Search Results (137)

Ovarian cancer (OC) remains one of the deadliest gynecological malignancies, largely due to late diagnosis and the emergence of resistance to platinum–based chemotherapy. Long non–coding RNAs (lncRNAs) have recently emerged as key regulators of tumor progression and therapeutic adaptation. In this study, we performed integrative transcriptomic profiling of patient–derived TCGA ovarian tumor samples and carboplatin–resistant A2780 (CBDCA–R–A2780) cells to identify lncRNAs whose dysregulation overlaps between a cell–line resistance model and patient tumors. Our analyses revealed extensive transcriptional remodeling across both datasets, with MNX1–AS1 consistently emerging as a strongly deregulated transcript. Differential expression analysis showed robust upregulation of MNX1–AS1 in resistant cells and tumor tissues, accompanied by correlations with epithelial–mesenchymal transition (EMT)–related transcription factors such as FOXA1 and SNAI2 and inverse associations with epithelial markers including CDH1. Computational predictions using RIblast identified specific MNX1–AS1 binding regions with candidate miRNAs and mRNAs, prioritizing EMT–related transcripts (e.g., SNAI2, FOXA1, ZEB1) with favorable hybridization energies for future validation. Additional prioritized interactors included genes linked to stress response (IER2, FOSB) and invasion (MMP11, MMP1). Because A2780 has been discussed as an endometrioid–like/non–serous ovarian cancer model, mechanistic inferences primarily apply to this in vitro context, while TCGA analyses provide associative support rather than mechanistic validation. Collectively, these findings highlight MNX1–AS1 as a candidate regulator associated with transcriptional reprogramming in OC and a promising prognostic biomarker warranting further functional testing. Full article

Background: Kölliker’s organ (KO) support cells undergo orderly, time-dependent degeneration that is essential for auditory development and is accompanied by precisely regulated autophagic activity; however, the molecular hierarchy linking autophagy to this remodeling remains obscure. This study aimed to elucidate the regulatory mechanisms connecting autophagic flux to lysosomal biogenesis and auditory function during cochlear development. Method: We established an Atg5^flox/flox; Sox2Cre+ mouse model with deletion of the autophagy gene Atg5 in cochlear-supporting cells. Auditory function was assessed via Auditory Brainstem Response (ABR) testing. Transcriptomic profiling of the neonatal basilar membrane was performed to screen for downstream targets. Mechanistic validation included spatiotemporal immunofluorescence mapping (E18–P30) and in vitro functional assays using siRNA-mediated knockdown and lysosomal tracking. Results: At 2 months of age, Atg5^flox/flox; Sox2Cre+ mice exhibited moderate-to-severe sensorineural hearing loss accompanied by significant outer hair cell loss. Bulk RNA-seq of the basilar membrane identified fork-head box A3 (Foxa3) as a significantly downregulated transcription factor within the lysosomal–autophagy network. Spatiotemporal immunolabelling from embryonic day 18 to postnatal day 30 revealed that FOXA3 expression becomes progressively restricted to KO cells during postnatal development, with ATG5 loss reducing FOXA3 protein levels by 62.4%. In vitro, deficiency of either Atg5 or Foxa3 in primary KO cells resulted in comparable reductions in LAMP1-positive puncta. Conclusions: These findings support a model wherein the ATG5-FOXA3 axis contributes to lysosomal biogenesis in developing KO cells, with implications for understanding mechanisms of congenital sensorineural hearing loss. Full article

Background/Objectives: TP53 mutation or deletion status is important for determining cellular responses to DNA-damaging drugs. Oxaliplatin (OXA) is combined with the fluoropyrimidine (FP) drug 5-fluorouracil (5-FU) in the FOLFOX regimen used to treat advanced colorectal cancer (CRC). However, the effects of TP53 deletion on 5-FU + OXA synergy are not well known. We investigated potential synergy between OXA and 5-FU and compared it with OXA synergy with a novel polymeric FP, CF10, in four cell lines harboring either wild-type (WT) or TP53-null status. Methods: Using CompuSyn and the highest single agent (HSA) models, we compared synergy between CF10 and OXA (COXA) and between 5-FU and OXA (FOXA). Cell cycle analysis was performed, as was Western blot quantification of canonical DNA damage pathway proteins. Likewise, immunofluorescent and confocal analysis allowed us to compare topoisomerase 1 cleavage complex and double-strand DNA break formation. Results: COXA synergy displayed minimal TP53 dependence with greatly improved potency compared to FOXA. COXA synergy resulted from OXA increasing: (i) Topoisomerase 1 (Top1) cleavage complex formation; (ii) DNA double-strand breaks (DSBs), and (iii) Checkpoint Kinase 1 and 2 (p-Chk1/2) phosphorylation, consistent with increased replication stress. Additionally, increased S-phase entry in TP53-null cells enhanced synergy between CF10, 5-FU, and OXA as S-phase drugs. Conclusions: Our results demonstrate that OXA synergizes with CF10 more effectively than with 5-FU through enhanced replication stress in both WT and TP53-null cells by causing greater Top1-mediated DNA double-strand breaks. Our studies provide a foundation for further testing of this combination in an orthotopic liver metastatic setting and eventual clinical development. Full article

Background/Objectives: CEU-938, an innovative antimicrotubule prodrug bioactivated by cytochrome P450 1A1 (CYP1A1), represents a promising targeted alternative for cancer cells overexpressing this enzyme. To optimize its clinical utility and minimize off-target effects in breast cancer (BC) patients, this study aims to identify predictive biomarkers of CEU-938 efficacy. Methods: The antiproliferative activity of CEU-938 was assessed across a panel of 39 human breast cancer and non-tumorigenic cell lines. Differential expression analyses were subsequently performed to distinguish CEU-938-responsive from non-responsive cell lines using a threshold of 1000 nM. Candidate biomarkers identified through this approach were then validated by RT-qPCR and Western blot analyses. Results: CEU-938 demonstrated marked and selective antiproliferative activity across molecular subtypes of human breast cancer, with efficacy observed in approximately 40% of triple-negative breast cancer (TNBC), 70% of estrogen receptor-positive (ER⁺), and 80% of human epidermal growth factor receptor 2-positive (HER2⁺) breast cancer cell lines, while sparing non-tumorigenic human breast cells (MCF 10A, MCF-12A, 184B5). Differential expression analysis identified five candidate biomarkers associated with CEU-938 responsiveness, namely, FOXA1 (log2-fold change (LFC) = 3.1), RAB25 (LFC = 3.8), RHOV (LFC = 2.9), PRKCH (LFC = 1.6), and HDAC9 (LFC = −1.7). Among these, FOXA1 and RAB25 robustly validated by RT-qPCR and Western blot analyses, showing strong inverse correlations with CEU-938 sensitivity (Spearman correlation coefficients of −0.82 and −0.61, respectively, at the protein level). The predictive value of FOXA1 and RAB25 was further confirmed by Western blot analyses in two independent breast cell line models, the non-responsive MCF-12A and the responsive MDA-kb2. Conclusions: Collectively, these findings identify FOXA1 and RAB25 as robust predictive biomarkers of response to CEU-938. Notably, FOXA1 and RAB25 are strongly implicated in breast cancer biology, and FOXA1 has been directly linked to the aryl hydrocarbon receptor (AHR), the main regulator of CYP1A1. These results position CEU-938 as a strong precision-therapy candidate that combines target selectivity, a favorable toxicity profile, and biomarker-enabled patient stratification, with potential clinical benefit in ER⁺ and HER2⁺ enriched tumors, as well as a subset of TNBC. Full article

During the periparturient period, negative energy balance (NEB) in dairy cows leads to increased concentrations of non-esterified fatty acids (NEFA) in the blood, which can induce fatty liver disease and ketosis. Forkhead box protein A3 (FOXA3) is a key transcription factor that regulates liver metabolism; however, its specific role in the pathogenesis of fatty liver in dairy cows remains unclear. This study aimed to investigate the mechanism by which FOXA3 regulates hepatic lipid metabolism. We collected liver samples from dairy cows with fatty liver (n = 10) and from healthy cows (n = 10). Bovine primary hepatocytes were isolated from the liver tissue of calves (n = 5), followed by NEFA treatment, and we utilized FOXA3 overexpression, immunofluorescence, and RNA sequencing (RNA-seq) to conduct our analysis. Our results demonstrated that FOXA3 expression in the livers of cows with fatty liver was significantly lower than in healthy cows. NEFA treatment resulted in the downregulation of FOXA3 protein levels in hepatocytes, promoting triacylglycerol (TAG) accumulation and the expression of lipogenesis-related genes. Conversely, FOXA3 overexpression mitigated NEFA-induced lipid accumulation, inhibited the expression of lipogenesis-related genes and proteins—particularly SREBP1—and affected cell proliferation, and the intracellular localization of FOXA3 and SREBP1. RNA-seq analysis suggested that FOXA3 may influence hepatic lipogenesis through pathways such as PI3K-Akt and the cell cycle. In summary, FOXA3 mitigates NEFA-induced hepatic lipid accumulation through a dual mechanism: regulating SREBP1 expression and inhibiting cellular proliferation. These findings highlight FOXA3′s potential as a novel target for the prevention and treatment of fatty liver disease in dairy cows. Full article

Forkhead box protein A3 (FOXA3), also known as hepatocyte nuclear factor 3g (HNF3g), is a member of the FOX family of transcription factors and regulates lipid and glucose metabolism and liver regeneration. Hepatic FOXA3 is reduced in obesity and patients with metabolic dysfunction-associated steatohepatitis (MASH). So far, it remains unknown whether hepatic FOXA3 is essential for regulating lipid metabolism or metabolic dysfunction-associated liver disease (MASLD). In this study, we first investigated whether genetic inactivation of hepatocyte Foxa3 affected the development of MASLD/MASH in C57BL/6 mice and then explored whether loss of hepatocyte Foxa3 regulated atherosclerosis development in Ldlr-deficient mice. Inactivation of Foxa3 in hepatocytes did not affect the development of Western diet-induced MASLD/MASH in C57BL/6 mice but attenuated MASH development in Western diet-fed Ldlr-deficient mice. Moreover, genetic loss of hepatocyte Foxa3 ameliorated hyperlipidemia and atherosclerosis in Ldlr-deficient mice. In Ldlr-deficient mice, loss of hepatocyte Foxa3 resulted in reduced expression of lipogenic, pro-inflammatory, or fibrogenic genes in the liver and reduced cholic acid levels in plasma and bile. Thus, hepatocyte FOXA3 loss confers protection against the development of MASH and atherosclerosis in hyperlipidemic Ldlr-deficient mice. Full article

Lung adenocarcinoma is the most common form of lung cancer with a 5-year survival rate of 15%, largely due to asymptomatic metastasis and late diagnosis. Overexpression of Polycomb group (PcG) proteins, particularly EZH2, the catalytic component of Polycomb Repressive Complex 2 (PRC2), has been associated with the pathogenesis of lung cancer, frequently showing correlation with cancer progression and poor prognosis. In this study, EZH2 levels were modulated by CRISPR/Cas9 gene editing and PRC2 activity was inhibited with EZH2 inhibitor EPZ6438 or EED inhibitor MAK683. EZH2 gene editing reduced cell proliferation, migration, invasion, and colony formation and reduced NFκ-B signaling activation, indicating an antitumoral effect in vitro. Moreover, EZH2 inhibition also increased the expression of differentiation-related genes, such as GATA5, FOXA2, and lung surfactants, indicating a pro-differentiation effect. However, EZH2-edited cells injected into an immunocompromised mouse model generated larger tumors compared to unedited cells. This was accompanied by increased expression of other PcG genes, including EZH1, CBX2, RING1, EED, and SUZ12, suggesting a compensatory interaction between PRC2 and PRC1 complexes. These findings provide significant clinical relevance, both in elucidating the mechanisms of novel molecular targets and in guiding treatment strategies for lung cancer when using epigenetic inhibitors. Full article

Bt Cry toxins remain the cornerstone of transgenic crop protection against Lepidopteran pests, yet field-evolved resistance, particularly in invasive species such as Spodoptera frugiperda and Helicoverpa armigera, can threaten their long-term efficacy. This review presents a comprehensive and unified mechanistic framework that synthesizes current understanding of Bt Cry toxin modes of action and the complex, multilayered regulatory mechanisms of field-evolved resistance. Beyond the classical pore-formation model, emerging evidence highlights signal transduction cascades, immune evasion via suppression of Toll/IMD pathways, and tripartite toxin–host–microbiota interactions that can dynamically modulate protoxin activation and receptor accessibility. Resistance arises from target-site alterations (e.g., ABCC2/ABCC3, Cadherin mutations), altered midgut protease profiles, enhanced immune regeneration, and microbiota-mediated detoxification, orchestrated by transcription factor networks (GATA, FoxA, FTZ-F1), constitutive MAPK hyperactivation (especially MAP4K4-driven cascades), along with preliminary emerging findings on non-coding RNA involvement. Countermeasures now integrate synergistic Cry/Vip pyramiding, CRISPR/Cas9-validated receptor knockouts revealing functional redundancy, Domain III chimerization (e.g., Cry1A.105), phage-assisted continuous evolution (PACE), and the emerging application of AlphaFold3 for structure-guided rational redesign of resistance-breaking variants. Future sustainability hinges on system-level integration of single-cell transcriptomics, midgut-specific CRISPR screens, microbiome engineering, and AI-accelerated protein design to preempt resistance trajectories and secure Bt biotechnology within integrated resistance and pest management frameworks. Full article

This study aimed to elucidate the oncogenic role of FOXA1(forkhead box A1) in ovarian cancer and to evaluate its potential as both a therapeutic target and a diagnostic biomarker. We further investigated whether FOXA1 inhibition could enhance responsiveness to immune checkpoint blockade and overcome chemoresistance. A total of seventy-six ovarian tissue samples were analyzed, including nine normal, thirty-four benign, and thirty-three malignant specimens. IHC (immunohistochemistry) staining was performed to assess FOXA1 expression and its correlation with tumor stage. Functional studies were conducted using FOXA1 siRNA in SK-OV3 and HEYA8 cell lines. Changes in cell proliferation, migration, invasion, and wound-healing ability were evaluated following FOXA1 silencing. Quantitative RT-PCR was used to measure the expression of FOXA1 and EMT (epithelial–mesenchymal transition)-related genes. The effects of FOXA1 inhibition on sensitivity to carboplatin and the immune checkpoint inhibitor atezolizumab were also examined. IHC analysis revealed significant differences in FOXA1 expression among normal, benign, and malignant tissues, with levels correlating with tumor stage. FOXA1 silencing significantly reduced proliferation and decreased migration and invasion by 60–80%, accompanied by marked downregulation of EMT-related genes. Moreover, FOXA1 inhibition enhanced atezolizumab responsiveness and reduced carboplatin resistance in ovarian cancer cells. In summary, FOXA1 acts as an oncogenic driver in ovarian cancer, promoting proliferation, invasion, and EMT activation. Its overexpression correlates with disease progression, supporting its potential as a biomarker and therapeutic target. Targeting FOXA1 could enhance immunotherapy efficacy and help overcome chemoresistance in ovarian cancer. Full article

Cancer patients are highly vulnerable to severe COVID-19, requiring models that capture tumor–virus interactions. We investigated tumor- and variant-specific effects of SARS-CoV-2 Gamma and Delta infections using patient-derived organoids (PDOs) from metastatic breast, lung, and colorectal cancers. Viral infection was quantified by Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR) 24 h post-infection, and morphological changes and immune mediators were profiled. Genomic analysis using whole-exome sequencing was performed to identify contributing host-related gene alterations. The Delta variant produced consistently higher viral loads in lung and breast PDOs, while colorectal PDOs showed variable susceptibility. Infection led to reduced area and perimeter and increased circularity across all tumor types. Immune profiling revealed distinct responses: Gamma decreased Interferon alpha (IFNα) in lung PDOs and increased E-selectin in colorectal PDOs. Delta broadly reduced inflammatory mediators in lung [10 kDa interferon gamma-induced protein (IP-10) and Intercellular adhesion molecule 1 (ICAM-1)] and breast [Interleukin-6 (IL-6), Interleukin-13 (IL-13), and Interleukin-17A (IL-17A)] PDOs, while increasing Macrophage inflammatory protein 1-beta (MIP-1β) in colorectal PDOs. Host gene variants involved in trafficking (FYCO1 and RAB7A) and immune signaling (FOXA2, SFTPD, STAT3, and TET2) were associated with differential infection profiles. These findings show that SARS-CoV-2 induces variant- and tumor-specific morphological and immunological changes in cancer PDOs, highlighting the potential of this model to unravel host–virus interactions and identify genetic factors that shape infection outcomes in cancer. Full article

The liver is the central organ in metabolism; however, modeling hepatic diseases remains limited by current experimental models. Animal models frequently fail to predict human liver physiology, while primary hepatocytes rapidly dedifferentiate in culture. We performed comprehensive transcriptomic profiling of induced pluripotent stem cells (iPSCs) differentiation into hepatocyte-like cells (HLCs) under two-dimensional (2D) and three-dimensional (3D) culture conditions. RNA sequencing analysis revealed the sequential activation of lineage-specific markers across major developmental stages: definitive endoderm (FOXA2, SOX17, CXCR4, CER1, GATA4), posterior foregut (PROX1, GATA6), and hepatoblasts (HNF4A, AFP). Comparative analysis demonstrated a markedly enhanced hepatic gene expression of 3D organoids, as demonstrated by a 33-fold increase in HNF4A expression and elevated levels of mature hepatocyte markers, including ALB, SERPINA1, and UGT2B15. However, the 3D cultures retained fetal characteristics (290-fold higher AFP expression) and exhibited significantly impaired metabolic function, with CYP3A4 expression levels reduced by 2000-fold compared to the adult human liver. This partial maturation was further supported by a moderate correlation with adult liver tissue (ρ = 0.57). We demonstrated high reproducibility across five biologically distinct iPSCs lines, including those derived from patients with rare monogenic disorders. The establishment of quantitative benchmarks provides a crucial tool for standardizing in vitro liver models. Furthermore, we delineate the specific limitations of the current model, highlighting the need for further protocol optimization to enhance metabolic maturation and P450 enzyme activity. Functional validation of metabolic activity (CYP enzyme assays, albumin secretion) was not performed; therefore, conclusions regarding hepatocyte functionality are based on transcriptomic evidence. Full article

Oct4 is well established as a core regulator of pluripotency, yet emerging evidence points to an additional role in lineage specification during the exit from the pluripotent state. Although Oct4 expression has been observed in early mesodermal progenitors, its precise function in this developmental context remains unclear. To investigate this, we employed embryoid bodies (EBs) as a model of spontaneous differentiation that recapitulates key aspects of early embryonic development in vitro. In accordance with previous studies, reporter assay revealed a distinct temporal pattern characterized by the strong, transient co-expression of Oct4 and the early mesoderm-specifying marker gene Brachyury within a narrow developmental window, consistent with the Oct4 role in early mesodermal progenitors. We further examined the consequences of the Oct4 loss at early stages of this differentiation. Conditional knockout of the Oct4 gene resulted in a significant reduction in EB size and accumulation of cells in the G0/G1 phase, indicating a critical requirement for Oct4 in maintaining cell proliferation. Despite this defect, cells retained the ability to initiate multilineage differentiation, albeit with reduced expression of Brachyury and elevated expression of endodermal markers FoxA2 and Sox17. Interestingly, the formation of beating cardiomyocyte-like structures was also diminished following Oct4 loss and could not be rescued by simply increasing cell numbers. Taken together, these findings highlight an important Oct4 function in mesodermal differentiation, mediated through the maintenance of proliferative capacity of early mesodermal progenitors. Full article

Non-syndromic cleft lip with or without cleft palate (ns-CL/P) is one of the most common craniofacial anomalies with a multifactorial etiology. To investigate the contribution of rare variants to disease risk, we performed whole-exome sequencing (WES) in 58 patients with ns-CL/P from a homogeneous Polish population, excluding from analysis 423 previously investigated cleft candidate genes. After stringent filtering, prioritization, and segregation analysis, we identified 31 likely pathogenic (LP) variants across 30 genes, significantly enriched in categories related to developmental processes. Notably, 29% of variants occurred in genes not previously linked to clefting, including AGO1, ARID1A, ATP1A1, FOXA2, GDF7, HOXB3, LRP5, MAML1, and ZNF319. Three were de novo: FOXA2_p.Arg260Pro, MAML1_p.Gln65Ter, and ZNF319_p.Gln64Ter. Most of the remaining variants were inherited from unaffected parents, suggesting incomplete penetrance and possible modifier effects consistent with the heterogeneous etiology of ns-CL/P. Additionally, analysis of common variants in the 30 loci harboring rare LP variants revealed nominal associations with ns-CL/P for NXN, EXT1, MAML1, and TP53BP2 loci. These results support the candidacy of these genes and suggest contributions from both rare and common variants. In conclusion, we report novel LP variants expanding the spectrum of candidate genes and providing new insights into the genetic landscape of orofacial clefts. Full article

Background: Individual differences in immune responses to African swine fever virus (ASFV), whether induced by vaccination or natural infection, may be linked to genetic variation in the genes involved in antigen presentation. Methods: A total of nine pigs from the 112-population were selected for RNA-seq analysis. To pinpoint key transcription factors (TFs) regulating gene expression in the lymph nodes, weighted Kendall’s Tau rank correlation analysis was performed to link the TF binding potential with the extent of differential expression of target genes. Results: CD8⁺ T cells expressing a specific epitope of the ASFV p72 protein (ACD8⁺) accounted for 41% of the total CD8⁺ T cells in peripheral blood. A total of 2062 transcripts were identified as differentially expressed across the nine pigs (q-value < 1 × 10⁻⁸). Differential expression levels of the target genes for MECP2, ETS1, ZBTB33, ELK4, and E2F4 were significantly correlated with their TF binding potential (p < 0.05). Six SNPs were identified in the promoter region of ELK4. Analysis of the 112-pig population revealed that SNPs at S.-404A>G and S.-668C>T loci were significantly associated with ACD8⁺ levels (q-value < 0.01). Individuals with the AA genotype at S.-404A>G had significantly higher ACD8⁺ counts compared to those with AG and GG genotypes (q-value < 0.05). At the S.-668C>T locus, ACD8⁺ levels were highest in the CC genotype, followed by CT and TT genotypes, with CC showing notably higher ACD8⁺ counts (q-value < 0.05). Notably, the S.-404A>G site overlaps with potential binding sites for TFs FOXA2, GATAs, and TRPS1, while the S.-668C>T site lies within the binding regions for NR1H3, RARA, VDR, and NR1I3. Conclusion: These mutations may disrupt TFs binding to the ELK4 promoter, potentially reducing ELK4 expression and impairing antigen processing and presentation. Full article

Background: Sodium/iodide symporter (NIS) is a membrane protein involved in iodide transport into cells, making it a key component of thyroid physiology and radioiodine therapy for thyroid cancer. Although NIS is expressed in many extrathyroidal tissues, including breast tumors, its functional role and prognostic significance in these contexts remain a subject of active investigation. Understanding the mechanisms regulating NIS, its influence on cellular processes such as migration and metastasis, and its connection with transcription factors like FOXA1 could contribute to the development of new therapeutic strategies for breast cancer treatment. This study aims to investigate the correlation between sodium/iodide symporter (NIS) expression and response to neoadjuvant chemotherapy in patients with triple-negative breast cancer (TNBC). Methods: The current retrospective study included 161 TNBC patients who received neoadjuvant chemotherapy followed by mastectomy. NIS expression was assessed via immunohistochemistry, graded semi-quantitatively from 0 to 3+. The Residual Cancer Burden (RCB) scale was used to evaluate the response to chemotherapy. Statistical analysis included Lilliefors tests and Kendall’s tau correlation coefficient. Publicly available Cancer Genome Atlas datasets were analyzed to assess the relationship between NIS and FOXA1 expression. Results: NIS immunopositivity was observed in 69.5% of TNBC samples compared to 63.3% GATA-3-positive and 31.0% of Mammaglobin-positive samples. While no significant correlation was found between NIS expression and age, TNM stage, or Ki-67, a statistically significant moderate positive correlation (τ = 0.481, p < 0.01) was identified between NIS expression and RCB index, indicating that higher NIS expression was associated with a poorer response to neoadjuvant chemotherapy. TCGA data analysis revealed a statistically significant increase in NIS mRNA expression in FOXA1-mutated TNBC samples compared to FOXA1-wild-type samples (p < 0.05). Younger patients exhibited higher Ki-67 levels (τ = −0.416, p < 0.05). Conclusions: Higher NIS expression correlates with chemoresistance to neoadjuvant chemotherapy in TNBC patients. This phenomenon may be linked to FOXA1 activity, suggesting that NIS may represent a potential biomarker for chemoresistance in TNBC. The inverse correlation between patient age and Ki-67 levels may be associated with a different mutational landscape in younger patients. Full article