International Journal of Molecular Sciences

20 pages, 10530 KiB

Open AccessArticle

Mitochondrial Transfer from Human Platelets to Rat Dental Pulp-Derived Fibroblasts in the 2D In Vitro System: Additional Implication in PRP Therapy

by Koji Nishiyama, Tomoni Kasahara, Hideo Kawabata, Tetsuhiro Tsujino, Yutaka Kitamura, Taisuke Watanabe, Masayuki Nakamura, Tomoharu Mochizuki, Takashi Ushiki and Tomoyuki Kawase

Int. J. Mol. Sci. 2025, 26(12), 5504; https://doi.org/10.3390/ijms26125504 (registering DOI) - 8 Jun 2025

Abstract

Platelet mitochondria have recently been increasingly considered “co-principal” along with platelet growth factors to facilitate tissue regeneration in platelet-rich plasma therapy cooperatively. To develop a convenient method to test this potential, we examined mitochondrial transfer using a simple two-dimensional culture system. Living human [...] Read more.

Platelet mitochondria have recently been increasingly considered “co-principal” along with platelet growth factors to facilitate tissue regeneration in platelet-rich plasma therapy cooperatively. To develop a convenient method to test this potential, we examined mitochondrial transfer using a simple two-dimensional culture system. Living human platelets were prepared from PRP obtained from 12 non-smoking healthy male adults (age: 28–63 years) and suspended in medium. Platelet lysates were prepared from sonicated platelet suspensions in PBS. After treatment with ultraviolet-C irradiation, a mitochondrial respiration inhibitor, or a synchronized culture reagent, rat dental pulp-derived fibroblasts (RPC-C2A) were co-cultured with platelets or platelet lysates for 24 h. Mitochondrial transfer was evaluated by visualization using a fluorescent dye for mitochondria or an antibody against human mitochondria. Ultraviolet-C-irradiated cells substantially lost their viability, and treatment with living platelets, but not platelet lysates, significantly rescued the damaged fibroblasts. Fibroblast mitochondria appeared to increase after co-culture with resting platelets. Although more microparticles existed around the platelets on the fibroblast surface, the activated platelets did not show significant increases in any parameters of mitochondrial transfer. This simple co-culture system demonstrated mitochondrial transfer between xenogeneic cells, and this phenomenon should be considered as an additional implication in PRP therapy. Full article

(This article belongs to the Section Molecular Biology)

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19 pages, 1445 KiB

Open AccessReview

Understanding the Immune System’s Intricate Balance: Activation, Tolerance, and Self-Protection

by Jui-Yun Chen, Li-Jane Shih, Min-Tser Liao, Kuo-Wang Tsai, Kuo-Cheng Lu and Wan-Chung Hu

Int. J. Mol. Sci. 2025, 26(12), 5503; https://doi.org/10.3390/ijms26125503 (registering DOI) - 8 Jun 2025

Abstract

Understanding the mechanisms of immune activation and deactivation is paramount. A host must initiate effective immunity against pathogenic infections while avoiding triggering immunity against self-antigens, which can lead to detrimental autoimmune disorders. Host immunological pathways can be categorized as Immunoglobulin (Ig)G-dominant eradicable immune [...] Read more.

Understanding the mechanisms of immune activation and deactivation is paramount. A host must initiate effective immunity against pathogenic infections while avoiding triggering immunity against self-antigens, which can lead to detrimental autoimmune disorders. Host immunological pathways can be categorized as Immunoglobulin (Ig)G-dominant eradicable immune reactions and IgA-dominant tolerable immune reactions. Eradicable immune reactions include Th1, Th2, Th22, and Thαβ immune responses against four different types of pathogens. Tolerable immune reactions include Th1-like, Th9, Th17, and Th3 immune responses against four different types of pathogens. Here, we try to determine the mechanisms of activation and deactivation of host immune reactions. The spleen and liver play contrasting roles in mediating immune responses: the spleen is primarily involved in immune activation, whereas the liver is responsible for immune deactivation. Similarly, the sympathetic and parasympathetic nervous systems have opposing functions in immune modulation, with the sympathetic system promoting pro-inflammatory responses and the parasympathetic system facilitating anti-inflammatory processes. Furthermore, adrenocorticotropic hormone (ACTH) and glucocorticosteroids exhibit contrasting effects on immune regulation: ACTH is involved in activating adaptive immunity while inhibiting innate immunity, whereas glucocorticosteroids activate natural IgM antibody associated with innate immunity while inhibiting adaptive immunity. Heat shock proteins, particularly molecular chaperones induced by fever, play pivotal roles in immune activation. Conversely, IgD B cells and gamma/delta T cells contribute to immune deactivation through mechanisms such as clonal anergy. Understanding these mechanisms provides insights into immunological pathways, aiding in the better management of infectious diseases and autoimmune disorders. Full article

(This article belongs to the Special Issue The Role of Cytokines in Health and Diseases)

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19 pages, 6864 KiB

Open AccessArticle

Co-Aggregation of Syndecan-3 with β-Amyloid Aggravates Neuroinflammation and Cognitive Impairment in 5×FAD Mice

by Fan Ye, Mingfeng Li, Min Liu, Xinghan Wu, Fan Tian, Yanju Gong, Yan Cao, Jingtai Zhang, Xueling Zhang, Chuan Qin and Ling Zhang

Int. J. Mol. Sci. 2025, 26(12), 5502; https://doi.org/10.3390/ijms26125502 (registering DOI) - 8 Jun 2025

Abstract

Abnormal deposition of β-amyloid (Aβ) is a core pathological feature of Alzheimer’s disease (AD). Syndecan-3 (SDC3), a type I transmembrane heparan sulfate proteoglycan (HSPG), is abnormally overexpressed in the brains of AD patients and model animals, specifically accumulating in the peri-plaque region of [...] Read more.

Abnormal deposition of β-amyloid (Aβ) is a core pathological feature of Alzheimer’s disease (AD). Syndecan-3 (SDC3), a type I transmembrane heparan sulfate proteoglycan (HSPG), is abnormally overexpressed in the brains of AD patients and model animals, specifically accumulating in the peri-plaque region of amyloid plaques. However, its regulatory mechanism in the process of Aβ deposition remains unclear. This study aims to clearly define the role of SDC3 in Aβ aggregation and neuroinflammation, two critical processes in AD pathogenesis. Specifically, we investigate how SDC3 modulates Aβ aggregation and its interaction with neuroinflammatory pathways, which may contribute to the progression of AD. By elucidating the mechanisms underlying SDC3’s involvement in these processes, we seek to provide new insights into potential therapeutic targets for AD. In this study, a 5×FAD mouse model with downregulated SDC3 expression was constructed. Behavioral assessments and synaptic function tests were performed to explore the effects of SDC3 on cognition in 5×FAD mice. Immunofluorescence co-localization technology was utilized to analyze the pathological co-deposition of SDC3 and Aβ in the hippocampus, cortex, and meningeal blood vessels. Quantitative assessments of pro-inflammatory cytokines such as Tnf-α and Cxcl10 in the brain were performed through histopathological analysis combined with qPCR. Western blotting was used to examine the phosphorylation status of STAT1/STAT3 and the expression changes of IBA1/GFAP to systematically analyze the molecular mechanisms through which SDC3 regulates AD pathology. This study revealed that SDC3 expression was significantly upregulated in the brain regions of the 5×FAD model mice and co-localized pathologically with Aβ. Cell lineage tracing analysis showed that the elevated SDC3 expression primarily originated from glial cells. Behavioral and pathological results demonstrated that downregulation of SDC3 significantly improved cognitive dysfunction in the model mice and effectively reduced the Aβ burden in the brain. Molecular mechanism studies showed that downregulation of SDC3 reduced the phosphorylation of STAT1 and STAT3, thereby inhibiting the activation of the JAK-STAT and cGAS-STING signaling pathways, reducing the activation of microglia/astrocytes and suppressing the expression of pro-inflammatory cytokines such as Tnf-α and Cxcl10. This study reveals that SDC3 co-localizes with Aβ pathology and synergistically exacerbates neuroinflammation. Knockdown of SDC3 can simultaneously reduce both Aβ deposition and the release of inflammatory factors from glial cells. Mechanistic research indicates that SDC3 drives a “glial activation–cytokine release” vicious cycle through the JAK-STAT and cGAS-STING signaling pathways. These findings suggest that SDC3 may serve as a key hub coordinating amyloid pathology and neuroinflammation in AD, providing new insights for the development of combination therapies targeting the HSPG network. Full article

(This article belongs to the Section Molecular Neurobiology)

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12 pages, 2774 KiB

Open AccessArticle

Sodium–Glucose Cotransporter 2 and Glucose Levels Affect Clear Cell Renal Cell Carcinoma Progression

by Yujiro Nagata, Ikko Tomisaki, Hisami Aono, Nguyen Thu Quynh, Eiji Kashiwagi and Naohiro Fujimoto

Int. J. Mol. Sci. 2025, 26(12), 5501; https://doi.org/10.3390/ijms26125501 (registering DOI) - 8 Jun 2025

Abstract

The biological significance of sodium–glucose cotransporter 2 (SGLT2) in clear cell renal cell carcinoma (ccRCC) has yet to be elucidated. In this study, we aimed to determine the role of SGLT2 in ccRCC tumor progression. The human ccRCC line KMRC-1, which contains a [...] Read more.

The biological significance of sodium–glucose cotransporter 2 (SGLT2) in clear cell renal cell carcinoma (ccRCC) has yet to be elucidated. In this study, we aimed to determine the role of SGLT2 in ccRCC tumor progression. The human ccRCC line KMRC-1, which contains a von Hippel–Lindau (VHL) gene mutation, was used to assess the effects of the SGLT2 inhibitor (SGLT2i) dapagliflozin on proliferation and migration in media containing different glucose concentrations (25, 12.5, or 5 mM). Dapagliflozin significantly reduced cell proliferation and migration in 25 mM glucose medium. Similarly, SGLT2 knockdown involving short hairpin RNA lentiviral transfection significantly decreased cell viability, migration, and colony formation compared with the control subline in 25 mM glucose medium. Moreover, tumor progression was inhibited in the media with low glucose concentrations. Remarkably, 2 µM dapagliflozin inhibited the progression of ccRCC at concentrations as low as 5 mM (normoglycemic model) glucose medium as well as 25 mM (severe glycemia model) glucose medium. In addition, dapagliflozin treatment significantly enhanced the apoptosis of ccRCC cells. Our findings demonstrate that SGLT2 impacts the progression of ccRCC with the VHL mutation. In light of the above findings, SGLT2is, which exert the dual effects of SGLT2 blockade and glycemic control, may represent a novel therapeutic agent, particularly in patients with ccRCC who suffer from concurrent diabetes mellitus. To the best of our knowledge, this is the first preclinical study demonstrating the impact of SGLT2 inhibition on the progression of ccRCC with the VHL mutation. Full article

(This article belongs to the Special Issue Molecular Biology of Cancer—Implications for Diagnosis and Treatment: 3rd Edition)

14 pages, 1475 KiB

Open AccessArticle

Structural Insights into the ADCC Mechanism and Resistance of Mogamulizumab, a First-in-Class Anti-CCR4 Therapy for Cutaneous T cell Lymphoma

by Seung Beom Choi, Hyun Tae Lee, Nahyeon Gu, Yu-Jeong Jang, Ui Beom Park, Tae Jun Jeong, Sang Hyung Lee and Yong-Seok Heo

Int. J. Mol. Sci. 2025, 26(12), 5500; https://doi.org/10.3390/ijms26125500 (registering DOI) - 8 Jun 2025

Abstract

Mogamulizumab is a humanized monoclonal antibody that targets C-C chemokine receptor 4 (CCR4) present on certain T cells in lymphomas and leukemias. This antibody-based therapy has demonstrated efficacy in treating various cutaneous T cell lymphomas (CTCLs), including mycosis fungoides and Sézary syndrome, through [...] Read more.

Mogamulizumab is a humanized monoclonal antibody that targets C-C chemokine receptor 4 (CCR4) present on certain T cells in lymphomas and leukemias. This antibody-based therapy has demonstrated efficacy in treating various cutaneous T cell lymphomas (CTCLs), including mycosis fungoides and Sézary syndrome, through the depletion of CCR4-expressing T cells by antibody-dependent cellular cytotoxicity (ADCC). However, the precise epitope and binding mode of mogamulizumab responsible for its augmented ADCC activity remain undisclosed. Here, X-ray crystallographic studies of mogamulizumab in complex with a 28-residue N-terminal peptide indicated that SIYSNYYLYES (residues 14–24) would constitute the antibody epitope. Another high-resolution structure, using a short core peptide of these 11 residues, has elucidated unambiguous electron density for the bound peptide, confirming consistent binding for both peptides. This linear epitope is located in the membrane-proximal region of CCR4, facilitating the Fc-mediated effector functions, including ADCC. The structures also provide insights into the molecular basis for the resistance of the CCR4 L21V variant to mogamulizumab, which is due to a lack of structural complementarity with mogamulizumab binding. Understanding the structural basis for the mechanism of action of mogamulizumab is crucial for optimizing anti-CCR4 therapeutics to improve treatment outcomes for patients with these challenging diseases. Full article

(This article belongs to the Section Molecular Pharmacology)

12 pages, 652 KiB

Open AccessArticle

Effects of Two Hormonal Protocols for FTAI on the Fertility of Repeat Cows

by Luis Miguel Vargas Ortiz, Verónica Cristina Andrade Yucailla, Juan Ramón García Díaz, Néstor Vicente Acosta Lozano, Ramón Gonzalo Aragadvay Yungán and Raciel Lima Orozco

Int. J. Mol. Sci. 2025, 26(12), 5499; https://doi.org/10.3390/ijms26125499 (registering DOI) - 8 Jun 2025

Abstract

This study, carried out from January to July 2022 in three provinces of Ecuador, aimed to evaluate the effect of two hormonal protocols for fixed-time artificial insemination (FTAI) on the follicular dynamics, hormonal profile and fertility of dairy cows affected by repeat cow [...] Read more.

This study, carried out from January to July 2022 in three provinces of Ecuador, aimed to evaluate the effect of two hormonal protocols for fixed-time artificial insemination (FTAI) on the follicular dynamics, hormonal profile and fertility of dairy cows affected by repeat cow syndrome (RCS). Two groups of Holstein cows with RCS were formed, G1 (conventional) and G2 (J-Sinch), with 26 and 24 animals, respectively. Gynaecological examinations and hormonal determinations in blood serum were carried out. Follicular diameter and concentrations of FSH, LH and P4 were compared by t-Student test for independent samples, estrus and pregnancy were compared by binomial comparison of proportions, and factors associated with pregnancy were determined by a model of logistic regression (LR). In G1, the diameter of the dominant follicle was greater (p < 0.05) in the left ovary on day 7 following intravaginal device implantation. However, it was similar (p > 0.05) in the right ovary on days 7, 8 and 9. The estradiol and LH concentrations at the time of FTAI and the P4 concentrations 15 days after FTAI, as well as the pregnancy rate, were higher in G1 (p < 0.05). The LR model explained 60.91% of pregnancies (p < 0.001), and the concentrations of estradiol, LH and P4 and the absence of estrus at the time of FTAI had an influence on the pregnancy rate (p < 0.05). It was concluded that the inclusion of estradiol benzoate increased the dominant follicle diameter and the concentrations of estradiol, LH and P4 and the pregnancy rate at the first FTAI. Full article

(This article belongs to the Special Issue Molecular Insights into Reproductive Biology and Related Diseases)

16 pages, 1583 KiB

Open AccessArticle

Microneurotrophin BNN27 Exerts Significant Anti-Inflammatory Effects on Murine T-Lymphocytes Following CFA-Induced Inflammatory Pain

by Smaragda Poulaki, Aikaterini Kalantidou, Ioanna Lapi, Achille Gravanis and Maria Venihaki

Int. J. Mol. Sci. 2025, 26(12), 5498; https://doi.org/10.3390/ijms26125498 (registering DOI) - 8 Jun 2025

Abstract

During tissue injury or infection, leukocytes are activated to produce proinflammatory mediators, which trigger the immune system to produce anti-inflammatory and analgesic molecules. Our previous studies provide evidence that synthetic microneurotrophins, like BNN27, exert significant analgesic and anti-inflammatory effects during Complete Freund’s Adjuvant [...] Read more.

During tissue injury or infection, leukocytes are activated to produce proinflammatory mediators, which trigger the immune system to produce anti-inflammatory and analgesic molecules. Our previous studies provide evidence that synthetic microneurotrophins, like BNN27, exert significant analgesic and anti-inflammatory effects during Complete Freund’s Adjuvant (CFA)-induced inflammation and pain. Thus, the aim of the present study was to examine if the effect of BNN27 on inflammatory pain is mediated at least in part by activation of T-lymphocytes. For this purpose, six hours following the injection of CFA, spleens were harvested in PBS and lymphocytes were collected and placed in medium containing concanavalin-A and IL-2 to prompt T-lymphocyte proliferation and differentiation. Cells were then treated with BNN27 at different concentrations and the media and cells were collected for ELISA and PCR assays. The proliferation rate of T-cells was also examined using the MTT assay. Our results showed that BNN27 significantly increased the proliferation of T-lymphocytes. In addition, BNN27 significantly decreased IL-6 and TNF-α protein levels, while it increased the mRNA expression of μ-opioid receptor and opioid peptides PENK and POMC at different time points. Our data demonstrate considerable anti-inflammatory and analgesic effects of BNN27, making it a promising molecule for inflammation and pain management. Full article

(This article belongs to the Special Issue Molecular Mechanisms of Alzheimer’s Disease)

14 pages, 2285 KiB

Open AccessArticle

Transglutaminase 2 Stimulates Cell Proliferation and Modulates Transforming Growth Factor-Beta Signaling Pathway Independently of Epithelial–Mesenchymal Transition in Hepatocellular Carcinoma Cells

by Hiromi Yamaguchi, Ramiro José González-Duarte, Xian-Yang Qin, Yuriko Abe, Ichiro Takada, Benjamin Charroy, Verna Cázares-Ordoñez, Shigeyuki Uno, Makoto Makishima and Mariko Esumi

Int. J. Mol. Sci. 2025, 26(12), 5497; https://doi.org/10.3390/ijms26125497 (registering DOI) - 8 Jun 2025

Abstract

Transglutaminase 2 (TG2) is a multifunctional protein and plays a role in cancer progression. We previously identified TG2 as an early-recurrence biomarker in hepatocellular carcinoma (HCC). TG2-knockdown (shTG2) and control (shCtl) HCC cell lines were used for comparative analyses to clarify the molecular [...] Read more.

Transglutaminase 2 (TG2) is a multifunctional protein and plays a role in cancer progression. We previously identified TG2 as an early-recurrence biomarker in hepatocellular carcinoma (HCC). TG2-knockdown (shTG2) and control (shCtl) HCC cell lines were used for comparative analyses to clarify the molecular mechanisms underlying the contribution of this protein to HCC malignancy. The proliferation of shTG2 cells was slightly but significantly decreased compared with that of shCtl cells. Differential gene expression profiling based on GeneChip arrays revealed the enrichment of the PI3K-Akt signaling pathway and showed that the expression of Dickkopf-1 and -3 (DKK1 and DKK3, respectively), inhibitors and modulators of the Wnt/β-catenin signaling pathway, was increased in shTG2 cells. The expression of epithelial–mesenchymal transition (EMT)-related genes was similar in both shCtl and shTG2 cells before and after TGF-β1 treatment, even though TGF-β1 markedly upregulated TG2. Thus, TG2 may contribute to cancer malignancy via the stimulation of cell proliferation signaling, such as PI3K-Akt and Wnt/β-catenin signaling, but not EMT. This effect might be further enhanced by humoral factors such as TGF-β1 from the tumor microenvironment. Full article

(This article belongs to the Special Issue Immunomodulatory Molecules in Cancer)

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17 pages, 1675 KiB

Open AccessArticle

Gene Copy Number Dictates Extracellular Vesicle Cargo

by Sumeet Poudel, Zhiyong He, Jerilyn Izac and Lili Wang

Int. J. Mol. Sci. 2025, 26(12), 5496; https://doi.org/10.3390/ijms26125496 (registering DOI) - 8 Jun 2025

Abstract

Extracellular vesicles (EVs) are membrane-surrounded vesicles that carry heterogeneous cellular components, including proteins, nucleic acids, lipids, and metabolites. EVs’ intravesicular and surface contents possess many biomarkers of physiological and pathological importance. Because of the heterogeneous cargo, EVs can mediate local and distal cell–cell [...] Read more.

Extracellular vesicles (EVs) are membrane-surrounded vesicles that carry heterogeneous cellular components, including proteins, nucleic acids, lipids, and metabolites. EVs’ intravesicular and surface contents possess many biomarkers of physiological and pathological importance. Because of the heterogeneous cargo, EVs can mediate local and distal cell–cell communication. However, the way in which the genome signature regulates EV cargo has not been well studied. This study aimed to understand how genetics impact EV cargo loading. EVs were isolated from vector copy number cells with a fluorescent reporter (GFP) with varying inserted transgene copies and from NIST SRM 2373 cells (MDA-MB-231, MDA-MB-453, SK-BR-3, and BT-474), which contain varying copies of the HER2 gene. Spectradyne nCS1 was utilized to count EVs and measure size distribution. Imaging Flow Cytometry was used to analyze the surface protein content of single EVs and for total EV counts. The RNA content of the EVs was measured using ddPCR. Our results from stable reporter cell lines and breast cancer cell lines suggest that the gene copy number dictates the protein cargo of the EVs but not the RNA content. Increasing copies of a reporter gene (GFP) or a naturally occurring gene (HER2) from breast cancer cells correlated with increasing EV counts positive for the protein cargo compared to total EV counts until a copy threshold was reached. This study has broad implications for understanding EV biology in the context of cancer biology, diagnostics, EV biology/manufacturing, and therapeutic delivery. Full article

(This article belongs to the Section Molecular Biology)

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20 pages, 3172 KiB

Open AccessArticle

Biphasic CAPA-IVM Improves Equine Oocyte Quality and Subsequent Embryo Development Without Inducing Genetic Aberrations

by Muhammad Fakhar-I-Adil, Daniel Angel-Velez, Emin Araftpoor, Qurratul Ain Amin, Mohamed Hedia, Marcel Bühler, Kris Gevaert, Björn Menten, Ann Van Soom, Susana Marina Chuva de Sousa Lopes, Dominic Stoop, Chloë De Roo, Katrien Smits and Björn Heindryckx

Int. J. Mol. Sci. 2025, 26(12), 5495; https://doi.org/10.3390/ijms26125495 (registering DOI) - 8 Jun 2025

Abstract

In vitro maturation (IVM) of oocytes retrieved from ovum pick-up (OPU) or ovarian tissue (OT) is a standard approach for patients with specific conditions where prior hormonal stimulation is contraindicated. However, the developmental competence of oocytes matured in vitro is still inferior to [...] Read more.

In vitro maturation (IVM) of oocytes retrieved from ovum pick-up (OPU) or ovarian tissue (OT) is a standard approach for patients with specific conditions where prior hormonal stimulation is contraindicated. However, the developmental competence of oocytes matured in vitro is still inferior to that of oocytes matured in vivo. Capacitation IVM (CAPA-IVM) includes an extra step of pre-maturation culture (PMC) with c-type natriuretic peptide (CNP) as a meiotic arrestor to better synchronize cytoplasmic and nuclear maturity in oocytes by allowing the cytoplasm additional time to acquire essential components critical for optimal competency. This study aims to evaluate the effect of CAPA-IVM on equine oocyte quality and developmental competence. Immature cumulus–oocyte complexes (COCs) were retrieved from slaughterhouse ovaries and matured in vitro either in CAPA-IVM (short 6 h, long 24 h pre-maturation) or standard IVM. Mature oocytes from each group were analyzed for calcium-releasing potential (n = 52) and single-oocyte proteomics (n = 44), and embryo development (n = 229) was assessed after fertilization with piezo-drilled intracytoplasmic sperm injection (ICSI). Genetic analysis of developed blastocysts (n = 41) was performed to detect chromosomal aberrations. Our findings demonstrate that CAPA-IVM of equine COCs yields significantly higher maturation rates than controls. Moreover, short CAPA-IVM with six hours pre-maturation culture showed substantially higher embryo development potential than the control group (20/69 vs. 9/63, respectively). Genetic analysis revealed a high euploidy rate in equine blastocysts regardless of the maturation conditions. Live calcium imaging of the fertilized oocytes demonstrated that the majority of oocytes displayed non-continuous calcium oscillation patterns, irrespective of maturation conditions. Single-oocyte proteomics reveals a comparable proteomic landscape between mature oocytes subjected to short CAPA-IVM and standard IVM. However, we identified four enriched gene sets with positive enrichment scores after short CAPA-IVM, related to cytoskeleton regulation, ribosomal function, and cytosolic components. Our findings indicate that CAPA-IVM holds the potential to improve oocyte quality and competence in horses. However, further fine-tuning of culture conditions would benefit the effective use of these IVM systems. Moreover, given that the mare serves as an excellent model for human reproduction, the molecular trends identified in this study could provide valuable insights for advancing human artificial reproductive technologies. Full article

(This article belongs to the Special Issue Molecular Research on Embryo Developmental Potential)

19 pages, 811 KiB

Open AccessArticle

Multi-Cohort Exploration of Repetitive Element Transcription and DNA Methylation in Human Steatotic Liver Disease

by Neil A. Youngson, Aikaterini Tourna, Timothy Chalmers, Kelly V. Prates, Josepmaria Argemi, Ramon Bataller, Koroush S. Haghighi, Lindsay E. Wu, Shilpa Chokshi, Peter Starkel, Patrick S. Western, Margaret J. Morris and Stephen M. Riordan

Int. J. Mol. Sci. 2025, 26(12), 5494; https://doi.org/10.3390/ijms26125494 (registering DOI) - 8 Jun 2025

Abstract

Transposable elements (TEs) make up around half of the human genome. Their transcription is repressed in most somatic cells to maintain genome integrity and function. The repression is chiefly maintained by a combination of epigenetic modifications such as DNA methylation and histone modifications. [...] Read more.

Transposable elements (TEs) make up around half of the human genome. Their transcription is repressed in most somatic cells to maintain genome integrity and function. The repression is chiefly maintained by a combination of epigenetic modifications such as DNA methylation and histone modifications. However, recent research suggests that liver steatosis is associated with extensive changes to the hepatocyte epigenome. Furthermore, studies in mice have reported diet- and drug-induced changes to TE transcript levels in liver. The confirmation of these effects in human liver has not previously been undertaken. Here, we examined TE transcription in liver tissue from three patient cohorts with histologically confirmed liver steatosis caused by alcohol consumption or metabolic dysfunction. The quantitation of the number of transcripts with TE-homology in RNA-Seq data from a cohort of 90 bariatric surgery patients with metabolic dysfunction-associated steatotic liver disease (MASLD) revealed a trend for the reduction in TEs of all classes due to increasing steatosis, but no effect of fibrosis. This pattern was also present in a separate cohort of MASLD and HCC patients, as RT-qPCR also showed a reduction in Alu element transcripts in advanced steatosis, but again, no effect of fibrosis. Contrastingly, in a cohort of alcohol-related liver disease patients, the reduction in LINE-1 transcripts was associated with either increased steatosis or increased fibrosis. Moreover, the examination of LINE-1 DNA methylation levels in the MASLD and HCC cohort indicated that DNA methylation was also negatively associated with LINE-1 transcription in MASLD. This study suggests that TE transcript levels in human liver are slightly reduced by steatosis, that DNA methylation is an influential epigenetic regulator of LINE-1 retrotransposon transcription in steatosis, and that Alu transcript levels in background liver could be a new biomarker for HCC in cirrhotic and non-cirrhotic MASLD. Full article

(This article belongs to the Special Issue Targeting Epigenetic Network in Cancer)

20 pages, 1477 KiB

Open AccessArticle

Anticancer Potential of Halogen Derivatives of Methyl 6-Acetyl-5-Hydroxy-2-Methyl-1-Benzofuran-3-Carboxylate

by Mariola Napiórkowska, Emilia Grosicka-Maciąg, Piotr Podsadni and Dagmara Otto-Ślusarczyk

Int. J. Mol. Sci. 2025, 26(12), 5493; https://doi.org/10.3390/ijms26125493 (registering DOI) - 8 Jun 2025

Abstract

The presented results are a continuation of our research on the synthesis and biological properties of halogen benzofuran derivatives, particularly their anticancer potential. We examined the cytotoxicity of two derivatives, methyl 4-chloro-6-(dichloroacetyl)-5-hydroxy-2-methyl-1-benzofuran-3-carboxylate (7) and methyl 6-(dibromoacetyl)-5-methoxy-2-methyl-1-benzofuran-3-carboxylate (8), in the [...] Read more.

The presented results are a continuation of our research on the synthesis and biological properties of halogen benzofuran derivatives, particularly their anticancer potential. We examined the cytotoxicity of two derivatives, methyl 4-chloro-6-(dichloroacetyl)-5-hydroxy-2-methyl-1-benzofuran-3-carboxylate (7) and methyl 6-(dibromoacetyl)-5-methoxy-2-methyl-1-benzofuran-3-carboxylate (8), in the following human cancer cell lines: SW480, SW620, HCT116, HepG2, PC3, A549, and MDA. The MTT assay results showed that compound 7 exhibited the most promising activity against A549 cells, while compound 8 demonstrated significant activity against both A549 cells and HepG2 cells. The biological activity of these compounds was evaluated by the trypan blue assay, reactive oxygen species generation, lipid peroxidation and IL-6 secretion. To investigate the proapoptotic activity of these compounds, the two following types of tests were performed: Annexin V Apoptosis Detection Kit I and Caspase-Glo 3/7 assay. Moreover, we checked the effect of both tested derivatives on the cell cycle and tubulin polymerization. The obtained results revealed that the presence of bromine and methoxy group in the structure has an influence on the biological properties of compound 8. This derivative exhibited stronger pro-oxidative effects and proapoptotic properties compared to those observed for derivative 7. Both compounds decreased IL 6 secretion in the tested cancer cell lines; however, the stronger effect was observed for HepG2 cells. Analysis of the cell cycle in the presence of the tested compounds revealed that compound 7 induced G2/M phase arrest in HepG2 cells, while compound 8 caused cell cycle arrest at the S and G2/M phases in A549 cells. On the other hand, both derivatives had a minimal effect on tubulin polymerization. These findings suggest that compounds 7 and 8 could serve as starting points for further development of anticancer agents. Full article

(This article belongs to the Section Biochemistry)

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10 pages, 599 KiB

Open AccessArticle

Clinical and Genetic Characteristics of Parkinson’s Disease Patients with Substantia Nigra Hyperechogenicity

by Łukasz Milanowski, Piotr Szukało, Małgorzata Kowalska, Alicja Sikorska, Dorota Hoffman-Zacharska and Dariusz Koziorowski

Int. J. Mol. Sci. 2025, 26(12), 5492; https://doi.org/10.3390/ijms26125492 (registering DOI) - 8 Jun 2025

Abstract

Hyperechogenicity of the substantia nigra (SN) is observed using transcranial ultrasonography in patients with Parkinson’s Disease. In this study, we investigated whether monogenic forms of PD are more prevalent in these patients and clinically defined their characteristics. Eighty-eight PD patients were part of [...] Read more.

Hyperechogenicity of the substantia nigra (SN) is observed using transcranial ultrasonography in patients with Parkinson’s Disease. In this study, we investigated whether monogenic forms of PD are more prevalent in these patients and clinically defined their characteristics. Eighty-eight PD patients were part of the analysis. All patients received clinical diagnoses from experienced movement disorder specialists. Each patient underwent transcranial ultrasonography and genetic testing for mutations in the SNCA, PRKN, LRRK2, DJ1, and PINK1 genes. SN hyperechogenicity was identified in 48 patients. Compared to the non-hyperechogenicity group, these patients did not have monogenic forms of PD more frequently, but they did have REM sleep behavior disorder significantly more often, lived in rural areas, and experienced a later age of disease onset. Our study indicated no association between substantia nigra echogenicity and the presence of mutations in the SNCA, LRRK2, DJ1, PRKN, and PINK1 genes. Hyperechogenicity of the substantia nigra, however, remains a common finding in patients with Parkinson’s Disease, correlating with certain features of the disease. Full article

(This article belongs to the Special Issue Parkinson’s Disease and Related Diseases: Mechanisms, Genetic Factors and Treatments)

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14 pages, 1230 KiB

Open AccessOpinion

The Anatomical and Evolutionary Impact of Pain, Pleasure, Motivation, and Cognition: Integrating Energy Metabolism and the Mind–Body BERN (Behavior, Exercise, Relaxation, and Nutrition) Framework

by George B. Stefano, Pascal Buttiker, Maren M. Michaelsen and Tobias Esch

Int. J. Mol. Sci. 2025, 26(12), 5491; https://doi.org/10.3390/ijms26125491 (registering DOI) - 8 Jun 2025

Abstract

In this manuscript, we highlight the evolutionary origins of mitochondria from bacterial endosymbionts and explore their contributions to health, energy metabolism, and neural–immune communication. Mitochondrial adaptability and the roles played by these organelles in promoting oxygen-dependent ATP production provide critical regulation of cognition, [...] Read more.

In this manuscript, we highlight the evolutionary origins of mitochondria from bacterial endosymbionts and explore their contributions to health, energy metabolism, and neural–immune communication. Mitochondrial adaptability and the roles played by these organelles in promoting oxygen-dependent ATP production provide critical regulation of cognition, motivation, and inflammation. Hypoxia has been identified as an important initiator of inflammation, neurodegeneration, and mitochondrial dysfunction, emphasizing the overall importance of oxygen homeostasis to health and well-being. The Behavior, Exercise, Relaxation, and Nutrition framework highlights these observations as tools that can be used to optimize mitochondrial efficiency. Interestingly, mitochondrial dysfunction may also be linked to psychiatric disorders (e.g., schizophrenia), a hypothesis that focuses on energy dynamics, a proposal that may extend our understanding of these disorders beyond traditional neurotransmitter-focused concepts. Collectively, these perspectives underscore the critical contributions of mitochondria to health and disease and offer a novel framework that may help to explain the connections featured in mind–body medicine. Full article

(This article belongs to the Section Molecular Endocrinology and Metabolism)

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23 pages, 8576 KiB

Open AccessArticle

Pterostilbene Reduces Cyclophosphamide-Induced Interstitial Cystitis by Facilitating Nrf2 Activation and Suppressing the NLRP3 Inflammasome Pathway

by Jiong Zhang, Jipeng Wang, Xinhao Wang, Zehao Yan, Lingfeng Meng and Yaoguang Zhang

Int. J. Mol. Sci. 2025, 26(12), 5490; https://doi.org/10.3390/ijms26125490 (registering DOI) - 8 Jun 2025

Abstract

Interstitial cystitis/bladder pain syndrome (IC/BPS) causes significant discomfort in patients and impairs the quality of urination. Pterostilbene (PTE), a natural polyphenol antioxidant, has demonstrated beneficial effects in mitigating inflammation, enhancing antioxidant capacity, and ameliorating organ dysfunction in various chronic nonspecific inflammatory conditions. The [...] Read more.

Interstitial cystitis/bladder pain syndrome (IC/BPS) causes significant discomfort in patients and impairs the quality of urination. Pterostilbene (PTE), a natural polyphenol antioxidant, has demonstrated beneficial effects in mitigating inflammation, enhancing antioxidant capacity, and ameliorating organ dysfunction in various chronic nonspecific inflammatory conditions. The aim of this study was to evaluate the efficacy of PTE in IC/BPS and elucidate its underlying mechanisms using a rat model of cyclophosphamide (CYP)-induced interstitial cystitis. In comparison, chronic pain progression, histopathological features, and cytokine levels demonstrated that PTE mitigated the severity of symptoms in CYP-induced rats by inhibiting the NLRP3 inflammasome in a dose-dependent manner. Further mechanistic investigations indicated that PTE intervention alleviated oxidative stress in CYP-induced IC in rats via activation of the Nrf2/HO-1 signaling pathway. Moreover, inhibitors of the Nrf2/HO-1 pathway effectively blocked PTE-mediated attenuation of oxidative stress. The suppression of NLRP3 inflammasome activation by PTE could also be reversed by inhibition of the Nrf2/HO-1 pathway. In vitro studies revealed that PTE enhanced the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and suppressed NLRP3 inflammasome activation in SV-HUC-1 cells exposed to lipopolysaccharide (LPS) and Adenosine Triphosphate (ATP). These findings collectively suggest that PTE treatment inhibits oxidative stress and suppresses NLRP3 inflammasome activation through modulation of the Nrf2/HO-1 pathway. Full article

(This article belongs to the Section Molecular Immunology)

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26 pages, 1815 KiB

Open AccessArticle

A High-Yield Recombinant Inactivated Whole-Virion Nasal Influenza A(H1N1)pdm09 Virus Vaccine with an Attenuated PB2 Gene

by Seung-Eun Son, Jin-Ha Song, Ho-Won Kim, Se-Hee An, Seung-Ji Kim, Chung-Young Lee, Hyuk-Joon Kwon and Kang-Seuk Choi

Int. J. Mol. Sci. 2025, 26(12), 5489; https://doi.org/10.3390/ijms26125489 (registering DOI) - 7 Jun 2025

Abstract

During the 2009 H1N1 pandemic (pdm09), the poor replication of PR8-derived vaccine strains in embryonated chicken eggs (ECEs) delayed vaccine production, necessitating costly adjuvants. To improve egg-based yield, we generated PB2-substituted H1N1 strains via reverse genetics, replacing PR8 PB2 with a PB2 lacking [...] Read more.

During the 2009 H1N1 pandemic (pdm09), the poor replication of PR8-derived vaccine strains in embryonated chicken eggs (ECEs) delayed vaccine production, necessitating costly adjuvants. To improve egg-based yield, we generated PB2-substituted H1N1 strains via reverse genetics, replacing PR8 PB2 with a PB2 lacking mammalian-adaptive mutations (dtxPB2), cognate pdm09 PB2 (19PB2), or avian PB2. All PB2-substituted strains achieved over tenfold higher titers than the conventional PR8 PB2-containing strain (rGD19), with rGD19/dtxPB2 and rGD19/19PB2 exhibiting significantly higher titers and reduced murine virulence. Among these, rGD19/19PB2 produced the highest hemagglutinin (HA) yield and, when administered intranasally as a binary ethyleneimine (BEI)-inactivated whole-virion vaccine, elicited a significantly stronger broncho-alveolar IgA response than rGD19. Both rGD19 and rGD19/19PB2 provided comparable protection against a homologous H1N1 challenge, yet only rGD19/19PB2 conferred full survival protection after a lethal heterologous H3N2 challenge. These findings show that incorporation of cognate PB2 enhances H1N1 replication in ECEs and antigen yield, reduces murine virulence, and confers robust homo- and heterosubtypic protection via intranasal immunization, underscoring the promise of PB2-modified H1N1 strains as inactivated mucosal whole-virion vaccines for future vaccine development. Full article

(This article belongs to the Special Issue Current Advances in Antivirals and Vaccines)

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11 pages, 799 KiB

Open AccessArticle

Endothelial Function and Matrix Metalloproteinase 9 (MMP9) in Women with Polycystic Ovary Syndrome (PCOS)

by Vaia Lambadiari, Sotirios Pililis, Stamatios Lampsas, Aikaterini Kountouri, John Thymis, Loukia Pliouta, Melpomeni Peppa, Sophia Kalantaridou, Evangelos Oikonomou, Gerasimos Siasos and Ignatios Ikonomidis

Int. J. Mol. Sci. 2025, 26(12), 5488; https://doi.org/10.3390/ijms26125488 (registering DOI) - 7 Jun 2025

Abstract

Polycystic ovary syndrome (PCOS) is a complex endocrine disease. This study investigates the relationship between endothelial function, insulin resistance, and hormonal profiles in women with PCOS. Forty women with PCOS were included: metformin (n = 20), GLP1-RAs (n = 10), and [...] Read more.

Polycystic ovary syndrome (PCOS) is a complex endocrine disease. This study investigates the relationship between endothelial function, insulin resistance, and hormonal profiles in women with PCOS. Forty women with PCOS were included: metformin (n = 20), GLP1-RAs (n = 10), and oral contraceptive pills (n = 10). A 75 g oral glucose tolerance test (OGTT) was performed, and the 0, 60, and 120 min insulin, glucose, and endothelial functions were evaluated. The postprandial and fasting state Matsuda Index and HOMA Index were measured. All measurements were performed at baseline and at a 6-month follow-up. At baseline, the percentage change in the Perfused Boundary Region (PBR) was associated with the percentage change in glucose at 120 min of the OGTT (r = 0.42, p < 0.05). The Matsuda Index, Homa Index, and testosterone levels were associated with the PBR (2.91 ± 0.1 μm) at 120 min of the OGTT (r = 0.41, r = 0.38 and r = 0.28, respectively). MMP9 levels were associated with the Matsuda and Homa Index (r = 0.45, p < 0.05 and r = 0.41, p < 0.05, respectively). At the 6-month follow-up, all the participants presented improvements of the Matsuda Index (7 ± 0.31 vs. 9.1 ± 0.2), Homa Index (5.3 ± 0.8 vs. 2.91 ± 0.1), MMP9 (210 ± 30 vs. 178 ± 28 ng/mL), and testosterone levels (44.2 ± 5 vs. 39.1 ± 2 ng/dL) compared to the baseline (p < 0.05 for all the comparisons). Patients who received GLP1-RA agonists presented the greatest improvement in MMP9 levels. Postprandial hyperglycemia, insulin resistance, and testosterone levels are associated with an impaired glycocalyx thickness in women with PCOS. Full article

(This article belongs to the Special Issue Cardiovascular Disease: Molecular Pathologies, and Therapeutic Strategies)

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21 pages, 13493 KiB

Open AccessArticle

Sponge bHLH Gene Expression in Xenopus laevis Disrupts Inner Ear and Lateral Line Neurosensory Development and Otic Afferent Pathfinding

by Karen L. Elliott, Clayton Gordy, Hannah Ingvalson, Charles Holliday, Jessica Halyko, Douglas W. Houston, Bernard M. Degnan and Bernd Fritzsch

Int. J. Mol. Sci. 2025, 26(12), 5487; https://doi.org/10.3390/ijms26125487 (registering DOI) - 7 Jun 2025

Abstract

Basic helix–loop–helix (bHLH) transcription factors, such as those in the atonal family, are important in cellular fate determination. The expression of the sponge ortholog of the atonal bHLH gene family, AmqbHLH1, in Xenopus laevis previously resulted in the formation of ectodermal ectopic [...] Read more.

Basic helix–loop–helix (bHLH) transcription factors, such as those in the atonal family, are important in cellular fate determination. The expression of the sponge ortholog of the atonal bHLH gene family, AmqbHLH1, in Xenopus laevis previously resulted in the formation of ectodermal ectopic neurons. However, the extent to which these neurons persist through development and the effects on the inner ear and lateral line, which require a critical level and timing of bHLH genes, remains unexplored. To test these long-term effects, we injected various concentrations of AmqbHLH1 mRNA into X. laevis embryos and assessed neurosensory development at developmental stages coinciding with fully developed neurosensory structures. The expression of AmqbHLH1 mRNA in X. laevis resulted in a dose-dependent reduction in or loss of ears and the lateral line system without eliminating ectopic neurons. At the lowest concentrations examined, we found that inner ear neurosensory development consisted sometimes of only a few scattered hair cells in a single-layer epithelium. Furthermore, low concentrations of AmqbHLH1 mRNA affected inner ear afferent guidance. Our data suggest that the AmqbHLH1 gene has some anti-neurosensory abilities in frogs and that the overexpression of a single gene may not be sufficient for stable long-term transdifferentiation in cells. Full article

(This article belongs to the Section Molecular Neurobiology)

39 pages, 1588 KiB

Open AccessReview

Gut Microbiota-Targeted Therapeutics for Metabolic Disorders: Mechanistic Insights into the Synergy of Probiotic-Fermented Herbal Bioactives

by Yue Fan, Yinhui Liu, Chenyi Shao, Chunyu Jiang, Lijuan Wu, Jing Xiao and Li Tang

Int. J. Mol. Sci. 2025, 26(12), 5486; https://doi.org/10.3390/ijms26125486 (registering DOI) - 7 Jun 2025

Abstract

Gut microbiota dysbiosis is intricately linked to metabolic disorders such as obesity, type 2 diabetes mellitus (T2DM), hyperlipidemia, and non-alcoholic fatty liver disease (NAFLD). Traditional Chinese medicine (TCM), particularly when combined with probiotic fermentation, offers a promising therapeutic strategy by modulating microbial balance [...] Read more.

Gut microbiota dysbiosis is intricately linked to metabolic disorders such as obesity, type 2 diabetes mellitus (T2DM), hyperlipidemia, and non-alcoholic fatty liver disease (NAFLD). Traditional Chinese medicine (TCM), particularly when combined with probiotic fermentation, offers a promising therapeutic strategy by modulating microbial balance and host metabolism. This narrative review synthesizes current research on probiotic-fermented herbal bioactives, focusing on their mechanisms in ameliorating metabolic diseases. Probiotic and bioactive compounds (e.g., berberine, polysaccharides) are highlighted for their roles in enhancing intestinal barrier function, regulating microbial metabolites like short-chain fatty acids (SCFAs), and reducing inflammation. Fermentation techniques improve the bioavailability of TCM components while reducing toxicity, as seen in fermented Salvia miltiorrhiza and Rhizoma Coptidis. Despite promising results, challenges include the complexity of microbiota–host interactions and variability in TCM standardization. Future directions emphasize integrating multi-omics technologies and personalized approaches to optimize probiotic-fermented TCM therapies. This review underscores the potential of combining traditional herbal wisdom with modern biotechnology to address metabolic disorders, which pose significant global health challenges, through a “gut microbiota–metabolism” axis. Emerging evidence highlights the critical role of gut microbiota dysbiosis in the pathogenesis of these conditions. TCM has shown promise in modulating gut microbiota to restore metabolic homeostasis. This review synthesizes current research on TCM-derived interventions, such as herbal compounds, probiotics, and fermentation techniques, that target gut microbiota to ameliorate metabolic disorders. We discuss mechanisms of action, including prebiotic effects, enhancement of intestinal barrier function, and regulation of microbial metabolites, while addressing the limitations and future directions of TCM-based therapies. Full article

(This article belongs to the Section Molecular Endocrinology and Metabolism)

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