Search Results (2,465)

The lipoate-protein ligase A (LplA) identified in Escherichia coli K-12 exhibits structural homomeric oligomerization and reversible lower critical solution temperature (LCST)-type phase separation in vitro. In this study, based on the ability of LplA to form condensates, it was utilized as a temperature-sensitive purification tag in the field of protein purification for the first time, and a novel and convenient one-step purification method was established. A universal vector was developed for the fusion expression of LplA and the target protein. The fusion protein forms condensates upon heating, separating from the solution, and redissolves in buffer at lower temperatures, enabling the purification of the target protein from cell lysates. Through exploration of phase separation temperatures, 30 °C was determined to be the optimal purification temperature. Subsequently, three enzymes of different molecular sizes (lipase EstA, endoglucanase BcsZ, and endoglucanase EglS) demonstrated the versatility of this condensate-based purification method. Furthermore, the specific activity and purification efficiency of the purified enzymes were comparable to those of enzymes purified by conventional affinity chromatography. This research contributes to the introduction of condensates into protein purification applications, offering potential support for the large-scale production and purification of functional proteins. Full article

In this study, we combine experimental and computational approaches to elucidate a density functional theory (DFT)-derived mechanism for superoxide scavenging by resveratrol, piceatannol, and oxyresveratrol. Using rotating ring–disk electrode (RRDE) hydrodynamic voltammetry, the superoxide radicals are generated in situ, allowing direct measurement of antioxidant activity. Data show that the catechol-containing piceatannol is approximately four times more active than resveratrol, while resveratrol and oxyresveratrol exhibit similar efficiencies, indicating that the additional 2′-OH group in oxyresveratrol has minimal impact. Vitamin C (ascorbic acid) facilitates scavenging by acting as a proton donor for resveratrol, piceatannol, and 4′-OH oxyresveratrol, but it is unable to deprotonate the 2′OH group of oxyresveratrol. The experimental results suggest a superoxide dismutase (SOD)-like mechanism, obtained from energetically feasible DFT calculations, in which these stilbenes convert two superoxide anions into H₂O₂ and O₂, helped by vitamin C. Mechanistically, the first superoxide is reduced by abstracting a hydroxyl-group hydrogen atom, while the second undergoes oxidation via π–π interaction with the aromatic system, releasing O₂. Notably, resveratrol can be regenerated through a catalytic cycle involving vitamin C. These data underscore the SOD-mimicking properties of dietary polyphenols and suggest a need to reevaluate resveratrol’s clinical utility regardless of its low bioavailability. Full article

In hepatocellular carcinoma (HepG2), aberrant histone modifications are closely linked to long non-coding RNA (lncRNA) expression. However, existing computational models lack physical interpretability at specific promoter coordinates. To address this, we developed a position-specific statistical scoring model based on adjacent and next-adjacent nucleotide frequencies. We trained two independent, position-specific matrices representing increased and decreased modification states across 600 bp promoter windows centered on the true signal summits. Finally, ten-fold cross-validation revealed that significant energy differences between sequences with increased and decreased histone signals enable excellent classification performance. These results indicted a strong correlation between the total energy of local DNA structures and histone modification signal. Full article

Non-thermal millimeter-wave (MMW) irradiation represents a promising non-invasive strategy for cancer therapy, yet its effects in physiologically relevant 3D systems remain poorly defined. Here, we evaluated the biological impact of MMW exposure in 3D non-small-cell lung cancer (NSCLC) spheroids (NCI-H1299, A549) and normal WI-38 fibroblasts under active cooling to suppress bulk heating. We demonstrate that cellular responses are governed primarily by power density (PD), irradiation geometry, and genotype-dependent susceptibility. High-PD pyramidal horn (PH) irradiation (~4.9 mW/cm²) induced rapid apoptosis, metabolic collapse, and near-complete loss of clonogenic survival, whereas lower-PD waveguide (WG) irradiation (~0.6 mW/cm²) produced depth-limited, cumulative cytotoxicity. Surviving cancer cells exhibited robust senescence-associated growth arrest, particularly in p53-deficient NCI-H1299 cells, indicating a dual apoptotic–senescent anti-proliferative response. In contrast, WI-38 fibroblasts showed minimal apoptosis and only transient stress-associated senescence, confirming selective tumor vulnerability. Mechanistic modeling suggests that MMW energy couples to glycan-rich membrane domains, generating localized electromagnetic hotspots that trigger calcium influx, mitochondrial dysfunction, and depth-dependent apoptosis. These findings establish a mechanistic basis for selective, non-thermal MMW-induced cytotoxicity in 3D NSCLC models and support further preclinical development of MMW-based therapeutic strategies. Full article

RelA/SpoT homologue family enzymes participate in controlling the cellular levels of the alarmone (p)ppGpp, thereby activating the stringent response and promoting survival under stress conditions. These proteins contain an N-terminal catalytic domain and a C-terminal regulatory domain. They catalyze both the synthesis of ppGpp/pppGpp from ATP and GDP/GTP and their hydrolysis to GDP/GTP and pyrophosphate. Here, we report the crystal structure of the N-terminal domain of Rel from Streptococcus equisimilis in complex with pppGpp at 3.2 Å resolution. The asymmetric unit contains a dimer with asymmetric ligation: pppGpp occupies only the synthetase site in one monomer, whereas in the other monomer, it is bound in both the hydrolase and synthetase sites. The two monomers exhibit distinct conformational states, with pronounced rearrangements of the flexible loops surrounding the binding pockets, including the α2/α3 and α8/α9 loops that act as steric gates. Molecular dynamics simulations support the dual binding arrangement and reveal additional probable transient binding sites, including a region in the linker between hydrolase and synthetase subdomains. These findings provide a structural framework for understanding how pppGpp binding modulates the opposing catalytic activities of bifunctional Rel enzymes and suggest possible mechanisms for (p)ppGpp-mediated autoregulation. Full article

Crohn’s disease (CD) is a chronic inflammatory bowel disease requiring accurate and timely diagnosis. Current diagnostic tools may be invasive, costly, or insufficiently specific. Surface-enhanced Raman spectroscopy may enable rapid, minimally invasive detection of disease-associated biochemical alterations in serum. This cross-sectional pilot study included age- and sex-matched patients with Crohn’s disease and healthy controls. Serum samples were analyzed using surface-enhanced Raman spectroscopy. Spectral data were preprocessed and analyzed using principal component analysis-linear discriminant analysis and partial least squares-discriminant analysis. Classification performance was evaluated using leave-one-out cross-validation. Variable importance in projection scores was used to identify discriminatory vibrational bands. Fifty-four participants fulfilled the clinical inclusion criteria, while 51 participant-level spectra were retained for final classification analysis. PCA-LDA differentiated CD from healthy controls with a sensitivity of 85.19%, specificity of 91.67%, accuracy of 88.24%, and AUC of 0.881. PLS-DA showed slightly higher performance, with a sensitivity of 88.89%, specificity of 95.83%, accuracy of 92.16%, and AUC of 0.937. Relevant discriminatory bands were observed at 498, 639, 728, 813, 1136, 1205, 1443, 1579, and 1657 cm⁻¹, suggesting alterations in purine metabolism, protein structure, lipid composition, and nucleic acid-associated signals. Serum-based SERS combined with multivariate analysis showed promising ability to distinguish patients with CD from healthy controls in this pilot cohort. Larger multicenter studies are required to validate these findings and assess clinical applicability. Full article

The interaction between proteins and salts in aqueous solutions represents a compelling scientific problem in both biophysical and medical research. One manifestation of the interaction in the bovine serum albumin (BSA)–NaCl–H₂O system is the formation of ordered patterns upon droplet drying. In our experiments, the topographic features of the deposited particles as well as their elemental composition were studied using optical microscopy and scanning electron microscopy (SEM). In this work, we experimentally investigated the stepwise change in the characteristic structures of the precipitate with an increase in the BSA concentration from 0.005 to 35 mg/mL. The formation of discrete BSA–NaCl ring deposits near the droplet edge and around crystallization centers in the interior at BSA concentrations of 0.05–1 mg/mL proved particularly interesting. We demonstrated the sequence of ring structure formation: the process primarily begins with the formation of BSA aggregates arranged in sectors around the circumference, which in turn serve as nucleation sites for NaCl crystallization. We propose a qualitative conceptual–phenomenological interpretation of the observed experimental effects. Concentration-dependent patterns in the emergence and development of other patterns (such as spikes, fractal structures, and chrysanthemum-like formations) were established. These results expand our understanding of protein behavior in aqueous-salt solutions. This can be used in medical diagnostics as biomarkers. The characteristic patterns presented in the work can serve as a useful experimental basis for further studies of the impact of physicochemical factors on proteins and other biopolymers. Full article

In prokaryotes, translation initiation orchestrates protein synthesis through a network of dynamic interactions among the ribosome, mRNA, initiator tRNA^fMet, and initiation factors (IFs). Traditional approaches that rely on radioactive labeling or surface immobilization are hindered by inherent safety risks and methodological constraints. We present a fluorescence-based analytical platform that integrates microscale thermophoresis (MST) as a unified, multiparametric toolkit for comprehensive interrogation of bacterial translation initiation at the molecular level. By systematically applying MST to a panel of fluorescently labeled components—initiator tRNA^fMet, mRNAs, and initiation factors—we quantify assembly pathways and equilibria as initiation progresses from simple bimolecular interactions to higher-order, multicomponent complexes. To broaden the fluorescence toolbox for ribosomal studies, we developed a robust BODIPY-labeling protocol for 70S ribosomes and confirmed preservation of structural integrity and function by nano differential scanning fluorimetry, stopped-flow kinetic assays, and peptide-synthesis activity tests. Our microscale fluorescent system facilitates probing initiation at a variety of steps, since the role of magnesium ions and initiation factors upon 30S initiation complex formation. The same platform can be applied to investigate the effects of different compounds on translation initiation, as demonstrated for a number of antibiotics, aptamers, and antimicrobial peptides. Using this approach, we determined the antibiotic streptomycin dissociation constant for both 30S and 70S ribosomes, which proved identical at 0.3 ± 0.1 μM, and demonstrated the effect of the antimicrobial peptide rumicidin-1 on translation initiation. Offering a cost-effective and high-sensitivity alternative to conventional methods, this approach advances mechanistic understanding of prokaryotic translation and provides a versatile framework for the discovery of novel protein synthesis inhibitors. Full article

Poly(ε-caprolactone) (PCL) is a biodegradable aliphatic polyester with applications in many areas. Lipase B from Moesziomyces antarcticus (CALB, previously known as Candida antarctica lipase B) is a well-characterize enzyme capable of hydrolysing several polyesters. In this study, the codon-optimized gene encoding CALB was cloned into the yeast Yarrowia lipolytica to enhance its natural capabilities toward polyesters biodegradation. PCL films biodegradation was conducted directly in the medium using the engineered yeast at 28 °C. Process optimization employing baffled flasks significantly improved degradation efficiency and reduced time to 24 h. This study showed that the engineered yeast Y. lipolytica is a promising host for polyester biodegradation. Full article

Left ventricular assist device (LVAD) implantation is a life-saving therapy for end-stage heart failure but may compromise immune integrity. Mechanical shear stress and surface-induced innate immune activation can trigger bleeding and thromboembolic complications. While thrombotic mechanisms are well characterized, the associated inflammatory response remains poorly studied. We investigated thromboinflammation in patients with terminal heart failure (n = 8) implanted with the HeartWare ventricular assist device. Blood samples were collected before and immediately after implantation, daily for five days, and three months post-implantation. Ten age- and sex-matched healthy individuals served as controls. Samples were analyzed for a broad panel of thromboinflammatory and cell damage biomarkers. Twenty-eight of 43 biomarkers were significantly elevated (p < 0.05) at patient baseline compared with controls, indicating a pre-existing low-grade inflammatory state prior to LVAD implantation. Complement activation products increased markedly immediately after implantation—C3bc, C3bBbP, and the terminal complement complex C5b-9 rose 2.8-, 8.9-, and 6.6-fold, respectively, compared with baseline (p < 0.0001), but returned toward baseline within 24 h. A similar transient increase was observed for TNF, IL-6, IL-8, IL-10, IFN-γ, MMP-8, MMP-9, tissue factor, and prothrombin fragment 1.2 (p < 0.05). LVAD implantation with cardiopulmonary bypass induces a strong but transient immune response, including robust complement activation. Targeting upstream complement pathways may help attenuate downstream thromboinflammatory processes during the acute post-implantation period. Full article

Antimicrobial photodynamic therapy is a method that utilizes photodynamic inactivation of microorganisms exposed to a photosensitizer irradiated by a specific wavelength, followed by the formation of reactive oxygen species and subsequent oxidative stress. In contrast to antibiotics, which are generally efficient against specific microorganisms, photodynamic inactivation exhibits efficacy against a wide range of bacteria, representing a promising and non-invasive alternative to treating infections caused by pathogens of different origins. This study compares the antibacterial efficacy of five different photosensitizers, including TMPyP, Protoporphyrin IX, PdTPPS₄, Methylene Blue, and ZnPCS₂, against eight representatives of various pathogens, including Gram-negative bacteria Escherichia coli, Pseudomonas aeruginosa, Gram-positive bacteria Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Enterococcus faecium, MRSA and Bacillus subtilis. An optimal irradiation protocol was developed based on growth curve measurements involving double irradiation. To induce the photodynamic effect, we utilized LED emitters with wavelengths of 414 nm and 660 nm, chosen to align with the photophysical properties of the photosensitizers. Additionally, the research included assessments of the radiation’s phototoxicity and the photosensitizers’ dark toxicity against specific microorganisms. The optical properties of the photosensitizers were analyzed using absorption spectrophotometry. The effectiveness of photodynamic inactivation was assessed by determining the minimum inhibitory and bactericidal concentrations. This study aimed to identify the most suitable photosensitizer for clinical application, considering the toxicity of the photosensitizer, the radiant exposure, and its efficacy in photodynamic inactivation. Full article

The protein folding problem has traditionally been defined by two complementary challenges: predicting the three-dimensional structure of a protein from its amino acid sequence and understanding the mechanism by which this structure is attained. While recent advances in artificial intelligence have largely addressed the former, the latter remains unresolved. Early studies showed that many small proteins fold in a cooperative two-state manner, shifting attention toward transition states and energy landscapes. Comparative analyses of protein families further revealed that folding mechanisms are often conserved among proteins sharing the same topology, suggesting a dominant role of structure in shaping folding pathways. However, this framework does not explain when and how a protein commits to a specific topology. Metamorphic proteins, in which highly similar sequences adopt distinct native folds, provide a powerful complementary approach. Studies of these systems show that closely related sequences can follow different folding mechanisms without sharing common intermediates. These findings indicate that folding pathways are determined at very early stages and are encoded within the denatured ensemble through subtle structural and energetic biases. Here, we review the evolution of protein folding studies and propose a unified view in which folding mechanisms are selected early, with the denatured state playing a central role in defining both folding pathways and final topology. Full article

Magnetic resonance techniques have evolved beyond conventional proton-based imaging, enabling access to a broader range of nuclei that provide complementary structural, functional, and molecular information. This review presents a comprehensive overview of multinuclear NMR and MRI in solid and soft materials as well as in biomedical applications, with particular emphasis on ¹H, ¹³C, ³¹P, ²³Na, and ¹⁹F nuclei. Proton-based methods remain the foundation of magnetic resonance due to their high sensitivity and widespread applicability, offering insights into molecular mobility, hydration, and microstructural heterogeneity. In contrast, heteronuclear approaches enable more specific characterization of chemical structure (¹³C), phosphorus-containing functional groups and membranes (³¹P), ionic homeostasis and transport (²³Na), and exogenous tracers with negligible biological background (¹⁹F). Together, these techniques extend magnetic resonance from primarily anatomical imaging toward functional, metabolic, and molecular-level analysis. The review further discusses key hardware aspects, including magnetic field strength and radiofrequency coil design, highlighting the trade-offs between low- and high-field systems and the growing importance of multinuclear coil architectures. For example, because ¹H, ²³Na, ³¹P, and ¹⁹F resonate at different Larmor frequencies, multinuclear experiments require dedicated or multi-tuned RF coils that balance sensitivity, field homogeneity, and decoupling between channels. Mechanisms of contrast generation are examined in detail, distinguishing between endogenous sources—such as water, ions, and metabolites—and exogenous contrast agents, including gadolinium-, manganese-, and fluorine-based compounds, as well as targeted and theranostic platforms. A comparative framework of endogenous and exogenous signals is presented, emphasizing their complementary roles in balancing safety, specificity, and sensitivity. Finally, the opportunities and challenges of multinuclear magnetic resonance are critically evaluated, including limitations in sensitivity, signal-to-noise ratio, data interpretation in heterogeneous systems, and technical complexity. Emerging directions such as ultrahigh-field imaging, advanced RF technologies, hyperpolarization, and artificial intelligence-assisted reconstruction are discussed as key drivers for future development. Overall, multinuclear NMR and MRI represent a powerful and expanding toolbox for probing complex material and biological systems, with the potential to significantly enhance diagnostic capabilities and deepen our understanding of structure–function relationships across multiple scales. Full article

How do ryanodine receptors (RyRs) open simultaneously to trigger the contraction of whole myofibrils within a large skeletal muscle cell? One possible answer is the uniformity of mechanosensitive RyRs, which is mechanically forced by the neighboring environment, including proteins. Here, we review papers addressing this proposed “mechanical sarcoplasmic reticulum (SR) paradigm”. Crystals of the molecular complexes comprising RyR and L-type voltage-gated Ca²⁺ channels were observed at the T-tubule/SR junction in situ using cryo-electron tomography. Observations of the SR vesicles isolated from rabbit and scallop cross-striated muscles using negative staining and transmission electron microscope raised a hypothesis of dynamic rearrangement of the Ca²⁺-ATPase (ATPase) molecules in response to cytoplasmic calcium concentration, as follows: (i) At a low calcium concentration where the ratio of operating ATPase molecules to the total molecules is at a submaximal level, the ATPase molecules form, at least in part, their cylindrical crystals in the SR membrane with the help of ATP; this results in the elongation of the SR vesicles. (ii) High concentrations of calcium, at which the ratio of operating ATPase molecules is maximal, reversibly collapse the ATPase crystals to transform the elongated vesicles into round forms comprising tightly attached crystal patches. These data further lead to the idea that the reversible growth of cylindrical ATPase crystals provides a dynamic crystalline network, which acts as an “SR membrane-endoskeletal motor” to manipulate the SR movement. The possibility of interactions between ATPase crystals and neighboring RyR crystals is also discussed. Full article

Big Bang theories are connected to gravity by force of attraction. Forced lengthening, like eccentric contractions, instigate proprioception as a result of working against gravity. Piezo2, as the principal mechanosensory ion channel responsible for proprioception, is theorized to fine-modulate these anti-gravitational contractions in order to provide system-wide ultrafast postural control. This mechanism may instantaneously emit energy and force through Piezo2 in order to offset gravity by anti-gravity entropic-spring-like stochastic mechanics and it is suggested to be propagated by quantum tunneling of protons (and electrons). However, a Piezo2-initiated wormhole-like mechanism with the contribution of cryptochromes should be considered as part of this ultrafast long-distance non-synaptic neurotransmission, although the quantum gravity concept is short of being unequivocally proven to be unified with quantum theory. The impairment of this theoretical ultrafast signaling is analogous to a Big Bang-like mechanism within a given compartment, or acquired Piezo2 channelopathy, leading to the principal gateway to pathophysiology. Full article