Biology subjects

Nguyen, D. T.

Publications and source records attributed to Nguyen, D. T..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Identification of diagnostic KASP markers for selection of crown rust resistance in oats

Oat crown rust, caused by Puccinia coronata f. sp. avenae, poses a significant threat to global oat production (Avena sativa L.). Molecular markers are essential to assist in the integration of multiple resistance genes into a single oat cultivar to achieve genetic resistance durability. Here, we validated previously reported markers for the race-specific resistance genes Pc39, Pc45/PcKM, Pc54, and Pc68, and developed new KASP markers closely linked to these loci. These markers were subsequently screened across a collection of 150 oat cultivars. Analysis of molecular marker data, pedigree information, and disease resistance profile indicated several oat cultivars likely carrying Pc39 and Pc68. Newly identified carriers of Pc68 include the cultivars Galileo and Graza80, while Warrego, Kowari, Possum, Forester, Drummond, Hokonui, Glider, and Culgoa were identified as potential carriers of Pc39. Discrepancies between the previous postulation of Pc gene carriers and our phenotyping and genotyping analysis were also found. For example, the oat line Glider was previously postulated to carry Pc58 or Pc59, but it was positive for the Pc39-associated marker and had a similar resistance profile to Pc39 carriers. These findings underscore the importance of the utilisation of molecular markers in tracking the resistance genes in breeding germplasm. Key messageThis study has developed and validated KASP molecular markers associated with crown rust resistance genes Pc39, Pc45/PcKM, Pc54, and Pc68 that could be used for marker-assisted selection in oats.

genetics↗

bioRxiv2024

Characterisation of virulence of Puccinia coronata f. sp. avenae in Australia in the 2023 growing season

Crown rust caused by the basidiomycete fungus Puccinia coronata f. sp. avenae (Pca) results in significant crop losses worldwide. Genetic solutions to protect against this disease require disease resistance gene discovery and introduction of resistance genes into elite germplasm by breeders. To inform disease resistance breeding activities, it is paramount to monitor changes of virulence in the Pca population and link those to genotypes of the pathogen. In 2023, a collection of 37 Pca isolates from diverse regions in Australia were gathered and their infection types across a commonly used set of oat differential lines determined to assign virulence pathotypes. We compared those virulence phenotypes to data collected in previous collections. While some of the virulence phenotypes had been reported in 2022, our analysis detected new virulence or an increase in frequency for virulence on some resistance genes. Notably, some of the frequency increases in virulence were recorded in Western Australia (WA), a region of interest due to its role in oat production for milling.

plant biology↗

bioRxiv2024

The Updated Mouse Universal Genotyping Array Bioinformatic Pipeline Improves Genetic QC in Laboratory Mice

The MiniMUGA genotyping array is a popular tool for genetic QC of laboratory mice and genotyping of samples from most types of experimental crosses involving laboratory strains, particularly for reduced complexity crosses. The content of the production version of the MiniMUGA array is fixed; however, there is the opportunity to improve arrays performance and the associated reports usefulness by leveraging thousands of samples genotyped since the initial description of MiniMUGA in 2020. Here we report our efforts to update and improve marker annotation, increase the number and the reliability of the consensus genotypes for inbred strains and increase the number of constructs that can reliably be detected with MiniMUGA. In addition, we have implemented key changes in the informatics pipeline to identify and quantify the contribution of specific genetic backgrounds to the makeup of a given sample, remove arbitrary thresholds, include the Y Chromosome and mitochondrial genome in the ideogram, and improve robust detection of the presence of commercially available substrains based on diagnostic alleles. Finally, we have made changes to the layout of the report, to simplify the interpretation and completeness of the analysis and added a table summarizing the ideogram. We believe that these changes will be of general interest to the mouse research community and will be instrumental in our goal of improving the rigor and reproducibility of mouse-based biomedical research.

genetics↗

bioRxiv2024

A β-catenin:MSI2 axis regulates the expression of LEF1 and subsequent human haematopoietic stem/progenitor cell proliferation

Wnt/{beta}-catenin signaling is important for normal hematopoietic stem/progenitor cell (HSPC) biology and heavily implicated in acute myeloid leukaemia (AML). The central mediator {beta}-catenin is an attractive therapeutic target in AML however its targeting has been hampered by poor characterisation of its molecular interactions in haematopoietic cells. Our previous {beta}-catenin interactome study identified the significant enrichment of RNA-binding proteins (RBP) implying post-transcriptional roles for {beta}-catenin in myeloid cells. To identify {beta}-catenin-associated mRNAs we performed {beta}-catenin RNA-immunoprecipitation coupled to RNA-sequencing (RIP-seq) and identified significantly enriched Wnt signalling pathway transcripts. Using {beta}-catenin cross-linking immunoprecipitation (CLIP) we demonstrated a limited capacity for {beta}-catenin to bind RNA directly implying dependence on other RBPs. {beta}-Catenin was found to interact with MSI2 in both myeloid cell lines and AML patient samples, where expression was significantly correlated. MSI2 knockdown reduced Wnt signalling output (TCF/LEF activity), through suppression of LEF-1 expression and nuclear localization. Through both RIP and CLIP we demonstrate MSI2 binds LEF1 mRNA in a partly {beta}-catenin dependent fashion, and may impact the post-transcriptional control of LEF-1 expression. Finally, we show that MSI2-mediated expansion of human HSPCs could be partly driven through LEF1 regulation. This is the first study to show functional crosstalk between MSI2 and Wnt signalling in human cells, and indicates potential novel post-transcriptional roles for {beta}-catenin in a haematological context.

cell biology↗

bioRxiv2024

Genomics 2 Proteins portal: A resource and discovery tool for linking genetic screening outputs to protein sequences and structures

Recent advances in AI-based methods have revolutionized the field of structural biology. Concomitantly, high-throughput sequencing and functional genomics technologies have enabled the detection and generation of variants at an unprecedented scale. However, efficient tools and resources are needed to link these two disparate data types - to "map" variants onto protein structures, to better understand how the variation causes disease and thereby design therapeutics. Here we present the Genomics 2 Proteins Portal (G2P; g2p.broadinstitute.org/): a human proteome-wide resource that maps 19,996,443 genetic variants onto 42,413 protein sequences and 77,923 structures, with a comprehensive set of structural and functional features. Additionally, the G2P portal generalizes the capability of linking genomics to proteins beyond databases by allowing users to interactively upload protein residue-wise annotations (variants, scores, etc.) as well as the protein structure to establish the connection. The portal serves as an easy-to-use discovery tool for researchers and scientists to hypothesize the structure-function relationship between natural or synthetic variations and their molecular phenotype.

bioinformatics↗

bioRxiv2023

The HIV-1 capsid serves as a nanoscale reaction vessel for reverse transcription

The viral capsid performs critical functions during HIV-1 infection and is a validated target for antiviral therapy. Previous studies have established that the proper structure and stability of the capsid are required for efficient HIV-1 reverse transcription in target cells. Moreover, it has recently been demonstrated that permeabilized virions and purified HIV-1 cores undergo efficient reverse transcription in vitro when the capsid is stabilized by addition of the host cell metabolite inositol hexakisphosphate (IP6). However, the molecular mechanism by which the capsid promotes reverse transcription is undefined. Here we show that wild type HIV-1 particles can undergo efficient reverse transcription in vitro in the absence of a membrane-permeabilizing agent. This activity, originally termed "natural endogenous reverse transcription" (NERT), depends on expression of the viral envelope glycoprotein during virus assembly and its incorporation into virions. Truncation of the gp41 cytoplasmic tail markedly reduced NERT activity, indicating that gp41 permits the entry of nucleotides into virions. Protease treatment of virions markedly reduced NERT suggesting the presence of a proteinaceous membrane channel. By contrast to reverse transcription in permeabilized virions, NERT required neither the addition of IP6 nor a mature capsid, indicating that an intact viral membrane can substitute for the function of the viral capsid during reverse transcription in vitro. Collectively, these results demonstrate that the viral capsid functions as a nanoscale container for reverse transcription during HIV-1 infection.

microbiology↗

bioRxiv2023

MFSD7c functions as a transporter of choline at the blood-brain barrier

Mutations of MFSD7c (also known as Flvcr2), which is an orphan transporter, are linked to Fowler syndrome 1, 2. Here, we use Mfsd7c knockout mice and cell-based assays to reveal that MFSD7c is a choline transporter at the blood-brain barrier (BBB). We performed comprehensive metabolomics and detected differential changes of metabolites in the brains and livers of Mfsd7c knockout (Mfsd7c-/-) embryos. Particularly, we found that choline-related metabolites were altered in the brains but not in the livers of Mfsd7c-/- embryos. Thus, we hypothesized that MFSD7c regulates the levels of choline in the brain. Indeed, expression of human MFSD7c in cells significantly increased choline uptake. Interestingly, we showed that choline uptake by MFSD7c is greatly increased by choline-metabolizing enzymes, leading us to demonstrate that MFSD7c is a facilitative transporter of choline. Furthermore, single-cell patch-clamp showed that the import of choline by MFSD7c is electrogenic. Choline transport function of MFSD7c is conserved in vertebrates, but not in yeasts. We show that human MFSD7c is a functional ortholog of HNM1, the yeast choline importer. Employing our transport assays, we showed that several missense mutations of human MFSD7c from Fowler patients had abolished or reduced choline transport activity. Mice lacking Mfsd7c in the CNS endothelial cells suppressed the import of exogenous choline from blood but unexpectedly had increased choline levels in the brain. Stable-isotope tracing study revealed that MFSD7c is required for exporting choline derived from lysophosphatidylcholine (LPC) in the brain. Collectively, our work identifies MFSD7c as a choline transporter at the BBB. This study suggests that defective export of choline in the brain may be a cause of Fowler syndrome.

biochemistry↗

bioRxiv2023

Mfsd7b facilitates choline uptake and missense mutations affect choline transport function

MFSD7b belongs to the Major Facilitator Superfamily of transporters that transport small molecules. Two isoforms of MFSD7b have been identified and they are reported to be heme exporters that play a crucial role in maintaining the cytosolic and mitochondrial heme levels, respectively. Mutations of MFSD7b (also known as FLVCR1) have been linked to retinitis pigmentosa, posterior column ataxia, and hereditary sensory and autonomic neuropathy. Although MFSD7b functions have been linked to heme detoxification by exporting excess heme from erythroid cells, it is ubiquitously expressed with a high level in the kidney, gastrointestinal tract, lungs, liver, and brain. Here, we showed that MFSD7b functions as a facilitative choline transporter. Expression of MFSD7b slightly but significantly increased choline import, while its knockdown reduced choline influx in mammalian cells. The influx of choline transported by MFSD7b is dependent on the expression of choline metabolizing enzymes such as choline kinase (CHKA), but it is independent from gradient of cations. Additionally, we showed that choline transport function of Mfsd7b is conserved from fly to man. Employing our transport assays, we showed that missense mutations of MFSD7b caused reduced choline transport functions. Our results show that MFSD7b functions as a facilitative choline transporter in mammalian cells.

biochemistry↗

bioRxiv2023

Genotypic and resistance profile analysis of two oat crown rust differential sets urge coordination and standardisation

Puccinia coronata f. sp. avenae (Pca) is the causal agent of the disease known as crown rust which represents a bottleneck in oat production worldwide. Characterisation of pathogen populations often involves race (pathotype) assignments using differential sets, which are not uniform across countries. This study compared virulence profiles of 25 Pca isolates from Australia using two host differential sets, one from Australia and one from the USA. These differential sets were also genotyped using DArT sequencing technology. Phenotypic and genotypic discrepancies were detected on eight out of 29 common lines between the two sets, indicating that pathogen race assignments based on those lines are not comparable. To further investigate molecular markers that could assist in the stacking of rust resistance genes important for Australia, four published Pc91-linked markers were validated across the differential sets and then screened across a collection of 150 oat cultivars. Drover, Aladdin, and Volta were identified as putative carriers of the Pc91 locus. This is the first report to confirm that the cultivar Volta carries Pc91 and demonstrates the value of implementing molecular markers to characterise materials in breeding pools of oat. Overall, our findings highlight the necessity of examining seed stocks using pedigree and molecular markers to ensure seed uniformity and bring robustness to surveillance methodologies.

plant biology↗

bioRxiv2023

Virulence patterns of oat crown rust in Australia - season 2022

Puccinia coronata f. sp. avenae (Pca) is an important foliar pathogen of oat which causes crown rust disease. The virulence profile of 48 Pca isolates derived from different locations in Australia was characterised using a collection of oat lines often utilised in rust surveys in the USA and Australia. This analysis indicates that Pca populations in Eastern Australia are broadly virulent, in contrast to the population in Western Australia (WA). Several oat lines/Pc genes are effective against all rust samples collected from WA, suggesting they may provide useful resistance in this region if deployed in combination. We identified 19 lines from the USA oat differential set that display disease resistance to Pca in WA, some in agreement with previous rust survey reports. We adopted the 10-letter nomenclature system to define oat crown rust races in Australia and compare the frequency of those virulence traits to published data from the USA. Based on this nomenclature, 42 unique races were detected among the 48 isolates, reflecting the high diversity of virulence phenotypes for Pca in Australia. Nevertheless, the Pca population in the USA is substantially more broadly virulent than that of Australia. Close examination of resistance profiles for the oat differential set lines after infection with Pca supports hypotheses of allelism or redundancy among Pc genes or the presence of several resistance genes in some oat differential lines. These findings illustrate the need to deconvolute the oat differential set using molecular tools.

plant biology↗

bioRxiv2023

An integrative framework for circular RNA quantitative trait locus discovery with application in human T cells

Molecular quantitative trait locus (QTL) mapping of genetic variants with intermediate molecular phenotypes has proven to be a powerful approach for prioritizing genetic regulatory variants and causal genes identified by Genome-wide association studies (GWAS). Recently, this success has been extended to circular RNA (circRNA), a potential group of RNAs that can serve as markers for the diagnosis, prognosis, or therapeutic targets of cancer, cardiovascular, and autoimmune diseases. However, the detection of circRNA QTL (circQTL) currently is heavily reliant on a single circRNA detection algorithm for circRNA annotation and quantification which implies limitations in both sensitivity and specificity. In this study, we show that circQTL results produced by different circRNA calling tools are extremely divergent, making difficulties in interpretation. To resolve this issue, we develop an integrative method for circQTL mapping and implement it as an automated, reproducible, and scalable, and easy-to-use framework based on Nextflow, named cscQTL. Compared to the existing approach, the new method effectively identify circQTLs with an increase of 20-100% circQTLs detected and recovered all circQTLs that are highly supported by the single method approach. We apply the new method to a dataset of human T cells and discover genetic variants that control the expression of 55 circRNAs. By collocation analysis, we further identify circBACH2 and circYY1AP1 as potential candidates for immune disease regulation. cscQTL is freely available at: https://github.com/datngu/cscQTL.

bioinformatics↗

bioRxiv2023

mRNA challenge predicts brain cancer immunogenicity and response to checkpoint inhibitors

To prospectively determine whether brain tumors will respond to immune checkpoint inhibitors (ICIs), we developed a novel mRNA vaccine as a viral mimic to elucidate cytokine release from brain cancer cells in vitro. Our results indicate that cytokine signatures following mRNA challenge differ substantially from ICI responsive versus non-responsive murine tumors. These findings allow for creation of a diagnostic assay to quickly assess brain tumor immunogenicity, allowing for informed treatment with ICI or lack thereof in poorly immunogenic settings.

cancer biology↗

bioRxiv2023

Bioinformatics-Guided Discovery of Biaryl-Tailored Lasso Peptides

Lasso peptides are a class of ribosomally synthesized and post-translationally modified peptides (RiPPs) that feature an isopeptide bond and a distinct lariat fold. A growing number of secondary modifications have been described that further decorate lasso peptide scaffolds. Using genome mining, we have discovered a pair of lasso peptide biosynthetic gene clusters (BGCs) that include cytochrome P450 genes. Here, we report the structural characterization of two unique examples of (C-N) biaryl-containing lasso peptides. Nocapeptin A, from Nocardia terpenica, is tailored with Trp-Tyr crosslink while longipepetin A, from Longimycelium tulufanense, features Trp-Trp linkage. Besides the unusual bicyclic frame, longipepetin A receives an S-methylation by a new Met methyltransferase resulting in unprecedented sulfonium-bearing RiPP. Our bioinformatic survey revealed P450(s) and further maturating enzyme(s)-containing lasso BGCs awaiting future characterization.

biochemistry↗

bioRxiv2023

Three-Dimensional Bioconjugated Liquid-Like Solid (LLS) Enhance Characterization of Solid Tumor - Chimeric Antigen Receptor T cell Interactions

Cancer immunotherapy offers lifesaving treatments for cancers, but the lack of reliable preclinical models that could enable the mechanistic studies of tumor-immune interactions hampers the identification of new therapeutic strategies. We hypothesized 3D confined microchannels, formed by interstitial space between bio-conjugated liquid-like solids (LLS), enable CAR T dynamic locomotion within an immunosuppressive TME to carry out anti-tumor function. Murine CD70-specific CAR T cells cocultured with the CD70-expressing glioblastoma and osteosarcoma demonstrated efficient trafficking, infiltration, and killing of cancer cells. The anti-tumor activity was clearly captured via longterm in situ imaging and supported by upregulation of cytokines and chemokines including IFNg, CXCL9, CXCL10, CCL2, CCL3, and CCL4. Interestingly, target cancer cells, upon an immune attack, initiated an "immune escape" response by frantically invading the surrounding microenvironment. This phenomenon however was not observed for the wild-type tumor samples which remained intact and produced no relevant cytokine response. Single cells collection and transcriptomic profiling of CAR T cells at regions of interest revealed feasibility of identifying differential gene expression amongst the immune subpopulations. Complimentary 3D in vitro platforms are necessary to uncover cancer immune biology mechanisms, as emphasized by the significant roles of the TME and its heterogeneity.

bioengineering↗

bioRxiv2022

SPNS1 is required for the transport of lysosphingolipids and lysoglycerophospholipids from lysosomes

Accumulation of sphingolipids, especially sphingosines, in the lysosomes is attributed to the pathogenesis of several lysosomal storage diseases. In search for a lysosomal protein that mediates the release of sphingosines, we identified SPNS1 which shares the highest homology to SPNS2, a sphingosine-1-phosphate (S1P) transporter. We generated knockout cells and mice for Spns1 and employed lipidomics and metabolomics to identify SPNS1 ligands. We found that knockouts of Spns1 resulted in the accumulation of sphingolipids, including sphingosines in embryonic brains and cell lines. These results suggest that deficiency of SPNS1 affects the clearance of sphingolipids in lysosomes. Biochemical assays demonstrated that sphingosines released from lysosomes required SPNS1. Furthermore, by performing a comprehensive analysis of metabolites from livers of postnatal Spns1 knockout mice (gSpns1-cKO), we detected a striking accumulation of lysoglycerophospholipids including LPC, LPE, LPG, and lysoplasmalogens. Interestingly, the release of these lysoglycerophospholipids also required SPNS1. Global knockout of Spns1 (gSpns1-KO) resulted in embryonic lethality between E12.5-E13.5 with developmental defects. Postnatal deletion of Spns1 in mice caused lipid accumulation in the lysosomes and pathological conditions reminiscent of lysosomal storage diseases. These results reveal a critical molecular role of SPNS1 as a transporter for lysosphingolipids and lysoglyerophospholipids from the lysosomes and link its physiological functions with lysosomal storage diseases. SignificancePhospholipids, including glycerophospholipids and sphingolipids, are delivered to the lysosomes for recycling. The hydrolysis of these lipids by lysosomal enzymes generates the corresponding lysoglycerophospholipids, such as lysophosphatidylcholine and lysosphingolipids, such as sphingosine, which are believed to be exported out of the lysosomes for recycling in the cytoplasm. However, it is unknown how these lysophospholipids are released from the lysosomes. The current study utilized genetic knockout models in combination with mass spectrometry analysis of complex phospholipids and sphingolipids to characterize the roles of an orphan lysosomal transporter, namely SPNS1. These findings show that deficiency of SPNS1 results in the accumulation of lysophospholipids in cells and animal tissues and that the transporter is required to transport both lysoglycerophospholipids and lysosphingolipids out of the lysosomes. SPNS1 is critical for early development in mice. Ablation of SPNS1 at postnatal life causes pathological conditions reminiscent of lysosomal storage diseases in mice. These findings reveal the molecular functions of SPNS1 as a lysophospholipid transporter and provide a foundation for studying the transport of these lysolipids in lysosomal storage diseases.

biochemistry↗

bioRxiv2022

A screen for modulation of nucleocapsid protein condensation identifies small molecules with anti-coronavirus activity

Biomolecular condensates formed by liquid-liquid phase separation have been implicated in multiple diseases. Modulation of condensate dynamics by small molecules has therapeutic potential, but so far, few condensate modulators have been disclosed. The SARS-CoV-2 nucleocapsid (N) protein forms phase separated condensates that are hypothesized to play critical roles in viral replication, transcription and packaging, suggesting that N condensation modulators might have anti-coronavirus activity across multiple strains and species. Here, we show that N proteins from all seven human coronaviruses (HCoVs) vary in their tendency to undergo phase separation when expressed in human lung epithelial cells. We developed a cell-based high-content screening platform and identified small molecules that both promote and inhibit condensation of SARS-CoV-2 N. Interestingly, these host-targeted small molecules exhibited condensate-modulatory effects across all HCoV Ns. Some have also been reported to exhibit antiviral activity against SARS-CoV-2, HCoV-OC43 and HCoV-229E viral infections in cell culture. Our work reveals that the assembly dynamics of N condensates can be regulated by small molecules with therapeutic potential. Our approach allows for screening based on viral genome sequences alone and might enable rapid paths to drug discovery with value for confronting future pandemics.

molecular biology↗

bioRxiv2022

Glucose modulates transcription factor dimerization to enable tissue differentiation

Glucose is a universal energy currency for living organisms, however, its non-energetic functions in processes such as differentiation are undefined. In epidermis, differentiating cells exhibit dynamic changes in gene expression1-4 driven by specific transcription factors (TFs)5-9. The interplay between such TFs and biomolecules that also change in this process is not understood. Metabolomic analyses revealed that increased intracellular glucose accompanies differentiation of epidermal keratinocytes. This elevation also occurred in differentiating cells from other tissues and was verified in epidermal tissue engineered with glucose sensors, which detected a glucose gradient that peaked in the outermost differentiated layers. Free glucose accumulation, unaccompanied by its increased metabolism, was essential for epidermal differentiation and required GLUT1, GLUT3, and SGLT1 transporters. Glucose affinity chromatography and azido-glucose click chemistry uncovered glucose binding to diverse regulatory proteins, including the IRF6 TF, whose epidermal knockout confirmed its requirement in glucose-dependent differentiation. Direct glucose binding enabled IRF6 dimerization, DNA binding, genomic localization, and induction of IRF6 target genes, including essential pro-differentiation TFs GRHL1, GRHL3, HOPX and PRDM1. The IRF6R84C mutant found in undifferentiated cancers was unable to bind glucose. These data identify a new role for glucose as a gradient morphogen that modulates protein multimerization in cellular differentiation.

molecular biology↗

bioRxiv2022

Imputation and polygenic score performances of human genotyping arrays in diverse populations

Regardless of the overwhelming use of next-generation sequencing technologies, microarray-based genotyping combined with the imputation of untyped variants remains a cost-effective means to interrogate genetic variations across the human genome. This technology is widely used in genome-wide association studies (GWAS) at bio-bank scales, and more recently, in polygenic score (PGS) analysis to predict and to stratify disease risk. Over the last decade, human genotyping arrays have undergone a tremendous growth in both number, and content making a comprehensive evaluation of their performances became more important. Here, we performed a comprehensive performance assessment for 23 available human genotyping arrays in 6 ancestry groups using diverse public, and in-house datasets. The analyses focus on performance estimation of derived imputation (in terms of accuracy and coverage) and PGS (in term of concordance to PGS estimated from whole genome sequencing data) in three different traits and diseases. We found that the arrays with a higher number of SNPs are not necessarily the ones with higher imputation performance, but the arrays that are well-optimized for the targeted population could provide very good imputation performance. In addition, PGS estimated by imputed SNP array data is highly correlated to PGS estimated by whole genome sequencing data in most of cases. When optimal arrays are used, the correlations of key PGS metrics between two types of data can be higher than 0.97, but interestingly, arrays with high density can result in lower PGS performance. Our results suggest the importance of properly selecting a suitable genotyping array for PGS applications. Finally, we developed a web tool that provide interactive analyses of tag SNP contents and imputation performance based on population and genomic regions of interest. This study would act as a practical guide for researchers to design their genotyping arrays-based studies. The tool is available at: https://genome.vinbigdata.org/tools/saa/

genomics↗