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20 pages, 4056 KiB

Open AccessArticle

The Polybrominated Diphenyl Ether Bromoxib Disrupts Nuclear Import and Export by Affecting Nucleoporins of the Nuclear Pore Complex

by Karina S. Krings, Anastasia Ritchie, Laura Schmitt, Judith Hatzfeld, Gudrun Totzke, Thomas Lenz, María José Mendiburo, Björn Stork, Nicole Teusch, Peter Proksch, Kai Stühler, Lisa Müller and Sebastian Wesselborg

Mar. Drugs 2025, 23(3), 108; https://doi.org/10.3390/md23030108 - 28 Feb 2025

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Abstract

Polybrominated diphenyl ethers (PBDEs) are natural products with potent antimicrobial and antineoplastic activity. We have previously shown that the polybrominated diphenyl ether bromoxib (4,5,6-tribromo-2-(2′,4′-dibromophenoxy) phenol), isolated from the marine sponge Dysidea species, exhibits a strong cytotoxic potential in leukemia and lymphoma cells by [...] Read more.

Polybrominated diphenyl ethers (PBDEs) are natural products with potent antimicrobial and antineoplastic activity. We have previously shown that the polybrominated diphenyl ether bromoxib (4,5,6-tribromo-2-(2′,4′-dibromophenoxy) phenol), isolated from the marine sponge Dysidea species, exhibits a strong cytotoxic potential in leukemia and lymphoma cells by targeting mitochondrial metabolism. Here, using a mass spectrometric thermal proteome profiling (TPP) approach, we observed that bromoxib induces a rapid reduction in the levels of 19 nucleoporins (NUPs) that are part of the nuclear pore complex (NPC). This apparently affected the functionality of the NPC, as evidenced by the bromoxib-mediated inhibition of the nuclear translocation and subsequent gene reporter activity of transcription factors such as nuclear factor of activated T cells (NFAT) and nuclear factor κB (NF-κB). In addition, bromoxib inhibited the nuclear export of the mRNA of the human immunodeficiency virus transactivator of transcription (HIV-Tat) and the subsequent import of the HIV-Tat protein into the nucleus as determined by the decrease in Tat-dependent gene reporter luciferase activity. Inhibition of nuclear mRNA-export also affected expression of the short-lived anti-apoptotic Bcl-2 protein Mcl-1, which has been shown to induce apoptosis. Thus, its ability to target both mitochondrial metabolism and the NPC renders bromoxib a promising anticancer agent. Full article

(This article belongs to the Section Marine Pharmacology)

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Figure 1
Structure of bromoxib (4,5,6-tribromo-2-(2′,4′-dibromophenoxy)phenol). Full article ">Figure 2
Mass spectrometric thermal proteome profiling (TPP) and immunoblot analysis of apparently downregulated selected NUPs. Mass spectrometry-based TPP data (the two lowest treatment temperatures with negligible melting effect, three biological replicates; see panel (D)) was used to analyze the abundance changes of proteins upon treatment of Ramos B-cell lymphoma cells with 40 µM bromoxib for a duration of 30 min versus diluent control (DMSO, 0.4% v/v). (A) Volcano plot of the statistical significance (expressed as the negative decadic logarithm of the Student’s t-test p-value, −log10(p-value)) versus the bromoxib-mediated abundance difference on the log2 scale (difference of the average log2 intensities of bromoxib-treated samples versus diluent controls) for each protein. Proteins with significant abundance decrease (cutoffs: -log10(p-value) > 2.5 and difference < 0) were selected for functional protein association analysis shown in panel (B). NUPs are labeled in red and other proteins above the cutoff in blue (difference < 0, abundance decrease) or green (difference > 0, abundance increase). The dashed line indicates the y-axis cutoff at -log10(p-value) > 2.5. (B) A functional protein association network (based on a STRING database enrichment analysis, <a href="https://string-db.org" target="_blank">https://string-db.org</a>, v12.0; accessed on 06.Dec.2024) of selected (cutoffs: -log10(p-value) > 2.5 and difference < 0) proteins from panel (A). NUPs are labeled in red and other proteins in blue. (C) Schematic diagram showing the structure of the nuclear pore complex (NPC), with NUPs exhibiting a significant abundance decrease (cutoffs: -log10(p-value) > 2.5 and difference < 0) depicted in red. Scheme adapted from [<a href="#B13-marinedrugs-23-00108" class="html-bibr">13</a>,<a href="#B14-marinedrugs-23-00108" class="html-bibr">14</a>]. (D) Five selected NUP proteins (NUP54, NUP93, NUP98, NUP133, and NUP153) are shown with their respective TPP melting curves (solid lines showing the major abundance change: differential abundances were fitted using a fixed melting point for bromoxib treatments and diluent controls, dashed lines showing only minor melting point shifts; differential melting points were fitted using fixed abundance for bromoxib treatments and diluent controls). Significant abundance decrease (cutoffs: -log10(p-value) > 2.5 and difference < 0) of these proteins was observed after treatment of Ramos cells with bromoxib (40 µM bromoxib for 30 min). The curved brackets indicate the range used for the differential abundance analysis shown in panel (A). (E) Exemplary immunoblots of selected NUPs from Ramos cells treated with 40 µM bromoxib at 37 °C (employing one of the three independent biological replicates used from the TPP). GAPDH (glyceraldehyde 3-phosphate dehydrogenase) served as loading control. (F) Quantitative analysis of immunoblots (see panel (B) for representative immunoblots) of the selected NUPs. The immunoblots of NUP54, NUP93, NUP98, NUP133, and NUP153 from three independent biological replicates (n = 3) were quantified. The normalized signal intensity is shown for DMSO in red and bromoxib in black. Error bars = mean ± SD of three independent biological replicate experiments (TPP 37 °C treatment temperature). Statistical analysis was carried out using two-way ANOVA, with significance levels of * p ≤ 0.05; *** p ≤ 0.001. Full article ">Figure 3
Bromoxib-mediated downregulation of NUPs does not induce nuclear permeability. HeLa cells were treated with DMSO (0.1% v/v; diluent control), 0.1 µM, 10 µM, or 40 µM bromoxib for 24 h. Subsequently, cells were treated with digitonin to permeabilize the plasma membrane (but not the nuclear membrane). Texas red dextran was then added, which can enter the cytosol (due to digitonin-mediated permeabilization of the plasma membrane) but not the nucleus. To induce nuclear membrane permeability, Triton X was used as a positive control (0.2%, 15 min incubation). Exemplary microscopy images are shown (red: Texas red dextran; blue: DAPI stained nuclei). Full article ">Figure 4
Bromoxib inhibits the nuclear import and the gene reporter luciferase activity of the transcription factor NFAT. (A) Overview scheme of NFAT activation. The transcription factor NFAT plays a crucial role in T cell activation and is activated upon an increase in intracellular Ca2+. This could be induced upon T cell receptor triggering but could also be mimicked by the Ca2+-ionophore ionomycin. In the cytosol, Ca2+ binds to calmodulin and the Ca2+/calmodulin complex then activates the phosphatase calcineurin. Activated calcineurin then binds and dephosphorylates NFAT, thereby revealing the nuclear localization signal (NLS) of NFAT. NFAT then translocates across the NPC to the nucleus, where it induces the expression of cytokines (such as interleukins (IL-2,-3,-4,-5,-8,-13), INFγ, GM-CSF, or TNFα). (B) Ramos cells were treated with the Ca2+-ionophore ionomycin (5 µM) or the combination of 5 µM ionomycin and 10 µM bromoxib in a kinetic for 2 h, 4 h, or 6 h. Subsequently, the cellular and nuclear fractions were prepared and subjected to immunoblotting for NFAT, GAPDH (control for cytosolic fraction), or lamin B1 (control for nuclear fraction). (C) Quantitative analysis of immunoblots (see (B) for representative immunoblots). (D) Jurkat cells stably expressing an NFAT-luciferase reporter gene construct were treated with 10 µM or 40 µM bromoxib, 10 µM ionomycin, or simultaneously with the combination of bromoxib and ionomycin for 6 h. The respective NFAT-luciferase activity from three independent experiments performed in triplicates is shown. (E) Jurkat cells stably expressing an NFAT-luciferase construct were pretreated with 10 µM ionomycin for 30 min before adding 10 µM or 40 µM bromoxib. Alternatively, Jurkat cells were pretreated with 10 µM or 40 µM bromoxib for 30 min before adding 10 µM ionomycin as indicated. The respective NFAT-luciferase activity from three independent experiments performed in triplicates was evaluated after 6 h. Full article ">Figure 5
Bromoxib inhibits the NF-κB gene reporter activity, reduces the nuclear export of Tat mRNA, and inhibits subsequent Tat-driven gene reporter activity. (A) Schematic diagram of NF-κB activation. In the classical (canonical) signaling pathway, NF-κB is activated by proinflammatory cytokines such as TNFα. Triggering of the TNFα receptor (TNF-R1) activates the IκB kinase (IKK) complex that phosphorylates the inhibitor of NF-κB (IκB) at two specific N-terminal serine residues. The subsequent polyubiquitination and proteasomal degradation of the IκB protein leads to the release of NF-κB (p50/RelA), which then translocates across the NPC to the nucleus to induce the expression of respective target genes (such as cytokines, chemokines, growth factors, cell adhesion molecules, regulators of apoptosis, or acute phase proteins). (B) HEK293 cells were transiently transfected with an NF-κB luciferase reporter gene construct together with a β-galactosidase plasmid and treated with 10 ng/mL TNFα with or without 10 µM or 40 µM bromoxib for 6 h. The luciferase activity was measured and normalized to β-galactosidase activity. The respective NF-κB-dependent luciferase activity from three independent experiments performed in triplicates is shown. (C) Schematic overview of the HIV-Tat system used. In this system, TZM-bl cells that stably express luciferase under a Tat-dependent LTR promotor are transiently transfected with an HIV-Tat expression plasmid. The transcribed nuclear HIV-Tat mRNA is subsequently transported across the NPC into the cytosol and translated into the HIV-Tat protein. The HIV-Tat protein then translocates across the NPC to the nucleus where it induces the expression of luciferase under the HIV-1 derived LTR promoter. (D) TZM-bl cells were incubated with 10 µM of the pan-caspase inhibitor QVD together with 18.6 µM bromoxib or 0.4% w/v DMSO (diluent control) for 16 h. Subsequently, immunoblots for Tat or actin (loading control) were performed. (E) TZM-bl cells transiently transfected with HIV-Tat were incubated with 10 µM QVD together with 18.6 µM bromoxib or 0.4% w/v DMSO (diluent control) for 16 h. The respective luciferase activity from three independent experiments performed in quadruplicate is shown. Full article ">Figure 6
Bromoxib inhibits the expression of the short-lived anti-apoptotic Bcl-2 protein Mcl-1. (A) Schematic illustration of the effect of RNA polymerase II inhibition by the CDK9 inhibitor AZD4573 on the subsequent protein expression of short-lived proteins such as Mcl-1. RNA polymerase II mediates the transcription of mRNA in the nucleus, which then translocates across the NPC into the cytosol. There, mRNA is translated at the ribosomes into respective proteins. Finally, proteins are degraded by the proteasome. One of the short-lived proteins is the anti-apoptotic Bcl-2 protein Mcl-1. Studies by Cidado et al. have shown that the selective CDK9-inhibitor AZD4573 inhibits the CDK9-mediated activation of RNA polymerase II and induces the downregulation of Mcl-1 mRNA, followed by a rapid downregulation of Mcl-1 at the protein level [<a href="#B25-marinedrugs-23-00108" class="html-bibr">25</a>]. (B) Ramos cells were treated with 10 µM or 40 µM of bromoxib, or 0.1 µM of the CDK9 inhibitor AZD4573 in a kinetic for 0 h, 2 h, 4 h, 6 h or 8 h. Treatment with 0.2% w/v DMSO for 8 h served as diluent control. Subsequently, immunoblots for the anti-apoptotic Bcl-2 protein Mcl-1, the caspase substrate poly(ADP-ribose) polymerase-1 (PARP), or vinculin (loading control) were performed. Solid arrowheads indicate the uncleaved form of PARP (p116); open arrowheads indicate the cleaved form (p85). (C) Quantitative analysis of immunoblots (see (B) for representative immunoblots) from three independent biological replicates (n = 3). (D) To avoid caspase-mediated protein degradation, Ramos cells were pretreated with 10 µM of the caspase-inhibitor QVD for 30 min. Subsequently, cells were treated as described in (B). (E) Quantitative analysis of immunoblots (see (D) for representative immunoblots) from three independent biological replicates (n = 3). Full article ">

12 pages, 1316 KiB

Open AccessArticle

Targeting Tumor Microenvironment Interactions in Chronic Lymphocytic Leukemia Using Leukotriene Inhibitors

by Laia Sadeghi, Magali Merrien, Magnus Björkholm, Anders Österborg, Birgitta Sander, Hans-Erik Claesson and Anthony P. H. Wright

Int. J. Mol. Sci. 2025, 26(5), 2209; https://doi.org/10.3390/ijms26052209 - 28 Feb 2025

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Abstract

Chronic lymphocytic leukemia (CLL) cells depend on microenvironment niches for proliferation and survival. The adhesion of tumor cells to stromal cells in such niches triggers the activation of signaling pathways crucial for their survival, including B-cell receptor (BCR) signaling. While inhibitors of Bruton’s [...] Read more.

Chronic lymphocytic leukemia (CLL) cells depend on microenvironment niches for proliferation and survival. The adhesion of tumor cells to stromal cells in such niches triggers the activation of signaling pathways crucial for their survival, including B-cell receptor (BCR) signaling. While inhibitors of Bruton’s tyrosine kinase (BTKi) have shown efficacy in patients with CLL by disrupting these interactions, acquired resistance and toxicity remain a challenge during long-term therapy. Thus, identifying additional therapeutic modalities is important. Previously, we demonstrated that 5-lipoxygenase (5-LOX) pathway inhibitors reduced mantle cell lymphoma (MCL) cell adhesion to stromal cells, motivating us to investigate their potential in the context of CLL. We employed an ex vivo co-culture model to study CLL cell adhesion to stromal cells in the absence and presence of 5-LOX pathway inhibitors (zileuton and MK886) as well as the BTKi ibrutinib that was included for comparative purposes. Our findings demonstrated that different CLL samples adhere to stromal cells differentially. We observed a variable decrease in CLL cell adhesion to stromal cells following the inhibition of the 5-LOX pathway across a spectrum of patient samples that was distinct to the spectrum for ibrutinib. Positive and negative correlations were shown between the clinical and genetic features of the CLL samples and their level of adherence to stromal cells in both the absence and presence of the tested inhibitors. These results suggest the 5-LOX pathway as a candidate for assessment as a new therapeutic target in CLL. Full article

(This article belongs to the Special Issue Recent Advances in Anti-Cancer Drugs)

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Heterogeneous mutation status and stromal cell adhesion capacity of patient-derived CLL cells ex vivo. (a) Principal Component Analysis (PCA) illustrating the distribution of CLL patient samples based on mutation status (IGHV mutation status, del (17p), del (13q;14), del (11q), and Trisomy 12), shown in <a href="#ijms-26-02209-t001" class="html-table">Table 1</a>. (b) Box plot showing the level of adhesion to stromal cells for CLL cells from ten patient samples using a cell–cell binding assay. The patient-derived CLL cells were cultured with a mono-layer of HS-5 stromal cells for 4 h prior to the assay. The JeKo-1 MCL cell line was included as a positive control. Experiments were performed in triplicate for two biological replicates, with point colors representing the different replicates. The Kruskal–Wallis test was used to assess whether there was a significant difference in the adhesion levels between the samples. Full article ">Figure 2
Primary CLL cells from different patients respond differently to small-molecule inhibitors. (a–c) Adhesion levels of CLL cells from different samples to stromal cells were assessed using a cell–cell binding assay in the absence and presence of 0.5 μM ibrutinib and 1 μM MK886 or 1 μM zileuton. Box plots illustrate the median value of data normalized to values for untreated cells, and individual values outside the central quartiles are shown. The hashed red line represents the normalized value for untreated cells. Statistical significance between the treated and untreated conditions was evaluated using the Wilcoxon test, with p-values indicating the levels of significance for differences. Experiments were performed in four technical replicates for each of the two independently conducted experiments. (d–f) Pearson’s correlation analysis was performed to compare the median effect of ibrutinib with MK886 (d), ibrutinib with zileuton (e), and MK886 with zileuton (f) on the adhesion levels. The Pearson correlation coefficient (r) represents the strength of the linear relationship between the effects of the treatments. Full article ">Figure 3
The mutation status of CLL samples is associated with the level of their adhesion to stromal cells in the absence and presence of small-molecule inhibitors (a) PCA clustering was performed using both experimental data (adhesion values calculated as the ratio of CLL cells to stromal cells in the treated and untreated samples) and mutation status (IGHV mutation status, del (17p), del (13q;14), del (11q), and Trisomy 12), as shown in <a href="#ijms-26-02209-t001" class="html-table">Table 1</a>. (b) A cross-correlation analysis was performed to evaluate the level of association between the studied variables and the capacity of lymphoma cells to adhere to stromal cells without added drugs and in the presence of 0.5 µM ibrutinib, 1 µM MK886, and 1 µM zileuton. In the heatmap, blue indicates a positive correlation, while red represents a negative correlation. Full article ">

12 pages, 4365 KiB

Open AccessArticle

Increased VA-ECMO Pump Speed Reduces Left Atrial Pressure: Insights from a Novel Biventricular Heart Model

by Anirudhan Kasavaraj, Christian Said, Laurence Antony Boss, Gabriel Matus Vazquez, Michael Stevens, Jacky Jiang, Audrey Adji, Christopher Hayward and Pankaj Jain

Bioengineering 2025, 12(3), 237; https://doi.org/10.3390/bioengineering12030237 - 26 Feb 2025

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Abstract

Background and aims: The effect of veno-arterial extracorporeal membrane oxygenation (VA-ECMO) on left atrial pressure (LAP) in the presence of interventricular interaction and the Frank–Starling mechanism is unknown. We developed and validated a mock circulatory loop (MCL) incorporating a novel, 3D-printed biventricular heart [...] Read more.

Background and aims: The effect of veno-arterial extracorporeal membrane oxygenation (VA-ECMO) on left atrial pressure (LAP) in the presence of interventricular interaction and the Frank–Starling mechanism is unknown. We developed and validated a mock circulatory loop (MCL) incorporating a novel, 3D-printed biventricular heart model and Frank–Starling algorithm, and used this model to assess the determinants of LAP during VA-ECMO support. Methods: The MCL was designed to allow a separate ventricle or biventricular configuration, with or without an active Frank–Starling mechanism. The biventricular model with Frank–Starling mechanism was validated in terms of (1) the presence and degree of ventricular interactions; (2) its ability to simulate Frank–Starling physiology; and (3) its capacity to simulate normal and pathological cardiac states. In the separate ventricle and biventricular with Frank–Starling models, we assessed the effect on LAP of changes in mean aortic pressure (mAoP), ECMO pump speed, LV contractility and ECMO return flow direction. Results: In the biventricular configuration, clamping RA inflow decreased RAP, with a concurrent decrease in LAP, consistent with direct ventricular interaction. With a programmed Frank–Starling mechanism, decreasing RAP was associated with a significant reduction in both LV outflow and LV end-systolic pressure. In the biventricular model with a Frank–Starling algorithm, the MCL was able to reproduce pre-defined normal and pathological cardiac output, and arterial and ventricular pressures. Increasing aortic pressure caused a linear increase in LAP in the separate ventricle model, which was attenuated in the biventricular model with Frank–Starling mechanism. Increasing ECMO pump speed caused no change in LAP in the separate ventricle model (p = 0.75), but significantly decreased LAP in the biventricular model with Frank–Starling mechanism (p = 0.039), with stabilization of LAP at the highest pump speeds. Changing the direction of VA-ECMO return flow did not affect LAP in either the separate ventricle (p = 0.91) or biventricular model with Frank–Starling mechanism (p = 0.76). Conclusions: Interventricular interactions and the Frank–Starling mechanism can be simulated in a physical, biventricular MCL. In their presence, the effects of VA-ECMO on LAP are mitigated, with LAP reduction and stabilization at maximal VA-ECMO speeds. Full article

(This article belongs to the Special Issue Engineered Analysis of Tissue Mechanics and Blood Flow for Cardiovascular Host-Device Interaction)

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Figure 1
Mock circulatory loop configurations. (A): Pre-existing mock circulatory loop in a separate ventricle configuration, adapted from [<a href="#B11-bioengineering-12-00237" class="html-bibr">11</a>]. (B): Mock circulatory loop incorporating biventricular heart model. RV = right ventricle, LV = left ventricle, PVC = pulmonary venous compliance, PAC = pulmonary arterial compliance, SVC = systemic venous compliance, AoC = aortic compliance, SVR = systemic venous resistance, PVR = pulmonary vascular resistance, ECMO = extracorporeal membrane oxygenation, HVAD = HeartWare Ventricular Assist Device. Full article ">Figure 2
Three-dimensional schematics of biventricular heart box. (A): Front view, external. (B): Top view, external. (C): Front view, internal. (D): Top view, internal. Full article ">Figure 3
Interventricular interactions following clamping of right atrial inflow. (A): Separate ventricle model. (B): Biventricular model. Black and green arrows indicate clamping and unclamping of right atrial inflow, respectively. SV = separate ventricle model, BV = biventricular model, LAP = left atrial pressure, RAP = right atrial pressure. Full article ">Figure 4
Simulation of Frank–Starling mechanism in the biventricular model via clamping of RA inflow. (A): Biventricular model without programmed Frank–Starling mechanism. (B): Biventricular model with programmed Frank–Starling mechanism. RAP = right atrial pressure, LVESP = left ventricular end-systolic pressure, LV = left ventricle, BV = biventricular model without Frank–Starling mechanism, BVFS = biventricular model with Frank–Starling mechanism. Full article ">Figure 5
Determinants of left atrial pressure in separate ventricle model (left) and biventricular model with Frank–Starling mechanism (right). (A,B): Relationship between aortic pressure and LAP while holding ECMO pump speed constant. (C,D): Relationship between ECMO pump speed and LAP while holding aortic pressure constant. (E,F): Effect of changes in ECMO pump speed and LV contractility on aortic pressure and LAP. (G,H): Effect of ECMO return flow direction on LAP. mAoP = mean aortic pressure, LAP = left atrial pressure, SV = separate ventricle model, BVFS = biventricular model with Frank–Starling mechanism, ECMO = extracorporeal membrane oxygenation, LVF = left ventricular failure, RVF = right ventricular failure, BVF = biventricular failure. Full article ">Figure 6
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23 pages, 6513 KiB

Open AccessArticle

Novel Camptothecin Derivative 9c with Enhanced Antitumor Activity via NSA2-EGFR-P53 Signaling Pathway

by Fu Du, Aotong Zhang, Xin Qi, Ruijuan Yin, Tao Jiang and Jing Li

Int. J. Mol. Sci. 2025, 26(5), 1987; https://doi.org/10.3390/ijms26051987 - 25 Feb 2025

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Abstract

Therapeutic challenges persist in the management of non-small cell lung cancer (NSCLC) in oncology. Camptothecins have demonstrated as crucial agents in tumor therapy; however, their efficacy is significantly hindered by adverse effects and drug resistance. Herein, we present a novel camptothecin derivative named [...] Read more.

Therapeutic challenges persist in the management of non-small cell lung cancer (NSCLC) in oncology. Camptothecins have demonstrated as crucial agents in tumor therapy; however, their efficacy is significantly hindered by adverse effects and drug resistance. Herein, we present a novel camptothecin derivative named 9c, which exhibits impressive anti-NSCLC potency surpassing the widely recognized camptothecin analog FL118 through a novel mechanism. Our findings demonstrated that 9c effectively inhibited tumor malignancy through cell cycle arrest and apoptosis induction with the transcriptional downregulation of anti-apoptotic genes including survivin, Mcl-1, Bcl-2, and XIAP. Mechanistically, 9c induced a wild-type p53 expression by destabilizing the NSA2-EGFR axis, thus delaying the cell cycle progression and ultimately triggering apoptosis. 9c significantly inhibited the growth of the NSCLC xenograft in vivo without observed side toxicity. Importantly, it complemented the therapeutic advantages of the novel drug AMG510 for addressing KRAS-mutant NSCLC. Collectively, these findings position 9c as a promising candidate with innovative approaches to combat NSCLC. Full article

(This article belongs to the Section Bioactives and Nutraceuticals)

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11 pages, 6934 KiB

Open AccessCase Report

Calcifications of the Knee’s Medial Compartment: A Case Report and Literature Review on the Adductor Magnus Tendon as an Uncommon Location and the Role of Ultrasound-Guided Lavage

by Elena Jiménez-Herranz, Joao Vitor de Castro Fernandes, Juan José Ramos-Álvarez, Federico Del-Castillo-Díez, André Pedrinelli, Sofia Alvariza-Ciancio, Cristian Solís-Mencía and Federico Del-Castillo-González

Diagnostics 2025, 15(5), 534; https://doi.org/10.3390/diagnostics15050534 - 22 Feb 2025

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Abstract

Background: This paper examines the diverse etiologies of medial knee pain, emphasizing the prevalence of calcification-related pathologies, such as Pellegrini–Stieda Syndrome (PSS), particularly in the medial collateral ligament (MCL) and adjacent structures. Furthermore, we present a case of calcification of the distal adductor [...] Read more.

Background: This paper examines the diverse etiologies of medial knee pain, emphasizing the prevalence of calcification-related pathologies, such as Pellegrini–Stieda Syndrome (PSS), particularly in the medial collateral ligament (MCL) and adjacent structures. Furthermore, we present a case of calcification of the distal adductor magnus tendon (DAMT) insertion into the femoral condyle of the knee and describe its treatment using ultrasound-guided percutaneous lavage (UGPL). A narrative review was conducted based on a single case; it underscores the importance of accurate diagnosis using magnetic resonance imaging (MRI) to differentiate between various calcific conditions, guiding appropriate treatment strategies. Case Presentation: A 70-year-old patient presenting with severe medial knee pain, with a duration of 4 days, and functional impotence underwent X-ray, ultrasound, and magnetic resonance imaging (MRI) examinations, revealing calcification in the DAMT. Treatment consisted of UGPL. The patient’s pain level was assessed using the visual analog scale (VAS) initially and after 30 days of treatment. Upon initial assessment, the patient reported a VAS score of 9 out of 10. After 30 days of completing the treatment, the symptoms ceased. Follow-up imaging (X-ray, ultrasound, and MRI) showed only very tiny fragments of calcification remaining. Conclusions: UGPL is an effective technique for treating calcific tendinopathy of the DAMT insertion into the medial femoral condyle of the knee, offering significant pain relief and functional improvement. This case highlights the importance of considering this rare condition in the differential diagnosis of medial knee pain. Full article

(This article belongs to the Section Medical Imaging and Theranostics)

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Diagram of a coronal section of the knee showing the area of insertion of the magnus adductor tendon in the internal femoral condyle and corresponding image in MRI (arrow). Full article ">Figure 2
(a) Anteroposterior radiograph of the right knee showing calcification adjacent to the internal femoral condyle (black arrow). (b) Coronal PD-FS image of the right knee showing a hypointense calcification adjacent to the internal femoral condyle (white arrow). Full article ">Figure 3
(a) Long-axis sonogram showing an echogenic focus with posterior acoustic shadow overlying or adjacent to the internal femoral condyle (black arrow). Black Arrow = needle. (b) Hypervascularization is observed in the color Doppler scan. Full article ">Figure 4
(A) Photograph showing patient and ultrasound transducer positioning for sonographically guided percutaneous lavage of the calcified tendinopathy, “X” denotes the needle skin entry site. (B) Long-axis sonogram showing the needle located over the calcification (black and white arrow). (C) Syringe with calcium material obtained in the lavage. Full article ">Figure 5
Anteroposterior radiograph at 1-month post-UGPL. Only small residual fragments of the calcification are visible (arrow). The patient was asymptomatic (EVA 0). Full article ">Figure 6
Anteroposterior plain radiograph at 6 months post-UGPL. No calcifications are visible. Patient was asymptomatic (EVA 0). Full article ">

18 pages, 10138 KiB

Open AccessArticle

Micheliolide Alleviates Hepatic Fibrosis by Inhibiting Autophagy in Hepatic Stellate Cells via the TrxR1/2-Mediated ROS/MEK/ERK Pathway

by Yi Liu, Ling Yao, Yuanyuan Liu, Yunheng Yang, Ailing Liang, Honglin He, Yao Lei, Wenfu Cao and Zhiwei Chen

Pharmaceuticals 2025, 18(3), 287; https://doi.org/10.3390/ph18030287 - 20 Feb 2025

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Abstract

Background: Hepatic fibrosis is a major global health issue without an optimal drug treatment, highlighting the urgent need to find effective therapies. This study aimed to clarify the role and mechanism of micheliolide in treating hepatic fibrosis. Methods: The efficacy of [...] Read more.

Background: Hepatic fibrosis is a major global health issue without an optimal drug treatment, highlighting the urgent need to find effective therapies. This study aimed to clarify the role and mechanism of micheliolide in treating hepatic fibrosis. Methods: The efficacy of MCL was evaluated in a mouse model of CCl₄-induced hepatic fibrosis. LX-2 cells were subjected to MCL treatment, and subsequent changes in fibrosis markers, autophagy, and the MEK/ERK pathway were analyzed using transcriptomics and Western blotting. The interaction between MCL and TrxR1 or TrxR2 were validated using cellular thermal shift assays (CETSA) and drug affinity responsive target stability (DARTS) assays. Results: Our findings indicated that MCL significantly alleviated CCl₄-induced hepatic fibrosis, improved liver function, and downregulated the expression of fibrosis markers. Additionally, MCL significantly inhibited LX-2 cell activation by suppressing cell proliferation, extracellular matrix (ECM) production, and autophagy, while activating the MEK/ERK pathway. Moreover, MCL elevated intracellular and mitochondrial reactive oxygen species (ROS) levels, reduced mitochondrial membrane potential, and altered mitochondrial morphology. The ROS scavenger N-acetylcysteine (NAC) attenuated MCL-induced MEK/ERK pathway activation and increased collagen type I alpha 1 (COL1A1) and fibronectin (FN) expression. Further analysis confirmed that MCL directly interacts with TrxR1 and TrxR2, leading to the inhibition of their enzymatic activities and the induction of ROS generation. Ultimately, MCL attenuated the fibrotic process and autophagic flux in LX-2 cells. Conclusions: The findings of our study confirmed that MCL has the potential to alleviate hepatic fibrosis, thereby introducing a novel candidate drug and therapeutic strategy for management of this condition. Full article

(This article belongs to the Section Pharmacology)

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9 pages, 199 KiB

Open AccessCommentary

Should We Accept the Epiligament Theory About the Differences in the Healing Potential of the Medial Collateral and the Anterior Cruciate Ligament?

by Georgi P. Georgiev, Lyubomir Gaydarski and Boycho Landzhov

Biomedicines 2025, 13(2), 522; https://doi.org/10.3390/biomedicines13020522 - 19 Feb 2025

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Abstract

The epiligament (EL), described in 1990 as a connective tissue layer distinguishable from the ligament proper, has only recently gained recognition for its critical role in ligament function and repair. Previously overlooked, the EL is now understood to be a dynamic structure, particularly [...] Read more.

The epiligament (EL), described in 1990 as a connective tissue layer distinguishable from the ligament proper, has only recently gained recognition for its critical role in ligament function and repair. Previously overlooked, the EL is now understood to be a dynamic structure, particularly in the context of medial collateral ligament (MCL) healing. Rat model studies demonstrate that the EL actively contributes to ligament repair by serving as a source of cells and blood vessels, findings later corroborated in human studies. The EL’s role in spontaneous MCL healing highlights its importance, raising the question of whether differences in EL morphology and activity contribute to the poor healing capacity of the anterior cruciate ligament (ACL). Comparative studies reveal significant disparities in EL cellularity and activity between the ACL and MCL. The EL of the MCL is hypercellular, with robust expression markers like α-smooth muscle actin (α-SMA) and collagen types III and V, essential for tissue remodeling and structural integrity. Conversely, the ACL’s EL is less vascularized and exhibits weaker expression of these markers. While vascular endothelial growth factor (VEGF) promotes angiogenesis, its effectiveness is limited in the ACL due to restricted vascularization. Similarly, CD34, a progenitor cell marker, is more prominently expressed in the MCL’s EL, further supporting its superior healing potential. These findings suggest that the EL’s distinct structural and functional attributes are key determinants of ligament healing. Targeting the EL’s regenerative properties offers a promising therapeutic strategy, particularly for improving ACL repair outcomes. Further research is necessary to validate and expand these findings. Full article

(This article belongs to the Section Molecular and Translational Medicine)

27 pages, 1169 KiB

Open AccessReview

Updates on the Biological Heterogeneity of Mantle Cell Lymphoma

by Andrew Ip, Maciej Kabat, Lindsay Fogel, Hassan Alkhatatneh, Jason Voss, Amolika Gupta, Alexandra Della Pia, Lori A. Leslie, Tatyana Feldman, Maher Albitar and Andre H. Goy

Cancers 2025, 17(4), 696; https://doi.org/10.3390/cancers17040696 - 19 Feb 2025

Viewed by 478

Abstract

Advancements in mantle cell lymphoma (MCL) have illuminated the disease’s molecular diversity, leading to a wide variation in the outcomes observed in MCL. Current prognostic risk scores are continuously revised to incorporate new updates in the mechanistic or biologic understanding of MCL. Nevertheless, [...] Read more.

Advancements in mantle cell lymphoma (MCL) have illuminated the disease’s molecular diversity, leading to a wide variation in the outcomes observed in MCL. Current prognostic risk scores are continuously revised to incorporate new updates in the mechanistic or biologic understanding of MCL. Nevertheless, key high-risk features of MCL associated with rapid disease progression and poor survival, such as TP53 mutations, complex karyotypes, and blastoid or pleomorphic morphologies, remain absent from available prognostic tools. The greater accessibility of genomic technologies, such as next-generation sequencing (NGS), has enabled clinicians to identify specific genetic alterations that serve as prognostic signals and disease monitoring parameters, cultivating accurate risk profiling that is illustrative of MCL heterogeneity. Through an increased understanding of distinct MCL behaviors, novel therapies that mechanistically target disease biology, including Bruton’s tyrosine kinase inhibitors, BCL-2 inhibitors, ROR1 inhibitors, and bispecific T-cell engagers, have broadened the treatment armamentarium for relapsed/refractory MCL cases. These interventions, in addition to chemoimmunotherapy and autologous stem cell transplantation mainstays, confer the individualization of treatment and improved survival outcomes. Further exploration of the considerable biological heterogeneity of MCL can enhance knowledge, management, and the treatment of this rare lymphoma subtype. Full article

(This article belongs to the Special Issue Mantle Cell Lymphoma: From Biology to Therapy)

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14 pages, 3219 KiB

Open AccessArticle

Vibrio sp. and Identification of the ctx Gene of Cholera Toxin in the Mandinga Coastal Lagoon, Veracruz, Mexico

by María del Refugio Castañeda-Chávez, Rosa Elena Aguilar-Muslera, Christian Reyes-Velázquez, Fabiola Lango-Reynoso, Rosa Elena Zamudio-Alemán and Magnolia Gricel Salcedo-Garduño

Microorganisms 2025, 13(2), 352; https://doi.org/10.3390/microorganisms13020352 - 6 Feb 2025

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Abstract

Coastal lagoons have undergone changes due to anthropogenic activities, the presence of wastewater discharges, and unsustainable practices that alter water quality, favoring the presence of pathogenic microorganisms such as Vibrio. This study identified the presence of the genes for zinc metalloproteinase (HA) [...] Read more.

Coastal lagoons have undergone changes due to anthropogenic activities, the presence of wastewater discharges, and unsustainable practices that alter water quality, favoring the presence of pathogenic microorganisms such as Vibrio. This study identified the presence of the genes for zinc metalloproteinase (HA) Vibrio sp. and choleric toxin (ctx) Vibrio cholerae, associated with the sources of contamination in the Mandinga Coastal Lagoon (MCL). During 2017, samplings were carried out in which sources of contamination associated with anthropogenic activities were identified. At the same time, water samples were collected from which DNA was extracted and the presence/absence of the HA and ctx genes was detected with a PCR analysis. The HA gene was identified in the three seasons of the year, while the ctx gene was only present in the dry and rainy seasons. The prevalence of both genes in the study area was independent of the presence of the pollution sources identified in the area. The absence of the ctx gene during the northern season is associated with the variability of the physicochemical parameters typical of the season. Full article

(This article belongs to the Section Molecular Microbiology and Immunology)

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17 pages, 3747 KiB

Open AccessArticle

The Association of Genetic Variants Within the Type XII Collagen and Tenascin C Genes with Knee Joint Laxity Measurements

by Samantha Beckley, Roopam Dey, Shaun Stinton, Willem van der Merwe, Thomas Branch, Alison V. September, Michael Posthumus and Malcolm Collins

Genes 2025, 16(2), 164; https://doi.org/10.3390/genes16020164 - 27 Jan 2025

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Abstract

Background/Objectives: Types I, V, and XI collagen gene variants have been reported to associate with measurements of knee joint laxity and/or absolute knee ligament length changes. Type XII collagen and tenascin C are also ligament structural proteins whose expression is regulated by mechanical [...] Read more.

Background/Objectives: Types I, V, and XI collagen gene variants have been reported to associate with measurements of knee joint laxity and/or absolute knee ligament length changes. Type XII collagen and tenascin C are also ligament structural proteins whose expression is regulated by mechanical loading. This study investigated whether COL12A1 and TNC variants are associated with knee laxity and/or ligament length changes. Methods: Genu recurvatum, anterior–posterior tibial translation, external–internal tibial rotation, and ligament length changes were measured in 128 healthy participants. They were genotyped for COL12A1 (rs970547) and TNC (rs1061494, rs2104772, rs1138545). Results: Both the COL12A1 AA and TNC rs1061494 TT genotypes were associated with decreased external (p = 0.007, p = 0.010) and internal (p = 0.025, p = 0.002) rotation, as well as slack (p = 0.033, p = 0.014), in the dominant leg. Both genotypes, together with sex, weight, and/or COL1A1 genotypes, explained 26% and 32% of the variance in external and internal rotation, respectively. The TNC genotype, sex, and BMI explained 23% of the variance in slack. The COL12A1 AA and the TNC rs1061494 TT genotypes were associated with smaller changes in the MCL (aMCL: COL12A1 p = 0.009, TNC p = 0.045; iMCL: COL12A1 p = 0.004, TNC p = 0.043; pMCL: COL12A1 p = 0.003, TNC p = 0.067; aDMCL: COL12A1 p = 0.007, TNC p = 0.020; pDMCL: COL12A1 p = 0.007, TNC p = 0.023) and/or LCL (COL12A1 p = 0.652, TNC p = 0.049) lengths within the dominant knee. The TNC rs1061494 CC genotype was associated with larger changes in the non-dominant anterior (p = 0.021) and posterior (p < 0.001) ACL bundle lengths. Conclusions: These findings suggest that COL12A1 and TNC variants are associated with internal–external tibial rotation and knee ligament length changes in healthy individuals. Full article

(This article belongs to the Section Molecular Genetics and Genomics)

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The COL12A1 rs970547 (A/G) genotype effects on the external and internal tibial rotation, as well as slack, measured using the robotic knee testing (RKT) device of participants uninjured dominant (a–c) and non-dominant (d–f) legs. Expcet for internal rotation of the non-dominant knee (e), which is expressed as median (IQR), all other measurements are expressed as average ± standard deviation. The rare GG (dominant leg n = 4, non-dominant leg n = 5) genotype and the GA genotype were combined for the analysis. The individual values are shown as light grey circles, and the number of participants in each group is indicated. Unable to genotype 1 participant for rs970547. Significantly different p values are indicated in bold. Full article ">Figure 2
The TNC rs1061494 (T/C) genotype effects on the external and internal tibial rotation, as well as slack, measured using the robotic knee testing (RKT) device of participants uninjured dominant (a–c) and non-dominant (d–f) legs. Expcet for external rotation of the non-dominant knee (d), which is expressed as average ± standard deviation, all other measurements are expressed as median (IQR). The individual values are shown as light grey circles, and the number of participants in each group is indicated. Significantly different p values are indicated in bold with post-hoc analysis differences indicated with asterisks. * p < 0.05 and ** p < 0.001. Full article ">Figure 3
The COL12A1 rs970547 (A/G) genotype effects on the absolute change in the calculated length of the (a,f) anterior bundle of the superficial layer of the MCL (aMCL), (b,g) inferior bundle of the superficial layer of the MCL (iMCL), (c,h) posterior bundle of the superficial layer of the MCL (pMCL), (d,i) anterior bundle of the deep layer of the MCL (aDMCL), and (e,j) posterior bundle of the deep layer of the MCL (pDMCL) during internal to external tibial rotation of the uninjured dominant (a–e) and non-dominant (f–j) legs. The dominant and non-dominant leg ligament length changes are expressed as average ± standard deviation and median (IQR) respectively. The rare GG (dominant leg n = 4, non-dominant leg n = 5) genotype and the GA genotype were combined for the analysis. The individual values are shown as light grey circles, and the number of participants in each group is indicated. Significantly different p values are indicated in bold. Full article ">Figure 4
The TNC rs1061494 (T/C) genotype effects on the absolute change in the calculated length of the (a,c) anterior bundle of the ACL (aACL) and (b,d) posterior bundle of the ACL (pACL) during internal to external tibial rotation of the uninjured (a,b) dominant and (c,d) non-dominant legs. The aACL length changes are expressed as median (IQR), while the pACL length changes are expressed as average ± standard deviation. The individual values are shown as light grey circles, and the number of participants in each group is indicated. Significantly different p values are indicated in bold with post-hoc analysis differences indicated with asterisks. * p < 0.05, ** p < 0.001, and *** p < 0.0001. Full article ">Figure 5
The TNC rs1061494 (T/C) genotype effects on the (a) anterior and (b) posterior ACL bundles lengths within the non-dominant leg from maximum (Max) external (ER) to maximum internal (IR) rotation. ACL bundle lengths were calculated at every 0.02 Nm increment in applied torque during each participant’s knee rotational angles from maximum external (−5 Nm torque) to maximum internal (5 Nm torque). The average (solid line) ligament length and standard deviations (shaded area) for the TNC rs1061494 CC genotype (CC) and combined TC and TT genotypes (T) are shown in blue and red, respectively, with areas of overlap represented in purple. The regions of significant differences in ligament bundle length are shaded. Full article ">

12 pages, 866 KiB

Open AccessArticle

Safety and Outcomes of Percutaneous Dilatational Tracheostomy in Patients with Hematologic Malignancies: A Retrospective Cohort Study

by Asaf Miller, Roee Noy, Omri Simchon, Natalia Gvozdev, Yotam Shkedy and Danny Epstein

J. Clin. Med. 2025, 14(2), 657; https://doi.org/10.3390/jcm14020657 - 20 Jan 2025

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Abstract

Background/Objectives: Patients with hematologic malignancy (HM) often experience high rates of thrombocytopenia, thrombocytopathy, anemia, leukopenia, and coagulopathy, which can significantly increase the risk of procedural and postoperative complications. This study aimed to evaluate the safety and outcomes of percutaneous dilatational tracheostomy (PDT) [...] Read more.

Background/Objectives: Patients with hematologic malignancy (HM) often experience high rates of thrombocytopenia, thrombocytopathy, anemia, leukopenia, and coagulopathy, which can significantly increase the risk of procedural and postoperative complications. This study aimed to evaluate the safety and outcomes of percutaneous dilatational tracheostomy (PDT) in critically ill patients with HM. Methods: This retrospective cohort study included patients with HM who underwent PDT between 2012 and 2023 at a tertiary academic center. The primary outcome was early (7-day) bleeding complications rate. Secondary outcomes included PDT-related mortality, and mortality at 1 week, 30 days, and 1 year. Analyses were performed using a propensity-matched cohort to ensure balanced comparisons between groups. Results: Of the 1627 patients included in the analysis, 65 (4%) had HM. Patients with HM had a significantly higher Charlson comorbidity index and exhibited significantly higher rates of thrombocytopenia (platelet count < 100,000/mcL) compared to those without HM (8.0 [IQR 5.0–11.3] vs. 5.0 [IQR 2.0–7.0], p < 0.001; and 49.2% vs. 5.0%, p < 0.001, respectively). After propensity score matching, the one-week mortality rate was significantly higher in the HM group (23.4% vs. 4.3%, p = 0.007). However, the rates of intraoperative and bleeding complications as well as one-year mortality rates were similar between the groups. Conclusions: PDT can be safely performed in critically ill patients with HM. However, these patients exhibit high early mortality rates following the procedure. Full article

(This article belongs to the Section Hematology)

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18 pages, 5240 KiB

Open AccessArticle

Assessing the Efficacy of Mitochondria-Accumulating Self-Assembly Peptides in Pancreatic Cancer: An Animal Study

by Ho Joong Choi, Seongeon Jin, Junghyun Park, Dosang Lee, Hee Jeong Jeong, Ok-Hee Kim, Ja-Hyoung Ryu and Say-June Kim

Int. J. Mol. Sci. 2025, 26(2), 784; https://doi.org/10.3390/ijms26020784 - 17 Jan 2025

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Abstract

Although pancreatic cancer presents with one of the most unfavorable prognoses, its treatment options are very limited. Mitochondria-targeting moieties, considered a new and prominent treatment modality, are expected to demonstrate synergistic anticancer effects due to their distinct mechanism compared to conventional chemotherapeutic approaches. [...] Read more.

Although pancreatic cancer presents with one of the most unfavorable prognoses, its treatment options are very limited. Mitochondria-targeting moieties, considered a new and prominent treatment modality, are expected to demonstrate synergistic anticancer effects due to their distinct mechanism compared to conventional chemotherapeutic approaches. This study evaluated the therapeutic potential of mitochondria-accumulating self-assembly peptides, referred to as Mito-FFs, utilizing both in vitro and in vivo pancreatic cancer models. Cellular viability assays revealed a concentration-dependent decrease in the survival of MIA-PACA2 pancreatic cancer cells upon exposure to Mito-FF treatment (p < 0.05). Subsequent in vitro Mito-FF treatments prompted the use of several molecular analyses, including Real-time PCR, Western blot analysis, and MitoSOX staining, which collectively indicated an upsurge in apoptosis, a concurrent reduction in the antioxidant enzyme expression, and an elevation in mitochondrial ROS levels (p < 0.05). In a murine xenograft model of pancreatic cancer, the intravenous administration of Mito-FF yielded a notable reduction in the tumor volume. Moreover, it upregulated the expression of pro-apoptotic markers, such as cleaved PARP and c-caspase 3, while concurrently downregulating the expression of an anti-apoptotic marker, MCL-1, as evidenced by both Western blot analysis and immunohistochemical staining (p < 0.05). It also resulted in the reduced expression of antioxidant enzymes like HO-1, catalase, and SOD2 within excised tumor tissues, as confirmed using Western blot analysis (p < 0.05). Cumulatively, the findings underscore the significant anticancer efficacy of Mito-FF against pancreatic cancer cells, predominantly mediated through the induction of apoptosis, suppression of antioxidant enzyme expression, and enhancement of mitochondrial ROS levels within the tumor microenvironment. Full article

(This article belongs to the Special Issue Mitochondrial Function in Health and Diseases)

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The effects of Mito-FF on MIA-PACA2 pancreatic cancer cells. (A) The Mito-FF molecular structure. Mito-FF, a mitochondria-accumulating self-assembly peptide, is composed of three essential components: diphenylalanine, triphenylphosphonium (TPP), and pyrene, which functions as a fluorophore. (B) MTT assay results demonstrating the effect of various concentrations of Mito-FF on the viability of human pancreatic stellate cells (PSCs). (C) A Real-time PCR analysis depicting the expression levels of pro-apoptotic and anti-apoptotic markers in PSCs treated with Mito-FF. The results indicate no significant changes in the marker expression, suggesting a lack of apoptotic induction by Mito-FF at the concentrations tested. (D) The impact of Mito-FF on the cell viability. Cell viability assays demonstrated a concentration-dependent reduction in the MIA-PACA2 pancreatic cancer cell viability following 48 h of Mito-FF treatment (p < 0.05). (E) The colony assay. Representative images show the effects of Mito-FF (0.25 to 5 μM) on the colony-forming ability of MIA-PACA2 cells after 24 h of treatment. The quantification of the colony formation (% of control) demonstrates a significant concentration-dependent reduction in colonies at all tested concentrations compared to the control (p < 0.05). The values are presented as the mean ± the standard deviation of three independent experiments. (F) Fluorescence imaging of the Mito-FF uptake in MIA-PACA2 cells. Images showing the intracellular uptake of Mito-FF by tumor cells, with fluorescent tagging facilitating visualization. Control cells (Ctrls) showed minimal fluorescence, while cells treated with 50 µM and 100 µM Mito-FF exhibited an increased fluorescence intensity, indicative of Mito-FF uptake. The values are presented as the mean ± the standard deviation of three independent experiments. Statistical significance is indicated as follows: * denotes p < 0.05 compared to the control (untreated) group after 24 h of treatment at each Mito-FF concentration. + denotes p < 0.05 compared to the control (untreated) group after 48 h of treatment at each Mito-FF concentration. Full article ">Figure 2
Effects of Mito-FF on apoptosis in MIA-PACA2 pancreatic cancer cells. (A) Real-time PCR analysis showing concentration-dependent changes in mRNA expression of apoptosis-related markers Bax and Mcl-1 in MIA-PACA2 cells treated with Mito-FF. (B) Western blot analysis demonstrating concentration-dependent alterations in markers related to apoptosis (cleaved PARP, PUMA, Bim, and Bax) and those associated with anti-apoptosis (Mcl-1, Bcl-xL, and Bcl-2) in response to Mito-FF treatment. Relative densities of individual markers were quantified using Image J software (<a href="https://imagej.net/ij/download.html" target="_blank">https://imagej.net/ij/download.html</a>, accessed on 14 January 2025). and then were normalized to that of β-actin in each group. (C) Effects of Mito-FF on apoptosis of MIA-PACA2 pancreatic cancer cells determined using annexin V/PI staining and flow cytometry. Apoptotic cells were expressed as total percentage of annexin V-positive/PI-negative cells. Number of annexin V-positive cells (early- and late-apoptotic cells) was dose-dependently increased according to increasing concentration of Mito-FF (ranging from 0.25 to 5 μM). Data acquired through flow cytometric analysis were processed and interpreted using Attune NxT Flow Cytometer Software v3.1.2. Values are presented as mean ± standard deviation of three independent experiments. Note: * p < 0.05. Full article ">Figure 3
Effects of Mito-FF on oxidative stress in MIA-PACA2 pancreatic cancer cells. (A) Real-time PCR analysis of antioxidant enzymes. Treatment of MIA-PACA2 cells with Mito-FF led to concentration-dependent reduction in mRNA expression of antioxidant enzymes, including SOD2 and GPx. (B) Western blot analysis and quantification of NRF2, catalase, SOD2, and GPx levels in MIA-PACA2 cells treated with increasing concentrations of Mito-FF (0 to 5 μM). Quantification was performed by normalizing intensity of each band to β-actin, with control (Ct) corresponding to 0 μM Mito-FF treatment. Values represent mean ± standard deviation from three independent experiments. * denotes p < 0.05 compared to control group. (C) Quantification of MitoSOX red fluorescence. Mito-FF treatment induced concentration-dependent elevation in mitochondrial ROS levels in MIA-PACA2 cells, as evidenced by quantifying MitoSOX red fluorescence. Percentages of immunoreactive areas were measured using NIH image J (<a href="https://imagej.net/ij/download.html" target="_blank">https://imagej.net/ij/download.html</a>, accessed on 14 January 2025). and expressed as relative values to those in control tissues. Values are presented as mean ± standard deviation of three independent experiments. Note: * p < 0.05. Full article ">Figure 4
The anticancer effects of Mito-FF in a xenograft model of pancreatic cancer. (A) Representative images depicting BALB/c nude mice with established pancreatic cancer xenografts, captured on the 35th day following the initial administration of the experimental treatment. The control group received no Mito-FF treatment, while the Mito-FF group received regular tail vein injections. (B) Images of tumors excised from both control and Mito-FF-treated mice, highlighting the difference in the tumor size. (C) A graph illustrating the change in the tumor volume over 35 days. The Mito-FF-treated group showed a significant reduction in the tumor size compared to the control group starting from the 25th day (p < 0.05). (D) A graph comparing the body weight of mice in the control and Mito-FF groups, indicating no significant weight difference between the groups. (E) A Western blot analysis of apoptosis markers in the excised tumor tissues. The Mito-FF group showed an increase in the pro-apoptotic marker cleaved PARP and a decrease in the anti-apoptotic marker Mcl-1 compared to the control group (p < 0.05). (F) A Western blot analysis of antioxidant enzymes in the excised tumor tissues. The Mito-FF group showed a significant reduction in the expression of antioxidant enzymes (HO-1, SOD2, catalase) compared to the control group (p < 0.05). The relative densities of individual markers were quantified using Image J software (<a href="https://imagej.net/ij/download.html" target="_blank">https://imagej.net/ij/download.html</a>, accessed on 14 January 2025) and then were normalized to that of β-actin in each group. The values are presented as the mean ± the standard deviation of three independent experiments. Note: * p < 0.05. Full article ">Figure 5
A histological analysis of Mito-FF’s impact on pancreatic cancer xenografts in mice. (A) Images showing Hematoxylin and Eosin (H&E)-stained sections of tumor tissues from the xenograft model. The Mito-FF-treated group exhibited a notably reduced tumor cell density compared to the control group. (B) An immunohistochemical analysis of apoptotic markers. Staining for c-caspase 3 (Top) and cleaved PARP (Middle) demonstrated a significant augmentation in the immunoreactive regions within the Mito-FF treatment group, indicative of enhanced apoptotic activity. Conversely, staining for the anti-apoptotic marker Mcl-1 (Bottom) revealed a pronounced decrease in immunoreactive areas in the same treatment group, suggesting an amplification of anti-apoptotic processes. The values are presented as the mean ± the standard deviation of three independent experiments. The percentages of immunoreactive areas were measured using NIH image J (<a href="https://imagej.net/ij/download.html" target="_blank">https://imagej.net/ij/download.html</a>, accessed on 14 January 2025) and expressed as relative values to those in control tissues. Note: * p < 0.05. Full article ">Figure 6
A possible mechanism by which Mito-FF leads to cell apoptosis through oxidative stress. The diagram depicts the proposed mechanism by which Mito-FF induces apoptosis in cancer cells through oxidative stress. The process begins with the mitochondrial targeting of Mito-FF, driven by its TPP moiety and the mitochondrial membrane potential. Once localized in the mitochondria, Mito-FF reaches the critical aggregation concentration (CAC), initiating self-assembly into nanostructures that disrupt the mitochondrial membrane integrity. This disruption impairs the electron transport chain (ETC), leading to excessive ROS production. Elevated ROS levels result in the depletion of antioxidant enzymes and cause oxidative damage to mitochondrial DNA (mtDNA) and proteins, exacerbating the mitochondrial dysfunction. As the mitochondrial damage progresses, the membrane permeability increases, allowing for the release of pro-apoptotic factors like cytochrome c. When cytochrome c is released from mitochondria, it activates the intrinsic apoptotic pathway, leading to caspase activation and apoptosis. Abbreviations: CAC, critical aggregation concentration; ETC, electron transport chain; MT, mitochondria; mtDNA, mitochondrial DNA; TPP, triphenylphosphonium. Full article ">

16 pages, 1055 KiB

Open AccessArticle

The Effects of the Mamanet Cachibol League Intervention Program on Perceived Health Status, Mental Health, and Healthy Lifestyle Among Arab Women

by Karin Eines, Riki Tesler, Ruth Birk, Ariela Giladi, Ayelet Dunsky, Nada Alian, Limor Gonen and Sharon Barak

Healthcare 2025, 13(2), 169; https://doi.org/10.3390/healthcare13020169 - 16 Jan 2025

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Abstract

Background: The Israeli Mamanet Cachibol League (MCL) is a community-oriented athletic program serving mothers through non-competitive recreational sports participation. This study aimed to assess the effects of the MCL on perceived health status, mental health (happiness, depression, social capital), and healthy lifestyle behaviors [...] Read more.

Background: The Israeli Mamanet Cachibol League (MCL) is a community-oriented athletic program serving mothers through non-competitive recreational sports participation. This study aimed to assess the effects of the MCL on perceived health status, mental health (happiness, depression, social capital), and healthy lifestyle behaviors (physical activity and nutrition). Methods: This is an experimental study with a sample of 231 women (174 in the experimental group and 57 in the control). Participants completed questionnaires in November 2023 (T1) and then in August 2024 (T2). The questionnaire included questions on sociodemographic characteristics, perceived health status, mental health (happiness, depression, social capital), and healthy lifestyle behaviors (physical activity and nutrition). Results: At T1, the MCL participants reported better mental health (higher subjective happiness and social capital and lower depressive symptoms) than the control group. Over time, participation in the MCL led to significant improvements in mental health (reductions in depression and increases in subjective happiness and social capital). The participants showed substantial improvements in healthy lifestyle behaviors, with moderate effect sizes (effects size > 0.5) observed across these areas. Sociodemographic factors influenced the outcomes, with variations in health perception and physical activity linked to marital status and education level. Conclusions: Participation in the MCL program was associated with better mental health at baseline and significantly improved over time compared to the control group. The MCL participants also showed gains in healthy lifestyle behaviors, highlighting the importance of tailored interventions. Full article

(This article belongs to the Special Issue Women’s Health Care: State of the Art and New Challenges)

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16 pages, 2571 KiB

Open AccessArticle

Sodium Butyrate: A Multifaceted Modulator in Colorectal Cancer Therapy

by Alexandra Laura Mederle, Alexandra Semenescu, George Andrei Drăghici, Cristina Adriana Dehelean, Nicolae-Valentin Vlăduț and Dragoş Vasile Nica

Medicina 2025, 61(1), 136; https://doi.org/10.3390/medicina61010136 - 15 Jan 2025

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Abstract

Background and Objectives: Sodium butyrate (NaB) is a potent modulator of cancer-related gene networks. However, its precise mechanisms of action and effects at elevated doses remain insufficiently explored. This study investigated the impact of NaB at physiologically relevant doses on key cellular [...] Read more.

Background and Objectives: Sodium butyrate (NaB) is a potent modulator of cancer-related gene networks. However, its precise mechanisms of action and effects at elevated doses remain insufficiently explored. This study investigated the impact of NaB at physiologically relevant doses on key cellular metrics (viability, confluence, cell number, morphology, nuclear integrity) and a comprehensive set of apoptosis and proliferation regulators (including underexplored genes) in colorectal cancer (CRC) cells. Materials and Methods: Human HCT-116 cells were treated with increasing NaB concentrations (0–20 mM). Cell viability, confluence, number, morphology, and nuclear integrity were assessed using MTT and imaging assays. RT-PCR was used to determine changes in the expression of critical pro-apoptotic players (BAX, CASP3, PUMA, TP53), anti-apoptotic facilitators (BCL-2, MCL-1), cell division regulators (PCNA, Ki-67, CDKN1), and inflammation genes (NF-κB). Results: This study provides the first exploration of MCL-1 and PCNA modulation by NaB in the context of CRC and HCT-116 cells, offering significant translational insights. All treatments reduced cell viability, confluence, and number in a dose-dependent manner (p < 0.0001). Gene expression revealed dose-related increases in most pro-apoptotic markers (BAX, CASP3, PUMA; p < 0.001), and decreases for the other genes (p < 0.001). BAX emerged as the most responsive gene to NaB, while TP53 showed minimal sensitivity, supporting NaB’s effectiveness in p53-compromised phenotypes. Nuclear condensation and fragmentation at higher NaB doses confirmed apoptotic induction. Conclusions: NaB can modulate critical apoptotic and cell cycle genes, disrupt tumor cell proliferation, and overcome resistance mechanisms associated with anti-apoptotic regulators such as MCL-1. By targeting both short-term and long-term anti-apoptotic defenses, NaB shows promise as a preventive and therapeutic agent in CRC, particularly in high-risk phenotypes with compromised p53 functionality. These findings support its potential for integration into combination therapies or dietary interventions aimed at enhancing colonic butyrate levels. Full article

(This article belongs to the Special Issue Intestinal Microbiome in Human Health and Disease—Recent Updates)

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(a) The viability of HCT-116 cells at 24 h post-treatment. The data were obtained via the MTT assay and are given as average percentage (normalized to control cells) with one standard deviation. Marked bars (*) indicate significant differences compared to controls (Dunnet’s tests, ****—p < 0.0001, ***—p < 0.001, **—p < 0.01, *—p < 0.05). (b) The corresponding dose–response curve fitted with the 4PL regression model. The sigmoidal curve indicates a gradual viability reduction, with an indicative IC50 of ≈26.85 mM. Error bars represent standard deviations, and the fitted curve reflects the overall trend. Full article ">Figure 2
The measured values for (a) the cell confluence area and (b) the cell number at 24 h post-treatment. The data are given as average percentage (normalized to control cells) with one standard deviation. Marked bars (*) indicate significant differences compared to controls (Dunnet’s tests, ****—p < 0.0001, ***—p < 0.001, **—p < 0.01, *—p < 0.05). Full article ">Figure 3
The morphological aspect of the colon cancer cell line HCT-116 after 24 h of treatment with NaB was observed under 20× magnification. Control,cells exhibit (top left corner) normal morphology, with intact cell membranes and uniform density across the field of view. At 5 mM (top right corner), mild alterations and reduced density were noted. At 10 mM (bottom left corner), cell shrinkage and apoptotic bodies became evident. Higher doses of 15 mM (middle bottom) and 20 mM (bottom right corner) led to severe cell fragmentation, condensed nuclei, and significant density reduction, indicating apoptosis. Scale bars represent 200 µm. Full article ">Figure 4
Morphological changes in nuclei after 24 h of NaB treatment were observed under 20× magnification. Control nuclei were intact (top left corner), while 10% of cells at 5 mM (top right corner) showed mild condensation. At 10 mM (bottom left corner), 25% displayed condensation and fragmentation, increasing to about 50% at 15 mM and 70% at 20 mM ((middle bottom) and (bottom right corner), respectively), where extensive nuclear disintegration was observed. Yellow arrows indicate apoptotic features; scale bars represent 100 µm. Full article ">

22 pages, 7210 KiB

Open AccessArticle

Single Dose of Attenuated Vaccinia Viruses Expressing H5 Hemagglutinin Affords Rapid and Long-Term Protection Against Lethal Infection with Highly Pathogenic Avian Influenza A H5N1 Virus in Mice and Monkeys

by Fumihiko Yasui, Keisuke Munekata, Tomoko Fujiyuki, Takeshi Kuraishi, Kenzaburo Yamaji, Tomoko Honda, Sumiko Gomi, Misako Yoneda, Takahiro Sanada, Koji Ishii, Yoshihiro Sakoda, Hiroshi Kida, Shosaku Hattori, Chieko Kai and Michinori Kohara

Vaccines 2025, 13(1), 74; https://doi.org/10.3390/vaccines13010074 - 15 Jan 2025

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Abstract

Background/Objectives: In preparation for a potential pandemic caused by the H5N1 highly pathogenic avian influenza (HPAI) virus, pre-pandemic vaccines against several viral clades have been developed and stocked worldwide. Although these vaccines are well tolerated, their immunogenicity and cross-reactivity with viruses of different [...] Read more.

Background/Objectives: In preparation for a potential pandemic caused by the H5N1 highly pathogenic avian influenza (HPAI) virus, pre-pandemic vaccines against several viral clades have been developed and stocked worldwide. Although these vaccines are well tolerated, their immunogenicity and cross-reactivity with viruses of different clades can be improved. Methods: To address this aspect, we generated recombinant influenza vaccines against H5-subtype viruses using two different strains of highly attenuated vaccinia virus (VACV) vectors. Results: rLC16m8-mcl2.2 hemagglutinin (HA) and rLC16m8-mcl2.3.4 HA consisted of a recombinant LC16m8 vector encoding the HA protein from clade 2.2 or clade 2.3.4 viruses (respectively); rDIs-mcl2.2 HA consisted of a recombinant DIs vector encoding the HA protein from clade 2.2. A single dose of rLC16m8-mcl2.2 HA showed rapid (1 week after vaccination) and long-term protection (20 months post-vaccination) in mice against the HPAI H5N1 virus. Moreover, cynomolgus macaques immunized with rLC16m8-mcl2.2 HA exhibited long-term protection when challenged with a heterologous clade of the HPAI H5N1 virus. Although the DIs strain is unable to grow in most mammalian cells, rDIs-mcl2.2 HA also showed rapid and long-lasting effects against HPAI H5N1 virus infection. Notably, the protective efficacy of rDIs-mcl2.2 HA was comparable to that of rLC16m8-mcl2.2 HA. Furthermore, these vaccines protected animals previously immunized with VACVs from a lethal challenge with the HPAI H5N1 virus. Conclusions: These results suggest that both rLC16m8-mcl2.2 HA and rDIs-mcl2.2 HA are effective in preventing HPAI H5N1 virus infection, and rDIs-mcl2.2 HA is a promising vaccine candidate against H5 HA-subtype viruses. Full article

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