ABSTRACTAlzheimer's Disease (AD) is a global health issue, affecting over 6 million people in the United States, with that number expected to increase as the population ages. As a neurodegenerative disorder that affects memory and cognitive functions, it is well established that AD is associated with cardiovascular risk factors beyond only cerebral decline. In this study, we measured hemodynamic parameters related to cardiovascular and cerebrovascular function in 5xFAD mice with AD and their littermates. Specifically, we measured cardiovascular pulse wave velocity parameters, a marker of arterial stiffness and cardiovascular risk, and cerebrovascular pulse wave velocity, a novel technique to measure cerebral arterial stiffness. Our results showed that while 5xFAD mice exhibited significant differences in ejection time, pulse pressure, and Tei index, many other cardiovascular and cerebral parameters were not different. Despite reports that amyloid plaque deposition begins at an early age of 1.5 months in 5xFAD mice, our results did not indicate significant cardiovascular changes. Studies to elucidate cardiovascular and cerebrovascular parametric changes should be done at later ages where the underlying changes are more profound.

SENP3, a member of the sentrin-specific protease family, plays a pivotal role in lipid metabolism and the pathogenesis of fatty liver disease by regulating the dynamic process of SUMOylation. It has previously been demonstrated that SREBP2 is SUMOylated. However, the function and regulatory mechanism of SENP3-mediated SREBP2 de-SUMOylation in hepatocyte steatosis is unclear. Bioinformatic analysis (proteomes from whole cell extract and nuclear extraction, integrated transcriptomes, RNA sequence) SENP3-driven SREBP2 de-SUMOylation in lipid metabolism; Oil red O, nuclear and cytoplasmic extraction, western blot and immunofluorescence suggest SENP3 vitally contributes to hepatocyte steatosis; NLS predictions, CO-IP, co-transfection of plasmids, confocal microscopy indicates nuclear SENP3 associates with SREBP2 to promote its nuclear translocation; molecular docking and mutation assay confirmed SENP3 is responsible for de-SUMOylation SREBP2 at R576. Treatment with OA increased the level of both SENP3 and its nuclear fraction in HepG2 cells. Interfering with the SUMOylation and deSUMOylation cycle induced hepatocyte steatosis by overexpressing SENP3 and using a SUMO inhibitor, GA. Mechanistically, it is observed that the co-localization between SREBP2 and SENP3 is increased. SENP3 is responsible for de-SUMOylation SREBP2 at R576. Moreover, SENP3-mediated de-SUMOylation may also decrease ZMIZ1-ligated SUMO3 binding to SREBP2 at K464 in the nucleus. RNA interference of SREBP2 cannot reverse SENP3-overexpressed cell steatosis under fatty acid treatment. Expression of SREBP2 in vitro could upregulate the nuclear locations of CCTα binding to DNA without altering the active forms. Our findings demonstrate that de-SUMOylation is an important regulatory mechanism that governs the lipid accumulation of SREBP2 in mammalian cells. Also, the critical role of the de-SUMOylation of SREBP2 by SENP3 to exacerbate steatosis may be a potential therapeutic target for metabolic diseases like MAFLD/MASH.

Breast cancer remains the most commonly diagnosed cancer and a leading cause of cancer-related mortality among women worldwide. Despite advancements in treatment, resistance and toxicity issues highlight the urgent need for novel, effective, and safer therapeutic agents. Natural compounds are increasingly explored as promising sources of anticancer candidates due to their structural diversity and bioactivity. Among these, polyols, a class of sugar alcohols, have been reported to influence cancer cell behaviour by modulating oxidative stress, metabolic pathways and apoptosis, though their precise mechanisms and therapeutic potential remain underexplored. In this study, the anticancer potential of dulcitol, a naturally occurring polyol, was investigated in breast cancer cell lines with different molecular profiles. The cytotoxic effects of dulcitol were assessed using the MTT assay in MCF-7 (ER-positive), MDA-MB-231 (triple-negative), and MCF-10A (non-tumorigenic) breast cell lines. Mechanistic studies including flow cytometry-based cell cycle analysis, apoptosis detection (Annexin V-FITC), mitochondrial membrane potential assessment, caspase activation, and DNA damage analysis were performed on MDA-MB-231 cells. The expression levels of MMP-2 and MMP-9 genes were also evaluated using qRT-PCR. Dulcitol exhibited selective cytotoxicity against MDA-MB-231 cells at concentrations ≥ 7.5 mmol/L, while showing no significant effects on MCF-7 and MCF-10A cells. In MDA-MB-231 cells, dulcitol induced G0/G1 phase cell cycle arrest and promoted apoptosis in a dose-dependent manner. Additionally, increased caspase activity and mitochondrial depolarization were observed, indicating activation of the intrinsic apoptotic pathway. No significant DNA damage was detected; however, a significant downregulation of MMP-2 and MMP-9 expression suggested potential antimetastatic activity. Although the effective in vitro concentrations were relatively high, it should be noted that such levels are commonly required to reveal mechanistic effects in cell-based systems, and pharmacokinetic data on dulcitol are currently unavailable. Therefore, the present findings should be regarded as exploratory and hypothesis-generating, emphasizing the need for in vivo pharmacokinetic and efficacy studies to evaluate translational feasibility. In conclusion, our findings demonstrate that dulcitol selectively targets triple-negative breast cancer cells without affecting normal or ER-positive breast cells. Its ability to induce apoptosis and suppress metastatic gene expression highlights its promise as a potential natural therapeutic candidate for aggressive breast cancer subtypes.

Due to their stable nature and medical applicability properties, coumarin derivatives have fascinated medicinal chemists in the discovery of novel therapeutics. In this study, the cytotoxic/anticancer properties of some newly synthesized coumarin derivatives were aimed at designing, synthesizing, and examining cultured human neuroblastoma cells. Moreover, molecular docking studies were carried out to determine the potential mechanism. In addition, ADMET properties were evaluated to examine the drug-likeness of newly designed coumarin derivatives. To detect the cytotoxic action of compounds, 3-(4,5-dimethylthiazol-2-yl)-2,5 2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) release assays were carried out. In addition, Hoechst 33258 staining was used to detect abnormal nuclear structures. In silico, the estimates for all compounds (3a-3c) used in the study revealed that they possessed desirable physicochemical properties for bioavailability. The results of our study showed that all tested compounds exhibited remarkable cytotoxic effects on human neuroblastoma cell lines (p < 0.05). Additionally, among the compounds tested, 3a and 3c showed selective effects on neuroblastoma cells effectively at all tested concentrations. However, it was found that the selective feature of 3b, unlike the others, was concentration-dependent. Our findings clearly showed that novel coumarin derivatives exerted potent and selective anticancer effects. Results of molecular docking studies were in parallel with in vitro studies. Unlike the majority of hybrid coumarin derivatives reported in anticancer research, the present study introduces minimalist, heteroatom-free coumarins bearing bulky aliphatic substituents. These compounds demonstrated selective cytotoxicity against SH-SY5Y neuroblastoma cells and a favorable multi-target binding profile, highlighting a distinct hydrophobic volume-based SAR. As a result, the obtained data exhibited that all used molecules may be good multitarget drug alternatives for the treatment of neuroblastoma.

Age-related neuronal loss is a critical feature of neurodegenerative disorders, including Alzheimer's disease (AD), but its underlying molecular mechanisms, particularly those involving epigenetic regulation, remain incompletely understood. To analyze DNA methylation patterns in early-stage AD pathogenesis, we employed female presenilin-1/presenilin-2 double knockout (PSEN1/PSEN2 dKO, hereafter referred to as dKO) mice as a model system, This animal model have the age-dependent progressive neurodegenerative changes characteristic of neurodegenerative disorders. Using reduced representation bisulfite sequencing (RRBS), we comprehensively profiled hippocampal DNA methylation patterns in 9-month-old dKO mice compared with age-matched wild-type controls. Based on RRBS detection results, subsequent bioinformatics analysis through Bismark (v0.7.4) and DAVID v6.8 revealed 1216 differentially methylated sites across multiple chromosomes, corresponding to 796 genes. Through stringent filtering criteria, we identified 50 candidate genes exhibiting significant methylation changes in dKO hippocampal tissue as shown in hierarchical clustering. These genes were functionally enriched in DNA-dependent transcriptional regulation (p < 0.01) and protein binding activities (p < 0.05), with pathway analysis highlighting their involvement in ErbB signaling (FDR = 0.03), melanogenesis (FDR = 0.04), and oncogenic pathways (FDR = 0.05). Our study identifies significant hippocampal DNA methylation changes during the early stages of neurodegeneration in dKO mice. These epigenetic alterations highlight pathways potentially contributing to presenilin-dependent neuronal loss and suggests potential targets for modulating neurodegeneration linked to presenilin dysfunction.

Individuals with type 1 diabetes mellitus (T1DM) experience an increased risk of cardiovascular disease (CVD). To improve early detection and prevention strategies, a better understanding of early vascular changes is needed. Although coronary artery (CA) damage is a known T1DM complication, its underlying proteomic basis remains unclear. This study used a proteomic approach to identify differentially expressed proteins in the CAs of T1DM rat models, with the goal of identifying novel proteins and pathways associated with early diagnosis and prevention of CA complications. We established a streptozotocin-induced T1DM model in male Sprague-Dawley rats and conducted tandem mass tag-based quantitative proteomics and bioinformatics analyses to investigate protein expression profiles in CAs. The analyses identified 443 differentially expressed proteins, with 229 upregulated and 214 downregulated proteins. Functional annotation and pathway enrichment analyses revealed that these proteins primarily participate in lipid metabolism, the peroxisome proliferator-activated receptor (PPAR) signaling pathway, peroxisome function, and butanoate metabolism. Validation experiments using Western blotting analysis and quantitative real-time PCR confirmed significant upregulation of 3-hydroxy-3-methylglutaryl coenzyme A synthase 2 (HMGCS2), fatty acid-binding protein 4 (FABP4), and platelet glycoprotein 4 (CD36) at the protein and mRNA levels in diabetic rat CAs, consistent with the proteomic results. Our findings indicate that HMGCS2, FABP4, and CD36 may serve as important molecular markers for the early diagnosis or therapeutic targeting of CA damage in T1DM. The observed molecular changes appear to be linked to the PPAR signaling pathway. Clinical trial registration. Not applicable.

ABSTRACTThe essential trace element zinc is a well‐known modulator of T cell activation. There have been contradictory findings for the impact of zinc supplementation on T cell activation. In our study, we aimed to analyze IL‐2 production in Jurkat T cells during zinc supplementation in response to different stimuli. We found that zinc strongly suppresses IL‐2 production in Jurkat cells stimulated with phorbol 12‐myristate 13‐acetate (PMA)/calcimycin or phytohemagglutinin (PHA)/calcimycin. In contrast, zinc had no impact on IL‐2 production after PHA stimulation alone, suggesting the inhibitory zinc‐effect was linked to high calcium influx. To distinguish if the observed IL‐2 suppression is due to either potential competing effects of zinc influx or simple elevation of intracellular zinc levels, we pretreated the Jurkat cells with the zinc ionophore pyrithione for an increase of intracellular zinc before the stimulation. It was sufficient to suppress IL‐2 expression even when the cells were not further supplemented with zinc during stimulation. We propose that zinc's inhibitory effects on phosphatases stabilize the phosphorylated NFAT and thus block IL‐2 expression. Our findings underline the importance of a balanced zinc status for proper immune functions and suggest a supporting effect of zinc during immunosuppressive treatments.

Myocardial infarction (MI) represents a critical cardiovascular emergency, standing as a leading cause of global mortality. ATP, a typical damage-associated molecular pattern, is stored in cells at high concentrations. Upon cellular injury, hypoxia, or necrosis, substantial quantities of ATP efflux into the extracellular space, activating P2X7 receptors, thereby initiating multiple signaling cascades. In vivo studies demonstrated coordinated upregulation of P2X7 and autophagy-related proteins in the infarcted border zone. Transcriptome sequencing revealed Nox4 overexpression in the myocardial tissue post-infarction; furthermore, administration of the P2X7 receptor antagonist A740003 effectively reduced both autophagy-related protein levels and Nox4 expression. In vitro experiments indicated that hypoxia induced upregulation of Nox4, p-PERK/PERK, ATF4, Beclin-1, and ATG5 in cardiomyocytes, A740003 could inhibit the expression of these proteins, while overexpression of Nox4 counteracted this effect. Collectively, our findings indicated that the P2X7 receptor expression was elevated in the infarcted border zone following MI and implicated its role in excessive autophagy induced by hypoxia in cardiomyocytes-at least partially through the Nox4/PERK/ATF4 pathway, thereby exacerbating myocardial injury following MI.

Aging is considered the contributory accumulation of abruptions occurring through cell signaling cascades, which ultimately cause changes in physical functions, cell fate, and damage across all organ systems. DNA damage response (DDR) also occurs through telomere shortening, tumor formation, mitochondrial dysfunction, and so forth. Cellular aging occurs through cell cycle arrest, which is the result of extended DDR cascade signaling networks via MDC1, 53BP1, H2AX, ATM, ARF, P53, P13-Akt, BRAF, Sirtuins, NAD + , and so forth. These persistent cell cycle arrests initiated by DDR and other associated stress-induced signals promote a permanent state of cell cycle arrest called senescence-associated secretory phenotype (SASP). However, cellular aging gets accelerated with faulty DNA repair systems, and the produced senescent cells further generate various promoting contributors to age-related dysfunctional diseases including SASP. Any changes to these factors contribute to age-related disease development. Therefore, this review explores anti-aging factors targeting DDR and SASP regulation and their detailed signaling networks. In addition, it allows researchers to identify anti-aging targets and anti-aging therapeutic strategies based on identified and nonidentified targets.