Biology subjects

Jones, R. B.

Publications and source records attributed to Jones, R. B..

28 records · Page 2Linked to original sources

bioRxiv2022

Dynamic in vivo mapping of the methylproteome using a chemoenzymatic approach

Dynamic protein post-translation methylation is essential for cellular function, highlighted by the essential role of methylation in transcriptional regulation and its aberrant dysregulation in diseases including cancer. This underscores the importance of cataloging the cellular methylproteome. However, comprehensive analysis of the methylproteome remains elusive due to limitations in current enrichment and analysis pipelines. Here, we employ an L-Methionine analogue, ProSeMet, that is chemoenzymatically converted to the SAM analogue ProSeAM in cells and in vivo to tag proteins with a biorthogonal alkyne that can be directly detected via LC-MS/MS, or functionalized for subsequent selective enrichment and LC-MS/MS identification. Without enrichment, we identify lysine mono-, di-, and trimethylation, histidine methylation, and arginine methylation with site specific resolution on proteins including heat shock protein HSPA8, for which methylation is implicated in human disease. With enrichment, we identify 486 proteins known to be methylated and 221 proteins with novel methylation sites encompassing diverse cellular functions. Systemic ProSeMet delivery in mice pseudomethylates proteins across organ systems with blood-brain barrier penetrance and identifies site-specific pseudomethylation in vivo with LC-MS/MS. Leveraging these pipelines to define the cellular methylproteome may have broad applications for understanding the methylproteome in the context of disease.

biochemistry↗

bioRxiv2022

ST6GAL1 sialyltransferase promotes acinar to ductal metaplasia and pancreatic cancer progression

The role of aberrant glycosylation in pancreatic ductal adenocarcinoma (PDAC) remains an under-investigated area of research. In this study, we determined that the ST6GAL1 sialyltransferase, which adds 2,6-linked sialic acids to N-glycosylated proteins, is upregulated in patients with early-stage PDAC, and further increased in advanced disease. A tumor-promoting function for ST6GAL1 was elucidated using tumor xenograft models with human PDAC cells. Additionally, we developed a genetically-engineered mouse (GEM) with transgenic expression of ST6GAL1 in the pancreas, and found that mice with dual expression of ST6GAL1 and oncogenic KRASG12D have greatly accelerated PDAC progression and mortality compared with mice expressing KRASG12D alone. As ST6GAL1 imparts progenitor-like characteristics, we interrogated ST6GAL1s role in acinar to ductal metaplasia (ADM), a process that fosters neoplasia by reprogramming acinar cells into ductal, progenitor-like cells. We confirmed that ST6GAL1 promotes ADM using multiple models including the 266-6 cell line, GEM-derived organoids and tissues, and an in vivo model of inflammation-induced ADM. EGFR is a key driver of ADM and is known to be activated by ST6GAL1-mediated sialylation. Importantly, EGFR activation was dramatically increased in acinar cells and organoids from mice with transgenic ST6GAL1 expression. These collective results highlight a novel glycosylation-dependent mechanism involved in early stages of pancreatic neoplasia.

cancer biology↗

bioRxiv2022

Epigenetic Memory of COVID-19 in Innate Immune Cells and Their Progenitors

Severe coronavirus disease 2019 (COVID-19) is characterized by systemic inflammation and can result in protracted symptoms. Robust systemic inflammation may trigger persistent changes in hematopoietic cells and innate immune memory through epigenetic mechanisms. We reveal that rare circulating hematopoietic stem and progenitor cells (HSPC), enriched from human blood, match the diversity of HSPC in bone marrow, enabling investigation of hematopoiesis and HSPC epigenomics. Following COVID-19, HSPC retain epigenomic alterations that are conveyed, through differentiation, to progeny innate immune cells. Epigenomic changes vary with disease severity, persist for months to a year, and are associated with increased myeloid cell differentiation and inflammatory or antiviral programs. Epigenetic reprogramming of HSPC may underly altered immune function following infection and be broadly relevant, especially for millions of COVID-19 survivors. One Sentence SummaryTranscriptomic and epigenomic analysis of blood reveal sustained changes in hematopoiesis and innate immunity after COVID-19. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=197 HEIGHT=200 SRC="FIGDIR/small/479588v1_ufig1.gif" ALT="Figure 1"> View larger version (54K): org.highwire.dtl.DTLVardef@1ffe42dorg.highwire.dtl.DTLVardef@dd4868org.highwire.dtl.DTLVardef@1bcae8borg.highwire.dtl.DTLVardef@674e85_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2021

Combined PI3K and MLL inhibition synergizes in models of HR+ breast cancer

The high frequency of aberrant PI3K pathway activation in hormone receptor-positive (HR+) breast cancer has led to the development, clinical testing, and approval of the p110-selective PI3K inhibitor alpelisib. The limited clinical efficacy of alpelisib and other PI3K inhibitors is partially attributed to the functional antagonism between PI3K and estrogen receptor (ER) signaling, which is mitigated via combined PI3K inhibition and endocrine therapy. We and others have previously demonstrated a chromatin-associated mechanism by which PI3K supports cancer development and antagonizes ER signaling through the modulation of H3K4 methylation. Here we show that inhibition of the H3K4 histone methyltransferase MLL1 in combination with PI3K inhibition impairs HR+ breast cancer clonogenicity and cell proliferation. While combined PI3K/MLL1 inhibition reduces AKT effector signaling and H3K4 methylation, MLL1 inhibition increases PI3K effector signaling and upregulates the expression of receptor tyrosine kinase signaling cascades upstream of and including AKT. These data reveal a feedback loop between MLL1 and AKT in which MLL1 inhibition reactivates AKT. We additionally show that combined PI3K and MLL1 inhibition synergizes to cause cell death in in vitro and in vivo models of HR+ breast cancer, which is enhanced by the additional genetic ablation of the H3K4 methyltransferase and AKT target MLL4. Together, our data provide evidence of a feedback mechanism connecting histone methylation with AKT and may support the preclinical development and testing of pan-MLL inhibitors. Significance StatementPharmacological inhibition of PI3K provides limited efficacy in PIK3CA-mutated, HR+ breast cancers. Here the authors leverage PI3K/AKT-driven chromatin modification to identify MLL histone methyltransferases as a therapeutic target. Dual PI3K and MLL inhibition synergize to reduce clonogenicity and cell proliferation while enhancing apoptosis in in vitro models, and induces tumor regression in xenograft models of PI3K-activated, HR+ breast cancer. Furthermore, MLL1 inhibition reveals a feedback loop leading to AKT hyperactivation, which is relieved with combined PI3K/MLL inhibition. These findings demonstrate the utility of MLL inhibitors for the treatment of some solid cancers, as patients with HR+ breast cancer characterized by PIK3CA mutation may derive clinical benefit from combined PI3K/MLL inhibition.

cancer biology↗

bioRxiv2020

HIV-specific T-cell responses reflect substantive in vivo interactions with infected cells despite long-term therapy

Antiretroviral therapies (ART) durably suppress HIV replication to undetectable levels - however, infection persists in the form of long-lived reservoirs of infected cells with integrated proviruses, that re-seed systemic replication if ART is interrupted. A central tenet of our current understanding of this persistence is that infected cells are shielded from immune recognition and elimination through a lack of antigen expression from proviruses. Efforts to cure HIV infection have therefore focused on reactivating latent proviruses to enable immune-mediated clearance, but these have yet to succeed in driving reductions in viral reservoirs. Here, we revisited the question of whether HIV reservoirs are predominately immunologically silent from a new angle, by querying the dynamics of HIV-specific T-cell responses over long-term ART for evidence of ongoing recognition of HIV-infected cells. We show that T-cell responses to autologous reservoir viruses persist over years, and that the maintenance of HIV-Nef-specific responses was uniquely associated with residual frequencies of infected cells. These responses disproportionately exhibited a cytotoxic, effector functional profile, indicative of recent in vivo recognition of HIV-infected cells. These results indicate substantial visibility of the HIV reservoir to T-cells on stable ART, presenting both opportunities and challenges for the development of therapeutic approaches to curing HIV infection.

immunology↗

bioRxiv2020

The gut microbiome regulates memory function

Emerging evidence highlights a critical relationship between gut microbiota and neurocognitive development. Excessive consumption of sugar and other unhealthy dietary factors during early life developmental periods yields changes in the gut microbiome as well as neurocognitive impairments. However, it is unclear whether these two outcomes are functionally connected. Here we explore whether excessive early life consumption of added sugars negatively impacts memory function via the gut microbiome. Rats were given free access to a sugar-sweetened beverage (SSB) during the adolescent stage of development. Memory function and anxiety-like behavior were assessed during adulthood and gut bacterial and brain transcriptome analyses were conducted. Taxa-specific microbial enrichment experiments examined the functional relationship between sugar-induced microbiome changes and neurocognitive and brain transcriptome outcomes. Chronic early life sugar consumption impaired adult hippocampal-dependent memory function without affecting body weight or anxiety-like behavior. Adolescent SSB consumption during adolescence also altered the gut microbiome, including elevated abundance of two species in the genus Parabacteroides (P. distasonis and P. johnsonii) that were negatively correlated with hippocampal function. Transferred enrichment of these specific bacterial taxa in adolescent rats impaired hippocampal-dependent memory during adulthood. Hippocampus transcriptome analyses revealed that early life sugar consumption altered gene expression in intracellular kinase and synaptic neurotransmitter signaling pathways, whereas Parabacteroides microbial enrichment altered gene expression in pathways associated with metabolic function, neurodegenerative disease, and dopaminergic signaling. Collectively these results identify a role for microbiota "dysbiosis" in mediating the detrimental effects of early life unhealthy dietary factors on hippocampal-dependent memory function.

neuroscience↗

bioRxiv2020

HIV Diversity Considerations in the Application of the Intact Proviral DNA Assay (IPDA)

Opening Paragraph (serves as abstract for submission) and BodyThe Intact Proviral DNA Assay (IPDA) was developed to address the critical need for a precise and scalable method for intact HIV reservoir quantification1. This duplexed droplet digital PCR (ddPCR) assay simultaneously targets the HIV Packaging Signal ({Psi}) and the Rev Responsive Element (RRE) within Envelope (env) to distinguish genomically intact proviruses against a large background of defective ones2. The IPDA requires less time, resources, and biological material than the gold standard for replication-competent HIV reservoir measurement, the Quantitative Viral Outgrowth Assay (QVOA)3, and is being adopted in research and clinical studies4-7. In our cohort of HIV-1 subtype B-infected individuals from North America however, the IPDA yielded a 28% failure rate due to HIV polymorphism. We further demonstrate that within-host HIV diversity can lead the IPDA to underestimate intact HIV reservoir size, which could negatively impact clinical trial results interpretation. While the IPDA represents an important methodological advance, HIV diversity should be addressed before its widespread adoption.

genetics↗

bioRxiv2019

Inference based PICRUSt accuracy varies across sample types and functional categories

BackgroundDespite recent decreases in the cost of sequencing, shotgun metagenome sequencing remains more expensive compared with 16S rRNA amplicon sequencing. Methods have been developed to predict the functional profiles of microbial communities based on their taxonomic composition, and PICRUSt is the most widely used of these techniques. In this study, we evaluated the performance of PICRUSt by comparing the significance of the differential abundance of functional gene profiles predicted with PICRUSt to those from shotgun metagenome sequencing across different environments.\n\nResultsWe selected 7 datasets of human, non-human animal and environmental (soil) samples that have publicly available 16S rRNA and shotgun metagenome sequences. As we would expect based on previous literature, strong Spearman correlations were observed between gene compositions predicted with PICRUSt and measured with shotgun metagenome sequencing. However, these strong correlations were preserved even when the sample labels were shuffled. This suggests that simple correlation coefficient is a highly unreliable measure for the performance of algorithms like PICRUSt. As an alternative, we compared the performance of PICRUSt predicted genes to metagenome genes in inference models associated with metadata within each dataset. With this method, we found reasonable performance for human datasets, with PICRUSt performing better for inference on genes related to \"house-keeping\" functions. However, the performance of PICRUSt degraded sharply outside of human datasets when used for inference.\n\nConclusionWe conclude that the utility of PICRUSt for inference with the default database is likely limited outside of human samples and that development of tools for gene prediction specific to different non-human and environmental samples is warranted.

bioinformatics↗

bioRxiv2018

Telescope: Characterization of the retrotranscriptome by accurate estimation of transposable element expression

Characterization of Human Endogenous Retrovirus (HERV) expression within the transcriptomic landscape using RNA-seq is complicated by uncertainty in fragment assignment because of sequence similarity. We present Telescope, a computational software tool that provides accurate estimation of transposable element expression (retrotranscriptome) resolved to specific genomic locations. Telescope directly addresses uncertainty in fragment assignment by reassigning ambiguously mapped fragments to the most probable source transcript as determined within a Bayesian statistical model. We demonstrate the utility of our approach through single locus analysis of HERV expression in 13 ENCODE cell types. When examined at this resolution, we find that the magnitude and breadth of the retrotranscriptome can be vastly different among cell types. Furthermore, our approach is robust to differences in sequencing technology, and demonstrates that the retrotranscriptome has potential to be used for cell type identification. Telescope performs highly accurate quantification of the retrotranscriptomic landscape in RNA-seq experiments, revealing a differential complexity in the transposable element biology of complex systems not previously observed. Telescope is available at github.com/mlbendall/telescope.\n\nAuthor SummaryAlmost half of the human genome is composed of Transposable elements (TEs), but their contribution to the transcriptome, their cell-type specific expression patterns, and their role in disease remains poorly understood. Recent studies have found many elements to be actively expressed and involved in key cellular processes. For example, human endogenous retroviruses (HERVs) are reported to be involved in human embryonic stem cell differentiation. Discovering which exact HERVs are differentially expressed in RNA-seq data would be a major advance in understanding such processes. However, because HERVs have a high level of sequence similarity it is hard to identify which exact HERV is differentially expressed. To solve this problem, we developed a computer program which addressed uncertainty in fragment assignment by reassigning ambiguously mapped fragments to the most probable source transcript as determined within a Bayesian statistical model. We call this program, \"Telescope\". We then used Telescope to identify HERV expression in 13 well-studied cell types from the ENCODE consortium and found that different cell types could be characterized by enrichment for different HERV families, and for locus specific expression. We also showed that Telescope performed better than other methods currently used to determine TE expression. The use of this computational tool to examine new and existing RNA-seq data sets may lead to new understanding of the roles of TEs in health and disease.

genomics↗

bioRxiv2018

Susceptibility to Neutralization by Broadly Neutralizing Antibodies Correlates with Infected Cell Binding for a Panel of Clade B HIV Reactivated from Latent Reservoirs

Efforts to HIV cure are obstructed by reservoirs of latently infected CD4+ T-cells that can re-establish viremia. Broadly neutralizing HIV-specific antibodies (bNAbs), defined by unusually high neutralization breadths against globally diverse viruses, may contribute to the elimination of these reservoirs by binding to reactivated cells, targeting them for immune clearance. However, the relationship between neutralization of reservoir isolates and binding to corresponding infected primary CD4+ T-cells has not been determined. Thus, the extent to which neutralization breadths and potencies can be used to infer the corresponding parameters of infected-cell binding is currently unknown. We assessed the breadths and potencies of bNAbs against 36 viruses reactivated from peripheral blood CD4+ T-cells of ARV-treated HIV-infected individuals, using paired neutralization and infected-cell binding assays. Single antibody breadths ranged from 0-64% for neutralization (IC80[≤]10g/ml) and 0-89% for binding, with two-antibody combinations reaching 0-83% and 50-100%, respectively. Infected-cell binding correlated with virus neutralization for 10 out of 14 antibodies (e.g. 3BNC117, r=0.87, p<0.0001). Heterogeneity was observed, however, with a lack of significant correlations for 2G12, CAP256.VRC26.25, 2F5, and 4E10. Our results provide guidance on the selection of bNAbs for interventional cure studies; both by providing a direct assessment of intra-and inter-individual variability in neutralization and infected cell binding in a novel cohort, and by defining the relationships between these parameters for a panel of bNAbs.\n\nImportanceAlthough anti-retroviral therapies have improved the lives of people who are living with HIV, they do not cure infection. Efforts are being directed towards harnessing the immune system to eliminate the virus that persists, potentially resulting in virus-free remission without medication. HIV-specific antibodies hold promise for such therapies owing to their abilities to both prevent the infection of new cells (neutralization), and also to direct the killing of infected cells. We isolated 36 HIV strains from individuals whose virus was suppressed by medication, and tested 14 different antibodies for neutralization of these viruses and for binding to cells infected with the same viruses (critical for engaging natural killer cells). For both neutralization and infected-cell binding, we observed variation both between individuals, and amongst different viruses within an individual. For most antibodies, neutralization activity correlated with infected cell binding. These data provide guidance on the selection of antibodies for clinical trials.

immunology↗