[Research progress on chemical constituents and pharmacological activities of small molecule compounds in scorpions].

Scorpions, a group of oldest animals with wide distribution in the world, have a long history of medicinal use. Scorpio, the dried body of Buthus martensii, is a rare animal medicine mainly used for the treatment of liver diseases, spasm, and convulsions in children in China. The venom has been considered as the active substance of scorpions. However, little is known about the small molecules in the venom of scorpions. According to the articles published in recent years, scorpions contain amino acids, fatty acids, steroids, and alkaloids, which endow scorpions with antimicrobial, anticoagulant, metabolism-regulating, and antitumor activities. This paper summarizes the small molecule chemical components and pharmacological activities of scorpions, with a view to providing valuable information for the discovery of new active molecules and the clinical use of scorpions.

Marine-Derived Peptides with Anti-Hypertensive Properties: Prospects for Pharmaceuticals, Supplements, and Functional Food.

Hypertension, a major health concern linked to heart disease and premature mortality, has prompted a search for alternative treatments due to side effects of existing medications. Sustainable harvesting of low-trophic marine organisms not only enhances food security but also provides a variety of bioactive molecules, including peptides. Despite comprising only a fraction of active natural compounds, peptides are ideal for drug development due to their size, stability, and resistance to degradation. Our review evaluates the anti-hypertensive properties of peptides and proteins derived from selected marine invertebrate phyla, examining the various methodologies used and their application in pharmaceuticals, supplements, and functional food. A considerable body of research exists on the anti-hypertensive effects of certain marine invertebrates, yet many species remain unexamined. The array of assessments methods, particularly for ACE inhibition, complicates the comparison of results. The dominance of in vitro and animal in vivo studies indicates a need for more clinical research in order to transition peptides into pharmaceuticals. Our findings lay the groundwork for further exploration of these promising marine invertebrates, emphasizing the need to balance scientific discovery and marine conservation for sustainable resource use.

Photocatalytic Functionalization of Dehydroalanine-Derived Peptides in Batch and Flow.

Unnatural amino acids, and their synthesis by the late-stage functionalization (LSF) of peptides, play a crucial role in areas such as drug design and discovery. Historically, the LSF of biomolecules has predominantly utilized traditional synthetic methodologies that exploit nucleophilic residues, such as cysteine, lysine or tyrosine. Herein, we present a photocatalytic hydroarylation process targeting the electrophilic residue dehydroalanine (Dha). This residue possesses an α,ß-unsaturated moiety and can be combined with various arylthianthrenium salts, both in batch and flow reactors. Notably, the flow setup proved instrumental for efficient scale-up, paving the way for the synthesis of unnatural amino acids and peptides in substantial quantities. Our photocatalytic approach, being inherently mild, permits the diversification of peptides even when they contain sensitive functional groups. The readily available arylthianthrenium salts facilitate the seamless integration of Dha-containing peptides with a wide range of arenes, drug blueprints, and natural products, culminating in the creation of unconventional phenylalanine derivatives. The synergistic effect of the high functional group tolerance and the modular characteristic of the aryl electrophile enables efficient peptide conjugation and ligation in both batch and flow conditions.

Egg Yolk Selenopeptides: Preparation, Characterization, and Immunomodulatory Activity.

In this study, egg yolk selenium peptides (Se-EYP) were prepared using double-enzyme hydrolysis combined with a shearing pretreatment. The properties of the selenopeptides formed were then characterized, including their yield, composition, molecular weight distribution, antioxidant activity, in vitro digestion, and immunomodulatory activity. The peptide yield obtained after enzymatic hydrolysis using a combination of alkaline protease and neutral protease was 74.5%, of which 82.6% had a molecular weight <1000 Da. The selenium content of the lyophilized solid product was 4.01 µg/g. Chromatography-mass spectrometry analysis showed that 88.6% of selenium in Se-EYP was in the organic form, of which SeMet accounted for 60.3%, SeCys2 for 21.8%, and MeSeCys for 17.9%. After being exposed to in vitro simulated digestion, Se-EYP still had 65.1% of oligopeptides present, and the in vitro antioxidant activity was enhanced. Moreover, Se-EYP exhibited superior immune detection indices, including immune organ index, level of immune factors in the serum, histopathological changes in the spleen, and selenium content in the liver. Our results suggest that Se-EYP may be used as selenium-enriched ingredients in functional food products.

Investigation of the peptides with calcium chelating capacity in hydrolysate derived from spent hen meat.

Calcium peptide chelates are developed as efficient supplements for preventing calcium deficiency. Spent hen meat (SHM) contains a high percentage of proteins but is generally wasted due to the disadvantages such as hard texture. We chose the underutilized SHM to produce peptides to bind calcium by proteolysis and aimed to investigate chelation between calcium and peptides in hydrolysate for a sustainable purpose. The optimized proteolysis conditions calculated from the result of response surface methodology for two-step hydrolysis were 0.30% (wenzyme/wmeat) for papain with a hydrolysis time of 3.5 h and 0.18% (wenzyme/wmeat) for flavourzyme with a hydrolysis time of 2.8 h. The enzymatic hydrolysate (EH) showed a binding capacity of 63.8 ± 1.8 mg calcium/g protein. Ethanol separation for EH improved the capacity up to a higher value of 68.6 ± 0.6 mg calcium/g protein with a high association constant of 420 M-1 (25°C) indicating high stability. The separated fraction with a higher amount of Glu, Asp, Lys, and Arg had higher calcium-binding capacity, which was related to the number of ─COOH and ─NH2 groups in peptide side chains according to the result from amino acid analysis and Fourier transform infrared spectroscopy. Two-step enzymatic hydrolysis and ethanol separation were an efficient combination to produce peptide mixtures derived from SHM with high calcium-binding capacity. The high percentage of hydrophilic amino acids in the separated fraction was concluded to increase calcium-binding capacity. This work provides foundations for increasing spent hen utilization and developing calcium peptide chelates based on underutilized meat.

The novel angiotensin-I-converting enzyme inhibitory peptides from Scomber japonicus muscle protein hydrolysates: QSAR-based screening, molecular docking, kinetic and stability studies.

Food-derived angiotensin-converting enzyme-inhibitory (ACE-I) peptides have attracted extensive attention. Herein, the ACE-I peptides from Scomber japonicus muscle hydrolysates were screened, and their mechanisms of action and inhibition stability were explored. The quantitative structure-activity relationship (QSAR) model based on 5z-scale metrics was developed to rapidly screen for ACE-I peptides. Two novel potential ACE-I peptides (LTPFT, PLITT) were predicted through this model coupled with in silico screening, of which PLITT had the highest activity (IC50: 48.73 ± 7.59 µM). PLITT inhibited ACE activity with a mixture of non-competitive and competitive mechanisms, and this inhibition mainly contributed to the hydrogen bonding based on molecular docking study. PLITT is stable under high temperatures, pH, glucose, and NaCl. The zinc ions (Zn2+) and copper ions (Cu2+) enhanced ACE-I activity. The study suggests that the QSAR model is effective in rapidly screening for ACE-I inhibitors, and PLITT can be supplemented in foods to lower blood pressure.

Novel Multitarget ACE Inhibitory Peptides from Bovine Colostrum Immunoglobulin G: Cellular Transport, Efficacy in Regulating Endothelial Dysfunction, and Network Pharmacology Studies.

In this study, we used traditional laboratory methods, bioinformatics, and cellular models to screen novel ACE inhibitory (ACEI) peptides with strong ACEI activity, moderate absorption rates, and multiple targets from bovine colostrum immunoglobulin G (IgG). The purified fraction of the compound proteinase hydrolysate of IgG showed good ACEI activity. After nano-UPLC-MS/MS identification and in silico analysis, eight peptides were synthesized and verified. Among them, SFYPDY, TSFYPDY, FSWF, WYQQVPGSGL, and GVHTFP were identified as ACEI peptides, as they exhibited strong ACEI activity (with IC50 values of 104.7, 80.0, 121.2, 39.8, and 86.3 µM, respectively). They displayed good stability in an in vitro simulated gastrointestinal digestion assay. In a Caco-2 monolayer model, SFYPDY, FSWF, and WYQQVPGSGL exhibited better absorption rates and lower IC50 values than the other peptides and were thereby identified as novel ACEI peptides. Subsequently, in a H2O2-induced endothelial dysfunction (ED) model based on HUVECs, SFYPDY, FSWF, and WYQQVPGSGL regulated ED by reducing apoptosis and ROS accumulation while upregulating NOS3 mRNA expression. Network pharmacology analysis and RT-qPCR confirmed that they regulated multiple targets. Overall, our results suggest that SFYPDY, FSWF, and WYQQVPGSGL can serve as novel multitarget ACEI peptides.

Bioactive peptides: an alternative therapeutic approach for cancer management.

Cancer is still considered a lethal disease worldwide and the patients' quality of life is affected by major side effects of the treatments including post-surgery complications, chemo-, and radiation therapy. Recently, new therapeutic approaches were considered globally for increasing conventional cancer therapy efficacy and decreasing the adverse effects. Bioactive peptides obtained from plant and animal sources have drawn increased attention because of their potential as complementary therapy. This review presents a contemporary examination of bioactive peptides derived from natural origins with demonstrated anticancer, ant invasion, and immunomodulation properties. For example, peptides derived from common beans, chickpeas, wheat germ, and mung beans exhibited antiproliferative and toxic effects on cancer cells, favoring cell cycle arrest and apoptosis. On the other hand, peptides from marine sources showed the potential for inhibiting tumor growth and metastasis. In this review we will discuss these data highlighting the potential befits of these approaches and the need of further investigations to fully characterize their potential in clinics.

In silico prediction aided preparation of antioxidant soybean peptides by enzymatic hydrolysis for ameliorating lead exposure-induced toxicity.

Food derived bioactive peptides are prominent dietary supplements to protect against oxidative stress induced by lead (Pb) exposure. This study aimed to develop a new strategy for rapid preparation of highly active antioxidant soybean polypeptides (ASPs) against Pb toxicity. In silico enzymatic hydrolysis simulation and antioxidant activity prediction showed that pepsin, chymotrypsin and bromelain can produce peptides with the highest activity. The preparation process was then optimized, and the obtained ASP showed good antioxidant and metal-chelating activities in vitro. An in vivo study showed that ASP exerted prominent protective effects against Pb-induced cognitive impairment and tissue damage in mice by reducing Pb deposition and enhancing the antioxidant capacity in tissues and was superior to Vc, DMSA or their combination in some aspects. ASP composition analysis demonstrated that its prominent antioxidant activity might be attributed to the high proportion of amino acid residues E, L, P and V in the peptide sequence and L, V and A at the C- and N-termini. In conclusion, in silico prediction could facilitate the preparation of ASP. And the ASP prepared with the new strategy exerted prominent protective effects against Pb toxicity.

Selenium Decipher: Trapping of Native Selenomethionine-Containing Peptides in Selenium-Enriched Milk and Unveiling the Deterioration after Ultrahigh-Temperature Treatment.

Selenopeptide identification relies on databases to interpret the selenopeptide spectra. A common database search strategy is to set selenium as a variable modification instead of sulfur on peptides. However, this approach generally detects only a fraction of selenopeptides. An alternative approach, termed Selenium Decipher, is proposed in the present study. It involves identifying collision-induced dissociation-cleavable selenomethionine-containing peptides by iteratively matching the masses of seleno-amino acids in selenopeptide spectra. This approach uses variable-data-independent acquisition (vDIA) for peptide detection, providing a flexible and customizable window for secondary mass spectral fragmentation. The attention mechanism was used to capture global information on peptides and determine selenomethionine-containing peptide backbones. The core structure of selenium on selenomethionine-containing peptides generates a series of fragment ions, namely, C3H7Se+, C4H10NSe+, C5H7OSe+, C5H8NOSe+, and C7H11N2O2Se+, with known mass gaps during higher-energy collisional dissociation (HCD) fragmentation. De-selenium spectra are generated by removing selenium originating from selenium replacement and then reassigning the precursors to peptides. Selenium-enriched milk is obtained by feeding selenium-rich forage fed to cattle, which leads to the formation of native selenium through biotransformation. A novel antihypertensive selenopeptide Thr-Asp-Asp-Ile-SeMet-Cys-Val-Lys TDDI(Se)MCVK was identified from selenium-enriched milk. The selenopeptide (IC50 = 60.71 µM) is bound to four active residues of the angiotensin-converting enzyme (ACE) active pocket (Ala354, Tyr523, His353, and His513) and two active residues of zinc ligand (His387 and Glu411) and exerted a competitive inhibitory effect on the spatial blocking of active sites. The integration of vDIA and the iteratively matched seleno-amino acids was applied for Selenium Decipher, which provides high validity for selenomethionine-containing peptide identification.

Identification, characterization, and insights into the mechanism of novel dipeptidyl peptidase-IV inhibitory peptides from yak hemoglobin by in silico exploration, molecular docking, and in vitro assessment.

Dipeptidyl peptidase IV (DPP-IV) inhibitory peptides were screened and identified from yak hemoglobin for the first time by in silico analysis, molecular docking, and in vitro evaluation. Results showed that yak hemoglobin had a high potential to produce DPP-IV inhibitory peptides based on the sequence alignment and bioactive potential evaluation. Furthermore, "pancreatic elastase + stem bromelain" was the optimal combined-enzymatic strategy by simulated proteolysis. Additionally, 25 novel peptides were found from its simulated hydrolysate, among which 10 peptides had high binding affinities with DPP-IV by molecular docking. Most of these peptides were also in silico characterized with favorable physicochemical properties and biological potentials, including relatively low molecular weight, high hydrophobicity, several net charges, good water solubility, nontoxicity, acceptable sensory quality, and good human intestinal absorption. Finally, six novel DPP-IV inhibitory peptides were identified via in vitro assessment, among which EEKA (IC50 = 235.26 µM), DEV (IC50 = 339.45 µM), and HCDKL (IC50 = 632.93 µM) showed the strongest capacities. The hydrogen bonds and electrostatic attractions formed with core residues within the S2 pocket of DPP-IV could be mainly responsible for their inhibition performances. This work provided a time-saving method and broadened application for yak by-products development as sources of functional foods.

A critical review of a typical research system for food-derived metal-chelating peptides: Production, characterization, identification, digestion, and absorption.

In the past decade, food-derived metal-chelating peptides (MCPs) have attracted significant attention from researchers working towards the prevention of metal (viz., iron, zinc, and calcium) deficiency phenomenon by primarily inhibiting the precipitation of metals caused by the gastrointestinal environment and exogenous substances (including phytic and oxalic acids). However, for the improvement of limits of current knowledge foundations and future investigation directions of MCP or their derivatives, several review categories should be improved and emphasized. The species' uniqueness and differences in MCP productions highly contribute to the different values of chelating ability with particular metal ions, whereas comprehensive reviews of chelation characterization determined by various kinds of technique support different horizons for explaining the chelation and offer options for the selection of characterization methods. The reviews of chelation mechanism clearly demonstrate the involvement of potential groups and atoms in chelating metal ions. The discussions of digestive stability and absorption in various kinds of absorption model in vitro and in vivo as well as the theory of involved cellular absorption channels and pathways are systematically reviewed and highlighted compared with previous reports as well. Meanwhile, the chelation mechanism on the molecular docking level, the binding mechanism in amino acid identification level, the utilizations of everted rat gut sac model for absorption, and the involvement of cellular absorption channels and pathway are strongly recommended as novelty in this review. This review makes a novel contribution to the literature by the comprehensive prospects for the research and development of food-derived mineral supplements.

Xanthan gum and pectin as beverage stabilizers reduce the digestive enzyme hydrolysis of antioxidant and antihypertensive peptides obtained from a brewery byproduct.

An acidic beverage was formulated with xanthan gum (XG), pectin (P) and brewer spent grain (BSG) peptides with antioxidant and antihypertensive properties. The impact of hydrocolloids levels on peptide bioaccessibility was studied. Peptides were obtained from BSG using Purazyme and Flavourzyme enzymes. BSG peptides were fractionated by ultrafiltration (UF) and four fractions were obtained: F1 (>10 kDa), F2 (10-5 kDa), F3 (1-5 kDa), and F4 (<1 kDa). F3 showed the highest protein purity, ferulic acid content, proportion of amphipathic peptides, and bioactive properties (ABTS+ radical scavenging and ACE-I inhibitory activity). The identified peptides from F3 by tandem mass spectrometry were 138. In silico analysis showed that 26 identified peptides had ABTS+ inhibitory activity, while 59 ones presented good antihypertensive properties. The effect of XG and P levels on bioaccessibility of F3 peptides in the formulated beverages was studied by a central composite experimental design. It was observed that F3 peptides interacted with hydrocolloids by electrostatic forces at pH of formulated beverages. The addition of hydrocolloids to formulation modulated the release of the antioxidant peptides and protected the degradation of ACE-I inhibitory peptides from F3 during simulated gastrointestinal digestion. Finally, the level of hydrocolloids that produced intermediate viscosities in the formulated beverages improved the bioaccessibility of the F3 peptides.

Self-Assembled Materials Based on Fully Aromatic Peptides: The Impact of Tryptophan, Tyrosine, and Dopa Residues.

Peptides are able to self-organize in structural elements including cross-ß structures. Taking advantage of this tendency, in the last decades, peptides have been scrutinized as molecular elements for the development of multivalent supramolecular architectures. In this context, different classes of peptides, also with completely aromatic sequences, were proposed. Our previous studies highlighted that the (FY)3 peptide, which alternates hydrophobic phenylalanine and more hydrophilic tyrosine residues, is able to self-assemble, thanks to the formation of both polar and apolar interfaces. It was observed that the replacement of Phe and Tyr residues with other noncoded aromatic amino acids like 2-naphthylalanine (Nal) and Dopa affects the interactions among peptides with consequences on the supramolecular organization. Herein, we have investigated the self-assembling behavior of two novel (FY)3 analogues with Trp and Dopa residues in place of the Phe and Tyr ones, respectively. Additionally, PEGylation of the N-terminus was analyzed too. The supramolecular organization, morphology, and capability to gel were evaluated using complementary techniques, including fluorescence, Fourier transform infrared spectroscopy, and scanning electron microscopy. Structural periodicities along and perpendicular to the fiber axis were detected by grazing incidence wide-angle X-ray scattering. Finally, molecular dynamics studies provided interesting insights into the atomic structure of the cross-ß that constitutes the basic motif of the assemblies formed by these novel peptide systems.

Preparation, characterization, and bioavailability evaluation of antioxidant phosvitin peptide-ferrous complex.

BACKGROUND: Iron deficiency anemia (IDA) is one of the commonest global nutritional deficiency diseases, and the low bioavailability of iron is a key contributing factor. The peptide-iron complex could be used as a novel iron supplement to improve iron bioavailability. RESULTS: In this study, antioxidant low molecular weight (<3 kDa) phosvitin peptide (named PP-4) was separated to prepare a phosvitin peptide-ferrous complex (named PP-4-Fe); then the structural conformation of PP-4-Fe was characterized and its bioavailability by in vitro digestion was evaluated. The results showed that PP-4 had good ferrous-binding activity with 96.14 ± 2.86 µg Fe2+ mg-1 , and had a strong antioxidant effect with 995.61 ± 79.75 µmol TE mg-1 in 2,2'-azinobis'3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 62.3 ± 3.95 µmol FeSO4 mg-1 in ferric ion reducing antioxidant power (FRAP). After ferrous binding, the FRAP activity of PP-4-Fe, enhanced by 1.8 times, formed a more ordered structure with an increase in α-helix and decrease in γ-random coil. The ferrous binding sites of PP-4 involved were the amino, carboxyl, imidazole, and phosphate groups. The PP-4-Fe complex displayed excellent gastrointestinal stability and antioxidant effects during digestion. The iron dialysis percentage of PP-4-Fe was 74.59% ± 0.68%, and increased to 81.10% ± 0.89% with the addition of 0.25 times vitamin C (VC). This indicated that PP-4-Fe displayed excellent bioavailability and VC in sufficient quantities had a synergistic effect on improving bioavailability. CONCLUSIONS: This study demonstrated that antioxidant phosvitin peptide was an efficient delivery system to protect ferrous ions and suggested that the phosvitin peptide-ferrous complex has strong potential as a ferrous supplement. © 2023 Society of Chemical Industry.

An Offset Patterned Cross-ß Structure in Assemblies of C3 -Symmetric Peptide Amphiphiles.

Developing peptide-based materials with controlled morphology is a critical theme of soft matter research. Herein, we report the formation of a novel, patterned cross-ß structure formed by self-assembled C3 -symmetric peptide amphiphiles based on diphenylalanine and benzene-1,3,5-tricarboxamide (BTA). The cross-ß motif is an abundant structural element in amyloid fibrils and aggregates of fibril-forming peptides, including diphenylalanine. The incorporation of topological constraints on one edge of the diphenylalanine fragment limits the number of ß-strands in ß-sheets and leads to the creation of an unconventional offset-patterned cross-ß structure consisting of short 3×2 parallel ß-sheets stabilized by phenylalanine zippers. In the reported assembly, two patterned cross-ß structures bind parallel arrays of BTA stacks in a superstructure within a single-molecule-thick nanoribbon. In addition to a threefold network of hydrogen bonds in the BTA stack, each molecule becomes simultaneously bound by hydrogen bonds from three ß-sheets and four phenylalanine zippers. The diffuse layer of alkyl chains with terminal polar groups prevents the nanoribbons from merging and stabilizes cross-ß-structure in water. Our results provide a simple approach to the incorporation of novel patterned cross-ß motifs into supramolecular superstructures and shed light on the general mechanism of ß-sheet formation in C3 -symmetric peptide amphiphiles.

Human colostrum in vitro protein digestion: peptidomics by liquid chromatography-Orbitrap-high-resolution MS and prospection for bioactive peptides via bioinformatics.

Breast milk is known to contain bioactive peptides that are released during digestion, being a major source of bioactive peptides to the new-born, some of which act against invading pathogens. However, the formation of bioactive peptides during digestion of human colostrum remains largely uninvestigated. This study aimed to investigate the formation of peptides during simulated digestion of human colostrum from adult women and to prospect antimicrobial peptides. For this purpose, we used high-resolution MS to monitor the release of peptides during in vitro digestion. Bioinformatics was used for the prospection of antimicrobial activity of peptides. During simulated digestion (oral, gastric and duodenal phases), 2318 peptide sequences derived from 112 precursor proteins were identified. At the end of simulated digestion, casein-derived peptide sequences were the most frequently observed. Among precursors, some proteins were seen for the first time in this study. The resulting peptides were rich in proline, glutamine, valine and leucine residues, providing characteristic traits of antimicrobial peptides. From bioinformatics analysis, seven peptides showed potentially high antimicrobial activity towards bacteria, viruses and fungi, from which the latter was the most prominent predicted activity. Antimicrobial peptides released during digestion may provide a defence platform with controlled release for the new-born.

Influence of the modification of the cosmetic peptide Argireline on the affinity toward copper(II) ions.

Argireline (Ac-EEMQRR-NH2 ), a well-known neurotransmitter peptide with a potency similar to botulinum neurotoxins, reveals a proven affinity toward Cu(II) ions. We report herein Cu(II) chelating properties of three new Argireline derivatives, namely, AN4 (Ac-EAHRR-NH2 ), AN5 (Ac-EEHQRR-NH2 ), and AN6 (Ac-EAHQRK-NH2 ). Two complementary experimental techniques, i.e., potentiometric titration (PT) and isothermal titration calorimetry (ITC), have been employed to describe the acid-base properties of the investigated peptides as well as the thermodynamic parameters of the Cu(II) complex formation. Additionally, based on density functional theory (DFT) calculations, we propose the most likely structures of the resulting Cu-peptide complexes. Finally, the cytotoxicity of the free peptides and the corresponding Cu(II) complexes was estimated in human skin cells for their possible future cosmetic application. The biological results were subsequently compared with free Argireline, its Cu(II)-complexes, and the previously studied AN2 derivative (EAHQRR).

Unambiguous discrimination of all 20 proteinogenic amino acids and their modifications by nanopore.

Natural proteins are composed of 20 proteinogenic amino acids and their post-translational modifications (PTMs). However, due to the lack of a suitable nanopore sensor that can simultaneously discriminate between all 20 amino acids and their PTMs, direct sequencing of protein with nanopores has not yet been realized. Here, we present an engineered hetero-octameric Mycobacterium smegmatis porin A (MspA) nanopore containing a sole Ni2+ modification. It enables full discrimination of all 20 proteinogenic amino acids and 4 representative modified amino acids, Nω,N'ω-dimethyl-arginine (Me-R), O-acetyl-threonine (Ac-T), N4-(ß-N-acetyl-D-glucosaminyl)-asparagine (GlcNAc-N) and O-phosphoserine (P-S). Assisted by machine learning, an accuracy of 98.6% was achieved. Amino acid supplement tablets and peptidase-digested amino acids from peptides were also analyzed using this strategy. This capacity for simultaneous discrimination of all 20 proteinogenic amino acids and their PTMs suggests the potential to achieve protein sequencing using this nanopore-based strategy.

Development of a novel squalene/α-tocopherol-based self-emulsified nanoemulsion incorporating Leishmania peptides for induction of antigen-specific immune responses.

Vaccination has emerged as the most effective strategy to confront infectious diseases, among which is leishmaniasis, that threat public health. Despite laborious efforts there is still no vaccine for humans to confront leishmaniasis. Multi-epitope protein/peptide vaccines present a number of advantages, however their use along with appropriate adjuvants that may also act as antigen carriers is considered essential to overcome subunit vaccines' low immunogenicity. In the present study, a stable self-emulsified nanoemulsion was developed and double-adjuvanted with squalene and α-tocopherol. The prepared nanoemulsion droplets exhibited low cytotoxicity in a certain range of concentrations, while they were efficiently taken up by macrophages and dendritic cells in vitro as well as in vivo in secondary lymphoid organs. To further characterize nanoformulation's potent antigen delivery capability, three multi-epitope Leishmania peptides were incorporated into the nanoemulsion. Peptide encapsulation resulted in dendritic cells' functional differentiation characterized by elevated levels of maturation markers and intracellular cytokine production. Intramuscular administration of the nanoemulsion incorporating Leishmania peptides induced antigen-specific spleen cell proliferation as well as elicitation of CD4+ central memory cells, supporting the potential of the developed nanoformulation to successfully act also as an antigen delivery vehicle and thus encouraging further preclinical studies on its vaccine candidate potency.