ORCID Profile
0000-0001-6184-0925
Current Organisations
Telethon Kids Institute
,
Australian National University
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Molecular Evolution | Conservation and Biodiversity | Evolutionary Biology | Biological Adaptation
Ecosystem Adaptation to Climate Change | Expanding Knowledge in the Biological Sciences |
Publisher: Cold Spring Harbor Laboratory
Date: 18-06-2021
DOI: 10.1101/2021.06.17.448908
Abstract: Progesterone receptor (PGR) plays erse roles in reproductive tissues and thus coordinates mammalian fertility. In the ovary, acutely induced PGR is the key determinant of ovulation through transcriptional control of a unique set of genes that culminates in follicle rupture. However, the molecular mechanisms for PGR’s specialised function in ovulation is poorly understood. To address this, we assembled a detailed genomic profile of PGR action through combined ATAC-seq, RNA-seq and ChIP-seq analysis in wildtype and isoform-specific PGR null mice. We demonstrated the unique action of PGR-A isoform in the ovary through a transcriptional complex involving physical interaction with RUNX and JUN/FOS transcription factors. The assembly of this unique complex directs targeting of PGR binding to proximal promoter regions and enables chromatin accessibility, leading to ovulatory gene induction. This PGR signalling mechanism is specific to ovulation and provides potential targets for infertility treatments as well as new contraceptives that block ovulation.
Publisher: Springer US
Date: 2022
DOI: 10.1007/978-1-0716-2429-6_15
Abstract: Plant DNA preserved in ancient specimens has recently gained importance as a tool in comparative genomics, allowing the investigation of evolutionary processes in plant genomes through time. However, recovering the genomic information contained in such specimens is challenging owing to the presence of secondary substances that limit DNA retrieval. In this chapter, we provide a DNA extraction protocol optimized for the recovery of DNA from degraded plant materials. The protocol is based on a commercially available DNA extraction kit that does not require handling of hazardous reagents.
Publisher: Springer Science and Business Media LLC
Date: 12-2014
Publisher: Online Ancient Genome Repository
Date: 2016
Publisher: Elsevier BV
Date: 08-2019
DOI: 10.1016/J.BBR.2019.111917
Abstract: Environmental enrichment (EE) has been shown to modulate behavior and hippoc al gene expression however, the currently available literature does not explain the differential effects that may relate to the duration of EE. To investigate the differential effects of short- and long-term EE on locomotion, anxiety-, depressive- and cognition-like behaviors, and hippoc al gene expression under physiological conditions. We assigned either short-term or long-term intervention with respective controls to healthy C57BL/6 mice (n = 12-16/group). The short-term EE group received EE for four weeks starting at eight months of age, while the long-term EE group received EE for six months starting at three months of age. Differential effects of the duration of EE on various behaviors and hippoc al gene expression at nine months of age were measured using an established behavioral battery and high-throughput RT-qPCR, respectively. Both short-term and long-term EE significantly enhanced locomotion in the home cage and reduced depressive-like behavior in the forced-swim test. Long-term EE, however, reduced locomotion in the open-field test. Additionally, short-term EE reduced the mean body weight and showed anxiolytic effects in the elevated-zero maze (EZM), while these effects were lost after long-term EE. There were no effects of either short-term or long-term EE on the expression of 43 hippoc al genes of interest tested at adjusted p < 0.05. Both short and long-term EE are equally beneficial for baseline locomotor activity and depressive-like behavior. However, long-term EE affects locomotion adversely in a threatening environment and is anxiogenic.
Publisher: Wiley
Date: 29-04-2019
Publisher: CSIRO Publishing
Date: 2007
DOI: 10.1071/AR06155
Abstract: Genome sequencing and the associated bioinformatics is now a widely accepted research tool for accelerating genetic research and the analysis of genome structure and function of wheat because it leverages similar work from other crops and plants. The International Wheat Genome Sequencing Consortium addresses the challenge of wheat genome structure and function and builds on the research efforts of Professor Bob McIntosh in the genetics of wheat. Currently, expressed sequence tags (ESTs ~500 000 to date) are the largest sequence resource for wheat genome analyses. It is estimated that the gene coverage of the wheat EST collection is ~60%, close to that of Arabidopsis, indicating that ~40% of wheat genes are not represented in EST collections. The physical map of the D-genome donor species Aegilops tauschii is under construction (wheat.pw.usda.gov/PhysicalMapping). The technologies developed in this analysis of the D genome provide a good model for the approach to the entire wheat genome, namely compiling BAC contigs, assigning these BAC contigs to addresses in a high resolution genetic map, filling in gaps to obtain the entire physical length of a chromosome, and then large-scale sequencing.
Publisher: Oxford University Press (OUP)
Date: 06-2010
Abstract: To improve our understanding of the organization and evolution of the wheat (Triticum aestivum) genome, we sequenced and annotated 13-Mb contigs (18.2 Mb) originating from different regions of its largest chromosome, 3B (1 Gb), and produced a 2x chromosome survey by shotgun Illumina/Solexa sequencing. All regions carried genes irrespective of their chromosomal location. However, gene distribution was not random, with 75% of them clustered into small islands containing three genes on average. A twofold increase of gene density was observed toward the telomeres likely due to high tandem and interchromosomal duplication events. A total of 3222 transposable elements were identified, including 800 new families. Most of them are complete but showed a highly nested structure spread over distances as large as 200 kb. A succession of lification waves involving different transposable element families led to contrasted sequence compositions between the proximal and distal regions. Finally, with an estimate of 50,000 genes per diploid genome, our data suggest that wheat may have a higher gene number than other cereals. Indeed, comparisons with rice (Oryza sativa) and Brachypodium revealed that a high number of additional noncollinear genes are interspersed within a highly conserved ancestral grass gene backbone, supporting the idea of an accelerated evolution in the Triticeae lineages.
Publisher: Research Square Platform LLC
Date: 28-12-2021
DOI: 10.21203/RS.3.RS-1175572/V1
Abstract: BackgroundGenome-wide association studies (GWAS) have enabled the discovery of single nucleotide polymorphisms (SNPs) that are significantly associated with many autoimmune diseases including type 1 diabetes (T1D). However, many of the identified variants lie in non-coding regions, limiting the identification of mechanisms that contribute to autoimmune disease progression. To address this problem, we developed a variant filtering workflow called 3DFAACTS-SNP to link genetic variants to target genes in a cell specific manner. Here we use 3DFAACTS-SNP to identify candidate SNPs and target genes associated with the loss of immune tolerance in regulatory T cells (Treg) in T1D. ResultsUsing 3DFAACTS-SNP we identified from a list of 1,228 previously fine-mapped variants, 36 SNPs with plausible Treg-specific mechanisms of action. The integration of cell-type specific chromosome conformation capture data in 3DFAACTS-SNP, identified 119 regulatory regions and 51 candidate target genes that interact with these variant-containing regions in Treg cells. We further demonstrated the utility of the workflow by applying it to three other SNP autoimmune datasets, identifying 17 Treg-centric candidate variants and 35 interacting genes. Finally, we demonstrate the broad utility of 3DFAACTS-SNP for functional annotation of all known common ( % allele frequency) variants from the Genome Aggregation Database (gnomAD). We identified 7,900 candidate variants and 3,245 candidate target genes, generating a list of potential sites for future T1D or autoimmune research. ConclusionsWe demonstrate that it is possible to further prioritise variants that contribute to T1D based on regulatory function and illustrate the power of using cell type specific multi-omics datasets to determine disease mechanisms. Our workflow can be customised to any cell type for which the in idual datasets for functional annotation have been generated, giving broad applicability and utility.
Publisher: Cold Spring Harbor Laboratory
Date: 07-08-2020
DOI: 10.1101/2020.08.06.240754
Abstract: The analysis and interpretation of datasets generated through sequencing large numbers of in idual genomes is becoming commonplace in population and evolutionary genetic studies. Here we introduce geaR, a modular R package for evolutionary analysis of genome-wide genotype data. The package leverages the Genomic Data Structure (GDS) format, which enables memory and time efficient querying of genotype datasets compared to standard VCF genotype files. geaR utilizes GRange object classes to partition an analysis based on features from GFF annotation files, select codons based on position or degeneracy, and construct both positional and coordinate genomic windows. Tests of genetic ersity (eg. d XY , π, F ST ) and admixture along with tree building and sequence output, can be carried out on partitions using a single function regardless of s le ploidy or number of observed alleles. The package and associated documentation are available on GitHub at github.com/CMWbio/geaR .
Publisher: Hindawi Limited
Date: 11-05-2021
DOI: 10.1002/HUMU.24214
Abstract: Gelsolin (GSN) variants have been implicated in amyloidosis of the Finnish type. This case series reports a novel GSN:c.1477T>C,p.(Trp493Arg) variant in a family with ocular and systemic features consistent with Finnish Amyloidosis. Exome sequencing performed on affected in iduals from two families manifesting cutis laxa and polymorphic corneal stromal opacities demonstrated the classic GSN:c.654G>A,p.Asp214Asn variant in single affected in idual from one family, and a previously undocumented GSN:c.1477T>C variant in three affected first-degree relatives from a separate family. Immunohistochemical studies on corneal tissue from a proband with the c.1477T>C variant identified gelsolin protein within histologically defined corneal amyloid deposits. This study reports a novel association between the predicted pathogenic GSN:c.1477T>C variant and amyloidosis of the Finnish type, and is the first to provide functional evidence of a pathological GSN variant at a locus distant to the critical G2 calcium-binding region, resulting in the phenotype of amyloidosis of the Finnish type.
Publisher: Wiley
Date: 15-02-2019
DOI: 10.1111/MEC.15022
Abstract: Dermal phototaxis has been reported in a few aquatic vertebrate lineages spanning fish, hibians and reptiles. These taxa respond to light on the skin of their elongate hind-bodies and tails by withdrawing under cover to avoid detection by predators. Here, we investigated tail phototaxis in sea snakes (Hydrophiinae), the only reptiles reported to exhibit this sensory behaviour. We conducted behavioural tests in 17 wild-caught sea snakes of eight species by illuminating the dorsal surface of the tail and midbody skin using cold white, violet, blue, green and red light. Our results confirmed phototactic tail withdrawal in the previously studied Aipysurus laevis, revealed this trait for the first time in A. duboisii and A. tenuis, and suggested that tail photoreceptors have peak spectral sensitivities between blue and green light (457-514 nm). Based on these results, and an absence of photoresponses in five Aipysurus and Hydrophis species, we tentatively infer that tail phototaxis evolved in the ancestor of a clade of six Aipysurus species (comprising 10% of all sea snakes). Quantifying tail damage, we found that the probability of sustaining tail injuries was not influenced by tail phototactic ability in snakes. Gene profiling showed that transcriptomes of both tail skin and body skin lacked visual opsins but contained melanopsin (opn4x) in addition to key genes of the retinal regeneration and phototransduction cascades. This work suggests that a nonvisual photoreceptor (e.g., Gq rhabdomeric) signalling pathway underlies tail phototaxis, and provides candidate gene targets for future studies of this unusual sensory innovation in reptiles.
Publisher: Association for Research in Vision and Ophthalmology (ARVO)
Date: 29-03-2022
DOI: 10.1167/IOVS.63.3.26
Publisher: Research Square Platform LLC
Date: 10-08-2022
DOI: 10.21203/RS.3.RS-1862932/V1
Abstract: The epigenome and transcriptome constitute a critical element of a tightly regulated, cell-type specific gene expression program, and subtle perturbations in the regulation of this program can result in pathology. Epigenetic features such as DNA accessibility dictate transcriptional regulation in a cell type- and cell state- specific manner, and mapping this in health vs. disease in clinically relevant material is opening the door to new mechanistic insights and new targets for therapy. Assay for Transposase Accessible Chromatin Sequencing (ATAC-seq) allows profiling of chromatin accessibility with low cell input, making it amenable to the clinical setting, such as peripheral blood from clinical trials, and this makes it applicable to rare cell populations, such as regulatory T (Treg) cells. However, there is little known about the compatibility of the assay on materials recovered from cryopreserved rare cell populations. In the context of tolerance or autoimmunity, regulatory T cells play a critical role in maintaining immune homeostasis, and loss of numbers or function is linked to many diseases, making them a clinically relevant population to analyse using genomic platforms. Here we demonstrate the robustness and reproducibility of an ATAC-seq protocol comparing fresh or cryopreserved primary Treg cells, and comparing their profile in the steady state and in response to stimulation. We extend this method to explore the feasibility of conducting simultaneous quantitation of chromatin accessibility and transcriptome from a single aliquot of 50,000 Treg cells from cryopreserved PBMCs. Profiling of chromatin accessibility and gene expression in parallel within the same pool of cells controls for cellular heterogeneity and will be particularly beneficial for experiments constrained by limited input material, such as biobanked PBMC from clinical trials. This approach will be complementary to single-cell experiments as libraries used to profile chromatin accessibility and transcriptome are derived from the same population of cells, controlling for stochastic gene fluctuation in different cells in a population at any given time. Overall, we observed a high correlation of accessibility patterns and transcription factor (TF) dynamics between fresh Treg cells and cells recovered from cryopreservation s les. The distribution of fragment size, enrichment of transcription start sites (TSS) and genomic features of thawed Treg cells recapitulate that of the fresh cells. Furthermore, highly consistent global chromatin and transcriptional changes in response to stimulation were observed in both fresh and frozen s les. Lastly, highly similar transcriptomic profiles were obtained from whole cells and from the supernatants recovered from ATAC-seq reactions. This report highlights the feasibility of applying these techniques to profile the epigenomic landscape of cells recovered from cryopreservation biorepositories. Implementation of this approach is suitable in biorepositories and will contribute to advances in the field of translational research and personalized medicine.
Publisher: Springer Science and Business Media LLC
Date: 16-08-2023
DOI: 10.1038/S41586-023-06424-7
Abstract: Cells undergo a major epigenome reconfiguration when reprogrammed to human induced pluripotent stem cells (hiPS cells). However, the epigenomes of hiPS cells and human embryonic stem (hES) cells differ significantly, which affects hiPS cell function 1–8 . These differences include epigenetic memory and aberrations that emerge during reprogramming, for which the mechanisms remain unknown. Here we characterized the persistence and emergence of these epigenetic differences by performing genome-wide DNA methylation profiling throughout primed and naive reprogramming of human somatic cells to hiPS cells. We found that reprogramming-induced epigenetic aberrations emerge midway through primed reprogramming, whereas DNA demethylation begins early in naive reprogramming. Using this knowledge, we developed a transient-naive-treatment (TNT) reprogramming strategy that emulates the embryonic epigenetic reset. We show that the epigenetic memory in hiPS cells is concentrated in cell of origin-dependent repressive chromatin marked by H3K9me3, lamin-B1 and aberrant CpH methylation. TNT reprogramming reconfigures these domains to a hES cell-like state and does not disrupt genomic imprinting. Using an isogenic system, we demonstrate that TNT reprogramming can correct the transposable element overexpression and differential gene expression seen in conventional hiPS cells, and that TNT-reprogrammed hiPS and hES cells show similar differentiation efficiencies. Moreover, TNT reprogramming enhances the differentiation of hiPS cells derived from multiple cell types. Thus, TNT reprogramming corrects epigenetic memory and aberrations, producing hiPS cells that are molecularly and functionally more similar to hES cells than conventional hiPS cells. We foresee TNT reprogramming becoming a new standard for biomedical and therapeutic applications and providing a novel system for studying epigenetic memory.
Publisher: Cold Spring Harbor Laboratory
Date: 04-04-2022
DOI: 10.1101/2022.03.27.22273016
Abstract: The abundance of cell-free microRNA (miRNA) has been measured in many body fluids, including blood plasma, which has been proposed as a source with novel, minimally invasive biomarker potential for several diseases. Despite improvements in quantification methods for plasma miRNAs, there is no consensus on optimal reference miRNAs or to what extent haemolysis may affect plasma miRNA content. Here we propose a new method for the detection of haemolysis in miRNA high-throughput sequencing (HTS) data from libraries prepared using human plasma. To establish a miRNA haemolysis signature in plasma we first identified differentially expressed miRNAs between s les with known haemolysis status and selected miRNA with statistically significant higher abundance in our haemolysed group. Given there may be both technical and biological reasons for differential abundance of signature miRNA, and to ensure the method developed here was relevant outside of our specific context, that is women of reproductive age, we tested for significant differences between pregnant and non-pregnant groups. Here we report a novel 20 miRNA signature (miR-106b-3p, miR-140-3p, miR-142-5p, miR-532-5p, miR-17-5p, miR-19b-3p, miR-30c-5p, miR-324-5p, miR-192-5p, miR-660-5p, miR-186-5p, miR-425-5p, miR-25-3p, miR-363-3p, miR-183-5p, miR-451a, miR-182-5p, miR-191-5p, miR-194-5p, miR-20b-5p) that can be used to identify the presence of haemolysis, in silico , in high throughput miRNA sequencing data. Given the potential for haemolysis contamination, we recommend that assay for haemolysis detection become standard pre-analytical practice and provide here a simple method for haemolysis detection.
Publisher: Elsevier BV
Date: 04-2020
DOI: 10.1016/J.BBR.2020.112501
Abstract: The known effects of aging on the brain and behavior include impaired cognition, increases in anxiety and depressive-like behaviors, and reduced locomotor activity. Environmental exposures and interventions also influence brain functions during aging. We investigated the effects of normal aging under controlled environmental conditions and in the absence of external interventions on locomotor activity, cognition, anxiety and depressive-like behaviors, immune function and hippoc al gene expression in C57BL/6 mice. Healthy mice at 4, 9, and 14 months of age underwent behavioral testing using an established behavioral battery, followed by cellular and molecular analysis using flow cytometry, immunohistochemistry, and quantitative PCR. We found that 14-month-old mice showed significantly reduced baseline locomotion, increased anxiety, and impaired spatial memory compared to younger counterparts. However, no significant differences were observed for depressive-like behavior in the forced-swim test. Microglia numbers in the dentate gyrus, as well as CD8+ memory T cells increased towards late middle age. Aging processes exerted a significant effect on the expression of 43 genes of interest in the hippoc us. We conclude that aging is associated with specific changes in locomotor activity, cognition, anxiety-like behaviors, neuroimmune responses and hippoc al gene expression.
Publisher: Elsevier BV
Date: 12-2019
Publisher: MDPI AG
Date: 09-02-2023
Abstract: The search for novel microRNA (miRNA) biomarkers in plasma is h ered by haemolysis, the lysis and subsequent release of red blood cell contents, including miRNAs, into surrounding fluid. The biomarker potential of miRNAs comes in part from their multicompartment origin and the long-lived nature of miRNA transcripts in plasma, giving researchers a functional window for tissues that are otherwise difficult or disadvantageous to s le. The inclusion of red-blood-cell-derived miRNA transcripts in downstream analysis introduces a source of error that is difficult to identify posthoc and may lead to spurious results. Where access to a physical specimen is not possible, our tool will provide an in silico approach to haemolysis prediction. We present DraculR, an interactive Shiny/R application that enables a user to upload miRNA expression data from a short-read sequencing of human plasma as a raw read counts table and interactively calculate a metric that indicates the degree of haemolysis contamination. The code, DraculR web tool and its tutorial are freely available as detailed herein.
Publisher: Springer Science and Business Media LLC
Date: 02-08-2011
DOI: 10.1007/S10142-011-0240-5
Abstract: Powdery mildew of wheat (Triticum aestivum L.) is caused by the ascomycete fungus Blumeria graminis f.sp. tritici. Genomic approaches open new ways to study the biology of this obligate biotrophic pathogen. We started the analysis of the Bg tritici genome with the low-pass sequencing of its genome using the 454 technology and the construction of the first genomic bacterial artificial chromosome (BAC) library for this fungus. High-coverage contigs were assembled with the 454 reads. They allowed the characterization of 56 transposable elements and the establishment of the Blumeria repeat database. The BAC library contains 12,288 clones with an average insert size of 115 kb, which represents a maximum of 7.5-fold genome coverage. Sequencing of the BAC ends generated 12.6 Mb of random sequence representative of the genome. Analysis of BAC-end sequences revealed a massive invasion of transposable elements accounting for at least 85% of the genome. This explains the unusually large size of this genome which we estimate to be at least 174 Mb, based on a large-scale physical map constructed through the fingerprinting of the BAC library. Our study represents a crucial step in the perspective of the determination and study of the whole Bg tritici genome sequence.
Publisher: Elsevier BV
Date: 04-2016
Publisher: Cold Spring Harbor Laboratory
Date: 19-02-2022
DOI: 10.1101/2022.02.17.22270919
Abstract: Acute lymphoblastic leukemia (ALL) is the most common childhood cancer and comprises multiple genetically distinguishable subtypes. To detect subtypes, current pipelines include fusion calling, polymorphisms, candidate gene copy numbers and cytogenetics but these approaches have limitations. RNA-seq provides a functional genome-wide snapshot that enables classification of ALL subtypes, however, typical mRNA-seq clustering analyses lack the rigor of quantitative modelling. Furthermore, high-dimensional gene expression data across cohorts and countries contain biases that previous transcriptomics studies have not addressed. Our aim was to integrate easy-to-interpret reliable transcriptome-wide biomarkers into subtyping pipelines. We analyzed 2,046 s les from two continents, carefully adjusted for biases and applied a rigorous machine learning design with independent replication. Six ALL subtypes that covered 32% of patients were robustly detected by mRNA-seq (PPV ≥ 87%). Five other frequent subtypes were distinguishable in 40% of patients, although overlapping transcriptional profiles led to lower accuracy (52% ≤ PPV ≤ 73%). Based on these findings, we developed the Allspice tool that predicts ALL subtypes and driver genes from unadjusted mRNA-seq read counts as encountered in real-world settings. Allspice also includes quantitative classification and safety metrics to help determine the most plausible genetic drivers for cases where other findings are inconclusive.
Publisher: Cold Spring Harbor Laboratory
Date: 31-03-2022
DOI: 10.1101/2022.03.27.22273019
Abstract: The search for novel microRNA (miRNA) biomarkers in plasma is h ered by haemolysis, the lysis and subsequent release of red blood cell (RBC) contents, including miRNAs, into surrounding fluid. The biomarker potential of miRNAs comes in part from their multi-compartment origin, and the long-lived nature of miRNA transcripts in plasma, giving researchers a functional window for tissues that are otherwise difficult or disadvantageous to s le. The inclusion of RBC derived miRNA transcripts in downstream analysis introduces a source of error that is difficult to identify post hoc and may lead to spurious results. Where access to a physical specimen is not possible, our tool will provide an in silico approach to haemolysis prediction. We present DraculR, an interactive Shiny/R application that enables a user to upload microRNA expression data from short read sequencing of human plasma as a raw read counts table and interactively calculate a metric that indicates the degree of haemolysis contamination. DraculR and its tutorial are freely available from ( mxhp75.shinyapps.io/shinyV / ). Code is available from ( xhp75/shinyV .git ).
Publisher: Wiley
Date: 2018
DOI: 10.1002/CTI2.1011
Publisher: Cold Spring Harbor Laboratory
Date: 05-09-2020
DOI: 10.1101/2020.09.04.279554
Abstract: Genome-wide association and fine-mapping studies have enabled the discovery of single nucleotide polymorphisms (SNPs) and other variants that are significantly associated with many autoimmune diseases including type 1 diabetes (T1D). However, many of the SNPs lie in non-coding regions, limiting the identification of mechanisms that contribute to autoimmune disease progression. Autoimmunity results from a failure of immune tolerance, suggesting that regulatory T cells (Treg) are likely a significant point of impact for this genetic risk, as Treg are critical for immune tolerance. Focusing on T1D as a model of defective function of Treg in autoimmunity, we designed a SNPs filtering workflow called 3 Dimensional Functional Annotation of Accessible Cell Type Specific SNPs (3DFAACTS-SNP) that utilises overlapping profiles of Treg-specific epigenomic data (ATAC-seq, Hi-C and FOXP3-ChIP) to identify regulatory elements potentially driving the effect of variants associated with T1D, and the gene(s) that they control. Using 3DFAACTS-SNP we identified 36 SNPs with plausible Treg-specific mechanisms of action contributing to T1D from 1,228 T1D fine-mapped variants, identifying 119 novel interacting regions resulting in the identification of 51 candidate target genes. We further demonstrated the utility of the workflow by applying it to three other fine-mapped/meta-analysed SNP autoimmune datasets, identifying 17 Treg-centric candidate variants and 35 interacting genes. Finally, we demonstrate the broad utility of 3DFAACTS-SNP for functional annotation of any genetic variation using all common ( % allele frequency) variants from the Genome Aggregation Database (gnomAD). We identified 7,900 candidate variants and 3,245 candidate target genes, generating a list of potential sites for future T1D or autoimmune research. We demonstrate that it is possible to further prioritise variants that contribute to T1D based on regulatory function and illustrate the power of using cell type specific multi-omics datasets to determine disease mechanisms. The 3DFAACTS-SNP workflow can be customised to any cell type for which the in idual datasets for functional annotation have been generated, giving broad applicability and utility.
Publisher: Oxford University Press (OUP)
Date: 10-2018
Abstract: Haemophilus influenzae and Streptococcus pneumoniae are known aetiologic agents of chronic otitis media, frequently as a multispecies infection. In this study, we show that the outcome of H. influenzae/S. pneumoniae interactions is dependent on the nutrient source. In continuous culture containing chemically defined media with lactose, S. pneumoniae was non-viable in mono-culture, and in co-culture remained non-viable until 288 h. With glucose, S. pneumoniae became non-viable in mono-culture, but uniquely existed in 3 distinct states in co-culture: parental cells (until 24 h), a dormant state until 336 h and its re-emergence as a non-mucoidal, small colony variant (SCV). The S. pneumoniae SCV was stable and whole genome sequencing showed three major single nucleotide polymorphisms in the SCV cells-cap3A (capsule biosynthesis pathway), fpg (DNA glycosylase of the DNA repair mechanism) and glutamate-5-kinase. Previously, fpg mutants have shown increased mutator rates, permitting bacterial survival against host-generated stresses. Transcriptomics showed these SCV cells up-regulated sugar transporters and toxin/antitoxin systems. An animal model revealed a reduced survival in the lungs and ear by SCV cells. This is the first study documenting the effect of carbon source and the development of a distinct S. pneumoniae cell type during H. influenzae/S. pneumoniae interactions.
Publisher: Springer Science and Business Media LLC
Date: 30-06-2022
DOI: 10.1186/S13072-022-00456-5
Abstract: Genome-wide association studies (GWAS) have enabled the discovery of single nucleotide polymorphisms (SNPs) that are significantly associated with many autoimmune diseases including type 1 diabetes (T1D). However, many of the identified variants lie in non-coding regions, limiting the identification of mechanisms that contribute to autoimmune disease progression. To address this problem, we developed a variant filtering workflow called 3DFAACTS-SNP to link genetic variants to target genes in a cell-specific manner. Here, we use 3DFAACTS-SNP to identify candidate SNPs and target genes associated with the loss of immune tolerance in regulatory T cells (Treg) in T1D. Using 3DFAACTS-SNP, we identified from a list of 1228 previously fine-mapped variants, 36 SNPs with plausible Treg-specific mechanisms of action. The integration of cell type-specific chromosome conformation capture data in 3DFAACTS-SNP identified 266 regulatory regions and 47 candidate target genes that interact with these variant-containing regions in Treg cells. We further demonstrated the utility of the workflow by applying it to three other SNP autoimmune datasets, identifying 16 Treg-centric candidate variants and 60 interacting genes. Finally, we demonstrate the broad utility of 3DFAACTS-SNP for functional annotation of all known common ( 10% allele frequency) variants from the Genome Aggregation Database (gnomAD). We identified 9376 candidate variants and 4968 candidate target genes, generating a list of potential sites for future T1D or other autoimmune disease research. We demonstrate that it is possible to further prioritise variants that contribute to T1D based on regulatory function, and illustrate the power of using cell type-specific multi-omics datasets to determine disease mechanisms. Our workflow can be customised to any cell type for which the in idual datasets for functional annotation have been generated, giving broad applicability and utility.
Publisher: Wiley
Date: 29-03-2023
DOI: 10.1111/AGE.13322
Abstract: Horns, a form of headgear carried by Bovidae, have ethical and economic implications for ruminant production species such as cattle and goats. Hornless (polled) in iduals are preferred. In cattle, four genetic variants (Celtic, Friesian, Mongolian and Guarani) are associated with the polled phenotype, which are clustered in a 300‐kb region on chromosome 1. As the variants are intergenic, the functional effect is unknown. The aim of this study was to determine if the POLLED variants affect chromatin structure or disrupt enhancers using publicly available data. Topologically associating domains (TADs) were analyzed using Angus‐ and Brahman‐specific Hi‐C reads from lung tissue of an Angus (Celtic allele) cross Brahman (horned) fetus. Predicted bovine enhancers and chromatin immunoprecipitation sequencing peaks for histone modifications associated with enhancers (H3K27ac and H3K4me1) were mapped to the POLLED region. TADs analyzed from Angus‐ and Brahman‐specific Hi‐C reads were the same, therefore, the Celtic variant does not appear to affect this level of chromatin structure. The Celtic variant is located in a different TAD from the Friesian, Mongolian, and Guarani variants. Predicted enhancers and histone modifications overlapped with the Guarani and Friesian variants but not the Celtic or Mongolian variants. This study provides insight into the mechanisms of the POLLED variants for disrupting horn development. These results should be validated using data produced from the horn bud region of horned and polled bovine fetuses.
Publisher: Springer Science and Business Media LLC
Date: 28-08-2021
DOI: 10.1186/S13072-021-00417-4
Abstract: Eukaryotic genomes are highly organised within the nucleus of a cell, allowing widely dispersed regulatory elements such as enhancers to interact with gene promoters through physical contacts in three-dimensional space. Recent chromosome conformation capture methodologies such as Hi-C have enabled the analysis of interacting regions of the genome providing a valuable insight into the three-dimensional organisation of the chromatin in the nucleus, including chromosome compartmentalisation and gene expression. Complicating the analysis of Hi-C data, however, is the massive amount of identified interactions, many of which do not directly drive gene function, thus hindering the identification of potentially biologically functional 3D interactions. In this review, we collate and examine the downstream analysis of Hi-C data with particular focus on methods that prioritise potentially functional interactions. We classify three groups of approaches: structural-based discovery methods, e.g. A/B compartments and topologically associated domains, detection of statistically significant chromatin interactions, and the use of epigenomic data integration to narrow down useful interaction information. Careful use of these three approaches is crucial to successfully identifying potentially functional interactions within the genome.
Publisher: Proceedings of the National Academy of Sciences
Date: 14-04-2021
Abstract: The placenta is the fetomaternal interface, which is essential for fetal growth and survival, playing a central role in the health of both the fetus and its mother. The dynamic gene expression during pregnancy dramatically contributes to the correct functionality of this temporary tissue. The epitheliochorial placenta of the horse is a valuable resource to understand parent-of-origin expression due to minimal bias associated with remnants of maternal tissue compared to other eutherian mammals. Here, we identified genes whose transcription is biased to either the paternal or maternal chromosome in the equine placenta. Overall, this study contributes to a better understanding of regulatory processes in placental function, evolution, and disease, using horses as a model for eutherian mammals’ placenta.
Publisher: Springer Science and Business Media LLC
Date: 09-11-2010
DOI: 10.1007/S00122-010-1482-7
Abstract: The stem rust resistance gene Sr2 has provided broad-spectrum protection against stem rust (Puccinia graminis Pers. f. sp. tritici) since its wide spread deployment in wheat from the 1940s. Because Sr2 confers partial resistance which is difficult to select under field conditions, a DNA marker is desirable that accurately predicts Sr2 in erse wheat germplasm. Using DNA sequence derived from the vicinity of the Sr2 locus, we developed a cleaved lified polymorphic sequence (CAPS) marker that is associated with the presence or absence of the gene in 115 of 122 (95%) erse wheat lines. The marker genotype predicted the absence of the gene in 100% of lines which were considered to lack Sr2. Discrepancies were observed in lines that were predicted to carry Sr2 but failed to show the CAPS marker. Given the high level of accuracy observed, the marker provides breeders with a selection tool for one of the most important disease resistance genes of wheat.
Publisher: Wiley
Date: 30-07-2020
DOI: 10.1111/BJH.17002
Publisher: Springer Science and Business Media LLC
Date: 28-08-2019
DOI: 10.3758/S13415-019-00743-X
Abstract: Physical exercise (PE) and environmental enrichment (EE) have consistently been shown to modulate behavior and neurobiological mechanisms. The current literature lacks evidence to confirm the relationship between PE and EE, if any, and whether short-term treatment with PE, EE, or PE+EE could be considered to correct age-related behavioral deficits. Three-, 8-, and 13-month-old C57BL/6 mice were assigned to either PE, EE, or PE+EE treatment groups (n = 12-16/group) for 4 weeks before behavioral testing and were compared to controls. Differential effects of the treatments on various behaviors and hippoc al gene expression were measured using an established behavioral battery and high-throughput qPCR respectively. Short-term EE enhanced locomotor activity at 9 and 14 months of age, whereas the combination of PE and EE reduced locomotor activity in the home cage at 14 months. Short-term EE also was found to reverse the age-related increase in anxiety at 9 months and spatial memory deficits at 14 months of age. Conversely, short-term PE induced spatial learning impairment and depressive-like behavior at four months but showed no effects in 9- and 14-month-old mice. PE and PE+EE, but not EE, modified the expression of several hippoc al genes at 9 months of age compared with control mice. In conclusion, short-term EE may help to alleviate age-related cognitive decline and increase in anxiety, without altering hippoc al gene expression. On the contrary, PE is detrimental at a young age for both affective-like behaviors and spatial learning and memory but showed no effects at middle and late middle age despite hippoc al gene expression alterations.
Publisher: Bioscientifica
Date: 07-2016
DOI: 10.1530/REP-16-0014
Abstract: Epigenetic modifications, and particularly DNA methylation, have been studied in many tissues, both healthy and diseased, and across numerous developmental stages. The placenta is the only organ that has a transient life of 9 months and undergoes rapid growth and dynamic structural and functional changes across gestation. Additionally, the placenta is unique because although developing within the mother, its genome is identical to that of the foetus. Given these distinctive characteristics, it is not surprising that the epigenetic landscape affecting placental gene expression may be different to that in other healthy tissues. However, the role of epigenetic modifications, and particularly DNA methylation, in placental development remains largely unknown. Of particular interest is the fact that the placenta is the most hypomethylated human tissue and is characterized by the presence of large partially methylated domains (PMDs) containing silenced genes. Moreover, how and why the placenta is hypomethylated and what role DNA methylation plays in regulating placental gene expression across gestation are poorly understood. We review genome-wide DNA methylation studies in the human placenta and highlight that the different cell types that make up the placenta have very different DNA methylation profiles. Summarizing studies on DNA methylation in the placenta and its relationship with pregnancy complications are difficult due to the limited number of studies available for comparison. To understand the key steps in placental development and hence what may be perturbed in pregnancy complications requires large-scale genome-wide DNA methylation studies coupled with transcriptome analyses.
Publisher: Cold Spring Harbor Laboratory
Date: 07-03-2022
DOI: 10.1101/2022.03.03.22271882
Abstract: The human placenta is a rapidly developing transient organ that is key to pregnancy success. Early development of the conceptus occurs in a low oxygen environment before oxygenated maternal blood begins to flow into the placenta at ∼10-12 weeks’ gestation. This process is likely to substantially affect overall placental gene expression. Transcript variability underlying gene expression has yet to be profiled. In this study, accurate transcript expression profiles were identified for 84 human placental chorionic villus tissue s les collected across 6-23 weeks’ gestation. Differential gene expression (DGE), differential transcript expression (DTE) and differential transcript usage (DTU) between 6-10 weeks’ and 11-23 weeks’ gestation groups were assessed. In total, 229 genes had significant DTE yet no significant DGE. Integration of DGE and DTE analyses found that differential expression patterns of in idual transcripts were commonly masked upon aggregation to the gene-level. Of the 611 genes that exhibited DTU, 534 had no significant DGE or DTE. The four most significant DTU genes ADAM10, VMP1, GPR126 , and ASAH1 , were associated with hypoxia-responsive pathways. Transcript usage is a likely regulatory mechanism in early placentation. Identification of functional roles will facilitate new insight in understanding the origins of pregnancy complications.
Publisher: Future Medicine Ltd
Date: 03-2017
Abstract: Aim: To determine whether dynamic DNA methylation changes in the human placenta can be used to predict gestational age. Materials & methods: Publicly available placental DNA methylation data from 12 studies, together with our own dataset, using Illumina Infinium Human Methylation BeadChip arrays. Results & conclusion: We developed an accurate tool for predicting gestational age of placentas using 62 CpG sites. There was a higher predicted gestational age for placentas from early onset preecl sia cases, but not term preecl sia, compared with their chronological age. Therefore, early onset preecl sia is associated with placental aging. Gestational age acceleration prediction from DNA methylation array data may provide insight into the molecular mechanisms of pregnancy disorders.
Publisher: MDPI AG
Date: 21-07-2022
Abstract: The abundance of cell-free microRNA (miRNA) has been measured in blood plasma and proposed as a source of novel, minimally invasive biomarkers for several diseases. Despite improvements in quantification methods, there is no consensus regarding how haemolysis affects plasma miRNA content. We propose a method for haemolysis detection in miRNA high-throughput sequencing (HTS) data from libraries prepared using human plasma. To establish a miRNA haemolysis signature we tested differential miRNA abundance between plasma s les with known haemolysis status. Using these miRNAs with statistically significant higher abundance in our haemolysed group, we further refined the set to reveal high-confidence haemolysis association. Given our specific context, i.e., women of reproductive age, we also tested for significant differences between pregnant and non-pregnant groups. We report a novel 20-miRNA signature used to identify the presence of haemolysis in silico in HTS miRNA-sequencing data. Further, we validated the signature set using firstly an all-male cohort (prostate cancer) and secondly a mixed male and female cohort (radiographic knee osteoarthritis). Conclusion: Given the potential for haemolysis contamination, we recommend that assays for haemolysis detection become standard pre-analytical practice and provide here a simple method for haemolysis detection.
Publisher: Springer Science and Business Media LLC
Date: 04-08-2010
DOI: 10.1007/S10142-010-0184-1
Abstract: The recent release of the genome sequences of a number of crop and model plant species has made it possible to define the genome organisation and functional characteristics of specific genes and gene families of agronomic importance. For instance, Sorghum bicolor, maize (Zea mays) and Brachypodium distachyon genome sequences along with the model grass species rice (Oryza sativa) enable the comparative analysis of genes involved in plant defence. Germin-like proteins (GLPs) are a small, functionally and taxonomically erse class of cupin-domain containing proteins that have recently been shown to cluster in an area of rice chromosome 8. The genomic location of this gene cluster overlaps with a disease resistance QTL that provides defence against two rice fungal pathogens (Magnaporthe oryzae and Rhizoctonia solani). Studies showing the involvement of GLPs in basal host resistance against powdery mildew (Blumeria graminis ssp.) have also been reported in barley and wheat. In this mini-review, we compare the close proximity of GLPs in publicly available cereal crop genomes and discuss the contribution that these proteins, and their genome sequence organisation, play in plant defence.
Publisher: Oxford University Press (OUP)
Date: 26-04-2023
DOI: 10.1093/NAR/GKAD271
Abstract: Progesterone receptor (PGR) plays erse roles in reproductive tissues and thus coordinates mammalian fertility. In the ovary, rapid acute induction of PGR is the key determinant of ovulation through transcriptional control of a unique set of genes that culminates in follicle rupture. However, the molecular mechanisms for this specialized PGR function in ovulation is poorly understood. We have assembled a detailed genomic profile of PGR action through combined ATAC-seq, RNA-seq and ChIP-seq analysis in wildtype and isoform-specific PGR null mice. We demonstrate that stimulating ovulation rapidly reprograms chromatin accessibility in two-thirds of sites, correlating with altered gene expression. An ovary-specific PGR action involving interaction with RUNX transcription factors was observed with 70% of PGR-bound regions also bound by RUNX1. These transcriptional complexes direct PGR binding to proximal promoter regions. Additionally, direct PGR binding to the canonical NR3C motif enable chromatin accessibility. Together these PGR actions mediate induction of essential ovulatory genes. Our findings highlight a novel PGR transcriptional mechanism specific to ovulation, providing new targets for infertility treatments or new contraceptives that block ovulation.
Publisher: Unpublished
Date: 2019
Publisher: Frontiers Media SA
Date: 13-10-2016
Publisher: Springer Science and Business Media LLC
Date: 08-03-2017
DOI: 10.1038/NATURE21674
Abstract: Recent genomic data have revealed multiple interactions between Neanderthals and modern humans, but there is currently little genetic evidence regarding Neanderthal behaviour, diet, or disease. Here we describe the shotgun-sequencing of ancient DNA from five specimens of Neanderthal calcified dental plaque (calculus) and the characterization of regional differences in Neanderthal ecology. At Spy cave, Belgium, Neanderthal diet was heavily meat based and included woolly rhinoceros and wild sheep (mouflon), characteristic of a steppe environment. In contrast, no meat was detected in the diet of Neanderthals from El Sidrón cave, Spain, and dietary components of mushrooms, pine nuts, and moss reflected forest gathering. Differences in diet were also linked to an overall shift in the oral bacterial community (microbiota) and suggested that meat consumption contributed to substantial variation within Neanderthal microbiota. Evidence for self-medication was detected in an El Sidrón Neanderthal with a dental abscess and a chronic gastrointestinal pathogen (Enterocytozoon bieneusi). Metagenomic data from this in idual also contained a nearly complete genome of the archaeal commensal Methanobrevibacter oralis (10.2× depth of coverage)-the oldest draft microbial genome generated to date, at around 48,000 years old. DNA preserved within dental calculus represents a notable source of information about the behaviour and health of ancient hominin specimens, as well as a unique system that is useful for the study of long-term microbial evolution.
Publisher: Cold Spring Harbor Laboratory
Date: 02-02-2022
DOI: 10.1101/2022.02.01.475276
Abstract: The basic-Helix-Loop-Helix Per-Arnt-Sim (PAS) homology domain (bHLH-PAS) transcription factor (TF) family comprises critical biological sensors of physiological (hypoxia, tryptophan metabolites, neuronal activity, and appetite) and environmental (diet derived metabolites and environmental pollutants) stimuli to regulate genes involved in signal adaptation and homeostasis 1 . bHLH TFs bind DNA as homo or heterodimers via E-box (CANNTG) response elements, however the DNA binding specificity of the PAS domain-containing bHLH subfamily remains unresolved 1 . We systematically analysed cognate DNA binding hierarchies of prototypical bHLH-PAS family members (ARNT, ARNT2, HIF1α, HIF2α, AhR, NPAS4, SIM1) and demonstrate distinct core (NNCGTG) specificities for different heterodimer classes. The results also show that bHLH-PAS TFs bind over a large footprint 12-15bp and recognise preferential DNA sequences flanking the core. For ex le, specificity beyond otherwise identical core binding by SIM1 and the HIFs is mediated through N-terminal HIFα-DNA interactions. We also reveal an intimate relationship between DNA shape and both core and flanking TF binding allowing motif sequence flexibility and underpinning TF binding specificity. Furthermore, DNA-shape affinity relationships revealed that novel downstream PAS-A-loop DNA interactions are associated with AT-rich sequences that lead to high-affinity binding, and that loss of this function underpins a monogenic cause of human hyperphagic obesity in a recapitulated SIM1.R171H knock-in mouse model. Importantly, models of protein-DNA binding accurately predict in vivo occupancy, while response element methylation blocks DNA binding and predicts cell type specific chromatin occupancy. These data provide a definitive and accurate map of bHLH-PAS TF specificity and target selectivity through novel flanking protein-DNA interactions that are crucial for in vivo biological function.
Publisher: Cold Spring Harbor Laboratory
Date: 18-04-2017
DOI: 10.1101/127977
Abstract: Fruit attributes that affect wine quality are thought to be largely driven by the interaction of grapevine’s genetic characteristics with environmental factors (i.e. climate, soil and topography) and vineyard management. All these variables, in conjunction with the wine making process, give a wine its distinctive character. Understanding how grapevines perceive and adapt to a changing environment will provide us with an insight into how to better manage crop quality. Mounting evidence suggests that epigenetic mechanisms are a key interface between the environment and the genotype that ultimately affect the plant’s phenotype. Moreover, it is now widely accepted that epigenetic mechanisms are a source of useful variability during crop varietal selection that could affect crop performance. While the contribution of DNA methylation to plant performance has been extensively studied in other major crops, very little work has been done in grapevine. Here we used Methylation Sensitive Amplified Polymorphisms to obtain global patterns of DNA methylation, and to identify the main drivers of epigenetic ersity across 22 vineyards planted with the cultivar Shiraz in six distinctive wine areas of a major wine zone, The Barossa, South Australia. The observed epigenetic profiles showed a high level of differentiation that grouped vineyards by their area of provenance despite the low genetic differentiation between vineyards and sub-regions. Furthermore, pairwise epigenetic distances between vineyards with similar management systems showed a significant correlation with geographic distance. Finally, methylation sensitive Genotyping By Sequencing identified 3,598 differentially methylated genes that were assigned to 1,144 unique GO terms of which 8.6% were associated with response to environmental stimulus. Taken together, our results indicate that the intensity and directionality of DNA methylation differentiation between vineyards and wine sub-regions within The Barossa are driven by management and local growing conditions. Finally, we discuss how epigenetic variability can be used as a tool to understand and potentially modulate terroir in grapevine.
Publisher: Springer Science and Business Media LLC
Date: 09-07-2020
DOI: 10.1038/S41598-020-68261-2
Abstract: Previous studies of global binding patterns identified the epigenetic factor, EZH2, as a regulator of the homeodomain-only protein homeobox (HOPX) gene expression during bone marrow stromal cell (BMSC) differentiation, suggesting a potential role for HOPX in regulating BMSC lineage specification. In the present study, we confirmed that EZH2 direct binds to the HOPX promoter region, during normal growth and osteogenic differentiation but not under adipogenic inductive conditions. HOPX gene knockdown and overexpression studies demonstrated that HOPX is a promoter of BMSC proliferation and an inhibitor of adipogenesis. However, functional studies failed to observe any affect by HOPX on BMSC osteogenic differentiation. RNA-seq analysis of HOPX overexpressing BMSC during adipogenesis, found HOPX function to be acting through suppression of adipogenic pathways associated genes such as ADIPOQ, FABP4, PLIN1 and PLIN4. These findings suggest that HOPX gene target pathways are critical factors in the regulation of fat metabolism.
Publisher: Springer Science and Business Media LLC
Date: 18-06-2018
Publisher: Oxford University Press (OUP)
Date: 06-2021
DOI: 10.1038/S41438-021-00572-5
Abstract: Transfer RNAs (tRNA) are crucial adaptor molecules between messenger RNA (mRNA) and amino acids. Recent evidence in plants suggests that dicistronic tRNA-like structures also act as mobile signals for mRNA transcripts to move between distant tissues. Co-transcription is not a common feature in the plant nuclear genome and, in the few cases where polycistronic transcripts have been found, they include non-coding RNA species, such as small nucleolar RNAs and microRNAs. It is not known, however, the extent to which dicistronic transcripts of tRNA and mRNAs are expressed in field-grown plants, or the factors contributing to their expression. We analysed tRNA–mRNA dicistronic transcripts in the major horticultural crop grapevine ( Vitis vinifera ) using a novel pipeline developed to identify dicistronic transcripts from high-throughput RNA-sequencing data. We identified dicistronic tRNA–mRNA in leaf and berry s les from 22 commercial vineyards. Of the 124 tRNA genes that were expressed in both tissues, 18 tRNA were expressed forming part of 19 dicistronic tRNA–mRNAs. The presence and abundance of dicistronic molecules was tissue and geographic sub-region specific. In leaves, the expression patterns of dicistronic tRNA–mRNAs significantly correlated with tRNA expression, suggesting that their transcriptional regulation might be linked. We also found evidence of syntenic genomic arrangements of tRNAs and protein-coding genes between grapevine and Arabidopsis thaliana , and widespread prevalence of dicistronic tRNA–mRNA transcripts among vascular land plants but no evidence of these transcripts in non-vascular lineages. This suggests that the appearance of plant vasculature and tRNA–mRNA occurred concurrently during the evolution of land plants.
Publisher: MDPI AG
Date: 20-04-2022
DOI: 10.3390/IJMS23094574
Abstract: RNA sequencing provides a snapshot of the functional consequences of genomic lesions that drive acute lymphoblastic leukemia (ALL). The aims of this study were to elucidate diagnostic associations (via machine learning) between mRNA-seq profiles, independently verify ALL lesions and develop easy-to-interpret transcriptome-wide biomarkers for ALL subtyping in the clinical setting. A training dataset of 1279 ALL patients from six North American cohorts was used for developing machine learning models. Results were validated in 767 patients from Australia with a quality control dataset across 31 tissues from 1160 non-ALL donors. A novel batch correction method was introduced and applied to adjust for cohort differences. Out of 18,503 genes with usable expression, 11,830 (64%) were confounded by cohort effects and excluded. Six ALL subtypes (ETV6::RUNX1, KMT2A, DUX4, PAX5 P80R, TCF3::PBX1, ZNF384) that covered 32% of patients were robustly detected by mRNA-seq (positive predictive value ≥ 87%). Five other frequent subtypes (CRLF2, hypodiploid, hyperdiploid, PAX5 alterations and Ph-positive) were distinguishable in 40% of patients at lower accuracy (52% ≤ positive predictive value ≤ 73%). Based on these findings, we introduce the Allspice R package to predict ALL subtypes and driver genes from unadjusted mRNA-seq read counts as encountered in real-world settings. Two ex les of Allspice applied to previously unseen ALL patient s les with atypical lesions are included.
Publisher: American Society of Hematology
Date: 16-06-2022
Abstract: Transcriptome sequencing has identified multiple subtypes of B-progenitor acute lymphoblastic leukemia (B-ALL) of prognostic significance, but a minority of cases lack a known genetic driver. Here, we used integrated whole-genome (WGS) and -transcriptome sequencing (RNA-seq), enhancer mapping, and chromatin topology analysis to identify previously unrecognized genomic drivers in B-ALL. Newly diagnosed (n = 3221) and relapsed (n = 177) B-ALL cases with tumor RNA-seq were studied. WGS was performed to detect mutations, structural variants, and copy number alterations. Integrated analysis of histone 3 lysine 27 acetylation and chromatin looping was performed using HiChIP. We identified a subset of 17 newly diagnosed and 5 relapsed B-ALL cases with a distinct gene expression profile and 2 universal and unique genomic alterations resulting from aberrant recombination-activating gene activation: a focal deletion downstream of PAN3 at 13q12.2 resulting in CDX2 deregulation by the PAN3 enhancer and a focal deletion of exons 18-21 of UBTF at 17q21.31 resulting in a chimeric fusion, UBTF::ATXN7L3. A subset of cases also had rearrangement and increased expression of the PAX5 gene, which is otherwise uncommon in B-ALL. Patients were more commonly female and young adult with median age 35 (range,12-70 years). The immunophenotype was characterized by CD10 negativity and immunoglobulin M positivity. Among 16 patients with known clinical response, 9 (56.3%) had high-risk features including relapse (n = 4) or minimal residual disease & % at the end of remission induction (n = 5). CDX2-deregulated, UBTF::ATXN7L3 rearranged (CDX2/UBTF) B-ALL is a high-risk subtype of leukemia in young adults for which novel therapeutic approaches are required.
Publisher: CSIRO Publishing
Date: 2019
DOI: 10.1071/RD17321
Abstract: Haemoglobin expression is not restricted to erythroid cells. We investigated the gene expression of the haemoglobin subunits haemoglobin, alpha adult chain 1 (Hba-a1) and haemoglobin, beta (Hbb), 2,3-bisphosphoglycerate mutase (Bpgm) and the oxygen-regulated genes BCL2/adenovirus E1B interacting protein 3 (Bnip3), solute carrier family 2 (facilitated glucose transporter), member 1 (Slc2a1) and N-myc downstream regulated gene 1 (Ndrg1) in the murine preimplantation embryo, comparing invivo to invitro gene expression. Relatively high levels of Hba-a1 and Hbb were expressed invivo from the 2-cell to blastocyst stage in contrast, little or no expression occurred invitro. We hypothesised that the presence of haemoglobin invivo creates a low oxygen environment to induce oxygen-regulated gene expression, supported by high expression of Slc2a1 and Ndrg1 in invivo relative to invitro embryos. In addition, analysis of an invitro-derived human embryo gene expression public dataset revealed low expression of haemoglobin subunit alpha (HBA) and HBB, and high expression of BPGM. To explore whether there was a developmental stage-specific effect of haemoglobin, we added exogenous haemoglobin either up to the 4-cell stage or throughout development to the blastocyst stage, but observed no difference in blastocyst rate or the inner cell mass to trophectoderm cell ratio. We conclude that haemoglobin in the invivo preimplantation embryo raises an interesting premise of potential mechanisms for oxygen regulation, which may influence oxygen-regulated gene expression.
Publisher: Springer Science and Business Media LLC
Date: 08-10-2019
Publisher: Elsevier BV
Date: 12-2019
Publisher: Public Library of Science (PLoS)
Date: 21-11-2013
Publisher: Wiley
Date: 12-12-2021
DOI: 10.1111/BJH.17995
Publisher: Proceedings of the National Academy of Sciences
Date: 29-06-2015
Abstract: Northern corn leaf blight (NCLB) is one of the most devastating fungal diseases of maize. The Htn1 disease resistance gene confers quantitative field resistance against most NCLB isolates. Here we show that Htn1 encodes a putative wall-associated receptor-like kinase (RLK). RLKs act as important components of the first tier of the plant innate immune system by perceiving pathogen- or host-derived elicitors on the cell surface. RLKs are often associated with resistance to nonadapted pathogens and are a component of nonhost resistance. Our work demonstrates that the Htn1-RLK plays an important role in host resistance against adapted fungal pathogens.
Publisher: Springer Science and Business Media LLC
Date: 27-05-2010
Abstract: Expansins form a large multi-gene family found in wheat and other cereal genomes that are involved in the expansion of cell walls as a tissue grows. The expansin family can be ided up into two main groups, namely, alpha-expansin (EXPA) and beta-expansin proteins (EXPB), with the EXPB group being of particular interest as group 1-pollen allergens. In this study, three beta-expansin genes were identified and characterized from a newly sequenced region of the Triticum aestivum cv. Chinese Spring chromosome 3B physical map at the Sr2 locus (FPC contig ctg11 ). The analysis of a 357 kb sub-sequence of FPC contig ctg11 identified one beta-expansin genes to be TaEXPB11 , originally identified as a cDNA from the wheat cv Wyuna. Through the analysis of intron sequences of the three wheat cv. Chinese Spring genes, we propose that two of these beta-expansin genes are duplications of the TaEXPB11 gene. Comparative sequence analysis with two other wheat cultivars (cv. Westonia and cv. Hope) and a Triticum aestivum var. spelta line validated the identification of the Chinese Spring variant of TaEXPB11 . The expression in maternal and grain tissues was confirmed by examining EST databases and carrying out RT-PCR experiments. Detailed examination of the position of TaEXPB11 relative to the locus encoding Sr2 disease resistance ruled out the possibility of this gene directly contributing to the resistance phenotype. Through 3-D structural protein comparisons with Zea mays EXPB1 , we proposed that variations within the coding sequence of TaEXPB11 in wheats may produce a functional change within features such as domain 1 related to possible involvement in cell wall structure and domain 2 defining the pollen allergen domain and binding to IgE protein. The variation established in this gene suggests it is a clearly identifiable member of a gene family and reflects the dynamic features of the wheat genome as it adapted to a range of different environments and uses. Accession Numbers: ctg11 =FN564426 Survey sequences of TaEXPB11ws and TsEXPB11 are provided request.
Publisher: Cold Spring Harbor Laboratory
Date: 28-06-2020
DOI: 10.1101/2020.06.25.20139709
Abstract: During early human placental development, extravillous cytotrophoblasts (EVT) invade the uterine vasculature to sequester a maternal blood supply. The impact of this on placental gene expression has not been established for normal pregnancy. Using RNA sequencing, we profiled placental chorionic villous tissues from 96 pregnancies at 6-23 weeks of gestation. We identified 1,048 genes that were differentially expressed between 6-10 weeks’ and 11-23 weeks’ of gestation. These are predominantly genes that are enriched in transcription factor signalling, inflammatory response and cell adhesion. Using a co-expression network and gene set enrichment analyses, we reveal three distinct phases of gene expression coincident with phases of maternal blood flow to the placenta that impact immune function and are likely driven by oxygen tension, potentially in a sex-specific manner. These data represent a comprehensive transcriptional profile of early placental development and point to significant environmental, genetic and regulatory triggers that drive gene expression.
Publisher: Springer Science and Business Media LLC
Date: 27-05-2010
Abstract: The complexity of the wheat genome has resulted from waves of retrotransposable element insertions. Gene deletions and disruptions generated by the fast replacement of repetitive elements in wheat have resulted in disruption of colinearity at a micro (sub-megabase) level among the cereals. In view of genomic changes that are possible within a given time span, conservation of genes between species tends to imply an important functional or regional constraint that does not permit a change in genomic structure. The ctg1034 contig completed in this paper was initially studied because it was assigned to the Sr2 resistance locus region, but detailed mapping studies subsequently assigned it to the long arm of 3B and revealed its unusual features. BAC shotgun sequencing of the hexaploid wheat ( Triticum aestivum cv. Chinese Spring) genome has been used to assemble a group of 15 wheat BACs from the chromosome 3B physical map FPC contig ctg1034 into a 783,553 bp genomic sequence. This ctg1034 sequence was annotated for biological features such as genes and transposable elements. A three-gene island was identified among % repetitive DNA sequence. Using bioinformatics analysis there were no observable similarity in their gene functions. The ctg1034 gene island also displayed complete conservation of gene order and orientation with syntenic gene islands found in publicly available genome sequences of Brachypodium distachyon , Oryza sativa , Sorghum bicolor and Zea mays , even though the intergenic space and introns were ergent. We propose that ctg1034 is located within the heterochromatic C-band region of deletion bin 3BL7 based on the identification of heterochromatic tandem repeats and presence of significant matches to chromodomain-containing gypsy LTR retrotransposable elements. We also speculate that this location, among other highly repetitive sequences, may account for the relative stability in gene order and orientation within the gene island. Sequence data from this article have been deposited with the GenBank Data Libraries under accession no. GQ422824
Publisher: Elsevier BV
Date: 10-2023
Publisher: Wiley
Date: 19-08-2022
DOI: 10.1002/MGG3.2023
Abstract: Corneal dystrophies describe a clinically and genetically heterogeneous group of inherited disorders. The International Classification of Corneal Dystrophies (IC3D) lists 22 types of corneal dystrophy, 17 of which have been demonstrated to result from pathogenic variants in 19 identified genes. In this study, we investigated the diagnostic yield of genetic testing in a well‐characterised cohort of 58 in iduals from 44 families with different types of corneal dystrophy. In iduals diagnosed solely with Fuchs endothelial corneal dystrophy were excluded. Clinical details were obtained from the treating ophthalmologist. Participants and their family members were tested using a gene candidate and exome sequencing approach. We identified a likely molecular diagnosis in 70.5% families (31/44). The detection rate was significantly higher among probands with a family history of corneal dystrophy (15/16, 93.8%) than those without (16/28, 57.1%, p = .015), and among those who had undergone corneal graft surgery (9/9, 100.0%) compared to those who had not (22/35, 62.9%, p = .041). We identified eight novel variants in five genes and identified five families with syndromes associated with corneal dystrophies. Our findings highlight the genetic heterogeneity of corneal dystrophies and the clinical utility of genetic testing in reaching an accurate clinical diagnosis.
Publisher: Elsevier BV
Date: 2022
DOI: 10.1016/J.GIM.2021.09.001
Abstract: Genetic variants causing aberrant premessenger RNA splicing are increasingly being recognized as causal variants in genetic disorders. In this study, we devise standardized practices for polymerase chain reaction (PCR)-based RNA diagnostics using clinically accessible specimens (blood, fibroblasts, urothelia, biopsy). A total of 74 families with erse monogenic conditions (31% prenatal-congenital onset, 47% early childhood, and 22% teenage-adult onset) were triaged into PCR-based RNA testing, with comparative RNA sequencing for 19 cases. Informative RNA assay data were obtained for 96% of cases, enabling variant reclassification for 75% variants that can be used for genetic counseling (71%), to inform clinical care (32%) and prenatal counseling (41%). Variant-associated mis-splicing was highly reproducible for 28 cases with s les from ≥2 affected in iduals or heterozygotes and 10 cases with ≥2 biospecimens. PCR licons encompassing another segregated heterozygous variant was vital for clinical interpretation of 22 of 79 variants to phase RNA splicing events and discern complete from partial mis-splicing. RNA diagnostics enabled provision of a genetic diagnosis for 64% of recruited cases. PCR-based RNA diagnostics has capacity to analyze 81.3% of clinically significant genes, with long licons providing an advantage over RNA sequencing to phase RNA splicing events. The Australasian Consortium for RNA Diagnostics (SpliceACORD) provide clinically-endorsed, standardized protocols and recommendations for interpreting RNA assay data.
Publisher: Elsevier BV
Date: 06-2015
DOI: 10.1016/J.FORSCIINT.2015.03.008
Abstract: Soil can serve as powerful trace evidence in forensic casework, because it is highly in idualistic and can be characterised using a number of techniques. Complex soil matrixes can support a vast number of organisms that can provide a site-specific signal for use in forensic soil discrimination. Previous DNA fingerprinting techniques rely on variations in fragment length to distinguish between soil profiles and focus solely on microbial communities. However, the recent development of high throughput sequencing (HTS) has the potential to provide a more detailed picture of the soil community by accessing non-culturable microorganisms and by identifying specific bacteria, fungi, and plants within soil. To demonstrate the application of HTS to forensic soil analysis, 18S ribosomal RNA profiles of six forensic mock crime scene s les were compared to those collected from seven reference locations across South Australia. Our results demonstrate the utility of non-bacterial DNA to discriminate between different sites, and were able to link a soil to a particular location. In addition, HTS complemented traditional Mid Infrared (MIR) spectroscopy soil profiling, but was able to provide statistically stronger discriminatory power at a finer scale. Through the design of an experimental case scenario, we highlight the considerations and potential limitations of this method in forensic casework. We show that HTS analysis of soil eukaryotes was robust to environmental variation, e.g. rainfall and temperature, transfer effects, storage effects and spatial variation. In addition, this study utilises novel analytical methodologies to interpret results for investigative purposes and provides prediction statistics to support soil DNA analysis for evidential stages of a case.
Publisher: Elsevier BV
Date: 05-2019
DOI: 10.1016/J.BRAINRESBULL.2019.02.014
Abstract: Regular exercise can reduce depression-, anxiety-, and impaired cognitive-like behaviours, and upregulate hippoc al genes associated with neuroplasticity. However, the effects of ceasing exercise on depression-, anxiety-, and cognitive-like behaviours, and hippoc al gene expression remain unknown. 12-week-old C57BL/6 mice (n = 12-16/group) were randomised to six months of exercise (exercise (EXC)), four months of exercise then two months of no exercise (exercise-cessation (EC)), or no-exercise control (CONT) until aged nine months. Depression-, anxiety-, and cognitive-like behaviours were tested with the forced swim test, open field and elevated zero maze, Y-maze, and Barnes maze. The expression of 75 hippoc al genes were investigated by high-throughput quantitative polymerase chain reaction (qPCR). Exercise cessation increased depression- and anxiety-like behaviours, and impaired spatial learning and cognitive flexibility compared to CONT and EXC mice. 10/75 hippoc al genes were differentially expressed in EC mice, including increased expression of neurogenesis associated genes (Ntrk1), and reduced expression of immune (Il10, Gfap) and monoamine related genes (Htr1a) compared to CONT mice. Altered expression of nine genes including increased Slc6a4 and reduced Sirt1 expression were shown in EC mice compared to EXC mice. Exercise cessation increased depression- and anxiety-like behaviours and impaired some cognition-like behaviours with altered neurogenic, monoaminergic, and immune hippoc al gene expression consistent with the pathogenesis of depression and related anxiety described by the neurogenic, monoaminergic, and immune hypotheses of depression. Mice and humans share mammalian physiology, so these findings could be relevant to humans. These results require replication and possibly translation into high-quality pilot clinical trials.
Publisher: Informa UK Limited
Date: 19-08-2021
Publisher: Springer Science and Business Media LLC
Date: 08-02-2021
DOI: 10.1038/S41413-020-00123-Z
Abstract: Overnutrition causes hyperactivation of mTORC1-dependent negative feedback loops leading to the downregulation of insulin signaling and development of insulin resistance. In osteoblasts (OBs), insulin signaling plays a crucial role in the control of systemic glucose homeostasis. We utilized mice with conditional deletion of Rptor to investigate how the loss of mTORC1 function in OB affects glucose metabolism under normal and overnutrition dietary states. Compared to the controls, chow-fed Rptor ob −/− mice had substantially less fat mass and exhibited adipocyte hyperplasia. Remarkably, upon feeding with high-fat diet, mice with pre- and post-natal deletion of Rptor in OBs were protected from diet-induced obesity and exhibited improved glucose metabolism with lower fasting glucose and insulin levels, increased glucose tolerance and insulin sensitivity. This leanness and resistance to weight gain was not attributable to changes in food intake, physical activity or lipid absorption but instead was due to increased energy expenditure and greater whole-body substrate flexibility. RNA-seq revealed an increase in glycolysis and skeletal insulin signaling pathways, which correlated with the potentiation of insulin signaling and increased insulin-dependent glucose uptake in Rptor -knockout osteoblasts. Collectively, these findings point to a critical role for the mTORC1 complex in the skeletal regulation of whole-body glucose metabolism and the skeletal development of insulin resistance.
Publisher: Springer Science and Business Media LLC
Date: 07-06-2022
DOI: 10.1038/S42003-022-03528-0
Abstract: Non-coding RNAs (ncRNAs) form a large portion of the mammalian genome. However, their biological functions are poorly characterized in cancers. In this study, using a newly developed tool, SomaGene, we analyze de novo somatic point mutations from the International Cancer Genome Consortium (ICGC) whole-genome sequencing data of 1,855 breast cancer s les. We identify 1030 candidates of ncRNAs that are significantly and explicitly mutated in breast cancer s les. By integrating data from the ENCODE regulatory features and FANTOM5 expression atlas, we show that the candidate ncRNAs significantly enrich active chromatin histone marks (1.9 times), CTCF binding sites (2.45 times), DNase accessibility (1.76 times), HMM predicted enhancers (2.26 times) and eQTL polymorphisms (1.77 times). Importantly, we show that the 1030 ncRNAs contain a much higher level (3.64 times) of breast cancer-associated genome-wide association (GWAS) single nucleotide polymorphisms (SNPs) than genome-wide expectation. Such enrichment has not been seen with GWAS SNPs from other cancers. Using breast cell line related Hi-C data, we then show that 82% of our candidate ncRNAs (1.9 times) significantly interact with the promoter of protein-coding genes, including previously known cancer-associated genes, suggesting the critical role of candidate ncRNA genes in the activation of essential regulators of development and differentiation in breast cancer. We provide an extensive web-based resource ( www.ihealthe.unsw.edu.au/research ) to communicate our results with the research community. Our list of breast cancer-specific ncRNA genes has the potential to provide a better understanding of the underlying genetic causes of breast cancer. Lastly, the tool developed in this study can be used to analyze somatic mutations in all cancers.
Publisher: Springer Science and Business Media LLC
Date: 14-12-2021
DOI: 10.1038/S41598-021-03425-2
Abstract: Embryo implantation is a key step in establishing pregnancy and a major limiting factor in IVF. Implantation requires a receptive endometrium but the mechanisms governing receptivity are not well understood. We have recently discovered that podocalyxin (PCX or PODXL) is a key negative regulator of human endometrial receptivity. PCX is expressed in all endometrial epithelial cells in the non-receptive endometrium but selectively down-regulated in the luminal epithelium at receptivity. We have further demonstrated that this down-regulation is essential for implantation because PCX inhibits embryo attachment and penetration. However, how PCX confers this role is unknown. In this study, through RNAseq analysis of Ishikawa cell line stably overexpressing PCX, we discovered that PCX suppresses expression of genes controlling cell adhesion and communication, but increases those governing epithelial barrier functions, especially the adherens and tight junctions. Moreover, PCX suppresses multiple factors such as LIF and signaling pathways including Wnt and calcium signaling that support receptivity but stimulates anti-implantation genes such as LEFTY2. Functional studies confirmed that PCX promotes epithelial barrier functions by increasing key epithelial junction proteins such as E-cadherin and claudin 4. PCX thus promotes an anti-adhesive and impermeable epithelium while impedes pro-implantation factors to negatively control endometrial receptivity for implantation.
Publisher: Cold Spring Harbor Laboratory
Date: 21-05-2020
DOI: 10.1101/2020.05.18.101022
Abstract: Colorectal cancer (CRC) is one of the leading causes of cancer-related deaths in the world. It has been reported that ∼10%-15% of in iduals with colorectal cancer experience a causative mutation in the known susceptibility genes, highlighting the importance of identifying mutations for early detection in high risk in iduals. Through extensive sequencing projects such as the International Cancer Genome Consortium (ICGC), a large number of somatic point mutations have been identified that can be used to identify cancer-associated genes, as well as the signature of mutational processes defined by the tri-nucleotide sequence context (motif) of mutated sites. Mutation is the hallmark of cancer genome, and many studies have reported cancer subtyping based on the type of frequently mutated genes, or the proportion of mutational processes, however, none of these cancer subtyping methods consider these features simultaneously. This highlights the need for a better and more inclusive subtype classification approach to enable biomarker discovery and thus inform drug development for CRC. In this study, we developed a statistical pipeline based on a novel concept ‘gene-motif’, which merges mutated gene information with tri-nucleotide motif of mutated sites, to identify cancer subtypes, in this case CRCs. Our analysis identified for the first time, 3,131 gene-motif combinations that were significantly mutated in 536 ICGC colorectal cancer s les compared to other cancer types, identifying seven CRC subtypes with distinguishable phenotypes and biomarkers. Interestingly, we identified several genes that were mutated in multiple subtypes but with unique sequence contexts. Taken together, our results highlight the importance of considering both the mutation type and mutated genes in identification of cancer subtypes and cancer biomarkers.
Publisher: Springer Science and Business Media LLC
Date: 19-04-2022
DOI: 10.1186/S12859-022-04652-8
Abstract: Colorectal cancer (CRC) is one of the leading causes of cancer-related deaths worldwide. Recent studies have observed causative mutations in susceptible genes related to colorectal cancer in 10 to 15% of the patients. This highlights the importance of identifying mutations for early detection of this cancer for more effective treatments among high risk in iduals. Mutation is considered as the key point in cancer research. Many studies have performed cancer subtyping based on the type of frequently mutated genes, or the proportion of mutational processes. However, to the best of our knowledge, combination of these features has never been used together for this task. This highlights the potential to introduce better and more inclusive subtype classification approaches using wider range of related features to enable biomarker discovery and thus inform drug development for CRC. In this study, we develop a new pipeline based on a novel concept called ‘gene-motif’, which merges mutated gene information with tri-nucleotide motif of mutated sites, for colorectal cancer subtype identification. We apply our pipeline to the International Cancer Genome Consortium (ICGC) CRC s les and identify, for the first time, 3131 gene-motif combinations that are significantly mutated in 536 ICGC colorectal cancer s les. Using these features, we identify seven CRC subtypes with distinguishable phenotypes and biomarkers, including unique cancer related signaling pathways, in which for most of them targeted treatment options are currently available. Interestingly, we also identify several genes that are mutated in multiple subtypes but with unique sequence contexts. Our results highlight the importance of considering both the mutation type and mutated genes in identification of cancer subtypes and cancer biomarkers. The new CRC subtypes presented in this study demonstrates distinguished phenotypic properties which can be effectively used to develop new treatments. By knowing the genes and phenotypes associated with the subtypes, a personalized treatment plan can be developed that considers the specific phenotypes associated with their genomic lesion.
Publisher: Springer Science and Business Media LLC
Date: 10-08-2021
DOI: 10.1038/S41698-021-00215-X
Abstract: Ruxolitinib (rux) Phase II clinical trials are underway for the treatment of high-risk JAK2 -rearranged ( JAK2 r) B-cell acute lymphoblastic leukemia (B-ALL). Treatment resistance to targeted inhibitors in other settings is common elucidating potential mechanisms of rux resistance in JAK2 r B-ALL will enable development of therapeutic strategies to overcome or avert resistance. We generated a murine pro-B cell model of ATF7IP-JAK2 with acquired resistance to multiple type-I JAK inhibitors. Resistance was associated with mutations within the JAK2 ATP/rux binding site, including a JAK2 p.G993A mutation. Using in vitro models of JAK2 r B-ALL, JAK2 p.G993A conferred resistance to six type-I JAK inhibitors and the type-II JAK inhibitor, CHZ-868. Using computational modeling, we postulate that JAK2 p.G993A enabled JAK2 activation in the presence of drug binding through a unique resistance mechanism that modulates the mobility of the conserved JAK2 activation loop. This study highlights the importance of monitoring mutation emergence and may inform future drug design and the development of therapeutic strategies for this high-risk patient cohort.
Publisher: Springer Science and Business Media LLC
Date: 19-08-2019
DOI: 10.1038/S41598-019-48333-8
Abstract: Progesterone receptor (PGR) co-ordinately regulates ovulation, fertilisation and embryo implantation through tissue-specific actions, but the mechanisms for ergent PGR action are poorly understood. Here we characterised PGR activity in mouse granulosa cells using combined ChIP-seq for PGR and H3K27ac and gene expression microarray. Comparison of granulosa, uterus and oviduct PGR-dependent genes showed almost complete tissue specificity in PGR target gene profiles. In granulosa cells 82% of identified PGR-regulated genes bound PGR within 3 kb of the gene and PGR binding sites were highly enriched in proximal promoter regions in close proximity to H3K27ac-modified active chromatin. Motif analysis showed highly enriched PGR binding to the PGR response element (GnACAnnnTGTnC), but PGR also interacted significantly with other transcription factor binding motifs. In uterus PGR showed far more tendency to bind intergenic chromatin regions and low evidence of interaction with other transcription factors. This is the first genome-wide description of PGR action in granulosa cells and systematic comparison of erse PGR action in different reproductive tissues. It clarifies finely-tuned contextual PGR-chromatin interactions with implications for more targeted reproductive medicine.
Publisher: Springer Science and Business Media LLC
Date: 08-10-2021
DOI: 10.1186/S12864-021-07951-1
Abstract: The seminal vesicles synthesise bioactive factors that support gamete function, modulate the female reproductive tract to promote implantation, and influence developmental programming of offspring phenotype. Despite the significance of the seminal vesicles in reproduction, their biology remains poorly defined. Here, to advance understanding of seminal vesicle biology, we analyse the mouse seminal vesicle transcriptome under normal physiological conditions and in response to acute exposure to the reproductive toxicant acrylamide. Mice were administered acrylamide (25 mg/kg bw/day) or vehicle control daily for five consecutive days prior to collecting seminal vesicle tissue 72 h following the final injection. A total of 15,304 genes were identified in the seminal vesicles with those encoding secreted proteins amongst the most abundant. In addition to reproductive hormone pathways, functional annotation of the seminal vesicle transcriptome identified cell proliferation, protein synthesis, and cellular death and survival pathways as prominent biological processes. Administration of acrylamide elicited 70 differentially regulated (fold-change ≥1.5 or ≤ 0.67) genes, several of which were orthogonally validated using quantitative PCR. Pathways that initiate gene and protein synthesis to promote cellular survival were prominent amongst the dysregulated pathways. Inflammation was also a key transcriptomic response to acrylamide, with the cytokine, Colony stimulating factor 2 ( Csf2 ) identified as a top-ranked upstream driver and inflammatory mediator associated with recovery of homeostasis. Early growth response ( Egr1 ), C-C motif chemokine ligand 8 ( Ccl8 ), and Collagen, type V, alpha 1 ( Col5a1 ) were also identified amongst the dysregulated genes. Additionally, acrylamide treatment led to subtle changes in the expression of genes that encode proteins secreted by the seminal vesicle, including the complement regulator, Complement factor b ( Cfb ). These data add to emerging evidence demonstrating that the seminal vesicles, like other male reproductive tract tissues, are sensitive to environmental insults, and respond in a manner with potential to exert impact on fetal development and later offspring health.
Publisher: Frontiers Media SA
Date: 25-03-2022
DOI: 10.3389/FIMMU.2022.807750
Abstract: Parturition signals the end of immune tolerance in pregnancy. Term labour is usually a sterile inflammatory process triggered by damage associated molecular patterns (DAMPs) as a consequence of functional progesterone withdrawal. Activation of DAMPs recruits leukocytes and inflammatory cytokine responses in the myometrium, decidua, cervix and fetal membranes. Emerging evidence shows components of the inflammasome are detectable in both maternal decidua and placenta. However, the activation of the placental inflammasome with respect to mode of delivery has not been profiled. Placental chorionic villus s les from women delivering at term via unassisted vaginal (UV) birth, labouring lower segment caesarean section (LLSCS, emergency caesarean section) and prelabour lower segment caesarean section (PLSCS, elective caesarean section) underwent high throughput RNA sequencing (NextSeq Illumina) and bioinformatic analyses to identify differentially expressed inflammatory (DE) genes. DE genes ( IL1RL1 , STAT1 , STAT2 , IL2RB , IL17RE , IL18BP , TNFAIP2 , TNFSF10 and TNFRSF8 ), as well as common inflammasome genes ( IL1B , IL1R1 , IL1R2 , IL6 , IL18 , IL18R1 , IL18R1 , IL10 , and IL33 ), were targets for further qPCR analyses and Western blotting to quantify protein expression. There was no specific sensor molecule-activated inflammasome which dominated expression when stratified by mode of delivery, implying that multiple inflammasomes may function synergistically during parturition. Whilst placentae from women who had UV births overall expressed pro-inflammatory mediators, placentae from LLSCS births demonstrated a much greater pro-inflammatory response, with additional interplay of pro- and anti-inflammatory mediators. As expected, inflammasome activation was very low in placentae from women who had PLSCS births. Sex-specific differences were also detected. Placentae from male-bearing pregnancies displayed higher inflammasome activation in LLSCS compared with PLSCS, and placentae from female-bearing pregnancies displayed higher inflammasome activation in LLSCS compared with UV. In conclusion, placental inflammasome activation differs with respect to mode of delivery and neonatal sex. Its assessment may identify babies who have been exposed to aberrant inflammation at birth that may compromise their development and long-term health and wellbeing.
Publisher: Frontiers Media SA
Date: 30-10-2017
Publisher: Springer Science and Business Media LLC
Date: 15-04-2022
DOI: 10.1186/S13072-022-00442-X
Abstract: Mitochondrial DNA (mtDNA) copy number in oocytes correlates with oocyte quality and fertilisation outcome. The introduction of additional copies of mtDNA through mitochondrial supplementation of mtDNA-deficient Sus scrofa oocytes resulted in: (1) improved rates of fertilisation (2) increased mtDNA copy number in the 2-cell stage embryo and (3) improved development of the embryo to the blastocyst stage. Furthermore, a subset of genes showed changes in gene expression. However, it is still unknown if mitochondrial supplementation alters global and local DNA methylation patterns during early development. We generated a series of embryos in a model animal, Sus scrofa , by intracytoplasmic sperm injection (ICSI) and mitochondrial supplementation in combination with ICSI (mICSI). The DNA methylation status of ICSI- and mICSI-derived blastocysts was analysed by whole genome bisulfite sequencing. At a global level, the additional copies of mtDNA did not affect nuclear DNA methylation profiles of blastocysts, though over 2000 local genomic regions exhibited differential levels of DNA methylation. In terms of the imprinted genes, DNA methylation patterns were conserved in putative imprint control regions and the gene expression profile of these genes and genes involved in embryonic genome activation were not affected by mitochondrial supplementation. However, 52 genes showed significant differences in expression as demonstrated by RNAseq analysis. The affected gene networks involved haematological system development and function, tissue morphology and cell cycle. Furthermore, seven mtDNA-encoded t-RNAs were downregulated in mICSI-derived blastocysts suggesting that extra copies of mtDNA affected tRNA processing and/or turnover, hence protein synthesis in blastocysts. We also showed a potential association between differentially methylated regions and changes in expression for 55 genes due to mitochondrial supplementation. The addition of just an extra ~ 800 copies of mtDNA into oocytes can have a significant impact on both gene expression and DNA methylation profiles in Sus scrofa blastocysts by altering the epigenetic programming established during oogenesis. Some of these changes may affect specific tissue-types later in life. Consequently, it is important to determine the longitudinal effect of these molecular changes on growth and development before considering human clinical practice.
Publisher: Springer Science and Business Media LLC
Date: 24-01-2020
DOI: 10.1038/S41467-019-14224-9
Abstract: Protein-protein-interaction networks (PPINs) organize fundamental biological processes, but how oncogenic mutations impact these interactions and their functions at a network-level scale is poorly understood. Here, we analyze how a common oncogenic KRAS mutation (KRAS G13D ) affects PPIN structure and function of the Epidermal Growth Factor Receptor (EGFR) network in colorectal cancer (CRC) cells. Mapping PPIs shows that this network is extensively rewired in cells expressing transforming levels of KRAS G13D (mtKRAS). The factors driving PPIN rewiring are multifactorial including changes in protein expression and phosphorylation. Mathematical modelling also suggests that the binding dynamics of low and high affinity KRAS interactors contribute to rewiring. PPIN rewiring substantially alters the composition of protein complexes, signal flow, transcriptional regulation, and cellular phenotype. These changes are validated by targeted and global experimental analysis. Importantly, genetic alterations in the most extensively rewired PPIN nodes occur frequently in CRC and are prognostic of poor patient outcomes.
Publisher: Cold Spring Harbor Laboratory
Date: 20-07-2021
DOI: 10.1101/2021.07.19.453012
Abstract: De novo somatic point mutations identified in breast cancer are predominantly non-coding and typically attributed to altered regulatory elements such as enhancers and promoters. However, while the non-coding RNAs (ncRNAs) form a large portion of the mammalian genome, their biological functions are mostly poorly characterized in cancers. In this study, using a newly developed tool, SomaGene, we reanalyze de novo somatic point mutations from the International Cancer Genome Consortium (ICGC) whole-genome sequencing data of 1,855 breast cancers. We identify 929 candidates of ncRNAs that are significantly and explicitly mutated in breast cancer s les. By integrating data from the ENCODE regulatory features and FANTOM5 expression atlas, we show that the candidate ncRNAs in breast cancer s les significantly enrich for active chromatin histone marks (1.9 times), CTCF binding sites (2.45 times), DNase accessibility (1.76 times), HMM predicted enhancers (2.26 times) and eQTL polymorphisms (1.77 times). Importantly, we show that the 929 ncRNAs contain a much higher level (3.64 times) of breast cancer-associated genome-wide association (GWAS) single nucleotide polymorphisms (SNPs) than genome-wide expectation. Such enrichment has not been seen with GWAS SNPs from other diseases. Using breast tissue related Hi-C data we then show that 82% of our candidate ncRNAs (1.9 times) significantly interact with the promoter of protein-coding genes, including previously known cancer-associated genes, suggesting the critical role for candidate ncRNA genes in activation of essential regulators of development and differentiation in breast cancer. We provide an extensive web-based resource ( ncrna.ictic.sharif.edu ), to communicate our results with the research community. Our list of breast cancer-specific ncRNA genes has the potential to provide a better understanding of the underlying genetic causes of breast cancer. Lastly, the tool developed in this study can be used in the analysis of somatic mutations in all cancers.
Publisher: Public Library of Science (PLoS)
Date: 11-03-2019
Publisher: MDPI AG
Date: 19-01-2023
Abstract: Chromosomal rearrangements involving the KMT2A gene occur frequently in acute lymphoblastic leukaemia (ALL). KMT2A-rearranged ALL (KMT2Ar ALL) has poor long-term survival rates and is the most common ALL subtype in infants less than 1 year of age. KMT2Ar ALL frequently occurs with additional chromosomal abnormalities including disruption of the IKZF1 gene, usually by exon deletion. Typically, KMT2Ar ALL in infants is accompanied by a limited number of cooperative le-sions. Here we report a case of aggressive infant KMT2Ar ALL harbouring additional rare IKZF1 gene fusions. Comprehensive genomic and transcriptomic analyses were performed on sequential s les. This report highlights the genomic complexity of this particular disease and describes the novel gene fusions IKZF1::TUT1 and KDM2A::IKZF1.
Publisher: Cold Spring Harbor Laboratory
Date: 13-04-2020
DOI: 10.1101/2020.04.13.039131
Abstract: Transfer RNAs (tRNA) are crucial adaptor molecules between messenger RNA (mRNA) and amino acids. Recent evidence in plants suggests that dicistronic tRNA-like structures also act as mobile signals for mRNA transcripts to move between distant tissues. Co-transcription is not a common feature in the plant nuclear genome and, in the few cases where polycistronic transcripts have been found, they include non-coding RNA species such as small nucleolar RNAs and microRNAs. It is not known, however, the extent to which dicistronic transcripts of tRNA and mRNAs are expressed in field-grown plants, or the factors contributing to their expression. We analysed tRNA-mRNA dicistronic transcripts in the major horticultural crop grapevine ( Vitis vinifera ) using a novel pipeline developed to identify dicistronic transcripts from high-throughput RNA sequencing data. We identified dicistronic tRNA-mRNA in leaf and berry s les from 22 commercial vineyards. Of the 124 tRNA genes that were expressed in both tissues, 18 tRNA were expressed forming part of 19 dicistronic tRNA-mRNAs. The presence and abundance of dicistronic molecules was tissue and geographic sub-region specific. In leaves, the expression patterns of dicistronic tRNA-mRNAs significantly correlated with tRNA expression, suggesting that their transcriptional regulation might be linked. We also found evidence of syntenic genomic arrangements of tRNAs and protein coding genes between grapevine and Arabidopsis thaliana , and widespread prevalence of dicistronic tRNA-mRNA transcripts among vascular land plants but no evidence of these transcripts in nonvascular lineages. This suggests that the appearance of plant vasculature and tRNA-mRNA occurred concurrently during the evolution of land plants.
Publisher: The Royal Society
Date: 22-11-2015
Abstract: Self-fertilization (selfing) favours reproductive success when mate availability is low, but renders populations more vulnerable to environmental change by reducing genetic variability. A mixed-breeding strategy (alternating selfing and outcrossing) may allow species to balance these needs, but requires a system for regulating sexual identity. We explored the role of DNA methylation as a regulatory system for sex-ratio modulation in the mixed-mating fish Kryptolebias marmoratus. We found a significant interaction between sexual identity (male or hermaphrodite), temperature and methylation patterns when two selfing lines were exposed to different temperatures during development. We also identified several genes differentially methylated in males and hermaphrodites that represent candidates for the temperature-mediated sex regulation in K. marmoratus . We conclude that an epigenetic mechanism regulated by temperature modulates sexual identity in this selfing species, providing a potentially widespread mechanism by which environmental change may influence selfing rates. We also suggest that K. marmoratus , with naturally inbred populations, represents a good vertebrate model for epigenetic studies.
Publisher: Frontiers Media SA
Date: 15-09-2020
Publisher: Springer Science and Business Media LLC
Date: 07-09-2022
DOI: 10.1038/S41598-022-17931-4
Abstract: Panicum miliaceum L. was domesticated in northern China at least 7000 years ago and was subsequentially adopted in many areas throughout Eurasia. One such locale is Areni-1 an archaeological cave site in Southern Armenia, where vast quantities archaeobotanical material were well preserved via desiccation. The rich botanical material found at Areni-1 includes P. miliaceum grains that were identified morphologically and 14 C dated to the medieval period (873 ± 36 CE and 1118 ± 35 CE). To investigate the demographic and evolutionary history of the Areni-1 millet, we used ancient DNA extraction, hybridization capture enrichment, and high throughput sequencing to assemble three chloroplast genomes from the medieval grains and then compared these sequences to 50 modern P. miliaceum chloroplast genomes. Overall, the chloroplast genomes contained a low amount of ersity with domesticated accessions separated by a maximum of 5 SNPs and little inference on demography could be made. However, in phylogenies the chloroplast genomes separated into two clades, similar to what has been reported for nuclear DNA from P. miliaceum . The chloroplast genomes of two wild (undomesticated) accessions of P. miliaceum contained a relatively large number of variants, 11 SNPs, not found in the domesticated accessions. These results demonstrate that P. miliaceum grains from archaeological sites can preserve DNA for at least 1000 years and serve as a genetic resource to study the domestication of this cereal crop.
Publisher: Springer Science and Business Media LLC
Date: 17-08-2018
Publisher: Cold Spring Harbor Laboratory
Date: 22-08-2020
DOI: 10.1101/2020.08.19.20177873
Abstract: MicroRNAs (miRNAs) are increasingly seen as important regulators of placental development and opportunistic biomarker targets. Given the difficulty in obtaining s les from early gestation and subsequent paucity of the same, investigation of the role of miRNAs in early gestation human placenta has been limited. To address this, we generated miRNA profiles using 96 placentas from presumed normal pregnancies, across early gestation, in combination with matched profiles from maternal plasma. Placenta s les range from 6–23 weeks’ gestation, a time period that includes placenta from the early, relatively low but physiological (6–10 weeks’ gestation) oxygen environment, and later, physiologically normal oxygen environment (11–23 weeks’ gestation). We identified 637 miRNAs with expression in 86 s les (after removing poor quality s les), showing a clear gestational age gradient from 6–23 weeks’ gestation. We identified 374 differentially expressed (DE) miRNAs between placentas from 6–10 weeks’ versus 11–23 weeks’ gestation. We see a clear gestational age group bias in miRNA clusters C19MC, C14MC, miR-17∼92 and paralogs, regions that also include many DE miRNAs. Proportional change in expression of placenta-specific miRNA clusters was reflected in maternal plasma. The presumed introduction of oxygenated maternal blood into the placenta (between ∼10–12 weeks’ gestation) changes the miRNA profile of the chorionic villus, particularly in placenta-specific miRNA clusters. Data presented here comprise a clinically important reference set for studying early placenta development and may underpin the generation of minimally invasive methods for monitoring placental health.
Publisher: Frontiers Media SA
Date: 07-01-2021
DOI: 10.3389/FMICB.2020.597944
Abstract: A wines’ terroir, represented as wine traits with regional distinctiveness, is a reflection of both the biophysical and human-driven conditions in which the grapes were grown and wine made. Soil is an important factor contributing to the uniqueness of a wine produced by vines grown in specific conditions. Here, we evaluated the impact of environmental variables on the soil bacteria of 22 Barossa Valley vineyard sites based on the 16S rRNA gene hypervariable region 4. In this study, we report that both dispersal isolation by geographic distance and environmental heterogeneity (soil plant-available P content, elevation, rainfall, temperature, spacing between row and spacing between vine) contribute to microbial community dissimilarity between vineyards. Vineyards located in cooler and wetter regions showed lower beta ersity and a higher ratio of dominant taxa. Differences in soil bacterial community composition were significantly associated with differences in fruit and wine composition. Our results suggest that environmental factors affecting wine terroir, may be mediated by changes in microbial structure, thus providing a basic understanding of how growing conditions affect interactions between plants and their soil bacteria.
Publisher: Springer Science and Business Media LLC
Date: 02-2018
DOI: 10.1038/S41598-018-19655-W
Abstract: Genotyping-by-sequencing (GBS) or restriction-site associated DNA marker sequencing (RAD-seq) is a practical and cost-effective method for analysing large genomes from high ersity species. This method of sequencing, coupled with methylation-sensitive enzymes (often referred to as methylation-sensitive restriction enzyme sequencing or MRE-seq), is an effective tool to study DNA methylation in parts of the genome that are inaccessible in other sequencing techniques or are not annotated in microarray technologies. Current software tools do not fulfil all methylation-sensitive restriction sequencing assays for determining differences in DNA methylation between s les. To fill this computational need, we present msgbsR , an R package that contains tools for the analysis of methylation-sensitive restriction enzyme sequencing experiments. msgbsR can be used to identify and quantify read counts at methylated sites directly from alignment files (BAM files) and enables verification of restriction enzyme cut sites with the correct recognition sequence of the in idual enzyme. In addition, msgbsR assesses DNA methylation based on read coverage, similar to RNA sequencing experiments, rather than methylation proportion and is a useful tool in analysing differential methylation on large populations. The package is fully documented and available freely online as a Bioconductor package ( ackages/release/bioc/html/msgbsR.html ).
Publisher: MDPI AG
Date: 19-04-2022
DOI: 10.3390/IJMS23094506
Abstract: The human placenta is a rapidly developing transient organ that is key to pregnancy success. Early development of the conceptus occurs in a low oxygen environment before oxygenated maternal blood begins to flow into the placenta at ~10–12 weeks’ gestation. This process is likely to substantially affect overall placental gene expression. Transcript variability underlying gene expression has yet to be profiled. In this study, accurate transcript expression profiles were identified for 84 human placental chorionic villus tissue s les collected across 6–23 weeks’ gestation. Differential gene expression (DGE), differential transcript expression (DTE) and differential transcript usage (DTU) between 6–10 weeks’ and 11–23 weeks’ gestation groups were assessed. In total, 229 genes had significant DTE yet no significant DGE. Integration of DGE and DTE analyses found that differential expression patterns of in idual transcripts were commonly masked upon aggregation to the gene-level. Of the 611 genes that exhibited DTU, 534 had no significant DGE or DTE. The four most significant DTU genes ADAM10, VMP1, GPR126, and ASAH1, were associated with hypoxia-responsive pathways. Transcript usage is a likely regulatory mechanism in early placentation. Identification of functional roles will facilitate new insight in understanding the origins of pregnancy complications.
Publisher: MDPI AG
Date: 11-03-2022
DOI: 10.3390/IJMS23063034
Abstract: Pathogenic fungi can lose virulence after protracted periods of culture, but little is known of the underlying mechanisms. Here, we present the first analysis of DNA methylation flux at a single-base resolution for the plant pathogen B. cinerea and identify differentially methylated genes/genomic regions associated with virulence erosion during in vitro culture. Cultures were maintained for eight months, with subcultures and virulence testing every month. Methylation-sensitive lified polymorphisms were performed at monthly intervals to characterise global changes to the pathogen’s genome during culture and also on DNA from mycelium inoculated onto Arabidopsis thaliana after eight months in culture. Characterisation of culture-induced epialleles was assessed by whole-genome re-sequencing and whole-genome bisulfite sequencing. Virulence declined with time in culture and recovered after inoculation on A. thaliana. Variation detected by methylation-sensitive lified polymorphisms followed virulence changes during culture. Whole-genome (bisulfite) sequencing showed marked changes in global and local methylation during culture but no significant genetic changes. We imply that virulence is a non-essential plastic character that is at least partly modified by the changing levels of DNA methylation during culture. We hypothesise that changing DNA methylation during culture may be responsible for the high virulence/low virulence transition in B. cinerea and speculate that this may offer fresh opportunities to control pathogen virulence.
Publisher: Public Library of Science (PLoS)
Date: 17-10-2022
DOI: 10.1371/JOURNAL.PGEN.1010300
Abstract: RNA-sequencing (RNA-seq) efforts in acute lymphoblastic leukaemia (ALL) have identified numerous prognostically significant genomic alterations which can guide diagnostic risk stratification and treatment choices when detected early. However, integrating RNA-seq in a clinical setting requires rapid detection and accurate reporting of clinically relevant alterations. Here we present RaScALL, an implementation of the k-mer based variant detection tool km , capable of identifying more than 100 prognostically significant lesions observed in ALL, including gene fusions, single nucleotide variants and focal gene deletions. We compared genomic alterations detected by RaScALL and those reported by alignment-based de novo variant detection tools in a study cohort of 180 Australian patient s les. Results were validated using 100 patient s les from a published North American cohort. RaScALL demonstrated a high degree of accuracy for reporting subtype defining genomic alterations. Gene fusions, including difficult to detect fusions involving EPOR and DUX4 , were accurately identified in 98% of reported cases in the study cohort (n = 164) and 95% of s les (n = 63) in the validation cohort. Pathogenic sequence variants were correctly identified in 75% of tested s les, including all cases involving subtype defining variants PAX5 p.P80R (n = 12) and IKZF1 p.N159Y (n = 4). Intragenic IKZF1 deletions resulting in aberrant transcript isoforms were also detectable with 98% accuracy. Importantly, the median analysis time for detection of all targeted alterations averaged 22 minutes per s le, significantly shorter than standard alignment-based approaches. The application of RaScALL enables rapid identification and reporting of previously identified genomic alterations of known clinical relevance.
Publisher: Springer Science and Business Media LLC
Date: 15-05-2011
DOI: 10.1007/S00122-011-1611-Y
Abstract: Sr2 is the only known durable, race non-specific adult plant stem rust resistance gene in wheat. The Sr2 gene was shown to be tightly linked to the leaf rust resistance gene Lr27 and to powdery mildew resistance. An analysis of recombinants and mutants suggests that a single gene on chromosome arm 3BS may be responsible for resistance to these three fungal pathogens. The resistance functions of the Sr2 locus are compared and contrasted with those of the adult plant resistance gene Lr34.
Publisher: Springer Science and Business Media LLC
Date: 04-04-2023
DOI: 10.1038/S41598-023-32256-6
Abstract: Epigenetic features such as DNA accessibility dictate transcriptional regulation in a cell type- and cell state- specific manner, and mapping this in health vs. disease in clinically relevant material is opening the door to new mechanistic insights and new targets for therapy. Assay for Transposase Accessible Chromatin Sequencing (ATAC-seq) allows chromatin accessibility profiling from low cell input, making it tractable on rare cell populations, such as regulatory T (Treg) cells. However, little is known about the compatibility of the assay with cryopreserved rare cell populations. Here we demonstrate the robustness of an ATAC-seq protocol comparing primary Treg cells recovered from fresh or cryopreserved PBMC s les, in the steady state and in response to stimulation. We extend this method to explore the feasibility of conducting simultaneous quantitation of chromatin accessibility and transcriptome from a single aliquot of 50,000 cryopreserved Treg cells. Profiling of chromatin accessibility and gene expression in parallel within the same pool of cells controls for cellular heterogeneity and is particularly beneficial when constrained by limited input material. Overall, we observed a high correlation of accessibility patterns and transcription factor dynamics between fresh and cryopreserved s les. Furthermore, highly similar transcriptomic profiles were obtained from whole cells and from the supernatants recovered from ATAC-seq reactions. We highlight the feasibility of applying these techniques to profile the epigenomic landscape of cells recovered from cryopreservation biorepositories.
Publisher: Springer Science and Business Media LLC
Date: 16-07-2011
DOI: 10.1007/S10142-011-0237-0
Abstract: The assembly of a 1.3-Mb size region of the wheat genome has provided the opportunity to study a recent nuclear mitochondrial DNA insertion (NUMT). In the present study, we have studied two bacterial artificial chromosomes (BACs) and characterized a 52-kb NUMT segment from the tetraploid and hexaploid wheat BAC libraries. The conserved orthologous NUMT regions from tetraploid and hexaploid wheat Langdon and Chinese Spring shared identical gene haplotypes even though mutations (insertions, deletions, and substitutions) had occurred. The 52-kb NUMT was present in hexaploid variety Chinese Spring, but absent in variety Hope, by sequence comparison of their corresponding region. Amplifying the NUMT junctions using a set of the wheat materials including diploid, tetraploid, and hexaploid lines showed that none of the diploid wheat carried the region and only some tetraploid and hexaploid wheat were positive for the NUMT. Age estimation of the NUMT displayed the mean ages of Langdon NUMT and Chinese Spring NUMT to be 378,000 and 416,000 years ago, respectively. Reverse transcription PCR and sequencing of the nad7 gene showed 28 C → U RNA editing sites and four partial editing sites, as expected for mitochondrial DNA expression. Specific SNPs discriminated between cDNA from the nucleus and the mitochondria and suggested that the nuclear copy was not expressed. The mitochondrial DNA studied was inserted into the genome quite recently within the wheat lineage and gave rise to the non-coding nuclear nad7 gene. The NUMT segment could be lost and acquired frequently during the wheat evolution.
Publisher: Elsevier BV
Date: 11-2021
Publisher: Cold Spring Harbor Laboratory
Date: 21-10-2022
DOI: 10.1101/2022.10.20.513112
Abstract: Current HBV in vitro model systems suffer from many physiological limitations that restrict understanding of complex viral-host interactions and thus prohibit prediction of disease in vivo . We developed and assessed adult stem cell (AdSC) derived liver organoids as a novel model system for characterisation of the HBV lifecycle, the cellular response to infection and demonstrate their utility in assessing antiviral and immunomodulator response. This model system has the potential to be used in predicting in idual HBV responses to antivirals and viral reactivation in the setting of immunosuppressive agents. Ductal stem cells were isolated from healthy tissue acquired from liver resections or biopsy (n=12). Wnt3a & RSPO-1 containing medium was used to stimulate ductal stem cell expansion into organoids which were subsequently differentiated into hepatocyte-like cells. Mature hepatocyte metabolic markers (albumin, CYP3A4) and HBV entry receptor (Na-taurocholate co-transporting polypeptide, NTCP) expression were evaluated throughout differentiation using qRT-PCR and confocal microscopy. We assessed the organoids culture conditions required for HBV infection and HBV life cycle using HepAD38 (genotype D) and plasma derived HBV (genotype B & C). HBV infection was confirmed using immunofluorescence staining (HBcAg), qRT-PCR (RNA, cccDNA, extracellular DNA) and ELISA (HBsAg and HBeAg). We also assessed drug responsiveness using antivirals and an immunosuppressive agent, and cellular responses (interferon-stimulated genes) using interferon-α and viral mimic (PolyI:C). Following differentiation, organoids underwent structural remodelling and changes in cellular polarity, accompanied with an increase in albumin, CYP3A4 and NTCP mRNA expression. Optimal HBV infection was achieved in well-differentiated organoids using spinoculation of at least 200 copies/cell of AD38 derived HBV. Infected organoids demonstrate time and donor dependent increase in HBV RNA, cccDNA, extracellular DNA, HBe and HBsAg consistent with viral replication and antigen secretion. Using these markers we assessed drug-responsiveness to the HBV entry inhibitor, Myrcludex B and the JAK inhibitor, Baricitinib. Despite having a very robust interferon stimulated gene response to interferon-α and PolyI:C stimulation, HBV infection in liver organoids did not reveal innate immune activation. AdSC derived liver organoids support the full life cycle of HBV with significant donor dependent variation in viral replication and cellular responses. These features can be utilised for development of personalised drug testing platform for antivirals. Human liver organoid culture provides a personalised assessment of HBV infection, replication and responsiveness to antiviral therapy. This model system has a robust innate immune response and could be used to assess novel immune-modulating curative therapy.
Location: Australia
Location: Australia
Start Date: 2018
End Date: 2021
Funder: Cerebral Palsy Foundation
View Funded ActivityStart Date: 2021
End Date: 2023
Funder: Juvenile Diabetes Research Foundation Australia
View Funded ActivityStart Date: 2021
End Date: 2025
Funder: National Health and Medical Research Council (NHMRC)
View Funded ActivityStart Date: 2016
End Date: 2020
Funder: Eunice Kennedy Shriver National Institute of Child Health and Human Development
View Funded ActivityStart Date: 2018
End Date: 2021
Funder: Australian Research Council
View Funded ActivityStart Date: 2017
End Date: 2020
Funder: National Health and Medical Research Council
View Funded ActivityStart Date: 2013
End Date: 2015
Funder: Australian Research Council
View Funded ActivityStart Date: 2021
End Date: 2022
Funder: Australian Dental Research Foundation
View Funded ActivityStart Date: 01-2018
End Date: 12-2023
Amount: $371,347.00
Funder: Australian Research Council
View Funded Activity