Biophysica, Volume 5, Issue 4 (December 2025) – 9 articles

Innate immunity is the body’s first line of defense for mounting robust antiviral signaling. However, the role of cytoskeletal prestress, a hallmark of cellular mechanotransduction, in regulating innate immune pathways such as retinoic acid-inducible gene I (RIG-I) signaling remains poorly understood. Herein, we show that cells on soft vs. rigid substrates elicit cytoskeletal prestress-dependent activation of RIG-I signaling, leading to differential type-I interferon (IFN) gene expression. Cells were cultured on soft (0.6 kPa) and stiff (8.5 kPa) substrates to modulate cellular traction and prestress, followed by transfection of Poly(I:C), a synthetic viral dsRNA mimic, to measure the RIG-I-mediated innate immune response. Cells on soft substrates show minimal activation of RIG-I signaling, resulting in low expression of IFN-β1 and other IFN-stimulated genes (ISGs), compared to cells on stiff substrates. We further demonstrate that activation of TANK Binding Kinase 1 (TBK1), a downstream effector of the RIG-I pathway, is inhibited in cells on soft substrates due to the cytoplasmic sequestration of the Yes-associated protein (YAP), a HIPPO pathway effector protein. In contrast, cells on stiffer substrates experienced decreased TBK1 inhibition due to the nuclear localization of YAP and exhibited elevated TBK1 activation and heightened IFN and ISG expressions. Together, we demonstrate that cytoskeletal prestress represents a key biophysical regulator of innate immune signaling. Full article

Large-scale computer simulations were employed to investigate the conformational response of the spike protein components S1 and S2 using a coarse-grained model. Temperature was systematically varied to assess the balance between stabilizing residue–residue interactions and thermal fluctuations. The resulting contact profiles reveal distinct segmental reorganization and self-assembly behaviors between S1 and S2. At lower, thermoresponsive temperatures, pronounced segmental globularization occurs in the N-terminal domain (NTD; M153–K202) and receptor-binding domain (RBD; E406–E471) of S1, whereas S2 exhibits alternating regions of high and low contact density. Increasing temperature reduces this segmental globularization, leaving only minor persistence at elevated temperatures. The temperature dependence of the radius of gyration (R_g) further demonstrates the contrasting thermal behaviors of S1 and S2. For S1, R_g increases continuously and monotonically with temperature, reaching a steady-state value approximately 50% higher than that at low temperature. In contrast, S2 displays a non-monotonic response: R_g initially rises to a maximum nearly sevenfold higher than its low-temperature value, then decreases with further temperature increase. Scaling analysis of the structure factor reveals that the globularity of S1 diminishes significantly upon heating, while S2 becomes modestly more compact yet retains its predominantly fibrous character. Full article

Microencapsulation is a fundamental technology for protecting active compounds from environmental degradation by factors such as light, heat, and oxygen. This process significantly improves their stability, bioavailability, and shelf life by entrapping an active core within a protective matrix. Therefore, a thorough understanding of the physicochemical interactions between these components is essential for developing stable and efficient delivery systems. The composition of the microcapsule and the encapsulation method are key determinants of system stability and the retention of encapsulated materials. Recently, the application of computational tools to predict and optimize microencapsulation processes has emerged as a promising area of research. In this context, molecular dynamics (MD) simulation has become an indispensable computational technique. By solving Newton’s equations of motion, MD simulations enable a detailed study of the dynamic behavior of atoms and molecules in a simulated environment. For example, MD-based analyses have quantitatively demonstrated that optimizing polymer–core interaction energies can enhance encapsulation efficiency by over 20% and improve the thermal stability of active compounds. This approach provides invaluable insights into the molecular interactions between the core material and the matrix, ultimately facilitating the rational design of optimized microstructures for diverse applications, including pharmaceuticals, thereby opening new avenues for innovation in the field. Ultimately, the integration of computational modeling into microencapsulation research not only represents a methodological advancement but also pivotal opportunity to accelerate innovation, optimize processes, and develop more effective and sustainable therapeutic systems. Full article

This study explored the use of physical methods, namely X-ray diffraction, atomic force microscopy, and energy-dispersive X-ray spectroscopy, to analyze the structure and composition of tear fluid desiccates. Tear samples were collected from patients with dry eye syndrome, glaucoma, and multiple sclerosis. Our results revealed significant differences in the crystallization patterns, chemical composition, and morphology of tear fluid among the disease groups compared to healthy individuals. XRD analysis identified variations in salt crystallization within tear fluid desiccates. AFM provided nanoscale morphological visualization. EDX determined the presence of key chemical elements. Our findings showed that changes in crystallization and unbalance of ionic composition in tear fluid may serve as potential markers for diagnosing ocular diseases. This study highlights the potential of these techniques for non-invasive diagnostics and contributes to the development of innovative strategies for monitoring structural properties in tear fluid desiccates of analyzed inflammatory, and neurodegenerative diseases. Full article

Human granulocyte colony-stimulating factor (hG-CSF) is primarily used to treat neutropenia induced by cancer chemotherapy and bone marrow transplantation. The current identification test for hG-CSF relies on Western blot (WB), a labor-intensive and technically demanding method. This study aimed to screen and prepare an anti-hG-CSF nanobody to identify and quantify hG-CSF, with the ultimate goal of developing colloidal gold-labeled nanobody test strips for rapid identification. An alpaca was immunized with hG-CSF, and the VHH gene sequence encoding the anti-hG-CSF nanobody was obtained through sequencing following phage display library construction and multiple rounds of biopanning. The nanobody C68, obtained from screening, was expressed by E. coli, and its physicochemical properties such as molecular weight, isoelectric point, and affinity were characterized after purification. WB analysis demonstrated excellent performance of the nanobody in identification tests in terms of specificity, limit of detection (LOD), applicability with products from various manufacturers, and thermal stability. Additionally, we established an ELISA method for hG-CSF quantification utilizing the nanobody C68 and conducted methodological validation. Finally, colloidal gold-based test strips were constructed using the nanobody C68, with a LOD of 30 μg/mL, achieving rapid identification for hG-CSF. This study represents a novel application of nanobodies in pharmaceutical testing and offers valuable insights for developing identification tests for other recombinant protein drugs. Full article

Post-COVID-19 syndrome (PCS) is a multifactorial disorder comprising different subgroups. Our study aimed to investigate the longitudinal changes in retinal microcirculation in PCS patients. Eighty PCS patients were recruited at the Department of Ophthalmology at the Friedrich-Alexander University of Erlangen-Nürnberg. Retinal microcirculation was measured twice using optical coherence tomography angiography (OCT-A) within the superficial vascular plexus (SVP), intermediate capillary plexus (ICP), deep capillary plexus (DCP), and peripapillary region. Vessel density (VD) was calculated using the Erlangen Angio Tool with an APSified and Bruch’s membrane opening-based analyses. The least-squares means (LS-Means) of VD were 30.4 (SE = 0.168) vs. 30.3 (SE = 0.166) (SVP), 22.4 (SE = 0.143) vs. 22.2 (SE = 0.141) (ICP), 23.9 (SE = 0.186) vs. 23.8 (SE = 0.185) (DCP), and 27.4 (SE = 0.226) vs. 27.0 (SE = 0.224) (peripapillary) in patients with PCS at visits 1 and 2, respectively. The study cohort showed physically stable PCS symptoms with PEM/fatigue and concentration disorders as major symptoms and only a slight, clinically irrelevant improvement of the Bell Score. The multivariate longitudinal model confirmed the clinical observations by showing that VD did not change significantly during follow-up (p = 0.46). Strong interdependencies between the macular layers (p < 0.001) were observed. The data of the present study suggests that while overall APSified macular VD and BMO-based APSified peripapillary VD were stable within a PCS cohort of physically stable PCS symptoms, individual patients may experience coordinated microvascular changes, particularly within the macular plexuses. Together, the results support a model of heterogeneous yet biologically consistent microvascular response in PCS pathophysiology. Full article

A systematic review was conducted to assess the effects of microgravity and space radiation on astronauts’ oral health. This review aimed to determine if these conditions increase the risk of dental and periodontal diseases, identify pre-mission dental care strategies, and specify relevant dental emergencies for astronauts to manage during missions. Following PRISMA guidelines, the review was registered on PROSPERO (CRD42023472765). Databases including PubMed, Scopus, Web of Science, Cochrane Library, and OVID Medline were searched. Of the 13 studies identified, 7 were eligible for qualitative synthesis. The included studies revealed that space conditions compromise oral health. Findings indicate changes in saliva composition, with a significant decline in salivary lysozyme levels during missions lasting 28 to 84 days. Salivary IgA levels also increased before and peaked after flights (microgravity alters fluid shear and protein folding). Viral reactivation was a key finding, with latent viruses such as Epstein–Barr virus (EBV), cytomegalovirus (CMV), and varicella zoster virus (VZV) being reactivated during missions (immune suppression and gene expression shifts under spaceflight stress). Data from a study found that 50% of crew members shed viruses in their saliva or urine, and 38% tested positive for herpesviruses. The included studies also documented alterations in the oral microbiome, including increased gastrointestinal and decreased nasal microbial diversity. This suggests alterations in salivary biomarkers, viral shedding, and microbiome changes in astronauts during long-duration missions. These changes appear associated with immune dysregulation and stress, but causality remains uncertain due to observational designs, small heterogeneous samples, and confounding factors. Although current evidence is indicative rather than definitive, these findings highlight the need for preventive dental measures prior to missions and preparedness for managing oral emergencies in-flight. Future studies should address the mechanistic separation of microgravity and radiation effects, with implications for upcoming Moon and Mars missions. Full article

Myelin is a membranous structure critically important for human health. Historically, it was believed that myelin remained largely unchanged in the adult brain. However, recent research has shown that myelin is remarkably dynamic, capable of adjusting axonal conduction velocity and playing a role in learning, memory, and recovery from injury, in response to both physiological and pathological signals. Axons are more efficiently insulated in myelinated fibers, where segments of the axonal membrane are wrapped by the myelin sheath. Although extensive data are available on the electrical properties of myelin, its structural and mechanical characteristics—as well as the role of its lipid composition—are also relevant, although much less explored. The objective of our review is derived from this point since alterations in lipid components can lead to axonal dysfunction, giving rise to neurological disorders such as multiple sclerosis and other demyelinating conditions. In this review, concerning the lipid composition of myelin, we focus on two distinct classes of lipids: sphingolipids and long-chain fatty acids, emphasizing the differential contributions of saturated versus polyunsaturated species. We analyze studies that correlate the mechanical vulnerability of myelin with its lipid composition, particularly sphingomyelin, thereby underscoring its role in protecting neurons against physical stress and providing a robust microstructural network that reinforces the white matter as a whole. From a biochemical perspective, we examine the susceptibility of myelin to oxidative stress, metabolic disorders, and extreme nutritional deficiencies in relation to the role of long-chain fatty acids. Both perspectives highlight that the aforementioned lipids participate in a complex biomechanical balance that is essential for maintaining the stability of myelin and, consequently, the integrity of the central and peripheral nervous systems. Full article

Humanized mouse models offer human-specific platforms for investigating complex host–pathogen interactions, addressing shortcomings of conventional preclinical models that often fail to replicate human immune responses accurately. This integrative review examines the intersection of advanced morphological phenotyping and artificial intelligence (AI) to enhance predictive capacity and translational relevance in infectious disease research. A structured literature search was conducted across PubMed, Scopus, and Web of Science (2010–2025), applying defined inclusion and exclusion criteria. Evidence synthesis highlights imaging modalities, AI-driven phenotyping, and standardization strategies, supported by comparative analyses and quality considerations. Persistent challenges include variability in engraftment, lack of harmonized scoring systems, and ethical governance. We propose recommendations for standardized protocols, risk-of-bias mitigation, and collaborative training frameworks to accelerate adoption of these technologies in translational medicine. Full article