Circulating tumor DNA (ctDNA) is characterized by low abundance and fragmentation, limiting the development of genetic variant detection technologies. In this study, we established a highly sensitive and specific assay by combining peptide nucleic acid (PNA)-mediated PCR clamping with CRISPR/Cas13a trans-cleavage detection. A PNA probe targeting the wild-type (WT) EGFR T790M allele was designed to suppress WT amplification during PCR, while minimally affecting mutant allele amplification. By combining the target specificity of Cas13a for mutant alleles with the WT-suppression capability of PNA-PCR, we achieved a dual-enrichment effect for mutant detection. When applied to EGFR T790M mutation detection, the assay reached an analytical sensitivity of 0.02%. We established a standard curve for T790M detection using cell-free DNA standards. Clinical validation in 20 plasma samples from lung adenocarcinoma patients demonstrated that the PNA-Cas13a assay achieved a diagnostic sensitivity of 93.3% (95% CI: 68.1%-99.8%) and a specificity of 100% (95% CI: 47.8%-100%), with detection concordance comparable to or improved over ARMS-PCR in this pilot cohort. The results suggest its preliminary diagnostic utility in liquid biopsy. In conclusion, the PNA-Cas13a assay enables sensitive and specific detection of EGFR T790M mutations in ctDNA, is readily adaptable to multiple gene loci, and holds promise for clinical monitoring of tumor drug resistance.

TRAF1 is an NF-κB inducible signaling adaptor protein that regulates immune responses. While it limits the NF-κB and MAPK pathways downstream of TLR in monocytes and macrophages, it promotes the survival of T and B lymphocytes downstream of 4-1BB and CD40, respectively. This multifaceted role requires genetic and pharmacological manipulation of TRAF1 expression and its interaction with its signaling partners. Key to such studies is the availability of reliable and specific antibodies that detect TRAF1 protein expression. Here, we systematically evaluate four different commercially available TRAF1 antibody clones (1F3, H3, E12, and 45D3) using wildtype (WT) and Traf1 knockout (KO) cells as definitive controls. We confirm robust induction of Traf1 mRNA in wild-type bone marrow derived macrophages (BMDMs) following stimulation with LPS, Pam3CSK4, or Resiquimod, and T cell activation by CD3 or CD3 + CD28 co-stimulation. Despite this, clones 1F3 and 45D3 produced non-specific signals in negative-control lysates, while clones E12 and H3 detected mouse TRAF1 only when overexpressed in HEK293FT cells and failed to detect endogenous TRAF1 in isolated BMDMs or T cells, even when lysates were intentionally overloaded or enriched by immunoprecipitation. Notably, clone H3 reproducibly detected TRAF1 in lysates of total splenocytes activated with anti-CD3 or anti-CD3 plus LPS, indicating that the assay itself is functional and that detection becomes possible in mixed cell populations where higher-expressing subsets are present. These findings reveal a critical sensitivity gap: currently available commercial antibodies are not adequate for measuring endogenous TRAF1 in isolated monocytes/macrophages or T cells, the very cell types most relevant to inflammation-driven and autoimmune diseases, and underscore the need for new, rigorously validated reagents to support cell-type specific studies of TRAF1 biology in mouse models.

Tumor-immune interactions in the cancer microenvironment have a major influence on the development of colorectal cancer (CRC), as well as immune evasion by the CRC tumor. Chemokine CXCL9 (which signals through the CXCR3 receptor) has been implicated in the immune recruitment of cells and progression of tumors; however, the role of CXCL9 in CRC is poorly understood. To explore the mechanistic role of CXCL9 in CRC cell proliferation, migration, invasion, survival, and immune modulation, we utilized CRC cell lines HCT116 and HT-29. Knocking down CXCL9 expression using siRNA decreased CRC cell proliferation, EdU incorporation, clonogenic survival, and anchorage-independent growth. The restoration of CXCL9 expression partially reversed these observations. In migration and invasion assays, CXCL9 increased motility and epithelial-mesenchymal transition (EMT) of CRC cells via the regulation of E-cadherin, N-cadherin, and vimentin expression. Silencing CXCL9 expression (due to the CXCL9 siRNA) resulted in increased apoptosis and G0/G1 cell cycle arrest, whereas restoring CXCL9 expression allowed CRC cells to transition to S-phase. Mechanistically, CXCL9 maintained redox homeostasis by preventing accumulation of reactive oxygen species (ROS), and activating pro-survival signaling pathways (AKT, ERK1/2, and NF-κB). Importantly, CXCL9 positively regulated immune checkpoint molecules (PD-L1 and IDO1) at both mRNA and protein levels. In co-culture experiments, CXCL9 promoted CD8+ T-cell chemotaxis through CXCR3 and enhanced T-cell-mediated cytotoxicity, effector molecule expression, and pro-inflammatory cytokine secretion under the tested in vitro conditions. Together, these in vitro findings suggest that CXCL9 contributes to CRC cell proliferation, migration, invasion, redox regulation, immune checkpoint-associated signaling, and CD8+ T cell responses in simplified co-culture models. The novelty of this study lies in the combined evaluation of tumor-cell phenotypes, PD-L1/IDO1-associated signaling, and CD8+ T cell responses within the same CXCL9-modulated CRC cell-line system. Further validation using patient-derived samples, in vivo models, and clinically annotated cohorts is required before broader conclusions regarding the CRC tumor microenvironment or therapeutic targeting can be made.

Celiac disease (CD) is a prototypical example of gluten-induced enteropathy characterized by a strong breakdown of mucosal tolerance. However, the spatial processes underlying intercellular communication across the epithelial-lamina propria barrier remain poorly known. Exosomes have evolved to be useful orchestrators of this autoinflammatory cascade. In contrast to their passive metabolic by-product role, gut-derived exosomes functionally mediate the non-canonical presentation of deamidated gliadin peptides and disease-associated HLA-DQ complexes, which directly prime pathogenic CD4[+] T cells independently of conventional cellular synaptic interaction. At the same time, dysregulated exosomal microRNA (miRNA) signatures undermine tight junction integrity and promote pro-inflammatory signaling loops systemically, providing a probable mechanistic link to extra-intestinal symptoms. In this review, the dual role of exosomes as pathogenic propagation vectors and real-time liquid biopsies in CD will be critically discussed.

Acute myeloid leukemia (AML) is a hematologic malignancy that necessitates the identification of new therapeutic targets. Recently, the low-density lipoprotein receptor (LDLR) has been linked to an unfavorable prognosis in AML. LDLR plays a crucial role in various signaling pathways, including the MAPK signaling pathway. Our data show high LDLR expression in the AML cell lines THP-1 and NB4. Knockdown of LDLR using shRNA reduced AML cell proliferation, induced apoptosis, and led to S-phase cell accumulation, accompanied by modulation of the MAPK pathway. Additionally, myosin regulatory light chain-interacting protein (MYLIP) is expressed at low levels in AML and regulates LDLR. Overexpression of MYLIP downregulated LDLR expression, accompanied by an influence on the MAPK signaling pathway. Functionally, MYLIP overexpression decreased AML cell proliferation, increased apoptosis, and potentially delayed S-phase progression. These findings suggest that MYLIP and LDLR may regulate AML cell expansion, potentially through effects on the MAPK pathway. Furthermore, the p38-MAPK activator PCS reversed the inhibitory effect of LDLR knockdown and MYLIP overexpression on cell proliferation. Consequently, targeting LDLR could be a promising approach for AML treatment.