The permeation of most antibiotics through the outer membrane of Gram-negative bacteria occurs through porin channels. To design drugs with increased activity against Gram-negative bacteria in the face of the antibiotic resistance crisis, the strict constraints on the physicochemical properties of the permeants imposed by these channels must be better understood. Here we show that a combination of high-resolution electrophysiology, new noise-filtering analysis protocols and atomistic biomolecular simulations reveals weak binding events between the β-lactam antibiotic ampicillin and the porin PorB from the pathogenic bacterium Neisseria meningitidis. In particular, an asymmetry often seen in the electrophysiological characteristics of ligand-bound channels is utilised to characterise the binding site and molecular interactions in detail, based on the principles of electro-osmotic flow through the channel. Our results provide a rationale for the determinants that govern the binding and permeation of zwitterionic antibiotics in porin channels.

The new di- and tetranuclear Fe(III) μ-oxido complexes [Fe4(μ-O)4(PTEBIA)4](CF3SO3)4(CH3CN)2] (1a), [Fe2(μ-O)Cl2(PTEBIA)2](CF3SO3)2 (1b), and [Fe2(μ-O)(HCOO)2(PTEBIA)2](ClO4)2 (MeOH) (2) were prepared from the sulfur-containing ligand (2-((2,4-dimethylphenyl)thio)-N,N-bis ((1-methyl-benzimidazol-2-yl)methyl)ethanamine (PTEBIA). The tetrairon complex 1a features four μ-oxido bridges, while in dinuclear 1b, the sulfur moiety of the ligand occupies one of the six coordination sites of each Fe(III) ion with a long Fe-S distance of 2.814(6) Å. In 2, two Fe(III) centers are bridged by one oxido and two formate units, the latter likely formed by methanol oxidation. Complexes 1a and 1b show broad sulfur-to-iron charge transfer bands around 400-430 nm at room temperature, consistent with mononuclear structures featuring Fe-S interactions. In contrast, acetonitrile solutions of 2 display a sulfur-to-iron charge transfer band only at low temperature (228 K) upon addition of H2O2/CH3COOH, with an absorption maximum at 410 nm. Homogeneous oxidative catalytic activity was observed for 1a and 1b using H2O2 as oxidant, but with low product selectivity. High valent iron-oxo intermediates could not be detected by UV-vis spectroscopy or ESI mass spectrometry. Rather, evidence suggest preferential ligand oxidation, in line with the relatively low selectivity and catalytic activity observed in the reactions.

The formation of neutrophil extracellular traps (NETs) is an immune defense mechanism of neutrophilic granulocytes. Moreover, it is also involved in the pathogenesis of autoimmune, inflammatory, and neoplastic diseases. For that reason, the process of NET formation (NETosis) is subject of intense ongoing research. In vitro approaches to quantify NET formation are commonly used and involve neutrophil stimulation with various activators such as phorbol 12-myristate 13-acetate (PMA), lipopolysaccharides (LPS), or calcium ionophores (CaI). However, the experimental conditions of these experiments, particularly the media and media supplements employed by different research groups, vary considerably, rendering comparisons of results difficult. Here, we present the first standardized investigation of the influence of different media supplements on NET formation in vitro. The addition of heat-inactivated (hi) fetal calf serum (FCS), 0.5% human serum albumin (HSA), or 0.5% bovine serum albumin (BSA) efficiently prevented NET formation of human neutrophils following stimulation with LPS and CaI, but not after stimulation with PMA. Thus, serum components such as HSA, BSA and hiFCS (at concentrations typically found in the literature) inhibit NET formation to different degrees, depending on the NETosis inducer used. In contrast, in murine neutrophils, NETosis was inhibited by FCS and BSA, regardless of the inducer employed. This shows that mouse and human neutrophils have different susceptibilities toward the inhibition of NETosis by albumin or serum components. Furthermore, we provide experimental evidence that albumin inhibits NETosis by scavenging activators such as LPS. We also put our results into the context of media supplements most commonly used in NET research. In experiments with human neutrophils, either FCS (0.5-10%), heat-inactivated (hiFCS, 0.1-10%) or human serum albumin (HSA, 0.05-2%) was commonly added to the medium. For murine neutrophils, serum-free medium was used in most cases for stimulation with LPS and CaI, reflecting the different sensitivities of human and murine neutrophils to media supplements. Thus, the choice of media supplements greatly determines the outcome of experiments on NET-formation, which must be taken into account in NETosis research.

Neutrophilic granulocytes are able to release their own DNA as neutrophil extracellular traps (NETs) to capture and eliminate pathogens. DNA expulsion (NETosis) has also been documented for other cells and organisms, thus highlighting the evolutionary conservation of this process. Moreover, dysregulated NETosis has been implicated in many diseases, including cancer and inflammatory disorders. During NETosis, neutrophils undergo dynamic and dramatic alterations of their cellular as well as sub-cellular morphology whose biophysical basis is poorly understood. Here we investigate NETosis in real-time on the single-cell level using fluorescence and atomic force microscopy. Our results show that NETosis is highly organized into three distinct phases with a clear point of no return defined by chromatin status. Entropic chromatin swelling is the major physical driving force that causes cell morphology changes and the rupture of both nuclear envelope and plasma membrane. Through its material properties, chromatin thus directly orchestrates this complex biological process.

We report an efficient synthesis route for the formation of gold/silver-core–PE-shell nanohybrids in a simple self-assembly approach using PE with strong aurophilicity and argentophilicity, via thiol- and trithiocarbonate terminated moieties. This united the unique properties of polyethylene (PE) with gold and silver nanoparticles, using the well-defined end-group design of PE. These nanocomposites showed a similar solubility as PE, as confirmed by dynamic light scattering, and could be fully incorporated into a polyethylene matrix with different particle contents, as visualized by transmission electron microscopy. Using UV/vis-spectroscopy, we observed reversible, thermoresponsive aggregation/deaggregation properties in the nanohybrids, validating the strong and effective anchoring of PE on gold/silver surfaces.

Reduction of the pincer complex [OsIIICl2(PNP)] (PNP = N(CHCHPtBu2)2) affords the isolation and full characterization of an osmium(II) complex with square-planar coordination geometry, i.e. [OsIICl(PNP)]. Spectroscopic, structural and magnetic data in combination with multireference computations indicate strong temperature independent paramagnetism, which arises from an energetically well separated ground state that mixes with excited states through spin-orbit coupling.