Designing Quantum Capacitive Peptide Interfaces for Electroanalytical Applications.

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-NH2 (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.

A novel insight on SARS-CoV-2 S-derived fragments in the control of the host immunity.

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.

Photobiomodulation Reduces the Cytokine Storm Syndrome Associated with COVID-19 in the Zebrafish Model.

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.

New Strategies for Novel Drugs: Antimicrobial Peptides Containing Ferrocene with Improved Antifungal and Antiplasmodial Biological Activity.

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.

Modular Label-Free Electrochemical Biosensor Loading Nature-Inspired Peptide toward the Widespread Use of COVID-19 Antibody Tests.

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 (AbS) 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 AbS 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-AbS 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.

Toxicological impact of SARS-CoV-2 on the health of the neotropical fish, Poecilia reticulata.

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 (H2O2), 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.

What different physical techniques can disclose about disruptions on membrane structure caused by the antimicrobial peptide Hylin a1 and a more positively charged analogue.

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-NH2) and its synthetic analogue K0Hya1 (KIFGAILPLALGALKNLIK-NH2), 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, K0Hya1 (+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.

Hesperetin targets the hydrophobic pocket of the nucleoprotein/phosphoprotein binding site of human respiratory syncytial virus.

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 Emergence of the New P.4 Lineage of SARS-CoV-2 With Spike L452R Mutation in Brazil.

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.

Recent advances in SARS-CoV-2 Spike protein and RBD mutations comparison between new variants Alpha (B.1.1.7, United Kingdom), Beta (B.1.351, South Africa), Gamma (P.1, Brazil) and Delta (B.1.617.2, India).

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.

Non-Toxic Dimeric Peptides Derived from the Bothropstoxin-I Are Potent SARS-CoV-2 and Papain-like Protease Inhibitors.

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-Cys11, Lys12,Lys13-(pBthTX-I)2K ((pBthTX-I)2K)) and derivatives against SARS-CoV-2 are reported. The lead peptide (pBthTX-I)2K and derivatives showed attractive inhibitory activities against SARS-CoV-2 (EC50 = 28-65 µM) and mostly low cytotoxic effect (CC50 > 100 µM). To shed light on the mechanism of action underlying the peptides' antiviral activity, the Main Protease (Mpro) and Papain-Like protease (PLpro) inhibitory activities of the peptides were assessed. The synthetic peptides showed PLpro inhibition potencies (IC50s = 1.0-3.5 µM) and binding affinities (Kd = 0.9-7 µM) at the low micromolar range but poor inhibitory activity against Mpro (IC50 > 10 µM). The modeled binding mode of a representative peptide of the series indicated that the compound blocked the entry of the PLpro 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.

Shedding light on toxicity of SARS-CoV-2 peptides in aquatic biota: A study involving neotropical mosquito larvae (Diptera: Culicidae).

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.

Toxicological insights of Spike fragments SARS-CoV-2 by exposure environment: A threat to aquatic health?

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.

On the role of surrounding regions in the fusion peptide in dengue virus infection.

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.

Nanostructured functional peptide films and their application in C-reactive protein immunosensors.

Peptides with an active redox molecule are incorporated into nanostructured films for electrochemical biosensors with stable and controllable physicochemical properties. In this study, we synthesized three ferrocene (Fc)-containing peptides with the sequence Fc-Glu-(Ala)n-Cys-NH2, which could form self-assembled monolayers on gold and be attached to antibodies. The peptide with two alanines (n = 2) yielded the immunosensor with the highest performance in detecting C-reactive protein (CRP), a biomarker of inflammation. Using electrochemical impedance-derived capacitive spectroscopy, the limit of detection was 240 pM with a dynamic range that included clinically relevant CRP concentrations. With a combination of electrochemical methods and polarization-modulated infrared reflection-absorption spectroscopy, we identified the chemical groups involved in the antibody-CRP interaction, and were able to relate the highest performance for the peptide with n = 2 to chain length and efficient packing in the organized films. These strategies to design peptides and methods to fabricate the immunosensors are generic, and can be applied to other types of biosensors, including in low cost platforms for point-of-care diagnostics.

Interaction of synthetic antimicrobial peptides of the Hylin a1 family with models of eukaryotic structures: Zwitterionic membranes and DNA.

Antimicrobial peptides (AMPs) have been appointed as a possible alternative to traditional antibiotics in face of pathogens increasing resistance to conventional drugs. Hylin a1 (IFGAILPLALGALKNLIK), an AMP extracted from the skin secretion of a South American frog, Hypsiboas albopunctatus, was found to show a strong cytotoxicity against bacteria and fungus, but also a considerable hemolytic action. Considering the toxicity of the peptide in eukaryotic cells, this work focuses on investigating the effects of the interaction of the Hylin a1 analogues W6Hya1, D0W6Hya1 and K0W6Hya1 with models of eukaryotic structures, namely zwitterionic liposomes of dipalmitoyl phosphatidylcholine (DPPC) and calf-thymus DNA (CT DNA). Through intrinsic Trp fluorescence we determined that the peptide affinity for fluid DPPC bilayers follows the decreasing order: D0W6Hya1 (+2) > W6Hya1 (+3) ¼ K0W6Hya1 (+4). Fluorescence data also indicate that the Trp residue in the more positively charged peptide, K0W6Hya1, is less deep in the bilayer than the residue in the other two peptides. This finding is supported by differential scanning calorimetry (DSC) data, which shows that both D0W6Hya1 and W6Hya1 disturb DPPC gel-fluid transition slightly more effectively than K0W6Hya1. DPPC DSC profiles are homogeneously disturbed by the three peptides, probably related to peptide-membrane diffusion. Surprisingly, the peptide that displays the lowest affinity for PC membranes and is located at the more superficial position in the bilayer, K0W6Hya1, is the most efficient in causing formation of pores on the membrane, as attested by carboxyfluorescein leakage assays. The three peptides were found to interact with CT DNA, with a deep penetration of the Trp residue into hydrophobic pockets of the double helix, as indicated by the significant blue shift on the Trp fluorescence, and the displacement of DNA-bound ethidium bromide by the peptides. The experiments of DNA electrophoresis confirm that Hylin peptides bind DNA in a concentration-dependent manner, inducing complete DNA retardation at the relative AMP/plasmid DNA weight ratio of ~17. These findings could help to better understand the AMPs toxic effects on eukaryotic cells, thus contributing to the design of healthier therapeutic agents.

Antimicrobial activity of RP-1 peptide conjugate with ferrocene group.

Parasitic diseases are a neglected and serious problem, especially in underdeveloped countries. Among the major parasitic diseases, Leishmaniasis figures as an urgent challenge due to its high incidence and severity. At the same time, the indiscriminate use of antibiotics by the population is increasing together with resistance to medicines. To address this problem, new antibiotic-like molecules that directly kill or inhibit the growth of microorganisms are necessary, where antimicrobial peptides (AMPs) can be of great help. In this work, the ferrocene molecule, one active compound with low levels of in vivo toxicity, was coupled to the N-terminus of the RP1 peptide (derived from the human chemokine CXCL4), aiming to evaluate how this change modifies the structure, biological activity, and toxicity of the peptide. The peptide and the conjugate were synthesized using the solid phase peptide synthesis (SPPS). Circular dichroism assays in PBS showed that the RP1 peptide and its conjugate had a typical spectrum for disordered structures. The Fc-RP1 presented anti-amastigote activity against Leishmania amazonensis (IC50 = 0.25 µmol L-1). In comparison with amphotericin B, a second-line drug approved for leishmaniasis treatment, (IC50 = 0.63 µmol L-1), Fc-RP1 was more active and showed a 2.5-fold higher selectivity index. The RP1 peptide presented a MIC of 4.3 µmol L-1 against S. agalactiae, whilst Fc-RP1 was four times more active (MIC = 0.96 µmol L-1), indicating that ferrocene improved the antimicrobial activity against Gram-positive bacteria. The Fc-RP1 peptide also decreased the minimum inhibitory concentration (MIC) in the assays against E. faecalis (MIC = 7.9 µmol L-1), E. coli (MIC = 3.9 µmol L-1) and S. aureus (MIC = 3.9 µmol L-1). The cytotoxicity of the compounds was tested against HaCaT cells, and no significant activity at the highest concentration tested (500 µg. mL-1) was observed, showing the high potential of this new compound as a possible new drug. The coupling of ferrocene also increased the vesicle permeabilization of the peptide, showing a direct relation between high peptide concentration and high carboxyfluorescein release, which indicates the action mechanism by pore formation on the vesicles. Several studies have shown that ferrocene destabilizes cell membranes through lipid peroxidation, leading to cell lysis. It is noteworthy that the Fc-RP1 peptide synthesized here is a prototype of a bioconjugation strategy, but it still is a compound with great biological activity against neglected and fish diseases.

Serological point-of-care and label-free capacitive diagnosis of dengue virus infection.

Dengue non-structural protein 1 (NS1 DENV) is considered a biomarker for dengue fever in an early stage. A sensitive and rapid assay for distinguishing positive from negative dengue infection samples is imperative for epidemic control and public health in tropical regions because it enables the development of instantaneous updatable databases and effective surveillance systems. Presently, we successfully report, for the first time, the use of the electrochemical capacitive method for the detection of NS1 DENV biomarker in human serum samples. By using a ferrocene-tagged peptide modified surface containing anti-NS1 as the receptor, it was possible to differentiate positive from negative samples with a p < 0.01 in a reagentless and label-free capacitive format. This capacitive assay had a cut-off of 1.36% (confidence interval of 99.99%); it therefore opens new avenues for developing miniature label-free electrochemical devices for infectious diseases.

Effect of analogues of cationic peptides on dentin mineralization markers in odontoblast-like cells.

OBJECTIVES: To evaluate the effect of analogues of cationic peptides on the viability and the expression of phenotypic and genotypic markers of dentin mineralization in MDPC-23 odontoblast-like cells. MATERIALS AND METHODS: Cells were exposed to serial dilutions of analogues of cationic peptides hBD-3-1CV and KR-12-a5 compared to peptide LL-37 and their viability was assessed by methyltetrazolium assay. Next, peptides (0.78-62.5 µg/mL) were applied on the MDPC-23 cells for evaluating the total protein (TP) production, alkaline phosphatase (ALP) activity and mineralized nodule deposition. Gene expression of mineralization markers (DSPP and DMP-1) was also determined by quantitative PCR. RESULTS: LL-37 and hBD-3-1CV treatment did not affect cellular viability at concentrations below 62.5 µg/mL. KR-12-a5 reduced cell viability above 31.25 µg/mL. TP production was similar for all groups compared with the control group, except by hBD-3-1CV (at 15.62 µg/mL). LL-37 (at 62.5 µg/mL) induced higher ALP activity than control and other experimental groups. LL-37 and hBD-3-1CV, at 62.5 µg/mL and KR-12-a5 at 31.25 µg/mL stimulated the highest deposition of mineralized nodule. Overall, no statistical differences were observed between the groups for DSPP-1 and DMP-1 expressions. CONCLUSIONS: LL-37 was the only peptide that induced both ALP activity and mineralized nodules deposition, without affecting cell viability. None of peptides tested induced the expression of DSPP or DMP-1, genes commonly involved in active dentin mineralization.