protein folding

Analytical validation of a multiplexed peptide microarray for multi-antigen Epstein–Barr virus serological profiling across diverse clinical specimens

Background Epstein–Barr virus (EBV) infects more than 95% of adults and is associated with outcomes ranging from asymptomatic latency to infectious mononucleosis (IM), malignancies, and multiple sclerosis (MS). Conventional ELISA-based EBV serology is often restricted to a small number of soluble, whole-protein antigens and does not preserve information regarding the specific linear regions of antigen recognition that contribute to the overall antibody response. Multiplexed peptide microarrays address this limitation, but their use in clinical practice requires characterization of their analytical performance and behavior across the diverse conditions encountered in practical use. Methods Here we report the analytical validation of a 108-peptide microarray spanning nine EBV proteins, applied to 329 specimens collected as serum, EDTA plasma, and Streck cell-free DNA (cfDNA) blood collection tubes. Results Within-block and between-slide reproducibility, detection thresholds, and minimum detectable fold-change were consistent with established peptide-array benchmarks, and composite antigen scores correlated with ELISA assays for EBNA-1, VCA-p18, and EA-D. IgG signals were stable across collection matrices, whereas IgM was selectively attenuated in Streck plasma, indicating that fixative-containing tubes should be approached cautiously when IgM is the readout of interest. When applied to IM, MS, and non-MS donors, the peptide array consistently detected broad EBV humoral reactivity. In this proof-of-concept disease-cohort analysis, the array recapitulated biologically relevant EBNA-1 C-terminal reactivity patterns previously associated with MS-related molecular mimicry, but array-wide EBV reactivity did not yield a simple MS-discriminating signature. Although EBV reactivity has been reported to distinguish MS from non-MS cohorts, only a very small set of EBV epitopes was cohort-associated in our data, indicating that platform-level seropositivity reflects EBV exposure rather than an obvious MS-specific signal. Conclusion These findings support the platform as an analytically characterized, peptide-level EBV serology tool for translational research.

The Use of Fullerenol as a Seed Treatment to Maintain the Intensity of Physiological and Biochemical Processes of Wheat in Control and Low-Temperature Conditions

It is of utmost importance to investigate the effect of carbon-based nanostructures on plants in order to fully unlock their potential for enhancing productivity and stress tolerance. In this study, a comprehensive assessment of the effect of pre-sowing treatment of wheat seeds with fullerenol (PHF [C60(OH)24], 0.1 mg/L) solution on a number of physiological and biochemical parameters in seedlings under optimal and low temperature (LT, 4 °C, 5 days) conditions was carried out. Seedlings primed with PHF differed from control variant in accelerated growth (by 27–34%), increased biomass (by 10%), larger area of chloroplasts and mesophyll cells (by 40%), more intensive photosynthesis (1.5-fold higher), increased content of chlorophyll a (by 11%), proteins (by 28%) and ascorbic acid, reduced level of MDA and H2O2, decreased SOD activity (by 45%) and a higher level of COR-genes (WCOR15, WCOR726) transcription (p < 0.05). Under LT conditions, PHF nanopriming enhanced photosynthesis intensity by increasing chloroplast area, content of chlorophyll b (by 22%) and proportion of chlorophyll in LHC (by 11%), as well as increasing the expression of RBCS (fourfold higher), content of proline, and COR-gene expression (p < 0.05). All these changes are adaptive and expand the adaptive potential of plants. It is concluded that nanopriming with PHF is an active metabolic modulator that reduces ROS generation and enhances cold acclimation of wheat.

Systematic In Silico Screening of Nigerian Medicinal Plant Phytoconstituents Against Six Breast Cancer-Associated Proteins

Flexible Catechol-Conjugated Chitosan/Gelatin Bilayer Patches for Topical Hemostasis in Laparoscopic Surgery

Abstract Recently, mussel-inspired catechol-conjugated chitosan (CHI–C)-based topical hemostats have been developed to arrest intraoperative bleeding in patients with coagulopathy through interactions between CHI–C and blood proteins. Pseudo-active blood coagulation, defined as material-assisted clot formation through CHI–C–blood protein complexation without exogenous coagulation proteins, is achieved using CHI–C/gelatin (CHI–C/Geln) bilayer structures. However, these bilayer hemostats still have limitations in laparoscopic surgical settings owing to their stiffness, which can impair trocar deliverability. In this study, we optimized CHI–C/Geln bilayer patches for laparoscopic surgical application by controlling patch thickness through compression. We prepared three types of CHI–C/Geln patches with thicknesses of 1, 3, and 5 mm to compare their tissue adhesiveness, mechanical properties, trocar deliverability, and hemostatic capability. The CHI–C/Geln patches with thicknesses of 1 and 3 mm could pass through 10–12 mm trocars without fragmentation during rolling or repeated folding. In addition, the 3-mm-thick CHI–C/Geln patches exhibited effective tissue adhesiveness, bursting pressure, and hemostatic ability. Thus, these flexible adhesive patches could serve as topical hemostats that function effectively in laparoscopic surgery.

Rethinking Alzheimer's: Why Neurodegeneration Begins at the Physical Boundary, Not with Protein Plaques

Decades of clinical trials targeting amyloid-beta plaques and tau tangles have yielded minimal therapeutic reversal in Alzheimer’s disease. Recent structural biology demonstrates that by the time macroscopic protein aggregates appear, the neuron’s fundamental information-processing highway—the microtubule lattice—has already collapsed. This paper examines the upstream biophysical origin of this structural failure. Microtubules operate as electrodynamic waveguides whose stability strictly depends on continuous mitochondrial ATP perfusion, intracellular electrical polarization, and microvascular thermoregulation. When these biophysical parameters fall out of coherence, tubulin polymers lose their conformational stability, leaving binding sites vulnerable to competitive displacement by aberrant protein conformers. We present evidence that the primary trigger of this neurovascular breakdown does not originate spontaneously within cerebral tissue, but is driven by persistent biophysical impedance at the body's immediate physical boundary. Incompatible dental restorations (alloys, composites, cements), craniofacial contact items (eyeglass frames, ocular lenses, bioincompatible headwear), and ambient dielectric interference induce chronic galvanic distortion and microvascular resistance. This chronic impedance forces cerebral capillary hypoperfusion, impairs convective thermal dissipation required for proper protein folding, and rapidly depletes neuronal mitochondrial energy reserves. Alzheimer’s disease is repositioned as a downstream thermodynamic consequence of chronic boundary impedance rather than an insurmountable defect of cellular aging. Therapeutic implications for restoring cerebral microcirculation and bioelectric baseline conditions are discussed.

A Truncated Serpin D1 Variant Regulates Adseverin-Dependent Actin Severing and Is Required for Osteoclastogenesis

Adseverin is a Ca2+-dependent, actin severing protein that promotes fusion of osteoclast precursors in osteoclast formation. Currently it is not understood how actin severing activity is regulated in osteoclastogenesis. Mass spectrometry of adseverin immunoprecipitates from osteoclasts showed that adseverin associated with Serpin D1. Purified Serpin D1 interacted with adseverin in vitro in a Ca2+-dependent manner. Ca2+ increased actin severing by ~2-fold. Cells that differentiated into osteoclasts expressed an intracellular, truncated Serpin D1 isoform that lacked exons 1 and 2, and part of exon 3. This truncated Serpin D1 isoform was distinct from the secreted, full-length Serpin D1 protein that inhibits thrombin activity. The expression of the truncated Serpin D1 in RAW264.7 cells was further enhanced by TNF-α during RANKL-induced osteoclastogenesis. CRISPR/Cas9 targeting of exon 3 of Serpin D1 in RAW264.7 cells caused increases of the abundance of subcortical actin filaments. This treatment also altered the spatial distribution of adseverin, inhibited the expression of osteoclast-specific genes and reduced the formation of multinucleated osteoclasts by >90% in vitro. We conclude that a truncated, intracellular variant of Serpin D1 interacts with adseverin to promote actin severing and osteoclast formation.

Title: Algorithmic Complexity of Parallel Simulations of Complex Systems

This paper investigates the algorithmic complexity of parallel simulations of complex systems, aiming to develop a novel algorithm that significantly reduces computational costs while maintaining accuracy. Complex systems, such as protein folding and climate modeling, demand substantial computational resources. Existing simulation techniques often struggle with scalability, limiting the size and complexity of these models. This research explores a new approach – "parallel branching" – designed to intelligently distribute computational tasks across multiple cores, minimizing memory requirements and accelerating simulation times. The core mechanism centers around strategically partitioning problem space to optimize performance. We analyze the algorithm's complexity using established metrics and demonstrate its potential to substantially improve simulation efficiency compared to traditional methods. The findings highlight the importance of algorithmic optimization in tackling complex system simulations, offering a potentially transformative step toward more realistic and computationally feasible models.

Single nucleus RNA sequencing and spatial transcriptomics reveal unique functionalities of gray matter versus white matter oligodendrocytes in aging and Alzheimer′s Disease

Oligodendrocyte (OL) dysfunction and white-matter (WM) vulnerability are increasingly recognized as important aspects of aging and Alzheimer′s Disease (AD), yet human WM–focused, cellular–resolution transcriptomic data remain limited. Here, we profiled prefrontal WM from 48 brain donors spanning young adulthood and late–life with low versus high AD Neuropathologic Change (ADNC) using single–nucleus RNA sequencing followed by spatial transcriptomics (CosMx) in a subset of matched donors. We integrated aged WM OLs with a reference dorsolateral prefrontal cortex grey–matter (GM) OL dataset (SEA–AD) to define region– and pathology–associated OL programs. Across modalities, GM OLs exhibited a robust synapse/neurotransmitter-associated transcriptional signature relative to WM OLs, whereas this program was reduced with aging and attenuated in high ADNC GM. In contrast, WM OLs showed stronger immune-associated programs with aging and further enhancement in high ADNC, including cytokine/chemokine signaling and antigen presentation–related pathways. High ADNC WM OLs also displayed amplified proteostasis and stress-adaptation signatures, including selective upregulation of chaperone/heat shock genes and ferritin subunits, consistent with increased protein–folding demand and altered iron handling. To resolve OL state organization beyond static differential expression, we annotated OL sub–states using marker panels and inferred pseudotime-guided directional state–to–state flows within each tissue/condition stratum. This analysis identified a conserved newly formed differentiating (NFOL)/differentiating → lipid remodeling (APOE/ABCA1/LPL+) → Stress/ISR-reactive architecture, with a pronounced expansion of the Stress/ISR-reactive compartment and altered transition-associated pathway enrichment in high ADNC WM. Together, these data define WM–specific OL programs linked to aging and ADNC and nominate a stress/immune-enriched OL state landscape consistent with a putative senescence-like phenotype in diseased WM.

Amino Acid Substitutions in the C-Terminal Domains of Vip3Aa Enhance Insecticidal Toxicity

Vip3 proteins secreted by the entomopathogenic bacterium Bacillus thuringiensis (Bt) have an important role in biological control against economically important lepidopteran pests. The elucidation of Vip3 protein structures has helped to address the roles of domains and amino acid positions involved in toxicity, especially in the N-terminal domains I and II, thereby supporting their more efficient utilization. In this study, we evaluated the impact of combinations of critical amino acid substitutions, selected from previous studies, in domains IV and V of the Vip3Aa90 protein on its insecticidal activity against three lepidopteran pests. The double mutant S543N/I544L, triple mutants S543N/I544L/E627A and S543N/I544L/S686R, and quadruple mutant S543N/I544L/E627A/S686R were constructed in Escherichia coli by site-directed mutagenesis. Among these, only the Vip3Aa mutant proteins S543N/I544L/E627A and S543N/I544L/E627A/S686R could be expressed and purified for bioassays. Both mutant proteins had similar toxicity against Spodoptera littoralis, showing higher insecticidal activity than the wild-type (WT) Vip3Aa90 at the LC90 level. However, at the LC50 level, only a slight improvement in toxicity was observed for the quadruple mutant. In the case of S. exigua, no significant difference in toxicity was observed for either of the two mutant proteins with respect to the WT at either LC level. Interestingly, for G. molesta, though the toxicity of the triple mutant did not differ significantly compared to that of the WT protein, that of the quadruple mutant showed a marked decrease in toxicity of over 10-fold. This study revealed that combining selected amino acid substitutions in domains IV and V can enhance Vip3Aa90 toxicity against some lepidopteran species but can be either neutral or even deleterious in others.

Self‐Assembly of Hierarchically Structured Materials From Liquid Crystalline Mesophases of Tobacco Mosaic Virus‐Like Particles

Many biological materials owe their impressive performance to the hierarchical organization of protein subunits across multiple length scales, motivating next generation bio-inspired material design. However, limited understanding of the mechanisms by which proteins self-assemble into higher-order structures hinders our ability to mimic nature. Here, we demonstrate that recombinantly produced Tobacco Mosaic Virus coat proteins (TMVcp) can self-assemble into hierarchically structured macroscopic materials using a lyotropic liquid crystal (LC) as a transient precursor phase. TMVcp helical rods were formed under controlled pH and ionic conditions, then processed into macroscale filamentous structures using a drying droplet technique, which effectively mimics fabrication of biological fibers combining mechanical shear and dehydration. Polarized light microscopy (PLM) of the fluid phase revealed birefringence, indicating liquid crystalline alignment, while polarized confocal Raman spectroscopy confirmed the retention of the native TMVcp folded structure within the assembled filaments. Synchrotron small-angle x-ray scattering (SAXS) further revealed tight hexagonal packing of TMVcp helical rods, which are aligned along the filament axis, indicating a highly ordered nanoscale organization. These results highlight the potential of virus-like particles (VLPs) as model systems for elucidating and engineering the self-assembly of hierarchical biomaterials, opening new avenues for the development of sustainable, tunable protein-based materials for advanced applications.

Title: Temporal Topology Optimization for Biological Systems

Temporal Topology Optimization (TTO) presents a novel approach to biological system optimization, moving beyond static parameterization to model the dynamic evolution of topological states. This paper introduces a system leveraging network theory and evolutionary dynamics to predict emergent behaviors within biological systems, specifically focusing on protein folding and gene regulatory network design. We propose a framework that translates biological complexity into a network of interconnected topological states, enabling a predictive model of system behavior. The core mechanism centers around utilizing temporal evolution to iteratively refine these topological states, ultimately generating novel and optimized configurations. This work addresses a key limitation of existing evolutionary modeling – the reliance on fixed parameters – and offers a framework for understanding and manipulating complex biological systems through the lens of topology.

Quantitative characterization of L-citrulline as a precursor of L-arginine in rats: pharmacokinetic basis for nitric oxide-mediated effects

L-Arginine (ARG) is the primary substrate for nitric oxide (NO) synthesis via nitric oxide synthase (NOS), and L-citrulline (CIT) is its endogenous precursor. Although oral CIT has been reported to increase systemic ARG levels, the quantitative basis for its in vivo conversion to ARG and contribution to NO-mediated effects remains unclear. Therefore, this study aimed to quantitatively characterize the pharmacokinetic relationship between ARG and CIT in vivo and to determine whether CIT-derived ARG exposure could explain the pharmacodynamic effects observed after oral supplementation. Sprague-Dawley rats received oral ARG (500 mg/kg, n = 6), CIT (500 mg/kg, n = 6), or an equidose L-alanine control (n = 6) daily for 30 days. After 30 days of supplementation, both ARG and CIT groups exhibited significantly elevated plasma NOx levels, approximately 1.6-fold higher eNOS protein expression in the aortic arch compared with controls (p < 0.05), and reduced blood glucose concentrations. Plasma concentration–time profiles of both analytes following intravenous and oral administration of each compound were simultaneously analyzed within a single population pharmacokinetic model. The model estimated higher oral bioavailability for CIT than for ARG (F CIT , 82.2% vs. F ARG , 52.6%). It also indicated that CIT elimination was predominantly mediated by conversion to ARG (F met = 0.999), resulting in greater systemic ARG exposure following oral CIT than following direct oral ARG administration. These findings provide a quantitative pharmacokinetic basis for the NO-mediated pharmacodynamic effects observed with both compounds and support the role of CIT as an effective oral precursor of ARG.