Background/Objectives: Marine microorganism metabolites are structurally unique secondary metabolites possessing therapeutic potential. The current study aims to identify a novel ATPase regulator using a newly established bidirectional activity evaluation system to screen for microbial metabolites that inhibit the activities of Na+-K+-ATPase or Ca2+-Mg2+-ATPase. Methods: A total of 1258 marine microbial strains were isolated from sea mud in Zhoushan, Zhejiang. Results: The extract of strain ZSDH2536 exhibited Na+-K+ and Ca2+-Mg2+-ATPase inhibitory activity and was identified as Sirastachys pandanicola based on morphological and molecular phylogenetic analyses. The secondary metabolite was tentatively identified in the ZSDH2536 strain as a bisindole compound, and named Pandanicoline based on 1H-NMR, 13C-NMR and high-resolution mass spectrometry analysis. The chemical formula of Pandanicoline is C51H68N2O10, with an isotopic mass of 868.4874 Da. The maximum inhibition rate of Pandanicoline on Na+-K+ and Ca2+-Mg2+-ATPase was 36.37% and 37.27%, respectively. Moreover, in silico analysis also showed the binding energy of Pandanicoline with Na+-K+-ATPase was -9.124 kcal/mol and with the Ca2+-Mg2+-ATPase complex was -10.47 kcal/mol. Conclusions: The strain ZSDH2536 represents a promising source of dual inhibitors targeting Na+-K+ and Ca2+-Mg2+-ATPase. Pandanicoline exhibits potential as a lead compound for regulating ion homeostasis, providing new opportunities for further investigation into its mechanism and therapeutic applications.

Marine by-products represent a promising source of bioactive peptides. This study aimed to isolate and characterize a low-molecular-weight peptide fraction with antioxidant activity from Argentine shortfin squid carcass by-products, and to evaluate in vitro its cytocompatibility and protective effects against corticosterone (CORT)-induced oxidative injury in rat adrenal pheochromocytoma (PC12) cells and human astrocyte (hACs) cells. Argentine squid antioxidant peptide (PASN) was obtained by size-exclusion chromatography and fractionation-based screening. PASN exhibited the strongest overall free-radical-scavenging activity and consisted predominantly of components below 1 kDa (211.73–1013.48 Da). Spectroscopic analyses indicated that enzymatic hydrolysis transformed its structure from a rigid triple-helix conformation to a more flexible conformation dominated by β-turns (50.78%) and random coils (17.38%). In addition, thermogravimetric analysis confirmed its excellent thermal stability, with an onset decomposition temperature as high as 244.81 °C, supporting its potential applicability in high-temperature food-processing matrices. In vitro assays demonstrated that PASN exhibited high biocompatibility and promoted proliferation of both PC12 cells and hACs, while significantly improving cell viability under CORT challenge. PASN also reduced lactate dehydrogenase (LDH) leakage (hACs: 38.31%; PC12: 31.17%) in both cell models and restored total superoxide dismutase (T-SOD) activity (hACs: 69.46%, PC12: 66.40%). Immunofluorescence further revealed that PASN rescued the expression of brain-derived neurotrophic factor (BDNF) (hACs: 35.23%, PC12: 12.50%) and glutamate decarboxylase (GAD1/2) (hACs: 102.66%, PC12: 31.31%), key markers associated with synaptic plasticity and GABAergic sleep regulation. Collectively, PASN is a thermally stable squid-derived peptide fraction that exerts antioxidant and cytoprotective effects in neural cell models in vitro and represents a promising sustainable candidate for nutraceutical development.

Impaired wound healing is often caused by persistent inflammation, bacterial infection, and insufficient extracellular matrix remodeling. Natural polymer-based hydrogels represent ideal wound dressings but often struggle to balance structural stability and biological activity. Herein, we report a dual-functional network regulation strategy enabled by highly soluble mussel foot protein (HMFP) that acts simultaneously as a structural crosslinking regulator and bioactive effector to fabricate synergistic hydrogels (CS-SH-H) from β-chitosan (CS) and sodium hyaluronate (SH). HMFP homogenizes the porous microstructure, strengthens intermolecular interactions, and significantly improves thermal and structural stability via multivalent non-covalent bonding. In vitro, CS-SH-H shows excellent cytocompatibility, significantly promotes fibroblast proliferation and migration, and exerts potent antibacterial activity against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). In a mouse full-thickness skin defect model, the hydrogel dramatically accelerates wound closure, reducing the residual wound area to 25% on day 7, outperforming the control groups. Immunohistochemistry confirms that HMFP suppresses TNF-α-mediated inflammation and enhances Ki-67-positive cell proliferation, leading to accelerated re-epithelialization and collagen deposition. This study establishes HMFP as a promising marine-derived dual-functional network regulator for designing high-performance hydrogel dressings. This strategy is scalable and translatable for treating infected and inflammatory wounds.

Porous alumina scaffolds were engineered and surface‑bioactivated with calcium phosphates for potential application in bone tissue regeneration. A mechanically and structurally optimized porous alumina substrate (T25) was fabricated by spark plasma sintering with a carbon sacrificial template, yielding 43.4

Although blood–brain barrier (BBB) models are of great value in investigating neurological diseases, the structural complexity and intricate function based on cell–cell interactions of the BBB bring various limitations to the applications of existing models. In this study, a novel BBB micro-organoid model was established by culturing neurovascular unit (NVU) cells on a decellularized squid mantle scaffold (DSMS) film to reconstitute a more authentic and reliable NVU microenvironment for in vitro research. The DSMS applied was obtained from squid mantle scaffolds via decellularization, followed by defatting, and showed good biocompatibility with no cytotoxicity. The DSMS film was finally prepared by lyophilization. The lyophilized film exhibited a void ratio and pore size suitable for the adhesion and growth of endothelial cells (hCMEC/D3) and astrocytes (hACs), which led to the formation of a BBB-like spatial structure. The BBB micro-organoid model exhibited functional barrier properties, including an effective transendothelial electrical resistance (TEER) of approximately 230 Ω/cm2, restricted permeability to macromolecules—with apparent permeability coefficients (Papp) of 6.3 × 10−7 cm/s for 10 kDa and 2.7 × 10−7 cm/s for 70 kDa FITC–dextran—and expression of tight junctional complex (TJC) proteins such as vascular endothelial cadherin (VE-cad) and Zonula Occludens-1 (ZO-1). Furthermore, low-density lipoprotein receptor-related protein 1 (LRP1), a key receptor stably expressed in these two NVU cell types, was utilized as a critical indicator to assess the integrity of the BBB micro-organ model and its responsiveness to pathophysiological stimuli, particularly under thrombotic conditions. This study not only validates the feasibility of constructing a functionally competent BBB micro-organ model using DSMS films integrated with NVU cells but also provides a promising in vitro platform for subsequent studies on the BBB-related pathological mechanisms and the evaluation of drug permeability across the BBB.

Bioinks represent the core of 3D bioprinting, as they are the carrier responsible for enabling the fabrication of anatomically precise, cell-laden constructs that replicate native tissue architecture. Indeed, their role goes beyond structural support, as they must also sustain cellular viability, proliferation, and differentiation functions, which are critical for applications in the field of regenerative medicine and personalized therapies. However, at present, a persistent challenge lies in reconciling the conflicting demands of rheological properties, which are essential for printability and biological functionality. This trade-off limits the clinical translation of bioprinted tissues, particularly for vascularized or mechanically dynamic organs. Despite huge progress during the last decade, challenges persist in standardizing bioink characterization, scaling production, and ensuring long-term biomimetic performance. Based on these challenges, this review explores the inherent trade-off faced by bioink research optimizing rheology to ensure printability, shape fidelity, and structural integrity, while simultaneously maintaining high cell viability, proliferation, and tissue maturation offering insights into designing next-generation bioinks for functional tissue engineering.

Protamine is a promising marine-derived bioactive compound that is highly arginine-rich and has demonstrated unique advantages in medical and biological research. This study, for the first time, investigates the molecular mechanisms underlying the immunomodulatory effects of Salmon Protamine Sulfate (SPS), Symplectoteuthis oualaniensis Protamine (SOP), and its polyethylene glycol (PEG) derivative (SOP-PEG) on RAW264.7 macrophages. The results demonstrate that both SOP and SOP-PEG significantly enhance the proliferation of RAW264.7 cells by promoting the secretion of pro-inflammatory cytokines and nitric oxide (NO), increasing ROS production, and improving antioxidant capacity, in comparison to SPS. Elevated ROS levels play a crucial role in enhancing macrophage immune activity, while the enhanced antioxidant defense mechanisms help maintain redox homeostasis and protect against oxidative stress-induced cellular damage. A Western blot analysis reveals that SOP and SOP-PEG notably regulate the expression of key proteins associated with the PI3K/Akt signaling pathway and anti-apoptotic mechanisms. Furthermore, a flow cytometry analysis indicates a significant increase in the G2/M-phase cell population in the treatment groups, which is corroborated by Western blot data showing alterations in critical regulatory proteins. Notably, SOP-PEG exhibits the strongest effects in regulating macrophage immune activity, which can be attributed to the enhanced stability and prolonged bioactivity resulting from the PEGylation of SOP. This comprehensive study reveals how SOP and SOP-PEG enhance macrophage immune function through multiple mechanisms, including PI3K/Akt activation, redox regulation, and cell cycle modulation. It provides valuable insights and a theoretical foundation for their potential applications in immunotherapy and immune regulation.

Mussel foot protein is a bioadhesive protein with potential biomedical applications, but its limited solubility and poor biological stability hinder its widespread use. In this study, highly soluble mussel foot protein (HMFP) was successfully extracted using a stepwise selective enzymatic digestion method, with a molecular weight in the range of 11-17 kDa. Furthermore, a dual-functional polyethylene glycol (PEG) derivative of HMFP, designated HMFP-PEG, was synthesized. FTIR analysis confirmed the successful modification of HMFP with PEG, while TGA analysis and SEM observations demonstrated that PEG modification significantly enhanced the stability of the protein. Both HMFP and HMFP-PEG effectively scavenged free radicals, enhanced antioxidant enzyme activity, and reduced MDA levels. Additionally, they activated the PI3K/Akt and Nrf2/HO-1 signaling pathways, inhibiting H2O2-induced cell apoptosis. Notably, HMFP-PEG exhibited superior antioxidant and cell-protective effects compared to HMFP, suggesting that PEG modification improves the functional stability of the protein. This study provides theoretical support for the potential application of HMFP in the biomedical and tissue engineering fields.

Chronic inflammation is closely associated with various diseases, underscoring the need for natural, biocompatible anti-inflammatory candidates. For this purpose, mussel foot protein could be an excellent candidate due to its diverse biological activities. Hence, this study systematically evaluates the anti-inflammatory effects of a highly soluble mussel foot protein (HMFP) and HMFP-PEG using LPS-stimulated RAW264.7 cells as an in vitro inflammation model. The results reveal that both HMFP and HMFP-PEG markedly reduced intracellular reactive oxygen species (ROS) levels and suppressed the secretion of pro-inflammatory mediators, including IL-1β, TNF-α, and NO, while promoting the production of anti-inflammatory cytokines such as IL-10 and TGF-β. Mechanistically, both agents markedly inhibited the LPS-induced phosphorylation of PI3K, Akt, NF-κB, and IκB, indicating that their anti-inflammatory effects are mediated via suppression of the PI3K/Akt and NF-κB signaling pathways. Furthermore, HMFP and HMFP-PEG downregulated the expression of the inflammatory marker iNOS and markedly upregulated the M2 macrophage marker CD206, suggesting a role in promoting macrophage polarization toward an anti-inflammatory M2 phenotype. Notably, NF-κB signaling was identified as a key mediator in the anti-inflammatory mechanisms of both HMFP and its PEG-modified form. Collectively, these findings demonstrate that HMFP and HMFP-PEG exert significant anti-inflammatory effects through dual inhibition of NF-κB and PI3K/Akt signaling and by promoting M2 macrophage polarization, indicating their potential as promising candidates for the treatment of inflammation-related diseases.

As the focus on green chemistry intensifies, researchers are progressively looking to incorporate biodegradable and environmentally friendly solvents. Given the prevalent use of inorganic solvents in conventional methods for detecting selenium content, this study utilized a mixture design approach to create four deep eutectic solvents (DESs). The elements of the DESs consisted of six different compounds: guanidine hydrochloride, fructose, glycerol, citric acid, proline, and choline chloride. The synthesized deep eutectic solvents (DESs) exhibited a uniform and transparent appearance. The ideal ratios for each DES were established based on their density and viscosity measurements, leading to the formulations of DES1 (34% guanidine hydrochloride, 21% fructose, 45% water), DES2 (23% guanidine hydrochloride, 32% glycerol, 45% water), DES3 (27.5% citric acid, 27.5% proline, 45% water), and DES4 (30% choline chloride, 25% citric acid, 45% water). The characterization of the deep eutectic solvents (DESs) was performed using nuclear magnetic resonance (NMR) spectroscopy and infrared (IR) spectroscopy, which confirmed the molecular formation of each DES. Following this, the DESs were applied as extraction solvents in a process involving ultrasonic-assisted microextraction (UAE) combined with inductively coupled plasma mass spectrometry (ICP-MS) to assess the selenium levels in selenium-rich rice. The results were benchmarked against traditional microwave-assisted acid digestion (TM-AD), revealing selenium recovery rates ranging from 85.5% to 106.7%. These results indicate that UAE is an effective method for extracting selenium from selenium-rich rice, thereby establishing a solid data foundation for the environmentally friendly analysis of selenium content in rice.

Carboxymethyl chitosan (CMC)-based hydrogels have emerged as promising candidates for wound dressing applications due to their excellent biocompatibility and tunable physicochemical properties. In this study, a novel hydrogel functionalized with hyaluronic acid (HA) and RGD peptides (RGD) was fabricated and evaluated for its structural characteristics and wound-healing potential. Using CMC as the base matrix and EDC/NHS as crosslinking agents, four hydrogel variants were fabricated: CMC gel, CMC-HA gel, CMC-RGD gel, and CMC-HA-RGD gel. The preliminary cell compatibility experiment identified the optimal formulation as 1% CMC, 0.9% HA, and 0.02 mg/mL RGD, crosslinked with 1 vol% EDC and 0.05 wt% NHS. Scanning electron microscopy showed a porous architecture (100–400 μm), conducive to fibroblast viability and proliferation. Zeta potential measurements (|ζ| > 30 mV) indicated colloidal stability of the hydrogel system. Fourier-transform infrared spectroscopy confirmed successful crosslinking and integration of HA and RGD via hydrogen bonding and electrostatic interactions, forming a stable three-dimensional network. Thermogravimetric analysis revealed enhanced thermal stability upon HA/RGD incorporation. CCK-8 assays demonstrated significantly improved cell viability with HA/RGD loading (p < 0.05), while Ki-67 immunofluorescence confirmed enhanced fibroblast proliferation, with the CMC-HA-RGD gel showing the most pronounced effect. In vitro scratch assay results demonstrated that the CMC-HA-RGD hydrogel dressing significantly enhanced cellular migration compared to other carboxymethyl chitosan-based hydrogel groups (p < 0.05). The observed statistically significant improvement in cell migration rate versus controls underscores the distinctive enhancement of synergistic HA and RGD modification in accelerating cellular migration and facilitating wound repair. Collectively, these findings suggest that the CMC-HA-RGD hydrogel possesses favorable physicochemical and biological properties and holds strong potential as an advanced wound dressing for the treatment of chronic and refractory wounds.

This study evaluates the influence of conventional versus simplified drilling protocols on bone remodeling after the osteointegration period, marginal bone loss (MBL), and primary implant stability. A randomized, double-blind clinical trial was conducted involving 44 implants in 37 patients over a two-year period. The primary outcome was peri-implant tissue stability, measured as MBL at baseline, 12 months, and 24 months. Secondary outcomes included implant stability, measured via insertion torque, and survival rates. The results indicated no significant differences in initial bone remodeling and MBL between groups after 24 months. Both protocols demonstrated high survival rates, with one implant failure recorded in the simplified protocol group. Although simplified drilling protocols may reduce surgical complexity, concerns about heat generation and reduced adaptability in osteotomy were described in the literature. This study concludes that drilling protocol choice does not significantly impact bone levels during osteointegration, crestal bone maintenance, or implant survival over 24 months, but further research is needed to explore long-term effects and prosthetic factors.

Carboxymethyl chitosan (CMC)-based hydrogels (HG) have gained significant attention for therapeutic applications due to their biomimetic properties and biocompatibility. This study explores, for the first time, the regenerative and osteogenic potential of CMC-HG incorporated with a marine fungi-derived fibrinolytic compound, FGFC-1. The inclusion of FGFC-1 did not significantly alter the crucial characteristics of the HGs, including secondary structure, thermal stability, protein adhesion, and in vitro degradation. However, incorporation of FGFC1 increased the swelling capacity (from 132.88 % to 157.11 %) and decreased the mineral adhesion (0.416 at 0.1 mg/ml) and porosity (from 72.95 % to 54.29 %). In general, the bacterial adhesion was decreased by 44.3 % in HG than control., Optimal culture conditions for mesenchymal stem cells (MSCs) were achieved with 2 % CMC and FGFC-1 concentrations of 0.01-1 mg/ml (Supplementary Fig. S2), supporting significant MSC growth. SEM image proved more interconnected dense fibrillar clustered morphology of MSCs on HGs than 2D. FGFC-1 accelerated osteogenic differentiation of MSCs by increasing mRNA expression levels of Runx2 (4.98), collagen-1 alpha-1 (3.4), osteocalcin (3.62), and ALP (4.20), which was further validated through enhanced staining for alizarin red, von Kossa, and alkaline phosphatase, as well as immunostaining for osteocollagen and osteocalcin in differentiated MSCs within the hydrogels. Notably, FGFC-1 significantly induced osteogenic differentiation along with supplements. These findings highlight FGFC-1-loaded CMC hydrogels as a promising strategy for stem cell-mediated bone regeneration in biomedical applications.

Trans fats are linked to numerous chronic diseases and cellular dysfunction; however, Spain has not implemented effective regulatory measures to restrict their presence in food products. This study addressed these gaps by analyzing trans fat content in commercial pastries sold in Spain and their biological impacts on mesenchymal stem cells, further examining its compliance with international guidelines. Also, a novel and scalable method for extracting fatty acids from pastry samples was developed and applied, enabling precise analysis using gas chromatography alongside sensory property assessments. The findings revealed significant variability in TFA levels across samples. To assess the biological implications of these TFAs, mesenchymal stem cells (MSCs) were cultured to perform dose–response experiments using two selected pastry samples with the highest TFA content. Cellular adhesion, cytotoxicity, and proliferation were evaluated through MTT assays, bright-field, and fluorescence staining using FITC and DAPI markers. Results demonstrated dose-dependent impacts of TFAs on MSC viability, including reduced adhesion and proliferation alongside increased cytotoxicity. This study underlines the need for stricter regulatory frameworks to monitor TFA levels worldwide, including in Spain’s food industry. Additionally, it highlights the potential health risks associated with excessive TFA consumption, particularly concerning cellular health and growth mechanisms, which provide insights into its potential bioaccumulation implications. These findings provide a foundation for further research into dietary guidelines and industrial practices aimed at minimizing TFA exposure while promoting public health safety.

The study focuses on developing and evaluating 3D biomimetic fibrous scaffolds to enhance osteoblast differentiation and bone tissue regeneration. Utilizing a synergistic approach, biological and chemical factors were compartmentalized within the fibrous scaffolds through co-axial electrospinning. Bioactive glass (BG) was used for osteo-conductivity, and Bone-Conditioned Medium (BCM) for osteoinduction. The BCM, derived from ovine bone chips, was investigated for its optimal concentration using pre-osteoblast cells. Comprehensive assessment of the scaffolds included physicochemical properties, drug release, cell viability, and osteogenic potential. The scaffold's architecture, confirmed by Scanning electron microscopy (SEM) analysis, effectively emulated the natural extracellular matrix (ECM). Energy Dispersive X-ray Spectroscopy (EDX) and Fourier Transform Infrared Spectroscopy (FTIR) analyses verified the successful integration of BG and BCM, while UV-Vis spectroscopy demonstrated controlled BCM release. Both BG and BCM scaffolds notably enhanced osteoblast differentiation, as evident with Alizarin red staining. The combined use of BG and BCM in scaffolds synergistically promoted osteogenic differentiation and viability of MC3T3-E1 cells. Furthermore, these scaffolds significantly increased the expression of Bone Sialoprotein (BSP), Osteocalcin (OCN), and Runt-related transcription factor 2 (RUNX2) which indicate increase in osteogenic differentiation. This study provides evidence for advanced scaffold systems that can guide cell responses for effective bone tissue regeneration.

Cellulose nanocrystals (CNC) have garnered significant attention in pharmaceutical and medical applications due to their biocompatibility, biodegradability, renewability, and strong surface reactivity. In this study, we designed 3D-printed bioactive composite scaffolds via fused deposition modeling (FDM), incorporating polycaprolactone (PCL), CNC derived from Ficus thonningii bark, and silver nanoparticles (AgNps) synthesized through in situ reduction of silver nitrate AgNO3. Energy-dispersive X-ray spectroscopy (EDX) confirmed AgNps incorporation, while scanning electron microscopy (SEM) revealed a highly porous, interconnected structure. The inclusion of CNC and AgNps enhanced PCL's biodegradability, hydrophilicity, and hydroxyapatite nucleation, all crucial for osteoconductivity. The scaffolds demonstrated mechanical properties suitable for bone regeneration, effective antibacterial activity against Escherichia coli, and cytocompatibility with Mesenchymal Stem Cells (MSCs). These findings highlight the potential of PCL/CNCx/AgNps scaffolds as advanced biomaterials for bone tissue engineering, since they offer enhanced resorbability, antibacterial protection, and structural adaptability.

Squid skin decellularized dermal matrix (SADM) is gaining attention in tissue engineering and regenerative medicine due to its mimicking of the extracellular matrix property. Hence, SADM was used to investigate mimicking the microenvironment of cellular growth, inducing cellular infiltration and angiogenesis, and facilitating the repair of acute craniofacial wounds. For this, tissue regeneration membranes from squid skin were prepared by decolorization, degreasing and decellularisation methods. The effect of SADM in guiding bone tissue regeneration was evaluated using the rabbit skull bone defect model. SEM images of SADM had a bilayer membrane architecture characterized by a reticulated porous structure on one side and a dense, non-porous surface on the opposite side. Notably, the water absorption capacity of SADM was approximately eight times higher than its weight, exhibiting a porosity of 58% and a peak average tensile stress of 10.43 MPa. Additionally, simulations of tissue fluid degradation indicated a degradation rate of 70.42% and 88.33% on days 8 and 12, respectively. Following 4 and 8 weeks of animal studies focused on repairing cranial bone defects in rabbits, the findings demonstrated that SADM served as an effective barrier against fibrous connective tissue, promoted the proliferation of osteoblasts, and supported bone regeneration. This was confirmed through micro-CT imaging, and sections were stained with senna solid green. In summary, SADM is capable of directing cell infiltration and bone tissue formation, modulating the expression and secretion of inflammatory and skin repair-related factors, thereby enhancing tissue healing.

In recent years, arginine-rich basic proteins have garnered significant attention due to their essential roles in various biological processes. However, the potential of marine-derived proteins in this domain remains largely unexplored. This study presents, for the first time, the isolation and purification of a 14.3 kDa protamine (SOP) from the mature spermatogonial tissues of Symplectoteuthis oualaniensis. Additionally, we obtained an 18.5 kDa PEGylated derivative, SOP-PEG. The physicochemical properties of both SOP and SOP-PEG were comprehensively characterized using SEM, FTIR, CD, and TGA. PEGylation markedly altered the surface morphology, secondary structure, and thermal stability of SOP. In vitro studies demonstrated that PEGylation significantly enhanced the biocompatibility of SOP, leading to improved proliferation of L-929 fibroblasts. Furthermore, both SOP and its PEGylated derivative (SOP-PEG) regulated the cell cycle, activated the PI3K-Akt signaling pathway, and modulated anti-apoptotic mechanisms, suggesting their potential to support cell survival and facilitate tissue regeneration. Notably, SOP-PEG exhibited superior bioactivity, likely attributable to its enhanced delivery efficiency conferred by PEGylation. Collectively, these findings underscore the promising applications of SOP and SOP-PEG in regenerative medicine and highlight the pivotal role of PEGylation in augmenting the bioactivity of SOP.