Biology subjects

Christie, M.

Publications and source records attributed to Christie, M..

At least 19 recordsLinked to original sources

bioRxiv2026

Migratory bird and marine mammal surveillance fails to find evidence for an HPAI H5N1 2.3.4.4b incursion into Australia in 2025

The panzootic caused by high pathogenicity avian influenza (HPAI) H5N1 clade 2.3.4.4b has been devastating for animals, globally. Despite global spread, the virus remains absent in Oceania. Herein we report the results of our fourth year of enhanced migratory bird surveillance, coinciding with the spring migration of wild birds in 2025; none of the 847 migratory wild birds or 38 marine mammals were positive for HPAI H5N1, although we did detect LPAI. Surveillance remains a critical tool for HPAI H5N1 response, with early detection and rapid response being critical to mitigate the impacts of this virus on animal, environment and human health.

microbiology↗

bioRxiv2025

Surveillance of migratory shorebirds and seabirds in 2024 in Australia reveals incursions of a diversity of low pathogenicity avian influenza viruses, but not high pathogenicity avian influenza H5N1

The current panzootic of high pathogenicity avian influenza (HPAI) H5N1 has been catastrophic for wildlife, and following a significant sweep, clade 2.3.4.4b is found in every region aside from Oceania. Herein, we report the results of our third year of targeted surveillance of incoming migratory seabirds and shorebirds into Australia. We did not find evidence of HPAI H5N1 in any of the birds tested, and there were no reports of HPAI H5N1 in wildlife tested through other surveillance schemes in 2024. Unlike previous years, we detected a diversity of low pathogenicity avian influenza (LPAI) viruses in shorebirds. Through phylogenetic analysis we revealed that the H3N7 and H4N7 viruses recovered from Red-necked Stints were complex mosaic viruses, comprising segments of Eurasian, Australian shorebird, and Australian waterfowl segments. A H1N7 virus detected comprised a wholly Eurasian introduction, confirming this route for avian influenza viruses into Australian ecosystems. These results provide further evidence for the key role of long-distance migratory shorebirds in introducing novel LPAI viruses into Oceania. While our focus on northern migration routes remained appropriate for HPAI H5N1 surveillance in 2024, the continued spread of HPAI H5N1 to sub-Antarctic Islands demands consideration of a potential southern incursion route for Oceania in future.

microbiology↗

bioRxiv2025

Single-molecule flow cytometry

Flow cytometry (FC) is a powerful tool for high-throughput characterisation of cell populations, yet its ensemble fluorescence detection and the autofluorescence of cells impose a sensitivity limit of [~]100-1,000 fluorophores per cell. This precludes detection of low-abundance proteins and weak signalling events with biological importance and therapeutic potential. We introduce single-molecule Flow Cytometry (smFC), integrating high-numerical aperture oblique plane microscopy (OPM) with microfluidics to achieve optical sectioning and photon collection efficiencies suitable for single-molecule detection on flowing cells. Using super-bright, large Stokes shift fluorophores, smFC can detect labelled membrane proteins on cells with digital precision down to [~]2 molecules per cell, compared to an unlabelled control. This represents an improvement in the detection limit of 10- to 80-fold depending on the probe. We apply smFC to quantify the distribution of the membrane receptor c-kit in triple-negative breast cancer cells with unprecedented sensitivity. This revealed a previously unidentified distribution of 60% c-kit negative cells and 40% with low-abundance but heterogenous (1-200 mol/cell) surface distribution that is undetectable by conventional FC. These results establish smFC as a robust platform for high-throughput, quantitative single-molecule analysis in cell populations, opening new avenues for detecting rare biomarkers and quantifying the presence of low-abundance membrane proteins and targetable surface molecules with digital precision.

biophysics↗

bioRxiv2024

Myeloid-instructed CD14+CD4+ T cells within the tumor microenvironment are associated with TNFα signaling and prolonged survival in non-small cell lung cancer

The tumor microenvironment (TME) harbors a diverse array of innate and adaptive immune cells that can either support anti-tumor immunity or facilitate tumor progression. Among these, myeloid cells are key drivers of immunosuppression, yet therapeutic strategies targeting their function have achieved limited clinical success. A deeper understanding of the mechanisms by which immunosuppressive myeloid cells promote tumor progression is needed to guide the development of more effective treatments. Using spatial multi-omics analyses, we identified a population of myeloid-instructed CD14+ T cells present in both the tumor core and adjacent non-malignant lung tissue of patients with non-small cell lung cancer (NSCLC). CD4+ T cells acquired CD14 from myeloid cells by trogocytosis, resulting in an atypical T cell phenotype. High infiltration of CD14+CD4+ T cells in the tumor core was associated with poor patient survival. Spatial transcriptomics profiling revealed that tumors enriched in CD14+CD4+ T cells exhibited increased TNF signaling. Functional assays demonstrated that TNF enhanced trogocytosis, promoting the transfer of myeloid membrane components to CD4{square} T cells and driving the accumulation of CD14{square}CD4{square} T cells. These findings uncover a novel TNF-mediated mechanism of immunosuppression in the TME, whereby TNF promotes aberrant myeloid-T cell interactions that may contribute to tumor progression. This work highlights a previously unrecognized axis of innate-adaptive immune crosstalk in NSCLC and suggests that targeting TNF could disrupt this pathway, restore effective T cell function, and improve therapeutic outcomes.

cancer biology↗

bioRxiv2024

Rapid, simultaneous increases in the effective sizes of adaptively divergent yellow perch (Perca flavescens) populations

Aquatic ecosystems are highly dynamic environments vulnerable to natural and anthropogenic disturbances. High-economic value fisheries are one of many ecosystem services affected by these disturbances and it is critical to accurately characterize the genetic diversity and effective population sizes of valuable fish stocks through time. We used genome-wide data to reconstruct the demographic histories of economically important yellow perch (Perca flavescens) populations. In two isolated and genetically divergent populations, we provide independent evidence for simultaneous increases in effective population sizes over both historic and contemporary time scales including negative genome-wide estimates of Tajimas D, 3.1 times more SNPs than adjacent populations, and contemporary effective population sizes that have increased 10- and 47-fold from their minimum, respectively. The excess of segregating sites and negative Tajimas D values likely arose from mutations accompanying historic population expansions with insufficient time for purifying selection, whereas linkage disequilibrium-based estimates of Ne also suggest contemporary increases that may have been driven by reduced fishing pressure or environmental remediation. We also identified parallel, genetic adaptation to reduced visual clarity in the same two habitats. These results suggest that the synchrony of key ecological and evolutionary processes can drive parallel demographic and evolutionary trajectories across independent populations.

evolutionary biology↗

bioRxiv2024

Population-level whole genome sequencing of Ascochyta rabiei identifies genomic loci associated with isolate aggressiveness

Ascochyta blight caused by the ascomycete Ascochyta rabiei poses a major biotic threat to chickpea (Cicer arietinum) industries worldwide and incurs substantial costs to the Australian multimillion-dollar chickpea industry both in disease control and yield loss. The fungus was introduced to Australia in the 1970s from an unknown source population, and within a few decades, successfully established in all Australian agroecological chickpea growing regions. Although genetically highly clonal, a broad range of phenotypic variation in terms of aggressiveness exists among the Australian A. rabiei isolates. More recently, highly aggressive isolates capable of causing severe disease symptoms on moderate to highly resistant chickpea cultivars have increased in frequency. To identify genetic loci potentially associated with A. rabiei aggressiveness on Australian chickpea cultivars, we performed deep genome sequencing of 230 isolates collected from a range of agroecological chickpea growing regions between 2013 and 2020. Population genetic analyses using genome-wide single nucleotide polymorphism data identified three main clusters of genetically closely related isolates in Australia. Phylogenetic analyses showed that highly aggressive phenotypes developed multiple times independently throughout the phylogeny. Results point to minor contribution of multiple genetic regions and most likely epigenomic variations to aggressiveness of A. rabiei isolates on Australian chickpea cultivars. IMPACT STATEMENTThis research introduces new knowledge on the Australian A. rabiei population structure, molecular pathogenicity drivers and evolution as a clonal pathogen through comprehensive whole-genome sequencing approach. The knowledge generated on the structure and origin of the Australian A. rabiei and existence of only one mating type continues to inform researchers, growers, breeders and the broader industry on the importance for continued tight biosecurity measures and inform development of accurate and informed disease management and resistance breeding strategies. This research provides a rare real-life example to the effect of genetic drift on a clonal pathogen population and the importance of biosecurity to protect introduction from non-endemic isolates through seed importation in the current era of international markets. DATA SUMMARYAn online dataset containing the data and code required to reproduce the results found in this publication have been deposited at Zenodo (DOI 10.5281/zenodo.12575659). Isolate aggressiveness and collection metadata are available in the Ascochyta dashboard at http://bit.ly/asco-dashboard. Raw sequencing data used in this study was deposited to the NCBI Short Read Archive (SRA) and is available through BioProject PRJNA1175002. The authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.

genomics↗

bioRxiv2024

Long distance avian migrants fail to bring 2.3.4.4b HPAI H5N1 into Australia for a second year in a row

There is an ongoing and profound burden of lineage 2.3.4.4b high pathogenicity avian influenza (HPAI) H5N1 on wildlife and poultry, globally. Herein we report the continued absence of HPAI and antibodies against lineage 2.3.4.4b HPAI from October - December 2023, in migratory birds shortly after their arrival in Australia. Given the ever-changing phenotype of this virus, worldwide studies on the occurrence, or here absence of the virus, are of critical importance to understand the virus dispersal and incursion risk and development of response strategies.

microbiology↗

bioRxiv2023

Genomic DNA extraction optimization and validation for genome sequencing using the marine gastropod Kellet's whelk

Next-generation sequencing technologies, such as Nanopore MinION, Illumina Hiseq and Novaseq, and PacBio Sequel II, hold immense potential for advancing genomic research on non-model organisms, including the vast majority of marine species. However, application of these technologies to marine invertebrate species is often impeded by challenges in extracting and purifying their genomic DNA due to high polysaccharide content and other secondary metabolites. In this study, we help resolve this issue by developing and testing DNA extraction protocols for Kellets whelk (Kelletia kelletii), a subtidal gastropod with ecological and commercial importance, by comparing four DNA extraction methods commonly used in marine invertebrate studies. We identified an optimized protocol that balances efficiency, cost, and DNA extraction quality; the Salting out protocol. We validated the protocol using an independent set of tissue samples, then applied it to extract high-molecular-weight (HMW) DNA from over three thousand Kellets whelk tissue samples. The protocol demonstrated scalability and, with added clean-up, suitability for RAD-seq, GT-seq, as well as whole genome sequencing using both long read (ONT MinION) and short read (Illumina Novaseq) sequencing platforms. Our findings offer a robust and versatile DNA extraction and clean-up protocol for supporting genomic research efforts on non-model marine organisms, to help mediate the under-representation of invertebrates in genomic studies.

genomics↗

bioRxiv2022

Brief report: Reclassifying SCLC-Y as SMARCA4 deficient malignancies - resolving the controversy

IntroductionThe classification of small cell lung cancer (SCLC) into distinct molecular subtypes defined by ASCL1, NEUROD1, POU2F3 or YAP1 expression, paves the way for the development of targeted therapeutics. However, the existence of a distinct YAP1-expressing SCLC subtype remains controversial. Here we have undertaken a detailed molecular and histological characterisation of YAP1 expressing SCLC-Y to understand the biology of this proposed subtype. MethodsThe mutational landscape of human SCLC cell lines was interrogated to identify pathogenic genomic alterations unique to SCLC-Y. Xenograft tumours generated from cell lines representing the molecular subtypes of SCLC (SCLC-A, -N, -P and -Y) were evaluated by a panel of pathologists. Diagnoses were validated by transcriptomic analysis of primary tumour and human cell line datasets. ResultsUnexpectedly, pathogenic mutations in SMARCA4 were identified in six of eight SCLC-Y cell lines and correlated with reduced SMARCA4 mRNA and protein expression. Pathologist evaluations revealed that SMARCA4-deficient SCLC-Y tumours exhibited features consistent with thoracic SMARCA4-deficient undifferentiated tumours (SMARCA4-UT). Similarly, the transcriptional profile SMARCA4-mutant SCLC-Y lines more closely resembled primary SMARCA4-UT, or SMARCA4-deficient non-small cell carcinoma, than SCLC. Combining clinical, pathological, transcriptomic, and genetic data we found little evidence to support a diagnosis of SCLC for any of the YAP1-expressing cell lines originally used to define the SCLC-Y subtype. ConclusionsSCLC-Y cell lines harbour inactivating SMARCA4 mutations and exhibit characteristics consistent with SMARCA4-deficient malignancies rather than SCLC. Our findings suggest that, unlike ASCL1, NEUROD1 and POU2F3, YAP1 is not a subtype defining transcription factor in SCLC.

cancer biology↗

bioRxiv2021

Single-molecule analysis of the entire perfringolysin O poreformation pathway

The cholesterol-dependent cytolysin perfringolysin O (PFO) is secreted by Clostridium perfringens as a bacterial virulence factor able to form giant ring-shaped pores that perforate and ultimately lyse mammalian cell membranes. To resolve the kinetics of all steps in the assembly pathway, we have used single-molecule fluorescence imaging to follow the dynamics of PFO on dye-loaded liposomes that lead to opening of a pore and release of the encapsulated dye. Formation of a long-lived membrane-bound PFO dimer nucleates the growth of an irreversible oligomer. The growing oligomer can insert into the membrane and open a pore at stoichiometries ranging from tetramers to full rings ([~]35-mers), whereby the rate of insertion increases linearly with the number of subunits. Oligomers that insert before the ring is complete continue to grow by monomer addition post insertion. Overall, our observations suggest that PFO membrane insertion is kinetically controlled.

biophysics↗

bioRxiv2021

Molecular basis for GIGYF-TNRC6 complex assembly in miRNA-mediated translational repression

The GIGYF proteins associate with 4EHP and RNA-associated proteins to elicit transcript-specific translational repression. However, the mechanism by which the GIGYF1/2-4EHP complex is recruited to its target transcripts remain unclear. Here we report the crystal structures of the GYF domains from GIGYF1 and GIGYF2 in complex with proline-rich sequences from miRISC-binding proteins TNRC6C and TNRC6A, respectively. The TNRC6 proline-rich motifs bind to a conserved array of aromatic residues on the surface of the GIGYF1/2 GYF domain, bridging 4EHP to Argonaute-miRNA mRNA targets. Our structures also reveal a phenylalanine residue conserved from yeast to human GYF domains that contributes to GIGYF2 thermostability. The molecular details we outline here are likely to be conserved between GIGYF1/2 and other RNA-binding proteins to elicit 4EHP-mediated repression in different biological contexts.

biochemistry↗

bioRxiv2021

Germline loss-of-function variants in the base-excision repair gene MBD4 cause a Mendelian recessive syndrome of adenomatous colorectal polyposis and acute myeloid leukaemia

Inherited defects in base-excision repair (BER) predispose to adenomatous polyposis and colorectal cancer (CRC), yet our understanding of this important DNA repair pathway remains incomplete. By combining detailed clinical, histological and molecular profiling, we reveal biallelic germline loss-of-function (LOF) variants in the BER gene MBD4 to predispose to adenomatous polyposis and -uniquely amongst CRC predisposition syndromes- to myeloid neoplasms. Neoplasms from MBD4-deficient patients almost exclusively accumulate somatic CpG>TpG mutations, resembling mutational signature SBS1. MBD4-deficient adenomas harbour mutations in known CRC driver genes, although AMER1 mutations were more common and KRAS mutations less frequent. We did not find an increased risk for colorectal tumours in individuals with a monoallelic MBD4 LOF variant. We suggest that this condition should be termed MBD4-associated neoplasia syndrome (MANS) and that MBD4 is included in testing for the genetic diagnosis of polyposis and/or early-onset AML.

genetics↗

bioRxiv2020

Discovery of Cyclic Peptide Ligands to the SARS-CoV-2 Spike Protein using mRNA Display

The COVID-19 pandemic, caused by SARS-CoV-2, has led to substantial morbidity, mortality and disruption globally. Cellular entry of SARS-CoV-2 is mediated by the viral spike protein and affinity ligands to this surface protein have the potential for applications as antivirals and diagnostic reagents. Here, we describe the affinity selection of cyclic peptide ligands to the SARS-CoV-2 spike protein receptor binding domain (RBD) from three distinct libraries (in excess of a trillion molecules each) by mRNA display. We identified six high affinity molecules with dissociation constants (KD) in the nanomolar range (15-550 nM) to the RBD. The highest affinity ligand could be used as an affinity reagent to detect spike protein in solution by ELISA, and the co-crystal structure of this molecule bound to the RBD demonstrated that it binds to a cryptic binding site, displacing a {beta}-strand near the C-terminus. Our findings provide key mechanistic insight into the binding of peptide ligands to the SARS-CoV-2 spike RBD and the ligands discovered in this work may find future use as reagents for diagnostic applications.

biochemistry↗

bioRxiv2019

Characterization of a Y-specific duplication/insertion of the anti-Mullerian hormone type II receptor gene based on a chromosome-scale genome assembly of yellow perch, Perca flavescens

BackgroundYellow perch, Perca flavescens, is an ecologically and commercially important species native to a large portion of the northern United States and southern Canada. It is also a promising candidate species for aquaculture. No yellow perch reference genome, however, has been available to facilitate improvements in both fisheries and aquaculture management practices.\n\nFindingsBy combining Oxford Nanopore Technologies long-reads, 10X genomics Illumina short linked reads and a chromosome contact map produced with Hi-C, we generated a high-continuity chromosome scale yellow perch genome assembly of 877.4 Mb. It contains, in agreement with the known diploid chromosome yellow perch count, 24 chromosome-size scaffolds covering 98.8% of the complete assembly (N50 = 37.4 Mb, L50 = 11). Genome annotation identified 41.7% (366 Mb) of repeated elements and 24,486 genes including 16,579 genes (76.3%) significantly matching with proteins in public databases. We also provide a first characterization of the yellow perch sex determination locus that contains a male-specific duplicate of the anti-Mullerian hormone type II receptor gene (amhr2by) inserted at the proximal end of the Y chromosome (chromosome 9). Using this sex-specific information, we developed a simple PCR genotyping test which accurately differentiates XY genetic males (amhr2by+) from XX genetic females (amhr2by-).\n\nConclusionsOur high-quality genome assembly is an important genomic resource for future studies on yellow perch ecology, toxicology, fisheries, and aquaculture research. In addition, the characterization of the amhr2by gene as a candidate sex determining gene in yellow perch provides a new example of the recurrent implication of the transforming growth factor beta pathway in fish sex determination, and highlights gene duplication as an important genomic mechanism for the emergence of new master sex determination genes.

genomics↗

bioRxiv2019

Rapid resistance to pesticide control is predicted to evolve in an invasive fish

Xenobiotic resistance is commonly found in species with short generation times such as bacteria, annual plants, and insects. Nevertheless, the fundamental evolutionary principles that govern the spread of resistance alleles hold true for species with longer generation times. One such example could occur with sea lamprey (Petromyzon marinus), a parasitic invasive species in the Laurentian Great Lakes that decimated native fish populations prior to its control with the pesticide 3-trifluoromethyl-4-nitrophenol (TFM). Since the 1950s, tributaries have been treated annually with TFM, where treatments effectively remove most, but not all, larval sea lamprey. We developed an eco-genetic model of sea lamprey to examine factors affecting the evolution of resistance and found that resistance alleles rapidly rise to fixation after 40-80 years of treatment, despite the species relatively long generation time (4-7 years). The absence of natal homing allows resistant individuals to spread quickly throughout the entire system, but also makes the early detection of resistance challenging. High costs of resistance and density independent reproduction can delay, but not prevent, the onset of resistance. These results illustrate that sea lamprey have the potential to evolve resistance to their primary control agent in the near future, highlighting the urgent need for alternative controls.

evolutionary biology↗

bioRxiv2019

RST1 and RIPR connect the cytosolic RNA exosome to the Ski complex in Arabidopsis

The RNA exosome is a key 3-5 exoribonuclease with an evolutionarily conserved structure and function. Its cytosolic functions require the co-factors SKI7 and the Ski complex. Here we demonstrate by co-purification experiments that the ARM repeat protein RESURRECTION1 (RST1) and RST1 INTERACTING PROTEIN (RIPR) connect the cytosolic Arabidopsis RNA exosome to the Ski complex. rst1 and ripr mutants accumulate RNA quality control siRNAs (rqc-siRNAs) produced by the post-transcriptional gene silencing (PTGS) machinery when mRNA degradation is compromised. The small RNA populations observed in rst1 and ripr mutants are also detected in mutants lacking the RRP45B/CER7 core exosome subunit. Thus, molecular and genetic evidence supports a physical and functional link between RST1, RIPR and the RNA exosome. Our data reveal the existence of additional cytosolic exosome co-factors besides the known SKI subunits. RST1 is not restricted to plants, as homologues with a similar domain architecture but unknown function exist in animals, including humans.

molecular biology↗

bioRxiv2019

Drug reformulation for a neglected disease. The NANOHAT project to develop a safer more effective sleeping sickness drug.

Human African trypanosomiasis (HAT or sleeping sickness) is caused by the parasite Trypanosoma brucei sspp. The disease has two stages, a haemolymphatic stage after the bite of an infected tsetse fly, followed by a central nervous system stage where the parasite penetrates the brain, causing death if untreated. Treatment is stage-specific, due to the blood-brain barrier, with less toxic drugs such as pentamidine used to treat stage 1. The objective of our research programme was to develop an intravenous formulation of pentamidine which increases CNS exposure by some 10-100 fold, leading to efficacy against a model of stage 2 HAT. This target candidate profile is in line with drugs for neglected diseases inititative recommendations. To do this, we evaluated the physicochemical and structural characteristics of formulations of pentamidine with Pluronic micelles (triblock-copolymers of polyethylene-oxide and polypropylene oxide), selected candidates for efficacy and toxicity evaluation in vitro, quantified pentamidine CNS delivery of a sub-set of formulations in vitro and in vivo, and progressed one pentamidine-Pluronic formulation for further evaluation using an in vivo single dose brain penetration study. Screening pentamidine against 40 CNS targets did not reveal any major neurotoxicity concerns, however, pentamidine had a high affinity for the imidazoline2 receptor. The reduction in insulin secretion in MIN6 {beta}-cells by pentamidine maybe secondary to pentamidine-mediated activation of {beta}-cell imidazoline receptors and impairment of cell viability. Pluronic F68 (0.01%w/v)-pentamidine formulation had a similar inhibitory effect on insulin secretion as pentamidine alone and an additive trypanocidal effect in vitro. However, all Pluronics tested (P85, P105 and F68) did not significantly enhance brain exposure of pentamidine. These results are relevant to further developing block-copolymers as nanocarriers, improving BBB drug penetration and understanding the side effects of pentamidine.

pharmacology and toxicology↗

bioRxiv2017

PI3K Activation In Neural Stem Cells Drives Tumorigenesis Which Can Be Suppressed By Targeting CREB

Hyperactivation of the PI3K signaling is common in human cancers, including gliomas, but the precise role of the pathway in glioma biology remains to be determined. Some limited understanding of PI3K signaling in brain cancer come from studies on neural stem/progenitor cells (NSPCs) where signals transmitted via the PI3K pathway cooperate with other intracellular pathways and downstream transcription factors to regulate NSPC proliferation. To investigate the role for the PI3K pathway in glioma initiation and development, we generated a mouse model targeting the inducible expression of a Pik3caH1047A oncogenic mutation and simultaneous deletion of the PI3K negative regulator, Pten, in NSPCs. We show that the expression of a Pik3caH1047A was sufficient to initiate tumorigenesis but that simultaneous loss of Pten, was required for the development of invasive, high-grade glioma. Mutant NSPCs exhibited enhanced neurosphere forming capacity which correlated with increased Wnt signaling. We also show that loss of CREB in Pik3ca-PTEN tumors led to a longer symptom-free survival in mice. Taken together, our findings present a novel mouse model for high-grade glioma with which we demonstrate that the PI3K pathway is important for initiation of tumorigenesis and that disruption of downstream CREB signaling attenuates tumor expansion.

cancer biology↗