Biology subjects

Rhodes, J.

Publications and source records attributed to Rhodes, J..

9 recordsLinked to original sources

bioRxiv2026

Temozolomide Induces Aberrant RNA Alkylation and Widespread Translational Repression

Temozolomide (TMZ) is a frontline alkylating chemotherapy, yet its direct impact on RNA modification and global translation dynamics remains poorly understood. Here, we demonstrate that TMZ induces pervasive RNA alkylation causing severe translational impairment. TMZ directly deposits aberrant methyl groups onto single-stranded mRNA in vitro, creating physical lesions that lower translational efficiency. In glioblastoma cells, acute TMZ exposure triggers a rapid, widespread accumulation of m7G on cellular RNAs, leading to the significant attenuation of global protein synthesis. Nanopore direct RNA sequencing identified distinct guanine-specific error signatures and sequence context preferences associated with TMZ-induced damage. Using a quantitative yeast spike-in ribosome profiling strategy, we mapped this translational repression at transcript-level, revealing a global downregulation of translational efficiency. This widespread repression disproportionately targets highly interconnected networks essential for cellular proliferation, specifically chromosome organization. We show that the severity of this translational repression is driven by a transcript's coding guanine density, stability and translation initiation speed. Together, our findings suggest that TMZ-induced alkylation targets stable, highly translated, guanine-rich transcripts. This establishes aberrant RNA methylation and subsequent translational arrest as a potential mechanism of temozolomide cytotoxicity.

biochemistry↗

bioRxiv2018

Transcriptional heterogeneity of Cryptococcus gattii VGII compared with non-VGII lineages underpins key pathogenicity pathways

Cryptococcus gattii is a pathogenic yeast of humans and other animals, which causes disease predominantly in immunocompetent hosts. Infection begins when aerosolized yeast or spores enter the body, triggering an immune response, including engulfment by macrophages. To understand the early transcriptional signals in both the yeast and its mammalian host, we performed a time-course dual RNA-seq experiment for four lineages of C. gattii (VGI-IV) interacting with mouse macrophages at 1hr, 3hr and 6hr post infection. Comparison of in vitro to ex vivo gene expression indicates lineage VGII is transcriptionally divergent to non-VGII lineages, including differential expression of genes involved in capsule synthesis, capsule attachment and ergosterol production. Various paralogs demonstrate sub-functionalisation between lineages including an upregulation of capsule biosynthesis-related gene CAP2, and downregulation of CAP1 in VGIII. Isolates also compensate for lineage-specific gene-losses by over-expression of genetically similar paralogs, including an over-expression of capsule gene CAS3 in VGIV having lost CAS31. Differential expression of one in five C. gattii genes was detected following co-incubation with mouse macrophages; all isolates showed high induction of oxidative-reduction functions and a downregulation of capsule attachment genes. We also show that VGII switches expression of two laccase paralogs (from LAC1 to LAC2) during co-incubation of macrophages. Finally, we found that mouse macrophages respond to all four lineages of C. gattii by upregulating FosB/Jun/Egr1 regulatory proteins at early time points. This study highlights the evolutionary breadth of expression profiles amongst the lineages of C. gattii and the diversity of transcriptional responses at this host-pathogen interface.\n\nImportanceThe transcriptional profiles of related pathogens and their response to host induced stresses underpin their pathogenicity. Expression differences between related pathogens during host interaction can indicate when and how these genes contribute to virulence, ultimately informing new and improved treatment strategies for those diseases. In this paper, we compare the transcriptional profiles of five isolates representing four lineages of C. gattii in rich media. Our analyses identified key processes including cell capsule, ergosterol production and melanin that are differentially expressed between lineages, and we find that VGII has the most distinct profile in terms of numbers of differentially expressed genes. All lineages have also undergone sub-functionalisation for various paralogs including capsule biosynthesis and attachment genes. Most genes appeared down-regulated during co-incubation with macrophages, with the largest decrease observed for capsule attachment genes, which appears coordinated with a stress response, as all lineages also upregulated oxidative stress response genes. Furthermore, VGII upregulated many genes that are linked to ergosterol biosynthesis and switched expression of the laccase LAC1 to LAC2 ex vivo. Finally, we saw a pronounced increase in the FosB/Jun/Egr1 regulatory proteins at early time points in bone marrow derived macrophages, marking a role in the host response to C. gattii. This work highlights the dynamic roles of key C. gattii virulence genes in response to macrophages.

genomics↗

bioRxiv2017

Rapid genome sequencing for outbreak analysis of the emerging human fungal pathogen Candida auris

BackgroundCandida auris was first described in 2009, and has since caused nosocomial outbreaks, invasive infections and fungaemia across 11 countries in five continents. An outbreak of C. auris occurred in a specialised cardiothoracic London hospital between April 2015 and November 2016, which to date has been the largest outbreak reported worldwide, involving a total of 72 patients.\n\nMethodsTo understand the epidemiology of C. auris infection within this hospital, we sequenced the genomes of outbreak isolates using Oxford Nanopore Technologies and Illumina in order to type antifungal resistance alleles and to explore the outbreak within its local and global context.\n\nFindingsPhylogenomic analysis placed the UK outbreak in the India/Pakistan clade, demonstrating an Asian origin. The outbreak showed similar diversity to that of the entire clade and limited local spatiotemporal clustering was observed. One isolate displayed resistance to both echinocandins and 5-flucytosine; the former was associated with a serine to tyrosine amino acid substitution in the gene FKS1, and the latter was associated with a phenylalanine to isoleucine substitution in the gene FUR1. These mutations are novel for this pathogen.\n\nInterpretationMultiple differential episodic selection of antifungal resistant genotypes has occurred within a genetically heterogenous population across this outbreak, creating a resilient pathogen and making it difficult to define local-scale patterns of transmission as well as implementing outbreak control measures.\n\nFundingAntimicrobial Research Collaborative, Imperial College London

epidemiology↗

bioRxiv2017

Scc2/Nipbl Hops Between Chromosomal Cohesin Rings After Loading

The cohesin complex mediates DNA-DNA interactions both between (sister chromatid cohesion) and within chromosomes (DNA looping) via a process thought to involve entrapment of DNAs within its tripartite ring. It has been suggested that intra- chromosome loops are generated through processive extrusion of DNAs through the lumen of cohesins ring. Scc2 (Nipbl) is essential for loading cohesin onto chromosomes but not for maintaining sister chromatid cohesion following DNA replication. It has therefore been assumed that Scc2 is involved exclusively in the cohesin loading process. However, it is possible that the stimulation of cohesins ABC-like ATPase by Scc2 also has a post-loading function, for example driving loop extrusion. Using fluorescence recovery after photobleaching (FRAP) and single-molecule tracking, we show that Scc2 binds dynamically to chromatin, principally through an association with cohesin. Scc2s movement within chromatin is consistent with a \"stop-and-go\" or \"hopping\" motion. We suggest that a low diffusion coefficient, a low stoichiometry relative to cohesin, and a high affinity for chromosomal cohesin enables Scc2 to move rapidly from one chromosomal cohesin complex to another, performing a function distinct from loading.

cell biology↗

bioRxiv2017

Population Genomics Of Cryptococcus neoformans var. grubii Reveals New Biogeographic Relationships And Finely Maps Hybridization

Cryptococcus neoformans var. grubii is the causative agent of cryptococcal meningitis, a significant source of mortality in immunocompromised individuals, typically HIV/AIDS patients from developing countries. Despite the worldwide emergence of this ubiquitous infection, little is known about the global molecular epidemiology of this fungal pathogen. Here we sequence the genomes of 188 diverse isolates and characterized the major subdivisions, their relative diversity and the level of genetic exchange between them. While most isolates of C. neoformans var. grubii belong to one of three major lineages (VNI, VNII, and VNB), some haploid isolates show hybrid ancestry including some that appear to have recently interbred, based on the detection of large blocks of each ancestry across each chromosome. Many isolates display evidence of aneuploidy, which was detected for all chromosomes. In diploid isolates of C. neoformans var. grubii (serotype A/A) and of hybrids with C. neoformans var. neoformans (serotype A/D) such aneuploidies have resulted in loss of heterozygosity, where a chromosomal region is represented by the genotype of only one parental isolate. Phylogenetic and population genomic analyses of isolates from Brazil revealed that the previously African VNB lineage occurs naturally in the South American environment. This suggests migration of the VNB lineage between Africa and South America prior to its diversification, supported by finding ancestral recombination events between isolates from different lineages and regions. The results provide evidence of substantial population structure, with all lineages showing multi-continental distributions demonstrating the highly dispersive nature of this pathogen.\n\nAuthor SummaryCryptococcus neoformans var. grubii is a human fungal pathogen of immunocompromised individuals that has global clinical impact, causing half a million deaths per year. Substantial genetic substructure exists for this pathogen, with two lineages found globally (VNI, VNII) whereas a third has appeared confined to sub-Saharan Africa (VNB). Here, we utilized genome sequencing of a large set of global isolates to examine the genetic diversity, hybridization, and biogeography of these lineages. We found that while the three major lineages are well separated, recombination between the lineages has occurred, notably resulting in hybrid isolates with segmented ancestry across the genome. In addition, we showed that isolates from South America are placed within the VNB lineage, formerly thought to be confined to Africa, and that there is phylogenetic separation between these geographies that substantially expands the diversity of these lineages. Our findings provide a new framework for further studies of the dynamics of natural populations of C. neoformans var. grubii.

genomics↗

bioRxiv2017

Cohesin Can Remain Associated With Chromosomes During DNA Replication

To ensure disjunction to opposite poles during anaphase, sister chromatids must be held together following DNA replication. This is mediated by cohesin, which is thought to entrap sister DNAs inside a tripartite ring composed of its Smc and kleisin (Scc1) subunits. How such structures are created during S phase is poorly understood, in particular whether they are derived from complexes that had entrapped DNAs prior to replication. To address this, we used selective photobleaching to determine whether cohesin associated with chromatin in G1 persists in situ after replication. We used unlabelled HaloTag ligand following fluorescent labelling to block newly synthesised Halo-tagged Scc1 protein from incorporating fluorescent dye (pulse-chase or pcFRAP). In cells whose cohesin turnover is inactivated by deletion of WAPL, Scc1 remains associated with chromatin throughout S phase. These findings suggest that cohesion might be generated by cohesin that is already bound to unreplicated DNA.

cell biology↗

bioRxiv2016

Architecture and dynamics of the jasmonic acid gene regulatory network

The phytohormone jasmonic acid (JA) is a critical regulator of plant growth and defense. To significantly advance our understanding of the architecture and dynamics of the JA gene regulatory network, we performed high-resolution RNA-Seq time series analyses of methyl JA-treated Arabidopsis thaliana. Computational analysis unraveled in detail the chronology of events that occur during the early and later phases of the JA response. Several transcription factors, including ERF16 and bHLH27, were uncovered as early components of the JA gene regulatory network with a role in pathogen and insect resistance. Moreover, analysis of subnetworks surrounding the JA-induced transcription factors ORA47, RAP2.6L, and ANAC055 provided novel insights into their regulatory role of defined JA network modules. Collectively, our work illuminates the complexity of the JA gene regulatory network, pinpoints to novel regulators, and provides a valuable resource for future studies on the function of JA signaling components in plant defense and development.

plant biology↗

bioRxiv2016

Simulations of CYP51A from Aspergillus fumigatus in a model bilayer provide insights into triazole drug resistance

1Azole antifungal drugs target CYP51A in Aspergillus fumigatus by binding with the active site of the protein, blocking ergosterol biosynthesis. Resistance to azole anti-fungal drugs is now common, with a leucine to histidine amino acid substitution at position 98 the most frequent, conferring resistance to itraconazole. In this study, we create a homology model of CYP51A using a recently published crystal structure of the paralog protein CYP51B. The derived structures, wild-type and L98H mutant, are positioned within a lipid membrane bilayer and subjected to molecular dynamics simulations in order improve the accuracy of both models. The structural analysis from our simulations suggests a decrease in active site surface from the formation of hydrogen bonds between the histidine substitution and neighbouring polar side chains, potentially preventing the binding of azole drugs. This study yields a biologically relevant structure and set dynamics of the A. fumigatus Lanosterol 14 alpha-demethylase enzyme and provides further insight into azole antifungal drug resistance.

biochemistry↗

bioRxiv2016

A population genomics approach to assessing the genetic basis of within-host microevolution underlying recurrent cryptococcal meningitis infection

Recurrence of meningitis due to Cryptococcus neoformans after treatment causes substantial mortality in HIV/AIDS patients across sub-Saharan Africa. In order to determine whether recurrence occurred due to relapse of the original infecting isolate or reinfection with a different isolate weeks or months after initial treatment, we used whole-genome sequencing to assess the genetic basis of infection in 17 HIV-infected individuals with recurrent cryptococcal meningitis. Comparisons revealed a clonal relationship for 15 pairs of isolates recovered before and after recurrence showing relapse of the original infection. The two remaining pairs showed high levels of genetic heterogeneity; in one pair we found this to be a result of infection by mixed genotypes, whilst the second was a result of nonsense mutations in the gene encoding the DNA mismatch repair proteins MSH2, MSH5 and RAD5. These nonsense mutations led to a hypermutator state, leading to dramatically elevated rates of synonymous and non-synonymous substitutions. Hypermutator phenotypes owing to nonsense mutations in these genes have not previously been reported in Cryptococcus neoformans and represent a novel pathway for rapid within-host adaptation and evolution of resistance to firstline antifungal drugs.

genomics↗