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Byrne, K.

Publications and source records attributed to Byrne, K..

12 recordsLinked to original sources

Disease investigation in a New Zealand high-health pig herd reveals a divergent porcine adenovirus

Porcine adenoviruses are common in domestic pigs and are generally regarded as low-pathogenicity viruses associated with mild or subclinical infections. However, their diversity, host associations and disease potential remain poorly understood. Here, we investigated a disease outbreak in an immunologically naive 320-sow high-health hysterectomy-derived pig herd in New Zealand. The outbreak was characterised by anorexia, lethargy, diffuse cutaneous erythema, death in neonatal piglets, and an increased occurrence of mummified fetuses. Histopathological examination of a piglet revealed widespread intranuclear inclusion bodies within endothelial cells, indicative of systemic viral infection. Total RNA sequencing was performed on tissues from an affected piglet and mummified fetuses. Metatranscriptomic analysis identified an abundant adenovirus in multiple piglet and aborted fetus tissues, and de novo assembly recovered a 27,685-nucleotide genome. Phylogenetic analysis placed the virus within the genus Mastadenovirus but it formed a divergent lineage distinct from all previously characterised porcine adenoviruses. Despite sequence divergence, the genome retained the characteristic organisation of mastadenoviruses. Aside from a single detection of porcine kobuvirus identified at low abundance in the small intestine and colon from the piglet, no other viral pathogens were identified. The high viral abundance and systemic tissue distribution observed in both the piglet and fetus, together with the presence of widespread adenoviral inclusion bodies in the piglet, support an association between this novel adenovirus and the disease outbreak. These findings expand the diversity of porcine adenoviruses and suggest that previously unrecognised adenovirus lineages may contribute to reproductive and neonatal disease when introduced into immunologically naive high-health herds.

microbiology↗

Tumor-intrinsic MHC-II activation in pancreatic ductal adenocarcinoma enhances immune response and treatment efficacy

Pancreatic ductal adenocarcinoma (PDAC) is characterized by an immunosuppressive tumor microenvironment (TME) and poor prognosis. While major histocompatibility complex class II (MHC-II) expression is traditionally associated with professional antigen-presenting cells, its role in PDAC malignant cells remains underexplored. Herein, we utilized single-cell RNA sequencing (scRNA-seq), spatial transcriptomics, bulk RNA sequencing, multiplex immunohistochemistry (mIHC) and ex vivo studies in culture with both human and murine models to investigate the prognostic relevance of MHC-II expression in malignant PDAC cells. Elevated MHC-II expression in malignant cells was strongly associated with increased infiltration of CD4+ T and CD8+ T cells in human PDAC, and pronounced co-localization with plasma cells, indicative of an antigen-activated immune microenvironment. In the KPC mouse model of PDAC, pharmacologic induction of MHC-II expression by cobimetinib treatment in malignant epithelial cells significantly enhanced the therapeutic response to immune checkpoint blockade (ICB). These findings highlight the role of malignant cell- intrinsic MHC-II expression in promoting antigen presentation and fostering an anti-tumor immune microenvironment. Our results position MHC-II as a promising prognostic biomarker and therapeutic target in PDAC, paving the way for novel immunomodulatory strategies. Summary of highlightsSingle-cell and spatial transcriptomic analyses reveal that elevated MHC-II expression in malignant PDAC cells correlates with increased infiltration of CD4 and CD8 T cells. Stimulating MHC-II expression in tumors effectively enhances immunotherapeutic responses to ICB in the PDAC KPC mouse model, including PDAC tumors previously resistant to therapeutic interventions. MHC-II serves as a prognostic biomarker and a promising target for immunotherapy in PDAC.

cancer biology↗

Pseudomonas aeruginosa uses kinases NahK and RetS to control the motile-biofilm switch

The multidrug-resistant bacterium Pseudomonas aeruginosa (Pa) poses a significant threat to public health. This Gram-negative bacterium establishes pathogenicity through formation of multicellular communities, known as biofilms, that result in significant resistance to antibiotics and host immune systems. In Pa, the motile-to-biofilm transition is regulated through an interconnected signaling network known as the Gac Multikinase Network (Gac-MKN). This network comprises two regulatory branches: the HptB signaling network and GacS/A signaling network. In the Gac-MKN, several histidine kinases converge to regulate the activity of the post-transcriptional regulator protein, RsmA. Although previous studies have assessed the role of individual kinases in this network, the role of each Gac-MKN kinase in regulating RsmA activity has not been quantitatively characterized and compared in side-by-side experiments in the same reference strain, which is presented here. In this study, we show that kinases NahK and RetS are the predominant regulators of the Gac-MKN. Through controlled testing of RsmA-dependent phenotypes, we demonstrate loss of nahK or retS leads to complete inactivation of RsmA, triggering biofilm formation. Our results support previous findings that RetS regulates RsmA through the GacS/A network but present the new finding that NahK is the central kinase involved in HptB phosphorylation; previous studies have attributed HptB phosphorylation to PA1611 and SagS. Our findings demonstrate that NahK signaling controls RsmA activity to rapidly transition between the motile and biofilm states. We anticipate the results of this study will facilitate the use of targeting the Gac-MKN to trigger biofilm dispersal for improved antibiotic treatment.

microbiology↗

Intracellular acidification by bacteria-derived valeric acid is a mechanism of trans-kingdom ecology against Candida parapsilosis colonization

In hematopoietic cell transplant patients, intestinal Candida parapsilosis expansion and translocation cause life-threatening candidemia, yet how commensal intestinal bacteria prevent Candida expansion remains incompletely defined. Here, we trained a machine learning model on supernatant metabolomic profiles of commensal bacteria to identify bacteria-derived inhibitors of fungal growth, with valeric and butyric acid as top hits. Experimental validation confirmed in silico predictions in three systems. First, in patient fecal samples, valeric and butyric acid levels inversely correlated with C. parapsilosis growth. Second, in vitro, valeric acid potently inhibited C. parapsilosis growth by causing intracellular acidification. Third, administration of glycerol valerate, and free or microencapsulated valeric acid blunted C. parapsilosis growth at murine intestinal sites where valeric acid could be detected. Thus, machine learning could identify a mechanistic driver of trans-kingdom ecology limiting C. parapsilosis intestinal expansion and may inform strategies to reduce patient risk of developing candidiasis during high-risk periods.

microbiology↗

Disabling PSGL-1 abrogates immune suppression and resistance to PD-1 blockade in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer for which there is a critical need to identify novel therapeutic targets. Herein we define PSGL-1 as a checkpoint inhibitor using a syngeneic orthotopic model of PDAC. As with PDAC patients, CD8+ T cells within murine PDAC tumors expressed high levels of PSGL-1. PSGL-1-/- mice displayed striking T cell-dependent control of primary tumors and lung metastases. Extensive spatial remodeling within PDAC tumors occurred in PSGL-1-/- mice with a dramatic loss of proliferating tumor cells and an increase in CD8+ T cell engagement of antigen-presenting cells. The prominent CD8+ T cell infiltrates included subsets of pre-exhausted T cells retaining hallmarks of stemness and multifunctional effector capacity. These changes enabled a near complete response of PDAC to therapeutic PD-1 blockade. Our findings identify PSGL-1 as a key regulator of anti-tumor immunity in PDAC, highlighting its potential as a therapeutic target to limit CD8+ T cell exhaustion and enhance immunotherapy response. SummaryHope et al describe a pivotal function of PSGL-1 in CD8+ T cell responses to pancreatic ductal adenocarcinoma. Genetic deletion of PSGL-1 elicits tumor control by increasing T cell infiltration and maintaining functional subsets, thereby promoting sensitivity to PD-1 blockade.

immunology↗

Multiomics plasticity in seed traits of pan-genome wheat cultivars

The molecular basis of cultivar-level variations in polyploid wheat that enables environmental adaptation while maintaining yield and quality in polyploid wheat remains poorly understood. We conducted a detailed phenotypic assessment and multiomics analysis of nine pan-genome polyploid wheat cultivars grown under control and drought conditions. We aimed to investigate the subgenome-level variations, cultivar differences and biochemical mechanisms affecting plant fitness under moderate drought stress. Intrinsic water use efficiency, grain yield, and grain protein content and quality differed among cultivars, supporting the plasticity of drought stress responses. Biased proteome and metabolome abundance changes in response to moderate drought stress during the vegetative stage indicate different strategies for the utilization of homeologous protein isoforms assigned to the A, B, and D subgenomes. Drought effects were detected at the protein level, but significant changes were observed in central carbon pathway metabolites and micronutrient profiles. The subgenomic localization of seed storage proteins highlight differences in nutrient reservoir accumulation and emphasizes the enhanced role of S-rich prolamins in the stress response. Subgenomic variations define cultivar phenotypes by producing molecules that accumulate and enable the underlying trade-offs between environmental adaptation and yield- or quality-related traits. These variations can be used to select crops with increased stress resistance without compromising yield.

plant biology↗

Pancreatic cancer cells overexpressing interleukin 6 induce T-cell-mediated tumor clearance and durable anti-tumor immune response

Background & AimsTumor immune resistance is recognized as a contributor to low survivorship in pancreatic ductal adenocarcinoma (PDAC). The inflammatory cytokine interleukin-6 (IL-6) promotes polarization of CD4 T cell populations away from immune tolerance, and induces differentiation of cytotoxic CD8 T cells. This work aims to test whether IL-6 could stimulate an anti-tumor response in PDAC MethodsWe overexpressed IL-6 in multiple KrasG12D/+, Tp53R172H/+, Pdx1-Cre (KPC) cell lines, which were orthotopically implanted in mice (OT-PDACIL6). We followed mouse survival and measured tumor growth, tumor histology, and plasma IL-6 at 5 and 10 days after tumor implantation. We measured tumor immune cell infiltration via flow cytometry and histology. We used antibody-based T cell depletion and secondary tumor implantation rechallenge to test the dependency of the durable immune reaction on T cells. We use lipid nanoparticle (LNP)-based delivery of IL-6 mRNA to the pancreas as an orthogonal approach for testing the effect of elevated IL-6 in the tumor microenvironment on anti-tumor T cell invasion. ResultsImproved survival occurred in all instances of OT-PDACIL6, with one cell line (KxPxCx) reproducibly resulting in long-term recurrence-free survival. With KxPxCx cells, circulating IL-6 was 100-fold higher in OT-KxPxCxIL6 than in OT-KxPxCxparental mice. Flow cytometry revealed increased T cells and NK cells, and decreased T regulatory cells, and we observed significantly increased lymphoid aggregates in OT-KXPXCXIL6 as compared to OT--KxPxCxparental tumors. Antibody-based CD4+ and CD8+ T cell depletion prevented tumor clearance and completely abolished the survival advantage in OT-KxPxCxIL6 mice. The anti-tumor immune response to OT-KxPxCxIL6 rendered mice immune to re-challenge with OT-KxPxCxparental tumors. LNP delivery of IL-6 to the pancreas elevated systemic IL-6 levels [~]50 fold, lowered tumor burden, and increased anti-tumor T cell phenotypes. ConclusionsLocally high IL-6 concentrations potently enhance the T cell-mediated anti-tumor response to PDAC. SYNOPSISInterleukin-6 induces rapid and durable T cell-driven immune clearance of pancreatic ductal adenocarcinoma. The anti-tumor immune microenvironment is hallmarked by increased lymphoid aggregate formation, increased CD4 T cell abundance, and decreased Treg abundance.

cancer biology↗

Machine learning links T cell function and spatial localization to neoadjuvant immunotherapy and clinical outcome in pancreatic cancer

Tumor molecular datasets are becoming increasingly complex, making it nearly impossible for humans alone to effectively analyze them. Here, we demonstrate the power of using machine learning to analyze a single-cell, spatial, and highly multiplexed proteomic dataset from human pancreatic cancer and reveal underlying biological mechanisms that may contribute to clinical outcome. A novel multiplex immunohistochemistry antibody panel was used to audit T cell functionality and spatial localization in resected tumors from treatment-naive patients with localized pancreatic ductal adenocarcinoma (PDAC) compared to a second cohort of patients treated with neoadjuvant agonistic CD40 (CD40) monoclonal antibody therapy. In total, nearly 2.5 million cells from 306 tissue regions collected from 29 patients across both treatment cohorts were assayed, and more than 1,000 tumor microenvironment (TME) features were quantified. We then trained machine learning models to accurately predict CD40 treatment status and disease-free survival (DFS) following CD40 therapy based upon TME features. Through downstream interpretation of the machine learning models predictions, we found CD40 therapy to reduce canonical aspects of T cell exhaustion within the TME, as compared to treatment-naive TMEs. Using automated clustering approaches, we found improved DFS following CD40 therapy to correlate with the increased presence of CD44+ CD4+ Th1 cells located specifically within cellular spatial neighborhoods characterized by increased T cell proliferation, antigen-experience, and cytotoxicity in immune aggregates. Overall, our results demonstrate the utility of machine learning in molecular cancer immunology applications, highlight the impact of CD40 therapy on T cells within the TME, and identify potential candidate biomarkers of DFS for CD40-treated patients with PDAC.

cancer biology↗

Combinatorial G x G x E CRISPR screening and functional analysis highlights SLC25A39 in mitochondrial GSH transport

The SLC25 carrier family consists of 53 transporters that shuttle nutrients and co-factors across mitochondrial membranes1-3. The family is highly redundant and their transport activities coupled to metabolic state. Here, we introduce a pooled, dual CRISPR screening strategy that knocks out pairs of transporters in four metabolic states -- glucose, galactose, OXPHOS inhibition, and absence of pyruvate -- designed to unmask the inter-dependence of these genes. In total, we screened 63 genes in four metabolic states, corresponding to 2016 single and pair-wise genetic perturbations. We recovered 19 gene-by-environment (GxE) interactions and 9 gene-by-gene (GxG) interactions. One GxE interaction hit illustrated that the fitness defect in the mitochondrial folate carrier (SLC25A32) KO cells was genetically buffered in galactose due to a lack of substrate in de novo purine biosynthesis. Another GxE interaction hit revealed non-equivalence of the paralogous ATP/ADP exchangers (ANTs) with ANT2 specifically required during OXPHOS inhibition. GxG analysis highlighted a buffering interaction between the iron transporter SLC25A37 and the poorly characterized SLC25A39. Mitochondrial metabolite profiling, organelle transport assays, and structure-guided mutagenesis suggests SLC25A39 is critical for mitochondrial glutathione (GSH) transport. Our work underscores the importance of systemetically investigating family-wide genetic interactions between mitochondrial transporters across many metabolic environments.

cell biology↗

An experimental workflow identifies nitrogenase proteins ready for expression in plant mitochondria

Industrial nitrogen fertilizer is intrinsic to modern agriculture yet expensive and environmentally harmful. We aim to reconstitute bacterial nitrogenase function within plant mitochondria to reduce nitrogen fertilizer usage. Many nitrogen fixation (Nif) proteins are required for biosynthesis and function of the mature nitrogenase enzyme, and these will need to be correctly processed and soluble within mitochondria as a pre-requisite for function. Here we present our workflow that assessed processing, solubility and relative abundance of 16 Klebsiella oxytoca Nif proteins targeted to the plant mitochondrial matrix using an Arabidopsis mitochondrial targeting peptide (MTP). The functional consequence of the N-terminal modifications required for mitochondrial targeting of Nif proteins was tested using bacterial nitrogenase assays. We found that despite the use of the same constitutive promoter and MTP, MTP::Nif processing and relative abundance in plant leaf varied considerably. Assessment of solubility for all MTP::Nif proteins found NifF, M, N, S, U, W, X, Y and Z were soluble, while NifB, E, H, J, K, Q and V were mostly insoluble. Although most Nif proteins tolerated the N-terminal extension as a consequence of mitochondrial processing, this extension in NifM reduced nitrogenase activity to 10% of controls. Using proteomics, we detected a [~]50-fold increase in the abundance of NifM when it contained the N-terminal MTP extension, which may account for this reduction seen in nitrogenase activity. Based on plant mitochondrial processing and solubility, and retention of function in a bacterial assay, our workflow has identified that NifF, N, S, U, W, Y and Z satisfied all these criteria. Future work can now focus on improving these parameters for the remaining Nif components to assemble a complete set of plant-ready Nif proteins for reconstituting nitrogen fixation in plant mitochondria.

synthetic biology↗

Engineering a functional NifDK polyprotein resistant to mitochondrial degradation

To engineer Mo dependent nitrogenase function in plants expression of proteins NifD and NifK will be an absolute requirement. Although mitochondria have been established as a suitable eukaryotic environment for biosynthesis of oxygen-sensitive enzymes such as NifH, expression of NifD in this organelle has proven difficult due to cryptic NifD degradation. Here we describe a solution to this problem. Using molecular and proteomic methods, we found NifD degradation to be a consequence of mitochondrial endoprotease activity at a specific motif within NifD. Focusing on this functionally sensitive region, we designed NifD variants comprising between one and three amino acid substitutions and distinguished several that were resistant to degradation when expressed in both plant and yeast mitochondria. Nitrogenase activity assays of these resistant variants in E. coli identified a subset that retained function, including a single amino acid (Y100Q) variant. The Y100Q variant also enabled expression of a NifD(Y100Q)-linker-NifK translational polyprotein in plant mitochondria, confirmed by identification of the polyprotein in the soluble fraction of plant extracts. The NifD(Y100Q)-linker-NifK retained function in E. coli based nitrogenase assays, demonstrating this polyprotein permits expression of NifD and NifK in a defined stoichiometry supportive of activity. Our results exemplify how protein design can overcome impediments encountered when expressing synthetic proteins in novel environments. Specifically, these findings outline our progress toward the assembly of the catalytic unit of nitrogenase within mitochondria.

synthetic biology↗

Novel engraftment and T cell differentiation of human hematopoietic cells in Art-/- IL2RG-/ SCID pigs

Pigs with severe combined immunodeficiency (SCID) are an emerging biomedical animal model. Swine are anatomically and physiologically more similar to humans than mice, making them an invaluable tool for preclinical regenerative medicine and cancer research. One essential step in further developing this model is the immunological humanization of SCID pigs. In this work we have generated T- B- NK- SCID pigs through site directed CRISPR/Cas9 mutagenesis of IL2RG within a naturally occurring DCLRE1C (Artemis)-/- genetic background. We confirmed Art-/- IL2RG-/Y pigs lacked T, B, and NK cells in both peripheral blood and lymphoid tissues. Additionally, we and successfully performed a bone marrow transplant on one Art-/- IL2RG-/Y male SCID pig with a bone marrow from a complete swine leukocyte antigen (SLA) matched donor without conditioning to reconstitute porcine T and NK cells. Next, we performed in utero injections of cultured human CD34+ selected cord blood cells into the fetal Art-/- IL2RG-/Y SCID pigs. At birth, human CD45+ CD3{varepsilon}+ cells were detected in peripheral blood of in utero injected SCID piglets. Human leukocytes were also detected within the bone marrow, spleen, liver, thymus, and mesenteric lymph nodes of these animals. Taken together, we describe critical steps forwards the development of an immunologically humanized SCID pig model.\n\nOne sentence summaryWe have generated a T- B- NK- SCID pig model through site directed mutagenesis of IL2RG in a naturally occurring Artemis null background and show successful engraftment of human T and B cells in blood and lymphoid organs after in utero injection of human hematopoietic stem cells.

immunology↗