N6-methyladenosine (m6A) modification constitutes a crucial layer of post-transcriptional regulations, but the landscape of its downstream readout effects remains less comprehensively understood. Therefore, we systematically assess the readout effects of m6A on mRNA half-life, translation efficiency, and alternative splicing across five cell lines (A549, HEK293T, HUVEC, JURKAT, and human embryonic stem cells (hESCs)) using actinomycin D-disrupted temporal transcriptome, ribosome sequencing, and ultra-high-depth transcriptome sequencing, respectively. Our analysis, coupled with the integration of public and newly profiled m6A methylome data, reveals high cell type specificity in m6A readouts where m6A level alone is insufficient to predict m6A readouts. Nonetheless, machine learning models focusing on RNA-binding protein (RBP) binding context can effectively predict the readouts and prioritize four novel m6A-associated proteins (FUBP3, FXR2, L1TD1, and DDX6). Their m6A-binding ability is validated by m6A RNA pull-down, transcriptome-wide binding site mapping, and electrophoretic mobility shift assay, while FUBP3 and L1TD1 are further suggested as m6A readers regulating mRNA stability based on half-life profiling of knockout cells. Finally, FUBP3, FXR2, and L1TD1 are demonstrated to regulate hESC differentiation without affecting self-renewal. Together, this study bridges the gap in understanding m6A functional readouts and lays the groundwork for future research on m6A-mediated stem cell fate decisions.

Core fucosylation of N-glycoproteins plays pivotal roles in regulating many cellular events such as receptor-ligand binding and cell adhesion. Here, we developed a chemoenzymatic method combining selective enrichment, enzymatic reactions, and multiplexed proteomics to systematically quantify the core fucosylation stoichiometries of glycoproteins in human cells. The results demonstrated that the core fucosylation stoichiometries vary dramatically in different subcellular compartments with the lowest in the lysosome and the highest in the extracellular matrix. Different core fucosylation stoichiometries were observed among glycosylation sites in various protein domains, and more aromatic and hydrophobic residues neighboring glycosylation sites are associated with lower core fucosylation stoichiometry. The method was applied to quantify the core fucosylation stoichiometry changes in the epithelial-to-mesenchymal transition (EMT), and some glycoproteins involved in extracellular matrix organization and ligand recognition displayed marked stoichiometry changes. Furthermore, the core fucosylation stoichiometries in embryonic human kidney cells (HEK293T) were compared with those in kidney cancer cells (A498). The average stoichiometry in HEK293T cells was much higher than that of A498 cells, indicating that core fucosylation may be a critical regulator in embryonic development. Without any sample restriction, this method can be extensively applied to investigate core fucosylation changes in various biological samples.

Current base editors act on a maximum of two base substrates and generate limited base conversions or transversions, hindering their applicability for inducing DNA sequence diversity. Here, we engineered a triple base editor (named ACG-BEs) using a fusion of adenine base editor with high A/C catalytic activity and evolved N-methylpurine DNA glycosylase. ACG-BEs enables efficient, multiplexed saturation mutagenesis across adenine (A), cytosine (C), and guanine (G), achieving conversion efficiencies of up to 80.5% for A-to-G/C/T, 75.8% for C-to-T/G/A, and 63.4% for G-to-C/T/A in HEK293T cells. Leveraging ACG-BEs, we identify novel mutations in the HBG1/2 promoter region that confer efficient activation of γ-globin expression in HUDEP-2 cells—a promising advancement for therapeutic strategies targeting hemoglobinopathies. These findings highlight ACG-BEs as a cutting-edge platform for multiplexed saturation mutagenesis, offering broad applications in genetic screening and therapeutic base mutation introduction through enhanced DNA sequence diversity.

Xiang pig is a native inbred line originating from a warm and humid mountainous region and is prone to infections by pathogens causing lung lesions. However, the underlying genetic factors still remain unclear. Our previous genomic resequencing comparisons found a deletion type of structural variation (SV) within the intron of gene cerebellar degeneration related protein 2 (CDR2) with a higher frequency in Xiang pigs compared with European pig breeds. This study is aimed to explore the relationship between the SV and the lung injury in Xiang pig populations. It demonstrated that the deletion genotype (MM) was dominant in Xiang pig herds and with a higher percentage of allele M than that in Xiang pig with pulmonary lesions and Large White breeds. By Sanger sequencing and RepeatMasker analysis, a SINE in 277bp (namely SINE-277) was detected and located in the opposite orientation of CDR2 gene. The inhibit effects of SINE-277 on transcription was confirmed by EGFP reporter gene after transfected into HEK-293T cells. And the expression levels of CDR2 was increased in the lungs with MM types (P < 0.05) via both RT-qPCR and Western blotting assays. Moreover, significant differences were estimated between the SV genotypes and the lung lesion severity scores, the antibody concentrations against pathogens, and expressions of nine inflammation factor genes including NFκB. It reinforced the effects of SINE on gene CDR2 expression and might be taken as a DNA marker for the resistance/susceptibility to lung diseases in pigs.

Abstract Introduction Although driver oncogenic fusions offer an appealing therapeutic target, there is a critical shortage of targeted therapies against many fusions. Customizable direct target inhibition would address this gap. We developed an individualized antisense oligonucleotide (ASO) targeting the NFIA::CBFA2T3 fusion for a patient with relapsed acute myelogenous leukemia (AML). Diagnosed at 5 months of age with isolated central nervous system (CNS) AML, the patient underwent surgical resection, radiation therapy, and multiple rounds of systemic and intrathecal (IT)/intraventricular chemotherapy over 18 months. Ultimately, widespread leptomeningeal and extra-CNS AML progression occurred, requiring extraventricular drain (EVD) placement prior to ASO administration. We hypothesized the ASO would eliminate NFIA::CBFA2T3 fusion expression and reduce viability of fusion-bearing cells. Methods CLIA-certified whole transcriptome RNA sequencing was performed at relapse. A panel of five unique ASO constructs were designed against the NFIA::CBFA2T3 fusion breakpoint. Constructs were tested in 3 model cell lines and 1 patient-derived cell line. Neurotoxicity of the lead ASO was evaluated in NOD scid gamma (NSG) mice. A single patient IND application with a dose escalation schema received IRB and FDA approval. GMP-grade ASO (2 mg) was administered via IT injection at disease progression after informed consent was obtained. Adverse events were reported per CTCAE v5.0. Pharmacokinetic analysis on cerebrospinal fluid (CSF) and peripheral blood was performed using liquid chromatography/mass spectrometry. Results The lead ASO diminished NFIA::CBFA2T3 transcript expression by 83% in HEK293T cells expressing NFIA::CBFA2T3 and reduced cell number by 57% (p&amp;lt;0.05) in the patient-derived cell line. There was no decrease of endogenous NFIA mRNA or growth-inhibitory effect to non-fusion-bearing cells. Animal models showed no signs of toxicity. Within 4 months of pre-clinical testing initiation, ASO was administered during a period of rapid disease progression. Surrounding administration, the patient experienced elevated CSF output (maximum 386 mL/day), cerebral edema, and elevated CSF cytokines IL-6 (18-905 pg/mL) and IL-8 (199-4039 pg/mL). Adverse events grade 3 or above with possible or probable attribution to ASO included depressed level of consciousness, cerebral edema, hydrocephalus, and seizure. Maximal ASO CSF concentration was 648 ng/mL at 48 hours and was undetectable by day 5 post ASO. The ASO was not detected in peripheral blood. The patient experienced further AML progression and died 21 days post ASO. Conclusion An NFIA::CBFA2T3 ASO was engineered and demonstrated decreased transcript expression preclinically. The successful clinical delivery demonstrates proof-of-principle for personalized ASOs in pediatric oncologic care. Toxicity attribution is complicated by rapid disease progression. Ongoing work will more deeply phenotype this patient’s clinical course and develop ASO platform trials. Citation Format: Monica Pomaville, Hyojeong Hwang, Alexis Boulter, Tina Glisovic-Aplenc, Praneeth Bommisetti, Katelyn Oranges, Brandi Nelson, Jeffrey Schubert, Feng Xu, Jinhua Wu, Gregory M. Podsakoff, Margaret Tartaglione, Olivia Caradonio, Ellen Maple, Johannes Van Der Loo, Aashim Bhatia, Michael LaRiviere, Madison Hollawell, Mateusz Koptyra, Marilyn Li, Theodore W. Laetsch, Peter Madsen, Jessica B. Foster, Richard Aplenc, Fange Liu. Personalized antisense oligonucleotide treatment in a patient with relapsed NFIA::CBFA2T3 acute myelogenous leukemia [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Fusion-Positive Cancer: From Discovery to Therapy; 2026 Jan 13-15; Philadelphia PA. Philadelphia (PA): AACR; Cancer Res 2026;86(1_Suppl):Abstract nr B008.

Lipid droplets (LDs) are dynamic organelles that exhibit cell-type-specific heterogeneity in size, composition, and abundance to support diverse cellular functions. However, the molecular mechanisms regulating this functional diversity remain poorly understood. Here, we identified a cell-type-specific truncated isoform of Plin2, which was present in macrophages but absent in adipocytes. Using N-terminal HA- and C-terminal FLAG-tagged Plin2 constructs combined with immunoprecipitation-mass spectrometry analysis in HEK293T cells or macrophages, we confirmed the N-terminal truncation and mapped the deletion site to residues 40-44. Ectopic expression of this truncated variant significantly reduced LD size in both macrophages and HEK293T cells. These findings reveal that macrophages modulate lipid storage by expressing distinct Plin2 protein variants, suggesting new therapeutic targets for lipid metabolism disorders.

The identification of SiMyD88 confirms the presence of a functional TLR-MyD88-IRAK signaling axis in the sea urchin Strongylocentrotus intermedius.

Hemophilia B (HB), an X-linked recessive disorder, results from variants in the coagulation factor IX gene (F9). The F9 c.520 + 13A > G variant is a recurrent intronic variant in HB patients, accounting for 15.05% of all documented F9 intronic variants. Despite prior predictions of its impact, the molecular mechanism associated with moderate to mild HB remains undissected. In silico predictions and splicing-competent cDNA constructs were used to assess the impact of F9 c.520 + 13A > G on mRNA splicing. Factor IX (FIX) variant (p.V174delinsGHNLM) expression in HEK293T cells was evaluated using activated partial thromboplastin time, enzyme-linked immunosorbent assay, Western blot analysis, and immunofluorescence analyses. Structural modeling and molecular dynamics simulations were performed to evaluate the structural impact of the variant. Engineered U1 small nuclear RNA (U1snRNA) was challenged with F9 full-length splicing-competent constructs to evaluate splicing correction. The F9 c.520 + 13A > G variant caused nearly complete aberrant splicing, producing the F9 c.520_521insGTCATAATCTGA insertion and the in-frame FIX p.V174delinsGHNLM variant. A small amount (approximately 10%) of wild-type FIX was also detected. We characterize the p.V174delinsGHNLM variant, which exhibited impaired secretion and increased intracellular accumulation. Interestingly, an engineered U1snRNA partially rescued aberrant splicing, restoring functional FIX levels to approximately 40%. This study elucidates the molecular mechanism of the F9 c.520 + 13A > G variant, which activates a cryptic 5' splice site in intron 5, leading to an in-frame FIX (p.V174delinsGHNLM) with secretion defects and loss of protein function. And Engineered U1snRNA partially rescued the splicing defect.

Abstract Introduction and aims Dystrophic epidermolysis bullosa (DEB) is an inherited blistering skin disease with over 800 causative mutations identified in the COL7A1 gene. eePASSIGE is a breakthrough DNA editing technology that pairs prime editing (PE) with serine integrase technology, allowing for targeted integration of large DNA constructs. We aim (i) to use eePASSIGE to integrate a full-length cDNA of COL7A1 into the AAVS1 safe harbour locus, and (ii) to develop lipid-based nanoparticles (LNPs) capable of delivering eePASSIGE machinery to human skin cells. Together, these two strategies form the basis for a proposed permanent ‘one-size-fits-all’ DEB cure. Methods We have used lipofection of HEK293T, N/TERT, and human fibroblast cells to provide proof of concept. For PE and eePASSIGE, lipofectamine was used to deliver plasmid DNA encoding editing constructs and pegRNAs. For PE, Sanger sequencing of target site amplicons was used to identify successful edits. For eePASSIGE, puromycin selection was used to isolate successfully edited cells. For LNP studies, fluorescent reporter mRNA was delivered to N/TERTs and fibroblasts; successful transfection was quantified with flow cytometry. Results In N/TERT cells, we achieved up to 17% PE efficiency in transfected cells, although only 1% of cells were successfully transfected. eePASSIGE was used to successfully integrate a puromycin resistance gene into HEK293T cells at the AAVS1 safe harbour locus. Lipid nanoparticles were used to successfully transfect human fibroblasts and keratinocytes in submerged culture with reporter constructs with up to 80% efficiency and with lower toxicity than lipofectamine. Conclusions Our experiments thus far have laid the groundwork for future study of eePASSIGE to integrate large coding constructs into disease-relevant cells. Our next steps are (i) to improve transfection of eePASSIGE machinery in cells of interest, and (ii) to optimize LNP formulations for use in three-dimensional culture and in vivo mouse skin.

Inborn errors of immunity (IEI) impairing brain-intrinsic immune defenses can underlie herpes simplex virus encephalitis. By whole-exome sequencing of cohorts of herpesvirus-associated recurrent lymphocytic meningitis and acute retinal necrosis, we identified two patients heterozygous for variants in interferon (IFN) regulatory factor 7 (IRF7). The expression of the Q185X (patient 1, P1) and A86Rfs23X (P2) IRF7 variants in HEK293T cells resulted in truncated IRF7 proteins that lacked IFN-transactivating ability. Peripheral blood mononuclear cells from P1 exhibited reduced type I IFN responses to HSV-2 infection. Genetic knock-in of the IRF7 Q185X variant in THP-1 cells and stem cell-derived plasmacytoid dendritic cells (pDC) confirmed the disrupted IFN expression, resulting in impaired paracrine antiviral protection of meningeal fibroblasts. Strikingly, genetically heterozygous index patient pDC, but not those of healthy carrier family members, showed expression of only the pathogenic IRF7 Q185X allele, resulting in a homozygous transcriptotype. Collectively, this study identifies genetically heterozygous but transcriptionally homozygous IRF7 deficiency as an IEI underlying herpesvirus central nervous system infection.

Dual cholinergic modulation in dementia: Quinuclidine carbamates targeting butyrylcholinesterase and α7 nicotinic receptor.

Combination of transfection incompetent lipids having strikingly different aliphatic chain lengths in a liposome demonstrates superior transfection and produces high titre lentivirus.

Nogo-A-cleaved amino-terminal fragment but not Nogo-B regulates STAT3 activation.

Vitexin alleviates atopic dermatitis-associated itch via TRPV4 inhibition in sensory neurons and MRGPRX2/MrgprB2 blockade in mast cells.

Highly sensitive luciferase-based assay with red fluorescent protein expression for accurate quantitative monitoring and real-time visualization of cell invasion.

Ethanol extract of Liriodendron tulipifera leaves displays anti-inflammatory activity by suppressing the Syk/Src/NF-κB pathway.

Controlled design of guanidinium-functionalized methacrylamides: tuning polyplex formation and transfection efficiency through hydrophobic and pH-responsive motifs.

Use of a Near Infrared Probe to Assess Intracellular Hydrogen Sulfide Production.

Antigen cross-presentation is a functional feature of some specialized antigen-presenting cells, allowing them to internalize and process antigens from extracellular sources and present them via major histocompatibility class I (MHC-I) molecules to CD8+ T lymphocytes. This process is critical for the mounting of protective immunity to tumors and intracellular pathogens. Antigen cross-presentation can be monitored through in vivo and in vitro assays that are key to evaluate the efficiency and the molecular and cellular mechanisms of cross-presentation by a given cell type. This chapter intends to complement published protocols by describing how to use MutuDC, a murine immortalized type 1 conventional dendritic cell-like line, and a "nonprofessional" human antigen-presenting cell, the human embryonic kidney HEK 293T cell line, endowed with the ability to cross-present immune complexes through overexpression of an Fc receptor, for in vitro antigen cross-presentation assays.

Polymeric nanogels hold strong promise for gene delivery, but their production is often limited by poor scalability and inconsistent control over physicochemical properties. To address this challenge, we present a scalable microfluidic strategy for engineering carboxymethyl chitosan-grafted branched polyethyleneimine plasmid DNA nanogels (CMC-bPEI-pDNA NGs) using a coaxial flow reactor. This continuous flow platform enables precise control over nanogel formation, offering tunability in particle size, surface charge, and encapsulation efficiency. Through systematic process development and parametric optimisation - including investigations into hydrodynamics, mixing, reactor geometry, and effect of reagent concentrations - we designed a novel process achieving high-throughput, reproducible nanogel production suitable for in vitro gene delivery. Optimised formulations, produced in as little as 3 s residence time, exhibited excellent monodispersity (polydispersity index, PDI < 0.2), sub-200 nm particle size, and pDNA encapsulation efficiency exceeding 90%. Fluorescence microscopy-based transfection assays confirmed effective intracellular delivery with high green fluorescent protein (GFP) expression in HEK293T cells 72 h post-transfection. We successfully scaled the process 100-fold by extending the reactor length, while maintaining similar physicochemical properties and biological performance. Nanogels produced at high throughput (1.14 L h-1) maintained a high GFP expression, confirming functional gene delivery and process scalability. We identified critical process parameters governing nanogel properties and scalability, including minimum residence time for nanogel formation, optimal flow rate ratios, reagent feeds configuration and reactor design for large-scale implementation. This work establishes a robust and scalable microfluidic process for producing functional polymeric nanogel gene delivery vectors, demonstrating its feasibility for translation from laboratory to larger-scale manufacturing, thereby serving as a proof of concept for future industrial-scale gene therapy applications.