The elegant hierarchical structures of biomacromolecules have promoted the pursuits of synthetic polymers with ordered monomer sequences and diverse topological architectures, which are in the initial stage. Here, a four-arm star-shaped segmented polyurethane (PU) with a controlled block sequence of amphiphilic seven-segment multifunctional arms containing a disulfide bond was prepared via a grafting-onto strategy, where the sequence-defined arms were synthesized by a diisocyanate-based liquid-phase iterative methodology and then conjugated onto four alkyne-functionalized pentaerythritol cores through click chemistry. The star-shaped PU can self-assemble into micelles in aqueous solution as the linear arm. Moreover, the four-arm star-shaped architecture endows self-assembled micelles with higher stability under various physiological conditions and enhanced redox-responsive performance, making them promising candidates for drug delivery. The experiments show that drug-loaded star-shaped PU micelles possess accelerated stimuli-responsive release profiles, optimized tissue distribution, and improved anticancer efficacy in vitro and in vivo in comparison with linear polymer micelles. This work demonstrates that advanced architectures of precise linear polymers hold tremendous potential for the structural regulation and performance adjustment of aggregation structures, opening the perspectives for the structural and functional design of next-generation materials.
Influenza causes 650,000 deaths, 3-5 million hospitalizations, and 1 billion cases worldwide each year. There is a limited repertoire of antivirals available to tackle this burden, highlighting the risk of developing resistance to current drugs. An effort to develop new influenza antivirals has been made, leading to the approval of baloxavir. Some mutations leading to reduced susceptibility to baloxavir have been reported, but information about their epidemiology is scarce. Thus, we aim to evaluate the prevalence of substitutions in the polymerase acidic (PA) subunit associated with baloxavir susceptibility over 16 epidemiological seasons in Europe. We evaluated 87,266 sequences collected from 2009 to 2025 in Europe, annotated the mutations, and identified all known amino acid substitutions related to changes in baloxavir susceptibility to assess their prevalence. A total of 149 (0.2%) sequences with at least one substitution were identified, 81 (0.09%) exhibiting reduced susceptibility. Overall, 17 different substitutions were detected, located both inside and outside baloxavir binding site. The number of substitutions detected ranged from 0 to 15 per season, with E23K, E23R, K34R, A36V, I38F, I38L, and E120D emerging after baloxavir approval in Europe. Double and triple concurrent substitutions were also identified. While the prevalence of sequences with substitutions that alter baloxavir susceptibility remains stable, the number of circulating substitutions increases over time. This implies the emergence of amino acid substitutions that did not circulate before and the concurrence of double and triple substitutions that might synergize their individual effects. These results highlight the need for virological surveillance and novel antiviral treatments.
The genus Sesamum (Pedaliaceae) comprises a wide range of cultivated and wild species. Sesame (Sesamum indicum) is recognized as one of the oldest oilseed crops cultivated worldwide, whereas S. schinzianum is a wild relative closely associated with the evolutionary history of cultivated sesame. Although the nuclear and chloroplast genomes of Sesamum species have been investigated in previous studies, mitochondrial genome evolution within the genus has received relatively limited attention. In this study, we assembled and comparatively analyzed the mitochondrial genomes of S. indicum and S. schinzianum by integrating BGI short-read sequencing and Oxford Nanopore long-read sequencing data. Genome assembly was performed using Flye and Unicycler, and annotation was conducted using PMGA together with manual curation. Comparative analyses were then carried out to examine genome organization, gene content, repetitive sequences, codon usage, RNA editing sites, chloroplast-derived sequences, phylogenetic relationships, and collinearity patterns. The mitochondrial genomes of S. indicum and S. schinzianum were assembled into one circular molecule and two major circular contigs, respectively, and both contained 36 conserved protein-coding genes. Abundant simple sequence repeats dominated by tetranucleotide motifs and notable repeat variation were detected. Codon usage showed moderate bias, and 478 and 455 RNA editing sites were predicted in S. indicum and S. schinzianum, respectively. Chloroplast-derived sequences accounted for 8.50% and 6.96% of the mitochondrial genomes, respectively. Phylogenetic and collinearity analyses supported a close relationship between the two Sesamum species and identified synteny-based structural differences between their mitogenomes. Comparative analysis of mitochondrial- and chloroplast-based phylogenies showed that the two datasets were largely congruent at the family level and consistently supported the close relationship between the two Sesamum species, although they differed in the placement of several deeper lineages. These results suggest that Sesamum mitogenomes retain conserved gene content while showing assembly- and synteny-supported structural differences. This study provides useful genomic resources for comparative and evolutionary studies of mitochondrial genomes in Pedaliaceae.
Dipolar recoupling techniques play a fundamental role in solid-state NMR spectroscopy, enabling precise structural characterization of solid materials under magic-angle spinning (MAS). Among these, the supercycled R41 2 (SR4) stands out for its efficiency and stability in recoupling heteronuclear dipolar interactions under fast MAS conditions (≥40 kHz). However, its performance significantly degrades at slow-to-moderate MAS rates (<35 kHz), which restricts its wide applications. To overcome this limitation, herein we introduce a practical modification of SR4 through the strategic incorporation of adjustable window delays between the recoupling π pulses, dubbed windowed SR4 (wSR4). This design introduces the variability of pulse duration and rf amplitude, which simplifies the recoupling optimization procedure, while enabling enhanced recoupling performance and improved tolerance to experimental imperfections. Theoretical analysis and NMR experiments collectively confirm that wSR4 substantially boosts heteronuclear dipolar recoupling efficiency while effectively suppressing undesired spin interactions, particularly under slow MAS conditions. The practical utility of wSR4 is further validated in in situ NMR experiments on SAPO zeolite, where the markedly improved 2D correlation spectroscopy provides deeper insights into local structures. This approach enables precise dipolar measurement, efficient polarization transfer, and the establishment of high-fidelity heteronuclear correlations, all of which are critical for structural elucidation in complex solid systems. Moreover, the windowed SR4 design strategy introduced here is expected to provide a generalizable framework for optimizing symmetry-based recoupling sequences across a wide range of MAS conditions.
The ability to access atomically tailored complex peptides and proteins provides powerful opportunities for dissecting molecular functions and advancing applications in chemical biology, therapeutics, and (bio)-materials science. Robust precision-engineering strategies are essential to construct well-defined protein architectures while preserving native folding and activity. To do so, chemoselective bioconjugation techniques have been developed to modify specific side chains of amino acids. This allowed for the selective introduction of functionalities on predetermined amino acids. However, ultimate control can be achieved only through site-selective modifications that precisely define both the nature of the linkage and the exact position of conjugation on elongated peptide sequences or fully assembled proteins. Cysteine residues are of particular interest, as their highly nucleophilic thiols offer excellent chemoselectivity and typically occur in low abundance in their reduced form. Here, we examine chemoselective transformations targeting cysteine residues that have been further refined to occur exclusively at predefined positions within a peptide or protein, thereby achieving a high degree of site-selectivity. This review focuses exclusively on chemical strategies for cysteine modification, offering guidance for future synthetic developments within the field of precision chemistry. Achieving this level of precision requires advanced chemical strategies that exploit the local environment of the targeted cysteine. One approach involves leveraging neighboring functional groups, for example, engaging the thiol together with the α-amine or carboxylate to enable selective N- or C-terminal modification, respectively. In such designs, the cysteine side chain may contribute through transient interactions, direct incorporation into the covalent linkage, or the stabilization of the desired product. Recently, a promising strategy has attracted increasing attention in which site-selectivity is enabled by temporary interaction with a proximal amine, thus being applicable to differentiate also between internal cysteines. Together, these strategies highlight that site-selective protein modification has evolved into a powerful tool for the rational design and functional control of complex biomolecules, redefining what is achievable in chemical biology, therapeutics, and biomaterials science. We anticipate that increasingly routine or user-friendly approaches such as the programmable TriTEx method will further accelerate the adoption of precision biomolecule conjugates in both research and industrial settings.
Four species of tardigrades have been found in the meiofauna communities of the seagrass meadows and adjacent areas off the coast of Vietnam. For two of them, Batillipes binhdinhicus sp. nov. and Batillipes ampullus sp. nov., integrative descriptions are presented. Batillipes binhdinhicus sp. nov. belongs to the B1 subgroup of species by having toe 3 shorter than toe 4 on legs IV, while Batillipes ampullus sp. nov. with toes 3 and 4 on legs IV of equal length belongs to the A group of species. Batillipes binhdinhicus sp. nov. can be distinguished from all previously described species by a set of features: very long cephalic cirri and leg sensory spines, both with swollen tips, presence of a single pair of conical lateral body projections between all pairs of legs and simple triangular caudal appendage. The distinctive features for Batillipes ampullus sp. nov. are short sensory organs on the fourth pair of legs, which consists of two parts and have swollen tips with a tuft of additional sensory filaments that are structurally similar to those found on the cephalic appendages. In addition, Batillipes ampullus sp. nov. does not have noticeable lateral body projections between the legs, and the body ends in a semi-circular tail appendage. Based on the obtained data on the nucleotide sequences of 18S rDNA and 28S rDNA, the distinction between the new species was confirmed.
CD1d-restricted invariant natural killer (iNK) T cells are innate T cells known for their ability to shape adaptive immunity toward inflammation or immune-suppression via the rapid production of Th1-, Th2-, and Th17-type cytokines from corresponding iNKT subsets such as NKT1, NKT2, and NKT17. IL-10-producing invariant NKT cells, termed NKT10 cells, are thought to play an immunoregulatory role, but their potential clinical use remains underexplored. We characterized human NKT10 cells from cord-derived iNKT cells and investigated their therapeutic utility in allogeneic stem cell transplantation. Cord and cord-derived iNKT cells contained a high frequency of CD4+CD25+CD161lowFoxP3+ iNKT cells and showed Th2/Th10-biased cytokine production upon antigenic stimulation. Accordingly, cord-derived iNKT cells displayed a distinct gene expression profile with upregulated genes related to NKT2, NKT10, and regulatory T cells compared with adult donor-derived iNKT cells. Furthermore, single-cell RNA sequencing analysis of cord-derived iNKT cells confirmed the presence of NKT10-like subset that was enriched with multiple immunoregulatory pathways and genes related to immune-checkpoints (NRP1, PD1, CLTA-4, and GITR) and NKT10 (MAF, HIF1A, and FoxP3), whereas the NKT1/17-like subset present in adult donor-derived iNKT cells showed upregulation of genes related to cytotoxicity (GZMA/B, KLRD1, and PRF1), NKR (KLRK1, KLRB1, KLRG1, and NKG7), NKT1 (EOMES and TBX21), and NKT17 (RORC). Lastly, cord-derived iNKT cells suppressed alloreactive T cell proliferation in vitro and ameliorated xenogeneic graft-versus-host disease where the immunodeficient NSG mice received human peripheral blood mononuclear cells supplemented with cord-derived iNKT cells. Thus, NKT10-enriched, cord-derived iNKT cells are candidate cell therapeutics for immune-modulation in allogeneic stem cell transplantation and other autoimmune diseases.
Breast cancer (BC) remains a leading cause of cancer-related mortality worldwide. Cancer-associated fibroblasts (CAFs) is a central stromal component of the tumor microenvironment (TME), critically influence BC progression and therapeutic resistance. However, the association between CAF heterogeneity and patient prognosis or response to immunotherapy remains poorly characterized. Here, we aimed to develop a CAF-associated gene signature by integrating single-cell RNA sequencing (scRNA-seq) and bulk RNA-seq data to predict clinical outcomes and immunotherapeutic response in BC. Gene expression profiles and clinical data from BC patients were sourced from TCGA and GEO databases. scRNA-seq data preprocessed (quality control, PCA, UMAP using Seurat) identified CAF-related genes. Prognostic genes were identified via univariate Cox, lasso, and multivariate Cox regression. Single-cell Gene Set Enrichment Analysis (scGSEA) assessed the signature's link to immune infiltration and immunotherapy genes. R tools evaluated signature characteristics and real-world applications. GO enrichment analysis was used to explore signaling pathways. CAF factor expression and CD8+ T-cell correlation in clinical BC samples were validated using qPCR, immunohistochemical (IHC), multiplex immunofluorescence (mIF), and Western blot. scRNA-seq analysis identified multiple CAF-specific marker genes that formed the core of our signature. Eight genes (ANXA5, APOD, CXCL14, GSN, IGFBP4, PPIB, TCF7L2, and TMEM98) were associated with favorable prognosis (low-risk), whereas three genes (SDC1, EMP1, and FAM114A1) conferred higher risk. A risk score model based on these 11 genes independently predicted overall survival (OS) across diverse BC pathological subtypes, demonstrating robust prognostic accuracy. Immune infiltration analysis revealed significantly reduced immune cell abundance in the high-risk group compared to the low-risk group, suggesting diminished responsiveness to immunotherapy. In tumor tissues relative to adjacent nontumor tissues, mRNA and protein levels of the high-risk genes (SDC1, EMP1, and FAM114A1) were consistently elevated (all p < 0.05). Moreover, both Western blotting and mIF showed significantly higher CAF abundance in high-risk samples (p < 0.01), concomitant with markedly lower CD8+ tumor-infiltrating lymphocyte counts (p < 0.05). GO enrichment analyses indicated that CAFs promote BC evolution and progression through complex signaling networks. Key pathways included extracellular matrix (ECM) remodeling, cell adhesion, tumor associated inflammation, and oncogenic cascades such as KRAS, WNT, IL-6/STAT3, and TNFα/NF-κB highlighting CAFs as pivotal regulators and potential therapeutic targets in BC. We present a novel CAF associated gene signature that robustly predicts prognosis and immunotherapy response in BC. As an independent prognostic indicator strongly correlated with immune infiltration, this model holds promise for guiding personalized therapeutic strategies. Future validation in large, multicenter cohorts and extension to other malignancies are warranted to facilitate clinical translation of CAF targeted biomarkers.
Acute myeloid leukemia (AML) is an aggressive hematologic malignancy associated with a high risk of relapse. Leukemia cutis is characterized by the presence of neoplastic leukocytes infiltrating the skin. Here, we present a previously treated AML patient who relapsed with isolated leukemia cutis without bone marrow involvement. Fluorescence in situ hybridization (FISH) and RNA sequencing of the skin lesion revealed a KMT2A::MLLT6 rearrangement, which enabled the use of revumenib. This case highlights the diagnostic complexity and therapeutic implications of isolated extramedullary AML relapse and underscores the need to perform genetic testing at the site of disease recurrence-especially without bone marrow involvement of relapsed disease. The authors have confirmed clinical trial registration is not needed for this submission.
Human gut microbial communities capable of degrading mucin are taxonomically unique and have a range of physiologically relevant metabolic outputs. To determine the feasibility of reviving mucin-degrading fecal microbial communities after cryopreservation, we employed 16S rRNA gene sequencing to characterize revived communities. Microbial communities were generally stable but small donor-dependent shifts in diversity, composition, and taxonomy were observed following revival. The revivability of these microbial communities is valuable for studying mucin-degrading microbial communities.
Background Patients with relapsed or refractory (R/R) follicular lymphoma (FL) after two or more prior lines of therapy can now be treated with mechanistically distinct modalities, including CD19-directed chimeric antigen receptor (CAR) T-cell therapy, CD20×CD3 bispecific antibodies, and a Bruton tyrosine kinase inhibitor plus anti-CD20 combination. Head-to-head data are lacking, and sequencing decisions are made with limited comparative evidence. We benchmarked durability outcomes across pivotal trials using reconstructed individual patient data (rIPD) derived from published Kaplan-Meier (KM) curves. Methods KM curves and numbers-at-risk tables from pivotal prospective studies in ≥2-line R/R FL were digitized and rIPD-reconstructed using a validated algorithm: axicabtagene ciloleucel (ZUMA-5), tisagenlecleucel (ELARA), lisocabtagene maraleucel (TRANSCEND FL), mosunetuzumab, epcoritamab (EPCORE NHL-1), and zanubrutinib plus obinutuzumab versus obinutuzumab (ROSEWOOD). Prespecified durability endpoints were landmark progression-free survival (PFS) at 12 and 24 months and restricted mean survival time to 24 months (RMST24). Overall survival (OS) landmarks were summarized where follow-up permitted. ROSEWOOD served as an internal validity check using a Cox model fit to reconstructed data. Results At 24 months, landmark PFS was 65.4% (95% confidence interval (CI) 52.0-82.4) for liso-cel, 62.0% (50.2-76.6) for axi-cel, 53.8% (45.2-63.9) for zanubrutinib plus obinutuzumab, 49.3% (39.3-61.7) for mosunetuzumab, 43.7% (31.3-61.1) for epcoritamab, and 24.7% (14.9-40.9) for obinutuzumab monotherapy. Twelve-month PFS ranged from 81.8% (74.5-89.8) with liso-cel and 77.9% (69.4-87.5) with axi-cel to 61.6% (53.6-70.8), 60.4% (50.8-72.0), and 59.1% (50.5-69.1) for zanubrutinib plus obinutuzumab, mosunetuzumab, and epcoritamab, respectively; tisagenlecleucel showed 12-month PFS of 68.3% (57.2-81.6) with shorter follow-up (maximum 18.2 months). PFS RMST24 estimates were 19.5 months for liso-cel, 18.6 for axi-cel, 16.5 for zanubrutinib plus obinutuzumab, 16.2 for mosunetuzumab, 14.8 for epcoritamab, and 11.9 for obinutuzumab. OS at 24 months was high across regimens (mosunetuzumab 87.3%, liso-cel 84.6%, axi-cel 82.8%, zanubrutinib plus obinutuzumab 77.3%, and epcoritamab 67.6%). In ROSEWOOD, reconstructed data reproduced the published PFS benefit for zanubrutinib plus obinutuzumab (hazard ratio (HR) 0.48, 95% CI 0.32-0.71; p < 0.001) with a weaker OS signal (HR 0.61, 0.35-1.07; p = 0.08). Conclusions In this rIPD-based durability benchmark for ≥2-line R/R FL, the estimated landmark PFS and RMST24 values varied across regimens, with numerically higher point estimates for the CAR T-cell therapies and the zanubrutinib plus obinutuzumab combination and numerically lower estimates for the bispecific antibodies and the obinutuzumab control; CIs overlapped substantially, and 24-month OS estimates were broadly similar across regimens. Because the source trials enrolled materially different populations, these side-by-side estimates are descriptive benchmarks and should not be read as head-to-head comparisons of efficacy. Faithful internal reproduction of the randomized ROSEWOOD effect supports reconstruction fidelity. The analysis provides a transparent, durability-focused reference framework to support sequencing discussions and hypothesis generation, not comparative effectiveness conclusions.
Background: Glioma is among the most malignant brain tumors, and its heterogeneity contributes significantly to treatment failure. Comprehensive profiling of cellular and molecular heterogeneity across different glioma stages and recurrence states is crucial for understanding therapeutic resistance and identifying novel targets. Accordingly, this study sought to systematically characterize the cellular and molecular heterogeneity of glioma across different stages and recurrence states using single-cell RNA sequencing, and to identify prognostic subtypes and potential therapeutic targets. Methods: We integrated public single-cell RNA sequencing data from glioma specimens, including lower-grade glioma (LGG), glioblastoma (GBM), and paired primary and recurrent tumors. Using these datasets, we identified distinct cellular subpopulations and their molecular signatures. Based on these glioma cell subpopulations, we reclassified gliomas from The Cancer Genome Atlas (TCGA) database into molecular subtypes and constructed a prognostic model. The functional role of a key candidate gene, insulin-like growth factor binding protein 2 (IGFBP2), was validated using in vitro knockdown experiments in mouse and human tumor cells and in vivo therapeutic studies in murine models, including combination therapy with anti-programmed cell death protein 1 (anti-PD-1) immune checkpoint blockade. Results: This analysis revealed that T cells in GBM and recurrent samples were predominantly exhausted, characterized by upregulation of PD-1 and T-cell immunoglobulin and mucin-domain containing-3 (Tim3), while myeloid cells exhibited an immunosuppressive phenotype with elevated expression of macrophage migration inhibitory factor (MIF) and cluster of differentiation (CD)276. We identified 12 distinct glioma cell subpopulations with varying proliferative and hypoxic signatures. Based on these subpopulations, TCGA gliomas were reclassified into two major subtypes. One subtype, enriched with myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs), was associated with poorer patient prognosis. A prognostic model was successfully established using differentially expressed genes between the subtypes. Furthermore, IGFBP2 was highly expressed in glioma cells, and its expression negatively correlated with T cell infiltration. In vitro knockdown of IGFBP2 downregulated programmed death-ligand 1 (PD-L1) expression on tumor cells. In vivo, IGFBP2 knockdown significantly suppressed tumor growth (p < 0.01) and extended survival in tumor-bearing mice (p < 0.05), and its combination with anti-PD-1 therapy markedly enhanced antitumor efficacy. Conclusions: This study provides deeper insights into the cellular ecosystem and heterogeneity of glioma, linking specific cellular features to patient prognosis. We identify IGFBP2 may play a role in regulating immunosuppressive tumor microenvironment and a potential therapeutic target.
[This corrects the article DOI: 10.3389/fimmu.2026.1682740.].
Differences in enhancer activity between species can help drive phenotypic diversity, yet enhancers often have conserved functions despite rapid sequence evolution, posing a challenge for quantifying their functional differences between species. Previous machine learning models have focused on the binary task of predicting differences in the presence of enhancers between species but have yet to demonstrate an ability to predict continuous differences in enhancer activity. Here, we trained convolutional neural networks on a regression task to predict chromatin accessibility-a proxy for enhancer activity-in the liver across five mammals, and we developed a novel framework to evaluate cross-species performance. We demonstrated that training on multiple species improves model generalization to both species used in training and held-out species. However, the models consistently achieved poor performance in predicting quantitative differences in accessibility between species at orthologous regions. Our study highlights the challenges in using regression models to predict chromatin accessibility changes between species. All data and code are available at http://daphne.compbio.cs.cmu.edu/files/azstephe/liver_regression_resource/ and https://figshare.com/projects/liverRegression/274293.
Lipoprotein lipase (LPL) is essential for plasma triglyceride (TG) hydrolysis, and biallelic LPL loss-of-function variants cause familial chylomicronemia syndrome. However, the clinical and functional significance of heterozygous LPL variants remains incompletely characterized. We investigated two heterozygous LPL missense variants, c.1187A>T (p.Glu396Val) and the novel c.1251G>C (p.Trp417Cys), in patients with severe hypertriglyceridemia (HTG). Two probands underwent comprehensive clinical, biochemical, and imaging assessments. Genetic analysis involved whole-exome sequencing and Sanger confirmation. Variant interpretation incorporated in silico prediction, evolutionary conservation, AlphaFold-based residue mapping, LPL-GPIHBP1 structural-context analysis, and NetCGlyc prediction. Functional effects were assessed in HEK293T cells transfected with wild-type or mutant LPL plasmids, measuring mRNA/protein expression and enzymatic activity. Clinically, Proband 1 exhibited recurrent pancreatitis with moderate hyperglycemia, whereas Proband 2 had extreme HTG without pancreatitis but with severe insulin resistance, reflecting variable expressivity. Two LPL missense variants, c.1187A>T (p.Glu396Val) and the novel c.1251G>C (p.Trp417Cys), both located in exon 8. Both affected residues were highly conserved and localized to the C-terminal domain by AlphaFold-based mapping. NetCGlyc predicted Trp417 as a potential C-mannosylation-related residue. Mutant LPL mRNA and protein expression were comparable to wild-type, whereas enzymatic activity was significantly reduced in cell lysates and culture medium. These findings provide functional evidence that p.Glu396Val and the novel p.Trp417Cys impair LPL enzymatic activity despite preserved protein abundance, supporting a qualitative functional defect. Our findings highlight the value of functional validation and metabolic assessment in interpreting heterozygous LPL variants in HTG.
Periostin (POSTN), an extracellular matrix protein secreted by cancer-associated fibroblasts (CAFs), critically shapes the tumor microenvironment. While POSTN contributes to tumor progression and immune regulation by modifying the microenvironment, its direct influence on T-cell activity and antitumor immunity remains unclear. In this study, single-cell and bulk RNA sequencing datasets were mined and combined with multiplex fluorescence immunohistochemistry to elucidate the role of POSTN+ CAFs. Primary CAFs were isolated and fibroblast lines with elevated POSTN expression were established to assess their direct impact on T-cells through functional assays. Transcriptome sequencing identified downstream regulatory pathways in POSTN+ CAFs. A mouse peritoneal metastasis model was used to evaluate the therapeutic potential of targeting POSTN+ CAFs in combination with immunotherapy. Integration of single-cell transcriptomic datasets enabled the construction of a predictive model for gastric cancer peritoneal metastasis (GCPM) based on T-cell exhaustion-associated genes. POSTN was found to be primarily expressed by CAFs in GCPM tissues, with elevated levels correlating with poor clinical outcomes. Functionally, POSTN+ CAFs promoted adhesion and spheroid formation by autocrine activation of the AKT-NF-κB-ICAM-1 signaling pathway, facilitating peritoneal dissemination. Co-culture and in vivo experiments suggested that POSTN+ CAFs may promote CD8+ T-cell exhaustion via ICAM-1-ETV3 signaling, resulting in diminished effector function and cytokine production. Inhibiting POSTN downstream signaling with the integrin receptor antagonist Cilengitide, especially together with immune checkpoint blockade (anti-PD-1 and anti-CTLA-4), markedly reduced T-cell exhaustion and suppressed GCPM. Our results indicate that POSTN+ CAFs are pivotal in maintaining an immunosuppressive microenvironment in GCPM. POSTN may facilitates CAFs-tumor and CAFs-T-cell interactions through ICAM-1, thereby promoting metastasis and promoting immune evasion. Targeting the POSTN-ICAM-1 axis may provide a therapeutic approach to enhance immunotherapy efficacy and enhance immunotherapy outcomes in patients with GCPM.
Multiple myeloma (MM) is a malignant plasma cell disorder characterized by clonal expansion of plasma cells in bone marrow. Genetic aberrations in MM are well‑established, while epigenetic alterations, particularly DNA methylation, are increasingly recognized as key regulators of gene expression. The modified bases 5‑methylcytosine (5‑mC) and 5‑hydroxymethylcytosine (5‑hmC) occur in gene promoters and regulatory regions. While the presence of 5‑mC in the promoter is associated with transcriptional silencing, 5‑hmC, generated by oxidation of 5‑mC through ten‑eleven translocation (TET), is associated with gene activation. The present study investigated promoter‑specific 5‑hmC and 5‑mC levels in TET1, TET2 and TET3 genes in patients with MM and myeloma cell lines. Quantification of methylation was performed using a glucosylation and enzyme digestion‑based method, bisulfite sequencing and nanopore sequencing. TET mRNA expression levels were assessed using reverse transcription‑quantitative PCR and interactions with specificity protein (Sp)‑1 Sp1 and Sp3 transcription factors were analyzed using chromatin immunoprecipitation. TET promoter‑wide 5‑mC and 5‑hmC profiles were compared in CD138+ sorted cells from newly diagnosed and relapsed patients with MM and in five myeloma cell lines treated with 5‑azacytidine and/or 5‑aza‑2'‑deoxycytidine. In newly diagnosed patients, TET1 mRNA expression levels were increased in comparison with TET2 and TET3, which corresponded to a reduced CCGG site methylation of the TET1 promoter (5‑10%) and increased TET3 methylation (21‑50%). Of the MM cell lines, the 5‑azacytidine‑treated KMS12‑PE cell line exhibited significantly higher TET2 gene expression levels when compared with TET1 and TET3. Subsequently, immunoprecipitation with Sp3 demonstrated increased TET2 recruitment, which suggested a potential interaction between Sp3 and TET2. The present findings indicated dynamic regulation of TET genes through CCGG site methylation and hydroxymethylation of their promoter in MM and demonstrated that demethylating agents selectively modulate TET gene expression and promoter occupancy, highlighting their potential impact on epigenetic gene activation. Notably, the divergent expression patterns observed suggest that TET1 acts as an oncogenic driver, while the inducible response of TET2 highlights its role as a potential tumor suppressor in myeloma biology. Consequently, the present results provide a rationale for the pharmacological restoration of TET2 expression as a strategic approach to suppress tumor progression through epigenetic reprogramming.
Backgrounds: Tertiary lymphoid structures (TLSs) are increasingly recognized as modulators of anti-tumor immunity, yet their clinical relevance in bladder cancer remains incompletely understood, partly owing to heterogeneity in their maturation states. Here, we demonstrate that germinal center (GC)-like TLS maturity, rather than TLS presence alone, is closely associated with immune activation and therapeutic response to Programmed Death-Ligand 1 (PD-L1) blockade in bladder cancer. The objective of this study was to systematically investigate the clinical significance, biological function, and therapeutic potential of tertiary lymphoid structure (TLS) maturation in bladder cancer. Specifically, we aimed to determine whether GC-like TLS maturity provides prognostic and predictive value beyond TLS presence alone, to elucidate the immune programs and tumor microenvironment remodeling associated with TLS maturation, and to explore whether TLS maturation can be therapeutically induced to enhance responsiveness to PD-L1 blockade. Methods: We performed an integrative analysis combining multi-cohort transcriptomics, spatially resolved histopathology, single-cell RNA sequencing, and functional murine experiments. TLS maturation states were defined using gene-expression-based GC-like TLS signatures and validated through multiplex immunohistochemistry. Clinical relevance was assessed in public immunotherapy cohorts and an independent neoadjuvant PD-L1-treated muscle-invasive bladder cancer (MIBC) cohort. Tumor immune microenvironment remodeling and chemokine-mediated cellular crosstalk were analyzed using deconvolution, Weighted Gene Co-expression Network Analysis (WGCNA), and CellChat. The therapeutic inducibility of TLS maturation was examined using a lymphotoxin-β receptor (LTβR) agonist in combination with PD-L1 blockade in a syngeneic bladder cancer model. Results: Across multiple transcriptomic cohorts, tumors enriched for GC-like TLS signatures exhibited significantly prolonged survival and higher objective response rates to anti-PD-L1 therapy, whereas less mature TLS phenotypes showed no consistent association with clinical association. These observations were independently validated in a neoadjuvant PD-L1-treated muscle-invasive bladder cancer cohort, in which high mature TLS density was associated with major pathological response and prolonged event-free survival, outperforming PD-L1 expression. Integrative histopathological and transcriptomic analyses indicated that GC formation marks a functional transition linking humoral immune programs with cytotoxic effector activity and shaping a memory-prone, pro-inflammatory tumor immune microenvironment. Chemokine signaling via the CC chemokine ligand 21 (CCL21)-C-C chemokine receptor type 7 (CCR7) and C-X-C motif chemokine ligand 12 (CXCL12)-C-X-C chemokine receptor type 4 (CXCR4) axes was strongly associated with TLS maturation and spatial organization. Finally, in a syngeneic bladder cancer model, pharmacological activation of lymphotoxin-β receptor signaling promoted TLS maturation and enhanced the antitumor efficacy of PD-L1 blockade. Conclusions: Together, these findings suggest that GC-like TLS maturity represents a clinically relevant biomarker and a potential therapeutic entry point for precision immunotherapy in bladder cancer. Therapeutic strategies that promote TLS maturation may convert immune-cold tumors into checkpoint-responsive states, providing a mechanistically grounded precision immunotherapy approach.
Caeconyx papso sp. nov. within the family Uristidae is described from the Porcupine Abyssal Plain, Northeast Atlantic Ocean. The new species differs from the only other member of the genus, Caeconyx caeculus, in possessing a triangular, sub-acute eye lobe, a convex and broadly rounded posterodistal margin of epimeron 1, and propodi of pereopods 3 and 4 that are not slender or elongate. Molecular sequence data for COI, H3, 16S, and 28S genes are provided for C. papso sp. nov. Phylogenetic analyses were conducted using this sequence data to assess the relationships between the new species, other members of the family Uristidae, and morphologically allied taxa within the family Tryphosidae. The new species is fully illustrated, and a key to species within Caeconyx is presented.
The multiplex analysis of reactive biomolecules is crucial in diagnostics and life science research. However, conventional methods using small-molecule-based fluorescent probes are limited in the number of simultaneously detectable targets because of the spectral overlap of their fluorescence wavelengths. Herein we present a sequencing-free multiplexed analysis platform using chemoresponsive DNA-based fluorescent probes. A target-responsive moiety was installed in the phosphate backbone of the probe to strategically destabilize the DNA duplex. Reaction with a target molecule such as hydrogen peroxide or nitroreductase cleaved this moiety, restoring duplex stability and thereby triggering the accumulation of fluorescent product DNAs on beads functionalized with a complementary sequence. By encoding beads with distinct fluorescence intensity ratios and sizes, we achieved the simultaneous and specific detection of multiple targets in a single sample. The system performed well even with complex biological samples. This modular "reaction-to-accumulation" strategy offers a generalizable approach for developing DNA-based multiplex detection systems tailored to different target molecules.