Biology subjects

Johnson, B. D.

Publications and source records attributed to Johnson, B. D..

4 recordsLinked to original sources

bioRxiv2026

Elevated isoform richness in males largely reflects transcriptional noise rather than proteomic complexity

Alternative splicing (AS) can generate multiple RNA isoforms from a single gene and is thought to contribute to phenotypic divergence, including differences between the sexes. Studies in several organisms have documented sex differences in splicing, however these have been largely reliant on short-read RNA sequencing which requires complex algorithms to assemble full-length transcripts and may underestimate both isoform diversity and sex differences in splicing. We used long-read, single molecule RNA-Seq to build a more complete catalog of sex-biased splicing in Poecilia reticulata, a focal species for studies of sexual dimorphism. Pairing long-read sequencing with isoform-level analyses, we identified a sixfold higher proportion of sex-biased splicing genes (37%) compared with short and long-read event-level approaches (6%, 11%). AS was common (70% genes) but only 54% of isoforms produced unique open-reading frames (ORFs). We found that males exhibited greater isoform richness than females in both tail and gonad tissues but produced a smaller proportion of isoforms with unique ORFs, suggesting that much of the increased isoform variation is unlikely to expand proteomic complexity and may instead reflect stochasticity during splicing rather than intentional transcriptional products intended for translation. Despite widespread AS, we found only 2.3% of genes exhibited sex-biased isoform switching, and only 52% of these switches generated distinct sex-biased ORFs. Together, our long-read data suggest that although isoform diversity is more extensive than previously appreciated, most alternative isoforms are unlikely to generate novel proteins. Instead, a relatively small number of sex-biased isoforms may disproportionately contribute to proteomic divergence between the sexes.

genomics↗

bioRxiv2025

Genome Report: Genome of the Amazon Guppy (Poecilia bifurca) reveals conservation of sex chromosomes and dosage compensation

The Amazon guppy, Poecilia bifurca, is a small live-bearing fish. Closely related species, such as P. reticulata, P. picta, and P. parae all share the same sex chromosome system, but with substantial diversity in the degree of Y degeneration and the extent of X chromosome dosage compensation. We built a female (XX) draft genome with 55X coverage of PacBio HiFi data, resulting in a 785 Mb assembly with 94.4% BUSCO completeness. Using this genome, we found that P. bifurca shares the same sex chromosomes as related species and shows substantial Y chromosome degeneration. We combined this with RNA-Seq data and find similar expression of X-linked genes between sexes, revealing that P. bifurca also exhibits complete X chromosome dosage compensation. We further identify 11 putative autosome-to-Y gene duplications, five of which show gene expression in guppy male germ cells.

genomics↗

bioRxiv2024

TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant

BackgroundHigh-risk neuroblastoma (HR-NBL) is an aggressive tumor of the sympathetic nervous system with high risk of relapse and poor overall survival. Allogeneic hematopoietic cell transplant (allo-HCT) has been used previously in HR-NBL patients; however, graft-versus-host-disease (GVHD) and disease progression have limited clinical application. Ex-vivo stimulated allogeneic natural killer (NK) cells represent a potential approach to enhance the graft-versus-tumor (GVT) effect without exacerbation of GVHD but have not shown efficacy in NBL. MethodsEx-vivo stimulated NK cells from C57BL/6NCr (B6) mice were expanded with soluble IL-15/IL-15R alone or with irradiated CD137L/CD54+ AgN2a-4P (15-4P) at a 1:1 ratio for 10-12 days. Allogeneic NK cells were then analyzed for activation, proliferation, cytokine production, and cytotoxicity against two murine NBL cell lines, Neuro2a and NXS2, in the absence or presence of anti-TIM-3. Lethally irradiated B6AJF1 Mice received allo-HCT from B6 donors followed by NBL challenge after 7 days to mimic tumor relapse. Select groups received anti-TIM-3 starting on day 9 for every 4 days with/without infusions of 15-4P B6 NK cells on days 14, 21, and 28. In select experiments, T cell and NK cells were selectively depleted to establish their contribution to the GVT effect. All groups were analyzed for tumor growth, GVHD and overall survival. ResultsCo-culturing NK cells with 15-4P results in 78-fold expansion with increased expression of Ki-67 and NKG2D, NKp46, TRAIL and TIM-3. 15-4P stimulated allogeneic NK cells showed enhanced cytotoxicity against NBL compared to IL-15 NK cells alone but was limited in part due to high expression of TIM-3 ligands on Neuro-2a compared to NXS2. The addition of TIM-3 blockade further enhanced NK cytotoxicity versus Neuro-2a, with enhanced 15-4P NK cell degranulation, Eomes, TRAIL and FasL expression observed. Analysis of RNA from 15-4P NK cells exposed to TIM-3 blockade showed gene expression of chemokines, NKG2D/DAP12 signaling, non-canonical NF-{kappa}b pathway and TRAIL signaling. Blockade of NKG2D, TRAIL or FasL on 15-4P NK cells abrogated cytotoxicity. In vivo, the combination of 15-4P stimulated allogeneic NK cells and TIM-3 blockade after allo-HCT resulted in prolonged survival against NBL with decreased tumor burden compared to NK cells or anti-TIM-3 alone, without inducing GVHD. Depletion of NK cells, but not T cells, abrogated the GVT effect. ConclusionAllo-HCT can be a platform for treating NBL using combination ex-vivo stimulated allogeneic NK cell therapy with TIM-3 blockade to enhance the GVT effect without inducing GVHD. Ethics StatementThe animal study M005915 was reviewed and approved by University of Wisconsin-Madison IACUC. WHAT IS ALREADY KNOWN ON THIS TOPICO_LIT cell depleted (TCD) allogeneic hematopoietic cells transplant (allo-HCT) has potential to be a salvage therapy for relapsed/refractory neuroblastoma (NBL) through the graft-versus-tumor (GVT) effect, however, the high incidence of graft-vs-host disease (GVHD) and disease progression has hindered widespread clinical application. Allogeneic NK cell therapy can be a safe and feasible treatment but has had limited efficacy in NBL. TIM-3 blockade has shown encouraging results for a variety of tumors but has not been explored for NBL nor used to enhance the GVT effect. C_LI WHAT THIS STUDY ADDSO_LIEx vivo stimulated allogeneic NK cells demonstrate robust expansion, proliferation, activation and cytotoxicity against NBL which is further augmented by the addition of TIM-3 checkpoint blockade. In an allo-HCT model of NBL relapse, mice treated with IL-15/IL-15R/AgN2a-4P (15-4P) stimulated NK cells and anti-TIM-3 exhibited prolonged overall survival and reduced tumor growth without exacerbating GVHD. C_LI HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICYO_LIRelapse of NBL after TCD allo-HCT can be successfully treated with the combination of adoptively transferred 15-4P stimulated allogeneic NK cells and TIM-3 checkpoint blockade, representing a novel therapeutic approach that can be translated to the clinic or used as a platform to treat other solid tumors with high TIM-3 ligand expression. C_LI

immunology↗

bioRxiv2023

Mesenchymal stem cells ameliorate inflammation in an experimental model of Crohn's disease via the mesentery

ObjectiveMesenchymal stem cells (MSCs) are novel therapeutics for treatment of Crohns disease. However, their mechanism of action is unclear, especially in disease-relevant chronic models of inflammation. Thus, we used SAMP-1/YitFc, a chronic and spontaneous murine model of small intestinal inflammation, to study the therapeutic effect and mechanism of human bone marrow-derived MSCs (hMSC). DesignhMSC immunosuppressive potential was evaluated through in vitro mixed lymphocyte reaction, ELISA, macrophage co-culture, and RT-qPCR. Therapeutic efficacy and mechanism in SAMP were studied by stereomicroscopy, histopathology, MRI radiomics, flow cytometry, RT-qPCR, small animal imaging, and single-cell RNA sequencing (Sc-RNAseq). ResultshMSC dose-dependently inhibited naive T lymphocyte proliferation in MLR via PGE2 secretion and reprogrammed macrophages to an anti-inflammatory phenotype. hMSC promoted mucosal healing and immunologic response early after administration in SAMP model of chronic small intestinal inflammation when live hMSCs are present (until day 9) and resulted in complete response characterized by mucosal, histological, immunologic, and radiological healing by day 28 when no live hMSCs are present. hMSC mediate their effect via modulation of T cells and macrophages in the mesentery and mesenteric lymph nodes (mLN). Sc-RNAseq confirmed the anti-inflammatory phenotype of macrophages and identified macrophage efferocytosis of apoptotic hMSCs as a mechanism of action that explains their long-term efficacy. ConclusionhMSCs result in healing and tissue regeneration in a chronic model of small intestinal inflammation. Despite being short-lived, exert long-term effects via macrophage reprogramming to an anti-inflammatory phenotype. Data Transparency StatementSingle-cell RNA transcriptome datasets are deposited in an online open access repository Figshare (DOI: https://doi.org/10.6084/m9.figshare.21453936.v1)

immunology↗