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17 pages, 3151 KiB  
Article
Towards a Consensus for the Analysis and Exchange of TFA as a Counterion in Synthetic Peptides and Its Influence on Membrane Permeation
by Vanessa Erckes, Alessandro Streuli, Laura Chamera Rendueles, Stefanie Dorothea Krämer and Christian Steuer
Pharmaceuticals 2025, 18(8), 1163; https://doi.org/10.3390/ph18081163 - 5 Aug 2025
Abstract
Background: With the increasing shift in drug design away from classical drug targets towards the modulation of protein-protein interactions, synthetic peptides are gaining increasing relevance. The synthesis and purification of peptides via solid-phase peptide synthesis (SPPS) strongly rely on trifluoroacetic acid (TFA) as [...] Read more.
Background: With the increasing shift in drug design away from classical drug targets towards the modulation of protein-protein interactions, synthetic peptides are gaining increasing relevance. The synthesis and purification of peptides via solid-phase peptide synthesis (SPPS) strongly rely on trifluoroacetic acid (TFA) as a cleavage agent and ion-pairing reagent, respectively, resulting in peptides being obtained as TFA salts. Although TFA has excellent properties for peptide production, numerous studies highlight the negative impact of using peptides from TFA salts in biological assays. Methods: Investigated peptides were synthesized via SPPS and the TFA counterion was exchanged for Cl via freeze-drying in different concentrations of HCl. Detection and quantification of residual TFA were carried out via FT-IR, 19F-NMR, and HPLC using an evaporative light-scattering detector (ELSD). A liposomal fluorescence assay was used to test for the influence of the counterion on the peptides’ passive membrane permeability. Results: All TFA detection methods were successfully validated according to ICH guidelines. TFA removal with 10 mM HCl was determined to be the optimal condition. No impact on peptide purity was observed at all HCl concentrations. Influences on permeability coefficients depending on peptide sequence and salt form were found. Conclusions: This study presents a systematic investigation of the removal of TFA counterions from synthetic peptides and their replacement with Cl counterions. Detected counterion contents were used to understand the impact of sequence differences, especially positive charges, on the amount and potential localization of counterions. Our findings emphasize the importance of counterion quantification and specification in assays with synthetic peptides. Full article
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11 pages, 972 KiB  
Article
Rapid and Accurate Detection of the Most Common Bee Pathogens; Nosema ceranae, Aspergillus flavus, Paenibacillus larvae and Black Queen Cell Virus
by Simona Marianna Sanzani, Raied Abou Kubaa, Badr-Eddine Jabri, Sabri Ala Eddine Zaidat, Rocco Addante, Naouel Admane and Khaled Djelouah
Insects 2025, 16(8), 810; https://doi.org/10.3390/insects16080810 - 5 Aug 2025
Abstract
Honey bees are essential pollinators for the ecosystem and food crops. However, their health and survival face threats from both biotic and abiotic stresses. Fungi, microsporidia, and bacteria might significantly contribute to colony losses. Therefore, rapid and sensitive diagnostic tools are crucial for [...] Read more.
Honey bees are essential pollinators for the ecosystem and food crops. However, their health and survival face threats from both biotic and abiotic stresses. Fungi, microsporidia, and bacteria might significantly contribute to colony losses. Therefore, rapid and sensitive diagnostic tools are crucial for effective disease management. In this study, molecular assays were developed to quickly and efficiently detect the main honey bee pathogens: Nosema ceranae, Aspergillus flavus, Paenibacillus larvae, and Black queen cell virus. In this context, new primer pairs were designed for use in quantitative Real-time PCR (qPCR) reactions. Various protocols for extracting total nucleic acids from bee tissues were tested, indicating a CTAB-based protocol as the most efficient and cost-effective. Furthermore, excluding the head of the bee from the extraction, better results were obtained in terms of quantity and purity of extracted nucleic acids. These assays showed high specificity and sensitivity, detecting up to 250 fg of N. ceranae, 25 fg of P. larvae, and 2.5 pg of A. flavus DNA, and 5 pg of BQCV cDNA, without interference from bee DNA. These qPCR assays allowed pathogen detection within 3 h and at early stages of infection, supporting timely and efficient management interventions. Full article
(This article belongs to the Section Insect Behavior and Pathology)
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17 pages, 6360 KiB  
Article
Integrating Lanthanide-Reclaimed Wastewater and Lanthanide Phosphate in Corn Cultivation: A Novel Approach for Sustainable Agriculture
by George William Kajjumba, Savanna Vacek and Erica J. Marti
Sustainability 2025, 17(15), 6734; https://doi.org/10.3390/su17156734 - 24 Jul 2025
Viewed by 330
Abstract
With increasing global challenges related to water scarcity and phosphorus depletion, the recovery and reuse of wastewater-derived nutrients offer a sustainable path forward. This study evaluates the dual role of lanthanides (Ce3+ and La3+) in recovering phosphorus from municipal wastewater [...] Read more.
With increasing global challenges related to water scarcity and phosphorus depletion, the recovery and reuse of wastewater-derived nutrients offer a sustainable path forward. This study evaluates the dual role of lanthanides (Ce3+ and La3+) in recovering phosphorus from municipal wastewater and supporting corn (Zea mays) cultivation through lanthanide phosphate (Ln-P) and lanthanide-reclaimed wastewater (LRWW, wastewater spiked with lanthanide). High-purity precipitates of CePO4 (98%) and LaPO4 (92%) were successfully obtained without pH adjustment, as confirmed by X-ray photoelectron spectroscopy (XPS) and energy-dispersive spectroscopy (EDS). Germination assays revealed that lanthanides, even at concentrations up to 2000 mg/L, did not significantly alter germination rates compared to traditional coagulants, though root and shoot development declined above this threshold—likely due to reduced hydrogen peroxide (H2O2) production and elevated total dissolved solids (TDSs), which induced physiological drought. Greenhouse experiments using desert-like soil amended with Ln-P and irrigated with LRWW showed no statistically significant differences in corn growth parameters—including plant height, stem diameter, leaf number, leaf area, and biomass—when compared to control treatments. Photosynthetic performance, including stomatal conductance, quantum efficiency, and chlorophyll content, remained unaffected by lanthanide application. Metal uptake analysis indicated that lanthanides did not inhibit phosphorus absorption and even enhanced the uptake of calcium and magnesium. Minimal lanthanide accumulation was detected in plant tissues, with most retained in the root zone, highlighting their limited mobility. These findings suggest that lanthanides can be safely and effectively used for phosphorus recovery and agricultural reuse, contributing to sustainable nutrient cycling and aligning with the United Nations’ Sustainable Development Goals of zero hunger and sustainable cities. Full article
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19 pages, 2472 KiB  
Article
Immunomodulation Through Fibroblast-Derived Extracellular Vesicles (EVs) Within 3D Polycaprolactone–Collagen Matrix
by Afsara Tasnim, Diego Jacho, Agustin Rabino, Jose Benalcazar, Rafael Garcia-Mata, Yakov Lapitsky and Eda Yildirim-Ayan
Biomimetics 2025, 10(8), 484; https://doi.org/10.3390/biomimetics10080484 - 22 Jul 2025
Viewed by 438
Abstract
Extracellular vesicles (EVs) have emerged as promising acellular tools for modulating immune responses for tissue engineering applications. This study explores the potential of human fibroblast-derived EVs delivered within a three-dimensional (3D) injectable scaffold composed of polycaprolactone (PCL) nanofibers and collagen (PNCOL) to reprogram [...] Read more.
Extracellular vesicles (EVs) have emerged as promising acellular tools for modulating immune responses for tissue engineering applications. This study explores the potential of human fibroblast-derived EVs delivered within a three-dimensional (3D) injectable scaffold composed of polycaprolactone (PCL) nanofibers and collagen (PNCOL) to reprogram macrophage behavior and support scaffold integrity under inflammatory conditions. EVs were successfully isolated from human fibroblasts using ultracentrifugation and characterized for purity, size distribution and surface markers (CD63 and CD9). Macrophage-laden PNCOL scaffolds were prepared under three conditions: macrophage-only (MP), fibroblast co-encapsulated (F-MP), and EV-encapsulated (EV-MP) groups. Structural integrity was assessed via scanning electron microscopy and Masson’s trichrome staining, while immunomodulatory effects were evaluated through metabolic assays, gene expression profiling, and immunohistochemistry for macrophage polarization markers (CD80, CD206). When co-encapsulated with pro-inflammatory (M1) macrophages in PNCOL scaffolds, fibroblast-derived EVs preserved scaffold structure and significantly enhanced macrophage metabolic activity compared to the control (MP) and other experimental group (F-MP). The gene expression and immunohistochemistry data demonstrated substantial upregulation of anti-inflammatory markers (TGF-β, CD163, and CCL18) and surface protein CD206, indicating a phenotypic shift toward M2-like macrophages for EV-encapsulated scaffolds relative to the other groups. The findings of this study demonstrate that fibroblast-derived EVs integrated into injectable PCL–collagen scaffolds offer a viable, cell-free approach to modulate inflammation, preserve scaffold structure, and support regenerative healing. This strategy holds significant promise for advancing immuno-instructive platforms in regenerative medicine, particularly in settings where conventional cell therapies face limitations in survival, cost, or safety. Full article
(This article belongs to the Special Issue Biomimetic Application on Applied Bioengineering)
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19 pages, 3398 KiB  
Article
Synthesis and Evaluation of [18F]AlF-NOTA-iPD-L1 as a Potential Theranostic Pair for [177Lu]Lu-DOTA-iPD-L1
by Guillermina Ferro-Flores, Myrna Luna-Gutiérrez, Blanca Ocampo-García, Nallely Jiménez-Mancilla, Nancy Lara-Almazán, Rigoberto Oros-Pantoja, Clara Santos-Cuevas, Erika Azorín-Vega and Laura Meléndez-Alafort
Pharmaceutics 2025, 17(7), 920; https://doi.org/10.3390/pharmaceutics17070920 - 16 Jul 2025
Viewed by 385
Abstract
Background/Objective: Programmed cell death ligand-1 (PD-L1), which is overexpressed in certain tumors, inhibits the body’s natural immune response by providing an “off” signal that enables cancer cells to evade the immune system. It has been demonstrated that [177Lu]Lu-DOTA-iPD-L1 (PD-L1 inhibitor [...] Read more.
Background/Objective: Programmed cell death ligand-1 (PD-L1), which is overexpressed in certain tumors, inhibits the body’s natural immune response by providing an “off” signal that enables cancer cells to evade the immune system. It has been demonstrated that [177Lu]Lu-DOTA-iPD-L1 (PD-L1 inhibitor cyclic peptide) promotes immune responses. This study aimed to synthesize and evaluate [18F]AlF-NOTA-iPD-L1 as a novel radiotracer for PD-L1 positron emission tomography (PET) imaging and as a potential theranostic pair for [177Lu]Lu-DOTA-iPD-L1. Methods: The NOTA-iPD-L1 peptide conjugate was synthesized and characterized by U.V.-vis, I.R.-FT, and UPLC-mass spectroscopies. Radiolabeling was performed using [18F]AlF as the precursor, and the radiochemical purity (HPLC), partition coefficient, and serum stability were assessed. Cellular uptake and internalization (in 4T1 triple-negative breast cancer cells), binding competition, immunofluorescence, and Western blot assays were applied for the radiotracer in vitro characterization. Biodistribution in mice bearing 4T1 tumors was performed, and molecular imaging (Cerenkov images) of [18F]AlF-NOTA-iPD-L1 and [177Lu]Lu-DOTA-iPD-L1 in the same mouse was obtained. Results: [18F]AlF-NOTA-iPD-L1 was prepared with a radiochemical purity greater than 97%, and it demonstrated high in vitro and in vivo stability, as well as specific recognition by the PD-L1 protein (IC50 = 9.27 ± 2.69 nM). Biodistribution studies indicated a tumor uptake of 6.4% ± 0.9% ID/g at 1-hour post-administration, and Cerenkov images showed a high tumor uptake of both [18F]AlF-NOTA-iPD-L1 and 177Lu-iPD-L1 in the same mouse. Conclusions: These results warrant further studies to evaluate the clinical usefulness of [18F]AlF-NOTA-iPD-L1/[177Lu]Lu-DOTA-iPD-L1 as a radiotheranostic pair in combination with anti-PD-L1/PD1 immunotherapy. Full article
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21 pages, 6281 KiB  
Article
Novel Compounds Featuring a Thiophene Carboxamide Scaffold: Synthesis, Characterization and Antiproliferative Evaluation
by Bogdan-Ionuț Mara, Alexandra Mioc, Livia-Nicoleta Deveseleanu-Corici, Codruța Șoica and Liliana Cseh
Int. J. Mol. Sci. 2025, 26(14), 6823; https://doi.org/10.3390/ijms26146823 - 16 Jul 2025
Viewed by 488
Abstract
Thiophene derivatives are particularly attractive for application in drug development for their versatile pharmacological properties. We synthesized a series of four compounds with thiophene carboxamide as a scaffold. The structures were established based on HR-MS and 1D- and 2D-NMR. The purity of the [...] Read more.
Thiophene derivatives are particularly attractive for application in drug development for their versatile pharmacological properties. We synthesized a series of four compounds with thiophene carboxamide as a scaffold. The structures were established based on HR-MS and 1D- and 2D-NMR. The purity of the compounds was established to be greater than 92% by thin-layer chromatography and NMR. The cytotoxic effects of the newly synthesized compounds were evaluated against the normal HaCaT cell line and A375, HT-29, and MCF-7 cancer cell lines. The cytotoxic assessment revealed that two compounds exhibit a significant cytotoxic effect on all cancer cell lines. To investigate their potential underlying mechanisms of action, several tests were performed: immunofluorescence imaging, caspase-3/7 assay, mitochondrial membrane potential (JC-1) assay, and 2′,7′–dichlorofluorescein diacetate (DCFDA) assay. MB-D2 proved to be the most cytotoxic and effective in terms of caspase 3/7 activation, mitochondrial depolarization and decrease in ROS production; these effects did not occur in normal HaCaT cells, revealing that MB-D2 has a high selectivity against A375 cancer cells. Full article
(This article belongs to the Section Biochemistry)
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19 pages, 3838 KiB  
Article
Identification of a Novel Antibacterial Function of Mammalian Calreticulin
by Yichao Ma, Jiachen Liu, Xinming Qin, Xiaojing Cui and Qian Yang
Biomolecules 2025, 15(7), 966; https://doi.org/10.3390/biom15070966 - 4 Jul 2025
Viewed by 431
Abstract
Calreticulin is a highly conserved and multifunctional molecular chaperone ubiquitously expressed in humans and animals. Beyond its well-established roles in calcium homeostasis, protein folding, and immune regulation, recent studies in aquatic species have suggested a previously unrecognized antimicrobial function of calreticulin. These findings [...] Read more.
Calreticulin is a highly conserved and multifunctional molecular chaperone ubiquitously expressed in humans and animals. Beyond its well-established roles in calcium homeostasis, protein folding, and immune regulation, recent studies in aquatic species have suggested a previously unrecognized antimicrobial function of calreticulin. These findings raise the question of whether calreticulin also exerts antibacterial activity in terrestrial mammals, which has not been systematically investigated to date. To address this knowledge gap, we successfully constructed and expressed recombinant goat calreticulin using the Pichia pastoris expression system, yielding a protein of over 99% purity that predominantly exists in dimeric form. Functional assays demonstrated that both recombinant goat and human calreticulin exhibited preliminary inhibitory activity against Escherichia coli, Salmonella typhimurium, and Pasteurella multocida. Calreticulin was capable of binding to these three bacterial species as well as bacterial lipopolysaccharides (LPS). Notably, in the presence of Ca2+, calreticulin induced bacterial aggregation, indicating a potential mechanism for limiting bacterial dissemination and proliferation. Given the high anatomical, genetic, and physiological similarity between goats and humans—particularly in respiratory tract structure and mucosal immune function—neonatal goats were selected as a relevant model for evaluating the in vivo antimicrobial efficacy of calreticulin. Accordingly, we established an intranasal infection model using Pasteurella multocida to assess the protective role of calreticulin against respiratory bacterial challenge. Following infection, calreticulin expression was markedly upregulated in the nasal mucosa, trachea, and lung tissues. Moreover, intranasal administration of exogenous calreticulin significantly alleviated infection-induced pathological injury to the respiratory system and effectively decreased bacterial loads in infected tissues. Collectively, this study systematically elucidates the antimicrobial activity of calreticulin in a mammalian model and highlights its potential as a natural immune effector, providing novel insights for the development of host-targeted antimicrobial strategies. Full article
(This article belongs to the Section Biomacromolecules: Proteins, Nucleic Acids and Carbohydrates)
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19 pages, 8164 KiB  
Article
A Novel PCR-Based Tool to Trace Oenological Saccharomyces cerevisiae Yeast by Monitoring Strain-Specific Nucleotide Polymorphisms
by Anna Baldisseri, Davide Santinello, Sara Granuzzo, Martina Frizzarin, Fabio De Pascale, Geppo Sartori, Paolo Antoniali, Stefano Campanaro and Raffaele Lopreiato
Foods 2025, 14(13), 2379; https://doi.org/10.3390/foods14132379 - 4 Jul 2025
Viewed by 353
Abstract
Saccharomyces cerevisiae plays a fundamental role in winemaking, not only driving alcoholic fermentation but also producing secondary metabolites that contribute to the organoleptic properties of wine. To ensure consistent quality and process efficiency, wineries commonly employ selected starter strains. Accordingly, the ability to [...] Read more.
Saccharomyces cerevisiae plays a fundamental role in winemaking, not only driving alcoholic fermentation but also producing secondary metabolites that contribute to the organoleptic properties of wine. To ensure consistent quality and process efficiency, wineries commonly employ selected starter strains. Accordingly, the ability to control strain purity and traceability is of critical importance. Currently, the inter-delta PCR method is widely used for the strain-specific genotyping of S. cerevisiae. However, its resolution diminishes when analyzing genetically similar strains, such as those isolated from related grape types or during genotyping of large yeast collections. To address this limitation, we developed a novel strategy that integrates computational and experimental approaches to identify highly specific allelic variants (single nucleotide polymorphisms, SNPs) within the S. cerevisiae genome. Comparative genomic analysis of twenty-eight different strains led to the identification of multiple strain-specific SNPs. From these, nine SNPs spanning five strains were selected and validated through targeted PCR assays. These assays confirmed the feasibility of using SNPs as reliable genetic markers for strain discrimination and traceability. Overall, our findings demonstrate that this SNP-based approach, implemented via multiplex allele-specific (AS) PCR assays, offers a rapid, cost-effective, and highly discriminatory alternative to current genotyping methods, particularly for differentiating closely related strains. Full article
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18 pages, 2427 KiB  
Article
Exploring the Therapeutic Potential of 177Lu-PSMA-617 in a Mouse Model of Prostate Cancer Bone Metastases
by Cheng-Liang Peng, Chun-Tang Chen and I-Chung Tang
Int. J. Mol. Sci. 2025, 26(13), 5970; https://doi.org/10.3390/ijms26135970 - 21 Jun 2025
Viewed by 627
Abstract
Prostate cancer is the second leading cause of cancer-related death in men, with metastatic castration-resistant prostate cancer (mCRPC) and bone metastases representing a critical clinical challenge. Although radium-223 (Ra-223) is approved for treating mCRPC with bone metastases, its efficacy remains limited, necessitating the [...] Read more.
Prostate cancer is the second leading cause of cancer-related death in men, with metastatic castration-resistant prostate cancer (mCRPC) and bone metastases representing a critical clinical challenge. Although radium-223 (Ra-223) is approved for treating mCRPC with bone metastases, its efficacy remains limited, necessitating the development of more effective therapies. This study investigates the therapeutic potential of 177Lu-PSMA-617, a PSMA-targeted radiopharmaceutical, in a murine model of prostate cancer bone metastases. To our knowledge, this is the first study to systematically evaluate 177Lu-PSMA-617 in an orthotopic bone metastatic prostate cancer model, providing a clinically relevant preclinical platform to assess both imaging and therapeutic performance. We conducted comprehensive preclinical evaluations, including synthesis, stability analysis, cell binding assays, nuclear imaging, in vivo biodistribution, pharmacokinetics, and antitumor efficacy. The synthesis of 177Lu-PSMA-617 demonstrated high radiochemical yield (99.2%), molar activity (25.5 GBq/μmol), and purity (>98%), indicating high product quality. Stability studies confirmed minimal release of free Lutetium-177, maintaining the compound’s integrity under physiological conditions. In vitro assays showed selective binding and internalization in PSMA-positive LNCaP prostate cancer cells, with negligible uptake in PSMA-negative PC-3 cells. In vivo biodistribution studies demonstrated efficient tumor targeting, with peak uptake in LNCaP tumors (23.31 ± 0.94 %IA/g) at 4 h post-injection. The radiopharmaceutical exhibited favorable pharmacokinetics, with high tumor-to-background ratios (tumor-to-blood, 434.4; tumor-to-muscle, 857.4). Therapeutic efficacy was confirmed by significant survival extension in treated mice (30.7% for 37 MBq and 53.8% for 111 MBq), with median survival times of 34 and 40 days, respectively, compared to 26 days in the control group. Radiation dosimetry analysis indicated a favorable safety profile with a calculated effective dose of 0.127 mSv/MBq. These findings highlight the novelty and translational relevance of using 177Lu-PSMA-617 in a clinically relevant bone metastasis model, reinforcing its potential as a dual-purpose agent for both targeted therapy and molecular imaging in advanced prostate cancer. Full article
(This article belongs to the Section Molecular Pharmacology)
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19 pages, 3955 KiB  
Article
Study on the Degradation of Aflatoxin B1 by Myroides odoratimimus 3J2MO
by Xue Wang, Yao-Yao Gao, Dun Wang, Qi Zhang, Hao-Ran Wang, Ting-Ting Zhang, Meng-Jie Zhu, Jing Dong, Dong Ling, Peng Feng, Xue-Hui Tang and Pei-Wu Li
Biology 2025, 14(6), 724; https://doi.org/10.3390/biology14060724 - 19 Jun 2025
Viewed by 477
Abstract
To address the issue of aflatoxin contamination, which poses a significant threat to food safety and human health, we have conducted extensive research. We have isolated a strain of Myroides odoratimimus (3J2MO) from the soil that exhibited remarkable efficiency in degrading various aflatoxin [...] Read more.
To address the issue of aflatoxin contamination, which poses a significant threat to food safety and human health, we have conducted extensive research. We have isolated a strain of Myroides odoratimimus (3J2MO) from the soil that exhibited remarkable efficiency in degrading various aflatoxin types, including AFB1, AFB2, AFG1, AFG2, and AFM1. SDS-PAGE analysis confirmed the purity of the enzymes to be over 95%. Through fluorescence assays, we quantified the enzymatic activity, with an AFB1 degradation rate of 95% achieved at 37 °C and a pH of 8.0. Further analysis using HPLC-MS/MS identified the degradation intermediates, revealing the mechanisms of lactone ring cleavage and epoxy group hydrolysis. GO/COG/KEGG annotations provided insights into the functions of these enzymes, with peroxidase linked to reactive oxygen species (ROS) generation and helicase associated with ATP-dependent conformational changes. Helicase, on the other hand, hydrolyzes ATP, driving conformational changes in AFB1 and facilitating its breakdown into non-toxic metabolites. The potential industrial-scale application of this discovery could significantly mitigate aflatoxin-related economic losses while minimizing chemical residues in the food chain. Full article
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20 pages, 8483 KiB  
Article
Comparative Efficacy of Exosomes Derived from Different Mesenchymal Stem Cell Sources in Osteoarthritis Models: An In Vitro and Ex Vivo Analysis
by Jaishree Sankaranarayanan, Hyung Keun Kim, Ju Yeon Kang, Sree Samanvitha Kuppa, Hong Yeol Yang and Jong Keun Seon
Int. J. Mol. Sci. 2025, 26(12), 5447; https://doi.org/10.3390/ijms26125447 - 6 Jun 2025
Viewed by 884
Abstract
Osteoarthritis (OA) is a prevalent and debilitating joint disorder that affects a substantial proportion of the global population, underscoring the urgent need for therapeutic strategies that extend beyond symptomatic management. Although mesenchymal stem cells (MSCs) have emerged as a promising therapeutic modality, their [...] Read more.
Osteoarthritis (OA) is a prevalent and debilitating joint disorder that affects a substantial proportion of the global population, underscoring the urgent need for therapeutic strategies that extend beyond symptomatic management. Although mesenchymal stem cells (MSCs) have emerged as a promising therapeutic modality, their clinical application remains constrained by several inherent limitations. This study explores a cell-free alternative by investigating the therapeutic potential of exosomes derived from bone marrow (BMSCs), adipose tissue (ADSCs), and umbilical cord (UMSCs) MSCs in mitigating OA pathogenesis, utilizing both in vitro and ex vivo models. Exosomes from each MSC source were isolated and characterized through nanoparticle tracking analysis, transmission electron microscopy, and Western blotting to confirm their identity and purity. Subsequently, their chondroprotective, anti-inflammatory, and regenerative properties were systematically assessed through evaluations of cell viability, expression profiles of inflammatory and chondroprotective markers, and chondrocyte migration assays. The results demonstrate that all three types of MSC-derived exosomes (MSC-Exos) exhibit low cytotoxicity while significantly suppressing proinflammatory markers and enhancing the expression of chondroprotective genes. Notably, BMSC-Exos and UMSC-Exos displayed superior efficacy in attenuating inflammation, promoting cartilage protection, and inhibiting chondrocyte apoptosis. Furthermore, all MSC-Exos markedly enhanced chondrocyte motility, a critical component of cartilage repair. Collectively, these findings support the therapeutic promise of MSC-Exos, particularly those derived from BMSCs and UMSCs, as a targeted, cell-free approach for the treatment of OA compared to ADSCs. By modulating inflammation, promoting cartilage regeneration, and preventing chondrocyte apoptosis, MSC-Exos may serve as a viable and scalable alternative to current MSC-based therapies for this widespread degenerative disease. Full article
(This article belongs to the Special Issue Molecular Advances and Perspectives in Rheumatic Diseases)
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23 pages, 3875 KiB  
Article
Chemical Composition, Quality, and Bioactivity of Laurus nobilis L. Hydrosols from the Adriatic Regions of Croatia: Implications for Dermatological Applications
by Lea Juretić, Valerija Dunkić, Ivana Gobin, Suzana Inić, Dario Kremer, Marija Nazlić, Lea Pollak, Silvestar Mežnarić, Ana Barbarić and Renata Jurišić Grubešić
Antioxidants 2025, 14(6), 688; https://doi.org/10.3390/antiox14060688 - 5 Jun 2025
Viewed by 634
Abstract
Laurus nobilis L., Lauraceae, bay laurel, has been traditionally used for its various therapeutic properties, and in recent years has been gaining interest for its potential applications in skincare products. However, the biological effects of bay laurel, particularly its hydrosols, a water fraction [...] Read more.
Laurus nobilis L., Lauraceae, bay laurel, has been traditionally used for its various therapeutic properties, and in recent years has been gaining interest for its potential applications in skincare products. However, the biological effects of bay laurel, particularly its hydrosols, a water fraction obtained during essential oil production, remain unexplored. The objective of this study was to identify the volatile compounds in L. nobilis hydrosols (LnHYs) from different coastal regions of Croatia (north, middle, and south Adriatic) and to evaluate their potential safety and efficacy for dermatological applications. Upon isolating LnHYs using microwave-assisted extraction, LnHY volatiles were identified and quantified using gas chromatography and mass spectrometry. Oxygenated monoterpenes were the dominant compounds in all LnHYs (61.72–97.00%), with 1,8-cineole being the most abundant component (52.25–81.89%). The physical and chemical parameters of LnHYs were investigated to assess their purity and quality. Biological activity (cytotoxicity and wound-healing effect) was tested on the human keratinocyte cell line (HaCaT), selected as the experimental model due to its relevance to skin biology. Additionally, contents of polyphenolic substances, antioxidative effects using the Oxygen Radical Absorbance Capacity (ORAC) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) methods, and the antimicrobial activity of LnHYs toward five skin microorganisms were determined. All tested hydrosols showed similar biological activity, with only minor differences. Cytotoxicity studies indicated the safety of the dermatological application of LnHYs, and the results of the wound-healing assay showed their neutral to mildly positive effect. Considering the growing use of bay laurel preparations in pharmaceutical and cosmetic applications, extensive studies on their biological activity, quality, and safety are essential to either support or regulate their use in humans. Full article
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18 pages, 3949 KiB  
Article
Biotechnological Utilization of Amazonian Fruit: Development of Active Nanocomposites from Bacterial Cellulose and Silver Nanoparticles Based on Astrocaryum aculeatum (Tucumã) Extract
by Sidney S. dos Santos, Miguel Ângelo Cerqueira, Ana Gabriela Azevedo, Lorenzo M. Pastrana, Fauze Ahmad Aouada, Fabrício C. Tanaka, Gustavo Frigi Perotti and Marcia Regina de Moura
Pharmaceuticals 2025, 18(6), 799; https://doi.org/10.3390/ph18060799 - 26 May 2025
Viewed by 562
Abstract
Background/Objectives: The rise of bacterial resistance and the search for alternative, biocompatible antimicrobial materials have driven interest in natural-based nanocomposites. In this context, silver nanoparticles (AgNPs) have shown broad-spectrum antibacterial activity, and bacterial cellulose (BC) is widely recognized for its high purity, hydrophilicity, [...] Read more.
Background/Objectives: The rise of bacterial resistance and the search for alternative, biocompatible antimicrobial materials have driven interest in natural-based nanocomposites. In this context, silver nanoparticles (AgNPs) have shown broad-spectrum antibacterial activity, and bacterial cellulose (BC) is widely recognized for its high purity, hydrophilicity, and biocompatibility. This study aimed to develop a bio-based BC–AgNP nanocomposite via green synthesis using Astrocaryum aculeatum (tucumã) extract and assess its antimicrobial performance for wound dressing applications. Methods: BC was biosynthesized via green tea fermentation (20 g/L tea and 100 g/L sugar) and purified prior to use. AgNPs were obtained by reacting aqueous tucumã extract with silver nitrate (0.1 mmol/L) at pH (9) and temperature (40 °C). BC membranes were immersed in the AgNPs dispersion for 7 days to form the nanocomposite. Characterization was performed using UV–Vis, DLS, TEM, SEM–EDS, FTIR, XRD, ICP–OES, and swelling analysis. Antibacterial activity was evaluated using the disk diffusion method against Staphylococcus aureus and Escherichia coli (ATCC 6538 and 4388). Results: The UV–Vis spectra revealed a gradual decrease in the surface plasmon resonance (SPR) band over 7 days of incubation with BC, indicating progressive incorporation of AgNPs into the membrane. ICP analysis confirmed silver incorporation in the BC membrane at 0.00215 mg/mL, corresponding to 15.5% of the initial silver content. Antimicrobial assays showed inhibition zones of 6.5 ± 0.5 mm for S. aureus and 4.3 ± 0.3 mm for E. coli. Conclusions: These findings validate the successful formation and antimicrobial performance of the BC–AgNP nanocomposite, supporting its potential use in wound care applications. Full article
(This article belongs to the Special Issue Therapeutic Potential of Silver Nanoparticles (AgNPs), 2nd Edition)
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14 pages, 1409 KiB  
Article
Production, Validation, and Exposure Dose Measurement of [13N]Ammonia Under Academic Good Manufacturing Practice Environments
by Katsumi Tomiyoshi, Yuta Namiki, David J. Yang and Tomio Inoue
Pharmaceutics 2025, 17(5), 667; https://doi.org/10.3390/pharmaceutics17050667 - 19 May 2025
Viewed by 542
Abstract
Objective: Current good manufacturing practice (cGMP) guidance for positron emission tomography (PET) drugs has been established in Europe and the United States. In Japan, the Pharmaceuticals and Medical Devices Agency (PMDA) approved the use of radiosynthesizers as medical devices for the in-house manufacturing [...] Read more.
Objective: Current good manufacturing practice (cGMP) guidance for positron emission tomography (PET) drugs has been established in Europe and the United States. In Japan, the Pharmaceuticals and Medical Devices Agency (PMDA) approved the use of radiosynthesizers as medical devices for the in-house manufacturing of PET drugs in hospitals and clinics, regardless of the cGMP environment. Without adequate facilities, equipment, and personnel required by cGMP regulations, the quality assurance (QA) and clinical effectiveness of PET drugs largely depend on the radiosynthesizers themselves. To bridge the gap between radiochemistry standardization and site qualification, the Japanese Society of Nuclear Medicine (JSNM) has issued guidance for the in-house manufacturing of small-scale PET drugs under academic GMP (a-GMP) environments. The goals of cGMP and a-GMP are different: cGMP focuses on process optimization, certification, and commercialization, while a-GMP facilitates the small-scale, in-house production of PET drugs for clinical trials and patient-specific standard of care. Among PET isotopes, N-13 has a short half-life (10 min) and must be synthesized on site. [13N]Ammonia ([13N]NH3) is used for myocardial perfusion imaging under the Japan Health Insurance System (JHIS) and was thus selected as a working example for the manufacturing of PET drugs in an a-GMP environment. Methods: A [13N]NH3-radiosynthesizer was installed in a hot cell within an a-GMP-compliant radiopharmacy unit. To comply with a-GMP regulations, the air flow was adjusted through HEPA filters. All cabinets and cells were disinfected to ensure sterility once a month. Standard operating procedures (SOPs) were applied, including analytical methods. Batch records, QA data, and radiation exposure to staff in the synthesis of [13N]NH3 were measured and documented. Results: 2.52 GBq of [13N]NH3 end-of-synthesis (EOS) was obtained in an average of 13.5 min in 15 production runs. The radiochemical purity was more than 99%. Exposure doses were 11 µSv for one production run and 22 µSv for two production runs. The pre-irradiation background dose rate was 0.12 µSv/h. After irradiation, the exposed dosage in the front of the hot cell was 0.15 µSv/h. The leakage dosage measured at the bench was 0.16 µSv/h. The exposure and leakage dosages in the manufacturing of [13N]NH3 were similar to the background level as measured by radiation monitoring systems in an a-GMP environments. All QAs, environmental data, bacteria assays, and particulates met a-GMP compliance standards. Conclusions: In-house a-GMP environments require dedicated radiosynthesizers, documentation for batch records, validation schedules, radiation protection monitoring, air and particulate systems, and accountable personnel. In this study, the in-house manufacturing of [13N]NH3 under a-GMP conditions was successfully demonstrated. These findings support the international harmonization of small-scale PET drug manufacturing in hospitals and clinics for future multi-center clinical trials and the development of a standard of care. Full article
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Article
Hydroxytyrosyl Eicosapentaenoate as a Potential Antioxidant for Omega-3 Fatty Acids: Improved Synthesis and Comparative Evaluation with Other Natural Antioxidants
by Natalia García-Acosta, Rosa Cert, Marta Jordán, Luis Goya, Raquel Mateos and Jose Luis Espartero
Biomolecules 2025, 15(5), 714; https://doi.org/10.3390/biom15050714 - 13 May 2025
Viewed by 655
Abstract
Hydroxytyrosol (HT), the primary phenolic compound in virgin olive oil, has notable cardiovascular benefits, particularly in preventing low-density lipoprotein (LDL) oxidation. However, its hydrophilicity limits its solubility and integration into lipid-based formulations. This study aimed to enhance its lipophilicity by synthesizing hydroxytyrosyl eicosapentaenoate [...] Read more.
Hydroxytyrosol (HT), the primary phenolic compound in virgin olive oil, has notable cardiovascular benefits, particularly in preventing low-density lipoprotein (LDL) oxidation. However, its hydrophilicity limits its solubility and integration into lipid-based formulations. This study aimed to enhance its lipophilicity by synthesizing hydroxytyrosyl eicosapentaenoate (HT-EPA), a derivative of HT and eicosapentaenoic acid (EPA), using a one-step enzymatic catalysis with lipase B from Candida antarctica (CALB). The reaction, performed as a suspension of HT in ethyl eicosapentaenoate (Et-EPA) (1:9 molar ratio) under vacuum, achieved higher yields and shorter reaction times than previously reported, with a purity exceeding 98%, confirmed by 1H-NMR. For the first time, the antioxidant capacity of HT-EPA in comparison with other natural antioxidants was assessed using the FRAP assay, while its oxidative stability in an omega-3-rich oil matrix was evaluated via the Rancimat method. HT-EPA and hydroxytyrosyl acetate (HT-Ac) displayed antioxidant activity comparable to HT but significantly higher than α-tocopherol, a common food antioxidant. Given the scarcity of effective lipid-soluble antioxidants, HT-EPA represents a promising candidate for omega-3 nutraceuticals, offering enhanced stability and potential health benefits. This study provides a simple, efficient, and scalable strategy for developing functional lipid-based formulations with cardioprotective potential by improving HT solubility while preserving its antioxidant properties. Full article
(This article belongs to the Section Lipids)
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