Background: Fermented milks are a source of bioactive peptides with different potential benefits on human health (mainly antioxidant and antihypertensive effects) and may be considered as functional foods. Furthermore, those made with sheep milk have higher nutritional value and higher concentrations of proteins, fats, minerals, and vitamins, if compared to milk of other species. Objectives: Aim of this study was to perform a biochemical characterization and a comparison between sheep fermented milk (SFM) made with commercial starter cultures and sheep milk YoAlp®, a fermented milk obtained using local strains of lactic acid bacteria (LABs) (Streptococcus thermophilus and Lactobacillus delbrueckii) which have been isolated during years in Aosta Valley mountain dairy cattle farm. Methods: Sheep milk and fermented milk (Sarda and Lacaune breeds) have been collected and immediately analyzed. Biochemical characterization, by a proteomic approach, GC/MS and microtiter plate assay methods, have been conducted to evaluate their peptide, fatty acid and aromatic profile, and to assess potential health promoting effects. Results: The comparison between SFM and YoAlp® peptide profile shows a higher number of peptides using autochthonous starter cultures (52) than commercial starter cultures (35). Among these peptides 20,78% and 29,87%, respectively, are supposed to be potentially bioactive (DPP - dipeptidyl peptidase-IV - inhibitor, ACE - angiotensin converting enzyme – inhibitor and antioxidant). In both products, the fatty acids profile was similar to that of origin sheep milk; concerning aromatic profile, YoAlp® shows yogurt typical aromatic assets, similar to the commercial one. As far as bioactivity is concerned, ACE inhibitor activity is high for both samples. Similar values, as expected by peptide profile analysis, have been obtained using commercial and autochthonous starters. Even in the case of antioxidant capacity, peptide profile bioactivity prediction has been confirmed by the assay showing a DPPH inhibition higher for SFM than for YoAlp®, but this difference is not statistically significative. Conclusions: Local strains of lactic acid bacteria from Aosta Valley seems to work as well as the commercial, preserving biodiversity and typicality. However, further analyses are needed to identify peptides generated by autochthonous LABs in order to understand microbial proteolytic activities and to investigate gastric digestion resistance of bioactive peptides which can have positive effects on human health.

Despite great strides in diagnosis and treatment, cardiac diseases remain the number one cause of mortality in the developed world, with rates catching up at an alarming rate in the developing world. Targeted delivery of therapeutics, specifically to cardiomyocytes, would open up new frontiers. Our prior work using a combinatorial in vitro and in vivo phage display methodology identified a 12-amino acid long peptide that targets cardiomyocytes selectively after an intravenous injection in as little as 5 mins, and hence was termed cardiac targeting peptide (CTP: APHLSSQYSRT). CTP has been used to deliver imaging agents, small drug molecules, photosensitizing nanoparticles, exosomes, and even miRNA to cardiomyocytes. As a natural extension for development of CTP as a clinically viable cardiac vector, we now present toxicity studies performed on the peptide. In vitro viability studies were performed in a human left ventricular myocyte cell line with 10µM of Cyanine-5.5 labeled CTP (CTP-Cy5.5). In vitro ion channel profiles were completed for CTP followed by more extensive studies performed by Eurofins in stably transfected cell lines for an extensive list of GPCR-coupled receptors. Positive data for GPCR-coupled receptors was interrogated further with RT-qPCRs performed on mouse heart tissue. In vivo studies consisted of pre- and post-blood pressure monitoring acutely after a single CTP (10 mg/Kg) injection. Further in vivo toxicity studies consisted of injecting 60, 6-week-old, wild-type CD1, male/female mice (1:1) injected with CTP (150 µg/Kg) intravenously. Mice were weighed immediately before injections and daily thereafter. Cohorts of mice were euthanized on day 0, 1, 2, 7 and 14 with inhalational CO2, followed by opening of the chest cavity, collection of blood via cardiac puncture, complete blood count analysis, metabolic profiling, and finally, liver, renal and thyroid studies. Lastly, mouse cardiac MRI was performed immediately before and after injections of CTP (150 µg/Kg) to assess changes in cardiac size or function. Human left ventricular cardiomyocytes showed no decrease in viability after a 30 min incubation with CTP-Cy5.5. In the Eurofins Cardiac Profiler Qube, no significant activation or inhibition of any of seventy-eight protein channels was observed other than OPRM1 and COX2 at the highest tested concentration, neither of which were expressed in mouse heart tissue as assessed by RT-qPCR. There was no change in blood pressures before and after injections with CTP at 10 mg/Kg. Blood counts and chemistries showed no evidence of significant hematological, hepatic or renal toxicities. Lastly, there was no significant difference in cardiac function, size or mass before and after CTP injections. Our studies with CTP showed no activation or inhibition of GPCR-associated receptors in vitro. We found no signals indicative of toxicity in vivo. Most importantly, cardiac functions remained unchanged acutely in response to CTP uptake. Further studies, in good laboratory practices fashion, are needed with more prolonged, chronic administration of CTP conjugated to a specific cargo of choice before human studies can be contemplated.

Spondyloarthritis comprises of a group of inflammatory diseases of the joints and spine with various clinical manifestations. The group includes ankylosing spondylitis, reactive arthritis, psoriatic arthritis, arthritis associated with inflammatory bowel disease, and undifferentiated spondyloarthritis. The exact etiology and pathogenesis of spondyloarthritis are still unknown, but five hypotheses explaining the pathogenesis exist. These hypotheses suggest that spondyloarthritis is caused by arthritogenic peptides, an unfolded protein response, HLA-B27 homodimer formation, malfunctioning endoplasmic reticulum aminopeptidases, and, last but not least, gut inflammation and dysbiosis. Here we discuss the five hypotheses and the evidence supporting each. In all of these hypotheses, HLA-B27 plays a central role. It is likely that a combination of these hypotheses, with HLA-B27 taking center stage, will eventually explain the development of spondyloarthritis in predisposed individuals.

Mosquitoes have distinct developmental and adult life history, and the vectorial capacity of females has been shown to be affected by the larval nutritional environment. However, little is known about the effect of developmental nutrition on insulin-signaling and nutrients storage. In this study, we used Aedes aegypti, the yellow fever mosquito, to determine whether larval nutrition affects insulin gene expression. We also determined the traits regulated by insulin signaling, such as stored-nutrients levels and fecundity. We raised mosquito larvae on two different diets, containing either high protein or high carbohydrates. Development on a high-carbohydrate diet resulted in several life-history phenotypes indicative of suboptimal conditions, including increased developmental time and decreased fecundity. Additionally, our data showed that insulin transcript levels are affected by a high-carbohydrate diet during development. Females, not males, reared on high-carbohydrate diets had much higher transcript levels of insulin-like peptide 3 (ILP3), a mosquito equivalent of human insulin, and these females more readily stored sugar from the meal into lipids. We also found that AaILP4, not AaILP3, transcript levels were much higher in the males after a sugar meal, suggesting sex-specific differences in insulin-signaling pathway. Our findings suggest a conserved mechanism of carbohydrate-mediated hyperinsulinemia in animals.

SORL1 gene encodes LR11/SorLA, a protein that binds -amyloid precursor protein (bAPP) and drives its intracellular trafficking. SORL1 mutations occurring frequently in a subset of familial cases of Alzheimer’s disease (AD) have been documented but their pathogenic potential is not yet clear and questions remain concerning their putative influence on the physiopathological processing of bAPP. We have assessed the influence of three SORL1 mutations that were described as likely disease-causing and that were associated with either benign (SorLA924) or severe (SorLA141 and SorLA511) AD phenotypes. We examined the influence of wild-type and mutants SorLA in transiently transfected HEK293 cells expressing either wild-type or Swedish mutated bAPP on bAPP expression, secreted Ab and sAPPa levels, intracellular Ab40 and Ab42 peptides, APP-CTFs (C99 and C83) expressions, a-, b- and g-secretases expressions and activities as well as Ab and CTFs-degrading enzymes. These paradigms were studied in control conditions or after pharmacological proteasomal modulation. We also established stably transfected CHO cells expressing wild-type SorLA and established the colocalization of wild-type SorLA and bAPP. Overall, although we mostly confirmed previous data concerning the influence of wild-type SorLA on bAPP processing, we were unable to evidence significant alterations triggered by our set of SorLA mutants, whatever the cells or pharmacological conditions examined. Our study however does not rule out the possibility that other AD-linked SORL1 mutations could indeed affect bAPP processing and that pathogenic mutations examined in the present study could interfere with other cellular pathways/triggers in AD.

Background: There are no known medicines or vaccines to control the COVID-19 pandemic caused by SARS-CoV-2 (nCoV). Antiviral peptides are superior to conventional drugs and may also be effective against COVID-19. Hence, we investigated the SARS-CoV-2 Spike RBD (nCoV-RBD) that interacts with hACE2 for viral attachment and entry. Methods: Three strategies and bioinformatics approaches were employed to design potential nCoV-RBD - hACE2 interaction-blocking peptides that may restrict viral attachment and entry. Firstly, the key residues interacting with nCoV-RBD - hACE2 are identified and hACE2 sequence based peptides are designed. Second, peptides from five antibacterial peptide databases that block nCoV-RBD are identified; finally, a chimeric peptide design approach is used to design peptides that can bind to key nCoV-RBD residues. The final peptides are selected based on their physiochemical properties, numbers and positions of key residues binding, binding energy, and antiviral properties. Results: We found (i) three amino acid stretches in hACE2 interact with nCoV-RBD; (ii) effective peptides must bind to three key positions of nCoV-RBD: Gly485/Phe486/Asn487, Gln493, and Gln498/Thr500/Asn501; (iii) Phe486, Gln493, and Asn501 are critical residues; (iv) AC20 and AC23 derived from hACE2 may block two key critical positions; (iv) DBP6 identified from databases can block the three sites of the nCoV-RBD interacting with one critical position Gln498; (v) seven chimeric peptides were considered promising among which cnCoVP-3, cnCoVP-4, and cnCoVP-7 are the top three; and (vi) cnCoVP-4 meets all the criteria and is the best peptide. Conclusion: All the ten peptides need experimental validation for their therapeutic efficacy.

Bacterial infections occur when wound healing fails to reach the final stage of healing, usually hindered by the presence of different pathogens. Different topical antimicrobial agents are used to inhibit bacterial growth due to antibiotic failure in reaching the infected site accompanied very often by an increased drug resistance and other side effects. In this review, we focus on antimicrobial peptides (AMPs), especially those with a high potential of efficacy against multidrug-resistant and biofilm-forming bacteria and fungi present in wound infections. Currently, different AMPs undergo preclinical and clinical phase to combat infection-related diseases. AMP dendrimers (AMPDs) have been mentioned as potent microbial agents. Various AMP delivery strategies, such as polymers, scaffolds, films and wound dressings, organic and inorganic nanoparticles, to combat infection and modulate the healing rate have been discussed as well. New technologies such as CRISPR-Cas are taken into consideration as potential future tools for AMP delivery in skin therapy.

Triphenylphosphonium (TPP) derivatives of a decapeptide related to sequences of bactenecin 7 (Bac7) and oncocin (Onc112) were synthesized and studied as potential antimicrobial compounds using various biochemical and microbiological assays. It was shown that while the reduction of the Bac7 length to 10 a.a. residues dramatically decreases the affinity to bacterial ribosomes, the modification of peptide with alkyl-TPP moieties leads to an increase in the affinity. New analogs which structure combines a decapeptide related to Bac7 and Onc112 — Bac(1-10, R/Y) — and TPP, attached to the C-terminal amino acid residue through alkylamide linkers, inhibit translation in vitro and are found to be more selective inhibitors of bacterial translation as compared with eukaryotic translation, than Onc112 and Bac7. Triphenylphosphonium analogs of a decapeptide related to Bac7 and Onc112 suppress the growth of both gram-negative bacteria, similar to Onc112 and Bac7, and gram-positive ones as alkyl-TPP derivatives, and also act against some resistant laboratory strains. Bac(1-10, R/Y)-C2-TPP, containing a short alkylamide linker between the decapeptide and TPP, is transferred into the E. coli cells via the SbmA transporter protein. TPP derivatives of the decapeptide Bac(1-10, R/Y) containing either decylamide or ethylamide linker cause B. subtilis membrane depolarization, similar to alkyl-TPP. The Bac(1-10, R/Y)-C2-TPP analog is proved to be non-toxic for mammalian cells by MTT-test.

Keywords: Antisense Oligonucleotides, Cell Penetrating Peptides, Delivery, DG9 peptide, Phosphorodiamidate morpholino oligomers (PMO), Pip, R6G.

Biomimetics, which are similar to natural compounds that play an important role in the metabolism, manifestation of functional activity and reproduction of various fungi, have a pronounced attrac-tion in the current search for new effective antifungals. Actual trends in the development of this area of research indicate that unnatural amino acids can be used as such biomimetics, including those containing halogen atoms; compounds similar to nitrogenous bases embedded in the nucleic acids synthesized by fungi; peptides imitating fungal analogues; molecules similar to natural sub-strates of numerous fungal enzymes and Quorum Sensing signaling molecules of fungi and yeast; etc. Most part of the review is devoted to the analysis of semi-synthetic and synthetic antifungal peptides and their targets of action. This review is aimed at combining and systematizing the cur-rent scientific information accumulating in this area of research, developing various antifungals with an assessment of the effectiveness of the created biomimetics and the possibility of combining them with other antimicrobial substances to reduce cell resistance and improve antifungal effects.

The precise molecular basis of anti-tumor immunity is not yet fully understood, although commensal Bifidobacterium (c-BIF) is expected to be one of the key players in cancer control via antigenic mimicry. We investigated the sequence similarity between c-BIF and cytotoxic T lymphocyte (CTL) epitope peptides against human tumor-associated antigens to better understand the molecular basis of antigenic mimicry. We used two different similarity analyses for this purpose, a linear sequence analysis and a similarity analysis of T cell receptor (TCR)-mediated recognition of CTL epitope peptides on antigen-presenting cells. The linear sequence analysis revealed 3,900 positive numbers of similarity sites between them with 126 mean per peptide and 107 median per peptide, while the TCR-mediated recognition analysis revealed 5,018 positive numbers with a mean of 162 and median of 132 per peptide. These results demonstrated the existence of durable and abundant sequence similarities between c-BIF and CTL epitope peptides, suggesting that the former play a pivotal in inducing cellular and humoral immunity against the latter in the absence of cancer cells via antigenic mimicry.

Small peptides are an important component of the vertebrate immune system. They are important molecules for distinguishing proteins that originate in the host from proteins derived of a pathogenic organism, such as a virus or bacterium. Consequently, these peptides are central for the vertebrate host response to intracellular and extracellular pathogens. Computational models for prediction of these peptides have been based on a narrow sample of data with an emphasis on the position and chemical properties of the amino acids. In past literature, this approach has resulted in higher predictability than models that rely on the geometrical arrangement of atoms. However, protein structure data from experiment and theory are a source for building models at scale, and, therefore, knowledge on the role of small peptides and their immunogenicity in the vertebrate immune system. The following sections introduce procedures that contribute to theoretical prediction of peptides and role in immunogenicity. Lastly, deep learning is discussed as it applies to immunogenetics and the acceleration of knowledge by a capability for modeling the complexity of natural phenomena.

The pharmaceutical market consists mainly of chemical and biological drugs. These drugs act on different types of targets and have distinct pharmacological properties. Generally, chemical drugs bind to the active site of target enzymes and easily penetrate the cell membrane owing to their small size; however, biological drugs can bind to the protein–protein interaction site but are less stable due to their protein properties. Cyclic peptides possess the pharmacological merits of both chemical and biological drugs, such as the ability to bind to the protein–protein interaction site and penetrate cell membranes. In this study, we developed a simple two-step system to generate a cyclic peptide library using the split intein of Npu DnaE and Gateway cloning. The first step is the PCR of Ready-to-use(R) template DNA having the coding sequences of random cyclic peptides between two split intein elements NpuC and NpuN and the recombination recognition site of Gateway cloning. The second step is the transformation of the PCR products via Gateway cloning to produce colonies with expression vectors to produce cyclic peptides comprising random amino acid sequences. The expression vectors in randomly chosen transformed colonies were confirmed to have random cyclic peptide sequences and all the clones, except ones having a stop codon in the cyclic peptide coding region, showed the expected protein splicing result. This simple two-step system for bacterial expression systems may be modified to suit various expression systems for cell-based assays.

Our skin epidermis constitutes the most important innate defense barrier against all pathogens. Therefore, even if skin lesions are common due to infections, scarring, genetic disorders, and other diseases, skin integrity is crucial for maintaining body homeostasis. Marine collagen peptides (MCPs) and Glycosaminoglycans (GAGs) have been described as potential wound healing (WH) agents. Recently, through an ex vivo clinical approach, we have demonstrated the nutricosmetic potential of fish cartilage hydrolysate (FCH), which presents a combination of collagen peptides and GAGs. Here, we coupled a clinical approach with in vitro experiments on human dermal fibroblasts (HDFs) and applied a Data Independent Acquisition - Parallel Accumulation Serial Fragmentation (diaPASEF) proteomic analysis, to assess the potential benefit of FCH in WH and its mode of action. Our results show that human serum enriched with circulating metabolites resulting from FCH ingestion, improved wound closure in scratch assays. In support, some proteins with anti-inflammatory and immunomodulatory properties or prone to promote hydration and extracellular matrix (ECM) stability showed increased expression in HDFs, after exposure to FCH-enriched serum. These findings suggest that FCH appears as a promising nutrient for WH and skin regeneration.

Celiac disease is an autoimmune disease triggered by oral ingestion of gluten, with certain gluten residues resistant to digestive tract enzymes. Within the duodenum, the remaining peptides incite immunogenic responses, including the generation of autoantibodies and inflammation, leading to irreversible damage. Our previous exploration unveiled a glutenase called Bga1903 derived from the Gram-negative bacterium Burkholderia gladioli. The cleavage pattern of Bga1903 indicated its moderate ability to mitigate the toxicity of pro-immunogenic peptides. The crystal structure of Bga1903, along with the identification of subsites within its active site, was determined. To improve its substrate specificity toward prevalent motifs like QPQ within gluten peptides, the active site of Bga1903 underwent site-directed mutagenesis according to structural insights and enzymatic kinetics. Among the double-site mutants, E380Q/S387L exhibits an approximately 38-fold increase in its specificity constant toward the QPQ sequence, favoring glutamines at the P1 and P3 positions compared to the wild type. Not only does the heightened specificity of E380Q/S387L improve the hydrolytic activity against pro-immunogenic peptides, but also holds this version of the enzyme as a promising candidate as an oral therapy enzyme for celiac disease.

Whether the different NR-AMPs could ever be utilized as drugs not only against prokaryotic (bacteria) pathogens but eukaryotic (fungal pathogens, and parasitic protists) depends on the side effects. To get experimental experience about the option of applying EPB-produced antimicrobials to pathogens, and parasites of veterinary significance, we present here the results of an in vitro, and an accompanying in vivo study on chicken. In the in vitro study, we tested the cytotoxic potential of the cell-free conditioned culture media (CFCM) of three entomopathogenic bacterium species, - X. budapestensis, DSM16342 (EMA); X. szentirmaii DSM16338 (EMC); Photorhabdus luminescens ssp. akhurstii TT01 - on chicken tissue culture cells, namely, on the Leghorn Male Hepatoma (LMH), [92] cells, (a permanent confluent hepato-carcinoma cell line). Each CFCM proved rather cytotoxic in this test. In the in vivo study, we fed freshly hatched male broiler chickens for 42 days with XENOFOOD [39] which contained autoclaved cultures of EMA, and EMC). These bacteria were grown on standard chicken (starter and grower) [HM3] [u4] feed, and the whole culture was used as a “food supplement”. [HM5]. It had been known that these EPB species cannot grow that is, not viable) atbody temperature (above 33 C).

Antimicrobial peptides provide a powerful toolkit for combating multidrug resistance. Combatting eukaryotic pathogens is complicated by the fact, the intracellular drug targets in the eukaryotic pathogen are frequently homologs of cellular structures of vital importance in the host organism. The entomopathogenic bacteria, (symbionts of entomopathogenic nematode species), release a series of non-ribosomal templated antimicrobial peptides, some may be potential drug candidates. The ability of an entomopathogenic nematode /entomopathogenic bacterium symbiotic complex to survive in a given polyxenic milieu is a coevolutionary product, explaining that those gene complexes which are responsible for the biosynthesis of different non-ribosomal templated antimicrobial protective peptides (including those which are potently capable to inactivate the protist mammalian -pathogen Leishmania and the gallinaceous bird pathogen Histomonas) are co-regulated. Our approach is based on comparative antimicrobial bioassays of the culture media of the wild-type and regulatory mutant strains. We concluded that Xenorhabdus budapestensis and X. szentirmaii are excellent sources of non-ribosomal templated antimicrobial peptides, that are efficient antagonists of the mentioned pathogens. Data on selective cytotoxicity of different cell-free culture media encourages us to forecast that the recently discovered “easy-PACId” research strategy is suitable for constructing EPB strains producing and releasing single, harmless, non-ribosomal templated antimicrobial peptides with considerable drug, (probiotic) -candidate potential.