Biology subjects

Johnson, N. M.

Publications and source records attributed to Johnson, N. M..

6 recordsLinked to original sources

bioRxiv2023

Extracellular domain, hinge, and transmembrane determinants affecting surface CD4 expression of a novel anti-HIV chimeric antigen receptor (CAR) construct

Chimeric antigen receptor (CAR)-T cells have demonstrated clinical potential, but current receptors still need improvements to be successful against chronic HIV infection. In this study, we address some requirements of CAR motifs for strong surface expression of a novel anti-HIV CAR by evaluating important elements in the extracellular, hinge, and transmembrane (TM) domains. When combining a truncated CD4 extracellular domain and CD8 hinge/TM, the novel CAR did not express extracellularly but was detectable intracellularly. By shortening the CD8 hinge, CD4-CAR surface expression was partially recovered and addition of the LYC motif at the end of the CD8 TM fully recovered both intracellular and extracellular CAR expression. Mutation of LYC to TTA or TTC showed severe abrogation of CAR expression by flow cytometry and confocal microscopy. Additionally, we determined that CD4-CAR surface expression could be maximized by the removal of FQKAS motif at the junction of the extracellular domain and the hinge region. CD4-CAR surface expression also resulted in cytotoxic CAR T cell killing of HIV Env+ target cells. In this study, we identified elements that are crucial for optimal CAR surface expression, highlighting the need for structural analysis studies to establish fundamental guidelines of CAR designs.

molecular biology↗

bioRxiv2023

The double life of trichomes: understanding their dual role in herbivory and herbicide resistance

External features of organisms often serve as the first line of defense in their immediate environments. Trichomes are hair-like appendages on plant surfaces that can defend against damaging agents such as pathogens, herbivores, and UV radiation. It is currently unknown if the variation observed in trichome traits represents dual or conflicting roles against different types of stressors. Here, we assess whether trichomes serve as an herbicide resistance trait and how it coincides with the conventionally studied defensive strategy of herbivory resistance. In a series of experiments, we exposed the annual invasive velvetleaf (Abutilon theophrasti) to glyphosate (active ingredient in the herbicide Roundup) to investigate whether trichome traits (shape and density) are linked to herbicide resistance and to test whether herbicide alters selection on plant trichomes. We found that an increased proportion of branched trichomes positively impacted herbicide resistance as well as chewing herbivory resistance. We also found evidence that glyphosate imposes positive selection on branched trichomes in velvetleaf. Overall, our results indicate that branched trichomes can contribute to both herbicide and herbivory resistance, serving as a dual structural form of resistance reducing plant injury. If our results are to be applied more broadly, it would suggest that herbicide exposure can alter the composition of plant trichomes, potentially impacting trichome-mediated defenses against various external stressors.

evolutionary biology↗

bioRxiv2022

Autophagic state prospectively identifies facultative stem cells in the intestinal epithelium

The intestinal epithelium exhibits a rapid and efficient regenerative response to injury. Emerging evidence supports a model where plasticity of differentiated cells, particularly those in the secretory lineages, contributes to epithelial regeneration upon ablation of injury-sensitive stem cells. However, such facultative stem cell activity is rare within secretory populations. Here we ask whether specific functional properties predict facultative stem cell activity. We utilize in vivo labeling combined with ex vivo organoid formation assays to evaluate how cell age and autophagic state contribute to facultative stem cell activity within secretory lineages. Strikingly, we find that cell age (time elapsed since cell cycle exit) does not correlate with secretory cell plasticity. Instead, high autophagic activity predicts plasticity and resistance to DNA damaging injury independently of cell lineage. Our findings indicate that autophagic status prior to injury serves as a lineageagnostic marker for the prospective identification of facultative stem cells.

cell biology↗

bioRxiv2021

Patient induced pluripotent stem cell-derived hepatostellate organoids establish a basis for liver pathologies in telomeropathies

Patients with dyskeratosis congenita (DC) and related telomeropathies resulting from premature telomere dysfunction suffer from multi-organ failure. In the liver, DC patients present with nodular hyperplasia, steatosis, inflammation, and cirrhosis. We model DC liver pathologies using isogenic human induced pluripotent stem (iPS) cells harboring a causal DC mutation in DKC1, or a clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-corrected control allele. Differentiation of these iPS cells into hepatocytes or hepatic stellate cells followed by generation of genotype-admixed hepatostellate organoids revealed a dominant phenotype in the parenchyma, with DC hepatocytes eliciting a pathogenic hyperplastic response in stellate cells independent of stellate cell genotype. Pathogenic phenotypes could be rescued via suppression of AKT activity, a central regulator of MYC-driven hyperplasia downstream of DKC1 mutation. Thus, isogenic iPS-derived admixed hepatostellate organoids offer insight into the liver pathologies in telomeropathies and provide a framework for evaluating emerging therapies.

cell biology↗

bioRxiv2021

Dicamba drift alters plant-herbivore interactions at the agro-ecological interface

Natural populations evolve in response to biotic and abiotic changes in their environment, which shape species interactions and ecosystem dynamics. Agricultural systems can introduce novel conditions via herbicide exposure to non-crop habitats in surrounding fields. While herbicide drift is known to produce a variety of toxic effects in plants, little is known about its impact on non-target wildlife species interactions. In a two-year study, we investigated the impact of herbicide drift on plant-herbivore interactions with common weed velvetleaf (Abutlion theophrasti) as the focal species. The findings reveal a significant increase in the phloem feeding silverleaf whitefly (Bermisia tabaci) abundance on plants exposed to herbicide at drift rates of 0.5% and 1% of the field dose. We also identified a significant phenotypic tradeoff between whitefly resistance and herbicide resistance in addition to whitefly resistance and relative growth rate in the presence of dicamba drift after increasing the populations grown in year two. In a follow-up greenhouse study, we found evidence that dicamba drift at 0.5% of the field dose significantly increased average chlorophyll content (mg/cm2) along with a positive correlation between whitefly abundance and chlorophyll content. Overall, these findings suggest herbicide exposure to non-target communities can significantly alter herbivore populations, potentially impacting biodiversity and community dynamics of weed populations found at the agro-ecological interface.

ecology↗

bioRxiv2020

Evolutionarily conserved regulation of embryonic fast-twitch skeletal muscle differentiation by Pbx factors

Vertebrate skeletal muscles are composed of both slow-twitch and fast-twitch fiber types. How the differentiation of distinct fiber types is activated during embryogenesis is not well characterized. Skeletal muscle differentiation is initiated by the activity of the myogenic basic helix-loop-helix (bHLH) transcription factors Myf5, Myod1, Myf6, and Myog. Myod1 functions as a muscle master regulatory factor and directly activates muscle differentiation genes, including those specific to both slow and fast muscle fibers. Our previous studies showed that Pbx TALE-class homeodomain proteins bind with Myod1 on the promoter of the zebrafish fast muscle gene mylpfa and are required for proper activation of mylpfa expression and the fast-twitch muscle-specific differentiation program in zebrafish embryos. Pbx proteins have also been shown to bind regulatory regions of muscle differentiation genes in mammalian muscle cells in culture. Here, we use new zebrafish mutant strains to confirm the essential roles of zebrafish Pbx factors in embryonic fast muscle differentiation. Furthermore, we examine the requirements for Pbx genes in mouse embryonic skeletal muscle differentiation, an area that has not been investigated in the mammalian embryo. Removing Pbx1 function from skeletal muscle in Myf5Cre/+;Pbx1fl/fl mouse embryos has minor effects on embryonic muscle development. However, concomitantly deleting Pbx2 function in Myf5Cre/+;Pbx1fl/fl;Pbx2-/- mouse embryos causes delayed activation and reduced expression of fast muscle differentiation genes. In the mouse, Pbx1/Pbx2-dependent fast muscle genes closely match those that have been previously shown to be dependent on murine Six1 and Six4. This work establishes evolutionarily conserved requirements for Pbx factors in embryonic fast muscle differentiation. Our studies are revealing how Pbx homeodomain proteins help direct specific cellular differentiation pathways.

developmental biology↗