Repurposing an antimicrobial peptide for the development of a dual ion channel/molecular receptor-like platform for metal ion detection.

The presence of non-essential metals in the environment as contaminants is prone to cause hazardous health problems following accumulation in the human body and the ensuing toxic effects. This calls for continuous discovery and innovation in the realm of developing easy-to-operate, cheap and sensitive sensors. Herein, we describe the proof of concept approach for designing a molecular receptor-like, chimeric sensor based on the pore-forming peptide alamethicin (Alm), tethered a linker with an ultrashort peptide nucleic acid (PNA) moiety, capable of generating functional ion channel oligomers in planar lipid membranes.

Controlling DNA Fragments Translocation across Nanopores with the Synergic Use of Site-Directed Mutagenesis, pH-Dependent Charge Tuning, and Electroosmotic Flow.

Biological and solid-state nanopores are at the core of transformative techniques and nanodevices, democratizing the examination of matter and biochemical reactions at the single-molecule level, with low cost, portability, and simplicity in operation. One of the crucial hurdles in such endeavors is the fast analyte translocation, which limits characterization, and a rich number of strategies have been explored over the years to overcome this. Here, by site-directed mutagenesis on the α-hemolysin protein nanopore (α-HL), sought to replace selected amino acids with glycine, electrostatic binding sites were induced on the nanopore's vestibule and constriction region and achieved in the most favorable case a 20-fold increase in the translocation time of short single-stranded DNA (ssDNA) at neutral pH, with respect to the wild-type (WT) nanopore.

Antimicrobial peptides: Opportunities and challenges in overcoming resistance.

Antibiotic resistance represents a global health threat, challenging the efficacy of traditional antimicrobial agents and necessitating innovative approaches to combat infectious diseases. Among these alternatives, antimicrobial peptides have emerged as promising candidates against resistant pathogens. Unlike traditional antibiotics with only one target, these peptides can use different mechanisms to destroy bacteria, with low toxicity to mammalian cells compared to many conventional antibiotics.

Correction: Considerable slowdown of short DNA fragment translocation across a protein nanopore using pH-induced generation of enthalpic traps inside the permeation pathway.

Correction for 'Considerable slowdown of short DNA fragment translocation across a protein nanopore using pH-induced generation of enthalpic traps inside the permeation pathway' by Loredana Mereuta , , 2023, , 14754-14763, https://doi.org/10.1039/D3NR03344A.

Integrin Facilitates the Internalization of TAT Peptide Conjugated to RGD Motif in Model Lipid Membranes.

In recent years, targeted drug delivery has attracted a great interest for enhanced therapeutic efficiency, with diminished side effects, especially in cancer therapy. Cell penetrating peptides (CPPs) like HIV1-TAT peptides, appear to be the perfect vectors for translocating drugs or other cargoes across the plasma membrane, but their application is limited mostly due to insufficient specificity for intended targets. Although these molecules were successfully used, the mechanism by which the peptides enter the cell interior still needs to be clarified.

Considerable slowdown of short DNA fragment translocation across a protein nanopore using pH-induced generation of enthalpic traps inside the permeation pathway.

A pressing challenge in the realm of nanopore-based sensing technologies for nucleic acid characterization has been the cheap and efficient control of analyte translocation. To address this, a plethora of methods were tested, including mutagenesis, molecular motors, enzymes, or the optimization of experimental conditions. Herein, we present a paradigm exploiting the manipulation of electrostatic interactions between 22-mer single-stranded DNAs (22_ssDNA) and low pH-induced charges in the alpha-hemolysin (α-HL) nanopore, to efficiently control the passage of captured molecules.

Synthetic Receptor Based on a Peptide Antibiotic-Functionalized Chimera for Hybridization-Based Polynucleotide Detection.

Nanopores offer highly sensitive, low-cost, and single-molecule sensing capabilities, and the societal impact of this approach is best captured by the advent of nanopore-based DNA detection and sequencing technologies, which extract genomic information without amplification. To address a critical difficulty plaguing such undertakings involving especially protein-based nanopores isolated in lipid bilayers, namely, the formation of a stable, long-lasting single nanopore, we pioneer herein an approach for generating functional nanostructures enabling small single-stranded DNA (ssDNA) detection. We designed a dynamic hybrid construct by appending extramembrane peptide nucleic acid (PNA) segments to the C-terminus of modified ion channel-forming alamethicin monomers.

Coronary Syndromes and High-Altitude Exposure-A Comprehensive Review.

The aim of this review is to identify a preventive strategy in order to minimize the risk of adverse events in patients with coronary syndromes and acute exposure to high-altitude. For this purpose we searched the electronic database of PubMed, EMBASE, and Web of Science for studies published in the last 30 years in this field. The conclusions of this review are: patients with stable coronary artery disease on optimal treatment and in a good physical condition can tolerate traveling to high altitude up to 3500 m; on the other hand, patients with unstable angina or recent myocardial infarction no older than 6 months should take less interest in hiking or any activity involving high altitude.

Confocal Laser Scanning Microscopy and Model Membranes to Study Translocation Mechanisms of Membrane Active Peptides.

Membrane active peptides hold great potential for targeted drug delivery systems and understanding their mechanism of uptake is a key step in the development of peptide based therapeutics and clinical use. Giant unilamellar vesicles are cell-sized model membranes that can be individually observed under the microscope. The lipid composition of these membranes can be controlled, and interaction with peptides and changes induced by the peptides can be directly followed.

Probing the Hepatitis B Virus E-Antigen with a Nanopore Sensor Based on Collisional Events Analysis.

Real-time monitoring, simple operation, and cheaper methods for detecting immunological proteins hold the potential for a solid influence on proteomics and human biology, as they can promote the onset of timely diagnoses and adequate treatment protocols. In this work we present an exploratory study suggesting the applicability of resistive-pulse sensing technology in conjunction with the α-hemolysin (α-HL) protein nanopore, for the detection of the chronic hepatitis B virus (HBV) e-antigen (HBeAg). In this approach, the recognition between HBeAg and a purified monoclonal hepatitis B e antibody (Ab(HBeAg)) was detected via transient ionic current spikes generated by partial occlusions of the α-HL nanopore by protein aggregates electrophoretically driven toward the nanopore's vestibule entrance.

A Nanopore Sensor for Multiplexed Detection of Short Polynucleotides Based on Length-Variable, Poly-Arginine-Conjugated Peptide Nucleic Acids.

Real-time and easy-to-use detection of nucleic acids is crucial for many applications, including medical diagnostics, genetic screening, forensic science, or monitoring the onset and progression of various diseases. Herein, an exploratory single-molecule approach for multiplexed discrimination among similar-sized single-stranded DNAs (ssDNA) is presented. The underlying strategy combined (i) a method based on length-variable, short arginine (poly-Arg) tags appended to peptide nucleic acid (PNA) probes, designed to hybridize with selected regions from complementary ssDNA targets (cDNA) in solution and (ii) formation and subsequent detection with the α-hemolysin nanopore of (poly-Arg)-PNA-cDNA duplexes containing two overhangs associated with the poly-Arg tail and the non-hybridized segment from ssDNA.

A Single-Molecule Insight into the Ionic Strength-dependent, Cationic Peptide Nucleic Acids-Oligonucleotides Interactions.

To alleviate solubility-related shortcomings associated with the use of neutral peptide nucleic acids (PNA), a powerful strategy is incorporate various charged sidechains onto the PNA structure. Here we employ a single-molecule technique and prove that the ionic current blockade signature of free poly(Arg)-PNAs and their corresponding duplexes with target ssDNAs interacting with a single α-hemolysin (α-HL) nanopore is highly ionic strength dependent, with high salt-containing electrolytes facilitating both capture and isolation of such complexes. Our data illustrate the effect of low ionic strength in reducing the effective volume of free poly(Arg)-PNAs and augmentation of their electrophoretic mobility while traversing the nanopore.

Atomic force spectroscopy with magainin 1 functionalized tips and biomimetic supported lipid membranes.

Antimicrobial peptides are molecules synthesized by living organisms as the first line of defense against bacteria, fungi, parasites, or viruses. Since their biological activity is based on destabilization of the microbial membranes, a study of direct interaction forces between antimicrobial peptides and biomimetic membranes is very important for understanding the molecular mechanisms of their action. Herein, we use atomic force spectroscopy to probe the interaction between atomic force microscopy (AFM) tips functionalized with magainin 1 and supported lipid bilayers (SLBs) mimicking electrically uncharged membranes of normal eukaryotic cells and negatively charged membranes of bacterial cells.

Teaching an old dog new tricks: A lipid membrane-based electric immunosensor for real-time probing of the spike S protein subunit from SARS-CoV-2.

Fast, cheap, and easy to implement point-of-care testing for various pathogens constituted a game changer in past years due to its potential for early disease diagnosis. Herein, we report on the proof-of-concept of a simple method enabling in vitro detection of a structural spike protein subunit from the SARS-CoV-2 (S ) in aqueous samples. At the core of this discovery lies the well-known paradigm of monitoring the capacitive current across a reconstituted zwitterionic lipid membrane subjected to a periodic transmembrane potential, followed by the real-time spectral analysis enabling the extraction of the second harmonic of the capacitive current.

Single-molecule, hybridization-based strategies for short nucleic acids detection and recognition with nanopores.

DNA nanotechnology has seen large developments over the last 30 years through the combination of detection and discovery of DNAs, and solid phase synthesis to increase the chemical functionalities on nucleic acids, leading to the emergence of novel and sophisticated in features, nucleic acids-based biopolymers. Arguably, nanopores developed for fast and direct detection of a large variety of molecules, are part of a revolutionary technological evolution which led to cheaper, smaller and considerably easier to use devices enabling DNA detection and sequencing at the single-molecule level. Through their versatility, the nanopore-based tools proved useful biomedicine, nanoscale chemistry, biology and physics, as well as other disciplines spanning materials science to ecology and anthropology.

The Nanopore-Tweezing-Based, Targeted Detection of Nucleobases on Short Functionalized Peptide Nucleic Acid Sequences.

The implication of nanopores as versatile components in dedicated biosensors, nanoreactors, or miniaturized sequencers has considerably advanced single-molecule investigative science in a wide range of disciplines, ranging from molecular medicine and nanoscale chemistry to biophysics and ecology. Here, we employed the nanopore tweezing technique to capture amino acid-functionalized peptide nucleic acids (PNAs) with α-hemolysin-based nanopores and correlated the ensuing stochastic fluctuations of the ionic current through the nanopore with the composition and order of bases in the PNAs primary structure. We demonstrated that while the system enables the detection of distinct bases on homopolymeric PNA or triplet bases on heteropolymeric strands, it also reveals rich insights into the conformational dynamics of the entrapped PNA within the nanopore, relevant for perfecting the recognition capability of single-molecule sequencing.

Peptides-based therapy and diagnosis. Strategies for non-invasive therapies in cancer.

In recent years, remarkable progress was registered in the field of cancer research. Though, cancer still represents a major cause of death and cancer metastasis a problem seeking for urgent solutions as it is the main reason for therapeutic failure. Unfortunately, the most common chemotherapeutic agents are non-selective and can damage healthy tissues and cause side effects that affect dramatically the quality of life of the patients.

Non-Receptor-Mediated Lipid Membrane Permeabilization by the SARS-CoV-2 Spike Protein S1 Subunit.

Due to the pressing need to generate specific drugs or vaccines for COVID-19 and management of its outbreak, detailed knowledge regarding the SARS-CoV-2 entry into host cells and timely, cheap, and easy-to-use detection methods are of critical importance for containing the SARS-CoV-2 epidemic. Through electrophysiology and fluorescence spectroscopy experiments, we show that even in the absence of the angiotensin-converting enzyme 2 receptor, the S1 subunit from SARS-CoV-2 spike protein binding to neutral phospholipid membranes leads to their mechanical destabilization and permeabilization. A similar cytotoxic effect of the protein was seen in human lung epithelial cells.

Author Correction: Sequence-specific detection of single-stranded DNA with a gold nanoparticle-protein nanopore approach.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Sequence-specific detection of single-stranded DNA with a gold nanoparticle-protein nanopore approach.

Fast, cheap and easy to use nucleic acids detection methods are crucial to mitigate adverse impacts caused by various pathogens, and are essential in forensic investigations, food safety monitoring or evolution of infectious diseases. We report here a method based on the α-hemolysin (α-HL) nanopore, working in conjunction to unmodified citrate anion-coated gold nanoparticles (AuNPs), to detect nanomolar concentrations of short single-stranded DNA sequences (ssDNA). The core idea was to use charge neutral peptide nucleic acids (PNA) as hybridization probe for complementary target ssDNAs, and monitor at the single-particle level the PNA-induced aggregation propensity AuNPs during PNA-DNA duplexes formation, by recording ionic current blockades signature of AuNP-α-HL interactions.

Bee venom-derived antimicrobial peptide melectin has broad-spectrum potency, cell selectivity, and salt-resistant properties.

Antimicrobial peptides have attracted attention as alternatives to conventional antibiotics. Previously, a novel antimicrobial peptide, melectin, consisting of 18 amino acids was isolated from the venom of a bee, Melecta albifrons. Here, we investigated the antibacterial activity of melectin against drug-resistant bacteria.

Unzipping Mechanism of Free and Polyarginine-Conjugated DNA-PNA Duplexes, Preconfined Inside the α-Hemolysin Nanopore.

In this work, comparative studies on DNA-PNA and polyarginine-conjugated DNA-PNA duplexes unzipping inside the α-hemolysin nanopore (α-HL) are presented. We identified significant differences in the blockade currents, as the applied voltage across the nanopore facilitated the duplex capture inside the nanopore's vestibule against the constriction region, subsequent cDNA strand insertion inside the nanopore's β-barrel past the constriction site, its complete unzip from the duplex, and translocation. We observed that inside the voltage-biased nanopore, polyarginine-conjugated DNA-PNA duplexes dehybridize faster than their DNA-PNA counterparts and proposed a model to describe the duplex unzipping.

Mechanism of action of antimicrobial peptide P5 truncations against Pseudomonas aeruginosa and Staphylococcus aureus.

Rates of microbial drug resistance are increasing worldwide; therefore, antimicrobial peptides (AMPs) are considered promising alternative therapeutic agents to antibiotics. AMPs are essential components of the innate immune system and exhibit broad-spectrum antimicrobial activity. P5 is a Cecropin A-Magainin 2 hybrid analog peptide with antimicrobial activity against Gram-negative and Gram-positive bacteria.

Single-Molecule Dynamics and Discrimination between Hydrophilic and Hydrophobic Amino Acids in Peptides, through Controllable, Stepwise Translocation across Nanopores.

In this work, we demonstrate the proof-of-concept of real-time discrimination between patches of hydrophilic and hydrophobic monomers in the primary structure of custom-engineered, macro-dipole-like peptides, at uni-molecular level. We employed single-molecule recordings to examine the ionic current through the α-hemolysin (α-HL) nanopore, when serine or isoleucine residues, flanked by segments of oppositely charged arginine and glutamic amino acids functioning as a voltage-dependent "molecular brake" on the peptide, were driven at controllable rates across the nanopore. The observed differences in the ionic currents blockades through the nanopore, visible at time resolutions corresponding to peptide threading through the α-HL's constriction region, was explained by a simple model of the volumes of electrolyte excluded by either amino acid species, as groups of serine or isoleucine monomers transiently occupy the α-HL.