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Search Results (10,239)

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Keywords = in vivo evaluations

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21 pages, 1526 KiB  
Article
Broad Spectrum Antimicrobial Activity of Bacteria Isolated from Vitis vinifera Leaves
by Camilla Mandorino, Flavia Angela Maria Maggiolini, Margherita D’Amico, Annalisa Prencipe, Antonella Salerno, Carlo Bergamini, Carlo Pazzani, Maria Francesca Cardone and Antonio Domenico Marsico
Microbiol. Res. 2025, 16(3), 55; https://doi.org/10.3390/microbiolres16030055 (registering DOI) - 26 Feb 2025
Abstract
It is known that plant-associated microorganisms must be considered as key drivers for plant health, productivity, community composition, and ecosystem functioning. Much attention has been paid to the exploration of the plant-associated microbiome in search of new microorganisms with antagonistic activity against various [...] Read more.
It is known that plant-associated microorganisms must be considered as key drivers for plant health, productivity, community composition, and ecosystem functioning. Much attention has been paid to the exploration of the plant-associated microbiome in search of new microorganisms with antagonistic activity against various phytopathogens. P. viticola represents one of the main fungal agents of grape yield losses during the pre-harvest stage. To date, the use of chemical pesticides is the main method to control downy mildew infection in vineyards. In recent years, innovative and more environmentally suitable control strategies, such as Biological Control Agents (BCAs), have been greatly improved. In this study, 16 new bacterial strains, isolated from the leaves of three table grape cultivars, were tested for their in vivo effectiveness against downy mildew of grape, resulting in one B. velenzensis (‘BLG_B4), one P. pleuroti (‘BLG_B5), one P. psychrotolerans (‘BLG_B1.3’) and one B. subtilis (‘BLG_B1.1.1’) bacterial strains that were marked as good BCAs. As possible mechanisms of action, a genetic analysis was performed to evaluate the ability of selected bacterial strains to reduce the expression of two P. viticola effectors. Finally, their effectiveness against a wide range of fungal pathogens and their safety for human health was also evaluated. Full article
14 pages, 504 KiB  
Article
Enhancing Photoprotection and Mitigating Ex Vivo Stratum Corneum Oxidative Stress: A Multifunctional Strategy Combining Rosmarinic Acid with UVB Filters
by Pedro Ivo de Souza Macedo, Claudinéia Aparecida Sales de Oliveira Pinto, Camila Faustino Hiraishi, Gabriela de Argollo Marques, Cassiano Carlos Escudeiro, Fabiana Vieira Lima Solino Pessoa, João Gregório, Catarina Rosado, Maria Valéria Robles Velasco and André Rolim Baby
Antioxidants 2025, 14(3), 274; https://doi.org/10.3390/antiox14030274 - 26 Feb 2025
Abstract
Exposure to ultraviolet (UV) radiation is a major contributor to skin injury, including sunburn, photoaging, and augmented risk of skin cancer, primarily through the generation of reactive oxygen species (ROS) that induce oxidative stress. Rosmarinic acid (RA), a natural phenolic compound with antioxidant [...] Read more.
Exposure to ultraviolet (UV) radiation is a major contributor to skin injury, including sunburn, photoaging, and augmented risk of skin cancer, primarily through the generation of reactive oxygen species (ROS) that induce oxidative stress. Rosmarinic acid (RA), a natural phenolic compound with antioxidant and several other biological properties, has shown promise in mitigating such damage when incorporated into sunscreens. We evaluated RA’s possible interactions and potential to enhance the efficacy of three worldwide known UVB filters—ethylhexyl methoxycinnamate (EHMC), octocrylene (OCT), and ethylhexyl salicylate (EHS). The performance of sunscreens with and without RA (0.1% w/w) was analyzed through in vitro and in vivo photoprotective assessments. The HPLC-TBARS-EVSC (high-performance liquid chromatography—thiobarbituric acid reactive substances—ex vivo stratum corneum) protocol, which quantified oxidative stress reduction in the human stratum corneum, was also used. The in vitro photoprotective assays showed that RA had distinct levels of interactions with the UVB filters. When associated with EHMC, RA exclusively acted in the UVB range (SPF-enhancing effect). Remarkably, for EHS, RA contributed to a higher efficacy profile in the total UV spectrum. OCT-RA was the sample that reached the highest critical wavelength value parallelly to OCT, boosting the in vivo SPF by more than 157% in comparison to OCT. However, its in vitro SPF performance was not affected by the RA addition, being comparable to OCT, EHS, and EHS-RA. Furthermore, the HPLC-TBARS-EVSC protocol highlighted RA’s ability to reduce lipid peroxidation, with OCT-RA exhibiting the most notable protective effect. These findings underscore RA’s potential as a multifunctional additive in sunscreen systems, enhancing both photoprotection and oxidative stress mitigation. Full article
(This article belongs to the Section Natural and Synthetic Antioxidants)
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Figure 1
<p>In vivo SPF (sun protection factor) the sunscreen systems (n = 10). EHMC—ethylhexyl methoxycinnamate; OCT—octocrylene; EHS—ethylhexyl salicylate; RA—rosmarinic acid.</p>
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15 pages, 1308 KiB  
Article
A2A Adenosine Receptor Antagonists and Their Efficacy in Rat Models of Parkinson’s Disease
by Andrea Spinaci, Michela Buccioni, Diego Dal Ben, Beatrice Francucci, Karl-Norbert Klotz, Gabriella Marucci, Nicola Simola, Micaela Morelli, Annalisa Pinna, Rosaria Volpini and Catia Lambertucci
Cells 2025, 14(5), 338; https://doi.org/10.3390/cells14050338 - 26 Feb 2025
Abstract
Parkinson’s disease (PD) represents a growing challenge to global health, as it involves millions of people. The high grade of disability is due to the loss of dopaminergic neuron activity, and levodopa is the gold-standard therapy used to restore dopamine in the dopamine-denervated [...] Read more.
Parkinson’s disease (PD) represents a growing challenge to global health, as it involves millions of people. The high grade of disability is due to the loss of dopaminergic neuron activity, and levodopa is the gold-standard therapy used to restore dopamine in the dopamine-denervated regions. Another therapeutic approach is the use of A2A adenosine receptor antagonists and, among them, istradefylline is the only one currently approved for therapy in association with levodopa. In this work, we synthesized A2A adenosine receptor antagonists represented by 9-ethyl-2,8-disubstituted adenine derivatives, which were tested at human adenosine receptors in binding and functional assays. These compounds showed A2A adenosine receptor-binding affinities in the low nanomolar range and 1, 4, and 5 exhibited good potency in the functional assays. Hence, they were evaluated in in vivo rat models of PD, where they were demonstrated to revert haloperidol-induced catalepsy and potentiate levodopa-induced contralateral rotations in 6-hydroxydopamine-lesioned rats. The most potent derivative, 4, was then evaluated in the tacrine model, where it reduced the tremulous jaw movements, therefore demonstrating an action on parkinsonian tremor. These data revealed 8-ethoxy-2-phenethoxy-9-ethyladenine (4) as an A2A adenosine receptor antagonist endowed with antiparkinsonian effects and as a good candidate to treat the disease. Full article
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Figure 1
<p>Known A<sub>2A</sub>AR antagonists and synthesized ligands.</p>
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<p>Effect of A<sub>2A</sub>AR antagonists on catalepsy induced by haloperidol. Effect of <b>1</b> (5 mg/kg i.p.; n = 5), <b>4</b> (5 mg/kg i.p.; n = 6), or <b>5</b> (5 mg/kg i.p.; n = 5) on catalepsy induced by haloperidol (0.2 mg/kg s.c.; n = 7) in rats, 10, 30, 60, and 90 min after drug administration. Results are mean ± S.E.M. of the intensity of catalepsy, measured as the time spent in cataleptic posture by each rat in the test section. Statistical significance was determined by one-way ANOVA followed by Newman–Keuls’ post hoc test. ** <span class="html-italic">p</span> &lt; 0.0001, * <span class="html-italic">p</span> &lt; 0.05 versus haloperidol + vehicle.</p>
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<p>Potentiation of L-dopa-induced contralateral turning by A<sub>2A</sub>AR antagonists. Effect of administration of L-dopa (3 mg/kg i.p.) + vehicle (n = 8), L-dopa (3 mg/kg i.p.) + <b>1</b> (5 mg/kg i.p.; n = 5), L-dopa (3 mg/kg i.p.) + <b>4</b> (5 mg/kg i.p.; n = 7), L-dopa (3 mg/kg i.p.) + <b>5</b> (5 mg/kg i.p.; n = 5). Ordinate indicates the total number of turns measured in 2 h; values represent contralateral rotations, respectively. Results are mean ± S.E.M. of total turns. Statistical significance was determined by one-way ANOVA followed by Newman–Keuls’ post hoc test. ** <span class="html-italic">p</span> &lt; 0.0001, * <span class="html-italic">p</span> &lt; 0.05 versus L-dopa alone.</p>
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<p>Effect of <b>4</b> on tacrine-induced parkinsonian-like tremors. Effect of <b>4</b> (5 mg/kg i.p.) pre-treatment on tremulous jaw movements and tremor bursts induced in rats by tacrine (2.5 mg/kg i.p.). Results are mean ± SEM of tremulous jaw movements (<b>a</b>) and mean ± SEM of tremor bursts (<b>b</b>) recorded for 60 min after tacrine administration. Compound <b>4</b> significantly reduced tremulous jaw movements, but not tremor bursts, as compared to the vehicle. Statistical significance was determined by Student’s <span class="html-italic">t</span>-test. * <span class="html-italic">p</span> &lt; 0.05 versus vehicle; (n = 4–6).</p>
Full article ">Scheme 1
<p>Synthesis of 9-ethyl-2-phenylethyloxy-8-substituted adenines. Reagents and conditions: a. NBS, DMF, r.t., 45′; b. EtOH, NaOH, 95 °C, 7 h; c. (i) LDA, THF dry, −70 °C, 1 h; (ii) I<sub>2</sub>, THF, −70 °C-r.t. 2.5 h; d. ArSnBu<sub>3</sub>, (Ph<sub>3</sub>P)<sub>2</sub>PdCl<sub>2</sub>, THF dry, 60 °C, 2.5 h; e. H<sub>2</sub> 13 atm, PdO, HCl, <span class="html-italic">i</span>PrOH, 65 °C, 7 h.</p>
Full article ">Scheme 2
<p>Synthesis of 9-ethyl-2-(4-methoxyphenethoxy)-8-substituted adenines. Reagents and conditions: a. (4-O-CH<sub>3</sub>)-Ph(CH<sub>2</sub>)<sub>2</sub>OH, NaOH, 85 °C, 4 h; b. NBS, DMF, r.t., 10 min; c. 2-FurylSnBu<sub>3</sub>, (Ph<sub>3</sub>P)<sub>2</sub>PdCl<sub>2</sub>, THF, 60 °C, 5 h; d. HBr, 100 °C; e. (4-OH)-Ph(CH<sub>2</sub>)<sub>2</sub>OH, NaOH, MeCN dry, reflux, 24 h.</p>
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18 pages, 5094 KiB  
Article
The GPR30-Mediated BMP-6/HEP/FPN Signaling Pathway Inhibits Ferroptosis in Bone Marrow Mesenchymal Stem Cells to Alleviate Osteoporosis
by Shuangliu Chen, Jiřimutu Xiao, Shijie Zhou, Taxi Wumiti, Zitong Zhao, Ruihua Zhao, Yalan Pan, Qing Wang, Yong Ma, Lan Wu and Yang Guo
Int. J. Mol. Sci. 2025, 26(5), 2027; https://doi.org/10.3390/ijms26052027 - 26 Feb 2025
Abstract
Dysregulated iron metabolism-induced ferroptosis is considered a key pathological mechanism in the development of osteoporosis (OP). G protein-coupled receptor 30 (GPR30, also known as Gper1) is an estrogen-binding receptor that has shown therapeutic benefits in patients with certain degenerative diseases. Moreover, several studies [...] Read more.
Dysregulated iron metabolism-induced ferroptosis is considered a key pathological mechanism in the development of osteoporosis (OP). G protein-coupled receptor 30 (GPR30, also known as Gper1) is an estrogen-binding receptor that has shown therapeutic benefits in patients with certain degenerative diseases. Moreover, several studies have demonstrated the anti-ferroptotic effects of estrogen receptor activation. However, its role in the prevention and treatment of OP remains unclear, and there are currently no reports on the anti-ferroptotic function of GPR30 in OP. Therefore, this study aimed to investigate the ferroptosis-related effects and mechanisms of GPR30 in the context of OP. In vivo and in vitro experiments were conducted using wild-type (WT) C57BL/6 female mice and GPR30-knockout (GPR30-KO) C57BL/6J female mice. The microarchitecture of the distal femur was assessed using micro-computed tomography (micro-CT), and histomorphological changes were analyzed via hematoxylin and eosin (H&E) staining. Bone marrow mesenchymal stem cells (BMSCs) were isolated and cultured to establish an iron overload model using ferric ammonium citrate (FAC). Interventions included GPR30 overexpression via transfection and BMP-6 inhibition using LDN-214117. Cell viability was evaluated with the CCK-8 assay, while osteogenic differentiation and mineralization levels were assessed using ALP and Alizarin Red S (ARS) staining. Iron accumulation was detected via Prussian blue staining, oxidative stress levels were evaluated using ROS staining, and mitochondrial membrane potential changes were analyzed using JC-1 staining. Transmission electron microscopy (TEM) was employed to observe mitochondrial ultrastructural changes. Additionally, key gene and protein expression levels were measured using immunofluorescence and Western blotting. The micro-CT analysis revealed significant bone microarchitecture deterioration and bone loss in the GPR30-KO mouse model. At the cellular level, GPR30 overexpression markedly reduced iron accumulation and oxidative stress in BMSCs, restored the mitochondrial membrane potential, and improved the mitochondrial ultrastructure. Furthermore, GPR30 enhanced osteogenic differentiation in BMSCs by promoting the activation of the BMP-6/HEP/FPN signaling pathway, leading to increased expression of osteogenic markers. The protective effects of GPR30 were reversed by the BMP-6 inhibitor LDN-214117, indicating that BMP-6 is a critical mediator in GPR30-regulated iron metabolism and ferroptosis inhibition. GPR30 inhibits ferroptosis in BMSCs and enhances osteogenic differentiation by activating the BMP-6/HEP/FPN signaling pathway. This provides new insights and potential therapeutic targets for the treatment of osteoporosis OP. Full article
(This article belongs to the Section Molecular Genetics and Genomics)
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Graphical abstract

Graphical abstract
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<p>(<b>A</b>): Gene expression levels of GPR30 in BMSCs from osteoporotic (OP) patients compared to non-OP patients; with data sourced from GSE35958). (<b>B</b>–<b>F</b>): Bone volume ratio (BV/TV, %), bone mineral density (BMD), trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp) for each group of mice. (<b>G</b>): CT images of mouse femurs, histological sections of mouse femoral tissue stained with hematoxylin and eosin (HE) (5× and 20× magnifications, The scales are 200 μm and 100 μm respectively), and histological sections stained for tartrate-resistant acid phosphatase (TRAP). * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001. <span class="html-italic">n</span> = 3 per group.</p>
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<p>(<b>A</b>–<b>C</b>): Immunofluorescence detection of GPR30 and GPX4 expression in WT and GPR30-KO mice. (<b>D</b>,<b>E</b>): Prussian blue staining to detect iron ions in BMSCs of WT and KO mice, along with quantification. (<b>F</b>): Measurement of serum ferrous ion levels. (<b>G</b>,<b>H</b>): Prussian blue staining of bone tissue in mice and corresponding quantification. The blue dot pointed by the arrow is where iron ions are deposited. ** <span class="html-italic">p</span> &lt; 0.01, **** <span class="html-italic">p</span> &lt; 0.0001. <span class="html-italic">n</span> = 3 per group.</p>
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<p>(<b>A</b>): CCK8 assay to assess cell viability after treatment with different concentrations of FAC. (<b>B</b>): CCK8 assay to evaluate cell viability in response to FAC and DFO treatments across different groups. (<b>C</b>): q-PCR analysis of GPR30 expression in cells after lentiviral transfection. (<b>D</b>–<b>F</b>): ROS detection, mitochondrial membrane potential analysis, and quantification. (<b>G</b>): Transmission electron microscopy (TEM) of mitochondrial ultrastructure (8000x, 20,000x magnification, The scales were 2 μm and 500 nm respectively). (<b>H</b>,<b>I</b>): Prussian blue staining of cells. (<b>J</b>,<b>K</b>): Immunofluorescence detection of GPX4 expression. (<b>L</b>): q-PCR analysis of GPX4 expression. (<b>M</b>): CCK8 assay to measure cell viability. (<b>N</b>–<b>P</b>): ALP and ARS staining to assess osteogenic differentiation and mineralization of cells. (<b>Q</b>–<b>S</b>): Western blot analysis of RUNX2 and GPX4 expression. * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001, **** <span class="html-italic">p</span> &lt; 0.0001. <span class="html-italic">n</span> = 3 per group.</p>
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<p>(<b>A</b>): Immunohistochemistry to detect BMP-6 and HEP in the liver, and FPN in the duodenum. The scale in the figure is 20 μm. (<b>B</b>–<b>F</b>): Western blot analysis of BMP-6, HEP, and FPN expression. (<b>G</b>–<b>J</b>): Immunofluorescence detection of BMP-6 and FPN expression. * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001. <span class="html-italic">n</span> = 3 for each group.</p>
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<p>(<b>A</b>,<b>D</b>): Prussian blue staining of cells and corresponding quantification. The scale is 100 μm. (<b>B</b>,<b>C</b>): ALP staining and quantification to assess osteogenic differentiation of cells. The scale is 50 μm. (<b>E</b>,<b>F</b>): Immunofluorescence detection of BMP-6 expression. (<b>G</b>,<b>H</b>): Immunofluorescence detection of FPN expression. * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001, **** <span class="html-italic">p</span> &lt; 0.0001. <span class="html-italic">n</span> = 3 per group.</p>
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25 pages, 10662 KiB  
Article
A Novel Liposomal In-Situ Hydrogel Formulation of Hypericum perforatum L.: In Vitro Characterization and In Vivo Wound Healing Studies
by Ahmet Arif Kurt and İsmail Aslan
Gels 2025, 11(3), 165; https://doi.org/10.3390/gels11030165 - 26 Feb 2025
Abstract
Hypericum perforatum L. (H.P.) is a species with a well-documented history of use in wound healing practices across the globe. The objective of this study was twofold: firstly, to evaluate the in vivo efficacy of liposomal in situ gel formulations in wound healing, [...] Read more.
Hypericum perforatum L. (H.P.) is a species with a well-documented history of use in wound healing practices across the globe. The objective of this study was twofold: firstly, to evaluate the in vivo efficacy of liposomal in situ gel formulations in wound healing, both clinically and histopathologically, and secondly, to determine the physicochemical characterization of liposomal in situ gel formulations. The in vitro studies will be assessed in terms of particle size, zeta potential, release kinetics, rheological behaviors, and antioxidant and antimicrobial properties. The in vivo studies will be evaluated in clinical animal experiments and pathology studies. The in-situ hydrogel formulations were prepared using the physical cross-linking method with Poloxamer 188, Poloxamer 407, Ultrez 21, and Ultrez 30. The liposome formulations phospholipid 90H and lipoid S100 were prepared using the thin film solvent evaporation method. The antioxidant activity of the samples was evaluated through in vitro studies employing the DPPH antioxidant activity, ABTS+ test, and FRAP test. The antimicrobial activity of the samples was evaluated through the determination of MIC and MBC values employing the 96-well plate method. In vivo, 36 male New Zealand rabbits aged 32–36 weeks were utilized, with six rabbits in each group. The groups were composed of six distinct groups, including conventional and in situ gel liposome formulations of HHPM, three different commercial preparations, and a control group (n = 6). The HHPM-LG8 formulation developed in this study was found to be applicable in terms of all its properties. The new liposomal in situ hydrogel formulation demonstrated notable wound healing activity, a result that was supported by the formulation itself. Full article
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Figure 1
<p>HPLC chromatograms of hypericin in <span class="html-italic">H. perforatum</span> L. macerate-containing liposome sample (HHPM-L4).</p>
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<p>HPM-L4 SEM and DLS images.</p>
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<p>The in-situ gels’ rheological behavior at different pH and temperatures of GF1 and GF2.</p>
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<p>The different release kinetic models of the HHPM-LG8. (<b>a</b>) Korsmeyer Peppas (<b>b</b>) Zero Order (<b>c</b>) First Order (<b>d</b>) Higuchi (<b>e</b>) Hixson-Crowell.</p>
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<p>Graph of % cumulative hypericin content from HHPM-LG8 versus time.</p>
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<p>DPPH, ABTS, and FRAP antioxidant activity results.</p>
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<p>Wound healing score of HHPM, HHPM-LG8, CP1, CP2, and CP3 in the skin defect area after 4, 8, and 12 days. **** means <span class="html-italic">p</span> &lt; 0.0001.</p>
Full article ">Figure 8
<p>Wound healing images of HHPM, HHPM-LG8, CP1, CP2, and CP3 in the skin defect area after 0, 4, 8, and 12 days.</p>
Full article ">Figure 9
<p>Histopathological appearance of wound healing in the skin defect area according to groups. (<b>A</b>) HHPM, (<b>B</b>) HHPM-LG8, (<b>C</b>) CP1, (<b>D</b>) CP2, (<b>E</b>) CP3, (<b>F</b>) Control; Bars = 50 µm.</p>
Full article ">Figure 10
<p>Immunohistochemical expression of cytokeratin according to groups. (<b>A</b>) HHPM, (<b>B</b>) HHPM-LG8, (<b>C</b>) CP1, (<b>D</b>) CP2, (<b>E</b>) CP3, (<b>F</b>) Control; Bars = 50 µm.</p>
Full article ">Figure 11
<p>Immunohistochemical expression of VEGF according to groups. (<b>A</b>) HHPM, (<b>B</b>) HHPM-LG8, (<b>C</b>) CP1, (<b>D</b>) CP2, (<b>E</b>) CP3, (<b>F</b>) Control; Bars = 50 µm.</p>
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<p>Appearance of Type III and Type I collagen formation according to groups. (<b>A</b>) HHPM, (<b>B</b>) HHPM-LG8, (<b>C</b>) CP1, (<b>D</b>) CP2, (<b>E</b>) CP3, (<b>F</b>) Control; Bars = 50 µm.</p>
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17 pages, 572 KiB  
Article
Synthesis, Characterization, and Docking Studies of Some New Chalcone Derivatives to Alleviate Skin Damage Due to UV Light
by Arun Kumar Mishra, Kamal Y. Thajudeen, Chandra Shekhar, Mhaveer Singh, Harpreet Singh, Arvind Kumar, Sarvesh Kumar Paliwal, Emdad Hossain and Shahana Salam
Molecules 2025, 30(5), 1057; https://doi.org/10.3390/molecules30051057 - 25 Feb 2025
Abstract
Increasing cases of sunburn is one of the serious problems across the globe. In this connection, there is an urgent requirement for some effective sun screening agents. In the search for the same, nanoemulsions of some new synthesized and characterized chalcone derivatives were [...] Read more.
Increasing cases of sunburn is one of the serious problems across the globe. In this connection, there is an urgent requirement for some effective sun screening agents. In the search for the same, nanoemulsions of some new synthesized and characterized chalcone derivatives were prepared and evaluated in vitro and in vivo. In order to meet the said objective, in the first step, vanillin was reacted with 4-aminoacetophenone in the presence of 15% sodium hydroxide and ethanol to synthesize the target compounds (C-1 to C-5). Progress of reaction was monitored using thin-layer chromatography (TLC). The crystals of purified compounds were characterized using spectroscopic techniques such as Infrared (IR) spectroscopy, 1H-NMR spectroscopy, 13C-NMR, and mass spectrometry. We prepared the nanoemulsions of the final compounds (C-1 to C-5) and subsequently evaluated them for in vitro sun protection factor activity. The concentration of the nanoemulsions, consistently ranging from 0.88 to 0.91 mg/mL across all formulations, demonstrated a high degree of consistency. The range of particle size varied from approximately 172 to 183 nm, with low polydispersity index values (approximately 0.11 to 0.15). The negative zeta potentials recorded for all the formulations (ranging from −35.87 mV to −39.30 mV) showed that the nanoemulsions are electrostatically stable enough to keep them from sticking together. The pH values of the nanoemulsions ranged narrowly from approximately 5.00 to 5.16, which indicated the compatibility of emulsion with biological systems and the potential to reduce irritation or instability during administration. The viscosity of the nanoemulsions varied between 2.00 and 2.12 cP. In silico studies were performed using MMP-I and MMP-2 as target receptors. For in vitro SPF evaluation, the Mansur equation was employed. COLIPA guidelines were compiled for in vivo SPF evaluation. The nanoemulsions derived from compounds C-3 and C-4, designated as C-3 NE and C-4 NE, were more effective as anti-aging agents. Findings suggested the possible scope of further synthesis of newer synthetic derivatives of chalcones for furfur development nanoemulsions for better SPF activity. Full article
(This article belongs to the Section Medicinal Chemistry)
18 pages, 8963 KiB  
Article
Influence of Different Cationic Polymer-Based Micelles on the Corneal Behavior and Anti-Cataract Effect of Diosmetin
by Jing Zhang, Min Zha, Anping Wan, Satya Siva Kishan Yalamarty, Nina Filipczak and Xiang Li
Pharmaceutics 2025, 17(3), 302; https://doi.org/10.3390/pharmaceutics17030302 - 25 Feb 2025
Abstract
Background Despite many studies on polymer-incorporated nanocarriers for ophthalmic drug delivery, few have thoroughly explored the relationship between coating composition and performance. This study aimed to evaluate the effects of three commonly used cationic polymers—distearoyl phosphatidylethanolamine-polyethylene glycol 1000-poly(amidoamine) (DSPE-PEG1000-PAMAM), trimethyl chitosan (TMC), and [...] Read more.
Background Despite many studies on polymer-incorporated nanocarriers for ophthalmic drug delivery, few have thoroughly explored the relationship between coating composition and performance. This study aimed to evaluate the effects of three commonly used cationic polymers—distearoyl phosphatidylethanolamine-polyethylene glycol 1000-poly(amidoamine) (DSPE-PEG1000-PAMAM), trimethyl chitosan (TMC), and (2,3-dioleoyloxypropyl) trimethylammonium chloride (DOTAP)—on the corneal behaviors and anti-cataract efficacy of diosmetin (DIO)-loaded micelles (D-M-P, D-M-T, and D-M-D, respectively). Methods The DIO-loaded micelles were prepared using the thin-film dispersion method and incorporated with the three polymers through hydrophobic interactions and electrostatic adsorption. Structural characterization was demonstrated by TEM imaging and particle size analyzer. In vitro release behavior was detected by the dialysis method. Cell viability of D-M-P, D-M-T, and D-M-D on L929 cells was detected by CCK-8 assays, with cellular uptake performed using coumarin 6 as the fluorescence indicator. Precorneal retention behaviors of these three vesicles were observed by In Vivo Imaging System. Transcorneal permeability was determined by modified Franz diffusion method and the permeation routes of the vesicles are investigated. Selenite-induced cataract model was established. The anti-cataract effects of three different DIO-loaded micelles were evaluated by the observation of lens opacity and antioxidant enzyme activities. Eye Irritation of the DIO in different preparations was estimated using the Draize test, along with H&E staining of the corneas. Results Structural characterization of DIO-loaded micelles revealed that the vesicles were spherical, with a uniform size distribution of around 28 nm, a similar surface potential of approximately 6.0 mV, and a high DIO entrapment efficiency of about 95%. Compared to the DIO suspension, all three formulations exhibited a significant sustained-release effect. They showed no signs of irritation and demonstrated increased IC50 values in L929 cells, indicating improved biocompatibility. Cellular uptake in human lens epithelial cells (HLECs) was assessed using confocal laser scanning microscopy. C-M-T displayed the highest fluorescence signals, with a cellular internalization 3.2 times greater than that of the solution group. Both C-M-T and C-M-P enhanced vesicle retention on the corneal surface by at least 47.8% compared to the Cou-6 solution. Furthermore, TMC facilitated the paracellular transport of vesicles into the deepest layers of the cornea and delivered DIO across the cornea, with a Papp value 3.11 times and 1.49 times those of D-M-D and D-M-P, respectively. In terms of therapeutic efficacy, D-M-T demonstrated the most significant attenuation of lens opacity, along with enhanced antioxidant enzyme activities and inhibition of lipid peroxidation. Conclusion The modification of micelle vesicles with different cationic polymers significantly influences their performance in ocular drug delivery. Among the tested formulations, D-M-T stands out due to its multiple advantages, including enhanced transcorneal drug delivery, therapeutic efficacy for DIO, and safety, making it the most promising candidate for ophthalmic applications. Full article
(This article belongs to the Special Issue Polymer-Based Delivery System)
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<p>(<b>A</b>) TEM images of D-M-T (1), D-M-P (2), and D-M-D (3) (Bar = 100 nm); (<b>B</b>) in vitro release of DIO from different preparations at scheduled time points (n = 3); (<b>C</b>) cell viability of different concentrations of DIO (1), D-M-T (2), D-M-P (3), D-M-D (4) on L929 cells.</p>
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<p>(<b>A</b>) Cellular uptake of vehicles in HLECs after incubation with different preparations (Bar = 50 μm); (<b>B</b>) intracellular fluorescence intensity of HLECs after incubation with different preparations (*, <span class="html-italic">p</span> &lt; 0.05; ****, <span class="html-italic">p</span> &lt; 0.0001; ns, not significantly); (<b>C</b>) precorneal fluorescence signals of each group in rabbit corneas under IVIS imaging at different time points within 20 min.</p>
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<p>Corneal transmission curve in vitro of each preparation.</p>
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<p>(<b>A</b>) Corneal penetration depth of different preparations at 2 h; (<b>B</b>) corneal penetration depth of different preparations at 4 h.</p>
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<p>Horizontal corneal imaging of rabbit treated with C-M-T for 2 h (1–7: 5–35 μm for every 5 μm depth). Bar equals 50 μm.</p>
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<p>Slit lamp view of a lens with selenium-induced cataract in rats from day 1 to day 7 in each group.</p>
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<p>Contents of CAT, MDA, and SOD in the lens of rats in each group (***, <span class="html-italic">p</span> &lt; 0.001; **, <span class="html-italic">p</span> &lt; 0.005; *, <span class="html-italic">p</span> &lt; 0.01).</p>
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<p>Histopathological sections of rabbit eyes in different preparation groups (Bar = 100 μm).</p>
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15 pages, 35957 KiB  
Article
Establishment of a Novel In Vitro and In Vivo Model to Understand Molecular Carcinogenesis of Endometriosis-Related Ovarian Neoplasms
by Hasibul Islam Sohel, Tohru Kiyono, Umme Farzana Zahan, Sultana Razia, Masako Ishikawa, Hitomi Yamashita, Kosuke Kanno, Shahataj Begum Sonia, Kentaro Nakayama and Satoru Kyo
Int. J. Mol. Sci. 2025, 26(5), 1995; https://doi.org/10.3390/ijms26051995 - 25 Feb 2025
Abstract
The molecular mechanisms through which endometriosis-related ovarian neoplasms (ERONs) develop from benign endometrioma remain unclear. It is especially a long-standing mystery why ovarian endometrioma has the potential to develop into two representative histological subtypes: endometrioid ovarian carcinoma or clear cell ovarian carcinoma. This [...] Read more.
The molecular mechanisms through which endometriosis-related ovarian neoplasms (ERONs) develop from benign endometrioma remain unclear. It is especially a long-standing mystery why ovarian endometrioma has the potential to develop into two representative histological subtypes: endometrioid ovarian carcinoma or clear cell ovarian carcinoma. This study aimed to investigate the molecular carcinogenesis of ERONs using newly developed in vitro and in vivo carcinogenesis models. Epithelial cells were isolated and purified from surgically removed benign endometrioma samples, followed by immortalization by overexpressing cyclinD1/CDK4 in combination with the human TERT gene. Immortalized cells were subjected to various molecular manipulations by combining knockout or overexpression of several candidate drivers, including ARID1A, KRAS, PIK3CA, AKT, and MYC, based on previous comprehensive genome-wide studies of ERONs. These cells were then inoculated into immunocompromised mice and evaluated for malignant transformation. Inoculated cells harboring a combination of three genetic alterations successfully developed tumors with malignant features in mice, whereas those with two genetic manipulations failed to do so. Especially, ARID1A gene knockout, combined with overexpressing the KRAS oncogenic mutant allele (or overexpressing AKT) and c-Myc overexpression led to efficient tumor formation. Of note, these three combinations of genetic alterations produced tumors that histologically represented typical clear cell carcinoma in SCID mice, while the same combination led to tumors with endometrioid histology in nude mice. A combination of ARID1A mutation, KRAS mutation or AKT activation, and c-Myc overexpression were confirmed to be the main candidate drivers for the development of ERONs, as suggested by comprehensive genetic analyses of ERONs. A tumor immune microenvironment involving B-cell signaling may contribute to the diverse histological phenotypes. The present model may help to clarify the molecular mechanisms of ERON carcinogenesis and understand their histological diversity and novel molecular targets. Full article
(This article belongs to the Special Issue Molecular Genetics in Ovarian Cancer)
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<p>Characterization of immortalized HMOsisEC7 cells. (<b>A</b>) Immunocytochemistry of HMOsisE7 cells with pan-cytokeratin confirming epithelial morphology. (<b>B</b>) Growth curve of HMOsisE7 cells. Population doublings are shown at each time point (days after culture). (<b>C</b>) Western blot analysis for pan-cytokeratin. SKOV3 cells are used as a positive control. (<b>D</b>) PAX8 expression indicates the absence of stromal cell contamination. The full-length Western blot gel image is available in <a href="#app1-ijms-26-01995" class="html-app">Supplementary Figure S4</a>. GAPDH, glyceraldehyde3-phosphate dehydrogenase.</p>
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<p>Genetic manipulations introduced into immortalized HMOsisEC7 cells. The order of the introduction of genetic mutation/overexpression is shown, with the combination of manipulations indicated. KO, knockout. MT, mutation.</p>
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<p>Western blot analysis confirms the expression of ARID1A or other genes in HMOsisEC7 cells with various genetic manipulations. (<b>A</b>) Expression of <span class="html-italic">ARID1A</span> in cells with <span class="html-italic">ARID1A</span> KO in comparison with parental HMOsisEC7 cells and the SKOV3 cell line. (<b>B</b>) Expression of various target genes in cells with overexpressing <span class="html-italic">KRAS</span> or <span class="html-italic">PIK3CA</span> mutant alleles or overexpressing constitutively activated AKT or c-Myc. The name of each cell type and the various genetic manipulations are listed above the gel images. The full-length Western blot gel image is available in <a href="#app1-ijms-26-01995" class="html-app">Supplementary Figure S5</a>. KO, knockout. MT, mutation; OCCC, ovarian clear cell carcinoma. OEC, ovarian endometrioid carcinoma, GAPDH, glyceraldehyde3-phosphate dehydrogenase.</p>
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<p>Anchorage-independent growth assay of HMOsisEC7 cells with various genetic manipulations. Photographs of representative colonies with various genetic manipulations by transmitted light microscopy. The corresponding quantification is shown in the bar graph, representing the number of colonies (&gt;50 µm) after 21 days of seeding. * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01. KO, knockout. MT, mutation; OCCC, ovarian clear cell carcinoma. OEC, ovarian endometrioid carcinoma.</p>
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<p>Tumor formation assay in immunocompromised mice. HMOsisEC7 cells with three genetic manipulations, <span class="html-italic">ARID1A</span> KO, <span class="html-italic">KRAS</span> MT, and c-Myc overexpression (<b>A</b>) or <span class="html-italic">ARID1A</span> KO, AKT, and c-Myc overexpression (<b>B</b>) can form tumors in SCID mice, representing histological features of OCCC with significant HNF1-β expression. In contrast, HMOsisEC7 cells with three genetic manipulations, <span class="html-italic">ARID1A</span> KO, <span class="html-italic">KRAS</span> MT, and c-Myc overexpression (<b>C</b>) can form tumors in nude mice, showing histological features of OEC lacking HNF-1β expression. The lack of ARID1A expression and overexpression of c-Myc in these mouse tumors were confirmed by IHC (<b>D</b>). KO, knockout. MT, mutation; OCCC, ovarian clear cell carcinoma. OEC, ovarian endometrioid carcinoma.</p>
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20 pages, 4477 KiB  
Article
Dapansutrile Regulates Mitochondrial Oxidative Stress and Reduces Hepatic Lipid Accumulation in Diabetic Mice
by Ying Wu and Jiaqiang Zhou
Curr. Issues Mol. Biol. 2025, 47(3), 148; https://doi.org/10.3390/cimb47030148 - 25 Feb 2025
Abstract
(1) Background: Hepatic lipid accumulation is the initial factor in metabolic-associated fatty liver disease (MAFLD) in type 2 diabetics, leading to accelerated liver damage. The NOD-like receptor protein 3 (NLRP3) inflammasome plays a critical role in this process. Dapansutrile (DAPA) is a novel [...] Read more.
(1) Background: Hepatic lipid accumulation is the initial factor in metabolic-associated fatty liver disease (MAFLD) in type 2 diabetics, leading to accelerated liver damage. The NOD-like receptor protein 3 (NLRP3) inflammasome plays a critical role in this process. Dapansutrile (DAPA) is a novel NLRP3 inflammasome inhibitor; however, its effect on ectopic lipid accumulation in the liver remains unclear. This study aimed to investigate the therapeutic effect of DAPA on hepatic lipid accumulation in a diabetic mouse model and its potential mechanisms. (2) Methods: The effects of DAPA on hepatic ectopic lipid deposition and liver function under metabolic stress were evaluated in vivo using db/db and high-fat diet (HFD) + streptozotocin (STZ) mouse models. Additionally, the role and mechanism of DAPA in cellular lipid deposition, mitochondrial oxidative stress, and inflammation were assessed in HepG2 cells treated with free fatty acids (FFA) and DAPA. (3) Results: Our findings indicated that DAPA treatment improved glucose and lipid metabolism in diabetic mice, particularly addressing liver heterotopic lipid deposition and insulin resistance. DAPA treatment also ameliorated lipid accumulation and mitochondrial-related functions and inflammation in HepG2 cells through the NLRP3-Caspase-1 signaling axis. (4) Conclusions: Targeting NLRP3 with DAPA may represent a novel therapeutic approach for diabetes-related fatty liver diseases. Full article
(This article belongs to the Section Molecular Medicine)
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<p>Dapansutrile improved glucose and lipid metabolism in the HFD + STZ mouse model. (<b>A</b>) Body weight monitoring in the HFD + STZ mouse model (<span class="html-italic">n</span> = 5 mice per group). (<b>B</b>) Plasma glucose levels in the HFD + STZ mouse model (<span class="html-italic">n</span> = 5 mice per group). (<b>C</b>) Insulin levels in the HFD + STZ mouse model (<span class="html-italic">n</span> = 5 mice per group). (<b>D</b>,<b>E</b>) OGTT and AUC in the HFD + STZ mouse model (<span class="html-italic">n</span> = 5 mice per group). (<b>F</b>,<b>G</b>) ITT and AUC of the HFD + STZ mouse model (<span class="html-italic">n</span> = 5 mice per group). (<b>H</b>) Liver index (liver weight/body weight). (<b>I</b>) Liver total cholesterol (<span class="html-italic">n</span> = 5). (<b>J</b>) Liver triglycerides (<span class="html-italic">n</span> = 5). (<b>K</b>) Serum AST (<span class="html-italic">n</span> = 5). (<b>L</b>) Serum ALT (<span class="html-italic">n</span> = 5). Data are expressed as means ± SEM and were analyzed using one-way or two-way ANOVA and appropriate post hoc analyses (Tukey’s multiple comparison test). * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001, ns means not significant. Abbreviations: CTR: control; DAPA: Dapansutrile; HFD: high-fat diet; STZ: Streptozotocin; OGTT: oral glucose tolerance test; AUC: area under the curve; ITT: insulin tolerance test.</p>
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<p>Dapansutrile improved glucose and lipid metabolism in the db/db mouse model. (<b>A</b>) Body weight monitoring in the db/db mouse model (<span class="html-italic">n</span> = 5 mice per group). (<b>B</b>) Plasma glucose levels in the db/db mouse model (<span class="html-italic">n</span> = 5 mice per group). (<b>C</b>) Insulin levels in the db/db mouse model (<span class="html-italic">n</span> = 5 mice per group). (<b>D</b>,<b>E</b>) OGTT and AUC in the db/db mouse model (<span class="html-italic">n</span> = 5 mice per group). (<b>F</b>) Liver index (liver weight/body weight). (<b>G</b>) Liver total cholesterol (<span class="html-italic">n</span> = 5). (<b>H</b>) Liver triglycerides (<span class="html-italic">n</span> = 5). (<b>I</b>) Serum AST (<span class="html-italic">n</span> = 5). (<b>J</b>) Serum ALT (<span class="html-italic">n</span> = 5). Data are expressed as means ± SEM and were analyzed using one-way or two-way ANOVA and appropriate post hoc analyses (Tukey’s multiple comparison test). ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001, ns means not significant. Abbreviations: CTR: control; DAPA: Dapansutrile; HFD: high-fat diet; STZ: Streptozotocin; OGTT: oral glucose tolerance test; AUC: area under the curve; ITT: insulin tolerance test.</p>
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<p>Dapansutrile alleviated liver lipid deposition. (<b>A</b>,<b>B</b>) Representative images of gross morphology, and liver tissues stained with H&amp;E and Oil red O (<span class="html-italic">n</span> = 3). The yellow arrow indicates lipid droplets, and the red arrow indicates inflammatory cell infiltration and focal necrosis.</p>
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<p>Dapansutrile improved lipid deposition in FFA-treated HepG2 cells by enhancing mitochondrial function. (<b>A</b>,<b>B</b>) Representative images of Oil Red O staining (<span class="html-italic">n</span> = 3). The yellow arrow indicates lipid droplets. (<b>C</b>) Representative images of TEM (<b>D</b>,<b>E</b>) Mito-Tracker Deep Red of HepG2 cells. (<b>F</b>,<b>G</b>) Measurements of mitochondrial ROS levels in HepG2 cells. (<b>H</b>,<b>I</b>) JC-1 staining of mitochondria in HepG2 cells. (<b>J</b>,<b>K</b>) Oxygen consumption rate (OCR) analysis of hepG2 cells (<span class="html-italic">n</span> = 3). CTR group: control; FFA group: 0.5 mM FFA; DAPA: 0.5 mM FFA + 10 μM DAPA. Data are expressed as means ± SEM and were analyzed using one-way or two-way ANOVA and appropriate post hoc analyses (Tukey’s multiple comparison test). * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001, ns means not significant. Abbreviations: CTR: control; FFA: free fatty acid; DAPA: Dapansutrile; NLRP3: NLR family pyrin domain-containing.</p>
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<p>Dapansutrile alleviated liver cell damage in FFA-treated HepG2 cells by targeting the NLRP3-Caspase-1 signaling axis. (<b>A</b>–<b>H</b>) Gene expression in mouse liver tissue of <span class="html-italic">NLRP3</span> (<b>A</b>), <span class="html-italic">Asc</span> (<b>B</b>), <span class="html-italic">Caspase-1</span> (<b>C</b>), <span class="html-italic">Gsdmd</span> (<b>D</b>), <span class="html-italic">IL-1β</span> (<b>E</b>), <span class="html-italic">IL-18</span> (<b>F</b>), <span class="html-italic">IL-6</span> (<b>G</b>), and <span class="html-italic">TNF-α</span> (<b>H</b>). (<b>I</b>–<b>L</b>) Gene expression in HepG2 of <span class="html-italic">NLRP3</span> (<b>A</b>), <span class="html-italic">Asc</span> (<b>B</b>), <span class="html-italic">Caspase-1</span> (<b>C</b>) and <span class="html-italic">Gsdmd</span> (<b>D</b>). (<b>M</b>–<b>R</b>) Western blot of NLRP3, ASC, Pro-Caspase-1, p20, and GSDMD in HepG2. (<b>S</b>,<b>T</b>) IL-1β and IL-18 release of cell supernatants. CTR group: control; FFA group: 0.5 mM FFA; DAPA: 0.5 mM FFA + 10 μM DAPA. Data are expressed as means ± SEM and were analyzed using one-way ANOVA and appropriate post hoc analyses (Tukey’s multiple comparison test). * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001. Abbreviations: CTR: control; FFA: free fatty acid; DAPA: Dapansutrile; NLRP3: NLR family pyrin domain-containing 3; CASP1: Caspase-1.</p>
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15 pages, 3109 KiB  
Article
An Immunoinformatics-Based Multi-Peptide Vaccine Provides Antibody-Mediated Protection Against Acinetobacter baumannii Infection
by Sean Jeffreys, Jadelynn Aki, Megan P. Tompkins, Nicolas D. Prather, Ashlesh K. Murthy, James P. Chambers, M. Neal Guentzel, Chiung-Yu Hung, Bernard P. Arulanandam and Jieh-Juen Yu
Vaccines 2025, 13(3), 236; https://doi.org/10.3390/vaccines13030236 - 25 Feb 2025
Abstract
Background/Objectives: Acinetobacter baumannii is an opportunistic nosocomial pathogen characterized by its multidrug-resistant (MDR) phenotype, increasing patient mortality and healthcare costs as a result. Previously, we constructed an immunoinformatics-based Acinetobacter Multi-Epitope Vaccine (AMEV2) candidate and demonstrated robust protection against this MDR pathogen. In this [...] Read more.
Background/Objectives: Acinetobacter baumannii is an opportunistic nosocomial pathogen characterized by its multidrug-resistant (MDR) phenotype, increasing patient mortality and healthcare costs as a result. Previously, we constructed an immunoinformatics-based Acinetobacter Multi-Epitope Vaccine (AMEV2) candidate and demonstrated robust protection against this MDR pathogen. In this study, we delineate the mechanisms of AMEV2-mediated protective immunity. Methods: In vivo passive immunization with AMEV2 antisera and in vitro opsonophagocytic killing assays (OPKAs) were used to assess the critical role of antibody-mediated protection induced by AMEV2 vaccination. Results: The passive transfer of AMEV2 immune sera to naïve mice afforded 67% protection in a pulmonary challenge mouse model. Although AMEV2 sera reacts with bacterial antigens, it is not bactericidal on its own and does not enhance the complement-mediated direct killing of A. baumannii. However, OPKAs demonstrate AMEV2 sera enhancement of the killing of A. baumannii in the presence of primary bone marrow-derived macrophages. This killing occurs via complement and Fc gamma receptor-mediated phagocytosis. A highly immunogenic AMEV2 component peptide, pTonB, elicits pTonB-specific antibodies and protection in vivo. The depletion of pTonB antibodies from AMEV2 immune sera by pTonB absorption significantly reduced the opsonophagocytic killing of A. baumannii in vitro. Conclusions: The data presented here demonstrate the importance of humoral immunity and its protective mechanisms against A. baumannii. These findings further expand the in vivo evaluation of in silico-designed vaccines as a viable alternative to combat the current global MDR pathogen health crisis. Full article
(This article belongs to the Section Vaccines against Tropical and other Infectious Diseases)
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<p>AMEV2 immune sera provides passive protection against pulmonary <span class="html-italic">Acinetobacter baumannii</span> infection. C57BL/6 (7–8-week-old) mice (<span class="html-italic">n</span> = 6 per group) were injected intraperitoneally with 100 µL of either control or AMEV2 sera. At 24 h post-injection, mice were challenged intranasally with 10<sup>8</sup> CFUs of Ci79 <span class="html-italic">A. baumannii</span> and monitored for 30 days for survival. Log-rank Mantel–Cox test between control and AMEV2 passively vaccinated mouse survival status over 30 days.</p>
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<p>AMEV2 vaccination generates antibodies to <span class="html-italic">A. baumannii</span> but does not enhance complement-mediated killing. (<b>A</b>) Control and AMEV2 sera (<span class="html-italic">n</span> = 5 per group) were diluted 1:100 and checked for reactivity with whole cell UV-inactivated BL21 <span class="html-italic">E. coli</span> and two <span class="html-italic">A. baumannii</span> strains by indirect ELISA. Data are presented as the mean ± SD. Two-way ANOVA (<span class="html-italic">p</span> = 0.0471) with Sidak’s multiple comparisons test. (<b>B</b>) Ci79 or ATCC 19606 <span class="html-italic">A. baumannii</span> was incubated with heat-inactivated control sera or heat-inactivated AMEV2 sera in the presence of baby rabbit sera containing either intact or heat-inactivated complement in quadruplicate. Following incubation (1.5 h) at 37 °C, wells were serially diluted and plated to enumerate viable bacteria.</p>
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<p>AMEV2 immune sera enhance the opsonophagocytic killing of <span class="html-italic">A. baumannii</span> and increase bone marrow-derived macrophage bacterial uptake. (<b>A</b>) Ci79 bacteria were opsonized in the presence of heat-inactivated naïve mouse sera or pooled control or AMEV2 sera and fresh baby rabbit sera as a complement source for 30 min at 37 °C. Then, 10<sup>4</sup> CFUs of opsonized bacteria were seeded into wells with or without 10<sup>5</sup> adhered BMDMs. Following a 1 h incubation, the remaining viable bacteria were diluted and plated on LB agar for enumeration. The reduction in bacterial counts by the control and AMEV2 sera treatments was compared to naïve serum treatment and presented as % relative killing. Two-way ANOVA (<span class="html-italic">p</span> &lt; 0.0001) with Sidak’s multiple comparisons test. (<b>B</b>) Phagocytosed bacteria were released from macrophages by lysing and enumerated by serial dilution and plating on LB agar. Student’s <span class="html-italic">t</span>-test. Both results are expressed as the mean ± SD and each is representative of two independent experiments with technical replicates.</p>
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<p>AMEV2 sera opsonophagocytic killing is mediated by enhancing classical complement activation and FCγ receptor-mediated phagocytosis. (<b>A</b>) Ci79 bacteria were opsonized in the presence of heat-inactivated naïve mouse sera or pooled control or AMEV2 sera with or without complement. Opsonized bacteria (10<sup>4</sup> CFU) were seeded into wells containing 10<sup>5</sup> BMDM. Following a 1 h incubation, the remaining viable bacteria were diluted and plated on LB agar for enumeration. The reduction in bacterial counts by the control and AMEV2 sera treatments was compared to naïve serum treatment and presented as % relative killing. Results are expressed as the mean ± SD and are representative of two independent experiments with technical replicates. Two-way ANOVA (<span class="html-italic">p</span> = 0.0429) with Sidak’s multiple comparisons test. (<b>B</b>) Ci79 (10<sup>4</sup> CFU) bacteria opsonized in the presence of heat-inactivated naïve mouse sera or pooled control or AMEV2 sera without complement were seeded into wells containing either 10<sup>5</sup> WT BMDM or FcγR<sup>−/−</sup> BMDM. Following a 1 h incubation, the remaining viable bacteria were diluted and plated on LB agar for enumeration. The reduction in bacterial counts by the control and AMEV2 sera treatments in WT or FCγR<sup>−/−</sup> BMDM was compared to naïve serum treatment and presented as % relative killing. The results are expressed as the mean ± SD. Two-way ANOVA (<span class="html-italic">p</span> = 0.029) with Sidak’s multiple comparisons test.</p>
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<p>AMEV2 vaccination induces Th2 cellular immune responses. Seven- to eight-week-old C57BL/6 mice (<span class="html-italic">n</span> = 3 per group) were vaccinated subcutaneously with either PBS + AddaS03 (mock control) or rAMEV2 (10 µg) +AddaS03 (AMEV2) on days 0, 14, and 28, and rested for 4 weeks prior to spleen removal. (<b>A</b>) Representative ELISpot readouts (each well was seeded with 5 × 10<sup>5</sup> splenocytes) for control or AMEV2-vaccinated splenocytes stimulated with media, α-CD3, nonspecific peptide (pNspec), and rAMEV2 component peptides. (<b>B</b>) Detection of IFNγ, IL-4, and IL-5 spot-forming units (SFU) to AMEV2 peptides. Data are presented as the mean <span class="html-italic">±</span> SD. * <span class="html-italic">p</span> = 0.0114, ** <span class="html-italic">p</span> = 0.0015, **** <span class="html-italic">p</span> ≤ 0.0001. Two-way ANOVA (<span class="html-italic">p</span> &lt; 0.0001) with Tukey’s multiple comparisons test between SFUs of indicated AMEV2 peptide and pNspec.</p>
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<p>pTonB-specific antibodies appear to be protective in <span class="html-italic">Acinetobacter baumannii</span> infections. (<b>A</b>) AMEV2 immune sera collected before <span class="html-italic">A. baumannii</span> challenge were screened for peptide reactivity with peptide ELISA. The antibody compositions of AMEV2-vaccinated mice that survived the challenge were compared to those that succumbed to infection. * <span class="html-italic">p</span> = 0.024, **** <span class="html-italic">p</span> &lt; 0.0001. Two-way ANOVA (<span class="html-italic">p</span> &lt; 0.0001) with Sidak’s multiple comparisons test. (<b>B</b>) Pooled AMEV2 sera were absorbed with either pNspec or pTonB peptides and their pTonB-specific antibody titer was determined by peptide ELISA. The dotted line indicates the endpoint titer determination threshold of an OD<sub>450 nm</sub> value greater than 0.1. (<b>C</b>) The unabsorbed and absorbed AMEV2 vaccinated sera killing were compared by the opsonophagocytic killing assay. The reduction in bacterial counts by AMEV2 sera treatments was compared to naïve serum treatment and presented as % relative killing. Results are expressed as the mean ± SD and are representative of two independent experiments with technical replicates. * <span class="html-italic">p</span> ≤ 0.05, ** <span class="html-italic">p</span> = 0.0038. One-way ANOVA (<span class="html-italic">p</span> = 0.003) with Tukey’s multiple comparisons test.</p>
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14 pages, 5937 KiB  
Article
Curcumin Microemulsions: Influence of Compositions on the Dermal Penetration Efficacy
by Muzn Alkhaldi, Soma Sengupta and Cornelia M. Keck
Pharmaceutics 2025, 17(3), 301; https://doi.org/10.3390/pharmaceutics17030301 - 25 Feb 2025
Abstract
Background/Objective: This study provided a comparison of the influence of each component of the microemulsion formulation and investigated the impact of varying concentrations of the microemulsion components on curcumin’s ability to penetrate the skin using an ex vivo porcine ear model. Methods [...] Read more.
Background/Objective: This study provided a comparison of the influence of each component of the microemulsion formulation and investigated the impact of varying concentrations of the microemulsion components on curcumin’s ability to penetrate the skin using an ex vivo porcine ear model. Methods: Curcumin microemulsions with different compositions were prepared and analyzed for their physicochemical properties. The dermal penetration efficacy of curcumin was evaluated from the different formulations and compared with non-microemulsion formulations. Results: Findings proved that microemulsion formulations improve the dermal penetration efficacy for curcumin when compared with non-microemulsion formulations. The composition of the microemulsion affects the penetration efficacy of curcumin and increases with decreasing oil content and increasing surfactant and water content. The best penetration for curcumin is achieved with a microemulsion that contained 7.7 g of medium-chain triglycerides as the oil phase, 6.92 g of Tween® 80 and 62.28 g of ethanol as the surfactant mixture, and 23.1 g water. Conclusions: The present study provides a foundational basis for further development of different microemulsion formulations for enhancing the dermal penetration of poorly water-soluble active compounds. Full article
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<p>Overview of the experimental procedure of dermal penetration testing using the ex vivo porcine ear model.</p>
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<p>Original image: epifluorescence microscopy image of skin cut, demonstrating the scaling of stratum corneum thickness (SCT), Inverted image: original image after applying automated threshold demonstrating the measurement of mean penetration depth (MPD) of the penetrated curcumin from a formulation (200-fold magnification).</p>
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<p>Pseudoternary phase diagram of microemulsion composed of MCT oil, surfactant mixture (Smix), and water. Red points represent the formulated CUR ME formulations (Top to bottom: CUR ME 1 to CUR ME 9).</p>
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<p>Macroscopic photos and microscopic images of the formulations. (<b>Left-upper</b>): microemulsions without curcumin, (<b>left-lower</b>): microemulsions with curcumin. (<b>Right-upper</b>): curcumin in the non-microemulsion formulations—A: CUR in ethanol, B: CUR in MCT oil, C: CUR in Smix, D: CUR in Tween<sup>®</sup> 80. (<b>Right-lower</b>): light microscopic images of all produced formulations in this study (400-fold magnification, scale bar represents 50 µm).</p>
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<p>Visualization of vertical skin cuts observed through inverted epifluorescence microscopy (200-fold magnification).</p>
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<p>Inverted epifluorescence microscopic images after digital image processing evaluating the penetration efficacy of the formulations.</p>
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<p>Curcumin penetration efficacies from various CUR ME formulations and non-microemulsion formulations, expressed as the penetration efficacy (penetration efficacy in MGV/px). Significant differences are indicated by asterisks (***: <span class="html-italic">p</span> &lt; 0.001), while n.s. indicates no significant differences.</p>
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<p>Influence of formulations of CUR ME, and solutions and suspensions of curcumin on the SCT and MPD.</p>
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<p>Heatmap of Spearman’s rank correlation coefficients (rho) between composition, physicochemical characteristics and penetration efficacy parameters and the physicochemical properties for all CUR ME formulations. ZP: zeta potential, PE: penetration efficacy. ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001.</p>
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20 pages, 3267 KiB  
Article
Carmofur Exhibits Antimicrobial Activity Against Streptococcus pneumoniae
by Wenting Lyu, Yuqing Zhang, Zhen Zhang and Hao Lu
Antibiotics 2025, 14(3), 231; https://doi.org/10.3390/antibiotics14030231 - 25 Feb 2025
Abstract
Background/Objectives: Streptococcus pneumoniae (S. pneumoniae) is a major pathogen causing severe infectious diseases, with an escalating issue of antimicrobial resistance that threatens the efficacy of existing antibiotics. Given the challenges in developing traditional antibiotics, drug repurposing strategies offer a novel [...] Read more.
Background/Objectives: Streptococcus pneumoniae (S. pneumoniae) is a major pathogen causing severe infectious diseases, with an escalating issue of antimicrobial resistance that threatens the efficacy of existing antibiotics. Given the challenges in developing traditional antibiotics, drug repurposing strategies offer a novel approach to address the resistance crisis. This study aims to evaluate the antibacterial and anti-biofilm activities of the approved non-antibiotic anticancer drug carmofur against multidrug-resistant S. pneumoniae, and investigate the mechanism of action, and assess therapeutic potential in vivo. Methods/Results: Antimicrobial tests revealed that carmofur exhibited strong antibacterial activity against multidrug-resistant S. pneumoniae strains, with minimum inhibitory concentrations (MICs) ranging from 0.25 to 1 µg/mL. In the biofilm detection experiments, carmofur not only inhibited the formation of biofilms, but also effectively removed biofilms under high concentration conditions. Mechanistic studies showed that carmofur disrupted bacterial membrane permeability and decreased intracellular ATP levels. Molecular docking and dynamics simulation assays indicated that carmofur could stably bind to thymidylate synthase through hydrogen bonding and hydrophobic interactions, thereby exerting antibacterial effects. Meanwhile, carmofur was able to repress the expression of the thyA gene at the mRNA level. In a mouse infection model, the carmofur treatment group showed a reduction of approximately two log levels in bacterial load in lung tissue and blood, a significant decrease in the levels of inflammatory cytokines TNF-α and IL-6, and an improvement in survival rate to 60%. Conclusions: In summary, carmofur demonstrated significant antibacterial and anti-biofilm activities against multidrug-resistant S. pneumoniae and showed good anti-infective effects in vivo, suggesting its potential clinical application as a therapeutic agent against drug-resistant bacteria. Full article
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<p>Effects of different concentrations of carmofur on the growth of <span class="html-italic">S. pneumoniae</span> strain 17426. (<b>A</b>) The 2D structural formula of carmofur; (<b>B</b>) bacterial growth curves based on optical density (OD 600 nm) over time. Carmofur concentrations were set at 0.25 µg/mL, 0.5 µg/mL, and 1 µg/mL. Untreated control was used as a reference; (<b>C</b>) changes in colony-forming units (CFUs/mL) over time in carmofur-treated groups. Concentrations of 0.5 µg/mL, 1 µg/mL, and 2 µg/mL were tested. Each group consisted of three biological replicates. Data are presented as mean ± standard deviation (Mean ± SD). *** <span class="html-italic">p</span> &lt; 0.001, and n.s. indicates not significant. The statistical analysis at the end of the experiment was performed via Student’s <span class="html-italic">t</span> test.</p>
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<p>Anti-biofilm effects of carmofur (0.25, 0.5, 1, 2 and 5 µg/mL) against <span class="html-italic">S. pneumoniae</span>. Auranofin (MIC = 0.5 µg/mL), a known reported drug, served as a positive control. (<b>A</b>) Inhibition of biofilm formation at varying concentrations of carmofur. (<b>B</b>) Eradication effect on pre-formed biofilms. (<b>C</b>) Total bacterial count in mature biofilms, evaluated with acridine orange fluorescence probe at different carmofur concentrations. (<b>D</b>) Reduction in metabolically active cells in mature biofilms, assessed by ATP levels. Each group consisted of three biological replicates. Data are presented as Mean ± SD. Significance levels are indicated as follows: *, <span class="html-italic">p</span> &lt; 0.05; **, <span class="html-italic">p</span> &lt; 0.01; ***, <span class="html-italic">p</span> &lt; 0.001; ‘n.s.’ indicates no significant difference. Statistical analyses were performed via one-way ANOVA.</p>
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<p>Antibacterial mechanism of carmofur. (<b>A</b>) PI uptake in <span class="html-italic">S. pneumoniae</span>. (<b>B</b>) Analysis of intracellular ATP levels in <span class="html-italic">S. pneumoniae</span> treated with various concentrations of carmofur. Daptomycin (8 µg/mL) served as a positive control. Each group consisted of three biological replicates. Data are presented as mean ± SD. Significance levels are indicated as follows: *, <span class="html-italic">p</span> &lt; 0.05; **, <span class="html-italic">p</span> &lt; 0.01; ***, <span class="html-italic">p</span> &lt; 0.001. The statistical analysis at the end of the experiment was performed via one-way ANOVA.</p>
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<p>Molecular docking and interaction analysis of carmofur with target protein. (<b>A</b>) Three-dimensional structure of the target protein, comprising typical α-helices and β-sheets; (<b>B</b>) superimposed results of carmofur’s four primary binding conformations, showing its spatial compatibility; (<b>C</b>) distribution of binding energies (Cdocker energy) across different conformations, with the lowest binding energy (around −25 kJ/mol) marked in the red box as the optimal conformation; (<b>D</b>) detailed interaction analysis of carmofur and 5-fluorouracil within the binding pocket; (<b>E</b>) two-dimensional spatial binding mode of carmofur in the binding pocket, showing multiple interactions.</p>
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<p>The effects of carmofur on thymidylate synthase. (<b>A</b>) The root mean square deviation (RMSD) as a function of time, indicating that the system gradually reached stability during the molecular dynamics simulation; (<b>B</b>) the solvent-accessible surface area (SASA) over time, reflecting fluctuations in protein solvent accessibility; (<b>C</b>) the radius of gyration (Rg) as a function of time, demonstrating the overall compactness of the protein conformation; (<b>D</b>) the root mean square fluctuation (RMSF) analysis, revealing the conformational flexibility of different protein residues; (<b>E</b>) the number of hydrogen bonds over time, illustrating the dynamic characteristics of hydrogen bonding interactions within the system; (<b>F</b>) the binding free energy decomposition results, showing the energetic contributions of different residues to ligand binding; (<b>G</b>) the effect of carmofur on relative mRNA expression levels. Data are presented as mean ± standard deviation (mean ± SD); mean ± SD was repeated three times. * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01; n.s. indicates no significant difference. The statistical analysis at the end of the experiment was performed Via Student’s <span class="html-italic">t</span> test.</p>
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<p>Therapeutic efficacy of carmofur in mice infected with <span class="html-italic">S. pneumoniae</span>. (<b>A</b>) Results of blood biochemical indexes to evaluate the effects of carmofur on liver and kidney functions in mice. (<b>B</b>) Survival rate of mice treated with carmofur 1 h post-infection with an inoculum dose of 5 × 10<sup>8</sup> CFU. (<b>C</b>) Bacterial load in blood and lung tissues of mice. (<b>D</b>) Levels of TNF-α and IL-6 in mouse serum. (<b>E</b>) Histopathological changes in lung tissue post-treatment. Data are presented as mean ± standard deviation (Mean ± SD). ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001; n.s. indicates no significant difference. The statistical analysis at the end of the experiment was performed Via Student’s <span class="html-italic">t</span> test.</p>
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15 pages, 2510 KiB  
Article
Silver Dimolybdate Nanorods: In Vitro Anticancer Activity Against Breast and Prostate Tumors and In Vivo Pharmacological Insights
by João Victor Barbosa Moura, Natália Cristina Gomes-da-Silva, Luciana Magalhães Rebêlo Alencar, Wellington Castro Ferreira, Cleânio da Luz Lima and Ralph Santos-Oliveira
Pharmaceutics 2025, 17(3), 298; https://doi.org/10.3390/pharmaceutics17030298 - 24 Feb 2025
Abstract
Background: The development of nanostructured materials for cancer therapy has garnered significant interest due to their unique physicochemical properties, including enhanced surface area and tunable electronic structures, which can facilitate targeted drug delivery and oxidative stress modulation. This study investigates the anticancer [...] Read more.
Background: The development of nanostructured materials for cancer therapy has garnered significant interest due to their unique physicochemical properties, including enhanced surface area and tunable electronic structures, which can facilitate targeted drug delivery and oxidative stress modulation. This study investigates the anticancer potential of monoclinic silver dimolybdate nanorods (m-Ag₂Mo₂O₇) against aggressive breast (MDA-MB-231) and prostate (PC-3) cancer cells and explores their in vivo pharmacokinetic behavior. Methods: m-Ag₂Mo₂O₇ nanorods were synthesized via a hydrothermal method and characterized using XRD, SEM, Raman, and FTIR spectroscopy. In vitro cytotoxicity was evaluated using MTT assays on MDA-MB-231 and PC-3 cell lines across concentrations ranging from 1.56 to 100 µg/mL. In vivo biodistribution and radiopharmacokinetics were assessed using technetium-99m-labeled nanorods in male Swiss rats, with gamma counting employed for tissue uptake analysis and pharmacokinetic parameter determination. Results: m-Ag₂Mo₂O₇ nanorods exhibited a modest cytotoxic effect on MDA-MB-231 cells, with 50 µg/mL reducing cell viability by 23.5% (p < 0.05), while no significant cytotoxicity was observed in PC-3 cells. In vivo studies revealed predominant accumulation in the stomach, liver, spleen, and bladder, indicating reticuloendothelial system uptake and renal clearance. Pharmacokinetic analysis showed a rapid systemic clearance (half-life ~6.76 h) and a low volume of distribution (0.0786 L), suggesting primary retention in circulation with minimal off-target diffusion. Conclusions: While m-Ag₂Mo₂O₇ nanorods display limited standalone cytotoxicity, their ability to induce oxidative stress and favorable pharmacokinetic profile support their potential as adjuvant agents in cancer therapy, particularly for chemoresistant breast cancers. Further studies are warranted to elucidate their molecular mechanisms, optimize combinatorial treatment strategies, and assess long-term safety in preclinical models. Full article
(This article belongs to the Special Issue Recent Advances in Nanotechnology Therapeutics)
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<p>(<b>a</b>) X-ray diffraction pattern of m-Ag<sub>2</sub>Mo<sub>2</sub>O<sub>7</sub> nanorods obtained by hydrothermal synthesis. (<b>b</b>) Representation of the crystal structure of the m-Ag<sub>2</sub>Mo<sub>2</sub>O<sub>7</sub> crystal (Mo—Green, Ag—Silver, and O—Red atoms), showing the [MoO<sub>6</sub>] clusters connected by oxygen atoms forming distorted/deformed octahedrons. (<b>c</b>) Low- and (<b>d</b>) high-magnification SEM images of m-Ag<sub>2</sub>Mo<sub>2</sub>O<sub>7</sub> nanorods.</p>
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<p>(<b>a</b>) Raman and (<b>b</b>) FTIR spectra of m-Ag<sub>2</sub>Mo<sub>2</sub>O<sub>7</sub> nanorods. Inset: Magnification of the spectral region of 550–100 cm<sup>−1</sup> where the Raman modes have low intensity.</p>
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<p>(<b>A</b>) MDA-MB-231 and PC-3: cell viability after treatment with different concentrations of m-Ag<sub>2</sub>Mo<sub>2</sub>O<sub>7</sub> nanorods using a human breast cancer cell line and a prostate tissue derived from a human, respectively. The results are expressed as the percentage of total cell viability (spectrophotometric readings at 450 nm) in the culture medium after the addition of a 1 mg/mL MTT solution (positive response). The height of the histogram bar is the mean ± SEM of three independent experiments. MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. (<b>B</b>) The representative images of the key concentrations of m-Ag<sub>2</sub>Mo<sub>2</sub>O<sub>7</sub> (control; 100 μg/mL; 50 μg/mL; and 1.5 μg/mL) obtained from an inverted light microscope with a 10× objective lens. Scale bar: 20 μm. * means statically different.</p>
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<p>Biodistribution assay showing the tissue accumulation of m-Ag<sub>2</sub>Mo<sub>2</sub>O<sub>7</sub>.</p>
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<p>The main pharmacokinetic data derived from temporal reconstruction based on serial blood sampling. ** mean statically different.</p>
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34 pages, 7476 KiB  
Article
Unveiling Pharmacological Mechanisms of Bombyx mori (Abresham), a Traditional Arabic Unani Medicine for Ischemic Heart Disease: An Integrative Molecular Simulation Study
by Doni Dermawan and Nasser Alotaiq
Pharmaceutics 2025, 17(3), 295; https://doi.org/10.3390/pharmaceutics17030295 - 24 Feb 2025
Abstract
Background: Ischemic heart disease (IHD), a leading cause of cardiovascular morbidity and mortality, continues to challenge modern medicine. Bombyx mori (Abresham), a traditional ingredient in Unani medicine, has shown promise in cardiovascular health, but its molecular mechanisms remain poorly understood. Methods: To [...] Read more.
Background: Ischemic heart disease (IHD), a leading cause of cardiovascular morbidity and mortality, continues to challenge modern medicine. Bombyx mori (Abresham), a traditional ingredient in Unani medicine, has shown promise in cardiovascular health, but its molecular mechanisms remain poorly understood. Methods: To explore the therapeutic potential of Bombyx mori for IHD, an integrative molecular simulation approach was applied. Network pharmacology was employed to identify the most favorable target receptor for the disease. Molecular docking simulations evaluated the binding affinities of chemical and protein-based compounds from Bombyx mori to the selected receptor. Molecular dynamics (MD) simulations confirmed the stability of these interactions under physiological conditions. Pharmacophore modeling identified key structural features critical for bioactivity, while in silico toxicity assessments evaluated the safety profiles of the compounds. Results: Key bioactive compounds from Bombyx mori, including Menaquinone-7, Quercetin, and Behenic acid, showed strong interactions with the target receptor, ACE2. The MD-based MM/PBSA calculations revealed the binding free energy values of Menaquinone-7 (−35.12 kcal/mol), Quercetin (−29.38 kcal/mol), and Behenic acid (−27.76 kcal/mol), confirming their strong binding affinity. Protein-based compounds, such as Chorion class high-cysteine HCB protein 13 (−212.43 kcal/mol), Bombyxin A-5 (−209.36 kcal/mol), and FMRFamide-related peptides (−198.93 kcal/mol), also displayed promising binding affinities. In silico toxicity assessments revealed favorable safety profiles for most compounds. Conclusions: This study positions Bombyx mori as a promising source of therapeutic agents for IHD. Future work should focus on experimental validation of these computational findings through in vitro and in vivo studies. Full article
(This article belongs to the Special Issue In Silico Approaches of Drug–Target Interactions)
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<p>Results of the network pharmacology analysis. <span class="html-italic">Bombyx mori</span> compounds-target network with 140 nodes and 268 edges. Target proteins are depicted as orange nodes, while the chemical bioactive compounds derived from <span class="html-italic">Bombyx mori</span> are represented by green-to-blue gradient nodes.</p>
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<p>Results of the network pharmacology analysis. ACE2 is identified as the most prominent target receptor, showing extensive interactions with proteins targeted by the <span class="html-italic">Bombyx mori</span>-derived chemical compounds.</p>
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<p>Results of molecular docking simulations. 3D representations of molecular poses and interactions between the ACE2 receptor and the top three performing compounds derived from <span class="html-italic">Bombyx mori</span>, alongside the standard inhibitor captopril. (<b>A</b>) Captopril_ACE2 complex, illustrating the reference binding interaction. (<b>B</b>) Menaquinone-7_ACE2 complex, showing its strong binding affinity and key interaction points. (<b>C</b>) Quercetin_ACE2 complex, highlighting its favorable hydrogen bonding network. (<b>D</b>) Behenic acid_ACE2 complex, demonstrating significant van der Waals interactions within the receptor’s active site.</p>
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<p>Results of molecular docking simulations. 2D interaction maps of the ACE2 receptor with the top three performing compounds derived from <span class="html-italic">Bombyx mori</span>, compared to the standard inhibitor captopril. (<b>A</b>) Captopril_ACE2 complex. (<b>B</b>) Menaquinone-7_ACE2 complex. (<b>C</b>) Quercetin_ACE2 complex. (<b>D</b>) Behenic acid_ACE2 complex.</p>
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<p>Results of molecular docking simulation. 3D binding poses of protein-based compounds derived from <span class="html-italic">Bombyx mori</span> interacting with the ACE2 receptor within the binding pocket. (<b>A</b>) DX600 peptide_ACE2 complex (standard inhibitor). (<b>B</b>) Chorion class high-cysteine HCB protein 13_ACE2 complex. (<b>C</b>) Bombyxin A-5_ACE2 complex. (<b>D</b>) FMRFamide-related peptides_ACE2 complex.</p>
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<p>Results of molecular docking simulations. (<b>A</b>) Binding affinity values (kcal/mol) of the top 10 chemical compounds derived from <span class="html-italic">Bombyx mori</span> docked to the active sites of ACE2. (<b>B</b>) Correlation matrix depicting the relationship between binding energy (kcal/mol) and individual energy components for the chemical compounds derived from <span class="html-italic">Bombyx mori</span>. (<b>C</b>) Binding affinity values (kcal/mol) of the top 10 protein-based compounds derived from <span class="html-italic">Bombyx mori</span> docked to the active sites of ACE2. (<b>D</b>) Correlation matrix illustrating the relationship between binding energy (kcal/mol) and individual energy components for the protein-based compounds derived from <span class="html-italic">Bombyx mori</span>. The correlation values span from −1 to 1, with 1 representing a perfect positive correlation, −1 indicating a perfect negative correlation, and 0 signifying the absence of any correlation.</p>
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<p>RMSF Profiles of ACE2-Ligand complexes. (<b>A</b>) RMSF profiles of chemical compounds derived from <span class="html-italic">Bombyx mori</span> (Menaquinone-7, Quercetin, and Behenic acid) compared to the standard inhibitor, Captopril, highlighting the fluctuations in key binding regions of ACE2. (<b>B</b>) RMSF profiles of protein-based compounds derived from <span class="html-italic">Bombyx mori</span> (Chorion class high-cysteine HCB protein 13, Bombyxin A-5, and FMRFamide-related peptides) compared to the standard protein inhibitor, DX600 peptide, showing similar fluctuations and disruption of hydrogen bonds in critical binding regions of ACE2.</p>
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<p>3D structure-based pharmacophore modeling results. (<b>A</b>) Pharmacophore model of the Captopril_ACE2 complex. (<b>B</b>) Pharmacophore model of the Menaquinone-7_ACE2 complex. (<b>C</b>) Pharmacophore model of the Quercetin_ACE2 complex. (<b>D</b>) Pharmacophore model of the Behenic acid_ACE2 complex. Hydrophobic interactions are depicted as yellow spheres, while hydrogen bond donors and hydrogen bond acceptors are represented by green and red arrows, respectively.</p>
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16 pages, 4439 KiB  
Article
Qualitative and Quantitative Evaluation of a Deep Learning-Based Reconstruction for Accelerated Cardiac Cine Imaging
by Junjie Ma, Xucheng Zhu, Suryanarayanan Kaushik, Eman Ali, Liangliang Li, Kavitha Manickam, Ke Li and Martin A. Janich
Bioengineering 2025, 12(3), 231; https://doi.org/10.3390/bioengineering12030231 - 24 Feb 2025
Abstract
Two-dimensional (2D) cine imaging is essential in routine clinical cardiac MR (CMR) exams for assessing cardiac structure and function. Traditional cine imaging requires patients to hold their breath for extended periods and maintain consistent heartbeats for optimal image quality, which can be challenging [...] Read more.
Two-dimensional (2D) cine imaging is essential in routine clinical cardiac MR (CMR) exams for assessing cardiac structure and function. Traditional cine imaging requires patients to hold their breath for extended periods and maintain consistent heartbeats for optimal image quality, which can be challenging for those with impaired breath-holding capacity or irregular heart rhythms. This study aims to systematically assess the performance of a deep learning-based reconstruction (Sonic DL Cine, GE HealthCare, Waukesha, WI, USA) for accelerated cardiac cine acquisition. Multiple retrospective experiments were designed and conducted to comprehensively evaluate the technique using data from an MR-dedicated extended cardiac torso anatomical phantom (digital phantom) and healthy volunteers on different cardiac planes. Image quality, spatiotemporal sharpness, and biventricular cardiac function were qualitatively and quantitatively compared between Sonic DL Cine-reconstructed images with various accelerations (4-fold to 12-fold) and fully sampled reference images. Both digital phantom and in vivo experiments demonstrate that Sonic DL Cine can accelerate cine acquisitions by up to 12-fold while preserving comparable SNR, contrast, and spatiotemporal sharpness to fully sampled reference images. Measurements of cardiac function metrics indicate that function measurements from Sonic DL Cine-reconstructed images align well with those from fully sampled reference images. In conclusion, this study demonstrates that Sonic DL Cine is able to reconstruct highly under-sampled (up to 12-fold acceleration) cine datasets while preserving SNR, contrast, spatiotemporal sharpness, and quantification accuracy for cardiac function measurements. It also provides a feasible approach for thoroughly evaluating the deep learning-based method. Full article
(This article belongs to the Section Biosignal Processing)
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<p>Reconstruction pipeline for Sonic DL Cine. The reconstruction pipeline for Sonic DL Cine takes a coil sensitivity map and multi-channel multi-phase under-sampled k-space cine data as inputs. The inputs go through multiple iterations of a data consistency (DC) step and a convolutional neural network (CNN) as regularization, until the final multi-phase cine images are reconstructed.</p>
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<p>Pipeline of generating multi-channel under-sampled cine datasets from the digital phantom. Single-slice cardiac images with different cardiac phases are extracted from the digital phantom. The cine images then go through multi-channel simulation, fast Fourier transform (FFT), and retrospective under-sampling in kt space via a variable-density (VD) sampling scheme to generate multi-channel under-sampled k-space data.</p>
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<p>Reconstructed multi-phase cine images from the digital phantom. Multi-phase fully sampled cine images (reference) were retrospectively under-sampled by 4-fold, 8-fold, and 12-fold and reconstructed using Sonic DL Cine. PSNR, SSIM, RMSE, and MAE were calculated for images from each cardiac phase, and the mean values across different cardiac phases were reported. PSNR = peak signal-to-noise ratio; SSIM = structural similarity index measure; RMSE = root mean square error; MAE = mean absolute error.</p>
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<p>Spatiotemporal profiles of cine images from the digital phantom. A reference line (red dashed line in (<b>a</b>)) was drawn across the heart from the cine image at the end-systole phase. Signal profiles along the reference line through all cardiac phases from the fully sampled and Sonic DL Cine-reconstructed cine images (acceleration factors = 4, 8, and 12) are shown in (<b>b</b>). In particular, signal profiles at the end-systole phase (green dashed line in (<b>b</b>)) from different cine images are compared in (<b>c</b>).</p>
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<p>Representative fully sampled and retrospectively under-sampled cine images from the short-axis plane. Retrospectively under-sampled cine images with ASSET (acceleration factor = 2) and Sonic DL Cine (acceleration factors = 4, 8, and 12) are compared to the fully sampled reference at both end-systole and end-diastole phases. Difference maps, amplified by a factor of 10, between the under-sampled and fully sampled cine images are also calculated.</p>
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<p>Representative fully sampled and retrospectively under-sampled cine images from the two-chamber, three-chamber, and four-chamber planes. Fully sampled and retrospectively under-sampled cine images with ASSET (acceleration factor = 2), and Sonic DL Cine (acceleration factors = 4, 8, and 12) on the two-chamber, three-chamber, and four-chamber planes are shown from top to bottom. Difference maps, amplified by a factor of 10, between the under-sampled and fully sampled cine images are also calculated.</p>
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<p>Quantitative evaluation of image quality for retrospectively under-sampled cine images from the short-axis, two-chamber, three-chamber, and four-chamber planes. PSNR and SSIM (with fully sampled cine images as reference) are calculated for retrospectively under-sampled cine images acquired on the short-axis, two-chamber, three-chamber, and four-chamber planes from all subjects. The central box represents the interquartile range (IQR), which contains the middle 50% of the data. It is bounded by the first and third quartiles. The line inside the box indicates the median value, and the lines extending from the box show the range of the data outside the IQR, which extend to the smallest and largest values within the range of 1.5 × IQR from the first and third quartiles. The stars are outliers outside the 1.5 × IQR range.</p>
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<p>Spatiotemporal profiles of cine images from healthy volunteers on the short-axis plane; (<b>a</b>) shows the reference line (red dashed line) drawn across the heart from the cine image at the end-systole phase. Signal profiles along the reference line across all cardiac phases for the fully sampled and Sonic DL Cine-reconstructed cine images (acceleration factors = 4, 8, and 12) are shown in (<b>b</b>). In particular, signal profiles at the end-systole phase (green dashed line in (<b>b</b>)) from Sonic DL Cine and reference images are compared in (<b>c</b>).</p>
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<p>Comparison of left ventricular cardiac function measured from Sonic DL Cine-reconstructed and fully sampled cine images on the short-axis plane. LVESV, LVEDV, and LVEF were measured from fully sampled and Sonic DL Cine-reconstructed cine images (acceleration factors = 4, 8, and 12), and Bland–Altman plots are drawn to assess the agreement between measurements from the two sets of data. Each point represents the bias between measurements from the Sonic DL Cine and fully sampled cine images for each subject. The dashed blue line shows the mean bias between measurements, and the dashed red lines represent the upper and lower 95% limits of agreement. LVESV = left ventricle end-diastolic volume; LVEDV = left ventricle end-systolic volume; LVEF = left ventricle ejection fraction.</p>
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<p>Comparison of right ventricular cardiac functions measured from Sonic DL Cine-reconstructed and fully sampled cine images on the short-axis plane. RVESV, RVEDV, and RVEF were measured from fully sampled and Sonic DL Cine-reconstructed cine images (acceleration factors = 4, 8, and 12), and Bland–Altman plots are drawn to assess the agreement between measurements from the two sets of data. Each point represents the bias between measurements from the Sonic DL Cine and fully sampled cine images for each subject. The dashed blue line shows the mean bias between measurements, and the dashed red lines represent the upper and lower 95% limits of agreement. RVESV = right ventricle end-diastolic volume; RVEDV = right ventricle end-systolic volume; RVEF = right ventricle ejection fraction.</p>
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