Biology subjects

Gordon, J.

Publications and source records attributed to Gordon, J..

At least 19 recordsLinked to original sources

bioRxiv2026

Disrupted RGS coupling underlies a clinically distinct subset of GNAO1-related disorder

Heterotrimeric G proteins are transducers for a wide variety of neurotransmitter receptors. Mutations in the G protein o subunit are implicated in an early-onset neurological condition, GNAO1-related disorder, that manifests as movement disorders, developmental delays, and/or epileptic seizures. In a screen of disease-linked mutations in Go, we identified a subset that had no obvious biochemical defects, warranting further investigation. By structural modeling, protein-protein binding experiments in vitro and in cells, as well as measures of downstream ion channel electrophysiology, we identified a subset that impairs interactions with regulator of G protein signaling (RGS) proteins. RGS proteins accelerate G protein GTPase activity and are therefore critical for proper neurotransmitter signaling. Further, synthesis of current clinical data revealed that patients harboring these variants have comparatively mild symptoms. Our study demonstrates the importance of RGS regulation in G protein function, cell signaling, and human neurobiology.

biochemistry↗

bioRxiv2026

Mice with impaired synaptic facilitation exhibit deficits in cognitive flexibility

Behavioral flexibility is crucial to animal survival in dynamic environments, and a failure to update actions in response to recent outcomes is a hallmark of many neuropsychiatric disorders. However, the cellular and circuit mechanisms in the brain that support behavioral flexibility remain poorly understood. Forms of short-term plasticity such as synaptic facilitation have been theorized to transiently maintain information in neural circuits, and flexibly modulate how circuits process information depending on recent activity. Despite theoretical support, there is no direct experimental evidence linking synaptic facilitation to flexible decision-making. Recently, the presynaptic calcium sensor Synaptotagmin-7 (Syt7) has been shown to be required for synaptic facilitation at many synapses in the mammalian brain. Here, we assess operant learning in male and female Syt7 KO mice to determine how facilitation contributes to learning both stable and dynamic reward contingencies. We find that Syt7 KO mice acquired stable contingencies similarly to wild-type controls. However, KO mice were impaired in learning dynamic contingencies, showed more perseverative responding, and were delayed in applying a new task rule to all trial types following reversal. Behavioral modeling revealed a reduced influence of recent trial history on decisions in KO mice compared to wild-type controls. The behavioral deficits could not be explained by differences in motivation or memory. These results suggest that synaptic facilitation supports adaptive decision-making and that disruptions of short-term plasticity impair animals ability to use recent outcomes to update behavior.

neuroscience↗

bioRxiv2026

lncRNA MANCR isoforms selectively mediate multiple levels of epigenomic and P53-responsive transcriptional control in triple negative breast cancer

Long noncoding RNAs (lncRNAs) are important regulators of gene expression and are frequently dysregulated in cancer. The mitotically associated lncRNA MANCR is highly expressed in aggressive cancers and contributes to genomic instability in triple-negative breast cancer (TNBC), but the molecular mechanisms underlying its activity remain poorly defined. Here we integrate computational and experimental approaches to examine the structure and regulatory interactions of MANCR isoforms. Analysis of transcriptomic datasets revealed tumor-type-specific expression patterns for seven MANCR isoforms in breast cancer cell lines. Computational prediction of RNA secondary structures identified conserved structural features across isoforms, suggesting potential functional specialization. We identify p53 as a MANCR-interacting protein through computational docking and RNA immunoprecipitation sequencing (RIP-seq) and demonstrate that MANCR depletion reduces p53-dependent transcriptional activity. Chromatin isolation by RNA purification sequencing (ChIRP-seq) revealed 1, 250 genomic regions associated with MANCR, including enrichment of p53 consensus motifs and GC-rich sequence elements. Motif analysis further identified candidate sequence features associated with MANCR-occupied chromatin regions. Computational prediction of RNA-miRNA interactions identified multiple potential miRNA binding sites across MANCR isoforms, including miR-6756-5p, which targets the androgen receptor (AR). Consistent with this prediction, AR expression decreased following MANCR knockdown in TNBC cells. Together, these results suggest that MANCR isoforms may contribute to transcriptional regulation in TNBC through interactions with chromatin, p53 signaling pathways, and potential miRNA regulatory networks. One Sentence SummaryMitotically-associated lncRNA (MANCR) is prevalent in aggressive cancers interacting with DNA, P53, and miRNAs, to mediate multiple levels of epigenetic transcriptional control in triple negative breast cancer.

cancer biology↗

bioRxiv2026

Acute degron-mediated RUNX1 loss reprograms enhancer activity to epigenetically drive epithelial destabilization and initiate cancer hallmarks

The RUNX1 transcription factor mediates cell-type specific gene expression. RUNX1 suppression and perturbations are recurrently associated with breast tumor initiation and progression. However, the mechanisms governing the dual roles of RUNX1 in sustaining the mammary epithelial phenotype while epigenetically suppressing initiation of cancer-compromised gene expression are poorly understood. To address this, we used the power of degron-mediated acute, selective, and complete RUNX1 ablation in human mammary epithelial cells. RUNX1 mediates promoter and distal enhancer-driven expression of a gene cohort. Dynamic epigenomic responsiveness upon RUNX1 ablation reveals a rapid and selective decrease in chromatin accessibility and H3K27ac at RUNX1-bound enhancers, but not promoters. While differentially initiated and expressed genes contacted by RUNX1-bound enhancers are enriched in pathways involved in epithelial maintenance and stemness, genes with RUNX1-promoter occupancy support DNA damage responsiveness. Modified cell morphology, metabolic control, increased breast cancer stemness, plasticity, anchorage-independent survival, chemoresistance, and perturbed DNA damage reactivity are observed upon RUNX1 ablation. Together, these findings define RUNX1 as an epigenetic tumor suppressor that maintains epithelial cell state by preserving enhancer activity and preventing gene expression associated with hallmarks of cancer.

cancer biology↗

bioRxiv2025

Beyond SOx: Mining Sulfur Bacteria Produce Less Acid due to Unidentified Enzyme Pathways

When thiosulfate is abundant, recent research indicates that the complete Sulfur Oxidation (cSOx) pathway is prevalent in oxic wastewater, while the incomplete Sulfur Oxidation and reverse Dissimilarity Sulfur Reduction (iSOx + rDSR) pathway has been found to be active under suboxic conditions, paired with nitrate reduction. However, both the activity of S4I (or other enzyme systems) and the suboxic sulfur reactions which occur when the rDSR pathway is not present in an SOB community remain unexplored. During this study, a series of 6 L benchtop microcosms were inoculated with Halothiobacillus spp.-dominated (containing cSOx and S4I pathways) sulfur oxidizing bacterial (SOB) communities preserved from an active tailing reservoir. The 16 microcosms were provided with high concentrations of the sulfur oxidation intermediate (SOI) compounds thiosulfate or tetrathionate under oxic and suboxic conditions to amplify the acid generating processes. The study identifies Halothiobacillus, Thiomonas, and Pandoraea, as the key SOB genera processing SOI. These microcosms demonstrate that protons yields resulting from thiosulfate oxidation by SOB are less than half of those predicted for thiosulfate oxidation, amongst the lowest reported for this genera. Despite adjustments for ion activity and PO42- buffering, this difference is indicative of mechanisms beyond cSOx. Further, when offered tetrathionate under oxic conditions, undescribed reactions unmatched to known sulfur enzymes were likely employed, since disproportionation is insufficient to explain observations. Meanwhile, suboxic depletion of thiosulfate, without changes in nitrate or nitrite concentrations sufficient to indicate their use as TEA requires further investigation.

biochemistry↗

bioRxiv2025

Using gnotobiotic mice to decipher effects of gut microbiome repair in undernourished children on tuft and goblet cell function

Studies have implicated perturbations in the postnatal development of the gut microbiome as a contributing factor to childhood undernutrition. Compared to a standard ready-to-use supplementary food, a microbiome-directed complementary food (MDCF-2) designed to repair these perturbations produced superior improvements in ponderal and linear growth in clinical trials of Bangladeshi children with moderate acute malnutrition. Here, reverse translation experiments are performed where intact fecal microbiomes collected from trial participants prior to and at the end of treatment are introduced into female gnotobiotic mice just after delivery of their pups. Pups received diets designed to resemble those consumed by children in the trials to recreate "unrepaired" and "repaired" gut ecosystems. Analyses of the abundances of bacterial strains (metagenome-assembled genomes), their expressed genes and metabolic products, combined with assessments of ponderal growth and intestinal epithelial lineage transcriptomes (single-nucleus RNA-Seq with follow-up immunocytochemistry) disclosed effects of MDCF-2 associated microbiome repair that cannot be determined, in part because no treatment control arms cannot be ethically incorporated into these trials. Specifically, microbiome repair in these mice produced significant increases in ponderal growth, changes microbial gene expression consistent with a less virulent gut ecosystem and changes in expression of (i) components of gut epithelial cell junctions in the enterocytic and goblet cell lineages, (ii) pathways for synthesis and secretion of eicosanoid immune effectors in chemosensory tuft cells, and (iii) goblet cell pathways involved in glycosylation and secretion of mucin. Experiments of the type described can help formulate and test hypotheses about how microbiome repair affects host biology. SIGNIFICANCE STATEMENTUndernutrition is a global health problem. Recent clinical trials of a gut microbiome-directed complementary food (MDCF-2) designed to repair the perturbed gut microbiomes of undernourished Bangladesh children produced superior growth outcomes versus a standard nutritional supplement. Given ethical considerations and tissue sampling constraints associated with these types of studies, we colonized gnotobiotic mice postnatally with microbiome samples obtained from trial participants before and after treatment to model "unrepaired" and "repaired" gut ecosystems. Using a multi-omics approach, we uncover heretofore unappreciated changes in expressed chemosensory tuft cell, mucus-producing goblet cell and absorptive enterocytic functions and interactions accompanying microbiome repair. Extending microbiome clinical trials back to preclinical models ( reverse translation) provides mechanistic insights that can inform design/interpretation of future interventions.

microbiology↗

bioRxiv2025

Systematic evaluation of metatranscriptomic differential gene expression in silico, in vitro, and in vivo enables elucidation of inter-species cross-feeding

Metatranscriptomic (MTX) sequencing quantifies gene expression from the collective genomes of microbial communities (microbiomes), enabling assessment of functional activity rather than functional potential. While differential expression testing is essential for RNA-sequencing analysis, current metatranscriptomic approaches have only been benchmarked on simulated data, resulting in a lack of standard practices for analysis of real datasets. Here, we use mock communities (defined mixtures of microbial cells with known properties) to quantitatively assess robustness and susceptibility of current approaches to various confounders including organisms low relative abundance, differential abundance, low prevalence, global transcriptional output changes, and compositional effects. We show that no current method is robust to all confounders and method performance on simulated data does not generalize to real datasets. We then apply the same approaches to MTX datasets generated from gnotobiotic mice colonized with defined consortia of human bacterial strains and show that the method nominated by the mock community comparisons successfully inferred cross-feeding dynamics that were subsequently validated in vitro. Finally, using metagenome-assembled genomes from a human clinical study, we leverage genome-level sequencing depth and detection of genes to exclude low information samples on a per-organism basis to overcome confounding low prevalence and enhance differential expression inference.

genomics↗

bioRxiv2025

Multiplex Gene Editing Suppresses Random Integration of Hepatitis B Virus DNA in Chronically Infected Liver

Gene editing technologies have opened the possibility of directly targeting viral DNA in therapeutic applications. In chronically infected hepatocytes with hepatitis B virus (HBV), covalently closed circular DNA (cccDNA) serves as the master template for viral transcripts and gene products. In the present study, we evaluated the outcomes of anti-HBV multiplex gene editing with the CRISPR-Cas9 endonuclease from Staphylococcus aureus (SaCas9) using primary human hepatocytes (PHHs) and HBV mouse models. Nonviral delivery of SaCas9-encoding mRNA and a pair of HBV-targeting guide RNAs (gRNAs) substantially reduced viral biomarkers and intrahepatic HBV DNA copies in vitro and in vivo, suggesting that fragmentation of HBV DNA primarily leads to its degradation. Hybridization capture sequencing analyses indicated that small insertions and deletions (indels) and structural variants including excisions and inversions of the viral sequences were accumulated in the residual HBV DNA. These assays also demonstrated that transient expression of the HBV-targeting SaCas9 significantly suppressed random integration of HBV DNA, while this therapeutic approach was unlikely to affect chromosomal translocations involving viral copies. Taken together, our results suggest that anti-HBV multiplex gene editing eliminates viral DNA from chronically infected hepatocytes, potentially reducing the risk of hepatocarcinogenesis associated with HBV DNA integration.

bioengineering↗

bioRxiv2025

Overcoming Immune Checkpoint Inhibitor Resistance via Potent and Selective Dual αvβ6/8 Inhibitors Based on Engineered Lasso Peptides

Integrins v{beta}6 and v{beta}8 in the tumor microenvironment (TME) have been shown to activate immunosuppressive TGF-{beta}, which serves as an important mechanism for immune checkpoint inhibitor resistance in a range of tumors. In this study, we demonstrate the utility of lasso peptides as versatile scaffolds for designing new therapeutics. A series of highly potent and selective dual v{beta}6/8 inhibitors were engineered through a combination of epitope scanning, computational design, and directed evolution. Several analogs, such as lassotides 36 and 47, were fully characterized and physicochemical, in vitro pharmacological, and in vivo data are reported. Lassotide 47, a half-life extended derivative of 36, was shown to strongly sensitize anti-mPD-1-resistant tumors in mice when dosed in combination with the checkpoint inhibitor. The 47/anti-mPD-1 combination was shown to halt tumor growth and regress tumors in mouse models of triple negative breast and ovarian cancers. Dual inhibition of v{beta}6/8 integrins expressed in the TME thus represents a promising tumor-specific strategy to overcome TGF-{beta}-driven resistance and enhance the anti-tumor efficacy of immune checkpoint inhibitors.

cancer biology↗

bioRxiv2024

Effects of intergenerational transmission of small intestinal bacteria cultured from stunted Bangladeshi children with enteropathy

Environmental enteric dysfunction (EED), a small intestinal disorder found at a high prevalence in stunted children, is associated with gut mucosal barrier disruption and decreased absorptive capacity1-4. To test the hypothesis that intergenerational transmission of a perturbed small intestinal microbiota contributes to undernutrition by inducing EED5, we characterized two consortia of bacterial strains cultured from duodenal aspirates from stunted Bangladeshi children with EED - one of which induced local and systemic inflammation in female gnotobiotic mice. Offspring of dams colonized with the inflammatory consortium exhibited impaired prenatal and postnatal growth, as well as immunologic changes phenocopying features of EED in children. Dam-to-pup transmission of the inflammatory consortium produced, in recently weaned offspring, alterations in inter-cellular signaling pathways related to intestinal epithelial cell renewal, barrier integrity and immune function. Cohousing of mice harboring the inflammatory or non-inflammatory consortia and subsequent screening of candidate disease-promoting bacterial isolates identified Campylobacter concisus, an organism typically found in the oral microbiota, as a contributor to enteropathy. The C. concisus strain induced, in a host nitric oxide synthase (NOS)-dependent manner, pro-inflammatory cytokine signaling. Moreover, host-derived nutrients generated by NOS augmented C. concisus growth. This preclinical model should facilitate identification of small intestinal microbiota-targeted therapeutics for (intergenerational) undernutrition.

systems biology↗

bioRxiv2024

Benchmarking open access publication rates for the pharmaceutical industry and research-intensive academic institutions

BackgroundIn biomedical and health sciences, many articles are published open access (OA). OA publication rates continue to grow, especially for pharmaceutical research. Previous analyses of pharmaceutical OA publication rates were labor-intensive and not easily automated. We aimed to create a free, live, online dashboard comparing OA publication rates between pharmaceutical companies and academic institutions. MethodsUsing the Lens - an online platform aggregating full-text content and metadata for over 280 million scholarly works - we built a dashboard showing OA publication rates for medical research articles by authors from the top 40 pharmaceutical companies (pharma) and 40 research-intensive academic institutions (academia). The dashboard details OA rates by model, license and therapy area across three time frames: 12-24 months; 0-12 months; 0-10 years. We downloaded data from the dashboard between 24 July and 4 August 2023 and performed further analysis. ResultsOf the articles published 12-24 months before data extraction, 76.6% by pharma and 69.5% by academia were OA. The most common OA models were gold (pharma, 37%; academia, 41%) and hybrid (pharma, 22%; academia, 11%). Oncology had lower rates of OA articles than other therapy areas. In the 10 years before data extraction, growth in the OA publication rate was generally more rapid for pharmaceutical companies than for academic institutions, regardless of field (change in overall % OA 2013-2023: pharma, 3.1; academia, 1.6). ConclusionsThis dashboard provides novel and regularly updated evidence on the comparative OA publication practices of pharmaceutical companies and academic institutions. In our snapshot analysis, the OA publication rate was higher for pharmaceutical companies than for academic institutions and continues to increase. Notable differences were observed in approaches, in terms of licenses and OA models, which may reflect different institutional practices and circumstances. We encourage others to use this open resource for new research and to report their results.

scientific communication and education↗

bioRxiv2024

Integrative genomic reconstruction of carbohydrate utilization networks in bifidobacteria: global trends, local variability, and dietary adaptation

Bifidobacteria are among the earliest colonizers of the human gut and are widely used as probiotics for their health-promoting properties. However, individual responses to probiotic supplementation may vary with strain type(s), microbiota composition, diet, or lifestyle conditions, highlighting the need for strain-level insights into the bifidobacterial metabolism of dietary and host glycans. Here, we systematically reconstructed 68 pathways involved in the utilization of mono-, di-, oligo-, and polysaccharides by analyzing the distribution of 589 curated metabolic functional roles (catabolic enzymes, transporters, transcriptional regulators) in 3083 non-redundant cultured Bifidobacterium isolates and metagenome-assembled genomes (MAGs) of human origin. Our analysis uncovered extensive inter- and intraspecies heterogeneity, including a distinct clade within the Bifidobacterium longum species capable of metabolizing -glucans. We also identified isolates of Bangladeshi origin that harbor unique gene clusters implicated in the breakdown of xyloglucan and human milk oligosaccharides. Thirty-eight predicted carbohydrate utilization phenotypes were experimentally validated in 30 geographically diverse Bifidobacterium isolates in vitro. Our large-scale genomic compendium expands the knowledge of bifidobacterial carbohydrate metabolism and can inform the rational design of probiotic and synbiotic formulations tailored to strain-specific nutrient preferences.

microbiology↗

bioRxiv2024

Assessment of Stiffness-Dependent Autophagosome Formation and Apoptosis in Embryonal Rhabdomyosarcoma Tumor Cells

Summary/AbstractRemodeling of the extracellular matrix (ECM) eventually causes the stiffening of tumors and changes to the microenvironment. The stiffening alters the biological processes in cancer cells due to altered signaling through cell surface receptors. Autophagy, a key catabolic process in normal and cancer cells, is thought to be involved in mechano-transduction and the level of autophagy is probably stiffness-dependent. Here, we provide a methodology to study the effect of matrix stiffness on autophagy in embryonal rhabdomyosarcoma cells. To mimic stiffness, we seeded cells on GelMA hydrogel matrices with defined stiffness and evaluated autophagy-related endpoints. We also evaluated autophagy dependent pathways, apoptosis, and cell viability. Specifically, we utilized immunocytochemistry and confocal microscopy to track autophagosome formation through LC3 lipidation. This approach suggests that the use GelMA hydrogels with defined stiffness represent a novel method to evaluate the role of autophagy in embryonal rhabdomyosarcoma and other cancer cells. Workflow O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=198 SRC="FIGDIR/small/583012v1_ufig1.gif" ALT="Figure 1"> View larger version (92K): org.highwire.dtl.DTLVardef@127453borg.highwire.dtl.DTLVardef@1325f8dorg.highwire.dtl.DTLVardef@1f2de0org.highwire.dtl.DTLVardef@fd0494_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2023

A consistent map in the medial entorhinal cortex supports spatial memory

The medial entorhinal cortex (MEC) is hypothesized to function as a cognitive map for memory-guided navigation. How this map develops during learning and influences memory remains unclear. By imaging MEC calcium dynamics while mice successfully learned a novel virtual environment over ten days, we discovered that the dynamics gradually became more spatially consistent and then stabilized. Additionally, grid cells in the MEC not only exhibited improved spatial tuning consistency, but also maintained stable phase relationships, suggesting a network mechanism involving synaptic plasticity and rigid recurrent connectivity to shape grid cell activity during learning. Increased c-Fos expression in the MEC in novel environments further supports the induction of synaptic plasticity. Unsuccessful learning lacked these activity features, indicating that a consistent map is specific for effective spatial memory. Finally, optogenetically disrupting spatial consistency of the map impaired memory-guided navigation in a well-learned environment. Thus, we demonstrate that the establishment of a spatially consistent MEC map across learning both correlates with, and is necessary for, successful spatial memory.

neuroscience↗

bioRxiv2023

Prevotella copri-linked effects of a therapeutic food for malnutrition

Preclinical and clinical studies are providing evidence that the healthy growth of infants and children reflects, in part, healthy development of their gut microbiomes1-5. This process of microbial community assembly and functional maturation is perturbed in children with acute malnutrition. Gnotobiotic animals, colonized with microbial communities from children with severe and moderate acute malnutrition, have been used to develop microbiome-directed complementary food (MDCF) formulations for repairing the microbiomes of these children during the weaning period5. Bangladeshi children with moderate acute malnutrition (MAM) participating in a previously reported 3-month-long randomized controlled clinical study of one such formulation, MDCF-2, exhibited significantly improved weight gain compared to a commonly used nutritional intervention despite the lower caloric density of the MDCF6. Characterizing the metagenome assembled genomes (MAGs) of bacterial strains present in the microbiomes of study participants revealed a significant correlation between accelerated ponderal growth and the expression by two Prevotella copri MAGs of metabolic pathways involved in processing of MDCF-2 glycans1. To provide a direct test of these relationships, we have now performed reverse translation experiments using a gnotobiotic mouse model of mother-to-offspring microbiome transmission. Mice were colonized with defined consortia of age- and ponderal growth-associated gut bacterial strains cultured from Bangladeshi infants/children in the study population, with or without P. copri isolates resembling the MAGs. By combining analyses of microbial community assembly, gene expression and processing of glycan constituents of MDCF-2 with single nucleus RNA-Seq and mass spectrometric analyses of the intestine, we establish a principal role for P. copri in mediating metabolism of MDCF-2 glycans, characterize its interactions with other consortium members including Bifidobacterium longum subsp. infantis, and demonstrate the effects of P. copri-containing consortia in mediating weight gain and modulating the activities of metabolic pathways involved in lipid, amino acid, carbohydrate plus other facets of energy metabolism within epithelial cells positioned at different locations in intestinal crypts and villi. Together, the results provide insights into structure/function relationships between MDCF-2 and members of the gut communities of malnourished children; they also have implications for developing future prebiotic, probiotic and/or synbiotic therapeutics for microbiome restoration in children with already manifest malnutrition, or who are at risk for this pervasive health challenge.

microbiology↗

bioRxiv2023

Adaptive planning depth in human problem solving

We humans are capable of solving challenging planning problems, but the range of adaptive strategies that we use to address them are not yet fully characterized. Here, we designed a series of problem-solving tasks that require planning at different depths. After systematically comparing the performance of participants and planning models, we found that when facing problems that require planning to a certain number of subgoals (from 1 to 8), participants make an adaptive use of their cognitive resources - namely, they tend to select an initial plan having the minimum required depth, rather than selecting the same depth for all problems. These results support the view of problem solving as a bounded rational process, which adapts costly cognitive resources to task demands.

neuroscience↗

bioRxiv2023

Incorporation of TRDV segments into TCR alpha chains

In human tumor models we tried to identify and clone the TCR of tumor-reactive T cells enriched in mixed lymphocyte tumor-cell cultures (MLTC). In a particular MLTC, we identified a predominant TCR beta chain using the Beta Mark Vbeta Kit, but a corresponding alpha chain could not be amplified via RT-PCR using TRAV-specific forward primers. Therefore, we applied 5RACE to obtain the TCR alpha chain sequences. The 5RACE product revealed an alpha chain that encompassed 89bp of the TRDV1 5UTR, followed by the TRDV1 coding sequence joined in frame to TRAJ24. The ORF reaching from the TRDV1 start codon to the TRAC segment was intact, suggesting a functional TCR. To analyze this MLTC population in greater depth we conducted 10X VDJ sequencing. CellRanger identified the beta chain known from the Beta Mark analysis, but no corresponding alpha chain in the filtered results. The corresponding TRDV-containing TCR alpha chain could, however, be detected in the "all_contig_annotations" files. In a separate project, we performed TCR sequencing of tumor-infiltrating lymphocytes (TILs) in a murine tumor model. Also here, a predominant clonotype contained a TCR alpha chain joining Trdv2-2 in frame to Traj49. Transfection of both TCR cDNAs resulted in cell surface localization of TCR and CD3 as validated by FACS. Tumor recognition of the human, TRDV1-containing TCR could be demonstrated by IFNgamma ELISpot whereas the murine TCR did not recognize a tumor-derived cell line. TRDV-containing alpha chains have been reported in the literature for two HLA I-restricted TCRs against HIV peptides (Ueno et al, Eur J Immunol, 2003). To determine whether such TDRV-containing TCRs are unique events or whether Vdelta segments are commonly incorporated into TCR alpha chains, we queried the NCBI Sequence Read Archive (SRA) for 10X VDJ data and analyzed 21 human and 23 murine datasets. We found that especially TRDV1, Trdv1 and to some extent Trdv2-2 are more commonly incorporated into TCR alpha chains than some TRAV genes, making the TRDV segments a relevant contribution to TCR alpha diversity. For apparently solitary beta chains in 10X VDJ datasets, we suggest to scrutinize the "all_contig_annotations" files as these may contain an accompanying, TRDV-containing alpha chain.

immunology↗

bioRxiv2022

Identification of tubulin polymerization inhibitors with a CRISPR-edited cell line with endogenous fluorescent tagging of β-tubulin and Histone 1

Tubulin is an essential protein to maintain the cellular structure and for the cell division process. Inhibiting tubulin polymerization has proven to be an effective method for slowing cancer cell growth. Traditionally, identifying tubulin polymerization inhibitors involved using pure tubulin for in vitro assays or procedures using cells that require cell fixing and anti-tubulin antibody staining. This study explores using a cell line developed via CRISPR genome editing as a cell model to identify tubulin polymerization inhibitors with live cells without using exogenous staining. The cell line has endogenous tagging with fluorescent proteins of {beta}-tubulin and a nuclear protein to facilitate image cellular segmentation by high-content imaging analysis (HCI). The cells were treated with known tubulin polymerization inhibitors, colchicine and vincristine, and the presence of phenotypic changes that indicate tubulin polymerization inhibition were confirmed via HCI. A library of 429 kinase inhibitors was screened to discover tubulin polymerization inhibitors and three compounds that inhibit tubulin polymerization were found (ON-01910, HMN-214, and KX2-391). Live cell tracking analysis confirms that depolymerization of tubulin occurs rapidly after compound treatments. These results suggest that CRISPR-edited cells with fluorescent endogenous tagging of {beta}-tubulin can be used to screen larger compound libraries containing diverse chemical families to identify novel tubulin polymerization inhibitors.

cancer biology↗