The understanding of Alzheimer's disease is shifting from a traditional focus on Aβ/tau pathology to an emerging consensus that positions immune dysregulation as a central synergistic driver in the early stages of the disease. However, the causal relationships between peripheral immune cells, plasma proteins, and Alzheimer's disease, as well as the mediating effects of plasma proteins on the disease, remain poorly understood. Moreover, there are no effective drug combination therapies targeting plasma proteins for Alzheimer's disease. This study investigated the causal associations between immune cells, plasma proteins, and Alzheimer's disease, with a focus on the role of Fc gamma receptor 3A in disease progression. Using a two-sample Mendelian randomization approach, we identified 59 plasma proteins and 65 immune cell types significantly associated with Alzheimer's disease. We performed data mining of a large Alzheimer's disease cohort and drug databases and established a biofactor-regulated neural network for rapidly screening and optimizing compound drug pairs. Among the immune cells, CD8 + T cells, particularly CD8 + CD28 + CD45RA. T cells, were found to have a protective effect against Alzheimer's disease. Furthermore, increased expression of Fc gamma receptor 3A (also known as CD16a, an activating receptors of NK cells) in plasma and the hippocampus correlated with enhanced CD8 + T-cell infiltration and accelerated Alzheimer's disease progression in 5×FAD mice. Mediation analysis revealed that Fc gamma receptor 3A mediates the effects of CD8 + T cells on Alzheimer's disease risk. Additionally, Fc gamma receptor 3A gene expression levels were significantly higher in patients with Alzheimer's disease compared with individuals with mild cognitive impairment and cognitively normal participants, as revealed by an analysis of the Alzheimer's Disease Neuroimaging Initiative database. These findings suggest that CD8 + T-cell infiltration and Fcγ receptor 3A expression play critical roles in the pathophysiology of Alzheimer's disease and may serve as therapeutic targets. Molecular docking analysis further identified 19 candidate drugs targeting Fcγ receptor 3A. This study proposes novel immune-based therapeutic strategies and introduces an omics-based intelligent drug discovery framework for repurposing existing drugs for the treatment of complex diseases. The key contributions of this study include the identification of potential immune-based therapeutic targets for Alzheimer's disease and demonstration of the utility of bioinformatics and drug repurposing approaches in addressing complex neurodegenerative diseases.
The formation of macrophage-derived foam cells is a critical factor in the development of atherosclerotic lesions. However, the mechanisms through which the cuproptosis pathway contributes to macrophage foam cell formation remain unclear. This study aimed to investigate the role and mechanisms of the cuproptosis inducer Elesclomol in atherosclerosis. Macrophages were exposed to ox-LDL to establish a foam cell model. Subsequently, CCK8, oil red O staining, and Western blot were used to investigate foam cell formation and the cuproptosis pathway in macrophages. Furthermore, proteomics analysis was employed to explore the molecular mechanisms underlying the effects of Elesclomol. The results demonstrated that Elesclomol promoted foam cell formation by increasing lipid accumulation in ox-LDL-treated macrophages, which was attributed to the inhibition of cholesterol efflux mediated by ATP-binding cassette transporters A1 and G1 (ABCA1 and ABCG1). Additionally, Elesclomol enhanced the cuproptosis pathway in macrophages, leading to increased intracellular ROS accumulation and consequent cell death. Mechanistically, the cytotoxic effects of Elesclomol were mediated through activation of the mitogen-activated protein kinase (MAPK) pathway and upregulation of metallothionein 2A (MT2A). Collectively, these findings underscore the critical role of cuproptosis in the pathogenesis of atherosclerosis and suggest that targeting this pathway may represent a promising therapeutic strategy for the disease.
Accumulating evidence suggests that SUMOylation plays a crucial role in the progression and resistance of secondary hyperparathyroidism (SHPT). However, the precise mechanism of SUMOylation in SHPT remains unclear. We identified the potential role of SUMOylation in SHPT based on RNA sequencing data obtained from Gene Expression Omnibus (GEO) datasets. Clinical samples were used to verify the expression of SENP1, SUMO1, SUMO2, GRHL2, and vitamin D receptor (VDR) in SHPT cells and tissues. Primary cells were extracted for subsequent experiments. Plasmid transfection and small interfering RNA (siRNA) were used to modulate SENP1 expression in SHPT primary cells. VDR relative expression was detected by Western blot (WB) and immunofluorescence. The effects of SENP1 on SHPT cell apoptosis and anti-proliferation were analyzed by flow cytometry and WB. Co-immunoprecipitation (Co-IP), chromatin immunoprecipitation (ChIP), and ChIP-quantitative polymerase chain reaction (ChIP-qPCR) were employed to explore the regulatory mechanisms of SENP1 in SHPT. We found that SUMOylation was significantly upregulated in SHPT and was closely related to calcitriol resistance. SENP1 and GRHL2 were downregulated. SENP1 was found to upregulate VDR and downregulate the SUMOylation of VDR, which mediates SENP1's regulation of SHPT cell apoptosis and anti-proliferation. Mechanistically Co-IP assays revealed binding between VDR, SENP1, SUMO1, and SUMO2. ChIP assays indicated that transcription of SENP1 was regulated by GRHL2, with binding sites identified by ChIP-qPCR. Additionally, we identified the potential binding pocket of SENP1 and screened 10 candidate small-molecule drugs approved by the US Food and Drug Administration (FDA). Our findings indicate a distinct mechanism of SENP1-mediated VDR SUMOylation and establish the critical role of the GRHL2/SENP1/VDR signaling axis in SHPT development.
L-dopa decarboxylase (DDC) is the biosynthetic enzyme of dopamine and serotonin. Although DDC has been originally studied for its role in neurotransmission, it has also been detected in peripheral organs, where it is implicated in cellular homeostasis. DDC has been identified by our research team as a negative regulator of dengue virus (DENV) replication in liver cells. The latter has been attributed, at least in part, to the physical interaction of DDC protein with phosphatidylinositol 3-kinase (PI3K), and its biosynthetic function. PI3K/AKT signaling and cell survival are manipulated by DENV to favor its replication. Based on the above, we investigated whether DDC exerts its antiviral activity against DENV propagation through modulation of DENV-induced cell death and especially apoptosis. Specifically, DDC silencing in Huh7.5 cells (shDDC) significantly reduced virus-induced cytopathic effect compared to the control cells (shControl). This finding was accompanied by suppression of both early and later stages of apoptosis in the silenced cells, as shown by Annexin V/PI staining and TUNEL assay, respectively. Accordingly, upon infection, shDDC cells showed suppressed activation of key caspases, BCL-2 family members and TRAIL-receptor genes that modulate the apoptotic cascade, compared to the control cells. Moreover, mitochondrial analysis in DENV-infected cells revealed that, upon DDC silencing, a less pronounced disruption of mitochondrial membrane potential and network integrity, higher respiratory capacity, lower ROS production, and reduced cytochrome c release were observed. As the PI3K/AKT pathway is known to be affected by both DENV and DDC, next we assessed whether DDC is involved in the virus-induced apoptosis through this axis. For this, we quantified the reduction of p-AKT and p-mTOR levels caused by DENV infection in the two cell lines, which was found greater in the shControl cells. Finally, chemical inhibition of AKT phosphorylation abolished the differences in cell viability and apoptosis between the two cell lines. In total, our findings highlight the suppressive role of DDC against DENV replication by modulating the PI3K/AKT-dependent apoptotic signaling.
Type 2 diabetes (T2DM), a chronic metabolic disorder characterized by pancreatic β-cell dysfunction and insulin resistance, is closely associated with oxidative stress. Interferon-stimulated gene 15 (ISG15), a ubiquitin-like modifier, has been implicated in redox regulation; however, its mechanistic role in T2DM progression remains unclear. To identify novel bio-factors contributing to T2DM, peripheral blood mononuclear cells (PBMCs) and serum samples were collected from T2DM patients and age-/BMI-matched healthy controls to quantify ISG15 expression. Complementary studies utilized a T2DM mouse model to assess β-cell-specific ISG15 expression within pancreatic islets. In parallel, MIN6 pancreatic β-cells were exposed to pathophysiological stressors, including hyperglycemic/hyperlipidemic (HG/PG) conditions and hydrogen peroxide (H2O2)-induced oxidative stress, to mimic the diabetic microenvironment. Protein-protein interactions were evaluated, and functional analyses were conducted following ISG15 genetic ablation. The results demonstrated that ISG15 expression was significantly elevated in PBMCs and serum from T2DM patients compared to healthy controls. Similarly, T2DM mouse models exhibited marked upregulation of ISG15 in islet β-cells. In vitro, exposure to HG/PG or H2O2 increased ISG15 levels, reduced MIN6 cell viability, and heightened reactive oxygen species (ROS) accumulation. Mechanistic studies revealed that ISG15 directly interacts with the PI3K regulatory subunit PIK3R2, suppressing its activity and thereby disrupting the PI3K/AKT/Nrf2 antioxidant signaling axis. This disruption led to exacerbated oxidative stress. Collectively, these findings indicate that ISG15 acts as a novel mediator of oxidative stress in T2DM by targeting and inhibiting the PIK3R2-dependent PI3K/AKT/Nrf2 pathway. These results uncover a previously unrecognized molecular mechanism driving β-cell dysfunction and identify ISG15 as a potential therapeutic target for mitigating oxidative damage in diabetes.
Inflammatory bowel disease (IBD) arises from dysregulated interactions between the immune system and the intestinal microenvironment. Finding new therapeutic targets could help to develop treatments that attenuate its severity. The present study investigated the immunomodulatory potential of bioaccessible sulforaphane (SFN, 0.100 μg/mL) from broccoli by-products. Interestingly, the main results evidenced that at this physiological concentration, SFN contributed to reducing the secretion of pro-inflammatory interleukins (IL-1β, IL-6, IL-17, IL-18, IL-23, TNF-α) by intestinal epithelium up to ~56%, whereas enhancing anti-inflammatory cytokines (IL-10, IL-4, IL-13) between ~24% and ~71%. These changes adjusted the proportion of CD86+ and CD206+ during macrophage differentiation, associated with the prevention of immune-mediated IBD. In addition, a reduction in the expression of macrophage-dependent pro-inflammatory cytokines and an augmentation of the tolerogenic classes were observed. The combined use of intestinal epithelial (Caco-2) and monocytic (THP-1) cell lines established an in vitro model of the epithelium-macrophage crosstalk, thereby enhancing the physiological relevance of our findings. These results were confirmed using a pure SFN-based model system, which demonstrated SFN's contribution to the anti-inflammatory properties of broccoli stalk and bridged the gap between in vitro findings and potential dietary/therapeutic applications. Thereby, this work demonstrated that dietary SFN contributes to a large extent to the reshaping capacity of the phytochemical burden of broccoli stalks, on the interleukin profile secreted by epithelium and macrophages, as well as the macrophage differentiation, thus supporting the valorisation of broccoli by-products for preventing and managing inflammatory conditions, such as IBD.
Parkinson's disease (PD) is a progressive neurodegenerative disorder. An essential early hallmark of PD is disrupted mitochondrial dynamics driven by impaired cellular energy homeostasis. Therapeutic interventions restoring mitochondrial function and biogenesis hold promise for neuroprotection in PD. In the present study, the neuroprotective effects of formononetin (FMN) were evaluated in both MPP+-induced SH-SY5Y cells and the MPTP-induced mouse model of Parkinson's disease, using concentrations of 5, 10, 20, and 40 μM in vitro and doses of 25 and 40 mg/kg in vivo. To evaluate its biological activity, we employed western blotting and immunofluorescence assay to quantify the expression of disease-relevant markers. Mitochondrial health was further assessed using Mitotracker, alongside reactive oxygen species (ROS) assessment. Motor behavior and molecular endpoint parameters were also measured. Our results demonstrated that FMN significantly attenuates MPP+/MPTP-induced neurotoxicity, improves motor function, and restores the expression of PGC-1α, tyrosine hydroxylase, and key mitochondrial proteins involved in mitochondrial DNA replication. Enhancement of mitochondrial fusion proteins and other transcriptional regulators was also observed in the administered groups. Flow cytometry and imaging analyses confirmed that FMN-mediated PGC-1α activation preserves mitochondrial integrity and reduces oxidative stress. Altogether, these findings provide evidence that formononetin exerts neuroprotective effects in PD by modulating the PGC-1α signaling axis.
Electrical stimulation (ES) has emerged as a promising technique in the field of bioengineering and biomedicine, particularly in bone regeneration and cell differentiation. ES using alternating current (AC) is based on the periodic reversal of current direction, which generates oscillating electric fields. The application of an electric field has effects on cell growth and differentiation, as well as on morphology and migration. This study aimed to explore the effect of applying AC electrostimulation within the proliferation, differentiation, and morphology process of osteoblastic cells. The electrical stimulation signals were daily applied for 3 h during 14 days. Different frequencies were tested (1 Hz, 10 Hz, 100 Hz, and 1 kHz), with amplitudes of 125, 250, 500, 750, 1000, and 1500 mV/mm. Cell viability was estimated using the AlamarBlue, and MC3T3-E1 differentiation levels were evaluated through alkaline phosphatase (ALP) activity. RUNX2, OSX, ALP, OPG, and RANKL gene expression was assessed by RT-PCR. Morphological analysis was performed through cell transfection followed by immunofluorescence. Statistical analysis was conducted by SPSS.23 and graphs generated through Graph-pad. Viability and ALP activity were optimal at 10 Hz. Once the frequency was defined, RUNX2, OSX, ALP, OPG, and RANKL gene expression revealed an increase in the differentiation and osteogenic activity levels at 10 Hz and 500-750 mV/mm. As well as, morphological studies showed an increase in the area, pseudopodia length, and numbers at 500 mV 10 Hz conditions. The optimal ES condition to differentiate MC3T3-E1 cells is 10 Hz 500-750 mV/mm. Electrostimulation has emerged as a promising technique in the field of bioengineering and biomedicine, particularly in bone regeneration and cell early maturation.
Glioblastoma (GB) is highly malignant with a median survival of 14 months despite conventional treatments like surgery, radiotherapy, and temozolomide. Resistance to these therapies necessitates innovative approaches, such as immune checkpoint inhibitors (ICIs) targeting cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), programmed cell death protein 1 (PD-1), and programmed death ligand 1 (PD-L1) to enhance T-cell-mediated tumor destruction. However, clinical trials have shown limited ICI efficacy in GB due to its immunosuppressive microenvironment and the blood-brain tumor barrier (BBTB), which impairs drug delivery. Emerging evidence highlights the gut microbiota as a pivotal modulator of ICI response, enhancing CD8+ and CD4+ T-cell function, antigen presentation, and immune modulation via the gut-brain axis in cancers. In addition, studies showed that gut-derived metabolites, including short-chain fatty acids, modulate immune responses and support blood-brain barrier integrity by regulating inflammatory signaling and tight junction proteins. Future GB research should prioritize clinical trials, mechanistic studies, and interventional strategies like fecal microbiota transplantation and probiotics to enhance ICI efficacy.
Dengue virus (DENV) transmission has greatly increased in the last decade, partly due to the geographical expansion of Aedes spp. mosquitoes. These arthropods are now found even in temperate climates, including Europe, where outbreaks have occurred. A better understanding of the life cycle is essential, as it may enable the development of specific treatments or therapies, which are currently lacking. Recent breakthroughs concerning the viral capsid (C) protein structure and function are encouraging. It is now clear that the C protein binds both to host lipid droplets and to the viral genome-interactions crucial for viral encapsidation and replication. Here, we investigate how DENV C associates with the viral genome. Using DENV C and single-stranded DNA sequences analogous to relevant genomic regions, we biophysically characterize their interaction. A decrease in fluorescence intensity and lifetime, as well as changes in the protein secondary structure, were observed upon interacting with single-stranded DNA. These results are consistent with molecular condensation, supporting a possible liquid-liquid phase separation contributing to DENV C-nucleic acid complex formation.
Glioblastoma is lethal brain tumor with dismal prognosis. ETV1, an ETS family transcription factor, has been implicated in multiple malignancies, yet its precise role and regulatory mechanisms in glioma progression remain incompletely defined. This study investigated the role of ETV1 in maintaining the malignant phenotype of high-grade gliomas through a novel regulatory axis involving microRNA-3175 (miR-3175) and STEAP2. ETV1 expression was analyzed in TCGA glioma datasets (n = 692) and 85 patient tissue specimens using bioinformatics, quantitative RT-PCR, Western blotting, and immunohistochemistry. ETV1 function was assessed through gain-of-function and loss-of-function studies in glioma cell lines (U251, A172) and validated in nude mouse xenografts with bioluminescence imaging. The ETV1-miR-3175-STEAP2 regulatory axis was characterized using chromatin immunoprecipitation, luciferase reporter assays, biotin-streptavidin pulldown, RNA immunoprecipitation, and rescue experiments. ETV1 was significantly upregulated in gliomas, with expression correlating to tumor grade and reduced overall patient survival. ETV1 overexpression promoted glioma cell proliferation, migration, and invasion in vitro and enhanced tumor growth while reducing survival in vivo, with elevated bioluminescence radiance indicating enhanced tumorigenicity, whereas ETV1 knockdown produced opposite effects and diminished bioluminescent signals. ETV1 transcriptionally activated miR-3175 through direct binding to its promoter. miR-3175 directly targeted and suppressed STEAP2 expression via conserved 3'UTR binding sites. STEAP2 functioned as a tumor suppressor; overexpression inhibited malignant phenotypes. Rescue experiments confirmed miR-3175 and STEAP2 mediate ETV1-driven glioma progression through a functional ETV1/miR-3175/STEAP2 regulatory axis. This study revealed a novel ETV1/miR-3175/STEAP2 regulatory axis driving glioma tumorigenesis and identified potential therapeutic targets for intervention.
Circulating tumor DNA (ctDNA) analysis has emerged as a pivotal minimally invasive tool for early detection, monitoring, and treatment stratification in cancer patients. However, the accuracy and reliability of ctDNA assays are profoundly influenced by preanalytical variables. This review discusses the impact of biological features (circadian rhythm, age, and sex), lifestyle factors (diet, smoking, and physical activity), as well as technical aspects such as hemolysis, leukocyte lysis, and delayed plasma separation on ctDNA integrity and concentration. Fluctuations in ctDNA levels driven by these factors highlight the need for clear guidelines regarding precollection timing, dietary restrictions, and sample processing. Furthermore, the adoption of harmonized protocols is essential to reduce variability and improve reproducibility across clinical and research settings.
Trifuhalol A (TFA), a phlorotannin derived from the edible brown seaweed Agarum cribrosum, has been reported to exert diverse physiological activities, yet its anti-diabetic mechanism remains unclear. This study systematically investigates the multi-targeted anti-diabetic effects of TFA, with a particular focus on enhancing glucose uptake and protecting pancreatic islets. In vitro enzyme inhibition assays demonstrated that TFA significantly inhibited the activities of α-glucosidase and α-amylase, indicating its potential to attenuate postprandial glycemic excursions by modulating carbohydrate hydrolysis. Additionally, TFA effectively suppressed the formation of advanced glycation end-products (AGEs), potentially reducing the risk of diabetes-associated complications. Mechanistically, TFA enhanced glucose uptake in C2C12 myotubes by activating the PI3K/Akt and AMPK signaling pathways, which in turn promoted the translocation of glucose transporter type 4 (GLUT4) to the plasma membrane, thereby facilitating cellular glucose utilization and insulin sensitivity. Furthermore, in vivo investigations using an alloxan-induced type 1 diabetic zebrafish further confirmed the bioefficacy of TFA, as evidenced by its capacity to reduce hyperglycemia, alleviate oxidative stress, and protect pancreatic islets, without eliciting observable systemic toxicity. Taken together, these findings provide both mechanistic and functional evidence supporting TFA as a safe and potent multi-target bioactive compound with promising applications in the development of functional foods and therapeutic strategies for diabetes management.
Plant-based food products have been developed from diverse plant sources as new food choices. Fermentation of plant matrices with lactic acid bacteria (LAB) has been shown to improve quality and bioactivity of the resulting product while proteolysis by the LAB in the plant-based matrix remains to be elucidated. In this study, a hazelnut-based matrix prepared for a plant-based product was fermented with four different starter cultures of LAB, and their effects on proteolysis, bioactivity and allergenicity were investigated. Sucrose supplementation of the hazelnut matrix stimulated fermentation and time to reach to the target pH of 4.5 was shortened. CH-1 was the fastest acidifying culture reducing pH to the target value after 5 h. While the cultures RSF-736 and CHN-11 required 18 h for fermentation, R-707 was co-cultured with CH-1 to reach the target pH within the same time. Bacterial counts were in the range of 5-8 log cfu/g without a significant change after 15 days of storage in the hazelnut-based products. Level of proteolysis as measured by changes in soluble protein and total free amino acid contents differed among the cultures. Reductions in the amounts of hazelnut proteins were also confirmed by SDS-PAGE analysis, especially in the products prepared with cultures R-707+CH-1 and RSF-736. Allergenicity of the hazelnut matrix, determined by a hazelnut-specific ELISA test, significantly decreased after fermentation with all the cultures. Fermentation also enhanced total phenolic content and antioxidant activity of the hazelnut matrix with CHN-11 demonstrating the highest values after storage. On the other hand, fermentation did not significantly alter α-amylase inhibitory activity compared to the activity of 10.2% in the unfermented control. In addition, fermentation resulted in no change or a slight reduction in ACE inhibitory activity compared to the activity of 46.9% in the unfermented control depending on the culture. These findings demonstrate that LAB species can degrade hazelnut matrix leading to a reduction in allergenicity and enhancement of antioxidant activity.
Myocardial infarction (MI) is a major cause of mortality. This study explores the cardioprotective effects of Coenzyme Q10 (CoQ10) and its molecular mechanisms in MI. A rat myocardial ischemia-reperfusion model was established, and infarct size was assessed via triphenyl tetrazolium chloride (TTC) staining. Hematoxylin and eosin (H&E) and Masson's staining analyzed histological changes, while terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) and flow cytometry assessed apoptosis. Western blot and immunofluorescence evaluated collagen I, collagen III, α-SMA, histone deacetylases (HDACs) (4,5,7,9,11), O-GlcNAc transferase (OGT), O-GlcNAcase (OGA), and O-GlcNAcylation. Enzyme-linked immunosorbent assay (ELISA) measured tumor necrosis factor (TNF)-α, interleukin (IL)-6, transforming growth factor (TGF)-β, suppression of tumorigenicity (ST)-2, galectin (Gal)-3, and growth differentiation factor (GDF)-15, while cell counting kit-8 (CCK-8), EdU, and Transwell migration assays assessed fibroblast proliferation and migration. O-GlcNAcylation of HDAC4 was analyzed using Click-iT labeling and immunoprecipitation. Cycloheximide chase assay and ubiquitination assay detected the impact of O-GlcNAcylation at Ser455 on HDAC4 protein stability. CoQ10 significantly improved cardiac function in rat models, reducing infarct size, apoptosis, fibrosis, and inflammation. CoQ10 treatment in MIRI rats increased ejection fraction (EF) and fractional shortening (FS), reduced infarct volume (TTC staining), and decreased collagen deposition (Masson's, Sirius Red staining). TUNEL and flow cytometry confirmed reduced cardiomyocyte apoptosis, while ELISA showed lower TNF-α, IL-6, and TGF-β, indicating suppressed inflammation. At the molecular level, CoQ10 reduced global O-GlcNAcylation together with decreased OGT and increased OGA expression. Western blot and immunofluorescence confirmed reduced HDAC4 levels, linked to O-GlcNAcylation suppression particularly at Ser455. CoQ10 promoted the degradation of HDAC4 through the precise inhibition of its stabilizing O-GlcNAc modification at Ser455. In vitro, CoQ10 enhanced oxygen-glucose deprivation and reperfusion (OGD/R) cardiomyocyte survival, reduced apoptosis (TUNEL, flow cytometry), and suppressed HDAC4 expression. In fibroblasts, CoQ10 inhibited TGF-β1-induced proliferation/migration (CCK-8, EdU, Transwell assays) and reduced collagen I, collagen III, and α-SMA expression, indicating anti-fibrotic effects. Furthermore, HDAC4 knockdown in vivo improved cardiac function, and in vitro reduced cardiomyocyte apoptosis and fibroblast proliferation/migration, reinforcing HDAC4's role in MI pathology. CoQ10 exerts cardioprotective effects by modulating O-GlcNAc modification and HDAC4 expression.
Renal cell carcinoma (RCC) ranks as the most prevalent form of urogenital cancer. This research aims to investigate the role of YTHDF2 in the RCC progression and identify new therapeutic targets for RCC. YTHDF2, E2F2, and CORO6 were assayed via qRT-PCR and Western blot. YTHDF2 was downregulated in RCC cells, while E2F2 and CORO6 were upregulated. After overexpressing YTHDF2 in RCC cells, cell viability, proliferation, invasion, and migration were measured. m6A levels were assessed. The binding of YTHDF2 to E2F2 was detected. The E2F2 mRNA stability was detected. The binding of E2F2 to the CORO6 promoter was analyzed. Overexpression of E2F2 or CORO6 was combined with YTHDF2 overexpression to validate the mechanism. Tumor growth and metastasis were observed. Results confirmed that YTHDF2 overexpression decreased cell proliferation, invasion, and migration. YTHDF2 bound to the m6A sites on E2F2 mRNA, promoted E2F2 degradation, and inhibited E2F2 expression. E2F2 bound to the CORO6 promoter to enhance CORO6 expression. Overexpression of E2F2 or CORO6 partially reversed the suppressive effects of YTHDF2 overexpression on RCC cell proliferation and invasion. YTHDF2 overexpression suppressed tumor growth and metastasis. In conclusion, YTHDF2 overexpression suppresses RCC progression by inhibiting E2F2 expression and reducing CORO6 expression via m6A modification.
Hydroxy-safflor yellow A (HSYA), a bioactive compound from Carthamus tinctorius, has reported anti-inflammatory and antitumor activities. However, its effects on breast cancer and the underlying immune mechanisms remain poorly defined. In this study, we investigated the immunomodulatory role of HSYA in breast cancer, with a focus on its impact on tumor-associated macrophages (TAMs) and T cell responses. Using in vitro assays, murine breast cancer models, and single-cell transcriptomic analysis, we found that HSYA significantly inhibited tumor growth and reshaped the tumor immune microenvironment. Flow cytometry and transcriptomic profiling revealed that HSYA treatment reduced tumor-associated macrophages (TAMs) infiltration and suppressed C-C motif chemokine ligand 5 (CCL5) expression, which was associated with enhanced recruitment and activation of tissue-resident memory T (TRM) cells. Co-culture and functional assays further supported a role for the TAM/CCL5 axis in mediating these immune effects. Collectively, our findings demonstrate that HSYA exerts antitumor activity, at least in part, by modulating the TAM/CCL5/TRM axis, highlighting its potential as an immunomodulatory therapeutic strategy in breast cancer.
The milk thistle plant (Silybum marianum) is known for its hepatoprotective properties. However, the poor water solubility of silymarin limits its dissolution in the intestinal tract and restricts its bioavailability following oral administration. To improve bioavailability, special formulations, in particular micellar solubilization, are explored. In this study, we examined the transport rate of silymarin in a krill oil-based formulation across a Caco-2 epithelial barrier after upstream digestion simulation in vitro. Furthermore, in a randomized cross-over design study the bioavailability of the krill oil-based formulation was investigated after single dose intake in fasting conditions in healthy participants. We could demonstrate that the apparent transport coefficient of silybin, measured as lead substance of milk thistle extract, across the epithelium is efficiently boosted by a krill oil formulation, resulting in a 28% increase compared to silymarin powder. Consistent with these findings, a significant enhancement of bioavailability (P < 0.0001) was demonstrated for the krill oil-based formulation in comparison to the milk thistle extract resulting in an 8.59-fold higher AUC0-8h and a 15.08-fold greater Cmax of silybin and faster uptake kinetic after single dose intake. These findings suggest that phospholipid-based delivery systems offer a promising strategy for improving the efficacy of lipophilic bioactives. Furthermore, the combination of krill oil with milk thistle extracts efficiently provides silybin, PUFAs, and choline, which are important nutrients contributing to liver and heart health.
Growing the hyperthermophilic archaeon Pyrococcus horikoshii OT-3 in medium supplemented with d-allo-Ile instead of l-Ile markedly upregulates the activity of broad substrate specificity amino acid racemase (BAR). In P. horikoshii genome, the BAR gene (PH0138) forms a cluster with PH0137, which encodes a putative transporter protein, and PH0140 that encodes a feast/famine regulatory protein (FFRP), involved in the transcriptional regulation of metabolic pathway genes. Here, we performed gene expression, protein-DNA interaction, and crystallographic analyses to elucidate the expression mechanism of the BAR cluster. Gene expression analysis revealed that d-allo-Ile simultaneously induces PH0138 and PH0137. Electrophoretic mobility shift assays demonstrated that the PH0140 protein binds to the PH0137 promoter in the presence of l-Ile, but this interaction is disrupted by d-allo-Ile, identifying PH0140 as a d-amino acid-responsive regulatory protein (DARP). The crystal structures of DARP bound to l-Ile and d-allo-Ile were compared with those of another FFRP family member, P. horikoshii FL11. Our findings reveal a dimeric arrangement of l-Ile-bound DARP resembling the DNA-bound (open) form of FL11, whereas d-allo-Ile-bound DARP corresponds to the DNA-unbound (closed) form. These conformational changes result from subtle alterations in hydrogen bonding around the coregulators. Furthermore, cultivation in d-allo-Ile substitution medium only impacted the expression of PH0138 and PH0137, and not the other genes, suggesting that DARP regulates a more limited gene set than FL11. Cumulatively, these results reveal a distinct mechanism by which an FFRP homolog controls d-amino acid utilization.
Critical-sized bone defects remain challenging to repair because successful regeneration requires both mechanical stability and the coordinated promotion of osteogenesis and vascularization. To address these needs, we developed a composite scaffold (GV@PHL) that integrates structural support with sustained pro-angiogenic signaling. A 3D-printed framework composed of polycaprolactone (PCL), nano-hydroxyapatite (nano-HA), and Laponite (PHL) was fabricated to form an interconnected porous architecture with intrinsic osteogenic potential and printability. A GelMA hydrogel was photo-crosslinked within the scaffold pores and covalently tethered to vascular endothelial growth factor (VEGF) to create a photo-embedded GelMA–VEGF phase, enabling sustained VEGF release. The composite scaffold (GV@PHL) was evaluated through in vitro and in vivo experiments to assess architecture stability, osteogenic differentiation, VEGF release behavior, endothelial cell responses, and vascularization. The GV@PHL scaffold maintained a stable porous architecture and exhibited synergistic performance combining structural integrity with biological activity. The PHL framework supported osteogenic differentiation, while the photo-crosslinked GelMA–VEGF hydrogel enabled controlled, sustained release of VEGF. Released VEGF promoted endothelial cell survival and enhanced vascularization in vitro and in vivo, demonstrating coordinated support for osteogenesis and angiogenesis. GV@PHL represents a practical strategy for integrating structural design and biological function in bone tissue engineering. By combining a mechanically stable, osteoinductive 3D-printed framework with sustained VEGF delivery to promote vascularization, this platform shows promise for treating critical-sized craniofacial and orthopedic bone defects. The online version contains supplementary material available at 10.1186/s12967-026-08090-5.