European Biophysics Journal最新文献

Quantitative metabolic profiling of bacterial communities provides biophysical insight into cellular respiration and energy transduction under controlled environmental conditions. The oxygen consumption rate (OCR) represents a direct, integrative readout of respiratory activity and has emerged as a sensitive phenotypic parameter for characterizing microbial physiology across different metabolic states. Here, we present an experimental protocol for measuring bacterial respiration using the Seahorse XFe96 Analyzer (Agilent), a microplate-based platform that enables high temporal resolution measurements of oxygen flux. Although originally developed for eukaryotic cell cultures, we adapted this technology to bacterial systems, allowing the simultaneous analysis of multiple strains in a medium-throughput format. The 96-well configuration, combined with sequential injection of up to four compounds, including metabolic substrates, stressors, and alternative electron acceptors, enables real-time perturbation of respiration and monitoring of dynamic OCR responses. To investigate bacterial respiration under physiologically and environmentally relevant oxygen tensions, OCR measurements were additionally performed under hypoxic conditions (5% O₂) by integrating the Seahorse XFe96 Analyzer with an oxygen-controlled glove box. This approach permits precise control of oxygen availability and extends OCR analysis to low-oxygen regimes relevant to chronic infections and aquatic or engineered environments. Overall, our results establish a robust and versatile biophysical platform for probing bacterial respiratory dynamics and for phenotypic screening applications.

In this review we propose a mechanism by which the antisecretory factor AF may decrease vertigo symptoms in Menière’s disease patients. This review is based on our previously published work and suggests a way of interpretation of the results obtained in view of the clinical effect of AF factor against vertigo caused by Menière’s disease. The Antisecretory Factor (AF) is an endogenous protein regulating water and ion transport across cellular membranes. AF was originally discovered and isolated from rat and pig pituitary gland but later it has been isolated from all mammalian and non-mammalian tissues examined. It has shown positive effects in patients with Mb Crohn, Colitis Ulcerosa and Mastitis with regression of clinical symptoms and inflammatory parameters. AF is also effective in suppressing traumatically induced increases of intra-cranial pressure (ICP). Another interesting activity of this protein is its anti-vertigo action in patients with Menière’s disease. We studied rat cerebellar granule cells in culture and demonstrated increases by AF of the inhibitory current activated by GABA at synapses. This implies strong inhibition of cerebellar granule cells in situ. The connection of cerebellum to other brain areas would finally result in activation of vestibular Deiters’ neurons in the brainstem, controlling antigravity neurons in the spinal cord. These actions may contribute to counteracting vertigo in Menière’s disease.

Proper folding of therapeutic monoclonal antibody (mAb) drugs is key to their efficacy and safety. Circular Dichroism (CD) spectroscopy is well suited for investigating the secondary structure of proteins, but the adequacy of the technique for antibody molecules has been severely impacted by the lack of suitable CD reference sets that include spectra of proteins of similar structure. Here we report on the performance of an expanded reference set that includes CD spectra of 14 mAbs, and uses structures derived from homology models. This enhanced reference set is part of a freely available Python-based software package, SSCalcPy-mAb, that includes the new SP-mAb178 reference set for secondary structure analysis using the SELCON3 method. We propose to employ both the secondary structure analysis results and a generated list of the proteins in the reference set that SELCON3 applies to derive the results that ascertain whether an antibody of interest is well-folded or not.

The inner surface of our airways is lined by a mucous fluid film that is continuously propelled towards the throat. The propulsion of this airway surface liquid is generated by the collectively coordinated oscillatory motion of a myriad of subjacent motile cilia. Inhaled particles are entrapped by the mucus layer and transported towards the pharynx where they are swallowed. Therefore, mucociliary clearance contributes to our airway’s primary defense mechanism by protecting our airways from inhaled particles. We developed a computational high-speed video reflection microscopy (CVRM) system that enables imaging and quantitative characterization of the mucociliary activity – particularly in terms of the cilia-caused dynamic modulation of the mucus surface – in air-liquid interface cell cultures. Nasal epithelial cells from healthy volunteers and people with primary ciliary dyskinesia (PCD) were differentiated at the air-liquid interface, representing a sophisticated model widely used to study the mucociliary clearance mechanism. The cell cultures were imaged by reflection microscopy using a high-speed camera and the videos were quantitatively analyzed using a newly extended version of our Cilialyzer software. The primary aim of this study was to develop and validate CVRM as a quantitative method for characterizing collective mucociliary dynamics under physiological air–liquid interface conditions. We found that the dynamic modulations of the mucus surface closely correspond to the underlying ciliary motion. By using specifically developed video processing methods, we quantitatively characterized the collective mucociliary activity in terms of ten space-time features. This allowed us to distinguish cell cultures derived from people with PCD from those derived from healthy volunteers and demonstrates that CVRM has clear potential for future diagnostic applications in PCD and beyond.

2,4-Dinitrophenol (DNP) is an artificial mitochondrial uncoupler that was used as an anti-obesity treatment until it was withdrawn due to high toxicity, a narrow therapeutic window, and a lack of an antidote. Despite these concerns, DNP’s efficacy surpasses that of many contemporary anti-obesity drugs, sparking renewed interest in its potential use under controlled conditions. However, the development of antidotes for their toxicity and advancement as therapeutic agents has been impeded by a critical gap in our understanding of the precise molecular mechanisms by which DNP and similar uncouplers operate within mitochondria. This review provides a brief historical overview of DNP use and explores various proposed molecular mechanisms of DNP action. It also summarizes recent results obtained using conductance measurements of artificial bilayer membranes, patch clamp of mitoplasts, in silico analysis, and cryo-electron microscopy. Continued integration of the electrophysiological, structural, and computational approaches is expected to clarify the molecular basis of DNP-mediated uncoupling, enabling the rational development of safer uncouplers and mechanism-based antidotes.

Fast and simple detection of diseases at their early stage and clinical monitoring of healing progress directly from body fluids, namely from urine, is a hot topic in modern personalized medicine. Functionalized nanofibers seem to be an ideal solution for the construction of specific and highly selective detection systems and microfluidic tools due to their extremely high ratio between their surface and volume, and also tunable sensitivity. The aim of this study is development of biosensors based on functionalized nanofibers leading to signal enhancement which would enable fluorescence detection and improve diagnostic accuracy in human fluids. Functionalized-PolyAcrylNnitrile (fPAN) membranes were fabricated and analyzed via SEM and fluorescence spectroscopy. Biotin-FITC binding was tested using a routine filtration setup and quantified with a Fluoromax4 spectrometer. Avidin-functionalized membranes showed strong, specific binding at picomolar levels Since the collected urine can be easily collected at volumes of more than 100 mL, and fluorescence measurement performed at a significantly smaller volume than 1 mL, our nanofiber-based system serves as an efficient specific enhancer and increases, thus, the detection limit by more than two orders of magnitude. This is already sufficient, for example, for a reliable fluorescence detection of urine markers in subpicomolar concentrations. Thus, this study describes a novel detection approach and application of fluorescence bionanosensors based on specifically functionalized polyacrylonitrile nanofibers for monitoring of subpicomolar concentrated markers in human fluids. These concentrations allow already monitoring of serious diseases at their early stage, which is crucial for distant and precision medicine.

Sedimentation velocity analytical ultracentrifugation (SV-AUC) has become a workhorse technique for characterization and quantitation of AAV-based therapeutics, providing information on distribution of various AAV capsid species. However, SV-AUC results can be compromised by cell misalignment. In this study, we used an optical alignment system to investigate the combined effects of cell misalignment and rotor speed on the sedimentation behavior and measured distributions of AAV species. Our findings indicate that at high speeds even minor deviations in cell alignment can introduce substantial artifacts, leading to inaccurate quantitation of relative levels of different AAV capsid species by SV-AUC and potentially to misinterpretation of product quality.

Despite its ubiquity in nature, some details of the animal olfactory system remain unclear. One such mystery is the mechanism by which olfactory receptors (ORs) recognize the olfactant molecules they bind to. Some evidence indicates that ORs can distinguish between molecules that differ only in isotopic composition, suggesting that olfactants’ vibrational modes may play a role in their recognition. In 2023, the first experimental structure of a human olfactory receptor—OR51E2—was produced, providing opportunity to shed additional light on this problem computationally. We simulate the infrared spectrum of the olfactant propionate ((text {C}_{2}text {H}_{5}text {COO}^{-})), as well as all its possible deuterations, in the OR51E2 binding site by quantum mechanics/molecular mechanics, with atomic positions taken at 25 time points over a 500 ns molecular dynamics simulation. The protein environment does not change the vibrational spectrum qualitatively, even at physiological temperature. The high-frequency C-H modes are about ({800,}{text {cm}^{-1}}) higher in energy than their deuterated counterparts, while the modes associated with the carboxyl group are almost unaffected by the C/H isotopic substitution. Because (text {C}_{2}text {H}_{5}text {COO}^{-}) binds to OR51E2 primarily via the carboxyl end, this result means that a vibrational component to propionate detection is unlikely.

Amyloid assembly is governed by a balance between intrinsic sequence determinants and environmental cues that modulate nucleation. The RIP homotypic interaction motif (RHIM) of receptor-interacting protein kinase 3 (RIPK3) provides a tractable model to dissect these principles. In solution, amyloid formation strictly requires the conserved VQVG RHIM core tetrad; a deliberately core-disrupted variant (VQVG→AAAA) fails to assemble under aggregation permissive conditions. Here, we use Thioflavin-T fluorescence assays and NMR spectroscopy to show that negatively charged lipidic interfaces unlock a latent amyloidogenic potential in this mutant. These results delineate two separable dimensions of amyloidogenesis, namely a sequence-encoded propensity and an environmental component that can catalyze nucleation by templating molecular proximity and orientation.

DNA-binding proteins from starved cells (Dps) are small multifunctional protein nanocages expressed by prokaryotes under oxidative stress or during starvation, acting as a key bacterial defense mechanism. Dps proteins protect DNA either through direct binding or by scavenging reactive oxygen species precursors. In most Dps homologs studied to date, DNA-binding is mediated by flexible, intrinsically disordered N- or C-terminal extensions. In this study, we investigated the interaction of Deinococcus grandis Dps (DgrDps) and a mutant variant, lacking the first 46 N-terminal residues, with supercoiled plasmid pUC19 using electrophoretic mobility shift assays (EMSA), DNase I protection assays, atomic force microscopy (AFM), and synchrotron radiation circular dichroism (SRCD). DgrDps binds supercoiled pUC19 with an apparent dissociation constant (K_D) of 5.2 ± 0.3 µM, exhibiting positive cooperativity. Our results indicate that DNA binding is primarily mediated by the flexible N-terminal tails of DgrDps. AFM imaging revealed that DgrDps binds to multiple sites on the plasmid, inducing DNA bridging and compaction. Furthermore, the presence of 96 Fe²⁺/dodecamer increased the compaction of these protein-pUC19 complexes. This organization confers physical protection against DNase I digestion. Additionally, SRCD spectroscopy provided insights into the structural features and thermal stability of the DgrDps-DNA complexes.