Membrane fluidity properties of lipid-coated polylactic acid nanoparticles.

Lipid coating is considered a versatile strategy to equip nanoparticles (NPs) with a biomimetic surface coating, but the membrane properties of these nanoassemblies remain in many cases insufficiently understood. In this work, we apply C-Laurdan generalized polarization (GP) measurements to probe the temperature-dependent polarity of hybrid membranes consisting of a lipid monolayer adsorbed onto a polylactic acid (PLA) polymer core as function of lipid composition and compare the behavior of the lipid coated NPs (LNPs) with that of liposomes assembled from identical lipid mixtures. The LNPs were generated by nanoprecipitation of the polymer in aqueous solutions containing two types of lipid mixtures: (i) cholesterol, dipalmitoylphosphatidylcholine (DPPC), and the ganglioside GM3, as well as (ii) dioleoylphosphatidylcholine (DOPC), DPPC and GM3. LNPs were found to exhibit more distinct and narrower phase transitions than corresponding liposomes and to retain detectable phase transitions even for cholesterol or DOPC concentrations that yielded no detectable transitions in liposomes. These findings together with higher GP values in the case of the LNPs for temperatures above the phase transition temperature indicate a stabilization of the membrane through the polymer core. LNP binding studies to GM3-recognizing cells indicate that differences in the membrane fluidity affect binding avidity in the investigated model system.

Immediate reuse of patch-clamp pipettes after ultrasonic cleaning.

The patch-clamp technique has revolutionized neurophysiology by allowing to study single neuronal excitability, synaptic connectivity, morphology, and the transcriptomic profile. However, the throughput in recordings is limited because of the manual replacement of patch-pipettes after each attempt which are often also unsuccessful. This has been overcome by automated cleaning the tips in detergent solutions, allowing to reuse the pipette for further recordings. Here, we developed a novel method of automated cleaning by sonicating the tips within the bath solution wherein the cells are placed, reducing the risk of contaminating the bath solution or internal solution of the recording pipette by any detergent and avoiding the necessity of a separate chamber for cleaning. We showed that the patch-pipettes can be used consecutively at least ten times and that the cleaning process does not negatively impact neither the brain slices nor other patched neurons. This method, combined with automated patch-clamp, highly improves the throughput for single and especially multiple recordings.

Effect-Directed Analysis Based on Transthyretin Binding Activity of Per- and Polyfluoroalkyl Substances in a Contaminated Sediment Extract.

Only a fraction of the total number of per- and polyfluoroalkyl substances (PFAS) are monitored on a routine basis using targeted chemical analyses. We report on an approach toward identifying bioactive substances in environmental samples using effect-directed analysis by combining toxicity testing, targeted chemical analyses, and suspect screening. PFAS compete with the thyroid hormone thyroxin (T4 ) for binding to its distributor protein transthyretin (TTR). Therefore, a TTR-binding bioassay was used to prioritize unknown features for chemical identification in a PFAS-contaminated sediment sample collected downstream of a factory producing PFAS-coated paper. First, the TTR-binding potencies of 31 analytical PFAS standards were determined. Potencies varied between PFAS depending on carbon chain length, functional group, and, for precursors to perfluoroalkyl sulfonic acids (PFSA), the size or number of atoms in the group(s) attached to the nitrogen. The most potent PFAS were the seven- and eight-carbon PFSA, perfluoroheptane sulfonic acid (PFHpS) and perfluorooctane sulfonic acid (PFOS), and the eight-carbon perfluoroalkyl carboxylic acid (PFCA), perfluorooctanoic acid (PFOA), which showed approximately four- and five-times weaker potencies, respectively, compared with the native ligand T4 . For some of the other PFAS tested, TTR-binding potencies were weak or not observed at all. For the environmental sediment sample, not all of the bioactivity observed in the TTR-binding assay could be assigned to the PFAS quantified using targeted chemical analyses. Therefore, suspect screening was applied to the retention times corresponding to observed TTR binding, and five candidates were identified. Targeted analyses showed that the sediment was dominated by the di-substituted phosphate ester of N-ethyl perfluorooctane sulfonamido ethanol (SAmPAP diester), whereas it was not bioactive in the assay. SAmPAP diester has the potential for (bio)transformation into smaller PFAS, including PFOS. Therefore, when it comes to TTR binding, the hazard associated with this substance is likely through (bio)transformation into more potent transformation products. Environ Toxicol Chem 2024;43:245-258. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Roadmap on optical sensors.

Optical sensors and sensing technologies are playing a more and more important role in our modern world. From micro-probes to large devices used in such diverse areas like medical diagnosis, defence, monitoring of industrial and environmental conditions, optics can be used in a variety of ways to achieve compact, low cost, stand-off sensing with extreme sensitivity and selectivity. Actually, the challenges to the design and functioning of an optical sensor for a particular application requires intimate knowledge of the optical, material, and environmental properties that can affect its performance. This roadmap on optical sensors addresses different technologies and application areas. It is constituted by twelve contributions authored by world-leading experts, providing insight into the current state-of-the-art and the challenges their respective fields face. Two articles address the area of optical fibre sensors, encompassing both conventional and specialty optical fibres. Several other articles are dedicated to laser-based sensors, micro- and nano-engineered sensors, whispering-gallery mode and plasmonic sensors. The use of optical sensors in chemical, biological and biomedical areas is discussed in some other papers. Different approaches required to satisfy applications at visible, infrared and THz spectral regions are also discussed.

Electrophysiological properties of layer 2/3 pyramidal neurons in the primary visual cortex of a retinitis pigmentosa mouse model (rd10).

Retinal degeneration is one of the main causes of visual impairment and blindness. One group of retinal degenerative diseases, leading to the loss of photoreceptors, is collectively termed retinitis pigmentosa. In this group of diseases, the remaining retina is largely spared from initial cell death making retinal ganglion cells an interesting target for vision restoration methods. However, it is unknown how downstream brain areas, in particular the visual cortex, are affected by the progression of blindness. Visual deprivation studies have shown dramatic changes in the electrophysiological properties of visual cortex neurons, but changes on a cellular level in retinitis pigmentosa have not been investigated yet. Therefore, we used the rd10 mouse model to perform patch-clamp recordings of pyramidal neurons in layer 2/3 of the primary visual cortex to screen for potential changes in electrophysiological properties resulting from retinal degeneration. Compared to wild-type C57BL/6 mice, we only found an increase in intrinsic excitability around the time point of maximal retinal degeneration. In addition, we saw an increase in the current amplitude of spontaneous putative inhibitory events after a longer progression of retinal degeneration. However, we did not observe a long-lasting shift in excitability after prolonged retinal degeneration. Together, our results provide evidence of an intact visual cortex with promising potential for future therapeutic strategies to restore vision.

Prevalent bee venom genes evolved before the aculeate stinger and eusociality.

BACKGROUND: Venoms, which have evolved numerous times in animals, are ideal models of convergent trait evolution. However, detailed genomic studies of toxin-encoding genes exist for only a few animal groups. The hyper-diverse hymenopteran insects are the most speciose venomous clade, but investigation of the origin of their venom genes has been largely neglected. RESULTS: Utilizing a combination of genomic and proteo-transcriptomic data, we investigated the origin of 11 toxin genes in 29 published and 3 new hymenopteran genomes and compiled an up-to-date list of prevalent bee venom proteins. Observed patterns indicate that bee venom genes predominantly originate through single gene co-option with gene duplication contributing to subsequent diversification. CONCLUSIONS: Most Hymenoptera venom genes are shared by all members of the clade and only melittin and the new venom protein family anthophilin1 appear unique to the bee lineage. Most venom proteins thus predate the mega-radiation of hymenopterans and the evolution of the aculeate stinger.

Survivin prevents the polycomb repressor complex 2 from methylating histone 3 lysine 27.

This study investigates the role of survivin in epigenetic control of gene transcription through interaction with the polycomb repressive complex 2 (PRC2). PRC2 is responsible for silencing gene expression by trimethylating lysine 27 on histone 3. We observed differential expression of PRC2 subunits in CD4+ T cells with varying levels of survivin expression, and ChIP-seq results indicated that survivin colocalizes with PRC2 along DNA. Inhibition of survivin resulted in a significant increase in H3K27 trimethylation, implying that survivin prevents PRC2 from functioning. Peptide microarray showed that survivin interacts with peptides from PRC2 subunits, and machine learning revealed that amino acid composition contains relevant information for predicting survivin interaction. NMR and BLI experiments supported the interaction of survivin with PRC2 subunit EZH2. Finally, protein-protein docking revealed that the survivin-EZH2 interaction interface overlaps with catalytic residues of EZH2, potentially inhibiting its H3K27 methylation activity. These findings suggest that survivin inhibits PRC2 function.

Structure and dynamics of the mitochondrial DNA-compaction factor Abf2 from S. cerevisiae.

Mitochondria are essential organelles that produce most of the energy via the oxidative phosphorylation (OXPHOS) system in all eukaryotic cells. Several essential subunits of the OXPHOS system are encoded by the mitochondrial genome (mtDNA) despite its small size. Defects in mtDNA maintenance and expression can lead to severe OXPHOS deficiencies and are amongst the most common causes of mitochondrial disease. The mtDNA is packaged as nucleoprotein structures, referred to as nucleoids. The conserved mitochondrial proteins, ARS-binding factor 2 (Abf2) in the budding yeast Saccharomyces cerevisiae (S. cerevisiae) and mitochondrial transcription factor A (TFAM) in mammals, are nucleoid associated proteins (NAPs) acting as condensing factors needed for packaging and maintenance of the mtDNA. Interestingly, gene knockout of Abf2 leads, in yeast, to the loss of mtDNA and respiratory growth, providing evidence for its crucial role. On a structural level, the condensing factors usually contain two DNA binding domains called high-mobility group boxes (HMG boxes). The co-operating mechanical activities of these domains are crucial in establishing the nucleoid architecture by bending the DNA strands. Here we used advanced solution NMR spectroscopy methods to characterize the dynamical properties of Abf2 together with its interaction with DNA. We find that the two HMG-domains react notably different to external cues like temperature and salt, indicating distinct functional properties. Biophysical characterizations show the pronounced difference of these domains upon DNA-binding, suggesting a refined picture of the Abf2 functional cycle.

Towards a realistic prediction of catalyst durability from liquid half-cell tests.

Liquid half-cell measurements provide a convenient laboratory method for determining relevant parameters of electro-catalysts applied in e.g. polymer electrolyte membrane fuel cells. While these measurements may be effective in certain contexts, their applicability to real-world systems, such as single-cells in a membrane electrode assembly (MEA) configuration, is not always clear. This is particularly true when assessing the stability of these systems through accelerated stress tests (ASTs). Due to different electrode compositions and operating conditions, nanoscale degradation proceeds differently. Nevertheless, given the high demands of MEA measurements in terms of time, testing equipment complexity, and amount of catalyst material, application-relevant predictions of catalyst durability from liquid half-cell tests are highly desirable. This study combines electrochemical and nanoparticle analysis based on transmission electron microscopy to conduct a typical voltage cycling AST for rotating disc electrode (RDE) measurements, showing that the loss of the electrochemically active surface area (ECSA) of the used Pt/Vulcan catalyst is strongly enhanced at 80 °C compared to room temperature, which goes along with increased nanoparticle coarsening. Additionally, a high ionomer/carbon mass ratio (I/C = 0.7) accelerates the ECSA loss, and further investigations of its influence suggest a combination of several factors, including the high local proton concentration and the presence of adsorbing anions. At the same temperature (80 °C) and I/C ratio (0.7), the ECSA loss vs. AST cycle number of the Pt/Vulcan catalyst is essentially identical for a voltage cycling AST conducted in either an RDE half-cell or an MEA configuration, suggesting that liquid electrolyte half-cell based ASTs can provide application-relevant results. Thus, our study points out a way for predicting the stability of electro-catalysts in MEAs based on RDE experiments that require less specialized equipment and only µg-quantities of catalysts.

EarGait: Estimation of Temporal Gait Parameters from Hearing Aid Integrated Inertial Sensors.

Wearable sensors are able to monitor physical health in a home environment and detect changes in gait patterns over time. To ensure long-term user engagement, wearable sensors need to be seamlessly integrated into the user's daily life, such as hearing aids or earbuds. Therefore, we present EarGait, an open-source Python toolbox for gait analysis using inertial sensors integrated into hearing aids. This work contributes a validation for gait event detection algorithms and the estimation of temporal parameters using ear-worn sensors. We perform a comparative analysis of two algorithms based on acceleration data and propose a modified version of one of the algorithms. We conducted a study with healthy young and elderly participants to record walking data using the hearing aid's integrated sensors and an optical motion capture system as a reference. All algorithms were able to detect gait events (initial and terminal contacts), and the improved algorithm performed best, detecting 99.8% of initial contacts and obtaining a mean stride time error of 12 ± 32 ms. The existing algorithms faced challenges in determining the laterality of gait events. To address this limitation, we propose modifications that enhance the determination of the step laterality (ipsi- or contralateral), resulting in a 50% reduction in stride time error. Moreover, the improved version is shown to be robust to different study populations and sampling frequencies but is sensitive to walking speed. This work establishes a solid foundation for a comprehensive gait analysis system integrated into hearing aids that will facilitate continuous and long-term home monitoring.

Thumb-domain dynamics modulate the functional repertoire of DNA-Polymerase IV (DinB).

In order to cope with the risk of stress-induced mutagenesis, cells in all kingdoms of life employ Y-family DNA polymerases to resolve resulting DNA lesions and thus maintaining the integrity of the genome. In Escherichia coli, the DNA polymerase IV, or DinB, plays this crucial role in coping with these type of mutations via the so-called translesion DNA synthesis. Despite the availability of several high-resolution crystal structures, important aspects of the functional repertoire of DinB remain elusive. In this study, we use advanced solution NMR spectroscopy methods in combination with biophysical characterization to elucidate the crucial role of the Thumb domain within DinB's functional cycle. We find that the inherent dynamics of this domain guide the recognition of double-stranded (ds) DNA buried within the interior of the DinB domain arrangement and trigger allosteric signals through the DinB protein. Subsequently, we characterized the RNA polymerase interaction with DinB, revealing an extended outside surface of DinB and thus not mutually excluding the DNA interaction. Altogether the obtained results lead to a refined model of the functional repertoire of DinB within the translesion DNA synthesis pathway.

Two-Color iSCAT Imaging of Ag Nanoparticles Resolves Size and Ambient Refractive Index Changes.

The ability to discern noble metal nanoparticles (NPs) with different sizes and in ambient media with different refractive indices has important applications in imaging and sensing. Here a two-color (405 nm, 445 nm) interferometric scattering (iSCAT) detection scheme is applied to characterize the wavelength-dependent iSCAT contrast of Ag NPs with nominal diameters of 10, 20, 40, and 60 nm and to distinguish between NPs of different sizes. The iSCAT contrast also depends on the ambient refractive index and the relative iSCAT contrast on both channels revealed a spectral red-shift for 40 and 60 nm Ag NPs when the ambient refractive index was increased from n = 1.3892 to n = 1.4328. With the selected wavelength channels, the spectral resolution of the two-color imaging strategy was, however, insufficient to resolve spectral shifts induced by refractive index changes for 10 and 20 nm Ag NPs.

Disulfide-Bond-Induced Structural Frustration and Dynamic Disorder in a Peroxiredoxin from MAS NMR.

Disulfide bond formation is fundamentally important for protein structure and constitutes a key mechanism by which cells regulate the intracellular oxidation state. Peroxiredoxins (PRDXs) eliminate reactive oxygen species such as hydrogen peroxide through a catalytic cycle of Cys oxidation and reduction. Additionally, upon Cys oxidation PRDXs undergo extensive conformational rearrangements that may underlie their presently structurally poorly defined functions as molecular chaperones. Rearrangements include high molecular-weight oligomerization, the dynamics of which are, however, poorly understood, as is the impact of disulfide bond formation on these properties. Here we show that formation of disulfide bonds along the catalytic cycle induces extensive µs time scale dynamics, as monitored by magic-angle spinning NMR of the 216 kDa-large Tsa1 decameric assembly and solution-NMR of a designed dimeric mutant. We ascribe the conformational dynamics to structural frustration, resulting from conflicts between the disulfide-constrained reduction of mobility and the desire to fulfill other favorable contacts.

Occurrence and tissue distribution of 33 legacy and novel per- and polyfluoroalkyl substances (PFASs) in Baikal seals (Phoca sibirica).

Per- and polyfluoroalkyl substances (PFASs) are bioaccumulative and associated with adverse effects in both wildlife and humans. The occurrence of 33 PFASs was assessed in the plasma, liver, blubber, and brain of 18 Baikal seals (Phoca sibirica) (16 pups and 2 adult females) from Lake Baikal, Russia (in 2011). Of the 33 congeners analysed for: perfluorooctanosulfonic acid (PFOS), 7 long chain perfluoroalkyl carboxylic acids (C8-C14 PFCAs) and 1 branched PFCA (perfluoro-3,7-dimethyloctanoic acid; P37DMOA) were most frequently detected. The PFASs in plasma and liver with the highest median concentrations were legacy congeners: perfluoroundecanoic acid (PFUnA; plasma: 11.2 ng/g w.w.; liver: 7.36 ng/g w.w.), PFOS (plasma: 8.67 ng/g w.w.; liver: 9.86 ng/g w.w.), perfluorodecanoic acid (PFDA; plasma: 5.13 ng/g w.w.; liver: 6.69 ng/g w.w.), perfluorononanoic acid (PFNA; plasma: 4.65 ng/g w.w.; liver: 5.83 ng/g w.w.) and perfluorotridecanoic acid (PFTriDA; plasma: 4.29 ng/g w.w.; liver: 2.55 ng/g w.w.). PFASs were detected in the brain of Baikal seals, indicating that PFASs cross through the blood-brain barrier. In blubber, the majority of PFASs were detected in low abundance and concentrations. In contrast to legacy PFASs, novel congeners (e.g., Gen X) were either detected infrequently or not found in Baikal seals. The worldwide occurrence of PFASs in pinnipeds was compared and lower median concentrations of PFOS were observed in Baikal seals relative to other pinnipeds. Conversely, similar concentrations of long chain PFCAs were found in Baikal seals compared to other pinnipeds. Furthermore, human exposure was assessed by estimating weekly intakes (EWI) of PFASs through Baikal seal consumption. Although PFASs concentrations were comparatively low relative to other pinnipeds, consumption of Baikal seal could exceed current regulatory guidelines.

Testosterone and persistent organic pollutants in East Greenland male polar bears (Ursus maritimus).

Legacy persistent organic pollutants (POPs) such as polychlorinated biphenyls (PCBs) are chemicals that undergo long-range transport to the Arctic. These chemicals possess endocrine disruptive properties raising concerns for development and reproduction. Here, we report the relationship between concentrations of testosterone (T) and persistent organic pollutant (POPs) in 40 East Greenland male polar bears (Ursus maritimus) sampled during January to September 1999-2001. The mean ± standard concentrations of blood T were 0.31 ± 0.49 (mean ± SD) ng/mL in juveniles/subadults (n = 22) and 3.58 ± 7.45 ng/mL in adults (n = 18). The ∑POP concentrations (mean ± SD) in adipose tissue were 8139 ± 2990 ng/g lipid weight (lw) in juveniles/subadults and 11,037 ± 3950 ng/g lw in adult males, respectively, of which Σpolychlorinated biphenyls (ΣPCBs) were found in highest concentrations. The variation in T concentrations explained by sampling date (season), biometrics and adipose tissue POP concentrations was explored using redundancy analysis (RDA). The results showed that age, body length, and adipose lipid content in adult males contributed (p = 0.02) to the variation in POP concentrations. However, although some significant relationships between individual organochlorine contaminants and T concentrations in both juveniles/subadults and adult polar bears were identified, no significant relationships (p = 0.32) between T and POP concentrations were identified by the RDAs. Our results suggest that confounders such as biometrics and reproductive status may mask the endocrine disruptive effects that POPs have on blood T levels in male polar bears, demonstrating why it can be difficult to detect effects on wildlife populations.

Preparation of Protein-Enriched Outer Membrane Vesicles from Escherichia Coli for In Situ Structural Biology of Outer Membrane Proteins.

Bacterial outer membrane vesicles (OMVs) can be selectively enriched with one or more outer membrane proteins to allow the biophysical characterization of these membrane proteins embedded in the native cellular environment. Unlike reconstituted artificial membrane environments, OMVs maintain the native lipid composition as well as the lipid asymmetry of bacterial outer membranes. Here, we describe in detail the steps necessary to prepare OMVs, which contain high levels of a designated protein of interest, and which are of sufficient homogeneity and purity to perform biophysical characterizations using high-resolution methods such as atomic force microscopy, electron microscopy, or single-molecule force spectroscopy.

Nanoplastics Weathering and Polycyclic Aromatic Hydrocarbon Mobilization.

Despite increasing efforts to recycle plastic materials, large quantities of plastics waste continue to accumulate in the oceans. Persistent mechanical and photochemical degradation of plastics in the oceans yields micro- and nanoscale plastic particles, which represent potential vectors for mobilizing hydrophobic carcinogens in an aqueous milieu. Yet, the fate and potential threats associated with plastics remain largely unexplored. Herein, we apply an accelerated weathering protocol to consumer plastics to characterize the effect of photochemical weathering on the size, morphology, and chemical composition of nanoplastics under defined conditions and validate that the photochemical degradation is consistent with plastics harvested from the Pacific Ocean. Machine learning algorithms trained with accelerated weathering data successfully classify weathered plastics from nature. We demonstrate that photodegradation of poly(ethylene terephthalate) (PET)-containing plastics produces enough CO2 to induce a mineralization process that results in the deposition of CaCO3 on nanoplastics. Finally, we determine that despite UV-radiation induced photochemical degradation and mineral deposition, nanoplastics retain their ability to sorb, mobilize, and increase bioaccessibility of polycyclic aromatic hydrocarbons (PAHs) in water and under simulated physiological gastric and intestinal conditions.

Restricting genomic actions of innate immune mediators on fetal hematopoietic progenitor cells.

Innate immune signaling protects against pathogens, controls hematopoietic development, and functions in oncogenesis, yet the relationship between these mechanisms is undefined. Downregulating the GATA2 transcription factor in fetal hematopoietic progenitor cells upregulates genes encoding innate immune regulators, increases Interferon-γ (IFNγ) signaling, and disrupts differentiation. We demonstrate that deletion of an enhancer that confers GATA2 expression in fetal progenitors elevated Toll-like receptor (TLR) TLR1/2 and TLR2/6 expression and signaling. Rescue by expressing GATA2 downregulated elevated TLR signaling. IFNγ amplified TLR1/2 and TLR2/6 signaling in GATA2-deficient progenitors, synergistically activating cytokine/chemokine genes and elevating cytokine/chemokine production in myeloid cell progeny. Genomic analysis of how innate immune signaling remodels the GATA2-deficient progenitor transcriptome revealed hypersensitive responses at innate immune genes harboring motifs for signal-dependent transcription factors and factors not linked to these mechanisms. As GATA2 establishes a transcriptome that constrains innate immune signaling, insufficient GATA2 renders fetal progenitor cells hypersensitive to innate immune signaling.