Estradiol (E2), a sex steroid hormone molecule, plays a key role in regulating the actin and shape dynamics of cells in a multitude of normal and pathophysiological conditions. While cytoskeletal rearrangements, membrane dynamics, and cellular protrusions are intimately involved in cell motility and invasiveness, little is known about the impact of E2 on these processes in estrogen-dependent epithelial cells. In this study, we quantified the impact of E2 on epithelial cell shape and actin dynamics. 12Z human endometriotic epithelial cells were transfected with LifeAct-GFP and observed with lattice lightsheet microscopy, a new imaging technique fast enough to capture 3D dynamics on second timescales. E2, when applied for 24 h, significantly decreased cell circularity, solidity, and rate of change of circularity, indicating a transition to a more elongated and less variable morphology. 24-h E2 treatment also induced the formation of large membrane protrusions reminiscent of invadopodia and led to a more disordered flow of actin within those protrusions. However, these effects were not seen after 15 min of E2 treatment, suggesting that longer-term signaling is required to drive these structural changes. Together, these results suggest that E2 modulates actin polymerization and membrane protrusion dynamics in endometriotic epithelial cells and may prime them for cell invasion. This work highlights a role for hormonal signaling in mediating cytoskeletal plasticity and migratory cell phenotypes.
Autosomal Dominant Polycystic Kidney Disease is caused by loss-of-function mutations in PKD1 or PKD2 genes, leading to reduced polycystin protein levels. Increasing PKD1 expression via CRISPR activation (CRISPRa) represents a promising therapeutic strategy; however, delivery of large CRISPRa plasmids into renal epithelial cells, and particularly primary cells, remains inefficient due to size-related barriers. We aimed to enable Pkd1 transactivation by miniaturizing CRISPRa plasmids into ~ 6 kb vectors using a one-pot method to enhance cellular uptake in mouse kidney epithelial cells. Using type IIS restriction enzymes, we excised the mammalian expression cassette from full-length large 9-11 kB plasmids. The excised cassette was engineered to have complimentary overhangs. Thermocycling with T4 DNA ligase promoted circularization of the excised cassette (forming ~ 6kB mini-CRISPRa vectors), and T5 exonuclease digestion removed residual backbone fragments. These mini vectors substantially enhanced nucleofection efficiency from 16.10% ± 0.53 to 54.17% ± 2.10 in Pkd1RC/- cells, and from 10.14% ± 1.40 to 31.27% ± 0.12 in primary Pkd1RC/Cond; Pkhd1Cre+ cells. Functionally, the mini-CRISPRa plasmid (mdCas9-VPR) with Pkd1-targeting sgRNAs induced robust endogenous Pkd1 upregulation compared with non-targeting controls: a 4.1-fold increase in Pkd1RC/- cells (p < 0.001) and a 2.9-fold increase in primary cells (p < 0.001). Full-length plasmids produced no significant activation in either cell type. Miniaturization of CRISPRa vectors with this one-pot approach overcomes delivery limitations in hard-to-transfect renal epithelial cells and enables efficient, functional Pkd1 activation, in vitro.
The clinical translation of prostate-specific membrane antigen (PSMA)-directed chimeric antigen receptor (CAR) T-cell therapy for metastatic castration-resistant prostate cancer (mCRPC) has reached a critical impasse. Despite compelling preclinical rationale and early biological activity, durable clinical responses remain scarce, constrained by three core solid tumor challenges: a profoundly immunosuppressive/metabolically hostile tumor microenvironment (TME), pervasive antigen heterogeneity driving immune escape, and intrinsic limitations in T-cell fitness and in vivo persistence. This review synthesizes the current translational landscape (updated to February 2026), and posits a tripartite synergistic framework to systematically deconstruct these barriers: (1) advances in CAR synthetic biology; (2) active TME reprogramming via armored CAR-T cells, stromal-targeting agents, and rational combinations; (3) next-generation cellular product paradigms, with a focus on stem cell-derived immune effectors. Emerging platforms, including induced pluripotent stem cell (iPSC)-derived CAR-T, CAR-natural killer (NK) cells, and CAR-macrophages, offer unprecedented opportunities to overcome autologous product limitations via off-the-shelf availability, enhanced persistence, and intrinsic TME resistance. We further delineate a translational roadmap emphasizing biomarker-driven adaptive trials, predictive humanized preclinical models, and accessibility strategies. All core claims are graded using the 2011 Oxford Centre for Evidence-Based Medicine (OCEBM) Levels of Evidence to ensure academic rigor. This work provides a strategic blueprint to advance PSMA-CAR-T therapy toward curative-intent mCRPC treatment, with insights broadly applicable to next-generation stem cell-derived immunotherapies.
Coffee extracts contain numerous bioactive compounds. Given the dietary link between coffee consumption and colorectal cancer, this study compared the effects of roasted and green (unroasted) coffee extracts on human colorectal cancer cells (HCT116) and non-cancerous fibroblasts (BJ-5ta) to evaluate how processing influences proliferation and molecular signaling. Real-time cell analysis (RTCA), qRT-PCR, and label-free quantitative proteomic analysis were performed to assess cellular responses. MTS and RTCA showed that in BJ-5Ta fibroblasts, coffee extracts increased proliferation in the order CNR < CAR < CAU < CNU, whereas the trend was reversed in HCT116 cancer cells. Proteomic analysis revealed that in BJ-5Ta cells, unroasted coffee extract caused downregulation of the ribosome pathway, and natural coffee extract caused downregulation of the gap junction pathway, indicating reduced protein synthesis and cell-cell communication as a potential stress-adaptive response. In contrast, in HCT116 cells, unroasted coffee extract upregulated the ribosome pathway. Also, natural coffee extract upregulated the pentose phosphate pathway in HCT116 cells, which may enhance NADPH production and reduce oxidative stress. Current evidence suggests coffee's bioactive compounds may have different effects varying by coffee extract type and their preparation.
The ovary is one of the first organs to lose functionality with age. We found that aging of the Drosophila ovary is characterized by an accumulation of phenotypes in the somatic compartment, including failure of the follicle cells to encapsulate germ-cell cysts, an extended S phase, and increased DNA damage. In aged ovaries, follicle encapsulation defects are associated with the lack of a germ-cell cyst checkpoint in early oogenesis. Single-cell RNA sequencing revealed that, across all cell types in the ovary, cells in the follicle lineage have the highest number of differentially expressed genes. Overexpression of Atg8a, a key autophagy machinery gene homologous to mammalian LC3, specifically in follicle cells prevents age-associated decline in the follicle epithelium and loss of reproductive capacity. Collectively, these findings demonstrate that genetic manipulation of a small population of ovarian somatic cells is sufficient to improve both cell-autonomous and non-autonomous features of reproductive aging.
The CHI3L1 protein supports various types of cancer progression and metastasis, where the background players were the upregulation of angiogenesis and microenvironment modulation. In glioblastoma (GB), high vascularisation is a key feature of these tumours, making anti-angiogenic therapy a pivotal treatment option. Autophagy, a dual-faced mechanism, may be useful as a target in GB treatment. This work presents the role of the CHI3L1 protein in autophagy in GB. U-87 MG glioblastoma cells and a GB spheroid model consisting of U-87 MG cells, macrophages and endothelial cells were used in the studies. A new tissue-like phantom was designed for the radiotherapy of spheroids. The role of CHI3L1 in autophagy regulation was analysed after cell starvation and treatment with G721-0282, a small molecule inhibitor of CHI3L1, as well as X-ray doses. The biological responses were evaluated using the Western blot method, immunohistochemical reactions, DHT (digital holographic tomography) and O-PTIR (optical phototermal infrared spectroscopy) to analyse biological responses in GB spheroids. Induction of autophagy in glioblastoma cells after CHI3L1 inhibition was observed, as well changes in CHI3L1 expression levels occurred after cell starvation and different X-ray radiation doses. Secondary structure changes in glycoproteins and decreases in nucleic acids were observed in O-PTIR. The expression of CHI3L1 in glioblastoma cells may be precisely regulated by an adverse environment, such as nutrient depletion or anti-tumour treatment. Inhibiting the CHI3L1 protein leads to upregulation of autophagy, meaning that CHI3L1 inhibitors could be used as a new therapy to upregulate autophagy in GB.
Durable CD4+ T cell memory is essential against mycobacterial infection, yet activation-induced cell death (AICD) limits the survival of activated clones after BCG vaccination. The upstream, cell-intrinsic brakes that govern this bottleneck remain incompletely defined. Combining transcriptomics, loss-of-function tests, and in vivo engineering, we identify Dapk1 as a pro-apoptotic regulator that is downregulated in memory CD4+ T cells and promotes activation-induced death in T cell models. We develop an activation-gated AAV platform in which an NFAT-IL-2 promoter drives Cre to flip a FLEXed U6-shRNA cassette, and package them into a single AIO vector. This design confines Dapk1 silencing to antigen-experienced T cells, preferentially within the CD4+CD44hi compartment. In BCG-vaccinated mice, transient activation-linked Dapk1 inhibition expands CD4+ TCM cells, enhances IL-2 and Th1-skewed recall responses, lowers pulmonary, and splenic bacterial burdens after M.tb challenge. These findings highlight a strategy to selectively modulate intrinsic death pathways during immune priming for strengthening vaccine-elicited T cell memory.
IgA nephropathy (IgAN) is characterized by deposition of IgA antibodies (Abs) in the glomerular mesangium. In IgAN, the mechanism by which IgA Abs are selectively deposited in the glomerular mesangial region remain unclear. Current study reported the presence of autoantibodies-IgA against β2 spectrin of mesangial cells in sera of IgAN model mice (gddY) and patients with IgAN, and identified β2-spectrin as the target antigen. And it found that IgA+ plasmablasts (PBs) secreting anti-β2-spectrin IgA Abs accumulated in the kidneys of gddY mice. These PBs exhibited a substantial accumulation of somatic hypermutations in both their heavy- and light-chain variable region genes. Lymphocytes from kidneys, renal lymph node, spleen, bone marrow and nasopharyngeal associated lymphoid tissue of 3-week-old ddY mice were analyzed by flow cytometry. To deplete CD4+ T cells in gddY mice, anti-CD4 monoclonal Abs (mAbs) were administered from 3 weeks of age. Renal phenotype (blood urea nitrogen (BUN), urinary albumin and renal pathology), the cell numbers of IgA+ PBs in the kidney and serum anti-β2 spectrin Abs were evaluated by Fuji-DRY-CHEM 7000V, enzyme-linked immunosorbent assay, microscopy, flow cytometry and western blotting, respectively. Depletion of CD4+ cells in gddY mice caused significant reduction of the number of IgA+ PBs spontaneously accumulating in the kidneys in the anti-CD4 treatment (29.0 ± 12.1/105 cells) than the control (154.0 ± 55.0/105 cells). However, The results showed the level of urinary albumin and BUN were no difference in in the anti-CD4 treatment (5627.0 ± 1316.2 μg/dL, 40.3 ± 4.5 mg/dL, respectively) compared to the control (4766.5 ± 809.2 μg/dL, 69.9 ± 33.2, respectively). And also the treatment did not affect the production of anti-β2-spectrin IgA Abs in sera, glomerular IgA deposition nor pathology. Our data suggest that the serum anti-β2-spectrin IgA Abs are produced in a CD4+ T cell-independent manner and the PBs infiltrating in the kidney are not the major source of serum anti-β2-spectrin IgA Abs at least in animal model. The present study will help to elucidate the mechanism of production of anti-β2-spectrin IgA Abs.
Chemotherapy-induced alopecia (CIA) remains one of the most distressing adverse effects of cancer therapy. Yet, no therapy is available to selectively protect healthy hair follicles (HFs) and their epithelial stem cells (eHFSCs) from chemotherapy-induced damage without awarding potential survival benefits to cancer cells. Here, we report how human HFs can be protected against 2 lead CIA-inducing chemotherapeutics by inducing selective transient cell cycle arrest. Pretreating scalp HFs before chemotherapy exposure ex vivo with ALRN-6924, a clinical-stage "stapled peptide" drug that binds with high affinity to key endogenous inhibitors of p53, selectively activated p53 signaling only in cells with wild-type TP53 genotype and upregulated p21. This led to temporary cell cycle arrest in healthy tissues without protecting TP53-mutant cancer cells and mitigated chemotherapy-induced HF damage on multiple levels, including excessive hair matrix apoptosis, premature catagen, pigmentary abnormalities, "mitotic catastrophe," and micronucleation. It also protected eHFSCs against DNA damage, apoptosis, and pathological epithelial-mesenchymal transition. Notably, even topically applied ALRN-6924 afforded relative chemotherapy protection ex vivo. These results provide proof of principle for a strategy to selectively protect rapidly proliferating healthy epithelial tissues and their stem cells in patients with TP53-mutant cancers, which promises to protect against acute and permanent CIA.
Progesterone production by the corpus luteum is essential for embryo implantation and early pregnancy maintenance and is acutely stimulated by luteinizing hormone (LH). While LH signaling through protein kinase A (PKA) is well established, downstream regulatory networks that constrain or shape luteal steroidogenesis remain incompletely defined. Here, we identify a previously unrecognized role for the Hippo signaling pathway in regulating luteal progesterone production. Using primary bovine luteal cells isolated from corpora lutea, we examined the relationship between LH/PKA signaling and Hippo signaling pathway activity. Expression, phosphorylation, and subcellular localization of Hippo pathway components were assessed by immunoblotting and nuclear fractionation. Progesterone production was quantified by ELISA. LH-induced transcriptional responses were analyzed using upstream regulator prediction from RNA sequencing. Functional roles of YAP1 and TAZ were evaluated using adenoviral overexpression of constitutively active mutants and siRNA-mediated knockdown. Hippo pathway components were enriched in luteal cells relative to follicular precursors. LH rapidly increased phosphorylation and cytoplasmic sequestration of YAP1 and TAZ in small luteal cells through PKA. Pharmacologic inhibition of LATS1/2 did not alter LH-stimulated progesterone production, suggesting that LH-induced steroidogenesis is not limited by LATS-dependent regulation of YAP1/TAZ. Sustained activation of YAP1 or TAZ suppressed LH-induced progesterone synthesis, whereas depletion of either factor enhanced progesterone output. Consistently, RNA-seq analysis identified YAP1/TAZ, and TEAD transcription factors as inhibited upstream regulators following LH stimulation in small luteal cells. Our findings support a model in which LH, via PKA and activation of Hippo signaling promotes progesterone synthesis by restraining YAP1/TAZ transcriptional activity in small luteal cells. This work identifies Hippo signaling as an unrecognized regulatory layer in luteal steroidogenesis and highlights YAP1/TAZ as potential therapeutic target for luteal insufficiency and infertility.
Targeting DNA payloads into human induced pluripotent stem cells (hiPSCs) typically requires multiple inefficient steps, slowing the testing of gene circuits and cell-fate programmes. Here we show that STRAIGHT-IN Dual enables simultaneous, allele-specific, single-copy integration of two DNA constructs efficiently within 1 week. STRAIGHT-IN Dual leverages the STRAIGHT-IN platform for near-scarless payload integration, facilitating the recycling of components for further modifications. Using STRAIGHT-IN Dual, we investigate how promoter choice and gene syntax influence transgene silencing and how these design features affect reporter expression and forward programming of hiPSCs into neurons, motor neurons and endothelial cells. We also incorporate a grazoprevir-inducible synthetic gene switch that complements tetracycline-inducible control, providing tunable and temporally controlled expression of different transcription factors within the same cell. STRAIGHT-IN Dual generates homogeneous engineered hiPSC populations, accelerating synthetic biology design-build-test cycles in stem cells and enabling controlled comparisons of circuit performances.
Increases in cytoplasmic free Ca2+ ions ([Ca2+]) are a critical signal in pancreatic islet beta-cells and are usually required for insulin secretion in response to glucose or other secretagogues. Changes in Ca2+, monitored using high-speed imaging across individual or multiple planes of the islet, can be used to explore the functional networks of beta-cells required for the precise regulation of insulin secretion. These networks are composed of functionally distinct beta-cell subpopulations: first-responders, highly connected hubs, and leader beta-cells, which initiate, connect, and dictate the pattern of spatially organized Ca2+ oscillations, respectively. Alterations in Ca2+ coordination among beta-cells contribute to defective insulin secretion, which underlies all forms of diabetes mellitus. Here, we provide a detailed protocol to perform Ca2+ imaging in isolated rodent islets, focusing on mouse islets expressing the genetic Ca2+ sensor, GCaMP6. We provide a step-by-step guide to evaluate general parameters of islet Ca2+dynamics, coordination, connectivity, and identification of specific functional subpopulations. This approach can be applied to investigate the role of Ca2+ dynamics and coordination in tissues where coordination is critical for normal function.
Colorectal cancer (CRC) remains one of the leading causes of cancer mortality, with a poor survival rate of less than 15%. Imatinib (IM) and Zebularine (ZEB) alone have shown potential effects in CRC treatment, but their combination has not been thoroughly studied. This study investigates the potential effects of IM and ZEB in colon cancer cells to provide a novel therapeutic agent for managing CRC. Cell growth inhibition, oxidative stress markers, and cell cycle progression were assessed in HCT-116 cells treated with IM, ZEB, and their combinations. ZEB uptake levels were analyzed by LC-MS/MS, apoptosis was quantified by flow cytometry, and gene expression changes were analyzed by qPCR. The expression of metastatic markers, apoptotic regulators, and EGFR was assessed. Both IM and ZEB inhibited cell growth in a concentration-dependent manner, and their combination showed synergistic effects. The combination significantly enhanced oxidative stress. The combination therapy increased apoptosis and necrosis. Furthermore, the combination induced significant S-phase arrest in the cell cycle. The combination treatment reduced metastatic markers (MMP9, MMP2), and the apoptotic marker Caspase-9 was upregulated. Additionally, the Bcl-2 protein, a key regulator of apoptosis, was significantly downexpressed. Remarkably, the combination treatment showed significant inhibition in EGFR levels. Both IM and ZEB combination showed promise in the management of CRC by inducing oxidative stress, promoting apoptosis, and modulating critical genes involved in metastasis and apoptosis. Further investigation will be needed to verify their application in preclinical and clinical settings.
Advanced biomaterials-based in vitro platforms are increasingly required to overcome the limited predictive power of conventional 2D cell cultures in colorectal cancer (CRC) drug screening. Herein, we report the development of a biomimetic, multicellular 3D CRC model based on gelatin methacrylate (GelMA) hydrogels, designed to recapitulate key structural and biological features of the tumor microenvironment. The platform integrates a vascularized hydrogel compartment with human endothelial cells, combined with cancer-associated fibroblasts and macrophages, enabling controlled tumor-stroma-vessel interactions within a physiologically relevant architecture. The GelMA hydrogels were comprehensively characterized, and their role in regulating endothelial viability, migration, and angiogenic marker expression was systematically evaluated. The drug screening capability of the platform was assessed using 5-fluorouracil (5-FU). Comparative analyses revealed that 3D cultures exhibited attenuated cytotoxicity, oxidative stress, and apoptotic responses relative to 2D monolayers, particularly at lower drug concentrations and prolonged exposure times. These findings demonstrate that GelMA-based microenvironment actively modulates cellular drug responses through multicellular interactions and diffusion-mediated effects, rather than acting as a passive scaffold. Overall, this study establishes a functional 3D in vitro platform that provides improved physiological relevance and predictive capability for preclinical CRC drug screening, while offering a human-relevant alternative aligned with the principles of the 3Rs.
Circulating tumor cell (CTC) is an evidenced biomarker for early cancer diagnosis and assessment of treatment response. This necessitates increasing attempt to develop new method for detection of CTCs with highly enriching and anti-interference capacity. Herein, we built a simple platform for CTC detection by combining aptamer-mediated specific recognition with affinity magnetic levitation (Maglev). The method involved modification of DNA tetrahedron (TDN) using SYL3C aptamer as tentacles, assembly of aptamer conjugated-TDN on microsphere by azide-alkyne click chemistry, enrichment of CTCs by the activated microspheres, and detection of CTCs by affinity Maglev. These yielded an aptamer sensor resembling octopus tentacles to enhance the capture selectivity and efficiency of CTCs. Once CTCs were captured, a microsphere@TDN@CTCs complex was formed, resulting in a change in the overall density. The number of CTCs was calculated by measuring the levitation height difference between microspheres pro- and post-binding of CTCs in affinity Maglev. An example study on MCF-7 cells exhibited a detection of 8.81 cells mL-1 in buffer solution, with repeatability characterized by a relative standard deviation of less than 4.48%, in comparison to 9.08 cell mL-1 in mimic clinical sample with a relative standard deviation of 6.98%. Analysis of mimic clinical samples demonstrated that the method was effective for the specific detection of CTCs with robust resistance to interference from co-existing substances in plasma. It is possible to serve as an early diagnostic alternative for clinically relevant cancers once large number of clinical trials are performed.
Human gingival fibroblasts (HGFs) are stromal cells that maintain periodontal tissue structure and extracellular matrix (ECM) dynamics. ECM stiffness serves as a physical cue that regulates HGF behavior and secretory profiles. This study investigated how substrate stiffness modulates the secretome of HGFs and observed the subsequent effects of this secretome on the osteogenic differentiation of human periodontal ligament cells (HPDLCs). HGFs isolated from healthy donors were cultured on polydimethylsiloxane substrates, representing soft or hard periodontal tissue under normal and lipopolysaccharide (LPS)-induced inflammatory conditions. The expression of cytokines and chemokines was analyzed using qRT-PCR and ELISA, with p38 MAPK inhibitors used to identify stiffness-associated signaling involvement. HPDLCs were treated with conditioned medium from HGFs (HGF-CM) under osteogenic induction, osteogenic marker expression was examined using qRT-PCR and immunofluorescence, with mineralization assessed by Alizarin red S staining. To establish mechanistic causality, functional blocking was conducted using a C-X-C motif chemokine receptor 4 (CXCR4) inhibitor. Hard substrates significantly increased the expression of anti-inflammatory cytokines and the C-X-C motif chemokine ligand 12 (CXCL12) in HGFs, whereas inhibition of p38 mitogen-activated protein kinase (MAPK) activity attenuated stiffness-associated CXCL12 expression. Under LPS-induced inflammatory conditions, hard substrates-maintained matrix metalloproteinase-9 suppression and tissue inhibitor of metalloproteinases 1 upregulation, although CXCL12 protein levels were decreased. Furthermore, HPDLCs treated with HGF-CM derived from hard substrates in osteogenic induction media exhibited elevated CXCR4 expression, increased osteogenic marker levels at days 14 and 21, and enhanced mineral deposition compared to those treated with HGF-CM from soft substrates. In addition, functional blocking with a CXCR4 inhibitor significantly reduced the expression of osteogenic markers (ALP, RUNX2, COL1A1, and OSX) and confirmed a subsequent decrease in matrix mineralization. Substrate stiffness modulated the paracrine behavior of HGFs, with CXCL12 serving as a representative example of a stiffness-responsive factor. These alterations in the HGF-derived secretome were associated with altered osteogenic and inflammatory responses in HPDLCs. These findings support the influence of the physical microenvironment on fibroblast-periodontal ligament cell interactions on anti-inflammatory response and periodontal tissue stabilization.
Ferroptosis is a regulated form of cell death driven by iron-dependent lipid peroxidation, with emerging relevance in cancer biology, particularly in therapy-resistant tumors such as colorectal cancer. Accurate and reproducible methods to monitor ferroptosis are essential for understanding its mechanisms and therapeutic potential. In this methodological paper, we present an optimized protocol for the evaluation of lipid peroxidation as a surrogate marker of ferroptosis in colon cancer cell models. Specifically, we describe an approach for quantifying malonylaldehyde and hydroxyalkenals (MDA+ 4-HDA), as key byproducts of lipid oxidation, using a colorimetric assay adapted for cell culture. This methodological framework provides a reliable basis for dissecting ferroptotic responses and for evaluating the activity of ferroptosis-inducing compounds in colorectal cancer research.
Aluminum (Al) in the form of γ-aluminum oxyhydroxide (γ-AlOOH) nanoparticles has been used as a vaccine adjuvant for nearly a century. At injection sites, animals and humans develop granulomatous inflammation, in which macrophages may contain intracellular crystalline-like structures, so-called crystalloid bodies (CBs), morphologically distinct from the injected adjuvant. This raised the hypothesis that CBs may originate from the in vivo crystallization of γ-AlOOH within macrophages. To test this, six sheep received two subcutaneous doses of a γ-AlOOH-adjuvanted viral vaccine, while six controls received the same antigen without an adjuvant. All injection sites (AlOOH: n = 12; controls: n = 12) were examined by hematoxylin and eosin, modified aluminum hematoxylin, and lumogallion fluorescence. Al and CBs occurred exclusively in AlOOH-vaccinated tissues. The adjuvant stock and vaccine formulations were characterized ex situ using a combination of structural and imaging techniques. Four granulomas containing CBs, collected 133 days postinjection, were further examined using in situ imaging and crystallographic approaches. No crystalline structures resembling CBs were identified in the injected products; only γ-AlOOH nanoparticles were present. In post-mortem granulomas, macrophages contained Al-positive vacuoles filled with nanoparticle aggregates compatible with γ-AlOOH, often adjacent to CBs. Crystallographic analyses demonstrated that CBs were highly ordered γ-Al2O3 microcrystals, and advanced SEM-EDS with elemental mapping similarly distinguished γ-AlOOH nanoparticles and CBs as separate phases. These findings support an in vivo formation of γ-Al2O3 microcrystals from γ-AlOOH nanoparticles within macrophages under physiological conditions. To our knowledge, these findings provide evidence consistent with Al crystallization within mammalian cells and suggest that macrophage phagolysosomal conditions may represent a previously unrecognized biomimetic pathway for γ-Al2O3 formation. This finding may offer a biological model for the development of green and low-energy synthesis strategies for γ-Al2O3, with potential industrial applications as well as relevance to immunotoxicology, pathology, and cell biology in the context of vaccine adjuvants.
Sex-based differences influence tumor biology, immune responses, and treatment outcomes in renal cell carcinoma (RCC), yet most computational models do not jointly incorporate sex hormones, immune composition, and tumor genetic evolution. Agent-based models (ABMs) effectively simulate tumor-immune interactions but are rarely extended to include sex-specific modulation or machine learning-based optimization. This study enhanced an agent-based learning model (ALM) to simulate RCC progression and treatment response by integrating hormonal effects, immune interactions, and tumor genetic adaptation with data-driven tuning. An RCC-specific ALM was developed incorporating immune agents (CD8+, NK, Treg, dendritic cells), hormone-sensitive mechanisms, tumor genetic modules, and effects of immune checkpoint inhibitors (ICIs) and tyrosine kinase inhibitors (TKIs). Tumor evolution was modeled using a genetic algorithm simulating promoter and gene mutations, with fitness defined by immune evasion and proliferation advantages. Model parameters were optimized using clinical outcomes from the ARON dataset via the Optuna framework, and performance was assessed using concordance index (CI) and mean squared error (MSE). Simulations reproduced sex-specific treatment responses. Female models showed delayed initial responses but stronger late immune activation and rapid tumor regression, whereas male models exhibited more stable early responses but greater tumor resilience driven by genetic adaptations. Adaptive learning showed capability of reducing prediction error with both fitness functions. This ALM offers an exploratory framework to provide preliminary insights into how sex hormones, immune dynamics, and tumor genetics may jointly contribute to shaping RCC treatment outcomes. Although the limited sample size constrains validation, the results suggest the potential of combining ABMs with biological data-driven optimization to support patient prediction and call for further investigation in larger cohorts.
Hepatocellular carcinoma (HCC) is a leading cause of cancer-related mortality worldwide, largely due to late diagnosis, molecular heterogeneity, and limited prognostic biomarkers. Aberrant protein phosphorylation plays a critical role in cancer progression by regulating DNA damage response, cell cycle control, and signaling pathways; however, the prognostic relevance of phosphorylation events in key DNA topology-related proteins remains incompletely understood. This study aimed to investigate the prognostic significance of phosphorylation of TOP1, TOP2A, TOP2B, and C1orf35 in HCC and to characterize their associated molecular features to identify potential diagnostic and therapeutic biomarkers. Publicly available HCC phosphoproteomic and proteomic datasets were analyzed to identify significantly upregulated phosphorylation sites of TOP1, TOP2A, TOP2B, and C1orf35. Integrated bioinformatics and machine learning approaches were applied, including Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), protein-protein interaction (PPI) network analysis, drug-gene interaction analysis, and survival analyses (overall and disease-free survival). A total of 11,547 phosphorylation sites corresponding to 4043 phosphoproteins were quantified from 159 HCC patients. Phosphorylation of TOP1, TOP2A, TOP2B, and C1orf35 was significantly upregulated. Enriched pathways included DNA damage response, homologous recombination repair, cell cycle regulation, SUMOylation, and TP53 signaling. PPI analysis identified these proteins as highly interconnected hub nodes. Elevated expression was significantly associated with poor clinical outcomes. Phosphorylated TOP1, TOP2A, TOP2B, and C1orf35 are strongly associated with HCC progression and poor prognosis, highlighting their potential as prognostic biomarkers and therapeutic targets. These insights not only enhance our understanding of the complex molecular mechanisms underlying HCC but also offer promising avenues for the identification of novel therapeutic targets.