Biology subjects

McNamara, S.

Publications and source records attributed to McNamara, S..

9 recordsLinked to original sources

bioRxiv2026

Reconstruction of FACS-partitioned Adaptive Immune Receptor Repertoires from FACS-partitioned B and T Cell Subsets

Adaptive immune-receptor repertoire sequencing (AIRRseq) is crucial for understanding immune system diversity and its relationship to disease dynamics. Partitioning of total B and T cells into their major subsets with distinct immunological functions - IgM+ vs. class-switched B cells (IgG+ > IgA+) and CD4+ vs. CD8+ T cells, respectively - allows for AIRRseq-based analysis of the unique contributions of each compartment to the overall immune response, a major advantage over traditional bulk sequencing workflows. However, data from these subsets is not directly comparable with the vast majority of publicly available AIRRseq data, which comes from unfractionated B and T cells, an important incompatibility. Here we investigate computational methods for reconstructing complete AIRRseq repertoires from partitioned B and T cell subsets in diverse individuals. Peripheral blood mononuclear cells (PBMCs) were partitioned via positive selection of IgM+ B-cell subsets and CD4+ T cells using immunomagnetic beads; genomic DNA was then extracted and B- and T-cell receptors were sequenced. Four reconstruction methods are introduced and evaluated for concordance with matching unpartitioned repertoires to assess preservation of key repertoire characteristics. Results show that these methods enable accurate estimates of overall immune-repertoire diversity from B- and T-cell subsets in a way that simply pooling the sequence data from sub-repertoires cannot.

immunology↗

bioRxiv2026

Adaptive variation in drought-related traits across southern and central European white oak (Quercus sect. Quercus) populations

European white oaks grow from the Mediterranean coast to Southern Scandinavia, a huge environmental gradient that has likely fostered environmental adaptation. In the face of climate change, leveraging adaptations through assisted gene flow could help improve drought tolerance and maintain forest health, but requires an understanding of the species-specific patterns of adaptation to be successful at the target location. In this study, three common gardens were established in Switzerland, Tuerkiye, and Austria for two European white oak species (Quercus robur, and Q. pubescens) using provenances from Central and Southern Europe. Almost 900 oak seedlings were measured at key water-use efficiency and life history traits for their first two year of life and genotyped with low coverage whole-genome sequencing. Trait heritability and environmental adaptation were then explored through pedigree-free animal models, while the genomic architecture of traits was mapped using a genome wide association study ("GWAS"). Across the species, the heritability of measured traits was moderate to high, but common garden had a strong impact, signalling an environmental effect on the phenotype. Adaptation to precipitation seasonality was detected in key productivity and growth traits for both species, but had a small effect on absolute trait values. The GWAS identified a striking 150 kbp association in the Cyclic Nucleotide-Gated Ion Channel gene family with leaf d13C values. This gene family is involved in stomata opening and likely impacts the intrinsic water use efficiency under stress. Together, the strong signals of phenotypic plasticity and rather weak signals of climatic adaptation in seedlings suggest that assisted gene flow in these two white oaks is relevant only for highly drought-sensitive populations, if conducted managers should focus on seeds sources with high precipitation seasonality and smaller leaf sizes.

evolutionary biology↗

bioRxiv2026

immunoPETE: A DNA-based integrated B-cell and T-cell receptor profiling platform

The vast diversity of B and T cell receptors generated through the recombination of Variable (V), Diversity (D), and Joining (J) gene segments plays a critical role in adaptive immunity. Profiling immune repertoires at the DNA level provides a robust and stable approach to capture the clonal composition of these receptors. immunoPETE is an assay designed to target recombined human T-cell Receptor Beta (TRB), T-cell Receptor Delta (TRD), and Immunoglobulin Heavy (IGH) chain genes directly from genomic DNA. Simultaneous profiling of B and T cell receptor chains in a single reaction provides internally normalized clone counts and facilitates the study of B-T cell interactions. Full-length amplicon consensus sequences representative of original template DNA molecules are accurately reconstructed using Unique Molecular Identifiers (UMIs). An in-house pipeline compiles VDJ rearrangements from the Complementarity-Determining Region 3 (CDR3) of TRB, TRD and IGH chains into comprehensive readouts at cell-level resolution. In this study, we describe the immunoPETE end-to-end workflow, followed by a comprehensive benchmarking of its performance in adaptive immune profiling. Where applicable, we used both natural and contrived samples and characterized the assays accuracy, linearity, and reproducibility across several metrics: retrieving CDR3 sequences, determining B and T cell ratios, total cell count, yield, fraction of functional rearrangements, clonal diversity, composition of dominant clones, pairwise similarity, and V/J gene usage frequencies. Furthermore, we assessed its quantitative limits concerning the total number of lymphocytes and the detection of rare clones. As an example of its applications, we show that adding immune biomarkers extracted from immunoPETE data to clinical factors improves prediction of progression-free survival in a cohort of non-muscle invasive bladder cancer (NMIBC) patients. Finally, we discuss the broad applications of immunoPETE in the study of aging, cancers, infections, and autoimmune disorders with reference to select published studies.

immunology↗

bioRxiv2025

The extracellular matrix in selective decussation of retinal ganglion cell axons: β2 laminins regulate the ipsilateral projection

AbstractIn binocular animals, retinal ganglion cell (RGC) axons selectively decussate at the optic chiasm. Selective decussation is directed by radial glia and midline neurons in the optic chiasm. Radial glia attach to the underlying pial basement membrane (PBM) that is rich in {beta}2 laminins. Here, we asked whether {beta}2 laminins in the PBM control the selective decussation of RGC axons. Genetic deletion of the {beta}2 subunit increased the proportion of non-decussating RGC axons in the ipsilateral tract. {beta}2 laminins are expressed in the PBM during the peak and late phases of RGC axon growth, and their deletion results in fragmentation of the PBM and dysmorphic radial glia. Consistent with the increase in the proportion of ipsilateral axons, we found persistent expression of the ipsilateral guidance cue EphrinB2 in radial glia during the late phase of axonal decussation, thus extending the developmental window for the development of the ipsilateral projection. Surprisingly, this increase in EphrinB2 was accompanied by an increase in the number of ipsilateral RGCs in the ventrotemporal retina. These results suggest that {beta}2 laminins regulate the size of the ipsilateral projection by providing cues that control the generation of ipsilateral RGCs and the expression of the ipsilateral cue EphrinB2 in the optic chiasm. Together, these findings position {beta}2 laminins as a novel regulator of the ipsilateral projection.

developmental biology↗

bioRxiv2025

HuBIE: The Human Blood Immunome Encyclopedia Of TCRs and BCRs in Bloodstream Infections and Cancer

T and B cells are central to adaptive immunity, where they identify and neutralize foreign antigens and cancer neo-antigens. Large-scale elucidation of T- and B-cell receptors (TCRs and BCRs) through immune-repertoire sequencing promises novel diagnostics, prognostic markers, and therapeutic strategies. However, progress is hampered by small cohort sizes, a lack of real-world patient diversity, and heterogeneous sample processing, impeding cross-study comparability. To overcome these limitations, here we introduce the Human Blood Immunome Encyclopedia (HuBIE), comprising immune-repertoire data from 2,614 samples collected from 1,941 participants. The cohort includes a range of bloodstream infections, several cancer types, and control participants, with many individuals providing longitudinal samples. We employed Roches immune receptor Primer Extension Target Enrichment (immunoPETE) platform to perform simultaneous targeted sequencing of T-cell receptor {beta} chains (TRB), T-cell receptor {delta} chains (TRD), and immunoglobulin heavy chains (IGH), thereby profiling TCRs and BCRs in all participants. We provide a comprehensive description of immune-repertoire diversity in cancer and bloodstream infections and examine variations across demographic variables such as age and race. We find significant differences in TRB and IGH composition across ethnic groups, and show that the fall in repertoire diversity with age follows distinct patterns for TRB, TRD, and IGH and is accompanied by age-related differences in VJ gene usage. Finally we demonstrate that greater immunological diversity is associated with improved survival but only for elderly participants. HuBIE thus constitutes a valuable resource for the immune-repertoire community, enabling large-scale mapping of the human immunome to accelerate development of diagnostics, prognostic biomarkers, and innovative therapeutic strategies.

immunology↗

bioRxiv2025

Tracking blaKPC Plasmid Dissemination within and between Enterobacterales across Michigan Over a Decade

AbstractblaKPC is endemic among Enterobacterales in the USA. While present on diverse plasmids, blaKPC burden is often associated with the clonal spread of multi-drug resistant (MDR) epidemic lineages. In this study we sought to determine the relative contributions of clonal spread and plasmid transfer to blaKPC burden across Michigan healthcare facilities over a decade. To this end we performed whole-genome sequencing of 1,058 KPC-producing isolates collected from 47 Michigan healthcare facilities between 2013 and 2022, including long-read sequencing for 527 isolates to enable precise plasmid tracking. Analysis with MOB-suite identified 64 distinct KPC plasmid types ("secondary clusters"), with the AK975 broad-host range plasmid being the most prevalent, found in 27% of isolates, spanning 20 species and 92 sequence types. Among genomes with AK975, 30% were from epidemic and 70% non-epidemic lineages, highlighting its broad role in regional blaKPC spread. Epidemic lineages of various species constituted 46% of the study population. Epidemic lineages differed in their primary plasmids, and even within epidemic lineages there were clonal expansions with distinct blaKPC plasmids, including in some cases AK975. These findings highlight two patterns of KPC spread: transmission of epidemic lineages harboring broad-range and lineage-specific KPC plasmids; and broader spread of AK975 among diverse species. Traditional surveillance studies often focus on common MDR lineages, potentially overlooking rare species and lineages that mediate the spread of plasmid-borne antimicrobial resistance (AMR) genes. Here we show how longitudinal studies tracking plasmids across species are essential to understand the pathways leading to AMR infections in hospitals. ImportanceThis decade-long longitudinal study highlights the persistence and spread of key KPC- carrying plasmid across multiple bacterial species in the region, including some uncommon ones. It also emphasizes the differences in KPC plasmids across lineages within the same species. While some lineages acquire multiple plasmids with resistance, they are unable to successfully maintain the plasmids. In contrast, clonal sub-populations of KPC-producing bacteria disseminate selected plasmids, establishing a stable host-plasmid combination. Comprehensive genomic surveillance that includes all pathogenic species and plasmids is crucial to understanding the regional transmission dynamics of plasmid-borne antimicrobial resistance (AMR). While outbreak studies define the blowup of a successful lineage and associated plasmids, longitudinal studies identify the reservoir-species and circulating plasmids in the context of plasmid- borne AMR.

genomics↗

bioRxiv2024

Monoclonal antibodies derived from B cells in subjects with cystic fibrosis reduce Pseudomonas aeruginosa burden in mice

Pseudomonas aeruginosa (PA) is an opportunistic, frequently multidrug-resistant pathogen that can cause severe infections in hospitalized patients. Antibodies against the PA virulence factor, PcrV, protect from death and disease in a variety of animal models. However, clinical trials of PcrV-binding antibody-based products have thus far failed to demonstrate benefit. Prior candidates were derivations of antibodies identified using protein-immunized animal systems and required extensive engineering to optimize binding and/or reduce immunogenicity. Of note, PA infections are common in people with cystic fibrosis (pwCF), who are generally believed to mount normal adaptive immune responses. Here we utilized a tetramer reagent to detect and isolate PcrV-specific B cells in pwCF and, via single-cell sorting and paired-chain sequencing, identified the B cell receptor (BCR) variable region sequences that confer PcrV-specificity. We derived multiple high affinity anti-PcrV monoclonal antibodies (mAbs) from PcrV-specific B cells across 3 donors, including mAbs that exhibit potent anti-PA activity in a murine pneumonia model. This robust strategy for mAb discovery expands what is known about PA-specific B cells in pwCF and yields novel mAbs with potential for future clinical use.

immunology↗

bioRxiv2021

Dual G9A and EZH2 inhibition stimulates an anti-tumour immune response in ovarian high-grade serous carcinoma

Ovarian high-grade serous carcinoma (HGSC) prognosis correlates directly with presence of intratumoral lymphocytes. However, cancer immunotherapy has yet to achieve meaningful survival benefit in patients with HGSC. Epigenetic silencing of immunostimulatory genes is implicated in immune evasion in HGSC and re-expression of these genes could promote tumour immune clearance. We discovered that simultaneous inhibition of the histone methyltransferases G9A and EZH2 activates the CXCL10-CXCR3 axis and increases homing of intratumoral effector lymphocytes and natural killer cells whilst suppressing tumour-promoting FoxP3+ CD4 T cells. The dual G9A/EZH2 inhibitor HKMTI-1-005 induced chromatin changes that resulted in the transcriptional activation of immunostimulatory gene networks, including the re-expression of elements of the ERV-K endogenous retroviral family. Importantly, treatment with HKMTI-1-005 improved the survival of mice bearing Trp53-/-null ID8 ovarian tumours and resulted in tumour burden reduction. These results indicate that inhibiting G9A and EZH2 in ovarian cancer alters the immune microenvironment and reduces tumour growth and therefore positions dual inhibition of G9A/EZH2 as a strategy for clinical development. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/443282v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@4c4323org.highwire.dtl.DTLVardef@cbd6fdorg.highwire.dtl.DTLVardef@8fb5e5org.highwire.dtl.DTLVardef@eef9c8_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2019

GEO2RNAseq: An easy-to-use R pipeline for complete pre-processing of RNA-seq data

In transcriptomics, the study of the total set of RNAs transcribed by the cell, RNA sequencing (RNA-seq) has become the standard tool for analysing gene expression. The primary goal is the detection of genes whose expression changes significantly between two or more conditions, either for a single species or for two or more interacting species at the same time (dual RNA-seq, triple RNA-seq and so forth). The analysis of RNA-seq can be simplified as many steps of the data pre-processing can be standardised in a pipeline.\n\nIn this publication we present the \"GEO2RNAseq\" pipeline for complete, quick and concurrent pre-processing of single, dual, and triple RNA-seq data. It covers all pre-processing steps starting from raw sequencing data to the analysis of differentially expressed genes, including various tables and figures to report intermediate and final results. Raw data may be provided in FASTQ format or can be downloaded automatically from the Gene Expression Omnibus repository. GEO2RNAseq strongly incorporates experimental as well as computational metadata. GEO2RNAseq is implemented in R, lightweight, easy to install via Conda and easy to use, but still very flexible through using modular programming and offering many extensions and alternative workflows.\n\nGEO2RNAseq is publicly available at https://anaconda.org/xentrics/r-geo2rnaseq and https://bitbucket.org/thomas_wolf/geo2rnaseq/overview, including source code, installation instruction, and comprehensive package documentation.

bioinformatics↗