Publications by authors named "Stephen Wilcox"

34 Publications

A single-cell RNA expression atlas of normal, preneoplastic and tumorigenic states in the human breast.

EMBO J 2021 Jun 5;40(11):e107333. Epub 2021 May 5.

ACRF Cancer Biology and Stem Cells Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, Vic, Australia.

To examine global changes in breast heterogeneity across different states, we determined the single-cell transcriptomes of > 340,000 cells encompassing normal breast, preneoplastic BRCA1 tissue, the major breast cancer subtypes, and pairs of tumors and involved lymph nodes. Elucidation of the normal breast microenvironment revealed striking changes in the stroma of post-menopausal women. Single-cell profiling of 34 treatment-naive primary tumors, including estrogen receptor (ER) , HER2 , and triple-negative breast cancers, revealed comparable diversity among cancer cells and a discrete subset of cycling cells. The transcriptomes of preneoplastic BRCA1 tissue versus tumors highlighted global changes in the immune microenvironment. Within the tumor immune landscape, proliferative CD8 T cells characterized triple-negative and HER2 cancers but not ER tumors, while all subtypes comprised cycling tumor-associated macrophages, thus invoking potentially different immunotherapy targets. Copy number analysis of paired ER tumors and lymph nodes indicated seeding by genetically distinct clones or mass migration of primary tumor cells into axillary lymph nodes. This large-scale integration of patient samples provides a high-resolution map of cell diversity in normal and cancerous human breast.
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http://dx.doi.org/10.15252/embj.2020107333DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8167363PMC
June 2021

Effective low-cost preservation of human stools in field-based studies for helminth and microbiota analysis.

Int J Parasitol 2021 Mar 26. Epub 2021 Mar 26.

The Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria, Australia; Faculty for Veterinary and Agricultural Sciences, The University of Melbourne, Melbourne, Victoria, Australia.

Molecular studies of gastrointestinal infections or microbiotas require either rapid sample processing or effective interim preservation. This is difficult in remote settings in low-income countries, where the majority of the global infectious disease burden exists. Processing or freezing of samples immediately upon collection is often not feasible and the cost of commercial preservatives is prohibitive. We compared fresh freezing (the 'gold standard' method), with low-cost chemical preservation in (i) a salt-based buffer consisting of DMSO, EDTA and NaCl (DESS) or (ii) 2.5% potassium dichromate (PD), for soil-transmitted helminth detection and microbiota characterisation in pre-school and school-aged children from north-western Thailand. Fresh frozen samples were frozen at -20°C on collection and maintained at -80°C within ~3 days of collection until molecular analysis, with international shipping on dry ice. In contrast, chemically preserved samples were collected and stored at ~4°C, transported on wet ice and only stored at -20°C on arrival in Australia ~8 weeks after collection, with international shipping on wet ice. DESS and PD provided better sensitivity for STH diagnosis, estimating higher infection rates (>80% for Ascaris lumbricoides and >60% for Trichuris trichiura; versus 56% and 15% for these parasites in fresh frozen samples) and egg abundance (inferred as gene copy number estimates). All methods performed similarly for microbiota preservation, showing no significant differences in alpha-diversity based on overall richness or inverted Simpson's Index. All three methods performed similarly for RNA and protein preservation in a small subset of samples. Overall, DESS provided the best performance, with the added benefit of being non-toxic, compared with PD, hence making it particularly applicable for studies in remote and resource-poor settings.
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http://dx.doi.org/10.1016/j.ijpara.2021.01.002DOI Listing
March 2021

Novel Pedagogical Training for Nursing Doctoral Students in Support of Remote Learning: A Win-Win Situation.

Nurse Educ 2021 Jan 29. Epub 2021 Jan 29.

Author Affiliations: Assistant Professor (Dr Burton), Doctoral Student (Mss Rodrigues, Jones-Patten, Ju, Abrahim, and Mr Saatchi), Instructional Design & Faculty Development Manager (Dr Wilcox), and Associate Professor (Dr Bender), Sue & Bill Gross School of Nursing, University of California, Irvine.

Background: The need for faculty to educate prospective nurses is urgent: without sufficient nursing faculty, schools regularly reject qualified applicants, despite an increasing need for nurses. At the same time, many graduate-prepared nurses lack preparation in teaching and pedagogical frameworks.

Problem: Literature on how PhD programs in nursing prepare graduates for teaching indicates that there is typically more emphasis on research than pedagogical learning.

Approach: With the shift to remote learning under the COVID-19 pandemic, the University of California Irvine created a Graduate Fellows program to provide support to faculty while offering graduate students education in pedagogy and remote learning.

Outcomes: Fellows were satisfied and reported increased understanding of challenges in teaching and increasing comfort with nurse faculty roles.

Conclusions: The collaborative efforts of fellows and faculty provided important resources at a critical time, and insights gained can inform similar projects in nursing faculty development.
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http://dx.doi.org/10.1097/NNE.0000000000000967DOI Listing
January 2021

Flexible Usage and Interconnectivity of Diverse Cell Death Pathways Protect against Intracellular Infection.

Immunity 2020 09 30;53(3):533-547.e7. Epub 2020 Jul 30.

The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia; Department of Medical Biology, University of Melbourne, Parkville, VIC, Australia. Electronic address:

Programmed cell death contributes to host defense against pathogens. To investigate the relative importance of pyroptosis, necroptosis, and apoptosis during Salmonella infection, we infected mice and macrophages deficient for diverse combinations of caspases-1, -11, -12, and -8 and receptor interacting serine/threonine kinase 3 (RIPK3). Loss of pyroptosis, caspase-8-driven apoptosis, or necroptosis had minor impact on Salmonella control. However, combined deficiency of these cell death pathways caused loss of bacterial control in mice and their macrophages, demonstrating that host defense can employ varying components of several cell death pathways to limit intracellular infections. This flexible use of distinct cell death pathways involved extensive cross-talk between initiators and effectors of pyroptosis and apoptosis, where initiator caspases-1 and -8 also functioned as executioners when all known effectors of cell death were absent. These findings uncover a highly coordinated and flexible cell death system with in-built fail-safe processes that protect the host from intracellular infections.
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http://dx.doi.org/10.1016/j.immuni.2020.07.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7500851PMC
September 2020

Novel drivers and modifiers of MPL-dependent oncogenic transformation identified by deep mutational scanning.

Blood 2020 01;135(4):287-292

The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia.

The single transmembrane domain (TMD) of the human thrombopoietin receptor (TpoR/myeloproliferative leukemia [MPL] protein), encoded by exon 10 of the MPL gene, is a hotspot for somatic mutations associated with myeloproliferative neoplasms (MPNs). Approximately 6% and 14% of JAK2 V617F- essential thrombocythemia and primary myelofibrosis patients, respectively, have "canonical" MPL exon 10 driver mutations W515L/K/R/A or S505N, which generate constitutively active receptors and consequent loss of Tpo dependence. Other "noncanonical" MPL exon 10 mutations have also been identified in patients, both alone and in combination with canonical mutations, but, in almost all cases, their functional consequences and relevance to disease are unknown. Here, we used a deep mutational scanning approach to evaluate all possible single amino acid substitutions in the human TpoR TMD for their ability to confer cytokine-independent growth in Ba/F3 cells. We identified all currently recognized driver mutations and 7 novel mutations that cause constitutive TpoR activation, and a much larger number of second-site mutations that enhance S505N-driven activation. We found examples of both of these categories in published and previously unpublished MPL exon 10 sequencing data from MPN patients, demonstrating that some, if not all, of the new mutations reported here represent likely drivers or modifiers of myeloproliferative disease.
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http://dx.doi.org/10.1182/blood.2019002561DOI Listing
January 2020

A novel metabarcoding diagnostic tool to explore protozoan haemoparasite diversity in mammals: a proof-of-concept study using canines from the tropics.

Sci Rep 2019 09 2;9(1):12644. Epub 2019 Sep 2.

Faculty of Veterinary and Agricultural Sciences, University of Melbourne, Parkville, VIC, 3052, Australia.

Haemoparasites are responsible for some of the most prevalent and debilitating canine illnesses across the globe, whilst also posing a significant zoonotic risk to humankind. Nowhere are the effects of such parasites more pronounced than in developing countries in the tropics where the abundance and diversity of ectoparasites that transmit these pathogens reaches its zenith. Here we describe the use of a novel next-generation sequencing (NGS) metabarcoding based approach to screen for a range of blood-borne apicomplexan and kinetoplastid parasites from populations of temple dogs in Bangkok, Thailand. Our methodology elucidated high rates of Hepatozoon canis and Babesia vogeli infection, whilst also being able to characterise co-infections. In addition, our approach was confirmed to be more sensitive than conventional endpoint PCR diagnostic methods. Two kinetoplastid infections were also detected, including one by Trypanosoma evansi, a pathogen that is rarely screened for in dogs and another by Parabodo caudatus, a poorly documented organism that has been previously reported inhabiting the urinary tract of a dog with haematuria. Such results demonstrate the power of NGS methodologies to unearth rare and unusual pathogens, especially in regions of the world where limited information on canine vector-borne haemoparasites exist.
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http://dx.doi.org/10.1038/s41598-019-49118-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6718641PMC
September 2019

Assessment of a metabarcoding approach for the characterisation of vector-borne bacteria in canines from Bangkok, Thailand.

Parasit Vectors 2019 Aug 8;12(1):394. Epub 2019 Aug 8.

Faculty of Veterinary and Agricultural Sciences, University of Melbourne, Parkville, VIC, 3052, Australia.

Background: Globally, bacterial vector-borne disease (VBD) exerts a large toll on dogs in terms of morbidity and mortality but nowhere is this more pronounced than in the tropics. Tropical environments permit a burgeoning diversity and abundance of ectoparasites some of which can transmit an extensive range of infectious agents, including bacteria, amongst others. Although some of these vector-borne bacteria are responsible for both animal and human diseases in the tropics, there is a scarcity of epidemiological investigation into these pathogens' prevalence. The situation is further exacerbated by frequent canine co-infection, complicating symptomatology that regular diagnostic techniques may miss or be unable to fully characterise. Such limitations draw attention to the need to develop screening tools capable of detecting a wide range of pathogens from a host simultaneously.

Results: Here, we detail the employment of a next-generation sequencing (NGS) metabarcoding methodology to screen for the spectrum of bacterial VBD that are infecting semi-domesticated dogs across temple communities in Bangkok, Thailand. Our NGS detection protocol was able to find high levels of Ehrlichia canis, Mycoplasma haemocanis and Anaplasma platys infection rates as well as less common pathogens, such as "Candidatus Mycoplasma haematoparvum", Mycoplasma turicensis and Bartonella spp. We also compared our high-throughput approach to conventional endpoint PCR methods, demonstrating an improved detection ability for some bacterial infections, such as A. platys but a reduced ability to detect Rickettsia.

Conclusions: Our methodology demonstrated great strength at detecting coinfections of vector-borne bacteria and rare pathogens that are seldom screened for in canines in the tropics, highlighting its advantages over traditional diagnostics to better characterise bacterial pathogens in environments where there is a dearth of research.
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http://dx.doi.org/10.1186/s13071-019-3651-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6686542PMC
August 2019

MOZ directs the distal-less homeobox gene expression program during craniofacial development.

Development 2019 07 24;146(14). Epub 2019 Jul 24.

Walter and Eliza Hall Institute of Medical Research, Melbourne, Parkville, VIC 3052, Australia

Oral clefts are common birth defects. Individuals with oral clefts who have identical genetic mutations regularly present with variable penetrance and severity. Epigenetic or chromatin-mediated mechanisms are commonly invoked to explain variable penetrance. However, specific examples of these are rare. Two functional copies of the (, ) gene, encoding a MYST family lysine acetyltransferase chromatin regulator, are essential for human craniofacial development, but the molecular role of MOZ in this context is unclear. Using genetic interaction and genomic studies, we have investigated the effects of loss of MOZ on the gene expression program during mouse development. Among the more than 500 genes differentially expressed after loss of MOZ, 19 genes had previously been associated with cleft palates. These included four distal-less homeobox (DLX) transcription factor-encoding genes, , , and and DLX target genes (including , , and ). MOZ occupied the locus and was required for normal levels of histone H3 lysine 9 acetylation. MOZ affected Dlx gene expression cell-autonomously within neural crest cells. Our study identifies a specific program by which the chromatin modifier MOZ regulates craniofacial development.
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http://dx.doi.org/10.1242/dev.175042DOI Listing
July 2019

The spread of resistance to imidacloprid is restricted by thermotolerance in natural populations of Drosophila melanogaster.

Nat Ecol Evol 2019 04 18;3(4):647-656. Epub 2019 Mar 18.

School of Biosciences, The University of Melbourne, Parkville, Victoria, Australia.

Imidacloprid, the world's most used insecticide, has caused considerable controversy due to harmful effects on non-pest species and increasing evidence showing that insecticides have become the primary selective force in many insect species. The genetic response to insecticides is heterogeneous across populations and environments, leading to more complex patterns of genetic variation than previously thought. This motivated the investigation of imidacloprid resistance at different temperatures in natural populations of Drosophila melanogaster originating from four climate extremes replicated across two continents. Population and quantitative genomic analysis, supported by functional tests, have revealed a mixed genetic architecture to resistance involving major genes (Paramyosin and Nicotinic-Acetylcholine Receptor Alpha 3) and polygenes with a major trade-off with thermotolerance. Reduced genetic differentiation at resistance-associated loci indicated enhanced gene flow at these loci. Resistance alleles showed stronger evidence of positive selection in temperate populations compared to tropical populations in which chromosomal inversions In(2 L)t, In(3 R)Mo and In(3 R)Payne harbour susceptibility alleles. Polygenic architecture and ecological factors should be considered when developing sustainable management strategies for both pest and beneficial insects.
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http://dx.doi.org/10.1038/s41559-019-0837-yDOI Listing
April 2019

PHF6 regulates hematopoietic stem and progenitor cells and its loss synergizes with expression of TLX3 to cause leukemia.

Blood 2019 04 12;133(16):1729-1741. Epub 2019 Feb 12.

The Walter and Eliza Hall Institute of Medical Research, Melbourne, VIC, Australia.

Somatically acquired mutations in () frequently occur in hematopoietic malignancies and often coincide with ectopic expression of However, there is no functional evidence to demonstrate whether these mutations contribute to tumorigenesis. Similarly, the role of PHF6 in hematopoiesis is unknown. We report here that deletion in mice resulted in a reduced number of hematopoietic stem cells (HSCs), an increased number of hematopoietic progenitor cells, and an increased proportion of cycling stem and progenitor cells. Loss of PHF6 caused increased and sustained hematopoietic reconstitution in serial transplantation experiments. Interferon-stimulated gene expression was upregulated in the absence of PHF6 in hematopoietic stem and progenitor cells. The numbers of hematopoietic progenitor cells and cycling hematopoietic stem and progenitor cells were restored to normal by combined loss of PHF6 and the interferon α and β receptor subunit 1. Ectopic expression of TLX3 alone caused partially penetrant leukemia. TLX3 expression and loss of PHF6 combined caused fully penetrant early-onset leukemia. Our data suggest that PHF6 is a hematopoietic tumor suppressor and is important for fine-tuning hematopoietic stem and progenitor cell homeostasis.
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http://dx.doi.org/10.1182/blood-2018-07-860726DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6695515PMC
April 2019

NLRP1 restricts butyrate producing commensals to exacerbate inflammatory bowel disease.

Nat Commun 2018 09 13;9(1):3728. Epub 2018 Sep 13.

Inflammation Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, 3052, Australia.

Anti-microbial signaling pathways are normally triggered by innate immune receptors when detecting pathogenic microbes to provide protective immunity. Here we show that the inflammasome sensor Nlrp1 aggravates DSS-induced experimental mouse colitis by limiting beneficial, butyrate-producing Clostridiales in the gut. The colitis-protective effects of Nlrp1 deficiency are thus reversed by vancomycin treatment, but recapitulated with butyrate supplementation in wild-type mice. Moreover, an activating mutation in Nlrp1a increases IL-18 and IFNγ production, and decreases colonic butyrate to exacerbate colitis. We also show that, in patients with ulcerative colitis, increased NLRP1 in inflamed regions of the colon is associated with increased IFN-γ. In this context, NLRP1, IL-18 or IFN-γ expression negatively correlates with the abundance of Clostridiales in human rectal mucosal biopsies. Our data identify the NLRP1 inflammasome to be a key negative regulator of protective, butyrate-producing commensals, which therefore promotes inflammatory bowel disease.
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http://dx.doi.org/10.1038/s41467-018-06125-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6137172PMC
September 2018

Inhibitors of histone acetyltransferases KAT6A/B induce senescence and arrest tumour growth.

Nature 2018 08 1;560(7717):253-257. Epub 2018 Aug 1.

Commonwealth Scientific and Industrial Research Organisation (CSIRO), Biomedical Program, Parkville, Victoria, Australia.

Acetylation of histones by lysine acetyltransferases (KATs) is essential for chromatin organization and function. Among the genes coding for the MYST family of KATs (KAT5-KAT8) are the oncogenes KAT6A (also known as MOZ) and KAT6B (also known as MORF and QKF). KAT6A has essential roles in normal haematopoietic stem cells and is the target of recurrent chromosomal translocations, causing acute myeloid leukaemia. Similarly, chromosomal translocations in KAT6B have been identified in diverse cancers. KAT6A suppresses cellular senescence through the regulation of suppressors of the CDKN2A locus, a function that requires its KAT activity. Loss of one allele of KAT6A extends the median survival of mice with MYC-induced lymphoma from 105 to 413 days. These findings suggest that inhibition of KAT6A and KAT6B may provide a therapeutic benefit in cancer. Here we present highly potent, selective inhibitors of KAT6A and KAT6B, denoted WM-8014 and WM-1119. Biochemical and structural studies demonstrate that these compounds are reversible competitors of acetyl coenzyme A and inhibit MYST-catalysed histone acetylation. WM-8014 and WM-1119 induce cell cycle exit and cellular senescence without causing DNA damage. Senescence is INK4A/ARF-dependent and is accompanied by changes in gene expression that are typical of loss of KAT6A function. WM-8014 potentiates oncogene-induced senescence in vitro and in a zebrafish model of hepatocellular carcinoma. WM-1119, which has increased bioavailability, arrests the progression of lymphoma in mice. We anticipate that this class of inhibitors will help to accelerate the development of therapeutics that target gene transcription regulated by histone acetylation.
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http://dx.doi.org/10.1038/s41586-018-0387-5DOI Listing
August 2018

DNA repair processes are critical mediators of p53-dependent tumor suppression.

Nat Med 2018 07 11;24(7):947-953. Epub 2018 Jun 11.

Molecular Genetics of Cancer Division, The Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria, Australia.

It has long been assumed that p53 suppresses tumor development through induction of apoptosis, possibly with contributions by cell cycle arrest and cell senescence. However, combined deficiency in these three processes does not result in spontaneous tumor formation as observed upon loss of p53, suggesting the existence of additional mechanisms that are critical mediators of p53-dependent tumor suppression function. To define such mechanisms, we performed in vivo shRNA screens targeting p53-regulated genes in sensitized genetic backgrounds. We found that knockdown of Zmat3, Ctsf and Cav1, promoted lymphoma/leukemia development only when PUMA and p21, the critical effectors of p53-driven apoptosis, cell cycle arrest and senescence, were also absent. Notably, loss of the DNA repair gene Mlh1 caused lymphoma in a wild-type background, and its enforced expression was able to delay tumor development driven by loss of p53. Further examination of direct p53 target genes implicated in DNA repair showed that knockdown of Mlh1, Msh2, Rnf144b, Cav1 and Ddit4 accelerated MYC-driven lymphoma development to a similar extent as knockdown of p53. Collectively, these findings demonstrate that extensive functional overlap of several p53-regulated processes safeguards against cancer and that coordination of DNA repair appears to be an important process by which p53 suppresses tumor development.
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http://dx.doi.org/10.1038/s41591-018-0043-5DOI Listing
July 2018

Chloroplast variation is incongruent with classification of the Australian bloodwood eucalypts (genus Corymbia, family Myrtaceae).

PLoS One 2018 18;13(4):e0195034. Epub 2018 Apr 18.

School of BioSciences, The University of Melbourne, Parkville, VIC, Australia.

Previous molecular phylogenetic analyses have resolved the Australian bloodwood eucalypt genus Corymbia (~100 species) as either monophyletic or paraphyletic with respect to Angophora (9-10 species). Here we assess relationships of Corymbia and Angophora using a large dataset of chloroplast DNA sequences (121,016 base pairs; from 90 accessions representing 55 Corymbia and 8 Angophora species, plus 33 accessions of related genera), skimmed from high throughput sequencing of genomic DNA, and compare results with new analyses of nuclear ITS sequences (119 accessions) from previous studies. Maximum likelihood and maximum parsimony analyses of cpDNA resolve well supported trees with most nodes having >95% bootstrap support. These trees strongly reject monophyly of Corymbia, its two subgenera (Corymbia and Blakella), most taxonomic sections (Abbreviatae, Maculatae, Naviculares, Septentrionales), and several species. ITS trees weakly indicate paraphyly of Corymbia (bootstrap support <50% for maximum likelihood, and 71% for parsimony), but are highly incongruent with the cpDNA analyses, in that they support monophyly of both subgenera and some taxonomic sections of Corymbia. The striking incongruence between cpDNA trees and both morphological taxonomy and ITS trees is attributed largely to chloroplast introgression between taxa, because of geographic sharing of chloroplast clades across taxonomic groups. Such introgression has been widely inferred in studies of the related genus Eucalyptus. This is the first report of its likely prevalence in Corymbia and Angophora, but this is consistent with previous morphological inferences of hybridisation between species. Our findings (based on continent-wide sampling) highlight a need for more focussed studies to assess the extent of hybridisation and introgression in the evolutionary history of these genera, and that critical testing of the classification of Corymbia and Angophora requires additional sequence data from nuclear genomes.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0195034PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5905893PMC
July 2018

Loss of NF-κB1 Causes Gastric Cancer with Aberrant Inflammation and Expression of Immune Checkpoint Regulators in a STAT-1-Dependent Manner.

Immunity 2018 03;48(3):570-583.e8

The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia; Department of Medical Biology, University of Melbourne, Parkville, Victoria 3010, Australia. Electronic address:

Polymorphisms in NFKB1 that diminish its expression have been linked to human inflammatory diseases and increased risk for epithelial cancers. The underlying mechanisms are unknown, and the link is perplexing given that NF-κB signaling reportedly typically exerts pro-tumorigenic activity. Here we have shown that NF-κB1 deficiency, even loss of a single allele, resulted in spontaneous invasive gastric cancer (GC) in mice that mirrored the histopathological progression of human intestinal-type gastric adenocarcinoma. Bone marrow chimeras revealed that NF-κB1 exerted tumor suppressive functions in both epithelial and hematopoietic cells. RNA-seq analysis showed that NF-κB1 deficiency resulted in aberrant JAK-STAT signaling, which dysregulated expression of effectors of inflammation, antigen presentation, and immune checkpoints. Concomitant loss of STAT1 prevented these immune abnormalities and GC development. These findings provide mechanistic insight into how polymorphisms that attenuate NFKB1 expression predispose humans to epithelial cancers, highlighting the pro-tumorigenic activity of STAT1 and identifying targetable vulnerabilities in GC.
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http://dx.doi.org/10.1016/j.immuni.2018.03.003DOI Listing
March 2018

Influence of fecal collection conditions and 16S rRNA gene sequencing at two centers on human gut microbiota analysis.

Sci Rep 2018 03 12;8(1):4386. Epub 2018 Mar 12.

The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, 3052, Australia.

To optimise fecal sampling for reproducible analysis of the gut microbiome, we compared different methods of sample collection and sequencing of 16S rRNA genes at two centers. Samples collected from six individuals on three consecutive days were placed in commercial collection tubes (OMNIgeneGut OMR-200) or in sterile screw-top tubes in a home fridge or home freezer for 6-24 h, before transfer and storage at -80 °C. Replicate samples were shipped to centers in Australia and the USA for DNA extraction and sequencing by their respective PCR protocols, and analysed with the same bioinformatic pipeline. Variation in gut microbiome was dominated by differences between individuals. Minor differences in the abundance of taxa were found between collection-processing methods and day of collection, and between the two centers. We conclude that collection with storage and transport at 4 °C within 24 h is adequate for 16S rRNA analysis of the gut microbiome. Other factors including differences in PCR and sequencing methods account for relatively minor variation compared to differences between individuals.
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http://dx.doi.org/10.1038/s41598-018-22491-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5847573PMC
March 2018

Biologically active constituents of the secretome of human W8B2 cardiac stem cells.

Sci Rep 2018 01 25;8(1):1579. Epub 2018 Jan 25.

St Vincent's Institute of Medical Research, Victoria, Australia.

The benefits of adult stem cells for repair of the heart have been attributed to the repertoire of salutary paracrine activities they appear to exert. We previously isolated human W8B2 cardiac stem cells (CSCs) and found they powerfully influence cardiomyocytes and endothelial cells to collectively promote cardiac repair and regeneration. Here, the complexity of the W8B2 CSC secretomes was characterised and examined in more detail. Using ion exchange chromatography to separate soluble proteins based on their net surface charge, the secreted factors responsible for the pro-survival activity of W8B2 CSCs were found within the low and medium cation fractions. In addition to the soluble proteins, extracellular vesicles generated from W8B2 CSCs not only exhibited pro-survival and pro-angiogenic activities, but also promoted proliferation of neonatal cardiomyocytes. These extracellular vesicles contain a cargo of proteins, mRNA and primary microRNA precursors that are enriched in exosomes and are capable of modulating collectively many of the cellular pathways involved in protein metabolism, cell growth, as well as cellular responses to stress and organisation of the extracellular matrix. Thus the W8B2 CSC secretome contains a multitude of bioactive paracrine factors we have now characterised, that might well be harnessed for therapeutic application for cardiac repair and regeneration.
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http://dx.doi.org/10.1038/s41598-018-19855-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5785502PMC
January 2018

A preliminary molecular phylogeny of shield-bearer moths (Lepidoptera: Adeloidea: Heliozelidae) highlights rich undescribed diversity.

Mol Phylogenet Evol 2018 03 8;120:129-143. Epub 2017 Dec 8.

School of BioSciences, The University of Melbourne, Parkville, Victoria 3010, Australia.

Heliozelidae are a widespread, evolutionarily early diverging family of small, day-flying monotrysian moths, for which a comprehensive phylogeny is lacking. We generated the first molecular phylogeny of the family using DNA sequences of two mitochondrial genes (COI and COII) and two nuclear genes (H3 and 28S) from 130 Heliozelidae specimens, including eight of the twelve known genera: Antispila, Antispilina, Coptodisca, Heliozela, Holocacista, Hoplophanes, Pseliastis, and Tyriozela. Our results provide strong support for five major Heliozelidae clades: (i) a large widespread clade containing the leaf-mining genera Antispilina, Coptodisca and Holocacista and some species of Antispila, (ii) a clade containing most of the described Antispila, (iii) a clade containing the leaf-mining genus Heliozela and the monotypic genus Tyriozela, (iv) an Australian clade containing Pseliastis and (v) an Australian clade containing Hoplophanes. Each clade includes several new species and potentially new genera. Collectively, our data uncover a rich and undescribed diversity that appears to be especially prevalent in Australia. Our work highlights the need for a major taxonomic revision of the family and for generating a robust molecular phylogeny using multi-gene approaches in order to resolve the relationships among clades.
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http://dx.doi.org/10.1016/j.ympev.2017.12.004DOI Listing
March 2018

Construction of developmental lineage relationships in the mouse mammary gland by single-cell RNA profiling.

Nat Commun 2017 11 20;8(1):1627. Epub 2017 Nov 20.

ACRF Stem Cells and Cancer Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, 3052, Australia.

The mammary epithelium comprises two primary cellular lineages, but the degree of heterogeneity within these compartments and their lineage relationships during development remain an open question. Here we report single-cell RNA profiling of mouse mammary epithelial cells spanning four developmental stages in the post-natal gland. Notably, the epithelium undergoes a large-scale shift in gene expression from a relatively homogeneous basal-like program in pre-puberty to distinct lineage-restricted programs in puberty. Interrogation of single-cell transcriptomes reveals different levels of diversity within the luminal and basal compartments, and identifies an early progenitor subset marked by CD55. Moreover, we uncover a luminal transit population and a rare mixed-lineage cluster amongst basal cells in the adult mammary gland. Together these findings point to a developmental hierarchy in which a basal-like gene expression program prevails in the early post-natal gland prior to the specification of distinct lineage signatures, and the presence of cellular intermediates that may serve as transit or lineage-primed cells.
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http://dx.doi.org/10.1038/s41467-017-01560-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5696379PMC
November 2017

Development of amplicon deep sequencing markers and data analysis pipeline for genotyping multi-clonal malaria infections.

BMC Genomics 2017 Nov 13;18(1):864. Epub 2017 Nov 13.

Swiss Tropical and Public Health Institute, Basel, Switzerland.

Background: Amplicon deep sequencing permits sensitive detection of minority clones and improves discriminatory power for genotyping multi-clone Plasmodium falciparum infections. New amplicon sequencing and data analysis protocols are needed for genotyping in epidemiological studies and drug efficacy trials of P. falciparum.

Methods: Targeted sequencing of molecular marker csp and novel marker cpmp was conducted in duplicate on mixtures of parasite culture strains and 37 field samples. A protocol allowing to multiplex up to 384 samples in a single sequencing run was applied. Software "HaplotypR" was developed for data analysis.

Results: Cpmp was highly diverse (H = 0.96) in contrast to csp (H = 0.57). Minority clones were robustly detected if their frequency was >1%. False haplotype calls owing to sequencing errors were observed below that threshold.

Conclusions: To reliably detect haplotypes at very low frequencies, experiments are best performed in duplicate and should aim for coverage of >10'000 reads/amplicon. When compared to length polymorphic marker msp2, highly multiplexed amplicon sequencing displayed greater sensitivity in detecting minority clones.
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http://dx.doi.org/10.1186/s12864-017-4260-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5682641PMC
November 2017

Aboriginal Australian mitochondrial genome variation - an increased understanding of population antiquity and diversity.

Sci Rep 2017 03 13;7:43041. Epub 2017 Mar 13.

Department of Biochemistry and Genetics, La Trobe Institute for Molecular Sciences, La Trobe University, Melbourne, Victoria, Australia.

Aboriginal Australians represent one of the oldest continuous cultures outside Africa, with evidence indicating that their ancestors arrived in the ancient landmass of Sahul (present-day New Guinea and Australia) ~55 thousand years ago. Genetic studies, though limited, have demonstrated both the uniqueness and antiquity of Aboriginal Australian genomes. We have further resolved known Aboriginal Australian mitochondrial haplogroups and discovered novel indigenous lineages by sequencing the mitogenomes of 127 contemporary Aboriginal Australians. In particular, the more common haplogroups observed in our dataset included M42a, M42c, S, P5 and P12, followed by rarer haplogroups M15, M16, N13, O, P3, P6 and P8. We propose some major phylogenetic rearrangements, such as in haplogroup P where we delinked P4a and P4b and redefined them as P4 (New Guinean) and P11 (Australian), respectively. Haplogroup P2b was identified as a novel clade potentially restricted to Torres Strait Islanders. Nearly all Aboriginal Australian mitochondrial haplogroups detected appear to be ancient, with no evidence of later introgression during the Holocene. Our findings greatly increase knowledge about the geographic distribution and phylogenetic structure of mitochondrial lineages that have survived in contemporary descendants of Australia's first settlers.
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http://dx.doi.org/10.1038/srep43041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5347126PMC
March 2017

Mitochondrial DNA diversity of present-day Aboriginal Australians and implications for human evolution in Oceania.

J Hum Genet 2017 Mar 1;62(3):343-353. Epub 2016 Dec 1.

Department of Biochemistry and Genetics, La Trobe University, Melbourne, VIC, Australia.

Aboriginal Australians are one of the more poorly studied populations from the standpoint of human evolution and genetic diversity. Thus, to investigate their genetic diversity, the possible date of their ancestors' arrival and their relationships with neighboring populations, we analyzed mitochondrial DNA (mtDNA) diversity in a large sample of Aboriginal Australians. Selected mtDNA single-nucleotide polymorphisms and the hypervariable segment haplotypes were analyzed in 594 Aboriginal Australians drawn from locations across the continent, chiefly from regions not previously sampled. Most (~78%) samples could be assigned to mtDNA haplogroups indigenous to Australia. The indigenous haplogroups were all ancient (with estimated ages >40 000 years) and geographically widespread across the continent. The most common haplogroup was P (44%) followed by S (23%) and M42a (9%). There was some geographic structure at the haplotype level. The estimated ages of the indigenous haplogroups range from 39 000 to 55 000 years, dates that fit well with the estimated date of colonization of Australia based on archeological evidence (~47 000 years ago). The distribution of mtDNA haplogroups in Australia and New Guinea supports the hypothesis that the ancestors of Aboriginal Australians entered Sahul through at least two entry points. The mtDNA data give no support to the hypothesis of secondary gene flow into Australia during the Holocene, but instead suggest long-term isolation of the continent.
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http://dx.doi.org/10.1038/jhg.2016.147DOI Listing
March 2017

Results of human factors testing in a novel Hemodialysis system designed for ease of patient use.

Hemodial Int 2016 10 19;20(4):643-649. Epub 2016 May 19.

Division of Nephrology, Department of Medicine, Stanford University School of Medicine, Stanford, California, USA.

Introduction Home hemodialysis has not been widely adopted despite superior outcomes relative to conventional in-center hemodialysis. Patients receiving home hemodialysis experience high rates of technique failure owing to machine complexity, training burden, and the inability to master treatments independently. Methods We conducted human factors testing on 15 health care professionals (HCPs) and 15 patients upon release of the defined training program on the Tablo™ Hemodialysis System. Each participant completed one training and one testing session conducted in a simulated clinical environment. Training sessions lasted <3 hours for HCPs and <4 hours for patients, with an hour break between sessions for knowledge decay. During the testing session, we recorded participant behavior and data according to standard performance and safety-based criteria. Findings Of 15 HCPs, 10 were registered nurses and five patient care technicians, with a broad range of dialysis work experience and no limitations other than visual correction. Of 15 patients (average age 48 years), 13 reported no limitations and two reported modest limitations-partial deafness and blindness in one eye, respectively. The average error rate was 4.4 per session for HCPs and 2.9 per session for patients out of a total possible 1,710 opportunities for errors. Despite having received minimal training, neither HCPs nor patients committed safety-related errors that required mitigation; rather, we noted only minor errors and operational difficulties. Discussion The Tablo™ Hemodialysis System is easy to use, and may help to enable self-care and home hemodialysis in settings heretofore associated with high rates of technique failure.
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http://dx.doi.org/10.1111/hdi.12430DOI Listing
October 2016

Deep Roots for Aboriginal Australian Y Chromosomes.

Curr Biol 2016 Mar 25;26(6):809-13. Epub 2016 Feb 25.

The Wellcome Trust Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridgeshire CB10 1SA, UK. Electronic address:

Australia was one of the earliest regions outside Africa to be colonized by fully modern humans, with archaeological evidence for human presence by 47,000 years ago (47 kya) widely accepted [1, 2]. However, the extent of subsequent human entry before the European colonial age is less clear. The dingo reached Australia about 4 kya, indirectly implying human contact, which some have linked to changes in language and stone tool technology to suggest substantial cultural changes at the same time [3]. Genetic data of two kinds have been proposed to support gene flow from the Indian subcontinent to Australia at this time, as well: first, signs of South Asian admixture in Aboriginal Australian genomes have been reported on the basis of genome-wide SNP data [4]; and second, a Y chromosome lineage designated haplogroup C(∗), present in both India and Australia, was estimated to have a most recent common ancestor around 5 kya and to have entered Australia from India [5]. Here, we sequence 13 Aboriginal Australian Y chromosomes to re-investigate their divergence times from Y chromosomes in other continents, including a comparison of Aboriginal Australian and South Asian haplogroup C chromosomes. We find divergence times dating back to ∼50 kya, thus excluding the Y chromosome as providing evidence for recent gene flow from India into Australia.
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http://dx.doi.org/10.1016/j.cub.2016.01.028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4819516PMC
March 2016

Antiquity and diversity of aboriginal Australian Y-chromosomes.

Am J Phys Anthropol 2016 Mar 30;159(3):367-81. Epub 2015 Oct 30.

Department of Biochemistry and Genetics, La Trobe Institute of Molecular Sciences, La Trobe University, Melbourne, VIC, Australia.

Objective: Understanding the origins of Aboriginal Australians is crucial in reconstructing the evolution and spread of Homo sapiens as evidence suggests they represent the descendants of the earliest group to leave Africa. This study analyzed a large sample of Y-chromosomes to answer questions relating to the migration routes of their ancestors, the age of Y-haplogroups, date of colonization, as well as the extent of male-specific variation.

Methods: Knowledge of Y-chromosome variation among Aboriginal Australians is extremely limited. This study examined Y-SNP and Y-STR variation among 657 self-declared Aboriginal males from locations across the continent. 17 Y-STR loci and 47 Y-SNPs spanning the Y-chromosome phylogeny were typed in total.

Results: The proportion of non-indigenous Y-chromosomes of assumed Eurasian origin was high, at 56%. Y lineages of indigenous Sahul origin belonged to haplogroups C-M130*(xM8,M38,M217,M347) (1%), C-M347 (19%), K-M526*(xM147,P308,P79,P261,P256,M231,M175,M45,P202) (12%), S-P308 (12%), and M-M186 (0.9%). Haplogroups C-M347, K-M526*, and S-P308 are Aboriginal Australian-specific. Dating of C-M347, K-M526*, and S-P308 indicates that all are at least 40,000 years old, confirming their long-term presence in Australia. Haplogroup C-M347 comprised at least three sub-haplogroups: C-DYS390.1del, C-M210, and the unresolved paragroup C-M347*(xDYS390.1del,M210).

Conclusions: There was some geographic structure to the Y-haplogroup variation, but most haplogroups were present throughout Australia. The age of the Australian-specific Y-haplogroups suggests New Guineans and Aboriginal Australians have been isolated for over 30,000 years, supporting findings based on mitochondrial DNA data. Our data support the hypothesis of more than one route (via New Guinea) for males entering Sahul some 50,000 years ago and give no support for colonization events during the Holocene, from either India or elsewhere.
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http://dx.doi.org/10.1002/ajpa.22886DOI Listing
March 2016

An inducible lentiviral guide RNA platform enables the identification of tumor-essential genes and tumor-promoting mutations in vivo.

Cell Rep 2015 Mar 26;10(8):1422-32. Epub 2015 Feb 26.

Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia; Department of Medical Biology, University of Melbourne, Parkville, VIC 3050, Australia. Electronic address:

The CRISPR/Cas9 technology enables the introduction of genomic alterations into almost any organism; however, systems for efficient and inducible gene modification have been lacking, especially for deletion of essential genes. Here, we describe a drug-inducible small guide RNA (sgRNA) vector system allowing for ubiquitous and efficient gene deletion in murine and human cells. This system mediates the efficient, temporally controlled deletion of MCL-1, both in vitro and in vivo, in human Burkitt lymphoma cell lines that require this anti-apoptotic BCL-2 protein for sustained survival and growth. Unexpectedly, repeated induction of the same sgRNA generated similar inactivating mutations in the human Mcl-1 gene due to low mutation variability exerted by the accompanying non-homologous end-joining (NHEJ) process. Finally, we were able to generate hematopoietic cell compartment-restricted Trp53-knockout mice, leading to the identification of cancer-promoting mutants of this critical tumor suppressor.
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http://dx.doi.org/10.1016/j.celrep.2015.02.002DOI Listing
March 2015

edgeR: a versatile tool for the analysis of shRNA-seq and CRISPR-Cas9 genetic screens.

F1000Res 2014 24;3:95. Epub 2014 Apr 24.

Molecular Medicine Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, 3052, Australia ; Department of Medical Biology, The University of Melbourne, Parkville, Victoria, 3010, Australia.

Pooled library sequencing screens that perturb gene function in a high-throughput manner are becoming increasingly popular in functional genomics research. Irrespective of the mechanism by which loss of function is achieved, via either RNA interference using short hairpin RNAs (shRNAs) or genetic mutation using single guide RNAs (sgRNAs) with the CRISPR-Cas9 system, there is a need to establish optimal analysis tools to handle such data. Our open-source processing pipeline in edgeR provides a complete analysis solution for screen data, that begins with the raw sequence reads and ends with a ranked list of candidate genes for downstream biological validation. We first summarize the raw data contained in a fastq file into a matrix of counts (samples in the columns, genes in the rows) with options for allowing mismatches and small shifts in sequence position. Diagnostic plots, normalization and differential representation analysis can then be performed using established methods to prioritize results in a statistically rigorous way, with the choice of either the classic exact testing methodology or generalized linear modeling that can handle complex experimental designs. A detailed users' guide that demonstrates how to analyze screen data in edgeR along with a point-and-click implementation of this workflow in Galaxy are also provided. The edgeR package is freely available from http://www.bioconductor.org.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4023662PMC
http://dx.doi.org/10.12688/f1000research.3928.2DOI Listing
February 2015

How can we eliminate error when we have no consistency?

Authors:
Stephen Wilcox

Biomed Instrum Technol 2013 ;Suppl:31

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http://dx.doi.org/10.2345/0899-8205-47.s2.31DOI Listing
January 2014

Genome sequences of six wheat-infecting fusarium species isolates.

Genome Announc 2013 Sep 5;1(5). Epub 2013 Sep 5.

Centre for Comparative Genomics, Murdoch University, Perth, Australia.

Fusarium pathogens represent a major constraint to wheat and barley production worldwide. To facilitate future comparative studies of Fusarium species that are pathogenic to wheat, the genome sequences of four Fusarium pseudograminearum isolates, a single Fusarium acuminatum isolate, and an organism from the Fusarium incarnatum-F. equiseti species complex are reported.
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http://dx.doi.org/10.1128/genomeA.00670-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3764410PMC
September 2013

Bread matters: a national initiative to profile the genetic diversity of Australian wheat.

Plant Biotechnol J 2012 Aug 9;10(6):703-8. Epub 2012 Jun 9.

Australian Centre for Plant Functional Genomics and University of Queensland, St. Lucia, Qld, Australia.

The large and complex genome of wheat makes genetic and genomic analysis in this important species both expensive and resource intensive. The application of next-generation sequencing technologies is particularly resource intensive, with at least 17 Gbp of sequence data required to obtain minimal (1×) coverage of the genome. A similar volume of data would represent almost 40× coverage of the rice genome. Progress can be made through the establishment of consortia to produce shared genomic resources. Australian wheat genome researchers, working with Bioplatforms Australia, have collaborated in a national initiative to establish a genetic diversity dataset representing Australian wheat germplasm based on whole genome next-generation sequencing data. Here, we describe the establishment and validation of this resource which can provide a model for broader international initiatives for the analysis of large and complex genomes.
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http://dx.doi.org/10.1111/j.1467-7652.2012.00717.xDOI Listing
August 2012