical and clinical considerations in the use of natriuretic peptide testing and the challenges and prospects of natriuretic-peptide-guided therapy in managing cardiovascular risk in patients with diabetes. We conclude with some reflections on future prospects for NPs. [ABSTRACT FROM AUTHOR]

Mitochondria provide cells with energy and regulate the cellular metabolism. Almost all mitochondrial proteins are nuclear-encoded, translated on ribosomes in the cytoplasm, and subsequently transferred to the different subcellular compartments of mitochondria. Here, we developed OptoMitoImport, an optogenetic tool to control the import of proteins into the mitochondrial matrix via the presequence pathway on demand. OptoMitoImport is based on a two-step process: first, light-induced cleavage by a TEV protease cuts off a plasma membrane-anchored fusion construct in close proximity to a mitochondrial targeting sequence; second, the mitochondrial targeting sequence preceding the protein of interest recruits to the outer mitochondrial membrane and imports the protein fused to it into mitochondria. Upon reaching the mitochondrial matrix, the matrix processing peptidase cuts off the mitochondrial targeting sequence and releases the protein of interest. OptoMitoImport is available as a two-plasmid system as well as a P2A peptide or IRES sequence-based bicistronic system. Fluorescence studies demonstrate the release of the plasma membrane-anchored protein of interest through light-induced TEV protease cleavage and its localization to mitochondria. Cell fractionation experiments confirm the presence of the peptidase-cleaved protein of interest in the mitochondrial fraction. The processed product is protected from proteinase K treatment. Depletion of the membrane potential across the inner mitochondria membrane prevents the mitochondrial protein import, indicating an import of the protein of interest by the presequence pathway. These data demonstrate the functionality of OptoMitoImport as a generic system with which to control the post-translational mitochondrial import of proteins via the presequence pathway. [ABSTRACT FROM AUTHOR]

Recently developed experimental and computational approaches to identify putative coding small ORFs (smORFs) in genomes have revealed thousands of smORFs localized within coding and non-coding RNAs. They can be translated into smORF peptides or microproteins, which are defined as less than 100 amino acids in length. The identification of such a large number of potential biological regulators represents a major challenge, notably for elucidating the in vivo functions of these microproteins. Since the emergence of this field, Drosophila has proved to be a valuable model for studying the biological functions of microproteins in vivo. In this review, we outline how the smORF field emerged and the nomenclature used in this domain. We summarize the technical challenges associated with identifying putative coding smORFs in the genome and the relevant translated microproteins. Finally, recent findings on one of the best studied smORF peptides, Pri, and other microproteins studied so far in Drosophila are described. These studies highlight the diverse roles that microproteins can fulfil in the regulation of various molecular targets involved in distinct cellular processes during animal development and physiology. Given the recent emergence of the microprotein field and the associated discoveries, the microproteome represents an exquisite source of potentially bioactive molecules, whose in vivo biological functions can be explored in the Drosophila model. [ABSTRACT FROM AUTHOR]

The synthetic peptide of lumican C-terminal 13 amino acids with the cysteine replaced by an alanine, hereafter referred to as lumikine (LumC13C-A: YEALRVANEVTLN), binds to TGFβ type I receptor/activin-like kinase5 (TBR1/ALK5) in the activated TGFβ receptor complex to promote corneal epithelial wound healing. The present study aimed to identify the minimum essential amino acid epitope necessary to exert the effects of lumikine via ALK5 and to determine the role of the Y (tyrosine) residue for promoting corneal epithelium wound healing. This study also aimed to determine the signaling pathway(s) triggered by lumican–ALK5 binding. For such, adult Lum knockout (Lum−/−) mice (~8–12 weeks old) were subjected to corneal epithelium debridement using an Agerbrush®. The injured eyes were treated with 10 µL eye drops containing 0.3 µM synthetic peptides designed based on the C-terminal region of lumican for 5–6 h. To unveil the downstream signaling pathways involved, inhibitors of the Alk5 and EGFR signaling pathways were co-administered or not. Corneas isolated from the experimental mice were subjected to whole-mount staining and imaged under a ZEISS Observer to determine the distance of epithelium migration. The expression of EGFR ligands was determined following a scratch assay with HTCE (human telomerase-immortalized cornea epithelial cells) in the presence or not of lumikine. Results indicated that shorter LumC-terminal peptides containing EVTLN and substitution of Y with F in lumikine abolishes its capability to promote epithelium migration indicating that Y and EVTLN are essential but insufficient for Lum activity. Lumikine activity is blocked by inhibitors of Alk5, EGFR, and MAPK signaling pathways, while EGF activity is only suppressed by EGFR and MAPK inhibitors. qRT-PCR of scratched HTCE cells cultures treated with lumikine showed upregulated expression of several EGFR ligands including epiregulin (EREG). Treatment with anti-EREG antibodies abolished the effects of lumikine in corneal epithelium debridement healing. The observations suggest that Lum/lumikine binds Alk5 and promotes the noncanonical Smad-independent TGFβ/TBRs signaling pathways during the healing of corneal epithelium debridement. [ABSTRACT FROM AUTHOR]

The advent of clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated nuclease 9 (Cas9) technology has revolutionized the field of genetic engineering, offering unprecedented potential for the targeted manipulation of DNA sequences. Advances in the mechanism of action of the CRISPR-Cas9 system allowed potential applicability for the treatment of genetic diseases. CRISPR-Cas9's mechanism of action involves the use of an RNA guide molecule to target-specific DNA sequences and the Cas9 enzyme to induce precise DNA cleavage. In the context of the CRISPR-Cas9 system, this review covers nonviral delivery methods for gene editing based on peptide internalization. Here, we describe critical areas of discussion such as immunogenicity, emphasizing the importance of safety, efficiency, and cost-effectiveness, particularly in the context of treating single-mutation genetic diseases using advanced editing techniques genetics as prime editor and base editor. The text discusses the versatility of cell-penetrating peptides (CPPs) in forming complexes for delivering biomolecules, particularly ribonucleoprotein for genome editing with CRISPR-Cas9 in human cells. In addition, it emphasizes the promise of combining CPPs with DNA base editing and prime editing systems. These systems, known for their simplicity and precision, hold great potential for correcting point mutations in human genetic diseases. In summary, the text provides a clear overview of the advantages of using CPPs for genome editing with CRISPR-Cas9, particularly in conjunction with advanced editing systems, highlighting their potential impact on clinical applications in the treatment of single-mutation genetic diseases. [ABSTRACT FROM AUTHOR]

The chemokine receptors CCR1 and CCR5 display overlapping expression patterns and ligand dependency. Here we find that ligand activation of CCR5, not CCR1, is dependent on N-terminal receptor O-glycosylation. Release from O-glycosylation dependency is obtained by increasing CCR5 N-terminus acidity to the level of CCR1. Ligand activation of CCR5, not CCR1, drastically improves in the absence of glycosaminoglycans (GAGs). Ligand activity at both CCR1 and CCR5 is boosted by positively charged/basic peptides shown to interact with acidic chemokine receptor N-termini. We propose that receptors with an inherent low N-terminus acidity rely on post-translational modifications (PTMs) to efficiently compete with acidic entities in the local environment for ligand capture. Although crucial for initial ligand binding, strong electrostatic interactions between the ligand and the receptor N-terminus may counteract following insertion of the ligand into the receptor binding pocket and activation, a process that seems to be aided in the presence of basic peptides. Basic peptides bind to the naked CCR1 N-terminus, not the CCR5 N-terminus, explaining the loss of boosting of ligand-induced signaling via CCR5 in cells incapable of glycosylation. [ABSTRACT FROM AUTHOR]

Pleiotrophin (PTN) is a secreted factor that regulates endothelial cell migration through protein tyrosine phosphatase receptor zeta 1 (PTPRZ1) and αvβ3 integrin. Genetic deletion of Ptprz1 results in enhanced endothelial cell proliferation and migration, due to the decreased expression of β3 integrin and the subsequent, enhanced cMet phosphorylation. In the present study, we investigated the effect of PTN and PTPRZ1 on activating the mTORC1 kinase and protein synthesis and identified part of the implicated signaling pathway in endothelial cells. PTN or genetic deletion of Ptprz1 activates protein synthesis in a mTORC1-dependent manner, as shown by the enhanced phosphorylation of the mTORC1-downstream targets ribosomal protein S6 kinase 1 (SK61) and 4E-binding protein 1 (4EBP1) and the upregulation of HIF-1α. The cMet tyrosine kinase inhibitor crizotinib abolishes the stimulatory effects of PTN or PTPRZ1 deletion on mTORC1 activation and protein synthesis, suggesting that mTORC1 activation is downstream of cMet. The mTORC1 inhibitor rapamycin abolishes the stimulatory effect of PTN or PTPRZ1 deletion on endothelial cell migration, suggesting that mTORC1 is involved in the PTN/PTPRZ1-dependent cell migration. The αvβ3 integrin blocking antibody LM609 and the peptide PTN112–136, both known to bind to ανβ3 and inhibit PTN-induced endothelial cell migration, increase cMet phosphorylation and activate mTORC1, suggesting that cMet and mTORC1 activation are required but are not sufficient to stimulate cell migration. Overall, our data highlight novel aspects of the signaling pathway downstream of the PTN/PTPRZ1 axis that regulates endothelial cell functions. [ABSTRACT FROM AUTHOR]

Antimicrobial peptides (AMPs) are emerging as a promising alternative to traditional antibiotics due to their ability to disturb bacterial membranes and/or their intracellular processes, offering a potential solution to the growing problem of antimicrobial resistance. AMP effectiveness is governed by factors such as net charge, hydrophobicity, and the ability to form amphipathic secondary structures. When properly balanced, these characteristics enable AMPs to selectively target bacterial membranes while sparing eukaryotic cells. This review focuses on the roles of positive charge, hydrophobicity, and structure in influencing AMP activity and toxicity, and explores strategies to optimize them for enhanced therapeutic potential. We highlight the delicate balance between these properties and how various modifications, including amino acid substitutions, peptide tagging, or lipid conjugation, can either enhance or impair AMP performance. Notably, an increase in these parameters does not always yield the best results; sometimes, a slight reduction in charge, hydrophobicity, or structural stability improves the overall AMP therapeutic potential. Understanding these complex interactions is key to developing AMPs with greater antimicrobial activity and reduced toxicity, making them viable candidates in the fight against antibiotic-resistant bacteria. [ABSTRACT FROM AUTHOR]

The skin is an essential organ that protects the body from external aggressions; therefore, damage from various wounds can significantly impair its function, and effective methods for regenerating and restoring its barrier function are crucial. This study aimed to mass-produce wound-healing exosomes using a fragment of the fibroblast growth factor 2 (FGF2)-derived peptide (FP2) to enhance cell proliferation and exosome production. Our experiments demonstrated increased cell proliferation when Wharton's jelly mesenchymal stem cells (WJ MSCs) were coated with FP2. Exosomes from FP2-coated WJ MSCs were analyzed using nanoparticle-tracking analysis, transmission electron microscopy, and Western blotting. Subsequently, fibroblasts were treated with these exosomes, and their viability and migration effects were compared. Anti-inflammatory effects were also evaluated by inducing pro-inflammatory factors in RAW264.7 cells. The treatment of fibroblasts with FP2-coated WJ MSC-derived exosomes (FP2-exo) increased the expression of FGF2, confirming their wound-healing effect in vivo. Overall, the results of this study highlight the significant impact of FP2 on the proliferation of WJ MSCs and the anti-inflammatory and wound-healing effects of exosomes, suggesting potential applications beyond wound healing. [ABSTRACT FROM AUTHOR]

This study aimed to explore the intricate relationship between mitochondrial dysfunction, infection, and neuroinflammation, focusing specifically on the impact of pathogenic epitopes of the Epstein–Barr Virus (EBV) nuclear antigen 1 (EBNA1) in a mouse model of mitochondrial dysfunctions. The investigation included female middle-aged PARK2−/− and C57BL/6J wild-type mice immunized with EBNA1386–405 or with active experimental autoimmune encephalomyelitis (EAE) induction by the myelin oligodendrocyte glycoprotein (MOG)35–55 peptide. The PARK2−/− mice developed more severe EAE than the wild-type mice. Following immunization with EBNA1386–405, only PARK2−/− exhibited symptoms resembling EAE. During the acute phase, PARK2−/− mice immunized with either MOG35–55 or EBNA1386–405 exhibited a similar infiltration of the T cells and macrophages in the spinal cord and decreased glial fibrillary acidic protein (GFAP) expression in the brain. However, the EBNA1386–405 -immunized PARK2−/− mice showed significantly increased frequencies of CD8a+ T cells and CD11c+ B cells, and distinct cytokine profiles in the periphery compared to the wild-type controls. These findings highlight the role of EBV in exacerbating inflammation, particularly in the context of mitochondrial deficiencies. [ABSTRACT FROM AUTHOR]

With the aim of improving the uncertainties associated with the correct diagnosis of seronegative rheumatoid arthritis (RA) and identifying those at risk of developing interstitial lung disease (ILD), we have designed new peptide antigens bearing three post-translational modifications (PTMs) (citrulline, homocitrulline and acetyl-lysine) related to RA that could complement existing tests based on anti-citrullinated peptide/protein antibodies (ACPAs). Several chimeric peptides were synthesized and comparatively tested as antigens in ELISAs with two cohorts of sera: 178 RAs and 110 healthy blood donors. The results indicated that although chimeric peptides containing all three PTMs and vimentin and enolase domains do not significantly outperform existing ACPA tests in terms of sensitivity and specificity, they show potential to complement current assays, especially when detecting antibodies in some seronegative patients. Furthermore, the presence of these autoantibodies significantly identified patients with RA and ILD. We can conclude that the identification of specific autoantibody profiles using synthetic antigens containing peptide domains derived from proteins present in the human joint could help in the early detection of the risk of ILD in patients with RA and be useful for adapting follow-up strategies and guiding decisions during treatment. [ABSTRACT FROM AUTHOR]

The Sam (Sterile alpha motif) domain of the lipid phosphatase Ship2 (Ship2-Sam) is engaged by the Sam domain of the receptor tyrosine kinase EphA2 (EphA2-Sam) and, this interaction is principally linked to procancer effects. Peptides able to hinder the formation of the EphA2-Sam/Ship2-Sam complex could possess therapeutic potential. Herein, by employing the FoldX software suite, we set up an in silico approach to improve the peptide targeting of the so-called Mid Loop interface of Ship2-Sam, representing the EphA2-Sam binding site. Starting from a formerly identified peptide antagonist of the EphA2-Sam/Ship2-Sam association, first, the most stabilizing mutations that could be inserted in each peptide position were predicted. Then, they were combined, producing a list of potentially enhanced Ship2-Sam ligands. A few of the in silico generated peptides were experimentally evaluated. Interaction assays with Ship2-Sam were performed using NMR and BLI (BioLayer Interferometry). In vitro assays were conducted as well to check for cytotoxic effects against both cancerous and healthy cells, and also to assess the capacity to regulate EphA2 degradation. This study undoubtedly enlarges our knowledge on how to properly target EphA2-Sam/Ship2-Sam associations with peptide-based tools and provides a promising strategy that can be used to target any protein–protein interaction. [ABSTRACT FROM AUTHOR]

Antimicrobial peptides represent a promising alternative to traditional drugs in relation to cost, toxicity, and, primarily, the growing problem of drug resistance. Here, we report on the activity against HSV-1 and HSV-2 of a previously described wide-spectrum synthetic decapeptide, Killer Peptide (KP). As determined by plaque reduction assays, treatment with KP at 100 μg/mL resulted in a reduction in the viral yield titer of 3.5 Logs for HSV-1 and 4.1 Logs for HSV-2. Further evaluation of KP antiviral activity focused on the early stages of the virus replicative cycle, including the determination of the residual infectivity of viral suspensions treated with KP. A direct effect of the peptide on viral particles impairing virus absorption and penetration was shown. The toxicity profile proved to be extremely good, with a selectivity index of 29.6 for HSV-1 and 156 for HSV-2. KP was also active against acyclovir (ACV)-resistant HSV isolates, while HSV subcultures in the presence of sub-inhibitory doses of KP did not lead to the emergence of resistant strains. Finally, the antiviral action of KP proved to be synergistic with that of ACV. Overall, these results demonstrate that KP could represent an interesting addition/alternative to acyclovir for antiviral treatment. [ABSTRACT FROM AUTHOR]

Neuroinflammation plays a vital role in neurodegenerative diseases and neuropsychiatric disorders, and microglia and astrocytes chiefly modulate inflammatory responses in the central nervous system (CNS). Toll-like receptors (TLRs), which are expressed in neurons, astrocytes, and microglia in the CNS, are critical for innate immune responses; microglial TLRs can regulate the activity of these cells, inducing protective or harmful effects on the surrounding cells, including neurons. Therefore, regulating TLRs in microglia may be a potential therapeutic strategy for neurological disorders. We examined the protective effects of GSP1-111, a novel synthetic peptide for inhibiting TLR signaling, on neuroinflammation and depression-like behavior. GSP1-111 decreased TLR2 expression and remarkably reduced the mRNA expression of inflammatory M1-phenotype markers, including tumor necrosis factor (TNF)α, interleukin (IL)-1β, and IL-6, while elevating that of the M2 phenotype markers, Arg-1 and IL-10. In vivo, GSP1-111 administration significantly decreased the depression-like behavior induced by lipopolysaccharide (LPS) in a forced swim test and significantly reduced the brain levels of M1-specific inflammatory cytokines (TNFα, IL-1β, and IL-6). GSP1-111 prevented the LPS-induced microglial activation and TLR2 expression in the brain. Accordingly, GSP1-111 prevented inflammatory responses and induced microglial switching of the inflammatory M1 phenotype to the protective M2 phenotype. Thus, GSP1-111 could prevent depression-like behavior by inhibiting TLR2. Taken together, our results suggest that the TLR2 pathway is a promising therapeutic target for depression, and GSP1-111 could be a novel therapeutic candidate for various neurological disorders. [ABSTRACT FROM AUTHOR]

Maurotoxin (MTX) is a 34-residue peptide from Scorpio maurus venom. It is reticulated by four disulfide bridges with a unique arrangement compared to other scorpion toxins that target potassium (K+) channels. Structure–activity relationship studies have not been well performed for this toxin family. The screening of Scorpio maurus venom was performed by different steps of fractionation, followed by the ELISA test, using MTX antibodies, to isolate an MTX-like peptide. In vitro, in vivo and computational studies were performed to study the structure–activity relationship of the new isolated peptide. We isolated a new peptide designated MTX1, structurally related to MTX. It demonstrated toxicity on mice eight times more effectively than MTX. MTX1 blocks the Kv1.2 and Kv1.3 channels, expressed in Xenopus oocytes, with IC50 values of 0.26 and 180 nM, respectively. Moreover, MTX1 competitively interacts with both 125I-apamin (IC50 = 1.7 nM) and 125I-charybdotoxin (IC50 = 5 nM) for binding to rat brain synaptosomes. Despite its high sequence similarity (85%) to MTX, MTX1 exhibits a higher binding affinity towards the Kv1.2 and SKCa channels. Computational analysis highlights the significance of specific residues in the β-sheet region, particularly the R27, in enhancing the binding affinity of MTX1 towards the Kv1.2 and SKCa channels. [ABSTRACT FROM AUTHOR]

The synthetic peptide TAT-I24 (GRKKRRQRRRPPQCLAFYACFC) exerts antiviral activity against several double-stranded (ds) DNA viruses, including herpes simplex viruses, cytomegalovirus, some adenoviruses, vaccinia virus and SV40 polyomavirus. In the present study, in vitro profiling of this peptide was performed with the aim of characterizing and improving its properties for further development. As TAT-I24 contains three free cysteine residues, a potential disadvantageous feature, peptide variants with replacements or deletions of specific residues were generated and tested in various cell systems and by biochemical analyses. Some cysteine replacements had no impact on the antiviral activity, such as the deletion of cysteine 14, which also showed improved biochemical properties, while the cyclization of cysteines 14 and 20 had the most detrimental effect on antiviral activity. At concentrations below 20 µM, TAT-I24 and selected variants did not induce hemolysis in red blood cells (RBCs) nor modulated lipopolysaccharide (LPS)-induced release of cytokines, such as interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), in human peripheral blood mononuclear cells (PBMCs). These data indicate that TAT-I24 or its peptide variants are not expected to cause unwanted effects on blood cells. [ABSTRACT FROM AUTHOR]