Biology subjects

Harris, J. C.

Publications and source records attributed to Harris, J. C..

7 recordsLinked to original sources

bioRxiv2025

Autocrine TGFβ2 enforces a transcriptionally hybrid cell state in Ewing sarcoma

Sub-populations of cancer-associated fibroblast (CAF)-like tumor cells deposit extracellular matrix (ECM) proteins that support Ewing sarcoma (EwS) progression and metastasis. We previously showed a hallmark of CAF-like EwS cells is their hybrid transcriptional state wherein the driver fusion oncogene, EWS::FLI1, maintains activation of proliferative programs but loses capacity to repress mesenchymal genes. Here, we studied primary patient tumors and cell line models to identify molecular drivers of this hybrid state. Our data reveal that hybrid EwS cells are induced and maintained by a TGF{beta} signaling positive feedback loop. Hybrid cells de-repress TGFBR2 and upregulate expression and secretion of TGF{beta}2 to sustain pathway activation and ECM deposition. While TGF{beta} ligands can potently induce growth arrest in cells of epithelial origin, we show that TGF{beta}1 and TGF{beta}2 promote cell invasion of EwS cells without affecting proliferation. Thus, stroma and tumor-derived TGF{beta} ligands induce and maintain hybrid EwS cells to promote pro-metastatic cell phenotypes.

cancer biology↗

bioRxiv2023

Tissue-specific increases in hypoxia-inducible factor 1 alpha protein are independent of mRNA levels during acute hypoxic exposure of the Gulf killifish, Fundulus grandis

The hypoxia inducible factor 1 (HIF1) is a central regulator of the molecular responses to low oxygen in animals. It has been extensively studied in mammals, where its tissue levels are regulated by stabilization of the alpha subunit (HIF1) when oxygen levels decrease. Consistent with this, the initial characterization of HIF1 in fish cells in culture indicated that protein abundance increased during hypoxia even when transcription was blocked. Subsequent studies, however, have reported an increase in HIF1 mRNA levels during hypoxia in certain tissues of selected species, raising the question whether hypoxic exposure brings about coordinated changes in HIF1 mRNA and protein in tissues when measured in the same fish. We have directly addressed this question by determining levels of HIF1 protein and mRNA in the tissues of Gulf killifish, Fundulus grandis, exposed to short-term hypoxia (24 h at 1 mg O2 l-1). HIF1 protein was higher in brain, ovary, and skeletal muscle from fish exposed to hypoxia compared with normoxic controls by 6 h, and it remained elevated in brain and ovary at 24 h. In contrast, HIF1 mRNA levels were unaffected by hypoxia in any tissue. Moreover, levels of HIF1 protein and mRNA in the same tissues were not correlated with one another, during either normoxia or hypoxia. These results suggest that, during the initial response to low oxygen, HIF1 protein levels increase as the result of post-translational protein stabilization, rather than new transcription, as predicted from studies in mammalian and fish cells in culture. Summary StatementParallel measurements of protein and mRNA of the hypoxia inducible factor support post-translational protein stabilization rather than new transcription in the initial response of fish to low oxygen

physiology↗

bioRxiv2023

Wound microbiota-mediated correction of matrix metalloproteinase expression promotes re-epithelialization of diabetic wounds

Chronic wounds are a common and costly complication of diabetes, where multifactorial defects contribute to dysregulated skin repair, inflammation, tissue damage, and infection. We previously showed that aspects of the diabetic foot ulcer microbiota were correlated with poor healing outcomes, but many microbial species recovered remain uninvestigated with respect to wound healing. Here we focused on Alcaligenes faecalis, a Gram-negative bacterium that is frequently recovered from chronic wounds but rarely causes infection. Treatment of diabetic wounds with A. faecalis accelerated healing during early stages. We investigated the underlying mechanisms and found that A. faecalis treatment promotes re-epithelialization of diabetic keratinocytes, a process which is necessary for healing but deficient in chronic wounds. Overexpression of matrix metalloproteinases in diabetes contributes to failed epithelialization, and we found that A. faecalis treatment balances this overexpression to allow proper healing. This work uncovers a mechanism of bacterial-driven wound repair and provides a foundation for the development of microbiota-based wound interventions.

microbiology↗

bioRxiv2023

The microbiota and immune system non-genetically affect offspring phenotypes transgenerationally

Summary paragraphThe host-microbiota relationship has evolved to shape mammalian processes, including immunity, metabolism, and development1-3. Host phenotypes change in direct response to microbial exposures by the individual. Here we show that the microbiota induces phenotypic change not only in the individual but also in their succeeding generations of progeny. We found that germ-free mice exhibit a robust sebum secretion defect and transcriptional changes in various organs, persisting across multiple generations despite microbial colonization and breeding with conventional mice. Host-microbe interactions could be involved in this process, since T cell-deficient mice, which display defective sebum secretion4, also transgenerationally transmit their phenotype to progeny. These phenotypes are inherited by progeny conceived during in vitro fertilization using germ-free sperm and eggs, demonstrating that epigenetic information in the gametes is required for phenotypic transmission. Accordingly, small non-coding RNAs that can regulate embryonic gene expression5 were strikingly and similarly altered in gametes of germ-free and T cell-deficient mice. Thus, we have uncovered a novel mechanism whereby the microbiota and immune system induce phenotypic changes in successive generations of offspring. This epigenetic form of inheritance could be advantageous for host adaptation to environmental perturbation, where phenotypic diversity can be introduced more rapidly than by genetic mutation.

microbiology↗

bioRxiv2022

Improving Memory via Automated Targeted Memory Reactivation during Sleep

A widely accepted view in memory research is that previously acquired information can be reactivated during sleep, leading to persistent memory storage. Recently, Targeted Memory Reactivation (TMR) has been developed as a technique whereby specific memories can be reactivated during sleep using a sensory stimulus linked to prior learning. TMR can improve various types of memory, raising the possibility that it may be useful for cognitive enhancement and clinical therapy. A major challenge for the expanded use of TMR is that experimenters must manually control stimulation timing and intensity, which is impractical in most settings. To address this limitation, we developed the SleepStim system for automated TMR in the home environment. SleepStim includes a smartwatch to collect movement and heart-rate data, plus a smartphone to emit auditory cues. A machine-learning model identifies periods of deep non-REM sleep and triggers TMR sounds within these periods. We tested whether this system could replicate the spatial-memory benefit of in-lab TMR. Participants learned the locations of objects on a grid, and then half of the object locations were reactivated during sleep over three nights. In an experiment with 61 participants, the TMR effect was nonsignificant but varied systematically with stimulus intensity; low-intensity but not high-intensity stimuli produced memory benefits. In a second experiment with 24 participants, we limited stimulus intensity and found that TMR reliably improved spatial memory, consistent with effects observed in laboratory studies. We conclude that SleepStim can effectively accomplish automated TMR and that avoiding sleep disruption is critical for TMR benefits.

neuroscience↗

bioRxiv2021

High Throughput Characterization of V(D)J Recombination Signal Sequences Redefines the Consensus Sequence

In the adaptive immune system, V(D)J recombination initiates the production of a diverse antigen receptor repertoire in developing B and T cells. Recombination activating proteins, RAG1 and RAG2 (RAG1/2), catalyze V(D)J recombination by cleaving adjacent to recombination signal sequences (RSSs) that flank antigen receptor gene segments. Previous studies defined the consensus RSS as containing conserved heptamer and nonamer sequences separated by a less conserved 12 or 23 base-pair spacer sequence. However, many RSSs deviate from the consensus sequence. Here, we developed a cell-based, massively parallel V(D)J recombination assay to evaluate RAG1/2 activity on thousands of RSSs. We focused our study on the RSS heptamer and adjoining spacer region, as this region undergoes extensive conformational changes during RAG-mediated DNA cleavage. While the consensus heptamer sequence (CACAGTG) was marginally preferred, RAG1/2 was highly active on a wide range of non-consensus sequences. RAG1/2 generally preferred select purine/pyrimidine motifs that may accommodate heptamer unwinding in the RAG1/2 active site. Our results suggest RAG1/2 specificity for RSS heptamers is primarily dictated by DNA structural features dependent on purine/pyrimidine pattern, and to a lesser extent, RAG:RSS base-specific interactions. Further investigation of RAG1/2 specificity using this new approach will help elucidate the genetic instructions guiding V(D)J recombination. Summary StatementPartially conserved recombination signal sequences (RSSs) govern antigen receptor gene assembly during V(D)J recombination. Here, a massively parallel analysis of randomized RSSs reveals key attributes that allow DNA sequence diversity in the RAG1/2 active site and that contribute to the differential utilization of RSSs in endogenous V(D)J recombination. Overall, these results will assist identification of RAG1/2 off-target sites, which can drive leukemia cell transformation, as well as characterization of bona fide RSSs used to generate antigen receptor diversity.

molecular biology↗

bioRxiv2021

Artemisinin-based hybrids produce intracellular proteasome inhibitors that overcome resistance in Plasmodium falciparum

Artemisinin resistant Plasmodium falciparum (Pf) is spreading despite combination chemotherapy (ACT). Here we report the design of artezomibs, single-molecule hybrids of an artemisinin and a Pf-selective proteasome inhibitor. Artezomibs exert a novel mode of action inside the malaria parasites. The artemisinin component covalently modifies parasite proteins, which become substrates of the Pf proteasome. The proteasomal degradation products that bear the proteasome inhibitor component of the hybrid then inhibit Pf proteasomes, including those with mutations that reduce binding affinity of the proteasome inhibitor component on its own. We demonstrated that artezomibs circumvent both artemisinin resistance conferred by Kelch13 polymorphism and resistance to the proteasome inhibitor associated with mutations in Pf proteasomes. This mode of action may enable the use of a single molecule with one pharmacokinetic profile to prevent the emergence of resistance.

microbiology↗