ORCID Profile
0000-0001-9330-887X
Current Organisations
University of New South Wales - Randwick Campus
,
Garvan Institute of Medical Research
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Publisher: Oxford University Press (OUP)
Date: 18-08-2015
Abstract: This article describes the cases of two patients with severe refractory adult respiratory syndrome (ARDS) who failed to improve after both standard life support measures, including mechanical ventilation, and additional measures, including extracorporeal ventilation (i.e., in a heart-lung machine). Unlike acute forms of ARDS (such in the current NIH-sponsored study of mesenchymal stromal cells in ARDS), recovery does not generally occur in such patients.
Publisher: Springer Science and Business Media LLC
Date: 03-02-2020
Publisher: Springer Science and Business Media LLC
Date: 31-10-2016
DOI: 10.1038/NBT.3701
Abstract: Little is known about the heterogeneity of small-RNA expression as small-RNA profiling has so far required large numbers of cells. Here we present a single-cell method for small-RNA sequencing and apply it to naive and primed human embryonic stem cells and cancer cells. Analysis of microRNAs and fragments of tRNAs and small nucleolar RNAs (snoRNAs) reveals the potential of microRNAs as markers for different cell types and states.
Publisher: Springer Science and Business Media LLC
Date: 21-07-2021
DOI: 10.1186/S12958-021-00801-5
Abstract: The period of time when the embryo and the endometrium undergo significant morphological alterations to facilitate a successful implantation—known as “window of implantation”—is a critical moment in human reproduction. Embryo and the endometrium communicate extensively during this period, and lipid bilayer bound nanoscale extracellular vesicles (EVs) are purported to be integral to this communication. To investigate the nature of the EV-mediated embryo-maternal communication, we have supplemented trophoblast analogue spheroid (JAr) derived EVs to an endometrial analogue (RL 95–2) cell layer and characterized the transcriptomic alterations using RNA sequencing. EVs derived from non-trophoblast cells (HEK293) were used as a negative control. The cargo of the EVs were also investigated through mRNA and miRNA sequencing. Trophoblast spheroid derived EVs induced drastic transcriptomic alterations in the endometrial cells while the non-trophoblast cell derived EVs failed to induce such changes demonstrating functional specificity in terms of EV origin. Through gene set enrichment analysis (GSEA), we found that the response in endometrial cells was focused on extracellular matrix remodelling and G protein-coupled receptors’ signalling, both of which are of known functional relevance to endometrial receptivity. Approximately 9% of genes downregulated in endometrial cells were high-confidence predicted targets of miRNAs detected exclusively in trophoblast analogue-derived EVs, suggesting that only a small proportion of reduced expression in endometrial cells can be attributed directly to gene silencing by miRNAs carried as cargo in the EVs. Our study reveals that trophoblast derived EVs have the ability to modify the endometrial gene expression, potentially with functional importance for embryo-maternal communication during implantation, although the exact underlying signalling mechanisms remain to be elucidated.
Publisher: Oxford University Press (OUP)
Date: 11-02-2022
Abstract: Is the composition of microRNAs (miRNAs) in uterine fluid (UF) of women with recurrent implantation failure (RIF) different from that of healthy fertile women? The composition of miRNAs in UF of women with RIF is different from that of healthy fertile women and the dysregulated miRNAs are associated with impaired endometrial receptivity and embryo implantation. It has previously been demonstrated that the miRNAs secreted from endometrial cells into the UF contribute to the achievement of endometrial receptivity. Endometrial miRNAs are dysregulated in women with RIF. In this descriptive laboratory case–control study, miRNA abundancy was compared between UF collected during implantation phase from healthy fertile women (n = 17) and women with RIF (n = 34), which was defined as three failed IVF cycles with high-quality embryos. Recruitment of study subjects and s ling of UF were performed at two university clinics in Stockholm, Sweden and Tartu, Estonia. The study participants monitored their menstrual cycles using an LH test kit. The UF s les were collected on Day LH + 7–9 by flushing with saline. S les were processed for small RNA sequencing and mapped for miRNAs. The differential abundance of miRNAs in UF was compared between the two groups using differential expression analysis (DESeq2). Further downstream analyses, including miRNA target gene prediction (miRTarBase), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis (g:Profiler) and external validation using relevant published data, were performed on the dysregulated miRNAs. Two miRNAs were technically validated with quantitative real-time PCR (RT-PCR). After processing of the sequencing data, there were 15 s les in the healthy fertile group and 33 s les in the RIF group. We found 61 differentially abundant UF miRNAs (34 upregulated and 27 downregulated) in RIF compared to healthy women with a false discovery rate of & .05 and a fold change (FC) of ≤−2 or ≥2. When analyzed with published literature, we found that several of the differentially abundant miRNAs are expressed in endometrial epithelial cells and have been reported in endometrial extracellular vesicles and in association with endometrial receptivity and RIF. Their predicted target genes were further expressed both in the trophectodermal cells of blastocyst-stage embryos and endometrial mid-secretory epithelial cells, as assessed by publicly available single-cell transcriptome-sequencing studies. Pathway analysis further revealed that 25 pathways, having key roles in endometrial receptivity and implantation, were significantly enriched. Hsa-miR-486-5p (FC −20.32 P-value = 0.004) and hsa-miR-92b-3p (FC −9.72 P-value = 0.004) were successfully technically validated with RT-PCR. The data are available in Gene Expression Omnibus (GEO) at eo/ with GEO accession number: GSE173289. This is a descriptive study with a limited number of study participants. Moreover, the identified differentially abundant miRNAs should be validated in a larger study cohort, and the predicted miRNA target genes and enriched pathways in RIF need to be confirmed and further explored in vitro. RIF is a major challenge in the current IVF setting with no diagnostic markers nor effective treatment options at hand. For the first time, total miRNAs have been extensively mapped in receptive phase UF of both healthy women with proven fertility and women diagnosed with RIF. Our observations shed further light on the molecular mechanisms behind RIF, with possible implications in future biomarker and clinical treatment studies. This work was financially supported by the Swedish Research Council (2017-00932), a joint grant from Region Stockholm and Karolinska Institutet (ALF Medicine 2020, FoUI-954072), Estonian Research Council (PRG1076), Horizon 2020 innovation (ERIN, EU952516) and European Commission and Enterprise Estonia (EU48695). The authors have no competing interests to declare for the current study.
Publisher: Cold Spring Harbor Laboratory
Date: 25-10-2019
DOI: 10.1101/817924
Abstract: Large-scale sequencing of RNAs from in idual cells can reveal patterns of gene, isoform and allelic expression across cell types and states 1 . However, current single-cell RNA-sequencing (scRNA-seq) methods have limited ability to count RNAs at allele- and isoform resolution, and long-read sequencing techniques lack the depth required for large-scale applications across cells 2,3 . Here, we introduce Smart-seq3 that combines full-length transcriptome coverage with a 5’ unique molecular identifier (UMI) RNA counting strategy that enabled in silico reconstruction of thousands of RNA molecules per cell. Importantly, a large portion of counted and reconstructed RNA molecules could be directly assigned to specific isoforms and allelic origin, and we identified significant transcript isoform regulation in mouse strains and human cell types. Moreover, Smart-seq3 showed a dramatic increase in sensitivity and typically detected thousands more genes per cell than Smart-seq2. Altogether, we developed a short-read sequencing strategy for single-cell RNA counting at isoform and allele-resolution applicable to large-scale characterization of cell types and states across tissues and organisms.
Publisher: American Society of Hematology
Date: 16-03-2023
Abstract: Myelodysplastic neoplasms (MDSs) and chronic myelomonocytic leukemia (CMML) are clonal disorders driven by progressively acquired somatic mutations in hematopoietic stem cells (HSCs). Hypomethylating agents (HMAs) can modify the clinical course of MDS and CMML. Clinical improvement does not require eradication of mutated cells and may be related to improved differentiation capacity of mutated HSCs. However, in patients with established disease it is unclear whether (1) HSCs with multiple mutations progress through differentiation with comparable frequency to their less mutated counterparts or (2) improvements in peripheral blood counts following HMA therapy are driven by residual wild-type HSCs or by clones with particular combinations of mutations. To address these questions, the somatic mutations of in idual stem cells, progenitors (common myeloid progenitors, granulocyte monocyte progenitors, and megakaryocyte erythroid progenitors), and matched circulating hematopoietic cells (monocytes, neutrophils, and naïve B cells) in MDS and CMML were characterized via high-throughput single-cell genotyping, followed by bulk analysis in immature and mature cells before and after AZA treatment. The mutational burden was similar throughout differentiation, with even the most mutated stem and progenitor clones maintaining their capacity to differentiate to mature cell types in vivo. Increased contributions from productive mutant progenitors appear to underlie improved hematopoiesis in MDS following HMA therapy.
Publisher: Cold Spring Harbor Laboratory
Date: 16-05-2020
DOI: 10.1101/2020.05.15.097519
Abstract: Multiple sclerosis (MS), a chronic inflammatory disease of the central nervous system (CNS), is associated with dysregulation of microRNAs (miRNA). We here analyzed all classes of small non-coding RNAs (sncRNAs) in matching peripheral blood mononuclear cells (PBMCs), plasma, cerebrospinal fluid (CSF) cells and cell-free CSF from relapsing-remitting (RRMS, n=12 in relapse, n=11 in remission), secondary progressive (SPMS, n=6) MS patients and non-inflammatory and inflammatory neurological disease controls (NINDC, n=11 INDC, n=5). We show widespread changes in small nuclear, nucleolar, transfer RNAs and miRNAs. In CSF cells, 133/133 and 115/117 differentially expressed sncRNAs are increased in RRMS relapse compared to remission and RRMS compared to NINDC, respectively. In contrast, 65/67 differentially expressed PBMC sncRNAs are decreased in RRMS compared to NINDC. The striking contrast between periphery and CNS suggests that sncRNA-mediated mechanisms, including alternative splicing, RNA degradation and mRNA translation, regulate the transcriptome of pathogenic cells primarily in the target organ.
Publisher: Springer Science and Business Media LLC
Date: 2014
Abstract: Emerging methods for the accurate quantification of gene expression in in idual cells hold promise for revealing the extent, function and origins of cell-to-cell variability. Different high-throughput methods for single-cell RNA-seq have been introduced that vary in coverage, sensitivity and multiplexing ability. We recently introduced Smart-seq for transcriptome analysis from single cells, and we subsequently optimized the method for improved sensitivity, accuracy and full-length coverage across transcripts. Here we present a detailed protocol for Smart-seq2 that allows the generation of full-length cDNA and sequencing libraries by using standard reagents. The entire protocol takes ∼2 d from cell picking to having a final library ready for sequencing sequencing will require an additional 1-3 d depending on the strategy and sequencer. The current limitations are the lack of strand specificity and the inability to detect nonpolyadenylated (polyA(-)) RNA.
Publisher: Springer Science and Business Media LLC
Date: 24-09-2018
DOI: 10.1038/S41596-018-0049-Y
Abstract: Small RNAs participate in several cellular processes, including splicing, RNA modification, mRNA degradation, and translational arrest. Traditional methods for sequencing small RNAs require a large amount of cell material, limiting the possibilities for single-cell analyses. We describe Small-seq, a ligation-based method that enables the capture, sequencing, and molecular counting of small RNAs from in idual mammalian cells. Here, we provide a detailed protocol for this approach that relies on standard reagents and instruments. The standard protocol captures a complex set of small RNAs, including microRNAs (miRNAs), fragments of tRNAs and small nucleolar RNAs (snoRNAs) however, miRNAs can be enriched through the addition of a size-selection step. Ready-to-sequence libraries can be generated in 2-3 d, starting from cell collection, with additional days needed to computationally map the sequence reads and calculate molecular counts.
Publisher: Oxford University Press (OUP)
Date: 12-04-2011
DOI: 10.1093/NAR/GKR214
Publisher: Wiley
Date: 30-09-2014
DOI: 10.1111/FEBS.13040
Abstract: Post-translational modification by the small ubiquitin-like modifier (SUMO) regulates the cellular response to different types of stress and plays a pivotal role in the control of oncogenic viral infections. Here we investigated the capacity of microRNAs (miRNAs) encoded by Epstein-Barr virus to interfere with the SUMO signaling network. Using a computational strategy that scores different properties of miRNA-mRNA target pairs, we identified a minimal set of 575 members of the SUMO interactome that may be targeted by one or more Epstein-Barr virus miRNAs. A significant proportion of the candidates cluster in a functional network that controls chromatin organization, stress, DNA damage and immune responses, apoptosis and transforming growth factor beta signaling. Multiple components of the transforming growth factor beta signaling pathway were inhibited upon upregulation of the BamHI-H rightward open reading frame 1 (BHRF1) encoded miRNAs in cells transduced with recombinant lentiviruses or entering the productive virus cycle. These findings point to the capacity of viral miRNAs to interfere with SUMO-regulated cellular functions that control key aspects of viral replication and pathogenesis.
Publisher: Springer Science and Business Media LLC
Date: 22-07-2012
DOI: 10.1038/NBT.2282
Publisher: Springer Science and Business Media LLC
Date: 04-05-2020
Publisher: Proceedings of the National Academy of Sciences
Date: 20-04-2021
Abstract: Dysregulation of microRNAs (miRNAs), a type of small noncoding RNAs (sncRNAs), has frequently been associated with multiple sclerosis (MS). However, most studies have focused on peripheral blood, and few investigated other classes of sncRNAs. To address this, we analyzed all classes of sncRNAs in matching peripheral blood mononuclear cells, plasma, cerebrospinal fluid (CSF) cells, and cell-free CSF from MS patients and controls. We demonstrate widespread alterations of small nuclear (snRNA)–derived RNAs, small nucleolar-derived RNAs (sdRNAs), transfer RNA–derived fragments, and miRNAs, particularly in CSF cells. The striking contrast between the periphery and central nervous system and between relapse and remission phases of disease highlights the importance of sncRNA-mediated mechanisms in MS, in particular alternative splicing and mRNA translation.
Publisher: Oxford University Press (OUP)
Date: 25-10-2006
DOI: 10.1093/NAR/GKL750
Publisher: Oxford University Press (OUP)
Date: 15-07-2008
DOI: 10.1093/NAR/GKN445
Abstract: Double-stranded RNA (dsRNA) is formed in cells as intra- and intermolecular RNA interactions and is involved in a range of biological processes including RNA metabolism, RNA interference and translation control mediated by natural antisense RNA and microRNA. Despite this breadth of activities, few molecular tools are available to analyse dsRNA as native hybrids. We describe a two-step ligation method for enzymatic joining of dsRNA adaptors to any dsRNA molecule in its duplex form without a need for prior sequence or termini information. The method is specific for dsRNA and can ligate various adaptors to label, map or lify dsRNA sequences. When combined with reverse transcription–polymerase chain reaction, the method is sensitive and can detect low nanomolar concentrations of dsRNA in total RNA. As ex les, we mapped dsRNA/single-stranded RNA junctions within Escherichia coli hok mRNA and the human immunodeficiency virus TAR element using RNA from bacteria and mammalian cells.
Publisher: Springer Science and Business Media LLC
Date: 2019
Publisher: Springer Science and Business Media LLC
Date: 28-02-2010
DOI: 10.1038/NCB2035
Abstract: The large tegument proteins of herpesviruses encode conserved cysteine proteases of unknown function. Here we show that BPLF1, the Epstein-Barr-virus-encoded member of this protease family, is a deneddylase that regulates virus production by modulating the activity of cullin-RING ligases (CRLs). BPLF1 hydrolyses NEDD8 conjugates in vitro, acts as a deneddylase in vivo, binds to cullins and stabilizes CRL substrates. Expression of BPLF1 alone or in the context of the productive virus cycle induces accumulation of the licensing factor CDT1 and deregulates S-phase DNA synthesis. Inhibition of BPLF1 during the productive virus cycle prevents cellular DNA re-replication and inhibits virus replication. Viral DNA synthesis is restored by overexpression of CDT1. Homologues encoded by other herpesviruses share the deneddylase activity. Thus, these enzymes are likely to have a key function in the virus life cycle by inducing a replication-permissive S-phase-like cellular environment.
Publisher: Cold Spring Harbor Laboratory
Date: 22-01-2022
DOI: 10.1101/2022.01.20.476890
Abstract: Progressively acquired somatic mutations in hematopoietic stem cells are central to pathogenesis in myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemia (CMML). They can lead to proliferative advantages, impaired differentiation and progressive cytopenias. MDS or CMML patients with high-risk disease are treated with hypomethylating agents including 5-azacytidine (AZA). Clinical improvement does not require eradication of mutated cells and may be related to improved differentiation capacity of mutated hematopoietic stem and progenitor cells (HSPCs). However, the contribution of mutated HSPCs to steadystate hematopoiesis in MDS and CMML is unclear. To address this, we characterised the somatic mutations of in idual stem, progenitor (common myeloid progenitor, granulocyte monocyte progenitor, megakaryocyte erythroid progenitor), and matched circulating (monocyte, neutrophil, naïve B cell) haematopoietic cells in treatment naïve and AZA-treated MDS and CMML via high-throughput single cell genotyping. The mutational burden was similar across multiple hematopoietic cell types, and even the most mutated stem and progenitor clones maintained their capacity to differentiate to mature myeloid and, in some cases, lymphoid cell types in vivo. Our data show that even highly mutated HSPCs contribute significantly to circulating blood cells in MDS and CMML, prior to and following AZA treatment. * Highly mutated HSPCs contribute significantly to circulating blood cells in MDS and CMML, prior to and following AZA treatment. * The mutational burden in matched bone marrow and peripheral blood cells in MDS and CMML was similar throughout myelopoiesis.
Publisher: Elsevier BV
Date: 08-2007
Abstract: Antisense agents that target growth-essential genes display surprisingly potent bactericidal properties. In particular, peptide nucleic acid (PNA) and phosphorodiamidate morpholino oligomers linked to cationic carrier peptides are effective in time kill assays and as inhibitors of bacterial peritonitis in mice. It is unclear how these relatively large antimicrobials overcome stringent bacterial barriers and mediate killing. Here we determined the transit kinetics of peptide-PNAs and observed an accumulation of cell-associated PNA in Escherichia coli and slow efflux. An inhibitor of drug efflux pumps did not alter peptide-PNA potency, indicating a lack of active efflux from cells. Consistent with cell retention, the post-antibiotic effect (PAE) of the anti-acyl carrier protein (acpP) peptide-PNA was greater than 11 hours. Bacterial cell accumulation and a long PAE are properties of significant interest for antimicrobial development.
Publisher: Springer Science and Business Media LLC
Date: 05-2015
DOI: 10.1038/NBT.3219
Publisher: Oxford University Press (OUP)
Date: 18-08-2016
Start Date: 2012
End Date: 2015
Funder: Swedish Research Council
View Funded ActivityStart Date: 2021
End Date: 2025
Funder: Vetenskapsrådet
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