Biology subjects

Warner, J.

Publications and source records attributed to Warner, J..

11 recordsLinked to original sources

bioRxiv2026

AGROtools: A comprehensive modular cloning toolset for Agrobacterium strain engineering

Agrobacterium-mediated transformation is a cornerstone of plant genetic engineering, yet many crops remain recalcitrant to transformation and its efficiency is highly species- and genotype-dependent. The limited diversity of disarmed agrobacterial strains available for biotechnology, and the technically challenging workflows typically required for strain modification, constrain efforts to develop improved plant transformation platforms. Here, we describe the development of AGROtools-a versatile, expandable, and user-friendly modular cloning plasmid collection comprising more than 75 individual components, that is designed to simplify genetic engineering of agrobacteria. Constructs assembled with this toolkit couple visual reporters with selectable and counterselectable markers to facilitate identification of recombinant clones, thereby reducing reliance on PCR-based screening. We constructed and tested reporter cassettes that are functional across diverse agrobacterial strains and genomic contexts. By linking a visual reporter combinatorially with various antibiotic resistance markers and orthogonal site-specific recombinase recognition sequences, AGROtools can enable marker removal and recycling for up to four successive genomic modifications. We demonstrate the utility of AGROtools by disarming 10 novel wild-type strains, and accomplishing serial modifications within individual strain backgrounds. In addition to conventional recA-mediated homologous recombination, the collection incorporates components for applying contemporary strain engineering approaches, including CRISPR/dCas9 base editing, INTEGRATE-mediated targeted insertion, and phage-derived recombineering systems. Constructs can be assembled and deployed rapidly, enabling isolation of strains carrying an intended modification in less than two weeks. AGROtools provides a broadly applicable resource for accelerating strain development and genetic studies in agrobacteria, with potential to advance plant biotechnology by aiding improvement of disarmed strains.

plant biology↗

bioRxiv2026

Malnourishment and expanded microbiome maintain superior efficacy of the recombinant tuberculosis vaccine BCG::ESAT-6-PE25SS

Tuberculosis (TB) remains the leading cause of infectious mortality. The limited efficacy of the only TB vaccine, Bacille Calmette-Guerin (BCG) against pulmonary disease necessitates improved vaccines. Host factors such as malnutrition and microbiome composition shape immune responses in humans, though these factors are overlooked in preclinical vaccine evaluation. Here we show that a recombinant BCG strain, BCG::ESAT-6-PE25SS, confers superior protection compared to BCG across murine models of malnutrition, antibiotic-induced dysbiosis and environmentally enriched microbiota. Unexpectedly, malnourished mice displayed reduced Mycobacterium tuberculosis (Mtb) burden, associated with altered host metabolism and immune composition. Microbiome disruption increased TB susceptibility, whereas diversification of microbiota enhanced resistance and immune heterogeneity. Vaccine efficacy correlated with enrichment of known immunomodulatory microbial taxa. These findings suggest diet-microbiome-immunity interactions as potential key determinants of TB pathogenesis and provide evidence for the importance of vaccine candidate evaluation under physiologically relevant co-morbid conditions

microbiology↗

bioRxiv2025

Candida albicans infection suppresses Lipopolysaccharide or Pseudomonas aeruginosa stimulated murine bone marrow derived macrophage (BMDM) responses

Candida albicans is a commensal fungus which populates most healthy individuals microbiota but can turn opportunistic in immunocompromised individuals and cause severe disease linked with high rates of mortality. With limited therapeutic options and increasing resistance to antifungals, novel treatment strategies for C. albicans infections is paramount. The exact immune response to C. albicans infections can be influenced by the surrounding microenvironment, for example, metabolic stresses or co-infection; although, knowledge on whether responses are enhanced or inhibited is lacking. Macrophages are a key immune cell in defence against C. albicans infection through phagocytic uptake and cytokine production that alerts other immune defence mechanisms. Here, we utilise a discovery screen approach using Data Independent Acquisition (DIA) based total proteomics to describe murine bone marrow derived macrophage (BMDM) response to C. albicans infection as well as in response to co-infection with gram-negative bacterial outer membrane component lipopolysaccharide (LPS) or live gram-negative bacteria Pseudomonas aeruginosa. We found C. albicans induced a surprisingly muted immune response in BMDMs as compared to LPS or P. aeruginosa. Moreover, upon co-infection with LPS or P. aeruginosa, C. albicans suppressed the BMDM proteome landscape and selectively suppressed BMDM secreted IL-6 and IL-12p40 cytokine responses to P. aeruginosa. Thus, C. albicans has significant suppressive capabilities in the host innate immune responses that could impact clinical outcomes during infection. Author SummaryHuman fungal pathogens are of increasing concern for global health due to infections in hospitalised patients, limited treatment options and increased resistance to antifungal treatments. The host inflammatory response to invasive infections like Candida albicans are important for trying to contain the pathogen. Pathogen evasion of this immune response poses a critical threat and can increase host morbidity and mortality. Macrophages are an important innate immune cell which recognises and responds to C. albicans infection in vivo. Here, we found that in vitro macrophage signalling, secreted cytokines and the total proteomic responses to C. albicans infection was much more subtle than the macrophage responses to live bacterial infection or the bacterial outer membrane component, lipopolysaccharide (LPS). Co-infection of C. albicans and bacteria suppressed selective macrophage protein expression including important inflammatory cytokines, IL-6 and IL-12p40. Thus, we describe an impressive suppressive response by C. albicans which poses a potential mechanism of enhanced immune evasion.

immunology↗

bioRxiv2023

Augmented gut hormone response to feeding in older adults exhibiting low appetite.

Age-related changes in gut hormones may play a role in anorexia of ageing. The aim of this study was to determine concentrations of ghrelin, PYY, and GLP-1 in older adults exhibiting an anorexia of ageing phenotype. Thirteen older adults with healthy appetite (OA-HA; 8f, 75{+/-}7 years, 26.0{+/-}3.2 kg{middle dot}m-2), fifteen older adults with low appetite (OA-LA; 10f, 72{+/-}7 years, 23.6{+/-}3.1 kg{middle dot}m-2), and twelve young adults (YA; 6f, 22{+/-}2 years, 24.4{+/-}2.0 kg{middle dot}m-2) completed the study. Healthy appetite and low appetite were determined based on BMI, habitual energy intake, self-reported appetite, and laboratory-assessed ad libitum lunch intake. Participants provided a fasted measure of subjective appetite and blood sample (0 minutes) before consuming a standardised breakfast (450 kcal). Appetite was measured every 30 minutes for 240 minutes and blood was sampled at 30, 60, 90, 120, 180 and 240 minutes. At 240 minutes, an ad libitum lunch meal was consumed. Relative energy intake at lunch (expressed as percentage of estimated total energy requirement) was lower for OA-LA (19.8{+/-}7.7%) compared with YA (41.5{+/-}9.2%, p<0.001) and OA-HA (37.3{+/-}10.0%, p<0.001). Ghrelin suppression was greater for OA-LA than YA at 90 minutes (-512{+/-}477 pg{middle dot}mL-1 vs. 174{+/-}182 pg{middle dot}mL-1, p=0.045) and 180 minutes (-502{+/-}147 pg{middle dot}mL-1 vs. -208{+/-}202 pg{middle dot}mL-1, p=0.049), and lower than OA-HA at 60 minutes (-447{+/-}447 pg{middle dot}mL-1 vs. -125{+/-}169 pg{middle dot}mL-1, p=0.039). GLP-1 concentration was higher for OA-LA compared with YA at 180 minutes (5.00{+/-}4.71 pM vs. 1.07{+/-}2.83 pM, p=0.040). Net AUC for PYY response to feeding was greater for OA-LA compared with OA-HA (p=0.052). No differences were seen in subjective appetite. These observations in older adults exhibiting an anorexia of ageing phenotype suggest augmented anorexigenic responses of gut hormones to feeding may be causal mechanisms of anorexia of ageing.

physiology↗

bioRxiv2023

Quantitative proteomics reveals tissue-specific, infection-induced and species-specific neutrophil protein signatures

Neutrophils are one of the first responders to infection and are a key component of the innate immune system through their ability to phagocytose and kill invading pathogens, secrete antimicrobial molecules and produce extracellular traps. Neutrophils are produced in the bone marrow, circulate within the blood and upon immune challenge migrate to the site of infection. We wanted to understand whether this transition shapes the mouse neutrophil protein landscape, how the mouse neutrophil proteome is impacted by systemic infection and perform a comparative analysis of human and mouse neutrophils. Using quantitative mass spectrometry we reveal tissue-specific, infection-induced and species-specific neutrophil protein signatures. We show a high degree of proteomic conservation between mouse bone marrow, blood and peritoneal neutrophils, but also identify key differences in the molecules that these cells express for sensing and responding to their environment. Systemic infection triggers a change in the bone marrow neutrophil population with considerable impact on the core machinery for protein synthesis and DNA replication along with environmental sensors. We also reveal profound differences in mouse and human blood neutrophils, particularly their granule contents. Our proteomics data provides a valuable resource for understanding neutrophil function and phenotypes across species and model systems.

immunology↗

bioRxiv2023

The glucocorticoid dexamethasone impairs the expression of anti-viral mediators in activated macrophages by inhibiting both expression and function of interferon beta.

Glucocorticoids potently inhibit expression of many inflammatory mediators, and have been very widely used to treat both acute and chronic inflammatory diseases for more than seventy years. However, they can have several unwanted effects, amongst which immunosuppression is one of the most common. Here we investigated effects of the synthetic glucocorticoid dexamethasone on the responses of primary mouse bone marrow-derived macrophages to the pro-inflammatory agonist lipopolysaccharide (LPS). At the mRNA level, dexamethasone inhibited the LPS-induced expression of more than 100 genes that are involved in cell-intrinsic defence against viral pathogens. Expression of most of the corresponding proteins was also reduced by dexamethasone. This antiviral disarmament occurred at two distinct levels. First, dexamethasone strongly and dose-dependently inhibited the expression of the type I interferon IFN{beta} by LPS-activated macrophages. IFN{beta} mediates an autocrine positive feedback loop in LPS-treated macrophages, promoting the expression of antiviral genes and other interferon-stimulated genes. Hence reduction of IFN{beta} expression contributes to impaired expression of antiviral genes. Dexamethasone also acted downstream of IFN{beta} to inhibit expression of a subset of interferon-regulated genes. We tested a number of hypotheses based on previous publications, but found that no single mechanism could account for more than a small fraction of the broad suppressive impact of dexamethasone on macrophage type I interferon signaling, underlining the complexity of this pathway. Preliminary experiments indicated that dexamethasone exerted similar inhibitory effects on primary human monocyte-derived or alveolar macrophages.

immunology↗

bioRxiv2022

Endogenous GDF15 and FGF21 additively alleviate hepatic steatosis and insulin resistance in obese mice.

Obesity in mice and humans is associated with elevated levels of at least two hormones responsive to cellular stress, namely GDF15 and FGF21. Over-expression of each of these is associated with weight loss and beneficial metabolic changes but where they are secreted from and what they are required for physiologically in the context of overfeeding remains unclear. Here we used tissue selective knockout mouse models to establish that, like FGF21, circulating GDF15 is primarily derived from the liver, rather than adipose tissue, muscle or macrophages in high fat fed mice. Combined whole body deletion of FGF21 and GDF15 does not result in any additional weight gain in high fat fed mice but is associated with significantly greater hepatic steatosis and insulin resistance. Collectively the data suggest that activation of the integrated stress response in hepatocytes is a major driver for GDF15 and FGF21 secretion in the context of overfeeding, and that they both act to alleviate this metabolic stress.

physiology↗

bioRxiv2021

A hemifused complex is the hub in a network of pathways to membrane fusion

Membrane fusion is a critical step for many essential processes, from neurotransmission to fertilization. For over 40 years protein-free fusion driven by calcium or other cationic species has provided a simplified model of biological fusion, but the mechanisms remain poorly understood. Cation-mediated membrane fusion and permeation are essential in their own right to drug delivery strategies based on cell-penetrating peptides or cation-bearing lipid nanoparticles. Experimental studies suggest calcium drives anionic membranes to a hemifused intermediate which constitutes a hub in a network of pathways, but the pathway selection mechanism is unknown. Here we develop a mathematical model that identifies the network hub as a highly dynamical hemifusion complex. Multivalent cations drive expansion of this high tension hemifusion interface between interacting vesicles during a brief transient. The fate of this interface determines the outcome, either fusion, dead-end hemifusion or vesicle lysis. The model reproduces the unexplained finding that calcium-driven fusion of vesicles with planar membranes typically stalls at hemifusion, and we show the equilibrated hemifused state is a novel lens-shaped complex. Thus, membrane fusion kinetics follow a stochastic trajectory within a network of pathways, with outcome weightings set by a hemifused complex intermediate. SignificanceCells use multicomponent machineries to fuse membranes for neurotransmitter and hormone release and other fundamental processes. Protein-free fusion using calcium or other multivalent cationic fusogens has long been studied as a simplifying model. Cation-mediated membrane fusion or permeation are key events for a number of current drug delivery strategies. However, the mechanisms determining outcomes are unknown. Here we develop a mathematical model that identifies a dynamic hemifusion complex as the decision hub that stochastically sets the outcome in a network of pathways. Cations transiently grow a high tension hemifusion interface between membrane-enclosed compartments, whose fate governs whether fusion, dead-end hemifusion or vesicle lysis occurs. The model provides a systematic framework to predict outcomes of cationic fusogen-mediated interactions between membrane-enclosed compartments.

biophysics↗

bioRxiv2018

Longitudinal Visual Analytics for Unpacking the Cancer Journey

Retrospective cancer research requires identification of patients matching both categorical and temporal inclusion criteria, often based on factors exclusively available in clinical notes. Although natural language processing approaches for inferring higher-level concepts have shown promise for bringing structure to clinical texts, interpreting results is often challenging, involving the need to move between abstracted representations and constituent text elements. We discuss qualitative inquiry into user tasks and goals, data elements and models resulting in an innovative natural language processing pipeline and a visual analytics tool designed to facilitate interpretation of patient summaries and identification of cohorts for retrospective research.

cancer biology↗

bioRxiv2018

A harmonized meta-knowledgebase of clinical interpretations of cancer genomic variants

Precision oncology relies on the accurate discovery and interpretation of genomic variants to enable individualized diagnosis, prognosis, and therapy selection. We found that knowledgebases containing clinical interpretations of somatic cancer variants are highly disparate in interpretation content, structure, and supporting primary literature, impeding consensus when evaluating variants and their relevance in a clinical setting. With the cooperation of experts of the Global Alliance for Genomics and Health (GA4GH) and six prominent cancer variant knowledgebases, we developed a framework for aggregating and harmonizing variant interpretations to produce a meta-knowledgebase of 12,856 aggregate interpretations covering 3,437 unique variants in 415 genes, 357 diseases, and 791 drugs. We demonstrated large gains in overlap between resources across variants, diseases, and drugs as a result of this harmonization. We subsequently demonstrated improved matching between a patient cohort and harmonized interpretations of potential clinical significance, observing an increase from an average of 33% per individual knowledgebase to 56% in aggregate. Our analyses illuminate the need for open, interoperable sharing of variant interpretation data. We also provide an open and freely available web interface (search.cancervariants.org) for exploring the harmonized interpretations from these six knowledgebases.

bioinformatics↗

bioRxiv2018

NvERTx: A gene expression database to compare Embryogenesis and Regeneration in the sea anemone Nematostella vectensis

For more than a century researchers have been comparing embryogenesis and regeneration hoping that lessons learned from embryonic development will unlock hidden regenerative potential. This problem has historically been a difficult one to investigate since the best regenerative model systems are poor embryonic models and vice versa. Recently however, the comparison of embryogenesis and regeneration has seen renewed interest as emerging models including the sea anemone Nematostella vectensis have allowed researchers to investigate these processes in the same organism. This interest has been further fueled by the advent of high-throughput transcriptomic analyses that provide virtual mountains of data. Unfortunately much of this data remains in raw unanalyzed formats that are difficult to access or browse. Here we present Nematostella vectensis Embryogenesis and Regeneration Transcriptomics - NvERTx, the first platform for comparing gene expression during embryogenesis and regeneration. NvERTx is comprised of close to 50 RNAseq datasets spanning embryogenesis and regeneration in Nematostella. These data were used to perform a robust de novo transcriptome assembly which users can search, BLAST and plot expression of multiple genes during these two developmental processes. The site is also home to the results of gene clustering analyses, to further mine the data and identify groups of co-expressed genes. The site can be accessed at http://nvertx.kahikai.org.

developmental biology↗