ABSTRACT
Short tandem repeat (STR) instability causes transcriptional silencing in several repeat expansion disorders. In fragile X syndrome (FXS), mutation-length expansion of a CGG STR represses FMR1 via local DNA methylation. Here, we find megabase-scale H3K9me3 domains on autosomes and encompassing FMR1 on the X chromosome in FXS patient-derived iPSCs, iPSC-derived neural progenitors, EBV-transformed lymphoblasts, and brain tissue with mutation-length CGG expansion. H3K9me3 domains connect via inter-chromosomal interactions and demarcate severe misfolding of TADs and loops. They harbor long synaptic genes replicating at the end of S phase, replication-stress-induced double-strand breaks, and STRs prone to stepwise somatic instability. CRISPR engineering of the mutation-length CGG to premutation length reverses H3K9me3 on the X chromosome and multiple autosomes, refolds TADs, and restores gene expression. H3K9me3 domains can also arise in normal-length iPSCs created with perturbations linked to genome instability, suggesting their relevance beyond FXS. Our results reveal Mb-scale heterochromatinization and trans interactions among loci susceptible to instability.
Subject(s)
Fragile X Syndrome , Humans , Fragile X Syndrome/genetics , Fragile X Syndrome/metabolism , Trinucleotide Repeat Expansion , DNA Methylation , Mutation , Fragile X Mental Retardation Protein/genetics , Fragile X Mental Retardation Protein/metabolismABSTRACT
Understanding the complexity of the long-lived HIV reservoir during antiretroviral therapy (ART) remains a considerable impediment in research towards a cure for HIV. To address this, we developed a single-cell strategy to precisely define the unperturbed peripheral blood HIV-infected memory CD4+ T cell reservoir from ART-treated people living with HIV (ART-PLWH) via the presence of integrated accessible proviral DNA in concert with epigenetic and cell surface protein profiling. We identified profound reservoir heterogeneity within and between ART-PLWH, characterized by new and known surface markers within total and individual memory CD4+ T cell subsets. We further uncovered new epigenetic profiles and transcription factor motifs enriched in HIV-infected cells that suggest infected cells with accessible provirus, irrespective of reservoir distribution, are poised for reactivation during ART treatment. Together, our findings reveal the extensive inter- and intrapersonal cellular heterogeneity of the HIV reservoir, and establish an initial multiomic atlas to develop targeted reservoir elimination strategies.
Subject(s)
HIV Infections , HIV-1 , Humans , HIV-1/physiology , CD4-Positive T-Lymphocytes , Virus Latency/genetics , HIV Infections/drug therapy , HIV Infections/genetics , Epigenesis, Genetic , Viral Load , Anti-Retroviral Agents/therapeutic useABSTRACT
Widespread changes to DNA methylation and chromatin are well documented in cancer, but the fate of higher-order chromosomal structure remains obscure. Here we integrated topological maps for colon tumors and normal colons with epigenetic, transcriptional, and imaging data to characterize alterations to chromatin loops, topologically associated domains, and large-scale compartments. We found that spatial partitioning of the open and closed genome compartments is profoundly compromised in tumors. This reorganization is accompanied by compartment-specific hypomethylation and chromatin changes. Additionally, we identify a compartment at the interface between the canonical A and B compartments that is reorganized in tumors. Remarkably, similar shifts were evident in non-malignant cells that have accumulated excess divisions. Our analyses suggest that these topological changes repress stemness and invasion programs while inducing anti-tumor immunity genes and may therefore restrain malignant progression. Our findings call into question the conventional view that tumor-associated epigenomic alterations are primarily oncogenic.
Subject(s)
Chromatin/metabolism , Chromosomes/metabolism , Colorectal Neoplasms/genetics , Colorectal Neoplasms/metabolism , DNA Methylation , Epigenesis, Genetic , Gene Expression Regulation, Neoplastic/genetics , Cell Division , Cellular Senescence/genetics , Chromatin Immunoprecipitation Sequencing , Chromosomes/genetics , Cohort Studies , Colorectal Neoplasms/mortality , Colorectal Neoplasms/pathology , Computational Biology , DNA Methylation/genetics , Epigenomics , HCT116 Cells , Humans , In Situ Hybridization, Fluorescence , Microscopy, Electron, Transmission , Molecular Dynamics Simulation , RNA-Seq , Spatial Analysis , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolismABSTRACT
Lymphocyte migration is essential for adaptive immune surveillance. However, our current understanding of this process is rudimentary, because most human studies have been restricted to immunological analyses of blood and various tissues. To address this knowledge gap, we used an integrated approach to characterize tissue-emigrant lineages in thoracic duct lymph (TDL). The most prevalent immune cells in human and non-human primate efferent lymph were T cells. Cytolytic CD8+ T cell subsets with effector-like epigenetic and transcriptional signatures were clonotypically skewed and selectively confined to the intravascular circulation, whereas non-cytolytic CD8+ T cell subsets with stem-like epigenetic and transcriptional signatures predominated in tissues and TDL. Moreover, these anatomically distinct gene expression profiles were recapitulated within individual clonotypes, suggesting parallel differentiation programs independent of the expressed antigen receptor. Our collective dataset provides an atlas of the migratory immune system and defines the nature of tissue-emigrant CD8+ T cells that recirculate via TDL.
Subject(s)
CD8-Positive T-Lymphocytes/cytology , CD8-Positive T-Lymphocytes/immunology , Animals , Cell Differentiation , Clone Cells , Cytotoxicity, Immunologic , Epigenesis, Genetic , Humans , Immunologic Memory , Lymph Nodes/cytology , Lymph Nodes/immunology , Macaca mulatta , T-Lymphocyte Subsets/immunology , Transcription, Genetic , Transcriptome/geneticsABSTRACT
The high mobility group (HMG) transcription factor TCF-1 is essential for early T cell development. Although in vitro biochemical assays suggest that HMG proteins can serve as architectural elements in the assembly of higher-order nuclear organization, the contribution of TCF-1 on the control of three-dimensional (3D) genome structures during T cell development remains unknown. Here, we investigated the role of TCF-1 in 3D genome reconfiguration. Using gain- and loss-of-function experiments, we discovered that the co-occupancy of TCF-1 and the architectural protein CTCF altered the structure of topologically associating domains in T cell progenitors, leading to interactions between previously insulated regulatory elements and target genes at late stages of T cell development. The TCF-1-dependent gain in long-range interactions was linked to deposition of active enhancer mark H3K27ac and recruitment of the cohesin-loading factor NIPBL at active enhancers. These data indicate that TCF-1 has a role in controlling global genome organization during T cell development.
Subject(s)
Chromatin , Enhancer Elements, Genetic , CCCTC-Binding Factor/genetics , CCCTC-Binding Factor/metabolism , Cell Cycle Proteins/metabolism , Enhancer Elements, Genetic/genetics , Gene Expression Regulation , T-Lymphocytes/metabolismABSTRACT
Immunological priming-in the context of either prior infection or vaccination-elicits protective responses against subsequent Mycobacterium tuberculosis (Mtb) infection. However, the changes that occur in the lung cellular milieu post-primary Mtb infection and their contributions to protection upon reinfection remain poorly understood. Using clinical and microbiological endpoints in a non-human primate reinfection model, we demonstrated that prior Mtb infection elicited a long-lasting protective response against subsequent Mtb exposure and was CD4+ T cell dependent. By analyzing data from primary infection, reinfection, and reinfection-CD4+ T cell-depleted granulomas, we found that the presence of CD4+ T cells during reinfection resulted in a less inflammatory lung milieu characterized by reprogrammed CD8+ T cells, reduced neutrophilia, and blunted type 1 immune signaling among myeloid cells. These results open avenues for developing vaccines and therapeutics that not only target lymphocytes but also modulate innate immune cells to limit tuberculosis (TB) disease.
ABSTRACT
Genome organization can regulate gene expression and promote cell fate transitions. The differentiation of germline stem cells (GSCs) to oocytes in Drosophila involves changes in genome organization mediated by heterochromatin and the nuclear pore complex (NPC). Heterochromatin represses germ cell genes during differentiation, and NPCs anchor these silenced genes to the nuclear periphery, maintaining silencing to allow for oocyte development. Surprisingly, we found that genome organization also contributes to NPC formation, mediated by the transcription factor Stonewall (Stwl). As GSCs differentiate, Stwl accumulates at boundaries between silenced and active gene compartments. Stwl at these boundaries plays a pivotal role in transitioning germ cell genes into a silenced state and activating a group of oocyte genes and nucleoporins (Nups). The upregulation of these Nups during differentiation is crucial for NPC formation and further genome organization. Thus, cross-talk between genome architecture and NPCs is essential for successful cell fate transitions.
Subject(s)
Cell Differentiation , Drosophila Proteins , Genome, Insect , Nuclear Pore , Oogenesis , Animals , Oogenesis/genetics , Drosophila Proteins/metabolism , Drosophila Proteins/genetics , Cell Differentiation/genetics , Nuclear Pore/metabolism , Nuclear Pore/genetics , Genome, Insect/genetics , Gene Expression Regulation, Developmental/genetics , Female , Drosophila melanogaster/genetics , Oocytes/metabolism , Transcription Factors/metabolism , Transcription Factors/genetics , Drosophila/genetics , Nuclear Pore Complex Proteins/metabolism , Nuclear Pore Complex Proteins/geneticsABSTRACT
The association of genomic loci to the nuclear periphery is proposed to facilitate cell type-specific gene repression and influence cell fate decisions. However, the interplay between gene position and expression remains incompletely understood, in part because the proteins that position genomic loci at the nuclear periphery remain unidentified. Here, we used an Oligopaint-based HiDRO screen targeting â¼1000 genes to discover novel regulators of nuclear architecture in Drosophila cells. We identified the heterochromatin-associated protein Stonewall (Stwl) as a factor promoting perinuclear chromatin positioning. In female germline stem cells (GSCs), Stwl binds and positions chromatin loci, including GSC differentiation genes, at the nuclear periphery. Strikingly, Stwl-dependent perinuclear positioning is associated with transcriptional repression, highlighting a likely mechanism for Stwl's known role in GSC maintenance and ovary homeostasis. Thus, our study identifies perinuclear anchors in Drosophila and demonstrates the importance of gene repression at the nuclear periphery for cell fate.
Subject(s)
Cell Differentiation , Cell Nucleus , Chromatin , Drosophila Proteins , Animals , Drosophila Proteins/metabolism , Drosophila Proteins/genetics , Chromatin/metabolism , Chromatin/genetics , Cell Nucleus/metabolism , Cell Nucleus/genetics , Female , Cell Differentiation/genetics , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Stem Cells/metabolism , Stem Cells/cytology , Gene Expression Regulation, Developmental/genetics , Drosophila/genetics , Germ Cells/metabolismABSTRACT
The human genome functions as a three-dimensional chromatin polymer, driven by a complex collection of chromosome interactions1-3. Although the molecular rules governing these interactions are being quickly elucidated, relatively few proteins regulating this process have been identified. Here, to address this gap, we developed high-throughput DNA or RNA labelling with optimized Oligopaints (HiDRO)-an automated imaging pipeline that enables the quantitative measurement of chromatin interactions in single cells across thousands of samples. By screening the human druggable genome, we identified more than 300 factors that influence genome folding during interphase. Among these, 43 genes were validated as either increasing or decreasing interactions between topologically associating domains. Our findings show that genetic or chemical inhibition of the ubiquitous kinase GSK3A leads to increased long-range chromatin looping interactions in a genome-wide and cohesin-dependent manner. These results demonstrate the importance of GSK3A signalling in nuclear architecture and the use of HiDRO for identifying mechanisms of spatial genome organization.
Subject(s)
Chromatin , Chromosome Positioning , Chromosomes, Human , Genome, Human , Glycogen Synthase Kinases , High-Throughput Screening Assays , Single-Cell Analysis , Humans , Chromatin/drug effects , Chromatin/genetics , Chromatin/metabolism , Chromosome Positioning/drug effects , Chromosomes, Human/drug effects , Chromosomes, Human/genetics , Chromosomes, Human/metabolism , DNA/analysis , DNA/metabolism , Genome, Human/drug effects , Genome, Human/genetics , Glycogen Synthase Kinases/antagonists & inhibitors , Glycogen Synthase Kinases/deficiency , Glycogen Synthase Kinases/genetics , High-Throughput Screening Assays/methods , Interphase , Reproducibility of Results , RNA/analysis , RNA/metabolism , Signal Transduction/drug effects , Single-Cell Analysis/methods , CohesinsABSTRACT
The majority of oncogenic drivers are intracellular proteins, constraining their immunotherapeutic targeting to mutated peptides (neoantigens) presented by individual human leukocyte antigen (HLA) allotypes1. However, most cancers have a modest mutational burden that is insufficient for generating responses using neoantigen-based therapies2,3. Neuroblastoma is a paediatric cancer that harbours few mutations and is instead driven by epigenetically deregulated transcriptional networks4. Here we show that the neuroblastoma immunopeptidome is enriched with peptides derived from proteins essential for tumorigenesis. We focused on targeting the unmutated peptide QYNPIRTTF discovered on HLA-A*24:02, which is derived from the neuroblastoma-dependency gene and master transcriptional regulator PHOX2B. To target QYNPIRTTF, we developed peptide-centric chimeric antigen receptors (PC-CARs) through a counter panning strategy using predicted potentially cross-reactive peptides. We further proposed that PC-CARs can recognize peptides on additional HLA allotypes when presenting a similar overall molecular surface. Informed by our computational modelling results, we show that PHOX2B PC-CARs also recognize QYNPIRTTF presented by HLA-A*23:01, the most common non-A2 allele in people with African ancestry. Finally, we demonstrate potent and specific killing of neuroblastoma cells expressing these HLAs in vitro and complete tumour regression in mice. These data suggest that PC-CARs have the potential to expand the pool of immunotherapeutic targets to include non-immunogenic intracellular oncoproteins and allow targeting through additional HLA allotypes in a clinical setting.
Subject(s)
Antigens, Neoplasm , Neuroblastoma , Oncogene Proteins , Peptides , Receptors, Chimeric Antigen , Animals , Humans , Mice , Africa/ethnology , Alleles , Amino Acid Sequence , Carcinogenesis , Cross Reactions , HLA-A Antigens/chemistry , HLA-A Antigens/immunology , Neuroblastoma/genetics , Neuroblastoma/immunology , Neuroblastoma/therapy , Oncogene Proteins/antagonists & inhibitors , Oncogene Proteins/immunology , Peptides/antagonists & inhibitors , Peptides/chemistry , Peptides/immunology , Receptors, Chimeric Antigen/immunology , Receptors, Chimeric Antigen/therapeutic useABSTRACT
Nuclear speckles are prominent nuclear bodies that contain proteins and RNA involved in gene expression. Although links between nuclear speckles and gene activation are emerging, the mechanisms regulating association of genes with speckles are unclear. We find that speckle association of p53 target genes is driven by the p53 transcription factor. Focusing on p21, a key p53 target, we demonstrate that speckle association boosts expression by elevating nascent RNA amounts. p53-regulated speckle association did not depend on p53 transactivation functions but required an intact proline-rich domain and direct DNA binding, providing mechanisms within p53 for regulating gene-speckle association. Beyond p21, a substantial subset of p53 targets have p53-regulated speckle association. Strikingly, speckle-associating p53 targets are more robustly activated and occupy a distinct niche of p53 biology compared with non-speckle-associating p53 targets. Together, our findings illuminate regulated speckle association as a mechanism used by a transcription factor to boost gene expression.
Subject(s)
Cell Nucleus/genetics , Gene Expression Regulation/genetics , Nuclear Proteins/genetics , RNA/genetics , Transcriptional Activation/genetics , Tumor Suppressor Protein p53/genetics , DNA/genetics , HEK293 Cells , Humans , Intranuclear Inclusion Bodies/genetics , Protein Binding/genetics , Transcription Factors/genetics , Transcription, Genetic/geneticsABSTRACT
Interactions between the genome and the nuclear pore complex (NPC) have been implicated in multiple gene regulatory processes, but the underlying logic of these interactions remains poorly defined. Here, we report high-resolution chromatin binding maps of two core components of the NPC, Nup107 and Nup93, in Drosophila cells. Our investigation uncovered differential binding of these NPC subunits, where Nup107 preferentially targets active genes while Nup93 associates primarily with Polycomb-silenced regions. Comparison to Lamin-associated domains (LADs) revealed that NPC binding sites can be found within LADs, demonstrating a linear binding of the genome along the nuclear envelope. Importantly, we identified a functional role of Nup93 in silencing of Polycomb target genes and in spatial folding of Polycomb domains. Our findings lend to a model where different nuclear pores bind different types of chromatin via interactions with specific NPC sub-complexes, and a subset of Polycomb domains is stabilized by interactions with Nup93.
Subject(s)
Chromatin/metabolism , Nuclear Pore/metabolism , Polycomb-Group Proteins/metabolism , Animals , Aquaporins/metabolism , Binding Sites/physiology , Cell Line , Drosophila/metabolism , Drosophila Proteins/metabolism , Female , Gene Expression Regulation/physiology , Genome/physiology , Male , Nuclear Envelope/metabolism , Nuclear Pore Complex Proteins/metabolismABSTRACT
The majority of oncogenic drivers are intracellular proteins, thus constraining their immunotherapeutic targeting to mutated peptides (neoantigens) presented by individual human leukocyte antigen (HLA) allotypes1. However, most cancers have a modest mutational burden that is insufficient to generate responses using neoantigen-based therapies2,3. Neuroblastoma is a paediatric cancer that harbours few mutations and is instead driven by epigenetically deregulated transcriptional networks4. Here we show that the neuroblastoma immunopeptidome is enriched with peptides derived from proteins that are essential for tumourigenesis and focus on targeting the unmutated peptide QYNPIRTTF, discovered on HLA-A*24:02, which is derived from the neuroblastoma dependency gene and master transcriptional regulator PHOX2B. To target QYNPIRTTF, we developed peptide-centric chimeric antigen receptors (CARs) using a counter-panning strategy with predicted potentially cross-reactive peptides. We further hypothesized that peptide-centric CARs could recognize peptides on additional HLA allotypes when presented in a similar manner. Informed by computational modelling, we showed that PHOX2B peptide-centric CARs also recognize QYNPIRTTF presented by HLA-A*23:01 and the highly divergent HLA-B*14:02. Finally, we demonstrated potent and specific killing of neuroblastoma cells expressing these HLAs in vitro and complete tumour regression in mice. These data suggest that peptide-centric CARs have the potential to vastly expand the pool of immunotherapeutic targets to include non-immunogenic intracellular oncoproteins and widen the population of patients who would benefit from such therapy by breaking conventional HLA restriction.
Subject(s)
Antigens, Neoplasm/immunology , HLA Antigens/immunology , Immunotherapy , Neoplasms/immunology , Neoplasms/therapy , Oncogene Proteins/immunology , Receptors, Chimeric Antigen/immunology , Animals , Antigens, Neoplasm/metabolism , Cell Line , Cell Line, Tumor , Cross Reactions , Cross-Priming , Female , HLA Antigens/metabolism , Homeodomain Proteins/immunology , Homeodomain Proteins/metabolism , Humans , Interferon-gamma/immunology , Mice , Neoplasms/metabolism , Oncogene Proteins/antagonists & inhibitors , Oncogene Proteins/metabolism , T-Lymphocytes/immunology , Transcription Factors/immunology , Transcription Factors/metabolismABSTRACT
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 3D organization of cancer genomes is largely undetermined. By mapping chromatin looping genome-wide in Notch-dependent triple-negative breast cancer and B cell lymphoma, we show that beyond the well-characterized role of Notch as an activator of distal enhancers, Notch regulates its direct target genes by instructing enhancer repositioning. Moreover, a large fraction of Notch-instructed regulatory loops form 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.
Subject(s)
Cell Transformation, Neoplastic/genetics , Chromatin/genetics , Lymphoma, B-Cell/genetics , Oncogenes , Receptors, Notch/genetics , Triple Negative Breast Neoplasms/genetics , Binding Sites , Cell Lineage/genetics , Cell Proliferation/genetics , Cell Transformation, Neoplastic/metabolism , Cell Transformation, Neoplastic/pathology , Chromatin/metabolism , Chromatin Assembly and Disassembly , Cyclin D1/genetics , Cyclin D1/metabolism , Enhancer Elements, Genetic , Gene Expression Regulation, Neoplastic , Gene Regulatory Networks , HEK293 Cells , Humans , Lymphoma, B-Cell/metabolism , Lymphoma, B-Cell/pathology , Mutation , Nucleic Acid Conformation , Promoter Regions, Genetic , Protein Binding , Proto-Oncogene Proteins c-myc/genetics , Proto-Oncogene Proteins c-myc/metabolism , Receptors, Notch/metabolism , Signal Transduction/genetics , Triple Negative Breast Neoplasms/metabolism , Triple Negative Breast Neoplasms/pathologyABSTRACT
Whole genome sequencing has increasingly become the essential method for studying the genetic mechanisms of antimicrobial resistance and for surveillance of drug-resistant bacterial pathogens. The majority of bacterial genomes sequenced to date have been sequenced with Illumina sequencing technology, owing to its high-throughput, excellent sequence accuracy, and low cost. However, because of the short-read nature of the technology, these assemblies are fragmented into large numbers of contigs, hindering the obtaining of full information of the genome. We develop Pasa, a graph-based algorithm that utilizes the pangenome graph and the assembly graph information to improve scaffolding quality. By leveraging the population information of the bacteria species, Pasa is able to utilize the linkage information of the gene families of the species to resolve the contig graph of the assembly. We show that our method outperforms the current state of the arts in terms of accuracy, and at the same time, is computationally efficient to be applied to a large number of existing draft assemblies.
Subject(s)
Algorithms , Bacteria , Genome, Bacterial , Bacteria/classification , Bacteria/genetics , High-Throughput Nucleotide Sequencing/methods , Sequence Analysis, DNA/methodsABSTRACT
The relationship between cohesin-mediated chromatin looping and gene expression remains unclear. NIPBL and WAPL are two opposing regulators of cohesin activity; depletion of either is associated with changes in both chromatin folding and transcription across a wide range of cell types. However, a direct comparison of their individual and combined effects on gene expression in the same cell type is lacking. We find that NIPBL or WAPL depletion in human HCT116 cells each alter the expression of ~2,000 genes, with only ~30% of the genes shared between the conditions. We find that clusters of differentially expressed genes within the same topologically associated domain (TAD) show coordinated misexpression, suggesting some genomic domains are especially sensitive to both more or less cohesin. Finally, co-depletion of NIPBL and WAPL restores the majority of gene misexpression as compared to either knockdown alone. A similar set of NIPBL-sensitive genes are rescued following CTCF co-depletion. Together, this indicates that altered transcription due to reduced cohesin activity can be functionally offset by removal of either its negative regulator (WAPL) or the physical barriers (CTCF) that restrict loop-extrusion events.
Subject(s)
Cell Cycle Proteins , Chromatin , Chromosomal Proteins, Non-Histone , Gene Expression Regulation , Humans , CCCTC-Binding Factor/genetics , CCCTC-Binding Factor/metabolism , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Chromatin/genetics , Genes, cdc , Genome , HCT116 Cells , CohesinsABSTRACT
We have developed AMRViz, a toolkit for analyzing, visualizing, and managing bacterial genomics samples. The toolkit is bundled with the current best practice analysis pipeline allowing researchers to perform comprehensive analysis of a collection of samples directly from raw sequencing data with a single command line. The analysis results in a report showing the genome structure, genome annotations, antibiotic resistance and virulence profile for each sample. The pan-genome of all samples of the collection is analyzed to identify core- and accessory-genes. Phylogenies of the whole genome as well as all gene clusters are also generated. The toolkit provides a web-based visualization dashboard allowing researchers to interactively examine various aspects of the analysis results. Availability: AMRViz is implemented in Python and NodeJS, and is publicly available under open source MIT license at https://github.com/amromics/amrviz .
Subject(s)
Genome, Bacterial , Genomics , Software , Genomics/methods , Drug Resistance, Bacterial/genetics , Phylogeny , Bacteria/genetics , Bacteria/drug effects , Anti-Bacterial Agents/pharmacologyABSTRACT
Whole genome analysis for microbial genomics is critical to studying and monitoring antimicrobial resistance strains. The exponential growth of microbial sequencing data necessitates a fast and scalable computational pipeline to generate the desired outputs in a timely and cost-effective manner. Recent methods have been implemented to integrate individual genomes into large collections of specific bacterial populations and are widely employed for systematic genomic surveillance. However, they do not scale well when the population expands and turnaround time remains the main issue for this type of analysis. Here, we introduce AMRomics, an optimized microbial genomics pipeline that can work efficiently with big datasets. We use different bacterial data collections to compare AMRomics against competitive tools and show that our pipeline can generate similar results of interest but with better performance. The software is open source and is publicly available at https://github.com/amromics/amromics under an MIT license.
Subject(s)
Genome, Bacterial , Genomics , Software , Workflow , Genomics/methods , Computational Biology/methods , Bacteria/genetics , Genome, Microbial , Drug Resistance, Bacterial/geneticsABSTRACT
Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare but dangerous side effect of adenoviral-vectored COVID-19 vaccines. VITT had been linked to production of autoantibodies recognizing platelet factor 4 (PF4). Here, we characterize anti-PF4 antibodies obtained from a VITT patient's blood. Intact mass measurements indicate that a significant fraction of these antibodies represent a limited number of clones. MS analysis of large antibody fragments (the light chain and the Fc/2 and Fd fragments of the heavy chain) confirms the monoclonal nature of this component of the anti-PF4 antibodies repertoire and reveals the presence of a mature complex biantennary N-glycan within the Fd segment. Peptide mapping using two complementary proteases and LC-MS/MS was used to determine the amino acid sequence of the entire light chain and over 98% of the heavy chain (excluding a short N-terminal segment). The sequence analysis allows the monoclonal antibody to be assigned to the IgG2 subclass and verifies that the light chain belongs to the λ-type. Incorporation of enzymatic de-N-glycosylation into the peptide mapping routine allows the N-glycan in the Fab region of the antibody to be localized to the framework 3 region of the VH domain. This novel N-glycosylation site is the result of a single mutation within the germline sequence. Peptide mapping also provides information on lower-abundance (polyclonal) components of the anti-PF4 antibody ensemble, revealing the presence of all four subclasses (IgG1-IgG4) and both types of the light chain (λ and κ). This case study demonstrates the power of combining the intact, middle-down, and bottom-up MS approaches for meaningful characterization of ultralow quantities of pathogenic antibodies extracted directly from patients' blood.
Subject(s)
Platelet Factor 4 , Humans , Platelet Factor 4/immunology , Platelet Factor 4/chemistry , COVID-19 Vaccines/adverse effects , COVID-19 Vaccines/immunology , COVID-19 Vaccines/chemistry , Autoantibodies/immunology , Autoantibodies/blood , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/chemistry , Amino Acid Sequence , Purpura, Thrombotic Thrombocytopenic/chemically induced , Purpura, Thrombotic Thrombocytopenic/immunologyABSTRACT
The Szeto-Schiller (SS) peptides are a subclass of cell-penetrating peptides that can specifically target mitochondria and mediate conditions caused by mitochondrial dysfunction. In this work, we constructed an iron-chelating SS peptide and studied its interaction with a mitochondrial-mimicking membrane using atomistic molecular dynamics (MD) simulations. We report that the peptide/membrane interaction is thermodynamically favorable, and the localization of the peptide to the membrane is driven by electrostatic interactions between the cationic residues and the anionic phospholipid headgroups. The insertion of the peptide into the membrane is driven by hydrophobic interactions between the aromatic side chains in the peptide and the lipid acyl tails. We also probed the translocation of the peptide across the membrane by applying nonequilibrium steered MD simulations and resolved the translocation pathway, free energy profile, and metastable states. We explored four distinct orientations of the peptide along the translocation pathway and found that one orientation was energetically more favorable than the other orientations. We tested a significantly slower pulling velocity on the most thermodynamically favorable system and compared metastable states during peptide translocation. We found that the peptide can optimize hydrophobic interactions with the membrane by having aromatic side chains interacting with the lipid acyl tails instead of forming π-π interactions with each other. The mechanistic insights emerging from our work will potentially facilitate improved peptide design with enhanced activity.