Biology subjects

Nguyen, S.

Publications and source records attributed to Nguyen, S..

45 records · Page 3Linked to original sources

bioRxiv2019

High-dimensional analysis reveals a distinct population of adaptive-like tissue-resident NK cells in human lung

Human adaptive-like “memory” CD56dimCD16+ NK cells in peripheral blood from cytomegalovirus-seropositive individuals have been extensively investigated in recent years and are currently explored as a treatment strategy for hematological cancers. However, treatment of solid tumors remains limited due to insufficient NK cell tumor infiltration, and it is unknown whether large expansions of adaptive-like NK cells that are equipped for tissue-residency and tumor-homing exist in peripheral tissues. Here, we show that human lung and blood contains adaptive-like CD56brightCD16− NK cells with hallmarks of tissue-residency, including expression of CD49a. Expansions of adaptive-like lung tissue-resident (tr)NK cells were found to be present independently of adaptive-like CD56dimCD16+ NK cells and to be hyperresponsive towards target cells. Together, our data demonstrate that phenotypically, functionally, and developmentally distinct subsets of adaptive-like NK cells exist in human lung and blood. Given their tissue-related character and hyperresponsiveness, human lung adaptive-like trNK cells might represent a suitable alternative for therapies targeting solid tumors.Competing Interest StatementThe authors have declared no competing interest.Abbreviations:EomesEomesoderminFITCFluorescein isothiocyanateHCMVHuman cytomegalovirusILCInnate lymphoid cellKIRKiller cell immunoglobulin-like receptorNKNatural killerPEPhycoerythrinROSReactive oxygen speciesView Full Text

immunology↗

bioRxiv2019

Multiple conformations facilitate PilT function in the type IV pilus

Type IV pilus-like systems are protein complexes that polymerize a fibre of pilins. They are critical for virulence in many pathogens. Pilin polymerization and depolymerization are powered by motor PilT-like ATPases thought to possess C2 symmetry. However, most PilT-like ATPases crystallize with either C3 or C6 symmetry and the relevance of these conformations is unclear. Here we determined the X-ray structures of PilT in four unique conformations and used these structures to classify the conformation of available PilT-like ATPase structures. Single particle electron cryomicroscopy (cryoEM) structures of PilT revealed condition-dependent preferences for C2, C3, and C6 conformations. The physiologic importance of these conformations was validated by co-evolution analysis and functional studies of point mutants, identifying a rare gain-of-function mutation that favours the C2 conformation. With these data we propose a comprehensive model of PilT function with broad implications for PilT-like ATPases.

microbiology↗

bioRxiv2019

Cohesin promotes interactions across domain boundaries to regulate gene expression

The mammalian genome can be segmented into thousands of topologically associated domains (TADs) based on chromosome conformation capture studies, such as Hi-C. TADs have been proposed to act as insulated neighborhoods, spatially sequestering and insulating the enclosed genes and regulatory elements through chromatin looping and self-association. Recent results indicate that inter-TAD interactions can also occur, suggesting boundaries may be semi-permissible. However, the nature, extent, and function, if any, of these inter-TAD interactions remains unclear. Here, we combine super-and high-resolution microscopy with Oligopaint technology to precisely quantify the interaction frequency within and between neighboring domains in human cells. We find that intermingling across domain boundaries is a widespread feature of the human genome, with varying levels of interactions across different loci that correlate with their differing boundary strengths by Hi-C. Moreover, we find that cohesin depletion, which is known to abolish TADs at the population-average level, does not induce ectopic interactions but instead reduces both intra- and inter-domain interactions to a similar extent. Reduced chromatin intermixing due to cohesin loss affects domain incorporation and transcriptional bursting frequencies of genes close to architectural boundaries, potentially explaining the gene expression changes observed in the cohesinopathy Cornelia de Lange syndrome. Together, our results provide a mechanistic explanation for stochastic domain intermingling, arguing that cohesin partially bypasses boundaries to promote alternating incorporation of boundary-proximal genes into neighboring regulatory domains.

cell biology↗

bioRxiv2019

High Throughput pMHC-I Tetramer Library Production Using Chaperone Mediated Peptide Exchange

Peptide exchange technologies are essential for the generation of pMHC-multimer libraries, used to probe highly diverse, polyclonal TCR repertoires. Using the molecular chaperone TAPBPR, we present a robust method for the capture of stable, empty MHC-I molecules which can be readily tetramerized and loaded with peptides of choice in a high-throughput manner. Combined with tetramer barcoding using multi-modal cellular indexing technology (ECCITE-seq), our approach allows a combined analysis of TCR repertoires and other T-cell transcription profiles together with their cognate pMHC-I specificities in a single experiment.

immunology↗

bioRxiv2019

Oncogenic Notch promotes long-range regulatory interactions within hyperconnected 3D cliques

Chromatin loops enable transcription factor-bound distal enhancers to interact with their target promoters to regulate transcriptional programs. Although developmental transcription factors, such as active forms of Notch, can directly stimulate transcription by activating enhancers, the effect of their oncogenic subversion on the 3-dimensional (3D) organization of the cancer genome is largely undetermined. By mapping chromatin looping genome-wide in Notch-dependent triple-negative breast cancer and B-cell lymphoma, we show that far beyond the well-characterized role of Notch as an activator of distal enhancers, Notch regulates its direct target genes through establishing new long-range regulatory interactions. Moreover, a large fraction of Notch-promoted regulatory loops forms highly interacting enhancer and promoter spatial clusters, termed "3D cliques". Loss-and gain-of-function experiments show that Notch preferentially targets hyperconnected 3D cliques that regulate the expression of crucial proto-oncogenes. Our observations suggest that oncogenic hijacking of developmental transcription factors can dysregulate transcription through widespread effects on the spatial organization of cancer genomes.

cancer biology↗

bioRxiv2018

Recurrent losses and rapid evolution of the condensin II complex in insects

Condensins play a crucial role in the organization of genetic material by compacting and disentangling chromosomes. The condensin I and condensin II complexes are widely considered to have distinct functions based on studies in a few model organisms, although the specific functions of each complex are yet to be fully understood. The condensin II complex is critical for genome organization in Drosophila, and is a key anti-pairing factor that separates homologous chromosomes in somatic cells. Intriguingly, the Cap-G2 subunit of condensin II is absent in Drosophila melanogaster, and this loss may be related to the high levels of homologous chromosome pairing in somatic cells seen in flies. Here, we find that this Cap-G2 loss predates the origin of Dipterans, and other CapG2 losses have occurred independently in multiple insect lineages. Furthermore, the Cap-H2 and Cap-D3 subunits have also been repeatedly and independently lost in several insect orders, and some taxa lack condensin II-specific subunits entirely. We used Oligopaint DNA-FISH to quantify pairing levels in ten species across seven orders, representing several different configurations of the condensin II complex. We find that all non-Dipteran insects display near-uniform low pairing levels, suggesting that some key aspects of genome organization are robust to condensin II subunit losses. Finally, we observe consistent signatures of positive selection in condensin II subunits across flies and mammals. These findings suggest that these ancient complexes are far more evolutionarily labile than previously suspected, and are at the crossroads of several forms of genomic conflicts. Our results raise fundamental questions about the specific functions of the two condensin complexes and the interplay between them in taxa that have experienced subunit losses, and open the door to further investigations to elucidate the diversity of molecular mechanisms that underlie genome organization across various life forms.

evolutionary biology↗

bioRxiv2018

Single tube bead-based DNA co-barcoding for cost effective and accurate sequencing, haplotyping, and assembly

Obtaining accurate sequences from long DNA molecules is very important for genome assembly and other applications. Here we describe single tube long fragment read (stLFR), a technology that enables this a low cost. It is based on adding the same barcode sequence to sub-fragments of the original long DNA molecule (DNA co-barcoding). To achieve this efficiently, stLFR uses the surface of microbeads to create millions of miniaturized barcoding reactions in a single tube. Using a combinatorial process up to 3.6 billion unique barcode sequences were generated on beads, enabling practically non-redundant co-barcoding with 50 million barcodes per sample. Using stLFR, we demonstrate efficient unique co-barcoding of over 8 million 20-300 kb genomic DNA fragments. Analysis of the genome of the human genome NA12878 with stLFR demonstrated high quality variant calling and phasing into contigs up to N50 34 Mb. We also demonstrate detection of complex structural variants and complete diploid de novo assembly of NA12878. These analyses were all performed using single stLFR libraries and their construction did not significantly add to the time or cost of whole genome sequencing (WGS) library preparation. stLFR represents an easily automatable solution that enables high quality sequencing, phasing, SV detection, scaffolding, cost-effective diploid de novo genome assembly, and other long DNA sequencing applications.

genomics↗

bioRxiv2018

Non-antibiotic antimicrobial triclosan induces multiple antibiotic resistance through genetic mutation

Antibiotic resistance poses a major threat to public health. Overuse and misuse of antibiotics are generally recognised as the key factors contributing to antibiotic resistance. However, whether non-antibiotic, anti-microbial (NAAM) chemicals can directly induce antibiotic resistance is unclear. We aim to investigate whether the exposure to a NAAM chemical triclosan (TCS) has an impact on inducing antibiotic resistance on Escherichia coli. Here, we report that at a concentration of 0.2 mg/L TCS induces multi-drug resistance in wild-type Escherichia coli after 30-day TCS exposure. The oxidative stress induced by TCS caused genetic mutations in genes such as fabI, frdD, marR, acrR and soxR, and subsequent up-regulation of the transcription of genes encoding beta-lactamase and multi-drug efflux pump, together with down-regulation of genes related to membrane permeability. The findings advance our understanding of the potential role of NAAM chemicals in the dissemination of antibiotic resistance in microbes, and highlights the need for controlling biocide applications.

microbiology↗

bioRxiv2017

Naturally-segregating Variation at Ugt86Dd Contributes to Nicotine Resistance in Drosophila melanogaster

Identifying the causative sequence polymorphisms underlying complex trait variation is a key goal of evolutionary and biomedical genetics. By knowing the precise molecular events that confer phenotypic changes we can gain insight into the pathways underlying complex traits and the evolutionary forces acting on variation. Genetic analysis of complex traits in model systems regularly starts by constructing QTL maps, but generally fails to identify causative nucleotide-level polymorphisms. Previously we mapped a series of QTL contributing to resistance to nicotine in a Drosophila melanogaster multiparental mapping resource, and here use a battery of functional tests to resolve QTL to the molecular level. One large-effect QTL resided over a cluster of UDP-glucuronosyltransferases, and quantitative complementation tests using deficiencies eliminating subsets of these detoxification genes revealed allelic variation impacting resistance. RNAseq showed that Ugt86Dd had significantly higher expression in genotypes that are more resistant to nicotine, and anterior midgut-specific RNAi of this gene reduced resistance. We discovered a naturally-segregating 22-bp frameshift deletion in Ugt86Dd, and overexpression of the insertion-containing allele in a range of tissues enhanced resistance. Accounting for the InDel event during mapping largely eliminates the QTL, implying the InDel explains the bulk of the effect associated with the mapped locus. Finally, we edited a relatively resistant genetic background to generate lesions in Ugt86Dd that recapitulate the naturally-occurring putative loss-of-function allele, and succeeded in radically reducing resistance. The putatively causative coding InDel in Ugt86Dd can be a launchpad for future mechanistic exploration of xenobiotic detoxification.\n\nARTICLE SUMMARYResolving the mutations that contribute to among-individual trait variation is a major goal of biomedical and evolutionary genetics. In general however, genetic mapping experiments do not allow immediate resolution of the underlying causative variants. Previous mapping work revealed several loci contributing to nicotine resistance in Drosophila melanogaster. We employed a battery of functional tests to demonstrate that the detoxification gene Ugt86Dd has a major phenotypic effect, and that a segregating frameshift mutation is likely causative. Editing the gene to introduce a premature stop codon led to a significant reduction in resistance, validating its role in xenobiotic detoxification.

genetics↗