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The occurrence of nosocomial infections has been on the rise for the past twenty years. Notably, infections caused by the Gram-positive bacteria Staphylococcus aureus represent a major clinical problem, as an increase in antibiotic multi-resistant strains has accompanied this rise. There is thus a crucial need to find and characterize new antibiotics against Gram-positive bacteria, and against antibiotic-resistant strains in general. We identified a new dermaseptin, DMS-DA6, produced by the skin of the Mexican frog Pachymedusa dacnicolor, with specific antibacterial activity against Gram-positive bacteria. This peptide is particularly effective against two multiple drug-resistant strains Enterococcus faecium BM4147 and Staphylococcus aureus DAR5829, and has no hemolytic activity. DMS-DA6 is naturally produced with the C-terminal carboxyl group in either the free or amide forms. By using Gram-positive model membranes and different experimental approaches, we showed that both forms of the peptide adopt an α-helical fold and have the same ability to insert into, and to disorganize a membrane composed of anionic lipids. However, the bactericidal capacity of DMS-DA6-NH2 was consistently more potent than that of DMS-DA6-OH. Remarkably, rather than resulting from the interaction with the negatively charged lipids of the membrane, or from a more stable conformation towards proteolysis, the increased capacity to permeabilize the membrane of Gram-positive bacteria of the carboxyamidated form of DMS-DA6 was found to result from its enhanced ability to interact with peptidoglycan.

Interleukin-6 (IL-6) belongs to the cytokine family and plays a vital role in regulating immune response, bone maintenance, body temperature adjustment, and cell growth. The overexpression of IL-6 can indicate various health complications, such as anastomotic leakage, cancer, and chronic diseases. Therefore, the availability of highly sensitive and specific biosensing platforms for IL-6 detection is critical. In this study, for the first time, epitope-mediated IL-6-specific magnetic molecularly imprinted core–shell structures with fluorescent properties were synthesized using a three-step protocol, namely, magnetic nanoparticle functionalization, polymerization, and template removal following thorough optimization studies. The magnetic molecularly imprinted polymers (MMIPs) were characterized using dynamic and electrophoretic light scattering (DLS and ELS), revealing a hydrodynamic size of 169.9 nm and zeta potential of +17.1 mV, while Fourier transform infrared (FTIR) spectroscopy and fluorescence spectroscopy techniques showed characteristic peaks of the polymer and fluorescent tag, respectively. Scanning electron microscopy (SEM) and high-resolution transmission electron microscopy (HRTEM) investigations confirmed the successful encapsulation of the magnetic core within the ca. 5-nm-thick polymeric shell. The MMIP-based electrochemical sensing platform achieved a limit of detection of 0.38 pM within a linear detection range of 0.38–380 pM, indicating high affinity (dissociation constant KD = 1.6 pM) for IL-6 protein in 50% diluted serum samples. Moreover, comparative investigations with the non-imprinted control polymer demonstrated an imprinting factor of 4, confirming high selectivity. With multifunctional features, including fluorescence, magnetic properties, and target responsiveness, the synthesized MMIPs hold significant potential for application in various sensor techniques as well as imaging.

Borrelia , spirochetes transmitted by ticks, are the etiological agents of numerous multisystemic diseases, such as Lyme borreliosis (LB) and tick-borne relapsing fever (TBRF). This study focuses on two surface proteins from two Borrelia subspecies involved in these diseases: CspZ, expressed by Borrelia burgdorferi sensu stricto (also named BbCRASP-2 for complement regulator-acquiring surface protein 2), and the factor H binding A (FhbA), expressed by Borrelia hermsii. Numerous subspecies of Borrelia , including these latter, are able to evade the immune defenses of a variety of potential vertebrate hosts in a number of ways. In this context, previous data suggested that both surface proteins play a role in the immune evasion of both Borrelia subspecies by interacting with key regulators of the alternative pathway of the human complement system, factor H (FH) and FH-like protein 1 (FHL-1). The recombinant proteins, CspZ and FhbA, were expressed in Escherichia coli and purified by one-step metal-affinity chromatography, with yields of 15 and 20 mg or pure protein for 1 L of cultured bacteria, respectively. The purity was evaluated by SDS-PAGE and HPLC and is close to about 95%. The mass of CspZ and FhbA was checked by mass spectrometry (MS). Proper folding of CspZ and FhbA was confirmed by circular dichroism (CD), and their biological activity, namely their interaction with purified FH from human serum (recombinant FH 15-20  and recombinant FHL-1), was characterized by SPR. Such a study provides the basis for the biochemical characterization of the studied proteins and their biomolecular interactions which is a necessary prerequisite for the development of new approaches to improve the current diagnosis of LB and TBRF. Key points • DLS, CD, SEC-MALS, NMR, HPLC, and MS are tools for protein quality assessment • Borrelia spp. possesses immune evasion mechanisms, including human host complement • CspZ and FhbA interact with high affinity (pM to nM) to human FH and rFHL-1 Graphical Abstract

Cell-penetrating peptides can internalize ubiquitously in many, if not all, cell types. To explore the specific targeting issue of cell-penetrating peptides (CPPs), we studied glycosaminoglycan (GAG)-binding peptides previously identified in Otx2 and En2 homeoproteins (HPs), alone or extended with the penetratin-like third helix (H3) of En2. HPs are indeed known to internalize in specific cells, thanks to their GAG-targeting sequence (Joliot et al. 2022; Cardon et al. 2023). We quantified the capacity of these peptides to enter into various cell lines known to express different levels and types of heparan sulfates (HS) and chondroitin sulfates (CS) GAGs. We also analyzed by calorimetry (DSC, ITC) and fluorescence spectroscopy, the binary and ternary interactions between heparin (HI), (4S, 6S)CS (CS-E), zwitterionic phosphocholine (PC) model membranes and those peptides. Altogether, our results demonstrate the existence of Ca2+-dependent interactions between CS-E or HI and PC lipid bilayers, the major phospholipid found in animal cell plasma membrane. Importantly, we show that CS-E can act as a Ca2+-dependent bridge with PC membranes that can be exploited by a chimeric CS-E-recognition motif-H3 peptide to bind and cross the membrane lipid bilayer and get access directly to the cytosol of cells. Altogether, this study brings further information uncovering the molecular mechanism of the translocation process of CPPs that implies specific GAGs at the cell-surface. It also shed light on the role of GAGs in the paracrine activity and cell specificity of HPs.