Correction for ‘Rapid detection of Candida albicans in urine by an Electrochemical Impedance Spectroscopy (EIS)-based biosensor’ by Tina D'Aponte et al. , Sens. Diagn. , 2023, 2 , 1597–1604, https://doi.org/10.1039/d3sd00209h.

Mono- and bis-guanyl hydrazone-functionalized tricyclic compounds were here designed and investigated as putative G-quadruplex ligands in the context of anticancer drug development. The G-quadruplex on Controlled Pore Glass (G4-CPG) assay, a fast and easy screening method based on affinity chromatography for identifying potential G-quadruplex binders, together with biophysical techniques such as circular dichroism and fluorescence spectroscopy, demonstrated a higher selectivity of mono- with respect to disubstituted derivatives in recognizing G-quadruplexes from telomeric and oncogenic DNA regions vs. duplexes. Among the mono-substituted compounds, higher G-quadruplex selectivity was found for those containing the pyrido [3,4-b]indole and dibenzofuran scaffolds compared to the 9H-fluorene, 9H-carbazole, and dibenzothiophene ones. Molecular docking studies suggested that the investigated ligands bound the hybrid telomeric G-quadruplex model by adopting a coplanar arrangement of the core and guanyl hydrazone moieties, both stacked on the 5'-G-quartet, while in the interaction with the parallel oncogenic G-quadruplex model the guanyl hydrazone moieties pointed towards the grooves/loops. Finally, biological assays highlighted the higher potential of mono-guanyl hydrazone-derivatized tricyclic compounds as selective anticancer agents, showing higher anticancer activity and selectivity of action than the bis-guanyl hydrazone derivatives.

In this study, we present novel nanosystems based on streptavidin-coated silica nanoparticles (sicastar® NPs) functionalized with the recently identified G-quadruplex(G4)-forming aptamer named L12, which specifically targets high mobility group box 1 (HMGB1), a key mediator in inflammatory diseases and cancer when extracellularly released. In our design, the L12 aptamer was conjugated with a biotin tag at one end (either 5′ or 3′) to allow its effective incorporation onto the NP surface, and with the Cyanine 5 fluorophore (Cy5) at the other available end, to monitor the functionalization efficiency on the sicastar® NPs. The two bis-conjugated L12 aptamers were first analyzed by spectroscopic and electrophoretic techniques, proving the same G4 conformational behaviour and higher thermal stability in comparison with those of unmodified L12, and then used to functionalize the selected NPs. The obtained aptamer-decorated nanosystems were characterized by fluorescence spectroscopy, monitoring the Cy5 signal, and DLS measurements, which showed quantitative incorporation yield and a mean hydrodynamic diameter of ca. 110 nm with size stability for at least one month. Both the L12-functionalized NP systems showed higher HMGB1 affinity and bioactivity compared to the original L12 aptamer and also to the free bis-conjugated L12 derivatives used as controls. Specifically, the L12-functionalized sicastar® nanosystems, at a 50 nM aptamer concentration, inhibited A549 cancer cell migration by more than 70% without inducing, at the same concentration, any cytotoxic effect on the same cells. Taken together, our results demonstrated that the bis-conjugated L12 aptamers are capable of markedly reducing the HMGB1-induced migration of lung carcinoma cells and that their incorporation onto sicastar® NPs substantially enhances this bioactivity, mainly leveraging multivalency effects. These aptamer-decorated NPs may thus represent advanced tools for effective HMGB1 inhibition.

The identification of reliable biomarkers is essential for improving breast cancer (BC) detection, prognosis, and treatment. This study explores a human telomeric G-quadruplex (G4) model, tel46, functionalized on Controlled Pore Glass (CPG) support, as a novel biomarker discovery tool. The oligonucleotide tel46 mimics multimeric G4 structures in telomeric overhangs. Using affinity purification-mass spectrometry, 93 proteins interacting with tel46 were identified starting from nuclear extract of MCF7 cells, linking them to pathways in DNA replication, repair, and genome stability, which are frequently altered in cancer. Integrating AP-MS data with quantitative proteomics comparing MCF7 to non-tumorigenic MCF10A cells, 27 tel46 interactors were identified among upregulated proteins. Functional analyses revealed enrichment in genome maintenance and repair pathways, while downregulated proteins were associated with fundamental cellular functions. Further bioinformatics analysis using public cancer proteomics database 19 were validated. Bioinformatic analysis based on transcriptomics and clinical data revealed MSH6, MSH2, ESRP1, and WDHD1 as the most promising potential biomarkers for breast cancer. Indeed, these proteins are highly expressed in BC and generally correlated to poor prognosis: in addition to their role as potential biomarkers for early diagnosis, these proteins might be used as targets for specific treatment, enhancing radiation sensitivity or decreasing tumour cell proliferation. As a proof-of-concept, this study proposes tel46-functionalized CPG as a potential tool for isolating cancer-related proteins and underscores the potential of G4-interacting proteins as biomarkers for BC diagnosis and therapy. Moreover, these findings establish a basis for further research into G4-mediated cancer mechanisms.

Fibrinogen (FIB), a key component of the coagulation cascade, is traditionally recognized for its role in hemostasis and tissue repair. However, due to its high plasma abundance and susceptibility to proteolytic cleavage during inflammation, it may also represent a previously unrecognized source of bioactive peptides. This study presents, for the first time, a comprehensive analysis of the antimicrobial, anti-inflammatory, and antiviral properties of six cationic antimicrobial peptides (AMPs) deriving from the C-terminal extremities of the three subunits of human fibrinogen (FIBα, FIBβ, and FIBγ), identified using a scoring function developed by our group. Antibacterial assays against Gram-positive and Gram-negative pathogens revealed different antimicrobial activity profile depending on their parent protein. Selected peptides displayed additive or synergistic effects when combined with conventional antibiotics or the thrombin-derived peptide (P)GKY20, highlighting their potential for combination therapies. Hemolytic assay confirmed the biocompatibility of fibrinogen-derived cryptic peptides with erythrocytes. Furthermore, the peptides significantly reduced LPS-induced nitric oxide release in murine macrophages Raw 264.7 cells, indicating anti-inflammatory activity. Notably, antiviral activity was observed against enveloped viruses (HCoV-229E and HSV-1) under various treatment conditions, while no activity was detected against the non-enveloped virus CVB3. Overall, these findings reveal human fibrinogen as a source of multifunctional cryptic peptides with broad-spectrum antimicrobial, antiviral, and immunomodulatory activities, supporting their potential as part of the innate immune system.

We here studied a focused set of covalent dimers of the G-quadruplex(G4)-forming aptamer L12, a 26-mer previously selected as inhibitor of High Mobility Group Box 1 (HMGB1), protein involved in various inflammatory and autoimmune diseases as well as in cancer. Inspired by the ability of L12 to form dimeric parallel G4 structures, proved to be highly bioactive towards HMGB1, we investigated several covalent dimeric analogues of L12, whose design exploited linkers of different nature and length, maintaining or inverting the polarity of the two L12 strands in the sequence. Several biophysical techniques were used to analyze the conformational behaviour, molecularity and thermal/serum stability of these dimers, which formed G4 structures of parallel or hybrid topologies. Their ability to interact with the target HMGB1 protein and inhibit HMGB1-induced cellular migration was tested in comparison with L12 non-covalent dimer. The best candidate was L12d1T3, containing a single thymidine as 3'-3' inversion of polarity motif in the junction between the two L12 strands. This oligonucleotide, forming a parallel G4 structure, showed strong affinity for the target protein (KDca. 40 nM), marked serum resistance (t1/2ca. 13 h), and excellent ability to hamper cell migration in A549 cells, with IC50 of 28 nM.

The synthesis and characterization of a mini-library of cyclic triimidazo triazine (TT) derivatives functionalized with one, two, or three ethynyl-N-methyl-pyridinium moieties are reported here. These compounds were designed with the aim of targeting cancer-related DNA G-quadruplex structures. The newly synthesized compounds were tested for their ability to bind G-quadruplexes from both telomeric and oncogene promoter sequences using an affinity chromatography-based assay, spectroscopic and electrophoretic techniques, as well as molecular docking analysis. The obtained results demonstrated the effective capacity of the investigated compounds to specifically recognize the selected G-quadruplex models, with their TT cores targeting the outer G-quartets and their positively charged N-methyl-pyridinium groups interacting with the top edge of G-quadruplex grooves. Notably, the trisubstituted cyclic triimidazole compounds showed higher stabilizing properties than the related disubstituted derivatives, which in turn were stronger binders than their monosubstituted analogs. However, the mono- and disubstituted derivatives showed higher G-quadruplex versus duplex recognition selectivity compared with the trisubstituted ones. Altogether, the biophysical experiments, also in agreement with the biological assays, underlined the advantage of introducing an alkyne linker between the triimidazole core and the methylpyridinium group, proving to be beneficial to increase both the stabilizing effects on the G-quadruplexes and the anticancer activity compared with the analogs of the same family lacking the alkyne linker.

Although natural antimicrobial peptides (AMPs) are endowed with excellent antimicrobial properties, only a few of them have been successfully translated to the market so far. This is mainly due to their short half-life, to their high susceptibility to protease degradation, and to the lack of appropriate strategies for their efficient targeted delivery. Hence, the development of an effective system to deliver AMPs to the site of infection is urgent. The system here selected is represented by bacterial cellulose nanoparticles (BCNPs). Nanocellulose has recently emerged as one of the most promising "green" materials, attracting great attention due to its unique features, including biodegradability, sustainability, biocompatibility, and special physicochemical properties. To produce BCNPs, Komagataeibacter xylinus has been selected as host producing strain. Once obtained BC macrofibers, the production of BCNPs was set up by enzymatic hydrolysis using a commercial mixture of cellulases from Trichoderma reesei to develop a sustainable green biotechnological process. The storage stability of produced BCNPs has been also evaluated. Obtained BCNPs have been functionalized through non-covalent bindings with an antimicrobial peptide previously identified in human apolipoprotein B and found to be endowed with strong antimicrobial properties in in vitro analyses and with good biocompatibility profiles when analyzed on human skin cells. This opens interesting perspectives to the applicability of the developed system in several biotechnological fields.

Milk is a primary nutrition source for newborns and adults and, in addition, is also a valuable reservoir of bioactive peptides. Many of these peptides are hidden as "cryptic" sequences in milk proteins and released in the bioactive form through protease digestions. Caseins, the most abundant proteins in bovine milk, host several cryptic bioactive peptides including those antimicrobials. In this study we report in-silico identification, production in recombinant form and extensive characterization of KNR50, a novel cationic antimicrobial peptide (CAMP) located at the C-terminus of bovine casein αS2. KNR50 shows antimicrobial activity against a large panel of bacteria and does not induce resistance development. In addition, KNR50 shows a remarkably wide spectrum of functional properties, as antibiofilm and antiviral activities, immunomodulatory and antioxidant properties as well as promising in-vivo anti-infective properties in a Caenorhabditis elegans model. These findings suggest that KNR50 could serve as a promising multifunctional agent with potential applications not only in combating infectious diseases and enhancing immune responses but also in non-clinical settings such as food preservation, where its antimicrobial properties could be exploited to extend shelf-life and improve food safety.

We report the synthesis and the antibacterial activity of a library of thioarylamide derivatives, which include N-19004, previously developed as Formyl Peptide Receptor 1 antagonist. These compounds were active against a broad spectrum of pathogens, including clinical strains of Staphylococcus aureus and Pseudomonas aeruginosa. N-19004 and its analog N-19004-S were proficient in biofilm growth inhibition and disruption. In a murine model of chronic Pseudomonas aeruginosa lung infection, N-19004 significantly reduced bacterial burden and inflammation at low doses. As revealed by Electron Paramagnetic Resonance and Scanning Electron Microscopy analyses, the antibacterial effect of N-19004 mainly derives from its interference with the bacterial membrane bilayer. The formation of long filamentous cells was also observed. With its structural simplicity, good in vivo tolerability, and simultaneous antibacterial and anti-inflammatory activity, N-19004 emerges as a promising candidate for treating multi-drug resistant infections, particularly in cystic fibrosis-related lung disease, where chronic inflammation plays an important role.

The human proteome represents a vast, largely untapped source of encrypted bioactive peptides with therapeutic potential. Here, we report the discovery and functional characterization of three antimicrobial encrypted peptides (EPs) derived from human matrix metallopeptidase-19 (residues 1-19, 1-33, and 247-279). These peptides exhibit potent, broad-spectrum activity against Gram-positive and Gram-negative bacteria, including clinical isolates and multidrug-resistant strains. Mechanistic studies reveal membrane depolarization and permeabilization as the primary mechanism of action. The peptides also inhibit biofilm formation, eradicate preformed biofilms, and exhibit selective antiviral activity against enveloped viruses. Importantly, they display negligible hemolysis and cytotoxicity toward mammalian cells while modulating inflammation through LPS neutralization. Synergy assays reveal synergistic or additive interactions with last-line antibiotics, and no resistance emerged after serial bacterial passaging. A fully d-amino acid analog of the lead peptide retained activity and exhibited cytocompatibility and in vivo efficacy in a murine skin infection model. These findings underscore the therapeutic promise of human protein-derived encrypted peptides and highlight proteome mining as a viable strategy for identifying host-compatible anti-infectives.

Benzofuran (BF) and benzodifuran (BDF) based compounds are known for their tunable optical and biological properties. Recently, their potential as chelating and anticancer agents has been examined and exploited. As dyes emissive in a physiological environment are in high demand for integrated therapeutic and imaging tools, BF and BDF scaffolds came up as possible theranostic probes. Here, we examined two series of BF and BDF based compounds with an extended conjugation pattern and a half-salen group producing a copper (II) ion-chelating O,N,O tridentate site. A combined experimental and theoretical approach was employed in the examination of spectroscopic and cytotoxic properties. The novel compounds are emissive according to an aggregation-induced emission mechanism and exhibit the DR/NIR (deep red/near-infrared) fluorescence required for penetration into living tissue. The results collected against HeLa, A431, and MCF7 human cells were discussed and compared, detecting in one case a very good performance against tumor cells as well as selectivity towards healthy HaCaT cells.

The employment of chemical agents in the food industry is raising several concerns by consumers and is leading to an increasing interest in natural food preservatives. Among alternatives, host defense peptides (HDPs) have attracted great interest for their ability to preserve food samples from contamination without altering their quality, taste, and organoleptic properties. Recently, we evaluated the applicability of ApoB-derived peptides as novel food bio-preservatives and demonstrated their ability to prevent chicken meat sample contamination when immobilized on chitosan films. To perform a further step towards the applicability of these peptides in the food field, here we evaluated peptides biosafety and digestibility. To do this, we used a multidisciplinary approach including the evaluation of the peptides' toxicity and antimicrobial activity, the analysis of resistance phenotype development, an in silico prediction of the peptides' susceptibility to proteases and the evaluation of the peptides' stability in simulated gastric and intestinal fluids. ApoB-derived peptides were found to be nontoxic when tested on human gastric carcinoma cells SNU-1 and on human colon-rectal adenocarcinoma cells HT-29, and not to induce resistance phenotype in Salmonella strains. Bioinformatic analyses showed that the peptides are susceptible to several proteases, as also confirmed by experiments in simulated gastric and intestinal fluids. Altogether these findings open interesting perspectives to the future applicability of ApoB-derived peptides as novel food biopreservatives.

We report the synthesis and in vitro evaluation of novel beta-sheet breaker peptides as promising Aβ fibrillogenesis inhibitors. Further 3D-GRID pharmacophore and metadynamics simulations rationalized the design of these peptides.

Human angiogenin (hANG) is the most studied stress-induced ribonuclease (RNase). In physiological conditions it performs its main functions in nucleoli, promoting cell proliferation by rDNA transcription, whereas it is strongly limited by its inhibitor (RNH1) throughout the rest of the cell. In stressed cells hANG dissociates from RNH1 and thickens in the cytoplasm where it manages the translational arrest and the recruitment of stress granules, thanks to its propensity to cleave tRNAs and to induce the release of active halves. Since it exists a clear connection between hANG roles and its intracellular routing, starting from our recent findings on heterologous ANG (ANG) properties in human keratinocytes (HaCaT cells), here we designed a variant unable to translocate into the nucleus with the aim of thoroughly verifying its potentialities under stress. This variant, widely characterized for its structural features and biological attitudes, shows more pronounced aid properties than unmodified protein. The collected evidence thus fully prove that ANG stress-induced skills in assisting cellular homeostasis are strictly due to its cytosolic localization. This study opens an interesting scenario for future studies regarding both the strengthening of skin defences and in understanding the mechanism of action of these special enzymes potentially suitable for any cell type.

New G-quadruplex-interactive and selective ligands are strongly required to evolve innovative, effective and minimally toxic anticancer agents. With this purpose, we have here synthesized and evaluated a mini-library of organic molecules featured by aromatic cores of different rigidity (naphthalene or bioxazole), decorated with pendant groups including positively charged moieties and/or H-bond donors/acceptors. By exploiting different biophysical techniques, we proved the ability of the bioxazole-based derivatives to strongly and selectively interact with telomeric and oncogenic G-quadruplexes, while the compound featured by a naphthalene core did not emerge as a good G-quadruplex ligand. Molecular docking studies demonstrated the ability of the bioxazole-based ligands to preferentially target the outer G-tetrads of both telomeric and oncogenic G-quadruplexes, by positioning their cores on the G-tetrads in a symmetrical or asymmetrical way respectively, with the pendant groups pointing towards or away from the grooves. All bioxazole-based ligands showed anticancer activity in the low micromolar range. Particularly, the bioxazole derivative bearing piperazine groups was the most active compound of the investigated series, whereas the derivatives bearing morpholine groups were the most selective ones on cancer cells, in full agreement with their ability to act as the strongest and most selective G-quadruplex ligands, respectively.

In this work, we present the case of the G-quadruplex(G4)-forming aptamers we recently identified for the recognition of HMGB1, protein involved in inflammation, autoimmune diseases and cancer. These aptamers were previously analyzed, without annealing them, after proper dilution of the stock solution in a pseudo-physiological buffer mimicking the extracellular environment where the protein exerts its pathological activity, and showed high thermal stability and nuclease resistance, good protein affinity and remarkable in vitro activity. These features were more marked for the aptamers forming dimeric, parallel G4 structures in solution. Herein, we fully characterized the same anti-HMGB1 aptamers after a standard annealing procedure performed on diluted samples. Notably, upon a thermal unfolding/folding cycle, these aptamers, and particularly the best ones in the not-annealed form, showed significant conformational switches compared to the same systems analyzed without annealing, forming exclusively monomeric G4 structures, featured by poor thermal and enzymatic stabilities, along with lower protein affinities. These results prove that, for these aptamers, analyzed in the chosen conditions, annealing at low concentration does not produce a beneficial effect in terms of favouring the most bioactive species.

The discovery of plant-derived compounds that are able to combat antibiotic-resistant pathogens is an urgent demand. Over years, Centaurea hyalolepis attracted considerable attention because of its beneficial medical properties. Phytochemical analyses revealed that Centaurea plant species contain several metabolites, such as sesquiterpene lactones (STLs), essential oils, flavonoids, alkaloids, and lignans.The organic extract of C. hyalolepis plant, collected in Palestine, showed significant antimicrobial properties towards a panel of Gram-negative and Gram-positive bacterial strains when the Minimal Inhibitory Concentration (MIC) values were evaluated by broth microdilution assays. A bio-guided fractionation of the active extract via multiple steps of column and thin layer chromatography allowed us to obtain three main compounds. The isolated metabolites were identified as the STLs cnicin, 11 beta,13-dihydrosalonitenolide and salonitenolide by spectroscopic and spectrometric analyses. Cnicin conferred the strongest antimicrobial activity among the identified compounds. Moreover, the evaluation of its antibiofilm activity by biomass assays through crystal violet staining revealed almost 30% inhibition of biofilm formation in the case of A. baumannii ATCC 17878 strain. Furthermore, the quantification of carbohydrates and proteins present in the extracellular polymeric substance (EPS) revealed the ability of cnicin to significantly perturb biofilm structure. Based on these promising results, further investigations might open interesting perspectives to its applicability in biomedical field to counteract multidrug resistant infections.

In-depth studies on the interaction of natural compounds with cancer-related G-quadruplex structures have been undertaken only recently, despite their high potential as anticancer agents, especially due to their well-known and various bioactivities. In this frame, aiming at expanding the repertoire of natural compounds able to selectively recognize G-quadruplexes, and particularly focusing on phenanthrenoids, a mini-library including dimeric (1–3) and glucoside (4–5) analogues of 9,10-dihydrophenanthrenes, a related tetrahydropyrene glucoside (6) along with 9,10-dihydrophenanthrene 7 were investigated here by several biophysical techniques and molecular docking. Compounds 3 and 6 emerged as the most selective G-quadruplex ligands within the investigated series. These compounds proved to mainly target the grooves/flanking residues of the hybrid telomeric and parallel oncogenic G-quadruplex models exploiting hydrophobic, hydrogen bond and π-π interactions, without perturbing the main folds of the G-quadruplex structures. Notably, a binding preference was found for both ligands towards the hybrid telomeric G-quadruplex. Moreover, compounds 3 and 6 proved to be active on different human cancer cells in the low micromolar range. Overall, these compounds emerged as useful ligands able to target G-quadruplex structures, which are of interest as promising starting scaffolds for the design of analogues endowed with high and selective anticancer activity.

Plants are considered a wealthy resource of novel natural drugs effective in the treatment of multidrug-resistant infections. Here, a bioguided purification of Ephedra foeminea extracts was performed to identify bioactive compounds. The determination of antimicrobial properties was achieved by broth microdilution assays to evaluate minimal inhibitory concentration (MIC) values and by crystal violet staining and confocal laser scanning microscopy analyses (CLSM) to investigate the antibiofilm capacity of the isolated compounds. Assays were performed on a panel of three gram-positive and three gram-negative bacterial strains. Six compounds were isolated from E. foeminea extracts for the first time. They were identified by nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS) analyses as the well-known monoterpenoid phenols carvacrol and thymol and as four acylated kaempferol glycosides. Among them, the compound kaempferol-3-O-α-L-(2″,4″-di-E-p-coumaroyl)-rhamnopyranoside was found to be endowed with strong antibacterial properties and significant antibiofilm activity against S. aureus bacterial strains. Moreover, molecular docking studies on this compound suggested that the antibacterial activity of the tested ligand against S. aureus strains might be correlated to the inhibition of Sortase A and/or of tyrosyl tRNA synthase. Collectively, the results achieved open interesting perspectives to kaempferol-3-O-α-L-(2″,4″-di-E-p-coumaroyl)-rhamnopyranoside applicability in different fields, such as biomedical applications and biotechnological purposes such as food preservation and active packaging.