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RIMap-RISC is a web-accessible database for transcriptome-wide modeling of human microRNA (miRNA) targeting. It computes plausible transcript-miRNA interactions and records their position, duplex secondary structure, free energy, site classification, dissociation constant, target accessibility, and evolutionary conservation. RIMap-RISC supports transcript-wide queries and allows users to explore and export interaction data through an interactive interface or RESTful API programmatic access. Unlike existing tools, RIMap-RISC integrates duplex-structure prediction within a biophysical framework modeling the bipartite architecture of RISC, accommodating a bridge between seed and supplementary pairing. A novel, unambiguous, miRNA-centric nomenclature for interaction types is also introduced.

BackgroundChimeric antigen receptor T cell (CAR-T)-based immunotherapies have reshaped the therapeutic landscape of cancer treatment, in particular for patients afflicted with leukemia. However, defects in CAR behaviors and clinical complications have hindered their widespread application across diverse cancer types. Chief among these defects is high tonic signaling, absent in native activating immune receptors, which accelerates T cell exhaustion and undermines treatment efficacy. We hypothesized that these limitations arise because current CAR architectures fail to replicate the modular design of native activating immune receptors, which integrate distinct receptor and signaling modules. This modular assembly is crucial for maintaining proper receptor regulation and function.MethodsTherefore, we set forth to develop a modular chimeric antigen receptor leveraging the same assembly principles found in native activating immune receptors to reestablish the intrinsic safeguards in receptor expression and signaling.ResultsThe resulting Modular Actuation Receptor Complex (MARC) displayed surface expression levels akin to its native immune receptor counterpart, the NK cell receptor KIR2DS3, while eliminating tonic signaling. In a clinically relevant mouse leukemia model, MARC-T cells exhibited remarkable long-term persistence and a less exhausted phenotype compared with conventional CAR-T cells.ConclusionsWith its modular architecture, the MARC offers unparalleled opportunities for optimization and broad applicability across different cell types, paving the way for transformative advancements in cell-based therapies. This innovation holds immense promise as a next-generation therapeutic tool in clinical settings.

The majority of cancer deaths are caused by solid tumors, where the four most prevalent cancers (breast, lung, colorectal and prostate) account for more than 60% of all cases (1). Tumor cell heterogeneity driven by variable cancer microenvironments, such as hypoxia, is a key determinant of therapeutic outcome. We developed a novel culture protocol, termed the Long-Term Hypoxia (LTHY) time course, to recapitulate the gradual development of severe hypoxia seen in vivo to mimic conditions observed in primary tumors. Cells subjected to LTHY underwent a non-canonical epithelial to mesenchymal transition (EMT) based on miRNA and mRNA signatures as well as displayed EMT-like morphological changes. Concomitant to this, we report production of a novel truncated isoform of WT1 transcription factor (tWt1), a non-canonical EMT driver, with expression driven by a yet undescribed intronic promoter through hypoxia-responsive elements (HREs). We further demonstrated that tWt1 initiates translation from an intron-derived start codon, retains proper subcellular localization and DNA binding. A similar tWt1 is also expressed in LTHY-cultured human cancer cell lines as well as primary cancers and predicts long-term patient survival. Our study not only demonstrates the importance of culture conditions that better mimic those observed in primary cancers, especially with regards to hypoxia, but also identifies a novel isoform of WT1 which correlates with poor long-term survival in ovarian cancer.

The majority of cancer deaths are caused by solid tumors, where the four most prevalent cancers (breast, lung, colorectal and prostate) account for more than 60% of all cases (1). Tumor cell heterogeneity driven by variable cancer microenvironments, such as hypoxia, is a key determinant of therapeutic outcome. We developed a novel culture protocol, termed the Long-Term Hypoxia (LTHY) time course, to recapitulate the gradual development of severe hypoxia seen in vivo, to mimic conditions observed in primary tumors. Cells subjected to LTHY underwent a non-canonical epithelial to mesenchymal transition (EMT) based on miRNA and mRNA signatures as well as displayed EMT-like morphological changes. Concomitant to this, we report production of a novel truncated isoform of WT1 transcription factor (tWt1), a non-canonical EMT driver, with expression driven by a yet undescribed intronic promoter through hypoxia-responsive elements (HREs). We further demonstrated that tWt1 initiates translation from an intron-derived start codon, retains proper subcellular localization, DNA binding, and its human ortholog negatively predicts long-term patient survival. Our study demonstrates the importance of culture conditions that better mimic those observed in cancers, especially with regards to hypoxia, and identifies a novel isoform of WT1 which correlates with poor long-term survival in ovarian cancer.

Random mutations followed by natural selection is one of the key mechanisms during natural evolution that results in the generation of new functions. This process is typically slow in nature because of low mutational frequency. In contrast, the B cells of mammalian immune systems have evolved somatic hypermutation (SHM) mechanisms that introduce mutations at the immunoglobulin genomic loci at a significantly higher frequency than the rest of the genome. SHM allows B cells to rapidly evolve new antibody sequences without compromising their fitness as a consequence of genome-wide mutations. In this work, we developed a continuous directed evolution platform in human B cell lines (CODE-HB) that recruits and repurposes the SHM mechanisms to rapidly evolve reporter proteins like the Green Fluorescent Protein. This approach uses a stable, non-immunoglobulin locus within the genomes of human B cell lines. To comprehensively characterize the mutational profile and breadth of this strategy, we performed single-molecule sequencing experiments. We illustrate the utility of the platform by rapidly evolving antibody fragments in a continuous manner by B cell surface display targeting avian subtypes of influenza hemagglutinin (e.g., H5) from emerging strains.Competing Interest StatementThe authors have declared no competing interest.Footnotes* We have significantly expanded on our manuscript and added several new results and figures to show the utility of this approach as well as fundamentally characterize this system in greater depth.

Bacteriophage auxiliary metabolic genes (AMGs) alter host metabolism by hijacking reactions. Previous studies used functional annotations to infer AMG impacts but neglected propagation effects on global metabolism and phage production. We demonstrate the first integration of AMGs and phage assembly into a genome-scale metabolic model, using a general method applied to the infection of Prochloroccocus marinus MED4 by P-HM2. We experimentally validate our approach to predicting AMG impact on growth using cp12 mutations in Syne-chococcus elongatus. We predict that 17 directly hijacked reactions substantially impact over 30% of the metabolism, including carbon fixation, photosynthesis, and nucleotide synthesis. We find that indirect impacts are synergistically and antagonistically coupled and are either phage-aligned—shifting feasible reaction velocities in accordance with maximal phage production—or phage-antialigned. Pareto optimization reveals that phage-aligned reactions limit host growth, while phage-antialigned reactions do not. We provide systems-level insight into AMG perturbations, highlighting how nontrivial cascading effects shape microbial functions.