Biophysica doi: 10.3390/biophysica6040058

Authors: Shainy Mathew Cheruvathur Krishna Prasad Nooralabettu

Developing advanced bioinspired photoprotective barrier from marine resources represents a critical frontier of bioprocessing. This study established a rational design and implementation of effective photoprotective surface-coating eumelanin from ink of an Indian squid (Uroteuthis duvaucelii). The Central Composite Design was developed to optimize extraction and functionalization parameters of eumelanin on quartz substrates, strategically developing the matrix for peak optical attenuation within the potential Far-UVC window (220 nm). Translational photoprotective efficacy of the surface, as well as finished eumelanin on quartz surface, was validated by subjecting them to a challenging macro-level biological assay using a hospital-grade 254 nm ultraviolet germicidal source (125 µWcm−2). Quantitative physical dosimetry established that the squid eumelanin coating (A254 = 1.00) reduced internal transmittance to approximately 10%, successfully dampening the incident fluence from 0.225 J cm−2 down to a heavily attenuated 0.0225 J cm−2 at the biological sample plane. While unshielded control indicator microbial strains suffered complete lethal inactivation, the eumelanin barrier maintained exceptional cell viability, yielding biological shielding efficiencies of 98% for Bacillus subtilis, 96% for Staphylococcus aureus, and 92% for Escherichia coli. Characteristic features from FE-SEM, FTIR, and XRD analysis established that this superior photoprotective property is governed by the extensively conjugated, π-π-stacked indolic architecture possessing a characteristic 3.4 Å interlayer d-spacing, which facilitates rapid, non-radiative energy dissipation. This work establishes an effective framework for translating squid biomass into high-value, transparent optical barriers, providing a potential sustainable alternative to synthetic ultraviolet absorbers.

Biophysica doi: 10.3390/biophysica6040057

Authors: Noah Drebing Naomi Watanabe

In this study, we investigate how lightning-derived electric-field influences nonlinear excitation dynamics in excitable systems. Cloud-to-ground (CG) lightning observations from the National Lightning Detection Network (NLDN), including event time, location, and peak current, were used to reconstruct realistic lightning-derived electric-field inputs. The electric field distribution was estimated from lightning peak current and propagation distance using a physical formulation, and discrete lightning events were converted into continuous time-dependent forcing signals through Gaussian kernel superposition while preserving their spatiotemporal organization. The resulting electric-field signals were then applied to the FitzHugh–Nagumo (FHN) model, where biologically inspired excitation dynamics were simulated and analyzed using normalized external inputs. The simulations demonstrate that temporally accumulated lightning-derived forcing induces nonlinear transitions between excitation regimes. Stronger peak-current inputs more readily exceed excitation thresholds and produce enhanced responses, including repeated excitation events, whereas weaker inputs generate limited or sub-threshold responses. These results show that excitation dynamics depend not only on electric-field amplitude but also on the temporal accumulation and organization of lightning activity. Furthermore, a spatially extended reaction–diffusion FHN model demonstrates that lightning-induced electric-field attenuation coupled with nonlinear dynamics can generate spatially propagating excitation structures. This physics-based framework provides a conceptual approach for linking naturally occurring electric-field environments with nonlinear excitable-system dynamics. Although the present model does not represent direct physiological coupling, it provides a foundation for exploring how structured environmental electric fields may influence threshold-dependent dynamical responses.

Biophysica doi: 10.3390/biophysica6040056

Authors: Rashad Hall To Dang Daniel B. Erenso Horace T. Crogman

Localized optical absorption by nanoscale inclusions can profoundly alter energy deposition in optical traps, giving rise to nonlinear and long-timescale dynamics. Recent experiments have reported the formation of expanding optically darkened regions and episodic plasma-like emission during pulsed near-infrared optical trapping of magnetic beads interacting with biological cells. Here, we develop a reduced-order mechanistic model to investigate whether absorber-driven optical–thermal feedback associated with Fe3O4 inclusions is sufficient to reproduce the observed pre-plasma darkening dynamics. The model is constructed progressively from first-principles electromagnetic absorption and pulse-scale thermal diffusion to nonlinear feedback mediated by an evolving optically modified region. Single-pulse and multi-pulse simulations demonstrate that isolated iron-oxide absorbers cool too rapidly to sustain long-timescale thermal accumulation through linear heating alone. However, incorporation of a bubble-mediated optical feedback channel produces bounded growth, partial optical darkening, and slow relaxation dynamics consistent with experimentally observed minute-scale evolution. Electromagnetic absorption was computed using full core–shell Mie theory, yielding absorption cross-sections sufficient to support strong localized optical attenuation under experimentally relevant trapping conditions. The resulting reduced-order feedback framework reproduces stable growth–relaxation cycles, finite transmission plateaus, and self-limited optical darkening without requiring runaway heating or catastrophic cavitation. To evaluate the model quantitatively, simulated transmission dynamics were compared against experimentally measured normalized transmission traces digitized from previously reported optical trapping experiments. The fitted model reproduced the observed finite transmission plateau and slow post-activation relaxation with good agreement (R2≈0.86, RMSE ≈1.3×10−2). These results support the interpretation that experimentally observed optical darkening arises from a feedback-regulated optical–thermal process involving slowly evolving structural modification of the trapping region rather than cumulative thermal storage within isolated absorbers. The present framework provides a quantitatively constrained reduced-order description of feedback-mediated optical darkening under pulsed optical trapping conditions and establishes iron-oxide absorption as a physically plausible ignition mechanism for dark-state formation in the pre-plasma regime.

Biophysica doi: 10.3390/biophysica6040055

Authors: Puja Jaishwal Satarudra Prakash Singh

The continuous evolution of the SARS-CoV-2 virus, marked by the emergence of new variants, poses a significant threat to the efficacy of existing vaccines. However, a promising approach to addressing vaccine failure caused by viral mutations (particularly in the spike protein) is the development of a variant-proof (conserved), non-spike, multiepitope universal nanostructure vaccine with multifunctionality, biocompatibility, self-adjuvanticity, and structural similarity to pathogens in terms of size and shape. This study aimed to design a self-assembled nanostructure vaccine (SANV) featuring pentameric and trimeric coiled-coil peptide motifs, as well as other functional motifs, including epitopes, TAT, PADRE, and adjuvant. The cytotoxic T lymphocyte (CTL), helper T lymphocyte (HTL), and B lymphocyte (BL) epitopes of SANV were screened from the IEDB with more than 50% individual predicted population coverage (PPC) and fused using linkers to enable self-assembly. The multimerization of the 24 SANV monomers was modeled using the GalaxyHomomer and AlphaFold web servers. Subsequently, the leading SANV constructs with (SANVa9) and without (SANVb6) adjuvant were analyzed for their physicochemical profiles and assessed for antigenicity, allergenicity, solubility, and antioxidant potential. Furthermore, the molecular interactions, specificity, and stability of SANVa9 and SANVb6 with the broadly neutralizing sarbecovirus antibody 5817 and toll-like receptors (TLR2, TLR3, and TLR7) were analyzed using molecular docking and simulation over a 100-nanosecond time scale. Finally, the comparative immune simulation profiles of SANVa9 and SANVb6 with controls indicated stronger, broad-spectrum immune responses that could be translated into in vitro and in vivo studies and warrant further evaluation before clinical use.

Biophysica doi: 10.3390/biophysica6040054

Authors: Ali Dahaghin Milad Salimibani Paria Jahansa

In therapeutic applications, ultrasound is widely used in physiotherapy, tissue repair, and cancer treatment. Regarding cancer treatment, as an emerging field for technology, significant research efforts have been devoted to the area of ultrasound therapy. The derived energy from beams can be deposited in tissues not only through heating but also through non-thermal mechanisms, whereby cancer cells are subject to cell death. Ultrasound-induced heating can generate localized temperature elevations within biological tissues, making it a subject of interest for thermal therapeutic applications. Nevertheless, excessive temperature elevations outside the primary exposure region may result in undesirable thermal effects within the surrounding tissue. In this study, we used continuous 3 MHz ultrasound waves at the powers of 0.4 to 1.4 W on ex vivo chicken breast tissue in a water bath to prevent fluctuations in temperature. The process was also numerically modeled with a maximum error of 0.4% from the measured data. Temperature measurements revealed a significant difference between the region of maximum acoustic pressure along the beam axis and deeper tissue locations (in some cases, above 3.5 °C). These findings indicate that temperature gradients can develop within homogeneous tissue during ultrasound exposure, emphasizing the importance of controlling acoustic power and exposure conditions. Moreover, increasing the temperature was significant during the first moments of treatment, which highlights the importance of precise controls for rate and precision in therapy. The numerical simulations also showed that increasing acoustic power elevates tissue temperature while simultaneously producing a less uniform temperature distribution. These observations may be useful for the optimization of future ultrasound-based thermal treatment strategies; however, direct clinical extrapolation requires further investigation using physiologically representative tissue models.

Biophysica doi: 10.3390/biophysica6030053

Authors: Igor Jerman Linda Ogrizek Jonatan Pihir

Lactose is widely used as a pharmaceutical excipient, yet little is known about how its physicochemical behavior may be influenced by pretreatment history and weak environmental magnetic conditions. In this pilot study, we investigated oxidation–reduction potential (ORP) and UV absorbance of 0.2% aqueous lactose solutions prepared from lactose powders with different pretreatment histories: Active water, Native water, and untreated control. Samples were exposed for 30 min to three static magnetic field conditions: weak geomagnetic field (~4 µT), ambient geomagnetic field (~30 µT), and elevated static field (~750 µT). UV/VIS spectroscopy was performed in the 200–400 nm range, with particular focus on the deep-UV absorption maximum near 200 nm. The strongest differentiation between pretreated samples and control occurred under weak geomagnetic conditions. In this weak-field regime, pretreated lactose solutions showed higher ORP values and a same-direction trend toward increased UV absorbance near 200 nm relative to untreated lactose. Across all samples, both ORP and UV absorbance decreased with increasing magnetic field strength, indicating a consistent field-dependent shift in the overall physicochemical state of the lactose solutions, particularly in redox balance and deep-UV optical response. The same-direction changes in ORP and increased 200 nm absorbance at the group level suggests that weak-field conditions may influence oxidation-related processes, potentially including the formation or stabilization of lactose oxidation products such as lactobionic acid. These findings indicate that lactose-containing aqueous systems may be sensitive to both pretreatment history and low-intensity magnetic environments, with potential implications for pharmaceutical formulation stability, quality control, and biotechnological reproducibility.

Biophysica doi: 10.3390/biophysica6030052

Authors: Katarina Žikić Dejan Žikić

Pulse wave propagation through blood vessels is affected by many biophysical parameters that change with aging. The aim of this study was to investigate both theoretically and experimentally how the pulse wave velocity changes in the vertical position and to introduce a new parameter in biophysics: pulse wave acceleration (PWA). Using a biophysical model of the cardiovascular system, placed in horizontal and vertical positions, pressure waveforms were measured along the arterial tree at several sites at different diastolic pressures and pump frequencies. Blood flow waveforms on the carotid and femoral arteries in the supine and standing positions were measured on the subjects. The results showed that the pulse pressure wave accelerates in the direction of gravity and decelerates in the opposite direction both in the model and in humans. A new biophysical parameter, PWA, was defined, and the experimental results are in agreement with the mathematical model. Due to the acceleration of the pulse wave, the reflected wave in the standing position arrives earlier in systole and contributes to the increase in pressure. This emerging biophysical parameter may contribute to a better understanding of the phenomenon of wave propagation of blood through blood vessels.

Biophysica doi: 10.3390/biophysica6030051

Authors: Evgenii Yu. Sherstoboev Marina G. Danilets Anastasia A. Ligacheva Evgenia S. Trofimova Natalia S. Selivanova

It has recently been shown that by sequential vibrational processing (named ‘crossing’) of two closely located vials with the initial substance in one of them and water or lactose in the other, it is possible to obtain novel products, or vibrational iterations. Vibrational iterations are, in certain cases, capable of exerting a modifying effect on the initial substance or its target, thereby altering its physicochemical and biological properties. Since water is the environment in which metabolic processes occur in cells, organs, and tissues, vibrational iterations obtained using water as initial substance can potentially affect their course. In this work, we assessed the effect of vibrational iterations of water on the metabolic activity of primary peritoneal macrophages of C57BL/6 mice and macrophage-like tumor cells of the RAW 264.7 line. It was shown that one vibrational iteration of water increased the metabolic activity of peritoneal macrophages and suppressed the metabolic activity of tumor cells. At the same time, another vibrational iteration of water did not influence the metabolic activity of peritoneal macrophages but decreased the metabolic activity of tumor cells. Probably, taking into account the modifying activity of vibrational iterations, their action is aimed at changing the properties (possibly, structuring) of intracellular water. The results obtained by us show the possibility of a fundamentally new differential effect on the metabolic activity of healthy and tumor cells, which opens up new prospects for antitumor therapy.

Biophysica doi: 10.3390/biophysica6030050

Authors: Osama S. Alothmnai Ali H. Alsaif Ragab E. Saif Tariq Abuhaimed Amna Y. Siddiqui

This study aimed to evaluate the surface changes depicted on Reciproc and Reciproc Blue R25 files after their immersion in different irrigants at different temperatures compared to the non-immersed controls, utilizing Atomic Force Microscopy (AFM). To measure the Root Mean Square (RMS) and Mean Roughness (Sa), eight R25 files (four/system) were divided into four groups (n = 2; one file/system): non-immersed (control), immersed in 17% ethylenediaminetetraacetic acid (EDTA) set at 37 °C, and immersed in 5.25% sodium hypochlorite (NaOCl) set at 37 °C or at 45 °C. Immersion time was 10 min after which AFM was conducted. There was no significant difference in mean RMS and Sa between the control and the 17% EDTA group (p > 0.05). Immersion in 5.25% NaOCl at 37 °C significantly increased surface irregularities on both files (p < 0.05). This increase was further accentuated by NaOCl’s temperature rise to 45 °C (p < 0.05). Reciproc exhibited significantly higher surface roughness compared to Reciproc Blue under all conditions (p < 0.05). Immersion in 5.25% NaOCl altered the surface topography of Reciproc and Reciproc Blue which was further accentuated by its temperature rise, while immersion in 17% EDTA had no significant effect on their surface changes. Reciproc demonstrated significantly higher surface roughness compared to Reciproc Blue under all tested conditions.

Biophysica doi: 10.3390/biophysica6030049

Authors: Yedukondala Rao Veeranki Luis R. Mercado-Diaz Hugo F. Posada-Quintero

Emotion recognition (ER) in human–computer interaction (HCI) holds immense potential for real-world applications, but traditional approaches based on electroencephalography (EEG) face challenges due to the complexity and impracticality of collecting and analyzing EEG data in ambulatory settings. This study explores electrodermal activity (EDA), a simpler measure of the sympathetic nervous system response that can be collected at multiple peripheral body sites, as a potential alternative for ER. We investigated the variable frequency complex demodulation (VFCDM) technique to analyze EDA and EEG signals and used deep learning models (ResNet50 and MobileNetV2) to classify arousal states (high arousal, HA vs. low arousal, LA). Our results show that EDA signals analyzed by VFCDM and classified by MobileNetV2 achieve promising performance, with an accuracy of 91.45%, comparable to the best EEG-based model (91.98%), in arousal classification. This suggests that EDA offers a viable and more practically accessible approach to ER in HCI compared to traditional EEG-based methods. Future work should explore larger and more diverse datasets, incorporate valence classification through multimodal fusion, and investigate the neural mechanisms underlying EDA-EEG interactions during emotional processing to further advance robust ER for HCI applications.

Biophysica doi: 10.3390/biophysica6030048

Authors: Nada M. Doleib Hend Maroof Tag Ragaa A. Hamouda

The Senegal tree (Sengalia senegal) is the primary plant source of Gum Arabic (GA), a natural secretion rich in soluble fiber and bioactive polysaccharides. It has longstanding uses in traditional medicine, nutrition, and pharmaceuticals. The present study aimed to evaluate the phytochemical profile, antimicrobial, anti-inflammatory, and anticancer activities of GA methanolic extract (GAME), supported by molecular docking analysis of its key compounds. The gas chromatography–mass spectrometry (GCMS) analysis of the GAME identified many compounds, such as 9-octadecenoic acid (38.29%), methyl ester (15.52), 1,2-benzenedicarboxylic acid, 3-nitro (9.8%), hexadecadienoic acid, methyl ester (8.5), and á-d-mannofuranoside, methyl (7.38). The molecular docking analysis showed that 9-octadecenoic acid had strong binding affinity with target proteins, which included xanthine oxidase (XO), lipoxygenase (LOX), and cyclooxygenase-2 (COX-2), with the highest affinity to XO (−137.03 kcal/mol) and lipoxygenase (−135.09 kcal/mol). GAME possessed broad-spectrum antibacterial activity against Salmonella typhimurium (S. typhimurium), Escherichia coli (E. coli), Pseudomonas aeruginosa (P. aeruginosa), and Staphylococcus aureus (S. aureus), with a zone of inhibition from 16.28 to 16.93 mm. B. subtilis was resistant to the tested extract. The extract also showed good membrane stability and potent inhibition of albumin, XO, LOX, and COX-2, with IC50 values of 31.62, 13.02, 27.6, and 28.99 μg/mL, respectively. The cytotoxic assessment demonstrated moderate, dose-dependent effects on the Caco-2 (colorectal adenocarcinoma) and HeLa (cervical carcinoma) cell lines. These findings highlight the therapeutic potential of GA as a natural plant source of antibacterial, anti-inflammatory and anticancer agents. The combination of molecular docking with in vitro assays provides strong evidence supporting its application in the development of plant-based pharmaceuticals. This research suggests that GA could be a useful ingredient in the creation of anti-inflammatory and antibacterial drugs derived from plants.

Biophysica doi: 10.3390/biophysica6030047

Authors: Shadrach C. Eze Stephen C. Nnemolisa Joy C. Onyesoro Toluwalope D. Ilori Victor S. Uche Augustine C. Madueke Wande M. Oluyemi Adeniyi T. Adewumi Salerwe Mosebi Innocent U. Okagu

Beta Secretase (BACE1) is a well-validated target for Alzheimer’s therapies, but there has been attrition in drug development. Herein, we leveraged machine learning (ML), virtual screening and molecular dynamics (MD) to identify novel compounds with potential activity against BACE1. We developed ML algorithms to distinguish active and inactive compounds from public databases. Molecular docking and dynamics were used to explore the inhibition mechanism, thermodynamic stability, and the flap dynamics of the BACE1-ligand complexes. Random Forest Classifier (RF) showed excellent metrics (accuracy: 0.9807; F1 score: 0.9804; specificity 0.9977), compared to other models. Molecular docking with predicted actives revealed compounds BA1, BA2, and BA3 with strong affinity for BACE1. Compound BA2, a cysteinyl sulfoxide derivative, showed good stability (RMSD) during simulations (1.307 ± 0.109 Å) compared to Verubecestat (1.602 ± 0.159 Å). MMGBSA-based binding free energy (ΔGbind; kcal/mol) showed that BA2 (−33.820 ± 4.254) had comparatively lower energy than Verubecestat (−21.090 ± 6.183). BA2 maintained electrostatic interactions with the catalytic dyad (Asp36 and Asp232) and Thr76 of the flap. BA2 also maintained the flaps in a semi-open conformation (d0: 11.807 ± 0.401 Å) throughout the simulation. Our study clearly demonstrates the utility of ML in prioritization of compounds before molecular docking and MD in early phases of drug discovery.

Biophysica doi: 10.3390/biophysica6030046

Authors: Sheng-Nan Wu Rasa Liutkevičienė Vita Rovite Chung-Hung Tsai Sheng-Che Lin

Telmisartan (TEL) is a non-peptide, orally administered antihypertensive agent primarily known as angiotensin II type 1 (AT1) blocker. In this review, we provide a detailed overview of how TEL modulates voltage-gated Na+ current (INa) and affects action potential (AP) firing behavior. TEL exerts differential stimulatory effects on the peak and late components of INa when subjected to brief depolarizing pulses across a range of cell types, such as mHippoE-14 hippocampal neuron, cultured dorsal root ganglion neurons, and HL-1 atrial cardiomyocytes. TEL can augment the non-inactivating (persistent) INa elicited by ascending long ramp pulse in mHippoE-14 cells. By using a parvalbumin-expressing interneuron-based modeled cell combined with bifurcation analysis, it is possible to predict how applied current influences subthreshold oscillations and the generation of somatic spiking in the presence of TEL. According to the Hodgkin-Huxley model, mimicking the action of TEL—characterized by an increased peak amplitude of INa and a slowed inactivation time course—leads to the emergence of periodic oscillations in membrane potential. Using a Markovian process, a separate model can also be mathematically constructed, showing that changes in certain rate constants can simulate the effect of TEL on INa in cardiac cells. The molecular docking prediction between TEL and the NaV1.7 channel was made by expected formation of hydrophobic interactions as well as hydrogen bonding. In addition to its antagonistic action at the AT1 receptor and its agonistic activation of peroxisome proliferator-activator-γ, TEL may also directly enhance INa, thereby modulating AP firing in a variety of excitable cells. Current evidence supports TEL’s modulatory impact on NaV channel activity and cellular excitability, while also acknowledging that the mechanism—whether direct or indirect—remains under investigation.

Biophysica doi: 10.3390/biophysica6030045

Authors: Enrique Canessa

Electroencephalographic (EEG) signals provide macroscopic observables of complex neural dynamics. We introduce a horizon-inspired framework in which measured EEG signals are modeled as projections of a complex wave-like representation constrained by an effective boundary analogous to an event horizon. In this formulation the signal amplitude obeys a renormalization-group scaling relation while EEG spectral entropy parameterizes the accessibility of observable modes. The resulting solutions generate oscillatory structures whose geometry and spectral signatures can be explored through signal analysis and sonification. This mapping between entropy-based neural observables and wave-like signal representations provides a physically motivated framework linking entropy measures, scale-dependent dynamics, and observable neural oscillations. The work is intentionally conceptual. It provides a falsifiable framework intended to stimulate future empirical investigations.

Biophysica doi: 10.3390/biophysica6030044

Authors: Deny Puriyani Azhary Yuda Prasetya Nugraha Rachmat Mauludin

A Fixed-dose combination (FDC) therapy of ezetimibe (EZT) and atorvastatin (ATV) is increasingly prescribed for high-risk hyperlipidemic patients with cardiovascular disease. However, the pharmaceutical production of FDC EZT/ATV tablets often results in poor ATV dissolution under acidic conditions, failing to meet regulatory requirements. This study aimed to improve ATV dissolution in acidic media through nanosuspension (NS) technology and microenvironmental pH modification. The experimental stages included preparation and characterization of ATV-NS, optimization of FDC EZT/ATV-nanocrystal tablets with pH modifiers, and evaluation of dissolution similarity (f2) against the innovator product Atozet®. ATV-NS was prepared via sonication and high-pressure homogenization using different stabilizers. Poloxamer 188-stabilized ATV-NS demonstrated optimal stability (particle size: 466.6 ± 8.9 nm; polydispersity index: 0.12 ± 0.10; zeta potential: −44.20 ± 0.06 mV) and significantly enhanced solubility (p < 0.05) compared with pure ATV. FDC EZT/ATV-nanocrystal tablets incorporating pH modifiers achieved f2 values for ATV of 52.36, 51.31, and 51.09 in NaCl/HCl pH 1.2, acetate buffer pH 4.5, and phosphate buffer pH 6.8, respectively. In contrast, EZT exhibited f2 values of 18.18, 16.72, and 14.66 (acceptable range: 50–100). Although complete profile similarity was not obtained for EZT, ATV dissolution in acidic media improved significantly (p < 0.05), supporting the feasibility of developing a bioequivalent generic FDC EZT/ATV tablet.

Biophysica doi: 10.3390/biophysica6030043

Authors: Abdurrahim Tekin Kemal Paksoy Enes Özlük Gülşah Öztürk

Deep brain stimulation (DBS) with implantable pulse generators (IPGs) is widely used in the treatment of movement disorders. Rechargeable IPGs (RC-IPGs) were developed to extend device longevity and address the limitations of battery life in non-rechargeable IPGs. However, data regarding patient compliance and device-related complications remain limited. Therefore, this retrospective observational study evaluated compliance, satisfaction, and complications in patients with RC-IPGs. Compliance in 42 patients with RC-IPGs was evaluated using the Timmermann questionnaire together with additional questions regarding device preference, complaints, and complications. Statistical analyses were performed using NCSS software (Number Cruncher Statistical System, version 2020; NCSS LLC, Kaysville, UT, USA). Although a substantial percentage (42.9%) of patients needed help during recharging, the overall satisfaction score was high (96% of the maximum possible score), and 95.2% of patients preferred RC-IPGs if a repeat DBS would be required, and the rate of RC-IPG complications (7.1%) was low. The patients rated the display screen with the lowest scores (54.05%), mainly those who underwent two or more DBS surgeries. The training subscore showed a statistically significant negative correlation with age (r = −0.531, p = 0.001), and dystonia patients, constituting the youngest group in the cohort, rated training with higher points. This study provides additional data on patient compliance and safety of RC-IPGs. These findings may contribute to a better understanding of patient experience and factors affecting compliance with rechargeable systems.

Biophysica doi: 10.3390/biophysica6030042

Authors: Shamima Akter Ghanim Ullah Aman Ullah

Alzheimer’s disease (AD) is the most common form of dementia, characterized by the progressive accumulation of amyloid β (Aβ) plaques and neurofibrillary tangles of tau protein in and around neurons. However, these markers appear relatively late in the disease, and their direct causality is incompatible with clinical observations. Extensive data suggest that dysregulation of Ca2+ signaling is an early event in the pathogenesis of AD. In familial AD (FAD), mutations in presenilin are shown to alter Ca2+ homeostasis by affecting the gating properties and/or the expression levels of inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) and ryanodine receptor (RyRs)—the main channels responsible for Ca2+ release from the endoplasmic reticulum (ER). Thus, understanding the mechanism through which these channels disrupt Ca2+ homeostasis at different spatiotemporal scales is crucial to determining their role in AD. Here, we use computational modeling to investigate how the gating kinetics of single IP3R in FAD-affected cells differ from those in wildtype (WT) cells and how these differences translate to impaired Ca2+ signaling at subcellular and whole-cell levels. Our detailed analysis reveals a significantly lower threshold for Ca2+ oscillations at the whole-cell level in terms of agonist concentration, with higher frequency and amplitudes in FAD-affected cells. These results shed new light on the observed Ca2+ hyperactivity in the pre-clinical stage of AD, reporting high-frequency Ca2+ oscillations in neurons.

Biophysica doi: 10.3390/biophysica6030041

Authors: Dominik Godlewski Michał Osuchowski Tomasz Kubrak Agnieszka Przygórzewska Sara Czech David Aebisher

Herein, we report a simple procedure regarding the photodynamic therapy (PDT) treatment as a minimally invasive modality for treating superficial bladder cancer that utilizes a photosensitizer, light, and oxygen to generate cytotoxic reactive oxygen species (ROS). This study evaluates the histopathological and morphological changes induced by PDT in an ex vivo model of low-grade (LG) pTa non-muscle-invasive bladder cancer (NMIBC). We investigated the efficacy of exogenous protoporphyrin IX (PpIX) and Rose Bengal (RB) by incubating tissue samples (n = 30) with an oxygen-saturated solution of PpIX (1–3 mM) or RB (0.3–0.5 mM) for one hour. Since the criticism of using frozen tissue in research already exists, this framing explains how to mitigate those limitations. Thus, we use oxygen-saturated solutions PpIX and oxygen-saturated solutions of RB. We discussed a few aspects related to the use of frozen tissue in PDT. Frozen tissue preserves lipids critical for assessing membrane damage and maintains higher levels of metabolic markers like antioxidant molecules like glutathione and more likely lack factors such as metabolic activity, intact cell membranes, and oxygenation. It is critical to differentiate between “artifactual” changes and the “pathological” death of cells. Thus, we used histopathological microscopy observation typically used in daily clinical investigations to characterize cells before and after PDT. Following irradiation with the light dose of 72 J/cm2 (410 nm or 532 nm at 300 mW for 15 min), hematoxylin–eosin staining revealed concentration-dependent apoptotic changes, including chromatin condensation, pyknosis, and nuclear fragmentation. While both agents induced cell death, RB demonstrated faster and more intense cytotoxicity than PpIX. These findings provide microscopic evidence of PDT-induced tumor destruction and suggest that RB is a potent candidate for further preclinical evaluation. At 410 nm (deep blue/violet), light penetration in biological tissue is very shallow, typically only around 0.3 to 1 mm; therefore, in a 2 mm thick tissue sample, most of the light would be absorbed within the first millimeter, with minimal light reaching the full depth of tissues. In this protocol, the generated ROS is used to destroy tumor tissue by attacking the cellular microenvironment directly. This led to immediate membrane disruption and lipid peroxidation. The proof-of-concept is an early-stage study designed to verify that a PDT treatment is feasible, safe, and biologically active in an ex vivo model of LG pTa NMIBC.

Biophysica doi: 10.3390/biophysica6030040

Authors: Emina Dervišević Zurifa Ajanović Muhamed Katica Lejla Dervišević Yanko Kolev Francesca Licitra Margherita Neri Angelo Montana

Climate change significantly affects human physiology and contributes to increased morbidity and mortality, with heat stress representing one of the most severe consequences of thermal imbalance. The aim of this study was to analyze morphological changes to leukocytes on the peripheral blood smears of Wistar rats exposed to hyperthermia using the geometric morphometrics method. A total of forty Wistar albino rats were divided into three experimental groups according to water temperature exposure (37 °C, 41 °C, and 44 °C). Peripheral blood smears were prepared, stained, and digitally recorded using Motic Images Plus 2.0 software, after which selected images were analyzed using geometric morphometric programs (tpsDig, tpsUtil, and MorphoJ) to evaluate leukocyte shape variations. Comparative analysis demonstrated statistically significant morphological changes in polymorphonuclear cell shapes between the control group (37 °C) and rats exposed to 41 °C (p = 0.009). Significant differences were also identified in mononuclear cell morphology between the antemortem and postmortem groups (p = 0.00307). The findings indicate that exposure to elevated temperatures induces measurable alterations in white blood cell morphology, confirming that hyperthermia produces significant structural changes in polymorphonuclear cells and mononuclear cells detectable through geometric morphometric analysis.

Biophysica doi: 10.3390/biophysica6030039

Authors: Eveline M. Bezerra Pedro N. Silva Junior Taciano A. Sorrentino Francisco A. M. Sales Alice M. C. Martins Ricardo P. Santos Ewerton W. S. Caetano Valder N. Freire Roner F. da Costa

Chagas disease remains a major neglected parasitic illness in Latin America and other endemic regions, and benznidazole (BZN) is still the primary trypanosomacidal drug despite its incompletely understood mechanism of action. This work provides a detailed biophysical characterization of the conformational behavior and vibrational properties of benznidazole (BZN), a first-line trypanocidal drug still widely used for the treatment of Chagas disease. Using density functional theory combined with relaxed potential energy surface scans in vacuum and implicit water, two low-energy conformers (BZN1 and BZN2) were identified, separated by moderate rotational barriers and a small energy difference, indicating that both are intrinsically accessible at room temperature. For each conformer, infrared and Raman spectra were calculated and assigned via vibrational mode analysis, then compared with FT-IR and FT-Raman spectra recorded for pharmaceutical-grade polycrystalline BZN. The theoretical and experimental spectra show excellent agreement, with a Raman band in the 1350–1400 cm−1 region emerging as a sensitive conformational marker: the experimental maximum at 1359cm−1 matches the most intense BZN1 mode, whereas the corresponding BZN2 band appears about 13cm−1 higher in frequency. This clear spectroscopic fingerprint demonstrates that the solid drug is overwhelmingly composed of the BZN1 conformer, despite the theoretical accessibility of BZN2. Overall, the study links the conformational landscape of benznidazole to its vibrational signatures and highlights Raman spectroscopy, supported by quantum chemical calculations, as a powerful tool for conformational and potential polymorphic control of this clinically important nitroimidazole.

Biophysica doi: 10.3390/biophysica6030038

Authors: Saranya Nallusamy Riswana Begam Mohamed Yousuf Nivetha Vadivel Rashmi Panigrahi

Cynodon dactylon (Bermuda grass) is a perennial medicinal grass widely distributed across tropical and subtropical regions and known for its antioxidant and anti-inflammatory properties. The present study aimed to identify bioactive metabolites from the leaves of C. dactylon and evaluate their potential interaction with PTEN-induced kinase 1 (PINK1), a crucial regulator of mitochondrial quality control implicated in neurodegenerative disorders, particularly Parkinson’s disease. GC–MS analysis identified a total of 95 phytochemicals, of which the top 20 metabolites were selected based on retention time and area percentage. These metabolites were subjected to virtual screening using PyRx, with ATP employed as the reference ligand. Among the screened metabolites, 5,8,11-eicosatrienoic acid was the high-affinity compound which predicted a binding affinity of −5.9 kcal/mol and forming two hydrogen bond interactions within the PINK1 active site. The docked complexes were further evaluated through a 100 ns molecular dynamics simulation in replicates that showed stable binding of the protein–ligand complex, as reflected by RMSD values, reduced residue fluctuations and stable radius of gyration and solvent-accessible surface area. These findings suggest that 5,8,11-eicosatrienoic acid from C. dactylon may act as a potential PINK1 modulator for Parkinson’s disease.

Biophysica doi: 10.3390/biophysica6030037

Authors: Uziel Jeffet Shir Dviker Shiri Livne Shira Akrabi Nir Sterer

Coaggregation by bridging bacteria such as Fusobacterium nucleatum is considered a key element in dental biofilm development and maturation. Previous studies showed that sublethal exposure to blue light caused damage to cell membrane integrity. The aim of the present study was to test the effect of blue light phototoxicity on this bacterium’s ability to coaggregate with the early colonizer Streptococcus sanguinis. Fusobacterium nucleatum bacterial cells were suspended in coaggregation buffer (CAB) and exposed to blue light (400–500 nm) for 0, 70, 140 and 280 s (i.e., fluences of 0, 96, 192 and 384 J/cm2, respectively). Following blue light exposure, samples were mixed with Streptococcus sanguinis suspensions and coaggregation was measured using a visual scale, spectrophotometric analysis and light microscopy. Results showed that blue light exposure significantly reduced the ability of Fusobacterium nucleatum to coaggregate with Streptococcus sanguinis. These results suggest that blue light antibacterial phototoxicity may be considered as a viable option in preventing dental biofilm-related conditions.

Biophysica doi: 10.3390/biophysica6020036

Authors: Jorge A. Vila

One of the most puzzling and unsolved challenges in molecular biology is understanding how proteins fold. Despite having advanced predictive tools that can accurately estimate the native structures of proteins, we still lack a comprehensive model that explains how amino acid sequences dictate folding pathways and trajectories. This manuscript introduces a novel treatment for the issue by employing the “principle of least action.” This approach enables us to explore an intriguing question: how does a protein achieve its native state at a constant folding rate and within a biologically plausible time frame? A response to this inquiry will help us understand why proteins must fold along specific pathways and identify the boundary conditions that limit their availability. Furthermore, the principle of least action—together with the effective trajectory conjecture—enables us to explain why different proteins could exhibit the same folding rate. Finally, it will enable us to provide an in-depth description of the genesis and solution of Levinthal’s paradox. Our results are expected to pave the way for a more profound understanding of how proteins fold, shedding light on how the amino acid sequence and its surrounding environment encode the protein’s folding pathways and, consequently, the protein’s three-dimensional structure.

Biophysica doi: 10.3390/biophysica6020035

Authors: Ashish Kabra

Deubiquitinases, or DUBs, have emerged as pivotal regulators of cellular homeostasis, coordinating the delicate balance between protein ubiquitination and deubiquitination. Their versatile roles span from controlling protein turnover to modulating signal transduction pathways, thereby influencing diverse cellular processes, including DNA damage repair, apoptosis, and immune responses. This review comprehensively explores the current understanding of DUBs, elucidating their structural diversity, catalytic mechanisms, physiological functions, and implications in human diseases. Moreover, we discuss the therapeutic potential of targeting DUBs in various pathological conditions, highlighting recent advancements and challenges in developing DUB-specific inhibitors.

Biophysica doi: 10.3390/biophysica6020034

Authors: Indu Passi Ram Kumar Sushant Salwan Pooja A. Chawla Nisha Bansal Bhupinder Kumar

Quality by design (QbD) is a systematic approach focused on achieving consistent, predictable quality based on predefined objectives. Unlike traditional methods, QbD prioritizes risk assessment and management, which significantly enhances the robustness of the analytical method. In this study, we initiated factor screening using a three-factor, two-level design to evaluate three independent variables: flow rate, pH, and mobile phase composition. To further investigate the interaction of these variables, we employed Central Composite Design (CCD). This allows us to apply response surface methodology to the Critical Analytical Attributes (CAAs), specifically retention time, peak area, and symmetry factor, by conforming to the method’s robustness. The combination of pregabalin and duloxetine hydrochloride (HCl) dosage form was determined using a straightforward, exact, specific, and accurate reverse-phase HPLC approach. The results showed retention times of 3.265 min and 4.318 min for duloxetine HCl and pregabalin, respectively. Pregabalin demonstrated linearity from 100 to 200 μg/mL (R2 = 0.998), whilst duloxetine HCl demonstrated linearity between 20 and 120 μg/mL (R2 = 0.997). Lower LOD values of 0.925 µg/mL and 0.853 μg/mL and LOQ values of 2.809 μg/mL and 2.587 μg/mL of pregabalin and duloxetine HCl, respectively, suggest good sensitivity for the technique. The drug content of the commercial formulation may thus be determined using the recommended method. This technique can be used for standard quality control studies to simultaneously estimate pregabalin and duloxetine HCl. The novelty of the present studies lies in the development of a robust RP-HPLC method for simultaneous estimation of pregabalin and duloxetine HCl using a systematic AQbD approach, enhancing robustness, reproducibility, and reliability, making it highly suitable for routine quality control and regulatory applications.

Biophysica doi: 10.3390/biophysica6020033

Authors: Sumedha Inukollu Shimantika Maikap Alexandra Lucaciu Prathyusha Yamarthi Anil Annamneedi Rajkumar Vutukuri

Ischemic stroke induces complex molecular responses that disrupt subcellular organelles’ function and contribute to brain injury, yet the temporal changes of organelle-specific transcriptomic remodeling remain to be investigated. In this study, we performed in silico analysis of publicly available transcriptomic data from isolated brain microvessels of transient middle cerebral artery occlusion (tMCAO) mouse model. Using in silico approaches, we analyzed differential gene expression at 24 h (acute phase) and 7 d (intermediate phase) post-stroke, focusing on mitochondria, endoplasmic reticulum (ER), and Golgi apparatus. Functional enrichment (Gene Ontology, KEGG) and protein–protein interaction network analyses were performed. Our analysis of the data revealed that at 24 h post-stroke, all three organelles exhibited marked transcriptional remodeling, where mitochondrial pathways showed disrupted metabolic and redox regulation; ER pathways indicated activation of biosynthetic processes, stress signaling, and ferroptosis; and Golgi-related genes reflected altered vesicular trafficking and glycosylation. By 7 d, mitochondrial alterations subsided, whereas ER and Golgi pathways displayed downregulation of metabolic and neuronal signaling processes, indicating persistent dysfunction and incomplete microvascular recovery. Phase-specific drug–gene interaction analysis will be useful to understand temporal organelle-associated transcriptional organization and to guide future investigations of neurovascular remodeling after ischemic stroke.

Biophysica doi: 10.3390/biophysica6020032

Authors: Ísis P. A. Perez Douglas G. Freitas Juliana Soares Marcos F. DosSantos Nathan B. Viana Bruno Pontes

The effects of static magnetic fields (SMFs) on fibroblast proliferation and migration remain debated, largely due to variability in field intensity, orientation, and exposure duration, as well as the predominant use of endpoint-based assays that may not fully capture the temporal dynamics of cellular responses. Thus, it remains unclear whether reported SMF effects reflect changes in proliferation, migration, or both. Here, we examined how SMFs with different field configurations affect NIH3T3 fibroblast behavior. Three setups were tested: a field generated by two neodymium magnets arranged in a face-to-face configuration on opposite sides of the culture dish (SMF1) and single-magnet setups with either the north (SMF2 and SMF2a) or south poles (SMF3 and SMF3a) facing the cells. SMF1 was associated with a 41% increase in proliferation relative to control, while single-cell migration velocities, directional persistence, and collective wound closure showed no detectable changes. In contrast, SMF2 and SMF3, as well as their low-field variants SMF2a and SMF3a, did not produce significant effects. Our results suggest that a specific SMF configuration is associated with increased fibroblast proliferation without detectable changes in migration parameters under the tested conditions. This integrative approach helps contextualize prior divergent findings by suggesting that SMF effects may be configuration-dependent, thereby contributing to a more rational application of magnetic stimulation in cellular and tissue engineering contexts.

Biophysica doi: 10.3390/biophysica6020031

Authors: Angela Albanese Manasi M. Natu Paul M. Seidler

Cellular protein quality control comprises the ubiquitin proteasome system, autophagy, and molecular chaperones, which maintain proteostasis in healthy tissues. The failure of these cellular and molecular pathways, which normally safeguard the proteome, can cause and even exacerbate amyloidoses, the abnormal accumulation of proteins into amyloid fibrils that drive neurodegeneration. Amyloidoses can also damage peripheral organs; examples include light chain amyloidosis, cardiac amyloidosis, and renal amyloidosis. Restoring proteostasis and preventing protein aggregation is therefore an active area of research, with several promising strategies under investigation. Among these approaches, small-molecule modulators that restore proteostasis are attractive candidates because they may simultaneously rescue multiple quality control mechanisms and remodel aggregates to improve their accessibility to endogenous degradation pathways. Here, we propose that amyloid pathology disrupts multiple proteostasis pathways simultaneously, creating a feedforward cascade in which the breakdown of interconnected proteostasis networks drives progressive protein aggregation, which in turn propels proteostasis collapse. Pharmacological interventions targeting protein aggregation offer opportunity to rescue interconnected proteostasis networks, which could, in turn, cooperatively manage or eliminate pathogenic amyloid burden.

Biophysica doi: 10.3390/biophysica6020030

Authors: Shih-Chu Jeff Liao Beniamino Barbieri Gerd Baumann Zeno Földes-Papp

This article addresses a current point of contention in the field of single-molecule/single-particle tracking, as well as the relevant literature, and supplements it with some published cell-based experiments to illustrate our conclusions and known theorems. We attempt to explain the controversy surrounding the differing biophysical and cell biological results of studies on the individual molecule and those “at the single-molecule level” as well as at the level of many molecules in such a way that even readers who are unfamiliar with the subject can understand it without having to read all the mathematical, physical, and biophysical references. Given this abundance of studies in the literature, it is obvious that genuine single-molecule studies are urgently needed, i.e., single-molecule studies that focus on increasing the sensitivity of the temporal resolution of single-molecule measurements and not just on spatial resolution.

Biophysica doi: 10.3390/biophysica6020029

Authors: Wafa Hourani Balakumar Chandrasekaran Sankar Muthumanickam Pandi Boomi

Background/Objectives: Until now, there have been no suitable medicines to treat infections caused by the Ebola virus. Cistus incanus, a traditional medicinal plant, contains several phytocompounds exhibiting antioxidant and anti-inflammatory properties. Methods: In this research, the molecular level interactions of the phytocompounds of Cistus incanus were investigated for their antiviral potential against the active site of VP40 protein of Ebola virus using in silico molecular docking. Further, the potential compounds were assessed for their stability in the protein using molecular dynamics (MD) simulations. Results: Methyl gallate, catechin, and quercetin showed excellent docking scores of −9.8, −8.8, and −7.7 kcal/mol, respectively, and favorable interactions with the target protein. These complexes showed good stability over the 100 ns MD simulation time. In addition, the phytocompounds displayed favorable pharmacokinetics and drug-like properties. Conclusions: Our study offers the antiviral potential of phytocompounds (methyl gallate, catechin, and quercetin) of Cistus incanus, suggesting their suitability as lead candidates for the treatment of Ebola viral infection.

Biophysica doi: 10.3390/biophysica6020028

Authors: Viacheslav V. Krylov Daniil A. Sizov Anastasia A. Sizova

The biological effects of weak low-frequency magnetic fields (LFMFs) remain controversial, particularly regarding frequency-specific resonance-like responses. Many previous studies tested different frequencies sequentially, potentially introducing uncontrolled environmental variability. This study aimed to evaluate frequency-dependent effects of LFMFs on the growth of juvenile Daphnia magna under strictly synchronized and temperature-controlled conditions. Genetically identical neonates from a single parthenogenetic brood were simultaneously exposed to sinusoidal 50 μT magnetic fields at 20, 25, 30, 35, or 40 Hz using spatially separated Helmholtz coils integrated into a closed-loop thermal stabilization system. Body length was measured after 48, 96, and 144 h of exposure. No significant growth differences were detected after 48 h. After 96 h, a significant biological effect was observed only at 30 Hz. The most pronounced responses occurred after 144 h, with significant growth stimulation at 25, 30, and 35 Hz and a maximal effect at 30 Hz. The frequency–response relationship exhibited a dome-shaped pattern that became less sharply peaked with prolonged exposure. These findings demonstrate duration-dependent and frequency-specific stimulation of juvenile daphnid growth with weak LFMFs. It suggests that exposure time critically influences the manifestation and breadth of resonance-like magnetobiological effects.

Biophysica doi: 10.3390/biophysica6020027

Authors: Lucien Bettendorff

Electron transfer is one of the most essential processes in biological systems. Redox reactions, either directly or indirectly, drive the main ATP-synthesizing pathways, especially those relying on a chemiosmotic mechanism, and as such, they are fundamental to photosynthesis and respiration. During biochemical redox reactions, electrons are transferred from a low-potential donor to a high-potential acceptor, mainly affecting the oxidation state of carbon atoms. The mechanism of electron transfer remains an intriguing enigma because of the wave-particle duality of subatomic particles. According to the biophysical conditions, electrons can be transferred by quantum tunneling or hopping from one redox site to another. While the driving force is always the electrochemical potential, a particularly interesting case is reversible electron bifurcation, where downhill (exergonic) redox reactions are coupled with uphill (endergonic) reactions by splitting the electrons of a two-electron donor. Here, we aim to discuss these different mechanisms in a comprehensive review accessible to students, teachers, and researchers in biological sciences.

Biophysica doi: 10.3390/biophysica6020026

Authors: Ixchel Ramírez-Camacho Fernando León Cedeño José Germán Vázquez Cuevas Eva Florencia Lejarazo Gómez Ulises Martínez-Ortega Mirthala Flores-García Ana María Mejía-Domínguez Aurora de la Peña-Díaz Fausto Alejandro Jiménez-Orozco

Novel pharmacological approaches advocate developing multitarget drugs, that is, molecules capable of simultaneously acting on two or more pharmacological targets to produce synergistic effects from a single compound in each disease. This strategy may help reduce required doses and prevent drug–drug interactions typically associated with polypharmacy. Coumarins are natural products with diverse pharmacological activities, including antioxidant, anti-inflammatory, anticancer, neuroprotective, cardioprotective, and antithrombotic effects. The pleiotropic actions of these molecules suggest that modifying the coumarin structure could yield new multi-target antiplatelet agents with greater efficacy and safety than those currently available in clinical practice. In this work, we began with a theoretical approach using molecular docking and designed three coumarins that simultaneously inhibited platelet aggregation induced by epinephrine, collagen, and ADP. Experimentally, we evaluated the structure activity relationship of three coumarins: (A) 6,7-dimethoxy-3-(1H-pyrrol-1-yl)-2H-chromen-2-one, (B) 7,8-dimethoxy-3-(1H-pyrrol-1-yl)-2H-chromen-2-one, and (C) 3-(1H-imidazol-1-yl)-6,7-dimethoxy-2H-chromen-2-one. In silico studies suggest that compounds B and C may exhibit antagonistic interactions at the α2-adrenergic, GPVI collagen, and P2Y12 ADP receptors. Additionally, molecular docking indicates essential interactions between the compounds and the GPIIb/IIIa fibrinogen receptor.

Biophysica doi: 10.3390/biophysica6020025

Authors: Ahmed Shahat Belal Gabriel Tchuente Kamsu Ahmed A. Al-Kubaisi Cromwel Tepap Zemnou

The common metabolic disorder, lactose intolerance, is often treated with oral lactase enzyme supplements, which can frequently cause gastrointestinal instability. This work utilizes Malbranchea cinnamomea’s AA9 lytic polysaccharide monooxygenase (LPMO) to target β-lactose (β-lactose) in an investigation of a new enzymatic approach for lactose breakdown. Potential possibilities for lactose breakdown are AA9 LPMOs, copper-dependent enzymes that oxidatively cleave glycosidic bonds in polysaccharides. We employed a combined in silico method that incorporated molecular docking, density functional theory (DFT) calculations, and molecular dynamics (MD) simulations. Docking studies revealed that β-lactose formed hydrogen bonds with key residues SER100, ASN54, and ARG56, exhibiting a greater binding affinity (−5.4 kcal/mol) toward LPMO compared to the control citric acid (−4.9 kcal/mol). Upon DFT analysis, (LPMO) showed excellent stability and appropriate reactivity for enzyme interaction. The higher stability of the LPMO-β-lactose complex was highlighted by MD simulation over 100 ns, which showed lower root mean square deviation (RMSD) and root mean square fluctuation (RMSF) values, greater structural compactness, and reduced solvent accessibility when compared to the control. These collective findings suggest that β-lactose interacts efficiently with the AA9 LPMO active site, supporting its potential as a novel enzymatic target for lactose degradation. This computational study provides a theoretical foundation for developing alternative therapeutic strategies for lactose intolerance, though further in vitro and in vivo investigations are required to validate these findings.

Biophysica doi: 10.3390/biophysica6020024

Authors: Hong Luo Jie He Zhichao Liu Chen Zeng

Molecular representation learning (MRL) has garnered significant attention due to its pivotal role in downstream applications such as molecular property prediction and drug discovery. In most MRL approaches, molecules are encoded into 2D topological graphs via graph neural network (GNN), which suffers from over-smoothing issues and limited receptive fields. Furthermore, most GNN models fail to utilize the 3D spatial structural information that determines molecular physicochemical properties and biological activity. To this end, here we propose multimodal contrast-enhanced molecular representation learning (MCMRL). This approach utilizes both the 2D topological information and 3D structural information of molecules for contrastive learning to enhance molecular graph representations. Further, it integrates additional molecular fingerprint information and feature fusion techniques to incorporate multimodal knowledge, yielding more reliable and generalizable molecular representations. MCMRL is pre-trained on ~10 million unlabeled molecules from PubChem, followed by various downstream benchmark tasks. Experimental results demonstrate that MCMRL achieves superior performance in 9 out of 13 benchmark tests for molecular property prediction, validating its effectiveness in molecular representation learning. Furthermore, potential molecular drugs binding to biological target protein DRD2 screened by MCMRL representation show promising affinity score, which also demonstrates the efficacy of the proposed method.

Biophysica doi: 10.3390/biophysica6020023

Authors: Joachim Storsberg Sophia Rosencrantz Ruben R. Rosencrantz

The interface between biomaterials and biological systems is crucial for medical implants and tissue engineering. Surface modifications are a key strategy for controlling interactions. Synthetic glycopolymers offer a versatile toolbox, mimicking the structure and function of natural glycoconjugates like mucins. This review highlights the significance of glycopolymers for targeted surface modifications of established biomaterials, such as silicones and poly(meth)acrylates. Controlled polymerization techniques, like the reversible-addition-fragmentation chain-transfer (RAFT) polymerization, enable the synthesis of well-defined glycopolymer architectures. Glycopolymeric surface functionalization creates tailored interfaces for different biological responses, from preventing protein and cell adhesion to promoting specific cell-type binding. The focus lies on using single, well-characterized polymeric base materials and tuning their surface properties through glycopolymer coatings to achieve various and specific functions. This approach opens new dimensions in the development of advanced biomaterials for applications like contact lenses, drug delivery systems, and biosensors and also possesses potential regulatory advantages by leveraging the safety profiles of existing materials.

Biophysica doi: 10.3390/biophysica6020022

Authors: Mariia Riabova Kelly C. Grayson Samantha Y. A. Terry

The accurate measurement of spheroid area and morphology is critical for the progression of the integration of 3D models in in vitro cancer research and is increasingly used to measure effective therapeutic efficacy of X-ray radiation. Current methods of measuring spheroids require labour-intensive manual analysis or the use of complex software tools. SphereMetrics was created as a user-friendly Shiny app with a straightforward interface designed to streamline the process of measuring the area and eccentricity of spheroids. It allows the upload and automated detection of spheroids across multiple file formats and generates robust and objective area and eccentricity measurements. Area measurements derived from SphereMetrics were compared to manual quantification with ImageJ and AnaSP for untreated and irradiated (0–20 Gy) human neuroendocrine BON-1 cancer spheroids. When compared to ImageJ and AnaSP, SphereMetrics was shown to provide fast, accurate data (R2 = 0.87 and 0.83, respectively). Spheroid analysis took 19.92 ± 8 s/image with SphereMetrics, approximately four times faster than ImageJ analysis (89.81 ± 11.52 s/image) and nine times faster than AnaSP (183.36 ± 31.62 s/image). SphereMetrics represents an accessible and efficient tool for spheroid analysis, facilitating data collection and analysis for routine in vitro model research, ideal for non-programmers.

Biophysica doi: 10.3390/biophysica6020021

Authors: Mutlay Keskin

Testicular metabolism can be non-invasively assessed using FDG-PET/CT, which provides insights into physiological and age-related changes. Understanding normal testicular FDG uptake is essential to distinguish between benign variation and pathological findings. In this retrospective study, 80 men (mean age: 54.7 years, range: 26–79) who underwent PET/CT for lung nodule evaluation were analyzed, excluding individuals with testicular disease, prior surgery, or elevated blood glucose (>180 mg/dL). FDG uptake (SUV_mean) and testicular volume were measured for each testis, and correlations with age, blood glucose, and volume were assessed using standard statistical methods. The mean testicular SUV_mean was 2.62 ± 0.50, showing a significant negative correlation with age and a weak positive correlation with testicular volume. After adjusting for volume, the negative association with age persisted, while no significant relationship with blood glucose was observed. These findings indicate that physiological testicular FDG uptake gradually declines with age, reflecting both metabolic and structural alterations. Recognizing these normal patterns is critical for accurate PET/CT interpretation and reducing the risk of false-positive findings.

Biophysica doi: 10.3390/biophysica6020020

Authors: Muthamil Balakrishnan Janardanan Kumar Jaison Jacob Mathunny Varshini Karthik Ashok Kumar Devaraj

Sarcopenia is a degenerative musculoskeletal condition recognised as the age-related decline in skeletal muscle mass, strength, and function. Traditional diagnostic methods are limited by cost, accessibility, and subjectivity. This study aimed to develop a non-invasive, AI-driven, video-based framework for early Sarcopenia detection using functional movement analysis. Participants with and without Sarcopenia were recorded performing functional movements such as level walking, stair climbing, and ramp walking. Ten representative frames were extracted from each participant, resulting in 300 images (150 Sarcopenic, 150 non-Sarcopenic) utilised for the study. The DeepSarcAE model is an integrated framework of an autoencoder and a CNN-based classifier. Its performance was benchmarked against pretrained architectures such as EfficientNet, ResNet, MobileNet, Inception, VGG16 and four custom CNN models. Evaluation metrics such as sensitivity, specificity, precision, negative predictive value (NPV), accuracy, and AUC were used to analyse the models. DeepSarcAE outperformed all other models, attaining 100% sensitivity, 83.33% specificity, 85.71% precision, 100% NPV, 91.67% accuracy, and an AUC of 0.96. VGG16 and MobileNet followed the performance of DeepSarcAE closely, while the Inception network exhibited the weakest results due to poor generalisation. TheDeepSarcAE framework offers a scalable, cost-effective, and non-invasive approach for Sarcopenia screening from the input gait image frames. Its promising preliminary performance highlights the potential of deep learning in early diagnosis and clinical decision support in preventive healthcare.

Biophysica doi: 10.3390/biophysica6020019

Authors: Hari Krishnareddy Rachamala Sreya Roy Srujan Marepally

Lipid-based delivery systems (LDS), including lipid nanoparticles (LNPs) and liposomes, have become indispensable tools in modern biomedicine owing to their biocompatibility, capacity to encapsulate diverse therapeutic agents, and potential for targeted delivery. Despite their clinical success, conventional batch-based manufacturing methods are hindered by variability, limited scalability, and complex processing steps, slowing their broader translation. Microfluidic technologies offer a transformative solution by enabling precise fluid handling, rapid mixing, and reproducible production of LDS with tunable physicochemical attributes such as particle size, lamellarity, and drug-loading efficiency. This review highlights advances in microfluidic design strategies, including hydrodynamic flow focusing, staggered herringbone mixers, and toroidal micromixers, and evaluates how critical parameters such as flow rate, solvent composition, and lipid concentration influence LDS performance. Furthermore, we discuss the application of microfluidics in drug delivery, nucleic acid therapeutics, and vaccine platforms, underscoring its role in improving scalability, quality control, and clinical translation. Finally, we examine current challenges, including throughput limitations and solvent handling, while outlining future directions for integrating emerging materials and additive manufacturing to optimize LDS fabrication. Collectively, microfluidic platforms provide a promising pathway for next-generation lipid nanomedicines with enhanced precision, reproducibility, and therapeutic efficacy.

Biophysica doi: 10.3390/biophysica6020018

Authors: Baatarmanlai Dorjgochoo Delgernaran Gomboragchaa Odonchimeg Munkhjargal Baatarjargal Purevdorj Enkhgerel Baljinnyam Idertungalag Sambuu-Yondon Amgalanzaya Dorjgochoo Enkhtaivan Erdene

Antimicrobial resistance represents not only a biological challenge but also a physicochemical limitation associated with antibiotic transport, membrane interaction, and local availability. In this preliminary study, a liposome-encapsulated doxycycline delivery system was developed using egg yolk-derived phospholipids, and its biophysical properties and release behavior were investigated. Phospholipids were isolated from egg yolk and used to prepare doxycycline-loaded liposomes via a thin-film hydration method combined with freeze–thaw processing. Liposome morphology was characterized by atomic force microscopy (AFM), while encapsulation efficiency was quantified by reversed-phase high-performance liquid chromatography (RP-HPLC). In vitro release kinetics were evaluated using a dialysis diffusion method, and antibacterial activity was assessed as a functional indicator of drug availability using minimum inhibitory concentration (MIC) assays against Staphylococcus aureus and methicillin-resistant S. aureus (MRSA). The prepared liposomes exhibited morphology with diameters of approximately 153 nm (PDI = 0.223). The encapsulation efficiency of doxycycline hyclate was 8.41%, and complete drug release was achieved within 48 h. Liposome-encapsulated doxycycline demonstrated a two-fold reduction in MIC values compared with free doxycycline. These findings offer preliminary insight to support further optimization and expanded investigation of liposome-encapsulated antibiotic delivery systems.

Biophysica doi: 10.3390/biophysica6020017

Authors: Héctor Enrique Trujillo-Ruiz Dania Guadalupe Leal-Rodríguez Hisila del Carmen Santacruz-Ortega Oliviert Martínez-Cruz Sandra Carolina De La Reé-Rodríguez Armando Burgos-Hernández Erika Silva-Campa Ángel Antonio Carbonell-Barrachina Carmen María López-Saiz

Cancer is the second leading cause of death worldwide, requiring more effective treatments. By-products from the white shrimp (Penaeus vannamei) are a promising source of bioactive compounds. Compounds with antiproliferative activity were isolated and identified in exoskeleton and cephalothorax extracts. The hexane extract of the exoskeleton reduced the viability of Human Prostate Carcinoma cell line (22Rv1) to 40.6% without toxicity in Adult Retinal Pigment Epithelium-19 (ARPE-19). Among the 19 fractions obtained, H3 reduced cell viability to 20.78%. Spectroscopic analysis identified bis(2-ethylhexyl) terephthalate, neoxanthin, and violaxanthin. Fluorescence microscopy showed morphological alterations. These findings demonstrate in vitro antiproliferative activity of compounds derived from shrimp by-products and support further studies to elucidate their mechanisms of action and evaluate their potential relevance in cancer prevention or therapeutic research.

Biophysica doi: 10.3390/biophysica6020016

Authors: Manuel Londa Vueba Ana Figueiras Luis Alberto Torres Goméz

Traditional drug design methods based on trial and error are costly and inefficient. The computational approach ToSS-MoDE (Topological Substructural Molecular Design) offers an alternative by linking molecular descriptors to biological activity. To develop a QSAR model to predict the anti-inflammatory activity of synthetic and natural compounds using weighted spectral moments. Spectral moments (µk) were calculated from the adjacency matrix between bonds for 410 compounds (180 active and 230 inactive). MODESLAB software (MICROSOFT OFFICE 365) was used to generate descriptors, and Linear Discriminant Analysis (LDA) was applied to classify activity. The model was validated with an external series of 62 compounds. Results. The model showed an overall classification of 91.59% in the training series and 90.2% in validation. The spectral moments µ0, µ3, µ4, and µ5 were the most significant. Diosgenin, a natural metabolite, showed potential anti-inflammatory activity (classification probability: 81%). The model showed strong training performance (91.7% accuracy) and promising external performance for confidently classified compounds. All datasets, descriptor-generation settings, coefficients, and posterior probabilities are fully described in the main text to ensure full reproducibility.

Biophysica doi: 10.3390/biophysica6010015

Authors: Ahlam Azalmad Mehdi El Ouartiti Mohamed Abour Mohamed Hilal

This retrospective planning study evaluated how arc number (AN) and control-point density (CP) affect VMAT quality, radiobiological endpoints, and workflow efficiency for locally advanced cervical cancer in a resource-conscious setting. Twenty-one patients (FIGO IIB–IIIB) were replanned in Monaco v5.51 (Monte Carlo) for 46 Gy using 6-MV beams (Elekta) with 1–4 coplanar arcs, and dual-arc plans were further analyzed using ≈250, 300, 350, and 400 CP per arc. Target coverage (D98/D95/V95/V98), conformity and homogeneity (CI, HI), and organs-at-risk (OARs) DVH metrics (including D2cc and Vx) were compared alongside monitor units, planning time, and delivery time. Increasing AN improved dose conformity and OAR sparing relative to single-arc plans, whereas increasing CP produced only modest dosimetric changes but substantially increased planning and treatment times. Radiobiological modeling using BED/EQD2 and EUD-based LKB NTCP indicated negligible bladder risk (<0.01%) and low rectal risk (<0.2%), but a higher small-bowel NTCP (~26%) driven by hotspot-sensitive descriptors; Niemierko TCP estimates were similar between leading dual-arc CP settings. Overall, a dual-arc strategy with ~250 CP per arc provided the most practical balance between plan quality, estimated biological effect, and deliverability.

Biophysica doi: 10.3390/biophysica6010014

Authors: Juan S. Jiménez

Protein folding displays a very narrow range of stability energy as measured by the ΔG° value associated with the process of protein folding from the unfolded sequence of amino acids defining a particular protein. We have recently reported the Gaussian distribution of ΔG° values obtained from a Data Base, constituted by more than five hundred data points from protein folding processes. An average value of ΔG° = −30.9 kJ/mol was obtained. Considering that a protein solution may include thousands of hydrogen bonds in water solution, this is an extremely low value, approximately equivalent to two hydrogen bonds. The enthalpy–entropy compensation has been repeatedly used as the main empirical mechanism, based in classical thermodynamics, to explain the low energy involved in stabilizing proteins. I present here a different approach, based on the thermodynamics of steady states in open systems, in an attempt to complement the achievements obtained through the EEC. The main conclusions explain the narrow range of ΔG° values reported previously as a consequence of environmental conditions, such as the protein solubility, and the plausible concentration, “in vivo,” of both protein forms, folded and unfolded. Particularly relevant is the condition of increasing the entropy of irreversible processes inside adiabatic systems. It is also worth noting the observation that, according to the results obtained from the theoretical model used, any protein could be folded with slightly different stability values of ΔG°, following the theorem of minimum entropy production rate.

Biophysica doi: 10.3390/biophysica6010013

Authors: Arturo Ponce

Ion channels are fundamental membrane proteins that mediate selective ion flow across biological membranes and thereby govern excitability, signaling, and homeostasis in virtually all cell types. Although channel function is determined by intrinsic structural features, the surrounding lipid milieu is now recognized as a decisive regulatory layer. Lipids tune ion channel activity through complementary mechanisms: they can bind directly to channel proteins, reshape bilayer physical properties, or act as signaling messengers that couple extracellular cues to channel gating. In addition, the organization of membranes into lipid microdomains such as rafts and caveolae can cluster channels with receptors and scaffolds, enhancing signaling specificity and efficiency. Recent advances in cryo-electron microscopy and molecular simulations have expanded our understanding of these lipid–channel interactions, revealing lipids as active modulators rather than passive structural components. This review provides a comprehensive overview of the principles by which lipids regulate ion channel function and highlights the biological and potential clinical significance of this fundamental interplay.

Biophysica doi: 10.3390/biophysica6010012

Authors: Indu Passi Nisha Bansal Thakur Gurjeet Singh Bhupinder Kumar

Neurodegenerative conditions such as Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, and amyotrophic lateral sclerosis (ALS) are devastating disorders with the gradual loss of neurons and cognitive or motor disability. This is a review article that develops the crucial functions of autophagy and proteostasis within the scope of the neurodegenerative disease. Autophagy is a very well-conserved cell process that poses a quality control checkpoint that allows for the degradation and recycling of damaged organelles and misfolded proteins. Chaperones, the ubiquitin–proteasome complexes, and endoplasmic reticulum-associated degradation (ERAD) are also referred to as proteostasis, which plays a key role in ensuring the correct protein folding properties and the prevention of toxic protein accumulation. This article offers a detailed discussion of the relationship between autophagy and proteostasis, illustrating the mechanisms of mutual cooperation of these processes, ensuring cellular homeostasis and inhibiting the formation of pathogenic protein aggregates. In addition, this review includes experimental data during preclinical studies and clinical trials and expounds on the therapeutic opportunities that show the potential of targeting autophagy and proteostasis to counter neurodegenerative disorders. Although research progress creates potential for new indicators, the issues and difficulties relating to the effects of regulating such complex cellular pathways are also discussed in the article. Finally, the review can add to the research of neurodegenerative disease mechanisms of autophagy and proteostasis as well as provide insights about the future of treatment and its implementation.

Biophysica doi: 10.3390/biophysica6010011

Authors: Anju Kumari Kuldeep Singh Seemasundari Yumlembam Hamidur Rahaman Mohd Saquib Ansari Laishram Rajendrakumar Singh

Peroxiredoxin 6 (Prdx6) is a bifunctional antioxidant enzyme with glutathione peroxidase and phospholipase A2 activities that plays an essential role in cellular redox regulation. However, the modulation of Prdx6 activity by endogenous small metabolites remains poorly understood. In this study, we investigated the effect of β alanine on Prdx6 structure and function using biochemical, biophysical, computational, and cellular approaches. Enzymatic assays revealed that β alanine enhances the peroxidase activity of Prdx6 in a dose-dependent manner. Spectroscopic analyses demonstrated β alanine-induced conformational stabilization of Prdx6, which was further supported by increased thermal stability. Molecular docking and molecular dynamics simulations identified a stable interaction of β alanine at a distinct allosteric site on Prdx6, accompanied by reduced local flexibility. In a proof-of-concept cellular system, β alanine treatment resulted in a significant reduction in intracellular reactive oxygen species, consistent with enhanced Prdx6-associated antioxidant activity. Collectively, these findings identify β alanine as a biochemical modulator of Prdx6 activity. The study is limited to mechanistic and cellular redox regulation and does not address tissue- or disease-specific physiology.

Biophysica doi: 10.3390/biophysica6010010

Authors: Shujie Yang Shuangling Zhang Min Chen Dongxiao Ma Yuxuan Sun Xiao Zhang Jing Zhang Xiaohang Zheng Han Zheng

Fish collagen is derived from processing residues of marine and freshwater fish (such as fish skin, scales, and bones), primarily composed of amino acids including glycine, proline, and hydroxyproline. It functions include maintaining tissue integrity and promoting cell proliferation and repair. Extraction methods primarily include acid, alkali, enzymatic, and physical approaches, while purification techniques involve gel filtration chromatography, ultrafiltration, and precipitation. Furthermore, thermal instability, insufficient mechanical strength, immunological concerns, and biocompatibility limitations restrict its application across various fields. This review summarizes the composition, extraction, purification, and existing challenges of fish collagen, proposing improvement strategies. It systematically addresses issues related to fish collagen’s biocompatibility, filling a gap in the literature. However, effectively enhancing its biocompatibility remains an urgent priority. Approaches such as nanotechnology and composite material development offer novel avenues for improving biocompatibility and future applications.

Biophysica doi: 10.3390/biophysica6010009

Authors: Gabriel Gonzalo Taco-Gárate Sandra Esther Loa-Guizado Corina Vera-Gonzales Herly Fredy Zegarra-Aragon Juan Aquino-Puma Carlos Alberto Arenas-Chávez

Cancer is one of the leading causes of mortality worldwide, with breast and colon cancers being among the most common neoplasms in men and women, respectively. Despite significant advancements in treatment, there is a pressing need to enhance specificity and reduce systemic side effects. Importantly, a distinctive feature of cancer cells is their acidic extracellular environment, which profoundly influences cancer progression. In this study, we evaluated the anticancer activity of a pH-sensitive nanocomposite based on silver nanoparticles and pegylated carboxymethyl chitosan (AgNPs-CMC-PEG) in breast cancer (MCF-7) and colon cancer (HCT 116) cell lines. To achieve this, we synthesized and characterized the nanocomposite using UV-Vis spectroscopy, Dynamic Light Scattering (DLS), Fourier-Transform Infrared Spectroscopy (FT-IR), and Scanning Electron Microscopy (STEM-in-SEM). Furthermore, we assessed cytotoxic effects, apoptosis, and reactive oxygen species (ROS) generation using MTT, DAPI, and H2DCFDA assays. Additionally, we analyzed the expression of DNA methyltransferases (DNMT3a) and histone acetyltransferases (MYST4, GCN5) at the mRNA level using RT-qPCR, along with the acetylation and methylation of H3K9ac and H3K9me2 through Western blot analysis. The synthesized nanocomposite demonstrated an average hydrodynamic diameter of approximately 175.4 nm. In contrast, STEM-in-SEM analyses revealed well-dispersed nanoparticles with an average core size of about 14 nm. Additionally, Fourier-transform infrared (FTIR) spectroscopy verified the successful surface functionalization of the nanocomposite with polyethylene glycol (PEG), indicating effective conjugation and structural stability. The nanocomposite exhibited a pH and concentration dependent cytotoxic effect, with enhanced activity observed at an acidic pH 6.5 and at concentrations of 150 µg/ml, 75 µg/ml, and 37.5 µg/ml for both cell lines. Notably, the nanocomposite preferentially induced apoptosis accompanied by ROS generation. Moreover, expression analysis revealed a decrease in H3K9me2 and H3K9ac in both cell lines, with a more pronounced effect in MCF-7 at an acidic pH. Furthermore, the expression of DNMT3a at the mRNA level significantly decreased, particularly at acidic pH. Regarding histone acetyltransferases, GCN5 expression decreased in the HCT 116 line, while MYST4 expression increased in the MCF-7 line. These findings demonstrate that the AgNPs-CMC-PEG nanocomposite has therapeutic potential as a pH-responsive nanocomposite, capable of inducing significant cytotoxic effects and altering epigenetic markers, particularly under the acidic conditions of the tumor microenvironment. Overall, this study highlights the advantages of utilizing pH-sensitive materials in cancer therapy, paving the way for more effective and targeted treatment strategies.

Biophysica doi: 10.3390/biophysica6010008

Authors: Salvatore Passarella

Mitochondria serve as central hubs of cellular metabolism, integrating catabolic and anabolic pathways through the controlled exchange of metabolites across their membranes. Although mitochondrial transport of several metabolites has been well documented, the mechanisms underlying the trafficking of fumarate, glutamine, and phosphoenolpyruvate as well as the role of the mitochondrial pyruvate kinase remain insufficiently represented in modern biochemistry textbooks. Here, we revisit the biochemical evidence supporting specific transport activities for these metabolites, discuss their physiological roles in major metabolic pathways, and highlight how foundational experimental studies have been overlooked in contemporary literature. Re-examining these mechanisms provides new insight into the dynamic interplay between mitochondrial function, cytosolic metabolism, and overall cellular homeostasis.

Biophysica doi: 10.3390/biophysica6010007

Authors: Yanara Alessandra Santana Moura Andreza Pereira de Amorim Maria Carla Santana de Arruda Marllyn Marques da Silva Ana Lúcia Figueiredo Porto Vladimir N. Uversky Raquel Pedrosa Bezerra

Thrombosis is a cardiovascular disease characterized by the pathological formation of a fibrin clot in blood vessels. Currently available fibrinolytic enzymes have some limitations, including severe side effects, high cost, short half-life, and low fibrin specificity. Proteins from microalgae and cyanobacteria have various biological effects and are emerging as promising sources for fibrinolytic enzymes. In this study, bioinformatics tools were used to evaluate the intrinsic disorder predisposition of microalgal fibrinolytic proteins, their capability to undergo liquid–liquid phase separation (LLPS), and the presence of disorder-based functional regions, and short linear motifs (SLiMs). Analysis revealed that these proteins are predominantly hydrophilic and exhibit acidic (pI 3.96–6.49) or basic (pI 8.05–11.0) isoelectric points. Most of them are expected to be moderately (61.4%) or highly disordered proteins (6.8%) and associated with LLPS, with nine proteins being predicted to behave as droplet drivers (i.e., being capable of spontaneous LLPS), and twenty-five proteins being expected to be droplet clients. These observations suggest that LLPS may be related to the regulation of the functionality of microalgal fibrinolytic proteins. The majority of these proteins belong to the blood coagulation inhibitor (disintegrin) 1 hit superfamily, which can inhibit fibrinogen binding to integrin receptors, preventing platelet aggregation. Furthermore, the SLiM-centered analysis indicated that the main motifs found in these proteins are MOD_GlcNHglycan and CLV_PCSK_SKI1_1, which can also play different roles in thrombolytic activity. Finally, Fisher and conservation analysis indicated that CLV_NRD_NRD_1, CLV_PCSK_FUR_1, CLV_PCSK_PC7_1, and MOD_Cter_Amidation motifs are enriched in intrinsically disordered regions (IDRs) of these proteins, showing significant conservation and suggesting compatibility with proteolytic activation and post-translational processing. These data provide important information regarding microalgal proteins with potential thrombolytic effects, which can be realized through protein–protein interactions mediated by SLiMs present in intrinsically disordered regions (IDRs). Additional analyses should be conducted to confirm these observations using experimental in vitro and in vivo approaches.

Biophysica doi: 10.3390/biophysica6010006

Authors: Liqun Zhang

ORF8 is the second most mutated protein in SARS-CoV-2. It can form oligomers such as trimers and can bind to the IL-17RA/RC receptor. To understand the possible role of ORF8 in SARS-CoV-2, the first step of this study involved predicting the ORF8 trimer structure and the complex structure of the ORF8 monomer bound to the IL-17RA receptor using docking and molecular dynamics simulation methods. It was found that ORF8 molecules bound to the central ORF8 molecule through covalent and noncovalent interactions exhibit similar RMSD and RMSF values as the central ORF8 molecule and form a similar buried surface area, but display different numbers of hydrogen bonds and varying dynamic correlations. Additionally, trimer formation increases the dynamic correlation of the noncovalently bound ORF8 unit. ORF8 can bind with the IL-17RA receptor stably. Regions on ORF8, including C25–I47, L60–S67, T80–C90, and S103–E110, and regions on IL-17RA, including L1–H63 and D122–M165, are involved in the binding interface of the complex. ORF8 becomes less rigid when bound to IL-17RA than in its monomer, dimer, and trimer forms. Based on dihedral angle correlation predictions, binding of ORF8 to IL-17RA reduces internal correlations within ORF8 while strengthening correlations within IL-17RA. The G50–T80 region of ORF8 appears to be critical for interaction with IL-17RA, and the L1–V150 region of IL-17RA should be critical for its dynamics once bound to ORF8. These results help elucidate the structure and dynamics of ORF8 in SARS-CoV-2.

Biophysica doi: 10.3390/biophysica6010005

Authors: Melissa L. Knothe Tate Sara McBride-Gagyi Eric J. Anderson Lucy Ngo

Here we present a retrospective, integrative review of the approaches and discoveries of our “collaboratory”, a meta-laboratory comprising cross-disciplinary collaborations across laboratories at fourteen different universities and clinics in seven different countries with shared lead investigators. By tying together insights from four decades of research and discovery, applied across cell types, as well as different tissues, organ systems, and organisms, we have aimed to elucidate the interplay between organisms’ movement and the physiology of their tissues, organs, and organ systems’ resident cells. We highlight the potential of increasing imaging and computing power, as well as machine learning/artificial intelligence approaches, to delineate the Laws of Biology. Codifying these laws will provide a foundation for the future, to promote not only the discovery of underpinning mechanisms but also the sustainability of our natural resources, from our brains to our bones, which serve as veritable “hard drives”, physically rendering a lifetime of cellular experiences and millennia of evolution.

Biophysica doi: 10.3390/biophysica6010004

Authors: William P. Heaps Anne Elise Packard Kristina M. McCammon Tyler P. Green Joseph P. Talley Bradley C. Bundy Dennis Della Corte

The glomerular filtration barrier poses a significant challenge for circulating proteins, with molecules below ~60–70 kDa facing rapid renal clearance. Endogenous proteins have evolved sophisticated evasion mechanisms including oligomerization, carrier binding, electrostatic repulsion, and FcRn-mediated recycling. Understanding these natural strategies provides blueprints for engineering therapeutic proteins with improved pharmacokinetics. This review examines how endogenous proteins resist filtration, evaluates their application in protein engineering, and discusses clinical translation including established technologies (PEGylation, Fc-fusion) and emerging strategies (albumin-binding domains, glycoengineering). We address critical challenges of balancing half-life extension with tissue penetration, biological activity, and immunogenicity—essential considerations for the rational design of next-generation therapeutics with optimized dosing and enhanced efficacy.

Biophysica doi: 10.3390/biophysica6010003

Authors: Sadia Tina Oussama Khibech Ali Zourif Samy Iskandar Kettani Halabi Mohamed Martin Ndayambaje Balouch Lhousaine Meryem El Jemli

Cedrus atlantica wood tar (CAWT) is traditionally used as a medicinal product, especially in low- and middle-income countries. Despite its traditional use, scientific support for its efficacy remains limited. This study evaluated the biological properties of CAWT using an integrated approach that combined qualitative and quantitative phytochemical analysis, disc diffusion and microdilution tests for antimicrobial assays (disc diffusion and microdilution), antioxidant activity (DPPH and ferric-reducing power assays), in silico ADMET/toxicity, docking, and MD/MMGBSA and provided a balanced comparison with reference antioxidants. This study demonstrated that CAWT is rich in secondary metabolites linked to biological activity, including polyphenols (307.39 ± 58.45 mg GAE/g), tannins (124.42 ± 6.14 mg TAE/g), and flavonoids (15.62 ± 2.53 mg QE/g). For free radical scavenging, CAWT inhibited DPPH with an IC50 of 19.781 ± 2.51 µg/mL and showed ferric-reducing activity with an IC50 of 83.7 ± 2.88 µg/mL for its antimicrobial activity against Pseudomonas aeruginosa; inhibition zones reached 35.66 ± 0.58 mm. In silico analysis, Swiss ADMET and pkCSM predicted ≥94% intestinal absorption, no cytochrome P450 liabilities, and low acute toxicity for six dominant terpenoids. Docking pinpointed trans-cadina-1(6),4-diene and α/β-himachalene as high-affinity ligands of LasR and gyrase B (ΔG ≈ −8 kcal mol−1). A 100 ns GROMACS run confirmed stable hydrophobic locking of the lead LasR complex (RMSD 0.22 nm), while MM/GBSA calculated a dispersion-dominated binding free energy of −37 kcal mol−1. Overall, CAWT showed in vitro antioxidant activity (DPPH and ferric-reducing assays) and inhibitory effects in disc diffusion assays, while in silico predictions for major terpenoids suggested favorable oral absorption and low acute toxicity. However, chemical composition analysis and bio-guided fractionation are necessary to confirm the antimicrobial activity and to validate the compounds responsible for the observed effects.

Biophysica doi: 10.3390/biophysica6010002

Authors: Adhikarimayum Priya Devi Seemasundari Yumlembam Kuldeep Singh Akshita Gupta Kananbala Sarangthem Laishram Rajendrakumar Singh

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder characterized by memory loss, cognitive decline, and oxidative stress-driven neuronal damage. Catalase, a key antioxidant enzyme, plays a vital role in decomposing hydrogen peroxide (H2O2) into water and oxygen, thereby protecting neurons from reactive oxygen species (ROS)-mediated toxicity. In AD, the catalase function is compromised due to reduced enzymatic activity and aggregation, which not only diminishes its protective role but also contributes to amyloid plaque formation through catalase-Aβ co-oligomers. Hence, therapeutic strategies aimed at simultaneously preventing catalase aggregation and enhancing its enzymatic function are of great interest. In this study, we screened twelve naturally occurring metabolites for their ability to modulate catalase aggregation and activity. Among these, dimethylglycine (DMG) emerged as the most potent candidate. DMG significantly inhibited thermally induced aggregation of catalase and markedly enhanced its enzymatic activity in a concentration-dependent manner. Biophysical analyses revealed that DMG stabilizes catalase by promoting its native folded conformation, as evidenced by increased melting temperature (Tm), higher Gibbs free energy of unfolding (ΔG°), and reduced exposure of hydrophobic residues. TEM imaging and Thioflavin T assays further confirmed that DMG prevented amyloid-like fibril formation. Molecular docking and dynamics simulations indicated that DMG binds to an allosteric site on catalase, providing a structural basis for its dual role in stabilization and activation. These findings highlight DMG as a promising therapeutic molecule for restoring catalase function and mitigating oxidative stress in AD. By maintaining catalase stability and activity, DMG offers potential for slowing AD progression.

Biophysica doi: 10.3390/biophysica6010001

Authors: Ossama Daoui Achraf Daoui Mohamed Yamni Marouane Daoui Souad Elkhattabi Samir Chtita Chakir El-Kasri

Making advancements in Quantitative Structure-Activity Relationship (QSAR) modeling is crucial for predicting biological activities in new compounds. Traditional 2D-QSAR and 3D-QSAR methods often face challenges in terms of computational efficiency and predictive accuracy. This study introduces a machine learning approach using 3D Discrete Tchebichef Moments (3D-DTM) to address these issues. The 3D-DTM method offers efficient computation, robust descriptor generation, and improved interpretability, making it a promising alternative to conventional QSAR techniques. By capturing global 3D shape information, this method provides better representation of molecular interactions essential for biological activities. We applied the 3D-DTM model to a dataset of 46 molecules derived from the Dihydrophenanthrene scaffold, screened against the enzymatic activity of 3-chymotrypsin-like protease, a key antiviral target. Principal Component Analysis and k-means clustering refined descriptors, followed by stepwise Multiple Linear Regression (step-MLR), Partial Least Squares Regression (PLS-R), and Feed-Forward Neural Network (FFNN) techniques for 3DTMs-QSAR model development. The results showed high correlation and predictive accuracy, with significant validation from internal and external tests. The step-MLR model emerged as the optimal method due to its balance of predictive power and simplicity. Validation through y-Randomization and applicability domain analysis confirmed the model’s robustness. Virtual screening of 100 novel compounds identified 32 with improved pIC50 values. This study highlights the potential of 3D-DTMs in QSAR modeling, providing a scalable and reliable tool for computational chemistry and drug discovery. A user-friendly software tool was also developed to facilitate 3D-DTM extraction from input 3D molecular images.

Biophysica doi: 10.3390/biophysica5040063

Authors: Asmaa Al-Failakawi Aishah Al-Jarallah Muddanna Rao Islam Khan

We investigated the roles and regulation of contractile and sodium ion transporter proteins in the pathogenesis of diarrhea in the acute ulcerative colitis. Acute ulcerative colitis was induced in male Sprague-Dawley rats using dextran sulfate sodium (DSS) in drinking water for seven days. The effects of nobiletin, a citrus flavonoid, were also examined. Increased myeloperoxidase activity, colon mass, and inflammatory cell infiltration were associated with damage to goblet cells and the epithelial cell lining indicating the development of acute ulcerative colitis. SERCA-2 calcium pump expression remained unchanged, whereas the phospholamban (PLN) regulatory peptide was reduced and its phosphorylated form (PLN-P) increased, suggesting a post-translational increase in SERCA-2 activity in the inflamed colon. Higher levels of IP3 were associated with a decrease in the Gαq protein levels without altering phospholipase C expression, suggesting that IP3 regulation is independent of Gαq protein signaling. In addition, the expression of sodium/hydrogen exchanger isoforms NHE-1, NHE-3 and carbonic anhydrase-1 and sodium pump activity were decreased in the inflamed colon. Nobiletin treatment of colitis selectively reversed the inflammatory and oxidative stress markers, including superoxide dismutase and catalase without restoring the expression of ion transporters. This study highlights alterations in the expression of ion transporters and their regulatory proteins in acute ulcerative colitis. These changes in the ion transporters are likely to reduce NaCl absorption and alter contractility, thereby contributing to the pathogenesis of diarrhea in the present model of acute ulcerative colitis. Nobiletin selectively ameliorates acute colitis in this model.

Biophysica doi: 10.3390/biophysica5040062

Authors: Sakib Mohammad Md Sakhawat Hossain Sydney L. Sarver

Mechanobiology explores how physical forces and cellular mechanics influence biological processes. This field has experienced rapid growth, driven by advances in high-resolution imaging, micromechanical testing, and computational modeling. At the same time, the increasing complexity and volume of mechanobiological imaging and measurement data have made traditional analysis methods difficult to scale. Artificial intelligence (AI) has emerged as a practical tool to address these challenges by providing new methods for interpreting and predicting biological behavior. Recent studies have demonstrated potential in several areas, including image-based analysis of cell and nuclear morphology, traction force microscopy (TFM), cell segmentation, motility analysis, and the detection of cancer biomarkers. Within this context, we review AI applications that either incorporate mechanical inputs/outputs directly or infer mechanobiologically relevant information from cellular and nuclear structure. This study summarizes progress in four key domains: AI/ML-based cell morphology studies, cancer biomarker identification, cell segmentation, and prediction of traction forces and motility. We also discuss the advantages and limitations of integrating AI/ML into mechanobiological research. Finally, we highlight future directions, including physics-informed and hybrid AI approaches, multimodal data integration, generative strategies, and opportunities for computational biophysics-aligned applications.

Biophysica doi: 10.3390/biophysica5040061

Authors: Zaria Booth Mazen Mezher Rudra Patel Venkat Maruthamuthu

Forces exerted by cells due to their internal contractility play fundamental roles in a host of processes, including adhesion, migration, survival and differentiation. Traction force microscopy (TFM) enables the determination of forces exerted by cells or cell collectives on their environment, which is typically taken to be an extra-cellular matrix (ECM)-coated substrate. Sample preparation for TFM involves the plating of cells onto an environment embedded with fiducial markers. The imaging of these fiducial markers in the presence and absence of the cells then enables calculation of the displacement of localized regions of the environment, and, consequently, the spatial distribution of forces exerted by the cells on their environment. Here, we consider the most widely used implementation of TFM (two-dimensional or 2D TFM) which enables the determination of in-plane forces exerted by cells plated on top of an elastic soft substrate. We present streamlined methods for preparing TFM substrates, with special consideration towards experimental steps involved in implementing it using an epifluorescence microscope. We highlight considerations involved in substrate choice between polyacrylamide (PAA) gels and soft silicones, fiducial marker (microbead) choice and distribution as well as microbead and ECM coupling to the substrate. We also point out caveats related to sub-optimal choices in the methodology which can affect the resultant traction force distribution, as well as further derived quantities such as inter-cellular forces in cell pairs computed using the traction force imbalance method (TFIM).

Biophysica doi: 10.3390/biophysica5040060

Authors: Deyvis Cabrini Teixeira Delfino Danton Diego Ferreira Margarete Marin Lordelo Volpato Vânia Aparecida Silva Renan Teixeira Delfino Christiano Sousa Machado de Matos Meline de Oliveira Santos

Water potential is an important indicator used to study water relations in plants, as it reflects the level of hydration in their tissues. There are different numerical variables that describe plant properties and can be acquired from leaf reflectance. The objective of this study was to estimate water potential in coffee plants using spectral variables. For this, a range of wavelengths that provided analytical flexibility was used. After this, machine learning techniques were employed to build data-driven models. The dataset used presents spectral characteristics (wavelength) of coffee plants, collected through the CI-710 Mini-Leaf Spectrometer equipment and also the water potential of each coffee plant, measured by the Scholander Chamber equipment. The dataset was divided into two crop management groups: irrigated and rainfed. Four machine learning techniques were implemented: Multi-Layer Perceptron (MLP), Decision Tree, Random Forest and K-Nearest Neighbor (KNN). The implementation of machine learning techniques followed two distinct strategies: regression and classification. The results indicate that the decision tree-based model demonstrated superior performance under irrigated conditions for regression tasks. In contrast, the KNN technique achieved the best performance for classification. Under rainfed conditions, the MLP model outperformed the other techniques for regression, while the Random Forest method exhibited the highest accuracy in classification tasks. While no hardware prototype was developed, the machine learning-based methods presented here suggest a possible pathway toward future intelligent, user-friendly, and accessible sensing technologies for coffee plantations.

Biophysica doi: 10.3390/biophysica5040059

Authors: Satoshi Migita

A rapid, colorimetric sensor for histamine detection is presented using citrate-stabilized gold nanoparticles enhanced with Cu2+ coordination. The sensing mechanism involves dual recognition: protonated histamine first adsorbs electrostatically onto AuNP surfaces at pH 5.5, followed by Cu2+-mediated coordination between imidazole rings that induces interparticle coupling, resulting in a characteristic shift of the localized surface plasmon resonance from 520 to 620 nm. The optical response, measured as the absorbance ratio A620/A520, exhibits excellent linearity over the range of 1.25–10 μM with a detection limit of 0.95 μM and total assay time under 30 min. The dual-recognition mechanism provides high selectivity for histamine over structural analogs, including L-histidine, imidazole, and L-lysine. The metal ion-mediated colorimetric approach described here achieves sub-micromolar sensitivity in simple buffer solutions, which is comparable to the histamine level used in in vitro cell assays and food-related studies. Thus, the present system is best viewed as a mechanistic model that can inform the design of future biosensing and analytical methods, rather than as a fully optimized sensor for direct clinical measurements in complex biofluids.

Biophysica doi: 10.3390/biophysica5040058

Authors: Andrew K. Morris Robert M. McCarrick Gary A. Lorigan

A topological analysis was performed by taking ESEEM measurements of site-specifically labeled E protein from SARS-CoV-2. The intensity of deuterium modulation arising from either deuterated solvent or deuterated lipid acyl chains revealed exposure to solvent or the bilayer hydrophobic region. Spin-labeled lipids and soluble spin labels were used as points of comparison. The data indicate that spin labels placed along the transmembrane helix of the E protein showed close contact with lipid acyl chains, but also substantial contact with solvent, while those placed on the C-terminal domain showed substantial but lower exposure to lipid acyl chains, with comparable solvent exposure. The results support the view that the C-terminal domain is in contact with the bilayer surface.

Biophysica doi: 10.3390/biophysica5040057

Authors: Xuanyi Zhang Zehua Wang Chenyang Wu Dongdong Lin

Protein droplets exhibit complex self-assembly and deposition behaviors driven by evaporation, which has attracted increasing attention in recent years. Under evaporation, limited volume and locally concentrated protein solutions can undergo liquid–liquid phase separation (LLPS) and liquid–liquid crystalline phase separation (LLCPS), inducing the formation of concentrated droplets and anisotropic structures. The combined effects of interfacial tension and internal flow field induce a variety of deposition patterns on the substrate, providing great significance for the development of functional biomaterials. This paper reviews the physical processes experienced by protein/fibril droplets during evaporation, focusing on the formation mechanism of evaporation and their phase separation behaviors. At the same time, the review systematically summarized the key factors affecting the deposition patterns, and a variety of methods were introduced to pattern deposition, such as external electric field and micro-structured substrates. Furthermore, the potential applications of proteins/fibrils droplet deposition were discussed in multiple fields. This review aims to provide systematic theoretical support and experimental reference for understanding and controlling the deposition behavior of proteins/fibrils droplets, and to promote their further application in functional materials and biomedical engineering.

Biophysica doi: 10.3390/biophysica5040056

Authors: Endris M. Endris Deresse A. Adem Horace T. Crogman Daniel B. Erenso

We present a proof-of-concept study using a laser trapping (LT) approach to characterize hemoglobin variants through controlled dielectric breakdown of red blood cell membranes. Using a 1064 nm infrared laser, we analyzed 62 cells from each of four hemoglobin types (Hb AS, Hb FA, Hb FSC, Hb AC), measuring the ionization time, cell area, and trap displacement to calculate the apparent threshold ionization energy (TIE*) and apparent threshold radiation dose (TRD*). Post-ionization trajectories and radiation intensity measurements provided charge distribution profiles for each variant. Our results indicate variant-specific differences in TRD* and charge-to-volume ratios across adults and infants (p < 0.05), while the TIE* values remained largely consistent. Charge analysis revealed statistically significant variation between some groups, suggesting that TRD* and charge-based parameters may offer sensitive markers of hemoglobin heterogeneity. This work demonstrates the feasibility of laser trapping as a complementary single-cell method for hemoglobin analysis. While limited in sample size, the approach highlights the potential of TIE* and TRD* measurements for differentiating hemoglobin variants and suggests future applications in hemoglobinopathy screening and diagnostic research.

Biophysica doi: 10.3390/biophysica5040055

Authors: Lyubov Filatova

The combination of antimicrobial agents with different mechanisms of action is an important step in the fight against drug-resistant microorganisms. In this study, the interaction of the lysozyme enzyme with ampicillin and colistin was investigated. These antibiotics are highly effective against Gram-positive (ampicillin) and Gram-negative (colistin) pathogenic microorganisms. Spectroscopic and kinetic methods and molecular docking were used in the research. The results of the spectroscopic analysis confirmed the intermolecular interaction of lysozyme with ampicillin or colistin. The formation of the lysozyme complex with ampicillin was accompanied by mixed quenching of the enzyme fluorescence and changes in its secondary structure (a slight decrease in the content of α-helices). The interaction of lysozyme with colistin was complemented by dynamic quenching of the enzyme fluorescence. The method of molecular docking established that the interactions of lysozyme with colistin were predominantly van der Waals, while hydrogen bonds predominated in the lysozyme complex with ampicillin. Despite the presence of interactions of ampicillin and colistin with amino acid residues from the active site of lysozyme, this did not affect its ability to cause destruction of bacterial cell walls. The results obtained can be used in the development of antibacterial drugs.

Biophysica doi: 10.3390/biophysica5040054

Authors: Chih-Hao Huang Feras A. Batarseh Aman Ullah

Early and accurate diagnosis of Alzheimer’s disease (AD) is critical for patient outcomes yet presents a significant clinical challenge. This study evaluates the effectiveness of four machine learning models—Logistic Regression, Random Forest, Support Vector Machine, and a Feed-Forward Neural Network—for the five-class classification of AD stages. We systematically compare model performance under two conditions, one including cognitive assessment data and one without, to quantify the diagnostic value of these functional tests. To ensure transparency, we use SHapley Additive exPlanations (SHAPs) to interpret the model predictions. Results show that the inclusion of cognitive data is paramount for accuracy. The RF model performed best, achieving an accuracy of 84.4% with cognitive data included. Without this, performance for all models dropped significantly. SHAP analysis revealed that in the presence of cognitive data, models primarily rely on functional scores like the Clinical Dementia Rating—Sum of Boxes. In their absence, models correctly identify key biological markers, including PET (positron emission tomography) imaging of amyloid burden (FBB, AV45) and hippocampal atrophy, as the next-best predictors. This work underscores the indispensable role of cognitive assessments in AD classification and demonstrates that explainable AI can validate model behavior against clinical knowledge, fostering trust in computational diagnostic tools.

Biophysica doi: 10.3390/biophysica5040053

Authors: Vladimir B. Krapivin Roman A. Stepanyuk Maria G. Khrenova

Fluorescent proteins find application as biocompatible, genetically encoded labels for visualization of living organisms tissues. Green fluorescent proteins (GFPs) are the most diverse, but proteins with red fluorescence have advantages, such as lower phototoxicity and better penetration into biological tissues. A promising approach is to obtain red fluorescent proteins (RFPs) from GFPs by introducing mutations that stabilize the oxidized chromophore state with an extended conjugated π-system. However, to date this remains a non-trivial task and experimental developments are carried out mainly by random mutagenesis. Development of descriptors obtained in molecular modeling can rationalize this field. Herein, we rely on experimental data on the AzamiGreen fluorescent protein and its variants that are oxidized to the red form. We perform classical molecular dynamics (MD) and combined quantum mechanics/molecular mechanics (QM/MM) simulations to determine structural and dynamic features that govern oxidation. We demonstrate that the red state is predominantly stabilized by interactions of polar lysine residues with chromophore oxygen atoms. Dynamic network analysis demonstrates that in red fluorescent proteins the chromophore motions are correlated with the movement of surrounding protein side chains to a higher extent than in green variants. The presence of different resonance forms of the chromophore determines the fluorescence band maximum value: a decrease in the phenolate form population leads to the red shift.

Biophysica doi: 10.3390/biophysica5040052

Authors: Kunal Bhattacharya Pukar Khanal Jagdish Chand Nongmaithem Randhoni Chanu Dibyajyoti Das Atanu Bhattacharjee

Alzheimer’s disease is driven by multiple molecular drivers, including the pathological behavior of two intrinsically disordered proteins, amyloid-β (Aβ) and tau, whose aggregation is regulated by sequence-encoded ensembles and liquid–liquid phase separation (LLPS). This review integrates recent advances in biophysics, structural biology, and computational modeling to provide a multiscale perspective on how sequence determinants, post-translational modifications, and protein dynamics regulate the conformational landscapes of Aβ and tau. We discuss sequence-to-ensemble principles, from charge patterning and aromatic binders to familial mutations that reprogram structural ensembles and modulate LLPS. Structural studies, including NMR, SAXS, cryo-EM, and cryo-electron tomography, trace transitions from disordered monomers to fibrils and tissue-level structures. We highlight experimental challenges in LLPS assays, emerging standards for reproducibility, e.g., LLPSDB, PhaSePro, and FUS benchmarks, and computational strategies to refine and condensate modeling. Finally, we explore the therapeutic implications, including condensate-aware medicinal chemistry, ensemble-driven docking, and novel insights from clinical trials of anti-Aβ antibodies. Together, these perspectives underscore a paradigm shift toward environment- and ensemble-aware therapeutic design for Alzheimer’s and related protein condensation disorders.

Biophysica doi: 10.3390/biophysica5040051

Authors: Arpan Roy Sydney Sarver Jarod Beights Sean Brennan Sazid Noor Rabi Sakib Mohammad Kyu Young Han Sabrina Nilufar Farhan Chowdhury

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.

Biophysica doi: 10.3390/biophysica5040050

Authors: Pornthep Sompornpisut Linh Truong Hoai Panisak Boonamnaj Brian G. Olson Ras B. Pandey

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 (Rg) further demonstrates the contrasting thermal behaviors of S1 and S2. For S1, Rg 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: Rg 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.

Biophysica doi: 10.3390/biophysica5040048

Authors: Daria Kondrakhova Vladimíra Tomečková Oleksandr Dobrozhan Ondrej Milkovič Hoydoo You Tatiana Kimáková Vladimír Komanický

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.

Biophysica doi: 10.3390/biophysica5040049

Authors: Karen Isela Vargas-Rubio Efrén Delgado Cristian Patricia Cabrales-Arellano Claudia Ivette Gamboa-Gómez Damián Reyes-Jáquez

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.

Biophysica doi: 10.3390/biophysica5040047

Authors: Qiang Ma Liuqiang Zhu Xiang Li Dening Pei Lei Yu Xinchang Shi Yong Zhou Zhihao Fu Chenggang Liang Xi Qin Junzhi Wang

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.

Biophysica doi: 10.3390/biophysica5040046

Authors: Cornelius Rosenkranz Marianna Lucio Marion Ganslmayer Thomas Harrer Jakob Hoffmanns Charlotte Szewczykowski Thora Schröder Franziska Raith Stephanie Zellinger Denzel Abelardo Jule Schumacher Merle Flecks Petra Lakatos Christian Mardin Bettina Hohberger

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.

Biophysica doi: 10.3390/biophysica5040045

Authors: Shahnawaz Khijmatgar Matteo Pellegrini Martina Ghizzoni Massimo Del Fabbro

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.

Biophysica doi: 10.3390/biophysica5040044

Authors: Marcela Ana Morini Viviana Isabel Pedroni

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.

Biophysica doi: 10.3390/biophysica5040043

Authors: Asim Muhammad Xin-Yu Zheng Hui-Lin Gan Yu-Xin Guo Jia-Hong Xie Yan-Jun Chen Jin-Jun Chen

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.

Biophysica doi: 10.3390/biophysica5030042

Authors: Giuseppe Maulucci

Metabolic imaging is undergoing a fundamental transformation. Traditionally confined to static representations of metabolite distribution through modalities such as PET, MRS, and MSOT, imaging has offered only partial glimpses into the dynamic and systemic nature of metabolism. This Perspective envisions a shift toward dynamic metabolic intelligence—an integrated framework where real-time imaging is fused with physics-informed models, artificial intelligence, and wearable data to create adaptive, predictive representations of metabolic function. We explore how novel technologies like hyperpolarized MRI and time-resolved optoacoustics can serve as dynamic inputs into digital twin systems, enabling closed-loop feedback that not only visualizes but actively guides clinical decisions. From early detection of metabolic drift to in silico therapy simulation, we highlight translational pathways across oncology, cardiology, neurology, and space medicine. Finally, we call for a cross-disciplinary effort to standardize, validate, and ethically implement these systems, marking the emergence of a new paradigm: metabolism as a navigable, model-informed space of precision medicine.

Biophysica doi: 10.3390/biophysica5030041

Authors: Paul R. Gardner

Heme enzymes that bind and reduce O2 are susceptible to poisoning by NO. The high reactivity and affinity of NO for ferrous heme produces stable ferrous-NO complexes, which in theory should preclude O2 binding and turnover. However, NO inhibition is often competitive with respect to O2 and rapidly reversible, thus providing cellular and organismal survival advantages. This kinetic paradox has prompted a search for mechanisms for reversal and hence resistance. Here, I critically review proposed resistance mechanisms for NO dioxygenase (NOD) and cytochrome c oxidase (CcO), which substantiate reduction or oxidation of the tightly bound NO but nevertheless fail to provide kinetically viable solutions. A ferrous heme intermediate is clearly not available during rapid steady-state turnover. Reversible inhibition can be attributed to NO competing with O2 in transient low-affinity interactions with either the ferric heme in NOD or the ferric heme-cupric center in CcO. Toward resolution, I review the underlying principles and evidence for kinetic control of ferric heme reduction via an O2-triggered ferric heme spin crossover and an electronically-forced motion of the heme and structurally-linked protein side chains that elicit electron transfer and activate O2 in the flavohemoglobin-type NOD. For CcO, kinetics, structures, and density functional theory point to the existence of an analogous O2 and reduced oxygen intermediate-controlled electron-transfer gate with a linked proton pump function. A catalytic cycle and mechanism for CcO is finally at hand that links each of the four O2-reducing electrons to each of the four pumped protons in time and space. A novel proton-conducting tunnel and channel, electron path, and pump mechanism, most notably first hypothesized by Mårten Wikström in 1977 and pursued since, are laid out for further scrutiny. In both models, low-energy spin-orbit couplings or ‘spintronic’ interactions with O2 and NO or copper trigger the electronic motions within heme that activate electron transfer to O2, and the exergonic reactions of transient reactive oxygen intermediates ultimately drive all enzyme, electron, and proton motions.

Biophysica doi: 10.3390/biophysica5030040

Authors: Constantinos Koutsojannis Athanasios Fouras Dionysia Chrysanthakopoulou

Medical physicists are transforming physiological measurements and electrophysiological applications by addressing challenges like motion artifacts and regulatory compliance through advanced signal processing, artificial intelligence (AI), and statistical rigor. Their innovations in wearable electrophysiology achieve 8–12 dB signal-to-noise ratio (SNR) improvements in EEG, 60% motion artifact reduction, and 94.2% accurate AI-driven arrhythmia detection at 12 μW power. In precision neurology, machine learning (ML) with evoked potentials (EPs) predicts spinal cord injury (SCI) recovery and multiple sclerosis (MS) progression with 79.2% accuracy based on retrospective data from 560 SCI/MS patients. By integrating multimodal data (EPs, MRI), developing quantum sensors, and employing federated learning, these can enhance diagnostic precision and prognostic accuracy. Clinical applications span epilepsy, stroke, cardiac monitoring, and chronic pain management, reducing diagnostic errors by 28% and optimizing treatments like deep brain stimulation (DBS). In this paper, we review the current state of wearable devices and provide some insight into possible future directions. Embedding medical physicists into standardization efforts is critical to overcoming barriers like quantum sensor power consumption, advancing personalized, evidence-based healthcare.

Biophysica doi: 10.3390/biophysica5030039

Authors: Sen Zheng

Cryo-electron microscopy single-particle analysis (cryo-EM SPA) has advanced three-dimensional protein structure determination, yet resolving intrinsically disordered proteins and regions (IDPs/IDRs) remains challenging due to conformational heterogeneity. This research evaluates cryo-EM’s capacity to map dynamic regions, assesses the adaptability of disorder prediction tools, and explores optimization strategies for dynamic structure prediction. Cryo-EM SPA datasets from 2000 to 2024 were categorized into different periods, forming a database integrating sequence data and disorder indices. Established prediction tools—AlphaFold2 (pLDDT), flDPnn, and IUPred—were evaluated for transferability, while a multi-level CLTC hybrid model (combining CNN, LSTM, Transformer, and CRF architectures) was developed to link local conformational fluctuations with global sequence contexts. Analyses revealed consistent advancements in average resolution and model counts over the past decade, although mapping disordered regions remained technically demanding. Both the adapted AlphaFold pLDDT and the CLTC model demonstrated efficacy in predicting structurally variable and poorly resolved regions. A subset of the cryo-EM missing residues exhibited intermediate conformational features, suggesting classification ambiguities potentially influenced by experimental conditions. These findings systematically outline the evolving capabilities of cryo-EM in resolving dynamic regions, benchmark the adaptability of computational tools, and introduce a hybrid model to enhance prediction accuracy. This study provides a framework for addressing conformational heterogeneity, contributing to methodological advancements in structural biology.

Biophysica doi: 10.3390/biophysica5030038

Authors: Ankit Monga Khush Jain Harvinder Popli Prashik Telgote Ginpreet Kaur Fariah Rizwani Ritu Chauhan Damandeep Kaur Abhishek Chauhan Hardeep Singh Tuli

Organs-on-Chips (OoC) technology has begun to be considered a pragmatic tool for drug evaluation, offering researchers an opportunity to move beyond the less physiologically relevant animal models. OoCs are microfluidic structures that imitate the functionalities of individual human organs, serving as mimicry tools for drug response and reproducibility studies. On the one hand, companies producing OoCs find managing and analyzing the large amounts of data generated challenging. This is where artificial intelligence (AI) can be deployed to address such problems. This paper will present the state-of-the-art of current OoC technology and AI, discussing the benefits and threats of combining these approaches. AI can be applied to optimize the process of OoC fabrication and operation, as well as for the big data analysis of OoC devices. By combining these technologies, scientists gain a powerful tool for drug development that is more efficient and accurate. However, processing the vast datasets generated by OoC systems often requires specialized AI expertise and computational resources. Despite the numerous possible benefits of amalgamating OoC technology with AI, several challenges and limitations need to be addressed. The large datasets generated by OoC systems can be difficult to process and analyze, which is a task that may require specialized AI expertise. Additionally, limitations of OoC systems include issues with reproducibility, as the devices are sensitive to perturbations in experimental conditions. Furthermore, the development and implementation of AI algorithms require significant computational resources and expertise, which may not be readily available to all research institutions. To overcome these challenges, interdisciplinary collaboration between biologists, engineers, data scientists, and AI experts is essential. Continued advancements in both OoC technology and AI will likely lead to more robust and versatile platforms for biomedical research and drug development, ultimately contributing to the advancement of personalized medicine and the reduction of reliance on animal testing.

Biophysica doi: 10.3390/biophysica5030037

Authors: Alireza Mirzaee Shantia Yarahmadian

Type 2 diabetes (T2D) is a chronic metabolic disorder requiring effective management to avoid complications. Metformin is a first-line drug agent and is routinely prescribed for the control of glycemia, but its underlying dynamics are complicated and not fully quantified. This paper formulates a control-oriented and interpretable mathematical model that integrates metformin dynamics into a classic beta-cell–insulin–glucose (BIG) regulation system. The paper’s applicability to theoretical and clinical settings is enhanced by rigorous mathematical analysis, which guarantees the model is globally bounded, well-posed, and biologically meaningful. One of the key features of the study is its global stability analysis using Lyapunov functions, which demonstrates the asymptotic stability of critical equilibrium points under realistic physiological constraints. These findings support the predictive reliability of the model in explaining long-term glycemic regulation. Bifurcation analysis also clarifies the dynamic interplay between glucose production and utilization by identifying parameter thresholds that signify transitions between homeostasis and pathological states. Residual analysis, which detects Gaussian-distributed errors, underlines the robustness of the fitting process and suggests possible refinements by including temporal effects. Sensitivity analysis highlights the predominant effect of the initial dose of metformin on long-term glucose regulation and provides practical guidance for optimizing individual treatment. Furthermore, changing the two considered metformin parameters from their optimal values—altering the dose by ±50% and the decay rate by ±20%—demonstrates the flexibility of the model in simulating glycemic responses, confirming its adaptability and its potential for optimizing personalized treatment strategies.

Biophysica doi: 10.3390/biophysica5030036

Authors: Radwan Ebna Noor Shahedul Islam Tracess Smalley Katarzyna Mizgalska Mark Eschenfelder Dimitra Keramisanou Aaron Joshua Astalos James William Leahy Wayne Charles Guida Aleksandra Karolak Ioannis Gelis Mildred Acevedo-Duncan

The overexpression of atypical protein kinase C-iota (PKC-ι) is a biomarker for carcinogenesis in various cell types, such as glioma, ovarian, renal, etc., manifesting as a potential drug target. In previous in vitro studies, ICA-1S and ICA-1T, experimental candidates for inhibiting PKC-ι, have demonstrated their specificity and promising efficacy against various cancers. Moreover, the in vivo studies have demonstrated low toxicity levels in acute and chronic murine models. Despite these prior developments, the binding affinities of the inhibitors were never thoroughly explored from a biophysical perspective. Here, we present the biophysical characterizations of PKC-ι in combination with ICA-1S/1T. Various methods based on molecular docking, light scattering, intrinsic fluorescence, thermal denaturation, and heat exchange were applied. The biophysical characteristics including particle sizing, thermal unfolding, aggregation profiles, enthalpy, entropy, free energy changes, and binding affinity (Kd) of the PKC-ι in the presence of ICA-1S were observed. The studies indicate the presence of domain-specific stabilities in the protein–ligand complex. Moreover, the results indicate a spontaneous reaction with an entropic gain, resulting in a possible entropy-driven hydrophobic interaction and hydrogen bonds in the binding pocket. Altogether, these biophysical studies reveal important insights into the binding interactions of PKC-ι and its inhibitors ICA-1S/1T.

Biophysica doi: 10.3390/biophysica5030035

Authors: Juan Antonio Noriega-Rodríguez Armando Tejeda-Mansir Hugo Sergio García

Protein purification is often performed for various applications. However, enzyme purification processes typically involve multiple steps that reduce yield and increase production costs. To overcome these challenges, we developed a novel three-step process to purify a lipase from whole sardine viscera (WSV), leveraging protein properties and the structural affinity of lipases for dye ligands. A crude extract of the viscera (CEV) was obtained by grinding the whole viscera in 50 mM phosphate buffer (pH 7.0, Solution B) followed by centrifugation (6000× g; 30 min, 0 °C). Lipolytic activity (3.3 U/mg) was recorded only in the supernatant. The purification process began with ammonium sulfate fractionation (30–50% saturation), increasing lipolytic activity in the precipitate (PF30-50) to 32.9 U/mg. PF30-50 was then ultrafiltered using a 30 KDa MWCO membrane, where 5% of semi-purified lipases (SPLSV) was retained with an activity of 156.5 U/mg (UF30). Finally, the SPLSV was injected into a column packed with dye ligand affinity adsorbent, pre-equilibrated with 1.0 M ammonium sulfate in buffer A. The WSV lipase was eluted using a step gradient to progressively reduce salt concentration. SDS-PAGE analysis revealed a single band of purified lipase from sardine viscera (PLSV) corresponding to a molecular weight of 123.4 kDa, with a specific activity of 266.4 U/mg. The combination of ammonium sulfate precipitation, ultrafiltration, and dye-ligand affinity chromatography provides a scalable and reproducible approach with potential industrial relevance, particularly in biocatalysis and waste valorization contexts.

Biophysica doi: 10.3390/biophysica5030034

Authors: Lisa Riedlsperger Heinz Anderle Andreas Schwaighofer Martin Lemmerer

Protein polarimetry has been evaluated as a simple and straightforward technique to detect the cryptic denaturation of exemplary proteins. The general rules of rotation vs. amino acid and structural composition and the respective knowledge gaps were reviewed, and the specific rotation of cystine was determined in 4 M NaCl solution as [α]D20 = –302.5°. The specific rotations at 589 nm and 436 nm and the ratio were measured for several model proteins, some purified plasma-derived proteins and for three monoclonal antibodies. The immunoglobulin G concentrates all showed a narrow ratio range likely characteristic for this protein class. Heat denaturation experiments were conducted at temperatures between 50 and 85 °C both for short-time (10 min) and for prolonged periods of heat exposure (up to 210 min). Denaturation by heat resulted not only in the known levorotatory shift, but also in a shift in the specific rotation ratio. The stabilizing effect of fatty acids in bovine serum could be demonstrated by this parameter. Polarimetry thus appears to be a particularly sensitive and simple method for the characterization of the identity and the thermal stability of proteins and should therefore be added again as a complimentary method to the toolbox of protein chemistry.

Biophysica doi: 10.3390/biophysica5030033

Authors: Pedro Ojeda-May

Protein enzymes are highly efficient catalysts that exhibit adaptability and selectivity under diverse biological conditions. In some organisms, such as bacteria, structurally similar enzymes, for instance, shikimate kinase (SK) and adenylate kinase (AK), coexist and act on chemically related ligands. This raises the question of whether these enzymes can accommodate and potentially react with each other’s ligands. In this study, we investigate the stability of non-cognate ligand binding in SK and explore whether external electric fields (EFs) can modulate this interaction, leading to cross-reactivity in SK. Using molecular dynamics simulations, we assess the structural integrity of SK and the binding behavior of ATP and AMP under EF-off and EF-on cases. Our results show that EFs enhance protein structure stability, stabilize non-cognate ligands in the binding pocket, and reduce local energetic frustration near the R116 residue located in the binding site. In addition to this, dimensionality reduction analyses reveal that EFs induce more coherent protein motions and reduce the number of metastable states. Together, these findings suggest that external EFs can reshape enzyme–ligand interactions and may serve as a tool to modulate enzymatic specificity and functional promiscuity. Thus, we provide computational evidence that supports the concept of using an EF as a tunable parameter in enzyme engineering and synthetic biology. However, further experimental investigation would be valuable to assess the reliability of our computational predictions.

Biophysica doi: 10.3390/biophysica5030032

Authors: Zhongbao Han Huizi Zheng Yimeng Qi Dilshadbek T. Usmanov Liyan Liu Zhan Yu

Icariin (ICA) is widely recognized for its health benefits. In this work, we examined the intermolecular interactions between ICA and proteinase K (PK) via multi-spectroscopic techniques and molecular simulations. The experimental findings revealed that ICA quenched the fluorescence emission of PK by forming a noncovalent complex. Both hydrogen bonding and van der Waals interactions are essential for the complex’s formation. Then Förster resonance energy transfer (FRET), competitive experiments, and synchronous fluorescence spectroscopy were adopted to verify the formation of the complex. Molecular docking studies demonstrated that ICA could spontaneously bind to PK by hydrogen bonding and hydrophobic interactions, which is consistent with the spectroscopic results. The PK-ICA complex’s dynamic stability was evaluated using a 50 ns molecular dynamics (MD) simulation. The simulation results revealed no significant structural deformation or positional changes throughout the entire simulation period. The complex appears to be rather stable, as seen by the average root-mean-square deviation (RMSD) fluctuations for the host protein in the PK-ICA complex of 1.08 Å and 3.09 Å. These outcomes of molecular simulations suggest that ICA interacts spontaneously and tightly with PK, consistent with the spectroscopic findings. The approach employed in this research presents a pragmatic and advantageous method for examining protein–ligand interactions, as evidenced by the concordance between empirical and theoretical findings.

Biophysica doi: 10.3390/biophysica5030031

Authors: Jack Pelzel Reese Anderson Darin J. Ulness Krys Strand

This study extends the application of photothermal spectroscopy to explore heat transfer dynamics in biological fluids, focusing on the examination of artificial vitreous humor (VH) models of human VH and an endogenous sample of cervine (deer) VH. The research integrates previously established methods for analyzing thermal lensing through photothermal deflection. By visualizing convective and conductive heat transfer processes in the artificial components of human VH, one gains insights into the dynamic behavior of heat transfer in the VH. Relevance extends to clinical cases where pathology requires replacement of endogenous VH with an artificial VH substitute. Several VH substitutes identified in the literature were chosen for this study based on their physical properties and relative abundance in the VH. Individual component fluids, and mixtures of these components, were analyzed at various concentrations based on their physiological concentration ranges in the human VH as they varied with age, sex, and certain disease states. By way of comparison to endogenous biological VH, a sample of VH obtained from a female white-tailed deer eye was analyzed, enhancing the understanding of heat transfer in artificial components of the VH compared to endogenous VH. There is a vast array of ophthalmological procedures that utilize an external heat source interacting with endogenous or artificial VH. The data found in this study will progress the understanding of heat transfer within artificial VH components in comparison to endogenous VH and contribute to the advancement of certain ophthalmological procedures.

Biophysica doi: 10.3390/biophysica5030030

Authors: Myeong Seon Jeong Han-ul Kim Mi Young An Yoon Ho Park Sun Hee Park Sang J. Chung Yoon-Sun Yi Sangmi Jun Young Kwan Kim Hyun Suk Jung

Recent major advancements in cryo-electron microscopy (cryo-EM) have enabled high-resolution structural analysis, accompanied by developments in image processing software packages for single-particle analysis (SPA). SPA facilitates the 3D reconstruction of proteins and macromolecular complexes from numerous individual particles. In this study, we systematically evaluated the impact of symmetry parameters and particle quantity on the 3D reconstruction efficiency using the dihydrolipoyl acetyltransferase (E2) inner core of the pyruvate dehydrogenase complex (PDC). We specifically examined how inappropriate symmetry constraints can introduce structural artifacts and distortions, underscoring the necessity for accurate symmetry determination through rigorous validation methods such as directional Fourier shell correlation (FSC) and local-resolution mapping. Additionally, our analysis demonstrates that efficient reconstructions can be achieved with a moderate particle number, significantly reducing computational costs without compromising structural accuracy. We further contextualize these results by discussing recent developments in SPA workflows and hardware optimization, highlighting their roles in enhancing reconstruction accuracy and computational efficiency. Overall, our comprehensive benchmarking provides strategic insights that will facilitate the optimization of SPA experiments, particularly in resource-limited settings, and offers practical guidelines for accurately determining symmetry and particle quantity during cryo-EM data processing.

Biophysica doi: 10.3390/biophysica5030029

Authors: Marie-Valérie Moreno Sid Ahmed Ben Mansour Frédéric Roscop

This study represents a first step toward improving the repeatability, reproducibility, and accuracy of a process designed to enhance dynamic water structuring. We aim is to investigate the optical reflectivity of a watery magnesium chloride solution treated with electromagnetic waves, we employ a novel methodology derived from human plethysmography (PPG) with three wavelengths spanning the visible and infrared spectra. We measured the reflectance of 17 flasks at 536 nm, 660 nm, and 940 nm before and after treatment, first using the succussion method (control) and second using a 50 Hz signal. The observed variability was acceptable, with repeatability errors below 0.15% and reproducibility errors below 3.5% across all wavelengths before and after treatment. Out of 51 samples dynamically structured using the succussion method, we obtained two false negatives, while one false negative was recorded out of 51 samples dynamically structured using the electromagnetic (EM) method. PPG appears to be a relevant sensor, as it correctly detected dynamically structured water in 99 out of 102 cases, using either the succussion or electromagnetic method. Our results show significant differences in reflectance (supposedly correlated with water’s structured status) at 536 nm between dynamically structured and dynamic non-structured samples (p < 0.001). Future improvements will include a validation protocol against gold-standard spectrophotometry with a larger sample size.

Biophysica doi: 10.3390/biophysica5030028

Authors: Dragan Nikolić Xu Zhang

We present a corrosion-resistant quadrupole ion trap mass spectrometer (QIT-MS) designed for trace detection of volatiles in sulfuric acid aerosols, with a specific focus on phosphine (PH3). Here, we detail the gas calibration methodology using permeation tube technology for generating certified ppb-level PH3/H2S/CO2 mixtures, and report results from mass spectra with sufficient resolution to distinguish isotopic envelopes that validate the detection of PH3 at a concentration of 62 ppb. Fragmentation patterns for PH3 and H2S agree with NIST data, and signal-to-noise performance confirms ppb sensitivity over 2.6 h acquisition periods. We further assess spectral interferences from oxygen isotopes and propose a detection scheme based on isolated phosphorus ions (P+) to enable specific and interference-resistant identification of PH3 and other reduced phosphorus species of astrobiological interest in Venus-like environments. This work extends the capabilities of QIT-MS for trace gas analysis in chemically aggressive atmospheric conditions.

Biophysica doi: 10.3390/biophysica5030027

Authors: Carmen Regina de Souza Maurício Ikeda Yoguim Nathalia Mariana Pavan Nelson Henrique Morgon Valdecir Farias Ximenes Aguinaldo Robinson de Souza

Coumarins are known for interacting with proteins and exhibiting diverse biological activities. This study investigates the interaction between coumarin-343 (C343) and human (HSA) and bovine (BSA) serum albumins. Fluorescence spectroscopy and theoretical simulations, including density functional theory (DFT) and molecular docking, were used to analyze the ligand–protein complex formation. The fluorescence quenching data revealed that C343 binds to both proteins, with binding constants of 2.1 × 105 mol·L−1 (HSA) and 6.5 × 105 mol·L−1 (BSA), following a 1:1 stoichiometry. Binding site markers identified drug site I (DS1), located in subdomain IIA, as the preferential binding region for both proteins. Computational results supported these findings, showing high affinity for DS1, with binding energies of −69.02 kcal·mol−1 (HSA) and −67.22 kcal·mol−1 (BSA). While complex formation was confirmed for both proteins, differences emerged in the induced circular dichroism (ICD) signals. HSA displayed a distinct ICD profile compared to BSA in both intensity and absorption maximum. Molecular Docking revealed that the C343 conformation differed between HSA and BSA, explaining the variation in ICD signals. These results highlight the importance of protein structure in modulating ligand interactions and spectral responses.

Biophysica doi: 10.3390/biophysica5030026

Authors: Ping Xie

An engineered molecular motor composed of an ATP-dependent kinesin-1 monomer and an ATP-independent diffusing microtubule-associated protein is proposed, and its dynamics are studied theoretically. It is shown that the engineered motor can move directionally on microtubules towards the plus end, bearing great potential for applications in therapeutics or nanorobotics. The engineered motor can have an unloaded velocity similar to the wild-type kinesin-1 dimer, can take a mechanical (either forward or backward) step by hydrolyzing an ATP molecule under any load, and can generate the maximum force that is about half of that generated by the wild-type kinesin-1 dimer.

Biophysica doi: 10.3390/biophysica5030025

Authors: Allen P. Minton

A brief summary of the effect of nonspecific interactions upon chemical equilibria in solutions containing a high total concentration of macromolecular solutes comparable to that found in biological fluid media is presented. Analyses of experimental measurements permitting relatively direct quantitation of the free energy of nonspecific intermolecular interaction in solutions of one or two macrosolutes are described, and a table listing published experimental studies of both homo- and hetero-interactions is provided. Methods for calculating the free energy of nonspecific interaction via theory and computer simulation are described. Recommendations for further progress in both measurement and calculation of interaction free energies are presented.

Biophysica doi: 10.3390/biophysica5030024

Authors: Jolanta Fertala Andrzej Steplewski Andrzej Fertala

Collagen II is a vital structural component in developing bones and mature cartilage. Mutations in this protein cause spondyloepiphyseal dysplasia, a disease characterized primarily by altered skeletal growth and manifesting with a range of phenotypes, from lethal to mild. This study examined transgenic mice harboring the R992C (p.R1124C) substitution in collagen II. Previous research demonstrated significant growth abnormalities and disorganized growth plate structure in these mice, and histological signs of osteoarthritic changes in the knee joints of 9-month-old mice with the R992C mutation. Our study focuses on detecting early structural changes in the articular cartilage that occur before histological signs become apparent. Through microscopic and spectroscopic analyses, we observed significant alterations in the distribution gradients of collagenous proteins and proteoglycans in the cartilage of R992C mutant mice. We propose that these early changes, eventually leading to articular cartilage degeneration in older mice, underscore the progressive nature of osteoarthritic changes linked to collagen II mutations. By identifying these early structural aberrations, our findings emphasize the importance of early detection of osteoarthritic changes, potentially facilitating timely, non-surgical interventions.

Biophysica doi: 10.3390/biophysica5020023

Authors: Aleksandar Simeonov Karo Michaelian

An ancient repertoire of ultraviolet (UV)-absorbing pigments which survive today in the phylogenetically oldest extant photosynthetic organisms, the cyanobacteria, point to a direction in evolutionary adaptation of the pigments and their associated biota; from largely UV-C absorbing pigments in the Archean to pigments covering ever more of the longer wavelength UV and visible regions in the Phanerozoic. Since photoprotection is not dependent on absorption, such a scenario could imply selection of photon dissipation rather than photoprotection over the evolutionary history of life, consistent with the thermodynamic dissipation theory of the origin and evolution of life which suggests that the most important hallmark of biological evolution has been the covering of Earth’s surface with organic pigment molecules and water to absorb and dissipate ever more completely the prevailing surface solar spectrum. In this article we compare a set of photophysical, photochemical, biosynthetic, and other inherent properties of the two dominant classes of cyanobacterial UV-absorbing pigments, the mycosporine-like amino acids (MAAs) and scytonemins. We show that the many anomalies and paradoxes related to these biological pigments, for example, their exudation into the environment, spectral coverage of the entire high-energy part of surface solar spectrum, their little or null photoprotective effect, their origination at UV-C wavelengths and then spreading to cover the prevailing Earth surface solar spectrum, can be better understood once photodissipation, and not photosynthesis or photoprotection, is considered as being the important variable optimized by nature.

Biophysica doi: 10.3390/biophysica5020022

Authors: Man Song Xiao Liu Jie Zhang Wuping Li Jingfen Pan Yanglei Jia

Ark shells are a group of bivalves that exhibit extraordinary adaptability to the dual environmental pressures of low oxygen and osmotic imbalance. These challenges are particularly pronounced in intertidal zones, where organisms are subjected to rapid and drastic changes in their surroundings. This research investigated the molecular mechanisms that underpin their survival and adaptive strategies, with particular focused on sodium–potassium ATPase (NKA), a pivotal enzyme responsible for maintaining cellular ion transmembrane gradients and ensuring cellular homeostasis under stress conditions. By utilizing genome assemblies and transcriptomics datasets from multiple ark shell species, we successfully identified two distinct NKA-α subunits and two NKA-β subunits, which are essential components of the NKA complex. Moreover, the discovery of a conserved NKA-interacting protein (NKAIN) highlights the complexity and evolutionary significance of the NKA-NKAIN system in ark shells. Phylogenetic analysis revealed a high degree of conservation in the NKA-α and NKA-β subunits across ark shells, suggesting strong selective pressures to preserve their functionality. However, the marked divergence observed between the two NKA-β subunits suggests that they may serve distinct roles in ion transport, potentially specialized for specific environmental conditions or stress responses. Comparative transcriptomic analysis further revealed the regulatory roles of NKA and NKAIN in the adaptive responses to hypoxia and osmotic stress, showing that these genes are dynamically modulated at the transcriptional level in response to environmental challenges. These findings provide a molecular foundation for understanding the osmotic adaptation mechanisms in ark shells and offer novel insights into their ability to thrive in mudflat habitats. This comprehensive exploration of the NKA-NKAIN system not only enhances our understanding of the resilience of ark shells but also provides valuable insights into the molecular and physiological strategies employed by bivalves in intertidal environments.