Biology subjects

Broach, J. R.

Publications and source records attributed to Broach, J. R..

9 recordsLinked to original sources

bioRxiv2026

Removing an S-layer enables endolysin-derived probes to detect Paenibacillus thiaminolyticus in mixed bacterial populations

The emerging neonatal bacterial pathogen Paenibacillus thiaminolyticus causes sepsis and postinfectious hydrocephalus, yet few tools are available to study or specifically detect it. We developed fluorescent probes based on cell wall-binding domains (CBDs) from bacteriophage-derived cell-wall hydrolases. CBDs can target structurally conserved cell-wall glycans, making them attractive affinity reagents for bacterial detection. None of the 11 probes we designed labeled intact cells under standard conditions, and we identified a surface layer (S-layer) that prevented access to their cell-wall targets. Brief acidic treatment (pH 2.0) released abundant high-molecular-mass proteins from the cell surface and enabled labeling by five distinct CBD probes, each containing a single C-terminal S-layer homology domain. Mass spectrometry identified the main released protein as an ortholog of the P. alvei SpaA S-layer protein, which we designate SlsA. Across 26 surveyed P. thiaminolyticus genomes, we identified four distinct SlsA variants with 58-79% pairwise amino acid identity, encoded within a conserved cluster homologous to the P. alvei S-layer-associated locus. After either the acidic treatment or a quick flame fixation, the lead probe mGL-BDPt9 labeled six P. thiaminolyticus strains, including three Ugandan clinical isolates, as well as a P. dendritiformis clinical isolate from a US infant, and three additional Paenibacillus species. mGL-BDPt9 also distinguished P. thiaminolyticus from five gram-positive sepsis pathogens in pairwise mixtures and detected it in a five-species suspension. Together, these findings establish the presence of an S-layer in P. thiaminolyticus and provide a rapid fluorescent labeling strategy for multiple Paenibacillus pathogens associated with neonatal paenibacilliosis.

microbiology↗

bioRxiv2024

Genome Instability Precedes Viral Integration in Human Papilloma Virus Transformed Tonsillar Keratinocytes

Approximately 70% of oropharyngeal squamous carcinomas (OPSCC) are associated with human papillomavirus (HPV). While HPV-positive (HPV+) OPSCC responds better to standard therapies and patients with HPV+ tumor generally have better outcomes than those with HPV-negative (HPV-) tumors, a subset of HPV+ patients do have poor outcomes. Our previous work suggested that tumors with integrated virus exhibit significantly greater genome wide genomic instability than those with only episomal viral genomes and patients with HPV+ OPSCC with episomal viral genomes had better outcomes. To explore the causal relation between viral integration and genomic instability, we have examined the time course of viral integration and genetic instability in tonsillar keratinocytes transformed with HPV16. HPV-infected human tonsil keratinocyte cell lines were continuously passaged and every fifth passage some cells were retained for genomic analysis. Whole genome sequencing and optical genomic mapping confirmed that virus integrated in five of six cell lines while remaining episomal in the sixth. In all lines genome instability occurred during early passages, but essentially ceased following viral integration but continued to occur later passages in the episomal line. To test tumorigenicity of the cell lines, cells were injected subcutaneously into the flanks of nude mice. A cell line with the integrated virus induced tumors following injection in the nude mouse while that with the episomal virus did not. We conclude that genomic instability is not the result of viral integration but likely promotes integration. Moreover, those transformants with episomal virus appear to be less tumorigenic than those with integrated virus.

cancer biology↗

bioRxiv2022

Type IV pili is a critical virulence factor in clinical isolates of Paenibacillus thiaminolyticus

Hydrocephalus, the leading indication for childhood neurosurgery worldwide, is particularly prevalent in low-and-middle-income countries (LMICs). Hydrocephalus preceded by an infection, or postinfectious hydrocephalus (PIH), accounts for up to 60% of hydrocephalus in LMICs. Since many children with hydrocephalus suffer poor long-term outcomes despite surgical intervention, prevention of hydrocephalus remains paramount. Our previous studies implicated a novel bacterial pathogen, Paenibacillus thiaminolyticus, as a contributor to PIH in Uganda. Here we report the isolation of three P. thiaminolyticus strains, Mbale, Mbale2, and Mbale3, from patients with PIH and the demonstration that the three clinical isolates exhibit virulence in mice while P. thiaminolyticus type strain, B-4156, does not. We constructed complete genome assemblies of the clinical isolates as well as the reference strain and performed comparative genomics and proteomics analyses to identify potential virulence factors. One candidate virulence factor is a cluster of genes carried on a mobile genetic element that encodes a type IV pilus and is present in all three PIH patient strains but absent in the type strain. Proteomic and transcriptomic data confirmed the expression of this cluster of genes in the Mbale strain, while CRISPR-mediated deletion of the gene cluster substantially reduced the virulence of this strain. Our comparative proteogenomic analysis also identified various antibiotic resistance loci in the virulent strains. These results provide insight into the mechanism of virulence of Paenibacillus thiaminolyticus and suggest avenues for the diagnosis and treatment of this novel bacterial pathogen. Author SummaryPostinfectious hydrocephalus (PIH), a devastating sequela of neonatal infection, is associated with increased childhood mortality and morbidity. Paenibacillus thiaminolyticus was recently identified as the dominant organism highly associated with PIH in an African cohort. Our whole-genome sequencing, RNA sequencing and proteomics of three clinical isolates and a type strain in combination with CRISPR editing has revealed the type IV pili (T4P), encoded in a mobile genetic element, as a critical virulence factor for P. thiaminolyticus infection. Given the widespread presence of T4P in pathogens, the presence of T4P operon could serve as an important diagnostic and therapeutic target in P. thiaminolyticus and related bacteria.

microbiology↗

bioRxiv2020

Vaginal Microbiome Topic Modelling of Laboring Ugandan Women With and Without Fever

The composition of the maternal vaginal microbiome may influence the duration of pregnancy, onset of labor and even neonatal outcomes. Maternal microbiome research in sub Saharan-Africa has focused on non-pregnant and postpartum composition of the vaginal microbiome. We examined the vaginal microbiome composition of 99 laboring Ugandan women using routine microbiology and 16S ribosomal DNA sequencing from two hypervariable regions (V1-V2 and V3-V4), using standard hierarchical methods. We then introduce Grades of Membership (GoM) modeling for the vaginal microbiome, a method often used in the text mining machine learning literature. Leveraging GoM models, we create a basis composed of a small number of microbial topics whose linear combination optimally represents each patient yielding more accurate associations. We identified relationships between defined communities and the presentation or absence of intrapartum fever. Using a random forest model we showed that by including novel microbial topic models we improved upon clinical variables to predict maternal fever. We also show by integrating clinical variables with a microbial topic model into this model found young maternal age, fever report earlier in the current pregnancy, and longer labors, as well as a more diverse, less Lactobacillus dominated microbiome were features of labor associated with intrapartum fever. These results better define relationships between presentation or absence of intrapartum fever, demographics, peripartum course, and vaginal microbial communities, and improve our understanding of the impact of the microbiome on maternal and neonatal infection risk.

genomics↗

bioRxiv2019

An integrative ENCODE resource for cancer genomics

ENCODE comprises thousands of functional genomics datasets, and the encyclopedia covers hundreds of cell types, providing a universal annotation for genome interpretation. However, for particular applications, it may be advantageous to use a customized annotation. Here, we develop such a custom annotation by leveraging advanced assays, such as eCLIP, Hi-C, and whole-genome STARR-seq on a number of data-rich ENCODE cell types. A key aspect of this annotation is comprehensive and experimentally derived networks of both transcription factors and RNA-binding proteins (TFs and RBPs). Cancer, a disease of system-wide dysregulation, is an ideal application for such a network-based annotation. Specifically, for cancer-associated cell types, we put regulators into hierarchies and measure their network change (rewiring) during oncogenesis. We also extensively survey TF-RBP crosstalk, highlighting how SUB1, a previously uncharacterized RBP, drives aberrant tumor expression and amplifies the effect of MYC, a well-known oncogenic TF. Furthermore, we show how our annotation allows us to place oncogenic transformations in the context of a broad cell space; here, many normal-to-tumor transitions move towards a stem-like state, while oncogene knockdowns show an opposing trend. Finally, we organize the resource into a coherent workflow to prioritize key elements and variants, in addition to regulators. We showcase the application of this prioritization to somatic burdening, cancer differential expression and GWAS. Targeted validations of the prioritized regulators, elements and variants using siRNA knockdowns, CRISPR-based editing, and luciferase assays demonstrate the value of the ENCODE resource.

genomics↗

bioRxiv2019

An Integrated Framework for Genome Analysis Reveals Numerous Previously Unrecognizable Structural Variants in Leukemia Patients' Samples

While genomic analysis of tumors has stimulated major advances in cancer diagnosis, prognosis and treatment, current methods fail to identify a large fraction of somatic structural variants in tumors. We have applied a combination of whole genome sequencing and optical genome mapping to a number of adult and pediatric leukemia samples, which revealed in each of these samples a large number of structural variants not recognizable by current tools of genomic analyses. We developed computational methods to determine which of those variants likely arose as somatic mutations. The method identified 97% of the structural variants previously reported by karyotype analysis of these samples and revealed an additional fivefold more such somatic rearrangements. The method identified on average tens of previously unrecognizable inversions and duplications and hundreds of previously unrecognizable insertions and deletions. These structural variants recurrently affected a number of leukemia associated genes as well as cancer driver genes not previously associated with leukemia and genes not previously associated with cancer. A number of variants only affected intergenic regions but caused cis-acting alterations in expression of neighboring genes. Analysis of TCGA data indicates that the status of several of the recurrently mutated genes identified in this study significantly affect survival of AML patients. Our results suggest that current genomic analysis methods fail to identify a majority of structural variants in leukemia samples and this lacunae may hamper diagnostic and prognostic efforts.

genomics↗

bioRxiv2017

Inhibition of the Platelet-activating factor receptor for the treatment of Amyotrophic Lateral Sclerosis?

Cerebrospinal Fluids (CSF) of Amyotrophic Lateral Sclerosis (ALS) patients have increased levels of the inflammatory cytokine IL-18. Because IL-18 is produced by dendritic cells stimulated by the Platelet-activating factor (PAF), a major neuroinflammatory mediator, it is expected that PAF is involved in ALS. Pilot experimental data on amplification of PAF receptor (PAFR) mRNA by RT-PCR show that PAFR is overexpressed, as compared to age matched controls, in the spinal cords of transgenic ALS mouse model SOD1-G93A, suggesting PAF mediation. Although anti-inflammatory drugs have been tested for ALS before, no clinical trial has been conducted using PAFR specific inhibitors. Therefore, we hypothesize that administration of PAFR inhibitors, such as Ginkgolide B, PCA 4248 and WEB 2086, have potential to function as a novel therapy for ALS, particularly in SOD1 familial ALS forms. Because currently there are only two approved drugs with modest effectiveness for ALS therapy, a search for novel drugs and targets is essential.

neuroscience↗

bioRxiv2017

Differential distribution of Neandertal genomic signatures in human mitochondrial haplogroups

Genetic contributions of Neanderthals to the modern human genome have been evidenced by comparison of present-day human genomes with paleogenomes suggesting that the Neanderthal introgression is higher in Asians and Europeans and lower in Africans. Neanderthal signatures in extant human genomes are attributed to intercrosses between Neanderthals and archaic Anatomically Modern Humans (AMH). Although Neanderthal signatures are well documented in the nuclear genome, it has been proposed that there is no contribution of Neanderthal mitochondrial DNA to contemporary human genomes. Here we show that modern human mitochondrial genomes contain potential 66 Neanderthal signatures, or Neanderthal single nucleotide variants (N-SNVs) being 36 in coding regions of which 7 are nonsynonymous. Also, 7 N-SNVs are associated with traits such as cycling vomiting syndrome, Alzheimers disease, Parkinsons disease and 2 N-SNVs are associated with intelligence quotient. Based on recombination tests, Principal Component Analysis (PCA) and the complete absence of these N-SNVs in 41 archaic AMH mitogenomes we conclude that convergent evolution due to homoplasy and not recombination, explains the presence of N-SNVs in present-day human mitogenomes.

genomics↗

bioRxiv2017

An Integrative Framework For Detecting Structural Variations In Cancer Genomes

Structural variants can contribute to oncogenesis through a variety of mechanisms, yet, despite their importance, the identification of structural variants in cancer genomes remains challenging. Here, we present an integrative framework for comprehensively identifying structural variation in cancer genomes. For the first time, we apply next-generation optical mapping, high-throughput chromosome conformation capture (Hi-C), and whole genome sequencing to systematically detect SVs in a variety of cancer cells.\n\nUsing this approach, we identify and characterize structural variants in up to 29 commonly used normal and cancer cell lines. We find that each method has unique strengths in identifying different classes of structural variants and at different scales, suggesting that integrative approaches are likely the only way to comprehensively identify structural variants in the genome. Studying the impact of the structural variants in cancer cell lines, we identify widespread structural variation events affecting the functions of non-coding sequences in the genome, including the deletion of distal regulatory sequences, alteration of DNA replication timing, and the creation of novel 3D chromatin structural domains.\n\nThese results underscore the importance of comprehensive structural variant identification and indicate that non-coding structural variation may be an underappreciated mutational process in cancer genomes.

genomics↗