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Kelly Limi Aida,1 Paula Fernanda Kreling,1 Karina Sampaio Caiaffa,2 Giovana Maria Fioramonti Calixto,3 Marlus Chorilli,3 Denise MP Spolidorio,4 Norival Alves Santos-Filho,5,6 Eduardo Maffud Cilli,5 Cristiane Duque1 1Department of Pediatric Dentistry and Public Health, Araçatuba Dental School, Sao Paulo State University (UNESP), Araçatuba, São Paulo, Brazil; 2Department of Endodontics, Araçatuba Dental School, Sao Paulo State University (UNESP), Araçatuba, São Paulo, Brazil; 3Department of Drugs and Medicines, School of Pharmaceutical Sciences, Sao Paulo State University (UNESP), Araraquara, São Paulo, Brazil; 4Department of Physiology and Pathology, Araraquara Dental School, Sao Paulo State University (UNESP), Araraquara, São Paulo, Brazil; 5Department of Biochemistry and Chemical Technology, Institute of Chemistry, Sao Paulo State University (UNESP), Araraquara, São Paulo, Brazil; 6Registro Experimental Campus, Sao Paulo State University (UNESP), Registro, São Paulo, Brazil Background: Anticaries agents must interfere with the adhesion of Streptococcus mutans and its proliferation in dental biofilm, without causing host toxicity and bacterial resistance. Natural substances, including cationic antimicrobial peptides (CAMPs) and their fragments, such as β-defensin-3 peptide fragment (D1–23), have been widely studied. However, the chemical and physical stability of CAMPs may be compromised by external factors, such as temperature and pH, reducing the period of antimicrobial activity. Methods: To overcome the aforementioned disadvantage, this study developed and characterized a drug delivery system and evaluated the cytotoxicity and effect against S. mutans biofilm of a D1–23-loaded bioadhesive liquid crystalline system (LCS). LCS was composed of oleic acid, polyoxypropylene-(5)-polyoxyethylene-(20)-cetyl alcohol, Carbopol® 974P and Carbopol® 971P. LCS was analyzed by polarized light microscopy (PLM), rheology (viscoelasticity and flow properties) and in vitro bioadhesion. The viability of epithelial cells was evaluated. Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) against S. mutans were determined for D1–23 for further evaluation of the effect against S. mutans biofilm after 4 and 24 h of exposure to treatments. Results: PLM, rheology, and in vitro bioadhesion tests showed that both viscosity and bioadhesion of LCS increased after it was diluted with artificial saliva. D1–23-loaded LCS system presented better activity against S. mutans biofilm after 24 h when compared to 4 h of treatment, showing a cumulative effect. Neither LCS nor D1–23-loaded LCS presented toxicity on human epithelial cells. Conclusion: D1–23-loaded LCS is a promising drug delivery system for the prevention of dental caries. Keywords: peptide fragments, drug delivery system, dental caries, biofilm, Streptococcus mutans

Introduction: Tritrpticin (TRP3) is a peptide belonging to the cathelicidin family and has a broad spectrum of antimicrobial activity. However, this class of biomolecules can be easily degraded in the body, making it necessary to use an efficient transport system. The ability to form stable nanostructures from the interaction of glycyrrhizin saponin with the pluronic polymer F127 was demonstrated, forming mixed biopolymeric micelles, highly promising as drug carriers., Objective: The present work sought to understand the physicochemical interaction of the antimicrobial peptide TRP3 with the mixed polymeric micelle made from pluronic F127 and the saponin glycyrrhizin., Methods: The interaction of tritrpticin with mixed nanostructured micelles was evaluated through fluorescence spectroscopy and fluorescence quenching with acrylamide. The experiments were performed at room temperature (25 ± 1°C), adopting an excitation wavelength set to 280 nm and emission between 300 and 500 nm, with a slit of 5 nm., Results: The interaction of the cationic peptide tritrpticin with the mixed biopolymeric micelles was observed through the blue shift of the fluorescence emission to shorter wavelengths, proving the change of tryptophan to a more hydrophobic environment. Through the fluorescence suppression technique, it was possible to indicate the location of the peptide in the mixed micelles, proving tritrpticin to be partially inserted inside them., Conclusion: It was concluded that tritrpticin interacted with mixed nanostructured micelles, forming a promising system for biotechnological applications., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Leishmaniasis is a neglected tropical disease caused by a protozoan of the genus Leishmania, which has visceral and cutaneous forms. The symptoms of leishmaniasis include high fever and weakness, and the cutaneous infection also causes lesions under the skin. The drugs used to treat leishmaniasis have become less effective due to the resistance mechanisms of the protozoa. In addition, the current compounds have low selectivity for the pathogen, leading to various side effects, which results in lower adherence to treatment. Various strategies were developed to solve this problem. The bioconjugation between natural compounds with antimicrobial activity and cell-penetrating peptides could alleviate the resistance and toxicity of current treatments. This work aims to conjugate the cell penetration peptide TAT to the guanidine GVL1. The GVL1-TAT bioconjugate exhibited leishmanicidal activity against Leishmania amazonensis and Leishmania infantum with a high selectivity index. In addition, the bioconjugate was more active against the intracellular enzyme CPP than the individual compounds. This target is very important for the viability and virulence of the parasite within the host cell. Docking studies confirmed the higher interaction of the conjugate with CPP and suggested that other proteins, such as trypanothione reductase, could be targeted. Thus, the data indicated that guanidines conjugated with cell-penetrating peptides could be a good approach for developing antileishmanial molecules.

Sticholysin I and II (St I/II) belong to the actinoporins family; these proteins form pores in host cell membranes by binding their N-terminal segment to the membrane, leading to protein-lipid (toroidal) pores. Peptides derived from actinoporins pore-forming domains replicate their folding properties and permeabilizing effects. Despite the advances in understanding how these proteins and peptides mediate pore formation, the role of different N-terminal segments in inducing membrane curvature is still unclear. Here we combine circular dichroism, electron paramagnetic resonance, and small-angle X-ray scattering to investigate how synthetic peptides encompassing the N-terminal segments of St I and II (StI 1-31 , StII 1-30 , StI 12-31 , and StII 11-30 ) interact with lipid bilayers and micelles as mimics of the topography of the initial membrane binding and of the subsequently formed positively curved pore. We investigate both the conformational changes and peptides' effects on membrane organization resulting from these interactions. According to the toroidal pore model, our results support that the actinoporins amphipathic α-helices rest at the membrane interface, forming pore walls with lipid head groups, while the 1-10 segment of St II penetrates the bilayer, acting as an anchor. We relate this ability to the higher hydrophobicity of this segment in St II, compared to St I. This unique feature of St II would contribute to enhanced pore formation, explaining St II's increased activity when compared to other actinoporins. Our results reinforce the notion that pore formation by actinoporins is a highly cooperative process where specific protein segments and the lipid bilayer mutually modulate their conformation and organization., Competing Interests: Declaration of competing interest All authors declare no conflicts of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Owing to fast SARS-CoV-2 mutations, biosensors employing antibodies as biorecognition elements have presented problems with sensitivity and accuracy. To face these challenges, antibodies can be replaced with the human angiotensin converting enzyme 2 (ACE-2), where it has been shown that the affinity between ACE-2 and the receptor binding domain (RBD) increases with the emergence of new variants. Herein, we report on Ni-doped ZnO nanorod electrochemical biosensors employing an ACE-2 peptide (IEEQAKTFLDKFNHEAEDLFYQS-NH 2 ) as a biorecognition element for detecting Spike (S) Wild-Type (WT) protein. The electrode was fully characterized in terms of electrochemical and physical properties. The sensor showed high cross reactivity with Spike protein B.1.1.7 and Spike protein B.1.351. Still, there was no cross reactivity with the Nucleocapsid protein WT, showing that the biosensor can identify ancestral WT S protein and S protein variants of concern. The device exhibited a LOD of 60.13 ng mL -1 across an S protein WT concentration range from 200 ng mL -1 to 1000 ng mL -1 and a LOQ of 182.22 ng mL -1 . The calculated sensitivity and specificity were 88.88 and 100 %, respectively. These results proved that the Ni-ZnO sensor has promising prospects for SARS-CoV-2 detection and diagnosis of other viruses, employing peptides as biorecognition elements., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025 The Author(s). Published by Elsevier B.V. All rights reserved.)

B-cell lymphoma 2 protein (Bcl-2) is an important regulator of cell apoptosis. Inhibitors that mirror the structural domain 3 (BH3) of Bcl-2 can activate apoptosis in cancer cells, making them a promising target for anticancer treatment. Hence, the present study aimed to investigate potential BH3-mimetic peptides from two vicilin-derived legume proteins from soybean and cowpea bean. The proteins were isolated and sequentially hydrolyzed with pepsin/pancreatin. Peptides < 3 kDa from vicilin-derived proteins from soybean and cowpea beans experimentally inhibited the growth of cultivated breast and prostate cancer cells. In silico analysis allowed the identification of six potential candidates, all predicted to be able to interact with the BH3 domain. The VIPAAY peptide from the soybean β-conglycinin β subunit showed the highest potential to interact with Bcl-2, comparable to Venetoclax, a well-known anticancer drug. Further experiments are needed to confirm this study's findings.

Leishmaniasis is recognized as a serious public health problem in Brazil and around the world. The limited availability of drugs for treatment, added to the diversity of side effects and the emergence of resistant strains, shows the importance of research focused on the development of new molecules, thus contributing to treatments. Therefore, this work aimed to identify leishmanicidal compounds using a peptide dimerization strategy, as well as to understand their mechanisms of action. Herein, it was demonstrated that the dimerization of the peptide TSHa, (TSHa) 2 K, presented higher potency and selectivity than its monomeric form when evaluated against Leishmania mexicana and Leishmania amazonensis . Furthermore, these compounds are capable of inhibiting the parasite cysteine protease, an important target explored for the development of antileishmanial compounds, as well as to selectively interact with the parasite membranes, as demonstrated by flow cytometry, permeabilization, and fluorescence microscopy experiments. Based on this, the identified molecules are candidates for use in in vivo studies with animal models to combat leishmaniasis.

Two new heptapeptides, [1-7-NαC]-crocaorbs A1 (1) and A2 (2), were isolated from the latex of Croton campanulatus. Their structures were determined using NMR spectroscopic techniques, ESI-HRMS data, Marfey's method, and further refined using molecular dynamics with simulated annealing (MD/SA). Molecular dynamics calculations of peptides 1 and 2 demonstrated greater stability in simulations using a biological solvent compared to those using DMSO. Compound 1, the most abundant peptide in latex, was assessed for NO production, antiplasmodial and cytotoxicity activities. The peptide significantly increased nitric oxide (NO) production at concentrations of 40, 20 or 10 μM (17.932 ± 1.1, 18.270 ± 0.9, 18.499 ± 0.7, respectively). Its antiplasmodial activity exhibited limited efficacy, with only 5% inhibition of Plasmodium falciparum 3D7 growth at a concentration of 50 μM. Also, it exhibited no cytotoxic effects in the J774A.1 murine macrophages cell line. This study represents the first report of a phytochemical investigation of the species C. campanulatus, which showed orbitides with distinctive sequences in contrast to other peptides described for the genus Croton and contributes to the study of structural diversity within this particular class of compounds., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Currently, the search for new alternatives to conventional antibiotics to combat bacterial resistance is an urgent task, as many microorganisms threaten human health due to increasing bacterial resistance to traditional medicines. Thus, new molecules such as antimicrobial peptides have emerged as promising alternatives because of their low induction of resistance and broad spectrum of action. In this context, in the past few years, our research group has synthesized and characterized a peptide derived from the C-terminal region of the Lys 49 PLA 2 -like BthTX-I, named p-BthTX-I. After several studies, the peptide (p-BthTX-I) 2 K was proposed as the molecule with the most considerable biotechnological potential. As such, the present work aimed to evaluate whether the modifications made on the peptide (p-BthTX-I) 2 K can be applied to other molecules originating from the C-terminal region of PLA 2 -like Lys 49 from snake venoms. The peptides were obtained through the solid-phase peptide synthesis technique, and biochemical and functional characterization was carried out using dichroism techniques, mass spectrometry, antimicrobial activity against ESKAPE strains, hemolytic activity, and permeabilization of lipid vesicles. The antimicrobial activity of the peptides was promising, especially for the peptides (p-AppK) 2 K and (p-ACL) 2 K, which demonstrated activity against all strains that were tested, surpassing the model molecule (p-BthTX-I) 2 K in most cases and maintaining low hemolytic activity. The modifications initially proposed for the (p-BthTX-I) 2 K peptide were shown to apply to other peptides derived from Lys 49 PLA 2 -like from snake venoms, showing promising results for antimicrobial activity. Future assays comparing the activity of the dimers obtained through this strategy with the monomers of these peptides should be carried out.

Antimicrobial peptides are an emerging class of antibiotics that present a series of advantageous characteristics such as wide structural variety, broad spectrum of activity, and low propensity to select for resistance. They are found in all classes of life as defense molecules. A group of peptides derived from the protein Bothropstoxin-I has been previously studied as an alternative treatment against multi-drug-resistant bacteria. The peptide p-BthTX-I (sequence: KKYRYHLKPFCKK) and its homodimer, linked by disulfide oxidation through the residues of Cys11 and the serum degradation product [sequence: (KKYRYHLKPFC) 2 ], were evaluated and showed similar antimicrobial activity. In this study, we synthesized an analogue of p-BthTX-I that uses the strategy of Fmoc-Lys(Fmoc)-OH in the C-terminal region for dimerization and tryptophan for all aromatic amino acids to provide better membrane interactions. This analogue, named p-BthW, displayed potent antibacterial activity at lower concentrations and maintained the same hemolytic levels as the original molecule. Our assessment revealed that p-BthW has a quick in vitro bactericidal action and prolonged post-antibiotic effect, comparable to the action of polymyxin B. The mode of action of p-BthW seems to rely not only on membrane depolarization but also on necrosis-like effects, especially in Gram-negative bacteria. Overall, the remarkable results regarding the propensity to develop resistance reaffirmed the great potential of the developed molecule., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

The aim of the study was to investigate the effect of antimicrobial peptides (AMPs) Hylin-a1, KR-12-a5, and Temporin-SHa in Candida albicans as well as the biocompatibility of keratinocytes spontaneously immortalized (NOK-si) and human gingival fibroblasts (FGH) cells. Initially, the susceptible (CaS-ATCC 90028) and fluconazole-resistant (CaR-ATCC 96901) C. albicans strains were grown to evaluate the effect of each AMP in planktonic culture, biofilm, and biocompatibility on oral cells. Among the AMPs evaluated, temporin-SHa showed the most promising results. After 24 h of Temporin-SHa exposure, the survival curve results showed that CaS and CaR suspensions reduced 72% and 70% of cell viability compared to the control group. The minimum inhibitory/fungicide concentrations (MIC and MFC) showed that Temporin-SHa was able to reduce ≥50% at ≥256 µg/mL for both strains. The inhibition of biofilm formation, efficacy against biofilm formation, and total biomass assays were performed until 48 h of biofilm maturation, and Temporin-SHa was able to reduce ≥50% of CaS and CaR growth. Furthermore, Temporin-SHa (512 µg/mL) was classified as non-cytotoxic and slightly cytotoxic for NOK-si and FGH, respectively. Temporin-SHa demonstrated an anti-biofilm effect against CaS and CaR and was biocompatible with NOK-si and FGH oral cells in monolayer.

Background: Peptide drugs are advantageous because they are subject to rational design and exhibit highly diverse structures and broad biological activities. The NS2B-NS3 protein is a particularly promising flavivirus therapeutic target, with extensive research on the development of inhibitors as therapeutic candidates, and was used as a model in this work to determine the mechanism by which GA-Hecate inhibits ZIKV replication., Objective: The present study aimed to evaluate the potential of GA-Hecate, a new antiviral developed by our group, against the Brazilian Zika virus and to evaluate the mechanism of action of this compound on the flavivirus NS2B-NS3 protein., Methods: Solid-phase peptide Synthesis, High-Performance Liquid Chromatography, and Mass Spectrometry were used to obtain, purify, and characterize the synthesized compound. Real-time and enzymatic assays were used to determine the antiviral potential of GA-Hecate against ZIKV., Results: The RT-qPCR results showed that GA-Hecate decreased the number of ZIKV RNA copies in the virucidal, pre-treatment, and post-entry assays, with 5- to 6-fold fewer RNA copies at the higher nontoxic concentration in Vero cells (HNTC: 10 μM) than in the control cells. Enzymatic and kinetic assays indicated that GA-Hecate acts as a competitive ZIKV NS2B-NS3 protease inhibitor with an IC 50 of 32 nM and has activity against the yellow fever virus protease., Conclusion: The results highlight the antiviral potential of the GA-Hecate bioconjugate and open the door for the development of new antivirals., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Chikungunya virus (CHIKV) belongs to the Alphavirus genus and is responsible for significant outbreaks worldwide. Currently, there is no approved antiviral therapy against CHIKV. Bioactive peptides have great potential for new drug development. Here, we evaluated the antiviral activity of the synthetic peptide GA-Hecate and its analogs PSSct1905 and PSSct1910 against CHIKV infection. Initial screening showed that all three peptides inhibited the CHIKV replication cycle in baby hamster kidney fibroblast cells (BHK-21) and human hepatocarcinoma epithelial cells (Huh-7). GA-Hecate and its analog PSSct1905 were the most active, demonstrating suppression of viral infection by more than 91%. The analog PSSct1905 exhibited a protective effect in cells against CHIKV infection. We also observed that the analogs PSSct1905 and PSSct1910 affected CHIKV entry into both cell lines, inhibiting viral attachment and internalization. Finally, all tested compounds presented antiviral activity on the post-entry steps of CHIKV infection in all cells evaluated. In conclusion, this study highlights the potential of the peptide GA-Hecate and its analogs as novel anti-CHIKV compounds targeting different stages of the viral replication cycle, warranting the development of GA-Hecate-based compounds with broad antiviral activity.

Redox-active moieties assembled on metallic interfaces have been shown to follow quantum mechanical rules, where the quantum capacitance of the interface (directly associated with the electronic structure of the redox-active moieties) plays a key role in the electron transfer dynamics of the interface. Modifying these interfaces with biological receptors has significant advantages (simplifying molecular diagnostics methods, reducing size, time, and cost while maintaining high sensitivity), enabling the fabrication of miniaturized electroanalytical devices that can compete with traditional ELISA and RT-PCR benchtop assay methods. Owing to their intrinsic characteristics, the use of peptide-based redox-active moieties is a promising chemical route for modifying metallic surfaces, resulting in a high quantum capacitive signal sensitivity. In the present work, different ferrocene-tagged peptides with a structure of Fc-Glu-XX-XX-Cys-NH 2 (XX = serine, phenylalanine, glycine) were used to form self-assembled monolayers on gold. The feasibility of using these interfaces in an electroanalytical assay was verified by detecting the NS1 DENV (Dengue Virus) biomarker to compare the efficiency of peptide structures for biosensing purposes. Parameters such as the formal potential of the interface, normalized electronic density of states (DOS), quantum capacitance, and electron transfer rate constants were obtained for Ser-, Phe-, and Gly-peptides. The Gly-peptide structure presented the highest analytical performance for sensing NS1 with a sensitivity of 5.6% per decade and the lowest LOD (1.4 ng mL -1 ) and LOQ (2.6 ng mL -1 ), followed by Phe-peptide, whereas Ser-peptide had the lowest performance. This work demonstrates that the use of peptides to fabricate a self-assembled monolayer as a biosensor component has advantages for low-cost point-of-care diagnostics. It also shows that the performance of the sensing interface depends strongly on how the chemistry of the surface is designed as a whole, not only on the redox-active group.

Leishmaniasis refers to a collection of diseases caused by protozoa from the Leishmania genus. These diseases, along with other parasitic afflictions, pose a significant public health issue, particularly given the escalating number of at-risk patients. This group includes immunocompromised individuals and those residing in impoverished conditions. The treatment of leishmaniasis is crucial, particularly in light of the mortality rate associated with nontreatment, which stands at 20-30,000 deaths per year globally. However, the therapeutic options currently available are limited, often ineffective, and potentially toxic. Consequently, the pursuit of new therapeutic alternatives is warranted. This study aims to design, synthesize, and evaluate the leishmanicidal activity of antimicrobial peptides functionalized with guanidine compounds and identify those with enhanced potency and selectivity against the parasite. Accordingly, three bioconjugates were obtained by using the solid-phase peptide synthesis protocol. Each proved to be more potent against intracellular amastigotes than their respective peptide or guanidine compounds alone and demonstrated higher selectivity to the parasites than to the host cells. Thus, the conjugation strategy employed with these compounds effectively contributes to the development of new molecules with leishmanicidal activity., Competing Interests: The authors declare no competing financial interest., (© 2023 The Authors. Published by American Chemical Society.)

The emergence of infections caused by microorganisms in the oral cavity and increasing concerns regarding the use of antibiotics have resulted in the development of novel antimicrobial molecules, such as antimicrobial synthetic peptides. The purpose of this study was to evaluate the antimicrobial and antibiofilm activities of the native peptide KR-12 and its derivative, the synthetic peptide [W7]KR12-KAEK, against planktonic and biofilms Enterococcus faecalis strains. The methods used to evaluate the antimicrobial activity in planktonic cultures include minimum inhibitory concentration and minimum bactericidal concentration assays. The effects of [W7]KR12-KAEK on biofilm formation and mature biofilms were evaluated by quantifying biomass (crystal violet staining) and counting colony-forming units. Structural assessments of the biofilms and cellular morphological changes were performed using scanning electron microscopy. Peptide [W7]KR12-KAEK showed potential antimicrobial activity against planktonic cells. Interestingly, the native peptide KR-12 showed no antimicrobial activity. Moreover, it inhibited biofilm formation and disrupted the mature biofilms of E. faecalis strains. These results suggest that [W7]KR12-KAEK may be a potential molecule for the development of auxiliary antimicrobial therapies against oral infections., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Candida albicans is considered one of the most important opportunistic fungi due to the large arsenal of virulence factors that help throughout the progress of the infection. In this sense, antimicrobial peptides (AMPs) appear as an alternative, with great antifungal action. Among these, aurein 1.2 has been widely explored, becoming the basis for the discovery of new AMPs, such as K-aurein (K-au). Thus, this study evaluated the anti-C. albicans potential of K-au against virulence factors, planktonic growth, and biofilm formation of clinical isolates. Firstly, K-au antifungal activity was determined by the microdilution method and time-kill curve. The inhibition of hydrolytic enzyme secretion (proteinase, phospholipase, and hemolysin) and germ tube formation was tested. Then, the antibiofilm potential of K-au was verified through biomass quantification and scanning electron microscopy (SEM). All tests were compared with the classical antifungal drug, amphotericin B (AmB). The outcomes showed fungicidal action of K-au at 62.50 µg mL -1 for all isolates, with a time of action around 150-180 min, determined by the time-kill curve. K-au-treated cells decreased by around 40% of the germinative tube compared to the control. Additionally, K-au inhibited the biofilm formation by more than 90% compared to AmB and the control group. SEM images show apparent cellular disaggregation without the formation of filamentous structures. Therefore, the findings suggest a promising anti-C. albicans effect of K-au due to its fungicidal activity against planktonic cells, or its ability to inhibit important virulence factors like germ tube and biofilm formation. Thus, this peptide could be explored as a useful compound against C. albicans-related infection., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

This study aimed to evaluate Attalea funifera seed oil with or without resveratrol entrapped in organogel nanoparticles in vitro against A375 human melanoma tumor cells. Organogel nanoparticles with seed oil (SON) or with resveratrol entrapped in the seed oil (RSON) formed functional organogel nanoparticles that showed a particle size <100 nm, polydispersity index <0.3, negative zeta potential, and maintenance of electrical conductivity. The resveratrol entrapment efficiency in RSON was 99 ± 1%. The seed oil and SON showed no cytotoxicity against human non-tumor cells or tumor cells. Resveratrol at 50 μg/mL was cytotoxic for non-tumor cells, and was cytotoxic for tumor cells at 25 μg/mL. Resveratrol entrapped in RSON showed a decrease in cytotoxicity against non-tumor cells and cytotoxic against tumor cells at 50 μg/mL. Thus, SON is a potential new platform for the delivery of resveratrol with selective cytotoxic activity in the treatment of melanoma.

Re-emerging arboviruses represent a serious health problem due to their rapid vector-mediated spread, mainly in urban tropical areas. The 2013-2015 Zika virus (ZIKV) outbreak in South and Central America has been associated with cases of microcephaly in newborns and Guillain-Barret syndrome. We previously showed that the conjugate gallic acid-Hecate (GA-FALALKALKKALKKLKKALKKAL-CONH 2 )-is an efficient inhibitor of the hepatitis C virus. Here, we show that the Hecate peptide is degraded in human blood serum into three major metabolites. These metabolites conjugated with gallic acid were synthesized and their effect on ZIKV replication in cultured cells was evaluated. The GA-metabolite 5 (GA-FALALKALKKALKKL-COOH) was the most efficient in inhibiting two ZIKV strains of African and Asian lineage at the stage of both virus entry (virucidal and protective) and replication (post-entry). We also demonstrate that GA-metabolite 5 does not affect cell growth after 7 days of continuous treatment. Thus, this study identifies a new synthetic antiviral compound targeting different steps of ZIKV replication in vitro and with the potential for broad reactivity against other flaviviruses. Our work highlights a promising strategy for the development of new antivirals based on peptide metabolism and bioconjugation.

Despite all efforts to combat the pandemic of COVID-19, we are still living with high numbers of infected persons, an overburdened health care system, and the lack of an effective and definitive treatment. Understanding the pathophysiology of the disease is crucial for the development of new technologies and therapies for the best clinical management of patients. Since the manipulation of the whole virus requires a structure with an adequate level of biosafety, the development of alternative technologies, such as the synthesis of peptides from viral proteins, is a possible solution to circumvent this problem. In addition, the use and validation of animal models is of extreme importance to screen new drugs and to compress the organism's response to the disease. Peptides derived from recombinant S protein from SARS-CoV-2 were synthesized and validated by in silico, in vitro and in vivo methodologies. Macrophages and neutrophils were challenged with the peptides and the production of inflammatory mediators and activation profile were evaluated. These peptides were also inoculated into the swim bladder of transgenic zebrafish larvae at 6 days post fertilization (dpf) to mimic the inflammatory process triggered by the virus, which was evaluated by confocal microscopy. In addition, toxicity and oxidative stress assays were also developed. In silico and molecular dynamics assays revealed that the peptides bind to the ACE2 receptor stably and interact with receptors and adhesion molecules, such as MHC and TCR, from humans and zebrafish. Macrophages stimulated with one of the peptides showed increased production of NO, TNF-α and CXCL2. Inoculation of the peptides in zebrafish larvae triggered an inflammatory process marked by macrophage recruitment and increased mortality, as well as histopathological changes, similarly to what is observed in individuals with COVID-19. The use of peptides is a valuable alternative for the study of host immune response in the context of COVID-19. The use of zebrafish as an animal model also proved to be appropriate and effective in evaluating the inflammatory process, comparable to humans., (© 2023. The Author(s).)

Chikungunya virus (CHIKV) and Zika virus (ZIKV) are important disease-causing agents worldwide. Currently, there are no antiviral drugs or vaccines approved to treat these viruses. However, peptides have shown great potential for new drug development. A recent study described (p-BthTX-I) 2 K [(KKYRYHLKPF) 2 K], a peptide derived from the Bothropstoxin-I toxin in the venom of the Bothrops jararacussu snake, showed antiviral activity against SARS-CoV-2. In this study, we assessed the activity of this peptide against CHIKV and ZIKV and its antiviral action in the different stages of the viral replication cycle in vitro. We observed that (p-BthTX-I) 2 K impaired CHIKV infection by interfering with the early steps of the viral replication cycle, reducing CHIKV entry into BHK-21 cells specifically by reducing both the attachment and internalization steps. (p-BthTX-I) 2 K also inhibited the ZIKV replicative cycle in Vero cells. The peptide protected the cells against ZIKV infection and decreased the levels of the viral RNA and the NS3 protein of this virus at viral post-entry steps. In conclusion, this study highlights the potential of the (p-BthTX-I) 2 K peptide to be a novel broad-spectrum antiviral candidate that targets different steps of the replication cycle of both CHIKV and ZIKV.

Although the exact mechanism of the pathogenesis of coronavirus SARS-CoV-2 (COVID-19) is not fully understood, oxidative stress and the release of pro-inflammatory cytokines have been highlighted as playing a vital role in the pathogenesis of the disease. In this sense, alternative treatments are needed to reduce the level of inflammation caused by COVID-19. Therefore, this study aimed to investigate the potential effect of red photobiomodulation (PBM) as an attractive therapy to downregulate the cytokine storm caused by COVID-19 in a zebrafish model. RT-qPCR analyses and protein-protein interaction prediction among SARS-CoV-2 and Danio rerio proteins showed that recombinant Spike protein (rSpike) was responsible for generating systemic inflammatory processes with significantly increased levels of pro-inflammatory ( il1b , il6 , tnfa , and nfkbiab ), oxidative stress ( romo1 ) and energy metabolism ( slc2a1a and coa1 ) mRNA markers, with a pattern similar to those observed in COVID-19 cases in humans. On the other hand, PBM treatment was able to decrease the mRNA levels of these pro-inflammatory and oxidative stress markers compared with rSpike in various tissues, promoting an anti-inflammatory response. Conversely, PBM promotes cellular and tissue repair of injured tissues and significantly increases the survival rate of rSpike-inoculated individuals. Additionally, metabolomics analysis showed that the most-impacted metabolic pathways between PBM and the rSpike treated groups were related to steroid metabolism, immune system, and lipid metabolism. Together, our findings suggest that the inflammatory process is an incisive feature of COVID-19 and red PBM can be used as a novel therapeutic agent for COVID-19 by regulating the inflammatory response. Nevertheless, the need for more clinical trials remains, and there is a significant gap to overcome before clinical trials can commence.

Recent studies have shown that the peptide [des-Cys 11 ,Lys 12 ,Lys 13 -(p-BthTX-I) 2 K] (p-Bth) is a p-BthTX-I analog that shows enhanced antimicrobial activity, stability and hemolytic activity, and is easy to obtain compared to the wild-type sequence. This molecule also inhibits SARS-CoV-2 viral infection in Vero cells, acting on SARS-CoV-2 PL pro enzymatic activity. Thus, the present study aimed to assess the effects of structural modifications to p-Bth, such as dimerization, dendrimerization and chirality, on the antibacterial activity and inhibitory properties of PL pro . The results showed that the dimerization or dendrimerization of p-Bth was essential for antibacterial activity, as the monomeric structure led to a total loss of, or significant reduction in, bacterial activities. The dimers and tetramers obtained using branched lysine proved to be prominent compounds with antibacterial activity against Gram-positive and Gram-negative bacteria. In addition, hemolysis rates were below 10% at the corresponding concentrations. Conversely, the inhibitory activity of the PL pro of SARS-CoV-2 was similar in the monomeric, dimeric and tetrameric forms of p-Bth. Our findings indicate the importance of the dimerization and dendrimerization of this important class of antimicrobial peptides, which shows great potential for antimicrobial and antiviral drug-discovery campaigns.

Cerato-ulmin (CU) is a 75-amino-acid-long protein that belongs to the hydrophobin family. It self-assembles at hydrophobic-hydrophilic interfaces, forming films that reverse the wettability properties of the bound surface: a capability that may confer selective advantages to the fungus in colonizing and infecting elm trees. Here, we show for the first time that CU can elicit a defense reaction (induction of phytoalexin synthesis and ROS production) in non-host plants ( Arabidopsis ) and exerts its eliciting capacity more efficiently when in its soluble monomeric form. We identified two hydrophobic clusters on the protein's loops endowed with dynamical and physical properties compatible with the possibility of reversibly interconverting between a disordered conformation and a β-strand-rich conformation when interacting with hydrophilic or hydrophobic surfaces. We propose that the plasticity of those loops may be part of the molecular mechanism that governs the protein defense elicitation capability.

Background: (p-BthTX-I) 2  K, a dimeric analog peptide derived from the C-terminal region of phospholipase A2-like bothropstoxin-I (p-BthTX-I), is resistant to plasma proteolysis and inhibits severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strains with weak cytotoxic effects. Complications of SARS-CoV-2 infection include vascular problems and increased risk of thrombosis; therefore, studies to identify new drugs for treating SARS-CoV-2 infections that also inhibit thrombosis and minimize the risk of bleeding are required., Objectives: To determine whether (p-BthTX-I) 2  K affects the hemostatic system., Methods: Platelet aggregation was induced by collagen, arachidonic acid, and adenosine diphosphate (ADP) in the Chronolog Lumi-aggregometer. The coagulation activity was evaluated by determining activated partial thromboplastin clotting time (aPTT) and prothrombin time (PT) with (p-BthTX-I) 2  K (5.0-434.5 µg) or 0.9% NaCl. Arterial thrombosis was induced with a 540 nm laser and 3.5-20 mg kg - 1 Rose Bengal in the carotid artery of male C57BL/6J mice using (p-BthTX-I) 2  K. Bleeding time was determined in mouse tails immersed in saline at 37 °C after (p-BthTX-I) 2  K (4.0 mg/kg and 8.0 mg/kg) or saline administration., Results: (p-BthTX-I) 2  K prolonged the aPTT and PT by blocking kallikrein and FXa-like activities. Moreover, (p-BthTX-I) 2  K inhibited ADP-, collagen-, and arachidonic acid-induced platelet aggregation in a dose-dependent manner. Further, low concentrations of (p-BthTX-I) 2  K extended the time to artery occlusion by the formed thrombus. However, (p-BthTX-I) 2  K did not prolong the bleeding time in the mouse model of arterial thrombosis., Conclusion: These results demonstrate the antithrombotic activity of the peptide (p-BthTX-I) 2  K possibly by kallikrein inhibition, suggesting its strong biotechnological potential., (© 2023. The Author(s).)

Background: Antimicrobial resistance is an emerging global health challenge that has led researchers to study alternatives to conventional antibiotics. A promising alternative is antimicrobial peptides (AMPs), produced as the first line of defense by almost all living organisms. To improve its biological activity, the conjugation of AMPs is a promising approach., Objective: In this study, we evaluated the N-terminal conjugation of p-Bt (a peptide derived from Bothrops Jararacuçu`s venom) with ferrocene (Fc) and gallic acid (GA). Acetylated and linear versions of p-Bt were also synthesized to evaluate the importance of N-terminal charge and dimeric structure., Methods: The compounds were obtained using solid-phase peptide synthesis. Circular dichroism, vesicle permeabilization, antimicrobial activity, and cytotoxicity studies were conducted., Results: No increase in antibacterial activity against Escherichia coli was observed by adding either Fc or GA to p-Bt. However, Fc-p-Bt and GA-p-Bt exhibited improved activity against Staphylococcus aureus . No cytotoxicity upon fibroblast was observed for GA-p-Bt. On the other hand, conjugation with Fc increased cytotoxicity. This toxicity may be related to the membrane permeabilization capacity of this bioconjugate, which showed the highest carboxyfluorescein leakage in vesicle permeabilization experiments., Conclusion: Considering these observations, our findings highlight the importance of adding bioactive organic compounds in the N-terminal position as a tool to modulate the activity of AMPs., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Leishmaniasis is a highly prevalent, yet neglected disease caused by protozoan parasites of the genus Leishmania . In the search for newer, safer, and more effective antileishmanial compounds, we herein present a study of the mode of action in addition to a detailed structural and biological characterization of LQOF-G6 [ N -benzoyl- N '-benzyl- N ″-(4-tertbutylphenyl)guanidine]. X-ray crystallography and extensive NMR experiments revealed that LQOF-G6 nearly exclusively adopts the Z conformation stabilized by an intramolecular hydrogen bond. The investigated guanidine showed selective inhibitory activity on Leishmania major cysteine protease Lm CPB2.8ΔCTE (CPB) with ~73% inhibition and an IC 50-CPB of 6.0 µM. This compound did not show any activity against the mammalian homologues cathepsin L and B. LQOF-G6 has been found to be nontoxic toward both organs and several cell lines, and no signs of hepatotoxicity or nephrotoxicity were observed from the analysis of biochemical clinical plasma markers in the treated mice. Docking simulations and experimental NMR measurements showed a clear contribution of the conformational parameters to the strength of the binding in the active site of the enzyme, and thus fit the differences in the inhibition values of LQOF-G6 compared to the other guanidines. Furthermore, the resulting data render LQOF-G6 suitable for further development as an antileishmanial drug.

In this study, the action of antimicrobial peptide (AMP) P5 and antimicrobial photodynamic therapy (aPDT) mediated by bixin and chlorin-e6 (Ce6) on Cutibacterium acnes (C. acnes) in planktonic phase and biofilm were evaluated both as monotherapies and combined therapies. Microbial viability after treatments were quantified by colony-forming units per milliliter of the sample (CFU/mL) and have demonstrated that all treatments employed exerted bactericidal activity, reducing the microbial load by more than 3 log 10 CFU/mL, also demonstrating for the first time in the literature the antimicrobial photodynamic effect of bixin that occurs mostly through type I mechanism which was proved by the quantification of superoxide anion production. Bacterial biofilm was completely eliminated only after its exposure to aPDT mediated by this PS, however, Ce6 proved to be a more efficient PS, considering that most of the photodynamic effect of bixin- aPDT was exerted by excitation of the endogenous C porphyrins of C. acnes with blue light. The combination of P5 with Ce6-aPDT showed a synergistic effect on the bacterial biofilm with a reduction in microbial load by more than 10 log 10 CFU/mL, in which the ability of P5 to permeabilize the polymeric extracellular matrix of the biofilm explains the obtained results, with greater internalization of the PS as shown by the Confocal Laser Scanning Microscopy. One-way ANOVA (Analysis of Variance) with Tukey's post-test and two-way ANOVA with Bonferroni's post-test were used to compare the values of continuous variables between the control group and the treatment groups., Competing Interests: Disclosure of author of conflicts of interest The authors declare that there are no conflicts of interest., (Copyright © 2022. Published by Elsevier B.V.)

Limitations of the recognition elements in terms of synthesis, cost, availability, and stability have impaired the translation of biosensors into practical use. Inspired by nature to mimic the molecular recognition of the anti-SARS-CoV-2 S protein antibody (Ab S ) by the S protein binding site, we synthesized the peptide sequence of Asn-Asn-Ala-Thr-Asn-COOH (abbreviated as PEP2003) to create COVID-19 screening label-free (LF) biosensors based on a carbon electrode, gold nanoparticles (AuNPs), and electrochemical impedance spectroscopy. The PEP2003 is easily obtained by chemical synthesis, and it can be adsorbed on electrodes while maintaining its ability for Ab S recognition, further leading to a sensitivity 3.4-fold higher than the full-length S protein, which is in agreement with the increase in the target-to-receptor size ratio. Peptide-loaded LF devices based on noncovalent immobilization were developed by affording fast and simple analyses, along with a modular functionalization. From studies by molecular docking, the peptide-Ab S binding was found to be driven by hydrogen bonds and hydrophobic interactions. Moreover, the peptide is not amenable to denaturation, thus addressing the trade-off between scalability, cost, and robustness. The biosensor preserves 95.1% of the initial signal for 20 days when stored dry at 4 °C. With the aid of two simple equations fitted by machine learning (ML), the method was able to make the COVID-19 screening of 39 biological samples into healthy and infected groups with 100.0% accuracy. By taking advantage of peptide-related merits combined with advances in surface chemistry and ML-aided accuracy, this platform is promising to bring COVID-19 biosensors into mainstream use toward straightforward, fast, and accurate analyses at the point of care, with social and economic impacts being achieved.

Cyclotides are mini-proteins with potent bioactivities and outstanding potential for agricultural and pharmaceutical applications. More than 450 different plant cyclotides have been isolated from six angiosperm families. In Brazil, studies involving this class of natural products are still scarce, despite its rich floristic diversity. Herein were investigated the cyclotides from Anchietea pyrifolia roots, a South American medicinal plant from the family Violaceae. Fourteen putative cyclotides were annotated by LC-MS. Among these, three new bracelet cyclotides, anpy A-C, and the known cycloviolacins O4 (cyO4) and O17 (cyO17) were sequenced through a combination of chemical and enzymatic reactions followed by MALDI-MS/MS analysis. Their cytotoxic activity was evaluated by a cytotoxicity assay against three human cancer cell lines (colorectal carcinoma cells: HCT 116 and HCT 116 TP53 -/- and breast adenocarcinoma, MCF 7). For all assays, the IC 50 values of isolated compounds ranged between 0.8 and 7.3 μM. CyO17 was the most potent cyclotide for the colorectal cancer cell lines (IC 50 , 0.8 and 1.2 μM). Furthermore, the hemolytic activity of anpy A and B, cyO4, and cyO17 was assessed, and the cycloviolacins were the least hemolytic (HD 50 > 156 μM). This work sheds light on the cytotoxic effects of the anpy cyclotides against cancer cells. Moreover, this study expands the number of cyclotides obtained to date from Brazilian plant biodiversity and adds one more genus containing these molecules to the list of the Violaceae family.

Jatropha mollissima is endemic to Brazil and is used for traditional medicinal purposes, including the treatment of snakebite. In this study, latex obtained from this plant was fractioned using reversed-phase chromatography, and the fractions were then screened for peptides. A 755 g/mol peptide was obtained, and MS/MS analyses indicated it had a cyclic sequence (Pro-Leu-Gly-Val-Leu-Leu-Tyr). This peptide sequence was present in the Jatropha genome database, and an identity value of 90.71%, an E-value of 0.0, and a score of 883 with NO-associated protein 1/chloroplastic/mitochondria of Jatropha curcas were obtained from the NCBI nonredundant protein sequence (nr) database. Molecular docking analyses performed with the peptide against a metalloendopeptidase belonging to Crotalus adamanteus snake venom suggested the cyclic peptide establishes favorable interactions with the catalytic site of the enzyme. Therefore, it could inhibit enzyme catalysis. This belief was corroborated by the formation of 6 hydrogen bonds with the linear form of the peptide. Tighter complexation of the cyclic form (41 kcal/mol more energetic) revealed better spatial blocking. The linear form outperformed the cyclic form in complexing the required energy, recruiting more catalytic residues (6/2), and in establishing more hydrogen bonds (6/3). However, cyclic folding provided a more significant spatial block within the catalytic site. The set of results suggests that the cycle peptide, here called Jatromollistatin, which was previously described as jatrophidin and pohlianin A in two other species of Jatropha, is a promising candidate to inhibit venom proteases. This belief is corroborated by the topical use of the latex for initial treatment of snakebites., (© 2022 John Wiley & Sons Ltd.)

The use of serological tests is valuable to diagnose Zika virus (ZIKV) infection and carry out epidemiological surveillance. However, ZIKV serological tests may result in false positives due to cross-reactivity between antibodies against other Flavivirus, especially dengue virus that worldwide disseminated. We used three online tools to predict amino acid sequences of B-cell epitopes. We selected and synthetized two epitopes that showed appropriate features in the molecular dynamic simulation and demonstrated to be suitable for serological assays., (Copyright © 2022 Elsevier B.V. All rights reserved.)

There have been significant impacts of the current COVID-19 pandemic on society including high health and economic costs. However, little is known about the potential ecological risks of this virus despite its presence in freshwater systems. In this study, we aimed to evaluate the exposure of Poecilia reticulata juveniles to two peptides derived from Spike protein of SARS-CoV-2, which was synthesized in the laboratory (named PSPD-2002 and PSPD-2003). For this, the animals were exposed for 35 days to the peptides at a concentration of 40 µg/L and different toxicity biomarkers were assessed. Our data indicated that the peptides were able to induce anxiety-like behavior in the open field test and increased acetylcholinesterase (AChE) activity. The biometric evaluation also revealed that the animals exposed to the peptides displayed alterations in the pattern of growth/development. Furthermore, the increased activity of superoxide dismutase (SOD) and catalase (CAT) enzymes were accompanied by increased levels of malondialdehyde (MDA), reactive oxygen species (ROS) and hydrogen peroxide (H 2 O 2 ), which suggests a redox imbalance induced by SARS-CoV-2 spike protein peptides. Moreover, molecular docking analysis suggested a strong interaction of the peptides with the enzymes AChE, SOD and CAT, allowing us to infer that the observed effects are related to the direct action of the peptides on the functionality of these enzymes. Consequently, our study provided evidence that the presence of SARS-CoV-2 viral particles in the freshwater ecosystems offer a health risk to fish and other aquatic organisms., (Copyright © 2022. Published by Elsevier B.V.)

Considering the challenges related to antimicrobial resistance, other strategies for controlling infections have been suggested, such as antimicrobial photodynamic therapy (aPDT) and antimicrobial peptides (AMP). This study aims to perform a systematic review and meta-analysis to obtain evidence on the antimicrobial effectiveness of aPDT associated with AMP and establish in vitro knowledge on this topic for further study designs. The PubMed, Scopus, Web of Science, Science Direct, Scielo, and Cochrane Library databases were searched. Two independent and calibrated researchers (Kappa = 0.88) performed all the systematic steps according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA). The odds ratio (OR) was used as the effect measure. The Peto method was used to perform the meta-analysis due to the sparse data. Twenty studies were included in the present review. The result was significant (OR = 0.14/ p = 0.0235/I-squared = 0%), showing better outcomes of aPDT associated with peptides than those of aPDT alone for controlling the microbial load. Only 20% of the studies included evaluated this approach in a biofilm culture. Combined treatment with aPDT and AMP highly increased the ability of microbial reduction of Gram-positive and Gram-negative bacteria. However, additional blind studies are required to evaluate the efficacy of this therapy on microbial biofilms.

The human Respiratory Syncytial Virus (hRSV) is one of the most common causes of acute respiratory diseases such as bronchiolitis and pneumonia in children worldwide. Among the viral proteins, the nucleoprotein (N) stands out for forming the nucleocapsid (NC) that functions as a template for replication and transcription by the viral polymerase complex. The NC/polymerase recognition is mediated by the phosphoprotein (P), which establishes an interaction of its C-terminal residues with a hydrophobic pocket in the N-terminal domain of N (N-NTD). The present study consists of biophysical characterization of N-NTD and investigation of flavonoids binding to this domain using experimental and computational approaches. Saturation transfer difference (STD)-NMR measurements showed that among the investigated flavonoids, only hesperetin (Hst) bound to N-NTD. The binding epitope mapping of Hst suggested that its fused aromatic ring is buried in the protein binding site. STD-NMR and fluorescence anisotropy experiments showed that Hst competes with P protein C-terminal dipeptides for the hRSV nucleoprotein/phosphoprotein (N/P) interaction site in N-NTD, indicating that Hst binds to the hydrophobic pocket in this domain. Computational simulations of molecular docking and dynamics corroborated with experimental results, presenting that Hst established a stable interaction with the N/P binding site. The outcomes presented herein shed light on literature reports that described a significant antireplicative activity of Hst against hRSV, revealing molecular details that can provide the development of a new strategy against this virus.

The present work monitors structural changes in anionic membranes (DPPG; 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol)) caused by the native antimicrobial peptide (AMP) Hylin a1 (Hya1; IFGAILPLALGALKNLIK-NH 2 ) and its synthetic analogue K 0 Hya1 (KIFGAILPLALGALKNLIK-NH 2 ), with an extra positive residue of lysine at the N-terminus of the peptide chain. Anionic membranes were used to mimic anionic lipids in bacteria membranes. Differential scanning calorimetry (DSC) evinced that both peptides strongly disrupt the lipid bilayers. However, whereas the native peptide (+3) induces a space-average and/or time-average disruption on DPPG bilayers, the more charged, K 0 Hya1 (+4), appears to be strongly attached to the membrane, clearly giving rise to the coexistence of two different lipid regions, one depleted of peptide and another one peptide-disrupted. The membrane fluorescent probe Laurdan indicates that, in average, the peptides increase the bilayer packing of fluid DPPG (above the lipid gel-fluid transition temperature) and/or decrease its polarity. Spin labels, incorporated into DPPG membrane, confirm, and extend the results obtained with Laurdan, indicating that the peptides increase the lipid packing both in gel and fluid DPPG bilayers. Therefore, our results confirm that Laurdan is often unable to monitor structural modifications induced on gel membranes by exogenous molecules. Through the measurement of the leakage of entrapped carboxyfluorescein (CF), a fluorescent dye, in DPPG large unilamellar vesicles it was possible to show that both peptides induce pore formation in DPPG bilayers. Furthermore, CF experiments show that Hylin peptides are strongly bound to DPPG bilayers in the gel phase, not being able to migrate to other DPPG vesicles. Here we discuss the complementarity of different techniques in monitoring structural alterations caused on lipid bilayers by Hylin peptides, and how it could be used to help in the understanding of the action of other exogenous molecules on biological membranes., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Background: Fungal and parasitic diseases are global health problems, and the available treatments are becoming ineffective, mainly due to the emergence of resistant strains of pathogens. Furthermore, the drugs currently in use exhibit high toxicity and side effects. The scarcity of efficient treatments for fungal and parasitic diseases has motivated the search for new drug candidates, including antimicrobial peptides. The chemokine class RP1 peptide shows inhibitory activity against bacteria, viruses, cancer cells and parasites. In addition, the organometallic compound ferrocene showed antiparasitic activity., Objective: Study aimed to assess the effect of conjugation of the RP1 peptide with ferrocene in terms of its structure, biological activity against fungi and parasites and toxicity., Methods: Peptides and conjugates were synthesized using solid phase peptide synthesis (SPPS). The Fc-RP1 peptide showed antifungal and antimalarial activities with low toxicity in the U87 and HepG2 cell lines., Results: The mechanism of action of these peptides, analyzed by flow cytometry in the fungus Cryptococcus neoformans, was through membrane permeabilization, with an emphasis on the Fc-RP1 peptide that presented the highest rate of PI-positive cell marking., Conclusion: In conclusion, ferrocene conjugated to antimicrobial peptide RP1 is an attractive biomolecule for drug discovery against fungal and parasitic diseases., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

This study evaluated the cytocompatibility and antimicrobial/antibiofilm effects of epigallocatechin-3-gallate (EGCG) associated with peptide LL-37 and its analogue KR-12-a5 against oral pathogens. The effect of the compounds on metabolism of fibroblasts was evaluated by methyltetrazolium assays. Antimicrobial activity of the compounds was evaluated on Streptococcus mutans, Enterococcus faecalis, Actinomyces israelii, and Fusobacterium nucleatum under planktonic conditions, on single- and dual-species biofilms and E. faecalis biofilms in dentinal tubules and analyzed by bacterial counts and confocal microscopy. Data were statistically analyzed considering p < 0.05. EGCG and peptide combinations were not toxic to fibroblasts. KR-12-a5 showed synergistic or addictive effects with EGCG and LL-37 against all bacteria tested. However, EGCG associated with KR-12-a5 demonstrated the highest bactericidal activity on all bacteria tested, at lower concentrations. In single-species biofilms, EGCG + KR-12-a5 eliminated S. mutans and A. israelii and reduced E. faecalis and F. nucleatum counts around 5 log CFU/mL. EGCG + KR-12-a5 reduced E. faecalis (-3.93 log CFU/mL) and eliminated S. mutans in dual-species biofilms. No growth of E. faecalis and significant reduction in A. israelii (-6.24 log CFU/mL) and F. nucleatum (-4.62 log CFU/mL) counts were detected in dual-species biofilms. The combination of EGCG and KR-12-a5 led to 88% of E. faecalis dead cells inside dentin tubules. The association of EGCG and KR-12-a5 was cytocompatible and promoted synergistic effect against biofilms of bacteria associated with endodontic infections., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Knowledge about how the COVID-19 pandemic can affect aquatic wildlife is still extremely limited, and no effect of SARS-CoV-2 or its structural constituents on invertebrate models has been reported so far. Thus, we investigated the presence of the 2019-new coronavirus in different urban wastewater samples and, later, evaluated the behavioral and biochemical effects of the exposure of Culex quinquefasciatus larvae to two SARS-CoV-2 spike protein peptides (PSPD-2002 and PSPD-2003) synthesized in our laboratory. Initially, our results show the contamination of wastewater by the new coronavirus, via RT-qPCR on the viral N1 gene. On the other hand, our study shows that short-term exposure (48 h) to a low concentration (40 μg/L) of the synthesized peptides induced changes in the locomotor and the olfactory-driven behavior of the C. quinquefascitus larvae, which were associated with increased production of ROS and AChE activity (cholinesterase effect). To our knowledge, this is the first study that reports the indirect effects of the COVID-19 pandemic on the larval phase of a freshwater invertebrate species. The results raise concerns at the ecological level where the observed biological effects may lead to drastic consequences., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The Spike protein (S protein) is a critical component in the infection of the new coronavirus (SARS-CoV-2). The objective of this work was to evaluate whether peptides from S protein could cause negative impact in the aquatic animals. The aquatic toxicity of SARS-CoV-2 Spike protein peptides derivatives has been evaluated in tadpoles (n = 50 tadpoles/5 replicates of 10 animals) from species Physalaemus cuvieri (Leptodactylidae). After synthesis, purification, and characterization of peptides (PSDP2001, PSDP2002, PSDP2003) an aquatic contamination has been simulated with these peptides during 24 h of exposure in two concentrations (100 and 500 ng/mL). The control group ("C") was composed of tadpoles kept in polyethylene containers containing de-chlorinated water. Oxidative stress, antioxidant biomarkers and AChE activity were assessed. In both concentrations, PSPD2002 and PSPD2003 increased catalase and superoxide dismutase antioxidants enzymes activities, as well as oxidative stress (nitrite levels, hydrogen peroxide and reactive oxygen species). All three peptides also increased acetylcholinesterase activity in the highest concentration. These peptides showed molecular interactions in silico with acetylcholinesterase and antioxidant enzymes. Aquatic particle contamination of SARS-CoV-2 has cholinesterasic effect in P. cuvieri tadpoles. These findings indicate that the COVID-19 can constitute environmental impact or biological damage potential., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Aims: This study aimed to evaluate the in vitro cytotoxicity and efficacy of synthetic host defence peptides (HDPs), alone or in combination with florfenicol (FFC), oxytetracycline (OTC) or thiamphenicol (TAP), against different pathogenic bacteria isolated from diseased fish., Methods and Results: Solid-phase synthesis, purification and characterization of several HDPs were performed manually, using the fluorenylmethyloxycarbonyl protecting group in different resins and via high-performance liquid chromatography-mass spectrometry, respectively. The in vitro cytotoxicity and antimicrobial activity of HDPs, FFC, OTC and TAP against Nile tilapia red blood cells (RBCs) and relevant fish pathogenic bacteria (Aeromonas, Citrobacter, Edwardsiella, Streptococcus, Lactococcus and Vibrio) was determined using the haemolysis assay and broth microdilution method, respectively. The checkerboard assay was used to evaluate the synergy between the most active HDPs and other antimicrobials against the tested strains. MUC 7 12-mer, FFC, OTC and TAP were not cytotoxic to Nile tilapia RBCs, in all tested concentrations. LL-37, (p-BthTX-I) 2 and Hylin-a1 were not cytotoxic at concentrations up to 78·13, 19·53 and 9·77 μg ml -1 , respectively. HDPs demonstrated potent antimicrobial activity (minimum inhibitory concentration ≤31·25 µg ml -1 ) against Aeromonas jandaei (KR-12-a5), Citrobacter freundii (Kr-12-a5; (p-BthTX-I) 2 ; LL-37; and Hylin a1), Streptococcus agalactiae (Hylin a1; (p-BthTX-I) 2 and LL-37), Lactococcus garviae (Hylin a1), and Vibrio fluvialis (KR-12-a5). The combinations of (p-BthTX-I) 2 with TAP and LL-37 with FFC showed synergistic activity against C. freundii (fractional inhibitory concentration index of 0·25 and 0·50, respectively)., Conclusions: Synthetic HDPs have the potential as a good treatment option for bacterial diseases in aquaculture., Significance and Impact of the Study: The in vivo effectiveness of synthetic HDPs such as KR-12-a5; LL-37; (p-BthTX-I) 2 and Hylin a1 can be tested alone or in combination with conventional antimicrobials as a treatment option to reduce the use of antimicrobials in aquaculture., (© 2021 The Society for Applied Microbiology.)

The emergence of several SARS-CoV-2 lineages presenting adaptive mutations is a matter of concern worldwide due to their potential ability to increase transmission and/or evade the immune response. While performing epidemiological and genomic surveillance of SARS-CoV-2 in samples from Porto Ferreira-São Paulo-Brazil, we identified sequences classified by pangolin as B.1.1.28 harboring Spike L452R mutation, in the RBD region. Phylogenetic analysis revealed that these sequences grouped into a monophyletic branch, with others from Brazil, mainly from the state of São Paulo. The sequences had a set of 15 clade defining amino acid mutations, of which six were in the Spike protein. A new lineage was proposed to Pango and it was accepted and designated P.4. In samples from the city of Porto Ferreira, P.4 lineage has been increasing in frequency since it was first detected in March 2021, corresponding to 34.7% of the samples sequenced in June, the second in prevalence after P.1. Also, it is circulating in 30 cities from the state of São Paulo, and it was also detected in one sample from the state of Sergipe and two from the state of Rio de Janeiro. Further studies are needed to understand whether P.4 should be considered a new threat., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Bittar, Possebon, Ullmann, Geraldini, da Costa, de Almeida, da S. Sanches, Nascimento-Júnior, Cilli, Artico Banho, Campos, Ferreira, Sacchetto, da Silva, Parra, Moraes, da Costa, Vasconcelos, Spilki, Nogueira, Rahal and Araujo Jr.)

New variants of SARS-CoV-2 Alpha (B.1.1.7); Beta (B.1.351) Gamma (P.1) and Delta (B.1.617.2) quickly spread in the UK, South Africa, Brazil and India, respectively. To address whether mutations in SARS-CoV-2 RBD spike protein could affect virus infectivity, peptides containing RBD amino acids mutations have been constructed and interacted with human ACE2 by computational methods. Our results suggest that mutations in RBD amino acids K417, E484, L452, T478 and N501 are expressively increasing the affinity of this protein with human angiotensin-converting enzyme 2 (ACE2), consequently, variants Alpha (B.1.1.7), Beta (B1.351), Gamma (P.1) and Delta (B.1.617.2) could be more infective in human cells compared with SARS-CoV-2 isolated in Wuhan-2019 and the Gamma and Delta variants could be the most infective among them., Competing Interests: The authors declare that there is no interest conflict., (© 2021 The Author(s).)

The COVID-19 outbreak has rapidly spread on a global scale, affecting the economy and public health systems throughout the world. In recent years, peptide-based therapeutics have been widely studied and developed to treat infectious diseases, including viral infections. Herein, the antiviral effects of the lysine linked dimer des-Cys 11 , Lys 12 ,Lys 13 -(pBthTX-I) 2 K ( (pBthTX-I) 2 K) ) and derivatives against SARS-CoV-2 are reported. The lead peptide (pBthTX-I) 2 K and derivatives showed attractive inhibitory activities against SARS-CoV-2 (EC 50 = 28-65 µM) and mostly low cytotoxic effect (CC 50 > 100 µM). To shed light on the mechanism of action underlying the peptides' antiviral activity, the Main Protease (M pro ) and Papain-Like protease (PL pro ) inhibitory activities of the peptides were assessed. The synthetic peptides showed PL pro inhibition potencies (IC 50 s = 1.0-3.5 µM) and binding affinities ( K d = 0.9-7 µM) at the low micromolar range but poor inhibitory activity against M pro (IC 50 > 10 µM). The modeled binding mode of a representative peptide of the series indicated that the compound blocked the entry of the PL pro substrate toward the protease catalytic cleft. Our findings indicated that non-toxic dimeric peptides derived from the Bothropstoxin-I have attractive cellular and enzymatic inhibitory activities, thereby suggesting that they are promising prototypes for the discovery and development of new drugs against SARS-CoV-2 infection.

Guided bone regeneration (GBR) technique helps to restore bone tissue through cellular selectivity principle. Currently no osteoinductive membrane exists on the market. Osteogenic growth peptide (OGP) acts as a hematopoietic stimulator. This association could improve the quality of bone formation, benefiting more than 2.2 million patients annually. The objective of this work was to develop membranes from ureasil-polyether materials containing OGP. The membranes were characterized by differential scanning calorimetry (DSC) and small angle X-ray scattering (SAXS). OGP was synthesized by the solid phase method. Sterilization results using gamma radiation at 24 kGy did not change the structure of the material, as confirmed by DSC. The SAXS technique revealed the structural homogeneity of the matrix. OGP was incorporated in 66.25 × 10 -10  mol and release results showed that the ureasil-PPO400/PEO500 and ureasil-PPO400/PEO1900 membranes released 7% and 21%, respectively, after 48 h. In vivo results demonstrated that the amount and quality of bone tissue formed in the bone defects in the presence of ureasil-polyether membranes with OGP were similar to commercial collagen material with BMP. The results allow us to conclude that membranes with OGP have characteristics that make them potential candidates for the GBR., (© 2021 John Wiley & Sons Ltd.)

Based on the antimicrobial activity of bothropstoxin-I (BthTX-I) and on the premise that a C-terminal peptide of Lys49 myotoxin can reproduce the antimicrobial activity of the parent protein, we aimed to study the mechanism of action of a peptide derived from the C-terminal region of the myotoxin BthTX-I [(p-BthTX-I) 2 , sequence: KKYRYHLKPFCKK, disulfide-linked dimer] against Gram-positive and Gram-negative bacteria. Fluorescence quenching technique showed that the carboxyfluorescein labeled-peptide [CF-(p-BthTX-I) 2 ] when incubated with E. coli displayed a superior penetration activity than when incubated with S. aureus. Cell death induced by the peptide (p-BthTX-I) 2 showed a loss of membrane integrity in E. coli and S. aureus; however, the mechanisms of cell death were different, characterized by the presence of necrosis-like and apoptosis-like deaths, respectively. Scanning electron microscopy studies in E. coli and S. aureus showed morphological changes in the cells, with superficial deformities, appearance of wrinkles and bubbles, and formation of vesicles. Our results demonstrate that the mechanism of action of the peptide (p-BthTX-I) 2 is different in Gram-negative (E. coli) and Gram-positive (S. aureus) bacteria. Knowledge of the mechanism of action of these peptides is important, since they are promising prototypes for new antimicrobial drugs., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Dengue virus infection depends on its fusion with the host membrane, where the binding occurs through interaction between proteins on the virus cell surface and specific viral receptors on target membranes. This process is mediated by the fusion peptide located between residues 98 and 112 (DRGWGNGCGLFGKGG) that forms a loop in domain II of dengue E glycoprotein. In this study, we evaluated the role of fusion peptide surrounding regions (88-97 and 113-123) of the Dengue 2 subtype on its interaction with the membrane and fusion activity. These sequences are important to stabilize the fusion peptide loop and increase fusion activity. Three peptides, besides the fusion peptide, were synthesized by SPPS using the Fmoc chemical approach. The first contains the fusion peptide and the C-terminal region of the loop (sequence 98-123); another contains the N-terminal region (88-112) and the larger peptide contains both regions (88-123). The peptides were able to interact with a model membrane. Differences in morphology of the monolayer promoted by the peptides were assessed by Brewster Angle Microscopy (BAM). Our data indicated that the C-terminal region of fusion peptide loop is more efficient in promoting fusion and interacting with the membrane than the N-terminal sequence, which is responsible for the electrostatic initial interaction. We propose a 2-step mechanism for the interaction of the dengue virus fusion peptide with the host membrane, where the N-terminal sequence docks electrostatically on the headgroups and then the C-terminal interacts via hydrophobic forces in the acyl chains., (Copyright © 2021 Elsevier Inc. All rights reserved.)