Epidermal Growth Factor Receptor (EGFR) is often present on the cell surface of a wide variety human malignancies, including non-small-cell lung cancer (NSCLC), glioblastoma (GBM), pancreatic ductal carcinoma (PDC), and castration-resistant prostate cancer (CRPC). Efforts to optimize immunotherapies targeting EGFR are limited by murine intolerance of human EGFR. To overcome this obstacle, we developed C57BL/6 mice in which a truncated variant of human EGFR (hEGFRt), lacking the ligand binding domain and cytoplasmic domain, is expressed from the CAG regulatory elements comprised of the CMV enhancer, β-actin promoter, and β-globin poly-adenylation signals. Cetuximab, a high-affinity, clinically available anti-human EGFR antibody, retains affinity for hEGFRt. The hEGFRt cDNA in the CAG-hEGFRt transgene is flanked by loxP sites to enable its excision thereby reducing interaction of hEGFRt-directed immunotherapies with normal tissues. The CAG-hEGFRt(f/f) transgene is abundantly expressed in hematopoietic lymphoid and myeloid cells, with low-level expression evident also in non-hematopoietic liver, lung, kidney, and brain. Mx1-Cre-mediated transgene excision in adult mice reduces hEGFRt ~ 5-fold in blood mononuclear cells. CAG-hEGFRt(f/f) adult mice are tolerant of syngeneic NSCLC, GBM, prostate, and PDC lines expressing hEGFRt. These mice retain hEGFRt tolerance after transgene deletion. CAG-hEGFRt(f/f) mice provide a new and important tool for the development of immunotherapies targeting hEGFR.
Evidence for causal associations between functional dyspepsia (FD) and human metabolites remains limited. Two-sample and multivariable Mendelian randomizations to detect the causal relationships between FD and human metabolites. Single nucleotide polymorphisms significantly associated with human metabolites were selected as instrumental variables, the inverse variance weighting method was used as the primary analysis method, and the results were tested for heterogeneity and horizontal pleiotropy. Genetically predicted levels of 26 circulating metabolites and 5 cerebrospinal fluid metabolites were associated with FD. Among them, Total cholesterol in large low-density lipoprotein (odds ratio [OR] = 0.985, 95% confidence interval [CI] 0.975-0.995, P = .003) and cholesteryl esters to total lipids ratio in intermediate-density lipoprotein (OR = 0.983, 95% CI 0.971-0.996, P = .008) were negatively associated with FD. It was also positively associated with the level of phenylalanine (OR = 1.037, 95% CI 1.009-1.065, P = .009) and phospholipids to total lipids ratio in small high-density lipoprotein (OR = 1.015, 95% CI 1.003-1.027, P = .017). After multivariable adjustment, FD was causally associated with phospholipids to total lipids ratio in small high-density lipoprotein (OR = 1.332, 95% CI 1.060-1.674, P = .014). Sensitivity analyses showed no evidence of heterogeneity and horizontal pleiotropy in the above results. Human metabolites may be associated with FD.
Zika virus (ZIKV) is primarily transmitted through mosquito bites, and the skin acts as the initial site of viral entry into the host. Consequently, resident skin cells are among the first targets of infection. The epidermis, mainly composed of keratinocytes, can mount an antiviral response against arboviruses through the production of interferons, interferon-stimulated genes, cytokines, and antimicrobial peptides (AMPs), including the Trappin-2/Elafin (Tr2/E) peptide. However, the antiviral activity of Tr2/E during ZIKV infection remains poorly understood, therefore, this study aimed to investigate the antiviral activity of Tr2/E in human keratinocytes during ZIKV infection. In this study, we evaluated the permissiveness of the human keratinocyte cell line HaCaT to infection with a Mexican isolate of ZIKV and observed that these cells support productive viral infection. We then assessed whether ZIKV infection induces endogenous expression of Tr2/E. Tr2/E transcripts were detected in infected cells and showed increased expression over time post-infection, which correlated with the presence of its corresponding protein. Furthermore, we evaluated the antiviral potential of this peptide through exogenous treatment of infected keratinocytes. A significant reduction in ZIKV infection following Tr2/E treatment was observed. Collectively, these findings provide additional insight into the involvement of AMPs in the antiviral response to ZIKV infection and highlight Tr2/E as a potential antiviral factor.
Microbial interactions shape the composition and stability of the human gut microbiome. Yet, few studies have systematically investigated species-species growth interactions and the mechanisms behind these. Here we show that among 36 representative gut bacterial strains, when two species interact, the interactions are mostly inhibitory. To provide biological insight into specific interactions, we further investigate the basis of a positive interaction, showing that Clostridium perfringens promotes the growth of Mediterraneibacter gnavus through extracellular vesicles. Additionally, we identify Veillonella parvula as a species capable of modulating environmental pH, thereby enabling the growth of Parabacteroides merdae, a strain highly sensitive to acidic conditions. This pH-increasing effect is enhanced by guanine supplementation and persists in multi-species communities containing different pH-lowering strains from diverse bacterial phyla. Although V. parvula is commonly present in human gut microbiomes, it is generally found at low levels. Given the spatial organization of bacteria in the gut, the local pH modulation by V. parvula might support the growth of acid-sensitive strains. Overall, the comprehensive dataset and mechanistic insights presented here provide a starting point to predict microbiome composition by integrating growth interactions.
Metronomic chemotherapy with oral capecitabine + vinorelbine (Cape + VNL) provides synergistic cytostatic activity and antiangiogenic and immunomodulatory effects, potentially offering prolonged disease control with limited toxicity in HER2-negative metastatic breast cancer (MBC). However, efficacy in the real-world (RW) setting, especially in late lines, and the impact of dihydropyrimidine dehydrogenase (DPYD) polymorphisms on dose reduction and safety remain limited. In this retrospective study, 200 patients with human epidermal growth factor receptor 2 (HER2)-negative MBC were treated at the ASST Cremona Hospital (2015-2023) with metronomic Cape (1000 mg twice daily, in normal metabolizers; 500 mg twice daily, in DPYD variant carriers) + VNL (20 mg/day, once daily, 5-days-on/2-days-off). All patients underwent pretreatment DPYD genotyping and dose adjustment. Treatment was administered in the second-to-fourth setting. The primary end point was time-to-next treatment or death (TNTD); secondary end points included overall survival (OS), disease control rate (DCR) ≥24 weeks, overall response rate (ORR), safety, and genotype-toxicity correlations. The median age was 61 years, and DPYD variants were present in 14.5% of patients; 34%, 41%, and 25% received therapy as second-, third-, and fourth-line treatment. The median TNTD was 22.0 weeks, and the OS was 64.0 weeks. The DCR was 38.5%, and the ORR was 22.0%. Efficacy was comparable between DPYD variant carriers and normal metabolizers (all P values > .05). In later lines, Eastern Cooperative Oncology Group performance status 2 and >2 metastatic sites were independent negative prognostic factors (all values P < .05). Overall, 7.5% grade 3 toxicities occurred, especially in variant carriers without dose reduction and grade 4-5 events. These RW data suggest that metronomic Cape + VNL may represent a clinically active and manageable option in heavily pretreated HER2-negative MBC. Our findings support prospective evaluation of DPYD-guided dose individualization as a strategy to optimize the benefit-risk balance of fluoropyrimidine-based metronomic regimens.
Human visceral leishmaniasis (HVL) in Algeria is primarily caused by Leishmania infantum. This study aimed to genetically identify the Leishmania species responsible for HVL in Algerian patients. This descriptive molecular case series included 13 patients diagnosed between 2010 and 2022. DNA from blood, bone marrow, and skin lesion smears was analyzed using ITS1-PCR, RFLP analysis and Sanger sequencing. The resulting sequences were compared with GenBank references, and phylogenetic analyses were performed. L. infantum was identified in 11 of 13 cases (84.6%). Most visceral leishmaniasis cases occurred in patients younger than 16 years, whereas adult cases were primarily immunocompromised patients, particularly those living with HIV/AIDS. Phylogenetic analyses revealed a high degree of similarity between Algerian L. infantum isolates and Mediterranean strains. In addition to L. infantum, L. tropica DNA and, surprisingly, L. major DNA was also identified. L. major DNA was detected in one immunocompetent child, while L. tropica DNA was detected in a skin lesion of an immunocompromised adult who presented with a concomitant relapse of HVL. These atypical molecular findings require cautious interpretation and further investigation. L. infantum remains the principal causative agent of HVL in Algeria. The atypical molecular findings should be interpreted cautiously and highlight the need for continued molecular surveillance, larger studies incorporating additional genetic markers, and investigations of vector dynamics and animal reservoirs.
Enhancer of zeste homolog 2 (EZH2) is a key epigenetic regulator implicated in tumor progression; however, its expression pattern and subcellular localization across different stages of endometrial carcinogenesis remain incompletely characterized. This study evaluated EZH2 expression in two biologically distinct endometrial carcinoma cell lines (Ishikawa and MFE-319) and in archived human endometrial tissues representing proliferative and secretory endometrium, hyperplasia, and Type I and Type II endometrial carcinomas. Histopathological evaluation was performed using hematoxylin and eosin staining. EZH2 expression was assessed by immunocytochemistry and Western blot analysis in the cell lines and by immunohistochemistry in tissue specimens, while apoptosis was evaluated by TUNEL assay in tissue samples. MFE-319 cells demonstrated significantly higher EZH2 expression than Ishikawa cells (H-score: 352.9 ± 78.9 vs. 137.6 ± 31.5, p < 0.0001), and Western blot analysis confirmed the same direction of change. Among tissue specimens, the highest EZH2 immunoreactivity was observed in Type II endometrial carcinoma (366.0 ± 63.7), with significantly higher expression than proliferative endometrium (231.5 ± 59.3), secretory endometrium (190.5 ± 52.4), and hyperplasia without atypia (261.0 ± 56.9), whereas no significant differences were detected among several intermediate histopathological groups. Nuclear localization of EZH2 became more prominent in atypical hyperplasia and carcinoma tissues. Apoptotic indices were significantly higher in both Type I and Type II carcinomas than in normal endometrium and hyperplasia groups, representing an association with increased EZH2 expression rather than evidence of a direct mechanistic relationship. These findings demonstrate that EZH2 expression differs across histopathological categories of endometrial lesions, with the highest expression observed in Type II endometrial carcinoma. The observed predominance of nuclear EZH2 in atypical hyperplasia and carcinoma further supports its association with aggressive tumor biology, although additional functional and clinicopathological studies are required to establish its clinical and biological significance.
AI tools have the potential to enhance personalized clinical care, particularly in radiology. However, their integration into clinical workflows remains complex, especially in pediatric oncology, where early cancer detection is critical. Children with Li-Fraumeni syndrome (LFS), a rare cancer predisposition disorder, undergo regular surveillance whole-body magnetic resonance imaging (wbMRI), which presents an opportunity for AI-assisted tumor detection. We evaluated the feasibility of an AI-assisted overlay for highlighting tumor-like regions in pediatric surveillance wbMRI and explored how access to the overlay influenced radiologist workflow, candidate-lesion marking behavior, follow-up recommendations, and perceived workload. We developed a patch-based AI segmentation model trained on augmented 2D slices from 675 surveillance wbMRI volumes of pediatric patients with LFS. The model was designed to highlight regions with high tumor probability. A reader study was conducted with 2 radiologists who independently reviewed wbMRI cases both with and without AI assistance. We measured evaluation time, number and location of reader-marked candidate lesions, type of follow-up recommendation, and subjective feedback using structured questionnaires. AI assistance altered interpretation workflows for both radiologists, with mixed effects. On average, the time required to evaluate each case increased when using the AI tool for both radiologists. However, one radiologist had an increase in the number of candidate lesion locations selected with the tool, and one had a decrease in the number of candidate lesion locations selected with the tool. Subjective feedback indicated that one of the radiologists reported lower mental demand with the AI tool, while both radiologists reported lower stress with the AI tool. Interrater variability was evident, underscoring the need for personalized calibration of AI tools. AI-assisted wbMRI interpretation can improve tumor detection in pediatric cancer surveillance by reducing false negatives. However, its influence on workflow efficiency and interradiologist variability highlights the importance of careful implementation. Successful integration requires addressing challenges such as improving the predictive precision of AI models, offering intuitive end-user designs and instructions, and building trust in AI outputs. AI outputs can influence workflow and behavior in reader-specific ways. Clinical translation will require larger, randomized, multireader studies and model refinement to reduce false positives and quantify lesion-level reader performance. This can help ensure better patient outcomes in addition to reduced clinician burnout.
Lyme disease, caused by the bacterium Borrelia burgdorferi (Spirochaetales: Spirochaetaceae) and transmitted by the blacklegged tick, Ixodes scapularis Say (Acari: Ixodidae), in the eastern United States, is the most common vector-borne disease in North America. Human disease risk depends on interactions among ticks and sylvatic hosts, and greater host biodiversity has been hypothesized to reduce risk through the dilution effect. However, the influence of biodiversity varies with community composition, species abundance, and habitat change. We conducted a literature review to compile biological parameter estimates for 15 common host species. These parameters were used in a mathematical model incorporating tick-to-host ratio-dependent host-finding success, allowing for incomplete redistribution of ticks, to assess transmission dynamics in the northeastern United States, where human Lyme disease incidence is high. We evaluated how species richness, evenness, and habitat changes affect nymphal infection prevalence (NIP) and density of infected nymphs (DIN). Our results indicate that many small mammal species play context-dependent roles, acting as dilution hosts in communities of highly competent hosts and as amplification hosts in lower-competence communities. Varying host density revealed "tipping points," where small abundance changes led to communities producing more infected than uninfected ticks. Habitat fragmentation simulations showed that biodiversity can decrease NIP but often increases DIN. Together, these findings provide insight into B. burgdorferi transmission cycles and emphasize the need to consider NIP and DIN jointly when assessing how biodiversity alters transmission dynamics.
Developing climate-resilient, lodging-resistant rice requires integrating strong culm and root related genetics, high-throughput and trait-based phenotyping, and optimized agronomic practices. Coordinated use of molecular breeding, gene editing, and management strategies offers sustainable solutions to mitigate lodging under variable environments. Lodging is a major abiotic stress that significantly reduces grain yield and quality in rice, while also increasing vulnerability to pests. Human-induced intensification, including high planting densities, excessive nitrogen application, off-season sowing, and cultivation in upland areas, further increases rice susceptibility to lodging, posing a major management challenge. Sole reliance on the sd1 gene has proven insufficient for ensuring effective lodging resistance. This review examines the diverse factors influencing lodging susceptibility and resistance in rice, including morphological, physiological, biochemical, genetic, agronomic, and environmental aspects, and integrates current advances to guide future research and breeding strategies for lodging-resistant rice. Among these factors, the stem has emerged as a key tissue influencing lodging resistance. Advances in molecular breeding have identified several QTLs and genes, such as SCM1, SCM2, SCM3, SCM4, APO1, and prl4, that are associated with lodging resistance, enabling the use of marker-assisted selection in breeding programs. The review also discusses integrated strategies that combine advanced phenotyping tools, optimized agronomic practices, mathematical modelling, and genetic approaches including molecular breeding and gene editing to effectively develop lodging-resistant rice varieties.
Orthohantavirus dabieshanense (DBSV) is a newly emerging hantavirus and poses a potential threat to human health. Current detection methods based on polymerase chain reaction (PCR) assays for other hantaviruses such as Hantaan virus (HTNV) and Seoul virus (SEOV) may carry the risk of false negatives. This study aims to address the technical bottlenecks of low accuracy and insufficient sensitivity in current detection of DBSV. We developed a signal amplification strategy mediated by a photoactivated magnetic 3D DNA walker, which enables light-controllable fluorescence detection. Specifically, this fluorescence sensing platform introduces the catalytic hairpin assembly (CHA) technique for dual signal amplification. This system requires dual activation by UV irradiation and target RNA. Then, exonuclease III (Exo III) provides propulsion for the walking process to release a specific trigger strand, subsequently activating the CHA cycle and producing a fluorescent signal. Experimental results confirm that the system displays excellent selectivity and anti-interference ability, successfully distinguishing target sequences with single-base mismatch (SBM), double-base mismatch (DBM), and triple-base mismatch (TBM). Furthermore, the platform achieves a DBSV-RNA detection limit (LOD) of 30 fM with a linear range spanning 10-1-106 pM. The applicability of the method has been rigorously validated using human serum specimens. This study provides a novel strategy for the application of spatiotemporally controllable DNA walkers in RNA detection, demonstrating considerable potential for early viral diagnosis and field monitoring.
Glioblastoma (GBM) is characterized by profound immunosuppression and limited responsiveness to conventional T cell-based therapies and immune checkpoint blockade. Here, we establish a human microglial cell line to explore the concept of a brain-adapted platform for chimeric antigen receptor (CAR) expression targeting GBM. We engineered human microglial cells with anti‑carbonic anhydrase IX (CAIX) CARs incorporating either CD28 or 4-1BB costimulatory domains, alone or co-expressing anti-programmed cell death ligand 1 (PD-L1) monoclonal antibodies (mAbs). Our results demonstrate that CAIX-targeted CAR-engineered microglia-like cells (CAR-MG) mediate potent, antigen-dependent tumor killing in vitro, with CD28-based constructs exhibiting superior antitumor efficacy and a robust pro-inflammatory cytokine profile. Furthermore, local secretion of anti-PD-L1 synergized with CAIX-targeting to significantly reduce tumor burden in vivo - an effect closely associated with the successful reprogramming of infiltrating myeloid cells toward an M1-like anti-tumoral state, evidenced by an increase in the frequency of cells expressing CD38, CD86, and HLA-DR. Notably, the anti-PD-L1 secretory platform downregulated tumor-derived PD-L1, effectively remodeling the immunosuppressive glioblastoma microenvironment. These findings suggest that this engineered microglial cell line platform has the potential to heat the immunologically "cold" GBM microenvironment. Together, these results identify CAIX-targeted engineered CAR-MG as an effective immunotherapeutic approach and support further development of CNS-targeted immune cells to overcome key barriers to cellular immunotherapy in GBM.
mRNA splicing represents a fundamental level of gene regulation that alters proteomic diversity and cellular state. Its dysfunction can profoundly rewire metabolism, yet underlying mechanisms remain elusive. Here, we investigate Verheij syndrome, caused by mutations in core splicing factor PUF60, using a Caenorhabditis elegans model, human cell lines, and patient-derived samples. We demonstrate that RNP-6/PUF60 deficiency disrupts splicing of genes governing one-carbon metabolism and phospholipid remodeling, impairing S-adenosylmethionine/S-adenosylhomocysteine cycling and phosphatidylcholine synthesis. These perturbations trigger the integrated stress response and compromise mTORC1 signaling, causing developmental growth defects. Vitamin B12 supplementation restores metabolic balance by reactivating S-adenosylmethionine-dependent phospholipid remodeling and mTORC1 activity, effectively rescuing Verheij-like phenotypes. Similar responses arise from perturbing another splicing factor, PRP-19. Mechanistically, intron retention of nhr-114/HNF4 transcription factor drives these phenotypes, while restoring its splicing rescues them. Our findings implicate vitamin B12-dependent one-carbon metabolism as a metabolic modulator with therapeutic potential to mitigate Verheij syndrome and other spliceosomopathies.
Liver disease in Alpha-1 antitrypsin deficiency (AATD) is caused by the toxic accumulation of mutant Z alpha-1 antitrypsin (Z-AAT) within the endoplasmic reticulum (ER) of hepatocytes. Livers from PiZ transgenic mice expressing the human Z-AAT and AATD patients were both found to have increased p62/SQSTM1, a multifunctional protein involved in protein homeostasis, consistent with previous reports. However, whether p62/SQSTM1 is a marker of Z-AAT globules or plays an active role in Z-AAT proteostasis is unclear. The goal of this study was to elucidate the involvement of p62/SQSTM1 in the formation of Z-AAT globules that are responsible for liver injury in AATD. In the present study, we found that p62/SQSTM1 decorated ubiquitin-positive, Periodic-Acid Shiff-diastase-resistant Z-AAT globules and interacted with Z-AAT at the ER-cytosol interface. Genetic ablation of p62/SQSTM1 in PiZ mice (PiZ;p62-/-) led to marked reduction in hepatic Z-AAT globules and polymers, and decreased serum Z-AAT, highlighting a central role for p62/SQSTM1 in disease pathogenesis. Moreover, hepatocyte-specific somatic deletion of the ubiquitin-association (UBA) domain of p62/SQSTM1 reduced Z-AAT aggregation. Furthermore, KEAP1 was identified as a binding partner of p62/SQSTM1-Z-AAT complex, leading to nuclear translocation and activation of NRF2. Inhibition of KEAP1-p62/SQSTM1 interaction reduced the abundance of p62 and phosphorylated p62, decreased intracellular Z-AAT, and redistributed NRF2 to the cytoplasm. In conclusion, this study identifies p62/SQSTM1 as a regulator of Z-AAT proteostasis and link Z-AAT/p62 accumulation to KEAP1 sequestration and NRF2 pathway activation in liver disease due to Z-AAT.
NK2 homeobox 1 (NKX2-1), a master regulator robustly expressed in lung, thyroid, and forebrain, is indispensable for specifying lung epithelial fate and serves as a definitive marker of lung progenitors. Here, we generated a human induced Pluripotent Stem Cell (iPSC) line harboring a doxycycline (dox)-inducible Cas9 and an NKX2-1-EGFP-puro reporter via CRISPR/Cas9-mediated homology-directed repair. This dual-function line combines inducible genome editing with real-time tracing of early lung progenitors, enabling their prospective isolation and screening for stage-specific maturation regulators. Therefore, this engineered iCas9-NKX2-1 EGFP line is a key resource for dissecting human lung development, modeling pulmonary disease, and advancing regenerative therapies.
Chronic wounds pose challenges due to delayed healing and infection risks. Advanced dressings that provide moisture, support, and bioactivity are critical for regeneration. Hydrogels offer high water content and biocompatibility, while the human amniotic membrane (hAM) provides proteins and growth factors that enhance healing. However, hAM's fragility limits direct use. This study developed a hydrogel of chitosan, hydroxypropyl methylcellulose (HPMC), and propylene glycol (PG), incorporating lyophilized hAM particles to combine the hydrogel's physicochemical properties with hAM's bioactivity. Chitosan hydrogels crosslinked with β-glycerophosphate were optimized for gelation, swelling, degradation, FTIR, rheology, and strength. The optimized formulation exhibited controlled degradation and stability. Incorporating hAM (5% w/v) enabled sustained protein release without disrupting the network (FTIR). Characterization included FTIR and Bradford protein assays. Biological evaluations assessed cytotoxicity and antibacterial activity. The hydrogel showed rapid gelation (<10 min at 37 °C), high swelling (166.07 ± 41.03%), strength (110 ± 60 Pa), >85% L929 fibroblast viability, and inhibition of gram-positive and -negative bacteria. The self-healing AM-loaded hydrogel shows promising properties for wound dressings. Further in vivo and clinical studies are warranted.
Genome-wide association studies identify cancer susceptibility loci, but downstream protein mechanisms remain incompletely defined. We integrate polygenic risk scores (PRSs) for 21 cancers with 4,955 plasma proteins measured in cancer-free Atherosclerosis Risk in Communities (ARIC) participants to prioritize cancer-related proteins and protein networks. The protein quantitative trait score (pQTS) approach assesses associations between cancer PRS and individual protein levels, while ARCHIE partitions cancer risk variants into trans-regulated protein-network components using sparse canonical correlation analysis. Across cancers, pQTS identifies 90 protein associations, including 53 distal trans associations, and ARCHIE identifies 19 components spanning 433 proteins. Downstream analyses connect prioritized proteins to cancer driver genes, somatic alterations, immune cell populations, CRISPR dependency, and cancer-relevant pathways. Cervical cancer and basal cell carcinoma illustrate immune, human papillomavirus (HPV)-related, pigmentation, and inflammatory mechanisms. These findings show that PRS-proteome integration can reveal circulating protein networks underlying inherited cancer susceptibility.
Mutations in the MEFV gene, which encodes pyrin, are associated with a spectrum of inflammatory conditions called pyrin-associated autoinflammatory diseases (PAADs). Of the 400 MEFV variants listed in the Infevers database, most are classified as variants of uncertain significance. Thus, genetic diagnosis of PAADs remains challenging, and the molecular mechanisms underlying pyrin activation remain poorly understood. Here, we used a cell-based pyroptosis assay to stratify 265 missense MEFV variants and identified previously uncharacterized pathogenic variants. We then characterized the interaction between the pyrin B30.2 domain and CDC42, a key regulator of pyrin intracellular trafficking and activation. We found that classical familial Mediterranean fever (FMF)-related variants bind tightly to CDC42 to induce pyrin hyperactivation, whereas certain non-FMF variants induce pyrin hyperactivation independently of CDC42, indicating involvement of multiple pathways in pyrin activation. Our approach provides a proof of concept for a genotype-first approach, which may advance our understanding of complex human diseases.
Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive lung disease characterized by persistent alveolar epithelial injury and aberrant tissue remodeling. Increasing evidence suggests that senescence of alveolar epithelial cells (AECs) contributes to impaired epithelial regeneration and maladaptive tissue repair by limiting reparative capacity and promoting profibrotic signaling. However, the molecular drivers of AEC senescence and their impact on the immune microenvironment in IPF remain incompletely understood. Here, we investigated senescence-associated genes involved in IPF pathogenesis and evaluated their diagnostic and therapeutic potential. IPF transcriptomic datasets were retrieved from the Gene Expression Omnibus (GEO). Senescence-related differentially expressed genes (SRDEGs) were identified by intersecting IPF-derived differentially expressed genes with a curated human senescence gene list. Functional enrichment analyses were performed to delineate SRDEG-associated biological processes. Hub genes were prioritized using machine-learning approaches, and a diagnostic model was constructed and assessed by receiver operating characteristic (ROC) analysis. Candidate genes were further validated through in vivo and in vitro experiments. Given the upstream regulatory role of CHEK2 in DNA damage response-associated cellular senescence, Fostamatinib was screened as a potential therapeutic agent, and its interaction with CHEK2 and functional effects were examined using molecular docking, molecular dynamics simulations, and experimental assays. Two senescence-associated hub genes, CHEK2 and TP53 BP1, were identified as key contributors to IPF pathology (FDR-adjusted P < 0.05), and a model incorporating both genes achieved high diagnostic performance. Experimental validation, however, indicated that only CHEK2 showed IPF-specific differential expression and was closely associated with AEC senescence and fibrotic progression. In silico analyses supported stable binding between Fostamatinib and CHEK2, and subsequent molecular and cellular experiments suggested that Fostamatinib may attenuate CHEK2-associated senescence and profibrotic responses. Collectively, these findings identify CHEK2 as a critical regulator of AEC senescence and IPF development, supporting its potential use as a diagnostic biomarker and therapeutic target. Fostamatinib may represent a candidate therapeutic strategy for IPF by modulating CHEK2-related senescence pathways.
The development of effective strategies for treating bone defects can be based on gene therapy methods aimed at regulating the differentiation of osteoprogenitor cells. One of the approaches is to use siRNA molecules in knockdown systems for genes inhibiting osteogenic cell differentiation. In this work, we aimed at developingapproaches to induce osteogenic differentiation of mesenchymal stem cells (MSCs) by siRNAs-mediated knockdown of GSK3β siRNAs in cultures of MSCs derived from human adipose tissue (AD-MSCs). For this purpose, we compared the transfection efficacy of lipoplexes and polyplexes formed with one of four siRNA molecules and five commercial transfection agents most commonly used in laboratory practice. The most effective transfection agent was found to be linear polyethylenimine (PEI) which demonstrated high cytocompatibility both in free form and in polyplexes (even when maximum concentrations were used). Using the polyplexes formed by the newly designed siRNA and PEI, we constructed a highly efficient GSK3β gene knockdown system, which showed effectiveness in AD-MSC cultures. As a result, we demonstrated the osteoinductive properties of GSK3β siRNA molecules in these cultures. These results provide a methodological basis for future siRNA-based strategies targeting GSK3β in osteogenic applications, with in vivo studies needed to establish its translational potential.